NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 291978

Publi8laed bll

THE NATIONAL INSTITUTE OF GENETICS
Mi8ima, Sizaoia·ken, Jallan
1979Annual Reportof theNational Institute of Genetics
No. 29, 1978

<Genetic Stocks Center)

Published by

The National Institute of Genetics, Japan
1979

CONTENTS
General statement
Staff ..
Council..............................................
Association for propagation of the knowledge of genetics
Projects of research for 1978
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Researches carried out in 1978
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Molecular Genetics
Interaction between bases involved in the 5'-terminal cap structure of eukaryotic messenger RNA. HATTORI, M. and
MIURA, K.............................................
Structure near the 5'-terminal of some eukaryotic mRNAs.
SHIMOTOHNO, K., HIDAKA, S. and MIURA, K.. . . . . . . . . . . . . .
The primary sequence of the late region of polyoma virus RNA:the expression of the late genes and comparison with DNA
sequences ofSV40 and BKV. SOEDA, E.. 0.......
A simple method for the identification of altered subunits in mutant
RNA polymerases of Escherichia coli. SUGIURA, M., ITO, N.
and SUZUKI, M
Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases
61-77 from Escherichia coli tRNAfMet. OHTSUKA, E.,NISHIKAWA, So, MARKHAM, A. F., TANAKA, S., MIYAKE,
T., WAKABAYASHI, T., IKEHARA, M. and SUGIURA, M
0"Cloning and characterization of tobacco chloroplast 23S and 16S
rRNA genes. SUGIURA, M. and KUSUDA, J. . . . . . . . . . . . . . . .
Gene controlling DNA initiation in Eschrichia coli: dna A + andsuppresser genes of dna A-. TAKEDA, Y. and HIROTA, Y.. .
0II.

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Microbial Genetics
Properties of penicillin binding protein (PBP) la, Ib and 3.
TAMURA, T., SUZUKI, H., NISHIMURA, Y. and HIROTA, Y.
Further genetical study of flagellar mutants in E. coli K-12.
KOMEDA, Y
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Expression of the hag gene in E. coli K-12: analysis of hag-lac
gene fusion. KOMEDA, Y.

III.

Biochemical Genetics and Immunogenetics
Electrophoretic and heat-stability variations of serum esterase(Es-l) isozymes in wild mice (Mus). AOTSUKA, T. and
MORIWAKI, K.
Genetic control of esterase in Japanese species of Trilium. IHARA,
M. and ENDo, T.
Maternal effect embryo lethal mutant in Drosophila melanogaster.
YAMADA, M. A. and NAwA, S.

IV.

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Developmental and Somatic Cell Genetics
Growth rate and cell cycle lengths of hydra. TAKANO, J., FUJISAWA, T. and SUGIYAMA, Too...
..
A mutant hydra strain (sf-I) containing temperature sensitive
interstitial cells. MARCUM, B. A., FUJISAWA, T. and SUGIYAMA, T.
Comparison of effective lethal phases of embryos from threecomplementation groups of the rudimentary of Drosophilamelanogaster. KURODA, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of conditioned medium on chondrogenesis of quail limb-bud
mesenchymal cells in culture. KURODA, Y. and MATSUTANI,
E.
Chondrogenesis of quail limb-bud mesenchymal cells in clonal
culture. MATSUTANI, E. and KURODA, Y............. ....
Induction of 8-azaguanine resistant mutations by sterigmatocystinin cultured embryonic human diploid cells. KURODA,
Y....................................................
Mutagenic activity of tryptophan pyrolysis products on embryonichuman diploid cells in culture. KURODA, Y... . . . . .. . . .. ..
Genetic study on highly teratocarcinogenic charactor of 129/ter-Sv
mice. NOGUCHI, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A multipotential teratocarcinoma (STT-2) derived from a male of
129Jter-Sv strain. NOGUCHI, T. and WADA, T.
Lethal hybrid rescue gene of Drosophila simulans. WATANABA,
T.K.

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CONTENTS

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v. Cytogenetics
The third scientific expedition for the study of rodents to islands
and coastal area of the Indian Ocean, I. Members, aims,
schedule and number of animals collected in the expedition.
YOSIDA, T. H.........................................
The third scientific expedition for the study of rodents to islands
and coastal area of the Indian Ocean, II. Distribution of Ceylonese type black rats. YOSIDA, T. H., MORlwAKI, K., TsuCHIYA, K., KATO, H., SABARATNAM, M., ARULPRAGASAM,
K. D. and FERNANDO, H. E.
The third scientific expedition for the study of rodents to islands
and coastal area of the Indian Ocean, III. Finding ofMauritius black rats with extra small acrocentrics due toRobertsonian fission. YOSIDA, T. H., MORlwAKI, K.,
KATO, H., TSUCHIYA, K., OCHIAI, Y. and MONTY, J..
The third scientific expedition for the study of rodents to islands
and coastal area of the Indian Ocean, IV. Karyotype of
black rats collected in Penang (Malaysia), Seychelles, andNairobi (Kenya). YOSIDA, T. H., KATO, H., TSUCHIYA, K.,
MORIWAKI, K., MENG, W. T., CHARLES, S. K., GILL, G. andHIRUMI, H.
The third scientific expedition for the study of rodents to islands
and coastal area of the Indian Ocean, V. Hemoglobin
types and chromosome C-band patterns of the mice collected
in Sri Lanka, Seychelles island and Mauritius island.
MORIWAKI, K., TSUCHIYA, K., KATO, H., YOSIDA, T. H.,
and MINEZAWA, M.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A new sporadic Robertsonian fusion of acrocentric pairs no. 7
and 10 in the black rats from Sapporo. YOSIDA, T. H.. . .. . .
Histological study on the hybrid embryos developed by artificial
insemination between Norway rats and black rats. TAYA,
C. and YOSIDA, T. H.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vivo sister chromatid exchange in cells of various organs of
the mouse. KANDA, N. and KATO, H.. . . . . . . . . . . . . . . . . . . .
Analysis of crossing over in the mouse meiotic cells by BrdU
labeling technique. KANDA, N. and KATO, H.
Comparison of karyotypes between the Japanese shrew mole

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

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and the furry snouted shrew mole. -HAMADA, T. and YOSIDA,
T. H.
Karyotypes of four filefishes. MUROFUSHI, M. and YOSIDA,
T.H-.........................................
Differential staining of inactive X chromosome in 25 rodent
species. KANDA, N. and YOSIDA, T. H...................
Comparative analysis of Japanese wood mice from Oki Islands
and mainland from view points of biochemical- and cytogenetics. HIRAI, H., UCHIDA, T. A. and MORIWAKI, K.........
An attempt on the cytological identification of chromosome insome linkage groups of the silkworm. MURAKAMI, A.,
OHNUMA, A. and IMAI, H. T.............
Mechanisms on the sterility in triploid silkworm. MURAKAMI,
A., OHNUMA, A. and IMAI, H. T.......
VI.

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Mutation and Mutagenesis in Animals
Studies on differential mutation response between Bombyx and
Drosophila to some indirectly acting chemical carcinogens.
(2) In vitro metabolic activity test of Drosophila microsomesfor N-dimethylnitrosamine (DMN) and N-acetyl-2-aminofluorene (AAF). KASTURI BAI, A. R., GOTO, M. and TAZIMA,Y.
Mutagenicity of dimethylbenzanthracene in the silkworm germcells. MURAKAMI, A., GOTO, M. and FUKASE, Y.
The mutagenic activity of mycotoxins in the silkworm. MURAKAMI, A., OZAWA, T. and FUKASE, Y.......
Genetic effects of tranquilizers on silkworm germ-cells. MURAKAMI, A., OZAWA, T. and TAZIMA, Y.
Induction of mutations in hamster embryonic cells from mothers
received total body irradiation of r-ray. KADA, T., ASANO,
T., YOKOIYAMA, A. and INUI, N.
Studies on the natural antimutagens in the environment. KADA,
T., OHTA, Y., HARA, M., YOKOIYAMA, A. and INOUE, T. . . . .

VII.

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Radiation Genetics and Chemical Mutagenesis in Micro-organisms
and Plants
Induction of mutation and phage by synchrotron orbital radiation

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CONTENTSv

in Bacillus subtilis spores. KADA, T. and OHTA, Y... .. . . . .
A desmutagenic factor from plant (Brassica oleracea) for mutagenic principle of tryptophan pyrolysate has peroxidase and
NADPH-oxidase activities. INOUE, T. and KADA, T.. . . . . .
Further characterization of a Bacillus subtilis endonuclease specific for apurinic sites in DNA. INOUE, T. and KADA, T.. . . . . .
Phenotypic expression of waxy starch mutants in cereals.
AMANa, E.............................................
Dose response of maize strains to chronic gamma-ray irradiation.
AMANa, E. and UKAI, Y.................................
Induction of waxy mutants in rice by reactor radiations. AMANa,E.
Test of environmental mutagens with a soybean strain, T-2l9.
FUJII, T...
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VIII.

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Population Genetics (Theoretical)

Change of gene frequencies by natural selection under population number regulation. KIMURA, M.. . . . . . . . . . . . . . . . . . . .
Stepwise mutation model and distribution of allelic frequencies
in a finite population. KIMURA, M. and OHTA, T.. . . . . . . . . .
Effect of overall phenotypic selection on genetic change at individual loci. KIMURA, M. and CROW, J. F.. . . . . . . . . . . . . . . .
Theoretical study of genetic variability, assuming stepwise production of neutral and very slightly deleterious mutations.
MARUYAMA, T. and KIMURA, M.........................
Sequence variability of immunoglobulins considered from thestandpoint of population genetics. OHTA, T... .. . . . . . . . . . .
Theoretical study on genetic variation in multigene families.
OHTA, T.
Polymorphism and loss of duplicate gene expression: A theoretical study with application to tetraploid fish. TAKAHATA,
N. and MARUYAMA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IX.

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Population Genetics (Experimental)
Seasonal change of inversion frequencies in a Mishima population of Drosophila melanogaster. INOUE, Y....... . . . . . . . .
Further genetic studies of the Katsunuma population of Drosophila melanogaster. WATANABE, T. K. and INOUE, Y... . . . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Evolutionary Genetics
Phylogeny of Drosophila based on the mating preference.
WATANABE, T. K. and KAWANISHI, M.....................

XI.

XII.

Human Genetics
Host resistance to the gene for retinoblastoma. MATSUNAGA,
E.
Recurrence risks to relatives of patients with retinoblastoma.
MATSUNAGA, E.
Quantitative analysis of C-positive variants in man. AZUMI,J., NAKAGOME, Y. and MATSUNAGA, E... ..
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Behavioral Genetics
Genetical analysis of noise sensItIvItIes of homozygous strainsof Drosophila melanogaster. OSHIMA, C. and TAKAMURA,T.
Actual activity at dusk and dawn is not needed for entrainmentof circadian activity rhythm of Drosophila melanogaster
under following continuous darkness. TAKAMURA, T. andOSHIMA, C.
Visual system mutants and light-dark cycle entrainment in activity
rhythm of Drosophila melanogaster. TAKAMURA, T. andOSHIMA, C.
Oviposition behavior of Drosophila melanogaster: Ethogramand hybrid analysis. TAKAMURA, T. and OSHIMA, C. . . . . . . . .
Different photo-preferences in Drosophila simulans and D.
melanogaster. KAWANISHI, M. and WATANABE, T. K... ....

XIII. Ecological Genetics
An observation of wild rice species in tropical Australia. OKA,
H. I.
Niche differentiation between two wild rice species observed in
Nigeria. MORISHIMA, H. and OKA, H. I.. . . . . . . . . . . . . . . . . .
Genetic diversity in rice populations of Nigeria: Influence ofcommunity structure. MORISHIMA, H. and OKA, H. I. . . . . . .
Variation studies in African wild and cultivated rice species.
SANO, Y. and MORISHIMA, H.

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Competition and copper tolerance in two sedges, Cyperus difformis
and Fimbristylis miliacea. MORISHIMA, H.. . . . . . .. . . .. . . ..
Neighborhood effects between rice and barnyard grass strains,
I. Performance in mixture and aggressiveness as influenced
by planting density. ASSEMAT, L. and OKA, H. I.. . .. .. . . . .
Neighborhood effects between rice and barnyard grass strains,
II. Experiments on the mechanisms of interaction between
plants. ASSEMAT, L. and MORISHIMA, H.. . . . . . . . . . . . . . . . . .
Increasing frequency of Drosophila simulans in Yamanashi-ken.
WATANABE, T. K. and KAWANISHI, M.....................
Different food preferences in Drosophila simulans and D. melanogaster. KAWANISHI, M. and LEE W. H...................

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XIV. Applied Genetics
The sporophytic pollen sterility in rice: Its genetic basis andintervarietal relationships as shown by F 2 sterility. OKA,
H.I.
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Character variations in backcross derivatives between Oryza
sativa and O. glaberrima: M-V linkage and key characters.
SANO, Y. and OKA, H. I...........
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Gene markers obtained in O. glaberrima. SANO, Y.. . . . . . . . . . . .
Sensitivity of barnyard grass, Echinochloa crus-galli to a herbicide.
IYAMA, S.............................................
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The effect of noise on egg laying performance of Japanese quails.
KAWAHARA, T.........................................
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A dark frayed feather nervous disorder mutant of Japanesequail. KAWAHARA, T................................. 100
Effects of noise on the learning performance of mouse strains
with different learning abilities and their hybrid. FUJISHIMA,
T.
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Nitrogen fixation in the rhizosphere of rice. FUJII, T., SANO,
Y., IYAMA, S. and HIROTA, Y.... '. . . . . . . . . . . . . . . . . . . . . . . . . 101

GENERAL STATEMENTThe National Institute of Genetics will greet the 30th anniversary of
the founding on June 1st of the next year. Thirty years old is, if comparedto man, the age to reach the full-fledged manhood. On this memorial
occasion we must consider deliberately how the Institute should be directedin the next decade.
Ever since its foundation, the Institute has been well-balanced in its
organization being consisted of laboratories of most fields of genetic
studies, and researches have been promoted by lively discussions in cooperative atmosphere among the research staffs of different disciplines.
Remarkable progress has been achieved recently in the fields of life science.
Furthermore, the scale of research has been becoming larger and larger.
If we stick to the conventional system and would not take an adequate
measure to modulate the situation, we shall be left behind the rapid progress of this science. In order to cope with such circumstances, it is necessary to strengthen the research power not only in the number of researchers
but also in their capacities. Far from that, due to the limitation in budgetary allotment, we are obliged to reduce the number of personnels every
year. It means a serious damage to the development of our science.
Looking back the calendar of this year, the following are worthy tonote. Kihara Institute for Biological Research put its Mishima branch
together with the main institute in Yokohama and Prof. H. Kihara,
who was the former director of the National Institute of Genetics, left
Mishima on February 10th, 1978. It was a great regret that we missed
a highly respected leader from Mishima. We had a farewell lunch at
the Institute on this occasion, inviting all other honorary members of
the Institute. In commemoration of the day Dr. Kihara gave us quite
an impressive lecture on his recent work. On the same day we held alsoa requiem mass to the memory of Dr. F. A. Lilienfeld who passed awaylast July.
On June 9th Dr. M. Kimura, head of the Department of Population
Genetics, was loaded a great honour. He was awarded by the Universityof Chicago the honorary degree of Dr. of Science for his distinguishedcontribution in the field of population genetics.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

In August the XIVth International Congress of Genetics was held in
Moscow. Prior to the Congress, the Genetics Society of America declared
to boycott the Congress against the discrimination enforced by the U. S. S. R.
Government in issuing visas to Israeli scientists. It caused a confusion
for a time. The Congress, however, ended with a great success as thebiggest one ever held, having 3,500 participants. From Japan more than
70 geneticists (10 from our Institute), including Professor Kihara, attended
the Congress, and I believe that we contributed a great deal to the successof the Congress.
The Institute received three visiting scientists from abroad in this fiscal
year, i.e., Mr. Louis Assemat from France (Dept. of Applied Genetics,
one year), Dr. A. R. Kasturi Bai from India (Dept. of Morphological
Genetics, 9 months), and Dr. B. A. Marcum from U.S.A. (Dept. ofBiochemical Genetics, 3 months).
The Institute had also several scientific visitors from Asian countries
in this year: in March parties of Science Foundation from Korea andfrom Thailand, in September a party of Academy of Science and in Octoberanother party of Food Science and Technology Investigation, both fromthe People's Republic of China. They made their efforts to materialize
scientific and personnel exchange programs between their own countries
and Japan.
The main building for Genetic Stocks Center was completed of its
construction in September. The budget for building annex laboratories
for breeding mice and rearing silkworms was approved by the government
at the end of this year. Thus, necessary arrangements have reached almost
completion for bringing the Genetic Stocks Center in its full operation.

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members1.

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

2.

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

3.

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

* Visiting researcher**

Deceased December 19, 1978

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

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. Ago, Head of the Laboratory
KAWAHARA, Takatada, D. Ago
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ago, Head of the Laboratory
The 3rd Laboratory
MORISHIMA-OKINO, Hiroko, Do Ago, Head of the Laboratory
6. Department of Induced Mutation
KAnA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TuTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Sc.
The 2nd Laboratory
KAnA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, D. Sc.
7. Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department

STAFFThe 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
AZUMI, Jun-ichi, B. Sc.
8. Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
YASUDA, Seiichi, D. Sc.
The 2nd Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.
YAMADA, Masao, D. Sc.
9.

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

10.

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

11.

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

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ANNUAL REPORlT OF NATIONAL INSTITUTE OF GENETICS NO. 29

SANO, Yoshio, D. Ag.
Animal Section
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.
INOUE, Yutaka, D. Sc.
Microbial Section
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
KOMEDA, Yoshibumi, D. Sc.

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

13. Department of Administration
OISHI, Tatsunori, Head of the Department
OHTSUKA, Haruichi, Chief of the General Affairs Section
KIMURA, Susumu, Chief of the Finance Section
Honorary Members

KIHARA, Hitoshi, D. SCo, Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
SAKAI, Kan-ichi, D. Ag.
TSUJITA, Mitsuo, D. Ag.
MORIWAKI, Daigoro, Do Sc., Managing Director of the Institute of Physicaland Chemical Research

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Professor of Univerisity of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, Keisuke, Member of Atomic Energy Commission
MORIWAKI, Daigoro, Managing Director of the Institute of Physical andChemical Research

COUNCIL

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MOROHOSHI, Seijiro, Professor of Tokyo University of Agriculture andTechnology
MUKAIBO, Takashi, President of University of TokyoNAGAKURA, Saburo, Professor of University of TokyoOOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, Director, National Institute for Environmental Studies
SHINOZAKI, Nobuo, Director of Insititute of Population Problems
TAKAHASHI, Ryuhei, Emeritus Professor of Okayama University
UMEZAWA, Hamao, Head of the Department of Antibiotics, National
Institute of Health
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Managing Director of the Institute of Physical and Chemical Research
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
KlHARA, 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 1978
Department of Morphological Genetics

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

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

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

PROJECTS OF RESEARCH FOR 1979

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)
Genetic analysis of developmental mechanisms in hydra (SUGIYAMA andFUJISAWA)

Department of Applied Genetics

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

Department of Induced Mutation

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

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Mutation and differentiation studies on 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

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

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

Theoretical studies of population genetics (KIMURA, MARUYAMA, OHTA, and
TAKAHATA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)PROJECTS OF RESEARCH FOR 1979

11

Department of Molecular Genetics

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

Studies and conservation of germplasm resources in rice and wheat species(FuJII, SANO and OKA)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII,
SANO, IYAMA and HIROTA)
Studies on genetic differentiation in rice (SANO)
Developmental genetic studies on mouse teratomas (NOGUCHI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Genetical study of flagellar formation in Escherichia coli K-12 (KOMEDA)
Basic studies on the gene purification and the construction of DNA banks(SUGIURA)RESEARCHES CARRIED OUT IN 1978I.

MOLECULAR GENETICS

Interaction between Bases Involved in the 5'-terminal
Cap Structure of Eukaryotic Messenger RNA
Masao HATIORI and Kin-ichiro MIURA

Most eukaryotic mRNAs and virus RNAs carry a so-called cap at the
5'-terminal as m7 G5'ppp5'N(m). This contains two pyrophosphate linkages
and 7-methylguanyilc acid carrying plus charge in its base moiety, being
quite different from inner part of an RNA molecule. This structure stabilizes mRNA for exonucleolytic attack and seemes to play any positive role
for an initial step in protein synthesis. Thus the knowledge on conformation of the cap structure is required. Here, we synthesized several kindsof the 5'-5' confronting nucleotide structure (cap in mRNA), including
methylated and non-methylated materials, and characterized these compounds by circular dichroism (CD) and ultraviolet absorption spectrum.
m7 GpppA and m7 GpppAm have UV maxima at 258 nm and 257 nm
respectively in 0.1 M Na+ (pH 7) at 25°. Molecular absorbances of thesecompounds are fairly lower than those which are calculated with each component. After the cleavage of pyrophosphate linkages by venom phosphodiesterase, absorbances of the intense peaks increase by 109 % in m7 GpppA
and 123 % in m7 GpppAm. This suggests that both m7 GpppA and m7 Gppp_
Am have base-stacked conformation in a neutral solution, and that thelatter stacks more strongly than the former.
CD spectrum of the 5'-5' confronting nucleotide structure shows typical
pattern. CD spectrum ofm7 GpppA has peaks at 235 and 265 nm, and troughs at 218 and 289 nm in 0.1 M Na+ (pH 7) at 26°. As shown in Fig. 1,
the spectrum is quite different from a summation spectrum of each component(AMP and m 7 GMP). The peak at 265 nm is opposite in sign andincreases in magnitude greatly compared to that of the summation spectrum.
The long wavelength negative cotton effect which sometimes is characteristic
for oligonucleotides containing a guanosine residue is also observed in thiscase. Except for this negative band, the peaks and troughs of m7 GpppA

RESEARCHES CARRIED OUT IN 1978

m7GpppA

1
10

x

0

CD

-1

13

: // ....•.........~/ \" .
'(f'? ,/:"
/,

....

\UMMATION OF AMP
ANO m7GMP:'• \ ,f'-2 220

240

260

280

300

320

WAVELENGTH (nm)are highly sensitive to temperature. By lowering temperature up to 2°,
the magnitude of peak at 256 nm increases significantly.
In the same condition (0.1 M Na+, pH 7, at 25°), CD spectrum ofm 7 GpppAm is quite similar in shape to that of m7 GpppA except for a longwavelength band. The magnitude of peaks at 265 nm increases appreciably
compared to that of m7 GpppA, suggesting that methylation of 2 /-hydroxyof adenosine in m7 GpppA strengthens the stacking interaction between
N7- methylguanine and adenine bases.
Structure near the 5'-terminal of Some
Eukaryotic mRNAs
Kunitada SHIMOTOHNO, Soh HIDAKA and Kin-ichiro MIURA

The protein synthesis starts at the initiation codon and proceeds in the
direction 5'--t3' on the template mRNA. An eukaryotic mRNA carries
a "cap" structure at the 5 -terminus, but it is not followed directly by aninitiation codon AUG. The part between the cap and the initiation codon
may participate with the initiation complex formation. Here we havestudied the nucleotide sequence of this region.
Messenger RNAs from cytoplasmic polyhedrosis virus (CPV), which contains 10 double-stranded RNA segments as a genome, were used for nucleotide sequence analysis around their 5'-termini. As the 5'-termini of these
RNAs are capped with 7-methylguanylic acid through pyrophosphate linI

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

kages [see this report No. 25 (1974)], the cap was deleted by tobacco pyrophosphatase before the 5'-terminal labeling with [UP] phosphate by
nucleotide kinase. The P2P] labeled CPV mRNAs were separated by gelelectrophoresis. Three smaller mRNAs were extracted from the gel withbuffer solution and analyzed. The mRNA was partially digested underlimited conditions with alkali, ribonuclease T 1 and U 2 • The digests wererun in parallel in a polyacrylamide gel and autographed on a X-ray film.
The arrangement of guanine, adenine and pyrimidine was deduced. The
arrangement of cytosine and uracil was analyzed by mobility shift in two
dimensional gel electrophoresis (pH 3.5 and pH 7).
The base arrangement of the 5'-terminal regions in three shorter RNA
segments are shown here. Common sequence for these RNAs are surrounded with box or underlined.
CPV mRNA

5

m

~

N

B

~

~

~

~

m7GpppAmG U A A A A GU C A GU ACe AU Ace G GA A A A A GIA U GIA A A A G GAY A A Y G'G A
5
m
~
N
~
~
~
~
m 7GpppAmG U A A A U C C C eGG C G U A A Ace G A A U A U C G eGA G Ace G Y A A A Y G
5

m

~

m

B

~

~

~

~

m7GpppAmGUAAA GU C CAGUACUAGUU AAA GAC AUUU GA C AAGGAY GAY Y AY G

Six bases arrangements continuing to the 5'-terminal cap were the same forthese RNAs, but the arrangements between these and the protein initiation
codon AUG were heterogeneous. There are some partially common
sequences (underlined) to two RNAs. In number 1 mRNA the nucleotide
sequences surrounding the first AUG consist of only purines, so that thesearea cannot construct a hair-pin structure.
Cucumber mosaic virus (CMV) contains 5 (4 in some strains) singlestranded RNA segments as genome. These viral RNA segments can function as messenger RNAs. These carry the cap structure at the 5'terminus. The smallest RNA segment (number 5) was analyzed as mentioned above. The base sequence near the 5'-terminus is shown here.
"w
m7 G 5 'pppG-U-U-U-U-G-U-U-U-G-A-U-G-G-A-G-A-A-U-U-G-C-G-U-A30

G-A-G-G-G-

RESEARCHES CARRIED OUT IN 1978

15

The sequence between the 5'-terminus and the initiation codon is quite
simple and V-rich. In this area a long common sequence to other mRNAs
so far analyzed was not found. A sequence complementary to the 3'terminal part of ribosomal RNA has been found in the sequence a head of
the initiation codon in many prokaryotic mRNAs and some eukaryotic
mRNAs. Shine and Dalgarno proposed that the mRNA is bound at firston ribosome by this sequence. However, such sequence was not detectedin CMV No.5 RNA. Thus the above-mentioned Shine-Dalgarno's idea
seems not to be common for every eukaryotic mRNA. (A part of thiswork was published in FEBS Letters 98 (1979) 115-118)
The Primary Sequence of the Late Region of Polyoma Virus DNA:the Expression of the Late Genes and Comparison with DNA
Sequences of SV40 and BKVEiichi SOEDA

The nucleotide seqeunce of the late region of the polyoma virus genome
has been deduced, which codes for the major capsid protein VPl and the
C-terminal region of the minor proteins VP2 and VP3. The amino acidsequence of VPI predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence and amino acid composition
of VPl (Hewick, R. M. et al. 1975, Virology 66: 408-419, Fey, G. andHirt, B. 1975, Cold Spring Harbor Symp. Quant. BioI. 39: 235-241).
When both nucleotide and amino acid sequences are compared with theircounterparts in the related viruses, SV40 (Rogiers, R. et al. 1978, Eur. J.
Biochem. 85: 205-223, Pan, J. et al. 1977, Nucleic Acids Res. 4: 25392547) and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum
homologies appear to occur in the regions which code for the C-terminal
of VPl, on the contrary of the result of heteroduplex analysis (Ferguson,
J. and Davis, R. W. 1975, J. Mol. BioI. 94: 135-149) with SV40 and polyoma
virus DNAs.
During the late lytic cycle of polyoma virus (Py), following commencement of viral DNA replication, three viral RNAs appear in cytoplasm ofcells. They have sedimentation coefficients of 16 s, 18 sand 19 s, each ofwhich directs in vitro synthesis of the viral capsid proteins, VPl, VP3 and
VP2, respectively. The main body of 16 s mRNA appears to be located16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

from 48 to 26 map units of Py genome counterclockwisely whereas thebodies of 18 sand 19 s mRNAs span completely the region of 16 s mRNA,
starting from the proximal part of the late gene. The genetic evidence
and peptide analysis have presented that the N-terminal of VPl and the Cterminals of VP2 and VP3 are closely located around 48 map units, possibly coding regions of these proteins overlap with each other with different
coding frames, by analogy with SV40. The evidence is consistent with the
topographical model of the late region of the physical map (Fig. I) whichshows positions of late mRNA and capsid proteins.
SV40 and Py have very similar growth cycle and genetic organization,suggesting they diverged from a common origin. Heteroduplex analysis
of DNAs between two viral species has revealed that the strongest sequencehomology occurred in the region where VPI and VP2/VP3 coding regionsof SV40 overlap each other with different coding frames. This led to
speculation that the same may be true for Py late gene and the overlap genewould be imposed severe restrictions on genetic drift, thus these region mustbe highly conserved.
Here we report the nucleotide sequence of the coding regions of VPI,
VP2 and VP3 of Py and compare with the counterparts of SV40 and BKV.
Conservation of overlap genes and divergency on the sequences of late genesof these viral species will be discussed from the aspect of molecularmap unit 10

Hpa II
Hint r

5

I

3

.0

50

II

30

{O

1
6

r9SRNA~Gl---V;,;,P,;,2----_UAiil-t-----------poryAIBSRNA-.--.------~:.jUG---V;,;,P.3--oiiiUAiiiA-----------POlyA
16SRNA..... -.. ---.-------------------------~~UG---..
V.P;,,1..UiiiiAApOlyA

Fig. 1. A topographical model of the polyoma virus late genes.
The cleavage maps of the restriction endonucleases Hpa II and HinfI are shownat the top on the linearised late region of polyoma virus DNA. The DNA has been
divided into 100 map units, taking the single cleavage site of the restriction endonuclease EcoRI as a zero reference point. The three known viral late messenger
RNAs (with polyadenylated tails) and their location in terms of the genomes areshown. Coding regions of the three virion proteins are shown using a heavy dottedline and the regions spliced out of the mature late mRNAs using a dotted line.

