NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 18

1967

PublUlred by

THE NATIONAL INSTITUTE OF GENETICS
Miaima. Sizuoka-ken. Japan1968

Annual Reportof the~r

National Institute of Genetics
No. 18, 1967

Published by

The National Institute of Genetics, Japan
1968

CONTENTS
General statement..................................................
Research member..................................................
Council............................................................
Projects of research for 1967.......................................
Researches carried out in 1967 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

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Cytogenetics
The use of chromosomal interchanges to test for crossing overand chromosome segregation. KIHARA, H. and SHIMOTSUMA,

M..........................................................
Characteristic aggregate formation of mouse plasma tumor cellswith different chromosome numbers. KURODA, Y...........
Selective sorting-out property of mouse plasma cell tumors for
specific types of normal cells. KURODA, Y..................
Cytogenetical and biochemical studies of 19 primary plasma
cell neoplasms induced in BALB/c mice. YOSIDA, T. H.,
IMAI, H. T. and MORIWAKI, K,..
.
Periodic ploidy changes in the mouse plasma cell tumor, MSPC1. IMAI, H. T., YOSIDA, T. H. and MORIWAKI, Koo...........
Cytological basis of tetraploidization mechanism found in mouseplasma cell tumor, MSPC-1. IMAI, H. T., YOSIDA, T. H.
and MORIWAKI, K.
Effect of gene dosage on the rate of protein synthesis in themouse plasma cell tumore, MSPC·1. MORIWAKI, K., IMAI,
H. T. and YOSIDA, T. Hoo..................................
Chromosome studies in 12 malignant hamster cell lines transformed in vitro by treatment with 4HAQO. YOSIDA, T. H.,
MASUJI, H. and KUROKI, Too................................
Chromosome abnormalities in bone marrow cells in several
strains of mice after injection with 4-nitroquinolin I-oxide.
YOSIDA, T. H. and TSURUTA,
Karyological studies of three species of insectivora collected inJapan. YOSIDA, T. H., MORIGUCHI, Y. and SONODA, J........
R . o o o o . . . . . . oo • • oooo . . . . . o o . o o

II. Physiological and developmental genetics
Effect of thermoshock and virus inoculation on cytoplasmic malesterility in Capsicum and Petunia. OHTA, Y................
Zymographic analysis of cytoplasmic male sterile Petunia andZea. OHTA, Y..............................................

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Histogenetic aggregation of dissociated imaginal disc cells of
Drosophila melanogaster larvae in rotation culture. KURODA,
Y.
Differentiation of imaginal discs of Drosophila melanogastercultured in synthetic medium and effects of some hormonal
substances. KURODA, Y. and MINATO, K....................
Distribution of auxin protectors and their kinetic study inJapanese morning glory. YONEDA, Y. and STONIER, T. .....
Distribution of IAA oxidase and peroxidase in Japanese morning glory stem. YONEDA, Y................................

III.

Biochemical genetics
Genetic effects of DNA in Ephestia. NAWA, S. and YAMADA,
M.A
Evidence of uptake of DNA by Ephestia eggs. YAMADA, M. A.
and NAWA, S.
Ribonucleic acid content in the hypodermis of the silkworm.
KOJIMA, K., TSUJITA, M. anp SAKURAI, S. .. . . . . . . . . . . . . . . . . .
Incorporation of 14C-amino acids into pteridine granules. SAKURAI,
S. and TSUJITA, M..........................................
Significance of the pteridine granule development in larval skincells of the silkworm. TSUJITA, M., KOJIMA, K. and SAKURAI,S..
Reconstitution of vesicular membrane of pteridine granules fromdegraded subunits. TSUJITA, M. and SAKURAI, S............
Formation of vesicle membranes of pteridine granules fromrepeated particles. TSUJITA, M. and SAKURAI, S.
A staining procedure of human serum lipoproteins on cellulose
acetate strips. OGAWA, Y.
Zymographic analyses of peroxidase-indoleacetate oxidase isozymes in several plants. ENDo, T. .•......................
Peroxidase and indoleacetate oxidase isozymes in Japanesemorning glory. YONEDA, Y. and ENDo, T.
Changes of peroxidase isozyme patterns by in vitro culture ofJapanese morning glory tissues. YONEDA, Y.
Effect of exogenous RNA on protein synthesis in L-M cell
culture. MORIWAKI, K......................................
Inhibitory effect of homologous RNA on protein synthesis incell-free system. MORIWAKI, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Myeloma protein synthesis in a cell-free system prepared frommouse plasma cell tumor, X-5563. MORIWAKI, K............

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CONTENTS

IV. Evolutionary genetics
Local differences in frequencies of No.1 chromosomal polymorphism of Rattus rattus. YOSHIDA, T. H.....................
Further studies on segregation of three chromosome types ofRattus rattus in the laboratory. YOSHIDA, T. H.. . . . . . . . . . . .
Serum transferrin polymorphism in Rattus rattus. MORIWAKI, K.
Migratory behavior of vestigial strains of Drosophila melanogaster. NARISE, T..........................................
Three intergeneric amphidiploids of the tribe Triticeae.
SAKAMOTO, S................................................
Phenetic variation and phyletic relationship of strains of Oryza
perennis, estimated by numerical taxonomic method.
MORISHIMA, H...............................................
Occasional escape from the isolating barrier surrounding Oryza
perennis subsp. barthii. CHU, Y. E. and OKA, H. I..........
Partial self-incompaticility found in Oryza penennis subsp. barthii.
CHU, Y. E. and OKA, H. 1...................................
Comparison of variations in peroxidase isozymes between Ferennis-Sativa and Breviligulata-Glaberrima series of Oryza. CHU,
Y. E. and OKA, H. 1.
V.

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57

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59

59

Mathematical and statistical studies on population genetics
Gene frequency cline in a habitat of finite length. KIMURA, M.
and MARUYAMA, T.
On the special functions of, population genetics. MARUYAMA, T.
Development of temporary overdominance associated with neutral alleles. MARUYAMA, T. and KIMURA, MOo...............
Effect of epistasis on the propability of joint fixation in 2 loci.
KIMURA, M. and OHTA, T...................................
Effects of initial linkage disequilibrium on fixation probabilities
for 2 loci. OHTA, T........................................
Distribution of matrimonial distance in Mishima district.
YASUDA, N..................................................
The use of surname for estimating Wright's F-statistics in man.
YASUDA, N..................................................
A gene counting method of maximum likeilihood for estimating
gene frequency. YASUDA, N................................

VI. Experimental studies on population genetics
Allelic rate between lethal genes extracted from Japanesenatural populations of Drosophila melanogaster. OSHIMA, C.
and WATANABE, T. K.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Viability of lethal heterozygotes under fluctuating environment.
OSHIMA, C. and WATANABE, T. K............................
Persistence of a lethal gene associated with SD in cage populations of Drosophila melanogaster. WATANABE, T. K.
Interference by a foreign species in the genetic constitution ofa Drosophila melanogaster population. NARISE, T...........
Aggressiveness of race 21B of wheat leaf rust. KATSUYA, K. ..

VII. Radiation genetics and chemical mutagenesis in animals
Comparison of repair ability of premutational damages among
different radiation sensitivity strains of the silkworm.
T AZIMA, Y. and ONIMARU, K.
Aerobic and anoxic X-irradiation to spermatids in different
radiation sensitivity strains of the silkworm. T AZIMA, Y.,
ONIMARU, K. and ONUMA, A.................................
Relation between character of diapausing and mutation response
to r-radiations observed in the silkworm. TAZIMA, Y.,TEULADE, P. and ONIMARU, K. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Difference in the proportion of mosaics among mutants inducedby 14 MeV neutrons, r-rays and some chemical mutagens
in silkworm spermatogenic cells. TAZIMA, Y., ONIMARU, K.
and FUKASE, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage of r-irradiation to the germ cells and survival of discovered mutants in the silkworm. T AZIMA, Y. and ONIMARU,
K.
..•....
Studies on strain differences in radiosensitivity of the silkworm
III. Comparison of radiosensitivity of a sensitive and a
resistant strain during early cleavage cycle. MURAKAI, A. .
Studies on strain differences in radiosensitivity of the silkworm
IV. Variation in radiosensitivity to different X-ray dose
during an early embryonic stage. MURAKAMI, A.
Studies on strain differences in radiosensitivity of the silkworm
V. Relation between X-ray and UV-sensitivity in early
embryonic stage. MURAKAMI, A. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Relative biological effectiveness of fast neutrons for the inductionof dominant lethals at various stages of male germ cells inthe silkworm. MURAKAMI, A. ... . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of X-irradiation on aggregate·forming activity of embryonic chick and quail liver and heart cells. KURODA,

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Y. •.......••••...•......•..................................

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Skeletal abnormalities in the F 1 offspring of neutron-irradiatedmale mice. TUTIKAWA, K.

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CONTENTS

v

VIII. Radiation genetics in microorganisms and plants
Selective growth of radiation sensitive mutants in Escherichia
coli K12. KADA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Radiosensitization by potassium iodate and related compounds.
KADA, Too..................................................
Photoreactivation of UV-induced mutations in maize. FUJII, T.
Endosperm mutations in maize induced by fast neutrons. FUJII,
T
Sectorial mutations induced by r-ray treated fertilized maize
endosperm. FUJII, T.......................................
Modification of killing efficiency in neutron irradiations ofArabidopsis seeds. FUJII, T.
Storage and water content effects on radiation damage in wheat.
MABUCHI, T. and MATSUMURA, S.............................
Wave length dependence of UV damage in maize pollen.
AMANO, E.
Comparison of photoreactivating activity on different chromosomes of maize. AMANO, E.
Dosimetry systems for the 14.1 MeV neutron generator. HAYASHI,
M. and AMANO, E.

101
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112

IX. Microbial genetics
Fingerprinting analysis of fiagellin of antigen type Salmonella
recombinants. YAMAGUCHI, S.
..............
Mapping of some jla and mot cistrons closely linked to Hi in
Salmonella. YAMAGUCHI, S. and IINo, Too...................
Behavior of conjugational hemizygotes of Salmonella typhimuriumin special reference to motility characters. IINO, T.
Flagellar characters of an F-his strain of Salmonella typhimurium. IINO, T..............................................
Newly isolated arginine sensitive mutants of Salmonella typhimurium. ISHIDSU, J.
Conversion to streptomycin-sensitivity induced by A-lysogenization accompanying increase in reversion frequencies toprototraphy in Escherichia coli K12. KADA, T..............

X.

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Human genetics
Sexual variation in finger pattern types and ridge counts.
MATSUNAGA, E. and MATSUDA, E
120
Increased finger ridge counts in Turner's syndrome. MATSUNAGA,
E., MATSUDA, E. and OISHI, H.
121
The complete amino acid sequence of a Atype Bence-Jones protein.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

SHINODA, T. .•........... . . . . .... ... ..... . . . .... . . . . . .... .. .
Cytogenetic study on familial Down's syndrome. KIKUCHI, Y.,OISHI, H., TONOMURA, A. and KURITA, T.
An incomplete No. 5 chromosome pair in two patients with the
cri du chat syndrome. OISHI, H., KIKUCHI, Y. and MATSUDA,E
Clinical conditions of patients with apparently normal chromosomes, IV. OISHI, H., KIKUCHI, Y. and SHIBATA, K.

XI.

123

124

125126

Applied genetics
General and differentiol relative growth rates of mutant lines
129
of rice. aKA, H.1. and MORISHIMA, H.
Further observations on the effects of the earliness gene, E, inthe genetic background of a rice variety, Taichung 65.
TSAI, K. H. and aKA, H. 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129Analysis of variance in diallel crosses with parents in nested,hierarchal, classification. FUJISHIMA, T..................... 130
Electrophoretic variation in enzymes of tobacco plants. NARISE,
S........................................................... 133
Interaction between sire genotype and photoperiodic treatment
in Japanese quails. KAWAHARA, T.
133
Experiment on bilateral asymmetry in wings of Drosophilamelanogaster. NARISE, T. and SAKAI, K. 1.. . . . . . . . . . . . . . . . .. 134Multidirectional selection in Japanese quails. INOUYE, T. and
SAKAI, K. 1.
135Biochemical relashionships among different organs of tobacco
plants. NARISE, S. and SAKAI, K. 1. .... 000... 00000..... 0. .• 136
Competition in relation to tree age in Cryptomeria japonica.
HAYASHI, S. and SAKAI, K. 1. 000 •...... 0..... 000.••.. 0. . . .. .• 137
Analysis of a clonal progeny in a natural forest of Cryptomeriao
SAKAI, K.1., HAYASHI, So, OHBA, K. and TOMITA, K.......... 138

0...
Books and papers published in 1967 by research members
Abstracts of diary for 1967
0000
00. . .. .. .. .. .
Foreign visitors in 1967 00
0......................
Acknowledgement.
00.........•••••.. 0.00. 0" 0...................
Author index
0.0
0.. o' 0.0 00 .. 0..•.••.. 0............

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GENERAL STATEMENT
According to our expectation, the budget for the main building was
allocated this year. The complete edifice is shown on the front pageof this report. It took eight years to renew the old wooden structurewith the present one built in concrete.
To our great regret, we have to announce that Dr. Seiji Matsumura,head of the Department of Induced Mutation died from leukemia on the
19th of February, 1967, at the age of 57.
Dr. Matsumura started his scientific career after graduating from Kyoto
University, majoring in genetics in 1931. His main interests in his earlydays were directed to the cytogenetics of pentaploid wheat hybrids. He
could establish seven nullisomics of the D-genome and he also found theprinciples governing the change of chromosome numbers in the offspringof those hybrids. With his fundamental study of triploid sugar beets a
successful triploidy breeding of this crop was established.
He joined our Institute as a staff member after its establishment in
1949. He became the head of the Department of Induced Mutation in
1955, where he showed his ability as research worker as well as administrator. He succeeded in selecting good collaborators and equipping
his laboratory with various instruments within a short time. His many
valuable contributions were published in several series. Among others
his efforts were directed to the study on the mechanisms of radiation
injury and to the application of induced mutations to plant breeding.
He was also associated with the Kihara Institute for Biological Research
as its secretary. Due to his efforts the institute could overcome the difficulties encountered during and after the War. With his untimely passing
away the National Institute has lost an excellent scientist and administrator and the Kihara Institute deplores the loss of the efficient manager.
Our staff members are busy in the preparation for the coming XIIInternational Congress of Genetics which will be held in Tokyo, 1968.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

RESEARCH MEMBER
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.
ONIMARU, Kimiharu
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi
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
IMAI, Hirotami T., D. Sc.; MASUJI,* Hiroshi;
TSURUTA,* Reiko; SEKIYA,* Kunio
The 2nd Laboratory
MORIWAKI, Kazuo, 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.; OHTA,* Yasuo, D. Ag.

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

RESEARCH MEMBER

3

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
TSUJIUA, 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, Do Ago, Head of the Laboratory
KAWAHARA, Takatada, D. Ag.; FUJISHIMA, Tohru, D. Ag.;NARISE, * Takashi, Do 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
aKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.; CHU,* Yaw-En; SHAHI,* B. B.
6. Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAMA, Kiyosi, Acting Head of the Laboratory
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
FUJII, Taro, D. Ag.; KATSUYA, * Keizo, Ph. D.
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo; SADAIE, Yoshito; HAYASHI, Masaru
7. Department of Human Genetics
MATSUNAGA, Ei, D. Medo, D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

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, * Kunihiko

8. 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, D. Sc.
The 2nd Laboratory
IrNO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.; ISHIDSU, Jun-ichi
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
MARUYAMA, Takeo, Ph. D.
The 2nd Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
YASUDA, Norikazu, Ph. D.
10. Experimental Farm
SAKAI, Kan-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira
Honorary 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

OYAUCHI, Toru, 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 InstituteTAzIMA, Yataro, Managing Director, Head of the Morphological Genetics
Department
OSHIMA, Chozo, Manager, Head of the Physiological Genetics
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 University of Tokyo
MORIWAKI, Daigoro, Vice Chairman, Professor of Tokyo Metropolitan
University
BABA, Isamu, Director of National Institute of Agricultural Sciences
FURUHATA, Tanemoto, Director of Scientific Research Institute of Police
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIKKAWA, Hideo, Professor of Osaka University
MAKINO, Sajiro, Professor of Hokkaido University
MATsuo, Takane, Professor of University of TokyoMISONOO, Keisuke, Director of National Institute of Radiological Sciences
OCHI, Yuichi, President of Azabu University of Veterinary Science
OGUMA, Kan, Emeritus Professor of Hokkaido University
SAKATA, Takeo, President of T. Sakata Company
T ACHI, Minoru, Director of Institute of Population Problems
UEMURA, Teijiro, Director of Institute of Applied Microbiology, Universityof Tokyo
WADA, Bungo, Emeritus Professor of University of TokyoWATANABE, Yasusi, President of Shizuoka University

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

PROJECTS OF RESEARCH FOR 1967
Department of Morphological Genetics

Genetics of the silkworm (TAZIMA and ONIMARU)
Repair processes in radiation mutagenesis (TAZIMA and ONIMARU)
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 and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture(KURODA)
Effects of radiation on cells in tissue culture (KURODA)
Department of Cytogenetics

Cytogenetical and biochemical studies on tumor cells (YOSIDA, MORIWAKI,
IMAI and MASUJI)
Mechanism of chromosomal abnormalitites by treatment with chemicals(YOSIDA, SEKIYA and TSURUTA)
Studies on chromosomal and biochemical polymorphism of Muridae (YOSIDA,
MORIWAKI and MORIGUCHI)
Experimental breeding and genetices of mice and rats (YosIDA, MORIWAKI,SAKAKIBARA, MORIGUCHI and SONODA)
Morphological and genetical studies on some plant tumors (YONEDA and
CHU)
Cytogenetical and biochemical studies on morning glory (YONEDA)
Department of Physiological Genetics

Population genetics of deleterious genes in nautral populations of Drosophila melanogaster (OSHIMA and WATANABA)
Mechanisms of persistence of some lethal genes (OSHIMA and WATANABE)
SD (segregation distorter) associated with lethal genes (WATANABE)
Analysis of fitness in a fluctuating environment (OSHIMA and WATANABE)
Nucleus substitution in wheat and related species (KIHARA, SAKAMOTO and
OHTA)
Basic studies on hybrid wheat breeding (KIHARA)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Genetic bases of ecological differentiation in Agropyron (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Studies on cytoplasmic inheritance in higher plants (OHTA)RESEARCHES CARRIED OUT IN 1967

7

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 retardede moulting (rm) in the silkworm (TSUJITA)
Studies on the pteridine granule formation in larval hypodermal cells of
the silkworm (TSUJITA, SAKURAI and KOJIMA)
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)
Genetical and biochemical studies of membrane proteins in the silkworm(SAKURAI and TSUJITA)
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 hAMA)Studies on competition in plants and animals (SAKAI, hAMA, 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)
Studies on the effects of X-ray irradiation on quantitative characters ofrice (hAMA)
Biochemical studies on development of higher plants (SAKAI, NARISE, S.
and HONDA)
Genetic studies of isolating barriers in Oryza (OKA and CHU)
Numerical taxonomic studies 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 in rice (MORISHIMA and OKA)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Department of Induced Mutation

Radiation genetics in mice (TUTIKAWA)
RBE and dose rate effect in higher plants (FuJII and AMANO)
Radiation genetics in Arabidopsis (FuJII)
Fine structure analysis in maize (AMANO)
Biological effects of ultraviolet radiation (KADA, FUJII, AMANO and HAYASHI)
Radiation-induced and chemical mutagenesis in microorganisms (KADAand SADAIE)Radiation dosimetry (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 acids and their constituents (SHINODA)
Department of Microbial Genetics

Genetic fine structure analysis on microorganisms (IINO and YAMAGUCHI)
Genetics of cellular regulatory mechanisms (SUZUKI and ISHIDSU)
Genetics of bacterial flagella (IINo, ENOMOTO and SUZUKI)
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)
Uses of computers in the theoretical studies of population genetics(KIMURA and MARUYAMA)
Studies on the genetic structure of human populations (YASUDA)RESEARCHES CARRIED OUT IN 1967I.

CYTOGENETICSThe Use of Chromosomal Interchanges to Test for Crossing
Over and Chromosome Segregation
Htoshi KIHARA and Minoru SHIMOTSUMA*

Tetrad pollen association was studied in the watermelon. It was foundto continue for a short time after the dehiscence of the anthers. According to the number of normal pollen grains within a tetrad, these wereclassified into five types (Fig. 1).
Type-I: 4 normal pollen grains in a tetrad.
Type-2: 3 normal, 1 empty in a tetrad.
Type-3: 2 normal, 2 empty in a tetrad.
Type-4: 1 normal, 3 empty in a tetrad.
Type-5: 4 empty in a tetrad.

'5,~~,(;t.,Type-!

Type-2Type-3Type-4Type-5

Fig. 1. Photomicrographs of 5 types of pollen tetrads in the chromosomalinterchange heterozygotes (@+9 II ).

All types except type-5 were found in the three normal vanetles
employed. Five types of pollen tetrads were observed in three interchange heterozygotes (@+9u) obtained from r-irradiation. Frequency ofthese five types was different in different strains. Type-I tetrads are
the result of alternate segregation of a ring of 4 chromosomes (@), while
type-5 tetrads are obtained either from adjacent-I or adjacent-2 segregation. Therefore the ratio of type-I to type-5 tetrads represents theratio of alternate to open segregation. Those ratios were respectively
1.0: 1, 2.6: 1 and 2.1: 1 for the three interchange heterozygotes, MT-I,
AT-1 and FT-1. The ratio of alternate, adjacent-I and adjacent-2 segre* Kihara Institute for Biological Research, Yokohama.

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Varieties
Miyako No.1
Asahi-Yamato
Fumin

Strains
MT-l
AT-l
FT-l

Alternate222

Adjacent-l

Adjacent-2

1

1

1
1

o
o

gation was estimated as follows: Type-3 pollen tetrads are the result of
single crossing over between the interstitial segments. Therefore thepercentage of type-3 tetrads corresponds roughly to the double numberof single crossover. In the three interchange heterozygotes, MT-l, AT-l
and FT-l, the percentage was 14.5, 59.8 and 61.3, respectively. A fairly
interesting detail of our work was the finding that from the distributionof normal and aborted pollen grains in a tetrad, we could recognize
whether a single crossing over had occurred or not.

Characteristic Aggregate Formation of Mouse Plasma Tumor
Cells with Different Chromosome Numbers l l
Yukiaki KURODA

A solid tumor of plasma cell neoplasm X5563, originally obtained fromDr. Michael Potter, National Cancer Institute, Bethesda, Maryland, andmaintained in this Institute by serial subcutaneous transplantations in
C3H mice, was examined for its specific aggregation pattern from dissociated single cells by rotation cultures.
It has previously been reported that this tumor had a modal chromosome number of 84 with one submedian metacentric and one subtelocentricchromosome as markers (Yosida et al., 1966).
The solid tumor was dissociated into single cells by treatment withtrypsin and cell suspensions each containing 106 cells in 3 ml culture
medium were rotated on a gyratory shaker by the standard procedure.
After 24 hours of rotation culture two aggregates were formed whichwere different in their morphology, namely one was large and compact
with a rough surface and the other was small, loose and amorphous.
Histologically the former consisted solely of degenerated cells in which
the nuclei were faintly stained and some had completely lost their stainability with hematoxylin, whereas the latter consisted solely of active
cells, some being in mitotic phase.
The ascites form of plasma cell tumor which developed in a BALB/cmouse after injection with complete Freund adjuvant was tested for its
aggregation pattern in rotation cultures. Cell suspensions each contain11 This work was supported by a Grant in Aid for Foundamental Scientific Research from the Ministry of Education in Japan.

RESEARCHES CARRIED OUT IN 196711

ing 106 cells in 3 ml culture medium were rotated by the standard
procedure. After 24 hours of rotation culture two or three sphericalaggregates were formed. It was found that histologically in these aggregates active cells in the center were surrounded by degenerating
peripheral cells.
From the results obtined from the rotation cultures of mouse plasma
cell tumors, both solid and ascites forms, it is concluded that the degenerating cells become different in their surface properties from theactively proliferating cells of the same cell population and the former
are sorted out from the latter in rotation cultures. The difference inaggregation pattern between solid and ascites plasma cell tumors, i.e.,
solid tumors forming separate aggregates, either of active cells onlyand degenerating cells only, and ascites cell tumors segregating active
cells from degenerating cells in the same aggregate, may reflect thedifference in the in vivo conditions under which solid and ascites celltumors were maintained in the mouse.
Two strains of the solid tumor of mouse plasma cell neoplasm developed
by injection with complete Freund adjuvand and maintained for 14 generations of subcutaneous transplantations in mice were found to have diploid(2s=40) and hypotetraploid (2s=73) chromosome numbers.
The relationship between chromosome number and aggregation patternin those two strains was examined. The solid tumors were dissociatedinto single cells by trypsinization and the cell suspensions were rotatedas described above for 24 hours. The hypotetraploid tumor cells formed
larger and more compact aggregates than did the diploid cells underidentical conditions employed here. Since under the subcutaneous con·dition in the host mouse the former showed higher proliferating activity
than the latter, the aggregate-forming activity of mouse plasma celltumors may have some relation to the proliferating activity of tumor
cells.

Selective Sorting-out Property of Mouse Plasma Cell Tumors
for Specific Types of Normal CeIls ll
Yukiaki KURODA

The solid tumors of mouse plasma cell neoplasm which have near·tetraploid chromosome number, were dissociated into single cells bytrypsinization, intermingled with a variety of normal cells from chick1) This work was supported by a Grant in Aid for Foundamental Scientific Research from the Ministry of Education in Japan.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

Behavior of mouse plasma tumor cells intermingled witha variety of normal cells from chick embryos
Embryonic chick cells

Behavior of mouseplasma tumor
cellsType of cells

Age of embryo

Ectodermal

Epidermis
Ectoderm of CAM*

7th day
7th day

SegregatedMesodermal

Heart
Sclerotic cartilage
Mesonephros
DermisMesoderm of CAM*
Mesenchyme

8th day
8th day
7th day
7th day
7th day
4th day

Not-segregatedEndodermal

Liver
Endoderm of CAM*

8th day
7th day

Segregated* CAM: chorioallantoic membrane

embryos, and tested in rotation cultures for their selective sorting-out
property in co-aggregates with normal cells from tissues of various
embryonic origin. In these experiments, chick normal cells were ofadvantage as they could be distinguished by size and staining abilityof their nuclei from mouse tumor cells in co-aggregates of both, considering that it was known that cells show their specific histogenetic behavior
though they are derived from genetically remote animals.
When dissociated mouse plasma tumor cells were intermixed with avariety of normal cells from chick embryos and cultured in rotation,
the former showed a selective sorting-out property for specific types ofnormal cells as shown in Table 1.
The results indicate that mouse plasma tumor cells became sorted out
with embryonic chick cells of ectodermal and endodermal origin but
formed chimaeric tissues interspersed with chick cells of mesodermal
origin. Such selective affinity of tumor cells to mesodermal cells has
been demonstrated by HeLa cells as reported previously by the author(Ann. Rep. 17: 18, 1966). It is not known whether this selective affinity
to mesodermal cells is the property restricted to some tumor cells, i.e.,mouse plasma cell tumors and HeLa cells, employed by the author orthis character may be revealed by all tumor cells which are derived
from mesodermal tissue. It is an interesting idea that the selective
affinity of tumor cells for a specific type of normal cells might be connected with the selective mechanism by which the original neoplastic
cells metastasize to some specific types of normal tissues or organs.

13

RESEARCHES CARRIED OUT IN 1967

Cytogenetical and Biochemical Studies of 19 Primary Plasma
Cell Neoplasms Induced in BALB/c Miceli
Tosihide H. YOSIDA, Hirotami T. IMAI and Kazuo MORIWAKI

Near-tetraploid karyotypes were observed almost in all tumor cells oftransplantable mouse plasma cell neoplasms obtained from Dr. M. Potter(Yosida et al. 1964 and 1966, this report 14; 42 and 16: 45). In order toascertain the chromosomal condition of the primary plasma cell tumors,
we have already examined five of them (MSPC-l to 5) (Yosida et al.
1967, this report 17: 11). The modal chrosome number of one (MSPC-l)
Table 1.

Tumor line
(MSPC)
1*
2

3*
4
5*
6
7

8
9
10
11
12
13

14
15
16
17
18
19*

Plasma cell tumors (MSPC) induced by Freundadjuvant in BALB/c mice
Specific protein

Sex
Ser.

As.

rA
rF

rF
rA

+

+

+
+

+

+

+
+

+

+

+

Ur.

Character ofprimarytumor

Chromosome
number(mode)

Sol.

81 (40)93 (86)35~ 94 (86)39~168 (78)35~ 94 (44)As.
Sol. +As.
As.
Sol.+As.
As.
As.
Sol.+As.
As.
As.
As.
Sol.+As.
As.
As.
Sol.
As.
As.
As.
Sol.+As.

38~

39~

84 (79)(86)
83~ 90 (87)40~ 79 (41)40~ 90 (84)39~ 84 (40)
76~160 (80)76~ 78 (82)39~

75~16482~

86 (85)75~157(78)* Tumors transplanted successfully. Ser. = serum. As. = ascites. Ur. = urea.
Sol. = solid tumor. + = r-globulin detected. - = r-globulin not detected.
11 This work was supported in part by a research grant from the National
Cancer Institute (CA 07798-04), Public Health Service, U.S.A. and by a Grant in
Aid for Foundament Scientific Research from the Ministry of Education in Japan.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

was 40, of another (MSPC-5) 44, and the remaining three (MSPC-2, 3
and 4) had hyper- and hypotetraploid karyotypes. Recently 14 primary
plasma cell tumors in BALB/c mice were obtained about half to one
year after injection of Freund adjuvant, and we observed their character, r-globulin, and chromosome constitution. All tumors obtained areshown in Table 1, in which 5 already published tumors are included.
r-globulin was checked by electrophoresis in serum and/or ascites oftumor bearing mice. Among the 19 tumors r-globulin was found inserum of ascites of 11 mice, but was not found in 6 mice. r-globulinof two mice was not examined. As to the type of tumor, 12 were
found to be ascites tumors, two were solid tumors and the remaining 5
were solid tumors with ascites. It is interresting that transplantationof tumors was successful only when they have developed as solid tumors
or solid with ascites and in tumors positive for r-globulin production.
In 15 primary tumors the chromosomes were successfully observed.
Among them the modal chromosome number in two tumors was characterized by 40 chromosomes, two tumors had hyperdiploid chromosome
numbers and the remaining 11 had near-tetraploid karyotypes. Relation
between the frequency of polyploid cells and the latent period after injection of Freund adjuvant it given in Table 2. As the table shows,cells with diploid chromosome number were observed in tumors developed
after a short latent period such as 6 to 8 months, while tumors developed
Table 2.

Relation between polyploidization and latent period to tumor
development after injection of Freund adjuvant

Latentperiod(in months)

Tumor
line*
(MSPC)

±2n

±4n

±8n

6

7

14%

86

0

50

7

10

54.5

45.4

0

11

12
11
8
9

83.3
6.7
0
0

16.7
93.3
96.7
100

0
0
3.3
0

30
30
30
30

10

13

0

91. 7

8.3

12

12

19
14
17

0
0
0

90
100
100

10
0
0

30
41
30

8

* Same as in Table 1.

PloidyNo. of cells
observed

RESEARCHES CARRIED OUT IN 196715

10 to 12 months after injection had only tetraploid or octoploid cells.
Based on the above investigations, it is suggested that tumors can develop from cells with diploid chromosomes but they may change totetraploid level by creating vigorous cell types.

Periodic Ploidy Changes in the Mouse Plasma Cell Tumor, MSPC-l
Hirotami T. IMAI, Tosihide H. YOSIDA and Kazuo MORIWAKI

Besides the characteristic tetraploidization found in this tumor (Ann.
Rep. 17: 12), changes of tetraploid cell population to a diploid one werealso observed. By these two repeatedly reversed alterations, the ploidyof this tumor altered periodically. Up to now, this tumor has beentransplanted for more than two years, during which the periodic ploidy
change has been repeated at least four times (Fig. 1).
_100

...e

~

~

..

...J

W

U

o

90<{

rr

•

lL

o

'0

elL-

i •• '''1
1.
0

>u

o

5

8'"

0

00

§

..'_

i

z

i

..
.''.. .
"• I

>->--

w

~ •

00
0

••

!Br08
o

00

10
15
20
25
TRANSPLANT GENERATION

Fig. 1. Alteration in the frequency of tetraploid cells during serialtransplantations of the mouse plasma cell tumor, MSPC-l
Open circle: Ascites form. Solid circle: Solid form.

It was suggested that the tetraploidization was mainly due to binucleatecell formation by a failure of cytokinesis (This Ann. Rep.). On the other
hand the recovery of a diploid population seemed to be caused by aperiodic activation of the proliferation of diploid cells which were present
in a tetraploid cell population in minority. In most cases this recovery
appeared when the cell population consisted of fifty or sixty percent of
tetraploid cells. In ascites form tetraploidization proceeded irreversibly,This work was supported in part by a Grant in Aid for Foundamental Scientific
Research from the Ministry of Education in Japan and by a research grant fromthe National Cancer Institute (CA 07798-04), Public Health Service, U.S.A.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

The effect of r-ray irradiation of the host mouse on the recovery
of diploid cell populations in MSPC-l

Pretreatmentof hostTetraploids ininoculum

Tetraploids ingrown tumort*Control
200R
300R
400R

6296
62
62
62

20.9±11.0%
17.6± 7.7
15.7± 5.1
13.2± 5.9

0.87 <2.07 (596, df=22)
1.44<2.10 (596, df=18)
2.20>2.07 (596, df=22)* Each t value was calculated against control percentage.
whereas a remarkable recovery of diploid was observed when the tumor
was transplanted subcutaneously in a solid form (See Fig. 1, transplant
generation 28).
To obtain some information on the condition of the host mouse inconnection with the recovery of diploid cells, ascites tumor cells (62percent tetraploid cell population) were injected into mice subcutaneously
after r-ray (200, 300 and 400 R) irradiations. As shown in Table 1, a
remarkable recovery of diploid cell population was observed in all grown
tumors and no significant difference was found between control and experiments, except for a minor feature, namely the radiation in recovery
rate was less in the experimental group. This finding might suggestthat an immunological or some other physiological condition of the hostmice does not allow for such an extensive recovery of diploid cells. It
is interesting that there is a slightly significant increase in recovery athigh dosage of r-ray irradiation (400 R).
A clearer result was obtained in a simultaneous transplantation of 6
and 62 percent tetraploid tumor lines to the same host mouse (Table 2).
As far as observed, the former diploid tumors did not change the modeof their diploid stem line, but in all latter, 62 percent tetraploid lines,
recovery of diploid cells was observed. These experiments strongly
suggest that the recovery mechanism of diploid cells in a tetraploidtumor line is an intrinsic character of the tumor cells.
Table 1. The effect of host mouse on the frequencyof tetraploid cells in MSPC-l
Inoculation
Subcutaneous, at left abdomen
Subcutaneous, at right abdomen

Tetraploids ininoculum

Tetraploids ingrown tumor

62%6%34.3±10.5%
3.8± 1.796

RESEARCHES CARRIED GUT IN 1967

17

Cytological Basis of Tetraploidization Mechanism Foundin Mouse Plasma Cell Tumor, MSCP-l
Hirotami T. IMAI, Tosihide H. YOSIDA and Kazuo MORIWAKIMSPC-I tumor which primarily had developed as a diploid line (s=40)
has been characterized by a frequent occurrence of tetraploidization(Ann. Rep. 17: 12). The diploid line has been maintained by strict
selection and it had the tendency to change to tetraploidy. In thetransient state from diploid to tetraploid, the tumor was composed of amixed population of diploid and tetraploid cells. The ratio of tetraploid
to diploid cells varied with the condition of host or tumor.
Three hypotheses are proposed for the mechanism of tetraploidizationin this tumor line; namely, (1) endoreduplication, (2) cell fusion and (3)failure of cytokinesis. As far as observed, no mitotic figure showing
endoreduplication was found. Therefore the first hypothesis seems to beuntenable. On the other hand, there were frequently observed binucleate
cells in this tumor (Fig. I-B). The following evidences make the 3rd
hypothesis more probable than the 2nd one. (1) Two nuclei in the
binucleate cells were labeled simultaneously by H 3 -thymidine. (2) Tetraploid cells had frequently a pair of marker chromosomes (metacentrics orsubmetacentrics) which suggests the occurrence of autopolyploidization.
(3) Anaphase figures in which a cytokinesis seemed to have failed wereobserved.

