NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 17

1966

Published by

THE NATIONAL INSTITUTE OF GENETICS
KUJi1lUl, Sizuoka-ken, Japan1967

Annual Reportof theNational Institute of Genetics
No. 17, 1966

Published by

The National Institute of Genetics, Japan
1967

CONTENTS
General statement.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council............................................................
Projects of research for 1966
Researches carried out in 1966
1.

II.

Cytogenetics
Induction of plasma cell neoplasms in BALB/c mice, theirkaryotypes and r-globulin specificity. YOSIDA, T. H., IMAI, H.
T., MORIWAKI, K. and MIGITA, S.
Change of ploidy in plasma cell tumors (MSPC) in early transplant
generations. IMAI, H. T., YOSIDA, T. H. and MORIWAKI. K. "Comparative study of karyotypes in sublines producing and nonproducing r-globulin in mouse plasma cell tumors. YOSIDA, T.
H., POTTER, M. and IMAI, H. T.
Karyotypes of mouse plasma cell tumor MOPC-31B before andafter in vitro cultivation. YOSIDA, T. H., IMAI, H. T., MASUDA,
T. and NAMBA, Y.
Comparative study of mouse leukemias developed by treatment
with chemicals and radiation. YOSIDA, T. H., TSURUTA, R. and
KURITA, Y.
Alteration of karyotypes in a mouse leukemia strain DML.
TSURUTA, R. and YOSIDA, T. H.............................
Changes in aggregate-forming activity of cells in carcinogenesis.
KURODA, Y.
Difference in aggregate-forming activity between normal andmalignant cells. KURODA, Y.
Physiological and developmental genetics
Behavior of nuclei in germinating pollen grains of wheat, riceand maize. KIHARA, H. and HORI, T.
Photoperiodic response of various Oryza species. IX. KATAYAMA,

T. C

,.

Anatomical studies on interior root found in root of rice plant.
KATAYAMA, T. C.
A genetic study on skeleton-length in Japanese quail. ISOGAI, I.,
KAWAHARA, T. and SAKAI, K. I.
Genetic changes in body weight caused by competition in chickens.
FUJISHIMA, T.

1
2
5
6
11

11
12

13

14

15
16
17
18

19
19
21
21
22

ii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

A genetical study on organ formation in Nicotiana tabacum L.
HIGUCHI, S. and SAKAI, K. 1.
Major gene and polygenes governing the rachis deficiency inrice. WASANO, K. and SAKAI, K. 1. . . . . . . . . . . . . . . . . . . . . . .
Estimation of genetic parameters in Chamaecyparis forests.
SAKAI, K. 1., HAYASHI, S. and MUKAIDE, H.
Developmental genetic study of panicle formation in rice. IyAMA,S.
A developmental genetic study in rice. BALAL, M. S. and SAKAI,
K. 1.
Electrophoretic comparison of soluble proteins from different
organs of tobacco plant. NARISE, S. and SAKAI, K. 1.
Analysis of genetic correlations between panicle, internode andleaf lengths among mutant strains of a rice variety, Norin 8.
MORISHIMA, H. and OKA, H. I.
Analysis of growth curves for panicle and internode elongationin mutant strains of a rice variety, Norin 8. MORISHIMA, H. and
OKA, H. 1.
Differentiation of aggregation-promoting materials from embryonicchick liver cells. KURODA, Y.
Characterization of tissue-specific materials with cell-binding
activity obtained from embryonic chick cells. KURODA, Y. ..
In vitro cultivation of single cells from Drosophila melanogasterlarvae. KURODA, Y.

III.

25
26
27
28
30
31

32

33
34
35
37

Biochemical genetics
Some notes on the chromogranules in hypodermal cells of silkworm
larvae. TSUJITA, M.
Development of chromogranules in the larval skin of the silkworm.
TSUJITA, M. and SAKURAI, S.
Some properties of nucleic acid in larval skin cells of the silkworm.
KOJIMA, K., TSUJITA, M. and SAKURAI, S.
Incorporation of 14C-glycine into chromogranules of larval skincells of the silkworm. SAKURAI, S. and TSUJITA, M.
Chemical characterization of chromogranule membrane in larvalskin cells of the silkworm. SAKURAI, S. and TSUJITA, M. ..
Genetic effects of DNA in Ephestia. NAWA, S. and YAMADA, M. ..
Peroxidase isozymes in leaves of Pharbitis nil. ENDO, T.
Hormonal enzyme regulation in the cultured hypocotyl of Pharbits
nil. ENDO, T.
Variation in peroxidase isozymes of Oryza perennis and O. sativa.
CHU, Y. E.

39
40
42
44
46
48
50
50
51

CONTENTS

Characterization of xanthine dehydrogenase from Drosophila.
SHINODA, T.
Multiple molecular forms of xanthine dehydrogenase in Drosophila.
SHINODA, T.

IV.

V.

Evolutionary genetics
An intergeneric hybrid between Eremopyrum orientale andHenrardia persica. SAKAMOTO, S. . . . . . . . . . . . . . . . . . . . . . . . .
Three intergeneric hybrids among Heteranthelium piliferum,
Eremopyrum buonapartis and Hordeum sp. SAKAMOTO, S. ..
Diallele crosses among Sikkimese rice types. III. KATAYAMA, T.
C.
Further studies on embryo transplantation in the genus Oryza.
KATAYAMA, T. C.
Geographical distribution of winter, intermediate and spring typesof common wheat. NAKAI, Y.
Embryosac sterility of F 1 hybrids between strains of Oryza
perennis. CHU, Y. E. and OKA, H. I.
Pattern analysis of character variations in Oryza perennis.
MORISHIMA, H. and OKA, H. I. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Population survey of No.1 chromosome polymorphism of blackrats (Rattus rattus) collected in Japan and Korea. YOSIDA, T.
H., MORIGUCHI, Y., KANG, Y. S. and SHIMAKURA, K.
Segregation of three chromosome types in black rats crossed in
the laboratory. YOSIDA, T. H. and MORIGUCHI, Y.
Mathematical and statistical studies on population genetics
Simulation studies on the number of neutral alleles maintainedin a finite population by mutation. KIMURA, M.
Two loci polymorphism as a stationary point. KIMURA, M.
The mutational load with epistatic gene interactions in fitness.
KIMURA, M. and MARUYAMA, T. . . . . . . . . . . . . . . . . . . . . . . . . . .
Eigenvalues in a genetics problem. MARUYAMA, T.
A diffusion process with heterosis. MARUYAMA, T.
An application of Kimura's formulae to define the evolutionary
load in a small population. MARUYAMA, T.
Dimensionality of human migration. YASUDA, N.
Dimensionality and distance in human migration. YASUDA, N. ..
A statistical singularity at the ABO blood group system. YASUDA,
N.

VI.

Experimental studies on population genetics
Deleterious genes in the second chromosome concealed in natural

iii

53
53

55
55
56
57
58
59
59

61
62

64
65
67
68
70
72

73
74
75

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

iv

populations of Drosophila melanogaster. OSHIMA, C. and
WATANABE, T. K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Distribution of persistent lethal genes in natural populations.
OSHIMA, C. and WATANABE. T. K.
Recessive visible mutant genes on the second chromosome
concealed in natural populations. OSHIMA, C. and WATANABE,
T. K.
Segregation distorter (SD) genes and their linked lethal genes in
Drosophila melanogaster. WATANABE, T. K. and OSHIMA, C. ..
A mechanism of persistence of some lethal genes in natural
populations of Drosophila melanogaster. WATANABE, T. K. ..
Further study on chromosomal polymorphism in the Kofu andKatsunuma natural populations. WATANABE, T. and OSHIMA,C.
Lack of chromosomal interaction with respect to overdominancein Drosophila melanogaster. MUKAI, T.
The detrimental load to the lethal load ratio (D: L ratio) of
newly arising mutations in Drosophila melanogaster. MUKAI,
T. and CROW, J. F.
Studies on the competition between races lA and 21B of wheat
leaf rust. KATSUYA, K.
Interaction among genotypes for migration in Drosophila melanogaster. NARISE, T.
The relation between migratory activity and competitive ability
in Drosophila melanogaster. NARISE, T.
Experimental induction of bilateral asymmetry in wings ofDrosophila melanogaster. NARISE, T. and SAKAI, K. I. ....

VII.

77
78

79
80
80

81
83

84
85
86
87
88

Radiation genetics and chemical mutagenesis in animals

Post-irradiation modification and mechanism of reverse dose-rate
effect on mutation induction in silkworm gonia. TAZIMA, Y.
and SADO, T.
Repair of radiation induced premutational damages revealed byfractionated irradiation of silkworm spermatids. TAZIMA, Y.
and ONIMARU, K.
Modification of r-ray-induced mutation frequencies in the silkworm
by post-treatment of spermatids and spermatozoa with nitrogengas. TAZIMA, Y. and ONIMARU, K.
Mechanisms of mutation induction by mitomycin-C in the silkworm.
T AZIMA, Y. and ONIMARU, K.
Mutagenicity of a nitrofuran derivative applied to silkworm germ
cells. TAZIMA, Y. and FUKASE, Y.

90

91

92
94
95

CONTENTS

v

Changes in the mutation response of post-meiotic silkworm germ
cells to r-rays with the progressing spermiogenesis. T AZIMA,Y.
97Studies on strain differences in radiosensitivity in the silkworm.
I. Screening of sensitive and resistant strains to embryonicradiation killing. MURAKAMI, A. and TAZIMA, Y.
98
Studies on strain differences in radiosensitivity in the silkworm.
II. Relation between sensitivity to embryonic killing andmutability. MURAKAMI, A. and TAZIMA, Y.
100
Relation between sensitivity to killing and mutation observed
during a mitotic cycle of silkworm cleavage nuclei. MURAKAMI, A.
102
The effect of 5-bromodeoxyuridine (BUDR) on the frequency of
14 MeV fast neutron induced mutations in the gonial cells of
the silkworm. MURAKAMI, A.
103
Relative biological effectiveness of 14.1 MeV neutrons in the
induction of dominant lethal mutations in the mouse. TUTIKAWA, K.
105

VIII. Radiation genetics in plants
RBE of radiations in E-1 hole of Kyoto University Reactor (KUR).
MATSUMURA, S., AMANO, E. and HAYASHI, M.
107
Comparison of mutagenic efficiency between EMS and r-rays.
MATSUMURA, S. and FUJII, T.
109
Comparison of the killing effect of i-rays and thermal neutrons.
FUJII, T.
110
On the determination of absorbed dose in heavy ionizing particles.
FUJII, T.
111
Photoreactivation of an UV-induced mutation in maize. MATSUMURA, S. and MABUCHI, T.
112
Photoreactivation of UV-induced damage in maize pollen. AMANO,
E. and MABUCHI, T.
114
On the somatic variations in corn and chrysanthemum underchronic r-irradiation. MATSUMURA, S. and FUJII, T......... 115
Endosperm mutations induced by UV in corn. FUJII, T.
117
IX. Microbial genetics
Genetic map of Hi gene III Salmonella. YAMAGUCHI, S. andIINO, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
A straight flagellar mutant in Salmonella. IINO, T. and MITANI,
M
120

vi

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Flagellin biosynthesis in Salmonella spheroplasts. SUZUKI, H.
and IINO, T.
121
Genetic fine structure of the mot loci in Salmonella typhimurium.
ENOMOTO, M.
121
Mapping of three mot loci in Salmonella by linkage analysis.
ENOMOTO, M.
122
Difference in frequencies of cotransduction of mot C with Hi
gene in Salmonella. ENOMOTO, M. and YAMAGUCHI, S.
123
Infection of bacteriophage-chi to Serratia marcescens. IINo,
124
T
Normal repressed level of ornithine transcarbamylase activity incrude extracts of an arginine sensitive mutant of Salmonella
typhimurium. ISHIDSU, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

x. Human genetics
Evaluation of the family planning programme in Japan. MATSUNAGA, E.
Association of ear-wax types with susceptibility to arteriosclerosis-A preliminary report. MIYAHARA, M. and MATSUNAGA, E.
Maternal age of mosaics with Down's syndrome. MATSUNAGA,
E., TONOMURA, A., OISHI, H. and KIKUCHI, Y.
Chromosome replication in Down's syndrome. KIKUCHI, Y. and
OISHI, H.
Phenotypes and sex chromosomes in five patients with Turner's
syndrome. OISHI, H., KIKUCHI, Y. and MATSUDA, E.
Clinical conditions of patients with apparently normal chromosomes. III. OISHI, H. and KIKUCHI, Y.
Amino acid sequence around cystine residues of a lambda typehuman Bence-Jones protein. SHINODA, T.
Cellulose acetate electrophoresis and a2-lipoprotein of humanserum. OGAWA, Y.
Books and papers published in 1966 by staff members..............
Abstracts of diary for 1966
Foreign visitors in 1966 .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment
Author index

126
127
129
130
131
133
134
135
136
142
143
145
146

GENERAL STATEMENT
During this fiscal year there was no significant progress toward thecompletion of our Institute except for the establishment of an additional
laboratory in the Department of Population Genetics. We still want to
add three departments, namely for molecular genetics, biophysics, andfine structure.
A budget for our main building was not allocated this year. However
we shall be able to complete the remaining one-third of the building by
the end of 1967.
This year Dr. T. H. Yosida received the award given by the Genetics
Society of Japan for his paper on the relation between chromosomal alteration and development of tumors.
To our regret, death removed from our ranks Dr. YO Takenaka, headof the Department of Cytogenetics, who died of stomach cancer on the
18th of March, 1966. He started his scientific career after graduatingfrom the University of Tokyo where he had majored in cytology under
Prof. K. Fujii in 1927. Immediately thereafter he went to Seoul University where he taught until 1946 botany courses for junior students. Afterthe end of World War II, he came back to Japan and became a memberof our Institute established in 1949. In 1953 he became the head of the
Department of Cytogenetics. His main contributions in plant cytology
were studies on Rumex, Lilium and Nicotiana. Later he concentrated on
Prunus (cherry trees) and became even well known among the citizens of
Misima as an enthusiastic investigator of cherry trees. According tohis opinion Prunus yedoensis, one of our most popular cherries, might bea hybrid between P. lannesiana and P. pendula. He also has bredseveral new strains. One of them, the Showa-Sakura cherry tree, is most
famous. One hundred and twenty plants of this strain were planted in
the Garden of the Imperial Palace and will produce beautiful flowers in
two to three years. The grateful citizens of Misima are planning to erecta monument in recognition of his contributions in front of our Institutebordered by cherry trees which he loved so much.
When he was in Seoul, Dr. Takenaka was a mountaineer and a traveler.
Almost all the high mountains were climbed by him. He has written abook entitled" The Mountains and Landscape of Korea (1938)." He traveled
twice to Inner Mongolia as leader of scientific expeditions.
Dr. Takenaka will not be forgotten by his pupils at the Seoul University
as a teacher and also by his colleagues at the National Institute of Genetics
for his endeavors in the early years of its establishment and development.

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

STAFF(At the End of 1966)
Director

KIHARA, Hitoshi, D. Sc., Member of Japan Academy, Emeritus Professorof Kyoto University
Members

1. Department of Morphological Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag.
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
SADO, Toshihiko, D. Ag.

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
MORIWAKI, Kazuo, D. Sc.; IMAI*, Hirotami; MASUJI*, Hiroshi;
TSURUTA*, ReikoThe 2nd Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
YONEDA, Yoshiaki, D. Sc.

3. Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department
The 1st Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
WATANABE, Takao K.
The 2nd Laboratory
KIHARA, Hitoshi, D. Sc., Head of the Laboratory
SAKAMOTO, Sadao, D. Ag.; KATAYAMA*, Tadao C., D. Ag.;NAKAI*, Yasuo; HORI*, Tadaaki* Research members under grant from other organizations or visiting researchers.

STAFF3

4. Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ag., Head of the Department

The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-aki

The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.

The 3rd Laboratory
TSUJITA, Mitsuo, D. Ag., Head of the Laboratory
SAKURAI, Susumu; KOJIMA*, Kunihiro

5. Department of Applied Genetics
SAKAI, Kan-Ichi, D. Ag., Head of the Department

The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag. (in U.S.A.); FUJISHIMA, Tohru, D. Ag.;NARISE*, Takashi, D. Sc.; INOUE*, Teruo

The 2nd Laboratory
lYAMA, Shin-ya, D. Ag., Head of the Laboratory
EL-BALAL*, Mohamed S.; HAYASHI*, Shigesuke; NARISE*, Sumiko,
D. Med.; TOMITA*, Koji; WASANO*, Kikuo; HIGUCHI*, Seiichiro;
KOIKE*, Tuneo

The 3rd Laboratory
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.; CHU*, Yaw-En

6. Department of Induced Mutation
MATSUMURA, Seiji, D. Ag., Head of the Department

The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
MUKAI, Terumi, Ph. D., D. Sc. (in U.S.A.)The 2nd Laboratory
MATSUMURA, Seiji, D. Ag., Head of the Laboratory
FUJII, Taro, D. Ag.

The 3rd Laboratory
MATSUMURA, Seiji, D. Ag., Head of the Laboratory
AMANO, Etsuo

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

The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

SHINODA, Tomotaka (in U.S.A.); MATSUDA, Ei

The 2nd Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
KIKUCHI, Yasumoto, D. Sc.; OISHI, Hidetsune, D. Sc.;SHIBATA*, Kunihiko8.

Department of Microbial Genetics
IINO, Tetsuo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi, D. Sc.; YAMAGUCHI*, Shigeru
The 2nd Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.; ISHIDSU, Jun-ichi; SUZUKI*, Yasuko

9.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
HIRAIZUMI, Yuichiro, D. Sc. (in U.S.A.); MARUYAMA, Takeo, Ph. D.
The 2nd Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
YASUDA, Norikazu, Ph. D.

10. Experimental Farm
MATSUMURA, Seiji, D. Ag., Head of the Farm
MIYAZAWA, Akira
I-Ionorary Members and Part-Time Staff

KOMAI, Taku, D. Sc., Member of Japan Academy, Emeritus Professor of
Kyoto University
KUWADA, Yoshinari, D. Sc., Member of Japan Academy, Emeritus Professor of Kyoto University
LILIENFELD, Flora A., Ph. D.
OGUMA, Kan, D. Ag., Emeritus Professor of Hokkaido University
TANAKA, Yoshimaro, D. Ag., D. Sc., Member of Japan Academy, Emeritus
Professor of Kyushu University
Department of Administration

MORINAGA, Norihiro, Head of the Department

COUNCIL

5

KANAMORI, Shigeru, Chief of the General Affairs Section
TANAKA, Mutsuo, Chief of the Finance Section
Association for Propagation of the Knowledge of Genetics

KIHARA, Hitoshi, President, Director of the Institute
TAZIMA, Yataro, Managing Director, Head of the Morphological Genetics
Department
MATSUMURA, Seiji, Manager, Head of the Induced Mutation Department
MATSUNAGA, Ei., Manager, Head of the Human Genetics Department
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Emeritus Professor of Tokyo University

COUNCILOKADA, YO, Chairman, Emeritus Professor of Tokyo University
MORIWAKI, Daigoro, Vice Chairman, Professor of Tokyo Metropolitan
University
FURUHATA, Tanemoto, Director of Scientific Research Institute of Police
IMAI, Tomizo, Director of National Institute of Agricultural Sciences
KAYA, Seiji, Emeritus Professor of Tokyo University
KIKKAWA, Hideo, Professor of Osaka University
MAKINO, Sajiro, Professor of Hokkaido University
MATSUO, Takane, Professor of Tokyo University
OCHI, Yuichi, President of Azabu University of Veterinary Science
OGUMA, Kan, Emeritus Professor of Hokkaido University
SAITO, Toshio, Governor of Sizuoka Prefecture
SAKATA, Takeo, President of T. Sakata Company
T ACHI, Minoru, Director of Institute of Population Problems
TSUDA, Kyosuke, Director of Institute of Applied Microbiology, Tokyo
University
TSUKAMOTO, Kempo, Director of National Institute of Radiological Sciences
WADA, Bungo, Emeritus Professor of Tokyo University

6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

PROJECTS OF RESEARCH FOR 1966
Depar~men~ of Morphological Gene~ics

Genetics of the silkworm (TAZIMA and ONIMARU)
Repair processes in radiation mutagenesis (TAZIMA, SADO and ON1MARU)
Genetic studies of radiosensitivity in the silkworm (MURAKAMI)
Chemical mutagenesis in the silkworm (TAZIMA and ONIMARU)
Genetic studies on insect cells in tissue culture (KURODA)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Biochemical genetics on tissue-specific materials with cell-binding activity(KURODA)
Depar~men~ of Cy~ogene~ics

Cytogenetical and biochemical studies on tumor cells (YosmA, MORIWAKI,
KURITA, MASUJI, OHARA, IMAI, FUKAYA and TSURUTA)
Mechanism of chromosomal abnormalities by treatment with chemicals(YosmA, KURITA and TSURUTA)
Studies on chromosomal polymorphism of Muridae (YosmA, MORIWAKI and
MORIGUCHI)
Experimental breeding and genetics of mice and rats (YosmA, MORIWAKI,
KURITA, SAKAKIBARA, MORIGUCHI and SONODA)
Correlation between taxonomy and karyology of ants (IMAI)
Morphological and genetical studies on some plant tumors (YONEDA and
CHU)
Cytogenetical and biochemical studies on morning glory (YONEDA)
Depar~men~of Physiological Gene~ics

Genetic studies on insecticide resistance in Drosophila pseudoobscura(OSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE, T. K.)
Studies on chromosomal aberrations of natural populations of Drosophilamelanogaster (OSHIMA and WATANABE, T. K.)
Nucleus substitution in wheat and related species (KIHARA and HORI)
Comparative gene analysis with reference to the origin of wheat (KIHARA
and TSUNEWAKI)
Geographical distribution of necrosis genes in wheat (TSUNEWAKI and
NAKAI)PROJECTS OF RESEARCH FOR 1966

7

Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Genetic bases of ecological differentiation in Agropyron (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Morphological studies of Oryza (KIHARA and KATAYAMA)
Investigation of .photoperiodic responses of Oryza species (KATAYAMA)
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA, YAMADA and
TSUJITA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA and TSUJITA)
Studies on a gene for retarded moulting (rm) in the silkworm (TSUJITA)
Studies on the chromogranule formation in larval hypodermal cells of thesilkworm (TSUJITA and SAKURAI)
Analysis of genetic action on cell differentiation in higher organisms(TSUJITA and NAWA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum proteins (OGAWA)
Comparative studies on seed proteins of rice plant by electrophoretic
analysis (SAKURAI)
Genetics on isozymes in plants (ENDO)
Enzyme regulation in cultured organ of morning glory (ENDO)
Department of Applied Genetics

Studies on developmental instability in poultry (SAKAI, KAWAHARA andFUJISHIMA)
Quantitative genetic studies in poultry (KAWAHARA, FUJISHIMA and INOUE)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Studies on competition in plants and animals (SAKAI, IYAMA, FUJISHIMA
and NARISE, T.)
Estimation of genetic parameters in forest trees (SAKAI, HAYASHI andTOMITA)
Developmental genetics of quantitative characters in plants (SAKAI, ELBALAL, WASANO and HIGUCHI)
Genetic studies on developmental instability in plants (SAKAI and SHIMAMOTO)
Studies on the effects of X-ray irradiation on quantitative characters ofrice (IYAMA)
Biochemical studies on development of higher plants (SAKAI, NARISE, S.

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

and HONDA)
Genetic studies of isolating barriers in Oryza (OKA and CHU)
Survey of geographical variation in Oryza perennis (MORISHIMA and OKA)
Experiments on natural selection in wild and cultivated rice forms (MORISHIMA and OKA)
Analysis of sterility genes in Oryza (OKA and MORISHIMA)
Analysis of genetic plant types (MORISHIMA and OKA)
Department of Induced Mutation

Radiation genetics of mice (TUTIKAWA)
Population genetics of Drosophila (MUKAI)
Studies on the effects of irradiation on populations (MUKAI)
Radiation genetics of cereals (MATSUMURA, FUJII and MABUCHI)
Radiation genetics of Arabidopsis (FuJII)
Radiation genetics and its practical application (MATSUMURA and MABUCHI)
Radiation genetics of corn (FUJII and AMANO)
Biophysical studies of radiation genetics (IKENAGA and KONDO)
Radiation dosimetry (IKENAGA, AMANO and HAYASHI)
Department of Human Genetics

Genetic consequences of population trends (MATSUNAGA)
Dermatoglyphics (MATSUNAGA and MATSUDA)
Down's syndrome in Japan (MATSUNAGA, OISHI and KIKUCHI)
Cytogenetics in man (OISHI, KIKUCHI and SHIBATA)
DNA replication in human chromosomes (KIKUCHI and OISHI)
Biochemical studies on plasma proteins and enzymes (SHINODA)
Chemical modification of ribonucleic acid and their constituents (SHINODA)
Department of Microbial Genetics

Genetic fine structure analysis on microorganisms (IINO and YAMAGUCHI)
Genetics of cellular regulatory mechanisms (SUZUKI, H., ISHIDSU andSUZUKI, Y.)
Genetics of bacterial flagella (IINO, ENOMOTO and SUZUKI, H.)
Genetics of motility in bacteria (ENOMOTO)
Genetics of host range in bacteriophages (IINO, ENOMOTO and YAMAGUCHI)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA)PROJECTS OF RESEARCH FOR 1966

9

Uses of computers in the theoretical studies of population genetics(KIMURA and MARUYAMA)
Effects of radiation-induced mutation on fitness (HIRAIZUMI)
Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)
Studies on the genetic structure of human populations (YASUDA)RESEARCHES CARRIED OUT IN 1966I.

CYT06E:NE:TICS

Induction of Plasma Cell Neoplasms in BALB/c Mice, Their
Karyotypes and r-Globulin Specificity I)
Tosihide H. YOSIDA, Hirotami T. IMAl, Kazuo MORlWAK[
and Shunsuke MlGITA21

13 plasma cell tumors induced by Dr. M. Potter of National Cancer
Institute, Bethesda, U.S.A., in BALB/c mice were characterized by neartetraploid stemline cells, except for one tumor which had a hyperdiploidkaryotype (Yosida et al1964 and 1966, this Ann. Rep. 14 and 16). Almost all
tumors were observed through many transplant generations from 10th to
70th. In order to ascertain the chromosomal condition of primary plasma
cell tumors, we have studied 5 primary plasma cell neoplasms induced in thislaboratory. Among 35 BALB/c mice injected with complete Freund adjuvant five developed plasma cell tumors. They were named MSPC-1 to
MSPC-5. The range of chromosome number distribution, modal chromosome number, ratio of cells at diploid(s), triploid(1.5s), tetraploid(2s) andoctoploid(4s) level, specificity of r-globulin in serum and urine in all fivetumors are given in Table 1. In all those neoplasms no marker chromosomes were observed in the primary state.
Table 1.

Karyological and biochemical characteristics of 5
plasma cell neoplasms (MSPC)% of polyploid cells*

Name oftumorsRange ofchromo no.

Mode

MSPC-lMSPC-2
MSPC-3MSPC-4MSPC-5

38-81
39-93
35-94
39-168
35-94

40
86
39
78
44

Is

1.5s

2s

4s

92
14
24
12

0
8
2
2
4

8
78
64
84
24

0
0
0
2
0

72

No. of Specific protein
cells observed Serum Urine
50
50
50
50
50

r-A
r-F
r-F

A-chainr-A* Is, 1.5s, 2s and 4s denote respectively near-di-, near-trio, near-tetra- and nearoctoploid chromosome numbers.
11 This work was supported by a research grant from the National Cancer Institute (CA 07798-03), Public Health Service, U.S.A.
2) Virus Institute, Kyoto University, Kyoto.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Change of Ploidy in Plasma Cell Tumors (MSPC) in Early
Transplant Generations l )Hirotami T. IMAI, Tosihide H. YOSIDA and Kazuo MORIWAKIIn mouse plasma cell neoplasm MSPC-1, the majority of cells (92 percent) were characterized by having near-diploid chromosome number (srange) in the primary state. Among them, 68 per cent cells showed
exactly diploid chromosome number (40). The tumor was transplanted to
3 mice, in 2 of them successfully. But the development of the transplanted
tumors was very slow. About four months after inoculation, a small
tumor was recognized in the site of inoculation. The chromosome number
in most cells of the tumor thus developed was reduced to 39. In the second
transplant generation, 3 mice were successfully transplanted. Amongthem, one mouse (2a) was killed 29 days after inoculation and its tumor
cells were examined. 48 per cent of them were characterized by having
near-tetraploid chromosome (2s) number. The mode was at 78 chromosomes. In another mouse (2b) which was killed 43 days after transplantation,
cells at 2s level were increased to 66 per cent. The mode, however, wasdecreased to 76 chromosomes. In the remaining mouse (2c) cells at 2s
level increased to 70 per cent, while the mode was reduced to 73 chromosomes.
Tumor 2a, one of the second transplant generation, was again successfully transplanted to three other mice (3a, 3b and 3c). Since this generation, two tumor lines, one with diploid and another near-tetraploid
chromosomes, were established separately. The diploid line of MSPC-1,
however, easily changed to tetraploid condition in the course of transplantations.
In the case of MSPC-3 plasma cell tumor, 14 and 34 per cent cells wereat diploid level in the primary solid and ascites tumors, respectively. In
the first transplant generation of the solid tumor frequency of diploidcells was reduced to 6 per cent. Frequency of near-diploid cells in another MSPC-5 line which was characterized by 44 modal chromosomes was
72 per cent in the primary tumor, but in the first transplant generation
cells at near-diploid level were remarkably decreased to about 9 per cent,and in the second transplant generation no cells at diploid level could beobserved in the tumor cell population.
Based on the above results, we conclude that plasma cells can develop
to malignancy in diploid condition, but they easily change to tetraploid
condition in the course of cell multiplication.
11 This work was supported by a research grant from the National Cancer Institute (CA 07798-03), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1966

13

Comparative Study of Karyotypes in Sublines Producing
and Non-Producing r-Globulin in Mouse
Plasma Cell Tumors!)
Tosihide H. YOSIDA, Michael POTTER and Hirotami T. IMAI

Chromosomes of r-globulin producing (positive) and non-producing (negative) sublines in three mouse plasma cell tumors, RPC-6A, RPC-20 and
MOPC-70, were compared in order to ascertain whether a karyotypic
difference could be found between them. All negative lines were derived
from their positive parental lines in the course of serial transplantations.
The RPC-6 positive and negative lines were examined at the 56th andthe 73rd transplant generations, respectively. In RPC-20 line, they wereat the 77th and 75th transplant generations, respectively, while, inthe MOPC-70A positive line cells at the 12th transplant generation wereobserved. 70A ·10A and 70A ·10E positive lines which were established by
10 cell transplantations of the 70A line were also observed. They wereat the 69th and 59th transplant generations, respectively. In the r-globulin
negative line, the 82nd transplant generation was used (Table 1). As
the table shows, karyotypes of all r-globulin non-producing (negative)
sublines were remarkably different from those of their parental positivelines.
In the RPC-20 line the change of karyotypes in the negative line fromthat of the positive parental line was clear. In the positive line, 77 and76 chromosomes, among them as markers one submetacentric (SM) , one
metacentric (M) and one minute (m), were observed most frequently. Thelong telocentric chromosomes (TC) with secondary constriction near thecentromere were another marker in this line. They have the appearanceof SAT-chromosomes. In the negative subline, however, 75 and 74
chromosomes, among them one SM-, one M- and two m-markers, wereusually observed. In the karyotype of this subline only one TC-element
was found. One new minute marker found in the negative line was similar
to the SAT-shaped element of TC-chromosome. Based on the above investigations, it is suggested that the karyotypes of the negative line had
developed by breakage at the secondary constriction of the TC-chromosome
included in the positive line karyotype, and the SAT-like element withcentromere which resulted from the breakage remained in the negativeline karyotype as a new minute, and then some telocentric chromosome
was lost, producing the negative line karyotype from that of the positiveline.
1) This work was supported by a research grant from the National Cancer Institute (CA 07798-03), Public Health Service, U.S.A.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table l.

Karyotypes in protein producing (positive) and non-producing(negative) sublines in mouse plasma cell tumors
Protein

Chromosomeno. (Mode)

Markerchromosome*"

Positive
Negative
Positive64-176 (68)
53-145 (73)67-154 (77)

Negative40-145 (75)r-A serum
None
Lambda
chain
None

56
70
69

"

Positive37-128 (73)

1M
2M,2SM
1M, ISM,
2TC, 1m
1M, ISM,lTC, 2mNone

r-F serum,
excess
kappa

12

"
"

"

69
59
82

Tumor line
RPC-6A
RPC-20MOPC-70A

MOPC·70A·10A"
MOPC-70A·lOE"
MOPC-70A
Negative

57-68
63-70
68-86

(66)
(68)
(77)

2L,lm

r-Globulin

Transplant
generation

None

70

* M=metacentric; SM = submetacentric; L=extremely long telocentric;
TC=telocentric with secondary constriction near the centromere;m=minute.

The relation of karyotype change between positive and negative sublines in the other two lines (RPC-6A and MOPC-70A) was not clearlyrecognized, because the difference between their karyotypes was toocomplicated.
Karyotypes of Mouse Plasma Cell Tumor MOPC-31B before
and after in Vitro Cultivation!)
Tosihide H. YOSIDA, Hirotami T. IMAI, Yujiro NAMBA2),
Toru MAsuDA2), and Sunsuke MIGITA2)

Mouse plasma cell tumor MOPC-31B which was obtained from Dr. M.
Potter of the National Cancer Institute, Bethesda, U.S.A., was characterized by producing r-F globulin and excess kappa chain. Number of chromosomes of the tumor cells at the 61st transplant generation ranged from76 to 81 showing the highest frequency at 80 (54 per cent). Among 50
cells 90 per cent had one long telocentric and one minute as marker
chromosomes, while only 8 per cent cells showed one metacentric marker
in addition to the above two. These tumor cells were cultivated in vitro
11 This work was supported by a research grant from the National Cancer Institute (CA 07798-03), Public Health Service, U.S.A.
2) Virus Institute, Kyoto University, Kyoto.

RESEARCHES CARRIED OUT IN 1966

15

by Y. Namba, one of the authors. After 10 culture generations, chromosomes in 50 tumor ceIls were again analysed. Among them 64 per cent
ceIls had three marker chromosomes (one long telocentric, one metacentric and one minute) which were observed rarely in the parental ascites
form. On the other hand, ceIls with two markers commonly observed inascites tumor were never found in the ceIls of the culture adapted line,
although they produced r-F globulin and kappa chain as weIl as the ascites form.
Comparative Study of Mouse Leukemias Developed byTreatment with Chemicals and Radiation!)
Tosihide H. YOSIDA, Reiko TSURUTA and Yoshinori KURITA

In order to find a relation between the chromosomal condition of mouse
leukemias and the source of carcinogenic agents, we have used methylchoranthren, DMBA, and r-radiation for the induction of leukemias. In
the present experiments chromosomes of 28 leukemias were observed.
Among them 11 leukemias were induced by treatment with methylchoranthren given to adult RF-strain mice, 3 by treatment with DMBA ofadult RF strain mice, 2 by DMBA given to newborn RF strain mice,
2 by DMBA given to newborn Swiss albino (SWM) mice, 4 by treatment
with r-rays of adult C57BL mice, and one by treatment with DMBA andr-rays of an RF adult mouse. Five spontaneous leukemias of RF-strain
mice were also observed. The chromosomes of all leukemias developed
primarily were observed in various organs, such as bone marrow, spleen,
thymus and lymphnodes. The results of observations are summarized asfollows:
1) Leukemias induced by chemicals and r-radiation showed a mode ofchromosome numbers varying from 39 to 48.
2) The modal chromosome number was different by the organ examined. Frequency (per cent) of cells with diploid 40 as modal chromosome
number to those with chromosomes over and under 40 as the mode differs markedly by the organ examined; namely, in bone marrow, spleen,
thymus, and lymphnodes it was 76.2, 66.7, 23.6 and 53.3 per cent,respectively. Cells with chromosome numbers outside of 40, mostly had
41 chromosomes.
3) Among leukemias developed by treatment with DMBA three deve11 This work was supported by a Grant in Aid for Foundamental Scientific Research from the Ministry of Education in Japan (No. 94002, 1966), and by a research
grant from the National Cancer Institute (CA 07798-03), Public Health Service,
U.S.A.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

loped by injection of the chemical at the adult stage, and other fourdeveloped by injection of the same drug to newborn mice. The former
showed normal diploid chromosome numbers in all organs examined, whilein the latter deviating chromosome numbers from normal karyotype were
found in many organs examined.
A clear relationship between chromosome alteration and developmentof mouse leukemias by various carcinogenic agents could not be found atpresent. The study will be continued.
Alteration of Karyotypes in a Mouse Leukemia Strain DMVIReiko TSURUTA and Tosihide H. YOSIDA

An ascites leukemia strain DML developed in an RF-strain mouse bytreatment with DMBA had 40 chromosomes in the primary tumor. All
chromosomes were rod-shaped like those of the normal karyotype of themouse. The chromosome number was reduced to 39 at the first transplant
generation and was again reduced to 37 (mode) and 38 in the 5th transplant generation. Cells with 37 chromosomes were characterized by having
two submetacentric elements, one large and one small, and those with 38
chromosomes had one small submetacentric chromosome. From the karyological analysis it is suggested that cells with 38 chromosomes havedeveloped from those with 37 chromosomes by breakage of the large submetacentric element at the centromere. After the 11th transplant generation cells with 38 chromosomes were observed most frequently in thecell population, and this condition was maintained until the present 20thtransplant generation.
In the 6th transplant generation the solid tumor developed at the siteof inoculation was accompanied by an ascites tumor in the peritoneal
cavity. The solid and ascites type tumors were transplanted separately
and two tumor lines were established. Karyotypes of the solid type tumor
did not change from those of the original ascites line. In mice bearing
solid type tumors, bone marrow, splean and mesentery lymphnodes were
examined karyologically. In these organs cells with typical DML karyotypes were usually observed. This result means that the DML-cells
transplanted subcutaneously invaded easily those organs.