RESEARCHES CARRIED OUT IN 1978

17

evolution.
The fine physical map of Py with several restriction enzymes has been
defined. The nucleotide sequence of Py late region was deduced by the
Maxam and Gilbert procedures, using 3'- and 5'-terminally labelled fragments generated by digestion of Py DNA with restriction enzymes.
In the nucleotide sequence with the same polarity of late mRNAs (Fig.2),
there are two long uninterrupted coding frames: One lies at the 5'-end of thesequence and contains the potential coding sequences for VP2jVP3. The
other contains 383 consecutive sense codons starting with the ATG andextends from 47.5 to 25.8 map units where the body ofVPI gene has located.
The partial N-terminal sequence of VPI synthesized in vitro has completely
matched up to the first 20 cycles of sequence with that predicted from the
DNA sequence.
The nucleotide sequence is furnished with several structural futures including the potential recognition sequences for ribosome binding and splicing of mRNA proceeding the VPI initiation site and AATAAA sequencenear the polyA tails of late mRNA. The region preceeding the VPI initiation site is characterized by a long inverted repeat sequence, which possibly forms a hairpin loop in the mRNAs. The similar sequence which canform a part of the loop is also found in the leader sequence late mRNAby 900 nucleotides upstream of the body. Such a structure might be utilized
in the recognition events involved in the processing which leads to mature
16 s mRNA.
When both the nucleotide and amino acid sequences of these viral
species are aligned, extensive homologies are found throughout most of
the VPl genes (Fig. 2). Heteroduplex analysis with covalently linked DNAsof SV40 and Py suggests that the strongest homologies are located aroundthe N-termini of the VPl genes. The present data, on the contrary, showthat most of the larger homologous nucleotides are found in regions of thegenes coding for the C-termini. This disparity may be a reflection of the twodifferent methods of analysis. The overlap region of VPl with VP2jVP3of polyoma virus shorter by 89 nucleotides than that of SV40 and shows
very limited homology with SV40. This leads to the suggestion that theoverlap segments both viruses have been free from stringency imposed on
drifting during evolution and that the proteins VP2jVP3 of polyoma virus
have possibly been truncated by the appearance of a termination codon
within the sequence.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

.

"
,""
.""

.....
CAG ciA eee Me Cel,j,AT C(T

GilA Ar-C erA AC' CAG Gr.;' r,~r. c.i.~ TAC TAr Gr.T TGG AGe ... c;~ GGr, AiT AAT TTG GeT Ae;' TCA GAT .,CG , •• TfiG
(jAT GA .. CAr cu• • "AtoA Gr.c TH. "roT AAA A('ie TTA C;CA GeT CiA" AI-A eAG TTT .eA GAT GAC~~~ r;:] ~~~ ~~: ~~? ~;~ ;~: ~~~~~: ~~~ ~t~ ~~; ~ ~:: ~~~ ~ ~~~illm ~~~ ~~~ ~~~ :~~ ~~~ :~~ ~~~ ~t~ ~~~ ~ :~: ~~:tTl i:.i.e C(.A, '';'1 A~T AC;" CTI (cr. /Ie.}., iGG Ai,;' ATl; ~CA ""G Tee AGe TTC CCA TGe erc ;'AT Gi.G .GA.C cTC ACe TGT GAt:" Ace CiA CAli
leT

«(~

(".e "loA 6"'" C.ll.~ (Tli (el lGC lAC AGT (;Tr; r.CT AI,' Art eeT TTl; «(1 AAT TTl. AliT G"r, GA' nA Ace HiT GGh "AT ATT TTG~~~ ~ :~~ ~~~ ~[.~ ~~~ l~~~ ~~~, ~~i~ ~~~rml :~~ ~ ;~~ ;i~ ~~~ ~~~ ~ ~~~ ~~~ :~~ ~t:: ~~~ t~~ T~~ ~~~ ~~~ ~~~ T~~ ~~~

27.
27.

""

>6,
>6.
110
110

'"
"0
1>0
1>0

,U,T AU(j(~~A Gr.,. "lor TeC ACT CC" GTG (,~~ G~C .. t,c c~. TAT CAT GTG TT;' GeT GGT GGC c,GG G'\~ CCG elT G~C CTC eAG GGA elT

....

,,

Gr." T,~ ,IlAT TTT ("T TTl TTl GCT CiTT (,(,l r.G(, GAA CCT TTG GAG CTG CAG GGT GTG;:: ~:: ~~~ ~~~ ~ :2~ ~~~ ~~~ ~ ~~~ ~~:: l~:: ~ ~~~l ~~~ ~~~ [0] ~~~ ~~~ A~~ e;~ 6~~ gt~ ~~G ~:d tE~ ~~0 tE~: ~t~ rot ~ ;:~,"1&.

r.Tr, ACi. c;;r Grc ~(, ... ;;'10 Ai..;' ;;<: .ur, (;H (,;,~ (,(,,, Gn. G~. ~CA ATe ~~~ ArA ATe ~c; 1oAr. ur. GAC ATe; GTe ,IlAe ,Il;U GAC CAA GTe:rA GCt. "~C He ",1", ~(r,,~ Hr CCT GCT CA"
A(T "TA ~cr: cc"" "AT GeT A(J. GT!
(iAe "'GT CAG (,IlG

63.

••• ••• r,H HT (,(".T (,(/ GI". .... ,..AJ. (ce "TT(,1,0\~~~ ~~~ :~::'I ~1¥~ j::::;, ~II:;; ~~~ ;~;;I ~'~'0 ~t~ ~t~1 ~~: ~:.~ ~~t ~I~~~ ~~6 ~ ~~~ l~i ~ ~:t ~:~ :~~ ~;~ ~:~ :~~ ~~~ ~~: ~t~ ~~~

630

21.
21.

..

120
72.

,,

81.
81.
27.
270

>,A (,1+t,r·/·
i,~.i. Ge;" LAG (,(,c eTII
icc T(;l GTA GAT ATA "T(:T(;G "'(;A Ci++ AiA A(,A ;, .... e t .... + GTe;
TCA
C;C:T (,T! (,r.r; ete TTC; l',e AllA GeT GIIC AGC nr, TAT (,TT Tcr (ieT GTT EAe jilT 1GT G(,(, CTG ••• TTT ACe-AAC ACT TeT (;GA ACA eAG

iic cTc If.r

TAc (.T(

6GC

rei..

~~~ ~~t ~:~~ ::;:g t~;: ~~~ ~ ~~ ~~.~ ~'~~ ~~~ ~~l: ~~~ ~~~ ~l ~:~~ ~:t :~p rtf ~~~ ~ ~~~ :~~ ~~~ ~ :~~ ~~~ ;~: ~~; i~: ~~~

'00
'00
>0'
300
99.
,,,

'"
'"
1170
11TO

'".

".

Fig. 2. Comparison of nucleotide and amino acid sequences of the VP1 geneand gene products of Py and SV40.
The VP1 sequences of Py and SV40 are aligned so as to maximise homology.
Identical peptides and nucleotides, at corresponding positions, are indicated by theboxes or dots, respectively.

A Simple Method for the Identification of Altered Subunits
in Mutant RNA Polymerases of Escherichia coli
Masahiro SUGIURA, Nozomu ITO and Mie SUZUKI

DNA-Dependent RNA polymerase from Escherichia coli is a multi-subunit
enzyme with a subunit composition of ad3f3'a. In order to study the

RESEARCHES CARRIED OUT IN 1978

19

structure, function, and genetics of this enzyme and its subunits, methods
for reconstitution of the active enzyme from its isolated subunits have beendeveloped. These methods are, however, laborious and time-consuming
and hardly applicable to unstable mutant RNA polymerases. During thecourse of isolating temperature-sensitive mutants of RNA polymerase, wedeveloped a simple procedure to identify altered subunits. The procedure
consisted of mixing a mutant enzyme with an excess of a wild-type subunit
followed by denaturation and renaturation. This gave active enzyme molecules in which the majority of a subunit species was replaced by a corresponding wild-type subunit. When enzyme containing temperaturesensitive fi' subunit was mixed with an excess of each separate wild-type
subunit followed by denaturation and renaturation, only the mixture containing wild-type fi' subunit was temperature resistant. Likewise, the rifampicin-resistant enzyme exchanged specifically with wild-type fi subunit
became rifampicin sensitive. Thus results indicated that the method wasapplicable to identify altered subunits in mutant RNA polymerases. The
subunit replacement experiments were also carried out successfully using
crude enzyme preparations. This work was published in Analytical Biochemistry 84, 337-339 (1978).

Joining of 3'-Modified Oligonucleotides by T4 RNA Ligase.
Synthesis of a Heptadecanucleotide Corresponding to theBases 61-77 from Escherichia coli tRNAfMet
Eiko OHTSUKA, Satoshi NISillKAWA, A. F. MARKHAM,
Seiji TANAKA, Tetsuo MIYAKE, Toshiaki WAKABAYASHI,
Morio IKEHARA and Masahiro SUGIURA

Chemically synthesized fragments corresponding to the 3' end of tRNAfMetfrom Escherichia coli were joined by T4-induced RNA ligase to yield aheptadecanucleotide (bases 61-77). The 3' terminus of C-C-A was modiified by introduction of the ethoxymethylidene group to prevent intra- andintermolecular self-joining reactions at the 3' end. The terminal trimer was
phosphorylated using polynucleotide kinase and joined to C-A-A with RNA
ligase. The hexamer [C-A-A-C-C-A (ethoxymethylidene)] corresponding
to bases 72-77 was obtained in a yield of 60%. An undecanucleotide (bases 61-71) which had been synthesized in a yield of 34 % by similar
enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

with the 5'-phosphorylated hexamer (bases 72-77) using an excess of RNA
ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-CC-A (bases 61-77). The product was identified by homochromatography
and nearest neighbor analysis. This work was published in Biochemistry17, 4894-4899 (1978).
Cloning and Characterization of Tobacco Chloroplast
238 and 168 rRNA Genes
Masahiro SUGIURA and Jun KUSUDA

Previously we have shown that tobacco chloroplast 23S and 16S rRNA
hybridized with two EcoRI fragments of tobacco chloroplast DNA, 1.9 X 106(I) and 2.8 x 106 (E) dalton fragments, and have cloned these two fragments
using the E. coli plasmid pMB9 (this report No. 28, 1977).
In order to know whether these two fragments are contiguous, we thenattempted to construct a recombinant DNA molecule containing bothfragments. The chloroplast DNA was digested partially with EcoRI in thepresence of low concentration of distamycin A. Hybridization of (5'32P)
rRNA to a Southern image of the partial EcoRI digests produced a seriesof radioactive bands. The partial EcoRI fragments ranged from 4.5 X 106
to 6 X 106 daltons were extracted from a preparative gel electrophoresis andcloned using pMB9 as a vector and E. coli HB101 as host bacteria. Twohundreds and seventy one transformants were obtained from 0.2 ftg each of
pMB9 and the chloroplast DNAs and 12 clones were found to containrRNA sequences. Ten out of the 12 clones contained both E and I fragments and an additional fragment of 0.46 x 106 daltons. One of the 10
clones, TCP6 was used for further studies. The plasmid pTCP6 DNA waspartially digested by EcoRI and analyzed by agarose gel electrophoresis.
It was found to contain the three fragments in the order of E - 1- 0.46 x 106
fragment. This clearly indicates that the E and I fragments are contiquous.
In order to locate 23S and 16S rRNA sequences on the E+I fragment,
the Southern image of pTCP6 EcoRI fragments was hybridized separately
to (5'32P) 23S and 16S rRNAs. The autoradiographs showed that 23SrRNA hybridized to E fragment whereas 16S rRNA hybridized mainly to
the I fragment and slightly to the E fragment. There results and molecularweights of the rRNAs could give the approximate location of the rRNA
genes within the fragments E and I.

21

RESEARCHES CARRIED OUT IN 1978
rCI

rC309

rCP6

Fig. 1. EcoRl restriction maps of recombinant plasmids containing tobacco
chloroplast rRNA genes. Arrows, EcoRl sites; Numerals, Molecular weights(x 10- 6 daltons)

This work was published in Molec. Gen. Genetics, 172, 137-141 (1979)
and was presented at the 8th ICN-UCLA Symposia on Molecular and Cellular Biology, "Extrachromosomal DNA", Keystone, U.S.A., March 1979.
Gene Controlling DNA Initiation in Escherichia coli:
dnaA + and Suppressor Genes of dnaAYutaka TAKEDA and Yukinori HIROTA

It has been known that the dnaA gene product is required in the initiation
step of Escherichia coli chromosome replication. In this communication,
we report the cloning of genes containing dnaA, and the genes which suppress the temperature sensitive (ts) mutation of dnaA. Restriction mapsof these chromosomal regions were also described.
Whole E. coli chromosomal DNA of a mutant of E. coli, W311O, and aplasmid DNA, pMB9 (Bolivar et at. Gene, 2, 75-93, 1977) or pCRl (Covey
et at. Molec. gen. Genet., 145, 155-158, 1976), were digested with EcoRIand ligated with T4 DNA ligase. The CaCl 2-treated cells of CRT46recA (adnaA ts mutant) were transformed with the ligation mixtures and the colonies which became thermoresistant and drug resistant were selected. Fromthese transformants, two plasmids were isolated: one was constructed by ligation with pMB9 and the other with pCRl, and those DNAs were designatedas pYT47 and pYT48, respectively. These plasmids had the same EcoRI

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

fragment of E. coli DNA whose molecular weight was 5.5 x 106 daltons.
After the transformation of CRT46recA with pYT47 or pYT48 DNA, thefrequency of appearance of drug resistant colonies at 30°C and at 42°C was
the same.
Another plasmid, pYT46, was isolated by selection of Kanamycin
resistant transformants at 30°C (ligated with pCRI). After the transformation experiment of CRT46recA with pYT46 DNA, Kanamycin resistant transformants appeared at 30°C, but not at 42°C. All the transformants thus obtained at 30°C showed temperature resistance. Therefore,
we thought that pYT46 had a supperssor of dnaA which required a longtime to express its temperature resistance. pYT46 had an EcoRI fragment of E. coli, whose molecular weight was 3.5 x 106 daltons. pYT46,
pYT47 and pYT48 could not complement tna, rbs, uncA, asn and pyrE
mutations.
A lambda phage carrying dnaA gene (A dnaA) was isolated by a conventional in vivo recombination from A i2ltna by Murakami and Yamagishi(personal communication). This phage also had tna gene which locatedclose to dnaA.
pYT46, pYT47 and A dnaA DNAs were digested with the following restriction enzymes; EcoRI, HindIII, BamHI, BglII, KpnI, PstI, SaU, XhoI,
HpaI and the digests were analyzed by agarose and polyacrylamide gelelectrophoresis. Restriction maps were constructed by digestions of theDNAs with various combinations of restriction endonucleases. The restriction maps of E. coli DNA region of pYT46, pYT47 and A dnaA wereshown in Fig. I. Compared with each other, we concluded that three
DNAs had no overlapping region. It is likely that these DNAs were originated from three different regions of E. coli chromosome. We thought that
A dnaA had the structure gene of dnaA, because A dnaA had also tna, alinked gene of dnaA on the chromosome map of E. coli. pYT46 and pYT47
had suppressor genes which were different from dnaA. The molecularnature of these suppressor gene products were not known. Increase of thegene dose might cause the suppression of dnaA ts mutation, because cloning
vectors, pMB9 and pCRI, had 10-20 copies per cell.
The suppressor gene product of pYT47 was characterized, as follows.
From pYT47, two deletion mutants, pYT62 and pYT63, were isolated after
the digestion of pYT47 DNA with KpnI or PstI and ligation with T4 DNA
ligase. These plasmids having different deletion could not suppress dnaA

RESEARCHES CARRIED OUT IN 1978
PYT46

ELI

HL1---L~_~...JJr

H

B

23

pYT47

)., Q..n.gA

I

I

B
I

Hp

I

HE
E
II
I

I

X
I

E
I

SeA)
l..

Hp

0

5

I

I

Fig. 1.

ts mutation. The deleted regions of pYT62 and pYT63 were shown in
Fig. 2. pYT47, pYT62 and pYT63 were introduced into a minicellproducing strain LC130. Minicells produced from these strains were purified through sucrose gradient centrifugations 3 times, and incubated at
37°C for 30 minutes to degrade host mRNA. Some proteins newly synthesized from plasmid DNA were labelled with [35 S]-methionine and analyzed
with two dimensional gel electrophoresis described by O'Farrell (J. BioI.
Chern., 250, 4007-4021, 1975). pYT62 could not synthesize two proteins
whose molecular weights were 68,000 and 20,000 daltons, and pYT63 could
not synthesize only one protein of 68,000 (Fig. 3).
Six independent mutants which could not suppress dnaA ts mutation wereisolated from pYT47 after mutagenesis with N-methyl-N'-nitro-N-nitrosopYT47

62- - - - - - -------- --63
Fig. 2.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

guanidine or hydroxylamine. The proteins coded by these mutant plasmids
were analyzed with two dimentional polyacrylamide gel electrophoresis.
Five of these mutants showed the same pattern as pYT47, while only
one mutant, designated pYT74, synthesized a protein having molecularweight of 68,000 daltons with altered isoelectric point. This change ofisoelectric point of 68,000 protein was recovered in pYT81, a spontaneous
revertant of pYT74 which could suppress a dnaA ts mutation (Fig. 3). We
concluded that the protein having molecular weight of 68,000 daltons wasa suppressor protein of dnaA ts mutation, because the suppression of dnaA~b as~c

7 .5

7.~

...,.

-.. ....
-•.... ...

- ....
pMB9.......

...,.

- .....
pYT63._------- ----._._-

.
pYT47

68,000

....

-

.....
....
pYT81

acidic75,000AY_"'-- pYT74

AY -

-+

f-

.

75,000."AT-

68,000-...

75,000

-

."

-

74+47

AW:- 68,000

Fig. 3. Pattern of two dimensional gel electrophoresis.
Purified minicells carrying plasmids were treated with Triton-X 100 after sonication. Soluble fractions were used as samples for electrophoresis. Electrophoresis
were done as described by O'Farrell. All proteins were stained with Coomassie
brilliant blue R250. Proteins newly synthesized from plasmed DNA were labelledwith 35S-methionine and detected by autoradiography.
In this figure, spots of proteins after staining were shown. Arrows indicate
mutant and wild type proteins having a molecular weight of 68,000. This spot wasradioactively labelled.

RESEARCHES CARRIED OUT IN 1978

2S

ts mutation was caused by the change of isoelectric point of the protein.
Recently, Murakami and Yamagishi identified the gene product of dnaA,
whose molecular weight was about 50,000 daltons (personal communication). Therefore, the gene product of a suppressor gene on pYT47 wasdifferent with the dnaA protein.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

II.

MICROBIAL GENETICS

Properties of Penicillin Binding
Protein (PBP) la, lb and 3
Toshihide TAMURA, Hideho SUZUKI, Yukinobu NISlllMURA
and Yukinori HIROTA

The function of PBPs of E. coli K12 in growth and division have been
speculated by analysing mutants defective in one or more PBPs (Suzuki
et al., Proc. Nat. A cad. Sci., 75, 664-668, 1978). Using membrane fraction of these mutants, attempts were made to search for possible enzymatic
activities of each PBPs in terms of murein synthesis in the subcellular system. The findings to date are that at least PBP-lb (the gene product of
ponB+) may playa principal role in murein synthesis in vitro, and that
other PBPs be auxiliary in directing murein synthesis to morphogenesis ordivision of a cell. As a consequence of these studies, purification of theindividual PBP was attempted in order to examine molecular characteristics of each PBP. For this purpose, we took advantage of utilizing thespecially constructed strains as a source of PBPs. In these strains, thecellular content of required PBP was amplified ten times or more by the
introduction of a ColE! plasmid carrying the relevant PBP-gene and theunwanted PBPs were genetically eliminated as far as possible by the combination of mutations leading to the loss of these PBPs. The constructedstrains were JE7434 (ponBlO85 ftsI730 dacAl191 dacB12 pLC29-47), JE5770(ponA980 ftsI730 dacBl2 dacA1191 pLC19-l9) and JE7430 (ponN 1104
ponB704 dacA1l91 dacB12 pLC26-6). Membrane fractions prepared by
Ribi-cell-disintegrator were solubilized by Triton X-IOO in the presence of
LiCl. The solubilized materials were desalted by dialysis and pre-fractionated by DEAE-Sepharose column chromatography, in which PBPs werepartially separated each other. The fractions containing the desired PBP
but almost free from other PBPs were pooled and subjected to covalent
Ampicillin-Sepharose affinity chromatography. The covalently bound PBPs
to Ampicillin residue of the resin were eluted by neutralized hydroxylamine,
desalted by dialysis and stored at 4°C.
In the last procedure, partial separation of PBPs could also be achieved
8

27

RESEARCHES CARRIED OUT IN 1978by eluting PBPs from the affinity resin after successive incubations forincreasing length of time at 37°C with neutralized I M hydroxylamine.
This step-wise elution method in the affinity chromatography to the material obtained after DEAE-Sepharose chromatography improved the purityof the final preparation of PBP-Ia & -lb. About 90% homogeneity of
PBPla, Ib, and 3 were obtained as examined in SDS-acrylamide gel electrophoresis. The preparation of PBP-3 obtained after the affinity chromatography was accompanied with a minor PBP which ran faster than theauthentic PBP-3 in electrophoreogram. Genetic experiments showed theclose relationship between this minor PBP and the PBP-3, suggesting
the possible precursor-product relationship. Neither activities for murein
synthesis in vitro nor those for D-D carboxypeptidase was detected in the
purified PBP-Ia, -I b or -3 as long as UDP-linked murein precursors, i.e.
UDP-MurNAc-L-Ala-o-Glu-meso-A 2pm-o-p'C]Ala-o-p'C]-Ala, were employed as substrates.
When a membrane fraction of the PBP-Ib defective mutant was employed in the in vitro system for murein synthesis, polymerization of murein
precursors was greatly suppressed, at the same time considerable accumulation of the lipid intermediate was observed. The addition of
purified PBP-I b to this PBP-I b defective system promoted murein synthesis with consumption of the accumulated lipid intermediate. Apparently,
the purified PBP-I b functioned to synthesize murein in compensation forthe defect of PBP-I b of the membrane in the reaction system. Verylittle stimulation or practically no stimulation in the murein synthesis
in vitro was observed by addition of the purified PBP-Ia or -3, respectively(Table I).
Table 1.

Murein synthetase activity ofPBP-la and lb, in vitro
Incorporation (cpm)

Addition

MureinLipid-intermediate

None
PBPla
PBPlb
PBP3

319
606
2999
381

1611
1602
228
1708

Substrates:
GIcNAcMembrane

UDP-MurNAc-L-Ala-D-Glu-meso-A 2 pm-D-Ala-D-Ala ; uC-UDP: ponAt'l104 ponB-1085 dacA-1191 dacB-12

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Further Genetical Study of Flagellar Mutants in E. coli K-12
Yoshibumi KOMEDA

Twenty nine flagellar genes in Escherichia coli K-12 have previously been
assigned to three regions of the genome. Flagellar region I is locatedbetween pyrc and ptsG, region II is located between aroD and uvrc,
and region III is located between uvrC and his. In this study flagellar
mutants in Escherichia coli K-12 were obtained by selection for resistance to. the flagellotropic phage X. They were analysed in complementation tests using PI phage-mediated transduction. In addition to the fla
genes already described, 9 more flagellar genes were identified. This analysis defined 6 more fla genes in region I (flaU, flaW, flaX, flaY, flaZ and
flbA), 1 more fla gene in region II (flbB) and 2 more flagellar genes inregion III (flbC and motD). Region I was shown to include at least 12
fla cistrons. Complementation analysis with polar Mu phage-induced Flamutants and with J..-fla phage, defined four transcriptional units in region
I. These were: flaU, flbA-flaW-flaV-flaK-flaX-flaL-flaY-flaM, flaZ, andflaS-flaT, with transcription proceeding from left to right. The flbB gene
was identified to be a member of an operon: flbB-flaI in region II.
In region III, a previously unidentified gene flbC was located between hag
andflaN. The motD gene was located betweenflaA andflaR and the operon was reoriented as flaA-motD-flaR-flaQ-flaP, with transcription proceeding from left to right. Now we have 39 genes (che, fla, flb, hag, mot)necessary· for the synthesis and function of the flagellar apparatus ofE. coli K-12. Out of these genes, 28 genes (fla, flb, hag) are responsible
for the completion of the flagellar structure.
Expression of the hag Gene in E. coli K-12:
Analysis of hag-lac Gene Fusion
Yoshibumi KOMEDA

Previous studies have defined 28 genes necessary for the synthesis of theflagellar apparatus of Escherichia coli K-12. Most flagellar defects resultin a Fla- phenotype which is not very informative biochemically. Most
structural and regulatory components of the organelle have no easily measurable activity and only manifest themselves when integrated into thewhole organelle. This makes it difficult to measure the synthesis of aflagellar gene product and to study the regulation of expression of the

RESEARCHES CARRIED OUT IN 1978

29

flagellar genes. The in vivo gene-fusion technique developed by M. Casadaban allows an analysis of the regulation of virtually any gene by coupling the lac genes to the gene (operon) in question. We decided to apply
this method in the study of fla regulation, and have been able to fuse
most flagellar genes to the lac genes by using novel Mu phage carrying
the lac genes. This study analysed the influence of the flagellar genes on theexpression of the hag gene (structural gene for flagellin). To this end, ahag: : Mud (Apr, lac) mutant which had the lac genes fused to thepromoter of the hag gene was constructed. This allowed the measurementof the hag gene expression by detection of beta-galactosidase activity.
The following observations were made: (I) The hag gene was shown to
be expressed constitutively in Fla + cells. (II) Hag-gene-expression was
positively regulated by flaA, flaB, flaC, flaD, f/aE, flaG, f/aH, f/aI, flaK,
f/aL, flaM, f/aN,flaO, flaP, flaQ,flaR,flaV,f/aW, flaX, f/aY, f/aZ, flbA
and flbB genes. Hag-lac expression was not observed in strains withthese fla mutations. (III) The hag gene was expressed in mutants with
f/aS, flaT, flaU and flbC defects. Therefore, these genes were not involved
in regulation of hag gene transcription.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICS
Electrophoretic and Heat-stability Variations of Serum
Esterase (Es-l) Isozymes in Wild mice (Mus)
Tadashi AOTSUKA'" and Kazuo MORIWAKI

We have found the heat-stability difference betwee two types of Es-l
isozymes (Es-l a and Es-l b) in 21 inbred mouse strains, and demonstrated
based on the results of cross experiments that the difference in the heatstability is due to the genetic alteration on the Es-l locus (no otherfactors are responsible for observed heat-stability variations in inbred mice:
Aotsuka and Moriwaki; Ann. Rep. Natl. Inst. Genet. (Japan) 24: 32, 1977).
In the present paper we report the isozyme variations at the Es- I locus
examined with 8 species of wild mice (Mus) collected from Japan andSoutheast Asia by using both electrophoretic mobility and heat-stability.
Sera were treated at 60°C for 0, 3 and 6 minutes before applying to acrylamide slab gel electrophoresis, by which we could staisfactorily discriminate
the difference of heat-stability between Es-l a and Es-l b. Three types ofheat-stabilities (stable, moderate and sensitibe) were identified based on theintensity of zymograms. For controls, sera of inbred mice, BIOBR(Es-l a)and C3HjHeJ(Es-l b ), were examined simulaneously. The results are summarized in Fig. 1. In a survey of 95 individuals of wild mice, a total of
7 electromorphs (differing in electrophoretic mobility, i.e., af, a, a-, b+,b+, b, b- and c) were detected. Additional variations for Es-l locus were
found by heat denaturation tests. The five electrophoretic classes (a, a-,b+, band b-) can further be divided into two or three classes differed inheat-stability; stable, moderate and sensitive. The heat-stability differences
within a electromorph occured at three levels of phylogenetic relationships,
within a species, among subspecies and species. A total of 14 variants(a doubling of the number of isozymes observed by electrophoresis) weredetected by using both electrophoresis and heat-stability tests. If the moderately heat-stable zymogeams are the heterozygotes of heat stable andsensitive alleles, the number of alleles of Es-l locus become 12. The present
survey of hidden genetic variabilities well supply a fruitful information about the genetic diversity within and between Mus species.
'"

Tokyo Metropolitan University

_stablefllll22 moderatec:::J sensitive
a+

+

:;l:l
tT1

~

'l///.m

a

b+
b

I

==:I

~.~-~_.I

W2llUIllJ--:J

1--------,

w;m. ::::J~ffi-J,-------.

'---:::J1a~----.

[-~':'~I-I~/////7/////Jbc
Control

M. MOL.

M. DOM.

M. BAC.

M. CAS.

M. URB.

CAL

LEG

CRY

Inbred
mouse

(33)*

(10)

(15)

(12)

(3)

(11)

(7)

(4)

Fig. 1. Electromorphs and their heat-stabilities of serum esterase (Es-!)
isozymes of eight wild species of Mus. Species abbreviations are; M. MOL,=Mus
musculus molossinus, M. DOM.=Mus musculus domesticus, M. BAC.=Mus musculus
bactrianus, M. CAS.=Mus musculus castaneus, M. URB.=Mus musculus urbanus,
CAL,=Mus calori, LEG.=Mus legada, CRV.=Mus cervicolor.
... ( ) No. of individuals examined.

!i1

~
~~

o
C

>-i

-Z
\0

-J

00

Y>

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Genetic Control of Esterase in Japanese Species of Trillium
Masaaki IHARA and Toru ENDO

We have examined the electrophoretic phenotypes of esterase (Est) ofseeds, whole body of the 2nd year juveniles and leaf blade of adultplants in three species as well as their F I hybrids. The Est zymogram isshown schematically in Fig. I.
The result indicates that there are two groups of isozymes preferentiallyreacting with either I-naphtyl acetate (a-specific in zone I) or 2-naphtyl
acetae (.a-specific in zone II) used as a substrate together with fast blue RRsalt as a dye coupler. The a-specific bandmorph consists of one cathodally moving isozyme and four anodally migrating ones, whereas the fjspecific bandmorph is composed of I or 2 anodally and rapidly migrating
isozymes. We designate respecitve bandmorphs in the a-specific isozymes
as Al to A. for anodal band and C I for cathodal one; those in the .aspecific isozymes are referred merely as isozymes in zone II because of no
species difference.
The Al and A 2 bands recovered in every cross; the A a band did notrecover in the cross of tschonoskiix smallii but it recovered in the crossesof kamtschaticum X tschonoskii and its reciprocal one. Therefore it is necessary to confirm F 2 segregation for the genetic control of Aa bandmorph.

+~--;)j

2

3

4

5

6

7

8

9

10

11