B
Fig. 1. Cells in the mixed cell population of mouse plasma cell tumor,
MSPC-1.
I-A. Diploid cell. I-B. Binucleate cell. I-e. Tetraploid cell.
This work was supported in part by a Grant in Aid for Foundamental Scientific
Research from the Ministry of Education in Japan and by a research grant fromthe National Cancer Institute (CA 07798-04), Public Health Service, U.S.A.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

Frequencies of binucleate cells of mouseplasma cell tumor, MSPC-l

Frequency oftetraploid cells

Frequency of
binucleate cells

Total
observed
cells

Binucleate cells
Tetraploid cells

296
15
30
38
54
68
84

1.2396 ( 34)
3.43
( 60)
3.16
(133)
3.78
(157)
3.85
( 41)
2.89
( 27)
0.63
( 6)

2764
2009
4212
4150
1065
934
946

61. 596
19.9
10.5
10.0
7.2
4.3
0.8

Numbers in the parentheses represent observed cell numbers.

As shown in Table 1, the frequency of binucleate cells increased from
one to four percent in parallel with the increase of the percentage oftetraploid cells. The maximum frequency of tetraploid cells was about
40 percent, but it decreased with the increase of diploid cells. As asynchronization of the mitotic phase of the two nuclei in binucleate cells
appeared as a tetraploid figure at metaphase. The ratio of binucleateinterphase cells against tetraploid metaphase was calculated (Table 1).
The ratio was high at the stage of only a few tetraploid cells present
and it decreased gradually following the increase of tetraploid cells.
This result may indicate that at the start of tetraploidization, most of
the tetraploid cells are represented by binucleate cells in which the twonuclei unite. One of the possible sequences of events leading to tetraploidization found in this tumor is considered to be as follows: Diploidcell (Fig. I-A) ~ Karyokinesis -) Failure of cytokinesis - ) Binucleate cell(Fig. I-B) ~ Union of binuclei -) Tetraploid cell (Fig. I-C). The same
alteration of nuclei as above were frequently observed in the otherprimary plasma cell tumors (Ann. Rep. 17: 11).

Effect of Gene Dosage on the Rate of Protein Synthesisin the Mouse Plasma Cell Tumor, MSPC·l ' }Kazuo MORIWAKI, Hirotami T. IMAI and Tosihide H. YOSIDA

A diploid plasma cell tumor, MSPC-I-D, has been induced by FreundThis work was supported in part by a Grant in Aid for Foundamental Scientific
Research from the Ministry of Education in Japan and by a rasearch grant fromthe National Cancer Institute (CA 07798-04), Public Health Service, U.S.A.
11

19

RESEARCHES CARRIED OUT IN 1967
Table 1. Comparison of the rates of total and myeloma protein
syntheses, cell volume and protein content between diploidand hypotetraploid mouse plasma cell tumors.
Tumor strains

Ploidy (Mod. No.)
Total protein synth.
(cpmj106 cellsjhr)
Cell volume(mm 3j10 6 cells)
Protein content(pgj10 6 cells)
MPjTP ratio

TjD

ratio

MSPC-1-D

MSPC-1·T

Diploid (40)Hypotetraploid (73)

1.83

13.300±2.200

18,600±5,100

1.40

12.3±1.815.5±1. 7

1.26

72.0±23.8

107.0±32.6

1.50

0.0107 ±0.0012

0.0073±0.0007

0.68

t

3.21>2.85(1%, df=20)
6.68>2.98(1%, df=14)
3.41>2.80(1%, df=24)
5.13>3.36(1%, df=8)

MP: Myeloma protein synthesis
TP: Total protein synthesis

adjuvant treatment of a BALB/c mouse in Misima, 1966. From thesecond transplant generation of this tumor, a hypotetraploid subline,
MSPC-1·T, was derived. For the purpose of finding out whether the
duplicated genes in the hypotetraploid cell have the same degree of expression as in the diploid cells, the two tumor lines were compared interms of the rates of total and myeloma protein synthesis per cell.
The rate of total protein synthesis was measured by incorporating
C 14 -leucine into a TCA-insoluble fraction of tumor cells, and that of
myeloma protein was estimated from the radioactivity of the myeloma
protein secreted into the incubation medium and separated by DEAE
cellulose column chromatography. As shown in Table 1, the rates ofboth protein syntheses, cell volume and protein content did not increasein parallel with the duplication of chromosome number. These results
could suggest that the duplicated genes in the hypotetraploid cells might
be partially suppressed including the gamma globulin locus.
In this case, however, another possibility can be considered, namelythat the elimination of chromosomes at the formation of hypotetraploids
from true tetraploids was accompanied by the loss of specific genes
regulating the total and the myeloma protein synthesis, which might
result in an apparent suppression of gene dosage effect. To rule out
this possibility, the rates of the protein syntheses were determined
employing the transient stage of the plasma cell tumor which had
a mixture of various ratios of diploids and tetraploids but no hypotetraploids. The rate of total protein synthesis increased in parallel

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

with the increase of tetraploid cells until 40 percent, beyond which
it reached a plateau (Fig. 1). Between the rate of myeloma protein
synthesis and the percentage of tetraploid cells an almost similar relationship was demonstrated.
In the transient phase from diploids to tetraploids, a considerable
number of binucleate cells was observed. Frequency of binuclei per••" ...... ---------~
I
I

.
I

I

.I / •

o

•

"'~
•

50

100

Tetraploid cells ("to)

Fig. 1. Changes in the rate of total protein synthesis according
to the increase of tetraploid celli in MSPC-1.

\.

0'0
5
0
0

.: .

~
u

:>

c

15

0

10

~

.

(5

CD

2
C"l

4

3

2

.

in
!!!.

C"l

~

2:

~

~

:>

r::r

OJ

It

50
Tetraploid cells("!.)

100

Fig. 2. Frequencies of binuclei per tetraploid cell and binucleate
cells in MSPC-l tumor during ploidy shift.

RESEARCHES CARRIED OUT IN 1967

21

tetraploid cell was calculated. They showed the highest frequency
simultaneously with the lowest percentage of tetraploid cells and became
gradually fewer according to the increase of the tetraploid cells (Fig. 2).
Biochemical and cytological findings presented in this study may leadto the assumption that diploid cells in this plasma cell tumor can bechanged to "mononucleate" tetraploid cells after passing through thetransient stage of binucleate "tetraploid" cells and that the suppressionof gene dose effect in the total and myeloma protein syntheses occurs
only in "mononucleate" tetraploid cells, not in the binucleate "tetraploid"
ones.

Chromosome Studies in 12 Malignant Hamster Cell Lines
Transformed in vitro by Treatment
with 4HAQO and 4NQOII
Tosihide H. YOSIDA, Hiroshi MASUJI and Toshio KUROKI21

The relation between chromosomal alteration and development of tumors
is an important problm. The senior author presented in 1966 (lap. Jour.
Genet. 41: 439) a hypothesis that a gradual change of karyotype by
sequential events of mutation and selection is a causative process in the
production of cells exhibiting vigorous malignant proliferation. In orderto demonstrate the relationship between chromosomes and tumors, weobserved the chromosomes of malignant cell lines of golden hamster
embryos transformed in vitro at several stages of cultivation after treatment with 4 hydroxyaminoquinoline N-oxide (4HAQO) and 4-nitroquinolinel-oxide(4NQO). Seven malignant transformed cell lines (HA-l, HA-2,
HA-4, HA-6, HA-7, HA-8 and HA-15) had developed by treatment with
4HAQO in vitro, 4 lines (NQ-2 to NQ-5) by treatment with 4NQO and oneline (ZEN-4) had developed spontaneously in the course of serial in vitro
cultivations. The chemicals were used by the following techniques.
The final concentrations of the carcinogens were 4 x 10- 6 M of 4NQO
and 10- 5 M of 4HAQO, and Eagle's MEM medium containing the above
drugs was added to a cell sheet after 7 day culture, kept for one day,and then changed to fresh medium. The treatment was repeated every
other day two to 7 times.
II This work was supported in part by a research grant from the National Cancer
Institute (CA 07798~04), Public Health Service, U.S.A., and by a Grant in Aid forFoundamental Scientific Research from the Ministry of Education in Japan.
21 Research Institute of Tuberculosis, Leprosy and Cancer, Tohoku University,
Sendai.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Normal somatic cells of the golden hamster embryos growing in vitro
had 44 chromosomes. Among 20 cells analysed no cells with chromosome numbers deviating from normal diploid were found. Among 7lines developed by treatment with 4HAQO, 4 lines (HA-l, HA-2, HA-4
and HA-7) showed a hypotetraploid chromosome number, while 3 lines(HA-6, HA-8 and HA-l5) showed diploid number as a mode. Chromosomes in tumors of two lines (HA-l and 8), which were transplanted
successfully to hamsters were analysed. Chromosome number distribution in HA-l tumor was almost the same as in HA-l in vitro cells, but
the number of cells at diploid level decreased in the tumor growing invivo. In the HA-8 tumor line cells grown in vivo, hyperdiploid chromosome numbers increased more than in the cells growing in vitro. Among
4 lines developed by treatment with 4NQO, one line (NQ-2) showed ahypotetraploid mode (82 chromosomes), one line (NQ-5) a diploid mode(44), and the remaining two lines (NQ-3 and NQ-4) had bimodal distribution with one diploid and one tetraploid mode. In ZEN-4 transformed
spontaneously in vitro, the modal number of chromosomes was 45.
All 8 tumor lines having hypotetraploid chromosome numbers strikingly
deviated from karyotypes of normal somatic cells. Among 4 tumor lines
having diploid chromosome numbers, two (HA-8 and NQ-5) had almost
the same chromosome constitution as normal somatic cells, but the other
2 (HA-6 and HA-l5) showed different karyotypes. It was noticeable thatboth tumors, HA-8 and NQ-5, with normal karyotypes have developed ashort time after treatment with the chemical. The former developed 28
days after treatment with 4HAQO, and the latter about 10 days after
treatment with 4NQO. In all other tumors, numerical aberrations suchas trisomy, tetrasomy, monosomy and also structural shanges, such as
breaks and translocation, were observed. The transformation of thesetumors occurred a long time (50-300 days) after treatment with thechemicals. Based on the above investigation, it can be concluded thatthe majority of malignant cell lines transformed a long time after treatment with 4HAQO and 4NQO in vitro had altered karyotypes, but a few
tumors developed a short time only after treatment had almost normal
karyotype, but were easily changeable by transplantations to animals or
serial cultivations in vitro. The results obtained from the present study
confirm the hypothesis that chromosome alteration may play an importantrole in creating more vigorous and malignant cell types.

23

RESEARCHES CARRIED OUT IN 1967

Chromosome Abnormalities in Bone Marrow Cells in
Several Strains of Mice After Injection
with 4-Nitroquinolin l-oxide u
Tosihide H. YOSIDA and Reiko TSURUTA

It is well known that 4-nitroquinolin I-oxide (4NQO) is one of stronger
carcinogenic agents. Leukemias have been induced in A strain miee bytreatment with the chemical (Nishizuka and Nakakuki 1964, Gann 55 : 495).
On the other hand, remarkable chromosome aberrations were induced in
Yoshida sarcoma cells by injection of the chemical into peritomeal cavityof rats bearing the tumor (Yosida et al. 1965, Gann 56: 253). The objectof the present study was to ascertain whether this chemical can induce
chromosome aberrations in bone marrow cells of mice injected by it
subcutaneously, and, if so, whether its effect on the chromosomes differs
by the strain employed.
0.2 ml of 10- 3 M 4NQO was injected subcutaneously to newborn mice,and the chromosomes of bone marrow cells were observed 8, 10, 14, 18,
24, 48 and 72 hours after injection. Chromosome breaks and translocations were observed most frequently 10 hours after injection, and thenthe frequency gradually decreased, but was markedly reduced 18 hours
after. Frequencies of chromosomal abnormalities in bone marrow cells
Tabel 1. Frequency of chromosome aberrations that occurred in bonemarrow cells of various strains of mice injected with 4NQO
Strainof mice
C57BL/6
RF
AKR
ADD
BALB/c
C3H
SWM

Total
aberrant
cells (%)

96 of cellswith multiple
breaksAberration
per cellNo. ofmice
observed

28.1
23.7
38.2
40.3
23.6
28.2
47.0
30.3

0
0
9.25
0
0
0.9
4.6

0.499
0.305

0

0.425

3
2
2
2
5
1
3
4

+++*
0.719
0.515

+++
+++

* + + + Calculation not possible due to multiple breaks.
1) This work was supported in part by a research grant from the National Cancer
Institute (CA 07798-04), Public Health Service, U.S.A. and by a Grant in Aid forFoundamental Scientific Research from the Ministry of Education in Japan.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

in 8 mouse strains are given in Table 1. As the table shows, the frequency of cells with abnormal chromosomes was 23 to 40 percent, andit did not vary much by the strain of mice used, but multiple breaks
Le. two or more breaks in one chromosome, were observed only in 3
strains, namely AKR and C3H at high frequency and BALB/c strain at
low frequency. Frequency of aberrationa per cell increased almost linearlywith increasing frequency of cells with chromosome aberration. This
result suggests that chromosome aberration of bone marrow cells inducedby treatment with 4NQO occur at random.

Karyological Studies of Three Species of Insectivora
Collected in Japan
Tosihide H. YOSIDA, Yukuo MORIGUCHI and jun SONODA

Karyotypes of three house shrews (Suncus murinus), dsinezumi shrew(Crocidura dsinezumi) and Horsfield's shrew (Crocidura horsfieldii) belonging to the Insectivora were analysed. The house shrews and Horsfield's shrews were collected in Okinoerabu island, Kagoshima-ken, anddsinezumi shrew in Misima, Sizuoka-Ken. Karyotypes of bone marrowcells were analysed by usual drying technique.
Table l.

Species

Suncus murinus
Crocidura dsinezumi
Crocidura horsfieldii

Karyotype analysis of three Insectivora species
No. of pairs of several
autosome types
A

ST

SM

M

14
13
0

1
3

2
2

2

2

4

6

1

Type of
Total
sex-chromosomes chromosome
number
Y
X
SM
ST
M

SM
ST
SM

40
40
26

A =acrocentrics, ST = subtelocentrics, SM = submetacentrics, M = metacentrics.

The number of chromosomes in the house shrew, the dsinezumi shrew
and the Horsfield's shrew was 40, 40 and 26, respectively. Karyotypic
formulae of these species are shown in Table 1. As the table shows,
karyotypes of house shrew and dsinezumi shrew are rather similar,
but that of Horsfield's shrew is markedly different from those of two
species. The chromosomes of Horsfield's shrew are markedly larger
than those of house shrew and dsinesumi shrew. It is interesting tocompare the karyotypes of house shrew and Horsfield's shrew, because
if meta-, submeta-, and subtelocentric elements had been produced by

RESEARCHES CARRIED OUT IN 1967

25

centromeric fusion of 14 acrocentric pairs of the house shrew, the chromosome number would be reduced to 26 and the karyotype would become
very similar to that of Horsfield's shrew. From the above karyologicalinvestigations, it can be said that the karyotypes of dsinezumi shrew issimilar to that of house shrew, but a morphological difference in a few
chromosomes was found between them, and the karyotype of Horsfield'sshrew is more closely related to that of the house shrew.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

II.

PHYSIOLOGICAL AND DEVELOPMENTAL
GENETICS

Effect of Thermoshock and Virus Inoculation on Cytoplasmic
Male Sterility in Capsicum and Petunia
Yasuo OHTA

A phenotypical similarity has been known between cytoplasmic andvirus-induced male sterility in certain plants.
Cytoplasmic male sterility in Capsicum annuum L. is controlled by asterile cytoplasm (5) and a male sterile gene (ms) (Peterson 1958). 5
msms plants are pollen sterile, 5 Msms or 5 MsMs plants are pollen
fertile, and those having normal (N) cytoplasm are all pollen fertile.
The normal counterparts, N msms plants, are called maintainers. 5
Msms x N msms and N msms selfed seeds of variety Fresno Chile were
furnished by Dr. P. A. Peterson. The former produced male sterile (5msms) and male fertile (5 Msms) plants in about 1: 1 ratio. 5 msms
plants were crossed with several Japanese varieties. A progeny test
proved that Fushimiamanaga is a fertility restorer (N MsMs) and Ojishi
is a maintainer (N msms) (Ohta 1961).
On the other hand, 5 Msms plants obtained from a cross 5 msms
Freseno ChilexN MsMs Fushimiamanaga and N MsMs plants (Fushimiamanaga) were inoculated with an ordinary strain of cucumber mosaic
virus (CMV 0), two strains of tobacco mosaic virus, and tobacco ringspot
virus (TRSV). 5 Msms plants inoculated with CMV 0 or TRSV showed
reduced pollen fertility which was 64 to 68%. Selfing of those inoculated
plants has been successfully made; the selfed progeny will be tested forpollen fertility in the coming season.
After exposing 5 msms seedlings (varieties Fresno Chile and Ojishi)
for 15-120 minutes to 55-66°C temperatures, they were cultured as usual.
Several of Ojishi produced some normal pollen grains. The stronger was
the treatment the more normal pollen grains were produced, suggestingvirus-like nature of a cytoplasmic agent.
Cytoplasmic male sterility in Petunia hybrida Vilm. is also controlled
by a sterile cytoplasm (5) and a male sterile gene (ms) (Edwardson et al.
1967). Cytoplasmic male sterile (5 msms) and normal counterpart (Nmsms) seeds were furnished by Dr. J. R. Edwardson. Leaf saps of both
5 msms andN msms plants were inoculated into indicator plants suchas Vigna sinensis, Phaseolus vulgaris, Nicotiana glutinosa, Chenopodium
amaranticolor, and Cucurbita moschata. It was found that the cytoplasmic
agent was not infectious.

27

RESEARCHES CARRIED OUT IN 1967

Zymographic Analysis of Cytoplasmic Male Sterile
Petunia and Zea
Yasuo OHTA

Zymographic analysis during microsporogenesis (pre-meiosis to preanthesis) of cytoplasmic male sterile plants and their normal counterparts(maintainers) in Petunia hybrida Vilm. and Zea mays L. (Fla F6T and
Fla F6) were made with starch gel electrophoresis in peroxidase, nonspecific esterase, acid phosphatase, catechol oxidase, and several otherenzymes. Certain differences in zymographic pattern were recognized
between the cytoplasmic male steriles and their normal counterparts inperoxidase (Zea) and non-specific esterase (Petunia and Zea).
Histogenetic Aggregation of Dissociated Imaginal Disc Cells
of Drosophila melanogaster Larvae in Rotation Culture
Yukiaki KURODA

To elucidate at a cellular level under strictly defined conditions themechanism by which cells of identical genetic constituents show various
phenotypic expressions in various organs and tissues, dissociated cells
from various imaginal discs of Drosophila melanogaster were tested fortheir ability to form characteristic histogenetic aggregates in rotation
culture.
Eye-antennal discs and wing discs were dissected from mature thirdTable 1.

Components of medium K-10 for cultivationof Drosophila cells

Ingredients

mg/IOO ml

NaCIKCl
CaCI 2 ·2H 20
MgCI 2 ·6H 20
NaHC0 3
NaHP0 4 ·2H 20
GlucoseSucrose
Lactalbumin hydrolyzate
Tryptophan

700
20
2

10
5
20
80
2,600
1,280
100

Ingredients
Thiamin·HCl
Riboflavin
Pyridoxine. HCl
NiaicnCa-pantothenate
BiotinFolic acid
Choline·HCl
Inositep-Aminobenzoic acidmg/100ml
0.002
0.002
0.002
0.002
0.002
0.001
0.002
0.02
0.002
0.002

pH was adjusted to 6.6. The medium was sterilized through a Seitz filterand supplemented by 10% calf serum.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

instar larvae (96 hours after hatching at 25°C) grown under sterile
conditions. They were incubated in calcium- and magnesium-free salt
solution for 15 minutes, then in 1 per cent trypsin solution for 15 minutes,
and were dispersed in the culture medium into single cells by flushingthe dissected materials through the tip of a fine pipette. Considerable
effeorts have been made to improve and simplify the culture medium
for Drosophila cells. Medium K-1O shown in Table 1 was better thanother initial media.
One hundred eye-antennal discs isolated from Oregon-R larvae yielded
about 4x 106 single cells, whereas one hundred wing discs from the samestrain produced about 4.5x 106 cells. One hundred eye-antennal discs
from Bar larvae yielded about 3x 106 cells by the same procedure.
When single cell suspensions each containing 106 cells in 0.3 ml medium
were introduced into tightly covered micro-beakers, rotated on a gyratory
shaker for 24 hours at 100 rpm at 28°C, tissue-like cell aggregates werereconstituted in the center of the micro-beakers. Cell-aggregates obtained
from eye-antennal disc cells of the Oregon-R strain had an average
diameter of 0.6 mm, whereas eye-antennal disc cells from the Bar strain
formed smaller and looser aggregates of an average diameter of 0.4 mm.
Wing disc cells from the Oregon-R strain formed after 24 hours ofrotation aggregates of 0.5 mm in diameter which showed a characteristic
structure different from that of eye-antennal disc cells.
The differences in histogenesis and organogenesis of different imaginaldiscs are now further studied.

Differentiation of Imaginal Discs of Drosophila melanogaster
Cultured in Synthetic Medium and Effects of Some
Hormonal Substances
Yukiaki KURODA and Kiyoshi MINATO

Eye-antennal discs were dissected from third-instar larvae of Drosophilamelanogaster grown under sterile conditions and were cultured in hanging
drops of synthetic medium K-1O at 28°C.
In the medium without any supplementations of hormonal substances
eye-antenal discs showed a pronounced increase in the eye-forming andantenna-forming portions after 24 hours of cultivation. Folded area of
eye disc extended and flattened out, but no distinct differentiation of theommatidia was observed.
When cephalic complexes involving the brain hemispheres, ventral
ganglion, and ring gland of third-instar larvae were put into the medium
in which eye-antennal discs were cultured, a marked differentiation of

29

RESEARCHES CARRIED OUT IN 1967

ommatidia was observed. Several cells which would develop into anommatidium formed after 24 hours of cultivation single cell clusters
which were clearly separated from each other. The eye-forming portion
was distinguished from other portion of the eye disc.
Recently four steroids having ecdysone activity have been isolated
from a plant, Podocarpus nakaii Hay by Nakanishi et at. (1966) and
named ponasterone A, B, C, and D. Takemoto et at. (1967) have also
isolated two kinds of steroids, inokosterone and iso-inokosterone (ecdysterone) having ecdysone activity from the roots of Achyranthes fauriei.
The chemical structures of ecdysone and other steroids having ecdysone
activity are shown in Fig. 1.

OH

HO
IiO

Ecdysone
Ecdysterone
Ponasterone A
Inokosterone

R1
H
OH
OH
OH

R2
OH
OH
H
H

Ra
CH a
CH a
CH a
CH 20H

Chemical structures of ecdysone and related compounds

When these ecdysone analogues of plant onglll were added to themedium in which eye-antennal discs were cultured, a pronounced differentiation of ommatidia was observed. The formation of distinguishable
cell clusters was found like in the cultures of eye-antennaI discs cultured
together with cephalic complexes.
It was also found that dodecyl methyl ether, a syethetic substancehaving juvenile hormone activity, accelerated slightly the outgrowth ofeye-antennal discs which were explanted in TD-7 flasks. Phytol, which
is a terpene having juvenile hormone activity, was tested for its activity
in accelerating the outgrowth of eye-antennal discs in culture. Slightacceleration was found when phytol was added to the medium. Glycerol,cholesterol and Raben-type hormone had no effect on eye-antennal discs
in culture.
The artificial controls of differentiation and proliferation of imaginal
disc cells by adding ecdysone and juvenile hormone, under in vitro
conditions will be studied.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Distribution of Auxin Protectors and Their Kinetic
Study in Japanese Morning Glory*
Yoshiaki YONEDA and Tom STONIER**

The existence of substances which inhibit the enzymatic destructionof auxin in shoots of a normal strain of Japanese morning glory(Pharbitis nil) was confirmed, as was the fact that these substances
are distributed in a gradient diminishing from apex to base in amanner indicating their regulatory role in internode elongation andtissue maturation.
In addition to the two auxin protector substances reported previously(protector-I (Pr-I), molecular weight 5,000 to 10,000 and protector-II CPrII), molecular weight 1,500 to 5,000) which appeared to account for mostof the inhibition of enzymatic auxin destruction in young elongatingstem tissue, a third substance, designated as protector-A CPr-A), hasbeen found to be highly active in seeds, and stem tips of mature plants.
In germinating seeds, no activity of Pr-I or II was observed; in stem
tips, no activity of Pr-II and only a slight one of Pr-I was observed.
In contrast, old tissue contained no detectable amounts of Pr-A, but did
contain Pr-I and Pr-II. Between these extremes along the shoot axis,
mixtures of the three substances were found. This result indicates that
Pr-A is degraded into Pr-I and II and perhaps translocated in this form.
Gel filtration studies indicate that Pr-A has a molecular weight exceeding 200,000 gm/mol.
In vitro studies on the mechanisms of auxin protection by Pr-I indicatethat 1) the Pr-I -induced characteristic lag preceding auxin destruction
by peroxidase is completely eliminated by a strong oxidant such as H 20 2 ;2) strong organic reductants such as glutathione mimick the Pr-I-induced
lag and 3) the length of the lag varies inversely with the reaction rate.
These results are best explained on the basis that Pr-I acts as a strong
reductant to protect IAA from the enzymatic oxidation.

Distribution of IAA Oxidase and Peroxidase inJapanese Morning Glory Stem
Yoshiaki YONEDA

Distribution of auxin protector substances in the shoot of the Japanese* This work was supported by a fellowship from the Damon Runyon Memorial
Fund (DRF-378) to the first author and U. S. Public Health Service grant CA-06957
and Damon Runyon Memorial grant DRG-933 to the second author.
** Laboratory of Plant Morphogenesis, Manhattan College, N. Y., U. S. A.

RESEARCHES CARRIED OUT IN 196731

morning glory suggests their important implication in the elongationand maturation processes of internodes. A kinetic study of auxin
protection revealed that the protector-induced lag was reduced by anything that speeds up the auxin oxidation reaction. One of such factors
is the quantity of enzyme. So far, the efficiency of protectors wasdetermined by the volume of extract required to get a lag of certain
duration preceding IAA destruction in the presence of a constant quantityof horseradish peroxidase, without consideration of the endogeneous
enzyme in the extract.
From this view point, the quantitative aspects of endogeneous enzymes
were examined in detail. Stem internodes of young, vegetatively growing plants of a normal strain, TKS, were homogenized and the resultant
water extracts were assayed for enzyme activity. IAA oxidase activity
was determined by the increase of optical density at 247 mp in the reaction mixture consisting of IAA (substrate), dichlorophenol and MnCl 2(co-factors) plus 0.04 mM H 20 2 , which was used to inactivate the protectors.
Peroxidase activity was measured photometrically by guaiacol test.
The results indicated that distribution of both peroxidase and IAA
oxidase in stem internodes from base to stem tip was rather uniform:in other words, the overall variations of these enzyme activities amonginternodes were rather small, though the young terminal internode had
the highest activities of both enzymes. Both enzymes varied parallelwith each other. When H 20 2 was omitted from the reaction mixture of
IAA oxidase, a protector-induced lag was observed, which had a gradientof high activity in the young elongating internodes and low activity inthe non-elongating ones, as already reported.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

III.

BIOCHEMICAL GENETICS

Genetic Effects of DNA in Ephestia
Saburo NAWA and Masa-Aki YAMADA

It was already reported that when larvae or eggs of a/a (red-eyed)
Ephestia were treated with DNA prepared from wild-type adults (black-eyed), black-eyed mutants were found in the treated generation and in
subsequent generations obtained by backcrosses to untreated a/a animals,
although the breeding behaviors of some of the mutants were unusual(this Annual Report, No. 17). All of the black-eyed mutants, whichwere obtained in the experiments either by injection of larvae or treatment of eggs and appeared either in the treated or subsequent generations, were back-crossed to a/a successively until segregation into blackand red-eyed occurred. After the segregation had occurred, the black-eyedanimals produced always black-eyed and red-eyed progeny in the proportion of 1 : 1 in backcrosses and 3: 1 in sister-brother matings in allcases. Furthermore, in successive sister-brother matings, black-eyedanimals were bred to each other and have been kept as mutant lines
ever since. In these lines, no contribution of the a+ allele had takenplace other than that made by a+-DNA. To date no red-eyed individual
has appeared for more than ten generations. These results indicate thatwhatever the nature of the effect of DNA was in the first generations
it behaves ultimately as a chromosomal gene. There have been manycases of non-specific mutations induced by DNA in Drosophila after
treatment by feeding or injection. The induced mutations were in somecases restricted to particular segments of the chromosome and usuallyconsisted of deletions. If DNA were so mutagenic in our experiments
as to cause an exchange of nucleotides or a small deletion, such mutations at most positions on the a+-Iocus may have resulted in the appearance of a-phenotype when the a+·gene was affected by DNA. If thiswere the case, frequency of the change of wild to a-phenotype shouldhave been much higher than that of a to wild-phenotype. The experiments where a+/a or a+/a+ eggs were treated with a-DNA or a+-DNA,gave no such indication (d. Nawa and Yamada (1968), Genetics 58).
In control experiments, larvae or eggs of a/a were treated either with
DNA prepared from a/a adults or with sonically disrupted wild typeDNA. No black-eyed mutant was obtained. Production of black-eyedmutants in overall experiments to date is given in Table 1. The appearance of mutants behaving like a+/a+ homozygotes in the progeny derived
from crosses of treated but phenotypically unchanged animals with

33

RESEARCHES CARRIED OUT IN 1967
Table 1.

Production of black-eyed mutants by treatment of ala
larvae or eggs with DNA
Treated generation

DNA

wild

ala
sonicated wild
heated wild
saline*Bl

No. of adults
examined

Black-eyedmutants

No. of adults
examined

Black-eyedmutants

7,913
2,572
4,607
1,637
600

5
0
0
0
0

111,678
25,172
43,16429,284
16,823

18
0
0
1*
0

produced only red-eyed progeny in the cross to ala.

untreated a/a was reported in the previous paper. Additional experiments have confirmed this to be a not rare phenomenon.

Evidence of Uptake of DNA by Ephestia Eggs
Masa-Aki YAMADA and Saburo NAWA

It has been undoubtedly demonstrated that hereditary changes could
be induced in Ephestia either by injection of DNA into larvae or byimmersion of eggs in DNA solution (see the preceding paper). In connection with this result, the purpose of the study presented here was todetermine whether donor DNA penetrates the recipient cells without
degradation. The 3H-Iabelled DNA (1.5x106 cpm/,ug) was prepared froma thymine-less Salmonella mutant cultivated on a medium with added
3H-thymidine. Eggs (O.1-0.2g) ranging from 0 to 16 hours after spontaneous egg-laying were dechorionated by immersing in a solution of
sodium hypochlorite. After washing with saline, the eggs were immersedin the 3H-Iabelled DNA solution for 2 hours and then washed withsaline. In order to remove DNA adhering to the surface of eggs, they
were treated with DNase and washed thoroughly with saline. DNA wasextracted from the treated eggs by SDS-phenol method. The DNA was
submitted to fractionation by centrifuge chromatography on columns of
DEAE-cellulose paper pulp, which could give us estimations about thedegree of polymerization of the foreign DNA incorporated into eggs.
An elution property of the extracted radioactivity (bound with DNA) is
given in Table 1. Most of the radioactivity was found in fractions ofhigh molecular weight, indicating that the foreign DNA had been incorporated into the eggs without significant depolymerization.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Table l.

Fractionation of the DNA extracted from eggs treated with 3H-DNA
of Salmonella on centrifuge DEAE-cellulose column(Average
Fraction molecularNo.
weight)
1.
2.
3.
4.
5.
6.

(5 x 102)(2 X 10 3)(5 X 104 )(5 x 105 )(1 x 106 )(5 X 106 )Salmonella DNA used intreatment

DNA extracted from eggs
treated with 3H-DNA

Radioactivity
(cpm)

%

Radioactivity
(cpm)

(O.D)

%

0
0
16
195
1220
119

0
0
1
12.5
78.7
7.7

2
2
32
54
6
5

(0.13)(0.28)(1.83)(0.34)(0.85)(0.33)

2
2
32
54
6
5

Ribonucleic Acid Content in the Hypodermis of the Silkworm
Kunihiro KOJIMA, Mitsuo TTUJITA and SUSUMU SAKURAI

Using larvae from the 4th moulting stage to the 5th day of 5th instar
of normal strains, i. e. C-124, Daizo and w-c, as materials, nucleic acidwas extracted by phenol method from hypodermis and pteridine granules
and analysed by methylated albumin column chromatography. The experimental results are as follows.
1. The amount of s-RNA and r-RNA per 19 of fresh hypodermis wasmeasured throughout the period from the 4th moulting stage to the 5th
day of 5th instar. A large amount of s-RNA an dr-RNA was detected atthe 4th moulting stage.
2. The amount of those two kinds of RNA, s-RNA and r-RNA, wasmarkedly decreased on the 1st day of 5th instar as compared with the
4th moulting stage, but it increased gradually again and attained the
maximum on the 3rd day of 5th instar, thereupon decreasing from its
4th to 5th day.
3. The largest amount of s-RNA and r-RNA per 1 g of fresh hypodermis was found on the 3rd day of 5th instar, suggesting the highest
synthetic protein activity at this stage.
4. Nucleic acid fractions extracted from the hypodermis on the 1st
and 3rd day of 5th instar by phenol method consisted of s-RNA, r-RNA
and DNA. Nucleic acid fractions extracted from pteridine granules on
the 3rd day of 5th instar also consisted of s-RNA, r-RNA, and DNA-like
substance. Thus it become clear that numerous minute particles surrounding the pteridine granules were ribosomal particles.

