NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 12

1961

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan1962

Annual Reportof theNational Institute of Gene~ics
No. 12,1961l~..

Published by

The National Institute of Genetics, Japan
1962

CONTENTS
General Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstracts of diary for 1961
Staff'................................................
Council............................................................
Projects of research for 1961.......................................
Researches supported by foreign grants............................
Foreign visitors in 1961............................................
Researches carried out in 1961.....................................

A. Genetics, cytology and biochemistry of animals
1. The persistence of some natural lethal chromosomes ofDrosophila melanogaster in experimental population(OSHIMA)...................................................
2. Relative frequency of the second chromosomes bearing deleterious genes in natural populations of Drosophila melanogaster (OSHIMA, FUWA and IMAI)............................
3. The pre-adult viability of heterozygous flies for lethal chromosome (OSHIMA, FUWA and MUKAI)
4. Seasonal changes in the frequency of chromosomes bearing alethal gene in natural populations of Drosophila melanogaster (MINAMORI, SASAKI and OSHIMA)......................
5. Dieldrin resistance in Drosophila pseudoobscura (OSHIMA) .. . . .
6. Spontaneous polygenic mutation rate and the genetic structureof natural populations in D. melanogaster (MUKAI and CHIGUSA)
7. Two cases of heterosis associated with the recessive genes
sepia (se) and expanded wing (ew) in Drosophila melanogaster(CHIGUSA and MUKAI) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Transfer of the "sex-ratio" condition from D. willistoni toother species of Drosophila (SAKAGUCHI and POULSON)
9. Comparative analysis of transmissive efficiency and pathological effects of "sex-ratio" agents of different origin in
Drosophila (SAKAGUCHI and POULSON) . . . . . . . . . . . . . . . . . . . . . . .
10. Lethality and low viability induced by the segregation distorter locus (symbol SD) in Drosophila melanogaster (RIRAIZUMI)
11. Cytological observation of the SD region in the salivary glandchromosome of Drosophila melanogaster (TONOMURA (Toyo-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

FUKU».....................................................
12. Migratory activity of Drosophila as a genetic character (NARISE
and SAKAI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13. Stimulating effect of a foreign genotype on migration in
Drosophila (NARISE and SAKAI).............................
14. Pterine metabolism in Drosophila melanogaster (TAIRA)
15. The fourth case of translocation between the W chromosome
and an autosome in the silkworm (ONIMARU and TAZIMA) ..
16. Studies on the manifestation mechanism of dilute lemon genein the silkworm (TSUJITA).... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17. Amount of isoxanthopterin in the larvae of the silkworm(TSUJITA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18. Pteridines in the compound eyes of the silkworm moth,Bombyx mori (TSUJITA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19. Influence of temperature on the syntheses of contractile proteins in Triturus embryo (OGAWA).........................
20. Protective action of sodium-glucuronate on the formation of
the contractile muscle protein, actin, inhibited by X-ray
irradiation in Triturus embryo (OGAWA)
21. Effects of glucuronic acid and related compounds on the chemodifferentiation of muscle tissue in regenerating hind limbof Triturus (OGAWA).......................................
22. Two factors influencing sensitivity to X-rays in the synthesisof myosin during early embryonal stages (OGAWA).........
23. Sensitivity of a day-old chicks to low and high temperatures(KAWAHARA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24. Electrophoretic hemoglobin patterns in inbred rats and chicken(MORIWAKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25. Further study on post-axial polydactylism in the house mouse(YOSlDA, SAKAMOTO and NAKAMURA).........................
26. Invasion of tetraploid cells of the Yoshida sarcoma into thelymph nodes of the rat (YOSlDA) . . . . . . . . . . . . . . . . . . . . . . . . . .
27. Karyological analysis of a metastatic tumor cells in MH 134
hepatoma of the C3H strain of the mouse (INUl and YOSlDA).
28. Ploidy in tumor cells after direct inoculation into variousorgans of the mouse (YOSlDA and KAMIOKA). . . . . . . . . . . . . . . .
29. Competition of diploid and tetraploid tumor cells in the mouse(YOSIDA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30. Inhibitory effect of the components of Swertiae japonica on
cell division in living plant tissue and on growth of rat
tumors (TAKENAKA and OGAWA)............................
31. Anti-cancer activity of extract from Glycyrrhizae Radix

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CONTENTS(OGAWA)

Genetics, cytology and biochemistry of plants
32. Cytoplasmic male sterility of common wheat (KIHARA).......
33. Pistillody of Triticum durum induced by an Aegilops cytoplasm (KIHARA and TSUNEWAKI)............................
34. Progressive necrosis of seedlings in hexaploid wheat (TSUNEWAKI)
35. Relation between chlorophyll content and free amino acids inmutants of Triticum monococcum (FuJII and ONO)
36. On the occurrence of chlorophyll mutations in clusters ineinkorn wheat (FuJII)
37. Crossing experiments with chlorophyll mutants of einkorn
wheat (FUJII). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38. Developmental anatomy of glume epidermis in the genus
Oryza (KIHARA and KATAyAMA).............................
39. Genome analysis in the genus Oryza III (KIHARA, KATAYAMA,
MATSUMURA and MABUCHI)
40. Variation in blast susceptibility in wild rices closely anddistantly related to cultivated rice (KATSUYA)..............
41. Some considerations on rice cultivation in New Guinea (KATAYAMA)
42. Some observations on the blooming of Oryza species (KATAYAMA)
43. Photoperiodic responses of Oryza species IV (KATAyAMA).....
44. Intra-plant variation in seed size in rice (SAKAI and SUZUKI)..
45. Variation in susceptibility to blast disease in wild and cultivated rice (GOTO and SAKAl)...............................
46. Rice varieties intermediate between wild and cultivated forms
and the origin of the Japonica type (OKA and CHANG) .....
47. Responses to various growing conditions of wild and cultivatedrice forms (OKA and CHANG)...............................
48. Intra-specific variations of Oryza breviligulata in West Africa(MORISHIMA and OKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
49. Differences in breeding behavior between wild and cultivatedrice species (HINATA, MORISHIMA and aKA)..................
50. Tetraploid F 1 hybrid between Oryza sativa and O. glaberrima(HINATA and OKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51. Hybrid-sterility relationships among Asian strains of Oryza
perennis and O. sativa (HINATA and aKA)
52. Investigations on the measuring method of the intercellularspaces on several plants (KATAYAMA) .. . . . . . . . . . . . . . . . . . . . .

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53. Arisaema triphyllum, Jack-in-the-Pulpit, in Minnesota, especially at the Cedar Creek Natural History Area (SAKAMOTO).
54. Fertility and crossability of certain wild brambles of Minnesota and Wisconsin (SAKAMOTO and WILCOX).............
55. The origin of Prunus yedoensis (TAKENAKA) . . . . . . . . . . . . . . . . . .
56. Cytogenetic studies in the genus Nicotiana XIV (TAKENAKA).
57. Alkaloid decomposition by tobacco extract (TANAKA).........
58. Inheritance of anthocyanin concentrations in Torenia flowers(ENDO)
59. Cytological studies on the nuclei of yeasts (YONEDA).........

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C. Mathematical genetics
60. A general formula for the probability of fixation of mutant
genes in a population (KIMURA).. . . . . . . . . . . . . . . . . . . . . . . . . . .
61. On circular mating systems (KIMURA)...... . . . . . . . . . . . . . . . . . .
62. The maintenance of supernumerary chromosome in wild
populations of Lilium callosum by preferential segregation(KIMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
63. Distorted segregation and substitutional load (HIRAIZUMI).....
64. Population dynamics of sex-linked recessive lethals (MUKAI) .
65. Equation for polygenic mutation rate per locus per unit dose
(KONDO)...................................................
D. Genetics and biochemistry of microorganisms
66. A temperature independent flagellation mutant in Salmonella(IINO)
67. A modified selective medium for the isolation of motility
mutants of Salmonella (ENOMOTO and IlNo)
68. H-antigen of heterozygous hybrids between Salmonella abony
and Salmonella typhimurium (HIROKAWA and IINO).........
69. Chi-phage resistance of the salmonella serotypes having gantigen (SASAKI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E. Radiation genetics in animals
70. Radiation-induced mutation rate of polygenes controlling thenumber of sternopleural bristles in Drosophila melanogaster(MUKAI and CHIGUSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71. Further radiation-genetical consideration concerning thegenetic structure of natural populations (MUKAI) .. . . . . . . . .
72. Further studies on two types of dose-rate dependence ofradiation-induced mutation rates in spermatogonia andoogonia of the silkworm (TAZIMA and KONDO)..... . . . . . . . . .

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CONTENTS

73. Comparison of acute and chronic irradiations in respect toradiation-induced lethal mutation rates in the silkworm(TAZIMA and KOBAYASHI)
74. Differences in response of silkworm gonia to acute and chronic
r-irradiation (SADO)........................................
75. Number of gonial cells of the silkworm at early stages aroundhatching (KOBAYASHI).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76. Induction of a presumably somatic mutation with 5-bromouracil
in the silkworm (NAKAJIMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
77. Theoretical estimation of "survival cell number versus dose"
curve from experimental frequency distribution of the number of mutants (KONDO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78. Deformities in chick embryos caused by r-irradiation (KAWAHARA)
79. Genetic effects of chronic low dose irradiation on mice (MURAMATSU, SUGAHARA and OKAZAWA)............................

F. Radiation genetics in plants
80. Boron effects upon gamma-ray and thermal neutron irradiations of einkorn wheat; RBE of heavy particles fromIOB(n, a)7Li reaction: 1. Effects on seedling growth, fertility
and chromosome aberrations (MATSUMURA and MABUCHI)....
81. Ibid.: II. Effects on chlorophyll mutations (MABUCHI and
MATSUMURA)
82. Ibid.: III. Calculation of absorbed dose due to IOB(n, a)7Li
reaction (KONDO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
83. Radiological study of wheat monosomics (TSUNEWAKI)
84. Changes in rust susceptibility due to partial irradiation ofwheat seedlings and its dose dependence (KATSUYA)........
85. A study on the relation between dose rates of gamma-rays andchromosomal aberrations (SAKURAI and MATSUMURA)........
86. Polyploids and aneuploids of Triticum dicoccum produced by
N 20-treatment (KIHARA and TSUNEWAKI)....................
87. Comparison of 32p f3 ray and 137CS r ray effects on seedlingheight of wheat (KONDO and ISHIWA).......................
88. Effect of surface dose change on small seeds exposed to Co60 r rays (KONDO and ISHIWA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
89. Relation between chlorophyll mutations and dose rates ofgamma-radiation in rice (MATSUMURA and MABUCHI)........
90. Occurrence of variants in developmental stability in seed sizein the X-rayed progeny of rice (SAKAI and SUZUKI). . . . . . . .
91. Mutations in Pharbitis Nil induced by gamma rays (KIHARA

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and LILIENFELD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 108
Irradiation experiments with cultivated chrysanthemum (FuJIIand MABUCHI)108

G. Human genetics
93. Prezygotic selection in ABO blood groups (MATSUNAGA andHIRAIZUMI)
94. Selection in ABO blood groups: Viability and its relation to
the proportion of prenatal deaths (HIRAIZUMI and MATSUNAGA).
95. Selection in P blood groups in man: A probable case of prezygotic selection operating in both sexes (HIRAIZUMI).. . . . . .
96. Genetic study on sporadic retinoblastoma in Japan (MATSUNAGA)
97. Dimorphism in human normal cerumen (MATSUNAGA)
98. The frequency distribution of the so-called "drumsticks" inthe polymorphonuclear neutrophil leucocytes in Japanese
female blood films (TONOMURA (TOYOFUKU) and TONOMURA)..
99. Chromosome conditions in twenty patients (TONOMURA) . . . . . .