1) This work was supported by a Grant in Aid for Foundamental Scientific Research from the Ministry of Education in Japan (No. 94002, 1966), and by a research
grant from the National Cancer Institute (CA 07798-03), Public Health Service,
U.S.A.

RESEARCHES CARRIED OUT IN 1966

17

Changes in Aggregate-Forming Activity of Cells
in Carcinogenesis!)
Yukiaki KURODA

Dissociated cells from lO-day embryonic chick liver and heart were
cultured in monolayer in standard culture medium. After 2 days of cultivation the cells were infected with Rous sarcoma viruses (RSV) for 50
minutes at 38°C, washed and cultured for another two or five days inmonolayer. The cells were collected by treatment with trypsin and cellsuspensions containing each 3 x 106 cells in 3 ml culture medium wererotated on a gyratory shaker by the standard procedure. After 24 hoursof rotation the aggregation patterns of RSV-infected cells were comparedwith those of non-infected control cells which had been cultured for corresponding day number in monolayer.
Aggregates formed from RSV-infected liver cells showed an increasein average diameter in comparison with those from non-infected liver cells.
RSV-infected heart cells also formed larger aggregates than those from
non-infected heart cells. These results indicate that embryonic chickcells transformed by infection with Rous sarcoma virus may have altered
their surface properties functioning in mutual cohesiveness in aggregate
formation. The increase in adhesiveness in transformed cells coincides
with the previous findings of the piling-up behavior of RSV-infected transformed cells and loss of contact inhibition of neoplastic cells.
A malignant tumor that appeared spontaneously in mammary glands ofthe ddl mouse was dissociated by treatment with trypsin. Cell suspensions
containing each 3 x 106 cells in 3 ml culture medium were rotated bystandard procedure. Aggregation patterns obtained from rotation culturesof mammary tumor cells were compared with those from control culturesof normal mammary gland cells of the mouse.
Mammary tumor cells formed after 24-hour rotation some large aggregates 0.5 mm in diameter and many small aggregates. This was in clear
contrast with the complete absence of such aggregates in 24-hour control
cultures of normal mammary gland cells.
It has been reported that the embryonic cells showed age-dependentchanges in aggregation patterns and that cells maintained in monolayer
cultures showed a decline in aggregate-forming activity. Changes in aggregation patterns shown in the neoplastic transformed cells may havesome relation to changes accompanying differentiation which take placein the cells.
I) This work was supported by a Grant in Aid for Foundamental
Scientific Research from the Ministry of Education in Japan.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Difference in Aggregate-Forming Activity between Normal
and Malignant Cells!)
Yukiaki KURODA

Human cervical carcinoma, strain HeLa cells, were intermingled witha variety of normal cells from chick embryos and tested in rotation cuItures for their selective sorting-out property in co-aggregates with normal
cells from tissues of various embryonic origins. It was previously foundthat cells of different histogenetic identities (heterotypic), when co-aggregates, tended to become sorted out into distinct, type-specific groupings,
while cells with similar histogenetic functions (isotypic), though fromgenetically remote animals, remained interspersed within composite aggregates and formed chimaeric, mosaic tissues (Moscona, 1957).
When HeLa cells were intermixed with epidermal cells from 9-day embryonic chick dorsal skin and rotated for 24 hours, spherical or oval aggregates were formed. Internally, the cells were found grouped according
to types; the aggregates consisted of a central distinct region of chickepidermal cells and of an outer region formed solely by HeLa cells. HeLa
cells intermixed with 7-day embryonic chick liver cells of endodermal
origin formed large aggregates with rough surface, which consisted solely
of HeLa cells. Chick liver cells in the mixed cell suspension formed aggregates of spherical shape, separated from HeLa-aggregates.
When HeLa cells were intermingled with dermal cells from 9-day embryonic chick dorsal skin or mesoblast cells from 6-day embryonic chicklimb-bud, aggregates formed after rotation for 24 hours consisted ofchimaeric structures of HeLa cells and either of chick cells of mesodermal
origin. In the aggregates HeLa cells and chick cells were interspersed andclosely associated with each other.
The fact that HeLa cells became sorted out with embryonic chick cellsof ectodermal and endodermal origin and formed chimaeric tissues interspersed by chick cells of mesodermal origin, suggests that HeLa cells
might have originated from some mesodermal tissue of human cervix andmight have maintain their original property after a long period of cultivation. Selective affinity of HeLa cells for a specific type of normal cells
found in the present experiment may explain the selective mechanism bywhich the original neoplastic cells metastasize to some specific types oftissues or organs, though in topographically remote sites of the animal body.

1) This work was supported by a Grant in Aid for Foundamental Scientific Research from the Ministry of Education in Japan.

RESEARCHES CARRIED OUT IN 1966

II.

19

PHYSIOLOGICAL AND DEVELOPMENTAL
GENETICS
Behavior of Nuclei in Germinating Pollen
Grains of Wheat, Rice and Maize
Hitoshi KIHARA and Tadaaki HORI

Our microscopical studies on the behavior of the tube nucleus and two
sperm nuclei in germinating pollen grains were carried out in three representative species of cereals, namely, Triticum aestivum, Oryza officinalis and Zea mays.
The germination of pollen grains was observed on self-pollinated stigmas. For staining of the tube nucleus and the male nuclei, acetocarmine
solution was used. This was easy for wheat pollen grains, but very difficult for those of rice and maize. However if we strongly heat the pollen
grains mounted in acetocarmine over the flame of an alcohol lamp, thetrinucleate condition can be clearly seen in all three materials.
Normal pollen grains of all three materials contain one tube nucleus
and two sperm nuclei. In general all three species follow the same pattern of behavior of the three nuclei, i.e., two sperm nuclei enter the pollen
tube and the tube nucleus follows. This regular sequence in the movement of pollen nuclei is rarely disturbed.
Germination of the pollen grains on the stigma starts after 3-5 minutes.
In wheat, the tube nucleus remains frequently in the pollen grain, while
it almost always emigrates in rice and maize.
It is likely that male gametes are transported passively by the cytoplasmic stream to the pollen tube during germination, since they lie nearer
to the germ pore than to the tube nucleus. It is suggested that an autonomous movement of male gametes may act as an auxiliary agent intransportation. The tube nucleus seems to be intimately connected withthe cytoplasm and is located far from the germ pore. This may be themain reason why the tube nucleus enters the pollen tube later than the
sperm nuclei.

Photoperiodic Response of Various Oryza Species.

IX

Tadao C. KATAYAMA

One of the factors influencing photoperiodic sensitivity is the so-called
accumulation effect. The accumulation effect is shown by the photoperiodic effectiveness of short day treatment interrupted by long day condition.
Accumulation effect of strains of O. sativa, O. sativa var. spontanea, O.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

perennis, O. glaberrima, O. breviligulata and O. stapfii was analyzed thisyear. A combination of 12h 30 m light+ 1P30 m dark periods was used as
short day condition, whereas natural day length from June to August wasused as long day treatment.
W0027, a strain of O. glaberrima, treated by 15S (15 short days followed
by long day condition) headed 3.7 days earlier than the control plot; plants
treated by 5S+5L+10S (5 short days plus 5 long days plus 10 short days,
followed by long day condition) headed 0.3 day earlier than the control
plot. This result indicates that the interposed 5 long days cancelled out
the effect of the first short day treatment. However, plants treated by
5S+5L+ 15S headed 26.0 days earlier than the control plot. Thus, posttreatment by a longer period of short days could bring about recovery
from the cancelling effects of the interposed long day condition. On theother hand, plants treated by 10S+5L+5S headed 16.0 days earlier thanthe control plot. In this case the effect of a long initial period of shortdays could not be cancelled out by an interposed long day condition.
From these results, the following conclusions are drawn. Even if thetotal number of given short days was the same, the longer was the initialshort day treatment, the larger was the accumulation effect. In otherwords, complete photoperiodic induction is easily achieved by an earlycontinuous short day treatment.
In order to clarify the differences between the accumulation effects invarious strains or species, the heading dates of each strain treated in thesame way were compared. Having received 15 short days as a total, fourplots, Le., 1) 15S, 2) 10S+5L+5S, 3) 5S+5L+ lOS and 4) 5S+5L+5S+5L+
5S, were compared. For example Kyoto Asahi, a Japanese cultivatedvariety of O. sativa, headed respectively 17.7 days, 17.7 days, 15.3 days
and 14.7 days earlier than the control plot in 1), 2), 3) and 4) (5 per centl.s.d. = 1.06). Even in combined treatment, the effect of pre-treatment
was not cancelled out.
WOlO6, an Indian strain of O. sativa var. spontanea, headed 23.3 days,
26.7 days, 19.0 days and 1.3 days earlier than the control plot, in theabove 1), 2), 3) and 4), respectively (5 per cent l.s.d. =4.84). In this strain,
the effect of pre-treatment was almost completely cancelled by the combined treatment as the result in plot 4) shows. It is clear that the accumulation effect varies widely by the strain. Moreover, the lower was
the latitude from where the strain was obtained, the smaller was theaccumulation effect. This finding indicates adaptation to low latitudes,
where the period of effective short days for photoperiodic induction islonger than in higher latitudes.

RESEARCHES CARRIED OUT IN 1966

21

Anatomical Studies on Interior Root Found in Root of Rice Plant
Tadao C. KATAYAMA

Frequently in rice root, a lateral root grows into the cortex and elongates downward when the aerenchymatous tissue is strongly developed.
Such root can be called "interior root." Interior roots are often recognizedat the base of seminal and adventitious roots, but are never found inlateral roots. In many cases lateral roots, differentiated in mature plants
or initiated from old main roots, remain as interior roots. They can bedivided into the following six groups according to their development andshape. 1) Interior root elongates into the middle part of the cortex of
the main root but not into exodermis; it bends there and extends downward. 2) It pushes into exodermis of the main root and remains there.
3) It reaches to the exodermis of the main root and further extends
downward along it. 4) It extends downward and then pushes into theexodermis of the main root. 5) It elongates straight and pushes into theexodermis of the main root, extending there downward. Shape of themain root is consequently changed. 6) It breaks the exodermis of themain root and extends downward. In all cases, the tip of an interiorroot never breaks out through the exodermis of the main root.
Interior roots differ from normal lateral roots in anatomical structure,especially that of a small central cylinder. In general, they have beenfound in old roots or in basal parts of a root. Formation of interior roots
may be caused by the following factors; decreased cell divisions, andhardening of cell membranes in old plants.
A Genetic Study on Skeleton-Length in Japanese QuailIwahiro ISOGAI, Takatada KAWAHARA and Kan-Ichi SAKAI

Eighty pairs, each of one male and one female bird, all selected at
random from a quail population maintained for three generations as aclosed flock in the National Institute of Genetics were propagated. Seven
hundred birds from those pairs were killed and their bone-samples were
stained with alizarin-red. The length was recorded of skull, cervicalvertebrae, thoracic vertebrae, synsacrum plus caudal vertebrae, humerus,
ulna, metacarpus, femur, tibia and tarsometatarsus.
By the analysis of variance and covariance, it was found that bones ofa female were always larger than those of a male except for skull length
which was not different. Heritability of bone length of posterior extremities (femur, tibia and tarsometatarsus) was 0.84 on the average, while
the bones of anterior extremities (humerus, ulna and metacarpus) had

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

the little lower value of 0.68. Lower heritabilities were found in trunk
bone length (thoracic vertebrae, synsacrum and caudal vertebrae) withthe value of 0.38, cervical vertebrae with the value of 0.26, while thelowest value was obtained with the skull, the heritability being 0.21.
Genetic correlations among different bones were generally higher thanphenotypic correlations. Comparison among trunk bones, anterior andposterior extremities showed that genetic correlations between the twolatter were very high with rg=0.87. The trunk tended to be more orless highly correlated with posterior than with the anterior extremities.
Bones within each body part were highly correlated with each other incomparison with correlations between different body parts.

Genetic Changes in Body Weight Caused byCompetition in Chickens
Tohru FUJISHIMA

Three breeds of domestic fowl, White Leghorn, Rhode Island Red andBarred Plymouth Rock, were crossed by each other following the schemeof 3 x 3 diallel crosses, and the progeny were used to investigate theeffects of competition on growth, and to divide the body weight, whencompetition occurred, into the components of body weight and competitive
ability.
At one week of age, the males and females obtained from those crosses
were divided into three paternal strain groups, three maternal straingroups and two mixed groups involving three different paternal strains.
Each paternal and maternal strain group contained 27 birds comprising
9 birds of each of three different dams in a paternal strain and threedifferent sires in a maternal strain respectively, while each mixed groupcontained a total of 27 birds consisting of 3 birds of each of three different dams in every paternal strain.
Therefore, it is probable that competition may occur among different
maternal strains in a paternal strain group, among different paternalstrains in a maternal strain group, and among both combined in a mixed
group. The experiments were conducted under restricted and full feed
conditions in the mixed groups, but only under restricted conditions in
the paternal and maternal strain groups. The same amount of feed
consumed by the full-fed group in the previous week on a per bird basis
was fed to the restricted group every week during the experimental
period. Under these experimental conditions, the birds were reared until
6 weeks of age and weighed every week.
The models for estimating the genetic parameters were as follows;

RESEARCHES CARRIED OUT IN 1966

23

Yij=M+Si+ Dj+(SD)ij+ Eij
where Yij=progeny mean of the cross of sire breed i and dam breed j.
And, in paternal strain groups,

Si=si ,

Dj=dj+Cdj,(SD)ij = (sd)ij + Cdij

In maternal strain groups,

Si=si+Csi,

Dj=dj,(SD)ij = (sd)ij + Csij ,and in a mixed strain group,

Si=si+Csi,

Dj=dj+Cdj,(SD)ij = (sd)ij + C(sd)ij ,where
si =effect of body weight of sire breed i,
Csi = effect of competitive ability of sire breed i,
dj =effect of body weight of dam breed j,
Cdj =effect of competitive ability of dam breed j,(sd)ij=effect of interaction between si and dj,
Cdij = effect of competitive ability of dam strain j within i paternalstrain group,
Csij = effect of competitive ability of sire strain i within j maternalstrain groups,
CCsd)ij = effect of interaction between Csi and Cdj,
Thus, in male progeny,

si=Ai+Li,

dj=Aj+Lj+Mj,

Csi=Aei+Lei,

Cdj=Aej+Lej+Mej ,Csi=Aei+Lci,

Cdj=Aej+Mej,

and in female progeny,

si=Ai+Li, dj=Aj+Mj,

where
Ai =cumulative additive effect of the autosomal genes for the bodyweight of breed i,
Li =cumulative additive effect of the sex-linked genes for the bodyweight of breed i,
Mj =average maternal effect of body weight of breed j,
Aei =cumulative additive effect of the autosomal genes of competitive
ability for breed i,
Lei =cumulative additive effect of the sex-linked genes for the competitive ability of breed i,
Mej = average maternal effect of competitive ability of breed j,

22 Ai= 22j Mj= 22
Li= 22 Aei= 22 Lei= 22 Mej=O
i i i
~

J.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

24

Table 1.

Variance components and heritabilities for body weight.
2 weeks of age.;;;.:
1-0
....,

mo.

a1
,,2

D

roo

"~D

I-ob.O

2
0 FJ

ro
0...

h~

-::I
.: 1-0

B

0.
::I
0

1-0

b.O
"0
<11

x

~

,,2
s

ob
2

(JSD

,,~

h 2s

Table 2.

6 weeks of age

Male

FemaleMale

Female

2.76
5.61
9.35
69.16
0.06

27.97

75.17
0.45

481.40441.19617.691,805.07
0.29

268.44133.82740.46
1, 785.62
0.18

18.78
2.44
13.32
49.29
0.45

101.96601.981,484.45
2,209.88
0.05

486.45
76.93

3.11
17.01

2.82
11.30
25.20
38.10
0.07

Autosomal, sex-linked, and maternal effects of bodyweight and competitive ability.
Body weight

Autosomal effectSex-linked effect
Maternal effect637.53
1,108.35
0.42

A
L
M

W.L.: White Leghorn,
RockCompetitive ability

W.L.

R.I.R.

B.P.R.

W.L.

R.I.R.

B.P.R.

-20.9
+68.4
-31.7

+98.1
-81.2
-61.2

-77.2 Ac - 1.7+12.8 Lc +13.0+92.9 Mc +35.8

-78.4
+52.7
+33.9

+80.1
-65.7
-69.7

R.I.R.: Rhode Island Red,

B.P.R.: Barred Plymouth

The results of the present experiment, as shown in Tables 1 and 2,
indicate that when competition occurs in a population, the body weightof birds is genetically changed according to their competitive abilities,
that is, although at 2 weeks of age when no competition yet occurs, theheritabilities for body weight in paternal strain groups are equal to thosein mixed groups, while at 6 weeks of age, in mixed groups in which
competition occurs they are considerably different from those in paternalstrain groups, and the heritability for body weight at 6 weeks of age
changes from 0.29 to 0.05 in males and 0.18 to 0.42 in females.
It is found from Table 2 that the autosomal and maternal effects oncompetitive ability are compensatory with each other, and the effects oncompetitive ability are compensatory with those on body weight.

RESEARCHES CARRIED OUT IN 1966

25

A Genetical Study on Organ Formation in Nicotiana
tabacum L.
Seiichiro HIGUCHI and Kan-Ichi SAKAIThis study aimed at finding the genetical basis of organ formation in
Hicks,
Connecticut Broad Leaf, Coker 139, Coker 316, Coker 319, Ibusuki, Daruma,
Nicotin-free tobacco, T. I. 448A and Ambalema. Five plants selected at
random from each variety were investigated for the size of vegetativeand reproductive organs. Genetic correlations among them are given in
Table 1.

Nicotiana tabacum L. Ten varieties were used for the study:

Table 1. Genetic correlation coefficients between the dimensions of vegetativeand reproductive parts of Nicotiana tabacum L.
StipuleLongfilament

0.94

Style

0.58 0.60

CorolladiameterCorollatube

0.69 0.63 0.77

Calyx

0.30 0.22 0.58 0.72 0.63

~

Upper
Inter.eo mediate
en Lower~Upper'\;l Inter-j

mediate
Lower

Leaf

0.59 0.65 0.90 0.85

-0.01 0.08 0.18 0.16-0.11 0.69
-0.16-0.02 0.32 0.26 0.19 0.56 0.96
0.03 0.17 0.55 0.33 0.54 0.20 0.56 0.87
0.26 0.46 0.64 0.19 0.62-0.14-0.11 0.19 0.58
0.09 0.30 0.38 0.01 0.39-0.32-0.22-0.13 0.29 0.87
-0.02 0.09 0.37 0.30 0.48- 0.28-0. 33~ 0.12 0.40 0.68 0.86

It is found from Table 1 that the dimensions of flower parts, i. e. filaments, style and corolla, are mutually highly correlated, but not withthose of stipules and leaves. Calyx is moderately correlated with either
flower organs or stipules. Stipules are highly correlated with each otherbut not with leaves, while leaves are highly correlated among themselves.
Thus, according to size, organs of a tobacco plant may be divided into

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

three groups: 1) flower parts, i.e. stamens, pistil and corolla, 2) stipulesand 3) leaves. No conclusion can be drawn at present for the calyx.
Within each group, genetic correlations were relatively high, whereas
those between different groups tended to be low. In addition, there wasa tendency to high correlation between adjacent parts. No definite tendency
could be seen in environmental correlations.
Major Gene and Polygenes Governing the Rachis
Deficiency in Rice
Kikuo WASANO and Kan-Ichi SAKAI

The present study aims at finding the role of polygenes affecting amajor gene-controlled character in rice. The character under consideration is the rachis deficiency controlled by a recessive sp-gene. The nature
Table 1.

Effect of major gene, sp, and polygenes affecting the sp phenotype,on some quantitative characters in ricesp

Character
Expressivity of sp gene:e.~~ r,nt
Panicles
::::

Straw

·r
r
'"' C'

nma~ Panicles

Z~~.....lNon-bearing tillers
Ulm
PanicleIn",""'"

2nd Internode
3rd Internode

sp+

0
40.45
18.36
22.41
11.23
11.04
0.19
102.65
21.14
38.15
26.21
21.92

L

H

45.00
42.16
6.32
35.83
12.41
11.37
1.03
99.33
15.89
32.85
26.43
23.10

56.65
35.66
3.99
31.67
11.98
9.95
2.02
90.76
14.12
31.48
25.97
20.87

m

sp effect Polygene(M)effect (P)50.83 50.83**
38.91 - 1.54
5.16 -13.20**
33.75 11.34**
12.19
0.96**
10.66
0.38
1.53
1.34**
95.04
7.61**
15.01 - 6.13**
32.17
5.98**
26.20
0.01
21.99
0.07

L:
H:
m:
**,*:

Normal phenotype.
Lines selected for low expressivity.
Lines selected for high expressivity.
(H+L)j2.
Significant at 1% and 5% level, respectively.

(M) :(H+L)j2-sp+.

sp+ :

Ratio
(P)j(M)

5.83**
0.11-3.25**
2.11-1.17**
0.09
-2.08
-0.18
-0.22** -0.23-0.72*
1.89
0.50**
0.37
0.56-4.29**
0.15-0.89**-0.69**
0.12-0.23 />10.00-1.12** />10.00(P) : (H-L)j2.
/>10.00 means that polygene effect is very large in comparison with the effectof major gene.

RESEARCHES CARRIED OUT IN 1966

27of the character is described briefly in the preceding issue, Ann. Rep. 16: 74.
In the F2 population between a wild and asp-strain, 88 plants of 323 intotal were rachis deficient, homozygous for the recessive sp locus. Of
these 88 segregants, selection for high as well as low expressivity was
conducted. The results are summarized in Table 1. From Table 1, we
find that, 1) the major gene, sp, not only governs the rachis deficiency
but also increases straw weight and number of immature tillers anddecreases weight of panicles per plant, panicle length and length of firstinternode or culm. 2) There are polygenes which affect the above
character in addition to the major sp-gene. These polygenes also affectother characters pleiotropically. The effects, however, are not always thesame as those of the major gene. 3) Polygenes enhancing rachis deficiency operate toward decreasing other quantitative characters. 4) The intensityof polygenes in comparison with that of the major gene, sp, is found in
the last colmn of Table 1. It is given in the form of a ratio of polygene
effect against the effect of the major gene. Of twelve ratios, straw weightand number of tillers show negative values indicating that the effect ofpolygenes is opposite to that of the major gene. It is also interestingthat 2nd and 3rd internodes are little affected by the sp-gene, whereas
they are more or less affected by polygenes.

Estimation of Genetic Parameters in Chamaecyparis
Forests
Kan-Ichi SAKAI, Shigesuke HAYASHI and
Hiromasa MUKAIDE

Hinoki, Chamaecyparis obtusa Sieb. et Zucc., is one of the most important
tree species in Japan. They are propagated only from seed. The present
study deals with investigations on inter-tree competition and estimationof genetic, environmental and competitional parameters in several forestsof the species.
The forests were artificially planted and were 20 to 60 years old.
The adopted method of study will be published in detail in the coming
issues of Silvae Genetica (1967). The results are presented in Table 1.
It is found from Table 1 that competition apparently occurs in Chamaecyparis forests. It is of interest to find that an amount of genetic variation
for growth (h 2) appears to be inversely correlated with the index ofcompetitive stress (c 2 ). This might suggest, though it is quite speculativeat present, that in this species the genotypic growth is negatively correlated with competitive ability.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table 1. Genetic, environmental and competitional variance, heritability (h 2 ),index of competitive stress (c 2) and correlation coefficient between
adjoining two trees (ri, i+l)-----

StrainOkayama (1) Okayama (2)Number of trees
Age
G
E

C
h2
c2

200
27
8.02±0.27
0.05±0.01
0.99+0.150r(i, i+l)

Gifu (1)

200
207
22
45
-2.35±1.61 6.83±0.12
2.30±0.49 1.20±0.04
3.62±1.31 0.20±0.09
0.83
0
0.61
0.02
-0.038-0.190Gifu (2)Oita (A)

191
246
60
26
11.82±6.57 -0.26±2.56
2.30±2.20 7.03±1.05
0.88±5.05 4.20±1. 74
0.79
0
0.37
0.06
-0.122-0.004Developmental Genetic Study of Panicle Formation in Rice
Shin-ya IYAMA

Two populations of rice variety Norin No.8, one irradiated by 20,000 r
X-rays and the other as untreated control, were maintained by one parentone offspring method for this investigation. At the fifth generation, 25
lines were derived from the control and 35 lines from the X-rayed population. Ten plants from each line were chosen at random, and the length
Table 1.

Mean lengths of the various parts of culm and panicle
in the control and the X-rayed population
Mean length (em)Character

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Third culm internode
Second culm internode
First culm internode
PanicleSecond rachilla
Third rachilla
Fourth rachillaFirst rachis internode
Second rachis internode
Third rachis internodeControl

X-rayed16.75819.96831.58118.245
6.610
7.015
7.199
2.473
1.316
1.818

16.36819.61630.20517.555
6.463
6.812
6.963
2.376
1.297
1.734

RESEARCHES CARRIED OUT IN 1966

29

of vaious parts of culm and panicle were measured. The culm internodes
were numbered starting from the top and the parts within panicles
starting from the base. The first rachilla was not dealt with because of
its occasional absence. Analyses of variance and covariance were madeand the genetic variances and covariances as well as the genetic correlations among those characters were computed. The characters investigated and the mean values of the two populations are given in Table 1.
Following results were obtained: 1) There were no significant differences between the means of the two populations as to all characters
investigated. 2) Genetic variances of all but the second and the third
panicle internodes were increased by the X-ray irradiation. 3) Genetic
correlations among various parts in the control and the X-rayed population
were computed as shown in Tables 2 and 3. Three groups of characters
Table 2.

Character*

12
3

Genetic correlations among various parts of culm
and panicle in control population

2

3

4

5

6

7

8

1.0000.990 0.317 -0.177 -0.048 -0.0400.141 -0.072
0.990 1.000 0.533
0.059 0.127 0.233 0.357 0.195
0.317 0.533 1.000
0.220 0.248 0.320 0.331 0.330

9100.259 0.321
0.542 0.803
0.500 0.391

4
5
6
7
8

-0.1770.059 0.220-0.048 0.127 0.248- 0.040 0.233 0.320
0.141 0.3570.331-0.072 0.195 0.330

1.000
0.890
0.934
0.915
0.920

0.890
1.000
0.903
0.942
0.869

0.934 0.915
0.963 0.942
1. 000 0.977
0.977 1.000
0.975 0.944

0.920
0.869
0.975
0.944
1.000

0.048 0.428
0.059 0.442
0.264 0.655
0.305 0.678
0.394 0.565

910

0.259 0.542 0.500
0.321 0.803 0.391

0.048
0.428

0.059
0.442

0.264 0.305
0.655 0.678

0.394
0.565

1.000 0.883
0.883 1.000* Same as in Table 1.

were found as enclosed with broken lines, the genetic correlations between
the two belonging to the same group being very high, while those between the two belonging to the different ones very low.
It was concluded from the results that 1) the variety itself had certain
amount of genetic variation in the characters investigated. 2) Mutations
induced by the X-ray irradiation occurred in both positive and negative
directions, having no final effect on the population means. 3) From thegenetic correlations (Tables 2 and 3), the genes concerning to the formation of various parts investigated in panicle and culm were divided
into three groups, one is controlling the culm internode lengths, a second

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table 3.

Character*

1

Genetic correlations among various parts of culm
and panicle in X-rayed population
2

3

4

5

6

7

8

9

10

1
2
3

1.000 0.689 0.802 ·0.539 0.524 0.476 0.513 0.176 . 0.151 0.346
0.689 1.000 0.783 .0.701 0.589 0.596 0.712 0.525 0.170 0.420
0.802 0.783 1.000 0.848 0.748 0.782 0.837 0.561 . 0.147 0.472

4
5
6
7
8

0.539
0.524
0.476
0.513
0.176

9
10

0.151 0.170 0.147
0.346 0.420 0.472

0.701
0.589
0.596
0.712
0.525

0.848
0.748
0.782
0.837
0.561

1.000
0.884
0.935
0.960
0.797

0.884
1.000
0.974
0.906
0.658

0.935
0.974
1.000
0.970
0.747

0.960
0.906
0.970
1.000
0.804

0.797
0.658
0.747
0.804
1.000

0.051
0.118
0.140
0.079
0.317

0.460
0548
0.579
0.559
0.634

0.051 0.118 0.140 0.079 0.317 . 1.000 0.718
0.460 0.548 0.579 0.559 0.634 ·0.718 1.000* Same as in Table 1.

controlling the lengths of panicle, the lower rachillas of panicle and thefirst rachis internode, and a third responsible for the lower internode
lengths of panicle. 4) In addition, it was suggested from the genetic
correlations of the X-rayed population that most new mutations would
have been induced more or less pleiotropically for all the characters inthe same direction, making correlations between groups in the X-rayed
population higher than those in the control population.

A Developmental Genetic Study in Rice
M. S. BALAL and Kan-Ichi SAKAI

Organ formation in rice was investigated from the standpoint of statistical-genetics. Materials used were 99 lines selected at random fromthe Norin No.8 variety established since 1959 and the same number of
X-irradiated lines of the same variety. Characters investigated werelength of panicle, first, second and third leaves counted from the top
and first, second, third and fourth culm internodes. Analysis of variance
between and within lines showed that both populations of 99 lines eachcontained significant genetic variation, indicating that in an establishedrice variety a certain amount of genetic variation was retained, on the
one hand, and that the genetic variance in the X-rayed population waslarger than in the control population, on the other hand. Estimation ofgenetic correlation and degree of pleiotropic effect of polygenes (seeSakai and Suzuki 1964, in Rad. Bot. 4: 141~151) suggested that organs of

RESEARCHES CARRIED OUT IN 196631

rice could be divided into two developmental groups. One is the upperorgan group which includes panicle, first and second leaves and first
culm internode, which show high positive correlation among each other.
The second is the basal organ group which includes third and fourth
culm internodes which are highly positively correlated. The remaining
two organs, third leaf and second culm internode, are moderately correlated with the organs of either upper or basal group. In general, genetic
correlation between neighbouring organs, regardless whether they wereleaves, panicle or culm internodes, tended to be higher than that among
remote organs.
A selection experiment for the length of third culm internode showedthat the fourth was also affected by that selection, though it had little
effect on panicle and first as well as second culm internode. The application of nitrogen at the time of panicle formation yielded about 40 per centincrease in length of third and fourth culm internodes but only a 10 per
cent or less increase in panicle and first culm internode. An anatomical
study indicated that the third and fourth culm internodes developed inadvance of panicle primordia, while the upper internodes (first and second)developed after them.
The foregoing evidences indicate that organs developing in rice are notcontrolled by the same genes but by genes locally controlling specificorgans.

Electrophoretic Comparison of Soluble Proteins fromDifferent Organs of Tobacco Plant
Sumiko NARISE and Kan-Ichi SAKAI

In order to inquire into the mechanism of gene control over the development of higher plants, an electrophoretic analysis was undertaken withsoluble proteins from different organs of Nicotiana tabaccum L.. Organs
investigated were stamens, pistils, corollas, calyxes, nerves and mesophyllsof stipules and also those of leaves. They were separately homogenizedwith the 0.1 M phosphate buffer (pH 7.2) and centrifuged. The supernatants were precipitated with saturated ammonium sulfate and the precipitate was used for analysis after dialysis against distilled water.
Electrophoresis was carried out in columns of polyacrylamide gel using amodified method of Orstein and Davis. Protein bands were stained withamido black. All protein bands separated were compared on the basisof their relative mobility measured as fractions to the distance the fastest
band manifested. The experiment was repeated five to ten times foreach organ. By the (-test, twenty-eight different kinds of bands in all

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

were identified. Of those 28 bands, 14 were found in leaves from the
middle part of stem, 22 in leaves from the top, 17 in stipules, 7 in calyxes,
10 in petals, 9 in pistils and only 5 in stamens. For the purpose ofcomparison among organs, it seemed appropriate to take into account not
only the distribution pattern of bands, but also quantitative measurements
of intensity of each band. The quantitative evaluation was made by
grading the intensity into six classes 0 to 5 by naked eye. Thus, theresults obtained from this combined comparison were as follows: There
was a great deal of similarity among leaves in the number and the concentration of proteins of identical mobility, whereas not much similarity
between leaves and reproductive organs. Furthermore, the similarity
between stipules and leaves was relatively high and the same tendency
was shown between stipules and calyxes. These results may help tosolve the puzzle of genetic control over the development of higher plants.

Analysis of Genetic Correlations between Panicle, Internodeand Leaf Lengths among Mutant Strains of a
Rice Variety, N orin 8
Hiroko MORISHIMA and Hiko-Ichi OKA

For a survey of genetic variations in the sizes of different organs, 33
mutant strains induced from a rice variety, Norin 8, were grown in anexperimental plot, and were measured regarding the lengths at maturityof the panicle, the first to fifth (from the top) internodes, and the first
to fourth leaf sheaths and leaf blades. Those strains, obtained from the
National Institute of Agricultural Sciences through the kindness of Dr.
T. Kawai, were normally fertile and their yields were comparable to thatof Norin 8.
Genetic correlations between the measured character were computed,
and to find an integrated picture of character variations, the matrix ofgenetic correlations was studied by the method of principal component
analysis. The first component appeared to represent the general size ofvarious organs, the vector having almost equal loadings on all the characters. The scores given by this vector showed the variation between
generally tall and generally short strains.
The second component vector had positive loadings on the panicle, theupper (first and second) internodes and the upper leaves (both sheath andblade; first and second), but negative loadings on the lower internodes
and lower leaves. This indicates that the strains vary between a type
having well-elongated upper organs and short lower organs, and the opposite type. These plant types may be called the "upward-elongation"

RESEARCHES CARRIED OUT IN 1966

33

and" basal-elongation" types. The former type appeared to have higher
yielding potential than the latter, when the scores given by the second
component vector were compared with grain yields of the strains, estimated as panicle number per plant x spikelet number per panicle x fertility
X mean grain weight.
We have formerly found from a segregating population of rice twolatent phases of character association which we considered as variation
axes of "genetic plant types" (genetically conditioned character association),
one representing the variation between the" panicle-number" and" paniclelength" types, and the other representing the variation between the" internode-length" and" internode-number" types (Ann. Rep. 16: 68~69). The
above-mentioned" upward-elongation" and" basal-elongation" types may
be compared with the" internode-length" and "internode-number" types,
respectively, as the former would have fewer but longer elongated-internodes
than the latter. The occurrence of this plant-type variation in different
materials seems to suggest that it might result from a general trend of
the developmental system to respond to genetic variations.
The third component vector seemed to distinguish between different
kinds of organs, i. e., internode, leaf sheath and leaf blade. There may
be a phase of correlation at which organs of the same kind are correlated
more intimately than those of different kinds. The first to third components thus extracted could represent a 86 per cent portion of the total
multi-dimensional variation.