RESEARCHES CARRIED OUT IN 1967

35

5. Ribosomal RNA was not detected in the chromatographic profilesof the nucleic acid of pteridine granules isolated from larval hypodermis
on the 5th day of 5th instar. It seems that this lack of r-RNA is due
to disintegration of ribosomal particles surrounding the pteridine granules
and to their detachment from the granules at this stage.
Incorporation of HC-amino Acid into Pteridine Granules
Susumu SAKURAI and Mitsuo TSUJITA

In oder to define pteridine granule function in hypodermal cells, HC_alanine, HC-serine and HC-tyrosine were injected into the the body fluidof silkworm larvae on the 3rd day of 5th instar. Afer 2 hours, fresh
cytoplasm of hypodermal cells was suspended in 0.25 M sucrose solution
and centrifuged at 900 g, 5,000 g, 12,000 g, 35,000 g and 78,000 g, andradioactivity of resultant particles was counted.
14C was rapidly incorporated into pteridine granules as it could be
expected from our previous experiments with incorporation of 14C-glycine.
The specific radioactivity of premature granules sedimented at 78,000 g
was higher than that of mature granules.
Pteridine granules were dissolved in 2 per cent solution of sodium
deoxycholate. The solution was eluted through a Sephadex G50 column
and separated into polypeptide and membrane protein fractions, and thenthe radioactivity of each fraction was determined.
HC was incorporated into polypeptides and membrane protein of bothmature and pre-mature granules. However, polypeptides and membraneprotein from premature granules showed higher incorporation activityof HC amino acid than the mature granules.
Morphology of pteridine granules during the developmental process
was already reported. The electron micrographs of mature granules
showed that the maturation process caused the detachment of ribosomesfrom their membrane and transformation from rough surface to smoothsurface type.
It may be suggested from those experimental results that ribosomes
attached to the granule membrane participate in the synthesis of polypeptides which are secreted and stored within the granular vesicles.
Significance of the Pteridine Granule Developmentin Larval Skin Cells of the Silkworm
Mitsuo TSUJITA, Kunihiro KOJIMA and Susumu SAKURAI

Pteridine granules produced in hypodermal cells of normal silkworm

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

larvae consist each of a granule membrane and content, i. e. polypeptide-pteridine pigment and polypeptide-uric acid complexes, with which
the granules are filled.
In order to make clear the mechanism by which pteridine metabolic
products are accumulated into pteridine granules, the activity of enzymes
concerned with pteridine metabolism such as pterine dehydrogenaseor pteridine reductase in adipose tissue and hypodermis of the larvae
was examined.
Normal (C-124) and E-lem(E/E; tem/tem) larvae in 5th instar were usedas materials. As we were afraid that the larval hypodermis used inprevious experiments (Tsujita 1963) had contained near the skin adipose
tissue, in the present experiments the larval skin was as thoroughly as
possible freed from adipose tissue which was used for the preparationof crude enzyme solution.
The experimental results indicate that the activity of those enzymes
was very strong in adipose tissue but weak or lacking in the hypodermisof normal larvae. In lemon larvae weak activity of pteridine reductase
was found in adipose tissue but it was scarcely detected in hypodermis.
Nevertheless, products of pteridine metabolism such as isoxanthopterin
and sepiapterin were abundantly accumulated in hypodermal cells but
could hardly be detected in adipose tissue.
It was described in our previous paper that the developmental processof pteridine granules follows the sequential phases of endoplasmic
reticulum, and that their complete development into mature granules isdue to normal development of granule membrane to which the ribosomes
are attached. In addition, our experimental results concerning incorporation of labeled amino acid into pteridine granules suggest that polypeptides are synthesized by the ribosomes attached to the granule
membrane.
According to the general belief, it seems that the larval skin cells of
the silkworm have the ability to absorb uric acid from the body fluid
and to store it within the cells.
On the basis of our experimental results the following hypothesis ispresented for the formation of pteridine granules.
The hypodermis of the silkworm has the ability to absorb uric acid aswell as pteridine compounds through the body fluid from other organs
in which those compounds are produced, and, combining with polypeptides produced around the granules, are secreted into granule vesicles
where they are stored. In the hypodermal cells of normal larvae thegranule vesicles have complete or sufficient ability to secrete or store
those products and thus abundant pteridine granules of high density are
yielded. Normal larvae are opaque owing to the presence of thoseRESEARCHES CARRIED OUT IN 1967

37

granules in hypodermal cells. However, in the hypodermal cells of
mutant larvae homozygous for wox , w-a, or w ol , the granular development is defective and no mature pteridine granules or a small amountof minute granules is produced, resulting in their highly transparent skin.
Thus, it is concluded that although endoplasmic reticulum can be ingeneral observed in various tissue cells of the silkworm the coarse surface endoplasmic reticulum found in larval skin cells shows a special
differentiation according to its cellular function, namely production of
pteridine granules.

Reconstitution of Vesicular Membrane of Pteridine Granules
from Degraded Subunits
Mitsuo TSUJITA and Susumu SAKURAI

Purified mature pteridine granules dissolved in 0.2 mol phosphate buffer(pH 8.0) were heated at 95°C for 3 minutes. Non-degraded granules
were sedimented by refrigerated centrifugation at 20,000 g for 60 minutes,

220
260
300~
Fig. 1. Ultraviolet absorption spectrum of membrane proteinfrom pteridine granules.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

and the supernatant was overnight dialysed against distiled water. For
the reconstitution of granule membranes from degraded subunits, 1
volume of dissolved membrane preparation was mixed with 1 volume of0.156 mol sodium chloride solution containing 0.01 mol magnesium chloride. Fibrous substances could be by electron microscope observed in thesediment obtained from the mixture.
At first, pH of the mixture was about 8, it was converted to pH 5 byadding acetic acid and the preparation was kept at 5°C for 12 hours,
the white sediments produced were separated by centrifugation at 20,000 g
for 60 minutes and studied by electron microscope. It was found thata number of granule vesicles surrounded by thin membranes were reconstituted from self-assembling membrane subunits. Although thosereconstituted vesicular membranes were of smooth surface type without
attached ribosomal particles, rarely rough surface type vesicular membranewith a small number of attached ribosome-like particles were found.
Thin sections of reconstituted membrane material showed the same
pattern as the original membranes of pteridine granules. For the reconstitution of membrane materials, the presence of divalent cation (MgCI 2 )Table 1.

Amino acid composition of membrane proteinfrom pteridine granules
Amino acids

Micro moles

Aspartic acidThreonine
SerineProlineGlutamic acid
Glycine
AlanineValineCystine
Methionine
Isoleucine
LeucineTyrosine
Phenylalanine
Ammonia
LysineHistidine
Arginine

1.36
0.97
0.82
0.18

1.92
1.13
1.29
0.91
0.06
0.27
0.68
1.10
0.36
0.43

0.97
0.70
0.14
0.79

RESEARCHES CARRIED OUT IN 1967

39

gave the best results, but CaCl 2 was as effective.
The granule membrane subunits from which the lipid had been extracted by treatment with chloroform and methanol no longer showed amembranous structure; reintroduction of lipid into the extracted subunits
restored the property of vesicular formation.
Absorption spectrum of the membrane protein solution resembles thatof a mixture of tryptophan, tyrosine. cysteine and phenylalanine (Fig. 1).
The membrane protein purified from pteridine granules separated fromthe larval hypodermis of a normal strain (YD 4 ) was hydrolysed by 6 N
HCI at nODc for 20 hours and the amino acid constitution was analysedusing Hitachi amino acid analyser KLA 3. The experimental results are
given in Table 1.

Formation of Vesicle Membranes of Pteridine Granules
from Repeated Particles
Mitsuo TSUJITA and SUSUffiU SAKURAI

Using pteridine granules separated from the hypodermis of larvae of
a normal silkworm strain (C-124) the following electron-microscopic study
was carried out.
After heating a suspension of granules in phosphate buffer (pH 8.0) at
95 C for 3 minutes the degrading granules were centrifuged and thesediments were examined by electron microscope. Fibrous substances(called "strings") about 40 A in thickness, sometimes bundles of such
substances which protruded from broken parts of granule membranes
were observed. In positively stained granule sections, fibrous substances
showing linear arrangement of 30-40A thick particles could be observedin the granule membranes.
The fibrous structure was confirmed in the reconstitution experimentsof vesicular membran~s; fibrous substances about 40 A thick, 0.5-1.0 f1
long, running closely and almost parallel to each other in one layer wereobserved in newly formed membranes. Furthermore, in each of thefibres linearly arranged particles, 3O-50A in diameter, were observed.
It seems from above-mentioned observations that the membrane ofpteridine granules is made up of strings running almost parallel in one
layer, each string being composed of particles arranged linearly.
As described in our previous paper, various abnormally shaped anddefective granules are produced in the hypodermis of larvae homozygous
for w-3 multi-allelic genes, i. e. w-3, w-a, w-b, wo~ or oZ. Especially, inthe hypodermis of larvae homozygous for oZ granules having abnormallythick membranes in which sparse short fibrous substances, 0.1-0.2 f1 long,
D

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

were entangled were observed. Small amounts of secretion products
were stored within the granules and from some of them all contents
were lost leaving behind abnormal membranes having sparse fibrous
structure. In addition, aggregates of short fibrous substances, 0.1-0.2 flin length, composed of linearly arranged particles were scattered here
and there around the granules. They seemed to be masses of defective
fibrous substances which failed to form vesicle membranes.
It was reported that protein and lipid are the principal components ofpteridine granule membrane (Tsujita and Sakurai 1966) and that theirmembrane protein consists of a homogeneous single protein (unit membrane protein) (Sakurai and Tsujita 1967).
Green and Perdue (1966) define the membranes as vesicular or tubular
systems, the continuum being made up of lipoprotein-repeating units (onelayer thick) which are the only structural elements. As it is consideredthat their repeating units correspond to particles linearly arranged instrings in the pteridine granule membrane, the following diagrammatic
representation of their vesicular membrane construction is shown in Fig. 1.

at

Fig. 1. Diagrammatic representation of the membrane of pteridine
granules as a fused continuum of repeating particles. A particle consists of a base, a stalk and a detachable head (omitted
in the figure). rp: repeating particle. st: string.

Vesicular membrane formation from subunits confirmed by our reconstitution experiments may be regarded as a case of vesicularization by
repetition of units, and strings may be an intermediate form.
Generally speaking, organelles have two or more component membranes
which have entirely different repeating units and functional attributes
and are rather systems of membranes than individual membranes.
However, it seems from our observations that the membrane of pteridine
granules produced in larval hypodermis of the normal silkworm is amonolayer membrane. Rarely, partly double membranes which are per-

RESEARCHES CARRIED OUT IN 1967

41

haps overlapping parts of the monolayer could be seen. It is highly
probable that the membrane of pteridine granules is an example of asingle membrane structure.

A Staining Procedure of Human Serum Lipoproteinson Cellulose Acetate Strips
Yoshito OGAWA

The purpose of this report is to present a rapid and simpler highresolution technique for the fractionation of human serum lipoproteinson cellulose acetate strips. It is essentially the same as the Straus andWurm's method reported in 1958 for paper electrophoresisll • But theanalytical result of the present technique has a remarkable advantage
over the original one due to the superior character of cellulose acetate
as the supporting medium of electrophoresis (Fig. 1).
Standard barbital buffer of pH 8.6 and ionic strength from 0.05 to 0.07
was used and a constant current of 0.6 rnA was passed per one cm.
width through the strips at room temperature. Serum should be appliedin volum of approximetely 1.2 pI per one cm. width of a strip. A staining
alb

.g

I\ ~

+

~

10----------

3.7 emFig. 1. Curves obtained by scanning a Fat red 7B (thick line) anda Poneeau 3R (fine line) stained Separax (1 x 6 em.) cleared withparaffin oil by using O.5x8.0mm'slit adapter and 500mp wavelength. Arrow: a2-lipoprotein fraction.
11

Straus, R. and Wurm, M.:

Am.]. Clin. Path., 29: 581 (1958).

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

solution was prepared by addition of 0.1 gm of Fat red 7B (Chroma
10725) and 7.5 gm of trichloroacetic acid to 175 ml of methanol, which
was brought to a boil under a condenser so that less of the solvent was
minimized. After that 75 ml of hot distilled water of about 60°C was
added and the mixture was kept at 40°C.
Immediately after the removal from the electrophoresis chamber, thestrips were immersed in the dye solution for a period of two hours at
40°C and then transferred into a decolorizing bath, consisting of a one
per cent solution of 5 per cent commercial sodium hydrochloride in two
per cent acetic acid, for 4 or 6 minutes at room temperature until the
separated patterns had clearly developed. After rinsing in tap water,
the strips were dried between paper toweling.
The above staining procedure showed satisfactory results on Membranfolin (Germany) and Serometrics (U. S. A.) as well as on Separax (Japanese), but was not practicable on Millipore (U. S. A.), Sepraphore III(U. S. A.), Oxoid (England), Sartorius (Germany) and Cellogel (Italy).
The relation between the analytical results of lipoprotein fractions stained
by Fat red 7B and serum protein fractions stained by Ponceau 3R obtained under the same experimental condition on Separax is shown in
Fig. 1 and the normal values of serum lipoprotein fraction in the Japanese
population age ranging from 30 to 40 years are given in Table 1.
TobIe 1.

Normal values of Japanese serum lipoproteins analysed on Separax(Ogawa & Sakamoto 1968)
Globulin

Blood donors

Albuminr

f3
Male
10 persons(% range)

17.48±3.18 9.98±1.38 27.52±6.22 15.79±2.06 29.23±3.50(10.64-25.00) (6.67-12.77) (10.00-40.43) (11.11-20.00) (20.59-38.00)

Female10 persons(% range)

16.90±4.26 9.33±2.96 25.63±3.58 18.41±3.24 29.73±5.36( 5.56-25.52) (2.64-16.67) (19.95-30.80) (10.53-24.00) (18.00-41.66)

Technicalerror* 21.47±3.64 7.57±1.4225.17±4.37

13.00±2.2732.79±3.20* Calculated from the results of five time analysis of the same sample

Zyrnographie Analyses of Peroxidase-Indoleaeetate Oxidase
lsozyrnes in Several Plants
Toru ENDO

Many authors have considered that both peroxidase and indoleacetate

RESEARCHES CARRIED OUT IN 1967

43

oxidase activities are due to the same enzyme species. This was, inthis study, zymographically proved by a new staining method developedfor indoleacetate oxidase activity. The experimental results indicate·that some, but not all, peroxidase isozymes have an indoleacetate oxidase
activity in several plants on starch gel zymograms.
Zymographic examinations were carried out of a commercial peroxidase
preparation of horseradish as well as exiracts from several plant materials
regarding dual catalytic preperties of single isozymes. A reacting mixture
for the oxidase stain contained 1 to 5 mM indoleacetate, 0.5 mM trichlorophenol, 2 mg per ml of Fast Blue BB salt and 0.2 It acetate buffer, pH
5.0, at final concentrations. An unidentified intermediate resulting fromthe enzymatic indoleacetate degradation was shown to be coupled withdiazonium salt and to form an insoluble complex. The starch gel was.
sliced transversely; the top half was subjected to oxidase stain and thebottom half to peroxidase stain. At least four cathodal and four anodal
peroxidase bands were detected in the horseradish preparation, the former
appearing as dark oxidase bands and the latter as very faint bands.
In morning glory root, several anodal peroxidase bands were observed
as dark oxidase bands, which appeared rapidly during the staining treatment, but one sharp cathodal peroxidase band was faintly stained as anoxidase band. A similar result was also obtained with sweet potato root.
One anodal band was located as very weak peroxidase band but quickly
stained as a strong oxidase band, while one faint cathodal peroxidase
band did not appear as an oxidase band. In both turnip and radish, aheavy anodal peroxidase band was shown as an appreciably stainedoxidase band.
The indoleacetate oxidase zymograms were obtained in the absence ofmanganese ion. It was noted, however, that many of rice and maize
peroxidase isozymes required around 10 mM manganese ion at a final
concentration of the reacting mixture for oxidase stain. The results.
clearly show that the requirement for the metal ion is considerably different in the oxidases of plant materials. Also, intensity of peroxidase
bands was not always correlated with that of the corresponding oxidase
bands on zymograms.

Peroxidase and indoleacetate Oxidase Isozymes inJapanese Morning Glory
Yoshiaki YONEDA and Toru ENDO

The evidences that indoleacetate oxidase plays a significant role in thedevelopmental processes in plants, directly or indirectly through an auxin

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

protector system, are being accumulated (Yoneda & Stonier, 1967). Ourinterest has been focused on the relation of auxin destroying enzyme
and auxin protector system to the elongation of stem internodes inmorning glory. In this study, we have started to analyze such enzymes
that could control tissue differentiation.
We have developed a staining method for the detection of indoleacetateoxidase isozymes separated electrophoretically on starch gel zymograms(Endo, in press). By this method, the oxidase isozymes were compared
for identification with peroxidase isozymes. The crude extracts fromvarious organs of a normal strain, TKS, were zymographically analysed,
using borate buffer, pH 8.5, system.
In root, at least 5 isozyme bands of peroxidase moving towards theanode (they were located as lA to 5A numbering from the start line)
were identified. TKS strain is divided into two substrains concerninga cathodal sharp band, lC; it appeared in T016 but not in T036 substrain.
This band was distributed in most of the organs irrespective of theirage. In hypocotyl, 3A and 4A bands were usually found. Very young
hypocotyls had 3A and 2A ll 2A 2 bands (numbered by their location between 2A and 3A). Band 5A appeared in older hypocotyls. The isozyme
patterns of stem are similar to those of hypocotyl, and the terminal,
young elongating internodes had 2A 1 and 2A 2 bands. Whereas the basal,
old internodes had 5A band in addition to 3A and 4A. Cotyledons andleaf blades had only 3A band, but in very old stages, 5A, lA and 2A
bands appeared.
Weak bands, 2A 1 and 2A 2 , appeared both in very young hypocotyl andstem internodes. In contrast, 5A band was always detected when thetissues became older. Such an age-specific change of peroxidase andindoleacetate oxidase patterns suggests a special role of some isozymes
in tissue differentiation, in addition to organ-specific distribution of
isozymes.
In relation to the auxin protector system, effects of hydrogen peroxide
were zymographically examined. Peroxidase bands, 3A to 5A, took
intense indoleacetate oxidase stain, whereas peroxidase bands, lA and
2A, appeared as very weak or almost negative oxidase bands. These
oxidase bands, however, became strongly stained ones in the presence ofa very small amount of hydrogen peroxide. This indicates that hydrogen
peroxide inactivates the natural auxin protectors which migrate to theregions of lA and 2A bands on zymogram.

RESEARCHES CARRIED OUT IN 1967

45

Changes of Peroxidase Isozyme Patterns by in vitro Cultureof Japanese Morning Glory Tissues
Yoshiaki YONEDA

In order to investigate the relation of peroxidase isozymes to morphological tisssue differentiation, the tissues from three different organs(root, hypocotyl and cotyledon) in a normal strain, T036, were cultured
in vitro on four different media (Linsmaier & Skoog's basal medium,and this basal medium plus 10 ppm indoleacetic acid (IAA) or 5 ppm
kinetin (KT) singly or in combination). After one month culture in thedark, each tissue or callus was frozen, macerated and the exudate, absorbed in a filter paper, was run on starch gel electrophoretically toobtain zymograms of peroxidase, which was detected by staining withbenzidine-H 20 2 •Tremendous callus growth was observed on KT and IAA-KT media
in all three kinds of tissue, and IAA promoted a small callus growth inhypocotyl and root tissue, whereas no signs of callus formation wereseen in cotyledonary tissue. IAA stimulated root formation, on thecontary, suppressed by kinetin. Callus growth on basal medium wasalmost nil and only a few roots elongated strongly.
As described elswhere, each tissue used in this study had a charcacteristic pattern of peroxidase isozymes in vivo; namely, cotyledon had
only 3A band, hypocotyl had 3A and 4A, and root had 5 bands (lA to 5A).
The most fundamental change of the isozyme pattern observed was
the appearance of 1A and 2A bands and a very intense 5A band in all
tissues regardless of the medium, though 1A and 2A, induced on KT or
IAA-KT media were generally much more intense than on basal and IAA
media. Another interesting change was that while the tissues on basal
medium, producing no callus, retained their original bands of 3A and 4A,
they generally became weak or faded away along with callus growth.
The third change observed was that in connection with callus growth,
one or two new bands appeared between 2A and 3A, which were named
2A 1 and 2A 2 by their location.
Briefly, the disappearance or reduction of tissue specific isozymes andappearance of new bands seem to correlate with callus growth or lossof specific morphological structure, in addition to the basic change due
to in vitro culture. Thus, the patterns of peroxidase isozymes in calli(on KT and IAA-KT media) became almost the same, whatever organ
they derived from. These results suggest a close correlation of peroxidase
isozymes with tissue differentiation, caused by hormone treatment.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Effect of Exogenous RNA on Protein Synthesis in L-M Cell Culture11
Kazuo MORIWAKIIn this study an attempt was made to transfer the ability of specificprotein synthesis from a mouse plasma cell tumor, X-55632I , which can
produce a specific gamma glubulin, to L-m cell culture 31 , using X-5563
tumor RNA.
L-M cells were cultured in 199 peptone medium and treated with 0.01
pg/ml actinomycin D for 12 hours, prior to incubation for 28 hours with
5 pc/ml C14 -valine and 0.8 mg/ml RNA extracted from X-5563 turner or
C3H mouse liver by hot SDS-phenol method and used with 30 pg/ml

J~lVk
Q.

n~
u

~ "'1

Actinomycin & L- RNA

~.~
=-

ii:

_ .....

u~III='--....;;:0,,-.

Actinomycin & X-5563- RNA

n-

eil

to

(-) -3 -2

-1

1

2

3

4

S

•

7

•

•

10

t . Electropho~tic fraction
Orlg,nFig. 1. Induction of new protein synthesis following treatment
with exogenous RNA in L-M cell culture.
11
This work was conducted in Mammalian Genetics Center, Department of
Zoology, The University of Michigan, Ann Arbor, Michigan, U. S. A. and was
supported by a PHS Grant 5 TI GM 71-08.
21
Kindly furnished by Dr. M. Potter, National Cancer Institute, U. S. A.
31
Generously supplied by Dr. D. J. Merchant, Microbiology Department, The
University of Michigan Medical School.

47

RESEARCHES CARRIED OUT IN 1967

protamine sulfate. After RNA treatment, the soluble fraction of thecultured cells was extracted by homogenization and centrifugation. This
fraction was mixed with the serum of X-5563 bearing mouse serving as
a carrier and was submitted to paper electrophoresis. Amino black 10 B
staining of the paper was followed by cutting off each protein fractionof the paper and weighing and counting the radioactivity.
As shown in Fig. 1, the depressed level of protein synthesis in L-M
cells by actinomycin D could be restored to control level with eitherliver RNA or X-5563 RNA treatment. Especially, the X-5563 RNA wascapable of inducing new fractions of protein synthesis different from theendogenous spectrum of L-M cells on the pattern of electrophoretic paper.
In order to ascertain whether or not this newly induced proteins
contain a specific globulin (myeloma protein), the radioactivity of theabove mentioned soluble fraction was assayed by a serological method.
As far as the ratio of myeloma protein synthesis to total soluble protein
synthesis is concerned, the X-5563 RNA treatment fairly accerelated thesynthesis of myeloma protein in L-M cells (Table 1), though the mechanism of the function of the used RNA remains unknown.
Table 1.

Effect of X-5563 RNA on myeloma protein synthesis
in L-M cell culture
C14-val. incorporated,

cpm/106 cells/40 hrs.

M.P.

T.S.P.

395

14,600
2.540

0.027

257

6,220

0.042

186

6,650

0.028

T.S.P.:

Total soluble protein.

Treatments
Control (199P medium)
Act·D (0.018 pg/ml)
Act-D & X-5563 RNA(0.8 mg/ml)
Act-D & Liver RNA(0.8 mg/ml)
M.P.:

Myeloma protein,

T.P·/T.S.P.
ratio

Inhibitory Effect of Homologous RNA on Protein
Synthesis in Cell-Free Systeml)
Kazuo MORIWAKIThe present study deals with the inhibition of total protein synthesis
in a cell-free system by the addition of RNA extracted from the same
II
This work was conducted in Mammalian Genetics Center, Department of
Zoology, The University of Michigan, Ann Arbor, Michigan, U. S. A. and was
supported by a PHS Grant 5 TI GM 71-08.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

Components of the cell·free protein synthesis system

This buffer, pH 7.0
NH 4-acetate
Mg·acetate
KCI
Mercaptoethanol
17 amino acids (-Val.)
K-ATP
K-PET

80 mM
50 /I

8
50
9

PEP-kinase
15 pgjml
Valine-H3
5 pc
S·RNA (Bovine liver)
100 pg0.03 mM
CTP
0.03 /I
UTP
0.2
/I
GTP
Liver, Ehrlich tumor
0.4ml
or X5563 S-30*(10 mg Protein Eq.)

/I
/I
/I

0.075/1 ea.
~.5/1

2.5/1

Total volume* S-30:2.0ml

A supernatant fraction obtained from 0.25 M sucrose homogenate ofeach tissue by a centrifugation at 30,000 rpm for 30 min.

Table 2.

Inhibitory effect of homologous RNA in cell-freeprotein synthesis systems
H3. Val. incorporation (cpm)jI9 mg protein eq. S-30 Cell-freesystems
Liver

Experimental No.

1

4,750
NO
1,310
Liver RNA (2 mg)
Ehrlich tumor RNA (2mg)
X-5563 tumor RNA (2mg) 5,500

Ehrlich tumor

X-5563 tumor

1

2

1

2

750

688
1,825
258
1,158

2,266
4,365
3,028
2,732

283
763
1,098
335

171
729

kind of tissue as that from which the cell-free system was prepared.
Mouse liver, Ehrich ascites tumor and mouse plasma cell tumor (X-5563)
were employed as the materials for RNA extraction and the preparationof a cell-free protein synthesis system. RNA extraction was performedby hot SDS-phenol method. Each component contained in the system isenumerated in Table 1. After incubation at 37°C for one hour, thereaction was stopped by adding TCA to a final concentration of 5%.
The protein fraction was spun down and washed twice by 5% cold TCA
with 1% valine, followed by washing with 70% ethanol, 100% ethanol
and 3: 1 ethanol-ether. The radioactivity of H 3 ·valine in this precipitate
was markedly reduced by addition of a homologous RNA into the system(Table 2). This tendency was strongly exhibited in the combination ofliver RNA with the liver cell-free system and also in the combination of

RESEARCHES CARRIED OUT IN 1967

49

Ehrlich tumor RNA with Ehrlich tumor cell-free system, suggestingsome tissue specificity in the reaction of RNA with the cell-free protein
synthesis system. A hot TeA treatment of the protein precipitate seemed
to abolish the inhibitory action of homologous RNA, though the precise
mechanism has not been studied yet.

Myeloma Proein Synthesis in a Cell-Free System Preparedfrom Mouse Plasma Cell Tumor, X-556311
Kazuo MORIWAKI

Myeloma protein synthesis of this tumor in a cell-free system had been
-once before undertaken by Askonas (1961) who obtained a poor yield of
this protein. In the present study several factors concerning the efficiency of a cell-free system were examined using the system preparedfrom X-5563 tumor. The components of this system were enumerated,elsewhere (This Ann. Rep. p. 48).
First, 0.8mg/ml RNA extracted from X-5563 tumor or normal liver by
hot SDS-phenol method, was supplemented to the cell-free system.
Although the synthesis of total soluble protein was considerably enhanced
either by X-5563 RNA or normal liver RNA, both RNA's resulted in no
significant increase of myeloma protein synthesis.
As it is already known that liver cell sap contains a potent inhibitor·of RNase (Roth, 1958), the supernatant (S-l00) fraction obtained from0.25 M sucrose homogenate of mouse liver by centrifugation at 100,000 g
for 30 minutes was used as a RNase inhibitor throughout the preparationof the cell-free system in the second experiment. No increased myeloma
protein synthesis, however, was observed in the system prepared with
the S-lOO fraction, suggesting that RNase contained in the system is nota major cause of the poor yield of myeloma protein in the present experiment.
Thirdly, the process of cell disruption was carefully re-examined.
Tissues of the tumor and mouse liver as a control were minced andground in the cold by Potter-Elvehjem glass homogenizer. As shown in
Table 1, the homogenization after ten rounds of hand operation gave a·cell-free extract having a much higher activity for myeloma protein
synthesis, whereas operation by motor for one minute only seemed almost
to destory it. This result allows to assume that there exists a delicate
11This work was conducted in Mammalian Genetics Center, Department of
Zoology, The University of Michigan, Ann Arbor, Michigan, U. S. A. and was
supported by a PHS Grant 5 TI GM 71·08.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

mechanism in tumor cells concerned with specific pretein synthesis.
Table 1.

Effect of homogenization procedures on myeloma protein sythesisin X-5563 cell-free system

Process ofhomogenization

Motor operationfor 1 min.

Hand operation
10 rounds

Cell-free
extract

H3-Val. incorporated

cpm/19 mg protein

equivalent S-30

M.P

T.S.P.

M.P./T.S.P. ratio

X-5563
S-30

32
35
42

2,300
2,750
2,630

0.014
0.013
0.016

Liver
S-30

16
27

1,310
1,910

0.012
0.014

X-5556
S-30

615
698
824

1,305
1,316
1,442

0.47
0.53
0.57

Liver
S-30

33
32

1,390
1,200

0.024
0.027

Abbreviations: S-30; supernatant fraction obtained from 0.25 M sucrose homogenateof the tissues by centrifugation at 30,000 g for 30 minutes. M.P.; Myeloma protein.
T.S.P.; Total soluble protein.

51

RESEARCHES CARRIED OUT IN 1967

IV.

EVOLUTIONARY GENETICS

Local differences in frequencies of No. 1 chromosomal
polymorphism of Rattu8 rattu8
Tosihide H. YOSIDAII

Differences due to different locality in the frequencies of the polymorphic largest No.1 chromosome pair, namely a telocentric homomorphic(TIT), a telocentric and subtelocentric heteromorphic (TIS) and a subtelocentric homomorphic pair (SIS), were already reported (Yosida et al.
1967, this Annual Report 17: 61). We newly obtained the animals 3 localities in Japan, namely Tottori, Ooizumi (Gunma) and Okinoerabu(Kagoshima) and also added to our collection animals from Niigata andSeoul (Korea). The frequenceies of the three chromosomal types in No.
1 pair are shown in Table 1. As the table shows, the rats collected in
Tottori were of all of TIT type, while the population collected in 00izumi showed TIS and SIS types only. The population in Okinoerabu
was similar to those in other southern parts of Japan.
A population map of chromosomal polymorphism of animals collected
in Japan and Korea is shown in Fig. 1. As the figure shows, animals
collected in Sapporo, Niigata, Obama and Tottori which are located in
the north and north-west of Japan were of TIT type, those collected in
Sugadaira, Hiratsuka, Misima, Hamamatsu, Ube, Nagasaki and Okinoerabu located in the east and south showed about 20 to 30 percent TIS
animals. The population of Ooizumi (Gunma), located in the east, however, was markedly different from the other populations, in having TIS
and SIS types. Frequency in the Korean population was also slightly
Table 1. Frequency of black rats (Rattu8 rattu8) with three chromosome
types of No. 1 chromosomes collected in Japan and Korea
Locality
Karyotypes
TottoriOoizumi(Gunma)Okinoerabu(Kagoshima)

Niigata

Seoul(Korea)

TIT
TIS
SIS

24
0
0

0
9(81.8)%2(18.1)18(81.8)4(18.1)43
0018(46.1)
17(43.5)4(10.3)Total

24

11(99.9)22(99.9)43

39(99.9)

II The author wishes to thank to Dr. Y. S. Kang for collection of animals in
Korea.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

uu

~'VU
o

Fig. 1. Population map of the frequency of chromosomal polymorphismin Rattu8 rattu8 collected in Japan and Korea. Localities collected
the animals were show by circle, in which white, shaded and black
areas show the frequency of animals with TIT, TIS and SIS chromosome pairs, respectively.

different from those of Japan; namely, it was characterized by considerably high frequencies of T/S and S/S types. It is diffecult to conclude
at present, why the chromosomal polymorphism is maintained in thisspecies, how and where did the polymorphism develop, and what do the
local differences in frequencies of the polymorphism mean for the differentiation of this species. These problems will be further studied cy-

RESEARCHES CARRIED OUT IN 1967

53

togenetically and statistically when a larger material collected at moredifferent localities is available.
Further studies on segregation of three chromosome types ofRattus rattus in the laboratory
Tosihide H. YOSIDA

Segregation of three chromosome types (TIT, TIS and SIS) of No. 1
chromosome in Rattus rattus bred in the laboratory was already reported(Yosida et aI., 1967. this report, No. 17: 61) and the deta on F 1 hybrids
TIT x TIT, TIT x TIS, TIS x TIS and TIS x SIS were given. Newly weobtained F 1 hybrids TIS x TIT, TSI x SIS, SIS x TIT, SIS x SIT and SIS X
SIS, and we wish to add the new data and correct the old ones published last year. Scheme of crosses and data on segregation of the three
chromosome types are shown in Table 1.
Table 1. Segregation of three chromosome types in Rattus rattus
bred by several combination of parents and average litter sizeof the hybrids
Combinationof parents

Segregation

Total no. Total no. Averageof litters of animals litter
size

X 2*

TIT

TIS

SIS

TITx TIT

89(69)**

0

0

17

89

5.3

TITx TIS

36
(39)

42
(39)

0

13

78

5.6

0.46
P>0.3

TIS x TIT

28
(36.5)

45(36.5)

0

14

73

5.2

6.5
P<0.02TIS x TIS

29
(25)

54
(50)

17
(25)

20

100

5.0

3.52
P>O.l

0

30
(26.5)

23(26.5)

9

53

5.8

0.46
P>0.92

0

12
(12)

0

3

12

4.0

0

5
(5)

5
(5)

3

10

3.3

0

0

8
(9)

2

8

4.0

TIS x SIS
SIS x TIT
S/Sx TIS
S/S x SIS

* X 2 value was calculated for the theoretical segregation.
** Figures in parenthese show theoretical segregation.

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

As the table shows, SIS males and SIS females are fertile in the laboratory. Average litter size of hybrids TIT x TIS( 0 ) and TIS( 9 ) x TIT
is similar (5.6 and 5.2). This means that fertility of TIS males is notdifferent from that of TIS females. When SIS females were crossed to
TIT, TIS and other SIS males, the litter size of the hybrids decreased
to 3.3-4.0, while the litter size of the hybrids TIS( 9 ) x SIS( (5) was very
high (5.8). From the above results it is suggested that females withSIS chromosomes have lower productivity than those with TIS and TIT
chromosomes. In the case of TIS x TIS, the ratio of segregation of thethree chromosome types did not deviate significantly from the theoretical
1: 2: 1 ratio, although SIS animals are somewhat few. On the other,hand, in all crosses more animals with TIS heteromorphic chromosome
pair were usually obtained than expected from the segregation ratio.
Hybrid vigour may be involved.
Serum Transferrin Polymorphism in Rattu8 rattu8
Kazuo MORIWAKI

An electrophoretic survey by starch gel of serum protein polymorphism
in natural population of Rattus rattus in Japan has revealed three typesof transferrin variants, R-, N· and RN-type. R type was characterizedby two fast moving bands, N type by two slowly moving bands and RN
type by four bands as a mixture of both types, though all of those bands
migrated into the diffused region between slow al-globulin and fast alglobulin. Electrophoretic patterns of those three types and that of Rattus
norvegicus serum as a reference are schematically shown in Figure 1.
The starch gel electrophoresis was carried out under the following
conditions: Hydrolyzed starch powder was prepared from potato starch(Wako Chemicals) by hydrolysis in 0.12 N HCI at 37°C for 135 minutes.
Starch gel for electrophoresis contained 12.7% (WIW) hydrolyzed starchin glycine-borate-NaOH buffer (25 mM glycine, 5 mM boric acid and 5.3
mM NaOH, pH 8.6). Borate buffer (300 mM boric acid and 50 mM NaOH,
pH 8.3) was used for electrode vessels. Gel tray was laid horizontallyin the refrigerator and connected with a constant current-power supply
for 14 hours at 3.6 mA/cm2 • Staining of the proteins on the gel was performed by amino-black 10 B.
Ninety four rats which were collected from five localities in Japan,
Sizuoka Pref. (21), Gunma Pref. (10), Niigata Pref. (20), Tottori Pref. (21)and Okinoerabu Island (22), consisted of 70.3% R type (66), 26.4% RN
type (24) and 4.3% N type (4). No marked difference in the frequenciesof these three types was observed among those localities. When a Hardy-

RESEARCHES CARRIED OUT IN 1967

Anode

55

t
•._-. Prealbumin·-----Albumin}..•-••Postalbumins------Fast (1'1 Globulin----·-Slow (1'1 Globulin

RN

N

R. norvegicus---···Origin
R ·······Transferrin Phenotypes

R. rattus········Species

Fig. 1. Schematic patterns of serum electrophoresis in transferrin
variants of Rattus rattus.

Table l.