~~~

~:.~~)':7~~.~

J...~.~~:,:::?;s~:};~]~¥iI}'!ii.gt:~~:g'tt

ID1~~

o

:J

l'J)

•• o. • • • ------• --• ---- CJA4• • CJ

0

N

•

A3N
0

A2 :Jl'J)
A1-

•

••

C1

Fig. 1. Esterase zymograms showing the migration rates of the seed isozymes
in three species and their F I hybrids. (1) T. kamtschaticum (K); (2) T. tschOlWSkii(T); (3) TxK; (4) KxT; (5) K; (6) KxS; (7) SxK; (8) T. smallii (S); (9) SXT;
(10) T x S, (11) T. "0" designates the origin.

RESEARCHES CARRIED OUT IN 1978

33

The C1 and A, seem to be active in the adult plants and they are probably
governed by respective loci.
If the Al and A z bandmorphs are controlloed by the same locus, itwould be explained as follows: F and F + S isozymes appear in T. kamtschaticum, and S isozyme does in T. tschonoskii and F+S isozyme does inT. smallii.
Four plants of T. Hagae(2n=15), which is presumed to be natural hybrid between T. kamtschaticum(2n=1O) and T. tschonoskii(2n=20), revealed
the same zymogram. The C1 and Al bands of T. Hagae are superimposedby those of T. tschonoskii; the A z and Aa bands are common with all these
species as to their migrating rates. Thus the putative hybrid may have descended from the above two entities.
Maternal Effect Embryo Lethal Mutant in Drosophila melanogaster
Masa-Aki YAMADA and Saburo NAWA

For the investigation of the interaction between egg cytoplasm andzygote nucleus during the development, a X-linked mutant (fs(l) MY-18)
was isolated with EMS treatment. The viability of this mutant was almost
normal (the ratio of homozygous females to heterozygous females was 0.9).
When the heterozygous females of the mutant were crossed to the mutant
males, they normally produced their progenies. The homozygous females
produced apparently normal eggs in shape and size. However, they wereunable to develop into larvae even when fertilized with wild type sperms.
The development of the eggs was stopped at various stages from invergination stage to moving stage. In the most of the dead embryos, the abnormalities were observed in the anterior region of embryo.
When small amouts (1-1.5% of egg volume) of the wild type egg
cytoplasm were injected into the eggs at cllavage stage laid by homozygous females, 1-2% of the treated embryos could develop into larvae.
In the control experiment where the eggs were injected wih the mutant
cytoplasm, no larvae were obtained. These results suggest that the fs(l)MY-18 gene affects the production of the specific component of the egg
cytoplasm which is essential for the early development of the embryos.
When the cytoplasm of the wild type eggs was kept at - 20°C for 5 days,
the rescue activity was unchanged, showing that the component is considerably stable. The biochemical analyses of the cytoplasmic component
are in progress.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS
Growth Rate and Cell Cycle Lengths of Hydra
Iun TAKAN01), Toshitaka FUJISAWA and Tsutomu SUGIYAMAThe epithelial and interstitial cell cycle lengths of 4 strains of Hydra
magnipapillata were determined by continuous labelling of the cells withtritiated thymidine followed by autoradiography.
It was found that the interstitial cell cycle lengths were nearly constantamong the 4 strains examined (about I day), although these strains hadgreatly different growth rates (ranging from the doubling time of 3.1 to 9.3
days). This suggests that hydra may lack mechanism to regulate the rateof interstitial cell proliferation (see Bode, Flick and Smith, 1976).
The epithelial cell cycle length was found to be nearly equal to the
doubling time of hydra in 3 out of the 4 strains examined (as previously
found in H. attenuata by David and Campbell (1972). In one strahl (L4),however, the epithelial cell cycle (4.5 days) was much shorter than the
doubling time of animals (9.3 days). This appears to suggest that thegrowth rate of normal hydra depends on the epithelial cell proliferation rate
(or vice versa), and that the mechanism involved for this is defective in L4.
A Mutant Hydra Strain (sf-I) Containing
Temperature Sensitive Interstitial Cells
Beverly A. MARCUM2), Toshitaka FUJISAWA and Tsutomu SUGIYAMA

Temperature sensitive interstitial cells have been observed in a sexuallyinbred mutant strain of Hydra magnipapillata. This strain, sf-l (selffeeder1), contains normal cell numbers when cultured at 18°C. Within 24 hours
after increasing the temperature to 23°C, over 99 % of the interstitial cells
are eliminated. Nevre cells, nematoblasts, nematocytes, and gland cells
decrease more gradually, and after one week at 23°C the entire population
loses the ability to capture and ingest prey. Cell counts of macerated non1)

2)

Faculty of Science, Nagoya University
Department of Developmental and Cell Biology, University of California, Irvine,
California.

RESEARCHES CARRIED OUT IN 197835

feeders (nf-l) indicate that the polyps consist almost entirely of epitheliomuscular cells and digestive cells, with a very small number of gland cells.
Chimeric hydra consisting of wild type epithelial cells and sf-l mutant
interstitial cells are also sensitive to the increase in temperature; however,
the reciprocal combination of sf-l mutant epithelial cells and wild type interstitial cells is not temperature sensitie. No wild type strain of H. magnipapillata shows this sensitivity to an increase in temperature. Electon
microscopic examination of sf-l tissue indicates that interstitial cells undergo
extensive deterioration coincident with the increase to 23°C and that theirelimination is not due to phagocytosis by other cell types.
Comparison of Effective Lethal Phases of Embryos fromThree Complementation Groups of the rudimentaryof Drosophila melanogasterYukiaki KURODA

The rudimentary (r; 1-54.5) has some complementation groups for a wing
abnormality and pyrimidine auxotrophy. In the previous report, it was
found that cells obtained from r 39k embryos showed a characteristic
defect in the maturation of epithelial cells in culture, and that some intermediates in the pyrimidine biosynthetic pathway were effective in repairing
the r 39k embryoinc cell defect.
In the present work, the effective lethal phases were compared for threecomplementation groups of the rudimentary locus, r 9 (=r 39k), r 3B (=r 56i)
and r 2 (=r X917 ). Eggs obtained from matings of rlr females with rlYmales were dechorionated and their effective lethal phases were determinedby examining the arrested stage of embryos in saline solution.
Among 1,076 embryos from the r 3B strain, 3.1 % died before gastrulation,
47.0% died by abnormal gastrulation, 29.9% died after head and trunk
segmentation, 6.7% died after sac-like midgut segmentation, 10.1 % diedafter entrance of air into trachea, 1.8 % died after the stage of active
movement and 1.4 % died immediately after hatching. This indicates thatabout half of r 3B embryos died in early phases of embryoinic development.
Embryos obtained from the r 2 strain also showed a similar pattern of theeffective lethal phase to that of the r 3B strain. Among 2,271 r 2 embryos,
58.2 % died by abnormal gastrulation. The early lethal phase of these rwo
strains of the r complementation groups was clearly contrasted with that

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

of the r V strain, in which only 9.3 % died by abnormal gastrulation and 40.7
% died after entrance of air into trachea.
Embryonic cells from the r 88 anti r Z strains were cultured to examine
their defective cell types. These cells, however, were difficult to be maintained in culture, because their membrane was fragile. This may accountfor the early death and abnormal gastrulation of embryos in these strains.
Effect of Conditioned Medium on Chondrogenesis of
Quail Limb-Bud Mesenchymal Cells in Culture
Yukiaki KURODA and Etsuya MATSUTANI

Mesenchymal cells from the limb-buds of quail embryos can differentiate
into cartilage cells in culture. The degree of chondrogenesis depends on
the initial number of cells inoculated on the petri dishes. The reason why
the chondrogenesis is affected by the cell density may be considered asfollows; cells inoculated with high cell density have more mutual cell contacts with each other which enhance the expression of chondrogenesis, and/or the chondrogenesis in vitro is stimulated by some factor(s) accumulated
in the culture medium during cultivation of cells. In the present experiment
the effect of conditioned medium (CM) on the chondrogenesis of mesenchymal cells was examined.
The mesenchymal cells were obtained from the hind limb-buds of quail
embryos at stages 22-23. After removal of epithelial layer mesenchymal
cells were dissociated into single cells. The CM was obtained by cultivationof 2.5 X 108 cells in 60-mm petri dishes for 2 days at 37°C and stored at-20°C. To assay the activity of CM, 106 cells were cultured in 2 ml of
medium containing 1.5 ml of CM in 35-mm petri dishes. The cell layer inpetri dishes was fixed and stained with toluidine blue. The number of cartilage nodules formed was scored and used as an index of chondrogenesis.
The original CM had no significant effect on chondrogenesis of mesenchymal cells. When the CM was dialyzed against culture medium to compensate for loss of nutrient, it stimulated slightly the chondrogenesis ofcells. Next, the CM was condensed to a quater of its original volume byuse of a collodion bag, dialyzed against culture medium, and its activity
was tested. It was found that a high moleculr weight fraction of CM had
a marked effect on chondrogenesis of cells. This culture method needs arelatively much amount of cells and CM. Therefore, a micro-drop culture

RESEARCHES CARRIED OUT IN 197837

method was developed to test the activity of CM. Inocula of 4 X 10' cells
were incubated in 10 pI drop of medium containing 7.5 pI of CM in 35-mm
petri dishes. Then 2 ml of culture medium were added to the petri dishes
after incubation for 2, 4 and 24 hours in micro-drops. By using thisculture method, the length of culture time required for manifestation of theCM activity on chondrogenesis was examined. On the fourth day of culture,
the number of cartilage nodules was scored. Results indicated that an
expression of chondrogenesis was almost completely established even whencells were incubated in CM for only first 2 hours. For details, see Cell
Structure and Function 3: 237-248, 1978.
Chondrogenesis of Quail Limb-Bud Mesenchymal Cells in
Clonal Culture
Etsuya MATSUTANI and Yukiaki KURODA

The in vitro chondrogenesis of mesenchymal cells depends on an initialcell density in culture. It suggests that mesenchymal cells may require theirmutual cell contacts as well as some factors released by conditioning the
medium. In the present experiment the effect of cell contacts on chondrogenesis was examined by clonal cultures of mesenchymal cells.
Mesenchymal cells were dissociated from the limb-buds of quail embryos
at stages 20-21. In the primary clonal cultures, distinct fibroblastic cellcolonies were formed, but differentiated cartilage colonies were not found.
To obtain the cartilage cell colonies from mesenchymal cells, cells werepre-incubated in the following two ways; 1) Cells were primarily cultured inmonolayer, then cells were dissociated again and secondarily cultured inclonal cultures. 2) Cells were incubated in the gyratory shakers to allow thecell contacts in aggregates, then cells were dissociated again and secondarily cultured in clonal cultures.
Cell suspensions containing 106 cells were incubated for various lengthsof time (4, 8, 12, 16,24 and 48 hours) in 2 ml of medium in monolayer, orin 3 ml of medium in rotation. In the secondary clonal cultures, various
numbers of cells (5 X 108, 2.5 X loa and 108 in 35-mm petri dishes) were
cultivated for 2 weeks and then fixed and stained.
Cartilage colonies were found when mesenchymal cells were cultured primarily in rotation, whereas no cartilage colonies were formed in cells primarily cultured in monolayer. The number of cartilage colonies increased

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

as the pre-incubation time in rotation culture increased. A maximum number of cartilage colonies was obtained when cells were pre-incubated for 24hours in rotation. The number of cartilage colonies decreased when cells
were pre-incubated for 48 hours. Limb-bud mesenchymal cells dissociatedfrom embryos at stages 20-21 fail in the expression of differentiatedcharacters in primary clonal cultures. They, however, can express theirpotency of chondrogenesis in clonal cultures if they are pre-incubated intheir mutual cell contacts for a relatively short period. In embryos at stages 24-25, the condensation of mesenchymal cells proceeds at the centerregion of the limb-buds, and leads to the chondrogenesis. This suggeststhat the cellular contacts in mesenchymal cells may be an important stepin the chondrogenesis in the limb-buds.
Induction of 8-Azaguanine Resistant Mutations by
Sterigmatocystin in Cultured Embryonic
Human Diploid Cells
Yukiaki KURODA

Sterigmatocystine (STC) is a strong mutagenic mycotoxin which induces
chromosome aberrations, DNA strand breaks and 8-azaguanine (8AG)
resistant mutations in mouse mammary carcinoma FM3A cells. In thepresent experiment, the activity of sterigmatocystine to induce 8AG resistantmutations in cultured embryonic human diploid cells was examined.
STC reduced the colony-forming activity of human diploid cells whencells were treated with STC for 4 hours at concentrations of 0.01 to 1.0 pgjml.
The Do value calculated from a concentration-survival curve was 0.63 pgjml.
Cells were treated with STC at various concentrations for 4 hours, cultured
in normal medium for 6 days of mutation expression time, treated with trypsin and replated on fresh dishes at a cell density of 5 X 104 cellsjdish.
After incubation of cells in the presence of 30 pgjml 8AG for 14 days, thenumber of 8AG-resistant mutant colonies was scored.
The frequency of 8AG-resistant mutations increased slightly when cells
were treated with STC at concentrations of 0.01 and 0.03 pgjml. At a concentration of 0.1 pgjml the induced mutation frequency was 10.2 per 10°
survivors. At higher concentrations more than 0.3 pgjml of STC, mutant
colonies were hardly detected bacause of severe cytotoxicity of STC.
This mutagenic activity of STC on human diploid cells is almost similar toRESEARCHES CARRIED OUT IN 1978

39

that in mouse FM3A cells previously reported.
Mutagenic Activity of Tryptophan Pyrolysis Products on
Embryonic Human Diploid Cells in Culture
Yukiaki KURODA

It has been recently found that typtophan pyrolysis products formed during cooking of foods were strong frame-shift mutagens for Salmonella
typhimurium with a metabolic activation. These products also inducedin vitro transformation in embryonic hamster cells. In the present experiment, the activity of tryptophan pyrolisis products, Trp-P-I and Trp-P-2,
to induce 8-azaguanine (8AG) resistant mutations in cultured embryonichuman diploid cells was investigated.
Trp-P-I had a concentration-dependent and severe cytotoxic effect on thehuman cells. The colony-forming activity of cells decreased as the concentrations of Trp-P-I increased. The Do value calculated from concentration-survival curves for 4 hour- and 15 day-treatments with Trp-P-I were0.53 flgfml and 0.35 flgfmI. Trp-P-2 had a weaker cytotoxic effect than
Trp-P-I on human diploid cells. The Do value of Trp-P-2 was calculated tobe 3.0 flgfmI.
Cells were treated with Trp-P-I or Trp-P-2 at various concentrations for
4 hours, cultured in normal medium for 7 to 9 days of mutation expressiontime, and treated with trypsin solution. Replated cells were selected with30 flgfml of 8AG for 14 days, The frequency of 8AG-resistant mutations
increased significantly compared with that in untreated control cultures.
The induced mutation frequency in cells treated with Trp-P-I at a concentration of 0.3 flgfml was 7.0 per 105 survivors, whereas that in cells treatedwith Trp-P-2 at a concentration of 1.0 flgfml was 2.8 per 105 survivors.
These results indicate that tryptophan pyrolysis products had severe cytotoxic effects on hyman diploid cells than AF-2 and phloxine tested in theprevious experiments, but the formers were weaker mutagenic for inductionof 8AG-resistant mutations than the latters at concentrations giving about
fifty percent of surviving fraction.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Genetic Study on Ingbly Teratocarcinogenic Charactor of
129/ter-Sv Mice
Takehiko Nooucm

An inbred subline of mice, 129/ter-Sv, has highest incidence of testicular
teratomas (33 %) out of 129 sublines. But the incidences observed in siblings vary from mating to mating. The range of variation was from about
80 % to less than 10 %. If siblings with a low incidence of teratomas weremated, the teratoma incidences of their offsprings nearly always remainedlow (personal communication with L. C. Stevens and D. S. Varnum). Teratoma incidence of this inbred mice decreased from generation to generation
unless selection was made. Another enigmatic point in the teratocarcinogenic charactor of this subline is that incidence of bilateral teratomas isabnormally high. In 129 sublines left testes have two times as high incidence as the right testes. The frequency of bilateral teratomas shouldbe the product of the frequencies of unilateral teratomas if teratocarcinogenesis in the right testes occur independently from that in the left. Theoretically calculated incidence of bilateral teratomas was 0.9 %. But the incidence
really observed was as high as that of the left testes (L. C. Stevens,
J. Natl. Cancer Inst. 50: 235-242, 1973).
Analytical studies on proliferation of primordial germ cells (PGC) of the developing testes of 129fter-Sv revealed that 129fter-Sv embryos were heterogenous in regard to the pattern of proliferation and the number of PGC.
The embryonic testes of 129fter-Sv could be divided into three groups. In
the testes of group (1), PGC number and their proliferative pattern werenormal as compared with the testes of other 129 sublines with much lower
incidences of teratomas. In the group (2) testes PGC were present at
normal level, but mitotically active stage was prolonged to a little, but asignificant extent. The testes of group (3) looked very abnormal; PGC
number was reduced extremely and the mitotically active stage was extended
considerably. Four out of 44 testes of group (1), 14 out of 22 testes ofgoup (2) and 9 out of 10 testes of group (3) had teratomas respectively.
Group (2) testes appeared in most cases bilaterally. Group (3) testes appearedalways bilaterally. These facts explain well the enigma that incidence ofbilateral teratomas is very high in this inbred mice.
Bilateral teratomas nearly always result in sterility. If the charactors ofgroups (2) and (3) are controlled by a genetic factor, mice having the factor

RESEARCHES CARRIED OUT IN 1978

41

will be removed from the colony due to their high tendency of sterility
unless they are retained by selection. This hypothesis coinsides with theregression phenomenon observed in the highly teratocarcinogenic charactorof this sublines.
Even in a litter of embryos, those having groups (1), (2) or (3) sometimes
coexisted. The hypothetical genetic factor would, therefore, be a recessive
mutation rather than a dominant one. This recessive mutation was tentatively named ter. Group (1) (2), and (3) were supposed to be ascribedto genotypes +/+, +/ter, ter/ter respectively. According to this assumption
129/ter-Sv mice could have three different kinds of genotypes. Matings
between mice with these genotypes would result in several kinds of
combination of the genes. This explains well the large variation in the incidences observed among siblings.
A muItipotential Teratocacinoma (STT-2) derived from a Maleof 129/ter-Sv Strain
Takehiko NOGucm and Yukika WADA

A multipotential teratocarcinoma having near 100% retransplantability
was established. This carcinoma named STT-2 was derived from a spontaneous testicular teratocarcinoma found in a male of 129/ter-Sv inbred
strain, and has been kept for more than 1 year by subcutaneous or
intraperitoneal transplantations. This is the first transplantable teratoma obtained from this inbred strain. Tissues or cell types hitherto
identified in STT-2 solid tumors were embryonal carcinoma cells, primitive
endoderm cells, embryonic mesodermal and ectodermal tissues, many kindsof epithelia, pigment cells, nervous tissues, striated muscles, adipose tissues,
cartilarges, bones with marrows, pancreatic cells. This means that STT2 is a typical multipotential teratocarcinoma.
In order to get embryoid bodies of this tumor, solid STT-2 was minced
and injected intraperitoneally in syngeneic hosts. Tumor tissues induced
accumulation of very bloody ascitic fluid. The fluid usually contained relatively a small number of floating bodies (at levels of two figures). The
sizes of those floating bodies sometimes reached to several milimeters in diameter. These were identified as cystic form embryoid bodies. They
contained a variey of differentiated tissues as well as embryonal carcinoma
cells. Tissues or cell types hitherto identified in those embryoid bodies

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

were proximal endoderm layers which were usually forming the outer layerof the bodies, embryonal carcinoma cells, trophoblastic giant cells, blood
islands, primitive mesodermal and ectodermal tissues, cardiac muscles, andso on.
Solid tumors formed at subcutaneous sites usually grew very large.
Necrotic parts were relatively small when compared to OTT6050 solid tumors.
STT-2 may, therefore, be useful as a source of embryonic substances.
Lethal Hybrid Rescue Gene of Drosophila simulans
Takao K. WATANABE

The unisexuality in the hybrid progeny between Drosophila melanogasterand D. simulans has been known since the discovery of the latter species(Sturtevant, Genetics 5: 488 1920). The hybrid offspring between melanogaster females and simulans males were females and those in the reciprocal
cross were males. In the case of the cross between attached-X melanogasterfemales and simulans males the offspring were only males. These abnormal
sex-ratios in hybrids are due to selective lethalities exerted during
larval stages. According to Sturtevant the interspecific hybrids develop to
the adults only if they carry a simulans X when the cytoplasm is ofmelanogaster.
A gene, Lethal hybrid rescue (Lhr 2-95), which rescues the lethal hybrids
between the above species was found on a second chromosome of D. simulans
from a natural (Kokura) population. The cross, melanogaster ~ X simulans d" which normally produced only female progeny, resulted in the production of both females and males at an equal frequency when thesimulans males carried Lhr. The reciprocal cross, simulans ~ X melanogasterd" which ordinarily produces only males, gave rise to about 14% females
and 86 % males. The cross, XXjY melanogaster ~ X simulans d' carrying
Lhr, produced a few percent females, although normal simulans produced
only hybrid males.
The cytoplasmic sex ratio (SR) factor and daughterless (da) gene killed
either hybrid males or hybrid females according to their usual actions, showing no specific interactions with the Lhr gene. All hybrids, those rescued
by Lhr as well as those produced normally, were completely sterile.

RESEARCHES CARRIED OUT IN 1978

v.

43

CYTOGENETICSThe Third Scientific Expedition for the Study of Rodents to Islands
and Coastal Area of the Indian Ocean, I. Members, Aims, Scheduleand Number of Animals Collected in the Expedition
Tosihide H. YOSIDA

The first and the second scientific expeditions with the financial aid of
the Ministry of education, science and culture, Japan, to survey therodents in Southwest and Central Asia and Oceania were orgainzed in 1968
and 1972. This is the third expedition of the same series with the following aims.
(1) Members:
Tosihide H. Yosida (Leader)
Kazuo Moriwaki
National Institute of Genetics
Hatao Kato"
Kimiyuki Tsuchiya
Hokkaido Institute of Public
Health(2) Aims and route of the expedition:
In the previous survey, we found that the black rats showed threegeographical variants due to the difference of the number and the shapeof chromosomes; Asian type (2n=42), Oceanian (2n=38) and Ceylonesetypes (2n=40). The Asian type distributed in East and Southeast Asia andthe northern India and Pakistan, while Oceanian type distributed widelyin the world except the above Asian district. The Ceylonese type was only
found in Kandy, Sri Lanka, in our second expedition. The main purposeof the third expedition is to know whether the Ceylonese type black rats were
distributed widely in Sri Lanka and also to find out which type of black rats,
Asian, Oceanian and Ceylonese types, distributes in islands, and coastal areaof the Indian Ocean. The other purpose of the expedition is to elucidate
the relationship between chromosomal changes and speciation in rodents
and other small mammals and to breed them in laboratory, if it is possible,
to estabilish new experimental naimals. For the above purpose the expedition was planned to the following areas from November 1st to December
14th, 1978; Penang (Malaysia), Sri Lanka, Seychelles, Kenya (Nairobi),
Mauritius, and Europe.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29(3) The number of animals collected in the third expedition:
Species names and the number of animals collected in the third expedition
are listed in Table 1.
Table 1. Animal species and number of speciemens collected in
the third expedition
SpeciesRodents
Rattus rattus
R. norvegicus
Bandicota
Mus museu/us
M./eggada
M. minutoides
ApodemusMastomys
C/ethryonomis
Insectivora
Suncusmurinus
TenrecPrimates
Tupia gUs

Penang Sri Lanka Seychelles Mauritius Kanya Europe
10
24
5

76
10
3
9
13

19

20
4

5

17

10

1
14
2
7

2
3

3
2

2
42

- - - - - - " - - - .. "---,-_._-_.