Analysis of Growth Curves for Panicle and Internode Elongationin Mutant Strains of a Rice Variety, Norin 8
Hiroko MORISHIMA and Hiko-Ichi aKA

The growth and development of plants may be investigated frem
different viewpoints. This study is concerned with multivariate analysis
of growth parameters separately obtained from different organs of ricestrains. In 23 mutant strains induced from Norin 8 (a part of the materials
mentioned in the previous report), the lengths of panicles and the first
to fourth internodes were measured five times before and after heading
at one week interval and at maturity. In each strain, growth curves ofthese organs were estimated by fitting the data to Robertson's equation,log (xIA-x)=k(t-t1/2), where x is the size at time t, A is the final size,
t1/2 is the time at which a half of the final size is reached, and k is aparameter for growth rate. The actual growth rate (dxldt) was computedfrom these values.
Regarding the variations in A, t1/2- H (number of days between the t1/2

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

time and heading time), and dx/dt of different organs, genetic variances andcorrelations were computed. To observe the pattern of association of thesevalues, principal components were extracted from phenotypic correlation
matrices of A, tl/Z- Hand dx/dt, respectively. Genetic contributions tothe components were then estimated in terms of the regression coefficientsof genetic values on the component axes by a method newly devised by
Hashiguchi et at. (in press). From the" genetic vectors" obtained, variation
patterns in the growth of those organs were estimated as follows: 1) In
the final size (A), the first component (AI) showed the variation in general
size of all the five organs, and the second component (All) showed thephase of variation between the "upward-elongation" and "basal-elongation"
types, in the same manner as set forth in our previous report. 2) The
first component from the interval between (l/Z time and heading time
represented the general earliness of panicle and internode elongation
relative to heading. 3) The first component from the growth rate (dx/dt)
represented variation in the "general growth rate", while the second
component represented a phase of variation at which the growth rates
of panicle and the third internode were negatively associated with thoseof the first and second internodes.
The component scores given by these vectors were computed in eachstrain. Correlations between the scores indicated that 1) strains having
generally long organs tended to have a generally high growth rate forthose organs, and 2) strains of the" upward-elongation" type tended to havea low rate of panicle elongation and a high rate of internode elongation,and vice versa. It seems that slow panicle growth and fast panicle emergence (due to fast internode elongation) bring about well developed
panicles, elongated upper internodes and relatively short lower internodes.
Such a growth type may have high yielding potential.

Differentiation of Aggregation-Promoting Materials
from Embryonic Chick Liver Cells
Yukiaki KURODA

Trypsin-dissociated cells from avian and mammalian embryos when
cultured by rotation-mediated procedure tend to aggregate and form
histoformative structures. Liver cells dissociated from chick embryos at
the ages of 7-, 8-, 10-, 14-, 18-, and 20-days were tested for their aggregability in rotation cultures. Liver cells dissociated from 7-day embryos
formed two or three large aggregates after 24 hours of cultivation.
Aggregates formed from liver cells of older embryos showed a gradualdecline in average diameter. Almost complete loss of aggregability was

RESEARCHES CARRIED OUT IN 1966

35

seen in liver cells dissociated from 20-day embryos.
Mixed suspension of liver cells from both younger and older embryos
produced some aggregates which had an average diameter intermediate
between those found for each of these ages separately. Histologically the
major part of those aggregates consisted of liver cells from younger
embryos.
Cell-free supernatants were obtained from reciprocation cultures ofembryonic chick liver cells, and tested at 28°C for their activity in promoting aggregation. Supernatants prepared from 7-day embryonic liver
cells were considerably enhancing aggregation of 7-day embryonic liver
cells, but had no effect on those from I8-day cells. Supernatants preparedfrom I8-day embryonic liver cells had no effect on liver cells either from
7-day or I8-day embryos.
Supernatants prepared from 7-day embryonic liver cells promoted theformation of aggregates from I8-day embryonic liver cells by rotation at
38°C for three or more days. The promoting activity of aggregate formation by the supernatant seems to be temperature-dependent, since at
28°C this supernatant had no effect on the dissociated cells from I8-day
embryo. It seems likely that non-aggregation of cells from older embryos
might be related to changes accompanying progressing specialization.
Incubation of such specialized cells with active supernatants may modify
the cells so that they produce some materials acting on cell cohesion.

Characterization of Tissue-Specific Materials with Cell-Binding
Activity Obtained from Embryonic Chick Cells
Yukiaki KURODA

It has been previously found that in the process of aggregation ofdissociated embryonic cells some substances released from the cells into
the culture medium promoted the adhesive property of each cell. These
aggregation-promoting substances were contained in cell-free supernatants
prepared from a culture medium in which dissociated cells were shaken
by reciprocating motion at 100 rpm for 2 hours at 38°C. Supernatants
prepared from 7-day embryonic liver cells showed an activity in enhancing
aggregation of 7-day embryonic liver cells at 28°C, but no effect on 7-day
embryonic heart cells, whereas supernatants prepared from embryonicheart cells acted only on embryonic heart cells in forming aggregates,
but had no effect on embryonic liver cells; the aggregation-promoting
activity of the supernatants was tissue- or organ-specific. In the medium
added with 0.5 flg/ml actinomycin D or 5 flg/ml puromycin dissociatedliver cells were inhibited from forming aggregates. These results suggest

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17that the production of aggregation-promoting materials by the cells might
be mediated by DNA-dependent RNA synthesis and protein synthesis.
Some informations on the composition of the supernatants from 7-day
embryonic liver and heart cells were obtained. Both those supernatants
had UV absorption spectra with a peak at 280 mp and some amount ofprotein was detected by chemical determinations with slight amounts of
DNA and RNA. The supernatants lost their aggregation-promoting
activity by heat-treatment at 80 a C for 10 minutes.
Amino acid composition of proteins contained in supernatants from 7day embryonic liver and heart cells was compared. For the purpose ofthis analysis Tyrode's solution was used in which the cells were shaken
on a reciprocating shaker at 38 a C for 2 hours. Molar ratios of aminoacid components in acid hydrolyzates of supernatant proteins are given
Table 1.

Molar ratios of amino acid components in proteins in supernatants
prepared from 7-day embryonic chick liver and heart cells

Amino acid

Liver cell supernatantHeart cell supernatantAcidic and neutral
AlanineAspartic acid
Glutamic acid
GlycineIsoleucine
Leucine
Methionine
Phenylalanine
ProlineSerineThreonine
Tyrosine
Valine

0.83
1.08
1.07
0.97
0.56
1.00
0.03
0.37
0.55
0.63
0.45
0.21
0.77

0.78
1.03
1.05
0.84
0.62
1.00
0.18
0.39
0.51
0.49
0.49
0.27
0.81

1.40
0.60
0.32
1.00

0.96
0.61
0.30
1.00

Basic
Ammonia
Arginine
Histidine
LysineSamples were hydrolyzed in 6N HCI for 40 hours at 105 a C. A Hitachi
KLA-2 type amino acid analyzer was used. Amino acid components
are expressed in molar ratios corresponding to 1.00 mole of leucine
or lysine for acidic and neutral or basic amino acids respectively.

37

RESEARCHES CARRIED OUT IN 1966

in Table 1.
Protein in supernatants prepared from 7-day embryonic liver cells contained more glycine and serine and less isoleucine, methionine, and tyrosine
than those from 7-day embryonic heart cells.

In Vitro Cultivation of Single Cells from Drosophilamelanogaster Larvae
Yukiaki KURODA

In vitro cultivations of single cells from various imaginal discs of D.
melanogaster larvae were carried out. Newly laid eggs of a wild strain
Table 1.

Components of synthetic medium for cultivationof Drosophila single cells

Ingredient

mg/l000ml

NaCI
KCI
CaC\z·2H zO
MgCl z·6H zO
NaHCO aNaH zPO,·2H zO
KzHPO,
GlucoseTrehalose
Glycogen
L-Arginine
L-Aspartic acid
L-Asparagine
L-Alaninep-Alanine
L-Cystine· HCI
L-Glutamic acid
L-Glutamine
L-Glycine
L-Histidine
L-Isoleucine
L-Leucine
L-Lysine·HCI

2,100
1,790
1,100
4,180
700
1,240
2,080
1,000
4,000
300
2;)0
120
630
990
900
430
170
1,460
1,080
920
70
140
430

Ingredient

mg/l000mI

L-Methionine
680
L-Proline
2,620
L-Phenylalanine
100
1,000
L-Serine
L-Tyrosine
380
L-Threonine
340
L-Valine
340
Thiamin· HCI
0.02
Riboflavin
0.02
Pyridoxine· HCI
0.02
Niacin0.02
Ca-pantothenate
0.02
Biotin0.01
Folic acid
0.02
Choline· HCI
0.2
Inosite0.02
p-Aminobenzoic acid
0.02
Malic acid600
a-Ketoglutaric acid350
Succinic acid60
Fumaric acid60
Penicillin G potassium 5,000 units/ml
Dihydrostreptomycin sulfate 5, OOOmg/ml

pH was adjusted to 6.6 with 1 N NaOH.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17(Oregon-R) were collected and sterilized in 70 per cent ethyl alcohol for 10
minutes. The eggs were transferred to autoclaved food medium containedin bottles. Mature third-instar larvae (96 hours after hatching at 25°C)
grown under those sterile conditions were dissected in order to isolate various
imaginal discs used for cultivation. Wing discs, eye-antennal discs, legdiscs, testes, and ovaries were cultured in a synthetic medium supplemented
with 10 per cent calf serum in TD-7 flasks at 25°C. The components of theculture medium are shown in Table 1. Detailed procedure for cultivation
will be described elsewhere.
Another attempt was made to dissociate the imaginal discs from maturelarvae into single cells by treatment with trypsin. Isolated imaginaldiscs were incubated in 1 per cent trypsin solution for 15 minutes. Afterrinsing with physiological saline the discs were dispersed into single cells
in the culture medium by flushing them briskly through the tip of a finepipette. The single cell suspension obtained in this waY,was distributed
into TD-7 flasks and incubated at 25°C. Establishment of cell lines having some distinct genetic markers and isolation of clones from a varietyof tissues or organs by colony formation procedure are carried out.

RESEARCHES CARRIED OUT IN 1966

III.

39

BIOCHEMICAL GENETICS

Some Notes on the Chromogranules in Hypodermal Cellsof Silkworm Larvae
Mitsuo TSUJITA

When a part of white turbid cytoplasm of the hypodermis of 5th instar
larvae is placed on a seat mesh covered with a collodion film and electronmicroscopically examined, a number of elliptical, oval or spindle shaped
chromogranules into which the electron-rays do not completely penetrate
can be observed. The chromogranules in larval skin cells of the w-estrain have almost uniform size (1 x 1.5 ,u). On the contrary, in C-1OS
strain those having the usual size are mixed with small ones. In somechromogranules their granule (vesicle) membrane can be clearly discriminated. Their density is 1.4~1.7 which is higher than of any other cellorganelles.
In general, in the cytoplasm surrounding the chromogranules no structure interrupting intense electron rays can be observed. This seems toindicate that the chromogranules produced abundantly in the cytoplasmof larval skin cells of the normal strain are the organelles which bringabout the non-transparency of larval skin.
Each mature chromogranule consists of a vesicle and a thin membranesurrounding it. In the course of development, ribosomal particles, 150~
200 A in diameter, arrange themselves in a uniform layer at the peripheryof the granules. It seems that they are intimately associated with the
granule membrane. Although those particles are apt to be lost after thematuration of the granule, it was often observed that most of them remain sticking to the membrane.
The chromogranules disintegrate after the loss of granule contents inthe following cases.
1) A part of chromogranules in hypodermal cells of larvae disintegrates
in each instar, especially at the moulting stage. The granule membraneloses its normal function, the stored secretion products come out and thenit disintegrates.
2) All of chromogranules in hypodermal cells of the larvae disintegrate
at the full grown larval stage: They begin to issue forth their secretion
products through their membrane subjecting it to some change. Losing thesecretion products, the ghosts, i.e. the membranes, remain and then theyalso disappear. The density of chromogranules is remarkably reduced byloss of granule contents.
3) The fact that disintegration of chromogranules can be artificially

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

induced was reported (Tsujita and Sakurai, 1963). This disintegration
process was electron-microscopically studied.
When mulberry leaves painted with 0.3 per cent melamine solution were
continuously fed to normal or lemon yellow larvae from the beginning of
their instar, disintegration of chromogranules started 2~3 days after thetreatment, namely, the contents of granules were lost through theirmembranes and later the membranes disappeared.
All chromogranules disintegrate and pteridine compounds and uric acidare lost from hypodermal cells. Thus, larvae treated with melamine
become highly transparent. Normal opaque hypodermis could develop inlarvae with transparent hypodermis that were artificially produced, when
they were fed for about two days with fresh mulberry leaves without
melamine. In this recovery process a number of minute particles first
appeared in the cytoplasm and they gradually increased in size. Although
the skin of larvae in this stage exhibited semi-transparency, when thoseminute particles in hypodermal cells grew to usual size, the larvae showed white non-transparent skin.
4) It is difficult to fix the complete content of chromogranules because
almost all of them are lost in the procedure of fixation and dehydration.
For example, after fixation with osmic acid, dehydration was carried out
through alcohol from low to high concentration. During this procedurethe materials were examined by electron microscope and it was foundthat the granule contents were almost all lost through the granule membranes during dehydration.

Development of Chromogranules in the Larval Skinof the Silkworm
Mitsuo TSUJITA and Susumu SAKURAI

The developmental process of the chromogranules in larval skin cellsof the silkworm was studied by electron microscopical observation of thinsections.
The larval skin cells of the silkworm larvae have generally the shapeof cylinders and the elliptical nucleus occupies a central position. At
first, numerous ribosomes, 150~200 A in diameter, assemble here and there
in the cytoplasm forming ribosomal aggregates. A small vesicle appears
in the center of each aggregate. The vesicles gradually increase in sizeand spherical, oval or elliptical granules are formed. The ribosomes arrange themselves uniformly in a layer surrounding the granule membraneand some proteinous substance can be seen inside the vesicle. It may be said
that the precursor of chromogranules is a type of endoplasmic reticulum.

RESEARCHES CARRIED OUT IN 1966

41

It was substantiated (Tsujita and Sakurai, 1964) that soluble proteinscombining either with pteridine pigments or with uric acid are present
in the chromogranules. It may be safely said that those compounds arecontained in the granule vesicles and squeeze out when the chromogranules are pressed upon.
The well delimited precursor of the chromogranules can be precipitated by a procedure leading to the separation of the mitochondrial fraction.
It was stated above that the process of chromogranule development frominitial to mature stage can be followed up in the cytoplasm of hypodermal cells. The early steps of the same developmental process can befollowed up on the sections through the mitochondrial fraction.
Gene dependent variations were electron-microscopically observed as toshape and size of chromogranules produced in larval skin cells of normal
strains and mutants with transparent larval skin.
It may be inferred that a large amount of uric acid found in the larval
hypodermis of normal strains is mostly contained in the chromogranules.
On the contrary, size and shape of chromogranules produced in the
hypodermal cells of larvae with transparent hypodermis are abnormal andthe amount of mature granules is decreased. The larval skin becomestransparent to varying degree in accordance with the shape and theamount of chromogranules. For example, in hypodermal cells of larvaehomozygous for WO" or w-a no mature chromogranules are found. Consequently, uric acid or pteridine compounds can be scarcely detected intheir hypodermis. As for other mutant strains, transparency of larvalskin and the amount of uric acid and pteridine compounds varies fromstrain to strain according to the shape and amount of chromogranules,
especially their amount.
In the course of granule formation, the development of vesicle (granule)
membrane with attached to it numerous ribosomal particles, which playsome important role in the secretion of polypeptides, is the most important process. When it proceeds normally the granules have completeor sufficient ability for secretion and storage, and abundant chromogranules
packed compactly with secretion products are formed in larval skin cells.
So far as our experimental results are concerned it seems that uricacid production and pteridine metabolism are normally carried out in theemployed mutant strains with transparent larval skin. Therefore, it isinferred that in hypodermal cells of those larvae, the function of the
granules, i.e. secretion and storage, is defective owing to the abnormal
development of the granules, especially of granule membrane.
It was found by us that the granule membrane protein consists of ahomogenous single unit protein. It seems that the granule membraneprotein is made up by polymerization of this unit protein which is the

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

most important constituent of granule membrane.
Some Properties of Nucleic Acid in Larval Skin Cellsof the Silkworm
Kunihiro KOJIMA, Mitsuo TSUJITA and Susumu SAKURAI

It was reported in our previous paper (1966) that quite a considerable
amount of RNA is contained in the chromogranules of the larval skin cellsof the silkworm and that the RNA is found in the ribosomal particles
surrounding the membrane of chromogranules.
The present experiments were undertaken to clarify some propertiesof the nucleic acid obtained from larval skin cells and from chromogranules.
Larvae at late moulting stage, on 3rd, 5th and 7th day of 5th instar, andearly and middle mounting stages of C-124 strain were used as materials.
In order to gather chromogranules, the larvae were cut open at thedorsal side and all their inner organs were removed. Then, the cytoplasmic layer containing the chromogranules of hypodermis was scraped
off and gathered by a pincette tip, and the chromogranules were separated
according to the procedure previously reported (Tsujita and Sakurai, 1966).
The nucleic acid was extracted from purified chromogranules or fromhypodermis by the following procedure.
First, the sample was homogenized gently with teflon homogenizer ina solution of 0.14 M NaCI-1 mM MgCb-0.05 M acetate buffer (pH 6.0)
containing 17 mg bentonite per ml (volume ratio of sample to solution
1 mg: 9 mI). The homogenate was stirred gently with an equal volume
of cold 90 per cent aqueous phenol for 30 minutes and centrifuged at
13,000 X g for 30 minutes. The aqueous phase was mixed with two volumes
Table 1.

C-I24 Nucleic acid content in the hypodermis
Nucleic acid

Larval stage

Late moulting stage of 4th instar
3rd day of 5th instar
5th day of 5th instar
7th day of 5th instar
Early mounting stage
Middle mounting stage

Soluble RNA

DNA

Ribosomal RNA

0.522
0.658
0.210
0.255
0.127
0.135

0.255
0.362
0.036
0.040
0.038
0.060

2.90
6.25
1.98
1.68
1.30
1.90

The amount of nucleic acid is shown by mg per g hypodermis including cuticle.

RESEARCHES CARRIED OUT IN 196643

of ethanol, and then CHaCOOK was added until the solution came to 2
per cent of this compound. In order to obtain purified RNA and DNA
this precipitate was dissolved in 0.2 M NaCI-0.05 M phosphate buffer (pH6.7) and dialysed against 2 per cent CHaCOOK for 12 hours. The dialyzate
was mixed with two volumes of ethanol, and CHaCOOK was added until
the solution came to 2 per cent of this compound. Then this mixture wascentrifuged at 3,000 rpm for 15 minutes and the precipitate was dissolved
in 0.2 M NaCl-0.05 M phosphate buffer (pH 6.7).
Nucleic acid fractions were chromatographed on a methylated albumin
Kieselguhr column by a simplified procedure and the content of solubleRNA, DNA and ribosomal RNA was measured.
Nucleic acid contents are given in Table 1. This table shows that thelargest amount of RNA was detected in the hypodermis in larvae on 3rd
day of 5th instar.

1.0
I~ibosomal Rr--;AE

60s
Soluble RNA

50

Tube number

100

Fig. 1. Chromatographic profiles of the nucleic acid fraction
from hypodermis on 3rd day of 5th instar.

Chromatographic profiles of RNA fraction obtained from hypodermis andchromogranules of larvae on 3rd day of 5th instar are shown in Figs. 1 and 2.
Those two chromatographic patterns indicate that both samples, of44

ANNUAL REPORT OF NA TIO~AL INSTITUTE OF GENETICS ~O. 17

Ribosomal RNA

1.0

~ 0.5

405

60s

-e

.§
-<

O.O''-----..,,50~-----:l~OO~----­

Tube number

Fig. 2. Chromatographic profiles of the nucleic acid fractions
from chromogranules on 3rd day of 5th instar.

hypodermis and chromogranules, contained 4 S soluble RNA, DNA and
40 S, 60 S ribosomal particles. Therefore, it may be said from these ex·perimental results that the numerous minute particles surrounding themembrane of chromogranules are ribosomal particles.

Incorporation of HC-Glycine into Chromogranulesof Larval Skin Cells of the Silkworm
Susumu SAKURAI and Mitsuo TSUJITA

In order to observe polypeptide synthesis in chromogranules, 0.3 pc of
He-glycine was injected into the body fluid of the silkworm at 4th
moulting stage, on 3rd day and 5th day of 5th larval instar, and at fullgrown larval stage. The cytoplasm of the hypodermal layer was scraped
off and gathered. The raw fresh cytoplasm was mixed with 0.25 M
sucrose solution (volume ratio of sample to sucrose solution 1: 10) andcentrifuged at 900xg for 10 minutes.
The chromogranules were sedimented by centrifugation at 900xg for 10
minutes. Microsomes and mitochondria were isolated as shown in Fig. 1.

45

RESEARCHES CARRIED OUT IN 1966
Raw fresh cytoplasm in 10 volumes of 0.25 M sucrose solution
900 X g for 10 min

I

I

I

Crude
chromogranules

Supernatant layer
5,000 X g for 10 min

IIi

Mitochondria

Supernatant layer
12,000 X g for 10 min

I
Pellet(microsomes I)

Supernatant layer
104,000xg for 60 min

I

I
I

Pellet(microsomes IV)
Fig 1. Isolation procedure of labeled cell fractions.

The supernatant layer was centrifuged at 5,000 X g for 10 minutes. The
resultant pellet was the fraction containing mitochondria and chromogranuleprecursors (Tsujita and Sakurai, 1966). The protein residues obtained fromthe labeled cell fractions according to the procedure of Schmidt-Thannhauser and Schneider (STS) were dissolved in 0.3 N-potassium hydroxide
and the radioactivity was measured in a windowless gas flow counter.
C.P.M.
200

B.F.

100

CH.G.

.........-....-

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

30

180
min.
Fig. 2. Time course of incorporating reaction of 14C-labeledamino acid into body fluid and soluble polypeptides ofchromogranules.
C.P.M.: count per minute
H.F.
body fluid
CH. G.: chromogranules
60

90

120

150

As shown in Fig. 2, maximum incorporation of 14e-glycine into chromogranules could be seen 2 hours after injection (Sakurai and Tsujita, 1966).

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

The chromogranules had higher radioactivity than the microsomes andmitochondria, and moreover HC was mostly distributed among polypeptide
components of secretion products within the granules after incorporationof HC-Iabeled amino acid into the granules.

Chemical Characterization of Chromogranule Membranein Larval Skin Cells of the Silkworm
Susumu SAKURAI and Mitsuo TSUJITA

Preparation and purification of insoluble protein from granule membrane-·Purified chromogranules were dissolved in 2 per cent deoxycholate. The
solution was dialysed against distilled water for overnight at 5°C. The
precipitate formed was collected by centrifugation at 5,000 x g for 10
minutes, suspended in 0.25 per cent sucrose solution and again centrifuged at 1O,000xg for 10 minutes. The precipitate was dissolved in 8 M
urea solution and 2-mercaptoethanol was added at pH 8.6 (volume ratioof sample to 2-mercaptoethanol 1 mg: 1 mI). After 1 or 2 hours, the reaction mixture was eluted through Sephadex G 25 1.4 x 100 em column which
had been equillibrated with 0.001 M EDTA-0.5 per cent methylamine- HCI
buffer at pH 8, to remove excess urea and 2-mercaptoethanol. The reduced membrane was then treated with p-chloromercuribenzoic acid(PCMB)at room temperature for 30 minutes and adjusted to pH 8 (Anfinsen 1961).
The final mixture was dialysed against 0.5 per cent methylamine- HCI
buffer at pH 8 and further dialysed against distilled water. The suspension of PCMB-membrane protein was lyophilized for storage.

Electrophoretic and ultracentrifugation analysis of peME-membraneprotein-After removal of the PCMB by suspension in 0.001 M EDTA0.5 per cent methylamine- HCI buffer at pH 8, 1 ml of mercaptoethanol per
mg of the derivative was added and the fully reduced membrane protein
was applied to the Sephadex column as described above. The reduced
sample thus obtained was examined for electrophoresis. Polyacrylamidegels were prepared with 6 per cent cyanogum 41 in Tris- HCI buffer at
pH 9. Electrophoresis was carried out in vertical position for 7 hours at
5 rna constant. Tris-glycine buffer at pH 8.7 was used as the electrolyte.
The gels were stained with 0.1 per cent amido black 10 B in a solvent ofmethanol-water-acetic acid (5: 4: 1). Excess dye was removed by overnight washing the gel in a 10 per cent acetic acid.
As the electrophoretic pattern of Fig. la shows, purified granule membrane protein migrated as one clear-cut single band.
The mercaptoethanol reduced sample was dissolved in 5 per centmethylamine-HCI buffer at pH 8 and ultracentrifuged at 56,000 rpm.

RESEARCHES CARRIED OUT IN 1966

47

-Origin- Spacer gel

+
(a)

+
(b)

Fig. 1. Disk electrophoresis of protein from granule
membrane of normal and oily mutant.
a. Protein from normal granule membrane.
b. Protein from granule membrane of oily mutant.

Fig. 2. Sedimentation diagrams of protein from normal granule
membrane. Ultracentrifugation at 56,000 rpm at 4°C, The 0.25
per cent solution of the protein in methylamine-HCI buffer (pH 8).
Photographs were taken at 8 minutes intervals.

As shown in Fig. 2, purified granule membrane protein of the normal
strain exhibited a single symmetrical boundary in the analytical ultracentrifuge in 5 per cent methylamine-HCI buffer.
Thus, it may be inferred from the above-mentioned experimental results that the membrane protein of the chromogranules is composed of ahomogeneous single unit protein.
According to our preliminary experimental results, the electrophoretic
pattern of the membrane protein, obtained from chromogranules of hypodermal cells of 60CO r-ray induced w-b mutant with oily larval skin, exhibited
two or three minor bands, not shown by normal granule membrane, andelectrophoretic mobility of the major band differed from that of normal

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

granule membrane (Fig. Ib). This fact suggests that there is some defectin the formation of granule membrane protein and some important error
occurs in the secretion and storage of polypeptides within the granules.
As to the constituent amino acids of the granule membrane protein,seventeen amino acids, lysine, arginine, histidine, aspartic acid, glutamic
acid, threonine, serine, proline, glycine, alanine, cystine, valine, methionine, leucine, isoleucine, tyrosine and phenylalanine were detected using
a Hitachi's amino acid analyzer.
Genetic and biochemical studies of the amino acid sequence in the unit
protein of the granule membrane found in the present investigation andof the mode of polymerization of the unit protein in the formation ofchromogranule membrane is now under way.

Genetic Effects of DNA in Ephestia
Saburo NAWA and Mas'laki YAMADA

1. The experiment has been designed for inducing DNA-mediatedgenetic transformation in Ephestia. An imago of the wild type strain(NCR, a+/a+) is black eyed, while a homozygote (a/a) for the recessive
gene a, which controls the enzyme tryptophan pyrrolase, is red eyed.
Larvae of a/a were injected with DNA extracted from NCR adults. The
injected larvae were permitted to develop into adults and were examinedfor eye-color. Some of them were crossed to untreated a/a to score thechange in eye color in their progeny and in subsequent generation. Blackeyed animals have been obtained either among the treated animals or insubsequent generations. The results are summarized in Table 1. When
Table l.

ExperimentFrequency of mutations among treated animals and in
subsequent generations

B,

Treated generation

B2

No. of adults Black-eyed No. of adults Black-eyed No. of adults Black-eyedmutants
examinedmutants
examined
examinedmutants

1
2
3
4
5
6

105
140
214
220
85

0
0
0
1
0
0

6,655
4,010
9,425
9,105
3,398
7,039

0
0
0
2
4
0

1,196
2,156
1,974
13,9804,168
14,699

1
2
1
4
0
1

Total

818

1

39,632

6

38,173954

RESEARCHES CARRIED OUT IN 196649

transmission of the newly acquired black eye color has been followed forseveral generations by mating mutants with untreated a/a, it became
evident that there were several types of mutants. 1) Most of the black-eyedmutants have been confirmed to be a+/a heterozygotes. 2) One mutant
male, which appeared in B 2 in Table 1, gave a+/a heterozygotes and a/a
homozygotes in the proportion of 6: 1, when crossed with an a/a female,showing that the genotype of the mutant was not simply a+/a. It may
have been a mosaic. 3) A few mutants behaved as being a+/a+ homozygotes, since they gave only a+/a progeny when crossed by a/a. Thesemutants appeared in the progeny (Bl and B 2 in Table 1) derived fromcrosses of treated animals with untreated a/a. It is difficult to imagine
the occurrence of a+/a+ homozygotes in the progeny from crosses witha/a. Special care has been taken to avoid contamination by migrating
animals. 4) An unusual mutant, a black-eyed female, was obtained in thetreated generation. When the female was crossed with an a/a male, all
the Bl animals were black, although only five were obtained, all females.
All those females were crossed with a/a males. One pair gave no progeny.
Three pairs gave in the total 356 imagos, all of which were black, but all
were a+/a heterozygotes. The fourth pair gave rise to 123 black-eyedoffsprings, half of which were a+/a heterozgotes, but the rest were not, andgave only black-eyed animals (a+/a) in crosses with a/a. These observations
could be interpreted on the assumption that a piece of DNA incorporated
into the cell behaved as a particle which could reproduce itself, although
its sudden disappearance cannot be plausibly explained. 5) One mutant
female, which was apparently black-eyed, gave no black-eyed animals in
subsequent generations when crossed with a/a. It seems likely that inthis female the mutation occurred in somatic cells but not in germ cells.
Treatment of eggs of a/a with NCR-DNA has also been carried out,
giving similar results.
In a reverse type of experiment, eggs of NCR (a+/a+) were treatedwith DNA extracted from a/a adults. When imagos developed from treated eggs were crossed with a/a, a red-eyed mutant has been obtained.
Further experiments are now in progress.
2. Some properties of DNA used in the experiments have been described
previously (Ann. Rep. No. 15). The molecular weight of the DNA wasestimated from its viscosity and sedimentation. The intrinsic viscosity[r;] ranged from 70 to 110 for different preparations, using a rotating
cylinder viscometer by Zimm and Crothers. The mean value of sedimentation coefficient (S~.,o) was 30. Using these values, we obtained 16'"'-'23 X 106 as molecular weights for DNA prepared by the same method,which was used to extract DNA in the transformation experiments.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Peroxidase Isozymes in Leaves of Pharbitis nil
Toru ENDO

Although the biological function of plant peroxidases is yet poorly
understood, it may be considered in relation to the role of indoleacetic
acid oxidases. Peroxidases of intact cell sap were squeezed out fromleaves of the Japanese mornning glory, Pharbitis nil, and were zymographically examined by starch gel electrophoresis, pH 8.5 borate buffer
system. The enzymes were stained by slight modification of a method
reported before (Endo, Rad. Bot. 7: 35). The final concentrations of thereactants were 0.03 per cent hydrogene peroxide, 0.1 per cent benzidine
acetate and 0.01 M tris-acetic acid buffer, pH 4.0. Several isozymes weredetected in six leaves of different age of a strain with normal leaves andblue flowers, TKS-046. The results showed that number and activity of
isozymes generally increased with age.
On the zymogram of the youngest leaf, two or three blurred anodal
bands were observed and one sharp as well as one weak cathodal band.
In the oldest leaf, at least six anodal and two cathodal bands, sharp andheavy, were found. The sharp cathodal band showed similar activity inthe course of development of the leaves examined. However, anothersimilar strain, TKS-036 lacked the sharp band. In F I hybrids, TKS-046x
TKS-036 and reciprocal, the isozyme band always appeared at the same
location on the zymogram as that of TKS-046. No hybrid enzymes weredetected.
Hormonal Enzyme Regulation in the Cultured
Hypocotyl of Pharbitis nil
Toru ENDO

The in vitro effects of plant hormones and antihormones on enzyme
systems were zymographically studied in cultured hypocotyls of Pharbitis
nil. The hypocotyls were excised from 7 day-old seedlings, cut into
pieces 15 to 25 mm long and cultured for three weeks on Linsmaier-Skoog
medium (RM-1964) with or without hormones. The slant media, except
for control, contained one of the following optional constituents; 10 ppm
of kinetin (KT), gibberellic acid (GA), indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) or
100 ppm of maleic hydrazide (MH), triiodebenzoic acid (TIBA), chlorocholine chloride (CCC), N-dimethylaminosuccinamide (B-9) and cyclohexaimide (CHI).
Morphologically, roots were formed on control, GA, CCC, B-9 and IAA

RESEARCHES CARRIED OUT IN 1966

51

medium. A large callus developed, especially at the base of the cutting,on KT and IAA medium. A comparatively smaller callus was also foundon NAA and 2,4-D, as well as, rarely, on control medium. No differentiation was observed on MH, TIBA and CHI media. Also, TIBA resulted
in a browing of the cuttings except the parts near the ends.
The isozymes examined were those of peroxidase and esterase. Esterases were stained by a reacting mixture which contained 1 mM 1naphthylacetate, 0.02 M phosphate buffer, pH 7.0, and 1 mg/ml of Fast
Blue RR salt. Activities of both enzyme species were very weak in intact
tissue, but in general increased considerably after culturing. The zymographic analyses showed, more or less, differences in enzyme pattern not
only among the hypocotyl parts, callus and root, but also among the media
containing different constituents. In the cultured hypocotyl separated
from roots or callus, TIBA, NAA, 2,4-D, IAA and MH accentuated, in
this order, the activity of several peroxidase isozymes, in comparison withthe control. For instance, a few cathodal bands were newly detected in
TIBA-cultured material. IAA was unique in that one sharp cathodal
band was considerably activated. The other optional constituents were
not very effective. Even CHI, inhibitor of protein systhesis, did not affectthe formation and/or activation of peroxidases. At least four anodal andone cathodal esterase bands were observed in the hypocotyl after culturing. MH and 2,4-D appreciably activated those isozymes. A significant
observation was made in TIBA-cultured material which did not show any
esterase isozymes.
Variation in Peroxidase Isozymes of Oryza
perennis and O. sativa
Yaw-En CHU

Peroxidase isozymes in various organs of a wild rice species, Oryza
perennis Moench, were investigated using the starch-gel electrophoresis
method. From leaf blade, leaf sheath, internode, basal node, and root,eight isozyme bands were found running toward the cathode and fivebands running toward the anode. Each organ showed a specific patternof band distribution, and gene dependent differences were found in thepresence or absence of certain bands. In the leaf blade, leaf sheath androot, the activities of the bands were found to differ with the age of theorgan.
In six perennis populations of different origin, within-populational variations in zymograms were examined. Populations of perennial habit, one
from India and three from Africa, each showed several :different zymo-

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

grams, but a population from the Amazon basin, supposedly of annual
habit, was not polymorphic.
Further, the zymograms of leaf blade and leaf sheath were observedin 110 perennis strains collected from the natural habitats in different
countries (each represented by one plant), 38 sativa strains from various
Asian countries, and the intermediate perennis-sativa strains from
Jeypore Tract, India. The zymograms of perennis strains were quite
variable, and the frequencies of certain bands markedly differed among
geographical strain-groups. Most African and American strains had bands
4A and 5C, which were not frequent in the Asian material. Of several
zymogram types of leaf blade found among Asian perennis strains, two,
peculiar to the Asian strain-group, were found to characterize sativa
varieties, as shown in Table 1. Most Indica varieties had band 5C, which
Table l.

Comparison of leaf-blade zymograms between Asian perennis
and sativa strains
Asian perennis

Type
no.

1
2
3
4
5
6
7
8
9

Intermediate perennis-sativa
from ]eypore

Zymogram
Perennial
2A
2A 5C
5C
2A 4C 5C4A
4A 5C
4A 4C 5C
2A 3A 4A5C
4C 5C

No. of strains

CultivatedIndica ]aponica

sativaIndica ]aponica

Annual

Wild

2
6

6
4

8
5
1
1

1
12

4
1

2
16

18
2

7
3

5
6

1

1
1

19

23

15

13

5

18

20

most Japonica varieties did not show. The semi-wild strains from Jeypore
Tract showed a similar variation pattern as found in sativa varieties.
These isozyme variations seem to suggest genic differences that may
have some bearing on populational variability as well as on racial differentiation. It may be inferred that cultivated rice might have arisen
from wild forms having certain peroxidase isozymes.