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H. Technical note
100. Measurements of r ray contamination in the reactor of JRR-1
and response of silver-activated phosphate glass containing
lithium and boron (KONDO and ISHlWA)..................... 120
101. Dose-rate distribution in the Cs-137 gamma ray room (KONDO
and ISHIWA). . . . .. . . . .. .
.. . . . . . . . . . . . . . . .
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GENERAL STATEMENT
From October 11 to November 7 of this year, we have had the Southeast Asian Regional Training Course on Genetics sponsored by the
Ministry of Education and the Japanese Commission for UNESCO. There
were 19 participants from 7 nations. Our staff members gave lectureson subjects from their own fields, covering biochemical, radiation, popu·
lation as well as human genetics.
We believed that this course was successful for the participants not
only in becoming acquainted with the principles of genetics but also indeveloping mutual understanding among themselves and the membersof our Institute.
It was suggested that an advanced course in special fields of genetics
would be organized in the future.
The main building of the Institute is not yet completed. It will takeat least one more year.
Dr. Oshima, head of the Department of Physiological Genetics and Dr.
Matsunaga, head of the Department of Human Genetics were awarded
the prizes of Genetics Society of Japan and of Japan Society of Human
Genetics, respectively.
To the Tenth Pacific Science Congress which was held in Honolulu
from August 21 to September 2 we have sent several members.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

ABSTRACT OF DIARY FOR 1961
Feb. 24.
March 18.
April 19.
May 12.
19.
June
6.
23.
July 14.
Sep. 15.
Oct.
3.
Nov. 24.
Dec. 18.
19.

90th meeting of Misima Geneticists' Club.
91st meeting of Misima Geneticists' Club.
92nd meeting of Misima Geneticists' Club.
40th Biological Symposia
93rd meeting of Misima Geneticists' Club.
41st Biological Symposia
94th meeting of Misima Geneticists' Club.
95th meeting of Misima Geneticists' Club.
96th meeting of Misima Geneticists' Club.
97th meeting of Misima Geneticists' Club.
98th meeting of Misima Geneticists' Club.
42nd Biological Symposia
99th meeting of Misima Geneticists' Club.

STAFF
Director
Hitoshi KIHARA, D. Sc., M.J.A.
Members
1. Department of Morphological Genetics:
Yataro TAZIMA, D. Ag., Head of Department
Bungo SAKAGUCHI, D. Ag.,
Toshihiko SADO and Kimiharu ONIMARU
2. Department of Cytogenetics:
YO TAKENAKA, D. Sc., Head of Department
Tosihide H. YOSIDA, D. Sc.,
Kazuo MORIWAKI, D. Sc., Tsuguo TATEOKA, D. Sc. and Yoshiaki YONEDA
3. Department of Physiological Genetics:
Chozo OSHIMA, D. Sc., Head of Department
Hitoshi HIHARA, D. Sc., M,J.A., Toshifumi TAIRA, D. Sc., Koichiro
TSUNEWAKI, D. Ag., Ph. D. and Sadao SAKAMOTO (in U.S.A.)
4. Department of Biochemical Genetics:
Mitsuo TSUJITA, D. Ag., Head of Department
Yoshito OGAWA, M.D., Saburo NAwA, D. Sc. (in U.S.A.)
Tetsuo IINo, Ph. D., Toru ENDO, and Seizo TSUDA, D. Ag. (in1U.S.A.)STAFF

35.

Department of Applied Genetics:
Kan-Ichi SAKAI, D. Ag., Head of Department
Hikoichi OKA, D. Ag.,
Yukio YAMADA, D.Ag.(in U.S.A.), AkiraMIYAZAWA, Takatada KAWAHARA
Shinya IYAMA, D. Ag. (in U.S.A.) and Hiroko OKINO
6. Department of Induced Mutation:
Seizi MATSUMURA, D. Ag., Head of Department
Sohei KONDO, D. Sc., Kiyoshi TUTIKAWA, Taro FUJII, Terumi MUKAI
Ph. D., and Hiromi ISHIWA
7. Department of Human Genetics:
Ei MATSUNAGA, M. D., D. Sc., Head of Department
Motoo KIMURA, Ph. D., D. Sc. (in U.S.A.)
Yuichiro HIRAIZUMI, D. Sc., Akira TONOMURA, D. Sc. and Yasuko
TONOMURA, D. Sc.

Part-time Staff and Research Associates
Yoshinari KUWADA, D. Sc., M.J.A., Emeritus Professor of Kyoto
University
Kan OGUMA, D. Ag., Ex-Director, Emeritus Professor of Hokkaido University
Yoshimaro TANAKA, D. Ag., D. Sc., M.J.A., Emeritus Professor of Kyushu
University
Taku KOMAI, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Saziro MAKINO, D. Sc., Professor of Hokkaido University

Research members under the Grant from the Rockefeller Foundation
Rice Research Group:
Hiroko OKINO, Keizo KATSUYA, Tomoo MABUCHI, Tadao KATAYAMA, Kokichi HINATA and Akio SUZUKI.

Research members under the Grant from the National Institute of Health
Salmonella Research Group:
Itiro SASAKI and Masatoshi ENOMOTO.
Drosophila Research Group:
Sadao CHIGUSA and Keiko FUWA.

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Department of Administration
Tanryu OTOMO, Head of Department
Katsutaro ANDO, Chief of General Affairs Section
Seiichi KOIZUMI, Chief of Finance Section
Hiroko NAKANO, Ryoji OYAMA, Kazuo ENOMOTO and Keiichi KAWASHIMA
Clerks, Librarian, Typists, Chauffeur, Field Laborers and Janitors ..... 9

Misima Branch.:of Hatano Tobacco Experiment Station
Masao TANAKA, Head
Assistants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

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Association for Propagation_of the Knowledge of Genetics
Hitoshi KIHARA, President
YO T AKENAKA, Managing Director
Yataro TAZIMA, Managing Director
Seizi MATSUMURA, Manager
Bungo WADA, Manager
Yoshito SHINOTO, Manager

COUNCIL
YO K. OKADA, Director of National Science Museum, Chairman
Kan OGUMA, Ex-Director, Emeritus Professor of Hokkaido University
Toshinobu ASAI, Professor of Tokyo University
Tanemoto FURUHATA, Director of Scientific Research Institute of Police
Hideo MATSUKUMA, President of Japan Monopoly Corporation
Toshitaro MORINAGA, Director of National Institute of Agricultural Sciences
Toshio SAITO, Governor of Sizuoka Prefecture
Takeo SAKATA, President of T. Sakata Company
Bungo WADA, Professor of Sizuoka University
Seizo KATSUNUMA, Ex-President of Nagoya University
Masataka OMASA, Professor of Tokyo University
Daigoro MORIWAKI, Professor of Tokyo Metropolitan University
Yakichi NOGUCHI, Professor of Tokyo University
Kenpo TSUKAMOTO, Director of National Institute of Radiological Sciences
Minoru TACHI, Director of Institute of Population Problems

PROJECTS OF RESEARCH FOR 1961

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PROJECTS OF RESEARCH FOR 1961
Department of Morphological Genetics
Genetics of the silkworm (TAZIMA)
Studies on food preference of the silkworm (TAZIMA)
Chemical mutagenesis in the silkworm (TAZIMA, SADO and NAKAJIMA)
Cytological study of silkworm germ cells (SADO)
Studies on dose-rate dependence of radiation-induced mutation rates(TAZIMA and ONIMARU)
Hereditary infections in Drosophila (SAKAGUCHI)

Department of Cytogenetics
Cytology and genetics of tumors (YOSIDA)
Experimental breeding and genetics of mice and rats (YoslDA and KURITA)
Biochemical study on genetical abnormalities of mice (MORIWAKI)
Determination and differentiation of sex in higher plants (TAKENAKA)
Induction of abnormal mitosis and inhibition of growth by substances
extracted from certain plants (TAKENAKA)
Interspecific hybridization in Nicotiana (TAKENAKA and LILIENFELD)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus yedoensis (TAKENAKA)
Cytological studies on the yeast cell (YONEDA)

Department of Physiological Genetics
Genetical studies on insecticide-resistance in Drosophila (OSHIMA)
Physiological studies on eye-pigment formation in Drosophila (TAIRA andOSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila (OSHIMA)
Studies on the origin of wheat (KIHARA and TSUNEWAKI)
Studies on nucleus substitution in wheat and related species (KIHARA)
Male sterility in wheat (KIHARA)
Production of polyploids and aneuploids by N 20 treatment (KIHARA and
TSUNEWAKI)
Monosomic analysis of 6x wheat (TSUNEWAKI)

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Department of Biochemical Genetics
Biochemical genetics of insects and microorganisms (TSUJITA and NAWA)
Embryological and biochemical studies in the silkworm (TSUJITA andSAKAGUCHI)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA)
Chemical research in anti-tumor substances (OGAWA)
Biochemistry of the mechanism underlying variations in flower color inplants (ENDO)
Genetics of virus (TSUJITA)
Immunogenetics of salmonella (IINO)
Genetics of multi-drug resistance in bacteria (IINO)

Department of Applied Genetics
Studies on breeding and genetics in poultry (YAMADA and KAWAHARA)
Theoretical studies on plant breeding techniques (SAKAI)Studies on competition and migration in plants and animals (SAKAI, IYAMA
and NARISE)
Genetic studies of alkaloid content in tobacco plants (SAKAI and IYAMA)
Biometrical study of cytoplasmic inheritance (SAKAI, IYAMA and NARISE)
Genetic studies on developmental stability in plants (SAKAI, NARISE andSUZUKI)
Polyploidy and sterility in fruiting plants (FURUSATO and MIYAZAWA)
Evolutionary-genetic studies in Oryza (OKA and OKINO (MORISHIMA»)

Department of Induced Mutation
Radiation genetics of mice (TUTIKAWA)
Radiation population-genetics of Drosophila (MUKAI and CHIGUSA)
Studies on polygenic mutation in Drosophila (MUKAI and CHIGUSA)
Triploidy breeding of sugar beets (MATSUMURA)
Relation between the quality of radiations and mutations (MATSUMURA
and KONDO)
Radiation genetics of cereals (MATSUMURA, FUJII, KATSUYA and MABUCHI)
Radiation genetics and its practical application (MATSUMURA, FUJII and
MABUCHI)
Biophysical consideration of radiation genetics (KONDO)
Radiation dosimetry (KONDO and ISHIWA)RESEARCHES SUPPORTED BY FOREIGN GRANTS

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Department of Human Genetics
Prezygotic selection in ABO blood groups (MATSUNAGA and HIRAIZUMI)
Genetic studies on sporadic retinoblastoma in Japan (MATSUNAGA)
Cytogenetics in man (TONOMURA)
Sexual dimorphism in resting nuclei (TONOMURA (TOYOFUKU) and TONOMURA)
Theoretical studies of population genetics (KIMURA)
Effects of radiation-induced mutations on fitness (HIRAIZUMI)
Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)RESEARCHES SUPPORTED BY FOREIGN GRANTS
1.

JOINT RESEARCHES ON THE ORIGIN OF RICE SUp·
PORTED BY A GRANT FROM THE ROCKEFELLER
FOUNDATION

Section 1.
a.
b.

Collection and preservation of Oryza species (KIHARA)

Strains of 27 species so far collected amount to more than 4,100
Collection-tour was made this year by KATAYAMA to Philippines
and New Guinea

Section 2.

Morphology and physiology of Oryza species (MATSUMURA)

Comparison of radio-sensitivity of Oryza species (FUJII)Genome-analysis of Oryza species (KIHARA, MATSUMURA, NEzu, KATAYAMA and MABUCHI)
c. Susceptibility of wild and cultivated rice strains to blast fungus(KATSUYA)
d. Surface structure of lemma, palea and leaves of Oryza species(KIHARA and KATAYAMA)e. Investigation of photoperiodic responses of Oryza species (KATAYAMA)a.
b.

Section 3.
a.

Population-genetics in wild and cultivated rice (SAKAI)

Estimation of genetic variability among and within populations ofwild rice (SAKAI, lYAMA and NARISE)
b. Comparative studies of seedling and flowering characters of wild
and cultivated rice (NARISE and SAKAI)c. Variation and inheritance of within-plant variability in seed size
in wild and cultivated rice (SAKAI, NARISE and SUZUKI)
d. Variation studies of blast disease resistance in wild and cultivatedrice (GOTOH and SAKAI)8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Section 4. Genetic studies in wild and cultivated rice (OKA)
a.
b.
c.
d.
e.