Progeny test of serum transferrin variants in Rattus rattus.
Progenies

No ofParent litter
28
RxR
5
RxN
1
NxN
RxRN 18
RNxRN 18

R

RN

N

Total

141
0
0
54
29

0
23
0
50
55

0
0
5
0
18

141
23
5
104
102

Litter
size
5.1
4.6
5.0
5.8
5.7

Theor. ratio
Chi·square

R

RN

N

1
0
0
1
1

0
1
0
1
2

0
0
1
0
1

0.15(df=I, P>0.5)
3.01(df=2, P>0.2)

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Weinberg test was applied to this material, no significant departure fromnumerical expectations was obtained (X 2 =2.35, df=2, P>O.3).
Distribution of transferrin phenotypes among progeny from the laboratory crosses of R-, RN- and N-type is summarized in Table 1. These
data are consistent with the hypothesis that the two transferrin types,
Rand N, <lre controlled by two codominant alleles at a single locus.

Migratory behavior of vestigial strains of
DrQsophila melanogasterTakashi NARISE

In the series of experiments on migration with wild and vestigial strains of Drosophila melanogaster, it was found that the migratory activity of a vestigial strain was accelerated by a coexisting wild strain.
The extent of acceleration seemed to be dependent upon the similarity
in genetic constitution between the vestigial and the mixed wild strain.
To make the point clear, an experiment was conducted with four wild
strains, MS-1, Niihama, Oshoro and Tosu, their substituted vg strains(see the last issue of this Annual Report) and the F 1 hybrids between
MS-1 and the remaining three wild strains. These wild type strains
were mixed with the substituted ones in a certain combination and theeffect of genetic closeness on the change in migratory activity of thesubstituted flies was examined. The experimental results are presented
in Table 1.
Table l.

Migratory activity of substituted vg strains
Migratory activity of vestigial strains

MS-lNiihama X MS·l
Oshoro X MS-l
TosuxMS-l
Niihama
OshoroTOBUMS-l(vg)

Niihama(vg)Oshoro(vg)Tosu(vg)

17.2
17.8
22.0
20.4
18.0
24.8
23.6

19.6
16.0

24.4

16.4

20.0
14.0
8.0
13.6
11.6

As shown in Table 1, the migratory activity of MS-1 (vg) was 17.2%
in the mixture with MS-1, while the activity increased to 20.4% and23.696 respectively in the mixture with F 1 (TosuxMS-1) and Tosu strain.

RESEARCHES CARRIED OUT IN 1967

57

From the experimental results, it is concluded that the vestigial flies
become active in migration if the coexisting flies are different from themin genetic constitution. In other words, the overall difference in genotypes between the coexistants is capable of accelerating migration.
Three Intergeneric Amphidiploids of the Tribe Triticeae
Sadao SAKAMOTO

Applying 0.5% colchicine solution to the basal parts of tillering clones
of various intergeneric hybrids of the tribe Triticeae, the following threeintergeneric amphidiploids were produced:(1) Eremopyrum orientale
Jaub. et Spach (2n=28; Iran) X Agropyron tsukushiense var. transiens
Ohwi (2n=42; Japan), (2) Er. orientale x Henrardia persica var. glaberrima(Hausskn.) C.E. Hubbard (2n=14; Iran) and (3) Er. buonapartis (Spreng.)
Nevski var. buonapartis (2n=14; Iran) X Hordeum sp. (2n=28; material"No. 66w" was received from the All-Union Institute of Plant Industry,
Leningrad, 1964).
As to morphological characteristics (1) was of Agropyron type, (2) wasof elongated Eremopyrum type and (3) was of Hordeum type, having in
addition two florets with two, three or four empty glumes at each spikelet node of the spike.
Average chromosome paring at MI of PMCs and seed fertility of theamphidiploids were in the order of above enumeration (1) 29.4n+l1.2I ;5.596, (2) 20.2n+2.2I ; 42.8% and (3) 0.2Iv+0.3m+17.8n+4.9I ; 34%. Low
seed fertility of (1) resulted from unexpected high frequency of univalent
formation at MI.
Phenetic Variation and Phyletic Relationship of Strains of

Oryza perennis, Estimated by Numerical Taxonomic Method
Hiroko MORISHIMA

Last year (Ann. Rep. 17, p. 59-61), we reported about an analysis ofvariations among strains of Oryza perennis Moench made from the "phenetic" (overall similarity between existing organisms) standpoint. This
year, the same set of data (24 charactersx65 strains) were analysed not
only phenetically, but also from a "phyletic" (phylogenic) standpoint. In
some parts of the computation, F 1 sterility data of the strains were treated together with the data for other 24 characters. For phenetic studies,
the technique of "cluster analysis" (Sokal and Sneath 1963) and "patternanalysis" (Hayashi 1956) were employed. The results consistently showed that the four geographical groups of strains, Asian, African, American

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

and Oceanian, formed separate clusters on a phenetic plane. Then,"cladograms" showing evolutionary branching sequences of the strains
by a tree-like diagram were computed by Camin and Sokal's (1965) method, which is based on the hypothesis of minimum evolution. It was
attempted, further, to introduce a time axis into the cladograms. The
time elapsing between two given branching points in a cladogram wasconsidered proportional to their phenetic distance and inversely proportional to the relative evolutionary rate of the plants, and the latter was
estimated from breeding characteristics, i. e., the duration of life cycle,
seed dispersing ability, and the rate of autogamy.
The derived cladograms suggested that the geographical groups might
have evolved independently from one another, and that in the Asiangroup various advanced forms might have differentiated with a highevolutionary rate. It was postulated that geographical isolation between
continents and variation in the breeding system might have been the
major factors conditioning phylogenic differentiation of this plant group.
The main part of this study was done at the Computation Center of the
University of Kansas, where the reporter worked under Dr. R. R. Sokal
for two months supported by the Rockefeller Foundation.

Occasional Escape from the Isolating Barrier Surrounding
Oryza perennis subsp. barthii
Yaw-En CHU and Hiko-Ichi OKA

The African race of Oryza perennis, called barthii, is partly isolated
from its wild and cultivated relatives (other races of O. perennis, O. sativa
O. graberrima and O. breviligulata) by a barrier that hinders the development of F i embryos with a certain frequency. Observations of the deterioration process were reported before (Ann. Rep. 16, p. 71). This,
year, an analysis of the genes controlling this embryo deterioration was
attempted. In triple crosses of the sativa X (sativa X barthii) design, thecross-pollinated seeds were found to segregate into normal and shrunken
ones, the ratio being 1 : 1. This suggests that a set of complementary
dominant lethals controls the deterioration of F i embryos. The penetrance of the gene effect may not be complete so that a few germinable
F i seeds are sometimes obtained.
Plants of a barthii population may also have different combinations of
the lethal genes. Those with double recessive combination map produce
normal F i seeds with sativa or glaberrima. In Africa, barthii often grows
in the fields of sativa and glaberrima. Some barthii plants, raised fromseeds collected from natural habitats, due to some particular characters,

RESEARCHES CARRIED OUT IN 196759

were suspected to be hybrids with sativa or glaberrima. Having crossed
them with true barthii and with sativa, they proved to have the lethal
gene combination either of the barthii or the sativa parent. These barthii
plants, we call "Obake", seem to be slipping out of the isolating barrierby spontaneous successful hybridization. Their peroxidase isozymes
showed bands peculiar to sativa and to glaberrima, indicating that the"Obake" may be of a complex hybrid origin. This work was supported
by a grant of The International Rice Research Institute.
Partial Self-incompatibillity Found in Oryza perennis
subsp. barthii
Yaw-En CHU and Hiko-Ichi OKA

It was suggested by Jachuck and Sampath (1966, Oryza 3: 49-57) that
the African strains of O. prennis, called barthii, could be self-incompatible, as the panicles when bagged set only a few seeds. An investigation on this point was made in three plants, collected from Guinea (Af9)and Mali (ML36-4 and ML36-6, belonging to the same population). The
plants grown in pots were, after heading, separated by a distance ofabout 20 m, and self- and cross-pollinations by hand were compared withnatural self-pollination. Bagging was not made. Cross-pollination brought
about an apparently higher percentage of seed set (65% to 83%) thanselfed, the pollen tubes penetrating into the stigma tended to have aswollen tip, resulting in a failure to fertilize. This work was supported
by a grant of The International Rice Research Institute.

Comparison of Variations in Peroxidase Isozymes between
Perennis-Sativa and Breviligulata-Glaberrima
Series of Orgza
Yaw-En CHU and Hiko-Ichi OKA

The two cultivated rice species, O. sativa and O. glaberrima, are thought to have independently evolved from an Asian form of O. perennis
and African O. breviligulata, respectively. The isozyme variations inleaf-blade peroxidase were investigated in strains of O. breviligulata andO. glaberrima, and were compared with those previously observed in O.
perennis and O. sativa. The breviligulata and glaberrima strains showed
only three zymogram types, and appeared to be much less variable thanthe perennis-sativa series that showed eleven different zymogram types.
Also the within-populational variability in isozymes was quite small in

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

o. breviligulata, in contrast to the polymorphic populations of O. perennis. The zymogram variations suggested the evolutionary paths of theplants. Observations of sativa-glaberrima F 1 hybrids and back-cross progenies indicated that the two species might have basically the samegenes for peroxidase isozymes, showing differences in the presence orabsence of certain bands. This work was supported by a grant of The
International Rice Ressarch Institute.

61

RESEARCHES CARRIED OUT IN 1967

V.

MATHEMATICAL AND STATISTICAL STUDIESON POPULATION GENETICS
Gene frequency cline in a habitat of finite length
Motoo KIMURA and Takeo MARUYAMA

The previous report (Annual Report, No. 15, pp. 95-97, 1965), was abouta study by the senior author on the gene frequency cline in a linear
habitat of finite length. U sing the method of differential equation,
Kimura derived a series solution for the case of genic selection assumingthat the length of the habitat is relatively short.
When the length of the habitat is longer, the analytical solution of thedifferential equation seems to be very hard to obtain, but the solution
may be tabulated by applying numerical integration.
In the present note, we will consider a case in which the habitat
extends from X=!,( < 0) to X=!2( > 0) and in which the equation giving thegene frequency p(x) at point x satisfies the differential equation~~ =xP(1-P){1+h(1-2P)} ,(1)

with boundary conditions
dp=O
dx

at

X= !1

and

(2 )

X=!2.

With the help of a high speed computer, we are carrying out an extensive
tabulation of the solution of the above equation for various values of theTable.

-0 5{P(l)

l-.

P/(O)

-1 o{P(l)l - . P/(O)_15{P(l)l - . P/(O)l =5.0{P(l)
P/(O)

pel)

l =00 {P/(O)Comparison of values obtained from power series (3)and numerical integrationfrom power series

from numerical integration

0.489586
0.031240.418432
0.122700.25
0.24992

0.489686
0.03125
0.418420
0.122700.267274
0.23900
0.00045
0.32340o0.3302

62

ANUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

parameter h. So far, we have nearly completed tabulation for the casesof h=O and h=l.
The accompanying table shows some results of the numerical integration for the case of h=O (no dominance) and -/ 1 =/ 2=1. Also, in thetable, the corresponding values obtained by the series solution of Kimura,namelyx3
B 2x5
P(x)=0.5 - Bx+ 24 - W+, .. ,(3)

where B=/ 2j8-11/ 8 j4608, are listed for 1 up to 1.5.

On the special functions of population genetics
Takeo MARUYAMA

The spectral representation of the operator (1), acting in a suitable
Hilbert space, and the elementary solution for the parabolic partial differential equation (2),2A=(1-x2~-[4x+sh+(1-2h)sx] ...!L_[(1-x )(1-2h)S shx
d~

dx

2

_ X(1+X)~1-2h)S +C(X)]

(1 )

and
oif1(t, x)

at

Aif1(t, x)(2 )have been studied by M. Kimura. Taking first the case s=O, and thenthe case h=0.5, though assuming c(x)=O in both cases, he has obtained
the explicit forms of Green's functions which seem to have an increased
importance in population genetics because various kinds of information
concerning the stochastic behavior of a population may be obtained fromthem. However for cases other than those treated by Kimura, no comparable works have been done. Therefore it is desired to construct the
spectral representation of the operator (1) and the elementary solutionfor (2), for general cases. Since the spectral set of the operator (1) iscountable real numbers such that 0> a1 > a2 >,' . " a" >,' .. - 00, the representation has a simple form. The proof of this fact will be given
elsewhere when an extensive tabulation of the functions are given.
However it should also be mentioned that if we add two more terms,(1-,u)(1-x)j2 and lJ(1+x)j2, which represent the reversible mutations, tothe second term of (1), the spectrum is still discrete countable real numbers

RESEARCHES CARRIED OUT IN 1967

63

but includes zero as the largest eigen-value.
If we change (1) into an appropriate self-adjoint form, it is easy toshow that the eigenfunctions of (1) are mutually orthogonal with respect
to the weight function w(x)=(l-~) exp [-s(1-x)(2h+1-x+2hx)j8]. For
we can verify the equalityai[1

w(x)Ei(x)Ej(x)dx=aj~ ~1w(x)Ei(x)Ej(x)dx

where Ei(x) and Ej(x) are two eigenfunctions associated with eigenvalues
ai and aj, respectively. Since the first part of the operator (1) admits
the Gegenbauer polynomials as eigenfunctions and the spectrum is a setof countable real numbers, it is natural to expand an eigenfuction of (1)in the form
E(x)= ~ CnT~1\X)

(3)

71=0

where T's are the Gegenbauer polynomials and are the bounded solutionsof (1-~)T"(x)-4xT'(x)+n(n+3)T(x)=O. Now we want to determine thecoefficients c's in (3) so that E(x) satisfies the equation
AE(x)=aE(x)and is finite at both singular points, x=l and X= -1, that is equivalent
to the condition that E(x) is a square integrable function with respect
to the weight w(x) mentioned above, over the interval (-1, 1). Upon
substitution of (3) into (1) and making use of the following recurrence
relationships
xT~lI =[(n+1)T~~1+(n+2)T~:2dj(2n+3)

and
dT

lli

(1-~~=(n+1)T~~1-(n+3)T~11

we have a system of equations that the coefficients c's must satisfy;(4)
where the capital letters of the alphabet are functions of n, s, hand c(x)in the operator (1), and in each equation only D contains a, the separation constant. Although there is a simpler way to determine a and c's
simultaneously, it works only when s is small. Here a method of continued fraction developed from (4) will be used in actual computations,because it has a great advantage when a high speed computer is used
and more importantly it works up to large values of s. We can develop
the continued fractions into two directions; one in descending order of

64

ANUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

n and the other in ascending order of n. Then we choose a such that
one fraction converges to the reciprocal value of the other fraction. As
soon as a computer becomes available in our institute, extensive tabulations of these special functions will be carried out.

Development of temporary overdominance associatedwith neutral alleles
Takeo MARUYAMA and Motoo KIMURA

Universality of overdominance has long been a subject of controversy
among geneticists. It is an attractive hypothesis for explaining themaintenance of genetic variation in populations and there seems to be
little doubt that some variations are due to overdominance. However it
is too naive to assume that most of the variations are maintained by
overdominance. In a finite population, 2Ns, that is twice the population
size times the selection coefficient, has to be considerably greater thanunity to make overdominance effective for maintaining the variation.
Moreover, if we assume that most of the existing variations are due to
overdominance, the number of offspring produced by a single female, insome organisms, is not sufficient to overcome the genetic death thatwould be caused by segregations.
In the present study, we intend to show that in a finite population
52 polygenes

marker,r-------~------

Lo.ool--+lCnromoso me{::O.OOl--r - - --f1tness
1.0

genotype

Fltne~smarker(neutral)\ :~:~l

A 2A 2{:~bb

polygene
Experiment
50

__

1.0
1.0

~:~95
0.9
recomb1nat1on(rate,~0.001)
mutat1on(rate~0.01)~==:.:.::....

B~w1ld typeb~mutant

~)

00

~

,."0'
00 zygotes

Fig. 1. Scheme of Monte Carlo Experiment.

gamete pool

65

RESEARCHES CARRIED OUT IN 1967

temporary overdominance is produced when a marker locus with neutral
alleles is linked with other loci affecting fitness, even though there are
no loci having heterozygous advantage by themselves. For this purpose,
we carried out the following computer simulation experiments. We
assumed a diploid organism having one pair of homologous chromosomeson which the marker locus with neutral alleles Al and A 2 , and 52
polygene loci affecting fitness were located at 0.001 crossover distance
between adjacent loci. In an experiment performed, population size was
50, mutation rates between alleles were 0.001 (same for all loci), and weassumed that at each polygenic locus the mutant gene reduces fitnessby 10% in homozygous and 0.5% in heterozygous condition. The schemeof the experiment is illustrated in Figure 1. In the starting population,
the marker alleles Al and A 2 were set with equal frequencies, and ateach polygene locus, the mutant gene and the wild type gene were set
according to Wright's distribution formula, but independently of other loci.
Table 1. Result of Monte Carlo Experiment.

o

5

ro

lli

W

~

W

~

~

~

W

0.5770.605 0.693 0.595 0.686 0.718 0.733 0.6970.6370.6640.680
0.6080.7230.7630.7700.771 0.788 0.756 0.732 0.703 0.733 0.685
0.611 0.670 0.621 0.6400.623 0.575 0.597 0.641 0.5960.7140.616
Generation

~
60
ffi
ro m 00 ~ W % ~0.638 0.5ffi 0.654 0.471 0.580 0.471 0.349 0.468 0.409 0.440
0.7360.7450.7330.768 0.767 0.778 0.734 0.753 0.755 0.787
0.394 0.558 0.597 0.605 0.598 0.571 0.593 0.669 0.650 0.669

The results of an experiment that was continued over 100 generations
using this scheme are summarized as follows: The heterozygotes forthe marker, A I A 2 , showed about 15% higher fitness than the homozygotes, AlAI or A 2 A 2 • Thus we have demonstrated the occurrence ofapparent overdominance at a locus with neutral alleles on a chromosome
segment where no loci are showing heterosis by themselves. Details of
the results are given in Table 1. More extensive investigations of theabove phenomenon are under porgress.
Effect of epistasis on the probability ofjoint fixation in 2 loci
Motoo KIMURA and Tomoko OHTA

Let us consider a haploid population of effective size N e and assume

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. IS

that fitnesses and frequencies are as follows:
genotype
AB
Ab
aB
ab

fitnessl+s i +S2+ e
l+si
1+s2

frequencypq
P(l-q)
q(l-P)(l-P)(l-q)

1Here, P and q stand for the frequencies of A and B respectively. If u
is the probability of ultimate fixation, then u satisfies the following partial
differential equation which is a steady state form of the Kolmogorov
backward equation.

a2u
a2u
au
P(l-P) ap2 +q(1-q)a;;-+2N,P(1-P)(sl +eq)ap
au+2N,q(1-q)(s2+eP~=0(l}.

For the special case of Sl=S2=0, Kimura obtained, from the above equation, the following formula for u(AB), i.e., the probability of joint
fixation of A and B,
1_e- 2N"pq(2}u(AB)= 1
2N'

-e

'

It is interesting to note that this formula coincides with that for the
fixation probability of a single locus case in which a single allele denotedby AB has selective advantage e and initial frequency pq. By Monte·
Carlo experiments, Ohta found that the formula (2) is valid. The fixation
probability was also studied by Monte Carlo simulation; assuming various.
recombination values it was found that intensity of linkage has not muchinfluence on u(AB) with initial linkage equilibrium.
Kimura also showed that when IN,sll, IN,s21 and IN,el are all small,the approximate solution of (1) is,u(AB)=pq{l+N,sl(1-PHN,s2(1-qHN,e(1-pqH···} .

However, the general solution of (1) is not known at present. For thecases in which the above parameters are not small, Monte Carlo studies.
were also carried out. Fixation probability of A is very much affectedby the frequency of B for large e and it was found that, even with50% recombination, u(AB»u(A)·u(B) for positive e. (submitted tOo
Ziichter)

67

RESEARCHES CARRIED OUT IN 1967

Effects of initial linkage disequilibrium on
fixation probabilities for 2 lociTomoko OHTA

Probabilities of joint fixation for 2 linked loci were studied by themethod of Kolmogorov backward equation and also by Monte Carlo simulation with special reference to initial linkage disequilibrium. Such a
study may particularly be important when selection starts with a population produced by crossing 2 strains in breeding experiments.
When there was no selection or the selection coefficients were small,the probabilities were obtained by solving steady state form of theKolomogorov backward equation. Let us assume 2 pairs of genes, A anda in the first and, Band b in the second locus. Let the initial frequencies
of gametes, AB, Ab, aB and ab be g" g2' g3 and g4 respectively. Also,
let N, be the effective size of the population, c the recombination fraction
between A and B loci, and let u be the fixation probability of a gamete
type. Then u satisfies following partial differential equation which is a
steady state form of a Kolmogorov backward equation.

+ ~ N,s,{g,(1-g,-g2)u/ +g2(1-g,-g2)u/ -g3(g, +g2)Us'}+ ~ N,s2{g,(1- g, - g2)U/ - glg, + g3)U/ + g3(1- g, - g3)Us'}-N,cD{u/ -u/ -us'}=O,( 1)

where S, and S2 are respectively the selective advantages of A and B
over a and b measured as the difference between 2 homozygotes (assuming no epistasis). In the above equation, ud' is the second derivativeof u with respect to gi and gj, and, u;' is the first derivative of u withrespect to gi'
The general solution of the above formula has not been obtained, butwhen IN,s,1 and IN,s21 are small, the approximate solution for the fixation
probabilities was obtained. They are:u(AB)=c-g,-d+N,s,(l-g,- g2)(g,-dHN,s2(1-g, -g3)(g,-d)U(Ab)=c-g2+d+N'Sl(1-g,-g2)(g2+dHNeS2{2~,c- (l-g,- g3)(g,-d)}

68

ANUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

u(aB)~ g3 +d+ N eS l{ 2~eC -(1-g1-g2)(gl -d)} + N es2(1- gl - g3)(g3 +d)u(ab)=1-u(AB)-u(Ab)-u(ab)(2)

where d=2NecD/(2Nec+1).
When there is no selection, Le., SI =S2=0, the above formulas reduce to,u(AB)=gl- du(Ab)=g2+du(aB)=g3+ d( 3)u(ab)=g4- dwhich are exact solutIOns of formula (1) for the case of no selection.
The results agree with the probabilities obtained by Kimura (1963) by adifferent approach. However, these results are slightly different fromthose obtained by Karlin and McGregor who used the method of probability transition matrix. The difference is due to the models used.
When INes l and INes 2 are large, the solution has not been obtained,
but Monte Carlo experiments were carried out. From the results ofMonte Carlo study with various degrees of initial linkage disequilibrium
and INecl, it was concluded that the effects of disequilibrium for the caseof large INes l and IN es 2 are usually larger than the selectively neutralcase because of the rapid approach to fixation. Therefore, in some cases,in order to reduce disequilibrium, keeping a population as a large randommating one before starting selection may be recommended in breeding
experiments. (submitted to Ziichter)
l

1

l

1

Distribution of matrimonial distance in Mishima district
Norikazu YASUDA

Distribution of matrimonial distance, that is, the distance between thebirthplaces of spouses, is important in the study of the pattern of humanmigration and geographical range of marriages. It also supplies a basic
information for the evaluation of the probability of consanguineous
marriages, effective size and heterogeneity of a population, and also forthe estimation of the inbreeding coefficient.
Let m(x, y) be the probability density that a male born in (0, 0) marriesa female born at (x, y), where x and yare two coordinates in a Cartesiansystem. Under isotropic migration, the distribution of matrimonial
distance r=Vx 2 +f may be expressed in a polar coordinate system; namely,m(x, y)dxdy=2rrm/(r)rdr .

69

RESEARCHES CARRIED OUT IN 1967

Nine hundred and forty-four couples who were born (both husband andwife) in Mishima and its neighboring towns were sampled from koseki
or household records in which addresses of birthplaces are given. The
birthplaces were identified on maps (scale Ij3,OOO~ Ij25,OOO) with the aidof khozu or terrier. Distances were measured on the map along thestraight line between birthplaces of husband and wife, and the angle (8)between the line and the longitude was also measured for a survey ofisotropic migration.
The observed distribution is given in Figure 1. Among the observed
couples two showed the same address of their birthplaces, but in one ofthem, it was found that husband and wife were actually born in two
neighbor houses. This is a significantly different finding from that ofthe previous studies. The present result is due to a more precise evaluation of short distances. In order to find a mathematical form of m/(r),the observed number at a given distance was divided by the corresponding distance, and the resulting values were plotted on a log-square root
coordinate system (Figure 2). The result fits remarkably well to astraight line. That is, log [m/(r)rjrl=C'
or

-kvr,

m/(r)r=Cre-kv';'(C=e c ')where C=k 4 j24rr and k is obtainable from the slope, glvmg 2.3x 1.24=
2.85jkm. The maximum likelihood estimate for k and its standard error
70

60

50

11

Fig. 1.

Distribution of matrimonial distance in Mishima district (T=944).

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
1.0

x x
I

:X
I

k'~1.24:I
1

x
x

x

X

x /xx X

I

:

Xi':

L----l~---~X\~"<<
'"

X

xx\
X

x\x

\ X\
;\lARITAL DISTANCE ikm.)-r

Figure 2. Fitting of function Cre-k ,I"; to the distribution of marital
distance in Mishima district.

are k=4T/I.vr and s=V4T/I.vr, respectively, in which T is thetotal number of couples. The present data gave k=2.52±O.04/km. The
estimated mean (r) and standard deviation (a) of the distance werer=3.16±O.26 km and a=3.31±O.27 km, respectively.
For the above consideration, we assumed an isotropic nature of migration. This was examined using the variance of angle, which is expected
to be rr 2 /3=3.2899 (in radians). If the dimensionality is defined by
d=l+(observed v.)/(expected v.), then we obtain d=1.98, since theobserved variance is [I.82-C.~8i/T]/(T-l)=3.2405. However, migration
may be one-directional for long distances. This hypothesis was also
tested by using the dimentionality for a given distance interval, and we
found that the d-values did not deviate significantly from two for the
distance less than 7 km. This covers 91.6% of the sample. It is therefore
concluded that migration is isotropic in Mishima.
The project will be continued to clarify the above points.

RESEARCHES CARRIED OUT IN 1967

71

The use of surname for estimating Wright's
F-statistics in man
Norikazu YASUDA

Isonymous marriages, that is marriages between persons having thesame surname, take place either due to mere chance or to consanguineous
marriages. In the latter case, a quarter will be isonymous (1). Let I
be the proportion of observed isonymous marriages, k the expected isonymy frequency due to inbreeding, qm the male proportion with a certain
surname and qf the corresponding female proportion. Then we have
1= ~qmqf+(I-~qmqf)kwhere ~qmqf is the contribution due to random matches, and (1- ~qmqf)k
to non-random marriages. Sicne the population is divided into subgroups
by surname, ~qmqf and k would correspond to the inbreeding coefficientsF ST and Frs, respectively. F ST is the coefficient for an individual A
whose common ancestor was a person having a different surname from
A, while if the surname of ancestor was the same as that of A, wehave Frs.
Therefore, as shown by Crow and Mange (1), the total inbreeding
coefficient FIT of the population can be obtained from the relation (2):FIT=FsT+(I-FsT)Frs

where
and
Frs = (I- ~qmqf)/4(1-~qmqf) •At Nakazato village of Mishima district, 1464 couples were sampled fromkoseki or household records, in which his or her parents' surnames aswell as his or her own are recorded. Among them, 1063 males and 614
females were born in the area in question during the last hundred years.
Table 1.

Nakazato
Tokyo

Proportion of isonymous marriages and estimated F-statistics
at Nakazato and in Tokyo
Isonymy (I)9/430(0.0209)
10,116/363,797(0.0278)

FIs

FST

FrT

a

a/FIT

0.00456

0.002760.007310.0044*0.60

0.006410.00051

0.00695

0.0029**0.45* Shizuoka study (4).
**a =0.049/16, first cousin only.

Furusho, personal communication.

72

ANUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Their main occupation is farming. Table 1 summarizes the result,including a study in Tokyo (3). We have found that 95 pairs wereisonymous marriages according to koseki, but only nine of them were
truly isonymous. In this connection, we define the proper surname of anindividual as his or her father's surname when he or she was born. The
above phenomenon is mainly due to Muko- Yoshi-Engumi, a Japanesecustom for maintaining the family name through generations. When acouple does not have a son but has a daughter, a bridegroom-to-be isadopted just before the marriage takes place. Consequently, this results
in an increase of isonymous marriages in the koseki.
It is still too early to attribute any significance to the figures reported
in Table 1, but they suggest that F ST caused by random genetic drift
would be much higher in rural than in urban societies, although it isinteresting to note that the total inbreeding coefficients (FIT) are almost
the same. The mean inbreeding coefficient (a) from pedigree study would
be FIT if the ascertainment of pedigrees is complete, but it is only about
one half of FIT in both areas.
Sampling bias on F ST may be checked by a comparison of the quantities '2.qm 2, '2.q/ and '2.q~+f: each corresponding to the proportion ofisonymous pairs when two males are drawn at random, to the corresponding proportion in females, and to the frequency of isonymous
pairs irrespective of sex. Table 2 shows the results in terms of F sT • The
difference between the second and the third columns may be due tosample size with regard to sex, since koseki is filed by hittosha or family
representative who is usually a husband; only in sixty of the 1464 couples
the wife was the family representative. The last column is taken from
Table 2.

FST

N

Random component (FST) estimated from various procedures"f:.Qmqf(4"f:. Qf 2(4"f:.Qm 2(4"f:.Qm+f2(4"f:.q2(40.002761677

0.00294614

0.004441063

0.003461677

0.004925928

N=sample size
Table 3.

The relationship between the proportion of isonymous
marriages and matrimonial distance in Mishima

Marital distance (r)(km)
II: isonymous
II: couples
Frequency (1)0-0.4

0.4-0.8

0.8-1.2

1.2-2.8

2.8-

570
0.07144111
0.03602
96
0.0208

3
212
0.014

0
455
0.0000RESEARCHES CARRIED OUT IN 196773

a telephone directory which covers the studied area, and it can becompared with the fourth column obtained from koseki.
An attempt was made to study isolation by distance with a relationshipbetween the frequency (1) of isonymous marriages and the matrimonial
distances (r). Adding some other data in Mishima to the above survey,
we found that the proportion of isonymous marriages decreases with
distance (Table 3). A mathematical form of the relationship is tentatively I=exp (_kr 1 / 4 ), where k=2.37jkm.
The study is still continued in order to clarify.
1.

2.
3.
4.

Crow, J. F. and Mange, A. P., 1965. Eugen. Quat. 12: 199.
Wright, S., 1951. Ann. Eugen. 15: 323.
Kamizaki, M. 1954. Seibutu-Tokei 2: 292. (In Japanese).
Tanaka, K. 1963. In The Genetics of Migrant and Isolated populations (Ed.
E. Goldschmidt). p. 148.

A gene counting method of maximum likelihoodfor estimating gene frequency
Norikazu YASUDA

Let us suppose that the observed number of the i-th phenotypic classk

and the corresponding expected number are respectively ni (I ni=N) andki=1

NO i (IOi=I), where Oi is a function of gene frequency pj (j=I,.'";=1

in which m is the number of alleles).

m

The log-likelihood of observation isk

InL=const.+ I ni1nOi .
i=1

Defining score Uj as the first derivative of In L with respect to h, thenumber of the representatives of the j-th allele in the population may
be given by(j=l, ... , m).

(1)

The gene frequency is thus calculated fromm

Pj=Ejj I E r(j=I, ... , m) ,

(2 )

r=l

where the denominator is the number of identified genes in the population. Since E j may be expressed in terms of ni and proportion of acertain genotype within a given phenotype, h, the gene frequency to be
estimated may be obtained by iteration starting from a rough value ofp or h. The final value obtained by this method is the solution of

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

maximum likelihood equations which are Ui=Uj (i, j=l, ... , m) in thepresent terminology, because for all j we have U j = I'" E r from (1) and (2).
"'=1

Example. If individuals are unselectively sampled, the total number,of genes,

I E is independent from p; that is 2N. Thus, for instance,r

r=1

suppose that the MNSs blood group system is described by the following notations:
phenotype
MS
MSs
Ms
MNS
MNSs
MNs
NS
NSs
Ns

genotype
MSjMS
MSjMs
MsjMs
MSjNS
MSjNs, MsjNS
MsjNs
NSjNS
NSjNs
NsjNs

Total

observed no.

expected freq.

(ni)

(Oi)

nl

Pl2
2PIP2
P2 2
2PIPS
2PIP4 + 2P2PS
2P2P4
PS2
2pSP4
P4 2

n2
ns

n4
ns

n6
n7
n6
ng
N

We have then simple formulae:
gene
gene frequencyMS
PI =al + f3h
Ms
P2=a2-f3hNS
ps=as-f3hNs
P4=a4+f3h,
where a'S and f3 are constant so thatandal =(2nl +n2+n4)/2N ,IX2=(n2+2ns+n6+nS)/2N,

as=(n4 +2n7+ns +ns)/2N
f3=n s/2N.

IX4=(n6 +n8 +2n g )/2N

The probability, h, that an individual with phenotype MNSs has genotype
MS/Ns can be determined as a solution (O~h~l) of a cubic equation:S2!(h)=h +ah +bh+c=Oin which
a=[(al +IX4)-(a2+aS)- 13]/213
b=[ala4 +a2a$ - f3(al +(4)]/2f3 2

andc= -ala4/2f32 •RESEARCHES CARRIED OUT IN 1967

15

We may choose -cjb as a trial value of h. Further improvement may
be made through iteration using hi+l=h;-f(h;)jf'(h;), where f'(h)=3h z+2ah+b.

The variances of estimates are obtained after some calculations as.
follows:

andV(P4)=A[P4(Pl +pz) +PS{D-P3PiPl +pz)}] .
2N PS+P4
D-P1PZ(P3+P4)-P3P4(Pl +pz)

where D=Pl +2PZPS-(Pl +PZ)(Pl +Ps).
The above technique may be applied to any type of data that arecomplicated by sampling procedure and blood-relation among individuals.
For detail, see Yasuda, N. and M. Kimura, 1968. Ann. Hum. Genet. 31:.
409-420, and Yasuda, N. 1968. Jap.]. Hum. Genet. 12: 226-245.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

VI.

EXPERIMENTAL STUDIES ON POPULATION GENETICS
Allelic Rate between Lethal Genes Extracted from Japanese
Natural Populations of Drosophila melanogaster
Chozo OSHIMA and Takao K. WATANABE

During the past seven years, a total of 2886 second chromosomes wereextracted by Cy-Pm technique each from a different male fly collectedfrom several natural populations of D. melanogaster, distributed at Kofu
and Katsunuma. About fifteen per cent contained recessive lethal genes
and most of the lethal chromosomes have been maintained by the Cy
balanced condition in our laboratory. Allelism tests between these lethal
genes isolated simultaneously and in successive years were performed
and the results are shown in Table 1.
All allelic rates except that of 1959, were quite high, because manygroups of allelic lethal genes where isolated simultaneously and someof them have persisted for a long time as shown in Table 2.
As two kinds of lethal genes, namely I 204 and I 207, had been extracted in 1959, they have persisted at least 7 years in the natural
Table 1.

Results of allelism tests between lethal genes

Year

1966

1959

1963

1964

1965

No. of extracted second
chhromosomes

144

688

826

905

(323+ a ) 2563

No. of lethal chromosomes

21

114

100

140

375

14.58

16.57

12.11

15.47

14.63

No. of lethal chromosomes
tested for allelism

21

114

83

132

61

411

No. of crosses (half diallel)

210

6441

3403

8646

1830

20530

No. of allelic crosses

0

170

143

169

37

519

Allelic rate (%)

0

2.64

4.20

1.95

2.02

2.53

81

132 124

61

Frequency (%)

No. of lethal chromosomes
tested for allelism
No. of crosses (diallel)

97

79

Total

7857

10428

7564

25849

No. of allelic crosses

184

284

85

518

Allelic rate (%)

2.35

2.38

1.12

2.00

77

RESEARCHES CARRIED OUT IN 1967
Table 2.