RESEARCHES CARRIED OUT IN 1966

53

Characterization of Xanthine Dehydrogenase from Drosophila!)
Tomotaka SHINODA

Purification and general characterization of xanthine dehydrogenase of
Drosophila were carried out employing wild types CORN] and PACN) andmutants (lxd and Pm; Sb/ Xa). Some thousandfold purification of the enzyme
was achieved by application of several methods and the purified enzyme
was submitted to general characterization. The following results haveso far been obtained (Table 1).
Table 1.

General characteristics of xanthine dehydrogenase of Drosophila
Strain
ORNJ

PACN

6 M

Km(AHP) x 10Km(MB) x 10- 5 M

7.4
4.6

Relative mobility

FastSpecific activity
Heat stability
Molecular weight x 10 5;?is inhibition with 4 M urea

8,000

8.1
6.8
Inter·
mediate
5,200

+

+

Pm:Sb
Xa
7.0
5.8

lxd
3.2

30,000Inter·
mediate
1,200

+

+

Slow

Comple·mentation
XDH

2.5-2.6 2.5-2.6 2.5-2.6 2.5-2.6
94.3
95.1
96.1
97.5

Fast
80

±
2.5-2.6

Many functional similarities were observed among the enzymes obtain·
ed from various strains. Both the electrophoretic mobility and the specificactivity of the enzyme varied from strain to strain. These results may
suggest that each enzyme of various strains has a different structure with
common functions.
:Multiple :Molecular Forms of Xanthine Dehydrogenase
in Drosophila!)
Tomotaka SHINODA

Xanthine dehydrogenase (XDH) of Drosophila melanogaster is affectedby mutations of at least three loci, ry, ma·l and lxd. Of these, ry+ isthe structural gene for XDH, while ma·l+ and lxd+ play less direct rolesin the regulation of XDH activity, probably by controlling a cofactor of
XDH. In addition to genetic electrophoretic variants XDH exists in two
forms (XDH·I, and XDH·II) which can be detected using DEAE column
11 This work was carried out at the University of North Carolina, Chapel Hill,
North Carolina, U.S.A.

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

chromatography or polyacrylamide gel electrophoresis. In 4 per cent gel
the XDH-I migrates approximately 8 per cent faster than the XDH-IL
The kinetic properties of these forms do not differ greatly. Sucrose
gradient centrifugation indicates that they sediment at slightly different
rates. Conversion of XDH-I to XDH-II, but not vice versa, can be accomplished by incubation with Drosophila extracts that are deficient in XDH.
Some properties of the enzyme are summarized in Table 1.
Table 1.

Comparison of some properties of XDH-I and XDH-I1
XDH-I

Km(AHP) x 10- 6 M
Km(MB) x 10- 5 M
Sp. activity
Mobility
Mol. weight% of remaining activity at 50°C, 1 h
Effect of ma-l+(or ry+) factor% inhibition with 4 M urea

10.1
6.2
4,700
F

260,000
68

XDH-I1
7.1
5.5
10,200S
250,000
61.2

+
97.1

95.1

RESEARCHES CARRIED OUT IN 1966

IV.

55

EVOLUTIONARY GENETICS

An Intergeneric Hybrid between Eremopyrum
orientale and Henrardia persica
Sadao SAKAMOTO

Henrardia is a monotypic genus in the tribe Triticeae represented by
one species, Hn. persica (Boiss.) C.E. Hubbard. Morphologically this genus
is different from other members of the tribe. In order to find phylogenetic
relationships of this genus with other genera of the tribe, a strain of Hn.
persica var. glaberrima (Hausskn.) c.E. Hubbard (2n=14, Iran) was crossedwith various species of Aegilops, Eyemopyrum, Heteranthelium, Taeniatherum
and Triticum in 1965. From these crosses, 25 triploid F 1 hybrids were
produced only when Er. orientale (Linn.) Jaub. et Spach. (2n=28, Iran)was used as the female parent.
Morphological characteristics of the Fl hybrids were of Eremopyrum
type, while the length of the spikes was intermediate. Disarticulation of
the ripe spikelets was characterized by wedge type as that of the Eremopyrum parent. Average chromosome pairing per cell of the Fl was O.OOm+
0.42 II +20.20 r . Bivalents ranging from 0 to 4 were all terminally associated.
Thus, no genomic homology was found between Er. orientale and Hn.
persica. Complete sterility was observed. Colchicine solution (0.5 per cent)
was applied to the tillering clones of the F 1 and 150 well-developed seeds
were obtained in 1966.
Three Intergeneric Hybrids among Heteranthelium piliferum,
Eremopyrum buonapartis and Hordeum sp.
Sadao SAKAMOTO

The genus Heteranthelium in the tribe Triticeae is monotypic with Ht.
piliferum (Banks et Soland.) Hochst., having quite a specific spike characteristic, i. e. mixture of fertile and rudimental spikelets in one spike. In
1965 a strain of Ht. piliferum (2n=14, Afghanistan) was crossed with various
species of Aegilops, Eremopyrum, Henrardia and Hordeum. The following
two intergeneric hybrids were obtained: 1) two diploid hybrids, Ht. PiliferumxEr. buonapartis (Spreng.) Nevski var. buonapartis (2n=14, Iran),and 2) a triploid hybrid, Ht. piliferum X Hordeum sp. (2n=28, material"No. 66w" was received from the All-Union Institute of Plant Industry,
Leningrad, 1964). In the same year, 3) 19 triploid hybrids, Er. buonapartis var. buonapartisxHordeum sp., were also produced.
Growth of hybrids under 1) was subnormal and the shape of the spikes

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. I7

was of Eremopyrum type having no rudimental spikelets which are thecharacter of Heteranthelium parent. Growth of the hybrid under 2) was
vigorous and the spike morphology was intermediate between the parents.
The spikelets had two empty glumes and two florets like the Heteranthelium
parent but no rudimental spikelets were found. Growth of all hybrids
under 3) was very vigorous and the shape of their spikes was of Hordeum
type. However, three spikelets with two florets. each and four empty
glumes were observed at most spikelet nodes. Thus, the numerical composition at the spikelet node of the Fl was intermediate between theparents. Sterility of all three hybrid combinations was complete.
Average chromosome pairing per cell of the Fl hybrids was in 1)
0.0411(0-2)+ 13.9 1(10-14), in 2) 0.00m(0-1)+5.06 n (2-8)+ 10.881(5-17) and in 3)
0.00Iv(0-1)+0.00m(0-1)+5.5011(1-8)+9.97rC5-19). The parentheses indicate
the range of respective pairing. Judging from the chromosome pairing in1), 2) and 3), 5.0 11 and 5.511 observed in 2) and 3) indicate autosyndesis ofchromosomes derived from the Hordeum parent. The conclusion may be
drawn that the Hordeum parent used in this experiment was a tetraploid
species having two partially homologous genomes.
The tillering clones of 3) were treated with 0.5 per cent colchicine
solution and 31 seeds were obtained in 1966.

Diallel Crosses among Sikkimese Rice Types. III
Tadao C. KATAYAMA

Diallel crosses were carried out using sixteen strains, namely, fourteen
Sikkimese, one indica and one japonica strains. All crosses were successful
and the FI seeds obtained were sown in April, 1965. This year, thecharacters of intact and husked F I seeds, such as length, width, thickness,ratio of length to width and grain weight, were compared with those of
the parents. Investigation of many other characters of the hybrids, suchas number of tillers, plant height, heading date, internode length, isunderway.
In the previous paper (Katayama 1966), the Sikkimese strains could be
divided into 10 indica type and 4 japonica type strains based on the pollen
fertility of the FI's produced by diallel crosses. However, from the results
obtained at present, 3 strains, C7725 , C7727 and C7734, formerly classified
as indica, were found to be of intermediate type between indica andjaponica.
Therefore, Sikkimese rice is composed of typical japonica, typical indica
types and an intermediate type. It is assumed that Sikkim might be oneof the differentiation centers of cultivated Oryza sativa into japonica

RESEARCHES CARRIED OUT IN 1966

57

and indica types.

Further Studies on Embryo Transplantation in the Genus Oryza
Tadao C. KATAYAMA

In order to investigate the compatibility relation between embryo andendosperm in Oryza species, embryo transplantations were made between
rice species. The method was described in a previous paper (Katayama
1965). In the present article, two experiments are reported.
Four strains of O. sativa (two indica and two japonica types) and one
strain of each of the following nine species were used, namely, O. officinalis,
O. minuta, O. eichingeri, O. latifolia, O. australiensis, O. granulata, O.
ridleyi, O. brachyantha and O. subulata. Seedlings from intact seeds developed in addition to co1eopti1e, 1st, 2nd and 3rd leaves. Seedlings of O. sativa
developed three to four roots and those of other species developed one totwo roots. Embryos alone without endosperm produced only a co1eopti1e
and no root appeared except in a japonica type of O. sativa.
In heterogeneous transplantation, embryos of o. sativa were grafted upon
the endosperms of other species. In all cases, only a coleopti1e has developed. In all reciprocal transplantations, also only a co1eopti1e developed.
This means that the grafted embryos failed to absorb nutrients from alien
endosperms which suggests a distant relation to O. sativa.
In the previous report, it was strongly indicated that nutrient absorption from endosperms of indica type strains by embryos of the other closely
related species employed, was much better than from endosperms ofjaponica type strains. In order to study the details on this particularpoint, 10 strains of O. sativa (five japonica and five indica strains) as
testers, and one strain of O. sativa var. spontanea, O. perennis, O. barthii,
O. glaberrima and O. breviligulata, were used. When embryos of japonica
strains were grafted upon the endosperms of other species, co1eoptile and1st leaf developed in most cases. In all reciprocal transplantations, i. e.,when embryos of other species were grafted upon the endosperms ofjaponica strains, also co1eoptile and 1st leaf developed. When embryos ofindica strains were grafted upon the endosperms of other species, co1eoptile
and 1st leaf appeared in most cases. However, in the reciprocal transplantation, i. e., when embryos of the other species were grafted upon theendosperms of indica strains, co1eoptile, 1st, 2nd and, in some cases, even
3rd leaf, developed.
From these results, the previous finding was confirmed which indicatedthat indica type is taxonomically nearer related to the other employedspecies than japonica type.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Geographical Distribution of Winter, Intermediate andSpring Types of Common Wheat
Yasuo NAKAIThe growing habit of about 1400 varieties of common wheat collectedfrom various parts of the world was examined using growth chambers
adjusted to a constant temperature, 20°C, with 16 hr illumination. After
three months from sowing, when all spring standard varieties were
heading, the growing habit of the tested varieties was determined; varietiesthat headed at that time were classified as spring, those with visible ears
inside as intermediate and those showing no ear formation as winter types.
The result is summarized in Table 1.
Table l.

Locality
Pakistan
Afghanistan
Iran
India
Tibet
China
Japan
Greece
Italy
IberiaRussiaSwedenNorwayFinland
U.S.A.
S. America
Australia
Total

Geographical distribution of three growth habit
types in common wheat

No. var. tested
38
39
147
58
19
180
169
9
54

96
89
34
13
15
260
43
79

SpringIntermediate

Winter

(%)

(%)

(%)

58
12
27
98
100
27
37
44
52
30
69
100
100
100
33
81
97

18
30
11
0
0
12
11
12
2
4
2
0
0
0
7
7
0

24
58
62
2
0
61
52
44
46
66
29
0
0
0
60
12
3

1342

As to the varieties from Pakistan, India, Tibet, Russia, South Americaand Australia, many were of spring or intermediate type. In China, Japan,
Mediterranean countries and U.S.A., both winter and spring types wereRESEARCHES CARRIED OUT IN 1966

59

equally frequent. Some strains of Afghanistan and most of eastern Iranwere of spring type. With respect to the Scandinavia strains used inthis examination they were of spring type. The distribution pattern ofdifferent growth habit types of Aegilops squarrosa in central Asia (Kihara
and Tanaka, 1958) is identical to that found here for common wheat.
Ernbryosac Sterility of F 1 Hybrids between
Strains of Oryza perennis
Yaw-En CHU and Hiko-Ichi OKA

Embryosacs were observed in F1 hybrids between strains of Oryza perennis Moench, O. sativa 1., O. breviligulata Chev. et Roehr, and O. glaberrima Steud., which are pollen sterile in varying degree (Ann. Rep. 14: 74,
15: 84 and 16: 71). An embryosac with an egg nucleus, two polar nuclei
and antipodes were regarded as normal; this estimation was verified byback-crossing. Most Fl hybrids showing high pollen sterility had an appreciable percentage of normal embryosacs. For instance, the F1's between
American and Asian strains of O. perennis showed 45-70 per cent normal
embryosacs, though their pollen fertility was almost zero.
In sterile F1 hybrids, the deterioration of embryosacs seemed to takeplace in three different stages, namely 1) deterioration of the tetrad immediately after meiosis, 2) failure of mitosis producing two to four nuclei,
and 3) failure of nuclear differentiation into egg and polar nuclei. The
F 1hybrids between the Oceanian strains and those of the other geographicalgroups of O. perennis gave relatively low embryosac fertilities, ranging
between 25 per cent and 65 per cent. When the Oceanian perennis strains
were crossed with O. glaberrima or O. breviligulata, the F1 embryosacs
showed deterioration immediately after meiosis which was normal, andhad little or no normal embryosacs at the flowering time.
Pattern Analysis of Character Variations in Oryza perennis
Hiroko MORISHIMA and Hiko-Ichi OKA

Oryza perennis Moench is distributed throughout tropical countries of
the world and comprises many varieties or forms. The Asian forms
tend to differentiate into the perennial (perennis or balunga) and annual(spontanea or fatua) types. Our collections from New Guinea are also
divided into perennial and annual types. From 1958 to 1965, a numberof strains of this species, obtained from their natural habitats in different
countries, were repeatedly grown in a greenhouse or in automaticallycontrolled short-day plots, and records were taken regarding 18 metric

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

and 6 code-described characters. In each strain, representative values of
metric characters were obtained by comparing the data with controls. In
all the metric characters, variations among strains were found to be continuous.
To examine the pattern of character association among the strains, thetechnique of "quantification" was applied to the data for 65 strains, which
had complete sets of records. This method, devised by Hayashi (1951)
basing on a "multi-dimensional constellation model", is to conduct quantification of the data so as to maximize the correlation between, in ourII••

Asian strain
African strain
A American. strain
x Oceanian strain

2

••

,.
e• ~

'. .

interm:,type.
, -1 •

-2

.'

,
•

.

X

-1

•

\

'II-

,

X

X

-2

-

Fig. 1. Strains of Oryza perennis scattered on the basis of patternanalysis of variations in 24 characters.

case, strains and characters. The strains are then arranged in order inaccordance with their overall similarity. They were scattered on the plane
defined by two vectors given by two largest latent roots, as shown in
Fig. 1. The strains of Asian, American origin formed different clusters,
partly overlapping one another.
The differentiation of perennis strains into these geographical groups

61

RESEARCHES CARRIED OUT IN 1966

could also be found from their Fl sterility relationships (Ann. Rep. 14: 74
and 15: 84). The above results of computation indicate that the geographical groups can be distinguished by character-association patterns, though
not by a single character. The figure also shows that in the Asian group,
the perennial and annual types were separated, and in the Americangroup, strains from Cuba, Surinam and the Amazon basin could be separated. It seems that the varieties of O. perennis distributed in different
countries have different patterns of character correlation, possibly in
relation to their different modes of adaptation to environment.
Population Survey of No.1 Chromosome Polymorphism of Black
Rats (Rattus rattus) Collected in Japan and Korea
Tosihide H. YOSIDA, Yukuo MORIGUCHI,
Yung Sun KANG1) ond Kyojiro SHIMAKURA2)

It was already reported that black rats (Rattus rattus) were characterized
by a polymorphism of the largest No.1 chromosome pair, present as atelocentric homomorphic pair (TIT), a telocentric and subtelocentric heteromorphic pair (TIS) and a subtelocentric homomorphic pair (SIS). The
frequency of these three types in 124 rats collected in 7 localities ofJapan and 9 animals from Seoul, Korea, was already reported in this annual report (No. 16, 1966). Later we collected in total 236 rats in 9
lacalities of Japan and 26 rats in Seoul, Korea. The results of observation are given in Table 1. As the table shows, all animals collected in
Sapporo, Niitsu (Niigata Pre£.) and Obama (Fukui Pref.) had the TIT pair
Table 1. Frequency of black rats (Rattus rattus) with three typesof No. 1 chromosomes, collected in Japan and Korea
Locality
Karyotype

MisimaHiratsuka(Kanagawa) (Sizuoka)

SapporoNiitsu(Niigata)

Obama(Fukui)

Sugadaira(Nagano)

100%

100

100

80

70

70

TS

0

0

0

20

30

30

SS

0

0

0

0

0

Total no. ofanimals

50

30

11

7

56

TT

11 Seoul University, Korea.
2) Hokkaido University, Sapporo.

15

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Locality
Karyotype

Total in
Japan

Seoul
(Korea)0200(84.8%)
35(14.8%)
1(0.4%)

9(34.6%)
13(50%)
4(15.4%)

49

236(100.0)26(100.0)Hamamatsu(Sizuoka)

Ube(Yamaguchi)

Kusudomari
and Nagasaki

TT

57%

82

82

TS

29

18

18

SS

14

0

Total no. ofanimals

711

while in the other places in Japan, namely Sugadaira (Nagano Pref.),
Hiratsuka (Kanagawa Pref.), Misima, Hamamatsu (Sizuoka Pref.) , Ube(Yamaguchi Pref.) and Nagasaki (included Kusudomari), animals with TIS
heteromorphic pair were observed at 18 to 33 per cent. Except for only
one animal with SIS homomorphic pair which was found in Hamamatsu
population, all the others showed the TIT homomorphic pair. Frequencyof animals with TIT, TIS and SIS chromosomes in all 236 was 84.8, 14.8
and 0.4 per cent, respectively. On the other hand, among 26 animals
collected in Seoul, Korea, about 50 per cent had the TIT pair, and about
15 per cent animals were of SIS type.
Based on the above investigations, it can be said that the frequencyof animals with No.1 TIT, TIS and SIS chromosomes was significantly
different in different localities of Japan and in Korea.
Segregation of Three Chromosome Types in Black
Rats Crossed in the Laboratory
Tosihide H. YOSIDA and Yukuo MORIGUCHI

Segregation of three types (TIT, TIS, and SIS) of No.1 chromosomesin black rats, Rattus rattus, which were crossed in the laboratory was
already reported for some animals (Ann. Rep. 16, 1966). We got moredata on the segregation of the three chromosome types from animals bredin our laboratory. The results of observations are shown in the following
table (Table 1). Animals with SIS chromosomes were bred in the laboratory
with success. From crossing a TIS female by an SIS male we obtained
four litters. Average litter size of the hybrids was four. Chromosomesof two litters were observed. They segregated into 5 animals with a TIS
chromosome type and 3 animals with an SIS type. On the other hand,by cmssing an SIS female by an SIT male only one baby rat was obtained,

63

RESEARCHES CARRIED OUT IN 1966
Table l.

Segregation of three chromosome types in black rats
(Rattus rattus) bred in the laboratory

Karyotypes ofparents
T/S(¥)xT/S(o)T/T(¥)xT/S(o)T/S(¥)xT/T(o)T/T(¥)x T/T(o)T/S(¥)x S/S(o)Segregation
T/T

T/S

SIS

18
37
59
48
0

43
39
61
0
3

12
0
0
0
2

Total no. ofanimals
73
76
120
48
5

but it died soon after birth. By mating an SIS female by a T/T male
two baby rats were born, but they also died within one day after birth.
Based on the above investigation, SIS females of the black rat may be
unfit to bear and nurse their brood.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

V.

MATHEMATICAL AND STATISTICAL STUDIESON POPULATION GENETICS
Simulation Studies on the Number of Neutral Alleles
Maintained in a Finite Population by Mutation
Motoo KIMURAThe number of isoalleles that may be maintained in a finite population
was studied theoretically for neutral and overdominant mutations by Kimura
and Crow (1964), who derived, for neutral mutations, the following formulafor the equilibrium distribution of allelic frequencies;(1)

where M=N,u in which N, stands for the effective population number andu stands for the mutation rate per generation. In deriving the above
formula it was assumed that each mutant represents an allelic state notpreexisting in the population.
The above distribution has the meaning that <]J(x)dx gives an approximation to the expected number of alleles whose frequencies in the population lie within the range x to x+dx(0<x<1).
Using the above formula, the average and the effective number ofalleles in a population of size N may be given as follows:
na=\l

J1/

<]J(x)dx= 4M
(2NJ

l

i
J

(1-X)4M- 1 X- I dx,(2)

1/ (2N)n,=1/~: x <]J(x)dx=4M+1,
2(3)

The average number of alleles is defined as the reciprocal of the meanfrequency of alleles contained in a population, while the effective numberof alleles is defined as the reciprocal of the sum of squares of allelicfrequencies.
In order to test the validity of the above formulae, simulation studies
were carried out by using computer IBM 7090. Fig. 1 illustrates a resultof Monte Carlo experiment, assuming a population consisting of 50 males
and 50 females, of which only 25 males and 25 females actually participate
in breeding (N=l00, N,=50). In the experiment, the process of mutation
and random sampling of gametes was simulated by generating pseudorandom numbers in the computer. In each generation, 100 male and 100
female gametes are randomly chosen respectively from 25 breeding males
and 25 breeding females to form the next generation. Mutation to a new(not pre-existing) allele is induced in each gamete with probability 0.005

65

RESEARCHES CARRIED OUT IN 1966.,
.9fJ'~'/~~\~04·~~~.~\ ..rii. ~ ~~ '~tf ~
bd

1.0
!l.O

21J0

100

600

a

0

SOl)

1(0)

8

I:..'fXJ_

iJ_t'.#L _ 6 _

l-lOO

.'

_

h.,,.obbo OQ 'llJi)Ol.'\1)(J:;\100Gt'l1lTMion

Fig. 1. Neutral alleles, Random mutation, N=I00, N e =50, u=O.OO5.
e-- Average (actual) number 0--- Effective number

prior to the formation of zygotes (u='0.005). The initial population (atthe Oth generation) was set up such that it contained 200 different alleles;
outputs both of average and effective numbers of alleles were given at
the interval of 20 generations and the experiment was carried out until
generation 2,100 (actually illustrated in the figure at the interval of 40
generations). The balance between mutation and random extinction ofalleles was found to be attained well before generation 100. From theoutputs over generations 120-2,100, the average and the effective numberof alleles were computed as follows:n a =6.05,

n e=2.07

The corresponding values derived from equations (2) and (3) aren a =5.30,

ne=2.00

Thus, a fairly good agreement was observed between the actual values
from the simulation experiment and the theoretical predictions based on
the diffusion approximation.
Two Loci Polymorphism as a Stationary Point
Motoo KIMURA

In a previous note (d. Ann. Rep. No. 16: 86) a remarkable property
inherent in the linked gene system was reported under the term quasi
linkage equilibrium. The purpose of the present note is to show therelevance of this concept in the study of two loci polymorphism.
;

Let Xl, X2, Xa and X 4 (2: X=1) be respectively the frequencies of four1

chromosome types AlBl, A2Bl, AlB2 and A2B2 in a population, in which
Al and A2 are a pair of alleles in the first locus and B l and B 2 are a pairin the second locus. It is convenient here to give these chromosomes the

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

numbers, 1,2,3 and 4, so that the frequency of chromosome i is Xi (i=l,2, 3, 4) and the fitness (in selective values) of the genotype formed by theunion of chromosomes i and j may be denoted by Wij. In a large randommating population, if c is the recombination fraction between the two loci,the amount of change in· one generation of the chromosome frequencies
may be given as follows:.1 Xl = {XI(WI. -w)-cDw}/W.1XF {X2(W2. - wHcDw}/w.1Xs={Xs(ws. -lv)+cDw}/w.1X4={X4(W4. -w)-CDw}/lV

where
4

Wi. = 2: WijXj ,l(1)

w= 2: WijXiXj ,
ij

j=l

and

Thus, at the genetic equilibrium in which .1Xi=O (i=l, 2, 3, 4), we have
XI(WI. -w)= -X2(W2. -w)= -Xs(ws. -w)=X4(W4. -w)=cDw .

(2 )The equilibrium chromosome frequencies Xi (i=l, 2, 3, 4) may be obtainedby solving the above set of equations.
I will now show that the equilibrium point (Xl, X2, Xs, X4) is a stationary
point of w with the two side conditions,4

2: Xi=l ,i=land(X IX 4)/(X2XS)=Ro ,where R o is a constant giving the ratio between the frequencies of coupling
and repulsion phases at equilibrium, i.e. RO=(XIX4)/(X2XS). The stationary point of w with these two restrictions is equivalent to the stationary
point ofifJ=w-2p (2: Xi-1)--2A (log XIX4_ log Ro) ,i
X 2 Xsin which no restrictions are imposed among Xi's (-2p and -2A are
Lagrange multipliers). Thus

RESEARCHES CARRIED OUT IN 1966

67

at/J/aXl=2wl. -2p-(ZA/X1)=O ,at/J/aX2=2w2. -2p+(2)./X2)=O ,etc., giving
Xl(Uh. -p)= -X2(W2. -p)= -Xa(Wa. -p)=X.(W•. -p)=). ,(3 )in which, it is easy to show that
p=iiJ,

and4).=(Wl. -W2 -Wa. +wd/ 2: (1/X;) .
i=l

Thus, the relation (3) together with
(XIX.)/(X2Xa)=Ro ,should be equivalent to (2).
The Mutational Load with Epistatic Gene
Interactions in Fitness
Motoo KIMURA and Takeo MARUYAMA

The mutational load in a large population was first calculated by
Haldane in 1937 without assuming an epistatic component in fitness.
Later, a similar but more detailed calculation was carried out by Kimura(1961). Also, the mutational load in a small population was studied by
Kimura, Maruyama and Crow (1963).
In the present work, the effect of epistasis on the mutational load wasstudied using a model which assumes that fitness is a function of thenumber of mutant genes in an individual. The results are summarized asfollows:
1) If the deleterious effect of the mutant genes is proportional to thesquare of their number, the load under random mating becomes roughly
half as large as in the case of no epistasis, provided that the average
number of such genes per individual is fairly large. Under asexual reproduction, however, the epistasis has no effect in reducing the load. The
situation is intermediate for a random mating population of a hypotheticalorganism having only one pair of chromosomes between which no crossing
over takes place.
2) The mutational load may also be reduced under random mating if
fitness is a threshold character.

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

3) Epistatic interaction in fitness among deleterious mutant genes may
be classified into two types, namely, the reinforcing type and the diminishing type. In the former, the deleterious effect becomes disproportionately
large as their number in an individual increases. On the other hand, inthe latter, the deleterious effect per mutant gene becomes smaller as their
number increases. Consideration of mutational load as well as developmental homeostasis involved suggests that the reinforcing type of epistasis
among deleterious mutant genes must be more common than the diminishing type in nature.
For the details, see Kimura and Maruyama (1966), Genetics 54: 13371351.

Eigenvalues in a Genetics Problem
Takeo MARUYAMA

The smallest and the second smallest eigenvalues in a genetics problem
are important, for they determine respectively the asymptotic rate of
fixation in a case with absorbing boundaries and the asymptotic rate of
approach to a steady state if it exists. Kimura has given a power series
expansion of the smallest eigenvalue for the general case with absorbing
boundaries. Miller also gave an analytic method and numerical examples
in cases of heterosis. The purpose of the present work was to show thatan analogous method to the perturbation method in quantum mechanics
can be used to obtain all eigenvalues in a genetics problem, including ofcourse the first and second eigenvalues, when we know the characteristic
pairs of unperturbed equation.
The mathematical model considered is
Genotype
Fitness
We assume no mutation and no migration, and that the effective size ofa population is equal to N. Then the diffusion equation isaif> = _1_ ~{X(I-x)if>}+s~{(2x-I-2)x(l-x)if>}at
4N ax 2ax.

Letting if>(x;t)=T·X be the solution of the above equation, we have2

1 [ dx2
d {x(l-x)X}+4Ns~{(2x-l-z)x(l-x)X}d] =-A,
4N T' = X

y

where

(1)

RESEARCHES CARRIED OUT IN 1966

69

Defined22)
H= -(1-z
dz 2

h = d: {2(z-z)(1-z 2 )}linear operators

J

Then the right hand side of (1) is equivalent to(2 )(H+ah)<Pm(z)=Em<Pm(z) ,

where
a=NS.

Kimura has solved all the characteristic pairs of H¢m=~m¢m.

They are~m= --(m+ 1)(m+2)¢m=Tr;.I(Z)(Gegenbauer polynomials).

We now expand Em and <pm(Z) in powers of a, that is
E m= ~ anE;;:1

and <Pm(Z)= ~ antlJ):) .

n=O

(3)

n=O

Substituting the above Em and <pm in (2), we obtainH ~ an tlJ',;," +h }2 a n+1tlJ';:l = £ an
n=O

n=O

n=O

i:. E;:/ tlJ;::-i) .

i=L1

(4)

Equating the coefficients of like powers in (4), we havetlJ':.I=¢m=Tr;.1

IE~) =~m=-(m+1)(m+2)(H-~m)tlJ;;,1 +h¢m=Er;.I¢m(5 )Letting tlJ~) = ~ amj¢j (this is permissible, because the Gegenbauer polyj=O

nomials are a complete set), substituting this tlJr;.) in the last equation of(5) and using the fact that H¢j=~j¢j, we have
H 2: amj¢j-~m 2: amj¢;=Er;.I¢m-h¢m .
j

(6)

j

Multiplying both sides of (6) by (1-z 2 )¢m, integrating over the interval(-1, 1), and using the orthogonality of ¢m, we haveEO) __ (¢m, h¢m)m -II¢m11 2'

where

II¢mIl 2 =L1 (1-z )¢m¢mdz and (¢m, h¢m)= L1 (1-z )¢mh¢mdz .
2

2

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

In a similar, but more complicated way, 2nd, 3rd, 4th terms in (3) can beobtained. They areE;;) =.2:.2: (¢m, h¢i)(¢i, h¢j)(¢j, h¢m) ,(~i-~m)(~j-~m)

ioem joemwhereh=h-(¢m, h¢m) ,andE~) = -.2: .2: .2: (¢m, h¢i)(¢i, h¢j)(¢j, h¢k)(¢k, h¢m)(~i-~m)(~j-~m)(~k-~m)

i,<m j'<m hm- E;;').2: .2: (¢m, h¢J)(¢j, h¢m)ioem joem(~i-~m)(~j-~m)

A Diffusion Process with Heterosis
Takeo MARUYAMAAs Wright has emphasized, finite populations may be important in thecourse of evolution because random drift in gene frequency can move thepopulation from one adaptive peak to another on a fitness surface diagram.
Although it is true that random drift can eventually shift a finite population from one peak to any other peak, we would like to know the timerequired for it. As a first attempt to solve the problem, a simple model
was constructed of a fitness surface with only one peak, representing a
polymorphism due to heterosis, and an approximate solution of the genefrequency distribution was obtained.
The diffusion equation corresponding to heterotic selection in a finite
population is(1)
where Va.=x(1-x)j2N and Ma.=x(1-x)(Sl-(Sl+S2)X). A solution to equation (1) with initial condition lim ¢J(P, x;t)=o(P-x) has not yet been solved.
t~O

However if we restrict the behavior of the process at the local neighborhood of XO=Sl!(Sl +S2) , the point where the process starts, we have roughly
Va.=xo(1-xo)!2N and Ma.=xo(1-xo){Sl-(Sl+S2)X}. Then (1) becomesRESEARCHES CARRIED OUT IN 1966

where Y=SI-(SI +S2)X, I'=SIS2/(SI +S2) and a=Sls2/4N.
solution of (2) to be ifJ= T(t) Y(y), then we have71

If we assume the2_1_ dT(t) = _l_{a d Y + f3 dY +I'Y}=J
T(t) dt
Y(y)
dy 2
Y dy
.
1

(3 )

It is easy to see that T(t) = cAt . The right hand side of (3) becomes2
a d y +I'Y dY +(I'-J)Y=O.

dy 2

dy

If we let Z=..j ~y and Z(z)=Y(z) , we have

J)

2
d Z
dZ+ ( 1 - - Z=O,
+zdz 2dz
I'(4 )equation (4) is a Hermite differential equation and its integrable solutions
are Zn=C- z2 / 4Dn(z) n=O, 1,2,'"

where -

~ =0,1,2,'" andHence the solution of (2) is
ifJ(t;x) = ~ ancl\ntc-z2/4Dn(z) .
n=O

Now we want to determine the coefficients an such that limifJ(t;x)=o(xo-x).
t~O

The principle in determining an is ) o(x-xo)f(x)dx=f(xo) for all function
f(x) and the orthogonatity of Dn(z).
rI.( X· t) ='t',

The final form of the solution is[N(SI +52)J exp {- (- SI - X
SISZtrc
SI +S2)2/ 2N(SIslsd+S2)2 } .

Although this solution is valid for only a short time, it has a pleasing
form; ifJ(x;t) is a normal distribution (in particular it is the Wiener transition function) with variance proportional to time and the inverse of the
population size.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

An Application of Kimura's Formulae to Define the
Evolutionary Load in a Small Population
Takeo MARUYAMA

Gene frequencies or the distribution of gene frequencies are determinedby selection coefficients, population size, mutation rate etc. If the environment changes the relative fitness of genotypes, a new equilibrium will beattained by natural selection. If we assume for this discussion that thechange is such that the previously advantageous gene becomes disadvantageous, the shift of gene frequencies will be large. This case iscalled a gene substitution. If we further assume that there is neither
mutation nor migration during the process, the substitution will be complete.
This change in the relative fitnesses causes a genetic load, called theevolutionary load. Symbolically if we let let) be the genetic load at timet, then the evolutionary load for the whole process is given by

r=o l(t)dt .
It has been shown by Kimura that the load in an infinitely large population iswhere h is the degree of dominance and Po is the initial gene frequency.
The main trouble with finite populations is that, even if we assume Po to
be positive, some populations will loose the advantageous gene and will
stay in the lost class until mutation occurs. Therefore it is not possible
to define the evolutionary load in analogy to the case of an infinite population. However we may measure the load as follows; for each sample
path we calculate the difference in fitnesses between the best genotype andthe average fitness at a given time, add them from time zero to infinity
and take the expectation with respect to the collection of all sample paths.
Because this is the expected total genetic death spent in the substitutionof the gene, we divide it by the fraction which went to fixation. We callit the evolutionary load. Applying Kimura's solution 1> (P, x; t) to the
diffusion equation in genic selection and his solution u(P) to the probabilityof ultimate fixation we can write the load in a finite population, in a caseof genic selection, as1)00 dt ~1

- (-)

uP

1=0

.=0

sx¢(P, x;t)dx .

(1)

73

RESEARCHES CARRIED OUT IN 1966

The explicit formulae for ¢(P, x;t) and uCP) due to Kimura are~¢(P, x;t)= 2: Cke-l.-kt+2CzVlklll(z) ,k=Owhere z=1-2x, c=Ns, and Ck and Ak are constants, Vlk llJ = 2:' Ink T~I\Z)n=O.l

where Ink are constants and T111 (Z) are Gegenbauer polynomials, and
1_e- 4N8P
u(P)= 1_e- 4N8•

Therefore using the relation
I nm = (I T~J(z) T';;.I(Z) = {O if m-n=odd
J-I(n+ 1)(n+2) if m;;:;'n and m-n=even(1) can be expressed as

where[I (1- z2)(1- z)eczT~\z)dz
IIT~)112dn =,IIT~1112 = [I (1-Z2){T~I)(Z)}2dZ=2{.(3t)-1)J-In+- n!