135
38
8
32
13
1
14
1
2
10

-

-~._-~-_._----

Total

Total

118

27

43

12

-

----------

17

259

-------~----The Third Scientific Expedition for the Study of Rodents to Islands
and Coastal Area of the Indian Ocean, II. Distributionof Ceylonese Type Black Rats
Tosihide H. YOSIDA, Kazuo MORIWAKI, Kimiyuki TsucillYA*, Hatao KATO,
M. SABARATNAM**, K. D. ARULPRAGASAM** and H. E. FERNANDO***In our second expedition during 1972, the Ceylonese type was obtained
only in Kandy, located in the Central highland of Sri Lanka (This report
23: 43, 1973). Although the Ceylonese type black rats were obtained inthis place, we did not know whether this type of black rats is distributedin the other parts of Sri Landa. In order to know this problem theblack rats were collected from the following 9 localities in Sri Lanka during* Hokkaido Institute of Public Health.
** Dept. Zoo!. Univ. Sri Lanka, Colombo Campus, Colombo, Sri Lanka.
*** Central Agricultural Research Institute, Peradeniya, Sri Lanka.

RESEARCHES CARRIED OUT IN 197845

Table 1. Karyotypes of black rats Collected in Sri Lanka in
the third expedition

2nLocalities

38

Central highland
Kandy·
Peradeniya
Nuwara Eliya
central lowland
Anuradhapura
Coastal area
Puttalam
ColomboHambantota
Galle
Trincomalee
Mannar--_.~._--_._-

3
11
10
7
10
11

40

Total (~ : 0-)

15
7

15
5
7

(8: 7)(3: 2)(4: 3)

42

6(4: 2)

2

3
15
10
7
11
11

(1: 2)

2

6
(6.6)

32
(35.5)

39(7: 8)(4: 6)(4: 3)(5: 6)(3: 8)

--

Total
(%)

52
(57.8)

90(43: 47)(99.9)* Collected in 1972.
the third expedition (Table 1); Peradeniya and Nuwara Eliya located in thecentral highland, Anuradhapura in central lowland, and Mannar, Puttalam,
Colombo, Galle, Hambantota and Trincomalee in the surrounding coastal
area. A total of 90 black rats was collected in 10 localities including Kandy
in 1972. Among them 52 (57.8 %) were of Oceaninan type, 34 (27.8 %) were
Ceylonese type and the remaining 4 (4.4 %) were hybrids between the above
2 types. The hybrids had 39 chromosomes just as seen in the F 1 in thelaboratory mating.
Interesting finding is that all 27 rats collected from the central highland(Kandy, Peradeniya and Nuwara Eliya) showed only Ceylonese type. In
Anuradhapura which is located in the central lowland, however, Ceyloneseand hybrid types were found. On the other hand, in the coastal area 57
black rats were collected and among them 52 (91.2 %) were of Oceanian type
and the other 5 were of Ceylonese (3) and hybrid types(2). The black rats
collected in Mannar, Puttalam, Hambantota and Galle located in the northern and southern coast were of only Oceanian type, but the Ceylonese andhybrid types were found in Colombo and Trincomalee located in the west
and east side of the island. Finding of these rats in these areas could be

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

due to the migration from the central highland by some communication.
On the origin of the Ceylonese type black rats we (yosida et al., Chromosoma 45: 99, 1974) suggested that they could have occurred in southern
India from Asian type by the first Robertsonian fusion. From this type thesecond Robertsonian fusion had been taken, and then the Oceaninan typeblack rats developed. The latter type with more stronger competitive ability
should have expelled the former rats from India to Sri Lanka. By thepresent survey the Ceylonese type seems to have been driven to the central
highland of the island by Oceanian type. Based on these survey it can besaid that the Ceylonese type rats are distributed only in the central
highland in Sri Lanka and they are isolated just as surrounded by the Oceanian type rats.
The Third Secientific Expedition for the Study of Rodents to Islands
and Coastal Area of the Indian Ocean, III. Finding ofMauritius Black Rats with Extra Small Acrocentrics
due to Robertsonian Fission
Tosihide H. YOSIDA, Kazuo MORIWAKI, Hatao KATo, Kimiyuki TsucffiYA,
Yuriko OCffiAI and J. MoNlY

Depending on chromosome numbers, the black rats, Rattus rattus, areclassified into three geographical types, such as Asia (42 chromosomes),
Ceylon (40 chromosomes) and Oceania (38 chromosomes) (Yoside et al.,1971, 1974). The Asian type black rats are distributed in East and Southeast Asia and northern part of Southwest Asia. The Ceylonese type is foundonly in Sri Lanka, but the Oceanian type is distributed widely throughout
southern part of Southwest Asia, Central Asia, Europe, Africa, Oceania,
North and South America. Recently we had an opportunity to observe thechromosomes of black rats inhabiting in Mauritius Island in the Indian
Ocean in the third expedition and found that the karyotypes of theseanimals were markedly different from the other three geographical types
described above. All seventeen black rats collected there were characterized by having many extra small acrocentric autosomes. Their basic karyotypes were of Oceania type, because of the presence of the large metacentric
M 1 and M z pairs, but chromosome numbers in 13 specimens among themwere 42, those of 3 specimens 43, and those of the remaining one specimen
44. Although the Oceanian type rat had 2 small acrocentric autosomes

RESEARCHES CARRIED OUT IN 1978

47

(pair no. 13), 16 Mauritius rats had 10 small acrocentrics, and the remaining
one had 8 samll acrocentrics. Comparative karyotype analysis between Oceanian and Mauritius type rats by G-banding staining showed that the extra
small acrocentrics found in Mauritius rats occurred due to Robertsonian fission of small metacentric pairs no. 14 and 18 of the original Oceanian typerat. Only one rat with 8 small acrocentrics showed the heteromorphic pairno. 18 consisting of one metacentric and two acrocentrics. The large metacentric M 1 chromosome in 13 of 17 rats examined showed homologous
pair, but two of them were heteromophic by involving one metacentric M 1and two acrocentrics. In the remaining two rats M 1 chromosome was notobserved, but acrocentric pairs no. 4 and 7 were included. These acrocentrics were also suggested to be originated from Robertsonian fission of
the large metacentric M 1 chromosome. Robertsonian fission seemed to
be one of the important mechanism in karyotype evoluton in this animal.
The Third Scientific Expedition for the Study of Rodents to Islands
and Coastal Area of the Indian Ocean, IV. Karyotype ofBlack Rats Collected in Penang (Malaysia),
Seychelles, and Nairobi (Kanya)
Tosihide H. YOSIDA, Hatao KATO, Kimiyuki TSUCIDYA, Kazuo MORIWAKI,
W. T. MENG*, S. K. CHARLES*, G. GILL** and Hiroyuki HIRUMI***To know the karyotype the black rats in Penang, Seychelles andNairobi were examined in our third expedition. In Peanng island (Malaysia) located in the Malacca Straits, 10 rats were collected. All of them were
the Asian type with 2n=42, and they were identified to be Malyan black rats,
Rattus rattus diarddi, from their external feature. As found in Malayan black rats by us (Yosida et al., Chromosoma 33: 252, 1971) the chromosome
polymorphism in pairs no. 1 and 9 were observed in these rats. They wereshown in Table 1.
The 19 black rats collected from Seychelles in Indian Ocean showed always a typical Oceanian type with 2n=38. The black rats in this island
were similar to R. rattus refescens in India. The Oceanian type occurredin India might have been migrated to this island. In Nairobi, Kenya, 10* Dept. BioI. Sci., Univ. of Sains Malaysia, Penang, Malaysia.
** Dept. Agriculture and Land Use, Republic of Seychelles.
*** International Laboratory for Research on Animal Diseases, Nairobi, Kenya.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29
Table. 1. Frequencies of acrocentric and subtelocentric polymorphismin pairs no. 1 and 9 of black rats collected in Pennag, Malaysia
Chromosome types
Pairs no.

1
9

AlA

AIS

SIS

2
1

3
5

3
2

Total no. ofrats observed
8

black rats were collected and chromosomes were observed. All these rats
were also characterized by the Oceanina type. The karyotype of black rats
in South Africa and Somalialnd has been observed to be Oceanian type byCapanna and Civitelli (Boll. Zool. 38: 151, 1971). From these studies distribution of black rats in the coastal area of Indian Ocean in Africa seems
to be Oceanian type.
The Third Scientific Expedition for the Study of Rodents to Islands
and Coastal Area of the Indian Ocean, V. Hemoglobin Typesand Chromosome C-band Patterns of the Mice Collected
in Sri Lanka, Seychelles Islandand Mauritius Island.
Kazuo MORIWAKI, Kimiyuki TSUCHIYA*, Hatao KATO, Tosihide H. YOSIDA and
Mitsuru MINEZAWA·*As an attempt to reveal the differentiation process of mouse species fromthe view points of biochemical- and cytogenetics, 37 wild mice collected in
the present expedition were examined as to the electrophoretic patterns ofbeta-hemoglobin (Hbb) and the chromosome C-band patterns as well.
Ten mice obtained from Sri Lanka were taxonomically identified as Mus
Table 1.
Place ofCollection
Sri Lanka
Seychelles Is.
Mauritius Is.

Hbb phenotype
Name of species
Mus musculus urbanus
Mus leggada
Mus musculus subsp.
Mus musculus subsp.

o OP P DS S
5

5

100

(unique bands)

40010
00025

* Hokkaido Institute of Public Health.
• * Primate Research Institution, Kyoto University.

No. of miceobserved
1016
5
7

RESEARCHES CARRIED OUT IN 1978

49

musculus urbanus, tentatively, 16 also from Sri Lanka as Mus leggada, 5from Seychelles island as Mus musculus subsp. and 7 Mauritius island as
Mus musculus subsp. The latter two subspecies were some what similar
to M. m. domesticus.
Table 1 summarizes the type of beta-hemoglobin found in those mice.
Those finding likely support the recent argument assuming wide distributionof Hbb P allele in the Asian mice (Minezawa et al., Jap. J. Genet. 54: 165,
1979). Chromosome C-band patterns of those mice were mostly similar to
those of the laboratory mice, M. m. domesticus.
A New Sporadic Robertsonian Fusion of Acrocentric Pairs no. 7
and 10 in the Black Rats from Sapporo
Tosihide H. YOSIDA

The black rats, Rattus rattus, have been found to have three geographicaltypes, Asian (2n = 42), Ceylonese (2n=40) and Oceanian (2n=38». The
Ceylonese type has been derived from the Asian type by Robertsonian fusion
between pairs no. 11 and 12. The Oceanian type was remarkable by having
the other Robertsonian fusion between the pairs no. 4 and 7. All about
one thousand black rats, so far the chromosomes were observed by thepresent author in Japan, were characterized by having always 42 chromosomes with the Asian type idiogram. Recently five black rats (R. rattus
tanezumi) were collected from Sapporo, Japan by courtesy of Dr. Kimiyuki
Tsuchiya. Among them four rats were found to possess a normal complement consisting of 42 chromosomes. One female among them, however,
was found to have 41 chromosomes in all 15 metaphase cells obtained
from the primary cultures of the tail tissue by the author's routin procedure.
Among them one large metacentric element was outstanding on accountof its consistent occurrence in their metaphase complement. By applying
the G- and C-banding techniques it was revealed that the metacentric element
was derived from Robertsonian fusion between each one of the acrocentric
pairs no. 7 and 10.
Interesting is that the Oceanian type black rat is characetrized by theRobertsonian fusion of pairs no. 4 and 7, and the pair no. 7 is also
involved in the chromosome fusion in the present material. This chromosome pair seems to be liable to fuse with the other chromosome. The
occurrence of a Sapporo black rat having one large metacentric element

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

due to the fusion will serve as a matter important for the possibility of
the karyotype evolution in the geographical variants of black rats, such as
Ceylonese and Oceanian types, through a mechanism of the Robertsonian
fusion.
Histological Study on the Hybrid Embryos Developed by Artificial
Insemination between Norway Rats and Black Rats
Choji TAYA and Tosihide H. YOSIDA

Histological examination of hybrid embryos developed by artificial insemination between female Norway rats (Rattus norvegicus, 2n=42) and male
Asian type black rats (Rattus rattus jlavipectus, 2n=42) was carried out
with comparison between the hybrid and control embryos which developedby artificial insemination between female and male Norway rats. Although
the delay of development of the hybrid embryo was ovserved, the endoedrm
and ectoderm differentiated well on the 7th and the 9th day embryos as seenin control ones. The development of the mesoderm, however, was poorin these stages in the hybrid embryos. Until the 10th day after insemination,
the hybrid embryos were clearly observed in the decidua although the development has markedly delayed. On the 13th days the embryo had degenerated
completely and the rudimental embryonic cells were only observed in thedecidua. Lethality of the hybrid embryos seemed to be due to the marked
delay of the development and differentiation.
In vivo Sister Chromatid Exchange in Cells of Various Organsof the Mouse.
Naotoshi KANDA and Hatao KATO

In vivo sister chromatid exchange (SCE) in mouse cells derived fromvarious organs Was studied by infusing BrdU from the tail vein. It was
found that at BrdU concentration ranging from 2.2-13.5,ug/g/h, the SCE
frequency in bone marrow cells seemed to stay at a constant level (1.5-2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded thisdose range. When BrdU within this dose range was infused continuously
from the tail vein for appropriate hours to label chromosomes in variousorgans, the average SCE frequencies per cell were found to be 1.64 in bonemarrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal

RESEARCHES CARRIED OUT IN 197851

cells and 3.69 in cells from adjuvant stimulated lymph nodes. It was suggested that the spontaneous level of the in vivo SCE frequency might beabout 1.5-2JcellJtwo cell cycles in the mouse. In cells derived from intestine
and adjuvant stimulated lymph node, some unknown factors might work
as a inducer of SCEs resulting in a signifiiant increase in the SCE frequency
in these organs.
Analysis of Crossing Over in the Mouse Meiotic Cells by BrdU
Labelling Technique.
Naotoshi KANDA and Hatao KATO

Sister chromatid differential staining of male mouse meiotic cells wasachieved by the continuous BrdU infusion from the tail vein and following
fluorescent plus Giemsa staining technique, Analysis of 296 bivalents including XY, and 21 univalents reveals that; 1) The visible crossing overcoincided exactly with the chiasmata. 2) No evidence was obtained in support of the chiasma terminalization. 3) Anormalous type of crossing over
was found in the monochiasmatic bivalent. 4) Some of the terminal
associated bivalents might be achiasmatic, and univalents might originatedfrom such bivalents. 5) Sister chromatid association of XY bivalents may
be under genetic control, for the chromatid association of 25 XY bivalents
were all between lightly and heavily stained chromatids. 6) The chromosomal site, at whcih crossing over occurred, were unstained, suggesting that
this portion may differ structurally from the rest. 7) Sister chromatid
exchanges might be rare event during meiosis.
Comparison of Karyotypes between the Japanese Shrew Mole andthe Furry Snouted Shrew Mole
Takashi HAMADA and Tosihide H. YOSIDA

Karyotypes of the Japanese sherw mole (Urotrichus talpoides) and thefurry snouted shrew mole (Dymecodon polirostris) belonging to Insectivora
were analysed and their evolutional relationship was considered. Both species are similar in external feature, but the latter is slightly smaller than theformer and their dental form is different. Chromosome numbers of these
species are already reported by Tuschiya and Yosida (This report 21: 54,1971), but detail of karyotype is not yet recorded.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Material used in the present study were collected in the foot of Mt. Fuji,
Shizuoka. The chromosomes were observed from cultured tail cells by ourroutine technique. Chromosome number in both species was 2n=34 andtheir idiograms were the same as the above report. They are consistedof 11 metacentric pairs (nos. 1 to 11),2 submetacentirc pairs (nos. 12 to 13)
and 3 subtelocentric pairs (nos. 14 to 16), and a large metacentric X and asmall minute Y. Although the species had similar karyotype, subtelocentric
pair no. 14 is different between them. In the furry snouted shrew mole thelength of the longer arm in this pair was longer than that in Japaneseshrew mole, and therefore the arm index in both species is very different;in the former species it was 4.5 but in the latter 1.7. Another difference
was found in submetacentric pair no. 12. In Japanese shrew mole the shortarm of the pair is longer than the furry snouted shrew mole. In the former
this chromosome looks metacentric. All other chromosomes in both
species are very similar. According to taxonomists the furry snouted shrew
mole is more primitive than the Japanese shrew mole. Based on ourobservations and the taxonomists suggestion, the Japanese shrew mole issuggested to be derived from the furry snouted shrew mole by the following
cytogenetical mechanism; the breakage of long arm of pair no. 12 in thefurry snouted shrew mole had occurred and the broken end wastranslocated to the short arm of the pairs no. 14, and then the karyotypeof the Japanese shrew mole was established.
Karyotypes of Four Filefishes
Makoto MUROFUSHI* and Tosihide H. YOSIDA

Karyological studies in four species of filefishes belonging to Balistidae(Stephenolepis cirrhifer, S.japonicus, Paramonacanthus oblongus, and Navodon
modestus) were carried out. All these fishes were obtained between Juneand September, 1978, off the west cosat of Izu Peninsula, Japan, and thechromosomes were observed directly from the kidney or the gill tissues.
Diploid chromosome numbers in Stephenolepis cirrihifer, S. japonicus andParamonacanthus oblongus were 34, but in Navodon modestus it was 40. All
chromosomes in these four species were acrocentrics ranging from large tosmall size. The later one species is identified from the others by different
chromosome numbers, but the former three are difficult to be identified by*

Misima Junior College, Nihon University, Misima.

RESEARCHES CARRIED OUT IN 1978

S3

the karyotype from each other. These three species, however, were discriminated by the secondary constrictioin in one chromosome pair, which was
found in pair no. 9 in the case of Stephanolepis cirrhifer, pair no, 6 in S.
japonicus and pair no. 2 in Paramonacanthus oblongus.
Differential Staining of Inactive X Chromosome in 25 Rodent Species
Naotoshi KANDA and Tosihide H. YOSIDA

Twenty-five rodent species, mainly obtained from breeding colonies at
the National Institute of Genetics and some were captured in the wild, wereused in the present studies. Bone marrow cells and/or cultured cells derived
from the tail tip and lung tissues were treated with a 50°C hypotonic solution
and stained with Giemsa. With this technique the facultative heterochromatic X chromosome or the facultative portion of large, composite type X
chromosomes is stained darker than the other chromosomes, allowing it
distinguished from the homologus euchromatic X chromosme in female
metaphase cells. Intense staining of the single X chromosome was notobserved in male metaphase cells. It is suggested that this differential
staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to difference in the degreeof chromosomal condensatin or in DNA base sequence.
Comparative Analysis of Japanese Wood Mice from Oki Islands
and Mainland from View Points of Biochemicaland Cytogenetics
Hirohisa HIRAI,I) Teru Aki UCHlDA2) and Kazuo MORIWAKIOki Islands are located about 100 km far from Simane Peninsula, consisting of Dozen and Dogo. Aoki (Zool. Soc. Tokyo:3l-40, 1915) andTokuda (Bio-geog. Soc. Japan 4:79 and 127, 1941) termed Japanese woodmice of Mainland as Apodemus speciosus speciosus and those of Oki Is.
as A. s. navigator by their morphological features. Thereafter, Hiraiwa
et al. (Sci. Bull. Fac. Agr. Kyushu Univ. 16:547, 1958) and Miyao et al.
(Mamma. Sci. 16:37, 1968) claimed that the animal from Dozen could be
distinguished from A. s. navigator of Dogo at the level of subspecies, com1)

2)

Department of Parasitic Diseases, Kumamoto University School of Medicine
Faculty of Agriculture, Kyushu University

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

paring either the tail ratios and the dead centre of their skull specimens.
On the other hand, Imaizumi (Aniaml-life 1:79, 1971) denominated theanimal from Oki Is. as a new species, A. navigator navigator.
We examined those two problems by estimating the genetic distance andby the comparison of chromosome C-band patterns.
Fifty three individuals were collected rom two localities in Oki Is. (Dozenand Dogo) and three in Mainland (Daisen, Tottri and Toyama). Electrophoresis of plasma, hemoysate and liver extract by using thin layer acrylamide gel (Hirai et al., This peport 28 :36, 1978) and cellulose acetate membrane was achieved in order to estimate the gene freuqencies of the following 15 loci, Alb, a1MG, azP, Es-l, Es-2, Es-3, Es-4, Es-lO, Gpi, Hb, Id-1,
Mod-I, Pgm, Pre-Alb, and Tf. Genetic distance was calculated using thosedata by Nei's formula (Am. Nat. 106:283, 1972). C-banding patterns inchromosome were observed by BSG method (Sumner, Exptl. Cell. Res.
75:304, 1972).
Genetic distance in the mice between Dozen and Dogo was 0.026 andthat between Oki Is. and Mainland, 0.064. Taking the data in rodents
by Nei (Molecular Population Genetics and Evoultion, 175-209, 1975) and
Britton and Thaler (Biochem. Genet. 16:213, 1978) into consideration, we
assumed that genetic distance between Dozen and Dogo was involved inlocal race and that between Oki Is. and Mainland was at subspecies level,although it might not be concluded based only on genetic distance.
No reports have ever referred of the C-banding patterns in chromosomesof Japanese wood mice hitherto. In the present study, we failed to show
any significant difference in the patterns among the mice of various geographic origins.
An Attempt on the Cytological Identification of Chromosome
in Some Linkage Groups of the Silkworm
Akio MURAKAMI, Akio OHNUMA and Hirotami IMAIIn the previous report [This report 28: 68, 1977], it was communicatedthe result of cytological identificaion of the Z-chromosome in the silkworm
with the hypotriploid (3A +ZZ) male and indicated that the Z-chromosome
is one of the member of the large chromosome group or the 3rd longer. Thismethod is applicable to other linkage group chromosomes, but it takes
time to construct such the hypotriploid individuals for the specific linkage

RESEARCHES CARRIED OUT IN 1978

55

group chromosome. Whereas, in this insect, it is fairly easy to make thetranslocated chromosome marked with a small piece of the chromosome
in a specific linkage group. Accordingly, it is expected that, with the use of
the translocated chromosomes and the highly improved chromosome observation technique (e.g., Imai et al., 1977), the specific chromosome would bepossible to cytologically identify and correspond to the linkage group.
The present communication will report a result of preliminary chromosome observations to cytologically confirm the genetically well analyedtranslocated or fused chromosome complexes.
In this experiment, T(Z:\00+), T(Z:~br..), T(~br ..) or known as thetranslocated zebea and a spontaneously fused chromosome complex, F(VI:
XIV), were chosen as sample materials. The T(Z:\00+) line has a complicated chromosome complex between the Z chromosome and W chromosome with a piece of the 5th chromosome including pe+ gene locus, theT(z:Webro.) line has a translocation between the Z and W chromosome
with a piece of the 3rd chromosome including the zebra locus, theT~bro.) line has a simple transolcation between the W chromosome and 3rd
chromosome marked with the zebra locus. The F(VI :XIV) line is generously supplied from Dr. M. Tsujita. The translocated zebra, which had
been constructed by the late Dr. H. Hashimoto (1948). is maintained in ourlaboratory. The others were recently constructed with the radiationtreatment method and all of them are maintained in our laboratory.
The chromosome observation was subjected to meiotic spermatocytes, except
for the translocated zebra. For this line, the chromosome observation wasmade with premeiotic larval oocytes.
In the T(z:W?e+) line, some of preparations were shown to be normal
figures at the first meiotic mataphase in spermatocytes with n=28, however,in the others the complicated chromosomeal configuration wth n=27 wereclearly observed. In a certain case, it was observed a minute chromosome
in meiotic spermatocytes with n=28. Occasionaly, it was observed a more
complicated chromosome figure at the first meiotic metaphase in spermatocytes with n=29: a minute chromosome links the chromosome complex
having a part of the Z chromosome translocated with a part of W chromosome to the 5th chromosome. There were often obesrved differential chromosome configurations from preparation to preparation, suggesting that
this might be resulted from sampling procedures.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29--

In the T(Z :wzebra) line, at the first meiotic metaphase in spermatocytes,
a translocated chromosome configuration between the Z and W chromosome
marked with a piece of the 3rd chromosome was clearly observed.
In the F(VI :XIV) line, the fused chromosome configuration between the
VI and XIV chromosomes was frequently detected at metaphase and diakinesis in the first meitoic spermatocytes.
It should be noted, however, that in the translocated zebra, it was notdetected the translocated chromosome configuration in the premeiotic oocytesby the air drying preparation method, indicating that chromosomes in the
meiotic oocytes are not competent for cytological observations.
Mechanisms on the Sterility in Triploid Silkworms
Akio MURAKAMI, Akio OHNUMA and Hirotami IMAI

It is well known that, in the silkworm, triploid individuals are highly
sterile regardless of highly viable as in other organisms. Knowledge on the'mechanisms of the sterility in the insect seems to be meagre, although its
description on such the phenomenon has been rather extensively reported.
The present report aimes at obtaining more information from cytologicaland genetical viewpoints on the relationship between the sterility and triplidy
in the silkworm.
The triploid silkworms used for the present experiments were obtainedby the X-ray-indiced method (Murakami, 1976) for females (3n=8IA+ZZW,
where "A" means autosomes and Z or Ware sex-chromosomes), and by
CO 2-nondisjunction method (Murakami et al., 1978) for hypotriploid males(3n-l=81A+ZZ).
In the hypotriploid males, twenty-seven trivalents (3A) and one bivalent(ZZ) were observed at zygoten and pacyten in spermatocytes, but one or
two chromosomes began to detach from some trivalent chromosome sets at
diploten and diakinesis. Consequently, a number ofunivalents and bivalents
as well as trivalents were appeared at the first meiotic metaphase. As is wellestablished, univalent chromosomes move to either pole by chance and theseparation of trivalents was also irregular. Even chromosome segregationis quite unbalanced at the first meiotic division, the division was completed
apparently without any abnormal behaviours. In contrast, the second division is remarkably delayed as compared with the normal diploid cells depending on the formation of "twin-spermatids" connected by the chromosome

RESEARCHES CARRIED OUT IN 1978

57

bridge. This situation remained to the mid-pupal stage, whereas the normal
spermiogenesis completes by the last larval instar. It is of interest to notethat some of the twin-spermatids transforms finally into spermatozoa, but
most of them are non-functional or apyren. The formation of the apyren
might be partly due to the spermiogenesis under abnormal physiological or
hormonal conditions as compared with the normal ones. It is a matter ofcourse, the sterility in the triploid males is mainly due to the genetical unbalance in chromosomal contsitutions by the abnormal chromosomal segregation in meiosis.
In the triploid females, there were laid two different types of eggs, apparently normal shape eggs varied with their size and flaccid eggs with a small
amount of yolk in a ratio of one to one. These normal eggs or oocytes
are fertilized normally with haploid sperm, but development of their zygotes
is arrested in embryonic stages being due to the unbalanced chromosome
constitution as the abnormal chromosome segragation in meiosis. While,the flaccid eggs are unable to fertilize with haploid sperm. In this connection, it is of worthwhile to note here that, in the silkworm, the egg-formationvitellogenesis and chorion formation-is completed prior to the commencement of the first meiotic division. Consequently, it can be imagined thatthe occurrence of the flaccid eggs seems to have relation to some defeat
at the chromosome (or gene) level in the process of egg-formation. Thisdefeat may be reseuted from the differential sex-chromosome pair in the
triploid (ZZW): the chromosome set of a (ZW)Z pair would be able toform the normal eggs, while that of a (ZZ)W would form the flaccid eggs.
In summary, it can be said that the sterility of the troploid silkworms
may be caused by the abnormal chromosome (gene) function in oogenesisor the abnormal environmental condition in spermiogenesis as well as theabnormal chromosome segregation on the both gametogenesis.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

VI.