Statistical-systematic studies of wild and cultivated rice strains(MORISHIMA and OKA)
Survey of variations between O. perennis and O. sativa f. spontanea(MORISHIMA, CHANG and OKA)
Crossing-experiments and sterility of hybrids between wild andcultivated rice strains (HINATA and OKA)
Studies of intermediate wild-cultivated forms in rice (OKA andCHANG)
Responses to growing conditions of wild and cultivated rice forms(OKA and CHANG)

Section 5. Cytogenetics of Oryza species (TAKENAKA)
a. Karyotype analysis of Oryza species (YONEDA and SHINOHARA)
b. Comparative observations of chromosomes in haploid plants of
Oryza species (C. H. Hu)
c. Embryological studies in Oryza species (DoIDA)ll.

STUDIES OF DELETERIOUS GENES IN DROSOPHILA
POPULATIONS SUPPORTED BY A GRANT FROM
NATIONAL INSTITUTE OF HEALTH, U.S.A.

1) , Direct analysis of natural populations of D. melanogaster (OSHIMA andFUWA)2) - Experimental analysis
a. The pre-adult viability of heterozygous flies for lethal or semilethal chromosome (OSHIMA and FUWA)
b. The persistence of some natural lethal genes in experimental populations (OSHIMA and FUWA)
c. The persistence of two recessive genes in experimental populations(MUKAI and CHIGUSA)
3) Studies on mutation rates
a. Radiation-induced mutation rate (MUKAI and CHIGUSA)
b. Spontaneous mutation rate (MUKAI and CHIGUSA)FOREIGN VISITORS IN 1961

ID.

9

STUDIES IN HUMAN GENETICS SUPPORTED BY A
GRANT FROM THE ROCKEFELLER FOUNDATION

1. Selective factors maintaining human blood group polymorphism(HIRAIZUMI and MATSUNAGA)
2. Studies on dimorphism in human normal cerumen (MATSUNAGA)
3. Chromosome studies in patients with different congenital anomalies(TONOMURA)FOREIGN VISITORS IN 1961
March 16. Dr. Charlotte Auerbach (Edinburgh Univ., England)
27. Dr. H. B. BIRNBAUM (National Science Foundation, U.S.A.)
28. Dr. RICHARD K. ANDERSON and Cleo H. ANDERSON (Rockefeller
Foundation, U.S.A.)
April
8. Dr. VICENTE C. RODRIGUES (Bureau of Plant Industry,
Philippines)
Miss Payow Yimcharoen (Dept. Biology, Chulalongkon Univ.,
Bangkok, Thailand)
27. S. H. Ou (FAO Regional Office, Bangkok, Thailand)
June
3. Dr. G. L. McNEW (Boys Thompson Institute, New York,U.S.A.)
Dr. A. W. NORDSKOG (Iowa State Univ., U.S.A.)
6. Dr. A. HOLLAENDER (Oak Ridge National Laboratory, U.S.A.)
July
18. Dr. H. BOROUGHS (Inter American Institute of Agricultural
Sciences, Costa Rica)
23. Dr. V. KOVDA (UNESCO, Paris, France)
Dr. L. MATTSSON (UNESCO, Djakarta, Indonesia)Aug.
3. Dr. L. R. HOUSE (Rockefeller Foundation, New Dehli, India)
Dr. T. S. DHILLON, (Dept. Botany, Univ. of Hong Kong)
15. Dr. S. UDAYACHALEM (Physics Dept., Chulalongkon Univ.,
Bangkok, Thailand)
Sep.
12. Dr. K. SUVATABANDHU (BOTANY Dept., Faculty of Science,
Chulalongkon Univ., Bangkok, Thailand)
Dr. H. D. JORDAN (West African Rice Research Station, Sierra
Leone)
Dr. H. PARTHASARATHY (FAO Foundation, Bangkok, Thailand)
Dr. N. MANGAMMAL (c/o FAO Foundation, Bangkok, Thailand)
Oct.
5. K. R. STINO (Minister of Supply & Professor, College ofAgriculture, Giza, Egypt, U.A.R.)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Nov.

Dec.

12. L. MATTSSON, (UNESCO, Djakarta, Indonesia)
17. J. A. PATIL (Dept. of Agriculture, Mahapashtra State,
Bombay, India)
B. B. CHAVDHARI (Dept. of Agriculture, Mahapashtra State,
Bombay, India)
7. V. G. PANSE (Institute of Agricultural Research Station, New
Delhi, India)
18. Dr. T. MAKINODAN (Biology Division, Oak Ridge National
Laboratory, U.S.A.)RESEARCHES CARRIED OUT IN 1961
A. GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF ANIMALS
1.

The persistence of some natural lethal chromosome of Drosophilamelanogaster in experimental populations. lI(By Chozo OSHIMA)

The heterozygous flies for each of several lethal chromosomes isolated
in 1959 from the large Katsunuma population or small Suyama population
were introduced into experimental populations. The method described by
Buzzati-Traverso (1947) was used. The decreasing frequencies of lethal
heterozygotes have been followed up in the course of forty generations.
To determine the frequency at an arbitrarily chosen generation, one
hundred chromosomes sampled from the population were examined, tofind out whether or not they have contained the lethal gene, by the test
cross to the original lethal strain. The results are shown in Figure 1.
J..O

...""
Ii,
,.,
H

o

...~

II

....
~ 0·5
....

.......---=::::: g ~:ig'ri~-..:::::a:=-;;--:.:-: ~ ~ 8

:i

,.,
~

,,:::::::~~-----------Kl'l

2-22

I.":

J.o

Fig. 1.

20

Generation

3°40

Decreasing curves of natural lethal genes in experimental populations.

When the declining curves were compared with the theoretical curve,
which was given by the no-selection coefficient (s=O), these six lethal
genes could be assumed to show heterosis in the heterozygous state inexperimental populations. The two lethal genes among them, SU 3-19
1)This work was supported by Grant RG-7836 from the National Institute of
Health, U.S.A.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

which was allelic to SU 2-1, and SU 3-24 were demonstrated in our previous paper (Oshima and Kitagawa, 1961) to have been maintained for atleast one year in the Suyama population. As described in anotherreport, two alleles of these two genes, respectively, were found repeatedly
among the new lethal genes extracted from the same natural populationin 1961. From this fact, these two lethal genes clearly showed the superiority of heterozygotes in fitness in both experimental and natural populations.

2.

Relative frequency of the second chromosomes bearing deleterious
genes in natural populations of Drosophila melanogaster. 1I(By Chozo OSHIMA, Keiko FuwA and Yoshiko IMAI)

Many flies were collected from natural populations in Suyama and Juriki
Town in Shizuoka Prefecture on September 30th 1961. The second chromosomes of these flies were isolated individually by using the method ofcompletely marked inversion. On the basis of the proportion of wild typeflies to total flies emerged in the F 4 generation, the chromosomes bearing
deleterious and normal genes were classified into four grades, namely nowild type flies: lethal, 0.0-16.7%: semi-lethal, 16.8-25.0%: subvital andmore than 25.1%: normal chromosome. Such experiments have been
repeated every year since 1959. The relative frequencies of chromosomes
belonging to each grade are given in Table 1. No change was detected
Table 1. Frequencies of deleterious and normal second
chromosomes isolated from natural populations
Percentage of wild type flies

Population
Suyama andJuriki (1960)

0(1)
23

I 0-16.7
116.8-25.0125.1-33.3133.4(sl)
(sv)
(n)
(n)
I 3 I 15 I 118 I 25

No. ofchromosomes
tested184

I
I ±1.630.93 I ±8.152.02 I 64.13±
I 13.59± 3.54± 2.53Frequency(%)

12.50± 2.44

Suyama andJuriki (1961)

I 17 I 38 I 212 I 49 I 377
16.18 I 4.51 1 10 . 08 I 56.23 I 13.00 I± 1.90± 1.07± 1.55± 2.56± 1.73Frequency(%)61(1): lethal, (sl): semi-lethal, (sv): subvital, (n): normal.
1) This work was supported by Grant RG-7836 from the National Institute of
Health, U.S.A.

RESEARCHES CARRIED OUT IN 196113

in the relative frequencies of deleterious chromosomes isolated from natural
populations in the same locality in 1959 and 1960 and they were maintained
also in 1961 without significant fluctuation.
The lethal chromosomes have been maintained in the Cy balanced
system in the successive generations. Diallel crosses between new 61
lethal strains and old 23 lethal strains are in progress for detecting any
lethal genes which have been retained in the natural population during
over one year. Four crosses among about four hundred crosses were
found to be allelic. From the results, two lethal genes could be assumed
to be retained for one year in the same natural population and other two
lethal genes, which were demonstrated to persist from 1959 to 1960 by
our experimental results, were observed to be allelic to two new lethal
genes respectively. From this fact, the two lethal genes could be assumed
to be retained for two years in the Suyama population.

3.

The pre-adult viability of heterozygous flies for lethal chromosome 1l(By Chozo OSHIMA, Keiko FUWA and Terumi MUKAI)

Twenty-three lethal chromosomes and twenty-two normal chromosomes
were isolated from a small natural population in 1960. The pre-adultviabilities of their heterozygotes were estimated with Cy-Pm techniquedeviced by B. Wallace (1956). This technique (mating scheme) was convenient to obtain heterozygous flies for two normal chromosomes in random
combination, for one lethal and one normal chromosomes and for two
different lethal chromosomes, and their strain-specific viability could be
estimated by comparing it with that of Cy/Pm flies. The experimental
results are shown in Table 1. The mean relative viability of natural
Table 1.
Genotype

+/+
+/l
l/l

Relative viability of lethal heterozygotes
Line basis

Pooled basis
No. ofcounted flies
127,944
369,129
160,118

I

Relative
viability

No. oflinse

1.3530 ±0.0097
1. 2643**±0. 0052
1. 2768**±0. 0080

231
506
242

I

Relative
viability
1.3677 ±0.0166
1.3217*±0.0125
1.3416 ±0.0181

The viability of Cy/Pm: 1.0000*significant at the 5% level, **significant at the 1% level.
1) This work was supported by Grant RG-7836 from the National Institute of
Health, U.S.A.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

lethal heterozygotes was significantly less than that of normal heterozygotes
on either pooled or line basis, and that of double lethal heterozygotes
within the same populations was significantly less only on the pooledbasis. The mean coefficient of selection (5) for lethal heterozygotes was
0.0336 on the line basis and the individual coefficients were distributed
between +0.1906 and -0.1089. The selection coefficients of 8 lines among
23 lines showed negative values. It means that one third of lethal heterozygotes were not at a disadvantage in pre-adult viability. The relative
viability of double lethal heterozygotes was rather better than that ofsingle lethal heterozygotes. In contrast with this finding, the magnitudeof decrease in the mean viabilities of single and double induced-lethal
heterozygotes, having a relatively homozygous genetic background, wasapproximately proportional to the number of lethal genes per strain.
Thus, it may be assumed that natural selection produces a genic system,
i.e., a co-adaptation system, in which the interaction of genetic backgrounds ordinarily reduces their detrimentality of deleterious genes.

4. Seasonal changes in the frequency of chromosomes bearing alethal gene in natural populations of Drosophila melanogaster(By Sumio MINAMORI,* Makoto SASAKI and Chozo OSHIMA)

A seasonal change in the frequencies of chromosomes bearing a lethal
gene in some Russian populations of D. melanogaster was observed by
Dubinin (1946), but such phenomena have not been detected by Ives (1954)
and Goldschmidt et al. (1955) in natural populations of America and Israel.
The effective size of natural populations in several Japanese localities wasestimated to be smaller than that of American populations by Oshima andKitagawa (1961) and Minamori and Azuma (1962). A number of second
chromosomes were extracted by using the method of marked inversion
from natural populations in Hiroshima locality in early summer andautumn of 1961. These chromosomes were classified into four grades;
lethal, semi-lethal, subvital and normal chromosomes by using ratios of
non-Curly and Curly flies in the Fa generation. In these small populations,
it was examined whether the frequencies of lethal genes would be increased or not at the last seasonal expansion of the population. The
results of testing the frequencies of lethal and semi-lethal second chromosomes in flies collected in late July and early October are presented in
the following table. From the results shown in Table 1, the frequencies* Assistant Professor of Hiroshima University and temporary researcher uponrecommendation of the Ministry of Education for one year from October, 1961.