Allelic groups found in lethal genes

1963

1964

1965

1966

No. of lethal chromosomes

114

83

132

61

No. of non-allelic lethal chromosomes

68

39

81

40

No. of allelic groups

13

14

16

10

No. of appearance

1399204- 76
6
3
3
3
207- 2
2
2
2
2
2

10
9
8
7
207- 6
5
204- 4
2
2
2
2
2
2
2
2
2

204- 54
4207- 3
3
3
3
2
2
2

Year

8
204- 66
5207- 4
4
3
2
2
2
2
2

populations. Another two kinds of lethal genes, namely I 201 and1 215, have been obtained every year since 1963. The loci of theselong term persistent lethal genes, I 215, I 204, I 201 and I 207, were
determined at 1.9, 32.1, 47.9 and 58.4 on the second chromosome,respectively.

Viability of Lethal Heterozygotes under Fluctuating Environment
Chozo OSHIMA and Takao K. WATANABE

Several factors have been assumed to be involved in the persistence()f some recessive lethal genes such as linkage with an epistatic genecomplex, association with a heterotic inversion and linkage with SD
gene (Oshima 1965, 1967). Another factor confirmed in our recent
experiment is dealt with in this report. 323 second chromosomes wereisolated each from a different male fly collected simultaneously from

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

natural populations at Kofu and Katsunuma at the end of October 1966.
The viability of homozygotes for those chromosomes was examined
individually and thereafter 9 D;/D j heterozygotes, 89 DIN heterozygotes
and 225 N;/Nj heterozygotes were obtained by random combinations of
those chromosomes. (D: lethal or semilethal chromosomes, N: subvitalor normal chromosomes). Viability of all those heterozygotes wasestimated by Cy-Pm technique under both constant and fluctuating
temperature environments. Under the former environment a temperature of 25°C was constantly kept in the cultural room and under thelatter it was fluctuating twice within 24 hours between 20 and 30°C in
an "Insectoron" having a controller. The results are given in Table 3.
Table 3.

Genotype

No. oflines

Viabilities of three genotypes under constantand fluctuating environments
Constant (25°C)

Fluctuating (20-30°C)

No. ofcounted Relative viability
flies

No. ofcounted Relative viability
flies

NilNj
NID
DilDj

225
89
9

59148
22958
2005

0.9940±0.01395
0·9794±0.01844
0.8775±0.08520

46088
18054
1682

1.0137±0.01245
1. 0431±0. 01616**
0.9884±0.09371

Total

323

84111

0.9869±0.01123

65824

1. 0211 ±O. 01007*

The mean viability of Cyl Pm, Cy and Pm flies is standard (1.0000)

The viability of all three genotypes increased under the fluctuating
environment as compared with the constant one, but the viability oflethal or semilethal heterozygotes increased significantly and became
better than that of normal heterozygotes under the fluctuating environment.
Table 4.

Genotype

LIN

No. oflines

128

Viabilities of lethal heterozygotes under constantand fluctuating environments
Constant (25°C)

Fluctuating (20-30°C)

No. ofcounted Relative viability
flies

No. ofcounted Relative viability
flies

89790

* 5 % level significance1.0068±0.00864

70664** 1 % level significance1.0353±0.01118 *79

RESEARCHES CARRIED OUT IN 1967

Similar experiments were performed with lethal and normal chromosomes extracted from flies collected from natural Viennese population by
Dr. D. SPERLICH. The same tendency as our above experiment wasrecognized in the results. Especially the viability of lethal heterozygotes
increased significantly under the fluctuating environment as shown in
Table 4. From those results, the lethal heterozygotes showed thehighest response to the fluctuating environment. This could be a contributing factor to the presence of some lethal genes in natural populations. The delicate effect of lethal genes on viability should be
repeatedly examined in fluctuating environments.

Persistence of a Lethal Gene Associated with SD in Cage
Populations of Drosophila melanogaster
Takao K. WATANABE

It was previously reported that a lethal gene 1401 (=/201) has been
maintained in natural populations associated with SD gene and that theSD action was not always effective when the SD chromosomes werepaired with many second chromosomes isolated from the natural populations. The segregation ratio k (k=SD-progeny/total-progeny) varied
from 1.0 (SD-sensitive) to 0.5 (SD-resistant).
Two large experimental populations containing SD and lethal chromosomes started with the following initial genetic constitutions: 100 %SD-/201/SD-sensitive heterozygotes in S population and 100 % SD1201/SD-resistant heterozygotes in R population. About 150 females
were sampled from each population several times during about 250 days,
and 1201 gene frequency and SD frequency was examined. The mean
Table 5. Lethal frequencies, SD frequencies and mean k valuesof chromosomes extracted from cage populations

Sampling

I.
II.
III.
IV.
V.

R population
S population
Days after
construction No. of Lethal SD Mean k No. of Lethal SD Mean kflies freq. freq. value flies freq. freq. value
0
20
92
142
190
247

800
126
114
143
132
91

50.0
57.9
31.6
30.1
25.0
27.5

50.0
58.7
29.0
33.6
28.0

0.996
0.839
0.815
0.928
0.853

800
118
134
129
133

50.0
29.7
16.4
13.2
13.5

50.0
29.7
15.7
14.7
12.1

0.496
0.570
0.567
0.522
0.522

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

k value was also determined. The results are given in Table 5.
The frequency of lethals decreased from 50 % to 25-30 % in the S
population and about 13 % in the R population. These frequencies seemto have reached in both cases an equilibrium state. The frequency ofSD gene decreased with the lethal gene frequency in both populations.
Mean k value in the S population was 0.996 at the initial state. But it
decreased to 0.853. In the R population the initial mean k was 0.496
and was fixed at 0.522 after about 140 days.

Interference by a Foreign Species in the Genetic Constitutionof a Drosophila melanogaster Population
Takashi NARISE

It was already known that the gene frequency of a half-pint milkbottle population of Drosophila melanogaster might be affected byconstantly invading flies of another species, D. simulans. The rate ofchange was more or less proportionate to the number of the invading
flies. The melanogaster fly is a strong competitor against simulans. It
is of interest to find how a small number of melanogaster flies that
initially invaded into a simulans population could propagate at the costof simulans, and what would occur as to its gene frequency. This isa report of the results of an experiment dealing with this problem.
A population consisting of some 500 flies of simulans was grown in amilk bottle. One or two pairs of heterozygous melanogaster flies, ss/ssG,
were every two weeks repeatedly introduced into a simulans population.
The experiment was conducted with three replications.
As was expected, the melanogaster flies soon took the place of simulans
flies in the next generation when two pairs of melanogaster wereintroduced and in the third generation by introduction of one pair ofmelanogaster flies.
The frequency of ssG gene of melanogaster flies was lowered to 0.33
when initiated from one pair or 0.39 when initiated from two pairs ofmelanogaster instead of expected 0.45 by the effect of simulans flies.
After the extinction of simulans, however, the frequency of ssG generecovered to 0.45 which was the equilibrial point for the heterogeneous
population of Drosophila melanogaster.

RESEARCHES CARRIED OUT IN 1967

81

Aggressiveness of Race 21B of Wheat Leaf Rust
Keizo KATSUYA

The aggressiveness of races 1 A and 21 B of wheat leaf rust, Pucciniarecondita Rob. ex Desm. f. sp. tritici Erikss., was estimated in thecondition of a growth chamber. About the same number of seedlingsof a wheat variety, Norin No. 16, inoculated singly with urediosporesof races 1 A and 21 B were kept in a growth chamber. The growthchamber was maintained at the temperature of 20°C and relative
humidity of about 60 96. Light was supplied for 16 hours per day bycool white fluorescent tubes. Three months later, new seedlings of
Norin No. 16 planted in clay pots were placed in the growth chamber.
The seedlings were naturally infected with urediospores from theinitially inoculated plants. Six months after the experiment started,
new seedlings were again used which were also naturally infected in
the growth chamber. Thereafter randomly selected nine uredia fromthe naturally infected plants were isolated and multiplied on seedlings of
Norin No. 16 for making subcultures. The sub-cultures were tested on
differential varieties for identification of races 1 A and 21 B. The resultshow that all sub-cultures tested belonged to race 21 B. At the sametime, three mass-cultures originated from urediospores on naturally
infected plants in the growth chamber were also examined on differential
varieties for race identification. One of the mass-cultures was race 21 B
and the two others were mixtures of races 1 A and 21 B. But the
proportion of race 1 A in the mixtures was very small. The proportionof each race in the mixtures was determined on Norin No. 55 bycounting the number of resistant (race 1 A) and susceptible infections(race 21 B). From the results, it seems that under the condition of thegrowth chamber, as to sporulating ability and propagating ability ofurediospores race 21 B may be superior to race 1 A. The conclusion isthat race 21 B of wheat leaf rust is more aggressive than race 1 A
under the condition employed.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

VII.

RADIAliON GENETICS AND CHEMICALMUlAGENESIS IN ANIMALS

Comparison of repair ability of premutationaI damages among
different radiation sensitivity strains of the silkworm
Yataro TAZIMA and Kimiharu ONIMARU

As reported previously (this Annual Report No. 17, pp. 91-92) a direct
evidence of repair of premutational damages was obtained by split-dose
irradiation to silkworm spermatids. When 1,000 R r-rays were administered in two fractions to full-grown larvae of strain C108 with 6 and 12
hour intervals a decrease in induced mutation frequency was clearly observed.
After confirming the results, comparisons were made among different
sensitivity strains with respect to the effect of split-dose irradiation on
mutation frequency. The strains used were rb (the most sensitive), Ascoli (intermediate) and Kansen (the most resistant) in Experiment 671
and Kojiki (sensitive), Aojuku (intermediate) and Kansen in Experiment
010-5

B

A

140~....-_R!_'_ _

~~

",100
~

~

/

ABOOl1

i,~

•

~.

~

___ rb

11

'~_.
0

o

Intervals

HI

in

:

3 6
hour

Fig. 1. Comparison of the effect of split-dose irradiation on induced mutation frequency among three strains having different radiation sensitivity.
A: Mutation at pe locus, B: Mutation at re locus.

RESEARCHES CARRIED OUT IN 1967

83

672. r-rays were administered to full-grown larvae in two fractions,
500 R +500 R, with intervals of 0, 3, and 6 hours. The method used for
estimation of mutation frequency was similar to that reported previously.
The results of the Experiment 671 are shown in Fig. 1.
Throughout Experiments 671 and 672, a decrease in mutation frequency
by split-dose irradiation was observed only in the resistant strain Kansen.
These results, together with those obtained with strain C108, seem toindicate that only resistant strains are capable to repair radiation induced premutational damages, while sensitive strains lack this ability.
Aerobic and anoxic X-irradiation to spermatids in different
radiation sensitivity strains of the silkworm
Yataro TAZIMA, Kimiharu ONIMARU and Akio ONUMA

While we were engaged in applying pre- and post-irradiation treatmentswith nitrogen, it was soon noticed that a dose modifying effect toward
a decrease was more pronounced in irradiation in N 2 gas than post-treatment with the same gas. Mutation frequency obtained after irradiationin N 2 was 0.46 times as high as that obtained after irradiation in the air.
Based on these observations, irradiation experiments both in aerobic
and anoxic atmospheres were carried out in combination with post-treatment with O2 and N 2 atmospheres using strains with different radiation
sensitivity. The method used for the detection of mutations was thesame as that reported previously. 1,000 R X-rays were administered tofull-grown larvae in specially designed flat irradiation chambers, whichwere rotating during irradiation. Dose reduction factor thus obtained
was 0.48 for Kansen, the most resistant strain, while it was 0.64 for rb,
a highly sensitive strain.
These findings seem to suggest that some of the mutations in Kansen
induced in anoxic atmosphere were reparable, and the amount of repair
was estimated to be approximately 0.16 (0.64-0.48). An alternative explanation may be that the protectiveness of nitrogen gas from mutationinduction is lower for strain rb than for strain Kansen.
Furthermore, anoxic irradiation of strain rb markedly increased the
proportion of whole body mutants to mosaics, whereas anoxic irradiationof strain Kansen kept this proportion almost at the same level as obtained from aerobic irradiation. In the former case it seemed as if afairly large part of mosaic mutants had been converted into whole bodymutants. Since nitrogen is a well known protector from radiation injury,
the observed high incidence of whole body mutants cannot be interpreted
by a "lethal-hit" hypothesis, which asserts that a proportion of mosaics

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

is transformed into whole body mutants by a concomitant lethal hit on
the complementary strand. It could be rather explained by assuming
incapability of rb strain to restrict, under anoxic atmosphere, the transmission of a lesion from one strand of DNA to the complementary strand.
Relation between character of diapausing and mutation response
to r-radiations observed in the silkworm
Yataro TAZIMA, Piere TEULADE and Kimiharu ONIMARU

It was assumed in our previous paper (Tazima, 1954) that radiationinduced mutation frequencies depend on the metabolic conditions of ir·
radiated cells. With the purpose of obtaining supporting evidences ofthis assumption, experiments have been carried out to examine the effectof the diapause hormone upon mutation induction.
The extraction of the hormone was performed following Hasegawa's
method from ca. 3,000 suboesophageal ganglia extirpated from the pupaeof strain C108, which had been incubated during the embryonic stageat 25°C under light condition. The extract was injected as a saline solution into the body cavity of the full-grown larvae. However, all injected individuals died out though every precaution was used with respect
to extraction and injection.
Under those circumstances we were obliged to investigate the effectof a modified diapausing character upon mutation induction by applying
low and high temperatures during incubation. It is well known that incubation at low temperature produces non-diapausing egg layers, whilehigh temperature produces diapausing egg layers. Five egg-batches ofstandard type strain C108 were each divided into two; one group of thehalf batches was incubated under 25°C (high temperature) and the other
under 20°C (low temperature). Both groups were exposed to r-radiation
simultaneously at two developmental stages; i.e. immediately after hatching and after emergence of the moths. Mutation frequencies induced inspermatogonia and mature sperm were estimated by specific loci method.
The group incubated at high temperature produced 10096 diapausing
egg layers among females, while in the low temperature group 32.2% offemales were non-diapausing egg layers. In the latter group moths which
emerged at early dates were almost all non-diapausing egg layers andthose which emerged at later dates were mostly diapausing egg layers.
With the assumption that a similar tendency holds true also in the male,
male moths were divided into two groups according to the date of emergence; early emerging and late emerging.
Mutation frequencies obtained for both groups are shown in Fig. 1. A

85

RESEARCHES CARRIED OUT IN 1967

Irradiation to mature sperm

Low

(67.)

10

(I) 181"17 ell8rgiD& aoth"

P8,+

8

.......

l"'I

I

0
.-4
II

'J

6

...
0

t:l

!t:

""
....,g •
i
~

re,+

2
P8

o

10

5

Dos.
(A)

(Idl)

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Irradiation to mature sperm
(674)

8

(II) Late emerging moths

p8,+,..... 6

""I
....
0

II
'-J

>.
0

J:l

3
...t:
a'

4

J:l

....0
1i

r.,+

i

2

LOW}
_----oH1gb~_._----

o

_--

10

5

pe

H1gb} l'8

Low

(kJl)

Do••

(B)

Fig. 1. Relation between incubation temperature and induced mutation frequency after r-irradiation to mature sperm in male moths. A: Early emerging moths and B: Late emerging moths.

and B.
As clearly seen from the figures, radiation induced mutation frequencies
were higher in the group incubated under low temperature and in moths
emerged early which might have been non-diapausing egg layers if they
had been female.
These results seem to agree with the observation of one of the authors,

RESEARCHES CARRIED OUT IN 1967

87

Teulade, that eggs are resistant to radiation during oestivation, but they
become sensitive after hibernation.
Difference in the proportion of mosaics among mutants inducedby 14 MeV neutrons, r-rays and some chemical mutagens
in silkw,orm spermatogenic cells
Yataro TAZIMA, Kimiharu ONIMARU and Yosoji FUKASE

In the previous note (this Annual Report No. 17, pp. 97-98) we reported
that mutation response of silkworm spermatogenic cells to r-radiation
varies with the progress of development and that the incidence of mosaics in relation to whole body mutations is very low before miotic metaphase, reaches to the unity around V-4,5 (instar V day 4.5) and then increases very rapidly with the progress of spermiogenesis up to 6-10 inmature sperm.
Since mosaics and whole body mutations were assumed to be mutations,the former occurring in one of the double helices of DNA and the latter:>ccurring in both helices, it was considered to be of interest to compare
the above values with those obtained with densely ionizing radiations
3upposed to produce fairly large lesions and those obtained with some:hemical mutagens which were known to act on DNA within a very small
molecular range. 14.1 MeV neutrons, EMS and mitomycin-C were chosen
for our pourpose. Materials and method used for the detection of mutations were similar to those reported previously.
14.1 MeV neutron beams, generated from Cockroft-Walton type machine,rvere irradiated to the insects at V-I (germ cells being just before meiotic
metaphase) and mid-pupal stages (most of the germ cells being at the;tage of young sperm). The irradiation doses were 500 and 1,000 rad.
The ratios of mosaics to whole body mutations thus obtained were0.1-0,5 at V-I and 2.3-2.7 at mid-pupal stage, both being more or lesslower than those obtained for r-rays. EMS and mitomycin-C were administered to the insects by injection at stages of IV-3, V-I, maturelarvae and early pupae. The doses (concentration and volume) of theinjected chemical solutions varied according to the developmental stageof the insect. The incidence of mosaics was surprisingly high regardlessof the injected stage. For instance, injection at IV-3, when germ cells
are at the stage of late spermatocytes, gave mosaic ratios of 4-8 for EMS
and 11-16 for mitomycin-C. By injection at stages later than V-I, thevalues became much higher. Namely, mutations induced by both chemicals at later stages appeared as mosaics and very few were whole bodymutants.

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Those observations were in good accord with our expectation that thelarger is the mutation lesion the smaller should become the ratio of mosaics to whole body mutations and that the chemical mutagens such as
EMS and mitomycin-C will attack selectively single helices of DNA ofcells at non-dividing phase.
Stage of r-irradiation to the germ cells and survival of
discovered mutants in the silkworm
Yataro TAZIMA and Kimiharu ONIMARU

Since egg color genes are used in our study as markers for the detection of mutation, induced mutants are discovered at an early embryonicstage. Using this advantage, comparisons have been made with respect
to the survival rate among mutants induced at different developmental
stages of the germ cell.
The materials used in this experiment were obtained from our study
on the variation in mutation response in accordance with the developmentof germ cells (this Annual Report No. 17, pp. 97-98). The stages compared were III-I, V-4, fully grown larvae and moths. Male larvae ofwild type strain C108 were irradiated with 1,000 R r-rays, while irradiation to male moths was performed with 5,000 R r-rays. For the detectionof induced mutations specific loci method using pe and re genes was applied. Irradiation experiments were carried out in 1966; in spring irradiation was given to fullgrown larvae, in summer to III-I, and V-4 larvae
and in autumn to moths. Mutant pe and re eggs were collected in thewinter of 1966 and were submitted to survival tests in the spring of 1967.
Table 1.

Relation between the stage of applied r·irradiation to the germ
cells and survival of the discovered mutants (Expt. 671)

Mu~~tion Plot Irrad.
stage Dosethe locus No.

pereNo. of Hatched eggs Fully grown larvae Pupae---'-----.
eggs----'---0/
No. /0testedNo.
No.
%%pe-l III-I r-lOOO R 164//
pe-2 V-4
609
pe-3 Full G.
//274
pe-4 Moth r-5000 R 250

41
236
148

re-l III-l r-lOOO R 39
re-2 V-4
ff
278
re-3 Full G.
//
111
re-4 Moth r-5000 R
80

14
138

119

75
66

25.0
38.8
54.0
47.6

32
178
135
110

19.5
29.2
49.3
44.0

23
165
134
108

14.0
27.1
48.9
43.2

35.9
49.6
67.6

10
89
72

82.5

62

25.6
32.0
64.9
77.5

8 20.581 29.1
71 64.0
61 76.3

RESEARCHES CARRIED OUT IN 1967

89

The survival was checked at the following three stages: Le. at hatching, mounting of fully grown larvae and pupation. The data are given
in Table 1.
As can be seen from the Table, 1) hatchability of pe mutants was lower
than that of re mutants, 2) mutants induced at later developmental stages
had higher hatchability than those induced at earlier stages, and 3) failure
to survive occurred mainly during the embryonic stage of the mutants.
The finding (3) seems to suggest that they comprise a fairly large partof gross chromosomal aberrations in addition to point mutations inducedby radiations. The finding (2) does not appear to be compatible with our
expectation that gross chromosomal aberrations would be unable to passthrough moisis. Instead, mutations induced at earlier gemetogenic stages,
especially those induced at premeiotic stage showed lower survival rate.
Presumablly some change in physical property of genetic material may
be responsible for this difference as mentioned in our previous note.
Studies on strain differences in radiosensitivity of the silkworm
III. Comparison of radiosensitivity of a sensitive anda resistant strain during early cleavage cycle
Akio MURAKAMI

Differences in sensitivity to X-rays were examined in about 70 silkworm
strains. After X-irradiation to overwintered eggs at an early embryonicstage, their sensitivity with respect to the killing effect was determinedin terms of hatchability. The average LD so for 70 strains examined wasapproximately 1,000 R. LD so of the most sensitive strain, Kojiki (170 R),
was one-tenth of that of the most resistant strain, Tenmon (1,710 R).
Since it is known that the radiosensitivity of silkworm embryo is markedly different according to the developmental stage of the embryo (HIROBE
and OHI), one must carefully avoid this source of error. In this experiment the sensitivity was compared within one cell cycle at very earlycleavage of eggs.
Expt. 1. Eggs from females of a resistant strain Kansen (LD so =1,51O R)
and a sensitive strain Kojiki (LD so =170 R) crossed to males of a marker
strain, (pe re), and the reciprocal crosses, were irradiated with 1,000 R
X-rays. The eggs were kept at 25°C, and irradiated at 10 min intervals
during the period from 60 min (Meiosis I) to 230 min (Cleavage II) after
oviposition. The results obtained are shown in Fig. I-a and -b. As canbe seen from Fig. I-a sixfold difference in radiosensitivity was observed
between Kojiki and Kansen at dividing phase, while no difference wasobserved at interphase. However, in the reciprocal crosses, (pe rex Kojiki)

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

100

..
c.,

...,
u

a.

...c
.
.........

50

>.

.0

x pere..''""

..c
u

:r

0

100

60

150

200

230 (min)Time after oviposition

Fig. I-a. Effect of X-rays on the hatchability during meiosis and early cleavage in oocytes and/or in eggs of different sensitive strains of silkworm.

100•..........".

..
.

.~.\.

--0--- pere x Kansan--v-- pera x KojikiI

o I'"=--------:-'I':-::--

60

100

-=-=,..,...--_ _---;~.;;---_-::-':'~150

200

230 (min)Time after oviposition
Fig. I-b.

and (pe rexKansen), the difference in sensitivity was almost nil between
the two crosses regardless of different radiosensitivity between the strains used as male parents (Fig. I-b). These findings suggest that thecytoplasmic factor is, at least in part, responsible for the differential

91

RESEARCHES CARRIED OUT IN 1967

radiosensitivity among different silkworm strains.
Expt. 2. Eggs from inbred strains of Kansen and Kojiki and fromhybrids of, in both directions, reciprocal crosses between the two strains
were used as material. The eggs were kept at 25°C after deposition.
100 min eggs and 270 min eggs, corresponding to the stage of cleavage
II and to cleavage III, were irradiated with 1,000 R of X-rays at 10 min intervals. The results obtained are presented in Figs. 2-a and -b for inbredand hybrid-eggs, respectively. As shown in Fig. 2-a Kansen was 7-8

100

b)

-)

+>

.::
Cl!

<.>

f.<

a
.::

·rl

>..

+>

·rl

......

·rl
,D

'"

.<::
<.>
+>

Kojiki sib

'"

::r::

200

180

200

250

210 (min)Time after oviposition
Fig. 2. Effect of X-rays on hatchability during early cell cleavage of inbredeggs of two different strains in sensitivity and of hybrid-eggs obtained fromthe reciprocal crosses.

times as resistant as Kojiki and the difference between the two inbred
strains was more pronounced at the resistant phase (or interphase). It
is also seen from the figure that the resistant strain, Kansen, shows
clearly a cyclic change in radiosensitivity, while no such change is observed in the sensitive strain Kojiki. (Fig. 2-b) Perhaps the latter strain
is too sentitive to radiation. At any rate it was concluded from theseexperiments that an intrinsic difference is actually involved in the observed difference in radiosensitivity at early embryonic stage, although
it may be modified to a great extent by the stage of embryonic development.

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Studies on strain differences in radiosensitivity of the silkworm
IV. Variation in radiosensitivity to different X-ray doses
during an early embryonic stage
Akio MURAKAMI

It was reported in my previous note that a marked cyclic change wasobserved in radiosentitivity in accordance with the division cycle of cleavage nuclei at an early developmental stage of the silkworm. Thesefindings indicated that cells in the dividing phase were markedly sensitive
to killing action of radiation, while those in interphase were not. These
conclusions were obtained only after irradiation with 1,000 R X-rays. In
order to obtain a better understanding of sensitivity changes with respect
to the cell cycle, the present work was carried out with X-ray doses of0, 500, 1,000, 1,500 and 2,000 R. Eggs were kept at 25°C from 210 min
through 300 min after oviposition (during this time the eggs proceededfrom cleavage I to cleavage III) and were irradiated with X-rays (180 kVp,
%
100

.t-

......
.....•
o

II

...

.. SO
o:l

~

i

o"':2::':1"':0:-------2-S-0--------2-00-Tima aftar o~poBitioD(miD)

Fig. 1. Cyclic change in radiation sensitivity observed in synchronized early cleavage cells of silkworm for four different dose levels.
The two highest peaks and the two lowest troughs for embryonicmortality correspond to cells at metaphase/anaphase and at interphase/prophase. The egg-age of 210 to 230. 240 to 290 and 300 minutes corresponds to the stage of 2, 4 and 8 nuclei respectively.
The standard error of the mean of each point is given.

RESEARCHES CARRIED OUT IN 1967

93

25 mA, 1.0 mm Al filter and dose-rate at 1,000 R/min) at 10 min intervals.
The eggs used as material for wild type were F 1 eggs between femalesof C108 strain and males of a marker strain pe re which permitted simultaneously the examination of radiation-induced mutation frequencies. The
eggs were collected every 10 min at 25°C. The radiosensitivity wasmeasured in terms of embryonic mortality.
The relation between egg age and mortality for four different doses
is shown in Fig. 1. Mortality increased with the does for the whole dose
range and a cyclic change in radiosensitivity was observed within approximately 60 min cycle. With medium doses (1,000 and 1,500 R) a marked difference appeared with respect to sensitivity, while no clear-cut difference was observed with doses lower than 500 R and higher than 2,000 R.
Such difference in sensitivity pattern expressed by the different doses
applied may be explained by a differential reparability of the cells. Namely when cells are treated with higher doses, even those in resistant phasecannot repair radiation lesions, while after irradiation with lower doses,
the cells in sensitive phase may be capable to repair them to in the same
degree as when they were in the resistant phase. The dose-killing curvedfor 230 and 290 min-old eggs (both corresponding to sensitive phase) exhibited narrower shoulders than those for 210 and 260 min old eggs (bothcorresponding to resistant phase). The wide shoulders observed for cells
at resistant phase seem to pepresent the existence of an active repair
system which becomes incapacitated at higher X-ray doses.
Studies on strain differences in radiosensitivity of the silkworm
V. Relation between X-ray and UV-sensitivity inan early embryonic stage
Akio MURAKAMI

In a previous note marked differences in X-ray sensitivity were reported
among strains with respect to the killing of the embryo. In the mostresistant strain LD 50 was approximately 7-8 times as high as that of themost sensitive strain. Recently a repair mechanism of UV-induced damage has been reported for microorganism. This posed the question
whether or not a repair system for UV damage may be common phenomenon or at least may function also in some damages caused by ionizing radiation. Hence, this experiment was carried out. Materials usedwere some silkworm strains of different X-ray sensitivity: Kansen (1,510 R)
and Aojuku (1,480 R) and Kojiki (170 R). LD50 values obtained for embryonic killing are given in parentheses.
UV-irradiation was applied to the eggs at an early cleavage stage with

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

a commercial germicidal lamp (National Electric Co.). Care was taken
to avoid direct photoreactivation by visible light and all irradiation experiments were done in a dark room. Doses were adjusted by changing
the duration of irradiation but keeping the distance to the lamp constant.
The dose·rate used was 143 ergs mm- 2 sec-i. Irradiation was applied to200, 210 and 220 min-old eggs (which corresponded to the 2nd cleavage)
obtained from females mated with pe re males. Eggs were collected at
10 min intervals at 25°C. The UV sensitivity was measured in termsof hatchability.
UV Dose

1

10

30

50

70 (min)KansenKojiki
'p
\

0.01

\

\

\

\

AojukuFig. 1. Dose-hatchability curves for silkworm eggs (220 min-280 min
after oviposition) irradiated with ultraviolet (dose-rate: 143 ergs
mm- 2 sec-i).

The dose-response curve at 220 min old-eggs is presented in Fig. 1.
As can be seen in the figure, the radioresistance of Kansen (LD50 : 1O.3x
104 ergs mm- 2 ) to UV-killing is approximately threefold higher than forboth strains, Kojiki (LD50 : 27.5x 104 ergs mm- 2 ) and Aojuku (LD 50 : 27.5x
104 ergs mm- 2 ). It is very interesting to note that the radiosensitivity of

RESEARCHES CARRIED OUT IN 1967

95

strain Aojuku to UV-light has quite the same value as that of Kojiki,
however, strain Aojuku has approximately 7 times higher radioresistanceto X-ray killing in comparison with strain Kojiki as described earlier.
There was the same situation observed for 200 and 210 min old-eggs even
if the shape of dose-response relation was considerably different from thetreated egg-age (or the time at the start of UV irradiation). This evidence suggests that the nature of UV-sensitivity to embryonic killing
depends on the cell phase at the time of irradiation.
It is clear from the present experiment that no phenomenon of crossresistancy between X-ray and UV light sensitivity to killing effects ofsilkworm embryos was observed. This suggests that the mechanism for
X-ray killing and repair of silkworm embryos may be different from thosefor UV-light.
Relative biological effectiveness of fast neutrons for the
induction of dominant lethals at various stages of malegerm cells in the silkworm
Akio MURAKAMIIn the present study, the effect of fast neutrons and r-rays on the induction of dominant lethal mutations was compared for various stagesof male germ cells.
The sources of fast neutrons used were, 14 MeV neutrons derived fromT(d, n)He reaction and 1.5 MeV neutrons from 235U fission reaction. r-rays
were obtained from 137 Cs.
Irradiation experiments were performed at three germ cell stages: primordial germ cells in the embryos, early and late spermatogonia in earlylarval stages and mature sperm in the oldest pupae.
The incidence of dominant lethals was expressed by the percentage of
non-hatched eggs of the total number of fertilized eggs. After exposure,the males of wild type strain CI08 were mated to females having themarker genes pe and reo
The dose-response curves for the mature sperm were linear for bothradiations, hence, the RBE was obtained very easily. However, forother germ cell stages, the relation was not linear and such straightdirect comparison was impossible. Consequently, RBE values are represented arbitrarily as an inverse ratio of the respective doses required forthe survival of 80% and 50%. They are given in Table 1.
The values for the relative efficiency of neutrons are larger for mature
sperm than for pre-meiotic cells, i.e., primordial germ cells and spermatogonia. In spermatogonia the incidence of dominant lethals was clear-

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1. Relative efficiency of 137Cs r-rays, 14 MeV fast neutrons and
1.5 MeV fission neutrons on the induction of dominantlethals in silkworm.
R.
Germ cells

Primordial germ cells(egg stage)

Radiations

14 MeV neutrons)(

Spermatagonia(larval stage)

Mature sperm(adult stage)

8096

1.6

1.3

4.4

3.6

11.2

9.5

X-rays
14 MeV nentrons)(

Survival level
5096

X-ray
1. 5 MeV neutrons)

(

E.

X-ray
14 MeV neutrons)

(

B.

8.2*

X-rays* Calculated from the slope of the regression lines.
ly dependent on LET, as was previously observed for recessive visible
mutations. However, the LET dependence of dominant lethals is more
pronounced than that of recessive mutations, suggesting that the target
size is larger for dominant lethals than for recessive mutations.
As to the interpretation of the stage difference in RBE, it may not be
unreasonable to assume that germinal selection operates far more severely
for dominant lethals produced in primordial germ cells than in sperm.
In spermatogonia, the RBE of 1.5 MeV fission neutrons was approximately 2 times as high as that of 14MeV neutrons.

Effects of X-irradiation on aggregate-forming activity ofembryonic chick and quail liver and heart cells
Yukiaki KURODA

Dissociated cells from embryonic tissues of organ rudiments, when
cultured in gyrating flasks, formed aggregates having morphologicallyand biochemically tissue-specific characteristics. The patterns of the aggregates depend on the initial cell concentration, speed and temperature
at which the rotation cultures are carried out. Under a constant condition, the cells show a consistent aggregation pattern; the effects on itof various external factors can be quantitatively estimated.

97

RESEARCHES CARRIED OUT IN 1967

Livers and hearts were obtained from embryonic chicks and quails at
various stages and were dissociated into single cells by treatment withtrypsin. Cell suspensions each containing 106 cells per ml were introduced
into Petri dishes and treated with OR, 200R, 400R, 6OOR, SOOR, 1,OOOR, and1,200R of X-rays (175 KVp, 25 rnA, distance 40 em, filter 1.0 mm AI, dose
rate 300 R/min). After irradiation 3 ml of each cell suspension treatedwith various X-ray doses were transferred into 25-ml Erlenmeyer flasks,
which were rotated on a gyratory shaker at 70 rpm at 38°C.
The effects of X-irradiation on aggregation of dissociated lO-day embryonic chick liver and heart cells are shown in Table 1.
Table 1. Effects of X-irradiation on aggregation of dissociated embryonicchick liver and heart cells in vitro. (3 x 106 cells were rotatedfor 48 hours at 38°C at 70 rpm).
X-ray
dose (R)
0
200
400
600
800
1,000
1,200

Liver cell aggregate

Heart cell aggregate

Average diameter (p)0//0Average diameter (p)

%

190.0
220.0
227.5
254.3
248.0
238.0
218.0

100
116
120
134
131
125
115

325.0
416.8
450.0
500.0
425.0
418.0
410.8

100
128
138
154
131
129
126

As shown in Table 1, aggregates obtained from rotation cultures ofdissociated chick liver and heart cells were gradually increasing in the
average diameter with the amount of X-rays until 6OOR, then decreasing
it with still higher X-ray doses.
When liver and heart cells dissociated from 7-day and 10-day old embryonic quails were irradiated with various doses of X-rays, then tested
for their aggregability in rotation culture, the average diameter of theresultant aggregates was increasing gradually with the X-ray dose until
400R, then decreasing with still higher doses.
It has previously been found that aggregate-forming substances whichwere produced from dissociated cells act as intermediates in the formation of aggregates and that such substances may be produced throughRNA and protein syntheses (Moscona, 1961, 1963).
The findings in the present experiments suggest that the low doses of
X-ray may stimulate the activation of aggregate-forming substances already present at the cell surface and the higher doses of X-ray may in-

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

hibit the new production of these substances through suppression of RNAor protein synthesis.
Skeletal abnormalities in the F 1 offspring of neutronirradiated male mice
Kiyosi TUTIKAWA

Ehling has recently reported that part of overall genetic damage from
irradiation can be detected by studies of skeletal abnormalities in thefirst generation of mice. He has also emphasized that the most sensitive
indication of mutations in skeletal studies is probably a statistically significant increase of Class-I multiple abnormalities and Class-l abnormalitiesof the bilateral type of the appendicular skelton.
The present experiment was designed primarily to obtain an estimateof the frequency of neutron induced presumably dominant mutations affecting the skeleton. The following different preparation methods wereused; the part of skeleton involving skull, cervical vertebrae and the first
three thoracic vertebrae were macerated by means of the proteolytic
enzyme papain, and the remaining parts were prepared by a modificationof Dawson's technique.
The irradiation with 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. Male CBA strain mice were irradiatedwith doses of 242.5 and 485 rad neutrons, and subsequently the males
were mated to PW strain females which were established as an inbred
strain in our laboratory and are homozygous for six recessive genes (a,b, cch, d, p, and se). The F 1 offspring were sacrificed when about six
weeks old, and the animals were examined by means of prepared skeletal
specimens.
The examination of skeleton is still in progress. So far, only a small
number of the papain-digested skeletons among all prepared specimens
have been examined in detail. Animals with Class-l single or multiple
abnormalities of the axial skeleton which probably correspond to Ehling's
classification, are briefly described as follows (single animals are identified
by Arabic numerals);
1) Control group: (~188)Malformation of the skull similar to hydrocephalus. 2) Offspring derived from irradiated males with 242.5 radneutrons in postspermatogonial stages: (~73) Malformation of the skull,
shape anormalies of the interfrontal bone and foramen ovale (both), presphenoid-basisphenoid fusion, small body size; (~117) abnormal frontals,
presphenoid-basisphenoid fusion, dyssymphysis of the C7 and Thl, small

RESEARCHES CARRIED OUT IN 196799

body size; (# 230) foramina transversaria imperfecta in the C3, C4, C5
on both sides and C6 on the right side, small body size. 3) Offspring

derived from irradiated males with 485 rad neutrons in postspermatogonialstages: (# 17) Malformation of the skull, shape anomalies of the interfrontal bone and foramen ovale (both), abnormal elongation of the incisor,
presphenoid-basisphenoid fusion, basisphenoid-occipital fusion, dyssymphysis of the C4, foramina transversaria imperfecta in the C3, C5 on bothsides and C4 on the right side, small body size; (# 23) malformation of
the skull, absence of the right tubercula anteria in the C6, small bodysize.
In the examination of these limited parts of the axial skeleton, the abnormality ratio per gamete for the 242.5 rad group was 0.0099 (3 out of
302 offspring), 0.0196 in the 485 rad group (2 out of 102 offspring), and
0.0033 in the control group (lout of 303 offspring). The excess of abnormalities per rad was 2.7 x 10- 5 for the 242.5 rad group and 3.3 x 10- 5
for the 485 rad group with respect to irradiation in postspermatogonialstages. A more precise estimate of the frequency of neutron induced
presumably dominant mutations affecting the skeleton will be possible
when the experiment is complete.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

VIII.