MUTATION AND MUTAGENESIS IN ANIMALSStudies on Differential Mutation Response between Bombyx and Drosophila
to Some Indirectly Acting Chemical Carcinogens: (2) In vitro
Metabolic Activity Test of Drosophila Microsomes for NDimethylnitrosamine (DMN) and N-Acetyl-2-Aminofloorene(AAF)
A. R. KASTURI BAI, Michiko, GOTO and Yataro TAZIMA

Bombyx and Drosophila respond differentially to some indirect carcinogens/mutagens with regard to mutation production. DMN is clearly effective
in producing recessive lethal mutations in Drosophila, but it is not so in
Bombyx when tested by specific loci method. In contrast, AAF is incapableof producing recessive lethal mutations in Drosophila, whereas positive
mutagenicity is reported for Bombyx.
In order to elucidate the mechanism(s) underlying the discordance, we
attempted to compare the metabolic activation potential of microsomes
between those two insects using back mutation system of Salmonella.
Procedures for preparation of microsome fraction, mutagenicity assay with
Salmonella, etc. were reported previously together with experimental results
obtained for Bombyx. The work was extended to Drosophila using OregonR strain and same procedure. Microsome fractions were prepared fromhomogenates of larva, adult whole body, adult abdomen only and adultwhole body that fed on 0.005 % DMN for two days.
Experimental results: (1) Microsome fractions extracted from Drosophila
were incapable, as observed for Bombyx, to activate DMN to substances
mutagenically positive to Salmonella suggesting that both insects lack,or are very weak in, the capacity of activating DMN. (2) Drosophila
microsome fractions were active in converting AAF to mutagenically positive substance to Salmonella, which showed also a good accord with thoseobtained for Bombyx.
Above results do not support our previous expectation that discrepancies
in mutation responses between two insects could be explained by thedifferential metabolic capacities which are detectable by Salmonella backmutation system.

RESEARCHES CARRIED OUT IN 1978

59

Since mechanism(s) concerned with mutation production in insects aredifferent from those known for Salmonella, metabolic intermediates that can
produce mutations in Salmonella might not have been effective on mutation
production in insects.
Mutagenicity of Dimethylbenzanthracene in the
Silkworm Germ-Cells
Akio MURAKAMI, Michiko GOTO and Yosoji FUKASE

The mutagenicity test in grem-cells of the silkworm with several heterocyclic hydrocarbons, benz(a)pyrene, benzanthracene and so on, has been
carried out for several years, the results obtained so far indicated that thehydrocarbons are not mutagenic. Then, the mutagenicity test of 7,12dimethylbenzanthracene (DMBA), one of known strong mutacarcinogens,has been done with the silkworm egg-colour specific locus mutation method.
DMBA was dissolved in 0.025 ml of 0.5 %carboxyl methyl cellulose (CMC)
solution immediately before use. Solution of CMC was prepared in 0.85 %NaCI or saline solution for the silkworm. The mutagenicity of DMBA
was tested in (CI08xAojuku) F 1 hybrid female or male silkworm pupae.
A single dose of 5, 10, 20 or 40 ftg per capita of DMBA was injected into
the ventral body-cavity of seven day-old pupae. The control pupae received 0.5 % CMC solution without DMBA. The weight of the hybrid
pupa was ca. 1 to 1.5 g for female and ca. 0.5 to 1.2 g for males. Afteremergence, treated pupae were mated with untreated marker moths homozygous for egg colour genes at pe and re on the chromosome 5. Oviposited F 1 eggs were scored on the visible phenotype with whole-body or mosaic
mutations.
The result of the experiments indicated, however, that no significant
increase of any type of the mutations at both loci in pupal silkworm oocytes and spermatozoa was observed. This finding and others suggest thatsilkworm pupae are unable to biotransform the heterocyclic hydrocarbons
including DMBA into their active forms. It is very possible to expect that
this defect would be cover up if the mutation test method were coupledwith a metabolic activation system which allow detection of mutagenicity ofsuch the promutagenic compounds are reported previously for the detectionof mutagenicity in pupal spermatozoa of the silkworm with 2-(2-Furyl)3-(5-nitro-2-furyl) acrylamide [This report 26: 45, 1975]. DMBA was in-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

cubated with the incubation mixture including the rat-liver microsome fractions and the relevant cofactor systems at 37°C for 20 min. The resultant
reaction mixture was immediately administered by injection corresponding
to a dose of ca. 10-50 pg per capita of DMBA at the time of incubationin the body-cavity of the mid-stage female pupae. They were then mated tothe marker males to check the occurrence of egg-colour specific locus mutations. The result of the experiments indicated that the incidence of mosaic
mutations was almost linearly increased with the treated dose of DMBA,
while no significant increase in the whole-body mutations was observed
regardless of the loci tested. Frequencies of DMBA-induced mosaic mutations in oocytes were higher at the pe locus than re locus as observed withthe known chemical mutagens for the silkworm.
This finding suggests that the silkworm/rat-liver microsome systemthe silkworm egg-colour specific locurs mutation test method coupled with
the in vitro rat-liver metabolic activation system-is available to the mutagenicity test for promutagenic heterocyclic hydrocarbons.
(This research was partly supported by research grants from the Ministry of Education, Science and Culture and the Agency for Science and Technology.)
The Mutagenic Activity of Mycotoxins in the Silkworm
Akio MURAKAMI, Toshiko OZAWA and Yosoji FUKASE

Aflatoxin B1(AFTB1), a metabolic product of Aspergillusf/avus and its closely related species, iswell known to be the most potent hepatocarcinogenic
substance in experimental animals and it is frequently found in a variety
of agricultural products subject to spoilage by the mould. Such being thecase, this mycotoxin has often been the subject of environmental mutagen
studies with a various type of biological materials. Consequently, AFTB1
is also shown to be higWy mutagenic in many biological test systems. Mostof the studies tend to indicated that AFTB 1 is not genetically active per se,
but do require metabolic activation. In view of the general lack on mutagenic effects of AFTB1 on germ-cells of the silkworm, we carried out to study
the potentials of this mycotoxin and Sterigmatocystine (STC), which is ametabolic product of A. versicolor, in causing recessive visible mutationsin the insect. It should be emphasized that A. versicolor grows on lrice aswell as other cereals.

RESEARCHES CARRIED OUT IN 1978

61

In the experiments reported here, both sex wild-type F 1 (C108xAojuku)
hybrid pupae were injected intra-abdominally with 0.025 ml of various
concentrations (0.1-10 ,ug/capita) of the AFTB 1 or STC in 0.5% carboxyl
methyl cellulose (CMC) solution immediately before use. The germ-cellstage at this stage of pupae are prophase I oocytes and spermatozoa for female and male, respectively. Control pupae received 0.025 ml CMC solution
without the mycotoxin. Recessive visible mutations were detected by thepe :re egg-colour specific locus method. The treated pupae were mated tothe marker stock moths. All F 1 individuals (or eggs) were scored on thevisible phenotype with whole-body or mosaic mutations.
It was found that both mycotoxins are highly mutagenic in silkworm pupal
oocytes and spermatozoa and that, as the properties of chemical mutagenesis,
the frequency of induced mosaic mutations is much higher than that of wholebody mutations. The incidence of whole-body mutations seemed to be
somewhat higher than that of he corresponding control. The dose-response
relations for the mosaic mutation at both loci of pupal oocytes or spermatozoa
were a nearly linear fashion over the dose-range applied from 1 to 10 ,ug/capita.
It should be noted that the dose-response curves of mosaic mutations inoocytes by STC was linear down to a dose at 0.12 ,ug/pupa or ca. 0.1 ,ug/g
body weight and suggesting whether a threshold dose is existent at the very
low dose-range or not. The mutagenicity of AFTB 1 was higher in spermatozoa than oocytes, whereas that of STC was somewhat less in sperm thanoocytes. The mutagenic activity of AFTB 1 was about 10 times higher thanthat of STC in spermatozoa, while it was observed AFTB 1 to be as active as
STC in oocytes. In any case, the mutagenic activity of these two mycotoxins in the germ-cells of this insect is regarded as one of the mostpotent chemicals among indirect mutagens tested and that is somewhat
weak as compared with those of N-ethyl-N'-nitro-N-nitrosoguanidine, Ethyl
methansulphonate or Mytomycin C.
The result of the present experiments also showed that in the silkworm
germ-cells the mutagenic effect of STC incubated in the presence of rat-liver
microsome fraction appeares to be significantly reduced as compared withthat in the absence of the liver microsome fraction, which suggesting thatthe silkworm is physiologically competent in the metabolic activation of
the mycotoxins. Accordingly, it seemed that the silkworm egg-colour specific locus mutation test system may be useful as a prescreening for such the
mycotoxins that require metabolic activation.

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29(This research was supported in part by a grant from the Ministry of
Education.)
Genetic Effects of Tranquilizers on Silkworm Germ-Cells
Akio MURAKAMI, Toshiko OZAWA and Yataro TAZIMA

Some ataractic drugs have been tested for their carcinogenicity and cytotoxicity in several biological systems on account of their chemical structure
and common usage in our daily life. However, an obvious carcinogenicity of such the drugs has not been reported in distinction to theircytotoxic effects on sme test systems. In order to determine whether or
not genetic toxicity were observed in germ-cells of the silkworm, the midstage wild-type pupae of F 1 (CI08xAojuku) hybrids were abdominally injected Chlorodiazepoxide (7-Chloro-2-methylamino-5-phenyl-3H-l,4-benzodiazepin-4-oxide) HCl dissolved at a concentration of 50, 100, 150 or 200,ug/pupa in 0.025 ml of 0.5% Carboxyl methyl cellulose (CMC) solution.
They were then mated to marker stocks homozygous for egg-colour genes,
pe and re loci, to check the occurrence of recessive visible mutatons.
Chlorodiazepoxide HCl used in this study was supplied from the National
Institute of Hygienic Sciences, Tokyo as a test sample (:11:78-20) for the
Cooperative Programme on Short-Term Assays for Carcinogenicity inJapan. For the control, pupae were injected with 0.025 ml of 0.5 % CMC
solution alone.
The results obtained indicate that the lower doses, 50 and 100 ,ug/pupa
of Chlorodiazepoxide did not induce a significant increase in the mumber ofboth complete and mosaic mutational events in females (or oocytes), whilethe 150 and 200 ,ug dose of the drug induced a significant increase in thenumber of both complete and mosaic mutational events. It is well established that, in the silkworm, the majority of mutants induced in oocytes
after treatment with typical mutagenic chemicals is mosaic types, but theincidence of complete ones is very rare. Consequently, it can be said that
this high incidence of complete mutations seemed to be a characteristicfeature of this drug mutagenesis. Neither dose of Chlorodiazepoxide increased the mutational frequency above control level in the treated malepupae. The cellular difference may be interpreted due to either differential
DNA conformation or physiological condition in the germ-cells. Dataobtained in the present experiment together with results obtained previously

RESEARCHES CARRIED OUT IN 1978

63

indicate, however, that Chlorodiazepoxide is certainly not the sort of strong
chemical mutagens. At the higher concentrations the sterility was more
greatly incrased in the treated female pupae than in the male ones. In any
case, there is a possibility that an ataractic drug Chlorodiazepoxide HCI iscapable to induce germ-cell mutations in eukaryotes.
Diazepam, 7-Chloro-1 ,3-dihydro-I-methyl-5-phenyl-2H-I ,4-benzodiazepin-2-one, is a structurally related compound to Chlorodiazepoxide and has
also been used as a tranquilizer. This drug (~77-22) has also been
selected as a test chemical for the evaluation of its mutagenicity andcarcinogenicity by the Ministry of Health and Walfare, Japan. Diazepam
was found to induce an increase in the number of egg-colour specificlocus mutations in the treated pupal oocytes, indicating that it seems tohave a positive mutagenic action for grem-cells of the silkworm female.
In the Diazepam-treated male pupae with a drug concentration of 100 f.lg
per capita, there was no apparent increase in the frequency of the specificlocus mutations over the frequency in control experiments. The mutagenic
activity of Diazepam seemed to be about the same as that of Chlorodizepoxide HCI. The marked sterility was also observed at the higher concentration doses in either female or male.
(This research was supported in part by a Grant-in-Aid for Cancer
Research from the Ministry of Health and Walfare, Japan.)

Induction of Mutations in Hamster Embryonic Cells from Mothers
Received Total Body Irradiation of r-Ray
Tsuneo KADA, Taiji ASANO, Akiko YOKOlYAMA and Naomichi INUI1)

Induction of mutations resistant to 8-azaguanine (8AG) has been successfully observed in embryonic cells that had been isolated and cultured inin vitro from pregnant hamster exposed to different chemical mutagens(N. Inui et af., Mutation Res. 41: 351, 1976). This method was applied inradiation studies. Syrian golden hamsters on the 11th or 12th day of pregnancy were exposed to r-ray of different doses from a 6000 Ci source of
mcs. Subsequently the animals were given standard laboratory chew andwater add libitum for 24 hours. Their fetuses were then excised, finely
minced and dijested with 0.25 per cent trypsin. Primary cultures of the1)

Biological Research Center, ITS, Hadano, Kanagawa 257

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

trypsinized cells were initiated by seeding 1-5 X 106 cells into 10 ml of culture
medium in 72 cm 2 Falcon plastic tissue culture flasks. Cells were grown in
MEM supplemented with 10 per cent FCS (GIBCO N.Y.) at 37°C under
5 per cent CO 2 in air for 72-120 hours. To induce 8AG-resistant mutations,cells were transferred into dishes containing medium with 8AG (l020 pg/mI). The medium containing 8AG was changed every day for the
first 3 days and every 3 days for further 12 to 17 days. The cells in the dishes
were fixed and stained with Giemsa and the number of mutant colonies
was scored. Induced frequencies increased linearly from 17 to 250 rads(from 10 to 220 mutations per 107 viable cells) then a plateau was fond forboth 10 and 20 pg/ml AG resistance. Killing effects were expecially prononuced at a dose of 1000 rads, otherwise the present data on mutation induction were obtained under moderate surviving rates.
Studies on the Natural Antimutagens in the Environment
Tsuneo KAnA, Yoshiko OHTA, Masako HARA, Akiko YOKOlYAMA andTadashi INOUE

We already reported tha certain bio-metalic compounds such as cobaltous
chloride possess potent antimutagenic capacities in bacteria (E. coli WP2and B. subtilis mut-1) against spontaneous an radiation (UV and r-ray)-induced mutabilities (Ann. Rept. NIG No. 28: 77, 1977; Proc. Japan
Acad. 54B: 234, 1978). Recent observations showed that similar antimutagenic effect exists also in cultured mammalian cells such as the strain V79(Chinese hamster) for mutations producing 8-azaguanine resistant cells by rirradiation. Two types of hypothesis are under examination of mechanisms
involved. In the first, the cobaltous ions may serve as a error-proofing cofactor in radiation-induced SOS enzyme(s) involved in error-prone DNA
repair and replication. In an alternative one, we assume that the radiations
might affect primarily genes of mutator function and the induced constitutive mutator clones are sensitive to the metal compound.
We are now studying extensively on antimutagenic activities in the naturalenvironment including food materials. Our first survey on vegetables andfruits showed that Shiitake mushrooms contain a potent antimutagen which
has difusib1e low molecular weight. An another example of plant antimutagens was found in the family of lily.

RESEARCHES CARRIED OUT IN 1978

VII.

65

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICRO-ORGANISMS AND PLANTS
Induction of Mutation and Phage by Synchrotron
Orbital Radiation in Bacillus suhtilis Spores
Tsuneo KADA and Yoshiko OHTA

It is interesting, from radiation-biological and photobiological standpoints,

to know how synchrotron orbital radiation (SOR) interacts with cellularDNA. Our present studies showed clearly that the SOR induces gene mutation as well as SP02 phage development in Bacillus subtilis by
exposing their spores.
For experiments of mutation induction, spores of B. subtilis NIG
1121 (his met) were prepared on Schaeffer's medium, treated with lysozyme
then with SDS and washed well with distilled water. They were placedon Millipore filter round disks (pore-size; 0.22,um; diameter 25 mm), put
in the SOR chamber (Nuclear Research Center, University of Tokyo) driedin vacuum and irradiated in high vacuum. The irradiated spores were resuspended in distilled water, and plated after appropriate dilutions on brothenriched minimal agar (MB plates) to determine the survival as well as induced inductions (His- -+ His +). At doses 1~O mA· min, the frequency
of induced mutations increased linearly (from 13 to 150 His+ per 106 survivals.
For experiments of phage induction, a B. subtilis strain Hl7 was lysogenized with phage SP02 and spores of lysogenic bacteria were prepared. The
spores were placed on Millipore filter and exposed to SOR of different doses
in vacuum. Each filter carrying irradiated spores (about 1.8 X 107 ) was
immersed in distilled wafer (0.3 ml) by shaking for 2 hours at room temperature, heated at 75°C for 20 minutes, combined with 3.5 ml of NY
medium and divided into 0.5 ml portions. They were incubated at 37°C
by shaking for different periods. After adding one drop of chloroform,
each tube was incubated at 37°C for 15 minutes and free phages weretitrated. The number of free phages increased during incubation of theirradiated spores and this effect was much greater for the SOR lightexposed spores, indicating clearly a inductive effect of SOR irradiation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

A Desmutagenic Factor from Plant (Brassica oleracea) for
Mutagenic Principle of Tryptophan Pyrolysate has
Peroxidase and NADPH-Oxidase Activities
Tadashi INOUE and Tsuneo KADA

A desmutagenic factor for Trp-p-2 which is a potent mutagen discoveredby Sugimura and his collegues in pyrolysis products of tryptophan, waspurified to near homogeniety from cabbage (Brassica oleracea) leaves. The
factor had a molecular weight of 43,000 and had 45.2 p.g of sugar per mg
of protein as determined by phenol/sulfonic acid method. The purified
factor exhibited a hemo-protein-like absorption spectrum with Soret bandat 404 nm, and a and fi bands at 640 nm and 497 nm, respectively.
When reduced with sodium hydrosulfite, the Soret and a bands shifted to
437 nm and 555 nm, respectively, and fi band disappeared. Treatment withcyanide resulted in shift of the Soret band to 421 nm. These spectral properties are very similar to those of peroxidase of other plants especiallyof horseradish which also has NADPH-oxidase activity.
We then examined whether the desmutagenic factor had peroxidase and
NADPH-oxidase activity. The peroxidase activity was determined usinghydrogen peroxide and o-dianisidine as substrates; and NADPH-oxidase
activity was determined by a decrease in absorbancy at 340 nm owing tothe oxidation of NADPH. The experiments yielded the results that thefactor had both peroxidase and NADPH-oxidase activities. The former
activity was fully dependent on both resorsinol and Mn, and the latter
activity was inhibited by catalase. These properties are also very similar
to those of horseradish peroxidase, but commercial horseradish peroxidase
had no desmutagenic activity for Try-P-2.

Further Characterization of a Bacillus suhtilis Endonuclease
Specific for Apurinic Sites in DNA
Tadashi INOUE and Tsuneo KADA

We have identified several kinds of enzymes which may function in the
repair of gamma-irradiated DNA in extracts of Bacillus subtilis cells using
a new assay system in which a capacity of cellular extracts to enhance thepriming activity of gamma-irradiated DNA for purified DNA polymerase
was measured. Preliminary experiments revealed that one of them was

RESEARCHES CARRIED OUT IN 1978

67

an endonuclease specific for apurinic sites in DNA (T. Noguti and T. Kada,
Biochim. Biophys. Acta 395: 294, 1975) and the other was 'cleaning' exonuclease (T. Inoue and T. Kada, Bicohim. Biophys. Acta 478: 234, 1977).
The former enzyme was purified to near homogeniety and had properties
described below.
The endonuclease was a monomeric protein and had a molecular weight ofaround 56,000. The enzyme was specific for apurinic sites in double-stranded
DNA and had a pH optimum at 8.0. It was slightly stimulated with 50 mM
NaCI but completely inhibited with 500 mM NaCl. It required no ~iva­
lent cations and was insensitive to EDTA; it had no associated exonuclease.
These properties are very similar to those of Escherichia coli endonuclease
IV, which is also insensitive to EDTA and has no exonuclease activity, andvery different from those of the main endonuclease for apurinic sites (endonuclease VI) of the same bacterium. Detailed properties of the Bacillus
enzyme has been described (T. Inoue and T. Kada, J. Bioi. Chem. 253: 8559,
1978).
Phenotypic Expression of Waxy Starch Mutants in Cereals
Etsuo AMANO

In waxy starch mutants, amylose fraction of starch is reduced. Someof the mutant lines showed intermediate phenotype between waxy (wx)and normal (Wx). Intending to show that they are really intermediatemutants and probably due to missense mutations, quantitative measurements
were made. The method was modification of the blue value method.
Each starch solution sample was made up from a single kernel by autoclaving. After staining by iodine, the sample soultion was diluted in a colorimetric measuring cell. Transmission value of the sample solution wasmeasured in two wave lengths. 430 nm blue light was used to monitor
the dilution by measuring the common and major faction of starch, amylopectine, which was stained reddish purple. When the solution was diluted
to 50 %transmission in 430 nm, an electric signal was generated to commanda digital printer to register the transmission value of 660 nm red light.
This value would reflect the content of amylose fraction which was stained
blue by iodine.
In this year, 27 waxy mutant lines of maize and 18 EMS induced waxy
mutants of rice were analyzed. In maize, three EMS induced mutants were

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

confirmed to be intermediate waxy. Other mutants, including two UV induced and eight EMS induced mutants were very close to standard waxyC(WX ) or deletion mutant (wx R ). Among the EMS induced waxy mutants
in maize, 27.3 % were intermediate.
In rice, 18 waxy mutants induced by EMS in Norin No.8 were analyzed
and nine mutants (50%) were considered to be the intermediate types.
They distributed rather continuously from complete waxy to nearly normal
phenotype. Higher ratio of intermediate type in rice and its relation to
dosage effect of waxy gene found in this Japonica rice variety are under
inverstigation.
Dose Response of Maize Strains to Chronic Gamma-Ray Irradiation*
Etsuo AMANO and Yasuo UKAI**

To compare the dose response of mutation to chronic gamma-rays, 19
strains of waxy maize (wx, Ae) were planted in Gamma Field of Instituteof Radiation Breeding, NIAS, Ohmiya, Ibaraki. The plants were transplanted into the field after three week's growth and irradiated for 20 hours
a day during whole growing stage. Plant height and leaf lenght-width ratio
at maturity were also recorded as a measure of the effects of radiations onplant growth.
Previous experiments indicated that if amylose in starch were suppressedby waxy (wx) gene, forward mutation from normal (Ae) to amylose
extender (ae) could be detected in pollen stained by iodine (Nelson's I 2-KI
solution). Although the frequency of mutation was at the order of 10-'at the highest, examinations of 106 pollen grains per plant were not solaborious and this genetic analysis seemed to be a very sensitive system.
The mutant pollen grain wx ae stains darker by iodine than wx Ae pollens.
The darker stain was favorable to distinguish mutant pollens from physiologically damaged pollens and to detect the mutant pollen out of a large
number of the light colored pollens of original strains. This darkly stainedpollen might include both mutation from Ae to ae and reverse mutationfrom wx to Wx. In the present experiments, various wx strains being used,reverse mutation might occur in some strains. However, as the radiation* Cooperative Research Project sponsored by the Agriculutre, Forestry and FisheryResearch Council, MAFF.
** Institute of Radiation Breeding, NIAS.

RESEARCHES CARRIED OUT IN 197869

might not induce point mutation, reverse mutation of wx to Wx seemed tobe very infrequent or unlikely. The wx strains being all homozygous foreach wx, normal recombinant could not be expected. The colorations of
the mutant pollen were intermediate, i.e., lighter than Wx pollen, so thatwind blown and oter contamination could be discriminated.
Plant height of the most sensitive strain decreased above 150R/day but
in the most resistant strain the height decreased above 230R/day.
Frequencies of mutant pollen were examined under a low power binocular
microscope using large cover and slide glasses. Total number of pollen
examined was estimated from counts of 15 positions on the slide. Thenthe entire area (50 x 75 mm 2) was scanned to score dark mutant pollen grains.
Fluctuation of the freuqency of the dark pollen among the slides was large
probably due to mutation sectoring, but pooled data showed linear increase
from 3.8R/day up to lIR/day. In the most resistant strain, the linear increase
continued up to 23R/day. Other strains showed saturation above 16R/day.
Correlation between leaf width-length ratio and pollen mutation was examined. The strains tested evenly distributed showing no correlation as a general trend. However the results could be interpreted as showing that themost sensitive strain in genetic damage was also sensitive to growth inhibition. The same correlation was seen in the most resistant strain. Amongthe intermediates, some showed higher sensitivity in mutation but wereresistant in growth inhibition, other strains showed the reverse reactions.
Overall difference in sensitivity between extreme cases was not large.
Leaf width-length ratio differed by a factor of 10. Other indices differedby factors of two to four.
Induction of Waxy Mutants in Rice by Reactor Radiations
Etsuo AMANO

In maize, the frequencies of viable waxy (wx) mutants in radiaton treatedprogeny were as low as 1/10 or less of the powerful chemical mutagen, EMS,
treatment. To compare with maize, and to examine the characteristicsof radiation induced waxy mutants, seeds of rice, Oryza sativa, var. Norin
No.8. were irradiated in the thermal neutron facility of Kyoto University
Reactor (KUR)*. Dry seeds were sealed in polyethylene bags and irradiatedin the D 20 Irradiator Tube for 3,4, or 5 hours. Reactor output during* Research Reactor Institute Kyoto University

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

the treatments was 5 MW. The biological effects of the reactor radiation
were compared to those of mcs gamma-rays by two weeks seedling height.
Suppressions of seedling heights which were about 60 % of the control werecomparable from 30KR to 40KR of the gamma-ray. To avoid confusionon the origin of mutants obtained due to chimeric sectoring, it is favorable to limit the number of panicles developed from a single seed. For
this reason, Xl plants were kept in the seedling flats in greenhouse until
harvest, so that only a few panicles developed from a single seed. After
harvest and drying each panicle was threshed and hulled separately.
Opaque or other waxy like grains were tested by iodine, after scraping off
small portion of endosperm surface using a small grind stone. Among 1306
panicles pooled, in which more than ten grains could be examined, six panicles (0.459 %) segregated wx mutant grains. These six mutations were independent in their origins. It was noticed that some of them showedintermediate phenotype, or in other words, they were leaky wx mutants.
In the present experiment, frequency of wx mutants induced was as high as
EMS treatment. This high frequency and the occurence ofleaky wx mutation
became a new problems to be investigated.
Test of Environmental Mutagens with a Soybean Strain, T-219
Taro FUJII

Strain T-219 of Glycine max, made available through the kindness of
Dr. B. K. Vig of the University of Nevada, possesses gene Y u . This geneimparts green color to Y u Y u plants while YuYu plants are light green and
YuYu plants are yellow. When somatic mutation occurred in YuYu seeds,
the first two simple leaves show yellow spots by forward mutation, green
spots by back mutations, and twin spots consisting of green and yellow
ones by somatic crossingover (B. K. Vig Mutation Res. 31: 49, 1975).
The mutagenicity of AF-2, ,8-propiolactone, DAPA and ICR-170, which
had been known as mutagens through microbial testing systems, were testedby the use of this soybean strain. The concentrations of these chemicals
appllied to the seeds were decided on the basis of those applied in microbial
tests and those of EMS through the kindness of Drs. Kada and Inoue.
The results showed 0.0002 f-lgfml of AF-2 as well as 1 f-lgfml of ,8propiolactone produced 1,000 revertants per plate or ca. 107 cells, and asimilar mutation frequency was observed with 20 f-lgfml of EMS. On the

RESEARCHES CARRIED OUT IN 197871

other hand, an appreciable increment in mutation frequency was observedwith 2 Itg/ml of EMS in the soybean system. Accordingly, about 1/10 ofconcentrations used in the microbial system were used in the present experiment.
About 130 seeds (25 g in dry weight) of T-219 strain were soaked in 30 ml
solution with given concentrations of a chemical for 24 hours at 20°C.
The solution was almost completely absorbed by the soaked seeds and theirweight increased to 53 g. Treated seeds were sown in wooden boxes filledwith field soil and kept in a greenhouse (20-30°C). Mutant spots appearing
on the first two simple leaves were scored, and the mutagenicity of eachchemical compound was measured by the increase of spots per leaf.
Significant increase in mutation frequency were observed with 0.00008 Itg/ml
of AF-2 or 0.2 Itg/ml of t9-propiolactone, and the frequency increased almost
linearly with incraesing concentration. An increment in mutation frequency
was also observed with 0.125 Itg/ml of DAPA and with 0.0025 Itg/ml of ICR170.
This preliminary experiment indicates that the soybean system is highly
sensitive to mutagene and is useful for testing the mutagenicity of various
environmental agents. Furthermore, this system can be used for checking
the mutational effect of long-term seed storage.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

VIII.