15

RESEARCHES CARRIED OUT IN 1961
Table I. Frequencies of lethal and semi-lethal chromosomes extracted fromthree natural populations in Hiroshima locality in July and October

Difference
Frequency of
I betweenMonth. of
No. of tested
lethal and semiI collectIOn
Ichromosomes I lethal chromosomes collectionstwo

Population
Midori St.
Tera St.
Shiwa Town

Total

July
OctoberJuly
OctoberJuly
October

222
155
216
169
236
200

14.0
20.6
12.5
17.2
5.5
7.0

0.20<P<0.30

July
October

674
524

10.5
14.4

I 0.025<P<0.05

0.05<P<0.10

0.50<P<0.70

of deleterious chromosomes in flies collected in October were higher thanthose in July in all three populations and the difference between the pooled
frequencies was significant. The accumulation of deleterious genes inthese populations was found at the last expansion.

5. Dieldrin resistance in Drosophila pseudoobscura ll(By Chozo OSHIMA)In natural populations of D. pseudoobscura in America, a geographicalcline from east to west has been observed in the relative frequencies of
their chromosomal arrangements. PP(Pikes Peak) chromosome had beenrarely found in California before 1947, but its occurrence has rapidly increased to about 10 per cent in 1957, and on the other hand CH (Chiricahua)chromosome has decreased from 20 per cent to 4 per cent during the ten
years. Among several environmental factors that could cause such achange in the genetic structures of the California populations, the effectof insecticides was proposed by Professor Dobzhansky.
Ten homozygous strains for each chromosomal arrangement, ST(Standard), AR(Arrowhead), CH(Chiricahua), PP(Pikes Peak) and TL(TreeLine), which were extracted from the natural population in Mt. San
Jacinto, California by Dobzhansky in 1959, were used in this experiment.
Ten adult flies were exposed in small vials for 1 hour to 0.8, 0.4, 0.2,
0.1 and 0.05 per cent Dieldrin test papers, prepared by WHO and their
1) This work was supported by the Scientific Research Grant from the Ministryof Education.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

mortality was established after 24 hours. Such test was replicated tentimes for each dose with male and female flies separately. The experimental materials were homozygous flies, which were the offspring fromcrosses between the same chromosomal strains, and heterozygous flies,
which were hybrids between two different chromosomal strains. The
mortalities were transformed into arc-sine units and analyzed statistically.
From the results of analysis of variance, the following conclusion was
obtained; 1. there is no significant difference between the levels of resistance of homozygous and heterozygous flies for any chromosomal arrangement, 2. significant differences were observed between different homozygous or heterozygous chromosomal strains, 3. females were significantly
more resistant than males, 4. the differences between mortalities at different doses were significant, 5. the interactions between dose and strain,
and sex and dose were significant for both homozygous and heterozygous
strains.
From these results, it follows that the increase of relative frequency
of PP chromosome in California could not be caused by the effect of insecticides such as Dieldrin and might be attributed to the appearance of
co-adapted gene complexes including the PP chromosome in the genepool.

6. Spontaneous polygenic mutation rate and the genetic structureof natural populations in D. melanogaster(Terumi MUKAI and Sadao CHiGUSA)

According to KIMURA'S (1959) considerations based upon the classicalhypothesis which assumes that most of the genetic load is mutational,
the following conclusion may be drawn. Suppose that the number of locicontrolling the fitness in D. melanogaster is 5000, and the mutation ratein them is 5xlO-4 /locus/generation (ca. 50 times as large as the spontaneous mutation rate of major genes), then the survival rate becomes e- 5 •This indicates that only 7 eggs out of 1000 become adults. This result
is inconsistent with the actual situation in natural populations. Consequently, a proof that the spontaneous mutation rate of polygenes controlling the fitness is extremely high would be unfavorable to the classicalhypothesis. Therefore, an experiment was started for the estimation of
the mutation rate of polygenes controlling the viability, which is one of
the major components of fitness. The experiment is still in progress but
the result at hand will be reported here.

RESEARCHES CARRIED OUT IN 1961

17

A single second chromosome was sampled from an isogenic line (BURDICK'S
W160) and multiplied to 104 by using Cy(Pm stock, whose genetic background had been completely substituted by W160, and 104 lines were

established. In each line, the second chromosome has been maintained
through a single male in the cross of Cy(Pm(!? )XCY(+i( 0) (i=l, 2, ...,
104) for the purpose of accumulating spontaneous mutations affecting theviability. In Generation 10, the homozygous viabilities of these lines
were tested by the use of Cy technique with 4 or 5 replications for eachline on the basis of counting about 150,000 flies. Among the 104 lines,
2 lethal and 3 subvital lines in homozygous condition were detected.
These were clearly distinguishable from the other quasi-normal lines, on
the basis of the distribution pattern of their viabilities.
The degree of differentiation due to polygenic mutations among 98
quasi-normal lines (1 line is missing) was tested in homozygous conditionby the aid of the analysis of variance. The result is presented in Table
1. Although it was impossible to find significant differences in theviabilities of the 98 lines by this technique, the probability value wasTable 1.

Analysis of variance with respect to the viabilities of 98 quasi.
normal second chromosomes in homozygous condition

Source

S.S.

d.f.

M.S.

F

Between lines
Error

0.13550.361297328

0.001397
0.001101

P~0.06

Total

0.4967425

1.27

very close to 0.05. Therefore a further analysis was tried.
X2 test was conducted to compare in respect to viability the distribution pattern of homozygously quasi-normal lines in Generation 0 withthat in Generation 10. In this test, it was assumed that the distributionin Generation 0 was normal with the mean of the mid-point between thehighest and the lowest lines in Generation 10 [=(0.3633+0.2814)(2], andthe variance (11 2) which was estimated from the error variance (a e 2) ofTable 1 by the following appropriate procedure;2_
2 X harmonic mean of fly number per one replication in one line
11 -l1eharmonic mean of fly number per one line as a whole
The value of X2 thus obtained, 26.72 for d.f. =4, is highly significant,or the difference between the two generation is statistically significant.
This indicates that there was a heterogeneity in the homozygous viabilities
among 98 quasi-normal lines, and this heterogeneity was caused by many
mutations having small effects on viability.
A

A

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

7.

Two cases of heterosis associated with the recessive genes sepia(se) and expanded wing (ew) in Drosophila melanogaster(Sadao CHIGUSA and Terumi MUKAI)

Two cases of single gene heterosis due to the recessive genes sepia
and expanded wing have been tested. The latter was discovered in MUKAI
and BURDICK'S (1959) Population 1 and located at about 3-49 ±.
Four artificial population (Pop. A-I, Pop. A-2 for se and Pop. B-1, Pop.
B-2 for ew) were established, and the gene frequencies of Pop. (A, B)-land Pop. (A, B)-2 were 0.5 and 0.1, respectively. The homozygosities of
the genetic backgrounds in Pop. A's are extremely low, while those ofPop. B's are high. The populations have been maintained for 13-23 generations by the so-called Pearl's method. The frequencies of recessive
homozygotes were estimated almost in every generation. Four populations
are reaching equilibria where the frequencies of recessive homozygotes
are about 0.07 in Pop. A's and about 0.05 in Pop. B's. These phenomena
are supposed to have been caused by the superiority of the heterozygote
over either homozygote.
The components of the fitnesses of heterozygotes were estimated andthe superiority of heterozygotes seems to be due to better female fecundities
of se/+ and ew/+ than those of the wild-type homozygotes, respectively.
The relative female fecundities of se/+ and ew/+ to those of the wildtypes were about 1.38 and 1.31, respectively. The experiments are stillin progress.

8.

Transfer of the "sex-ratio" condition fromD. willistoni to other species of Drosophila(By Bungo SAKAGUCHI and Donald F. POULSON*)The maternally transmitted abnormal sex-ratio condition in D. willistoni
was demonstrated by Malogolowkin, Poulson and Wright (1959) to betransferable to previously normal strains of this species. Furthermore
Malogolowkin et al. (1960, 1961) demonstrated that the "sex-ratio" (SR)condition of D. willistoni can be transferred to other species, such as D.
equinoxialis and D. nebulosa.
This has been accomplished for the female recipients of two otherspecies, D. melanogaster and D. pseudoobscura. In the case of D. melanogaster two wild type strains, Oregon-R inbred and Sevelen inbred, a* Osborn Zoological Laboratory, Yale University, New Haven, Conn. (Supported
by grant 6017 from N.S.F.).

RESEARCHES CARRIED OUT IN 1961

19

triploid strain carrying attached-X chromosomes and a diploid strain withthe same attached-X chromosomes (y2scw a ec/ Y!? x y 31d sc 8 dmB 0) were used
as recipients females. Hemolymph (O.05~O.08,ul) from SR females of D.
willistoni was injected by micropipette into the abdomens of I-to 2-day
old virgin females of the recipient strains. The injected females weremated individually to males from their own strains and transferred to
new culture medium every two days.
Unisexual progenies began to appear in many instances even in theearly broods of each experimental series in the case of D. melanogaster.
From these, lines of SR have been established by mating with males
from the corresponding normal strains. The maintenance of SR condition in some of these lines proved to be much more stable than in others.
One of the best is a line derived from the attached-X strain demonstratingthat the Y chromosome is not of primary importance in the lethal disturbances associated with the SR agent. That the persistance of the SR
agent in D. melanogaster is dependent on the genotype of the flies isclearly demonstrated; for example, transfers to an inbred strain of OregonR took well and have shown a high level of stability in subsequent generations, while transfers into an inbred strain from Sevelen are much lessstable suggesting a high degree of host resistance. The same results
were obtained when the chromosomes of the attached-X strain and OregonR or Sevelen strain were exchanged.
The persistance of the SR agent of D. willistoni in D. pseudoobscura
has shown a high level of stability in subsequent generations.
It is indicated from this and other results (in this Annual Report) thatthe genotypes both of host and SR agent are of prime importance in theestablishment, stabilization, and persistence of SR in the hosts.

9. Comparative analysis of transmissive efficiency and pathologicaleffects of "sex-ratio" agents of different origin in Drosophila(By Bungo SAKAGUCHI and Donald F. POULSON*)

A maternally transmitted condition known as "sex-ratio" (SR) has been
demonstrated in a number of species from different geographical regionsof Drosophila, in particular D. bifasciata, D. equinoxialis, D. nebulosa, D.
paulistorum, D. prosaltans, and D. willistoni.
That the SR condition may be affected and even cured by elevatedtemperatures was first shown by Magni (1954) for the SR of D. bifasciata* Osborn Zoological Laboratory, Yale University, New Haven, Conn. (Supported
by grant 6017 from N.S.F.).

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

and by Malogolowkin (1959) for the SR of D. equinoxialis. However, theSR of D. willistoni and D. paulistorum showed no such temperature sensitivity and none was apparent in the SR of D. nebulosa. Thus differences among SR agents of different origin are suggested, although thehost species may also be involved in the temperature response.
In the present report the differences between the SR agents of different
origin have been examined from the view points of transmissive efficiency
into subsequent generations of the host flies and pathological effects of
the agents. The SR strains of the four species, D. bifasciata, D. equinoxialis, D. nebulosa and D. willistoni, were used as the donors of SR agent
and the inbred strain, Oregon-R, of D. melanogaster was used as a recipient
for testing.
The SR agent of D. nebulosa is readily transferred and incubation fromthe time of injection to the manifestation of the SR agent is very shortrequiring only a few days. The killing effect of the agent against malezygotes is remarkably strong and has also proved to be very stable andpersistent in subsequent generations in the females of the new host. The
SR agent of D. willistoni is readily transferred having the same incubation time as the agent of D. nebulosa but is somewhat less stable andpersistent in subsequent generations. But the agent of D. equinoxialis
is much more difficult to transfer and shows very considerable instability
and is very hard to maintain in D. melanogaster hosts. Furthermore
other effects produced by the SR agents of the three species have demonstrated that there are differences in the patterns of developmental
disturbances produced by each of them when transferred to new hosts.
In the case of the SR agent of D. bifasciata there has so far been no
success in transferring the condition within the same species or to D.
melanogaster. Poulson and Sakaguchi (1960,1961) were able to show that
the SR agents are small spirochetes, presumably treponemata, in each of
the three species which they studied, D. equinoxialis, D. nebulosa and D.
willistoni. In each case the transfer of the SR condition into a different
strain of the same or of a different species was correlated with the presence of transferred treponemata in the hemolymph of SR females of therecipient strain and its descendants. On the other hand in the case of
the SR agent of D. bifasciata it has not so far been possible to demonstrate the presence of typical spirochetes in the hemolymph of SR females.
This suggests a wholly intra-cellular SR agent, highly integrated withthe developmental system of the host.
These facts indicate differences among the SR agents of different speciesof Drosophila and from different geographical regions. It may then be
possible to make clear the origin of SR agent and the significance of its
biological evolution from a more detailed analysis of the differences among

RESEARCHES CARRIED OUT IN 1961

21

the SR agents derived from various species of Drosophila collected fromdifferent geographical regions.