2

Dimensionality of Human Migration
Norikazu YASUDA

A pattern of human migration has been considered as two-dimensional,
that is, man moves in all directions from where he was born. However,this is not true in a population with barriers.
Suppose that an individual moves from his birthplace to a place to where
he is attracted by job or mate. This exploration range will be proportional
to the inverse of dimensional power of marital distance (x), defined as the
distance between the birthplaces of consorts. An acceptable frequency
distribution is m(x)=ab/(l +ax)b+l, where b+ 1 represents dimension anda is a scale parameter or "attraction index".
Examples of estimated parameters are given as follows:

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Population
Brazil (grandparent)
Brazil (parent)
Japan (Ohdate)
Italy (M area)
Italy (P area)

Dimension

Attraction index

(b+l)

(a)

1.90±O.06
1.90±O.06
1.49±O.02
1.60±O.03
1.99±O.04

O.09±O.OI
O.06±O.OI
1.62±O.15
2.86±O.32
O.56±O.03

The estimated dimensionality in Italian samples agrees impressively
with the results of Cavalli whose approach was completely different.
Whether one or two in dimensionality is one of the important parameters
in a discussion of the relationship between the inbreeding coefficient anddistance. The above results then suggest that dimensionality in man is
two and deviation from it may be understood to be due to physical or
sociological barriers.
In addition, the attraction index may be used for a characterization of
a population: the smaller is the value of a, the more migration takes
place.
Dimensionality and Distance in Human Migration
Norikazu YASUDA

Direction of human migration may depend upon the geography of theplace where people live. By introducing the isotropic index, a method wasdevised to ascertain the effect of distance on dimensionality in man.
Suppose that an individual was born at a place P(XI, YI) and moved intoa place Q(X2, Yz) (both points might be same). The isotropic index (j isdefined as:

The expected variance of (j is zero if a person migrates in one direction
while it will be 1r 2 /48 (measured in radians) if isotropic migration takes
place. It may not be zero variance even if one dimensional migration isobserved, since the direction is often curvilinear. This will be serious
when the line makes a right angle. The method, however, is still applicable in such a case by dividing the population into two parts.
Dimension (d) is thus evaluated from d=1+s2/a2, wheres2=[22 (j2_(L. (j)2/ n l/(n-l) , n is the number of pairs and a2 =1r 2/48.
Applying the method in two studies on distribution of ABO blood groups

75

RESEARCHES CARRIED OUT IN 1966
Distance (KM)
.50 100.
2.0

300

200

400

500

600

700

•• -........-....:.:::.. . .
S\viss•••••••(3087 communities).2~u

Sweden(1071 communities)

";:;;

c:

"§ 1.05

C<

1.01

Fig. 1.

Dimensionality and distance in man.

in Switzerland (Rosin, 1956) and in Sweden (Beckman, 1959) where individuals are grouped by their birthplace so that the distribution ofcommunities may be considered approximately as an expected distributionof human migration, and making all possible pairwise combinations of 3087
communities in Switzerland and of 1071 in Sweden, we found that theisotropic distances are about 100 km. in Switzerland and about ZOO km. in
Sweden, reflecting the geography of each country. Dimension becomes
one for further remote distances (see figure).

A Statistical Singularity at the ABO Blood Group System
Norikazu YASUDA

Suppose that the observed and the expected numbers of individuals inthe standard ABO blood group classification are given by:

Phenotype

Genotype

0

00

A
B
AB

AA,AO
BB,BO
AB

Observed
no
nA
nB
nAB

ExpectedN[r 2+(r-r 2)a]N[P2 + Zpr + {P - (P2 + Zpr)}a]N[q2+Zqr+{q-(q2+Zqr)}a]
N[Zpq-Zpqa]

where p, q and r are gene frequencies of A, Band 0, respectively, a is
the inbreeding coefficient, and N=nA+nB+n..IB+no. Then the maximum
likelihood scores under the null hypothesis that a=O are

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

up=n{- ~]+nA~2~~p;]+nB[q~~r]+nAB[~]'
uq=no[- 22r ]+nAB [~]r]+nA [~2P+ r]+nB rLq +2qr
q,
2

and

Ua=n{l-;r]+nA[;~;r]+nB[:;;r]+nAB[ -1].

It is easily verified thatindicating that no reliable estimate of a can be obtained from this material
alone although one degree of freedom is left for it. The situation is not
improved either by subtyping A into Al and Az and AB into AlB and
AzB or B, AlB and AzB in the same manner as A and AB. Furthermore, if there exist m alleles which are codominant to each other, but
are dominant to the silent allele 0, we found that

Ua=- 2:.PiUp./2.
m

i=l

~

An example of m=3 was observed in the esterase-2 system in Drosophila
virilis (Ohba, personal communication).
No such difficulty, however, was observed from mating type frequencies
in the ABO system.

RESEARCHES CARRIED OUT IN 1966

VI.

77

EXPERIMENTAL STUDIES ON
POPULATION GENETICS

Deleterious Genes in the Second Chromosome Concealed in
Natural Populations of Drosophila melanogaster 1 )Chozo OSHIMA and Takao K. WATANABE

Among 905 second chromosomes isolated from natural populations at Kofu
and Katsunuma territory in late October 1965, the frequency of lethal
chromosomes was 15.47 per cent. The frequencies of lethal chromosomes
did not fluctuate to a remarkable extent during the past several years, but
those of semilethal and subvital chromosomes in the extracted chromosomes
in 1965 were decreased as compared with their earlier frequencies, as
shown in Table 1.
Table 1.

Frequencies of the second chromosomes carrying deleterious genes
in natural populations of the Kofu and Katsunuma territory

No. of the second
Year chromosomes
isolated

Frequency ofLethals

Semilethals

Subvitals

Normals

1959
1963
1964

144
668
740

13.19±2.82 19.44±2.30 16.67±3.11 50.69±4.17
20.21±1.55 18.11±1.49 25.15±1.68 36.53±1.8615.54±1.33 19.59±1.46 7.57±0.98 57.30±1.82

1965

905

15.47±1.20

6.85±0.84

3.76±0.63 73.92±1.46

A tester strain, having the dominant marker genes Cy and Pm on eachsecond chromosome, was used in the analysis, but its genetic background(X and third chromosomes) was previously substituted with normal chromosomes originated from the same natural population. Consequently, thegenetic background was assumed to be highly heterozygous. On thecontrary, the genetic background of the tester strain, which had been usedin the former experiments, was assumed to be homozygous, as it was
substituted with homozygous chromosomes of an isogenic Samarkand strain.
The frequencies of chromosomes carrying deleterious genes except forcomplete lethals, could have been influenced by the state of genetic
background of the tester strain. Therefore, the frequencies of semilethals
and subvitals, found in the past years (1959, 1963, 1964), would be nearerI) The following six investigations were supported by a grant from the Japan
Society for the Promotion of Science as part of the Japan-U.S. Cooperative Science
Program.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

to the real frequencies in the natural populations than those obtained in 1965.

Distribution of Persistent Lethal Genes in Natural Populations
Chozo OSHIMA and Takao K. WATANABE

From six different populations, distributed over the Kofu and Katsunuma
territory (15 X 3 km 2 ), 132 lethal chromosomes were isolated, and allelism
tests between lethal genes were performed. The results are represented
in Table 2.
Table 2.

Allelism tests between lethal genes of 132 lethal chromosomes
Whole
populations

WithinKofu
populations

WithinKatsunuma
populations

Between
Kofu & Katsunuma
populations

No. of lethals
No. of crosses
No. of alleliccrosses

132
8646

63
1953

69
2346

132
4347

169

54

38

77

Allelic rate (%)

1.95

2.76

1.62

1. 77

Seventy-nine lethal chromosomes, isolated from the same populations in
1964, have been maintained individually by the Cy-balanced condition.
Allelism tests between newly isolated 132 lethal chromosomes and the oldones were completely performed by the diallel cross. Among the total of
10428 crosses, 248 were allelic and allelic rate was determined to be 2.38
per cent, being similar to that (2.35 per cent) between lethals isolated in
1963 and 1964.
Table 3.

Frequency of persistent lethals among all isolated lethals
and their persistence periods

PeriodNo. of lethals
testedNo. of persistentlethals

Frequency(%)

1 Year1963-1964
1964-1965

100
140

35
52

35.0
37.1

2 Years

1963-1965

140

29

20.7

4 Years
5 Years
6 Years

1959-1963
1959-1964
1959-1965

114
100
140

10
9
10

8.8
9.0
7.1

RESEARCHES CARRIED OUT IN 1966

79

From the results of such successive allelism tests, some lethal genes wereconfirmed to have persisted for a long time in the Kofu and Katsunuma
populations, as shown in Table 3.
Eighty lethal genes among 132 lethals were assumed to be newly arisen
lethals and the allelic rates between them were very low, as shown in
Table 4.
Table 4.

Allelic rates between newly arisen lethal genes

Wholepopulation

WithinKofu
population

WithinKatsunuma
population

Between
Kofu & Katsunuma
population

No. of lethals
No. of crosses
No. of alleliccrosses

80
3160

38
703

42

861

80
1596

15

3

7

5

Allelic rate (%)

0.47

0.43

0.81

0.31

Recessive Visible Mutant Genes on the Second Chromosome
Concealed in Natural Populations
Chozo OSHIMA and Takao K. WATANABE

Among 905 chromosomes, 89 (9.8 per cent) had a recessive visible mutant
gene. A mutant gene, making bristles slender and short, was named
tentatively 'reduced' (rd) and similar mutant genes were found in 63
chromosomes. Another mutant gene, manifesting brown eye, was identified
to be the bzv gene (2-104.5); it was found in 12 chromosomes in addition
to five kinds of mutant genes (tentatively named spread wing, excisedwing, upturned wing, reduced wing and small body) observed in a few
chromosomes.
For the past two years, many chromosomes, having the rd gene, wereisolated from the Kofu and Katsunuma populations. By the results ofallelism tests between them, these rd genes could be divided into fivegroups and the loci of rd-1 and rd-4 were determined to be 66.9± and
51.8± respectively. The manifestation of rd gene is very similar to thatof Minute genes, of which about 20 loci have been recorded on the second
chromosome, most of them clustering near the centromere. It may be
assumed that by some mechanisms a fairly number of rd genes have been
maintained in the natural populations in spite of lower viability of heterozygous flies (rd/ + ).

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Segregation Distorter (SD) Genes and Their Linked
Lethal Genes in Drosophila melanogaster
Takao K. WATANABE and Chozo OSHIMA

Among 420 second chromosomes, which were extracted from natural
populations at Kofu and Katsunuma in late October of 1965,15 chromosomes(3.6 per cent) were found to have segregation distorter genes, as a result ofchecking segregation ratios in heterozygous males for each second chromosome and the cinnabar brown chromosome. Seven chromosomes among
them had each a lethal gene. The lethal gene on five of them was clarified
to be a persistent one (1401, locus: 47.9), which had been maintained in highfrequency in the Kofu and Katsunuma populations for at least two years.
Most SD chromosomes (86.7 per cent) had In~2R)C on the right arm,
whose equilibrium frequency in the natural populations was about 25 percent. The SD gene was assumed to be located near the proximal end of
the right arm. Then, the linkage between SD and the inversion could bestrong, but that between SD and [401 was weaker, because the lethal
gene was located on the left arm.
An SD chromosome was marked with Lobe (locus: 72.0) gene and wasused to analyze the varying segregation ratio in heterozygous male flies
in about 390 chromosomes extracted from the natural population. The
mean (k) segregation ratio was estimated to be 0.77, but it varied widelyfrom 0.4 to 1.0. At 0.5 as k value, the effect of the segregation distorter
was suppressed and at 1.0 as k value, complete expressibility of the gene
was assumed.
The segregation of homozygous male flies for SD genes was foundnormal, but in the offspring the number was reduced. Several hypotheses
illustrating the mechanism of segregation distortion have been proposed,
but the problem still remains unsolved.
A Mechanism of Persistence of Some Lethal Genes in
Natural Populations of Drosophila melanogaster
Takao K. WATANABE

Two lethal genes (1404, [407) on the second chromosome have been
maintained at high frequency for at least 6 years in the same natural
populations. One hundred twenty female flies, heterozygous for Cy andthe persistent lethal chromosomes (Cy/[404 or Cy/[407) and the same
number of female flies heterozygous for Cy and normal chromosomes (Cy/ni)
were mated with male flies having two normal chromosomes in heterozygous condition.

81

RESEARCHES CARRIED OUT IN 1966

Each female was mated with three males in a vial and transferred every
day to a new vial containing the culture medium. In the course of ex·
periments, the longevity of the two kinds of flies was examined, and thenumber of their progeny was compared as to productivity. The ratio of
non·Cy flies was estimated. Then, the viability of lethal heterozygotes(llni) could be compared with that of normal heterozygotes (ndm).
The mean longevity of Cy·lethal heterozygous females was about 6 days
shorter than that of Cy-normal heterozygous females, but the former
produced 509 flies on an average during 10 days after emergence and thelatter produced only 460 flies. The viability of lethal heterozygotes andnormal heterozygotes is given in Table 5.
Table 5.

Comparison of viabilities (pooled basis) of lethal andnormal heterozygous flies
Period

1-5 days
No. offlies

Viability*

11-41 days

6-10 days
No. offlies

28351 1.0035±0.01192 22861
llni
ndnj 27244 0.9519±0.01154 25383

Viability

No. offlies

1.0174±0.01346 20772
1.0021±0.01258 30370

Viability*
1.0377±0.01440
0.9867 ±0.01132* 1% level significance.

The data were divided into three parts according to the period of egg
laying. From the results, significantly more lethal heterozygous flies thannormal heterozygous flies were not only produced by younger parents but
also by older parents. This could be an effective mechanism of thepersistence for those lethal genes.

Further Study on Chromosomal Polymorphism in
the Kofu and Katsunuma Natural Populations
Taishu WATANABEll and Chozo OSHIMA

About 800 male flies were captured from two natural populations in the
Kofu and Katsunuma territory in late October of 1965. These male flies
were individually mated with several virgin females of an isogenic Sam·arkand strain. The salivary chromosomes of 8 or 10 Fl larvae of each
culture were observed. The chromosomal types of 573 male flies could bedetermined and the frequency of each kind of chromosomes was recorded.
The expected numbers of all possible homozygous and heterozygous com·1)

Department of Biology, Faculty of Science, Kyushu University.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

binations of the standard second and third chromosomes and various
inversion chromosomes were calculated on the basis of Hardy-Weinberg'slaw.
The observed numbers and expected numbers of three groups; homozygotic, single inversion heterozygotic and double inversions heterozygotic,
were compared with each other as shown in Table 6.
Table 6. Frequencies of nine combinations of homozygotic, single
inversion heterozygotic and double inversions heterozygotic
second and third chromosomes
Combination

No. of flies

Chromosome 2

Chromosome 3

Obs.

Exp.

Deviation

Homozygotic

Homozygotic

66

64.27

+ 1.73Single heterozygotic Homozygotic
Single heterozygotic
Homozygotic
Single heterozygotic Single heterozygotic
Total of single heterozygotic

85
100
155
340

92.20
109.52156.98358.70

7.20
9.52
1.98
-18.70

Double heterozygotic Homozygotic
Double heterozygotic Single heterozygotic
Double heterozygotic
Homozygotic
Single heterozygotic Double heterozygotic
Double heterozygotic Double heterozygotic
Total of double heterozygotic

34
55
32
31
15
167

30.86
52.55
22.87
32.79
10.95
150.02+ 3.14+ 2.45+ 9.13- 1.79+ 4.05+16.98Grand total

573

572.99+ 0.01

The heterotic effect of inversions, which have been maintained in fairly
high frequencies (6~42 per cent) in the natural populations, could berecognized at least in the presence of two or three inversions on one armof one chromosome.

83

RESEARCHES CARRIED OUT IN 1966

Lack of Chromosomal Interaction with Respect toOverdominance in Drosophila melanogaster ll
Terumi MUKAIAs previously reported (Genetics 50: 1-19, 1964), spontaneous mutant
polygenes controlling viability have been accumulated independently in
104 second chromosome lines which originated from a single normal
chromosome. Based upon the test for the heterozygous effects of thesemutant polygenes located only in one of the homologous chromosomes inGenerations 32, 60, and 78, we have proposed that an optimum level ofheterozygosity exists for manifestation of overdominance (Genetics 52:
493-501, 1965; Ann. Rep. 16). Furthermore, it was reported that the
optimum level of heterozygosity in the second chromosomes was independent of the heterozygosity Df the third chromosomes (Sbj -T- vs. +! +third chromosome genetic background) (Ann. Rep. 16). In order to confirm
the second finding, an experiment was conducted in Generation 85.
Homozygous and heterozygous viabilities of 50 chromosome lines selectedfrom the above 104 lines were tested both in homozygous and heterozygous
genetic backgrounds (SbjUbx third chromosome). The outline of the result
is presented in Table 1. Forty-six lines out of 50 showed overdominance
Table 1. Means of the standardized viabilities of homozygotes
and heterozygotes and their correlation coefficients
Homozygous
viability

Heterozygous
viability

Correlation coeff.
between homo- andheterozygotes

Homoz>:gous
0.3329*** +0.0455
genetIc background-1.0611*** ±O. 0045

-0.1180

Heteroz,Ygous
0.3113*** +0.0468
genetIc background-1.1007***±0.0074-0.2443

Notes 1. Number of chromosome lines=50.
2. Control viability=1.0000.
*** Significantly different from 1.0000 (P<0.001).

in both genetic backgrounds, two showed it only in homozygous genetic
background, and the remaining two only in heterozygous genetic background. Thus, it is confirmed that the manifestation of overdominancein the second chromosome is independent of the heterozygosity in the1) This work was conducted in the Department of Medical Genetics,
Universityof Wisconsin, Madison, Wisconsin, U.S.A. and was supported by a PHS Grant
GM-07666.

8t

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

third chromosome, contrary to the interaction among genes located within
chromosomes. If the second and the third chromosomes are not independent, overdominance is not to be expected in the second chromosomes
under the optimum heterozygosity hypothesis, because the level of heterozygosity of the third chromosomes (Sb/Ubx) is probably much higher thanthe optimum level for the second chromosomes.
The Detrimental Load to the Lethal Load Ratio (D:L Ratio) of
Newly Arising Mutations in Drosophila melanogaster ll
Terumi MUKAI and James F. CROW

Greenberg and Crow (1960) have proposed a method for testing thedegree of dominance of detrimental genes by comparing the D:L ratio in
equilibrium population and that for newly arising mutations. The former
ratio has been estimated to be 0.5~1.0 by Greenberg and Crow (1960),
Band (1964), and Temin (1966).
In order to estimate the D:L ratio for newly arising mutations, spontaneous mutations controlling viability have been accumulated at a minimumpressure of natural selection in 150 second chromosomes which originated
Table 1.

Summary of experimental results in Generations 10 and 20(CH)**(PQ)**(RT)**

Gen.O Gen.10 Gen.20 Gen.O Gen.10 Gen.20
Number oflethal lines

o

1

3

o

1

2

Gen.O Gen.10 Gen.20

o

2

6

Mean viability
including (34.10) 32.08
lethals

29.48(33.51) 31.64

29.82(28.11) 25.02

21.20

Mean via·
bilityexcluding (34.10) 32.73
lethals

31.36(33.51) 32.28

31.05(28.11) 26.06

24.01

D:L ratio

1.35

1.89

1.27

The number of chromosome lines is 50 in each group; each originated from asingle chromosome extracted from a natural population.
** Stands for the original Madison chromosomes.
II This work was conducted in the Department of Medical Genetics, Universityof Wisconsin, Madison, Wisconsin, U.S.A. and was supported by a PHS Grant
GM-07666.

RESEARCHES CARRIED OUT IN 1966

85

from 3 Madison chromosomes (50 from each Madison chromosome). Homozygous viability of each chromosome was tested in Generations 10 and 20.
The result is presented in Table 1. The D: L ratio was calculated for eachchromosome group which originated from a single Madison chromosome.
The results are 1.27 ~ 1.89 which are larger than those in equilibrium
populations. This indicates that the degree of dominance of detrimental
genes is larger than that of lethal genes which was estimated to be ii=0.02~0.04 by several workers (e.g. Crow and Temin 1964; Hiraizumi
and Crow 1960; Stern, et at. 1952).

Studies on the Competition between RaceslA and 21B of Wheat Leaf Rust
Keizo KATSUYA

Physiologic races of various plant pathogenic fungi continuously vary inprevalence in nature. The change in prevalence of races 1A and 21B ofwheat leaf rust, Puccinia rubigo-vera Winter f. sp. tritici Mains, was investigated by comparing in growth cabinets aggressiveness of the two races
and the factors that might influence them.
When a mixture of urediospores of the two races was used to inoculate twosusceptible wheat varieties, Norin No. 61 and Shinchunaka, for five uredial
generations race 21B quickly became predominant at 15° and 20°C, andafter four uredial generations race 1A was nearly eliminated from the
mixtures. Relative competitive abilities were not found to be influenced
by the density of pustules on infected leaves. The rate of growth ofpustules on the two wheat varieties grown at 15° and 20°C was determinedby measurement with a microscope. Pustules of race 21B grew fasterthan those of race 1A at 15°C, and at twelve days after inoculation pustule
size of race 21B was about 1.6 times larger than that of race 1A. At
20°C, however, pustules of race 21B grew almost the same as those ofrace 1A. Experimentation showed that the urediospores of race 21B weremore infective than those of race 1A on the two wheat varieties and thatthe incubation period of race 21B was remarkably. shorter than that ofrace 1A at 15°C. The predominance of race group 21 of wheat leaf rustin north Japan may have resulted from the greater aggressiveness ofthat race.

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Interaction among Genotypes for Migration in

Drosophila melanogasterTakashi NARISE

In the previous migration experiment with wild and vestigial flies ofDrosophila melanogaster it was found that the migratory activity of thevestigial flies increased due to mixing them with wild flies (see this Annual
Report No. 12). It then become the immediate task to find out if the genevestigial itself was responsible for making sensitive to the stimulation, or
was an interaction between the gene and its genetic background responsible. The present experiment was conducted to resolve this problem.
Employed were five wild strains, MS-1, Niihama, Oshoro, Gifu, and Tosu,
and their substitution vg strains. They were mixed one with another ina manner shown in Table 1. One hundred flies were introduced into themigration-tube in the either mixing or control experiment. The mixture
was composed of 50 flies of a wild strain and 50 flies of the vg-substituted
strain. The experimental results are presented in Table 1 and Table 2.
Table l.

Effect of vestigial genotype on migratory activity of five wild strains
Migratory activity of wild type strains

MS-l (vg)
Tosu (vq)
Gifu (vg)
Niihama (vg)
Oshoro (vg)SingleTable 2.

MS-l

Tosu

Gifu

Niihama

Oshoro

13.2
16.0
19.8
21.2
20.8
26.8

22.0
10.0
12.4
16.0
13.2
28.4

25.6
35.4
22.0
37.2
27.2
21.6

20.8
15.2
16.0
22.0
19.2
28.8

31.2
20.8
22.4
21.6
19.6
24.6

Effect of wild type on migratory activity of five vestigial strains
Migratory activity of vestigial substituted strains

MS-l
Tosu
Gifu
Niihama
OshoroSingleMS-l (vg)

Tosu (vg)

Gifu (vg)

Niihama (vg)

Oshoro (vg)

17.2
23.6
21.6
18.8
24.8
11.0

16.4
11.6
20.8
27.6
14.4
11.0

16.4
16.4
19.2
16.8
18.8
18.8

19.6
12.0
17.6
8.0
15.6
5.8

24.4
20.0
22.8
18.8
13.6
13.5

RESEARCHES CARRIED OUT IN 1966

87

In these tables, MS-l (vg) means the MS-l strain substituted with thevestigial gene into the genetic background of MS-l and so on.
It is found from Table 1 and Table 2 that the high migratory activityof wild type flies generally decreases by being mixed with a vestigial
strain except for the Gifu strain. The activity of vestigial strains, however,increases due to being mixed with a wild strain except for Gifu (vg) strain.
It is noteworthy that the migratory activity of a strain increased when
the strain was mixed with strain having different genetic background incomparison with that having the same genetic background except for
Niihama and Gifu (vg) strains.

The Relation between Migratory Activity and Competitive
Ability in Drosophila melanogasterTakashi NARISE

In order to find if migratory activity is in some way or other related
to competitive ability in Drosophila melanogaster, an experimental study
was conducted with a wild strain, MS-l. A selection experiment started
with two hundred flies of the strain for high migratory activity. The
selection was conducted using the migration-tube set, and the descriptionof the apparatus and method of experiment have been repeatedly reported
in the preceding issues of this Annual Report (see Annual Reports No.7
and No.8).
The selection was continued through eight generations, when the migratory activity of the selected strain attained 34.8 per cent in comparison
with the original population with 20.5 per cent migration.
Competitive ability of the selected strain was tested in the 8th selection
generation. Five pairs of flies of the selected strain were mixed with fivepairs of the tester, a wild type of D. simulans, and that were kept five daysin a half-pint milk bottle. Only emerged male flies of both species werecounted because of the difficulty in distinguishing female flies between
the two species. The number of all emerged male flies per bottle in asingle culture of selected and original melanogaster strains was 193.53 and
193.68, respectively, suggesting that the two strains were not different
with regard to propagating capacity. The capacity of D. simulans inthe single culture was as low as 68.20. Table 1 presents the number of
emerged male flies under mixed condition.
The findings from this experiment are : 1) the competitive ability ofthe selected strain was far lower than that of the original one, and 2) the
number of emerged flies in the next generation was not different between
the original and the selected strain suggesting that the fitness of these two

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table 1.

Number of all emerged male flies per bottle under mixed condition

Original
Selectedmelanogaster

simulans

72.40

0.73
5.96

49.24

strains had little changed.
From this experiment, the conclusion can be drawn that migratory
activity and competitive ability are negatively associated at least in thepresent fly population of D. melanogaster. For generalization of this fact,
however, more study is without doubt required.

Experimental Induction of Bilateral Asymmetryin Wings of Drosophila melanogasterTakashi NARISE and Kan-Ichi SAKAIThis study deals with an experimental induction of bilateral asymmetrty
in the wings of Drosophila melanogaster. It has been observed that thewings of wild flies are almost completely symmetrical. Accordingly, there
seemed to be little hope to obtain flies having asymmetrical wings bymere selection in wild populations. Finding, however, that the vestigial
flies are almost more or less asymmetrical in their wings, selection forhigh as well as for low asymmetry was attempted in the population ofvestigial stock. It was found that the selection was successful only forhigher, but unsuccessful for lower asymmetry. The selected vestigial strain
with high asymmetry in wings or, for simplicity, the vg-As strain, wascrossed with wild type flies whose wings are completely symmetrical. The
crossing was made between 25 wild type males and 125 vg-As females or
25 sets were set up each of 1 wild male and 5 vg-As females, yielding
125 families in total. The experiment was conducted at 25 ± 1°C.
Of the 25 sets of the hybridization experiment, 15 showed no sign ofasymmetry. In ten, however, one or more than two families gave rise
to bilateral asymmetry in wings of some flies. Those ten sets are shownin Table l.
It is found from Table 1 that the complete symmetry in wings of normal
wild flies can be upset by the effect of genetic system favoring asymmetry,
integrated by selection in the vestigial flies.
0RESEARCHES CARRIED OUT IN 1966

89

Table l. The sets of one wild 0 x 5 vg-As ~ ~, in which flies with wing
asymmetry arose in at least one family. o means that no F 1 had asymmetrical wings. The two figures without and with brackets stand fornumber in per cent of flies with asymmetrical wings and average value
in 0.1 mm of asymmetry, respectively
Female No.
Set No.
1

2
0

3

30.00
(0.058)
0

0

6

0

0

9

0

0

10

0

12
14

2.7
(0.100)
0

15

0

21

23.50
(0.075)11.80(0.050)

1

24

4

5

0

0

0

0

7.7
(0.150)19.00(0.088)00

38.40(0.090)24.30(0.083)
0

0

0

0

92.90
(0.069)020.00(0.088)
0

0

0

0

0

0

0

0

14.70
(0.080)
0

0

0

0

13.60
(0.067)
0

3

0

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

VII.

RADIATION GENETICS AND CHEMICALMUTAGENESIS IN ANIMALS

Post-Irradiation Modification and Mechanism of Reverse DoseRate Effect on Mutation Induction in Silkworm Gonia
Yataro TAZIMA and Toshihiko SADOAn enhancing effect on radiation-induced mutation was observed in
the silkworm after fractionated exposure of gonial cells. As a possible
interpretation of this phenomenon, some kind of modification in cellular
metabolism was assumed. The modified state, which becomes detectableby increased mutation frequency, persists at least for 48 hours and ischaracterized by a peak appearing 18-24 hours after the initial exposure.
The non-decay type curve suggested that this was not due to a biochemical
disturbance but presumably to cell synchronization.
The results of an experiment, where two split doses were given to two
experimental series in reciprocal order (250 R-L750 R and 750R+250R) 6
hours up to 48 hours apart, showed that the peak appears earlier for the
initial 250 R than for the initial 750 R series. By labelling S phase cellswith H3-thymidine, Sado observed that irradiated cells are blocked intheir progression at Gz and their proportion increases remarkablly in thecell population. This observation favours the hypothesis of cell synchronization. If this were the case, a decrease in mutation frequency should
also be detected when second irradiation were given at an appropriate
interval after the initial exposure. This, however, was not observed up
to 72 hour interval, suggesting that synchronization, if any, is destroyed
immediately in the subsequent cell cycle. Presumably the cells are released
from the blockade at a reduced rate.
Based on those results, we examined the validity of the same mechanism
for the interpretation of the reverse dose-rate effect. Cytological observations revealed that the mitotic process of gonia at a late stage is inhibited,being blocked at G, and Gz, during chronic exposure. Furthermore, it
was confirmed by a new series of chronic exposure experiments lasting
three days which were started every 24 hours that the chronic treatment
becomes more effective for mutation induction than the acute one as early
as one day after hatching, coinciding with the initiation of cellular activity.
This observation explains why the dose-rate effect is reversed within avery short period.
(Abstract of a paper presented at the third International Congress ofRadiation Research, Cortina D' Ampezzo)

RESEARCHES CARRIED OUT IN 1966

91

Repair of Radiation Induced Premutational Damages Revealed
by Fractionated Irradiation of Silkworm Spermatids
Yataro TAZIMA and Kimiharu ONIMARU

The results of our study, for past six years, on dose-rate effect ofradiation, strongly suggested the existence of a mechanism of repair of
premutational damages. Furthermore, the assumption of a repair phenomenon allowed a consistent interpretation of several kinds of experimental
data obtained so far. Our knowledge, however, was rather scanty as tothe mechanism of repair of premutational damages in higher organisms.
A new project has, therefore, been undertaken in our laboratory for theinvestigation of such a mechanism. As a first step of this project efforts
have been made to find out good evidences for the repair of premutational
damages which could be utilized as an experimental system for further
studies.
For the elucidation of repair, two methods can be applied. One is a
split dose irradiation with an appropriate time interval. The other is apost-irradiation treatment with various agents. In both cases, repair of
premutational damages, if any had occurred, would be detected by a decrease in observed mutation frequency.
As reported previously, a remarkable enhancing effect has been observedwith regard to mutation frequency when we irradiated silkworm spermatogonia with two split doses of 500 R+500 R. The mechanism was soonrevealed in a cytological study by Sado (1966). Namely, after initial exposure the proportion of G2 cells increased among the germ cell populationand if the second exposure was given to those cells an enhancement inmutation response was the result. Accordingly, those cases can not be
regarded as due to true repair.
In order to obtain conclusive evidences for the repair, fractionation
experiments have been carried out with spermatids. Silkworm spermatids
appear most abundantly in full-grown larvae. Furthermore, at this stage
the cells are fairly homogeneous with respect to their developmental stage.
Total dose of 1000 R was given to full-grown larvae in two fractions: 500 R+500 R. Intervals between the two exposures were 0, 6 and 12 hours.
The results are given in Fig. 1.
It can be seen from the figure that split dose irradiation clearly reduces
the induced mutation frequency in comparison with the same dose given
singly.
Similar experiments were carried out at V-4.5 (fifth instar day 4.5) andmid-pupal stage, but the results were less conclusive for both stages.
Since irradiation of full-grown larvae almost completely excludes the

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Fractionation (500 R+500 R)

80t

661 Full-grown larvae~ ::.[.----------- :_!'--

mO'

!::~~
,

~

3O~__

_~

............ ,p'..

------:'tremos

20l ---------...--------------..

lOt"
o

6

•

i2

Interval in hours
Fig. 1. Results of split dose irradiation of full-grownlarvae: 500 R + 500 R with 0, 6 and 12 hour intervals.
Heavy lines, solid and broken, represent the split dose
irradiation and light lines denote single dose irradiation.

risk of contamination with cells irradiated at other stages than spermatids,
the observed reduction in mutation frequency can be regarded as thereflection of a repair of premutational damages produced by radiation.
Modification of r·Ray-Induced Mutation Frequencies in the Silkworm
by Post-Treatment of Spermatids and Spermatozoa
with Nitrogen Gas
Yataro T AZIMA and Kimiharu ONIMARU

As reported in the foregoing article (this Annual Report), a direct evidence of a repair of premutational damages was obtained by fractionatedirradiation of spermatids. Another evidence has been obtained from thecomparison of the effects of post-irradiation treatment between nitrogenand oxygen.

RESEARCHES CARRIED OUT IN 1966

93

Both spermatids and spermatozoa were chosen as the object of thisstudy. Irradiation of spermatids was performed when the larvae attained
maturity and irradiation of spermatozoa was carried out after emergence.
The material used was strain C108. For the detection of mutation, specificloci method was used by employing pe and re genes as markers. The
effect of N2 post-treatment was examined both for N2 pre-treated andnon-pretreated cells. For N 2 pretreatment, the wild type males wereplaced in a nitrogen gas chamber 25 minutes before irradiation and there
they were exposed to r-rays. In the non-pretreated group, irradiation ofwild type males was carried out in the air. Irrespective of the pretreatment, the irradiated insects were divided into two groups: one was subjected to N 2 post-treatment for 25 or 50 minutes and the other to 02
post-treatment for the same duration.
The results of experiments obtained for spermatids are shown in Fig. 1
IUU

With
Nz-pretreatment

(662)

1000R
;:;

Without
pretreatment

(663)

2000R

(663\

2000R

50

(-)

50
_ Nz
=Oz
A: pe
B: pe mas
C: re
D: re masj100

Fig. 1. Effect of post-treatment with N2 as compared to thatwith O2 • r-irradiation of spermatids. Details in the text.

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

In this figure cases in which post-treatment with N2 was mutagenicallymore effective than with O2 are shown in the upward histogram and thosewhere N2 was less effective are represented in the downward histogram.
Our experiments show that post-treatment with N2 is in most cases
mutagenically more effective than that with 02 for spermatids irradiatedunder anaerobic condition. However, the effect becomes reverse when
the spermatids are irradiated under aerobic condition.
In contrast, N2 post-treatment of spermatozoa resulted in a lower
mutagenic effectiveness than 02 post-treatment when the insects had been
pretreated with N2 and irradiated under N2.
Those results almost agree with those obtained by Sobels et al. for
Drosophila and may be mentioned as another evidence of a repair of
premutational damages.

Mechanisms of Mutation Induction by Mitomycin-C in the Silkworm
Yataro T AZIMA and Kimiharu ONIMARU

During the course of our experiments of post-irradiation treatment ofsilkworm spermatogonia with various metabolic inhibitors, we observedthat mitomycin-C enhanced markedly the mutagenic effect of r-rays. The
enhancing effect was more pronounced when the chemical had been administered 12 hours after irradiation.
Concerning the effect of mitomycin-C upon biological systems, numerous
papers have been published by many workers and at least three different
mechanisms have been assumed: i.e., 1) inhibition of DNA synthesis, 2)degradation of DNA and 3) cross-linking of DNA.
Since a strong mutagenic effect upon silkworm spermatozoa has been
elucidated for some alkylating agents which cross-link DNA (Nakao, 1961),
experiments have been carried out to examine if mitomycin-C is mutagenically effective when given to mature spermatozoa.
The chemical was injected as a saline solution into wild type male pupaeof C108 strain two or three days before emergence. The administereddose of the chemical were 0.02~0.05 mg per capita. Mutation frequencies
were estimated by specific loci method as usual. The results are shownin Table 1.
As may be clearly seen from the table, injection of mitomycin-C resulted
in a high incidence of mosaic mutations as well as whole type recessive
mutations. The most striking was an extraordinarily high incidence ofmosaics. Their frequency was from 10 to 20 times as high as that of
the whole type.
Assuming that the incidence of mosaics was due to a single helix (ofRESEARCHES CARRIED OUT IN 1966
Table 1.