POPULATION GENETICS (THEORETICAL)

Change of Gene Frequencies by Natural Selection
under Population Number Regulation
Motoo K1MuRA

By incorporating a population number regulating mechanism into theformulation of genic selection involving a pair of alleles (AI and A z) withrespective frequencies x and I-x, it is shown that the change of x in one
generation is given by Jx=sx(l-x)/W, in wihch W is the mean absolute
selective value (in Wright's sense). It is also shown that, in the process inwhich advantageous allele (say AI) increases from a low frequency to ahigh frequency, quasi-equilibrium is rapidly attained where J W ~O. In
this state we have W~ 1+(sz/c)x(l-x) in the case of logarithmic population
number regulation, and W ~ 1+szx(l-x)/(cN) in the case of logistic regulation. In these expressions, s is the selective advantage of Al over A z,
and c is a coefficient relating to the total population number regulation.
It is pointed out that the approximation formula Jx=sx(l-x) is valid underwider circumstances than usually suggested by the conventional treatmentof genic selection. For details, see Proc. Natl. Acad. Sci. USA 75: 1934,1978.

Stepwise Mutation Model and Distribution ofAllelic Frequencies in a Finite Population
Motoo KIMURA and Tomoko OHTA

A mathematical theory is developed that enables us to derive a formulafor the equilibrium distribution of allelic frequencies in a finite population when selectively neutral alleles are produced in stepwise fashion(stepwise mutation model). It is shown that the stepwise mutation model
has a remarkable property that distinguishes it from the conventional
infinite-allele model (Kimura-Crow model): as the population size increases
indefinitely while the product of the effective population size and themutation rate is kept at a fixed value, the mean number of different
alleles contained in the population rapidly reaches a plateau which is not

RESEARCHES CARRIED OUT IN 197873

much larger than the effective number of alleles (reciprocal of homozygosity).
For details, see Proc. Natl. Acad. Sci. USA 75: 2868, 1978.
Effect of Overall Phenotypic Selection on
Genetic Change at Individual Loci
Motoo KIMURA and James F. CRow

The selective advantage of an allele G, (relative to the mean of alleles atthis locus) is given by

L:
L:

si=-A,=A i

W(X)F'(X)dXjW

W'(X)F(X)dXjW,

in which A, is the average excess of the allele on the character, X; W(X)
is the fitness fuction; F(X) is the frequency function; W is the mean fitness;and the prime denotes differentiation. With truncation selection s,=
A,F(C)jw, in which F(C) is the ordinate at the culling level and w is the
proportion saved; this does not depend on any assumption about the distribution of F(X). If the character is normally distributed, s,=AJja 2, in
which I is the selection differential and a 2 is the variance of the character
distribution. Finally, if the logarithm of the fitness is proportional to thesquared deviation from the optimum and the character is distributed normally, s,=A,K(Xop-m), in which X op is the optimum value of thecharacter, m is the mean value, and K is a constant determined by thevariances of the fitness function and the frequency function. Truncation is
the most efficient form of directional selection in the sense of producing
the maximum gene frequency change for a given effect of the gene on thecharacter, but fitness functions can depart considerably from sharp
truncation without greatly reducing the efficiency. For details, see Proc.
Natl. Acad. Sci. USA 75: 6168, 1978.
Theoretical Study of Genetic Variability, Assuming Stepwise Productionof Neutral and Very Slightly Deleterious Mutations
Takeo MARUYAMA and Motoo KIMURA

A mathematical theory is developed that enable us to compute the amount74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

of genetic variability maintained in a finite population, assuming that mutations occur in stepwise fashion ("Stepwise Mutation" or "OhtaKimura" Model) and that both selectively neutral and slightly deleterious
alleles are involved. Two numerical examples show that, if very slightly
deleterious mutations are prevalent, the amount of genetic variability increases much more slowly as the population number increases than is thecase when all the mutations are strictly neutral. For details, see Proc.
Natl. Acad. Sci. USA 75: 919, 1978.
Sequence Variability of Immunoglobulins Consideredfrom the Standpoint of Population Genetics
Tomoko OHTA

The gene family of variable region of immunoglobulins is an interesting
example to apply the theory of gene diversity of multigene families. The
variable region contains about 110 amino acid sites and these sites
are classified into the hypervariable and the framework regions.
The former is known to constitute antigen binding sites. The theoreticalresults on the equilibrium and transient behavior of the identity coefficient were applied to the analyses of sequence variability in the variable
regions ofimmunoglobulins. The probability ofamino acid identity between
corresponding residue sites at different but homologous immunoglobulin
sequences was computed both within and between species from data compiled by Kabat and his associates. The average identity coefficients of aminoacid was obtained for the hypervariable regions and also for the framework
regions. From statistical analysis based on population genetics theory it isconcluded that somatic mutations cannot be the major cause of hypervariability and that the results can readily be explained by the germ line theory.
For details, see Proc. Natl, Acad. Sci. USA 75: 5108, 1978.
Theoretical Study on Genetic Variationin Multigene Families
Tomoko OHTA

Genetic variation contained in a multigene family was theoretically investigated from the standpoint of population genetics. Unequal crossoveris assumed to be responsible for the coincidental evolution of mutant genes

RESEARCHES CARRIED OUT IN 197875

in a chromosome. When the allowed latitude of the duplicated or deleted
number of gene units at unequal crossover is 10-15% of the total gene number in one family, the arrangement of gene lineage in a chromosome isshown to be roughly random. The equilibrium properties of genetic variation or the probability of identity of two genes within a family (identity
coefficient) were studied under mutation, unequal crossover, interchromosomal crossover and sampling of gametes. The identity coefficient of amultigene family within a chromosome is shown to be approximately

in which a=2k/n 2 with k=effective number of cycles of unequal crossover
and with n=number of gene units in a family, v is the mutation rate pergene unit, fi is the rate of interchromosomal crossover per family and Ne
is the effective size of the population, all measured by the rate per
generation. The identity coefficient of a gene family between two different
chromosomes becomes approximately C 1 = Co/{l +4Ne v).
The equilibrium properties of the means, the variances and the covarianceof the two measures of identity coefficient are investigated by using the
diffusion equation method under the assumption of constant number ofgene units in the multigene family. Some models of natural selection basedon identity coefficient are considered. The possible significance of thevariance and covariance of identity coefficient among the chromosomes on
the adaptive differentiation of gene families such as those producing antibodies is discussed. For details see Genet. Res. 31: 13, 1978 and Genetics
88: 845, 1978.

Polymorphism and Loss of Duplicate Gene Expression: A Theoretical
Study with Application to Tetraploid Fish
Naoyuki TAKAHATA and Takeo MARUYAMA

We studied the fixation of null alleles at independent duplicate loci,assuming that wild-type active alleles mutate irreversibly to nonfunctionalnull alleles and that the population is finite and panmictic. Solving thetwo-dimensional Kolmogorov backward equation numerically, we obtained
the rate at which one of the active genes is lost and the amount of

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ANNUAL REPORt OF NATIONAL INSTITUTE OF GENETICS NO. 29

heterozygosity at specified times. Previously harmful genes, including recessive lethals, can be fixed at one of the duplicate loci, which would not
happen with a single locus. Examination of data from several fish families
showed that the rate of fixation of null alleles is too slow and theamount of heterozygosity too small to be compatible with complete recessivity at all loci. Our conclusion differs in this regard from that of Bailey
et 01., Proc. Natl. Acad. Sci. USA 75: 5575, 1978. They also reported thatthe time taken for 50 % of the loci to be fixed for null alleles is approximately 15N+v- 3 / ' where N and v are the effective population size and mutation rate when the alleles are completely recessive and the double homozygote is lethal. We found that the fixation rate depends not only on N,
but also on Nv. For details see Proc. Natl. Acad. Sci. USA 76: 4521,1979.

RESEARCHES CARRIED OUT IN 1978

IX.

77

POPULATION GENETICS (EXPERIMENTAL)

Seasonal Change of Inversion Frequencies in a Mishima
Population of Drosophila melanogasterYutaka INOUE

Drosophila flies were monthly collected from a population of Mishima.
Several banana traps were placed for a week at a shrine backyard prior
to collection. D. melanogaster as well as other domestic species were collected from June to November with a temporal decrease of population sizein mid-summer. The average frequency of polymorphic inversions washigher in summer and lower in spring and fall; 8.0% (June), 9.8% (July),
15.5% (Aug.), 14.4% (Sept.), 9.1 % (Oct.), 5.8% (Nov.). Four common
cosmopolitan inversions (2Lt, 2RNS, 3LP, 3RP) showed the same seasonalchange in frequency. Two inversions (In(2L)W and In(3L)Y), endemic
to the Eastern Japan and the frequencies were very low, appeared onlyin summer and early fall. The seasonal change suggests that the polymorphic inversions prefer hot to cool. This phenomenon is very consistent with the high frequency of inversions in the Ishigaki-jima(Okinawa) population and the low frequency in the northern (Sapporo)
population.

Further Genetic Studies of the Katsunuma Population of
Drosophila melanogaster
Takao K. WATANABE and Yutaka INOUE

Genetic variations in the second chromosone of D. melanogaster havebeen studied for 16 years in the Katsunuma population. The frequencychanges of deleterious genes and chromosome inversions are shown in Fig. 1.
Lethal and male-sterile chromosomes increased in frequency drastically inthe early seventies. After a while their frequencies reduced a little andbecame a constant level as about 25% and 10% respectively. Femalesterile chromosomes, on the contrary, increased in frequency gradually
after 1975 which overwhelmed male-sterile freauency in 1978.
Polymorphic inversion chromosomes such as In(2L)t and In(2R)NS had

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

40

UJ

6 30

II)

0

~

0

~ 20

u

lL

0

.. 10

0

'65

'75

'70

40
w

~
0

(/)

0
~
0

0::

:r:
u
lJ..
0

;!.

~:f

~---,----,-~~~--,-~n_(2~L)W_~:
'65

'70

1

:-j

'75

YEAR
Fig. 1. Frequency changes of deleterious genes and chromosome inversions
in the Katsunuma population.

been highly maintained in the Katsunuma during the sixties. However,their frequencies became a third or a half after the early seventies.
A new inversion, In(2L)W (28C-32C), appeared in the Katsunuma in 1972
and thereafter it became a member of polymorphic inversions though thefrequency was about 3-4 %.

RESEARCHES CARRIED OUT IN 1978

x.

79

EVOLUTIONARY GENETICS

Phylogeny of Drosophila Based on the Mating Preference
Takao K. WATANABE and Masaoki KAWANISHI

One-side mating preference among sibling species of Drosophila has beeninterpreted as follows: Females of a derived (new) species do not mate wellwith males of the ancestral (old) species whereas females of the ancestral
species readily mate with males of the derived species. This hypothesis
has come from the assumption that isolation between the females of new species and the males of old species allow a favorable situation for an incipient species to maintain its population, and that the species specific mating
behaviors have been conservatively maintained by each species.
The evolutionary sequence drawn by the asymmetrical mating preferences
only suggests the 'relative age of each species', which resulted in a unidirectional phylogeny of each sibling species group of Drosophila. Then, thephylogeny is revised by seeking the immediate ancestor of each species
from the degree of genetic closeness. For example, the evolutionary sequenceof the virilis group is (1) virilis, (2) littoralis, (3) novamexicana, (4) americana,(5) texana. But the most genetically close species of novamexicana isvirilis instead of littoralis among the older species than novamexicana.
Therefore the phylogeny of the group is branched as virilis~littoralis, andvirilis~novamexiacna~americana~texana. This phylogeny is very consistent with those of chromosomal (Stone, UTP 6205: 507, 1962) and electrophoretical (Nei, Am. Nat. 105: 385, 1971) evolution.
This method is also useful for understanding of phylogenetic relationsof other sibling species group of Drosophila whenever data on the reciprocal mating preferences are available. The evolutionary sequence ofthe melanogaster subgroup is determined by the asymmetrical matings asfollows; (1) melanogaster, (2) simulans, (3) yakuba, (4) erecta, (5) teissieri,(6) mauritiana. The phylogenetic relation revised by seeking the immediateancestor is as follows; melanogaster~simulans~yakuba~erecta, simulans~teissieri, and simulans~mauritiana. D. melanogaster is the ancestral species
and D. simulans evolved from it. From D. simulans three African endemics
evolved: D. yakuba first, D. teissieri second and D. mauritiana third.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Again, this phylogeny is very consistent with those of species distribution(Bock and Wheeler, UTP 7213: 1, 1972) and chromosomal (Lemeunierand Ashbumer, Proc. Roy. Soc. B. 193: 275 (1976) evolution.

RESEARCHES CARRIED OUT IN 1978

XI.

81

HUMAN GENETICS

Host Resistance to the Gene for Retinoblastoma
Ei MATSUNAGA

Retinoblastoma is a malignant neoplasm of the eye affecting one perca. 20,000 infants. There are two forms, hereditary and nonhereditary.
The nonhereditary form, which comprises most sporadic unilateral cases,
represents about 60 % of all retinoblastomas. The remaining 40 % are hereditary and due to an autosomal dominant gene with incomplete penetrance
and expressivity. While all bilateral cases, whether sporadic or familial,
are to be regarded as hereditary, a gene carrier can be unilaterally
affected or remain unaffected. By analyzing data from 261 pedigrees with
two or more cases of retinoblastoma, we arrived at a simple conclusion: the three distinct phenotypes of the gene carriers, i.e., unaffected,
unilaterally and bilaterally affected, are determined largely by inherited
host resistance to the retinoblastoma gene.
Segregation analysis of the data revealed that penetrance and expressivity
in children who received the gene from a carrier parent declined consistently with decreasing degree of parental expressivity. For example, if
the parent was bilaterally affected, the proportion of affected children was
0.49 and the disease was bilateral in 90 % of the cases; if the parent wasan unaffected carrier, the proportion decreased to 0.31 and 54 % wereaffected bilaterally. The possibility of multiple alleles with different penetrance at the retinoblastoma locus could be ruled out by the variability inpenetrance in different sibships in the same family. The observed distribution of the three phenotypes among the gene carriers was consistent witha multifactorial model with two thresholds for host resistance, but not witha Poisson distribution of tumors as assumed by Knudson (Proc. Nat!. Acad.
Sci. 68: 820,1971). Furthermore, with the host resistance model there wasno need to postulate delayed mutation in order to account for the reported
pedigrees with two or more collateral relatives affected with retinoblastoma.
The heritability of host resistance was estimated at approximately 90 %.
For details, see Am. J. Hum. Genet. 30: 406, 1978.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Recurrence Risks to Relatives of Patientswith Retinoblastoma
Ei MATSUNAGA

Prior to modern ophthalmologic care, retinoblastoma had been a lethal
condition, with rare exceptions of spontaneous regression. Owing to increasing success in the medical treatments, however, cases inherited fromthose who survived retinoblastoma are gradually increasing in many countries.
Appropriate genetic counseling is therefore ofgreat importance to the patientsand their family members.
Estimates for recurrence risks of retinoblastoma have been given usuallyon the assumption that the hereditary form is due to a dominant gene with
80 % penetrance. However, our studies demonstrated that penetrance andexpressivity of the gene in children declined with decreasing expressivity
in the carrier parent. Accordingly, using the segregation ratios of 0.49,
0.42 and 0.31 in children from bilaterally affected, unilaterally affected andunaffected carrier parents respectively, and the proportion of sporadic bilateral and unilateral cases that were inherited from an unaffected carrier parent
estimated at 0.10 and 0.07 respectively, recurrence risks to first- and seconddegree relatives of patients with retinoblastoma were calculated. The risk
to a first child of an unaffected person having a bilaterally affected parent
is as low as about 1 %, in contrast to the usual estimate of 6.7% derived fromthe assumption of constant penetrance of 80 % of the retinoblastoma gene.
For details, see Jpn. J. Ophthaimoi. 22: 313, 1978.

Quantitative Analysis of C-Positive Variants in Man
Jun-ichi AZUMI, Yasuo NAKAGOME and Ei MATSUNAGA

In the Paris Conference (Suppl., 1975), the use of numerical expression,
1 through 5, was recommended for the purpose of describing the size of avariant. However, no specific difinitions were given for the individual classes (numerals). As to the qh regions of nos. 1, 9 and 16, the use of thesize relative to either a long arm of chromosome 21 (21 q) (Muller et ai.,
Cytogenet. Cell Genet. 15: 239, 1975) or a short arm of chromosome 16(16p) (patil and Lubs, Human Genet. 38: 35,1977) was proposed. The ratios
thus obtained were assigned more or less arbitrarily into five classes.
In the present report, area of C-positive qh regions of chromosomes 1, 9

RESEARCHES CARRIED OUT IN 197883

and 16 was measured using a Nikon-Vickers M85 microdensitometer. A
few different methods including those of Muller et af. (1975), PatH and Lubs
(1977) and a new system were compared.
The mean area of C-positive 1qh, 9qh and 16qh segments were 1.98±0.31(,um 2 ±SD), 1.39±O.28 and O.83±O.17, respectively. It appeared that all
16qh segments were smaller than both a 21q and a 16q, while almost all
1qh segments were larger than either of them. When results were expressedin the numerical system, the mean scores for lqh segments were 3.8 and 3.5,
while those for 16qh were 2.3 and 1.9 respectively, by Muller et af. and PatH
& Lubs' system.
A new system ranks a qh segment into one of 5 classes based on how
different they are from an average of each lqh, 9qh and 16qh region in termsof standard deviation (SD). It is proposed that a variant with in ± ISD
of an average be assigned to the intermediate class (class 3) in the
Paris Conference system (Suppl., 1975), one within ± 2SD to either smallor large class (calss 2 or 4) and all other to either very small or very large(class 1 or 5). The present system compares favorably with any othermethods so far tested as to the detection of a qh variant. For details, see Jpn.
J. Human Genet. 24: 99, 1979.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

XII.

BEHAVIORAL GENETICS

Genetical Analysis of Noise Sensitivities of Homozygous Strains ofDrosophila melanogaster
Chozo OSHIMA and Tsuguhiko TAKAMURA

Many homozygous strains for different second chromosomes, have been
made by Cy/Pm method using many male flies, collected in a Katsunuma
natural population in 1975. Seven homozygous strains among them wereused in the experiment. Locomotor activities of individual fly in a small
glass cell (4x lOx 50 mm) under LD 12 : 12 with dusk and dawn of threedays and continued DD of three days under constant 25°C were recordedby the electronic actograph. Locomotor activities of flies of each strain
showed two high peaks at dusk and dawn and continued at low level during
the subjective daytime with individual circadian rhythm under DD environment. The pure sound (2000 Hz, 100 phon) was projected for 5 hours(0.00-5.00 AM) on resting flies and the grade of sensitivity was determinedby the activity occured for the period.
Five flies of each seven strains were examined and a strain numbered
59 was sensitive and a strain 83 was unsensitive. Other five strains wereintermediate between them, but their sensitibities were close to unsensitive
strain 83. Then, sensitive strain 59 was crossed with other six strains
reciprocally and locomtor activities of each hybrid strain under noise environment were recorded as the same method mentioned above. From the result,the gene(s) manifesting the noise sensitivity of strain 59 was recessive togene(s) of strain 83, and incomplete recessive to gene(s) of other strains.
This result was very similar with the result ontained in the previous year andthen, those results were put together. Only two of three strains among twelve strains had sensitive nervous center, bioclock controlling the locomotor
activity.

RESEARCHES CARRIED OUT IN 1978

85

Actual Activity at Dusk and Dawn is not Needed for Entrainment of
Circadian Activity Rhythm of Drosophila me/anogaster under
Following Continuous Darkness.
Tsuguhiko T AKAMURA and Chozo OSHIMA

Under LD 12 : 12 with dusk and dawn environment for three days, wild
typed flies of D. malanogaster became most active at dusk and dawn and they
were less active during day and night. When they were continuously put
into darkness (DD) for following three days, they moved continuously attheir subjective daytime and it was showed that they were diurnal insects,
and that they could memorize the time of dusk and dawn period in the previous three days.
To memorize the time, that is, to entrain the bioclock, is the actual
movement under LD daily cycle needed? We kept flies under theenvironment of LD 12 : 12 with dusk and dawn and of 12°C for three days
and some flies did not move at dusk and dawn becasue of the low temperature. At five hours after the onset of continuous darkness, the temperature
shifted from 12°C to 25°C and the locamoter activity of flies was recordedby actograph. Some flies, having no experience of actual movement at dusk
and dawn for previous three days, moved continuously during the sunjective
daytime for following three DD days. This fact indicates that the actual
locomotor activity at dusk and dawn of daily LD cycle is not needed toentrain the circadian rhythm of bioclock under following DD environment.

Visual System Mutants and Light-Dark Cycle Entrainment in
Activity Rhythm of Drosophila melanogaster.
Tsuguhiko TAKAMURA and Chozo OSHIMA

Ability of mutant flies to adjust locomotor activity rhythm to daily lightdark (LD) cycle was examined, which had such various defects in the visualsystem as w, en bw (no eye pigment), eym, eyN (no compound eye), so (nocompound eye and no ocelli), e, t, nonA (no on-off transients in electroretinogram (ERG)), rdgA, rdgB, norpA (no receptor potensial in ERG), sev (seventh rhabdomeres absent), and ora (outer rhabdomeres absent).
One double mutant, sev nonA, and one triple mutant, sev nonA;ora, among
these mutants could not entrain the daily rhythm of locomotor activity
to LD cycle and showed free running circadian rhythm. Considering our86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

results of mutants in ERG and rabdomeres in the light of Pak's review
on mutants affecting visual systems (in Handnook of Genetics. Vol. 3, ed.
R. C. King, New York, Plenum, 1976), it is concluded that the input of LD
information to medulla either through peripheral retinula cells (R l _ 6) or
central retinula cells (Rr,B) may be enough to synchronize the locomotor
activity with outer LD cycle.
All morphological mutants have normal entrainment ability of LD daily
cycle and it is confirmed that compound eye and ocelli are not necessary
to catch the change of light and dark environment. However, there aresome difficulties in explaining the results of ERG mutants and the morphological mutants at the same time.
Double mutant sev nonA showed normal eclosion rhythm under daily
LD cycle, and it was assumed that photoreceptors for locomotor activity
rhythm and for eclosion rhythm were different. Our data also suggeststhat the photoreceptor for locomotor activity rhythm itself is not the circadianoscillator because sev nonA flies showed normal free running period.
Oviposition Behavior of Drosophila melanogaster:
Ethogram and Hybrid Analysis.
Tsuguhiko TAKAMURA and Chozo OSHIMA

Two strains of D. melanogaster, which differ as to the preference ofoviposition site were set up by artificial selection. One prefers paper placedon the medium (P line), the other lays eggs on the medium avoiding thepaper (M line). To analyze these behavioral difference, the ethogram wasmade by observing directly the egg-laying behavior. A cycle of oviposition of the species is a chain of four behavioral components. Oviposition(D. melanogaster lays one egg at a time), intensive extension of abdomen
and its contraction soon after, walking and food intaking, searching with
egg-guide bending abdomen downward, then again oviposition. The searching behavior seems to be induced by the stimuli through tarsi because
there is difference in the frequency of the induction of searching behavior
between on the paper and on the medium. Actual judgment, if the place
is appropriate for oviposition, is made with egg-guide. This was demonstrated by the fact that flies, whose tips of egg-guides were cauterized, lost
discriminating ability of oviposition sites.
The difference between P and M lines was explained by the difference inRESEARCHES CARRIED OUT IN 1978

87

egg-inserting tendency. M line flies could insert eggs into the soft medium
but they could not insert eggs into the paper. On the other hand, P line
flies could lay eggs both on the paper and on the medium because they
dropped eggs. A new method to investigate only egg-inserting tendency
was established. after counting the number of eggs laid on the medium,the medium surface was treated with 2M NaOH solution for 20 minutes,
then the surface was washed with water current. With this treatment
eggs dropped and glued to the surface of the medium were washed awayand eggs inserted into the medium were left. Percentage of eggs left on
the surface is the measure of egg-inserting tendency. Using this method,both P and M lines and their F 1 hybrids were examined individually.
The distribution pattern of F 1 flies was almost between the parents and indicated that the charactor was controlled by polygenes.
Different Photo-preferences in Drosophila simulans and D. melanogaster
Masaoki KAWANISHI and Takao K. WATANABE

Photo-preferences of the sibling species, Drosophila simulans and D. melanogaster, were studied using an apparatus and population cage having a
gradient of light intensities. D. simulans preferred to stay and to lay eggs
at light places whereas D. melanogaster did not show such a strong light
preference. Selections of eggs in the light gradient cages made it possible
to segragate a mixed species population into different species populations;selection for photopositive flies soon eliminated D. melanogaster and selection for photo-negative or photo-neutral flies finally eliminated D. simulans.
The difference of the photo-preference seems to be a cause of coexistenceof the sibling species in nature. For ditails, see Japan. J. Genetics 53:
209, 1978.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

XIII.

ECOLOGICAL GENETICSAn Observation of Wild Rice Species in Tropical Australia
Hiko-Ichi OKA

Two wild rice species, the Oceanian type of Oryza perennis complex and
O. australiensis, are distributed in tropical Australia. The germ plasms ofthese taxa so far made available for us were quite limited and were notenough for variation studies. Also, there was no publication on the conditions of their natural habitats. I had a chance to visit Darwin andKununurra in the early April of 1978, and observed wild rice populations
at 11 sites.. Of them, one was of O. australiensis, eight were of O. perennis,
and the remaining two had both species growing sympatrically.
Both of the two species were found in marshes and river flood plains,which are inundated (50 cm or less) in the rainy season but are dry in thedry season. The Oceanian type of O. perennis is an annual grass. It formed
big populations in river flood plains. A hill or a small clump consistedof many plants each with one or a few tillers. Some plants standing inwater showed floating habit. The naturally shed seeds were found sticking
in the mud with their strong awns. At three sites, a part of the population
was in the shade of trees. The plants under trees were small in size andlate-maturing. Four populations were in pasture or in roadside ditch,while others were in the natural state. Plants growing in a rice field asa weed were also found.
O. australiensis had short rhizomes,and appeared to be of perennial habit.·
But the populations consisted of scattered small clumps each with several
panicles, suggesting that reproduction was mainly by seed. When this species was sympatric with O. perennis, it occurred in a somewhat elevatedplace as compared with the latter in lower places. On the basis of theseobservations and those from our previous study-tours in other parts of theworld, the conditions of the habitats of different wild species were summarized in a table.