10. Lethality and low viability induced by the segregation distorter
locus (symbol SD) in Drosophila melanogaster(By Yuichiro HIRAIZUMI)In a heterozygous SD male, SD breaks its partner chromosome in somestage of meiosis and thus more than 50% (usually 95% or more) SD-bearing
chromosomes are transmitted to the next generation. Here the question
arises whether the SD+-bearing chromosomes found in the F 1 -generation
are 1) those which were not affected by SD action or 2) those which
recovered from the break. If 2) is the case, then we may expect somechanges, perhaps viability reduction, in the SD+-bearing chromosomesfrom the heterozygous SD males. Accordingly, SDjen bw (and SD+jen bw
as a control) males were crossed to en bw/In(2L) Cy en bw females. The en
bw chromosomes in the heterozygous SD and SD+ males in P-generation
were derived from a single lethal-free chromosome and the remaining
genetic background had been made uniform before experiments. From
the F 1 of these matings en bwjln(2L) Cy en bw males were chosen to crossindividually to en bwjln(2LR) Cy females, and the F s en bwjln(2LR) Cy sibs
from each F 1 mating were mated to test the homozygote's viabilities ofthe en bw chromosomes in comparison with their Cy heterozygotes. For
the significance test the observed percentage of the en bw homozygotes(= r) found in the F 3 in each culture vial was transformed according to
the relation r=sinsR. The results are summarized in Table 1. Figures
Table 1.

R

Exp. setNo.

No. ofNo. of
I OriginalSDnant SD I cultures I SD+
I cultures
I Recombi-

1

31.16
(26.8)
31.69(27.6)
31.80(27.8)

2
3
T ot a I

31.62±0.341(27.5)

1

8(+ 1 lethal)
12
16

31.49
(27.3)
32.50(28.9)
32.42(28.8)
32.30±0.281
1 (28.6)20(+1 lethal)38
52(+1 lethal)

34.34(31.8)
33.52(30.5)
33.38(30.3)

No. ofcultures1837
3133.64±0.271
1 (30.7)

in brackets indicate the percentages of en bw homozygotes corresponding
to each R (=average of R) value. The lethal-bearing en bw chromosomes(indicated as +1 lethal etc.) were excluded from computing R. The

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

number of chromosomes examined was very small but it is worth notingthat 3 lethals were found among 148 chromosomes in the experimental
sets while nqne appeared among 86 chromosomes in the control sets.
Each experimental set was made at a different time, but in each set thecn bw chromosomes from the SDlcn bw male showed, on the average,
reduced viabilities (p<O.OI). It is interesting to note that the original
SD lines (=SD-72 and SD-5; strong SD) caused more viability reduction
than the recombinant SD (weak SD) lines did, although the difference
was not statistically significant. Results of mapping tests suggested
that all of the SD-induced lethals were located near the SD+ locus(within 5% crossover units around SD+).
The detailed mechanism for lethality and low viability induction is
not fully understood, but a small deletion accompanied by the breakagereunion event could be responsible.

11.

Cytological observation of the SD region in the salivary glandchromosome of Drosophila melanogaster(By Yasuko TONOMURA (TOYOFUKU»

It has been reported by Sandler and Hiraizumi that the segregation
distorter of D. melanogaster is a complex locus (symbol SD) which locatesclose to the centromeric heterochromatin of chromosome II (probably inthe right arm). The subject of the present report is a study of structureof the SD region by means of the salivary gland chromosome analysis.
(1) Inversions outside the SD region.
Sandler et al. (1959) reported that there were two types of the original
SD-bearing chromosome, i.e., 1) singly inverted (=SD-72) and 2) doubly
inverted (=SD-5), but they didn't indicate the positions of these inversions.
The present investigation shows that the inversion in SD-72 includes
sections from 51E to 55F in the right arm of chromosome II. In addition,SD-5 line carries one more inversion covering sections from 46A to 48Fin the same chromosome.
(2) Structural abnormality at the SD region.
In addition to the inversion mentioned under 1), the SD-72 line carries
a small aberration involving sections from 40A(?) (left arm) to 42A(rightarm) in chromosome II. On the other hand no such aberration was observed in the SD-5 line. Since both SD-72 and SD-5 lines exhibit strong
SD action, it is evident that this aberration is independent of SD action.
The nature of the aberration has not yet been fully understood, but it issuggested that this is either 1) a small pericentric inversion or 2) a dupli-

RESEARCHES CARRIED OUT IN 196123

cation. Since the aberrant segment is separated into two complementary
recombinants by crossing over (Sandler and Hiraizumi, 1960), the second
possibility, duplication, seems to be more likely. If this aberration isan inversion, then the "recombination" phenomenon within this region
is not due to the usual crossing over, but has to involve some othercomplex mechanisms. Further cytological observations are in progress.

12. Migratory activity of Drosophila as a genetic character(By Takashi NARISE and Kan-Ichi SAKAI)

A brief account of conclusions drawn from our genetic study on the
migratory activity of Drosophila melanogaster is given as follows:
1) Genetic effect of chromosome substitution.
The second and third chromosomes of a weak migratory Samarkand
strain were substituted by the corresponding chromosomes of IG-28 or
IG-25. IG-28 is characteristic of high mass-migratory activity while IG-25
is high in random-migratory activity.
The genetic effect of the second and third chromosomes of IG strains
on mass-migratory activity of Samarkand strain was quite apparent. The
third chromosome was genetically more effective than the second. Randommigratory activity of Samarkand was also increased either by the secondor the third chromosome of IG strain, the third chromosome being again
more effective than the second.
2) Observation of migratory activity in F 1 hybrids.
A number of strains differing in respect of mass- or random-migratory
activity were reciprocally hybridized.
i) Mass-migratory activity of F 1 hybrids was in most cases intermediate
between the two parents, though in some specific hybrids high heterotic
effect was observed.
ii) Random-migratory activity was also found to show a partial dominance toward the side of the active parent. A remarkable heterotic effect was observed in some specific combinations of strains. Reciprocal
difference was detected in all cross-combinations.
Thus, it has been concluded that (1) tendency to migration, either
random or mass, is controlled by genes of rather polygenic nature, (2)the third chromosome contained more effective genes than the secondand (3) a reciprocal difference was observed in the case of random-migration.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

13. Stimulating effect of a foreign genotype on migration
in Drosophila(By Takashi NARISE and Kan-ichi SAKAI)

Flies of the mutant vg-strain of Drosophila melanogaster are very slow
in migratory activities when they are in crowds by themselves. The
present report deals with the discovery of a stimulating effect of wildflies, introduced into a population of vg-flies, on their migrating rate.
In this experiment, a mixture of vg and wild flies was used in proportion of 0: 100, 10: 90, 20: 80, 30: 70, 40: 60, 50: 50, 60: 40, 70: 30, 80: 20,
90 : 10 and 100: O. The total number of flies in each mixture was madeconstant at 100. Migratory activity was measured in terms of the number of migrated flies from the original tube to the surrounding tubes.
For the details about the method of experiment, the reader may refer toprevious issues of this Annual Report. From this experiment, it was
found that an admixture even of 10% of wild flies caused a sudden increase in migratory activity of otherwise inactive vg flies.
The migration of wild flies which is naturally higher than that of vg
flies slightly increased in every mixture. The reason for the unexpectedchange in the migration rate of flies is not yet known.

14. Pterine metabolism* in Drosophila melanogaster(By Toshifumi TAIRA)

On the basis of our recent data, the biochemical relationships among
pteridines found in D. melanogaster are summarized in the following
scheme:Reductase+ TPNH

Intramolecullarhydrogen-transfer,j,Di~ydro-.
t sepIapterm(pa~i~lIY) I +0

Sepiapterin (SP) - - >,j,-2H- - > [DihYdrobiopterinl~ Biopterin (BP)

+2H

2

,j,

2-amino-4-hydroxypteridine (AHP) - - > Isoxanthopterin (IXP)t
Dehydrogenase + DPN

At the present time, it is obvious, from our previously published reports, that the activities of reductase and dehydrogenase of these path-* "Tetrahydrosepiapterin" expressed in our previous papers was corrected
into "Dihydrosepiapterin".

25

RESEARCHES CARRIED OUT IN 1961

ways are genetically controlled. The reductase step is restricted by
Hn r - 3 gene and the dehydrogenase step is suppressed by ry and ml-l
genes. Isoxanthopterin in the above scheme seems to be the final product of pteridine metabolism, but biopterin is assumed to be an importantintermediate rather than a final metabolite. The latter opinion is supported by two data, namely 1) the evidence that biopterin (possibly aderivative) is firstly revealed in the early drosophila embryo and 2) theevidence obtained by Ziegler and Nathan (1961) who found that a biopterin derivative (possibly riboside) found in Drosophila is converted into
a sepiapterin-like substance through a tetrahydrobiopterin derivative by
the supernatant extracted from Critidia, which requires biopterin as a
growth factor. Therefore, they have suggested that the tetrahydrobiopterin derivative might be a precursor of sepiapterin. However, we havefound that the supernatant prepared from Drosophila can not catalyze
the reduction of free biopterin. Such contradictory results might becaused by the difference of the substrates and/or the experimental organisms in these two experiments, and this contradiction may be explainedby clarifying whether or not the biopterin derivative is actually produced
from a mixture of free biopterin and a monosaccharide.
The biosynthesis of drosopterin (DP) is our main project. As mentioned
in our previous reports, the biosynthetic pathway of drosopterin is completely inhibited by se gene and also partially inhibited by d, Hnr-l and
Hn r - 3 genes, as summarized in Table 1.
Table 1.

Relative contents of pteridines in the eyes ofD. melanogaster

Strains

SP

BP

AHP

IXP

DP

Ore-R
v and enseel
Hn r - 1
Hn r - 3

±
±

±
±

±

+++
+++

+++
++
+
++

+++
+++
++
+++

++'
+++
++

±
±
±

±

++
++
+

~~

±

±

-

±

From this table and the given above scheme, it is assumed that atetrahydro-form of pteridine might be the precursor of drosopterin.
However, the chemical structure of drosopterin is not yet clarified.
Therefore, the determination of the chemical structure of drosopterin
is the immediate object of our study with the close cooperation of Dr.
Matsuura (Dept. Chem., Nagoya Univ.).

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

15.

The fourth case of translocation between the W chromosome
and an autosome in the silkworm(By Kimiharu ONIMARU and Yataro TAZIMA)

Three translocations involving the W chromosome have been known inthe silkworm, i.e., W·II (Tazima, 1941), W·III (Hasimoto, 1948) and W·X(Tazima et al., 1951). In all these cases a translocated piece of theautosome behaves as if it were a part of the W chromosome in its
transmission.
In the course of our study of spontaneous mutations, a new translocation was discovered between Wand the fifth chromosome that bears the+re locus. The most characteristic feature of this translocation is thatthe translocated piece behaves as the fifth chromosome in contrast withthe hitherto reported three cases.
Among 863,872 observed heterozygotes both for pe and re, + +Ipe re,87 pe individuals were found as presumable mutants for egg color. They
were raised and 35 attained to maturity, among them 16 !? !? and 19 0 o.
In order to discriminate the mutation-bearing chromosome from its homologue (pe re), these pe females were crossed to +re males. In F 1 segregation occurred as expected with respect to egg color at the ratio of 1
normal: 1 red. After discarding the red eggs, only normal eggs wereleft in each batch for further raising. Among 8 mutant lines thus raised
7 showed the normal sex-ratio of 1: 1, whereas in the remaining one allindividuals were female. This clearly suggested that translocation musthave occurred in the ancestor of this line.
Regarding the occurrence of translocation two possibilities may be considered: (A) translocation of a small piece of the fifth chromosome to W
and (B) translocation of a small piece of W that contains the female determining gene to the fifth chromosome. The breeding experiments withthis translocated chromosome led us to assume that the latter possibility
is more plausible than the former. If this proves true, the importantconculsion might be drawn that the female determining gene or genes
are located in a very limited segment of the W chromosome.