Injected dose
per capita
0.05mg0.02mg95

Results of injection of mitomycin-C into male pupae (654) of
the silkworm
pe re 'i' X treated + + 0
Observed number of eggs*
Unferti- Early
lized dying
3,174
(5.8)
3,517
(4.9)

Sur·
vivingTotal

2,225 49,005 54,404(4.1) (90.1)
2,401 65,788 71,706(3.4) (91.7)

Mutants detected**
pe

pe, +
mos

46
471
(94)
(951)
135
823
(205) (1,251)

re

re, +
mos

661
30
(61) (1,349)
959
71(108) (1,458)

Numerals in parentheses are (*) percentage of eggs observed and (**) mutation
frequency per surviving eggs represented in the order of 10- 5 •DNA) type mutation and that of whole type mutations to a simultaneous
occurrence of two single helix type mutations at the same locus, theprobability of the incidence of whole type mutations could have been
expected to be the square of the probability of the mosaics. The observed
frequency for the whole type was about one order higher than such expectation. This suggests that whole type mutants were also produced
by a single mutational event provoked on a double helix of DNA.
Anyhow, the above results indicate that mitomycin-C can act on oneof the double helices of spermatozoa DNA, where DNA synthesis is no
more in progress.
Mutagenicity of a Nitrofuran Derivative Applied to
Silkworm Germ Cells
Yataro T AZIMA and Yosoji FUKASE

It has been reported by Endo et al. (1963, 1966) that a nitrofuran derivative, 3-amino-6[2(5-nitro-2 furyl) vinyl]-l, 2, 4,-triazin hydrochloride,
NFT, exhibits in several respects a similar action to that of mitomycin-C.
According to their report, the chemical is a potent phage inducer inEscherichia coli K12 (i.) cells and inhibits DNA synthesis without affecting
RNA and protein synthesis in non-lysogenic bacteria.
Since mitomycin-C has been proved to have a strong mutagenicity inthe silkworm, we thought that it would be interesting to examine themutagenic effect of NFT in comparison with that of mitomycin-C.
NFT used in this experiment was kindly supplied by Prof. H. Endo,
Cancer Institute, Kyushu University. Two series of experiments havebeen carried out. Materials and methods used were almost the same as
those reported in the foregoing article if not otherwise specified.

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

1) Feeding experiment with third instar larvae
NFT was administered to the third instar larvae by painting an aqueous
solution of the chemical on mulberry leaves. The administered doses
are given in the foot notes of Table 1.
Table l.

Results of feeding NFT to the third instar larvae (664)

No. of eggs

pe re !f x treated ++ 0
Mutation frequency
No. of mutants detected(x 10- 5)

Treatment
observed

pe

pe, +
mas

re

re, +
mas

pe

pe, +
mas

re

re, +
mas

134,116
123,483
126,031

31
28
51

6
10
14

0
1
3

2
4
3

23
23
41

5
8
11

0
1
2

2
3
2

None
*NFT (L)**NFT (H)*NFT (L):**NFT (H):

10 mg of NFT was given to 1,000 larvae at one time.
50 mg of NFT was given to 1,000 larvae in five times.

It may be clearly seen from Table 1 that an increased dose of thechemical increases the mutation frequency both for whole and mosaic
mutants.
2) Injection experiment at post pupal stage
The chemical was injected as a saline solution to wild type male pupaeof C108 strain two or three days before emergence. Injections with known
mutagenic agents, nitromine (NM) and ethyl methane sulphonate (EMS),
were performed in parallel. The results are given in Table 2. In thisexperiment the mutagenic effect of NFT could be again observed. The
Table 2.

Results of injection of NFT into pupae (663)

No. of eggs

pe re !f x treated ++ 0
Mutation frequency
No. of mutants detected(x 10- 5)

Treatment

Control
NFT 0.01%" 0.1%
NM 0.025%
EMS 0.01%
0.1%

"

pe, +
mas

re

re, +
mas

pe

pe, +
mas

re

re, +
mos

12
15
11
25
50
55
59 1,065

2
1
4
4
4
3

2
5
5
9
43
602

28
8
15
56
66
9
22
51
44
40
57 1,029

1
1
3
4
3
3

1
5
4
8
34
582

observedpe149,908
98,540121,026
114,869
135,386
103,516

42
55
80
59

* Injected amount was 0.05 mL of physiological salt solution of the chemical at
the concentration specified.

RESEARCHES CARRIED OUT IN 1966

97

induced mutation frequencies were clearly enhanced for whole mutations,while those for mosaics were not significantly increased.
Those results are in sharp contrast to those obtained for NM, EMS aswell as mitomycin-C, which produced a high incidence of mosaics. NFT
seems to have a specific action by attacking both DNA helices at one
event.

Changes in the Mutation Response of Post-Meiotic Silkworm
Germ Cells to r-Rays with Progressing Spermiogenesis
Yataro TAZIMAThe double helix model of DNA suggests that there could be two typesof mutations concerning the site of occurrence, i.e. those occ*ring in
one of the double helices and those occurring in both helices. Identification of those two types may be possible if post-meiotic germ cells areirradiated. When mutants are obtained as mosaics, they may be attributed
to the single helix type. Whereas, mutants in whose whole body a recessive character is expressed may correspond to the double helix typemutation.
It has often been observed in the silkworm that irradiation of spermatozoa resulted in the production of a higher proportion of mosaics thanwhen early spermatids were irradiated.
In order to know if mutation response varies according to progressing
spermiogenesis, data obtained from different experiments have beenarranged in accordance with the development of the insects. Although
the extent of mutation response varies depending on the irradiated stage,
those data can be compared so far as the ratio of mosaics to whole mutants
is concerned.
In Fig. 1 calculated ratios of mosaics to whole mutants at each locus
are plotted against the progression of the developmental stages of theinsect. In the male, meiosis takes place most frequently around V-2(instar V day 2), and thereafter the germ cells enter into the spermatid
stage. When the larva attains maturity (Full G.) fully formed spermatozoa
appear in the testis and increase in number progressively thereafter un·
til the mid-pupal stage (M.P.).
As can be clearly seen from the figure, the incidence of mosaics inrelation to whole mutation is very low before meiosis, reaches to theunity around V-4.5 and then increases rapidly with the progress ofspermiogenesis.
The relation can be more intuitively understood from the curves marked
with rightward arrows. Those curves represent changes in induced fre-

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

I 10

7

7

10
rtl

I::

n:l

6

a 5
CI)

"0

..c:

:::

4

~

E
rtl
U

"@

,/

~/

,

3

5

,/

rtl

0

a 2

-

.....0,," re--------'-'c:::·· __--,,--- J

0

'';:

ro

Ct::

lJI-o

V-4.5 Full G.

Pupa

M.P.

Moth

Developmental stage
Fig. 1. Changes in the mutation response of silkworm spermatogenic cellswith the progress of development. Curves with rightward arrows are to
be read against the right ordinate and vice versa.

quencies per R for whole and mosaic mutants, in an average for two loci.
It can be inferred from those results that the site of mutation response
becomes reduced in size with the progression of spermiogenesis. Presumably this change might be correlated with a decreasing facility to repair
premutational damages.

Studies on Strain Differences in Radiosensitivity in the Silkworm
I. Screening of Sensitive and Resistant Strains to
Embryonic Radiation Killing
Akio MURAKAMI and Yataro TAZIMA

A comparison of radiation effects between strains of different radiosensitivity may help to understand better the mechanism of radiation
effects on living organisms. From this view point, experiments have been
carried out in order to select sensitive and resistant strains to embryonicradiation killing. A worldwide collection of silkworm strains has been
maintained for nearly 50 years at the Sericultural Experiment Station,
Tokyo. Among 250 strains, 30 representative strains were chosen andtheir radiosensitivity to embryonic killing was examined. X-irradiation

99

RESEARCHES CARRIED OUT IN 1966

was applied to over-wintered eggs on the 30th of March, 1966. Irradiation
conditions were: 173kVp, 25 rnA and 1.0 mm Al filter. Four different doses,
800, 1,200, 1,600 and 2,000 R, were given at dose-rate of 250 R/min. After
hatching of the surviving embryos, hatchability was calculated and LD 50
was estimated from dose-response curves. The results are shown in
Table 1. The average LD 50 for 30 strains was 910 R. It may be clearly
Table l.

Strain

LD 50 (R)AojukuAkajuku
KojikiTadamisanMatamukasi
Chusu
K6rai-sanmin
Ryukyu-tasanken
KansenKin-6
SeikyoSekko
Daient6
Hik6
Sy6k6

1,460
1,320
230
940
1,320
890
650
1,110
1,580
800
1,160
800
800
1,340
1,080

Race

Japanese

KoreanRyukyuanChineseDifferences in radiosensitivity among 30 strains
Race

Strain

LD 50 (R)fDai-anky6
Dai-k6jy6, Ben-y6
IS an t o-sanmm
I

860
1,050
800
800
980
930
670
1,100
140
520
610
760
1,020
1,280
1,500

I

ChineseA•\ Sisen-sanmin
China-southern f Daiz6
Asian\ Ringetu

IToruko-6ken
EuropeanAscoliBiohneMaruke-6ken
Maiera-zebra
Rosa
Var\ Kokugaseen from the table that there were striking differences among strains
with regard to radiosensitivity. The LD 50 values for the most radioresistant three strains were approximately 1,500 R. In contrast, twostrains were extremely sensitive, i.e., LD 50 was 140 R for Ascoli and 240 R
for Kojiki. These differences in radiosensitivity might have been, in part,due to different dosages of the relevant genes accumulated in those strains.
Table 2.

LD 50 's obtained for 16 combinations of diallelic crosses
FemaleMale
KansenAojukuKojiki
Ascoli(1,580)(1,460)
(230)
(140)

KansenAojukuKojiki

Ascoli

1,580
1,750
1,750
1,750

1,700
1,540
1,460
1,625

1,480
1,060
230
1,600

1,750
1,660
1,600
140

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

In order to investigate the mechanism, the radiosensitivity of F 1 eggs
was examined in all combinations of diallelic crosses between some resistantand sensitive strains. Those eggs were irradiated one day after Hel
treatment for artificial hatching. The results obtained are given in Table
2. LDso values thus obtained for self-bred batches were almost the sameas that observed for over-wintered eggs. In cross-bred batches, however,LDso's were roughly the same as those observed in inbred batches of theresistant strains. Those results suggest that the radiosensitivity of silkworm embryos is controlled by two major genes at least, and that theeffect of maternal or cytoplasmic factors is relatively small.
Studies on Strain Differences in Radiosensitivity in the Silkworm
II. Relation between Sensitivity to Embryonic Killing and Mutability
Akio MURAKAMI and Yataro T AZIMAAs reported in the foregoing article, some strains sensitive or resistantto radiation were discovered with regard to embyronic killing. Using
those strains, an investigation has been carried out to ascertain if sensitivity to mutation runs parallel to sensitivity to killing. The strains used
were: (resistant) Kansen, Aojuku and Hiko, (moderate) Sekko and (sensitive)
Ascoli and Kojiki. The mutability was measured by the specific locus
method, using egg colour markers. Irradiation conditions of X-rays were:
173kVp, 25mA and 1.0 mm Al filter throughout this study. Doses delivered
were 250 R per minute.
Irradiation of gonial cells: Both spermatogonia and oogonia were irradiated in the larvae after hatching. The dose applied was 1,000 R only.
The results are given in Table 1 a and b, separately for spermatogonia
and oogonia, respectively.
a.

Table 1. Mutation rates for the gonia I cells in five representative strains
Spermatogonia
Mutation-rate
StrainWhole
KansenAojukuSekko
Ascoli
Kojikire locus

pe locus

xlO
37.4

58.5
471.3

232.1
135.4

Mosaics
8

x10 8
1.6

5.8
3.1
2.2
15.6

Whole

Mosaics

x10 8
6.2
13.0

0.0

144.9
49.1
41.6

0.0
10.4

xlO 8
1.3

4.1

RESEARCHES CARRIED OUT IN 1966b.

101

Oogonia
Mutation-rate

pe locus

StrainWhole

X 10- 8KansenAojukuSekko
Ascoli
Kojikire locus
Mosaics

Whole

X 10- 8

19.0
173.5

9.0
12.7

35.0

7.0

X 10- 8

2.4
1.4
122.5
109.8
19.6

Mosaics

x10- 8
0.0
0.0
7.8
3.9
1.4

It can be seen from the table that the mutation rates are higher for
spermatogonia than for oogonia in all strains studied. It is of interest tonote that the mutation rate of a Chinese strain, Sekko, was extraordinarily
high, although as to embryonic killing it belonged to the moderate group.
Except for Sekko, it may be roughly inferred that sensitive strains toembryonic killing are also sensitive to mutation in both sexes.
Irradiation of oiJ"cytes: The sensitivity of oocytes and mature spermatozoa
was tested after irradiation of newly emerged adults. The results formature oocytes are given in Table 2. In this experiment X-ray doses
Table 2.

Mutation rates for the mature oJcytes in five representative strains
Mutation-rate

Strainpe locus
Whole
X 10- 8KansenAojukuHiko
Ascoli
Kojiki

470.6
386.0
634.8
1,012.4
1,111.7

re locus
Mosaics

Whole

Mosaics

x10- 8
28.4
30.2
35.5
130.3
72.3

x10- 8
500.6
372.3
766.1
447.9
732.0

27.0
14.9
23.1
77.7
55.6

X 10- 8given were 1,000 Rand 2,000 R. The mutation-rates are shown in an
average for the two doses. It appears that sensitive strains for embryonickilling, Ascoli and Kojiki, were also sensitive with respect to mutation.
Irradiation of mature spermatozoa: The results of the experiment withmature spermatozoa are presented in Table 3. In this experiment X-ray
doses given were 3,000 Rand 6,000 R. The mutation rate is expressedas an average of the two doses. No distinct correlation was found be-102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table 3.

Mutation rates for the mature sperms in five representative strains
Mutation·rate

StrainWhole
KansenAojukuHiko
Ascoli
Kojikire locus

pe locus
Mosaicsx 10 B
16.7
16.1
22.8
26.9
19.9

x10 B
37.6
26.3
34.3
24.2
38.8

Whole
x10 8
8.9
8.7
4.7
8.9
5.8

Mosaics
x10 8
12.4
14.2
18.7
22.8
18.2

tween radiosensitivity to embryonic killing and mutability. This finding
is in sharp contrast to those described above for the three types of germ
cells. The discrepancy might be due to either to the lack of recovery ofradiation damage or to a different repair system in mature spermatozoa.

Relation between Sensitivity to Killing and Mutation Observed
during a Mitotic Cycle of Silkworm Cleavage Nuclei
Akio MURAKAMI

Cyclic change in radiosensitivity to killing has been revealed for earlycleavage nuclei of the silkworm (Murakami, 1966). The most sensitive
phase within a cell cycle was found to be the metaphase/early telophase,whereas the least sensitive was the interphase. This finding suggested
that dividing chromosomes are sensitive.
The purpose of this study was to investigate the change in mutation
response in relation to the progress of the mitotic cycle during earlycleavage. Materials used were eggs laid by ++/++ females mated with
pe re/pe re males. These eggs were collected every 10 minutes and allowed
to develop for a definite duration until they were irradiated. From 170
minute to 230 minute old eggs after oviposition were irradiated at 10 minuteintervals. Radiation dose of 1,000 R was delivered in one minute. Mutation frequencies were estimated by specific locus method using the pe andre genes. The lethality was also examined at the same time. The mitotic
phase of the irradiated cells was determined cytologically. The results
are shown in Fig. 1. Mutations most frequently found in this experimentwere fractionals. The highest peak for embryonic killing was observed
at 190 minute age. In contrast, the highest mutation frequency wasobserved at 200 minute age. Cytological observation indicated that the

103

RESEARCHES CARRIED OUT IN 1966

('·;0

.";>

~c

E

I

<10·
3000

J

C
c
2000

"'"

0-

!;

"

C

0

".:3

"",..,.,

c

.0

E

'"

1000

30

170

180

190 200 210 220
Time after oviposition

~

"

:>l

(min)

Fig. 1. Changes in sensitivity to killing and mutation during amitotic cycle as expressed after oviposition as time function.

phase of maximum embryonic killing corresponded to metaphase/early
telophase (or M +Gl) and the highest mutation response appeared at late
telophase/early interphase (or Gl + early S). The discrepancy between thepeak stages for cell killing and for mutation response suggests that themechanisms involved are different. Presumably we may assume that thetarget or targets responsible for cell killing are chromosomes and thosefor mutation response are DNA strands.

The Effect of 5-Bromodeoxyuridine (BUDR) on the Frequency of
14 MeV Fast Neutron Induced Mutations in
the Gonia} Cells of the Silkworm
Akio MURAKAMI

It was reported in our previous paper (Murakami and Tazima, 1964)
that a post-irradiation treatment of silkworm spermatogonia with BUDR
or 5-Bromouracil (BU) increases the frequency of specific locus mutations
by about two or three times at the maximum (Murakami and Tazima,1964). This enhancing effect was assumed to be caused by an inhibition
with such chemicals of the recovery process of pre-mutational lesions.
It was thought, therefore, to be of interest to study whether the radiation lesions produced by higher LET (Linear Energy Transfer) radiations
could be modified by a similar treatment with those chemicals. Hence,the effect of BUDR treatment of 14 MeV fast neutron induced mutations
was studied in gonial cells of the silkworm.
Materials and method used were the same as described in the previous
paper (Murakami and Tazima, 1964). BUDR was administered to newly104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

ha tched larvae of a wild type by feeding them on leaves supplemented
with the chemical (20 mg per 1,500 heads) immediately after 14 MeV
neutron irradiation.
The results are given in Table 1 a and b, for males and females, separately.
Table l.
a.

Effect of BUDR on 14 MeV fast neutron-induced mutation
frequency in silkworm gonia

Males

re locus

pe locus
Treatment

Total No.
observed

Whole

Mosaics

Whole

Mosaics

Mut. Mut. Mut. Mut. Mut. Mut. Mut. Mut.
dltd. freq. dltd. freq. dltd. freq. dltd. freq.

48

xl0 5
32.9
33.1

3
5

xl0 5
1.7
3.4

4
4

xl0 5
2.3
2.8

0
1

xl0 5
0.0
0.7

172

78.4

6

2.7

33

15.0

2

0.9

133

89.9

8

5.4

40

27.0

2

1.4

212,420
177,34621
8xl0- 5
9.9
4.5

8
8

xl0- 5xl0- 5
6.6
3.8 14
1.7
4.5
3

0
1

xl0- 50.0
0.6

244,530

68

27.8

9

3.7

28

11.5

2

0.8

147,212

17

11.6

14

9.5

22

14.9

2

1.7

Control
176,245
BUDR*
145,184
14 MeV neutrons
219,5211,000 rad14 MeV neutrons
1,000 rad+BUDR* 147,976b.

58

Females
Control
BUDR*
14 MeV neutrons
1,000 rad14 MeV neutrons
1,000 rad+BUDR** BUDR:

20 mg per 1, 500 heads.

The mutagenicity of BUDR treatment alone is not clear. However,when combined with 14 MeV neutrons, mutation frequencies were slightly
enhanced in the experiment with spermatogonia. Whereas, no enhancing
effect was observed in the experiment with oogonia. In the former theenhancing effect was higher at the re locus than at the pe locus. As
to the enhancing effect of BUDR, neutron induced mutations responded
to a lesser extent than X-ray induced mutations. This might have been
due to the fact that neutron induced pre-mutational damages are lessresponsive to repair than those produced by low LET radiation.

RESEARCHES CARRIED OUT IN 1966

105

Relative Biological Effectiveness of 14.1 MeV Neutrons in the
Induction of Dominant Lethal Mutations in the Mouse
Kiyosi TUTIKAWA

The effectiveness of fast neutrons was compared with that of acute
X-rays in the induction of dominant lethal mutations following irradiationof spermatozoa.
The irradiation of 14.1 MeV neutrons from T(d, n)He reaction was
carried out at the Research Institute of Nuclear Medicine and Biology,
Hiroshima University, Hiroshima. Two to three months old male miceof CBA strain were irradiated with doses of 242.5 and 485 rad neutrons,
respectively. Other males of the same strain were given whether dosesof 222 or 445 rad X-rays (dose rate 47.5 r/min.), for comparison of dominantlethality with that for neutrons.
One day after the irradiation each male was outcrossed to two adultfemales of the ICR stock. Matings were detected by daily examinationof all females for vaginal plugs. Those females becoming pregnant were
autopsied on or about 14th day of gestation, when number of corpora
lutea, and of live and dead implants were recorded.
The dominant lethality was estimated from the live embryo/corpus
luteum ratio, the induced dominant lethality being given by l-(ratio in
irradiated series/ratio in control series). The comparisons of fast neutrons
and X-rays results to be considered here was based on the early matings
during first one week after irradiation.

.....

0.8

0

0.6

oS

..

:;3

.:l

., .-

::I

e

iii
~

0.4

.!l

j

0.2

~

,,

0

, .-

,,

,,

,,

100

,,

,

.-

~

/"iI'"
200

300

/I'/

,

400

500

Dose in rad

Fig. 1. Estimated dominant lethal mutation rates per
gamete induced by 14.1 MeV neutrons and X-rays inmouse spermatozoa.
• : Neutrons
8: X-rays

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Using-loge (l-P) as an estimation of the dominant lethal mutation
rates per gamete induced by two kinds of radiation, it was shown thatthe mutation frequency from neutrons is higher than that from X-rays,
and the dose-frequency curves are in linear relationship (Fig. 1). If weequate the mutation rate of 445 rad X-rays with that of 242.5 rad neutrons
around the 50 per cent level of dominant lethality, results suggest that14.1 MeV neutrons are about 1.8 times as effective as acute X-rays forthe induction of dominant lethal mutations in mouse spermatozoa.

RESEARCHES CARRIED OUT IN 1966

VIII.

107

RADIATION GENETICS IN PLANTS

RBE of Radiations in E-I Hole of Kyoto University
Reactor (KUR)
Seiji MATSUMURA, Etsuo AMANa and Masaru HAYASHITo obtain basic data for future use of Kyoto University Reactor (KUR),
dry seeds of rice plant and Einkorn wheat were irradiated in E-l exposure
hole of KUR under nitrogen gas or air atmosphere. As preliminary experiments, in June, 1966, hulled seeds of Oriza sativa were sealed inpolyethylene vials after being flushed with nitrogen gas so that the desired atmospheres were obtained, and, three days later, they were irradiated in E-l hole for 180 minutes at 1,000 kW. Radiation dosage of eachsample was adjusted by varying the distance from the core side end, andthermal neutron doses were calculated from activation of gold foils andatomic composition of the material. r-ray from 6,000 Ci mcs source of
National Institute of Genetics was used as standard radiation.
Results of seedling height measurement of the reactor radiations irradiated seeds showed that 50 per cent seedling height reducing doses
were obtained at 21 cm for nitrogen gas and 22 cm for air atmosphere,
from core side end of the E-l sample holding boat, revealing distinct
protective effect of nitrogen gas. Existence of such oxigen effect strongly
support the heavy contamination of r-rays measured by physical dosimetry(d. KURRI-TR-16).
In October, 1966, seeds of Triticum monococcum were prepared as samples sealed in specially designed two celled air tight acryl resin containers.
Nitrogen atmospheres were achieved by several times flushings of nitrogengas after reducing gas pressure by a vacuum pump each time, and sealedin nitrogen gas stream. Exposure to reactor radiation was done threedays later in E-l hole of KUR. As T. monococcum seeds are more radiation sensitive than O. sativa seeds, 90 minutes of exposure at the same
1,000 kW gave 50 per cent seedling height reducing dose at about 24 cm
for air preparation and 22 cm for nitrogen atmosphere, from the core endof the boat. Seedling height measurement on seventh day after sowing
again revealed oxigen effect as shown in Figure 1 in which E-l radiationdose was shown by thermal neutron component. Calculations of RBE of
the reactor thermal neutrons free of contaminating r-rays were tried at
the 50 per cent seedling height reducing dose, assuming that the contaminating r-rays in E-l radiations might have the same RBE as that of 137CS
r-ray, and that the difference of dosages of the two r-rays might be
attributed to thermal neutrons.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

100

\
10'

\
10 3
Radiation dose (rad)

10"

10'

Fig. 1. Effect on seedling height of T. monococcum of KUR E-1
thermal neutrons and 137Cs r ray.
Under air atmosphere, the difference in r-rays was 2,000 rads, effect ofwhich was comparable to 131 rads of thermal neutrons, giving RBE value
15.3. Since efficiency of the r-rays might be reduced under nitrogen gas
atmosphere the difference in r-rays was 5,000 rads and equivalent effectmight be brought about by 181 rads of thermal neutrons, giving RBE value
28.3. Although fluctuation of r-ray contamination and difficulty of its
dosimetry are the problems still to be worked out about the reactor radiations, following calculations were tried further. With the same assumption
about efficiency of r-rays, it might be considered that 131 rads of thermal
neutrons reduced final 13 per cent of the seedling height to reach 50 per
cent height of control in case of air, and 181 rads reduced final 17 percent in case of nitrogen atmosphere, to get to 50 per cent height, suggesting
the same efficiency of thermal neutrons in seedling height reduction regardless of the atmosphere.
As for RBE values of overall E-1 radiations to 187Cs r-ray, 1.12 for
exposure in air and 1.35 in nitrogen atmosphere were obtained.
Since contribution of thermal neutrons to the effect on the botanicalmaterials used was only 13.6 per cent (air) or 26.7 per cent (nitrogen),RBE being considered, the values estimated may subject to be corrected
when advanced dosimetry for contaminating r-rays can supply further
information.

RESEARCHES CARRIED OUT IN 1965

109

Comparison of Mutagenic Efficiency between EMS and r-Rays
Seiji MATSUMURA and Taro FUJII

Seeds of a normal strain and heterozygotic F, seeds from a cross between normal and chlorina mutant of Triticum monococcum were soaked
in water for two hours and then treated with 0.1, 0.3 and 0.5% EMS
sulutions for 22 hours. For comparison, the seeds soaked for 24 hours
were subjected to 0.5, 1.0 and 1.5 kR of r-rays. Moreover, EMS andr-ray treatments were combined to examine the synergestic effect of both
treatments.
In the normal strain, several types of stripes were observed in many
plants during the growth period of EMS treated lots, while they appearedvery seldom in r-ray lots. About 20 per cent survived plants had chlorinalike yellowish green stripes in 0.3% lot, and a similar frequency was also
observed in EMS 0.3% +r-ray 1.0 kR lot, while such type of stripes wasnever seen in the three r-ray lots, as shown in Table 1.
Table l.
StrainNormalF,

Appearance of chlorina-like streaks in the treated generation

Treatment

No. of Total
seeds No. ofsown spikes

Chlorina or chlorina-like streaks
No. ofplantsNo. ofspikes(%)

Control
EMS 0.1%" 0.3%
r-ray 0.5 kR" 1.0 kR
1.5 kREMS 0.3% +r·ray 1 kR

300
200
200
200
200
200
200

7183
4185
7990
5887
5226
4270

0
2
23
0
0
0
18

3
42

(0.042)(1. 004)42(0.984)Control
EMS 0.1%" 0.3%
r-ray 0.5 kR" 1.0kRII
1.5 kREMS 0.3% +r·ray 1 kR

300
400
400
348
400
400
400

7447
4703
6965
7138
5987
1607

0
20
1
2
2
9

46
3
4
6
24

(0.978)(0.043)(0.056)(0.100)(1.493)

Seeds from some tillers with stripes and from normal green tillers of thesame individual were sown and segregation of chlorina seedlings was
examined. Albina or other kinds of chlorophyll mutants were segregatedamong these X 2 seedlings, but no chlorina-like segregants was observed.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

From this fact, we may say that no somatic mutations from dominant
green to recessive chlorina occurred in F 1 by EMS treatment.
In the F 1 strain, number of chlorina or chlorina-like stripes in EMS 0.3%
lot amounted to 0.98 per cent and the frequency was almost the same asin the normal strain. Rates of striping in r-ray lots increased with increase of dosage but they were much lower than those of EMS 0.3% lot.
The rate in the EMS 0.3% +r-ray 1.0 kR lot was 1.49 per cent which ishigher than the sum from EMS 0.3% and r-ray 1.0 kR lots.
Segregation of several kind of chlorophyll mutants were observed in
the X2 generation of the normal strain. Chlorophyll mutation rate wassimilar in EMS 0.1% and r-ray 1.0 kR lot, but the in EMS 0.3% lot wasvery high. These results show that the mutagenicity of EMS is very
high in high dosages when the mutability is compared on the basis of
their survival rates.

Comparison of the Killing Effect of r-Rays
and Thermal Neutrons
Taro FUJII

Dry and 48 hour steeped seeds were used and exposed to thermal neutrons were done by the pneumatic tube which was provided in the reactorof Kyoto University; the length of irradiation time were 7 sec in minimumand 180 sec in maximum at the out put of 1 MW. The 7 sec irradiationof dry seeds showed merely 19 per cent of survival rate, and no survival
plants were observed in other lots.
From the above experience, out put of reactor was diminished to 50 kW
in the second experiment and similar observation was repeated; the detailsof irradiation and survival rates were given in Table 1 with the resultsof r-ray irradiations. r-ray irradiations for dry seeds had almost no effect,
but slight decrease of survival rate was observed in 100 kR lot. Decreaseof survival rate was marked in wet seeds lots. In the neutron lots, killing
effects were severe and no survival plant was observed by 180 sec irradiation of dry seeds while contaminated r-ray was 45 kR. Higher killing
effects in the wet seeds were also seen in the neutron lots, but it might
be the results of contaminated r-rays because the environmental modificationof radiation effect is very small in high LET radiations, while it is rather
large with sparsely ionizing radiations. These results show that the killing
effect of thermal neutrons was very high. RBE of thermal neutrons insomatic mutations will be examined in the next step of experiment andRBE versus LET relations should be determined.

111

RESEARCHES CARRIED OUT IN 1966
Table 1.

Dose
(sec)

0
5
10
20
30
40
60
90
120
180
300

Killing effects of thermal neutrons andT-rays for dry and wet seeds

Total neutron f1ux*(Nth/cm 2)

Contaminated T-ray(kR)**

Survival rate (%)
Dry

Wet

93.9
1.39x10 12
1/
2.44
1/
4.58
1/
7.48
1/
9.48
!I
14.5
1/
22.4
1/
26.6
1/
51.5
II
71.9

1.25
2.5
5.0
7.5
10.0
15.0
22.5
30.0
45.0
75.0

97.1
69.5
88.9
71.9
70.6
59.0
17.2
0.0
0.0

80.6
68.0
63.5
37.8
43.3
0.0
0.6
0.0
0.0
0.0

91.6
90.8
92.8
93.3
82.4

92.7
75.6
21.7
0.0
0.0

Dose of T-rays from CS'37 (kR)

10
30
50
70
100
* Estimated from the activation of Au-foil.
** Calculated from the measurement of T-rays at 1 MW.

On the Determination of Absorbed Dose in Heavy
Ionizing Particles
Tarc~ FUJIIDry F 2 seeds obtained from F 1 hybrids between a hairless mutant andthe wild strain of Arabidopsis were subjected to r-rays from a CS 137 source
and to He', 0 2 and Ar'o ions from the Hilac. From the experiments
we could recognize a severe killing effect of heavy ionizing particles, andalso dramatically high mutation rates and marked growth inhibition. RBE
for somatic mutation at 0.5 per cent frequency was roughly estimated as
10,35 and 5 for He-, C- and Ar-ions, respectivelyll. The range of heavy
ionizing particles is very short. The average seed volume was calculatedroughly from the weight and measurements of length and width of the11

Fujii, T., M. Ikenaga, and J. T. Lyman, 1966.

Rad. Bot. 6: 297-306.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

seeds, to be 20.5 x 10- 3 mm 3 • From this calculation, it was concluded that
Ar-ions must be stopped within the seeds but He- and C-ions certainly
could penetrate and pass through seed tissue. Moreover the energy
transfer varies according to the position of the range or tissue which
may be seen from the Bragg Curve 21 •Further experiment about killing efficiency of heavy ionizing particles
was done according to above questions. Dry seeds were irradiated with
C-ions in five different lots. Each lot received the same number of parTable 1.

Relation cetween the survival rates ard
the thickness of acsorbers--_._----

Acsorber(mg/cm 2 )Range(micron)

-

~

.

~

_

.

_

~

Survival rate(%)
- - - - - - - _ .. -Control
Lot 1
2
3
4
5

71.6
0
10.2
19.7
29.9
39.3

525
400
315
215
125

66.0
64.8
57.6
46.7
46.8

ticles per cm 2 , but the energies of the ions were varied for the different
lots by different thickness of absorbers and lot 1 received a surface doseof 400 rads (Table 1). Survival rates decreased with the increase of absorber's thickness. The results suggest that the irradiation of C-ions withmaximum stopping power will show more severe killing effect, because
the maximum of the stopping power is 6 X 10 3 Mev/g/cm 2 at the range of
45 mg/cm 2 • Namely, RBE value of C-ions should be more larger than thatobtained in the previous experiment when the embryo was attacked by
the ions with maximum stopping power. Further studies are now under
examination.

Photoreactivation of an UV-Induced Mutation in Maize
Seiji MATSUMURA and Tomoo MABUCHI

A normal maize strain (Su) concerning the sugary endosperm gene ofa local variety and a recessive line (su) of Golden Cross Bantam wereused in this study. Table 1 summarizes the data obtained from the experiments with UV- or r-ray induced mutations at Su locus after visible
21 Brustad, T., P. Ariotti and J. T. Lyman, 1950.
9454.

Lawrence Rad. Lab. Rep. UCRL-113

RESEARCHES CARRIED OUT IN 1966
Table 1.

Irradiation

Control
UV

Frequencies of mutations from Su to 8U in maize pollen
grains induced by UV and r-rays treated with
VL and dark after irradiation

Dose

No. of endosperm mutants
Whole
Obs.

Chimeras
Obs.

(%)

(%)

% of thewholemutations

0
0

VL
Dark

4164
4023

134
108

1(0.024)
1 (0.025)3(0.072)
1 (0.025)

25.0
50.0

2.7 X 10 3 ergs
5.4x 103 ergs

VL
VL
Dark
VL
Dark

3420
4556
2333
913
1557

148
85
41
43
45

6(0.17)18(0.39)22(0.94)
5 (0. 55)
25 (1.60)

15 (0 .44)
35 (0. 76)
23(0.98)
11 (1.20)
19 (1.22)

28.5
33.9
48.8
31.2
56.8

VL
Dark
VL
Dark

4122
5579
2134
4154

132
143
152
122

25(0.60)35(0.62)44(2.06)
122 (2.40)

11(0.27)13(0.23)
18 (0.84)
22 (0. 52)

69.4
72.9
70.9
81.9

/I

8.1x10 3 ergs
/I

r-raysPostNo. of No. ofirradiation seeds seeds
settreatment set
per ear

550R
/I

HOOR

light (VL) and dark post treatments. The percentage of seed set washigh after the two doses of r-rays, while it decreased with the increaseof UV-dose to about 50 per cent of the control lot at the highest dose.
Induced whole (endosperm wholly deficient for the dominant character)and chimeral mutants (endosperm consisting partially of su and Su phenotypes) were scored separately for VL and dark posttreatment. No marked differences in the frequency of whole and chimeral mutants between
VL and dark posttreatments were observed. As to the frequency of thetwo types, whole mutation was more frequent than the chimeral one.
The frequency of chimeras fits roughly a linear relation with increasing
doses, while it is rather exponential than linear for whole mutation.
It is noted that the frequency of mutations induced by r-irradiation
was not affected by subsequent VL treatment either for whole or chimeralmutants. On the other hand, the frequency of whole and chimeral mutations from UV-irradiation showed almost linear relation. The frequencyof whole mutation was considerably decreased to about one third by exposure to visible light, while there was no marked difference in the frequency of chimeras between VL and dark posttreatment.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Photoreactivation of UV-Induced Damage in Maize Pollen
Etsuo AMANa and Tomoo MABUCHIAs the positive proofs for existence of photoreactivation of ultraviolet(UV) induced mutation in maize pollens had been obtained (d. Ann. Rep.
No. 16: 112-114), further experiments to study the type of repairable
damages were tried using linked endosperm marker genes on chromosome
9, C, sh, bz and wx which located on short arm of chromosome 9 in thisorder being C the most distal.
The experimental procedures were basically the same as reported before (Ann. Rep. ibid.). Pollens of a multiple dominant stock, CI Sh Bz
Wx, were irradiated in a single layer in Petri dish with UV light from a
Toshiba germicidal lamp at a dose rate of 136 ergs/mm 2 /sec for 30, 60 or
90 seconds. Immediately after the UV treatments, half of the pollens
were exposed to visible light from two 400 W high pressure mercury fluorescent lamps (6,400 lux) for 30 minutes, and the other half were kept in
dark. They were pollinated in dark room under a natrium lamp illumination to a corresponding multiple recessive tester stock. Potted tester
plants were transferred to field after wrapping the pollinated silk and ear
with aluminum foil to shield from outside light. The multiple dominant. : Dark, entire kernel
0: Light, entire kernel
t: Dark, fractional kernel
D: Light, fractional kerneli, .~.~;
!~~/
ly

o~

•

o

30

,
60

90

UV irradiation time (sec)Fig, 1. Effect of post-irradiation exposure to visible lighton UV induced endosperm mutation in maize.