RADIATION GENETICS IN MICROORGANISMS
AND PLANTS
Selective Growth of Radiation Sensitive Mutantsin Escherichia coli K12
Tsuneo KADA

Among many auxotrophic strains regarding threonine obtained from
Escherichia coli K12 RfrR after ultraviolet irradiation, a strain named
Rfr 1014 had some particularities. When cells grown in broth were
washed and plated densely on threonine-free synthetic medium, somefast-growing colonies appeared very frequently (more than 10- 3). It was
found that bacteria purified from these colonies had stronger sensitivities
to either ultraviolet light or r-rays than the parent strain Rfr 1014. Cells
formed long filaments after very mild doses of radiations. Survival curves
were very similar to those of fil+ or lon- strains.
The following experiments suggest that the strain 1014 has a deficiency
at the usual threonine locus and an inactive suppressor gene for thestrain which is linked to the leucine locus. This is indicated by thr;o14(Su+). First, Rfr R (prototroph except B 1-, SmS ) was crossed to PA 309(thr- leu- try- his- arg- B 1- lac- gal- man- Sm r ) for 10 minutes and athreonine-independent strain witdout modification in other characters was
isolated (PA 309 thr+). Threonineless strains were then obtained bydirect selection from a 5 minute conjugational culture between Rfr 1014
and PA 309 thr+. These thr~14 strains were not leaky and their spontaneous reversion frequencies were between 10- 6 and 10- 7 • In the next
experiment, Rfr 1014 was crossed to one of thr;o14 recombinants named
PA 309 thr;o14 (thrw14 leu- try- his- arg- B 1- lac- gal- man- Sm r ) for 20
minutes and leu+ Smr recombinants were selected. It was found thatabout 80% of them became concomitantly prototrophs as to threonine.
Production of threonine prototrophs is not expected if both donors andrecipients were complete threonine auxotrophs. It is reasonable to suppose that a leucine-linked suppressor gene exists in the Rfr 1014 andthat it might have been transmitted into PA 309 thrlo14 and suppressed
this time actively thr~14'
Because suppressed threonineless bacteria (thr;o14 Su+) grow scarcely inthe absence of threonine, fast growing colonies were easily selected withhigh frequencies by plating these bacteria on threonine-free plates. The
possibility that they reverted to thr+ state involving true mutation in
the threonine locus may be improbable because we know that frequenciesof spontaneous true reversion in thr:014 Su- strains are low (between 10- 6

RESEARCHES CARRIED OUT IN 1967101

and 10- 7 ). On the other hand it seems more reasonable to suppose thatthe frequent production of fast-growing colonies must be related to somedivision abnormalities and that the genetic determinant(s) responsible forfilamentation may promote specifically the growth of Su+ cells. Present
studies on mapping our fg (fast-growing) gene(s) indicated that they arelocated in the gal region of the chromosome.

Radiosensitization by Potassium Iodate andRelated Compounds
Teuneo KADA

Potassium idoate and certain related compounds were found to be very
efficient radiosensiltizers; their presence at non-toxic levels during irradiation increased radiation-induced cellular lethality. The test-strain usedin these experiments was a threonineless and thiamineless derivative ofEscherichia coli Kl2 Hfr H. The two auxotrophic characters of thisstrain were prepared for other purposes and are of no significance forthe results described; the wild Kl2 strain behaves quite similarly.
Bacteria were grown exponentially in liquid broth, washed twice with
M/l5 phosphate buffer and resuspended in the same solution. Potassium
iodate and other related compounds were dissolved in distilled waterat diverse concentrations and sterilized by filtration. Usually one volumeof reagent solution was combined with nine volumes of air-saturated
bacterial suspension just before irradiation in order to obtain the desired
drug concentrations. The bacterial suspension contained approximately
I X 106 cells per ml at the time of irradiation. Air-saturated suspensions
held at the melting temperature of ice were irradiated with r-rays of
137es at a dose rate of 667 rads/min. Survivors were estimated by thesurface count technique of living bacteria on nutrient broth agar.
The effects of several chemicals on radiation inactivation of test bacteria were indicated in Table 1. Among the compounds tested, iodate,periodate anh paraperiodates possessed marked radio-sensitizing capacities at concentrations at which they were not toxic to non-irradiatedbacteria. When the iodine atom was replaced in iodate by other halogen
atoms, no sensitizing capacity was observed as was found in cases withchlorates or bromates. This also indicates that alikali ions are notresponsible for the sensitization. Detalied studies with potassium iodate
showed that the sensitizing capacity was pH-dependent and the maximum
effect was found between pH 5 and pH 6. Because periodates are known
to be mild oxidizing agents, the possibility is under examination if theirreaction with cellular sulfhydryl key molecules might be involved under102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1. Sensitizing effects of potassium iodate and related compounds on
r-ray inactivation of colony-forming capacity in Escherichia celi K12
pH ofbacterial
suspension

Radiation
dose(R)

No. ofviable cells
per ml

7.0
7'0
7.0
6.0
5.0

0
1290
1460
1290
1290

1.0 X 106
8.6x105
7.7 X 105
8.1x105

7.0
7.0
7.0
6.0
5.0

0
1290
1460
1290
1290

0.9 x 103

Potassium periodate (0.2 mM)

7.0
7.0

0
2000

1.0 X 106
5.2x10'

Trisodium paraiodate (0.2 mM)

7.0
7.0

0
2000

9.3 X 105

Potassium chlorate (2 mM)

7.0
7.0

0
1460

1.1x106
7.4 x 105

Potassium bromate (2 mM)

7.0
7.0

0
1460

9.6x1059.1x105

Reagnt present
during irradiation(concentration)

None

Potassium iodate (2 mM)

6.4 x 105
1.1 X 106
3.8x10'5.9 x 10 3
1.0x103

7.1 X 10'

irradiation.

Photoreactivation of UV-Induced Mutations in Maize
Tar6 FUJII

Photoreactivation of UV-induced endosperm mutations (Bz-gene) was
already reported by the present author (d. Ann. Rep. 17: 117-118).
Besides the Bz-gene the materials used in that experiment had the dominant and the recessive Yg 2 and yg2 gene (yellow green character) in maleand female stocks, respectively. Mutation from Yg to yg and its photorecovery was examined in F 1 seedlings. The results are given in Table
1. Very narrow stripes of yellowish-green color were observed in several
seedlings but partial mutations which appeared as sectors on a half or

RESEARCHES CARRIED OUT IN 1967

103

one-fourth of the leaf area were very seldom. Most of these narrowsectors might be caused without Yg~yg mutation because their frequency
is much lower than that of whole mutation (in Bz-gene, the proportion
Table l.

Treatment

Control
r-ray

UV

Dark
PR
250 R Dark
PR
500 R Dark
PR
750 R Dark
PR
15 sec Dark
PR
30 sec Dark
PR
60 sec Dark
PR
90 sec Dark
PR

Frequency of yg-seedlings

No. ofseeds

No. of ygseedlings(%)

No. ofseedlings
with narrow
stripes (%)

3479
2516
4971
3944
6134
6437
3596
6569
2420
6182
4106
675
549
5904
4557
4490

6 (0.17)10 (0.40)
15 (0.30)
28 (0.71)
41 (0.67)
42 (0.65)
46 (1.28)
71 (1.08)
6 (0.25)
11 (0.18)
29 (0.71)
1 (0.15)5 (0.91)
12 (0.20)
12 (0.26)
20 (0.45)

1 (0.03)0
6 (0.12)1 (0.03)9 (0.17)3 (0.05)
1 (0.03)4 (0.06)
1 (0.04)
1 (0.02)00
1 (0.18)02 (0.04)
6 (0.13)

Others*(%)
2 (0.06)0
4 (0.08)
3 (0.08)8 (0.13)3 (0.05)
1 (0.03)
16 (0.24)
3 (0.12)4 (0.06)00
1 (0.18)
3 (0.05)
2 (0.04)
1 (0.02)* No. of seedlings with white stripes or other kinds of chlorophyll deficiency.

of partial mutation to whole mutation was comparable). Further, white
stripes or other kinds of chrolophyll deficiency were also observed, but
the occurrence of these mutant seedlings is left out of consideration.
In the gamma-ray lots, mutation frequency after dark-treatment showedsome fluctuations according to the dosage but it was possible to judge
from the results that the post-treatments had no effect on gamma-ray
exposures. In the UV-exposures, on the other hand, the saturation effectof mutation frequency at the highest dosage lot after dark-treatment anda clear decrease in mutation frequency after PR-treatment were again
observed. Namely, a photorecovery phenomenon was observed only in
UV lot as was observed in endosperm mutations.
Endosperm Mutations in Maize Induced by Fast Neutrons
Taril FUJIIFor the promotion of RBE study, 14 MeV neutron generator was

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

installed last year. Irradiated by 14 MeV neutrons and gamma-rays
maize pollen with Bz-gene was crossed on the recessive stock. Mutationfrom Bz to bz could be observed in the F 1 seeds with bronzy aleuron
color and the results are given in Table 1. Neutron flux was calculatedwith S, P reaction, and the neutron doses applied to pollen were decided
Table 1.
Treatment
ControlGamma-rays
300 R
600 R
900 R
1500 R
Neutrons
154 rad284 rad295 rad545 rad2034 rad3700 rad10735 radEndosperm mutations after pollen irradiations

No. ofseeds

AverageNo. of
seeds

2927

146.4

1 (0.034)

2 (0.068)

2821
8642
5230
14231

111.8
96.0*
118.9*
151.4

1 (0.065)
17 (0.197)
46 (0.880)
256 (1. 799)

3 (0.106)
14 (0.162)
36 (0.688)
219 (1.539)

4631
6973
5346
7470
3058
1157
72

178.1
120.2*
130.4*
138.6
68.0
19.3
3.6

31 (0.669)
104 (1.491)
65 (1.216)
170 (2.406)
163 (5.330)
85 (7.347)
1 (1.389)

24 (0.518)
79 (1.133)
92 (1.721)
234 (3.312)
268 (8.704)
99 (8.557)
9(12.500)

No. of mutant seeds (per cent)
Whole

Partial* Some of the seeds were obtained from potted plants, therefore their seed set
was more reduced than in plants grown in the experimental field.

by the first collision dose calculated on the basis of a chemical pollen
analysis. Average seed set was not reduced in gamma-ray lots at the
dosages used, but it clearly decreased in the neutron lots of more than2000 rad. Two types, whole and partial mutation, were induced by bothradiations. Frequency of the latter was slightly higher than of theformer in gamma-ray lots, and the proportion of whole to partial mutations did not depend on the dosage. In the neutron lot, the proportion
was the same as for gamma-rays in lower dosage lots, however thereverse relation was observed at dosages higher than 295 rad. Moreover,the increase in frequency of whole mutations became flattened in higher
dosage lots in spite of the clear increase in partial mutations in theselots. Whole or partial mutations may arise from radiation damage ofchromosome or chromatid units, respecitvely. If this assumption iscorrect, whole mutations should be associated with semilethal lesions or

RESEARCHES CARRIED OUT IN 1967

105

feeble viability. Therefore, pollen grains which suffered radiation damage
leading to whole mutations may have less survivals than those withpartial mutations.
It is obvious that the mutation frequency after neutron irradiation isvery much higher than that after gamma-ray irradiation; by gamma-ray
irradiation 1.8% of whole mutation was induced at 1500 R, while it could
be induced at less than 545 rad by neutron irradiation. As to partial
mutation frequency, almost the same frequency was observed at 1500 R
of gamma-rays and 295 rad of neutrons. The material employed in thepresent experiment had also the yg2-gene and mutation from dominant
to recessive could be observed in the F 1 seedlings. Examination is now
underway and RBE value in the mutation frequency by pollen irradiation will be assessed after the experiments.

Sectorial Mutations Induced by Gamma-Ray TreatedFertilized Maize Endosperm
Taro FUJII

Mutation frequency of whole and partial mutations was investigatedafter pollen irradiation. In this experiment, no partial mutations of yg2gene (plant color) in the F 1 seedlings was observed while both wholeand partial mutations of Bz-gene (aleuron color) were found in the F 1
seeds (d. pp 102 in this issue and Ann. Rep. 17: 117-118). It is assumed
that the differentiation into root and leaf occurred in the fertilized egg
immediately after fertilization, and therefore, no partial mutations which
are induced by the damage of chromatid units, could be observed. Pollen
having Bz and Yg-genes was applied to a recessive stock, and the female
plants were 24, 48, and 72 hours after pollination exposed to 1 kR ofgamma-rays. Frequency of whole and partial mutations and relation
between the frequency and exposure time were investigated in F 1 seeds
and seedlings; the results are given in Table 1. Apparently, there were
no appreciable radiation effects on their maturing and fertility.
In endosperm mutations, frequeny of whole mutations (Bz-gene) was
clearly lower than after pollen irradiation even in the 24 hr lot, and it
further decreased to about one half in 48 hr lot. The same frequency
was observed in the 72 hr lot. On the contrary, frequency of partial
mutation markedly increased in the 24 hr lot and about one half or moreof the examined seeds had partial mutations in 48 and 72 hr lots. Mostof these seeds had two or more mutated sectors per seed. The decreaseof whole mutations was related to the increase of the partial kind.
In the F 1 seedlings, frequency of wholly mutated seedlings (yg-gene)106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

Relation between the time from pollination to irradiation andfrequency of whole and partial mutations
Time between
pollinationand irradiation

No. ofseeds orseedlings

Endosperm(bz-gene)

Pollen*
24 hr
48 hr
72 hr

Seedling(yg-gene)

Pollen*
24 hr
48 hr
72 nr

Number of mutants (%)
Whole

Partial

8858
5898
6079
6888

72 (0.81)
2 (0.03)1 (0.02)
1 (0.02)51 ( 0.58)
1744 (29.57)
3986 (65.57)
4100 (59.52)

7689
4110
4539
5614

89 (1.16)
65 (1.58)
42 (0.93)
25 (0.45)

3** (0.04)
28 (0.68)
74 (1.63)
389 (6.93)* Results from the gamma-ray 900 R irradiation to pollen grains.
** These seedlings had very narrow yellowish-green stripes; see text.

in the 24 hr lot is the same as in pollen irradiations (the difference indosage between two lots seems to be out of the question: it is merely
100 R), and it decreased to about one half and more according to thelapse of time after the pollination, i. e. 48 and 72 hours. But mutation
frequency of the partial kind increased with passing time and that of
the 24 hr lot was ten times higher than that of pollen irradiations.
(Yellowish green stripes on seedlings after pollen irradiation could not
be determined as partial mutation because of very narrow sectors; partial
mutation could be expected to have sectors occupying a half or onefourth of the leaf area). The frequency in the 48 hr lot was about twiceas that in the 24 hr lot; it was inversely proportional to the whole type,
and that in the 72 hr lot was about four times higher in spite of thedecrease to one half of whole mutations. Thus decrease of whole andincrease of partial mutations both in Bz- and Yg-genes with increasingtime between pollination and irradiation showed a reciprocal relation in
24 and 48 hr lots, but the relation was complicated in 72 hr lot; thephenomenon observed in that lot could be the result of a large numberof target cells according to repeated cell divisions.
In the present procedure, partial mutation could be observed in F 1
seedlings, but it could not be considered to be on effect of the damage
suffered by chromatid units as was observed in pollen irradiations.

107

RESEARCHES CARRIED OUT IN 1967

Modification of Killing Efficiency in Neutron
Irradiations of Arabidopsis Seeds
Tar6 FUJII

Dry and 24 hour steeped seeds of Arabidopsis thaliana were used.
Thermal neutron exposures were carried out by the pneumatic tube
Kyoto University Reactor at the output of 50 kW, the total neutron flux
being 3.9 x 1012 at the minimum and 7.1 x 10 13 at the maximum. The
neutrons consisted of about 90% thermal and 10% fast neutrons, and theneutron field was strongly contaminated by gamma-rays. The same materials were also exposed to monoenergetic 14 MeV neutrons from T(d, n)reaction neutron generator in our institute. Total neutron flux was 3.8 X
13lOll ~ 1.8 X 10 • Decrease of survival rates after irradiation is shown in
Table 1 together with the results from gamma-ray experiment for comparison with three kinds of radiation. In the experiment with thermal
neutrons, severe killing effect was again observed like in our previous
experiment: almost all plants died in the 71.5x 1012 neutron lot of dry
seeds and a similar survival rate was observed in the 35.6x 1012 lot of
wet seeds, while 37.5 kR of gamma-rays alone had no effect in the formercase and 16.5 kR might have had a only slight effect in the latter case
Table l.

Killing effects of gamma-rays and neutrons for dry
and wet seeds of Arabidopsis

Gamma-rays

Thermal neutrons

Fast neutrons

Dosage
(kR)

Dry

Wet

DosageDry

Wet

Dosage

Dry

(%)

(%)

(x 1012 )*

Wet

(%)

(96)

(xl011 )

(%)

(%)

10

91.6

92.7

80.7

76.7

3.8

85.8

83.9

30

90.8

75.6

85.2

39.7

4.6

80.6

78.6

50

92.8

21.7

72.5

6.0

15.5

73.9

51.0

70

93.3

0.0

72.5

2.1

19.0

66.2

32.4

100

82.4

0.0

3.9
(2.5)
15.4
(7.5)
26.6
(12.5)
35.6(16.5)
43.0(22.5)
53.1(27.5)
60.8(32.5)
71.5(37.5)

56.3

0.0

35.0

64.2

34.6

40.9

0.0

176.0

1.1

0.0

14.1

0.0

1.9

0.0

* Figures in parentheses show the contaminating gamma-rays in kR.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

when compared with the results with gamma-rays. Thus the killing
effect of thermal neutrons was very high, and it seems to have been theeffect of high LET radiations modified by the water content of thematerial. But the assessment of thermal neutrons' RBE is very complicated because of its multienergetic condition and abundance of contaminating gamma-rays.
Fast neutron irradiations to dry and wet seeds also showed the effectof water content. Survival rates in wet seeds at 19 or 35 X lOll neutron
lots were about half of those obtained from dry seeds. Fast neutron doses
given to seeds were calculated from the first collision dose converted fromthe given tissue dose by using chemical seed analysis, and dosage in radunit was reckoned from the multiplid total neutron flux by 5.98 X 10- 9 •From the calculations 3.8 and 35.0x 1010 neutrons are equivalent to 2.27
and 20.93 krad, respectively. When the results were compared with thatof gamma-rays, fast neutrons were nearly 10 times more effective in dry
seeds and about 5 times more effective in wet seeds at LD so level.
Namely, modification of radiation damage due to water content was also
observed in 14 MeV neutron exposures. Nevertheless, the modification
factor is too large in the present experiment, though the environmental
modification of radiation effect is, as a general rule, very small in highLET radiation. The problem is now to examine somatic mutations withspecific locus method in order to throw a light on the modification ofhigh LET radiation's damage due to water content.

Storage and Water Content Effects on Radiation
Damage in Wheat
Tomoo MABUCHI and Seiji MATSUMURA

Dry seeds of diploid wheat containing 6.0, 12.5 and 15.0% water weresealed in ampules filled with oxygen, nitrogen or air and exposed toacute and chronic gamma-rays under room temperature (20°C) or dry ice
temperature (ca. -80°C). In general, chronic irradiations were found tocause more inhibition than the acute ones, especially at higher irradiation doses (15 kR). There was no clear difference in the results between
air and oxygen treatments after either acute or chronic irradiation. On
the other hand, nitrogen treatments clearly showed a protective effectagainst the inhibition of seedling growth, especially under acute irradiation. Unexpectedly, post-irradiation storage had no effect on growth
inhibition under acute irradiations. As to seed fertility, there was nomarked difference between acute and chronic irradiations, and also
between air and oxygen trea,tments. In respect to mutation, chronic

RESEARCHES CARRIED OUT IN 1967

109

irradiations were found to be more effective than acute ones. On theother hand, in acute irradiations, a clear intensification of mutation induction due to storage was found only in nitrogen treatments. Comparisonof air, oxygen and nitrogen clearly showed that nitrogen had a protectiv
effect on mutation frequencies.
Seeds with 12.5% water content were somewhat inhibited by a 15 kR
dose in air-filled ampules. In dry seeds (6.0%), a clear difference due toradiation dose and dose rate as well as post-irradiation storage andnitrogen treatment was observed. Seedling growth was markedly inhibited by acute gamma-irradiation at room temperature. However,there was a slight inhibition by exposure at dry ice temperature ascompared with the non-irradiated control. A protective effect against
gamma-ray damage was also observed both at room and dry-ice temperature. It should be noted that the storage effect could not be completely
eliminated by combined treatments of low temperature and nitrogen
under our experimental conditions. From experiments arranged with
the intention to show the effects of varying temperature during treatment and storage, it was found that temperature during irradiations is
the primary factor and storage temperature is of secondary importanceas far as seeding growth is concerned.

Wave Length Dependence of UV Damage in Maize Pollen
Etsuo AMANO

As a preliminary experiment for future detailed investigation of photoreactivation in maize pollen (d. Ikenaga and Mabuchi 1966, Rad. Botany
6: 165-169), monochromatic ultraviolet (UV) lights were tested. Linked
endosperm marker genes on chromosome 9 were used as the genetic
system. Pollen of a multiple dominant stock, C I Sh Bz W x, Y, wasirradiated in a single layer by monochromatic output of UV monochrometer (Bausch and Lomb, 500 mm, 1200 grooves/mm, F: 4.4). A 100
W short arc super high pressure mercury lamp (Ushio USH-100 D) wasused as light source. The exit lens was replaced by a quartz plate andthe output light was reflected vertically downward by a surface mirror,
thus about 27 X 27 mm 2 field of irradiation was obtained for stainless steel
sample pans of 25 mm diameter. Single layer of pollen grains was madeby careful sieving the pollen into the pans. Both entrance and exit slits
were set at 10 mm for high intensity irradiation. Because of the characteristic spectrum of the light source, UV intensity as measured by athermopile (Eppley) and a microvoltmeter (Ohkura) varied 11.8 ergs/mm 2 /sec
at 250 mp to 125 ergs/mm 2/sec at 365 mp, but a constant dose of 4.15±

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

O.02x 103 ergs/mm 2 was delivered at each wave length tested. The
irradiated multiple dominant pollen was used to pollinate a multiple
recessive tester stock in darkroom. All the procedures were done underyellow light of sodium lamp and pollinated ears were wrapped in aluminum
foils to prevent photoreactivation. Results are shown in Fig. 1 in which• - - -

Fractional kernel• - - Entire kernel

~ 1.0
>,
0

<::

"0'" '
'k"

•

'H

..,
<l
..,al 0.5

"

::<::

Controllevel

250(1/2 dose)

Wave length(mu)Fig. 1. Wave length dependenc of UV induced mutation. UV doses were4000 erg/mm 2 constant except at 250 mu where the dose was 1/2 (see text).

dose of UV irradiations at 250 mp were that of 1/2 at other wave lengths,
since very poor seed set introduced by the full dose at this wave length
made the mutation detection unreliable. The mutations scored were thetotal of single- and multiple-loci mutations and mosaics, and, in case of
the fractional ones, mutations which occupied more than 1/8 of theendosperm surface were scored. The results indicated that among thetested wave lengths the most, and probably the only, effective wave
length was 250 mp. Since the wave lengths tested were not many andrather apart from each other, comparison with optical absorption characteristics of DNA may not be appropriate, but the experimental procedure
toward this problem and study of photoreactivation will be improved infuture.

RESEARCHES CARRIED OUT IN 1967

111

Comparison of Photoreactivativating Activity on
Different Chromosomes of Maize
Etsuo AMANa

Existence of photoreactivation of ultraviolet light (UV) irradiated maizepollen has been demonstrated by Ikenaga and Mabuchi (1966, Rad. Botany
6: 165-169), who used su mutation as an index of UV damage. Amanoand Mabuchi (Ann. Rep. No. 17: 114-115) tested it further using otherlinked endosperm marker genes, C I Sh Bz W x, and found that evendamages due to chromosome aberration could be reduced by post-treatment with visible light. However, in the results of these two experiments,
there seemed to be a difference in the spectrum of the photoreactivation.
Ikenaga and Mabuchi reported more photoreactivation in whole kernel
mutation than in partial mutation, but in Amano and Mabuchi's experiment with linked marker genes, more photoreactivation was observed in
fractional (partial) mutation (more than 1/8 of endosperm surface). The
present experiments were arranged to find out the cause of the discrepancy.
Pollen used was of the same stock as used in Amano and Mabuchi's experiment and only female testers were compared. The female stocks wereselfed progenies of the previous experiments. A little modification wasmade in UV intensity (4x4 W germididal lamps, 200 ergs/mm 2 /sec), andpollinations in su experiments were done in the field after dark with aflashlight. Other experimental procedures were basically the same aspreviously. Because of technical limitations, each dosage group wastreated on a different day and this might be the reason for the fluctuating data. However, the results were basically the same for the twosystems, indicating photoreactivation on both entire and fractional mutations. In the experiments with multiple recessive markers, most of theaberrant kernels showed loss of all marker genes or B-F-B mosaic type,in both entire and fractional kernel mutations. In case of loss of all
marker genes in entire kernel mutants, fertilization by the treated pollen
could be confirmed by the Y gene present in the pollen in contrast to
the female. Such simultaneous losses of dominant genes or initiationsof B-F-B cycles might involve chromosome breakage in some form. Some
B-F-B type mosaics were also found in the su system. Photoreactivation
were observed in both genetic systems, both mutation kinds, and in thechromosome breakage type UV damages. As for single locus mutations,the number of the mutants was not large enough for a definite conclusion,
but some decrease of UV damages was observed in the groups treatedby visible light.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Dosimetry Systems for the 14.1 MeV Neutron Generator
Masaru HAYASHI and Etsuo AMANO

In 1967, a Cockcroft-Walton accelerator type neutron generator (Toshiba
NT-200-2) was installed in a newly built well shielded room in theRadioisotope Laboratory, National Institute of Genetics. This machineis to produce 14.1 MeV monochromatic fast neutrons by 3H(d, n)4He
reaction for radiation biology experiments. Specifications of the machineare; maximum acceleration voltage: 200 kV, maximum ion current: 1 mA,
and neutron output: more than 1 x 1010 n/sec.
Several dosimetrical methods were tested.
1) Proton measurement by plastic scintillator: A 2/f X 2/f if> plastic
scintillation detector of known efficiency set at 3.2 m from the target
was used for both calibration and monitoring purposes. The signal pulses
were analysed by a single channel pulse height analyzer (RCL) to obtainproton signals generated by 14.1 MeV fast neutrons. Since some problems
were found in respect to the resolving time of the system, improvements
are in progress.
2) Activation of sulfur: A few grams of sulfure were placed aroundthe target and 32p generated in sulfur by 32S(n, p) 32 P reaction wasmeasured after processing, by a GM counting equipment of known
efficiency for 32p. This method can be used as simultaneous monitoringof actual radiation dosage delivered to the samples.
243) Activation of aluminum: 27AI(n, 1X) Na reaction was tested sincealuminum can be used as metal and 24Na, a gamma ray emitter, can bemeasured without processing. Accompanying product, 27Mg, decays faster(1/2 life: 9.45 min.) than 24Na. This will be a very convenient and fastmeans when simultaneous monitoring of delivered dose is needed as in
the cases of radiation biology experiments.
4) Other methods tried or in progress: Silver activated glass rods invarious casings have been tested with success to separate radiation
components (contaminating gamma rays etc.) in radiation meaurement.
Designing of an IX particles counting apparatus to measure nuclear reactions 3H(d, n)4He is in progress. Though this is a promissing andorthodox method in radiation physics, good fit with the sample holder
and other radiobiological set-ups must be taken into consideration in
mounting the IX particle detector.

RESEARCHES CARRIED OUT IN 1967

IX.

113

MICROBIAL GENETICS

Fingerprinting Analysis of Flagellin of Antigen Type
Salmonella Recombinants
Shigeru YAMAGUCHIThe isolation of eleven types of antigen type recombinants obtained
probably through intra-Hi recombination from transductions between
different g-complex antigenic Salmonella strains has been previously
reported (Yamaguchi & lino, 1967). In the present study, tryptic
peptide maps of flagellins of each of these eleven types of recombinants
were prepared and were compared with those of their parental typestrains.
Flagella detached from cells by shaking (about 750 strokes/min, 25 mm
amplitude) were purified by repeating low and high speed centrifugation, and were disintegrated into flagellin monomers by treatment with
0.01 N HCI for 30 min at room temperature. A sample of freeze-dried
flagellin dissolved in 0.05 M phosphate buffer (pH 8.0) was digested withtrypsin (2 % by weight) at 25°C for 24 hr. An aliquot of the digest wasapplied to a sheet (40x30cm) of Toyo No. 50 filter paper and subjected
to electrophoresis in pyridine/acetic acid/water at pH 6.65 for 3.5 hr at
15 V/cm and twice to ascending chromatography with n-butanol/acetic
acid/water (3: 1 : 1). Peptide spots were developed with ninhydrin
dissolved in acetone (0.25 % w/v).
The approximate number of spots was 35 to 36 for every specimen
and almost all the spots except a few seem to be common to all parental
and recombinant type strains. Five spots in total were distinguishable
among the maps of the parental type strains: two spots (A and B) were
specific for strain Tr6 (g" g2' g3' t: (e, n, x)), two (C and D) for Tr16
(g3' g4' f: (e, n, x)), and one (E) for Tr17 (g" g2' g4' gs, m: (e, n, x)).
These differing spots were found in various combinations on the mapsof the antigen type recombinants. For example, three spots were foundbesides the common spots on the map of SJl744, a recombinant between
Tr6 and Tr16: one was the same as B specific for Tr6 and the othertwo were the same as C and D specific for Tr16. No other spot thanthose found on the maps of the parental type strains was detected.
On the basis of the assumption that the antigen type recombinants
had resulted from a single crossover within Hi, a structural gene forflagellin, in transduction, we have mapped out the location of theantigenic specificity-determining sections within Hi as follows: g2- g,-g4-t-(g3' gs, m) (Yamaguchi & lino, 1967). The differing peptides

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

disclosed here were now mapped out as additional markers. As theresult, the following locations were assigned to sections A to E corresponding to peptides A to E: somewhere between g4 and (g3' g5' m)for A, C and D, somewhere between gl and g4 for B, and between gl and(g3' go, m) for E.

Mapping of Some fla and mot Cistrons Closely Linked
to HI in Salmonella
Shigeru YAMAGUCHI and Tetsuo IINO

By the use of deletion mutants and three-factor reciprocal crosses, theorder of fla and mot cistrons closely linked to Hi was determined.
Nine Hi-linked stable fla- mutants were used: one of them derived
from SJ241 (a: (enx», a phase-1 stable strain of Salmonella abortus-equi,and the other eight from the derivatives (g: (enx» of SJ241 , given Hlg-alleles from g-complex antigenic strains.
Following experiments were carried out: 1) measurement of co-transduction frequency of their fla- sites with Hi, 2) recombination andcomplementation tests by transduction in all their pairwise combinations,
3) recombination and complementation tests between them and themutants of the already known fla and mot cistrons derived from S.
typhimurium TM2 (Ii no & Enomoto, 1966; Enomoto, 1966), and 4) threefactor cross tests, in which reciprocal transductions between fla-306,
the fla- mutant of SJ241, and each of the other mutants were carriedout and Hi was used as the unselected marker.
The order of the co-transduction frequencies were fla-204, -263, -351,-373, -227, -306, -251 and -211. Mutational sites of fla-220, -227
and -251 were found to be in flaA, and that of fla-211 was in flaD.
Fla-373 was found to be a deletion mutant covering flaA, D, Band
motC, and (la-306 was also a deletion mutant covering a part of both
flaA and motC. Three mutants, fla-204, -263 and -351, constituted a
new complementation group designated as flaL. Three-factor crosstests showed that the relative order of flaA, D, L and Hi is flaD-flaAHl-flaL. From these results and those reported previously (Iino &Enomoto, 1966; Enomoto, 1966), the following order can be inferred:
flaB-flaD-flaA-motC-Hl-flaL. Further, three-factor cross tests between
a mutant in flaL and those in flaC, E, K and J originated from TM2
were also conducted, and it was shown that these cistrons are all on
the same side of Hi and flaL being the nearest to it.