16. Studies on the manifestation mechanism of dilutelemon gene in the silkworm(By Mitsuo TSUJITA)

A silkworm strain showing dilute yellow larval color was derived fromthe F 2 generation of the cross between YD 4 yellow lethal (temll +) andRESEARCHES CARRIED OUT IN 1961

27

lemon (tern/fern) individuals. The results of mating this mutant (d-fem)with another dilute lemon mutant which was reported by Chikushi (1961)
have proved that these two genes are allelic and that this character isdue to the presence of gene d-fem on chromosome II in addition to lemon
gene on chromosome III. Thus, the genetic constitution of the lemon
strain was confirmed as fern/fern; +d-lem/ +d-lem and that of dilute lemon
strain as fern/fern; d-fem/d-fem. For the purpose of studying the manifestation mechanism of dilute lemon gene the present experiments werecarried out.
1.

Measuring of the amount of pteridines

For the separation of yellow pigment and isoxanthopterin two-dimensional paper chromatography was used. Full grown larvae in the fifth
instar of normal, lemon and dilute lemon were used as materials. The
hypodermis taken from 5 larvae of each strain was dried and groundinto powder. 100 mg of the powder and 0.7 ml of 0.5 N perchloric acid
were mixed, and heated for two minutes on a water bath at 80 a C. Aftercentrifugation, 0.3 ml of each supernatant was placed on filter paper (Toyoroshi #51 40x40 em) and chromatographed by the solvent of a mixture ofn-propanol and 1% aqueous ammonia (2: 1) for the first and 5% aqueous
acetic acid for the second dimension. Each separated pteridine was eluted
with 5% sodium bicarbonate solution. The relative amounts of pteridinesof each strain were estimated by the intensity of fluorescence. The
standard fluorescence was determined by a solution of 0.5 g. of synthesized isoxanthopterin in 1 ml of 0.5% sodium bicarbonate solution. As
the yellow pigment is very unstable in alkali solution, the amount of
this substance showing yellow fluorescence was represented by the quantity of its photo-decomposed pteridine, 2-amino-4-hydroxypteridine-6-carboxylic acid, showing blue fluorescence. Measuring of each sample wasrepeated 3 times.
i)Amount of yellow pigment

The amount of yellow pigment extracted from lemon and dilute lemon
larvae in full grown stage of 5th instar is given in the following table.

pg/lOO mg dry weight
Genotype

lem/lem; +d-lem / +d-lem

13.5 ± 1.0

lem/lem; d-lem/d-lem

4.5 ± 0.5

The amount of yellow pigment obtained from lemon larvae is about
three times larger than that from dilute lemon larvae.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12ii) Amount of isoxanthopterin
The relative amount of isoxanthopterin extracted from normal, lemonand dilute lemon larvae in full grown stage of 5th instar is shown in thefollowing table.

~mPles

1

2

3

4

5

50
15
13

52
16
12

51
17
10

49
14
12

51
15
10

Total Mean

Genotype+/+
lem/lem; +/+lem/lem; d-lem/d-lem

253
77
57

50.6
15.4
11.4

flg/1OO mg
dryweight16±2.55±0.5
4±0.5

The amount of isoxanthopterin obtained from the lemon larvae as well
as from the dilute lemon larvae is far smaller than that obtained fromthe normals.
2. Injection of AHP into the body cavity of 5th instar larvae
E4Io

Value.

l~m/lem; d.-lem/ d.- tern

)80

"0
.100

.09
~8

.0'1

.06
05

.0

01

10

mf~utes

~o

40

1>0

Fig. 1. Enzyme activity of adipose tissue of normal
and dilute lemon larvae.

A small amount (0.2 cc per individual) of 2-amino-4-hydroxypteridine
CARP) was injected into the body cavity of each of 30 normal and 30
dilute lemon larvae on the 3rd day of 5th instar and the amount of
isoxanthopterin produced in their hypodermis was periodically measured

RESEARCHES CARRIED OUT IN 1961

29

after 1, 2, 4, 8 and 24 hrs. However, no significant change in the amountof isoxanthopterin could be observed in the larvae of both strains. It
seems, therefore, that the permeability of the hypodermal cells of dilutelemon larvae to AHP or isoxanthopterin is not different from that of thenormals.
3. The activity of pteridine reductase
Various tissues taken from larvae, pupae and imagoes of normal, lemon,and dilute lemon were used as materials. 0.6-1.0 g of fresh tissue was£':4-10 Value

180

lerrv'lem; d-lem/d-lel11

./70
16

./'1()
./30
120

+/+

./10
~.o

010
08
07
.06
0';0
01()
.030
020
.010

10

20

30

'1-0

minutesFig, 2. Enzyme activity of integument ot normal anddilute lemon larvae.

mixed with a phosphate buffer solution three times the tissue weight,
homogenized under ice cooling and centrifuged at 12,000-18,000 r.p.m. according to the nature of the tissue cells. The supernatant was used asa crude enzyme solution. The yellow pigment purified from the lemon
larvae having genotype lem/lem! and purified sepiapterin from the sepia
strain of Drosophila melanogaster were used as substrate. The activityof pterine reductase of the enzyme solution mentioned above was measured
according to the procedure described in my previous paper (Tsujita 1961).
No enzyme activity could be detected in several tissues of the larvae,
pupae and adults of dilute lemon. But, adipose tissue and integument
exhibited a weak or very weak enzyme activity. Some of the experi-

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

mental results are shown in Figs. 1. and 2. Thus, no significant difference
has been found in the enzyme activity between lemon and dilute lemon
strains.
4. Function of d-lem geneIn the dilute lemon larvae the activity of pterine reductase acting on
the step from yellow pigment (dihydropterin) to tetrahydropterin is very
weak just like that of lemon larvae. In spite of this fact the amount ofyellow pigment accumulated in the hypodermis of the former is verysmall, amounting to about one third of that of the latter. To explain
this small accumulation of yellow pigment in dilute lemon larvae two
hypotheses can be assumed. One of them is that owing to the defectivefunction of d-lem gene the amount of the precursor of yellow pigment
is small. The other hypothesis is that though the yellow pigment isnormally produced, a large part of it is lost from the hypodermal cells
owing to the defective system caused by the d-lem gene which keeps
this compound within the cells. If the first hypothesis were correct, theproduction of isoxanthopterin in the hypodermal cells of the larvae withthe genotype +l,m/+l,m; d-lem/d-lem should be far smaller than that in
the larvae with the genotype +lem/+l,m; +d-l,m/+d-lem. To test this possibility a cross between normal and dilute lemon strain was carried out
in order to find out whether or not segregation occurs as to the amountof isoxanthopterin among the F 2 individuals. However we could not getclear-cut results. It is inferred from the experimental results so far obtained that the second hypothesis is more reasonable than the first one.
Therefore, we are now planning to study the exact mechanism according
to the former hypothesis.

17. Amount of isoxanthopterin in the larvae of the silkworm(By Mitsuo TSUJITA)

Glassman and Mitchell (1959) who studied a mutant of Drosophila
deficient in xanthine dehydrogenase suggested that the ma-l+/ma-l+
females are passing a substance which is not produced in ma-l flies
but can be transmitted by this mutant to the progeny with the egg
plasma. They thought that this substance may be a precursor of xanthine
dehydrogenase. We succeeded in showing a clear-cut maternal effect of+lem on pterine reductase in the silkworm. In order to obtain information on the maternal effect of the substances pertaining to pteridine metabolism, studies of the amount of isoxanthopterin, an end product ofpteridine metabolism, produced by single larvae, were carried out.

RESEARCHES CARRIED OUT IN 1961

31

Materials and methods: Normal larvae (Daizo, P zz ) and larvae of F 1
and F z of a hybrid between A strain producing a large amount of isoxanthopterin and B strain producing a small amount of this compound
were used as materials. To measure the amount of isoxanthopterin contained in the hypodermis of single individuals, each of the larvae wascut open from the dorsal side and almost all of the inner organs were
removed such as digestive tube, Ma1pighian tubules, adipose tissue, silk
glands and others.
The remaining hypodermal tissue was spread on a filter paper anddried. After drying the weight of each individual hypodermis was determined. Each larval skin was cut into two or three pieces, which wereplaced in a small test tube with 2 cc distilled water and heated for 20
minutes on a water bath at the temperature of 9So-100oe in order toextract isoxanthopterin. The extraction was repeated twice and the total
amount of isoxanthopterin in the extracted solution was measured by
Beckman's spectrophotometer. This procedure is relatively simple sothat a large number of larvae can be examined for the amount of isoxanthopterin.
Experimental results: 1. Difference of the amount of isoxanthopterin
among races or strains
It was found that the amount of isoxanthopterin varied from race to raceor from strain to strain. An example is shown in the following table.
Table 1.

Amount of isoxanthopterin in the hypodermis of a full grown larva~
StrainsDaizo
P zz
S-strain

Weight of
hypodermis(average)mg
45.9±7.052.5±6.5
48.6±4.3

Relative amount of R. amount of IXP
isoxanthopterin(average)
Weight of
hypodermis (a verage)
50~6r
76.6±15.565.7±12.0
101.6± 7.1

1.7±O.4
1.3±O.2
2.1±O.3

2. Change in the amount of isoxanthopterin in development stages
Using P zz and a hybrid the amount of isoxanthopterin contained in eachlarva was determined in the course of development from the third dayof the third instar to the end of the fifth instar. There was variationin the size of larvae at the same stage, so that the weight of hypodermis
of each individual was ascertained and the ratio isoxanthopterin amount/
hypodermis weight was calculated. The experimental results are shownin Fig. 1.
Fig. 1 shows that the absolute amount of isoxanthopterin increases day
by day with the development from the beginning to the end of each of
the 4th and 5th instars, but decreases to some extent during the 4th

32

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,

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

1

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moulting stage and increases again markedly immediately after moulting.
Although the weight of the hypodermis is smallest at the beginning of

RESEARCHES CARRIED OUT IN 196133

the fourth and fifth instars it gradually increases with the development
and reaches the maximum at the end of these instars, and then it slightly decreases immediately after the fourth moulting. The ratio isoxanthopterin amount/hypodermis weight in single individuals is highest at the
beginning of the fourth and fifth instars and gradually decreases withthe development and attains the minimum at the end of these instars.
It is concluded from the experimental results so far obtained that theappropriate stage for the comparison of races or strains, or for surveying
segregation of F z larvae in the cross between A and B strains withrespect to isoxanthopterin amount and its relation to hypodermis weight,
is the beginning and middle stages of the fourth or fifth instar.
2. Results of the reciprocal crosses between A and B
Reciprocal crosses were made between A strain producing large amounts
of isoxanthopterin and B strain producing small amounts of this compound. Three batches of each of the reciprocal crosses were reared andusing 30 larvae of the fifth instar in each batch the amount of isoxanthopterin was determined. It was observed that the mean value of isoxanthopterin amount and its ratio to hypodermis weight in F 1 larvae,
when A was the female parent, tended to be larger than in the reciprocal cross. Furthermore, the same tendency was confirmed for the larvaeof the F z generation of the same crosses. However, whether or not
cytoplasmic effects are involved is not clear. The study will be continued.