RESEARCHES CARRIED OUT IN 1966

115

stock used as male parent carried also a dominant yellow endosperm
gene Y on chromosome 6 besides the aforementioned genes, and this Y
was useful to confirm fertilization by the treated pollen to y (white endosperm) carrying tester, in case of loss of all the four chromosome 9 marker
phenotypes.
Results of experiment are shown in Figure 1, in which B-F-B typemosaic kernels were included in either whole or fractional (more than 1/8
of surface area of the kernel) kernel mutation, according to the area ofmosaics. In this experiment, photoreactivations of UV induced damages
were observed as lowered mutation frequencies in the groups which weretreated with visible light after UV irradiations compared to the groups
without the post exposure to visible light.
Contrary to the previously reported results on su locus, in which wholekernel type showed more photoreactivation, present results showed more
photoreactivation in fractional or sectorial loss .of the dominant phenotypes
than in whole kernel type. This discrepancy might be due to the difference of the stocks used, commercial variety vs. genetical pure strain, or
the systems used, single specific locus or multiple loci, but, for a conclusive explanation, this should be studied further.
As for the type of damages which could be repaired by the post exposure
to visible light, simultaneous loss of a few neighboring marker genes and
B-F-B type mosaics were reduced by the post exposure. This strongly
suggests that some of the UV induced damages which would result inchromosome breakage might be repaired by post exposure to visible light.
Since the number of mutated kernels at a single locus were not many
enough, conclusion about repairability of the damages which would resultin so called point mutations should be reserved until further data will be
accumulated.
Distribution of initiating points of the B-F-B cycles seemed to be proportional to map distances.

On the Somatic Variations in Corn and Chrysanthemum
under Chronic r-Irradiation
Seiji MATSUMURA and Taro FUJII

Heterozygotic F 1 seeds from the cross between normal green (Yg 2) andyellow green mutant (yg2) of corn, and white flowering variety of chrysanthemum were planted in the r-greenhouse with four different lots on
July 18, and relation between the frequency of somatic mutation or variagation and dose rate effect were examined. Radiation intensity was 0.293.06 Rjhr, total irradiation time being 1741 hours; therefore, total dosage

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

in each lot was 430, 910, 2370 and 4500 R.
In corn, Yg2-7yg2 mutation was detected by yellow green stripes onleaves. Lengths of mutated stripes were different according to the developmental stage and occurrence of mutation; the number of the stripes
longer than 1 cm was counted in the present experiment. Dose versus
mutation frequency shows almost linear relations when the results shownin Table 1 are plotted on the figure.
Table 1. Somatic mutation frequency in corn underchronic r -irradiations
Dose rate(R/h)

Total dcsage

0.29
0.62
1.38
3.06

430
910
2470
4500

No. of examinedleaves(R)o

No. of stripes(frequency)

o

109
294
223
318
199

5(0.050)
9 (0.132)
30 (0.266)
28 (0.429)

Somatic variegation in chrysanthemum from white to yellow was observed in many flowers. When the variegation occurred in early stage,
yellow sectors appeared on several ray flowers and when it occurred inlate stage streak appeared on single ray flower. Dose versus variegation
curve also showed almost linear relation like that in corn. From theseresults, no dose rate effect was observed in the present experiment,
namely 0.29-3.06 R/hr, because when the recovery of radiation damage
occurred at low dose rate range, effect at higher dose rate range mustbe much higher than the present results.
Table 2.

Somatic variegations in chrysanthemum underchronic r -irradiations

Dose rate(R/h)

Total dosage

0.29
0.62
1.38
3.06

430
910
2470
4500

(R)

o

No. of examinedflowers

No. of streaks(frequency)

46
50
50
50
50

5 (0.11)18(0.36)48 (0.96)
50(1.00)
153(3.06)

No. of sectors(frequency)o4(0.08)3(0.06)4(0.08)29 (0.58)

117

RESEARCHES CARRIED OUT IN 1965

Endosperm Mutations Induced by UV in Corn
Taro FUJII

A wild strain (Bz) with respect to the bronz color of endosperm and arecessive homozygotic strain (bz) was used. Pollen grains of the domim.nt
line were irradiated by UV-rays, emitting primary 2537 A, at a dose rateof about 140 ergs/mm 2 /sec. Just after the irradiation, a half of the irradiated pollen grains was exposed to visible light of 6500 lux for 30
minutes, the pollen was crossed with recessive strain under the sun light(PR treatment). The other half of the irradiated pollen was held for 30
minutes in darkness and pollinations with recessive strain were done indark room with natrium lamp (Dark treatment). Plants of the darktreatment lot were kept under the dark condition for 24 hours and removed to the field. For comparison, r-ray irradiated pollen grains werepollinated after PR or dark treatments with the same manners as those
Table 1.

Treatment

Frequencies of Bz---.bz mutations induced by UV andr -rays with PR and dark treatment

Dose

Control
UV

15 sec30 sec60 sec9J sec

r-ray

300R
600R
900R

No. of mutation(frequency)

Postirradiation
treatment

No. of
seeds

Whole

PR
Dark

1246
5052

0
0

0
0

PR
Dark
PR
Dark
PR
Dark
PR
Dark

6673
2733

1 (0.02)4 (0.15)8 (0.12)17 (0. 62)

371
4289
389
4007
3035

2(0.54)2(0.05)5 (1. 29)2(0.05)20(0.66)

4 (1. 08)
5 (0.12)5 (1. 29)17(0.42)64(2.11)

PR
Dark
PR
Dark
PR
Dark

3211
4200
4823
4680
5017
3841

5(0.16)6(0.14)30 (0.62)
21 (0.45)
47 (0.94)
25 (0. 65)

2 (0.06)
8 (0.19)
16(0.33)7(0.15)23 (0.46)
28(0.72)

Fractionalof UV-irradiations. Results are summarized in the Table 1; the wholemutation means that the endosperm is wholly deficient for the dominant

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

character, and fractional mutation means that the endosperm has partiallyBz and bz characters. In the r-ray treated lots, mutation rate at higher
dosage lot was rather higher than the linear relation. Frequency of
fractional and whole mutations were almost same in each lot and thesefrequencies were not much different between PR and dark treatment. On
the other hand, dose versus mutation frequency was almost linear in UV
treated lots, and frequency of fractional mutation was higher than thatof the whole one in each lot in dark treatment. Frequency was markedly
decreased according to the PR treatment but rate of fractional mutationis still high. From the experiments, photorecovery of UV-damage could
be observed in Bz gene like that of Su gene by Matsumura and Mabuchi,
and further detailed experiments in relation to the sector size of fractional
mutation are now underway.

RESEARCHES CARRIED OUT IN 1966

IX.

119

MICROBIAL GENETICS

Genetic Map of HI Gene in Salmonella
Shigeru YAMAGUCHI and Tetsuo IINO

Flagellar protein, flagellin, of Salmonella has complex antigenicities; thatis, there are several serologically different antigenic determinants on one
flagellin molecule. The set of the specificities of this complex antigen
is determined as a whole by one of the allelic genes at the two loci,HI and H2, which are the structure genes for flagellin. Based on theassumption that the antigenic specificity-determing regions corresponding
to the respective antigenic determinants distribute linearly within H gene,it was attempted to assign the positions of these regions on the genetic
map of H gene, HI in the present experiment, by establishing the system
of intra-HI recombination. Three derivatives of 8J241, a stable phase-1
strain of Salmonella abortus-equi, given different alleles of HI-g.. bytransduction were used. Their designations and their compositions of theantigenic specificities are as follows; Tr6 gl, g2, gs, t, Tr16 gs, g.,j, and Tr17
gl, g2, g., gs, m.
The establishment of the system of intra-HI recombination was achievedby use of HI-linked fia- mutants. The principle of the system is that
occurrence of a fia+ transductant in transduction between a pair of fiamutants, whose mutation sites are closely linked to HI and are on the
opposite sides of it, should require a crossover at the region between the two
fia- sites which includes HI locus; therefore the closer the fia- sites are
to HI, the larger is the relative frequency of the occurrence of intra-HI
recombinants among the fia+ transductants.
About fifty fia- mutants from each of Tr6, Tr16, and Tr17 were isolated
by selection in which phage M8, a host-range mutant of bacteriophagechi, was used as selecting agent. In several mutants showing high frequency
of co-transduction of fia- with HI, the relative positions of their fia- sites
with respect to HI locus were examined by carrying out the reciprocaltransductions with a standard HI-linked fia- strain, a fia- mutant of 8J241 ,and then comparing the ratios of the number of fia+ transductants having
the donor-type flagellar antigen to that of fia+ transductants having therecipient-type between each pair of reciprocal crosses. Two classes of fiamutants which have their fia- sites on the opposite sides of HI locus were
found among the mutants originated from each of Tr6, Tr16, and Tr17.
Transductions were then carried out in all pair-wise combinations among
these mutants which have different alleles of HI-g.. each, and the fiasites are on the opposite sides of HI. Antigenic compositions of fia+

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17transductant clones were examined by use of anti-factor(s) serums. Amon.g
1266 fla+ transductant clones examined, 26 clones showed the antigenic
compositions of recombinant-types. From the consideration of the antigenic
compositions of these intra-HI recombinants it was shown that each of
antigenic specificity-determining sites corresponding to respective antigenic
determinants locates as a unit and as a whole they are in a linear arraywithin Hl. The arrangement established so far is as follows.
~(f)~---g2--g1--g4--t--(g3, g5, m)-'--

HI

--'

f is somewhere right of g4. Relative order of the regions in parentheses
has not yet been determined.

A Straight Flagellar Mutant in Salmonella
Tetsuo IINO and Michiko MITANIA mutant of Salmonella typhimurium was found to produce straightflagella in phase-2 (antigen-l.2) and normal flagella in phase-1 (antigen-i).
The straight flagella were observed by both light and electron microscopy
either with or without formalin fixation. Flagellar bundles of the mutant
bacteria prepared in 0.25 per cent methylcellulose (w/v) and examined bydark field microscopy were also found to be straight. It was shown byelectron microscopy that the component flagella of the straight flagellar
bundles were in most instances irregularly twisted around each other.
Heteromorphous cells which had both straight flagella and either normal
or mini-smaIl-amplitude flagella were seen at the frequency of about 10
per cent among the bacteria in phase-2.
The bacteria with straight flagella were non-motile but they weresensitive to bacteriophage-chi, which has been known to infect motile
bacteria of salmonella species.
In transduction with P22 phage from a normal flagellar strain to thephase-2 straight strain, transductional clones with normal flagella in bothphase-1 and phase-2 were obtained. The transductional clones showed
the antigen of the recipient in phase-1 and that of the donor in phase-2.
This indicates that the straight mutant originated by a mutation of thestructural gene of phase-2 flagellin.
In absorption-agglutination experiments with antisera prepared against
flagena of either normal-I.2 or straight-l.2, no antigenic difference between normal and straight flagella could be detected.

RESEARCHES CARRIED OUT IN 1966

121

Flagellin Biosynthesis in Salmonella Spheroplasts
Hideho SUZUKI and Tetsuo lINO

Salmonella typhimurium grown in a modified 3XD medium (Na2HP04
14.3 mM, KH 2P0 46 mM, NH 4CI 20 mM, MgS04 2 mM, CaCh 0.2 mM, glycerol
300 mM, Casamino acid 1.5 per cent, Gelatin 0.002 per cent) was washedwith 5 mM tris-buffer (adjusted to pH 8 at 30 with HCI) and suspended
in 50 mM tris-buffer containing 10 per cent sucrose at a concentration of
2~4 X 109 cells/m!.
The cell suspension was treated with lysozyme(100 pg/ml) for ten minutes and then EDTA (5 mM) was added. After
another ten minutes, not less than 99 per cent of cells were converted tospheroplasts as determined by microscopic observation and viable counts.
Then BSA (2 mg/m!) was added and the spheroplasts were centrifuged,
suspended in a small amount of saline containing 10 per cent sucrose anddispersed into minimal medium supplemented with 10 per cent sucroseand 2 mM amino acids to give a concentration of approximately 5 X 10 8cells/m!. At starting incubation, HC-alanine (0.2 pc/ml) or 3H-Ieucine wasadded. Flagellin fraction was prepared from cells which incorporated
radio active amino acid for thirty to sixty minutes, purified through reconstitution and analyzed with starch-gel electrophoresis. The results
indicated that the spheroplasts do synthesize flagellin to an extent comparable to the control cells. A microscopic observation of stained preparation, however, revealed that the spheroplasts still retained flagella. As
the intracellular pool is considered to be extremely small, if any, in
Salmonella, active flagellin synthesis of the spheroplasts could be interpreted as a reflex of intactness of flagella forming apparatus on thepersistent cell wall rather than an accumulation of flagellin in the cytoplasmof the spheroplasts.
0

Genetic Fine Structure of the mot Loci in
Salmonella typhimurium
Masatoshi ENOMOTO

Flagella of mot (motility) mutants which though flagellated are nonmotile
do not differ from those of the wild-type motile cell in antigenicity, number
per bacterium, shape under electron microscope and configuration observedby X-ray diffraction. Both the wild type and the mot mutants show flagellar
antigenic phase variation. These facts suggest that the paralysis is notdue to any defect of flagella themselves, but to a defect of the flagellumactivating mechanism within the bacteria. As a part of investigations
to clarify this mechanism, genetic analysis of mot loci was carried out.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Ninety-seven mot mutants derived from S. typhimurium strain TM2 weredivided into three complementation groups by abortive transduction tests:
motA with 64; motB with 30; and motC with 3. motA and motB were
found to constitute adjoining functional units because of the occurrenceof joint transduction of motA and motB, and of a deletion mutation covering the whole motB and a part of motA. motC was transduced jointlywith HI, a phase-l flagellin gene, at a fairly high frequencies and theorder of the three mutational sites within motC was determined: HI-C244C272-C279. The order of the representative mutational sites in motA andmotB was also determined as shown in Fig. 1 by deletion mapping andtwo-factor transduction tests.

...,...,.,.. ::B CISTRONA CISTRON

Fig. 1. Genetic map of motA and motB. The results of three seriesof transduction tests are arranged as follows: crosses between A -246
and motA mutants (top, right); between A-246 and motB mutants(top, left); and between A-246 and weak-complementing mutants(bottom). The scale iildicates recombination frequencies (precent).
The upper and lower maps are comparable with each other except
in respect of close mutational sites. A-238, -277, and -282 of theupper map, and A-294 and -297 of the lower map are deletions.

Mapping of Three mot Loci in Salmonella by Linkage Analysis
Masatoshi ENOMOTO

The chromosomal location of motA and motB and the position of motC
relative to HI were investigated. In order to analyse the linkage relationship between mot loci and other genes, systems of sexual recombination were adopted. Rfr mutant SWl39l (met- Hib H2enx) derived from
Salmonella abony strain SW803 and nonmotile F- mutants, SJ694 (leuhis- ser- motB- Hib H2enx), SJ703 (leu- his- ser- motB- Hli H2enx) ,SHOO (leu- motA- Hli H2enx) , and SJ608 (motC- Hli H21.2) , were used.
Selection of recombinants was made for the donor mot+ and for the recipient met+. In some crosses HI and H2 were added to the selection
markers. The motile recombinants thus obtained were examined for theirunselected auxotrophic markers. The order of the mot and other loci

RESEARCHES CARRIED OUT IN 1966123

involved in this work was inferred to be met-ser-H2-his-motC-HI-motBmotA-leu.

Difference in Frequencies of Cotransduction of motewith HI Gene in Salmonella
Masatoshi ENOMOTO and Shigeru YAMAGUCHIIn P22 mediated transduction from a motile wild-type strain to nonmotile
mutants of Salmonella, motC (motility) and some fla (flagellation) genes aretransduced jointly with HI (phase-1 flagellin) gene. When motile donor
strains having HI which was introduced from a certain Salmonella species
are used in transduction to a nonmotile mutant serotypically different fromthe donor, frequencies of cotransduction (percent fraction of motile transductant with donor HI allele) are different from each other among the
combinations, though the donors are phenotypically indistinguishable. In
order to elucidate this phenomenon, the following experiment was carriedout. Salmonella dublin strain SJll (HIgp motC+ flaK+), mutants motC-244(HIi) and flaK-48 (latent HIi) derived from S. tYPhimurium strain TM2
were used. The order of the genes in this chromosomal region is flaKHI-motC. By means of transduction from SJll to motC-244 and flaK-48,
motile transductants motC+ HIgp and flaK+ HIgp were isolated. The
former transductants received at least the chromosomal region between HI
and motC-244 from SJ11 and the latter received the region between flaKand HI. Using these transductants as donors and motC-244 as the recipient,transduction tests were carried out and the frequencies of cotransduction
were examined. With donors obtained from motC-244 the frequencies ofcotransduction were 32 to 44 per cent and with those obtained from flaK48 they were 4.5 to 16 per cent, whereas the frequency with SJll was
19 per cent. In the cotransduction of motC with HI, a crossover between
flaK and HI is required as well as a crossover at the right region of
motC (This Ann. Rept. 16: 91). It is inferred that the decreased frequency
of cotransduction with the donors obtained from flaK-48 is the result of
reduced crossover between flaK and HIgp, due to the heterogeneity of
the chromosomal region between S. tYPhimurium and S. dublin, and thatthe increased frequency with the donors obtained from motC-244 is theresult of relative increase in the crossover between flaK and HIgp, due
to the reduced crossover between HI and motC.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Infection of Bacteriophage-Chi to Serratia marcescens
Tetsuo lING

It was found that bacteriophage-chi, which has been known to attack
motile cells of both Salmonella and Escherichia species, is also able toinfect motile strains of Serratia marcescens. The Serratia strains used wereB-181-2 through B-181-10, B-182-1 through B-182-9, B-183-2 and B-183-3.
One of these 20 strains, B-181-6, was non-flagellate and consequently nonmotile. The remaining 19 strains were motile having peritrichous flagellawith normal curvature.
Sensitivities of those Serratia strains to chi and its host range mutant
M8, which is able to attack Salmonella possessing the g-complex antigen,were examined by the spotting method of E. W. Meynell. The bacteriophageused was propagated on Salmonella abortus-equi strain SJ241 which issensitive to both chi and M8. The non-motile strain, B-181-6, was completely resistant to both chi and M8, whereas the remaining 19 motile
strains were lysed by both chi and M8. The plaques formed with thelatter strains were clear and indistinguishable in size and shape fromthose formed with Salmonella strain SJ241.
A spontaneous non-flagellate mutant was isolated from motile strain B181-8 by screening on semi-solid nutrient agar medium. The mutant was
found to be resistant to both chi and M8. Chi-phage can be used as aselective agent for the isolation of non-motile mutants from actively motile
strains of Serratia by the same procedure which has been applied to
Salmonella and Escherichia coli.
Chi-phages were propagated in S. marcescens B-181-7, S. abortus-equi
SJ241 and E. coli W3110, and the stocks were designated chi (Ser), chi (Sal)and chi (Kc) respectively. The combinations of chi (Sal) and Serratia, andchi (Ser) and Salmonella, gave efficiencies of plating (EOP) not significantly
different from those of the homospecific combinations, Le. the combinations
in which the indicator strain used was the same as the propagating one.
However, when chi (Sal) or chi (Ser) were tested with Escherichia, theEOP decreased markedly. The infection by chi (E.c) of either Salmonella
or Serratia also resulted in decreased EOP as compared with the homospecific combinations. When chi (E.c) was grown on S. abortus-equi SJ241
or S. marcescens B-181-7, the progeny phages then exhibited high EOP :onthese bacteria while the EOP on E. coli W3110 decreased markedly. Thissuggests the presence of host induced modification in the system.
Anti-chi serum, prepared by immunization of a rabbit with a phagestock of chi (Sal), was absorbed with the propagating strain, diluted 1000
fold and neutralization :velocity constants with chi (Sal), chi (Ser) and125

RESEARCHES CARRIED OUT IN 1966

chi (E.c) were measured at 37°C. The constants found were 495±69,
497±61 and 521±73 respectively (95 per cent confidence interval). Thus
the efficiency of inactivation of chi (E.c) by anti-chi (Sal) serum was not
significantly different from those of both chi (Sal) and chi (Ser).

Normal Repressed Level of Ornithine Transcarbamylase
Activity in Crude Extracts of an Arginine Sensitive
Mutant of Salmonella typhimurium
Jun-ichi ISHIDSU

Among various enzymes having a relation to argmme and uracil biosyntheses, the activity of ornithine transcarbamylase which couples ornithineand carbamyl phosphate to citrulline was measured using crude extracts
prepared from arg-s-l cells grown under various culture conditions andcompared with the activity of wild type cells.
Arg-s-l was first grown in a synthetic minimal medium (Ishidsu, Ann.
Rep. No. 14, 1963) supplemented with 10- 3 M arginine and 10- 3 M uracil
to middle log phase. Cells were collected, washed and transferred withoutchanging original titer to the following four media: 1) minimal with nosupplementation (full derepression), 2) minimal with 10- 3 M uracil (derepression for the arginine synthesizing system), 3) minimal with 10- 3 M
arginine (derepression for the uracil synthesizing system) and 4) minimal
with 10- 3 M uracil and 10- 3 M arginine (continuous repression for both
systems). After 2 hours' incubation, cells of each culture were collected,
washed, resuspended in a small amount of potassium phosphate buffer(0.05 M, pH 7.0, containing 10- 4 M EDTA) and sonicated. After the removal of cell debris by centrifugation, the supernatants were subjected
to enzyme assay as crude extracts.
Results are summarized in the following table.
% Repression

Culture condition

Specific activity

Minimal10- 3 M uracil
10- 3 M arginine

0.807
0.713
0.00173

99.8

10- 3 M uracil +10- 3 M arginine

0.00161

99.8

o
11.6

These results are completely comparable with those obtained with wild
type cells.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17x.

HUMAN GENETICS

Evaluation of the Family Planning Programme in JapanEi MATSUNAGA

An official programme on family planning in Japan was launched in
1952, in order to replace the increasing practice of artificial abortion bycontraceptive methods. Placing the emphasis on the guidance of low income
families, local health centers have been playing a leading and supervisingrole in the programme, with the aid of some ten thousand public healthnurses and private work midwives. Besides, a number of voluntary organi·zations have assisted the government's programme, and mass communication by women's magazines has contributed greatly towards the
popularization of the knowledge about contraception. As a result, present
users of some kind of contraceptive measures have been rapidly increasing
among every social stratum during a short period.
On the other hand, the decline in births has still been accompanied bya tremendous number of notified abortions. Taking into account the likewise
tremendous number of unnotified ones-presumably some 60 to 100 per centof the notified cases-, a question has occasionally been raised as to theeffectiveness of the family planning programme as described above.
However, when measured per 1,000 women aged 15-49, the number of
notified abortions has steadily been decreasing from the maximum of 50 in
1955 to 30 in 1965, while the number of births dropped to 65 in 1957-justhalf the number of 1948-and remained thereafter almost constant, implying
this being the number of "wanted births" of the present Japanese.
Under these demographic trends, a method was devised to evaluate therespective roles in the lowering of birth rate of 1) induced abortions, 2)
contraceptive practice, 3) legalized sterilization, and 4) postponement ofmarriage. The result showed that, while the family planning programme
had scarcelly been successful during the first few years, the abortions havebeen, even allowing for as many unnotified as for the notified ones, increasingly replaced by contraception since 1957. It should be noted, however,that even in 1965 about two thirds of the reduction in births was presumably
based on induced abortions.
Details of this work will be published elsewhere.

RESEARCHES CARRIED OUT IN 1966

127

Association of Ear-Wax Types with Susceptibility toArteriosclerosis-A Preliminary Report!)
Mitsuo MIYAHARA' and Ei MATSUNAGA

It is now well known that the ear-wax types in man are determined bya simple genetic mechanism, the wet type being inherited dominantly overthe dry type. There is a marked variation in the incidence of the respective
types among various ethnic groups; more than 80 per cent of Japanesebelong to dry type, whereas this type of cerumen is exceptional among
both Caucasians and Negroes. Since there is an apparent association
between wet cerumen and development of apocrine sweat glands or axillaryodour, it may be assumed that the effect of the allele pair responsible for
ear-wax types is concerned with certain metabolic patterns presumably of
lipid material. It has been hoped that investigations will be carried out
to seek for an association between ear-wax types and some internaldiseases, such as arteriosclerosis and other related diseases (Matsunaga,"11962).
Data on ear-wax types have been collected from in- and outpatients inthe Department of Internal Medicine of the Sapporo Medical College
Hospital, Sapporo, during the last two years from January 1964 to December
1966. The diagnosis of "arteriosclerosis" of our patients was made, inmost instances, if they were affected with coronary arterial sclerosis or
ischemic heart disease, and in the remaining cases, if they had a historyof either apoplectic stroke or encephalomalacia, or they showed calcification of the arc of the aorta as revealed by roentgenography. The
conditions were then divided into two groups, i.e., arteriosclerosis withand without hypertension. Patients with other diseases were also includedin the study. In the classification of the disease entries, those with uncertain diagnosis were all included in the "miscellaneous" group.
Of 2,448 patients so far examined, there were 39 or 1.6 per cent whose
ear-wax type could not be determined with certainty. Although ourexperience suggests that ambiguous cases are most likely to be of wet
type, they were excluded from the subsequent analysis. The incidenceof wet cerumen among the remaining 2,409 was 16.9 per cent, which was
not significantly different from the 19.0 per cent (X'=2.92, d.f.=I, P>0.05)
previously obtained by a survey (Matsunaga et al. 41 , 1954) of 1,639 pupils
II This work was supported by a grant from the Toyo Rayon Foundation for the
Promotion of Science and Technology.
21 Department of Internal Medicine, Sapporo Medical College, Sapporo.
31 Matsunaga, E., 1962.
The dimorphism in human normal cerumen. Ann.
Human Genet. 25: 273-286.

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

of a middle school in Sapporo.
The distribution of the patients differed between sexes according to thedisease, but for each disease entry there was no significant difference inthe distribution of ear-wax types according to sex, so that the data werepooled for male and female patients, and the results are summarized inTable 1.
Table I.

Association of wet cerumen with a variety of diseases

Groups of patientsaffected with
I.

2.
3.
4.
5.
6.
7.
8.
9.
10.
II.

12.

Arteriosclerosis
Arteriosclerosis withhypertension
Essential hypertension
Diabetes meIlitus
Endocrine disorders
Heart diseases
Kidney diseases
Liver diseases
Blood diseases
Gastrointestinal diseases
Lung diseases
Miscellaneous
Total
Control%

Relativerisk forwet type

X2

29

30.2

1.85

7.05*

19

13.8

0.68

2.26

47
80
122
70
90
30
429
45
972

50
4
16
17
13
15
3
77
6
158

17.2
8.5
20.0
13.9
18.6
16.7
10.0
17.9
13.3
16.3

0.89
0.40
1.07
0.69
0.97
0.85
0.47
0.93
0.66
0.83

0.49
3.08
0.05
1.89
0.01
1.89
1.49
0.24
0.90
3.08

2,409
1,638

407
311

16.9
19.0

Wet typeNo.
examined

No.

96
138
29J

* Significant at the 1 per cent level.

From the table it can be seen that the incidence of wet cerumen among
the patients with arteriosclerosis, not accompanied by hypertension, wasstrikingly high-as high as 30.2 per cent-, whereas it was only 13.8 percent among the arteriosclerotic patients with hypertension, the difference
being significant at the 1 per cent level (x 2 =9.39, d.f.=I, P<O.01). In
other groups of patients the incidences of wet cerumen were all below
20 per cent. Using the figure of 19.0 per cent as control, the risks of the
wet type relative to the dry in the susceptibility to various diseases havebeen calculated by means of Woolf's methodS I (1955). The item for which
41 Matsunaga, E., S. Itoh, T. Suzuki, and S. Sugimoto, 1954. Incidence andinheritance of the ear-wax types. Sapporo Med. J. 6: 1-4.
SI Woolf, B., 1955. On estimating the relation between blood group and disease.
Ann. Human Genet. 19: 251-253.

RESEARCHES CARRIED OUT IN 1966

129

significance was shown was only arteriosclerosis without hypertension;for this condition the risk of individuals with wet cerumen was apparently
increased as much as 85 per cent as compared with individuals with dry
cerumen.
It has been widely recognized that there is a variation in the incidenceof arteriosclerosis among various ethnic groups; for example, coronary
arterial sclerosis is much more common in the United States than inJapan. Since the Japanese immigrants in the United States show higher
incidence of coronary arterial sclerosis than the Japanese in Japan, environmental factors such as dietary habits seem to predominate in the causationof the disease, but genetic factors underlying racial differences cannot be
excluded. The present study indicates that, while there is no association
between ear-wax types and arteriosclerosis accompanied by hypertension,individuals with wet cerumen are more susceptible to arteriosclerosis
without hypertension. This association may account, to some extent, forthe observed difference in the incidence of arteriosclerosis among various
ethnic groups.
This work is still in progress and the data will be analyzed with reference
to cholesterol level of the blood in the patients.

Maternal Age of Mosaics with Down's Syndrome
Ei MATSUNAGA, Akira TONOMURAl) Hidetsune OISHI
and Yasumoto KIKUCHI

The overwhelming majority of cases with Down's syndrome is now known
to be primary G-trisomics, and the etiological importance of maternal age
is well established. For the cases with translocations, maternal age is
generally accepted to be of no significance, while less attention has been
paid to maternal age of the mosaics.
In order to test for a possible effect of maternal age upon mitotic
non-disjunction, data have been collected from the literature, including
our own cases, concerning maternal age of 31 cases of mosaicism withvarying degree of clinical signs of Down's syndrome. When they weredivided into two classes, class A with predominantly normal cells andclass B with predominantly G-trisomic or tetrasomic cells as revealed byeither blood or skin culture, the mean maternal age was 35.1±1.6 forclass B mosaics, while it was 29.4±2.0 for class A mosaics, the difference
being significant at the 5 per cent level (t=2.24, d.f.=29, P<O.05). Sinceclass A mosaics are most likely to have developed from fertilized eggs
with normal karyotype, it appears that mitotic non-disjunction may not
1)

Department of Cytogenetics, Tokyo Medical and Dental University.

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

be affected by maternal age.
definite conclusion.

However, more data are needed to draw a

Chromosome Replication in Down's Syndrome
Yasumoto KIKUCHI and Hidetsune OISHI

Autoradiographic techniques have been used mainly for the study of
DNA replication pattern of human chromosomes. This technique may
be utilized also for chromosome identification. We report here a preliminary study of autoradiographic analysis of the G group chromosomes
in patients with Down's syndrome and in normal subjects.
Four female patients with the standard type of G-trisomy, two normal
females, and two normal males were studied. Leucocytes obtained fromvenous blood of each subject were cultured according to standard technique.
Chromosome replication at the end of the DNA synthesis period (S period)
was analysed by means of autoradiography, tritiated thymidine and continuous labeling technique.
Metaphase plates selected for analysis were scored according to thenumber of small acrocentrics labeled per cell. The results are shown in
Table 1. In normal subjects, cells with 4 labeled G chromosomes occurred
Table 1.

Number of cells showing the distribution of labelingamong acrocentrics of the G group
No. of G chromosomes labeled

Subjects
Normal males
Normal females
Female patients with
Down's syndrome

0

1

2

3

4

12
5

11
14

24
27

22
18

31
37

10

8

18

32

19

5

Total No. ofcells examined
100
100

18

100

most frequently (31 per cent in males and 37 in females); cells with 2labeled G chromosomes were found less frequently, while the frequenciesof cells with 1 or 3 labeled G chromosomes were still lower. This indicatesthat most of the homologous pairs of G group replicate synchronously.
In female patients with Down's syndrome, 32 out of 100 cells were observed
to have 3 labeled and 2 unlabeled G chromosomes, while there were only
18 cells with 2 labeled and 3 unlabeled G chromosomes. The results may
suggest that the sequence of replication of the extra chromosome is similar
to the original homologous pair.
For detail analysis of grain distribution, 20 metaphase plates wereRESEARCHES CARRIED OUT IN 1966

131

observed from a normal female and the same number from a female
patient with Down's syndrome. In 12 cells from the normal female, 2 G
chromosomes were generally labeled over the long arm (late replicatingpair), but chromosomes of another pair were completely unlabeled or
lightly labeled only over the centromere region or the short arm (early
replicating pair). The remaining cells did not show a consistent pattern.
In the patient with Down's syndrome, 12 cells had 3 late replicating G
chromosomes and 2 early replicating ones. On the other hand, 5 cells had
2 late and 3 early replicating G chromosomes. In the remaining cells,
different patterns were observed.
In the autoradiographs of chromosomes from each patient other thanthe G group, the characteristic pattern of late replication was observedin 1 X chromosome, in 2 of the D group (Nos. 13-15) and in Nos. 16 and18.
The above results suggest that the extra chromosome associated with
Down's syndrome belongs to the late replicating pair of G group, andthat its presence does not affect DNA synthesis of chromosomes other thanG group.
Phenotypes and Sex Chromosomes in Five Patientswith Turner's Syndrome
Hidetsune OISHI, Yasumoto KIKUCHI and Ei MATSUDA

It is now widely accepted that monosomy of X or! X deletion is responsible for clinical conditions called Turner's syndrome. However,individuals with this chromosome aberration do not always show all of
the classical signs of Turner's syndrome (shortness of stature with sexualinfantilism, cubitus valgus, and webbing of the neck). During our current
survey, in collaboration with some University Hospitals in Tokyo, ofhuman chromosomes in various congenital disorders and sex anomalies,
we examined the chromosomes in four cases of XO patients and in one
with XO/XXX mosaicism. The results of clinical and laboratory examinations are summarized in Table 1. In all cases, short stature, shieldchest, cubitus valgus without webbing of the neck, lacking or scanty pubic
hair, and a small, scarcely palpable uterus were noticed.
Analyses of dermal patterns of fingers and hands were performed on
three patients, and the formulas are given in Table 2. Since the total
ridge counts of the finger tips in Japanese females are on the average
137.70±41.09 (Matsunaga), our patients seem to have higher ridge counts.
The characteristic t"s were observed in the left palms of cases Nos. 4
and 5, while the axial triradius was absent in both hands of case No.3.

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
Table 1.

Some clinical data in five patients under study

Case No.

1

2

3

4

5

Sex chromosome constitution
DrumstickMaternal age at the birth ofthe patient
Paternal age at the birth ofthe patient
Birth weight (kg)Age examined (years)
Height (cm)Weight (kg)
Arm span (cm)
IQ (or intelligence)
Short neck
Webbed neck
Shield chest
Cubitus valgus
Menstruation
Development of the breasts
Pubic and axillary hairs
Small uterus
Gonadotropin (unit/day)
17·KS (mg/day)

XO

XO

XO

XO

XO/XXX+(14/500)*

37

27

30

24

24

45

35

35

28

28

17
143
41.5
141
99

3.10
16
134
34.7
123.7
Normal

3.18
15
130
40.0
131.8
Normal

2.15
10
106
23.5
94.7
49

2.45
10
121
25.5
120
123

+

+

+

+

+
+

+
+

+
+

+
+

+
<4
0.61

+
<4
1.0

+
+

+
Scanty
+
>32
4.37

+
>32
1.6

+
<32
0.9

* Including two cells with two drumsticks.
Table 2.
Finger patterntypes (I-V)*

Case No.
3{Ri g ht
Left

U.

U. U.

Dermatoglyphics of the patients
Total
ridge
Palmar pattern formulae**
count

U. Lp

atd angle

l1.X. 7.1- 0- Ar.O.O.l.O
200

W. U. U. U. W

4{Ri g ht
Left

U. At. U.

5{Ri g ht
Left

U. Ucp. Ucp. W. W

U.