RESEARCHES CARRIED OUT IN 1967

115

Behavior of Conjugational Hemizygotes of Salmonella
typhimurium in Special Reference
to Motility Characters
Tetsuo IINO

Conjugation experiment was carried out between an HfrB2·metA22·
mot strain, SJl788, and an F-·athA,pro·flaA strain, SL481, of S.
typhimurium. When the mixture of Hfr and F- bacteria (2x10B cells/ml)
was inoculated on semi-solid plates and cultivated at 37°C, branchedtrails and swarms appeared on the plates. The number of branchedtrails was ca. 1 to 5 % of the inoculated bacteria, while the number ofswarms was 196 of branched trails at 24 hours' incubation. On further
cultivation, swarms developped from minute colonies of branched trails,
their number increasing up to 20 %. The clones which have grown asswarms were all stable fla+·mot+, and 91 % of them were mer and therest mer· ath- .pro-. The bacterial clones grown from the minute colonies
were all fla- ·ath-· pro-; that is the same as the F- strain.
Those results are summarized as follows. After conjugation of SJl788
and SL481 , hemizygotes were produced. The hemizygotes currently
segregate parental type cells at a considerably high frequency ascompared with the combination of S]457 and SJl52 (lino and Suzuki,
Annual Report No. 15, 1964), producing branched trails on the indicatorplates.
The stable motile recombinants appear, forming swarms,
throughout the period of persistence of hemizygotes.
By counting the number of minute colony clusters of each branchedtrail, it was shown that the number of branched trails with different
cluster number distribute in such a way that P=2- c , where P is thefrequency of the branched trails with cluster number c. This equation
indicates that 5096 of the hemizygotes segregate non-motile parental typecells in each cell division cycle.
Flagellar Characters of an F-his Strainof Salmonella typhimurium
Tetsuo IINO

S11795 (F-his~/his-712·ser·arg) is a Salmonella strain carrying an
episomal F factor which attaches the his-operon of Salmonella chromosome. The strain was originally derived from S. tYPhimurium LT2(i: 1.2) by G. Fink, Department of Genetics, Cornell University, Ithaca,
N. Y., U. S. A. It was found that SJl795 is non-flagellate in phase-1 andproduces curly flagella in phase-2. Therefore the bacteria cannot spread116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

on semi·solid plates in either phase. Transduction was carried out fromthe phase-2 culture of a diphasic S. abony (b : enx) strain, SW803. Motile
transductional recombinants were selected for on semi·solid plates. The
normal type transductional clones obtained were either normal-b inphase-l and curly-1.2 in phase-2 or non·flagellate in phase-l and normalenx in phase-2.
Possibility that the F factor of SJl795 carries the Hl-fla gene cluster
together with the his-operon was examined by conjungation analysis.
SJl795 was mated with a fla- mutant of strain SJl780 (his-486, b: 1.2)
and his'" recombinants were selected for on minimal plates. The his+recombinants appeared at the frequency of 2 to 0.2 % of the inoculated
bacteria. Twenty five such recombinants examined were all fla-. Whenthe recombinants were selected for from the same bacterial mixture on
semi-solid plates, the motile recombinants, appearing at the frequencyof 10- 2 to 10- 3 % of the inoculated bacteria, were all b: 1.2 type, and
56 % of them were his+ and the rest his-. Further conjugation analysis,
in which a multiple auxotrophic mutant of S. abony was used as a female,indicated that ser, leu and gal markers were transferred together withfla and H2 from SJl795 to the female upon conjugation.
From these results it is concluded that the F factor of SJl795 does
not carry the Hl-fla gene cluster but the strain can transfer its chromosomal markers, including the Hl-fla cluster, with the same efficiency
as in ordinary male and female conjugation.

Newly Isolated Arginine Sensitive Mutantsof Salmonella typhimurium
jun-ichi ISHIDSU

Mutation to arginine sensitivity was induced by N-methyl-N'-nitro-Nnitrosoguanidine in a wild type LT -2 strain of S. typhimurium and sevenmutants were isolated after penicillin screening (arg-s-2~8). Amongthem, arg-s-5 and 7 were sensitive only to arginine as previously isolatedarg-s-l (Ishidsu, Ann. Rep. No. 14, 1963). The other five mutants (args-2, 3, 4, 6 and 8) were found to be sensitive also to uracil, though
the response to uracil was weaker than that to arginine and as much as
1 millimole uracil per liter could not completely prevent their growth.
They grew slowly in the minimal medium where arg-s-l, 5 or 7 cannormally grow. In either case, however, they showed quite normal
growth when the two substances, arginine and uracil, were added
together to the minimal medium.
When arg-s-3 was cultivated in a medium containing arginine and117

RESEARCHES CARRIED OUT IN 1967

uracil combined in various concentrations, growth of the mutant wascompletely inhibited immediately after either of the supplements was
exhausted while the other still remained in a considerable amount. This
indicates that these two substances repress the biosynthesis of each otherin this mutant.
The precursors included in the biosynthetic pathway of arginine or
uracil were tested separately or in various combinations to find out
whether they have the ability to inhibit the growth or to remove theinhibition caused by the chemicals of the opposite pathway. The results
are summarized in Table 1. It is noticeable from the table that chemicals
which can inhibit the growth of arg-s-3 when given alone have also theability of neutralizing the toxic effect of inhibitors belonging to theother pathway, and that, in either pathway, the precursors before
transcarbamylation (ornithine or aspartic acid) have neither the ability
described above. It is also seen from the table that ureidosuccinic acidand dihydroorotic acid, though they enter the uracil synthesizing pathway
after the transcarbamylation step, behave somewhat exceptionally andaccelerate the growth when given together with ornithine, which never
happens in the case of other precursors of uracil. Those observations seem
Table 1.

Strain

~
none
ASP
~*

URE
~

DHO
~

ORO
~

URA
CYT
THY

Growth responses of some strains to various arginine
and uracil precursors or related substancesarg-s-3org-s-lpyr-150

none ORN.'!,CIT --->ARG none ORN.'!,CIT--->ARG none ORN.'!,CIT--->ARG

-H+
-H+
-H+

-H+
-H+
-H+

* * * ±

-H+

+
+
+
+

-

-

-

-

-

-

-

-

-

-

-

-

-

* ±*-

-

-

-

-

-

-

-

-

-

-

-H+

-H+

-

-

+
+

-H+

-H+

+

+

-H+

-H+

-

* *
* *

-H+

-H+

-

-

-H+

-H+

-

-

-H+

-H+

-H+

-

-

-H+

-H+

-

-

-H+

-H+

-

+

+

-

-

-

-

-

-

-H+

-H+

-H+

*

* *

ORN: ornithine, CIT: citrulline, ARG: arginine, ASP: aspartic acid,
URE: ureidosuccinic acid, DHO: dihydroorotic acid, ORO: orotic acid,
URA: uracil, CYT: cytosine, THY: thymine.
pyr-150: a mutant requiring both arginine and uracil for growth.
* steps of transcarbamylation.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

to confirm the assumption that growth inhibition by arginine or by uracil
is a result of inhibition of carbamyl phosphate formation which acts as
a common substrate of ornithine- and aspartate-transcarbamylase (Ishidsu,
Ann. Rep. No. 16, 1965; No. 17, 1966).
It is interesting to note that the spontaneous reversibility to nonsensitivity to arginine is quite different from that to uracil (very much higher
in the latter case) in those mutants which are sensitive to both arginine
and uracil, although they are considered to have acquired the character
most likely by a single point mutation.

Conversion to Streptomycin-Sensitivity Induced by A-Lysogenization
Accompanying Increase in Reversion Frequency
to Prototrophy in Escherichia coli K 12
Tsuneo KADA

The author has been studying the effects of episomal infections on themutability at specific loci in bacteria. This report describes a pleiotropic
modification observed by lysogenization with 2-phage of certain polyauxotrophic streptomycin-resistant F- strains of Escherichia coli K12.
They were isolated as thr-;014 recombinants by a 30 minutes conjugation
between E. coli K12 Hfr 1014 (thr-;014 B 1 - 5m B ) derived from HfrH after
ultraviolet irradiation and PA309 thr+ (F- thr+ leu- try- his- arg- B 1 Sm r ) obtained by crossing HfrH with the original PA309. The typicalstrain utilized in the following experiments was named F- 5-1014 (thr;"14
leu- try- his- arg- B 1 - Sm r ), possessing some UV-modified genes
originated from Hfr 1014.
Many lysogenic strains were isolated by infecting free phages fromthe culture of E. coli K12 (2) on r S-1014. They were purified bysingle-colony isolations and their genetic characters were studied. Among
19 lysogenic independent strains thus obtained, no observable change
was found in 5 (type I), whreas the remaining 14 (type II) changed
drastically their genetic behavior. While the original non-lysogenic
strain F- S-1014 and lysogenic strains of type I were able to grow inbroth containing 2,000 flg/ml of streptomycin, type II strains were no
more able to proliferate in the presence of more than 2 flg/ml of theantibiotic.
Other modifications were concerned with reversion frequencies of auxotrophic characters. In Table 1, frequencies of spontaneous reversions as to several auxotrophic loci in three typical strains (FS-1014(2)-, r S-1014C2t type I and r S-1014(2t type II are shown.
Though no marked modification exists between F- S-1014(2)- and FS-1014(2t type I, reversion frequencies were increased in F- S-1014(At119

RESEARCHES CARRIED OUT IN 1967

Table 1. Frequencies of spontaneous reversions in strains of Escherichia coli
K12. Lysogenic strains were derived from F- S-1014. Bacteria weregrown exponentially in broth and washed cell suspensions wereappropriately diluted and plated on appropriate synthetic
media to count revertants and on broth agar todetermine the total cell number
Frequency of spontaneous reversions
Reversions
studiedF- S-1014().)(Streptomycinresistant)

F- S-1014().)+ type I(Streptomycinresistant)

F- S-1014().)+ type II(Streptomycinsensitive)

thr--.thr+
leu--.leu+
gal--.gal+
try--.try+
his--.his+arg--.arg+

2.5 X 10- 6<1 X 10-9
4.0 X 10- 8
1.2 X 10- 7

2.0x 10- 6> 10-1<5

X 10- 8

3.3 x

10-8

X 10- 9<1
6 X 10- 9
1.8 X 10- 7<5 X 10- 8
7 X 10- 8

5.0 X 10-5
3.8 X 10-7

> 10-1> 10-1
2.6 X 10- 6

type II. When many lysogenic strains were isolated from P A309, nosimilar observation was possible.
Introduction of extragenetic elements into the cell by episomes ortheir insertion into the chromosomes may cause modifications in thegenetic character of the host. It is primarily supposed that, according
to Gorini's finding of a suppression mechanism involving ribosomalchange, auxotrophic characters might have been suppressed by a Aintroduced or A-induced gene, thus producing complete or leaky prototrophic character. From the strain F- 5-1014, kanamycin-resistant or
neomycin-resistant strains were isolated and lysogenized with A. Lysogenic
strains thus obtained became also sensitive to these antibiotics whose
action sites are considered to reside in ribosomes. On the other hand,revertants regarding many loci should be again mutated to original
auxotrophic state by means of a single event at the suppressor locus.
Trials to demonstrate this possibility by PI-transduction experiments
rather favour the assumption of a mutator acting non-specifically on
auxotrophic loci. The actual relationship between the possible ribosomalchange and the increased mutability regarding auxotrophic characters is
not known. Further examinations are in progress to gain an insight
into the mechanisms involved. The initial and important parts of theseobservations were done at the Institute of Physical and Chemical Research(Laboratory of Microbiology), Komagome, Tokyo.

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18x.

HUMAN GENETICS

Sexual Variation in Finger Pattern Types and Ridge Counts
Ei MATSUNAGA and Ei MATSUDA

The dermal ridge patterns on finger tips can be studied by two different
approaches, 1. by classification of the patterns into some types such asarches, loops (radial and ulnar) and whorls, and 2. using the pattern size
as measured by ridge count. Arches are defined to have no ridge count,
while loops have usually lower counts than whorls. Thus, pattern type
and pattern size are not independent from each other. These two aspects,
however, have hitherto been investigated quite separately, leaving some
gaps to be clarified.
It is known that there is a sexual variation in the distribution of patterntypes as well as of total ridge counts, i.e., the sum of the ridge counts
for each of ten fingers of a given individual. Table 1 illustrates theresult of our study based on 200 males and 200 females, both healthy andunrelated subjects. As is shown in the table, males have, on the average,
significantly larger total ridge counts than females, and whorls are significantly more common among males than among females, whereas thereverse is true with respect to the frequency of ulnar loops.
The larger total ridge count of the males than that of the females
may be attributed mainly to two causes; one is the lower frequency ofloops and higher frequency of whorls among the males than among thefemales, and the other is possibly a larger ridge count within the same
pattern type on the same digit among the males than among the females.
In order to check the latter point, mean ridge counts of a whorl and aloop were compared between males and females. Table 2 clearly shows
Table 1.

Mean total ridge counts and percentages of different pattern types
in 200 males and 200 females
Pattern types

Total ridge count

Loop

Sex
Mean

S.D.

Arch

Males
Females

150.8
139.2

45.69
42.04

Diff. (M-F)+11.6** P<O.OI,(%)** P <0.001.

Whorl
Radial

Ulnar

1.7
1.9

3.3
2.6

45.6
53.2

49.5
42.4

-0.2

+0.7

-7.6**+7.1**121

RESEARCHES CARRIED OUT IN 1967
Table 2.

Digits (left and right combined)

Pattern
type

Whorl

Ulnar
loop

Radialloop

Mean ridge count of a whorl and a loop

II

III

IV

V

All
digits

18.5

Sex

Male
Female

21.0
19.3

16.8
15.8

16.9

19.0
18.2

16.5
15.4

18.6
17.4

Diff. (M-F)

1.7

1.0

1.6

0.8

1.1

1.2*

Male
Female

15.4
14.4

10.3
10.4

11.1
11.1

11.8
12.6

12.1
11.4

11.9

Diff. (M-F)

1.0

-0.1

o

-0.8

0.7

0.2

12.1

Male
Female

9.8
10.2

9.8
9.5

Diff. (M-F)

-0.4

0.3

* P<O.OOl.
that whorls on every digit of the males have on the average larger ridge
counts than those of the females, the difference in the overall mean being
highly significant. On the other hand, no difference is found between
the sexes in the mean ridge counts of loops, although there is more orless variation according to digits which may be due to chance fluctuation.
The above finding seems to be rather unexpected because the larger
total ridge count among males than among females has generally beentaken to imply that males had larger pattern size than females irrespectiveof the type, whorl or loop. Thus the mechanism for sexual variation in
total ridge count appears to be more complicated than has been thought.
The mean difference in the total ridge counts between males and females
was found to be 11.6±4.4 in our Japanese sample. Using the figures ofTables 1 and 2, it may be shown that about half of this difference isaccounted for by the different ridge counts of whorls between the sexes,
while the remaining half is due to the larger ridge counts of whorls thanloops seen in both sexes multiplied by the different frequencies of these
two types between the sexes.

Increased Finger Ridge Counts in Turner's Syndrome
Ei MATSUNAGA, Ei MATSUDA and Hidetsune OISHI

Recent advances in human cytogenetics have renewed the interest for122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

dermal ridge patterns that my be affected by chromosome aberrations.
Of particular interest is the negative correlation of the finger patternsize in terms of total ridge count with the number of sex chromosomespresent in various types of aneuploidy. Since there is an evidence thatthe total ridge count is controlled by autosomal genes, the above correlation has been interpreted to be due to non-specific dosage effect ofwhole sex chromosomes (Penrose, 1967). However, whether or not thevariation between normal males and females is also caused by the samemechanism, had to be clarified.
In this connection, we carried out ridge counting of the finger tips infive cases of Turner's syndrome with reference to pattern types. The
sex chromosome complements and the total ridge counts of these cases
are given in Table 1. The mean total ridge count was 189.6, which isvery high in agreement with the reports by previous workers. The
percentage frequencies of arches, radial loops, ulnar loops and whorls
were 2.0, 2.0, 44.0 and 52.0, respectively. When compared with the control series used in our previous report, these frequencies are similar to
those found in normal males rather than to normal females, but thedifference between our Turner cases and normal females was not
significant.
Table 2 compares mean ridge counts per digit of our Turner cases
Table 1.

Sex chromosome complements and total ridge counts
in five cases with Turner's syndrome
Sex chromosome complements

Total ridge count

XO
XO

151
200
150
264
184

Case 1Case 2
Case 3
Case 4
Case 5XOjXXXXO
XOjXXrMean: 189.6

Combined
Table 2.

Mean ridge count of different pattern types

Pattern types

Turner cases

Normal males

Normal females

Whorl
Ulnar loop
Radial loop

20.4±1.4*
18.1± 1.2**
21.0

18.6±0.2
12.1±0.2
9.8±0.817.4±0.2
11.9±0.2
9.5±0.8* Significantly larger than normal females (P < 0.05).
** Significantly larger than normal males and females (P<O.OOI).

123

RESEARCHES CARRIED OUT IN 1967

with those of the control series according to different pattern types. It
is to be noted that in Turner's syndrome the pattern size is substantially
increased irrespective of the pattern type. The mean ridge count of awhorl was significantly larger than in normal females, and that of anulnar loop was significantly larger than in normal males and females.
As to radial loops, there was only one such pattern, which had 21 ridges.
Since sexual variation in pattern size among normal subjects was foundfor whorls but not for loops, the above finding suggests that the genetic
mechanism whereby changes in pattern size are produced in aneuploidcases of the sex chromosomes may differ from that responsible for thevariation between XY and XX individuals.

The Complete Amino Acid Sequence of a A TypeBence-Jones Protein
Tomotaka SHINODA

Attempts were made to test the hypothesis that all light chains of
immunoglobulin, IC and A, have a variable amino-terminal half and aninvariant carboxyl-half and likewise to test the relationships between thegenetic factor and the chemical structure of the protein. With regard
to these points of view, the complete amino acid sequence of a A typehuman Bence-Jones protein, designated Ra, has been determined, including the assignment of the amide groups.
The results are as follows:(1) The examined protein has 217 amino acid residues, including three
extra residues which are inserted between Asn-28 and Gly-31. (2) Manyamino acid interchanges in the amino-terminal half (residue number from
1 to 112) are found to be random. (3) Approximately the half of themolecule with initiation of Gln-113 is found to be constant with the exception of lysine-arginine interchange, which is associated with a genetic
factor. (4) The protein has the arginine residue at position 195, suggesting that it is of type Oz( - ). (5) The protein has a blocked aminoterminal, presumably a pyrrolidonecarboxylic acid. (6) From sequencecomparison, there are some evidences for conservation of certain areasin the primary structure of light chains, that is, the positions that arevariable in A-chains are generally variable in the counterpart positionsof IC-chains, and vice versa.
The evidences seem to support the concept that the light chains ofPenrose, L. S.:
300, 1967.

Finger-print pattern and the sex chromosomes.

Lancet i:

298-

124

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

immunoglobulins have a variable amino-terminal half and an invariant
carboxyl-terminal half that carries the genetic factors. (The entire work
was performed at Indiana University, Indiana, U.S.A.)

Cytogenetic Study on Familial Down's Syndrome
Yasumoto KIKUCHI, Hidetsune OISHI, Akira TONOMURA"
and Takehiko KURITA21

It is now known that both in Europeans and Japanese more than 9096
of patients with Down's syndrome are of 21-trisomy, while the remaining
small portion consists of cases with translocation and mosaicism. In order
to find the incidence of chromosome mutation leading to translocation
Down's syndrome, 508 patients were examined by means of leucocyte
culture. Of the 508 cases, 26 (5.12%) were found to be of translocation
type; i.e., 12 t(GqGq), 13 t(DqGq) and 1 was a mosaic of 45,XY,D-, G-,t(DqGq)+j46,XY,D-, t(DqGq)+. In cases with t(DqGq), 5 (including
the mosaic) were inherited, 6 were sporadic, and the parents of theremaining 3 have not yet been examined. In cases with t(GqGq), 2 were
inherited, 7 were sporadic, and the parents of the other 3 were notexamined.
The family members of the patients with inherited translocation were
investigated. In the first family, the patient had (DqGq) translocation;
the father was a mosaic of 46,XYj47,XY, t(DqGq) +, and was phenotypically normal; the mother had apparently normal karyotype; a phenotypically normal sib of the patient had not yet been examined (Tonomura
et al. 196231 ). In the second family, the patient was a mosaic of 45,XY,
D-,G-, t(DqGq)+j46,XY,D-, t(DqGq)+; one cell line was of a balanced
translocation type and the other was of a translocation-trisomic type; themother was a carrier of translocation in the balanced form, and has
previously had 2 stillbirths; the father had apparently normal karyotype;a sib of the patient has not yet been examined but had normal phenotype.
In the third family, the patient had t(DqGq); the mother and the thirdsib of the patient were carriers; the father, the first sib and the maternal
grandparents had apparently normal karyotype; the second sib died atearly infancy. In this family, the translocation occurred de novo in oneof the parents of the carrier mother. In the remaining 4 families, theDepartment of Cytogenetics, Tokyo Medical & Dental University.
21 Pediatric Clinic, Kohnodai National Hospital.
31 Tonomura, A., T. Honda, and T. Kurita, 1962. Chromosome abnormalities ina child with Down's syndrome and in its father. Proc. jap. Acad. 38: 526-530.
11

RESEARCHES CARRIED OUT IN 1967

125translocation chromosomes of the patients were inherited from the carriermothers.
The above data indicate that, excluding the unexamined cases, theapproximate frequencies of inherited and sporadic translocation among
Down's syndrome were 1.8% and 3.3%, respectively, and that the population incidences of these two translocation types are of the order of
3x 10- 5 and 5.5x 10- 5 , respectively.
Maternal age at the patient's birth has also been subjected to statisticalanalysis. Mean maternal ages for the patients with inherited and sporadictranslocation were 30.5±2.69 years and 27.9±3.66 years, respectively.
These mean ages are not different from that of the control population(28.5±4.27 years).

An Incomplete No.5 Chromosome Pair in Two Patientswith the Cri du Chat Syndrome
Hidetsune OISHI, Yasumoto KIKUCHI and Ei MATSUDA

An incomplete chromosome pair in group B has been found in the cri
du chat syndrome (Lejeune et al.,ll 1963). Chromosome analyses revealed
that the deficiency consisted in the deletion of the short arm of no. 5.
However, it seems that the magnitude of the deletion varies somewhat
from case to case, and this could be reflected in the clinical features ofthe patients with the syndrome. From this point of view, the present
study deals with two cases of that deficient chromosome.
Case 1 was a 4 months old boy and his cry reminded of the cri duchat. The gestation lasted normally for 40 weeks and the birth weightwas 2000 g. At the time of his birth, the age of the mother and father
was 28 and 32 years, respectively. On both hands of the patient wereobserved transverse palmar creases and whorls on all fingers except thefirst with a lateral pocked loop, though the dermal ridges on the fingers
were poorly developed. The patient died of pneumonia 7 months after
birth. A 3 years old sister was phenotypically normal.
Case 2 was an 18 months old girl who was the first-born child of a
26 years old mother and 30 years old father. The gestation was 40 weeks
and the birth weight was 2060 g. The patient exhibited an odd cat-like
cry, and the parents said that the characteristics cry became less pronounced as the child grew older. The dermatoglyphics of the patient
1) Lejeune, J., J. Lafourcade, R. Berger, J. Vialatte, M. Boeswillwald, P. Seringe,
and R. Turpin, 1963. Trois cas de deletion partielle du bras court d'un chromosomes. C. R. Acad. Sci. Paris 257: 3098-3102.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

were as follows, and transverse palmar creases were also noted on her
hands:
Right hand.-Palmar formula, 7·5" ·5' .4-t' -Au/N· U/O·O·O·L,
at'd angle=64°; digits=Ucp, W, W, W, W, total ridge count=59.
Left hand.-Palmar formula, 7·5" ·5' ·3-t' -Au/N·L/L·O·O·L,
at'd angle=58°, digits=U, W, W, W, W, total ridge count=64.
The clinical features, such as mental and growth retardation, microcephaly, round face, hypertelorism, antimongoloid slant of palpebral
fissures, low-set ears and short neck which are frequently associated withthis syndrome, were found in both cases. However, there were also
epicanthus, micrognathia and congenital heart disease in case 1, but notin case 2.
Chromosome analyses of these patients revealed that one of the chromosomes of group B was lacking a portion of the short arm. The chromosomes of group B of ten cells were magnified about 20,000 times fromphotomicrographs by a photographic enlarger. The figures were thenmeasured by a planimeter. The lengths of the chromosomes were also
recorded in the same cells. The deficient chromosome corresponded tochromosome no. 5 in the size of the long arm, and the deleted portion
amounted to 64.7% of the short arm by area measurement and 49.4% bylength measurement in case 1, and respectively 61.8% and 43.8% in case
2. The parents had in both cases apparently normal chromosome sets.
Dumar and Kitzmiller 21 (1964) assumed that the deletion comprised
about the mass of a chromosome of group G, and McCracken and Gordon 31(1965) reported similarly that almost all of the short arm was absent intheir case. Ricci et al. 41 (1965) also observed a deletion of more than ahalf short arm in this syndrome. However, no exact measurements ofthe deletion, in comparison with a variable appearance of clinical features,
were recorded so far.

Clinical Conditions of Patients with Apparently
Normal Chromosomes, IV
Hidetsune OISHI, Yasumoto KIKUCHI and Kunihiko SHIBATA

Chromosome analyses performed on cultured leucocytes of patients
21 Dumars, K., Jr., and N. Gaskill, 1964.
Le cd du chat (crying cat) syndrome.
Amer. J. Dasease Child. 108: 533-537.
31 McCracken, J., and R. Gordon, 1965.
"Cd du chat" syndrome. Lancet i:23-25.
41 Ricci, N., B. Bentimiglia, B. Dallapiccola, F. Franceschini, and G. Preto, 1965.
"Cri du chat" syndrome. Lancet i: 1278-1279.

RESEARCHES CARRIED OUT IN 1967

127

having various pathological conditions showed that they had apparently
normal chromosome assortments of 46 chromosomes. Cases of someinterest are listed below.

Name

Age

Legal sex

Clinical conditiona) Multiple deformations with mental retardation
64 Y.M. 3 months M
Prominent occiput; malformed and low-set
ears; cleft palate; congenital heart disease;transverse palmar creases; overlapped fingers; hemangioma
Cleft palate; hare-lip; malformed ears;
65 LS.
M
hydrocephalus
1 yearFlat occiput; hypertelorism; strabismus;
66 H.E.
F
high-arched palate; micrognathia; shortneck; cubitus valgus
67 A.K.
Arnold-Chiari malformation; lacunal skull;
5 months Fhydrocephalus; myelomeningocele; bladderrectal incontinency; paraplegia of the leg
Oblique palpebral fissures; aplasia of the68 H.K.
3 yearsF
XII rib; spasmophilia; logopathy
Microphthalmia; cataract; low-set and mal1 month M
69 H.K.
formed ears; micrognathia; short neck70 K.S.
2 months M
Exophthalmos; low-set ears; hydrocephalus
F
Moebius's singn; strabismus; high-arched
71 M.Y. 1 year
palate; short neck; cubitus valgus; syndactyly
72 S.H.
Flat nasal bridge; short upturned nose;
2 months F
low-set ears; high-arched palate; webbed
neckM
73 J.M.
6 years
Malformed ears; cleft palate; micrognathia;
hypoplasia of the XII rib; transverse
palmar creases
74 E.N.
2 years
F
Congenital heart disease; narrow palpebral
fissures;transverse
palmarcreases;
logopathy
75 S.N.
3 years
M
Transverse palmar creases; presence of the
XIII rib; incurved fifth fingers; increased
iliac index (94°); spasmophilia
76 J.Y. & T.Y.
(twin) 4 months M
Hirschsprung's disease
4 years
M
77 Y.K.
Oblique palpebral fissures; incurved and128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS ON. 18

78 Y.O.

2 years

Mb) Sex anomalies
79 S.1.
2 months M
80 Y.1.
16 years F
81 O.T.
3 years
M

82 T .K.

21 years

Mc) Hereditary disease
83 H.A.
6 yearsM

short fifth fingers with clinodactyly; aplasiaof the XII rib
Hypertelorism; antimongoloid slant eyes;
epicanthus; strabismus; malformed ears;
micrognathia; short neckMale pseudohermaphroditism
Male pseudohermaphroditism
Slender body; short neck; shield chest;
retentio testes; flat occiput; high-archedpalate; logopathy; mental retardation(Klinefelter's syndrome?)
Male pseudohermaphroditism
Congenital ichthyosis; spastic paraplegia;
logopathy; mental retardation

RESEARCHES CARRIED OUT IN 1967

XI.

129

APPLIED GENETICS

General and Differential Relative Growth Rates of
Mutant Lines of Rice
Hiko-Ichi OKA and Hiroko MORISHIMA

Last year (Ann. Rep. 17, p. 33-34), we reported an observation of variations in growth pattern among 23 mutant lines radiation-induced from arice variety, Norin 8, though the results were incomplete. Further studyof the data gave the following results. Between-line variations in the
final size, the time at which half of the final size was attained (t1/2), andthe growth rate at t l l2, were studied for the panicle and first to fourth(from the top) internode. For each of the above three values, principal
components were extracted from correlations between the five organs,and the "genetic vectors" showing genetic contributions of the components
to the respective organs were computed by "Hashiguchi's method (Hashiguchi and Morishima, in press). In the final size, two latent phases ofgenetic variation were distinguished, one showing the general size, andthe other showing an allometric pattern that brought about variation
between "upper-elongation" and "lower-elongation" types. This patternis the same as reported last year. Variations in the growth rate of organs
similarly comprised an isometric and an allometric phase. In the allometric phase, lines whose panicle and third internode elongated relatively
slowly had a relatively high growth rate for the first and second internodes. Such lines tended to be the "upper-elongation" types at maturity.
As the panicle and third internode elongate some days before the elongation of first and second internodes that results in heading, the allometricpattern of growth rate suggests that the lines vary in the rhythm of development. This was proved by an observation of the change of relative growth rate in the period from panicle elongation to heading. It
was suggested that genes modify the rhythm of development and bringabout the size variations observed at maturity. This work was supported
by a grant of the Ministry of Education, Government of Japan.
Further Observations on the Effects of the Earliness Gene, E, in
the Genetic Background of a Variety, Taichung 65
Kuo-Hei TSAI* and Hiko-Ichi OKA

Taichung 65, a representative Ponlai rice variety, and its early isogenic* Chung-Hsing University, Taichung, Taiwan, Republic of China.

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

lines, A3 and B96, were compared regarding the growth pattern of organs,temperature response and yielding capacity in varying environment, inorder to estimate the effects of the E gene-block carried by the isogeniclines. A comparison of lines derived from BC 7 and BC lo generations
showed that three additional back-crosses with Taichung 65 slightly reduced the effect of the E gene-block on heading date. Perhaps E might
be linked with genes exaggerating its effect. The primary effect of the
E gene-block seemed to promote flower initiation and subsequent growthof certain flower organs. This resulted in increase or decrease of growthrate, growth duration and final size of various organs that develop after
flower initiation. For instance, the E gene increased the growth rate of
the panicle and reduced its growth duration, but decreased the growthrate of the first leaf (from the top) and extended its growth duration.
Further, it increased sensitivity of the plants to temperatures in the flowerinitiation period. But the E gene did not seem to affect the seasonal
and regional adaptabilities of the original genotype. In yielding capacity,
the early isogenic lines (about 10 days earlier than Taichung 65) werecomparable to other early Ponlai varieties, and when grown in the northern region of Taiwan in the second crop, they gave better yield thanTaichung 65. As to the pleiotropic effects of the E gene, as many as
32 were listed. They were considered to suggest the developmental paths
through which "ripples provoked by a stone" were transmitted. The experiments on adaptability and yield were conducted in Taiwan, supported
by a grant of the Science Development Council of the Republic of China.
Analysis of variance in diallel crosses with parents innested, hierarchal, classification
Tohru FUJISHIMA

The purpose of this study was to provide analytical procedures of diallel
crosses in which the parental lines employed were further classified bybreeds or races, nested or hierarchal classification. According to whether purebreds are included or not, there are three different analyticalmethods. The mathematical model common to those methods for Xijklm
which is the mth observation in crosses between jth maternal parent
within ith breed and Ith paternal parent within kth breed is as follows:
Xijklm=p+gi+gk+Sik+gj(i) +g!(kl +Sjlilk+Si/(kl +Sj(i)l(kl +mi + mj(i) +rik+
rj(ilk+ril(kl+rj<ill(k)+eijklm, m=l, 2, ... , n, where the g is the general com-bining ability, the S is the specific combining ability, the m is thematernal effect and the r is the reciprocal effect, and in Method 1 i,k=l, 2, ... , a, j(i), l(k)=l, 2, ... , k, in Method 2 i, k=l, 2, ... , a (i*k), j(i),131

RESEARCHES CARRIED OUT IN 1967
Table 1.
Method 1
Source of variation
Sums of squares

df

G.C.A. for breed(a)(b)
G.C.A. for line within breed(c)
S.C.A. for breed
S.C.A. for cross between line and
breed
(d)
(e)
S.C.A. for line within breed
M.E. for breed(f)M.E. for line within breed(g)R.E. for breed(h)R.E. for cross between line and
breed
(i)
R.E. for line within breed
(i)
Error
Total

1j2a(a-1)a(a-1)(k-1)
1j2a(k-1)(ak-a+ 1)
a-I
a(k-1)
1j2(a-1)(a- 2)
a(a- 1 )(k-1)

a(k-1)
1j2a(a-3)a(a- 2 )(k-1)
1j2a(a- 1)(k-1)2

11-21'
III- II'
IV-II+I"
V -21V -III-II'
VI-V+IVVIIa(k-1)
VIII- VII'1j2(2-1)(a-2)
IX-VIIa(a-2)(k-1)
X-2IX- VIII + VII'
1/2a(a- 1)(k-1)2
XI-X+lXak 2(a-1)(n- 1)
XIII-XII
ak 2n(a-1)-1
XIII-I
a-I

G.C.A
M.E
S.C.A
R.E

General combining ability
Meternal effectSpecific combining ability
Reciprocal effect

XI-X+lX
XIII-XII
XIII-Ia 2k 2(n- 1)

a 2 k 2 n-1

Method 3

Sums of squares

a-I

VI-V+IV
VII
VIII- VIIIX- VII
X- VIII-2IX+ VII

1j2a(k-1)(ak-a- 1)

Method 2
df

11-21
III-II
IV-II+I
V -2IV -III+IIa-I
a(k-1)

df

Sums of squaresa-Ia(k-1)
1j2a(a- 1 )a(a-1)(k-1)

11-21'

III-II
IV -11+1"
V-2IV'-III+II

1j2a{(k-1)(ak-a-1)-2}

VI-V +IV"VIIa(a-1)
VIII- VII1j2(a-1)(a-2)
IX- VIIq(a-1)(k-1)
X-2IX-VIII+VII
1j2a(k-1)(ak-a-1)
XI-X+IX
ak(ak-1)(n- 1 )XIII-XII
akn(ak-1)-1
XIII- Ia-I

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

l(k)=1,2, ... , k, and in Method 3 i, k=1, 2, ... , a, j(i), l(k)=1, 2, ... , k (j(i)*l(k) when i=k).