18. Pteridines in the compound eyes of the silkworm moth,Bombyx mori
(By Mitsuo TSUJITA)Five strains of the silkworm with the following genetic constitutions
were used as materials for the detection of pteridines in the compoundeyes of the moths.
Materials/

Genotype134+lem/+lem;+d-Iem/+d-lem ;+Wl/+Wl
lem/lem;+d-lem/+d-l em ;+W , /+Wllem/lem;d-lem/d-lem; +Wl/ +Wllem/lem; +d-Iem / +d-lem ;W';Wl5

lem/lem; +d-Iem / +d-Iem ;W2/W2

2

I

Color ofcompound eyes

egg color

black
black
black
yellowlight reddish
brown

black
black
black
white
light reddish
white

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Two-dimensional paper-chromatography was used for the separation ofseveral pteridines found in the compound eyes of Bombyx mori. The
compound eyes of 70 moths of each strain ~ere removed from the heads
and homogenized with 0.5 ml of 30% acidic ethanol (pH 2.0) and heated
for two minutes on a water bath at 80 a C. After centrifugation, 0.05 mlof each supernatant was put on filter paper (Toyo-roshi #51, 40x40 cm)
and chromatographed by the solvent of a mixture of n-propanol and 1%
aqueous ammonia (2: 1) for the first and 5% aqueous acetic acid for thesecond dimension. The experimental results are as follows:(1) Normaf compound eyes (+lem/+lem; +d-lem/+d-l,m; +w1/+"'1). Several spots were separated and some of them identified as isoxanthopterin,
biopterin and as xanthopterin-like substance. Besides, a large greenishblue spot and a small bluish green spot were observed.
(2) Compound eyes of the moths with the genotyPe fern/fern; +d-lem/+<1 lem; +w1/+w1. Spots of isoxanthopterin, biopterin and xanthopterinlike substance were observed. A clear-cut purplish-blue spot was observed near the spot of xanthopterin-like substance. This may be a characteristic of the fern/fern strain lacking the gene for white egg color.
(3) The compound eyes of the moths with the genotype fern/fern; dfem/d-fem. Separation of isoxanthopterin, biopterin and xanthopterin-

like substance was made. A purplish-blue spot, smaller than that foundin material (2), was observed.
( 4 ) Yellow cofored compound eyes of the moths with the genotype
fern/fern; WI/W,. In addition to the spots of isoxanthopterin, biopterin andxanthopterin-like substance a large spot appeared showing yellow fluorescence. This spot showed yellow color in visible light but in day lightit changed into its photo-decomposed pteridine, 2-amino-4-hydroxypteridine-6-carboxylic acid. A large spot of kynurenine and a small spot of
3-hydroxy-kynurenine appeared. The purplish-blue spot noticed in theabove-mentioned two materials (2) and (3) could not be observed.
(5)

Compound eyes of the moths with the genotype (tern/fern; UJ2/W2)'

Although almost the same pattern of chromatogram as that of material(4) was observed, the spot of yellow pigment was smaller. The reason
why the eggs with this genotype show light reddish brown color is notclear. It may be due to the presence of some other similar pigment inaddition to dihydropterin. Further studies on the eye pigments of thistype are necessary.
Briefly, it may be said that each of the five strains has its own featurein respect to the pteridines of the compound eyes. The yellow pigment
which is accumulated in the larval hypodermis can be scarcely detectedin the compound eyes of the genotype fern/fern; +"'1/+"'1. However, inthe presence of the gene for white egg color the compound eyes show

RESEARCHES CARRIED OUT IN 1961

35

distinctly yellow or light reddish brown color owing to the accumulationof large amounts of yellow and some other similar pigments.

19. Influence of temperature on the syntheses of contractile
proteins in Triturus embryo*(By Yoshito OGAWA)

The influence of temperature on the syntheses of contractile proteins,
actin and myosin, in early embryos of Triturus pyrrhogaster, BOlE wasexamined.
Approximately 1,000 embryos were divided into five equal groups whichwere raised separately at five different temperatures (10, 15, 18, 20 and
25°C). The determination of the first detectable trace of actin and myosinformation in embryos was carried out by serological technique as before
in the period of 12 hrs. after fertilization.o ,21 Anti-sera against G-Actinand myosin were prepared by injecting intravenously into rabbits a solution of G-Actin and myosin, isolated from the skeletal muscle tissue of
Triturus pyrrhogaster by Szent-Gyorgy's method, and were raised to theirserological specificity by resorption tests with saline extracts of liver,spleen and skin tissue of Triturus. Titres of both sera were adjusted to
1: 512 before the precipitin reaction with a saline extract of embryos.
Constant volumes of sera and progressively decreasing amounts of antigen
were used in order to avoid an excess of the latter.
It was found that actin first became detectable 84, 120, 132, 144 and156 hrs. after fertilization in embryos raised at 25, 20, 15 and 10°C.,
respectively. As to myosin formation the first detectable trace was found
108 hrs. after fertilization in embryos raised at 25°C., 168 hrs. when raised
at 20°C., 180 hrs. at 18°C., then again 168 hrs. at 15°C. and 132 hrs. at
10°C., as shown in Fig. 1. Thus, actin formation in early embryos wasmarkedly suppressed with dropping tempe~ature under the above experimental conditions. On the contrary, myosin formation was most strikingly suppressed at 18°C. and from there on was clearly promoted independently of falling or rising temperature.
This finding is unexpected but the result is noteworthy, because thetemperature of the natural breeding season of Triturus is 18°C. It may be
imagined that many metabolic pass-ways are in competition for the materials they need to keep a balance necessary for the growth of theembryo. For instance, there may be a metabolite present which at the* This work was supported by a Grant-in-Aid for Fundamental Scientific Research (No. 710267) from the Ministry of Education in Japan.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

25

U

~

..e
....<I)" ""-,

20
18

::3

tl
0.

~

"

15

8
<I)

"

Eo-<

10

1l10&in

84

108

132

156

180

Hra. after fertilization

Fig. 1. The influence of temperature on the syntheses of muscle proteins in
the early embryo of Triturus pyrrhogaster.

optimum temperature for growth appropriates the materials necessary
for myosin formation.
The order of actin and myosin formation in embryos raised at lOoe is
opposite to that found in natural development. This result furnishes
another evidence of the independence from each other of actin and myosinformation as already pointed out in another report by this writer. 2) ,8)
1) OGAWA, Y. Nature 182 (4645): 1312. 1958
2) OGAWA, Y. Nature 186 (4718): 77. 1960
3) OGAWA, Y. Med. and BioI. 58: 185. 1961

20. Protective action of sodium-glucuronate on the formationof the contractile muscle protein, actin, inhibitedby X-ray irradiation in Triturus embryo*(By Yoshito OGAWA)

When the effect of sodium-glucuronate on the growth of animal embryos
was studied, it was found that those raised in the solution of sodium-* This work was supported by a Grant-in-Aid from Tokyo Biochemical Researoeh
Foundation.

RESEARCHES CARRIED OUT IN 1961

37

glucuronate were generally not influenced by the environment. The effectof sodium-glucuronate protecting the skeletal muscle tissue of X-irradiatedTriturus embryos from abnormal differentiation was therefore examined.
The qualitative and quantitative analyses of the developing skeletal
muscle protein, actin, during early embryonal stages under various experimental conditions were carried out by the same serological technique
as before.'1,21
G-Actin was isolated from the skeletal muscle tissue of adult Triturusby Szent-Gyorgy's method. This protein (100 mg) was injected intravenously into rabbits and the obtained rabbit anti-serum was heated withsaline extracts of liver, spleen and skin tissue of Triturus to eliminate
non-specific antibodies. The titre of anti-serum was adjusted to 1: 512
before carrying out the precipitin reaction with saline embryo extract.
Immediately after fertilization, the embryos were raised at 20°C. in
0.01% solution of sodium-glucuronate (most effective concentration for
promoting the growth of Triturus embryos31), and X-irradiation of theembryos with 50 r, 200 rand 500 r was carried out 108 hrs. after fertilization. After irradiation, the treatment with sodium-glucuronate wascontinued whereupon the analyses of actin were immediately carried out.
Non-treated and irradiated, treated and non-irradiated, and normal (nontreated and non-irradiated) groups were prepared for control.
In normal embryos, actin first became detectable 132 hrs. after fertilization. In the case of irradiation, actin was detected 156 hrs. after ferTreated ///Trea7dI

TreaydTreated/L"," . /. ._" . /r.. . J.."_" .....
1I

I/

/

II

1

Lowest ant igenconcentration giving
positive results

I

JOOr
28

204
IrrFldiatedHr. after fertilization

Non-irradiated control

Fig, 1.

Effect of sodium-glucuronate on the synthesisof actin in X-irradiated Triturus embryos.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

tilization with 50r, 180 hrs. with 200r and 204 hrs. with 500r. 21 Thus,
the synthesis of actin is markedly suppressed not only as to the rate offormation but also as to its amount and the decline is proportional to theirradiated X-ray dosage as shown in Fig. 1.
In the treated embryos with sodium-glucuronate, the influence of Xirradiation could be scarcely observed either in the rate of actin formation or in its increasing amount. (Fig. 1.)
It was therefore proved that sodium-gJllcuronate markedly protectsfrom a decline of actin synthesis inhibited by X-irradiation in early embryonal stages.
1) OGAWA, Y. Nature 182 (4645): 1312, 1958
2) OGAWA, Y. Nature 186 (4718): 77, 1960
3) OGAWA, Y. Ann. Rep. Nat!. Inst. Genet. 10: 150, 1959

21. Effects of glucuronic acid and related compounds
on the chemo-differentiations of muscle tissue
in regenerating hind limb of Triturus*(By Yoshito OGAWA)

The effects of sodium-glucuronate, glucosamine and glucuronolacton on
the synthesis of the skeletal muscle proteins, actin and myosin, in theregenerating hind limb tissue after amputation at the knee were examined in Triturus pyrrhogaster, Boie.
About 800 of good-sized adult Triturus specimens were divided into
four equal groups. One was used as the non-treated control. The remaining three were subcutaneously injected, respectively, with solutionsof sodium-glucuronate, glucosamine and glucuronolacton at a dosage of
1.4 mg per 1 g body weight an 21 hours intervals immediately after theamputation until the removal of the granula. The animals were kept at
18°C. From 10 to 30 days after operation, the granulated tissue of theregenerating limb was removed at intervals of two-three days, and asaline extract was made. The identification of actin and myosin was
carried out by means of serological technique,uIn the non-treated control, actin and myosin first became detectable in
the regenerating limb tissue 20 and 27 days after the operation, respectivelyY As to the treated group with sodium-glucuronate, actin and* This work was supported by 3 Grant-in-Aid from Tokyo Biochemical Research
Foundation.

RESEARCHES CARRIED OUT IN 1961

39

myosin were first detectable 14 and 20 days, respectively, after operation.
Thus, sodium-glucuronate remarkably promoted the development ofmuscle tissue. When a solution of glucosamine was injected, both proteins were investigated 16 days after operation. In another treated group,
injected with a solution of glucuronolacton, the formation of actin was
found 24 days and that of myosin 16 days after operation. Therefore,
glucuronolacton supresses the synthesis of actin and promotes that ofmyosin.
The differentiation of muscle tissue is not accomplished until bothactin and myosin formations coincide. The time of chemo-differentiationof muscle tissue in the regenerating limb tissue treated with sodiumglucuronate, glucosamine and glucuronolacton were 20, 16 and 24 days
after operation, respectively. Above results show a similar tendency tothose found in the e2'periments on chemo-differentiation of muscle tissue
in early embryonal stages of Triturus. 21
When treated with glucuronolacton, the order of development of actin
and myosin is reversed to that found in the normal control. Such areversal was already found also in regenerating limb tissue after X-ray
irradiation with 2,500r, 18 days after amputation at the knee. 31 The
order of synthesis of actin and myosin is therefore easily reversible.
These results support the view that the processes of synthesis of actin
and myosin in regenerating tissue are independent of each other as they
were found to be also in early embryonal stage. 4 )1) OGAWA, Y. Nature 182 (4645): 1312, 1958
2) OGAWA, Y. Ann. Rep. Nati. Inst. Genet. 11 : 40, 1961
3) OGAWA, Y. Ibid. 11 : 38, 1961; Med. and BioI. 58 (1): 8, 1961
4) OGAWA, Y. Nature 186 (4718): 77, 1960

22.

Two factors influencing sensitivity to X-ray in the synthesisof myosin during early embryonal stages*(By Yoshito OGAWA)

Last year, two factors (A and B) were reported influencing during
early embryonal stages the sensitivity to X-ray in the synthesis of themuscle protein, actin. lI This paper deals with myosin formation, anotherimportant contractile protein in muscle tissue, with respect to X-irradiation at various stages of development of Triturus pyrrhogaster, Boie.