RESEARCHES CARRIED OUT IN 197889

Niche Differentiation between Two Wild Rice Speices Observed in Nigeria
Hiroko MORISHIMA and Hiko-Ichi OKA

Two wild rice species, Oryza breviligulata and O. perennis subsp. barthii(=O.longistaminata) are commonly found in marshes and depressions in
the inland of Nigeria. The former is an annual species while the latter isperennial and propagates itself mainly by rhizomes. From our fieldobservation in 1977 (Report: Oka et al., 1978) and 1978 experiments at
Misima, we found the following points.
Both O. breviligulata and O. longistaminata are distributed in varying waterconditions and under varying degrees of habitat disturbance. In 23 percentof the sites observed, these two species coexisted side by side. However,examining populations of one species separated from the other, we foundthat O. breviligulata tended to be associated with annual herbs and O. longistaminata with perennial herbs. Further, the former species was distributedin the area where the dry season continued longer and rainfall was moreunpredictable than was the latter species. Although the two species canbe sympatric in the same habitat, these differences indicate that they are
differentiated in niche requirement.
Most of sympatric populations of the two species were found instrongly disturbed sites like fields under cultivation or roadside depressions.
The populations of O. longistaminata sympatric with O. breviligulata orwith its domesticated type, O. glaberrima, when tested at Misima, produced
many plants with a high seed productivity as shown by large number ofseeds per plant, heavy seed weight, and high seed fertility. The reproductive
effort evaluated in terms of percent seed weight to total plant weight was
6.3 percent on the average (ranging 0 to 25 %) in sympatric populations,but it was 0.5 percent on the average (0 to 7 %) in populations of this species
separated from those of other species. O. breviligulata did not show sucha difference between sympatric and separated populations. The data foracid phosphatase isozymes suggested the possibility of gene flow to occur
from O. breviligulata to O. longistaminata in sympatric populations.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

Genetic Diversity in Rice Populations of Nigeria:
Influence of Community Structure
Hiroko MORISHlMA and Hiko-Ichi OKA

Two rice species, Oryza glaberrima and O. sativa, are often mix-planted
in west Africa, and their populations, particularly those of O. glaberrima,
are highly polymorphic in seed type. It was found that the seed-typediversity within glaberrima population as shown by the "amount of information," H = - I: Pt log. Pi' was correlated with species diversity in the field(also shown by H), and gave a curvilinear regression on the proportion ofglaberrima plants to the total number of rice plants. The highest diversity
occurred when the proportion was 50-60 percent. A similar trend was also
found for sativa populations. These relationships suggest that the complexityof the biotic environment as shown by species diversity is related to the
genotypic diversity in rice population. (Agro-Ecosystems 5: 263-269)

Variation Studies in African Wild and Cultivated Rice Species
Yoshio SANO and Hiroko MORISHlMA

A total of 122 accessions of rice species were collected from natural populations or from farmers fields in our visit to Nigeria in 1977 (Ann. Rep. 28:
101). An accession consisted of seeds sampled at random from 20 to 30
plants of a population, either on a single plant basis or in bulk. Out of them,
572 plants from 46 populations were tested in concrete beds with automatic
shortday control. The results are summarized as follows:a) Cultivated species. In a majority of rice fields observed in Nigeria,
the two species, Oryza sativa and O. glaberrima were mix-planted in varying
proportions. Records on a single plant basis were taken for 242 plantsfrom 11 populations regarding culm length, numbers of tillers and panicles
per plant, ligule length, degree of seed shedding, numbers of primary andsecondary panicle branches, seed fertility, reproductive effort or harvest
index, and other 13 characters. The two species showed distinct differencesin ligule length and some other characters, and most of the plants wereclearly distinguishable into the two species. However, the populations
from Kaduna (K-2) and Danbatta (D-3) contained a few plants withintermediate characters which were not classifiable into the two species.
The selfed progeny of such intermediate plants segregated for ligule length

RESEARCHES CARRIED OUT IN 197891

and showed a low seed fertility (55-78 %). In fact, a few plants which appeared to be F 1 hybrids were found in the field at Kaduna (K-2). These datasuggest that introgression occurs between the two rice species when they
are mix-planted in a certain condition. This was also recognized from an
observation of acid phosphatase isozymes.
b) Wild species. Nine populations of O. perennis subsp. barthii (=0.
longistaminata), 11 populations of O. breviligulata, and 5 populations of
O. punctata (330 plants in total) were tested. Records were taken for culm
length, tiller number, panicle number, seed weight, awn length, seed fertility,
reproductive effort, etc. O. breviligulata, an annual species, showed a highreproductive effort. The strains from eastern Nigeria tended to have higher
reproductive effort than those from western Nigeria (65-35 %). Strains ofO. glaberrima were also scattered on the same regression line of reproductive
effort on longitude. The other wild species showed lower values of reproductive effort (1-25 %). O. perennis subsp. barthii appeared to allocate
photosynthetic product mainly into vegetative organs including rhizomes.
In O. punctata, diploid (2n=24) and tetraploid (2n=48) forms were found.
Seven strains collected in the Sahel zone near Lake Chad were diploids,
while another strain obtained in a humid forest near Ibadan was tetraploid.
All diploid strains were annual while the tetraploid strain was perennial.
The diploid strains had short anthers, long awns, and a high reproductive
effort which characterized predominantly self-pollinated annuals, as compared with the perennial strain. This suggests that the mode of differentiationin O. punctata is similar to that in O. perennis in which the occurrence ofa perennial-annual continuum represents the main direction of differentiation.
Competition and Copper Tolerance in Two Sedges, Cyperus difformis
and Fimbristylis miliacea
Hiroko MORISHIMA

Among major weed species of rice fields, Cyperus difformis (abbrevaited C)
is most tolerant to copper while Fimbristylis miliacea (F) is least tolerantso far as their vegetative growth is concerned. Examinations of buried seedpopulations showed, however, that soil samples from copper-polluted fields
produced more F plants than those from control fields (Morishima and Oka,
Agro-Ecosystems 3: 131, 1977). To see how their competitive relation in
reproduction is affected by copper pollution, an experiment was carried out

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

as follows:
The seeds of C and F strains obtained from copper-polluted (P) and control (N) fields were mix-sown (1 : 1) to make the following four combinations, C(P)+F(P), C(N)+ F(N), C(P)+ F(N), and C(N)+ F(P). They
were sown on normal and toxic (200 ppm copper) soils filled in plastic trays
with two replications, in 1977 spring. At maturity, plant number per tray
and total dry weight were recorded for respective species. The seeds naturally sown on the same trays germinated in the next season (1978).
The number and total dry weight of the second generation plants were recorded again at maturity.
On normal soil, F generally showed in different strain-combinations higher
survivorship than C in the first generation. In the second generation, F
exclusively dominated over C, its frequency ranging from 83 to 100 percent.
On toxic soil, however, C dominated in all strain-combinations in thefirst generation. In the second generation, C was dominant in C(P)+F(P)as well as in C(N)+ F(N). F was dominant in C(P)+ F(N) and C(N)+ F(P).
The data thus suggested that F was a strong competitor with C on normal
soil and could replace C even on toxic soil in certain conditions.
Neighborhood Effects between Rice and Barnyard Grass Strains,
I. Performance in Mixture and Aggressiveness as
In8uenced by Planting Density
Louis ASSEMAT'" and Hiko-Ichi OKA

In order to look into the effect of neighboring plants on growth performance at varying planting densities, two strains of rice (Oryza sativa L.) and twoof barnyard grass (Echinochloa crus-galli Beauv.) were tested in a mixturediallel experiment with three replications. Seedlings of the four strains
were transplanted in rows radiating from a center in pure and mixed (alternate
planting) stands in all possible combinations so as to test them under changing densities. In both pure and mixed stands, the effect of density on single
plant dry weight and panicle weight could be represented by linear regressions of performance on density when the data were transformed into logarithms. A regression equation derived from Kira's equation, Wij<dl =W,;b,;d, was used, where W,; stood for single plant performance of strain i neigh'"

Present address: centre d'Etudes Phytosociologiques et Ecologiques, Route de
Mende, B. P. 5051, 34033 Montpellier-cedex, France

RESEARCHES CARRIED OUT IN 197893

bored by j and d was the deviation of a given density from mean density(plant number per unit area), both in logarithms. The model used for estimating neighborhood effect was: Wt;=,u+g,+ht;, where gj stood for genotypic effect in pure stand and h'i for the increment or decrement in i dueto the aggression of j (h,j=O). As the regression coefficient was modified
by neighbors, it was shown by bj+qjj, where b, stood for the coefficientin pure stand and q(j for its change in mixture. When these models werecombined, the neighborhood effect at given density was shown by hii-qt;d,where hij was the mean for all densities and -qijd represented its densitydependent portion.
Analysis of variance of the data proved that the neighborhood effects
estimated were in a greater part significant. The data showed that hij andq,j were correlated, resulting in a tendency of neighborhood effects to decrease with increasing density. But there were a few exceptions to this trend.
In many strain-combinations, hij and hii had inverse signs, but they were
not always inversely correlated and the data indicated that mode of interference could change according to the combination of strains and planting
density. Generally, the aggressiveness of barnyard grass was high at low
densities at which the resistance of rice was lowered. The competition effectbetween barnyard grass strains more sharply decreased with increasing density than that between rice strains. Cooperative association effect was detected in a few strain-combinations at high densities.
Nieghborhood Effects between Rice and Barnyard Grass Strains, II.
Experiments on the Mechanisms of Interaction between Plants
Louis AssEMAT and Hiroko MORISHIMA

Two strains of rice (A and B) and two of barnyard grass (C and D) were
tested in pure and mixed stands in all possible combinations, with and without
plastic pipes placed in the ground to keep the roots of each plant separate.
After the data were transformed into logarithms, the neighborhood effects
produced by the interaction of shoots and by that of roots were estimatedon the basis of an additive model. Both shoot and root interactions appeared to play important roles in producing neighborhood effects. Generally,root interaction was cooperative while shoot interaction was antagonistic,
with the exception of a case between rice strains in which root interaction
was also antagonistic.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

The neighborhood effects estimated from the planting density experimentwith the same four strains (foregoing report) showed strong correlations withthe percent cover by the leaves measured in pure stands 57 to 71 days after
seed-soaking. This suggested that the above-ground growth rate as shownby percent cover was a determinant of neighborhood effects and root growthwould be correlated with shoot growth.
A mix-planting experiment with early-sown and late-sown seedlings of ricestrains (A and B) proved that the interference between the early and lateplants of the same strain was magnificient. This suggested the role ofvariation in germination time in plant populations.
Increasing Frequency of Drosophila simulans in Yamanashi-ken
Takao K. WATANABE and Masaoki KAwANIsm

Since 1975, we have continued the collection of Drosophila flies around
Mt. Fuji area to know the mode of invasion of D. simulans to
Katsunuma (Yamanashi-ken) from the Pacific coast. Banana traps wereplaced for a week in early November to collect domestic or semidomestic
species of Drosophila. The percents of D. simulans among D. melanogasterand D. simulans are listed in Table 1, since the two species seemed to compete
the same niche.
Table 1. Percent of D. simulans among D. melanogaster and D. simulans (N)
1975

1976

1. Mishima
2. Susono
3. Gotenba
4. Fujiyoshida
5.0tsuki
6. KatsunumaA) VineyardB) Shrine
7. Kofu
8. Nirazaki
9. Kajikazawa
10. Minobu
11. Tomizawa
12. Fujinomiya

1978

1977

Locality

%

N

%

N

%

N

%

N

95.4
75.7
50.0
0
0

486
74
2
10
8

94.9
43.6
50.0
0
0.5

275
55
4
6
412

95.1
19.4
2.3
0
0.4

122
108
132
38
231

98.7
56.9
25.0
17.4
2.4

231
174
8
23
208

0

850

0

1375

0
0
0
0
38.5
98.5

25
21
15
27
13
199

0
0
0
0
0
98.9

56
163
142
16
9
189

0
0.7
3.2
0
1.3
1.1
4.3
91.8

1332
288
126
450
301
355
137
355

0.1
7.0
1.7
0
1.7
6.9
33.0
97.3

2294
431
118
365
423
628
176
111

RESEARCHES CARRIED OUT IN 1978

95

In 1975 no simulans was collected in the northern and western areas of
Mt. Fuji (4-10). But the invasion of simulans has occurred in the recent
two years from the eastern side of Mt. Fuji from Mishima (1 to 4), the western side from Fujinomiya (12 to 9), and the west-bound route from Tokyo(5 to 7). Only Nirazaki (8) is now free from simulans.
An interesting difference in the frequecy was observed in Katsunuma (6).
Two places (A and B) are a few hundred meters apart but the ecologicalniches are remarkably different. Vineyard (A) is surrounded by a wineryand human houses with a lot of debris of grapes. Shrine (B) is nearby
a highway surrounded by a lot of deciduous trees. Drosophila melanogaster
was found predominantly in the former place, but D. simulans seemed toincrease its population in the latter place.
Different Food Preferences in Drosophila simulans and D. melanogaster
Masaoki KAWANIsm and Won Ho LEE

Three kinds of fruit juice, orange (OR), grape (GR) and apple (AP), wereused for collection of Drosophila in nature .and for culture in laboratory.
D. simulans was remarkably attracted by apple and grape juice while
D. melanogaster preferred orange juice. On the other hand, relative numberof simulans progeny (simulansfmelanogaster) emerged from the mixed species vials containing each fruit juice medium was as AP>GR>OR whichsuggested that the food preferences of adults and larvae were basically
identical in the present species.
Apple juice food was relatively advantageous to D. simulans in early daysof laboratory cage life of mixed species population as well as in vials.
However, D. simulans had been mostly eliminated from the mixed cage
population within 100 days prObably due to other factors than food preference. For details, see Jap. J. Eco!. 28: 231, 1978.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

XIV.

APPLIED GENETICSThe Sporophytic Pollen Sterility in Rice: Its Genetic Basis andIntervarietal Relationships as Shown by F 2 Sterility
Hiko-Ichi OKA

The partial pollen sterility of true-breeding lines derived from a varietal
hybrid of rice (Oryza sativa L.) was attributable to duplicate genes causing
sporophytic sterility in certain homozygous combinations, in the same manneras set forth by Oka and Doida (1962) for seed sterility. The sporophyticpollen sterility was characterized by an instability of pollen development
resulting in a large variance of pollen fertility among spikelets of the sameplant. To examine the distributions of gametophytic and sporophytic sterilities in varietal hybrids, the F I and F 2 plants from crosses of 38 ricestrains with 3 test-strains (an Indica, a Japonica, and an Asian annual strainof o. perennis) were observed for pollen fertility. The cytoplasmic malesterility did not seem to be involved in the material. The magnitude ofsporophytic F 2 sterility was estimated by subtracting from the mean F 2 sterility an estimate of gametophytic F I sterility possibly transmitted to theF 2 population. Both the gametophytic and sporophytic sterilities of hybrids
were involved in the Indica-Japonica differentiation of rice varieties, butthey were not correlated among individual crosses. The crosses with thetest-strain of o. perennis showed little sterility in both the F I and F 2 generations. This suggests that the wild progenitor-the Asian form of o. pere·nnis would have dominant genes at many of duplicated loci and recessive
mutations accumulated with domestication might have resulted in the
differentiation of varietal groups like the Indica and Japonica types. (Jpn.
J. Genetics 53: 397, 1978)
Character Variations in Backcross Derivatives between Oryza sativa andO. glaherrima: M-V Linkage and Key Characters
Yoshio SAND and Hiko-Ichi OKA

To look into the mode of association of parental characteristics in the
hybrid progeny of the two rice species, character variations were observed

RESEARCHES CARRIED OUT IN 1978

97

in B1F1 plants and B1Fe (including a few B1Fe) lines obtained from backcrosses between a sativa (Ace. 108, an Indica from Taiwan) and a glaberrima(W025, from Guinea) strain. The backcross derivatives had different coefficients of relationship to glaberrima (or to sativa) parent ranging from 1/8
to 7/8. Ligule length, regenerating ability of excised stem segments, andvarious other characters were recorded. The measurement of a plant, i,for a character was evaluated by xi=(Xi-/-lld, where /-l= (X. +Xg )/2,
d=(X.-Xg )/2, and X. and X g stood for the parental sativa and glaberrima
values, respectively.
The distributions of x values showed appreciable transgressiveness inpanicle axis diameter and three other characters, particularly in the BzF 6
population, as a result of gene recombination. Awn length also served asa measure of gene recombination since both parents were awnless. The
standard deviation of x values for different characters was used as an index
showing recombination of parental characteristics or "character discordance". These data suggested that parental genes could be recombined almost freely in this species hybrid. In ligule length, secondary branch number, and some other characters, however, BzF 6 lines showing parental values(x.=l; x g =-l) were larger in number than those showing an intermediate
value. It was also found that the lines showing interdemiate values tended
to have a low seed productivity. Awn length was inversely correlated withseed productivity. Further between two of three independent marker genes
involved in the cross, a tendency to restriction of recombination was detected.
These facts were considered as indications of so-called M-V linkage.
Among the characters investigated, the probability of misclassification
between sativa and glaberrima estimated from intra- and inter-specific variations was correlated with transgressiveness in the hybrid. Ligule length andsecondary branch number per primary panicle branch, which seemed to
be useful as key characters, were practically non-transgressive and were
subjected to M-V linkage more obviously than other characters.
Gene Markers Obtained in O. glaherrima
Yoshio SANO

Among more than 500 strains of Oryza glaberrima maintained in ourstock, only a few striking morphological differences useful for genic analysis are found. In order to more gene markers and compare their loci and98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

effects with those of similar genes known in o. staiva, the seeds of a strainof O. glaberrima selected for colorless pericarp and insesitivity to photoperiod were treated with EMS (Ann. Rep. 27: 97). From about 7000 M aplants, a total of 96 mutants expressing characters like glutinous endosperm,"Daikoku" dwarfism, ligulelessness, long empty glume, and glabrousness
were selected (Ann. Rep. 27: 101). These characters were found to be eachcrossed with a strain of o. staiva (T 65) having a corresponding marker
gene. The complementation test showed that the mutant strains of o. glaberrima carried genes similar to wx, db 19, g, and gl known in O. sativa,
respectively. There are 10 mutants showing "Daikoku" dwarfism. All ofthem had the same recessive gene, which was symboled dl . The same wasthe case with 12 strains showing ligulelessness.
In O. sativa, the dl mutant is known to have the second internode (fromthe top) remaining non-elongated. In the glaberrima dl mutant, however,the second internode elongated. The F 1 plants between sativa dl andglaberrima dl strains also showed elongation of the second internode. Thissuggests either that the dl in sativa and dl in glaberrima differ in action, orthat their effect is modified by the genetic background.
Mutant characters like brown leaf spots, narrow leaf, small grain, andbrittle culm were also found to be controlled each by a recessive gene in
the asme manner as found in O. sativa.
Sensitivity of Barnyard Grass, Echinochloa crus-gall; to a Herbicide
Shinya IYAMA

Four lines of barnyard grass were selected out of the material collectedfrom various parts of Japan regarding the sensitivity to the herbicide conTable 1. Sensitivity of parent lines and Fa progenies ofbarnyard grass to propanil.
Parent line

% SurvivalFa

93-5 (Rl)81-27 (R2)
52-8 (SI)51-4 (S2)

58.1
61.8
19.8
38.6

RlxSI

S1xR2
RlxS2

Mid-parent% survival% Survival
1
2
3
1
2

33.4
37.8
35.3
40.8
55.5
47.1

}

38.9
40.8

}

48.4

RESEARCHES CARRIED OUT IN 197899

taining propanil. Crosses were made between lines showing high and low
sensitivities and their F z seedlings were examined with regard to sensitivity.
In percent survival after a spray of propanil, the F z means were almost equal
to mid-parent values as shown in Table I suggesting that the sensitivity wascontrolled by genes with additive effects.
The Effect of Noise on Egg Laying Performanceof Japanese Quails*
Takatada KAWAHARA

A wild strain of Japanese quail, Coturnix coturnix japonica, propagatedfor 12 generations in a domestic condition after being captured in the field,
showed changes in various productive and behavioral characters toward
the domesticated type. The wild strain was compared with a domestic
strain with regard to their sensitivity to noise. The birds, 208 wild and
19 domestic, were reared under a noise treatment with buzzer, 95 phons,working one hour, and 12 times a day for 20 days. The daily photoperiod
applied was 18-hour light and 6-hour darkness. Air temperature (25° ±3°q,
humidity (about 55 %), and other environmental conditions were kept as conNoise: buzzer treatment(95 phon every other hour I- - : Treatment

100

£

.
~

~

(n:208)....... ; Non-treatment (n:251)

l

I

90
80 .

.2

~

70

.

60

..

"0

w

50

T-,.

A

,
~

28 days

_c_

J,

...

0

5 days,..
0

19 days
I

0

or

.,o

b

,

Days from the begining of examination

Fig. 1. Egg production curves in noise treated and noiseless control groupsof wild strain.
A, egg production in the noiseless period before treatment; B, during noise treatment;
C, in the noiseless period after treatment.

* This work was supported by a grant from the Environment Agency.

,

.....

"0

100

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

stant as possible. The egg laying performance of the wild strain was reduced
under the noise treatment, but the domestic strain showed no depression.
The egg production rate of the wild strain decreased by 11.4 percent (66.9 %in the noiseless period before treatment, A in Fig. 1 and during noise
treatment, B in Fig. 1), while that of the domestic strain did not change(84.8 % in the noiseless period befoe treatment and 85.3 % during noise
treatment).
In the wild strain, the egg production rate was recovered in 9 days eventhough the noise tratment continued for 20 days. Its egg production rate
was recovered by 14.8 percent (55.5 % during noise traetment, B in Fig. 1
and 70.3 % in the noiseless period after tratment, C in Fig. 1). Heritabilityof the reactions of wild strain to noise treatment, estimated from the components of variance, was 0.08 in the reduction rate of egg production asshown by the difference between periods A and B; it was 0.11 in the recovery
rate of egg production as shown by the difference between periods C andB. In both reactions, genetic variance was much smaller than environmental
variance.
A Dark Frayed Feather Nervous Disorder
Mutant of Japanese Quail
Takatada KAWAHARA

During the course of propagation of a domestic foundation stock ofJapanese quail, Coturnix coturnix japonica, which has a plumage patternand other characters of the wild type. A female variant was found in theearly summer of 1975 from the foundation stock. The mutant birds obtained from the F 2 of the female variant had following characteristics.
1) Dark down and adult plumage; 2) frayed flight feathers; 3) a nervous
disorder characterized by a tremor of small amplitude. These characters
appeared in chicks of one day old, but the condition of nervous disorder
was variable among individuals and differed according to growing stages
and environmental conditions. The mutation was semi-lethal and viability
up to 20 weeks after hatching was about 40 %. Mortality was caused by dislocation of the hip joint. The mutant birds had lower values in body growth,
fertility, hatchability, age at sexual maturity and egg production rate than
the normal birds.
The results of various matings indicate that this mutant is due to a new

RESEARCHES CARRIED OUT IN 1978

101

autosomal recessive gene, symboled dn. Changes in these three characters,i.e., dark plumage, frayed feathers and variable magnitude of nervous
disorder indicate some change in the development of organs of ectodermal
origin. The recessive gene may be a chromosomal dificiency or translocation
although it is not detected yet.
Effects of Noise on the Learning Performance of Mouse Strains withDifferent Learning Abilities and their Hybrid*
Tohru FUJISHIMATo examine the effect of noise on the avoidance learning performance ininbred strains and hybrids, crosses were made between two inbred strains,
C3HjHeMs and SWMjMs. The former showed the highest discriminatory
ability while the latter showed the highest avoidance avility (Ann. Rep. 25:
82). These strains and their F 1 mice were exposed to one-hour "pink" noiseof 100 phons six times at one-hour intervals every night for a period of
one or three weeks, and they were tested with an automated Y-maze apparatus in which a buzzer and a lamp were used as the conditioned stimuli
and an electric shock as the unconditioned stimulus.
In the first training session after a one-week or three-week exposure tonoise, the mice showed no remarkable depression in the avoidance learning
performance. However, an additional one-week exposure to noise resulted
in an inhibition of consolidation of avoidance learning effects in the parental
inbred strains. The hybrid mice showed no such response. The data also
showed no acclimatization to noise in the activity and memory effect.