7.7.5".1-0- Ar.O.O.O.L/L
l1.X. 7.3- t-Au.O.O.V.O

U

U. R. Lp. Lp. U

151
9.7.5".3-tt" - LUV.V/V.O.O.V
11.9.8.5' -t- V.V/V.O.L.O
150

U. W. W. W. W

9.9.5".1-tt" - Ws.V/V.O.L.V* At; tented arch, W; whorl, R; radial loop,
U; ulnar loop, Lp; lateral pocket loop.
** Cummins-Midlo's formula.

42°
78°
43°
45°
75°
45°

g"
g"

Ucp; ulnar central pocket loop,

RESEARCHES CARRIED OUT IN 1966

133

Thenar patterns as seen occasionally in patients with this syndrome were
noticed in cases Nos. 4 and 5. In addition, all three patients had palmartransverse creases.
Further studies are required for the discrimination of some metricalcharacters genetically controlled by sex chromosomes.

Clinical Conditions of Patients with Apparently Normal
Chromosomes, III
Hidetsune OISHI and Yasumoto KIKUCHI

Based on leucocyte cultures of peripheral blood, patients with various
pathological conditions were found to have 46 chromDsomes of apparently
normal karyotype. Cases of some interest are listed below.

Name

Age

Legal sex

Clinical conditionsa)Multiple deformations with mental retardation
47 N.1.
3 yearsF
Hypertelorism; antimongoloid slant eyes;
low-set ears; webbing of the neck48 M.O. 4 years
Microcephaly; exophthalmos; low-set ears
F
49 Y.M. 3 years
Microcephaly; hypertelorism; epicanthus;M
micrognathia (" Cri du chat" syndrome?)
50 S.K.
Exophthalmos; large deformed lobes of the
5 months M
ear; bifid tongue; high-arched palate
51 T.O.
2 years
Oblique palpebral fissures; low-set ears;
M
polydactyly
Congenital glaucoma; aphasia
52 A.O.
1 yearF
53 Y.M. 1 year
Osteogenesis imperfecta; hydrocephalus;F
small deformed lobes of the ear; congenital
heart disease
54 S. T.
2 years
Hypertelorism; antimongoloid slant eyes;Mhydrocephalus
55 Y.S.
5 months F
Hypertelorism; syndactyly; antimongoloid
slant eyes; epicanthus
Hypertelorism; antimongoloid slant eyes
56 H. T. 2 yearsMb)

Sex anomalies
57 H. S. 36 years

M

58

F

E. M.

5 years

Slender body; funnel-shaped chest; small
penis; cataract; low excretion of 17KS;
high excretion of gonadotropin (Klinefelter's syndrome?)
Female pseudohermaphroditism

134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

59 -.S.
60 K.K.

c)

11 days
9 yearsF
F

61 A.G. 16 years
62 Y.K.
6 years

F
F

Hereditary disease
63 T. I. 23 years

M

Female pseudohermaphroditism
Primordial dwarfism;
cubitus valgus(Turner's syndrome?)
Male pseudohermaphroditism
Webbing of the neck; shield chest; shortstature; hypertelorism (Turner's syndrome?)
Recklinghausen's disease

Amino Acid Sequence Around Cystine Residues of a
Lambda Type Human Bence·Jones Protein ll
Tomotaka SHINODA

A complete location of disulfide bridges of type L human Bence-Jones
protein was carried out using a specimen, Sh. A peptic digest of Sh
protein was purified several times by high voltage electrophoresis at pH
6.5 and 3.5. The cystine peptides were oxidized with performic acid vapour.
Amino acid sequence of cystine peptides thus obtained was determined by
the subtractive Edman degradation method. Three disulfide bridges havebeen found; Two of these were the intrachain bridges-a bridge which
located in the variable part of the molecule and the other in the invariant
part-, and the remainder formed an interchain bridge at the C-terminal,
which showed why the Sh protein existed predominantly in the dimer
21
Val-Arg-ILu-Thr-Cys-Gln-Gly-Asp-Ser-Leu

I

Tyr-Cys-Asn-Ser-Arg
86
135
Val-Cys-Leu

I

Ser-Tyr-Ser-Cys-Gln
194
Fig. 1.

212
Pro-Thr-Glu-Cys-Ser

I

Pro-Thr-Glu-Cy s-Ser

212

Amino acid sequence around cystine residues.

form. The results are shown in Fig. 1.
It is suggested that the genetic conservation of secondary and tertiary
structures may be reflected in the location of the disulfide bridges of lightchains of immunoglobulin, and that a general manner of linkage may also
be a common feature of the heterogeneous population of light chains.
1)

This work was carried out at Indiana University, Bloomington, Indiana, U.S.A.

RESEARCHES CARRIED OUT IN 1966

135

Cellulose Acetate Electrophoresis and az-Lipoprotein of Human Serum
Yoshito OGAWA

When electrophoretic analysis of human serum is carried out using
cellulose acetate strips as a supporting medium, a fine new band often
appears in the part of IXz-globulin pattern (Fig. 1, arrow). The nature of+

Fig. 1. Separation pattern of human serum on cellulose acetate strip(Separax).

this new fraction was not yet studied. The present writer has thoroughly
examined this fraction and succeeded to confirm its identity with IXz'
lipoprotein fraction because of its affinity to Sudan Black B (Swahn's
method) and the results of immunochemical examination, immunoelectrophoresis and absorption test, applying anti IXz-lipoprotein rabbit serum(Behring-Werke).
Details of this works had been published in Physico-Chemical Biology
Vol. 12.

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

BOOKS AND PAPERS PUBLlS~ED IN 1966
BY STAFF MEMBERS
ASAKURA, S., G. EGUCHI and T. IINO, 1966. Salmonella flagella: in vitro reconstitution and over-all shapes of flagellar filaments. J. Mol. BioI. 16: 302-316.
CAVALLI-SFORZA, L. L., M. KIMURA and I. BARRAI, 1966. The probability of consanguineous marriages. Genetics 54: 37-60.
CHUNG, C. S., N. E. MORTON and N. YASUDA, 1966. Genetics of interracial crosses.
Ann. N. Y. Acad. Sci. 134: 666-687.
ENDO, T., 1966. Cytolasm and conformation of proteins (in Japanese). Nucleus andCytoplasm 9: 6-10.
ENDO, T., 1966. Application of zymography to plant sciences (in Japanese). SABCO
J. 2: 120-126.
ENDO, T. and D. SCHWARTZ, 1966. Tissue specific variations in the urea sensitivity of the E , esterase in maize. Genetics 54: 233-239.
ENDO, T. and D. SCHWARTZ, 1966. Regulation of cathecol oxidase. Maize Genetics
Coop. News Letter 40: 165-166.
ENOMOTO, M., 1966. Genetic studies of paralyzed mutants in Salmonella. I. Genetic finestructure of the mot loci in Salmonella typhimurium. Genetics 54: 715-726.
ENOMOTO, M., 1966. Genetic studies of paralyzed mutants in Salmonella. II. Mapping of three mot loci by linkage analysis. Genetics 54: 1069-1076.
ENOMOTO, M. and T. IINO, 1966. The comparison of normal and curly flagella in
Salmonella abortus-equi by two-dimensional separation of peptides. Japan. J.
Genetics 41: 131-139.
FUJII, T., M. IKENAGA and J. T. LYMAN, 1966. Radiation effects on Arabidopsis
thaliana. II. Killing and mutagenic efficiencies of heavy ionizing particles.
Radiation Botany 6: 297-306.
FUJII, T. and S. MATSUMURA, 1966. Comparison of mutagenic efficiency between
ethyl methanesulfonate and ionizing radiations in dipliod wheat (Preliminary
report). Seiken Zih6 18: 23-31.
GALSSMAN, E., T. SHINODA, E. J. DUKE and J. F. COLLINS, 1966. Interconversion ofmolecular forms of xanthine dehydrogenase from Drosophila. Ann. N. Y. Acad.
Sci. 130: (in press).
GALSSMAN, E., T. SHINODA, H. M. MOON and J. D. KALAM, 1966. In vitro complementation between non-allelic Drosophila mutants deficient in xanthine dehydrogenase. IV. Molecular weight. J. Mol. BioI. 20: 419-422.
IINO, T., 1966. Bacterial flagella (in Japanese). Protein Nucleic Acid Enzyme(Tokyo) 11: 41-49.
IINO, T., 1966. Morphogenesis of bacterial flagella (in Japanese). Kagaku To
Seibutsu 4: 345-350.
IINO, T. and M. ENOMOTO, 1966. Genetical :studies of non-flagellate mutants ofSalmonella. J. Gen. Microbiol. 43: 315-327.
IINO, T. and M. MIT ANI, 1966. Flagella-shape mutants in Salmonella. J. Gen.
Microbiol. 44: 27-40.

BOOKS AND PAPERS PUBLISHED IN 1966 BY STAFF MEMBERS

137

IINO, T., M. MIT ANI and S. ASAKURA, 1966. Formation of bacterial flagella (inJapanese). Inst. Appl. Microbiol. Tokyo Univ. Symp. 7: 113-128.
IKENAGA, M. and T. MABUCHI, 1966. Photoreactivation of endosperm mutations
induced by ultraviolet light in maize. Radiation Botany 6: 165-169.
KATAYAMA, T. C., 1966. Anatomy of the root of Oryza sativa with emphasis on
developmental processes. Seiken Ziho 18: 6-22.
KIHARA, H., 1966. Factors affecting the evolution of common wheat. Indian J.
Genetics Plant Breeding 26A (Special Symp. No.): 14-28.
KIHARA, H. and T. HORI, 1966. The behavior of nuclei in germinating pollen grainsof wheat, rice and maize. Der Ztichter 36: 145-150.
KIHARA, H. and K. TSUNEWAKI, 1966. Basic studies on hybrid wheat breeding,carried out at the National Institute of Genetics. Seiken Ziho 18: 55-63.
KIKUCHI, Y., 1966. The pattern of chromosome replication in man (in Japanese).
Kagaku 36: 65-69.
KIKUCHI, Y., 1966. Analysis of chromosomes in newborn infants (in Japanese). Clin.
Gynecol. Obstet. 20: 887-892.
KIMURA, M., 1965. Evolutionary considerations on spontaneous mutation rates(Preliminary report). Gamma Field Symp. 4: 85-96.
KIMURA, M. and T. MARUYAMA, 1966. The mutational load with epistatic geneinteractions in fitness. Genetics 54: 1337-1351.
KIRITANI, K., S. NARISE and R. P. WAGNER, 1966. The reductoisomerase of Neurospora crassa. J. BioI. Chern. 240: 2047-2051.
KIRITANI, K., S. NARISE and R. P. WAGNER, 1966. The dihydroxy acid dehydrataseof Neurospora crassa. J. BioI. Chern. 241: 2042-2046.
KURODA, Y., 1966. Sorting-out mechanisms of animal tissues and cells (in Japanese).
Inst. Appl. Microbiol. Tokyo Univ. Symp. 7: 42-55.
KURODA, Y., 1966. Tissue organization from single cells (in Japanese). Heredity(Tokyo) 20: 8-12.
KURODA, Y., 1966. Differentiation and tissue specificity of aggregation-promoting
materials from chick embryos (in Japanese). Japan. J. Exptl. Morphol. 20: 93.
KURODA, Y., 1966. Sorting-out phenomena of animal tissues and cells (in Japanese).
Kagaku To Seibutsu 4: 394-400.
MABUCHI, T. and S. MATSUMURA, 1966. Oxygen and storage effects on radiation
damage in einkorn wheat seed. W. I. S. 21: 10.
MABUCHI, T. and S. MATSUMURA, 1966. Radiosensitivity in pollen grains of Triticum
and Aegilops. W. I. S. 22: 20.
MATANO, Y. and T. H. YOSIDA, 1966. Alteration of stemline chromosomes in hypotetraploid Ehrlich carcinoma. Proc. Japan Acad. 42: 404-407.
MATSUMURA, S., 1966. Differences in effects of r-rays and fast neutrons on wheat.
W. I. S. 21: 3-6.
MATSUMURA, S., 1966. Radiation genetics in wheat. IX. Differences in effects of
gamma-rays and 14 MeV, fission and fast neutrons from Po-Be. Radiation
Botany 6: 275-283.
MATSUMURA, S. and T. FUJII, 1966. Radiosensitivity of wheat seeds. W. I. S.
22: 21.
MATSUMURA, S. and T. MABUCHI, 1966. Photoreactivation of an ultraviolet light138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17
induced mutation in maize pollen. Seiken Ziho 18: 1-5.
MATSUNAGA, E., 1966. Genetic epidemiology of sporadic retinoblastoma (in Japanese).
Igaku No Ayumi 59: 208-212.
MATSUNAGA, E., 1966. Causes of Down's syndrome (in Japanese). Heredity (Tokyo)
20(11): 33.
MATSUNAGA, E., 1966. Possible genetic consequences of family planning. J. Amer.
Med. Assoc. 198: 533-540.
MATSUNAGA, E., 1966. Down's syndrome and maternal inbreeding. Acta Genet.
Med. Gemell. 15: 224-230.
MATSUNAGA, E., 1966. Some remarks on the biology of twins. Japan. J. Human
Genet. 11: 227-228.
MORTON, N. E., M. P. MI and N. YASUDA, 1966. Bivalent alleles. Amer. J. Human
Genet. 18: 233-242.
MORTON, N. E., M. P. MI and N. YASUDA, 1966. A special theory of hemagglutination. Vox. Sang. 11: 12-20.
MORTON, N. E., M. P. MI and N. YASUDA, 1966. A study of the SIt alleles in northeastern Brazil. Vox. Sang. 11: 194-208.
MUKAI, T., 1966. Further studies on the optimum heterozygosity hypothesis in
Drosophila melanogaster. Genetics 54: 350-351.
MUKAI, T., I. YOSHIKAWA and K. SANO, 1966. The genetic structure of natural
populations of Drosophila melanogaster. IV. Heterozygous effects of radiationinduced mutations on viability in various genetic backgrounds. Genetics 53:
513-527.
MURAKAMI, A., 1966. Relative biological effectiveness of 14 MeV neutrons to gamma-rays for inducing mutations in mature sperm of the silkworm. Japan. J.
Genetics 41(1): 17-26.
MURAKAMI, A., 1966. Relationship between radiosensitivity and mitotic cycle during early developmental stages of silkworm eggs (in Japanese). Radiation BioI.
Res. Comm. 1(1): 29-42.
NARISE, T., 1965. The effect of relative frequency of species in competition.
Evolution 19: 350-354.
NARISE, T., 1966. The mode of migration of Drosophila ananassae under competitive
conditions. Univ. Texas Publ. 6615: 121-131.
NARISE, S. and J. L. HUBBY, 1966. Purification of esterase-5 from Drosophila
pseudoobscura. Biochim. Biophys. Acta 122: 281-288.
OGAWA, Y., 1966. Cellulose acetate electrophoretic technique (in Japanese). Japan.
J. Clin. Pathol. 11: 46-63.
OGAWA, Y.. 1966. Ponceau 3R staining of serum proteins on Separax (in Japanese).
Physico-Chern. BioI. (Tokyo) 11: 224-225.
OGAWA, Y., 1966. Cellulose acetate electrophoresis of human serum proteins. I.
(in Japanese). Japan. J. Med. Tech. 15: 147-153.
OGAWA, Y., 1966. Cellulose acetate electrophoresis of human serum proteins. II.
(in Japanese). Japan. J. Med. Tech. 15: 197-204.
OGAWA, Y., 1966. Human serum protein electrophoresis on cellulose acetate strips(in Japanese). Pathol. Bacteriol. (Tokyo) 48: 5-12.
OGAWA, Y., 1966. The cellulose acetate electrophoretic technique. III. Ponceau 3R

BOOKS AND PAPERS PUBLISHED IN 1966 BY STAFF MEMBERS

139

staining of albumin in human serum on Separax (in Japanese). Med. BioI.
(Tokyo) 73: 125-127.
OGAWA, Y., 1966. Growth, differentiation and regeneration. XLII. Effect of glucurono-Iactone on the development of skeletal myosin in X-irradiated Triturusembryos (in Japanese). Med. BioI. (Tokyo) 73: 130-133.
OGAWA, Y., 1966. The cellulose acetate electrophoretic technique. V. Ponceau 3R
staining of p- and r-globulin in human serum on Separax (in Japanese). Med.
BioI. (Tokyo) 74: 169-172.
OGAWA, Y., 1966. The cellulose acetate electrophoretic technique. VI. On theconcentration of trichloroacetic acid in staining and acetic acid in washingsolutions (in Japanese). Med. BioI. (Tokyo) 73: 198-202.
OGAWA, Y., 1966. The cellulose acetate electrophoretic technique. VII. Conditionsof current supply (in Japanese). Med. BioI. (Tokyo) 73: 228-231.
OGAWA, Y. and H. MIYAUCHI,1966. The cellulose acetate electrophoretic technique.
IV. Ponceau 3R staining of a-globulin in human serum on Separax (in Japanese)Med. BioI. (Tokyo) 73: 134-136.
OGAWA, Y., M. ABE, M. KITAMURA, N. KOSAK AI, K. SHIMAO, H. TOMITA H. HIRAI
and K. MOMMA, 1966. On the standard procedure for the analysis of serum
protein fractions by cellulose acetate electrophoresis (in Japanese). PhysicoChern. BioI. (Tokyo) 11: 351-356.
OGAWA, Y. and T. HASEGAWA, 1966. Normal value of serum protein fractions ofJapanese population on Separax, a new cellulose acetate membrane for electrophoretic analysis (in Japanese). Physico-Chern. BioI. (Tokyo) 11: 319.
OISHI, H., 1966. Human chromosomes and its analysis (in Japanese). Japan. J.
Clin. Pathol. 14: 780-784.
OISHI, H., 1966. Human diploid cell strains (in Japanese). Heredity (Tokyo) 20(7):55-57.
OKA, H. I., 1966. Growing season of rice in the tropics (in Japanese). Japan. J.
Trop. Agr. 9: 195-199.
OSHIMA, C., 1966. "Mechanisms of micro- and meso-evolution" in Modern Biology
9: 185-215, "Ecology and Evolution" (in Japanese). Iwanami-Shoten, Tokyo.
OSHIMA, C., 1966. Persistence of some recessive lethal genes in natural populationsof Drosophila melanogaster. Proc. Symp. held in Prague, August 9-11, 1965,
Mutation in Population: 41-48.
SAKAI, K. I. and S. IYAMA, 1966. Studies on competition in plants and animals.
XI. Competitive ability and density response in barley. Japan. J. Breeding
16: 1-9.
SAKAMOTO, S., 1966. Cytogenetic studies in the tribe Triticeae. IV. Natural hybridization among Japanese Agropyron species. Japan. J. Genetics 41: 189-201.
SAKAMOTO, S., 1966. Intergeneric hybrids between two Eremopyrum and Agropyron
species. W. I. S. 21: 22.
SAKAMOTO, S., 1966. An intergeneric hybrid between Eremopyrum orientale (Linn.)
Jaub. et Spach. and Aegilops squarrosa Linn. W. I. S. 22: 6.
SAKAMOTO, S. and M. MURAMATSU, 1966. Cytogenetic studies in the tribe Triticeae.
II. Tetraploid and hexaploid hybrids of Agropyron. Japan. J. Genetics 41: 155168.

140 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

SAKAMOTO, S. and M. MURAMATSU, 1966. Cytogenetic studies in the tribe Triticeae.
III. Pentaploid Agropyron hybrids and genomic relationships among Japaneseand Nepalese species. Japan. J. Genetics 41: 173-187.
SCHWARTZ, D. and T. ENDO, 1966. Alcohol dehydrogenase polymorphism in maize-Simple and compound loci. Genetics 53; 709-715.
SHINODA, T. and E. GLASSMAN, 1966. Multiple molecular forms of xanthine dehydrogenase in Drosophila (submitted for publication).
SUZUKI, H. and T. IINO, 1966. An assay for newly-synthesized intracellular flagel·lin. Biochim. Biophys. Acta 124: 212-215.
TANAKA, Y. and F. GOTO, 1965. Growth-accelerating action of K (Knobbed) genein the silkworm. III. Size and number of knobs with respect to the growthaccelerating action (in Japanese). Rept. Silk Sci. Res. Inst. 14: 12-16.
TANAKA, Y. and F. GOTO, 1965. Single gene action of some dominant charactersin the silkworm. I. (in Japanese). Rept. Silk Sci. Res. Inst. 14: 17-19.
TANAKA, Y. and F. GOTO, 1965. Experimental studies on the characteristics ofvarious genes in the silkworm. I. (in Japanese). Rept. Silk Sci. Res. Inst. 14:
20-25.
TAZIMA, Y., 1966. How mutations occur? (in Japanese). Section II, Chapter 5 in
"Gendai Seibutsugaku Taikei" (D. MORIWAKI and K. ONO, ed.) pp. 248-276.
Nakayama-shoten, Tokyo.
T AZIMA, Y., 1966. Radiations and Radiosotopes in Sericulture (in Japanese). Kagaku
To Gijitsu 5(1): 48-51, (2): 38-41, (3): 36-39, (4): 62-65, (5): 26-29, (6): 30-33,(7): 38-41, (8): 48-52, (9): 48-52, (10): 64-67, (11): 22-25, (12): 34-37, 6(1): 6265, (2): 34-37, (3): 52-55. Zenkoku Yosan Kyodokumiai Rengokai, Tokyo.
TAZIMA, Y., 1966. A report from the Third International Congress of Radiation
Research (in Japanese). Rad. BioI. Res. Comm. 1(1): 61-68.
TAZIMA, Y., 1966. A report from the Conference on Space Radiation Biology (inJapanese). Iden (Tokyo) 20(1): 45-46.
TONOMURA, A., H. OISHI, E. MATSUNAGA and T. KURITA, 1966. Down's syndrome:
A cytogenetic and statistical survey of 127 Japanese patients. Japan. J. Human
Genet. 11: 1-16.
TSAI, K. H. and H. I. OKA, 1966. Genetic studies of yielding capacity and adaptability in crop plants. 2. Analysis of genes controlling heading time in Taichung 65 and other rice varieties. Bot. Bull. Acad. Sinica 7: 54-70.
TSUJITA, M. and S. SAKURAI, 1966. Structure of chromogranules in the hypodermal
cells of silkworm larvae. Proc. Japan Acad. 42: 950-955.
TSUJITA, M. and S. SAKURAI, 1966. Chemical composition of chromogranules produced in the hypodermal cells of silkworm larvae. Proc. Japan Acad. 42:
956-959.
TSUJITA, M. and S. SAKURAI, 1966. Development of chromogranules in the larvalskin of the silkworm. Proc. Japan Acad. 42: 960-965.
YONEDA, Y. and T. STONIER, 1966. Elongation of stem internodes in the Japanesemorning glory Pharbitis nil in relation to auxin destruction. Physiol. Plant.
19: 977-981.
YOSIDA, T. H., 1966. Chromosomal alteration and the development of tumors.
XIV. Comparative idiogram analysis of several sublines of mouse lymphocytic

BOOKS AND PAPERS PUBLISHED IN 1936 BY STAFF MEMBERS

141

neoplasm, P388, growing in vivo and in vitro. Japan. J. Genetics 41: 43-58.
YOSIDA, T. H., 1956. Chromosomal alteration and the development of tumors. XV.
Change of chromosome pattern in 8-azaguanine and amethopterin-resistant
sublines of mouse lymphocytic neoplasm, P388, cultured in vitro. Japan. J.
Genetics 41: 59-74.
YOSIDA, T. H., 1966. Relation between chromosomal alteration and developmentof tumors (Prize Lecture of the Genetics Society of Japan) (in Japanese).
Japan. J. Genetics 41: 439-451.
YOSIDA, T. H., 1956. Method for chromosome analysis in Muridae, with special
regards to the chromosomal polymorphism (in Japanese). SABCO J. 2: 1-7.
YOSIDA, T. H., 1966. Part of chromosomes to induction of tumor and its growth(in Japanese). Igaku No Ayumi 59: 205-207.
YOSIDA, T. H., 1966. Karyology of malignant growths (in Japanese). Japan. J.
Cancer Clin. 12: 431-437.
YOSIDA, T. H., H. IMAI and H. SATO, 1966. Chromosomal alteration and the development of tumors. XII. Differentiation of karyotypes in mouse ascites
hepatoma MH-134 and MH-129P maintained in different locations. Gann 57:
9-17.
YOSIDA, T. H., Y. KURITA and K. MORIWAKI, 1966. Chromosomal alteration andthe development of tumors. XIII. Karyotype difference in solid and ascites typetumors of mouse plasma cell neoplasm X5563, with special regard to the serum
electrophoretic pattern of gamma globulin. Japan. J. Genetics 41: 9-19.
WATANABE, T. K. and C. OSHIMA, 1966. Distribution of natural lethal genes on
the second chromosomes of Drosophila melanogaster. Japan. J. Genetics 41;
367-378.

142 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

ABSTRACTS OF DIARY FOR 1966
January
21
January
25
January
26
February 18
March
1
March
24
April
8
April
20
April
22
May
20
June
4
June
24
July
18
July
29
September 12
September 30
October27, 28
November 2
November 4, 5November 10
November 11
November 25
December 16

141st Meeting of Misima Geneticist' Club
69th Biological Symposium
70th Biological Symposium
142nd Meeting of Misima Geneticist' Club
71st Biological Symposium
143rd Meeting of Misima Geneticist' Club
16th Meeting of the Society of Electrophoresi::;
72nd Biological Symposium
144th Meeting of Misima Geneticist' Club
145th Meeting of Misima Geneticist' Club
26th Meeting of the Board of Councillors
146th Meeting of Misima Geneticist' Club
73rd Biological Symposium
147th Meeting of Misima Geneticist' Club
4th Meeting of the Organizing Committee of the 12th
International Congress of Genetics
148th Meeting of Misima Geneticist' Club
Meeting of Directors of the National Institutes
74th Biological Symposium
18th Meeting of the Tokai Branch of the Sericultural
Society of Japan
75th Biological Symposium
Public Lectures on Genetics (at the Daiichi-Seimei Hall,
Tokyo)
149th Meeting of Misima Geneticist' Club
150th Meeting of Misima Geneticist' ClubFOREIGN VISITORS IN 1966

143

FOREIGN VISITORS IN 1966
2

STROMBERGER, H. G., Pasadena High School and JuniorCollege, Pasadena, California, U.S.A.
TOKIN, B. P., Department of Zoology, University of Lenin24
Jan.
grad, Leningrad, U.S.S.R.
NEWCOMBE, H. B., Biology Branch, Atomic Energy of Canada
Feb. 28
Limited, Chalk River, Ontario, Canada
Apr.
2
KANG, Y. H., Department of Botany, Yonsei University,
Seoul, Korea
Apr. 16-21 SCHMIDT, J, W., Department of Agronomy, University of
Nebraska, Lincoln, Nebraska, U.S.A.
Apr. 19
WAYLAND, H., Department of Engineering Science, California Institute of Technology, Pasadena, California, U.S.A.
May
4JORDAN, H. 0., Economics Development Institute, Universityof Nigeria, Enugu, East Nigeria
TAKAHASHI, T., Department of Geology, University ofJun. 30
Rochester, Rochester, New York, U.S.A.
DEGENHARDT, K. H., Institut fur Humangenetik und Ver18
JuI.
gleichende Erbpathologie der UniversWit Frankfurt am
Main, Germany
DAGG, C. P., The Jackson Laboratory, Bar Harbor, Maine,
19
JuI.
U.S.A.
GAFFNEY, L., c/o A. A. Tegel Pty. Ltd., Leppington, New
29
JuI.
South Wales, Australia
CARTER,O., Department of Agronomy, University of Sydney,
Aug.
9
New South Wales, Australia
CHO, W. K., Department of Zoology, Seoul National UniAug. 15
versity, Korea
CHAKRAVARTTI, M. R., Indian Statistical Institute, Calcutta,
Aug. 25
India
MAVALWALA,]., Department of Anthropology, Universityof California, U.S.A.
SWAMINATHAN, M. S., Division of Botany, Indian Agricultural
Research Institute, New Delhi, India
KIM, Y. C., College of Agriculture, Korea University, Seoul,Aug. 28
Korea
SHAHI, B. B., Seed Testing Laboratory, Department of Agriculture, H. M. G., Kathmandu, Nepal
YO, C. J., Department of Botany, College of Science,
National Taiwan University, Taipei, Taiwan, ChinaJan.

144 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

Aug.

Aug.
Aug.
Aug.
Aug.

Sep.

Sep.

28

HOUSE, L. R., The Rockefeller Foundation, New Delhi, India
CHAKRABANDHU, M.C., Department of Agriculture, Bangkok,
ThailandCARANGAL, A. R. Jr., Department of Research and Development, San Miguel Corporation, Manila, Philippines
Ku, Y., College of Agriculture, National Taiwan University, Taipei, Taiwan, China
CREECH, J. L., New Crops Research Branch, Beltsville,
Maryland, U.S.A.
CALDECOTT, R. S., Department of Agronomy and Plant
Genetics, University of Minnesota, Minnesota, U.S.A.
NATARAJAN, A. T., Institute of Radiobiology, Stockholm
University, Stockholm, Sweden
SIGURBJORSSON, B., Plant Breeding and Genetics Section,
I. A. E. A., Vienna, Austria
29
CLAUSEN, J., Department of Plant Biology, Carnegie Institution of Washington, Stanford, California, U.S.A.
30
LIDICKER, W. Z., Jr., Museum of Vertebrate Zoology, University of California, Berkeley, U.S.A.
31
KANG, Y. S., Department of Zoology, Seoul National University, Seoul, Korea
31-Sep. 1
BROCK, R.O., Division of Plant Industry, Commonwealth
Scientific and Industrial Research Organization, Canberra,
Australia
FRYXELL, P. A., Soil Crop Sciences Department, Texas A.
and M. University (U.S. Department of Agriculture),
Texas, U.S.A.
LEWIS, H., University of California, Los Angeles, California, U.S.A.
GRANT, W. F., Department of Genetics, McGill University,
Montreal, Canada
ASTAUROV, B. L., Department of Experimental Embryology,
A.N. Severtzov Institute of Animal Morphology, Moscow,
U.S.S.R.
CARSON, H. L., Department of Zoology, Washington Uni1
versity, St. Louis, Missouri, U.S.A.
LOVE, A., and LOVE, D., Department of Biology, Universityof Colorado, Boulder, Colorado, U.S.A.
2-3 HUNZIKER, J. H" Department of Biological Sciences, University of Buenos Aires, Buenos Aires, Argentina

FOREIGN VISITORS IN 1966

Sep.
Sep.

Sep.
Oct.
Oct.

Oct.
Nov.

Nov.
Nov.

145

6

MEYER, G. F., Max-Plank-Institute fUr Biologie, Tiibingen,Germany9
PAIK, Y. K., Department of Biology, School of Science,
Yonsei University, Seoul, Korea
GIESBRECHT, P., Molecular Biology Division, Robert Koch
Institute, Berlin, West Germany
20-21 BHATIA, C., Indian Agricultural Research Institute, New
Delhi, India
27
LEJEUNE,]., Institut de Progenese, Faculte de Medecine,
Universite de Paris, Paris, France
28-29 LAW, L. W., Carcinogenesis Section, National Cancer Institute" N. I. H., Bethesda, Maryland, U.S.A.
ROOSA, R. A., Wistar Institute, Philadelphia, Pennsylvania,
U.S.A.
STUDZINSKI, G., Jefferson Medical College, Philadelphia,
Pennsylvania, U.S.A.
PLEIBEL, N., Wistar Institute, Philadelphia, Pennsylvania,
U.S.A.
29-31 BORGHESE, E., Istituto di Anatomia Topografica, Dell'
Universita di Napoli, Napoli, Italy
2
KOLLER, P. C., Department of Cytogenetics, Chester Beatty
Research Institute, Institute of Cancer Research, Royal
Cancer Hospital, London, England
POGGENDORFF, W. H. G., Department of Agriculture, Faner
Place, Sydney, New South Wales, Australia
10
BAPAT, C. V., and BHISEY, A. N., Biology Division, IndianCancer Research Centre, Parel, Bombay, India
11
GERHARTZ, H., Medizinische Klinik der Freien UniversiUit,
Berlin, Germany

ACKNOWLEDGMENT
The editor wishes to express his sincere gratitude to Dr. F. A. LILIENFELD
for her kindness in reading the original manuscripts. (Yukiaki KURODA)

146 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 17

AUTHOR INDJ:X
107,114BALAL, B. S. . . . . . . . . . . . . . . . . . . . . . . .. 30
CHU, Y. E
51, 59CROW, J. F ••.......................• 84
ENDO, T ••.......................... 50
ENOMOTO, M
121, 122, 123
FUJII, T. .
109, 110, 111, 115, 117
FUJISHIMA, T.
22
FUKASE, Y
95
HAYASHI, M ••...................... 107
HAYASHI, S •........................ 27
HIGUCHI, S. •. . . . . . . . . . . . . . . . . . . . . . .. 25
HORI, T ••.......................... 19
IINO, T
119, 120, 121, 124
IMAI, H. T ••.............. 11, 12, 13, 14
ISHIDSU, J. .
125
ISOGAI, 1. •.......................... 21
IYAMA, S •.......................... 28
KANG, Y. S.
61
KATAYAMA, T. C •......... 19,21,56,57
KATSUYA, K •....................... 85
KAWAHARA, T
21
KIHARA, H. •. . . . . . . . . . . . . . . . . . . . . . .. 19
KIKUCHI, Y •.......... 129,130, 131, 133
KIMURA, M. .
64, 65, 67
KOJIMA, K. •.•...................... 42
KURITA, Y. •..................... . .. 15
KURODA, Y •............ 17,18, 3i, 35, 37
MABUCHI, T
112,114MARUYAMA, T •............ 67,68,70,72
MASUDA, T •••...................... 14
MATSUDA, E. .
131
MATSUMURA, S ••. '" .. 107, 109, 112, 115
MATUNAGA, E. ..........• 126, 127, 129
MIGITA, S.
11, 14MITANI, M. • •...................... 120
MIYAHARA, M. .
127
AMANO, E

MORIGUCHI, Y •.................• 61,62
MORISHIMA, H. .
32, 33, 59
MORIWAKI, K
11,12
MUKAI, T ••....................• 83,84
MUKAIDE, H.. . . . . . . . . . . . . . . . . . . . . . .. 27
MURAKAMI, A. .
98, 100, 102, 103
NAKAI, Y •.......................... 58
NAMBA, Y •......................... 14
NARISE, S.
31
NARISE, T
86,87,88
NAWA, S
48
OGAWA, Y
135
OISHI, H
129, 130, 131, 133
OKA, H.1. ................• 32, 33, 59, 60
ONIMARU, K •................. 91, 92, 94
OSHIMA, C •...........• 77, 78, 79, 80, 81
POTTER, M.
13
SADO, T •........................... 90
SAKAI, K. 1. ..... 21, 25, 26, 27,30, 31, 88
SAKAMOTO, S. •. . . . . . . . . . . . . . . . . . . . .. 55
SAKURAI, S •............... 40, 42, 44, 46
SHIMAKURA, K
61
SHINODA, T
53,134SUZUKI, H. •121
TAZIMA, Y •.. 90,91,92,94,95,97,98,100
TONOMURA, A
129
TSUJITA, M
39, 40, 42,44,46

15,16
.,105
W ASANO, K. .. . . . . . . . . . . . . . . . . . . . . .. 26
WATANABE, T •..................... 81
WATANABE, T. K
77,78,79,80
YAMADA, M. . . . . . . . . . . . . . . . . . . . . . . .. 48
YAMAGUCHI, S. . ..............• 119, 123
YASUDA, N
73, 74, 75

TSURUTA, R
TUTIKAWA, K.

YOSIDA, T. H.

.......... 11, 12, 13, 14, 15, 16, 61, 62

fjJftJlta~liif~miF~m 17 i}

1lH;fll42:¥ 7 ~ 20 El i=[HilU
1lH;fll42 :¥ 7 ~ 25 El ~rr*~••~ m.

ill

ffi AilU~:tt:~rr:lf~m3ZiI~~liif~mr*.J

fi

lIE

m3ZlI~~liif~mr*.J~.*

JkJjl;fi~ml8:llI~1IlT

ffiJilUm

Jm
184:~ mt~~~!=IJAilUM:

JkJj(fi~ml8:llI~1IlT184~ rr mI~

IE iL iI iii; ~ if 3't fiJi

"1Ml~=:Szm~EE 1,111'i[~·(=:Sz) (75) 0771, 0772, 4228(<<rd'l) 3492

,
q 100 ~..-...

"

.

'00

eqo

eoo

IC\OOnt