The tables of analysis of variance corresponding to those methods are
summarized in Table 1, where for Method 1,
I=(X..... )2a2k 2n, II= 'i.(Xi .... + X .. i .. )2j2ak2n,

III = 'i.(Xii ... +X.. ij .)2j2akn,ij
2IV = 'i.(Xi .k.. +Xkoi .. )2j4k n, V = 'i.(Xi .k1 . +Xk1i .. )2j2kn, VI= 'i.(Xijk1 . +
ik
ikt
~;jkl
2
X klii .)2j4n, VII = 'i. (Xi ....
'i ..f j2ak n, VIII= 'i. (Xij ... - X .. ij .)2j2akn,
i

i

-x.

ij

IX= 'i.(Xi'koo-Xk.i .. )2j4k2n, X= 'i. (Xi.kl,-Xkli .. )2j2kn, XI= 'i.(Xiik1 .ik

ikt

ijkl

X k1ii .)2j4n, XII = 'i.X2iikl.jn, XIII= 'i.X2iiklm
ijkl

ijklm

for Method 2,
2
I=(X..... )2jak 2 n(a-1), I' =(X..... )2jak2n(a-2), I" =(X..... )2jk n(a-1)(a-2),
II= 'i. )Xi .... +X.. i ..)2j2k 2n(a-2), II' = 'i.(Xi .... +X.. i .. )2j2k2n(a-1),ii
III = 'i.(Xii ... +X.. ij .)2j2kn(a-1), IV = 'i.(Xioko ' +Xk.;..)2j4k2n, V =ikij'i. (Xi .kl . + X kli oo)2j2kn, VI= 'i. (Xijkl . + X k1ii .)2j4n, VII = 'i. (Xi .... - X .. i .. )2j

ikt

ijkl

i

2ak 2n, VII' = 'i.(Xi .... -X.. i .. )2j2k2n(a-l), VIII= 'i.(Xii ... -X.. ii .)2jiij

2kn(a-1), IX= 'i.(Xi'k,,-Xk'i .. )2j4k2n, X= 'i.(;'kl,-Xkli .. )2j2kn, XI=
ik

ikt'i. (Xiik1 . -Xk1ij .)2j4n, XII = 'i. X2iikl.jn, XIII = 'i. X2ijklm

ijkl

ijkl

ijklm

for Method 3,
I=(X .....fjakn(ak-1), I' =(X ..... )2jakn(ak-2), I" =(X..... )2jn(ak-1)(ak-2),

II= 'i. (Xi .... +X.. i .. )2j2kn(ak-2), III = 'i. (Xii ·.. +X ..ij .)2j2n(ak-2),iij

IV = 'i. (X i . i oo)2jkn(k-1)+ 'i. (Xi .k·.. + X k'.;..)2j4k 2n, IV ' = 'i. (Xi . i ..)2j
i*k'

i

i

2
kn(k-2)+ 'i. (Xi .k·.. +Xk·. i ..)2j4k n, IV" = 'i. (Xi . i ..)2jn(k-1)(k-2)
i*k'i+ 'i. (Xi.k·oo + X k'.;..)2j4k 2n, V = 'i. (Xi .il . +X ili ..)2j2n(k-2)
i*kit+ i*kl'i. (Xi .k·1. + X k•1i ..)2j2kn, VI= 'i. (Xijk1 . + X k1ii .)2j4n,
ijkl

VII= 'i.(Xi .... -X.. i ..)2j2ak2n, VIII = 'i.(Xij oo.-Xoo ij .)2j2akn,iij

IX= 'i. (Xi.koo - X koi oo)2j4k2n, X= 'i. (i'kl. - X k1i .. )2 j2kn,
ik

ikt

XI= 'i. (Xijk1 .- X k1ii .)2j4n, XII = 'i. X 2 iikl.jn, XIII= 'i. X 2 iiklm
iikl

iikl

ijklm

RESEARCHES CARRIED OUT IN 1967

133

Electrophoretic variation in enzymes of tobacco plants
Sumiko NARISE

Our local varieties of tobacco are apparently different from the fluecured ones in many respects, either physiological or morphological. The
purpose of this experiment was to find the differences, if any, between
them on the basis of variation in enzymes, i.e. variation in isozyme constitution.
Three of Japanese varieties, Matsukawa, Kirigasaku and Ohdaruma,
and the same number of flue-cured varieties, Bright Yellow, Hicks andCoker 139, were used. The buffer-extract from leaf-nerves, leaf-mesophylls, calyxes, corollas, stamens, pistils and ovaries from six strains
were separately subjected to electrophoresis on acrylamide, and esterase
and acid phosphatase come into view by using appropriate reagents.
The results obtained were: 1) Seven different bands of esterase were
found in the Japanese strains, while nine appeared in the fluecured tobaccos. Among them, one was common to all organs of different strains,
whereas two were common to different organs of flue-cured strains only.
2) There were five different bands of acid phosphatase, two of which were
specific to the flue-cured strains, while one of the remaining three wasspecific to the Japanese strains.
Interaction between sire genotypes and photoperiodic
treatment in Japanese quails
Takatada KAWAHARA

The results described in the present report were obtained from 31 sirefamilies of Japanese quail reared in Auburn University. The experiment
was carried out with three photoperiodic treatments. Photoperiods applied were: (1) 24 hour light with no dark period (24L:OD), (2) 18 hour
light with 6 hour darkness (18L:6D) and (3) 12 hour light with 12 hour
darkness (12L:12D) per day. Other environmental conditions including
air temperature and humidity were kept as constant as possible. Observed traits were body weight at various stages of development, sexual
maturity, testis weight, egg weight, hen-day egg production rate fromfirst egg to 16 weeks of age. Egg production rate was adjusted according to sexual maturity and ordinary egg production rate from 12 to 16
weeks after hatching. Analysis of variance of the data obtained revealed that the interaction between sire genotypes and photoperiodic treatment was significant for sexual maturity and ordinary egg production
rate (Table 1). These interactions are understood to have occurred through134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Table 1.

Analysis of variance for sexual maturity, ordinary andadjusted egg production rates
Mean square

Source of variation

Degreesoffreedom

30
Sire genotype (G)2
Photoperiod (P)
24L:OD vs. 18L:6D and
12L:12D (P l )18L:6D vs. 12L:12D (P 2 )60Interaction
GxP l
GxP 2
Error

1
1
30
30

111

Sexualmaturity

Ordinaryegg production rate l )Adjusted
egg production rate l )116.17*8587.36**258.30*1209.39**

182.12666.57**4148.53**13026.18**
118.23**25.84
210.61**67.99

2352.43**66.35
227.35*186.09268.61*160.221259.32**
73.82
164.44140.66188.22147.54*, **: Exceed the 5% and 1% significance levels, respectively.
1): Arc sin v'Percentage transformation.

different responses of genotypes to light period of 18 and 12 hours perday. The interaction, however, was not significant in egg production
rate adjusted according to sexual maturity. This means that the statistically significant interaction in ordinary egg production rate is the effectof differences in the pheses of egg production.
Experiment on bilateral asymmetry in wings ofDrosophila melanogasterTakashi NARISE and Kan-Ichi SAKAIIn the experimental study on bilateral asymmetry in wing length inDrosophila melanogaster, it was observed that some flies, though infrequently, of the natural population had asymmetrical wings. Therefore,
it was expected that the genetic system producing asymmetrical wings
persists in natural populations. The present paper describes the resultsof an inbreeding experiment with three wild strains of D. melanogaster,MS-l, Suyama and Katsunuma. The initial number of inbred lines examined was 100 per strain. The number (%) of lines yielding offspring
having asymmetry in wing length is presented in Table 1.
It was found that inbreeding disclosed the presence of genes controlling asymmetrical development of wings, and the number (%) of pathological lines increased up to different generations. Conclusions drawn

135

RESEARCHES CARRIED OUT IN 1967
Table 1.

The number (%) of inbred lines yielding offsprmg
having asymmetry in wing length

Inbreeding
generation

1

MS-1
SuyamaKatsunuma

2.0 7.6 6.2 7.4 8.3 10.2 17.6 12.0 23.1 6.0 0.0 0.0
1.0 2.0 3.0 5.3 13.0 12.5 5.6 0.0 0.0 0.0 0.0 0.0
2.0 4.1 4.4 2.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

2

3

4

5

6

7

8

10

9

12

11

from this experiment are: 1) There is a concealed pool of genes responsible for asymmetrical development of wings in wild populations of D.
melanogaster which is disclosed by the effect of inbreeding. 2) The rateof disclosure varies from population to population due either to differencein gene constitution or buffer system in organic development.
Multidirectional selection in Japanese quails
Teruo INOUYE and Kan-Ichi SAKAI

It is known that the body size and the shank length of Japanese quails
are genetically positively and closely correlated. This report deals withthe result of a multidirectional selection for body size in various combinations with shank length. The selection was conducted in four directions: for larger body size and longer shanks and for smaller body sizeand shorter shanks, on the one hand, and for larger body size and shorter
Table 1. Cumulative selection differential and gen~tic gain obtained
after four generations of consecutive multidirectional selectionin Japanese quail1

Selection group-+- + *1

2
+-*1

3
-+*1

4
--*1

Cumulative selection
differential

Body weight, g.
Shank length, mm.

34.80
3.16

9.85
-2.31

-3.70
2.48

-22.10-2.72

Genetic gain

Body weight, g.
Shank length, mm.

17.95
2.32

0.95
-1.47

2.50
2.05

-11.40-1.81

Selective
efficiency (%)**

Body weight
Shank length

51.58
73.42

9.64
63.77

-67.57
82.46

51.58
66.54

*)

**)

The first positive or minus sign indicates the direction of selection forlarger or smaller body weight respectively, while the second is for shanklength; + -, for example, means selection for heavier body weight in
combination with shorter shank length.
Genetic gain divided by cumulative selection differential X 100.

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

shanks and for smaller body size and longer shanks, on the other. The
result of the consecutive selection is presented in the table. Data showthat the selection in favor of or against the two correlated characters
was quite effective whereas that in favor of one but against the otherwas less effective.
Fertility, hatchability, viability up to 8 weeks of age and number ofdays to first egg were investigated in connection with the effects of theselection. It was observed that the (+ +) selection and the (- -) selection were in general weaker than (+ -) and (- +) selection in relation
to the fitness of the bird.

Biochemical relationships among different organsof tobacco plants
Sumiko NARISE and Kan·lchi SAKAI

It was demonstrated in the previous issue of this Annual Report (No.
17) that an electrophoretic method was useful for biochemical comparisonof different organs in a plant. This paper describes a further technique
improvement yielding higher distinctness and repeatability in the patternof protein bands than before. The improvement consisted of a fraction·
ating procedure of protein into five fractions by the aid of ammonium
sulfate at pH 7.0 of different concentration: 0 to 30, 30 to 45, 45 to 50,
60 to 75 and 75 to 100% saturation. The five fractions thus separated
were subjected to electrophoretic analysis. In order to measure the similarity among organs, an index was constructed by summing up the squaresof differences between different organs, in respect of staining intensityof bands. It was called similarity index and had the following formula;where SYiX) is the similarity index comparing Y-th with X·th organ. Yi
and Xi stand for the rating of staining intensity of i-th band of Y·th andX-th organs. v is the number of ratings: six ratings from 0 to 5, andn is the number of bands found in either of the two organs compared.
The S value becomes unity if two organs are quite identical with regard
to protein band distribution but zero if no common band is detected between the two organs. The S values among different organs are given
in Table 1.
The findings according to Table 1 are as follows: 1) All organs, ir-

137

RESEARCHES CARRIED OUT IN 1967
Table 1. Similarity indices among different organs
Flower parts
Stamen Petal~~~~er

Calyx

O 773
:::::en .

::;:: 0.734 ::;:: 0.696
0.669

Petal

Leaf
Upper Intermediate

Bract leaf:::~:)0.676 ::::: 0.652 :::::\0.658
0.614

0.623

0.600

0.649

0.691

0.692

0.723

0.655

Calyx
Bract
leaf

0.691

Leaf Upper

respective of whether they are floral or foliar, have more than fifty percent
of protein constituents in common. 2) Organs constituting flower parts
exhibit satisfactory high values among themselves, but lower values withcalyx, bract leaf or leaf. 3) It is interesting to take note of the relationship between leaves, on the one hand, and calyx and bract leaf, on theother. The biochemical relationship between the calyx and either theupper or the intermediate leaf are not particularly close. The relationship between the bract leaf and the upper leaf is apparently high, despiteof the low value between upper and intermediate leaf.
Competition in relation to tree age in Cryptomeria japonica
Shigesuke HAYASHI and Kan-Ichi SAKAI

Transverse disks of 196 trees were collected from an artificial plantation some fifty years old of Cryptomeria japonica at Kagoshima U niversity. Position of each tree in the forest was recorded on a section paper.
The disks were measured for yearly growth in eight directions and every
five years the mean diameters of stem were used for the calculation ofcorrelations between adjacently growing trees. Mean diameter and thecorrelation coefficients were as follows:
Age in years

5

10

15

20

25

30

Diameter (cm)

2.72
0.423
0.485
0.448

4.44
0.622
0.596
0.608

7.68
0.162
0.221
0.192

11.36
-0.063
0.128
0.032

14.38
-0.078
0.117
0.021

16.64
-0.157-0.004-0.080rij

1""',,1'horizontal*
pooled138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18
Age in years

35

40

45

50

Diameter (em)18.26
-0.226-0.082-0.15419.44-0.245-0.136-0.19020.18-0.243-0.159-0.201

20.92-0.246-0.183-0.214rij(""'' ,.

horizontal*
pooled* Vertical and horizontal stand for r's computed from pairs of trees growing in vertical and horizontal rows to the slope, respectively.

It has been published recently that rio H1 of stem diameters between
two adjacently growing Cryptomeria trees tells the status of intertreecompetition: positive and high values indicate little or no competition
while zero or negative values show intense competition (Sakai, Mukaide
and Tomita, 1968 in Silvae Genetica). It is found from the above tablethat for the first 10 years, intertree competition appeared to be nil, but
was apparent in later years. It is thus concluded that in the forest investigated, trees younger than 10 years were free from intertree competition, but older trees 10 and 15 years of age started to compete witheach other and the competitive effect was accumulative year after year.
Analysis of a clonal progeny in a natural forest of
Cryptomeria
Kan-Ichi SAKAI, Shigesuke HAYASHI, Kihachiro OHBA
and Koji TOMITA

It is known that Crytomeria japonica propagates either vegetatively
or by seed in nature. This study aimed at distinguishing a clonal progeny from others in a natural forest of Cryptomeria by means of an
identity index which estimates the degree of similarity between a given
tree and other trees. It is computed on the basis of several needle characters:
hi=ln

and +l::2:D::2: -I,

where h i is the identity index comparing j-th tree with a given i-thtree. Dm(j, i) is the difference in moth character between j-th and i-thtree, n being the number of characters to be compared.
In this study, 226 Cryptomeria trees in a natural forest of about 750 m 2
were measured for five needle characters: twistedness, angle of insertion,
stiffness, density and intensity of color. They were classified into 39

139

RESEARCHES CARRIED OUT IN 1967

groups including each more than 2 trees besides 13 single trees. The
distribution of group size is presented in the following table.
Total
Mean
number number
1 2-45-78-1011-1314-1617-1920-2223-2627-29
ofof treesgroups per group
Number of trees per group

Number ofgroups13 19 11

2

3

o

3

o

o

1

52

5.12

At least, some of the group are regarded as clones, and the varianceof some quantitative characters measured within them was apparently
smaller than the total variance of the forest. It thus becomes possible
to make a genetic study of quantitative characters in a natural forest of
Cryptomeria japonica.

140 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

BOOKS AND PAPERS PUBLISHED IN 1967
BY S1AFF MEMBERS(1) CHU, Y. E., 1967. Variations in peroxidase isozymes of Oryza perennis andO. sativa. Japan. J. Genet. 42(4): 233-244.
(2) CHU, Y. E., 1967. Pachytene analysis and observations for chromosome association in haploid rice. Cytologia 32: 87-95.
(3) ENDO, T., 1967. Comparison of the effects of gamma-rays and maleic hydrazide
on enzyme systems of maize seed. Radiation Bet. 7: 35-40.
( 4) ENOMOTO, M., 1967. Nucleic acid and protein (in Japanese). Heredity (Tokyo)
21: 17-23.
(5) ENOMOTO, M., 1967. Composition of chromosome fragments participating inphage P22-mediated transduction. Virology 33: 474-482.
(6) FUJII, T., 1967. Comparison of the killing effect of gamma·rays and thermal
neutrons. Arabidopsis Information Service 4: 59.
(7) FUJII, T., 1967. On the determination of absorbed dose in heavy ionizing
particles. Arabidopsis Information Service 4: 60.
(8) FUJII, T., M. IKENAGA and J. T. LYMAN, 1967. Killing and mutagenic efficiencies of heavy ionizing particles in Arabidopsis thaliana. Nature 213:
175-176.
(9) FUJII, T. and S. MATSUMURA, 1967. Somatic mutations induced by chronic
gamma-irradiation. Seiken Ziho 19: 43-46.
(10) IINO, T. and MITANI, M., 1967. Infection of Serratia marcescens by bacteriophage chi. J. Virol. 1: 445-447.
(11) IINO, T. MITANI, M., 1967. A mutant of Salmonella possessing straightflagella. J. gen. Microbiol. 49: 81-88.
(12) IMAI, H. T., 1966: The chromosome observation techniques of ants and thechromosomes of Formicinae and Myrmicinae. Acta Hymenop. 2: 119-13l.
(13) KAWAHARA, T., 1967. Wild coturnix quail in Japan. The Quail Quartely
4: 62-63.
(14) KIHARA, H., 1967. Cytoplasmic male sterility in relation to hybrid wheat
breeding. Der Ziichter 37: 86-93.
(15) KIHARA, H., 1967. Cytoplasmic male sterility in relation to hybrid wheat
breeding (in Russian). Agricultural Biology (Moscow) 2: 214-225.
(16) KIHARA, H. and M. SHIMOTSUMA, 1967. The use of chromosomal interchanges
to test for crossing over and chromosome segregation. Seiken Zihil 19: 1-8.
(17) KIHARA, H. and K. TSUNEWAKI, 1967. Genetic principles applied to thebreeding of crop plants. Heritage from Mendel: 403-418.
(18) KIMURA, M., 1967. On the evolutionary adjustment of spontaneous mutation
rates. Genetical Research 9: 23-34.
(19) KIMURA, M., 1967. Recent advances in the theory of population genetics.
Proc. of the World Population Conference, 1965, 2: 478-480.
(20) KITANO, Y. and Y. KURODA, 1967. Effects of thyroxine, vitamin A and HeLa
cells on differentiation of aggregates of dissociated embryonic chick skin cells

BOOKS AND PAPERS PUBLISHED IN 1967 BY STAFF MEMBERS

141

in vitro. Exptl. Cell Res. 48: 350-360.
(21) KURODA, Y., 1967. A guide to developmental biology (in japanese). Heredity(Tokyo) 21(12): 55-59.
(22) KURODA, Y., 1967. Answers to various problems in developmental biology(in japanese). Seitai-no-kagaku 18(4): 214.
(23) Lu, Y. C., K. H. TSAI and H.!. OKA, 1967. Studies on soybean breeding in
Taiwan. 1. Growing seasons and adaptabilities of introduced varieties. Bot.
Bull. Acad. Sinica 8(1): 37-53.
(24) Lu, Y. C., K. H. TSAI and H.!. OKA, 1967. Studies on soybean breeding in
Taiwan. 2. Breeding experiments with successive hybrid generations grownin different seasons. Bot. Bull. Acad. Sinica 8(1): 80-90.
(25) MATSUMURA, S. and T. MABUCHI, 1967. Storage and nitrogen effects onradiation damage in einkorn wheat seeds with different water content. Seiken
Ziho 19: 9-17.
(26) MATSUNAGA, E., 1967. Parental age, live-birth order and pregnancy-freeinterval in Down's syndrome in japan. Ciba Foundation Study Group No. 25:
Mongolism, j. & A. Churchill, London.
(27) MATSUNAGA, E., 1967. Genetic characteristics of japanese populations (injapanese). jap. j. Physiol. 29: 1-11.
(28) MATSUNAGA, E., 1967. Genetic identification of interchanged children (injapanese). Heredity (Tokyo) 21(5): 30-34.
(29) MATSUNAGA, E., 1967. Application of cytogenetics to clinical medicine (injapanese). Modern Medicine (Tokyo) 22: 2606-2612.
(30) MATSUNAGA, E., 1967. Genetic causes of congenital anomalies (in japanese).
japanese Medicine in 1967. 1: 928-933.
(31) MITANI, M. and IINO, T., 1967. Phenocopies of a heteromorphous flagellar
mutant in Salmonella. j. Bacteriol. 93: 766-767.
(32) MORISHIMA, H., H.!. OKA and T. T. CHANG, 1967. Analysis of genetic variations in plant type of rice. 1. Estimation of indices showing genetic planttypes and their correlations with yielding capacity in a segregating population.
japan. j. Breed. 17(2): 73-84.
(33) MORISHIMA, H., H.!. OKA and T. T. CHANG, 1967. Analysis of genetic variations in plant type of rice. 2. Seasonal change in genetic plant type. japan.
j. Breed. 17(4): 251-261.
(34) OGAWA, Y., 1967. Cellulose acetate electrophoretic technique (in japanese).
"Electrophoretic technique" The 4th Edition. Ed. by The Society of Electrophoresis. Bunkodo, Tokyo. p. 111-150.
(35) OGAWA, Y., 1967. Standard procedure for the electrophoretic analysis ofhuman serum proteins on cellulose acetate strip (in japanese). j. Miyagi
Med. Technol. 16(1): 33-46.
(36) OGAWA, Y., 1967. Electrophoretic analysis of a2-lipoprotein in human serumon cellulose acetate strip (in japanese). Physico-Chern. BioI. 12(3): 122.
(37) OGAWA, Y., 1967. Sepraphore III, a new American cellulose acetate strip
for electrophoresis (in japanese). Physico-Chern. BioI. 12(3): 122-123.
(38) OGAWA, Y., 1967. Cellulose acetate electrophoretic technique (in japanese).
j. Kanagawa Med. Techno!. 1(1): 6-11.

142 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18(39) OGAWA, Y., 1967. Studies on the cellulose acetate electrophoretic technique
8. Clearing of cellulose acetate strip and scanning error (in Japanese). Med.
and BioI. 75(4): 128-130.
(40) OGAWA, Y., 1967. Studies on the cellulose acetate electrophoretic technique
9. Elution method of separation pattern of human serum proteins (in Japanese).
Med. and BioI. 75(4): 141-144.
(41) OGAWA, Y. and T. HASEGAWA, 1967. Studies on the cellulose acetate electrophoretic technique 10. Normal values of serum protein fractions on Separax
in Japanese adults (in Japanese). Med. and BioI. 75(5): 186-188.
(42) OGAWA, Y., 1967. Growth, differentiation and regeneration 43. Effect of
glucosamine on the development of skeletal muscle proteins in X-irradiatedTriturus embryos (in Japanese). Med. and BioI. 75(6): 204-207.
(43) OGAWA, Y., 1967. Studies on the cellulose acetate electrophoretic technique
11. Comparison of analytical results of serum protein fractions on paper andcellulose acetate strips by DISC electrophoresis (in Japanese). Med. and BioI.
75(6): 208-211.
(44) OISHI, H., 1967. Analysis of human chromosomes (in Japanese). J. Med.
Tech. (Tokyo) 11: 22-28.
(45) OISHI, H., 1967. Staining techniques of human chromosomes (in Japanese).
Jap. J. Clin. Path. 13: 94-100.
(46) OKA, H.1. and H. MORISHIMA, 1967. Variations in the breeding systems ofa wild rice, Drym perennis. Evolution 21(2): 249-258.
(47) OSHIMA, C., 1967. Fitness of flies having a lethal gene (in Japanese) The
Heredity 21(3): 39-43.
(48) OSHIMA, C., 1967. Fundemental evidence of microevolution (in Japanese)
Koko Kyoiku Kenkyu 3(35): 15-16.
(49) OSHIMA, C., 1967. Persistence of some recessive lethal genes in natural
populations of Drosophila melanogaster. ClItNCIA E CULTURA 19(1): 102110.
(50) PUTNAM, F. W., T. SHINODA, K. TITANI and M. WIKLER. 1967. Immunoglobulin structure: Variation in amino acid sequence and length of humanlambda light chains. Science, 157: 1050-1053.
(51) SAKAI, K.1. and H. MUKAIDE, 1967. Estimation of genetic, environmental,
and competitional variances in standing forests. Silvae Genetica 16: 149-152.
(52) SAKAMOTO, S., 1967. Genome analysis of the genus Eremopyrum. W. 1. S.
23-24: 21-22.
(53) SAKAMOTO, S., 1967. Cytogenetic studies in the tribe Triticeae. V. Intergeneric hybrids between two Eremopyrum species and Agropyron tsukushiense.
Seiken Zihll 19: 19-27.
(54) SHINODA, T., 1967. Red cell acid phosphatase types in a Japanese population.
Jap. J. Hum. Genet., 11: 252-256.
(55) SHINODA, T. and E. MATSUNAGA 1967. Serum alkaline phosphatase variants
and their association with ABO blood groups in a Japanese sample. Jap. J.
Hum. Genet., 12: 170-176.
(56) SOFUNI, T., Y. KIKUCHI, and A. A. SANDBERG, 1967. Chronology and pattern

BOOKS AND PAPERS PUBLISHED IN 1967 BY s1' AFF MEMBERS

\'.

~.

(57)

(58)
(59)

(60)
(61)
(62)

(63)

(64)

(65)

(66)

(67)
(68)

(69)

(60)

(71)

143

of human chromosome replication. V. Blood leukocytes of chronic myelocytic
leukemia. J. Nat. Cancer Inst. 38: 141-156.
STONIER, T. and YONEDA, Y., 1967. Stem internode elongation in the Japanesemorning glory (Pharbitis nil Choisy) in relation to an inhibitor system ofauxin destruction. Physiologia Plantarum 20: 13-19.
STONIER, T. and YONEDA, Y., 1967. Auxin destruction and growth in Japanesemorning glory stems. Annales of New York Academy of Sciences 144: 129-135.
TAZIMA, Y., 1967. The substantials contained and problems involved in the
1966 Report of the United Nations Scientific Committee on the Effects ofAtomic Radiation. (in Japanese) Kagaku 37(8): 411-414.
TAZIMA, Y., 1967. Radiations and radioisotopes in sericulture (in Japanese).
Kagaku to Gijutsu 6(1): 62-65, (2): 34-37, (3): 52-55.
TAZIMA, Y., 1967. Dr. TOYAMA's contribution to the Silkworm Genetics.
(in Japanese). Iden (Tokyo) 21(11): 4-8.
TAZIMA, Y., 1967. Biological effectiveness of radiations of different quality
at the cellular level. (in Japanese). Proceedings of the Symposium on Radiation
Quality in Radiation Actions and Microdosimetry (in Japanese): (10): 1-25.
TAZIMA, Y. and A. ONUMA, 1967. Experimental induction of androgenesis,
gynogenesis and polyploidy in Bombyx mori by treatment with CO 2 gas. J.
Sericult. Sci. Japan. 36(4): 286-292.
TSAI, K. H., Y. C. Lu and H. I. OKA, 1967. Studies on soybean breeding in
Taiwan. 3. Yield stability of strains obtained from disruptive seasonal
selection of hybrid populations. Bot. Bull. Acad. Sinica 8(2): 209-220.
TSUJITA, M. and S. SAKURAI, 1967. Genetic variations in shape and size of
pteridine granules of a normal silkworm strains and several other strains
with transparent skin. Proc. Japan. Acad. 43(10): 997-1002.
TSUJITA, M. and S. SAKURAI, 1967. Pterine granules in hypodermal cellsof the silkworm larva causing non-transparency of larval skin. Proc. Japan.
Acad. 43(10): 991-996.
WATANABE, T. K., 1967. Persistence of lethal genes associated with SD innatural populations of Drosophila melanogaster. Jap. J. Genet. 42: 375-386.
WIKLER, M., K. TITANI, T. SHINODA and F. W. PUTNAM, 1967. The completeamino acid sequence of a }. type Bence-Jones protein. J. BioI. Chern., 242:
1668-1670.
YASUDA, N. and N. E. MORTON, 1967. Studies on human population structure.
Proc. III. Inter. Congress Hum. Genet., J. F. Crow and N. V. Neel (ed.) Johns,
Hopkins Press pp. 249-265.
YONEDA, Y. and STONIER, T. 1967. Distribution of three auxin protector
substances in seeds and shoots of the Japanese morning glory (Pharbitis nil).
Plant Physiology 42: 1017-1020.
YOSIDA, T. H., OHARA, K. and L. W. LAW, 1967. Chromosomal alteration andthe development of tumors, XVI. Karyological studies on sensitive andresistant sublines of the mouse lymphocytic leukemia, L-1210, to several
antitumor agents. Jap. J. Genetics 42: 339-347.

144 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

ABSTRACTS OF DIARY FOR 1967
February
March
April
April
May
May
June
June
July
August151st Meeting of Misima Geneticist' Club
76th Biological Symposium
77th Biological Symposium
3
78th Biological Symposium
21
152nd Meeting of Misima Geneticist' Club12
79th Biological Symposium
22
153rd Meeting of Misima Geneticist' Club927th Meeting of the Board of Councillors
17
154th Meeting of Misima Geneticist' Club1415, 16 The 6th Summer Seminar on Genetics for High School
Teachers of Biology
155th Meeting of Misima Geneticist' Club
September 22
November 11
Public Lectures on Genetics (at the National Science
Museum, Ministry of Education, Tokyo)
156th Meeting of Misima Geneticist' Club
November 28
December 9
Japanese Poultry Breeder's Association
December 14
157th Meeting of Misima Geneticist' Club24
27

FOREIGN VISITORS IN 1967

145

FOREIGN VISITORS IN 1967
Jan.
Mar.

Mar.
Mar.
Apr.
Apr.
Apr.
Apr.
Apr.
May
June

June
June
June

July
July
July
Aug.

15

SCHEIN, M. W., National Science Foundation, Washington,
D. C., U.S.A.
23
TCHAN, Y. T., Department of Agricultural Microbiology,
University of Sydney, Sydney, Australia
QUISENBERRY, K. S., Agricultural Research Consultant,
Sarasota, Florida, U.S.A.
27
YAMAMOTO, N., Felis Institute, Temple University, Philadelphia, U.S.A.
.28]ESENA, C. C., Department of Agronomy, University ofPhilippines, The Philippines
KANAGIR, D., Department of Biochemistry, Institute of3
Nuclear Sciences "Boris Kidrich", Yugoslavia
5-6 GARCIA, A., World Seeds, Inc., California, U.S.A.
20-22 GOTTSCHALK, W., Institute of Genetics, Bonn University,
West Germany
26
ROBERTS, W., Boyce Thompson Institute for Plant Research,
N. Y., U.S.A.
TOKUNAGA, C., Department of Zoology, University of Cali29
fornia, Berkeley, California, U.S.A.
2-17 STICH, H. F., Department of Biology, McMaster University,
Hamilton, Ontario, Canada
6
EVERSON, E. H., Department of Crop Science Wheat Breeding and Genetics, Michigan State University, East
Lansing, Michigan, U.S.A.
11
SOEKARNA, D., Research Institute of Pest and Diseases of
Crop Plants, Bogor, Indonesia
26
NAVARATNA, S. K., Agricultural Officer, Division of Botany,
Central Agricultural Research Institute, Ceylon
30
SOEKARNA, D., Research Institute for Pest and Diseases of
Crop Plants, Bogor, Indonesia
MOSKOWITZ, M., Department of Biological Sciences, Purdue
University, Lafayette, Indiana, U.S.A.
11
KOJIMA, K., Department of Genetics, Texas University,
U.S.A.
17
LAMM, R. D., Colorado State Representative, Denver, Colorado, U.S.A.
19
HODGE, W. H., Tokyo Office, National Science Foundation,
U.S.A.
4
O'NELL, E., CSIRO, Division of Plant Industry, Canberra146 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

Aug.

7-8

Aug.

19

Aug

26

City, A.C.T., Australia
SUEOKA, N., Department of Biology, Princeton University,
Princeton, N. ]., U.S.A.
GARBER, B., College of Medicine, Department of Biochemistry, State University of New York, U.S.A.
BULL, H. B., Department of Biochemistry, University of
Iowa, U.S.A.
FEJER, D., Department of Biochemistry, Nutition and Food
Science, University of Ghana, Ghana
GERBAUT, L., Laboratorie de Virologie, Faculte de Pharmacie, Paris, France
LUSTING, B., Clairol Research Laboratories, Stanford,
U.S.A.
GORDON, M. ]., Spinio Division Beckman Instruments,
California, U.S.A.
ROBINSON, F. A., Twytord Laboratoires, Ltd., Twytorad
Abbey Road, London, N. W., U. K.
TUJIMOTO, H. Y., Department of Cell Physiology, Universityof California, U.S.A.
VESTLING, C. S., Department of Biochemistry, Universityof Iowa, Iowa, U.S.A.
BERG, C. P., Department of Biochemistry, Medical ResearchCenter, University of Iowa, Iowa, U.S.A.
BOVE, ]. M., Station Centrale de Physiologie Vegetale,
Centre National de Recherche Agronomique, Routes,
Versailles, France
DARROW, R. A., Massachusetts General Hospital, Boston.
Massachusetts, U.S.A.
FOWLER, A., Department of Biochemical Chemistry, University of California, School of Medicine, Los Angels,
California, U.S.A.
HARCRY, I., Laboratory of Nucleic Medicine and Radiological Biology, Colifornia, U.S.A.
LINKER, A., Department of Biochemistry and Pathology,
University of Utah, Utah, U.S.A.
MARK, K. K., New Asia College Chinese, University ofHong Kong, Hong Kong
ZABIN, I., Department of Biochemical Chemistry, Schoolof Medicine, University of California, California, U.S.A.
BARTLETT, G. R., Laboratory for Comparative Biochemistry,
California, U.S.A.

FOREIGN VISITORS IN 1967

Oct.

2

Oct.
Nov.

9
20

Dec.

18

147

FINLAY, K. W., Waite Agricultural Research Institute,
Glen Osmond, South Australia
HSIEH, S. C.,
Taiwan Agricultural Research Institute,
TaiwanPAPAZOV, N., Bulgarian Ambassador at TokyoSCOSSIROLI, R. E., Instituto di Genetica, Universita di
Bologna, Italia
COYNE, B. ]., c/o Committee on Mathematical Biology,
University of Chicago, U.S.A.
KOMING, D. R., N. I. H. Pacific Office, National Institute of
Health, U.S.A.
ANGELONE, L., N. I. H. Pacific Office, National Institute of
Health, U.S.A.

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

.•

148

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 18

AUTHOR INDEXAMANO, E
109, 111, 112
CHU, Y. E
58, 59, 59
ENDO, T
.42, 43
FUJII, T •............ 102, 103, 105, 107
130
FUJISHIMA, T.
FUKASE, Y•.......................... 87
HAYOSHI, M.
112
HAYASHI, S
137, 138
IINO, T. ..
114, 115, 115
IMAI, H. T
13, 15, 17, 18
INOUYE, T......................... 135
ISHIDSU, J
116
KADA, T
100, 101, 118
KATSUYA, K
81
KAWAHARA, T
133
KIHARA, H.••........................ 9
KIKUCHI, Y
124, 125, 126
KIMURA, M. ..
61, 64, 65
KOJIMA, K
34, 35
KURITA, Y•........................ 124
KURODA, Y
10, 11, 27, 28, 96
KUROKI, T
21
MABUCHI, T. .. . . . . . . . . . . . . . . . . . . .. 108
MARUYAMA, T
61, 62, 64
MASUJI, H
21
MATSUDA, E
120, 121, 125
MATSUMURA, S..................... 108
MATSUNAGA, E
120, 121
MINATO, K•.......................... 28
MORIGUCHI, Y
24
MORISHIMA, H.
57, 129MORIWAKI, K
13, 15,17,18,46,47,49,54
MURAKAMI, A
89, 92, 93, 95
NARISE, S
133, 136

NARISE, T
56, 80, 134
NAWA, S32, 33
OGAWA, Y
41
OHBA, K•.......................... 138
OHTA, T65, 67
OHTA, Y.,26, 27OISHI, H........•.... 121, 124, 125, 126
OKA, H. I•........ 58, 59, 59, 129, 129
ONIMARU, K
82, 83, 84, 87, 88
ONUMA, A
83
OSHIMA, C.,
76, 77SAKAI, K. I. .. 134, 135, 136, 137, 138
SAKAMOTO, S
57
SAKURAI, S
34, 35, 35, 37, 39
SHIBATA, K. . . . . . . . . . . . . . . . . . . . . .. 126
SHIMOTSUMA, M . .,9
SHINODA, T
123
SONODA, J
24
STONIER, T. .
30
TAZIMA, Y
82, 83, 84, 87, 88
TEULADE, P
84
TOMITA, K......................... 138
TONOMURA, A.
124
TSAI, K. H.. . . . . . . . . . . . . . . . . . . . . . .. 129
TSUJITA, M
34, 35, 36, 37, 39
TSURUTA, R
23
TUTIKAWA, K
98
WATANABE;T. K.,76, 77, 79
YAMADA, M. A.,
32, 33
YAMAGUCHI, S
113, 114
YASUDA, N. ..
68, 71, 73
YONEDA, Y•............ 30, 30. 43, 45
YOSIDA, T. H.
.. 13, 15, 17, 18, 21, 23,24,51,53

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