*

This work was supported by a grant from the Ministry of Environment Agency

Nitrogen Fixation in the Rhizosphere of RiceTaro Fum, Yoshio SAND, Sinya IYAMA and Yukinori HIROTA(a) Nitrogen fixation in rice strains.
We have proved nitrogen fixation in rice due to association of certain bacteria in the rhizosphere by the use of an improved experimental technique(Ann. Rep. 28: 114). For more reliable evaluation of nitrogen fixing
activities, we observed the relation between nitrogen fixing activity andplant age and diurnal variation, and recognized the following points.
(I) Nitrogen fixing activity was evident even at an early stage of growth and102

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

increased gradually with aging. The activity reached the maximum at thehaeding stage, and after holding a high value for about 2 weeks it decreased
with ripening. (2) Nitrogen fixation by algae in the paddy field is often included in that due to rhizosphere bacteria resulting in over-estimation ofbacterial nitrogen fixing activity. In our experiment, we covered soil sufrace
in the pot with alminum foil to hinder algal growth by shading. Yet, weobserved appreciable nitrogen fixing activities. (3) Nitrogen fixing activity
showed diurnal variation. Activity was highest in the early morning andevening, and was lowest around noon and mid night. (4) To observe thegenetic control of nitrogen fixing ability in rice strains, strain C5444 showing
a high activity was crossed with a standard strain T65, which was analyzed
for different genes and had a low or moderate nitrogen fixing activity.
Eighty Fa lines from the cross were assayed for acetylene-reducing activity.
The lines showed a wide variation in the activity which appeared to betransgressive. The data suggested that the nitrogen fixing ability was controlled by more than one gene and that new strains having higher nitrogen
fixing activity would be selected from varietal hybrids.
(b) Nitrogenjixing bacteria in the rhizosphere of rice
A 250 g sample of soil in which rice roots extended was stirred in
200 ml of sterilized water, kept for 2 days at 30°C, and 1 ml of supernatant
was suspended into 10 ml of nitrogen-free liquid medium containing 10 gilof glucose and 2 pg/ml of biotin. The medium was incubated at 30°C
for 2 days in darkness either in aerobic or in anaeroic condiaion. An appreciable acetylene reducing (nitrogen fixing) activity was observed in both conditions showing the presence of nitrogen fixing bacteria in the medium.
In corraboration with Dr. K. Komagata of Institute of Applied Microbiology, Tokyo University, nitrogen fixing bacteria were isolated on a nitrogenfree medium or on a malate medium. By this method, a Clostridium strain
was obtained previously. This year, several nitrogen fixing bacteria of
spiral, filamentous, or rod shapes were isolated and the strans were purified.
The experiment indicated that at least six species of nitrogen fixing
bacteria were present in the rhizosphere of rice.
An inoculation experiment was carried out with rice plants raized in nonsterilized and germ-free soil conditions in test tubes into which a Clostridium
strain was added. The non-sterilized plants showed an appreciable increasein acetylene reducing activity after the inculation of Clostridium. This indicated the contribution of this bacterium to nitrogen fixation in the rice

RESEARCHES CARRIED OUT IN 1978

103

rhizosphere. The germ-free plants showed no acetylene reducing activity
even of the bacterium was inoculated. This suggested that the soil became
unfavorable fQr the bacterial growth as the results of sterilization at 120°C.
In order to look for bacterial species capable of more efficient nitrogen
fixation in the rice rhizosphere, the germ-free plants grown on nitrogenfree agar medium in test tubes were inoculated with bacteria isolated fromthe rhizosphere. Clostridium could grow very well under this condition,so far examined.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

BOOKS AND PAPERS PUBLISHED IN 1978
BY STAFF MEMBERS
ABE, T., S. MISAWA, K. NISHIOKA, T. OKUNO, Y. NAKAGOME 1978: Formation of a ring
chromosome 14 subsequent to the de novo 13/14 reciprocal translocation: A new
cytogenetic evidence obtained by the nucleolus-organizer staining. Ann. Genet.
21: 109-112.
ABE, T., K. KAWAI, S. MISAWA, and Y. NAKAGOME 1978: Silver staining for the analysis of rearrangements of human acrocentric chromosomes. Proc. Jap. Acad. 54(Ser. B): 451-454.
AZUMI, J., G. KOHAMA, and M. SASAKI 1978: Cytogenetic studies in patients with cleft
lip and/or cleft palate (IV). (Screening studies of chromosomes, from August
1973 to August 1977). Jap. J. Hum. Genet. 23: 161-166.
Fum, T. 1978: Studies in neutron mutagenesis in maize--effects of dose fractionationand cell moisture status on mutation induction. Radioisotopes 27: 642-647.
FUJISAWA, T. and T. SUGIYAMA 1978: Genetic analysis of developmental mechanisms in
hydra. IV. Characterization of a nematocyst-deficient strain. J. Cell Sci. 30:
175-185.
HARADA, M. and T. H. YOSIDA 1978: Karyological study of four Myotis bats (Chiroptera, mammalia). Chromosoma (Berl.) 65: 283-291.
HATTORI, M., MIURA, K. YAMAGUCHI, H. OTANI and T. HATA 1978: Interaction between
bases involved in the 5'-terminal cap structure of eukaryotic mRNA. Nucleic
Acids Research, Special Publication No.5: 391-394.
HIROTA, Y., T. Fum, Y. SANa and S. IYAMA 1978: Nitrogen fixation in the rhizosphereof rice. Nature 276: 416-417.
IMAI, H. T. 1978: On the origin of telocentric chromosomes in mammals. J. theor.
BioI. 71: 619-637.
IMAI, H. T. and T. MARUYAMA 1978: Karyotype evolution by pericentric inversion asa stochastic process. J. theor. Bio. 70: 253-261.
INOUE, Y. and T. K. WATANABE 1978: Toxicity and mutagenicity of cadmium andfuryl-furamide in Drosophila melanogaster. Jap. J. Genet. 53: 183-189.
ISONO, S., K. !saNa and Y. HIROTA 1978: Mutations affecting the structural genes andthe genes coding for modifying enzymes for ribosomal proteins in Escherichia coli.
Molec. gen. Genet. 165: 15-20.
KAnA, T. and N. KANEMATSU 1978: Reduction of N-methyl-N'-nitrosoguanidine-induced mutations by cobalt chloride in Escherichia coli. Proc. Jap. Acad. 54 (Set. B):
234-237.
KAnA, T., K. MORITA and T. INOUE 1978: Anti-mutagenic of vegetable factor(s) on
the mutagenic principle of trytophan pryrolysate. Mutation Res. 53: 351-353.
KANEMATSU, N., M. HACHINOHE, D. HATANAKA, M. MORI, K. SHIBATA, Y. KURODA,
and H. KAWAHARA 1978: Effect of metal ions on tissue culture cells. II. Effect of

RESERCAHES CARRIED OUT IN 1978

105

metal salts on adhesion of Chinese hamster Don cells (In Japanese) Japan. J.
Oral BioI. 20: 270-278.
KAWAHARA, T. 1978: Body traits and behaviour in wild Japanese quail, Coturnix coturnix japonica. (In Japanese) Tori (Bull. Ornithol. Soc. Japan) 27: 105-112.
KAWANISHI, M. 1978: An ecological note on Drosophila oshimai. Jap. J. Ecol. 28:
97-99.
KAWANISHI, M. and W. H. LEE 1978: Food preferences of Drosophila simulans and D.
melanogsster. Jap. J. Ecol. 28: 231-235.
KAWANISHI, M. and T. K. WATANABE 1978: Difference in photo-preferences as a causeof coexistence of Drosophila simulans and D. melanogaster in nature. Jap. J. Genet.
53: 209-214.
KIMURA, M., S. EMURA, T. KAWAHARA, S. ITO, I.IsOGAI, I. SATO, M.ISHIGURO and K.
OHASHI 1978: Individual variation in cerebral esterase electrophoretic pattern of
quails, Coturnix c.japonica. (In Japanese) Jap. Poult. Sci. 15: 184-188.
KIMURA, M. 1978: Change of gene frequencies by natural selection under population
number regulation. Proc. Nat. Acad. Sci. USA 75: 1934-1937.
KIMURA, M. and J. F. CROW 1978: Effect of overall phenotypic selection on genetic
change at individual loci. Proc. Nat. Acad. Sci. USA 75: 6168-6171.
KIMURA, M. and T. OHTA 1978: Stepwise mutation model and distribution of allelicfrequencies in a finite population. Proc. Nat. Acad. Sci. USA 75: 2868-2872.
KOMEDA, Y., M. SILVERMAN and M. SIMON 1978: Identification of the structural genefor the hook subunit protein of Escherichia coli flagella. J. Bacteriol. 133: 364-371.
KOMEDA, Y., M. SILVERMAN, P. MATSUMURA and M. SIMON 1978: Genes for the hookbasal body proteins of the flagellar apparatus in Escherichia coli. J. Bacteriol.
134: 655-667.
KURODA, Y. 1978: Studies on mutagenicity testing for chemicals using reverse mutations in cultured human diploid cells. Mutation Res. 54: 217.
KURODA, Y. 1978: Pilot studies on genetic monitoring of environmental mutagens byusing somatic mutations in primary cultures of human embryonic cells. Mutation
Res. 54: 241-242.
KURODA, Y. and K. SUGIURA 1978: Dose-rate effects of ethyl methanesulfonate on
induction of 6-thioguanine-resistant mutations in cultured Chinese hamster cells.
Mutation Res. 53: 215-216.
KURODA, Y. 1978. Experimental procedure for testing gene mutations in cultured
cells. (In Japanese) "Procedures for Testing Genetic Toxicity and Mutation"(ed. by T. Kada) 157-164. Nihon-Eiseigijutsu-Kenkyukai (Tokyo).
KURODA, Y. 1978: The present status and prospective view of tissue culture studies
in the field of genetics. II. (In Japanese) Tissue Culture (Tokyo) 4: 191-200.
KURODA; Y. 1978: Development and differentiation of insect imaginal discs. (In Japanese)
Heredity (Tokyo) 32(3): 28-33.
KURODA, Y. 1978: Tissue culture studies in differentiation. I. (In Japanese) Tissue
Culture (Tokyo) 4: 366-374.
LEE, W. H. 1978: Genetic variation of walking and flying ability in Drosophila melanogaster. Jap. J. Genet. 53: 327-337.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

LEE, W. H. 1978: Temperature sensitive viability of hybrid between Drosophila melanogaster and D. simulans. Jap. J. Genet.53: 339-344.
MARUYAMA, T. and M. KIMURA 1978: Theoretical study of genetic variability, assuming
stepwise production of neutral and very slightly deleterious mutations. Proc. Nat.
Acad. Sci. USA 75: 919-922.
MATSUDA, H., T. GOJOBORI, N. TAKAHATA 1978: Theoretical study on protein polymorphism and its bearing on the evolution of protein molecules. "Evolution of Protein
Molecules" (ed. by H. Matsubara and T. Yamanaka), 89-100, Japan Scientific
Societies Press (Tokyo).
MATSUHASm, M., I. MARUYAMA, Y. TAKAGAKI, S. TAMAKI, Y. NISlllMURA and Y. HIROTA
1978: Isolation of a mutant of Escherichia coli lacking penicillin-sensitive D-alanine
carboxypeptidase IA. Proc. Nat. Acad. Sci. USA 75: 2631-2635.
MATSUTANI, E. and Y. KURODA 1978: Enhancement of chondrogenesis of cultured quaillimb bud mesenchymal cells by cellophane films. Cell Structure and Function
3: 237-248.
MORITA, K., M. HARA and T. KADA 1978: Studies on natural desmutagens: screening
for vegetable and fruit factors active in inactivation of mutagenic pyrolysis products
from amino acids. Agric. BioI. Chern. 42 (6): 1235-1238.
MORIWAKI, K. and H. T. IMAI 1978: Mechanism of ploidy shift from diploidy to tetraploidy in MSPC-l mouse myeloma. Exp. Cell Res. 111: 483-489.
MATSUNAGA, E., A. TONOMURA, H. OISm and Y. KIKucm 1978: Reexamination of paternal age effect in Down's syndrome. Hum. Genet. 40: 259-268.
MATSUNAGA, E. 1978: Hereditary retinoblastoma: delayed mutation or host resistance?
Am. J. Hum. Genet. 30: 406-424.
MATSUNAGA, E. 1978: Recurrence risks to relatives of patients with retinoblastoma.
Jpn. J. OphthalmoI. 22: 313-319.
MOWA, N. R., E. P. L. ASDOURIAN, Y. HIROTA and M. INOUYE 1978: Gene dosage
effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.
J. BacterioI. 133: 81-84.
NAKAGOME, Y., S.OKA, and M. HIGURASm 1978: Quinacrine and acridine-R banding
without a fluorescence microscope. Hum. Genet. 40: 171-176.
NAWA, S., M. A. YAMADA and M. TsunTA 1978: DNA-induced transformation in silkworm. Jap. J. Genet. 53: 375-379.
NOGucm, S., Z. YAMAIZUMI, T. OHGI, T. GOTO, Y. NISmMURA, Y. HIROTA and S.
NISHIMURA 1978: Isolation of Q nucleoside precursor present in tRNA of an E. coli
mutant and its characterization as 7-(cyano)-7-deazaguanosine. Nucleic Acids
Research 11: 4215-4223.
NOGucm, T.1978: Topics in mouse teratomas. (In Japanese) Chern. and BioI. 16:
2-9.
ODAKA, T., H. IKEDA, K. MORIWAKI, A. MATSUZAWA, M. MIZUNO and K. KONDO 1978:
Genetic resistance in Japanese wild mice (Mus musculus molossinus) to an NBtropic Friend murine leukemia virus. J. Nat. Cancer lost. 61: 1301-1306.
OKA, H. I. 1978: Phylogenetic differentiation of cultivated rice, 21. The sporophyticpollen sterility: Its genetic basis and intervarietal relationships as shown by F 1

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107

sterility. Jap. J. Genet. 53: 397-410.
OKA, H. I. 1978: An observation of wild rice species in tropical Australia. Special
Report under a Grant of Ministry of Education. 24pp.
OKA, H. I., H. MORISHIMA, S. SANO and T. KOIZUMI 1978: Observations of rice species
and accompanying savanna plants on the southern fringe of Sahara desert. Special
Report under a Grant of Ministry of Education. 94pp.
OKA, S., Y. NAKAGOME, T. HONDA and M. AR1MA 1978: A case of distal4q trisomy due
to familial (4; 5) (q31; pIS) translocation. Jap. J. Hum. Genet. 23: 167-172.
OHTA, T.1978: Theoretical study on genetic variation in multigene families. Genet.
Res. 31: 13-28.
OHTA, T. 1978: Theoretical population genetics of repeated genes forming a multigene
family. Genetics 88: 845-861.
OHTA, T.1978: Sequence variability of immunoglobulins considered from the standpoint of population genetics. Proc. Nat. Acad. Sci. USA 75: 5108-5112.
OHTSUKA, E., S. NIsmKAwA, A. F. MARKHAM, S. TANAKA, T. MIYAKE, T. WAKABAYASm,
M.IKEHARA and M. SUGIURA 1978: Joining of 3'·modified oligonucleotides by
T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases
61-77 from Escherichia coli tRNAtllet. Biochem. 17: 4894-4899.
PLusCHKE, G., Y. HIROTA and P. OVERATH 1978: Function of phospholipids in Escherichia coli. J. BioI. Chern. 253: 5048-5055.
ROGreRs, R., A. VAN VOORDE, E. SOEDA and W. FreRS 1978: Nucleotide sequence ofsimian virus 40 Hind K restriction fragment. Eur. J. Biochem. 85: 205-224.
SANO, Y. and F. KITA 1978: Reproductive barriers distributed in Melilotus species andtheir genetic bases. Canad. J. Genet. Cytol. 20: 275-289.
SANO, Y. and F. KITA 1978: Genes for reproductive isolation located on rearranged
chromosomes. Heredity 41: 377-383.
SHlMOI, N., K. TUTIKAWA and Y. YAGI 1978: Search for metabolic activation and depression of in vivo effect of chemical mutagens by host-mediated ree-assay. Mutation
Res. 54: 226-227.
SHINOZAKI, K. and T. OKAZAKI 1978: T7 gene 6 exonuclease has an RNase H activity.
Nucleic Acids Res. 5: 4245-4261.
SmoTA, K. and E. MATSUNAGA 1978: A genetic and epidemiologic study of polydactyly
in human embryos in Japan. Jap. J. Human Genet. 23: 173-192.
SIMON, M., M. SILVERMAN, P. MATSUMURA, H. RIDGWAY, Y. KOMEDA and M. Hn.MEN
1978: Structure and function of bacterial flagella. Sym. Soc. Gen. Microbiol.
28: 271-284.
SoEDA, E. and B. E. GRIFFIN 1978: Sequences from the genome of a non-transforming
mutant of polyoma virus. Nature 276: 294-298.
SoEDA, E., M. KIMuRA and K. MIURA 1978: Similarity of nucleotide sequences aroundthe origin of DNA replication in mouse polyoma virus and simian virus 40. Proc.
Natl. Acad. Sci. USA 75: 162-166.
SONNTAG, I., H. SCHWARZ, Y. HIROTA and U. HENNING 1978: cell envelope and shapeof Escherichia coli: multiple mutants missing the outer membrane lipoprotein
and other major outer membrane proteins. J. Bacteriol. 136: 280-285.

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SUGIURA, K., M. GOTO and Y. KURODA 1978: Dose-rate effects of ethyl methanesulfonate
on survival and mutation induction in cultured Chinese hamster cells. Mutation
Res. 51: 99-108.
SUGIURA, M., N. ITO and M. SUZUKI 1978: A simple method for the identification ofaltered subunits in mutant RNA polymerases of Escherichia coli. Anal. Biochem.
84: 337-339.
SUGIYAMA, T. and T. FUJISAWA 1978: Genetic analysis of developmental mechanisms in
hydra. II. Isolation and characterization of an interstitial cell-deficient strain.
J. Cell Sci. 29: 35-52.
SUGIYAMA, T. and T. FUJISAWA 1978: Genetic analysis of developmental mechanisms in
hydra. V. Cell lineage and development of chimera hydra. J. Cell Sci. 32: 215-232.
SUZUKI, H., Y. NISIDMURA and Y. HIROTA 1978: On the process of cellular divisionin Escherichia coli: A series of mutants of E. coli altered in the penicillin-binding
proteins. Proc. Nat. Acad. Sci. USA 75: 664-668.
SUZUKI, H., Y. NISffiMURA, S. YASUDA, A. NISIDMURA, M. YAMADA and Y. HIROTA 1978:
Murein-lipoprotein of Escherichia coli: A protein involved in the stabilizationof bacterial cell envelope. Molec. gen. Genet. 167: 1-9.
TAZIMA, Y.1978: Low dose-rate experiment with tritiated thymidin as a simulator ofchemical mutagens using silkworm oocyte system. Mutation Res. 53: 274-275.
TAZIMA, Y.1978: Analysis of chemically induced recessive visible mutants at locimarked with egg color gene in Bombyx mori. XIV Int. Congo Genetics Abst. II:
290.
TAZIMA, Y. 1978: Consequences of the AF-2 incident in Japan. Environmental Health
Perspectives 29: 183-187.
TAZIMA, Y.1978: Mutagens, comutagens and antimutagens of natural origin. XIV
Int. Congo Genetics Proceedings (in press).
TAZIMA, Y.1978: Radiation mutagenesis of the silkworm. "Silkworm, an importantlaboratory tool" (ed. by Y. Tazima), 213-245 Kodansha, Tokyo.
TAZIMA, Y. 1978: Mutagenicity testing of environmental chemicals. "Silkworm, an
important laboratory tool" (ed. by Y. Tazima), 247-268. Kodansha, Tokyo.
TAZIMA, Y. 1978: Fundamental problems in toxicology -Mutagenicity. (In Japanese)"Toxicology" (ed. K. Uraguchi, Y. Ueno, H. Kitagawa, Y. Kasuya and B. Sakai):
125-140, Chijin Shokan (Tokyo)
TAZIMA, Y.1978: Methods for detection on injuries-Mutagenicity. (In Japanese)"Toxicology" (ed. K. Uraguchi, Y. Ueno, H. Kitagawa, Y. Kasuya and B. Sakai):
1336-1348, Chijin Shokan (Tokyo)
TANAKA, K., K. C. CHuNG, H. HAYATSU and T. KAoA 1978: Inhibition of nitrosamine
formation in vitro by sorbic acid. Food and Cosmet. Toxicol. 16: 209-215.
TUTIKAWA, K. and K. HARADA 1978: The effects of chemical teratogens on the experssionof genes responsible for sacrovertebral abnormalities in mice. Teratology 18:
142-143.
TUTIKAWA, K., N. SHIMOI and Y. YAGI 1978: Mutagenicity of the products generated
by a reaction between chloroquine and nitrite. Mutation Res. 54: 230.
VOLKAERT, G., R. CONTRERAS, E. SOEDA, A. VAN DE VOORDE, and W. FmRS 1977: Nuc-RESEARCHES CARRIED OUT IN 1978

109

leotide sequence of the simian virus 40 Hind H restriction fragment. J. Mol. BioI.
110: 467-510.
WATANABE, T. K. and M. KAWANISID 1978: Geographical distribution of Drosophila
simulans in Japan. Zool. Mag. (Tokyo) 87: 109-116.
YAMAnA, M., M. MATSUHASID and M. TORn 1978: Effect of novobiocin and ethylenediamine tetraacetate on formation of cell packet in Micrococcus luteus. J. Gen. Appl.
Microbiol. 24: 307-315.
YAMAGISHI, H., H. MORISlllMA and H. I. OKA 1978: An experiment on the interaction
between cultivated rice and barnyard grass at different planting densities. AgroEcosystems 4: 449-458.
YASUO, K., Y. FUJIMOTO, M. KATO, Y. KnWCID and T. KAnA 1978: Mutagenicity ofbenzo-trichloride and related compounds. Mutation Res. 58: 143-150.
YOSIDA, T. H. 1978: An XXY male appeared in the F 2 hybrids between Oceanianand Ceylonese type black rats. Proc. Jap. Acad. 54 (Ser. B): 121-24.
YOSIDA, T. H. 1978: Robertsonian fusion of the acrocentric chromosomes in the blackrat from Chichijima, Japan. Proc. Jap. Acad. 54 (Ser. B): 167-172.
YOSIDA, T. H. 1978: A preliminary note on silver stained nucleolar organizer regionsin the black and Norway rats. Proc. Jap. Acad. 54 (Ser. B): 353-354.
YOSIDA, T. H. 1978: Some genetic analysis of supernumerary chromosomes in theblack rat in laboratory matings. Proc. Jap. Acad. 54 (Ser. B): 440-445.
YOSIDA, T. H. 1978: A new Robertsonin fusion of chromosomes found in a black rat
from Sapporo. Proc. Jap. Acad. 54 (Ser. B): 522-527.
YOSIDA, T. H. 1978: Experimental breeding and cytogenetics of the soft-furred rat.
Millardia meltada. Lab. Anim. (London) 12: 73-77.
YOSIDKAWA, K., H. KURATA, S.IwAHARA and T. KAnA 1978: Photodynamic actionof fluorescein dyes in DNA-damage and in vitro inactivation of transforming DNA
in bacteria. Mutation Res. 56: 359-362.
YOSHIMURA, K., H. AlBA and T. H. YOSIDA 1978: Reaginic antibody production in ten
inbred rat strains following immunization with ovalbumin. Jap. J. Veter. Sci. 40:
31-40.

110

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

ABSTRACTS OF DIARY FOR 1978
14
23
24
29
June
July
10
11
24
August10September 19
October4
November 21
December 6
February

249th Meeting of Misima Geneticists' Club
250th Meeting of Misima Geneticists' Club
137th Biological Symposium
251st Meeting of Misima Geneticists' Club
252nd Meeting of Misima Geneticists' Club
138th Biological Symposium139th Biological Symposium
140th Biological Symposium
141st Biological Symposium
253rd Meeting of Misima Geneticists' Club
142nd Biological Symposium
143rd Biological Symposium

RESEARCHES CARRIED OUT IN 1978

111

FOREIGN VISITORS IN 1978
January

122831-Feb. 1

February

5-March 628-Mach 6

March

7

March

13-21
14

March

24

April

27

May

II

II

12-Feb. I, '79
18
23
23
June

18-Sept. 12

July

20

RAJAN, S. S., c/o United Nations, Rangoon,
Burma.
JAIN, Ashok, Embassy of India, Tokyo.
BROCK, R. D., Plant Breeding and Genetics
Section, FAO IAEA, Austria.
SCHWARZ, Uli, Max-Planck-Institut, Germany.
SHARMA, G. P., Panjab University, India.
Lee, SE-YONG, Korea Atomic Energy Res.
Inst. Korea.
CROW, James F., University of Wisconsin,
U.S.A.
LAMSEEJAN, S., Atomic Energy Lab., Kasetsart
University, Bangkok, Thailand.
MANEEPHONG, Chairerg, Kasetsart University, Thailand.
BALAL, M. S., Field Crops Res. Inst., Agricutural Research Center, Giza, Egypt.
KLINGBERG, M. A., Israel Inst. for BioI.
Res., Ness-Ziona & Tel-Aviv University
Medical School, Israel.
KLINGBERG, W., Israel Inst. for BioI. Res.,
Ness-Ziona & Tel-Aviv University Medical
School, Israel.
KASTURI BAI, A. R., Bangalore University,
India.
SHADAKSHARASWAMY, M., Bangalore University, India.
KAo Nan Kuo, Prairie Regional Lab., National Research Council of Canada, Canada.
SORIANO, J. D., University of the Philippines, Philippines.
MARCUM, Beverly A., University of California, Irvine, U.S.A.
MANKAD, P. G., Embassy of India, Tokyo.

112

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 29

August

7-8
8
10-11

28-29
September 17

17

18
26-27
Ocotober

11

19
November 18-22
December

8

SHELDON, Bruce, C.S.I.R.O., Sydney,
Australia.
WARID, Warid A., University of Cairo, Egypt.
KING, R. C., Northwestern University,
U.S.A.
JUKES, T. H., University of California,
U.S.A.
KRISHNASWAMI, S., Central Sericulture
Research and Training Institute, Mysore,
India.
NARASIMHANNA, M. N., Central Tasar
Research Station, Ranchi, India.
A representative team of Chinese biologists,
Academia Sinica, Peking, China.
SHAHI, B. B., Parwanipur Agriculture
Station, Nepal.
HSUEH, Yu-Ku & others, Chinese Delegation of Food Science 8 Technology, China.
BAKER, Herbert G., University of California,
U.S.A.
STEVENS, Leroy C, Jakson Laboratory,
U.S.A.
BRENNER, S., University Postgraduate
Medical School, Cambridge, England.

RESEARCHES CARRIED OUT IN 1978113

AUTHOR INDEXAMANO, E •.................... 67, 68, 69

30, 67, 68, 69
ARULPRAGASAM, K. D. .
.44
ASANO, T •...•........•.....•....••. 63
ASSEMAT, L.
92, 93AZUMI,J82
CHARLES, S. K •...................•.. 47
CROW, J. F ••........................ 73
ENDo, T •..........•............••.. 32
FERNANDO, H. E .................•... 44
FUJII, T
70, 101
FUISAWA, T • . . . . . . . . . . . . . . . . . . . . . . . . 34
FUlISHIMA, T . . . . . . . . . . . • . . . . . . . . . . . 101
FUKASE, Y •..........•........... 59, 60
AOTSUKA, T.

.

GILL, G . . . . . . . . . . . • . . . . . . . . . . . . . . . .47
GOTO, M58, 59HAMADA, T ....•.....•.•.........•.. 51
HARA, M . . . . . . . . . . . . • . . . . . . . . . . . . . . 64
HATTORI, M • . . . . . . . . . . . . . . . . . . . . . . . . 12
HIDAKA, S ...........•............... 13
HIRAI, H
53
HIROTA, Y • . . . . . . . . . . . . . . . . . . 21,26,101
HIRUMI, H. .
.47
IHARA, M
32
IKEHARA, M ........•••.............. 19

IMAI, H. T. .

54, 56, 63

INOUE, T. . ........•..•.......... 64, 66
INOUE, Y ...............•........•... 77
INUI, N ...........•.••...........••. 63
ITo, N •••..........••...•..••....•.. 19
IYAMA, S ............•••.•.•...•. 98,101

KADA, T. .

63, 64, 65, 66

KANDA, N .......•.•.......... 50, 51, 53
KASTURI BAl, A. R. . .•.••............ 58
KATO, H •.•........• 44, 46, 47, 48,50,51
KAWAHARA, T •.......•.......•.. 99,100
KAWANISHI, M ..•....•..... 79,87,94,95
KIMURA, M •..........••.......•.• 72, 73
KOMEDA, Y •......•.•...........•••. 28
KURODA, Y ........••.•....... 35, 37, 39
KUSUDA, J •..•.....••.....•......••. 20

LEE, W. H

95

MARCUM, B. A. . ...•..•..•••••••.•.. 34

MARKHAM, A. F •...•.•.........•...• 19
MARUYAMA, T. . ..••...•.••.....• 73, 75
MATSUNAGA, E .....•••..•......... 81,82
MATSUTANI, E. .
36, 37

MENG, W. T •.•...•..•..•.•.•........ 47
MINEZAWA, M ••..•.....•............ 48
MIURA, K • . . . • . . . . . • . . • . . . . . . . . . 12, 13
MIYAKE, Too ..•....•..•...•........•. 19
MONTY, J •.......•.....•...•........ 46
MORISHIMA, H.

. ........•.. 89, 90, 91, 93

MORIWAKI, K • . . . . . . 30,44,46,47,48,53
MURAKAMI, A •...•.•.•.. 54, 56, 59, 60, 62
MUROFUSHI, Moo ........•.......•.... 52
NAKAGOME, Y. . ......•...........•.. 82
NAWA, S . . • . • . . . . . . . . . . . . . . . . . . . . . . . 33
NISHIKAWA, S. . ..•..•.•............. 19
NISHIMURA, Y. . •.......•.....••..... 26
NOGUCHI, T. . •...•..•.••.......• 40, 41
OCHIAI, Y ••........••••........•.... 46
OHNUMA, A. ..
54, 56, 63
OHTA, T. . .•....•..•.•.•........ 72, 74
OHTA, Y64, 65OHTSUKA, E. . ........•.•.........•. 19
OKA, H. I.
88, 89, 90, 92, 96
OSHIMA, C. . •...•...•.••....•• 84, 85, 86
OZAWA, T •••••.•••.....•.••...•.• 60, 62
SABARATNAM, M. .
44
SANO, Y •••••.•..•.....•.. 90,96,97,101
SHIMOTOHNO, K •.••..•...•••......•.. 13
SOEDA, E •.••........................ 15
SUGIURA, M. ..
19,20
SUGIYAMA, T • . . . . . • . . . • . . . . . . . . . . . . . 34
SUZUKI, H. • •.........•..........•.. 26
SUZUKI, M . . . . . . . . . . . . . . • . . . • . . . . . . . 19
TAKAHATA, N. . ........•......•..... 75
TAKAMURA, T •...•...........• 84, 85, 86
TAKANO, J •.•.•...........•.....•.. , .34
TAMURA, T •..••.•...........•......• 26
TANAKA, S •...........•......•.•.... 19
TAYA, C •............•...••..••..••. 50
TAZIMA, Y. . ..•................. 58, 62
TAKEDA, Y •.•..•.....•.....•.•.•.... 21
TSUCHIYA, K •.•.....•.•.•.•..• 46, 47, 48
UCHIDA, T. A •.......•.•.....•.•.•.. 53
UKAI, Y •.•.....•.....•....•........ 68
WADA, Y ••..•.........•.•.....•...• 41
WAKABAYASHI, T •••...•.•.•.•••.•...• 19
WATANABE, T. K.
.42, 77, 79, 87, 94
YAMADA, M. A. .
33
YOKOIYAMA, A •...•••••.•••.••••• 63, 64
YOSIDA, T. H ••. 43,46,47,48,49,50,51,52,53

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