* This work supproted by a Grant-in-Aid for Fundamental Scientific Research(No. 710267) from the Ministry of Education in Japan.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Approximately 15,000 embryos at different development stages were
exposed to single doses of X-rays of 5r, lOr, 30r, 50r, 200r, 500r, 1,OOOr
and 2,500r and examination for the first detectable trace of myosin after
fertilization was carried out in periods of 24 hrs. after irradiation byusing the same serological technique as before. 2 ) ,3) Myosin, preparedfrom adult Triturus, was injected into rabbits and the obtained antiserum was heated with saline extracts of liver, spleen and skin tissue of
Triturus to eliminate non-specific antibodies. The titre of anti-serum wasadjusted to 1 : 512 before carrying out the precipitin reaction with saline
extracts of X-irradiated embryos.
In normal embryos, myosin first became detectable 176 hrs. after fertilizations as already reported by the present author. 2) (In the present

204

'..."
~

180

0

.::. 156
c
0

.~

~

.~

'".'"..
...
.~

132
108

~

al

"

""'"

84
60
36

500r lOr 5r
1,000r 30r 50r
2,500r 200r60

108

Non-irr diated control

156

204

252

300

348

Age at myosin formation (Hours)
Fig. 1. Synthesis of myosin in early Triturus embryos irradiated by X-rays
applying doses from 5r to 2,500r.

experiment, the myosin formation in the non-irradiated control was firstrecognized 180 hrs. after fertilization, because the examinations werecarried out in periods of 24 hrs.) The time of myosin formation after
fertilization in the treated embryos is shown in Fig. 1. A dose of 5 r
given 36 hrs. after fertilization changes the time of myosin formation to
108 hrs. after fertilization (Fig. 1*) but in the case of irradiation 156 hrs.
after fertilization with a heavy dose of 2,500r, no myosin formation wasrecognized before the necrosis of the embryos (300 hrs. after fertilization) (Fig. 1**).

RESEARCHES CARRIED OUT IN 1961

41

The curves shown in Fig. 1 suggest a biochemical similarity of the
response to X-rays in actin and myosin syntheses, indicating the presenceof two factors sensitive to X-rays in the synthesis of myosin as well as
actin during early embryonal stages. One (A) suppresses the synthesisof muscle proteins and the other (B) markedly promotes it after X-irradiation. The points found on the right of the vertical control line indicate the effect of the X-ray inhibiting factor A (Fig. 1 0), and those on
the left refer to the X-ray promoting factor B (Fig 1 ~). But the two
factors of actin synthesis (Aa, Ba) may be different from those of the
myosin synthesis (Am, Bm), because the processes of synthesis of actin
and myosin formation are independent of each other. 3 ) Factor A in
myosin formation (Am) does not react to X-rays until doses over 500 r
are applied. On the contrary, factor Bm is very sensitive to X-rays and iseasily detectable with only 5r doses. Factor Am is detectable by irradiations between 108 and 156 hrs. after fertilization. The most effective
time of irradiation for factor Am is 156 hrs. in the above experimental
conditions. Exposures between 36 and 132 hrs. after fertilization show
clearly the reaction of factor Bm to X-rays. The most effective time of
X-irradiation for factor Bm is 36 hrs. after fertilization.
204

.,

..

.::."

180

0

--

156

"
"B
..,

132

.::

108

..

84

~

..
al

....al

to

«

I
I

60
36

5r

Non-irr1diated control

I
60

108

-156

204

'.!52

300

Age at lIluscle-"tisl3ue i1ifreren~iation (Hours)

Fig. 2. Chemo-differentiation of muscle tissue in early Trituru8 embryos
irradiated by X-rays applying doses from 5r to 2,500r.

The differentiation of muscle tissue is not accomplished before both

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

actin and myosin formations coincide. The chemo-differentiation processof muscle tissue of X-irradiated Triturus embryos is represented in Fig. 2.
1) OGAWA, Y. Ann. Rep. of Natl. Inst. Genet. 11 : 37. 1961
2) OGAWA, Y. Nature 182 (4645): 1312. 1958
3) OGAWA, Y. Nature 186 (4718): 77. 1960

23. Sensitivity of a day-old chicks to low and hightemperatures(By Takatada KAWAHARA)

The present study deals with a comparison between purebreds and theirhybrids with regard to tolerance for unusually high or low temperatures.
A day-old chicks of White Leghorns, Barred Plymouth Rocks and theirreciprocal crossbreds were used. A number of chicks belonging to thesame full-sib group from each strain were divided into two groups, one
for high-temperature exposure (42.5 C, 100 minutes), and the other for
low-temperature treatment (IDe, 120 minutes). After exposure to thoseabnormal temperatures the chicks were transferred to an incubator controlled at 37.6 D C and kept for 14 hours for observation.
The results of analysis of the obtained data are summarized as follows:1). F 1 chicks were more tolerant to low temperature than the purebreds,
the lethality of the hybrid chicks being 55.5% in contrast to 71.7% forpurebreds. The difference of 16.2% was statistically significant at the
1% level. 2). F 1 chicks, however, were less tolerant than the purebreds
to high temperature treatment. The lethality of treated hybrid chicks
was 61.8%, being 6.8% higher than that of 55.0% of the purebreds.
The difference was statistically significant at the 5% level. 3). The
correlation between low and high-temperature tolerance was -0.427±0.110
in the purebreds, while in the crossbreds, it was +0.185+0.129.
D

24. Electrophoretic haemoglobin patterns
in inbred rats and chicken(By Kazuo MORIWAKI)

Recently several investigators have contributed valuable investigationsof human haemoglobins in terms of genetics and protein chemistry. It
was also revealed by means of paper electrophoresis that the haemoglobinof mice occurs in two types, single and diffuse, which are considered to
be Mendelian characters.

RESEARCHES CARRIED OUT IN 1961

43

The present study is an attempt at finding by the method of electrophoresis technique a difference in haemoglobin type among inbred rat
strains. The strains used were as follows; Albany, Castle Black, Fischer,Long-Evans, Wayne pink-eyed hooded, Wistar and Wistar-King A. At
an ionic strength of 0.05 of verona I buffer (pH 8.6), two bands were observed on the anodic side of the paper; a faster major one and a slower
minor one. When the buffer was diluted to 0.025 of ionic strength, fourbands appeared; two major bands on the anodic side and two minor bandson the cathodic side. However, at these two concentrations of the buffer
no difference in the haemoglobin patterns could be observed between thestrains.
Between the species some difference in the patterns was found. At anionic strength of 0.05, Rattus rattus caught in a field showed one anodic
band, and Rattus norvegicus (d. Wistar) showed two anodic bands, asmentioned above. The mode of inheritance of these two types is notknown, because these two rats could not mate.
Further, an electrophoretic analysis of haemoglobin was performed inchicken inbred strains. The haemoglobins moved to the cathodic side at
an ionic strength of 0.025 of veronal buffer (pH 8.6), accompanied by one
faster and one slower band, but no difference was detected between thestrains. On the other hand, electrophoretical analysis has been carriedout on an agar plate containing 1% agar and 0.05 M citrate buffer (pH8.6). The presence of two components in the haemoglobin could be confirmed by this method for all the strains. For those experiments thefollowing chicken strains were used; White Leghorn, Plymouth Rock,
Rhode Island Red and Nagoya.

25.

Further study on post-axial polydactylism
in the house mouse(By Tosihide H. YOSIDA, Hitoshi SAKAMOTO
and Akira NAKAMURA)

A mutation, causing post-axial polydactylism of the front feet of thehouse mouse was found in this Institute and its genetics have been studied
by the authors. It was found that this character depended on the interaction of several genes; one major gene, and some minor genes which
probably modify the manifestation of the character.
Further genetical studies showed that the major gene was dominant
and two minor genes were recessive; the latter suppressed the action of
the major gene.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

26. Invasion of tetraploid cells of the Yoshida sarcoma
into the lymph nodes of the rat(By Tosihide H. YOSIDA)

In ascites tumor cell populations of the Yoshida rat sarcoma tetraploidcells are rare. ·However, in previous studies, the present author foundthat the tetraploid cells predominantly invaded lung, kidney, liver andspleen, but did not invade to a large extent either thymus or mediastinal
lymph nodes.
In the present study, another form of metastasis was investigated.
Cells of the Yoshida sarcoma were injected subcutaneously under the
tail. Following this procedure, regularly metastasis to the lumbar node
took place. Metastatic tumor cells in the lumbar node, in this situation,
revealed a distribution of tetraploid cells, similar to that found in the
ascites.

27. Karyological analysis of a metastatic tumor cells in
MH134 hepatoma of the C3H strain of the mouse(By Naomichi INUI and Tosihide H. YOSIDA)

In 1956 Sato converted a hepatoma (MH134 of strain C3H) to the ascites(form. This neoplasm readily metastasizes to lymph nodes. In the present
experiment the chromosome constitution of the metastatic tumor cells in
lymph nodes was compared with that found in ascites. Metastases were
induced experimentally in the lymph nodes in the mouse by injecting
ascites tumor cells under the skin of tails of 10 animals. Seven to tendays after transfer the tail was amputated at the base, and about 20
days later the mice were killed. The lymph node was greatly enlarged:at the base of the tail in six animals and contained metastatic tumor
cells. The ratio of diploid to tetraploid cells in the lymphatic glands. did not differ from that found in the ascites.

28. Ploidy in tumor cells after direct inoculation
into various organs of the mouse(By Tosihide H. YOSIDA and Toshiharu KAMIOKA)

Following a peritoneal injection of tumor-ascites cell population of ELD,
only a few (about 2 or 3%) tetraploid cells were found in ascites, but distinct metastases contained predominantly tetraploid cells. Two possible

RESEARCHES CARRIED OUT IN 1961

45

explanations were considered. (1) Tetraploid cells have a higher ability
to invade lung, liver, spleen and other organs. (2) These organs provide
better conditions for the multiplication of tetraploid tumor cells.
An experimental approach to this problem was to inject tumor cells
directly into the various organs. A population of the Ehrlich ascites
tumor (ELD), which had a ratio 27 : 100 of tetraploid: diploid cells wasused for the present study. Approximately 100 tumor cells were inoculated directly into brain, lung, liver and under the skin of mice. With
the exception of the lung, tumors developed in those sites. The ratio of
tetraploid to diploid tumor cells in various tumors was examined.
In the ascites population as control, the T/D ratio was 18/100, in thebrain 32/100, liver 20/100, skin 22/100. Thus, it was shown that whentetraploid cells were artificially introduced into the organs, they could
grow. These results indicate that at least the two above mentioned
factors are involved in the selective metastasis of tetraploid cells froma mixed population, and that both greater invasiveness of tetraploid cells.
and a better metastasis environment play a role.

29. Competition of diPloid and tetraploid tumor cells
in the mouse(By Tosihide H. YOSIDA)

In the previous studies by the present author and his collaborators concerning the invasiveness of tumor cells, it was found that polyploid cells
can multiply more readily in some organs, such as brain, liver, kidney,lung and spleen than the diploid ones, but they cannot increase as muchas to become preponderant in the lymphatic glands. It is suggested thatthe metastatic tumor cells in the above mentioned organs receive betteror more suitable nutrition than in the lymphatic glands.
To find out whether the tetraploid cells require better nutrition tomultiply in the peritoneal cavity than do the diploid cells, the following
experiment was performed: (1) Walker solution for tissue culture, whichcontained various amino acids and vitamins, was injected every day into
the peritoneal cavity after transplantation of Ehrlich ascites tumor. (2)As control, physiological salt solution was injected into the peritoneal
cavity by the same method. (3) As another control, a dilute solution of
4-NQO (a carcinostatic agent) was injected. A remarkable increase in
tetraploid tumor cells occurred in the ascites tumor in mice which were
injected with Walker solution (1). No remarkable change in the frequency of the tetraploid cells was found in the control (2), while a de-

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

crease of tetraploid cells occurred in the other control experiment (3) inwhich injection with a carcinostatic drug was used. These results strongly suggest that tetraploid tumor cells can multiply better in the peritoneal
cavity, when the conditions are made to suit their development.

30. Inhibitory effect of the components of Swertiae japonica
on cell division in living plant tissue
and on growth of rat tumors(By Yo TAKENAKA and Yoshito OGAWA)

The three components of Swertia japonica, swertiamarin, gentiopicrin
and amarogenin were investigated regarding their effect on cell divisions
in root tips of Allium scorodoprasum var. viviparum and on the growthof Yoshida sarcoma.
Water solutions of various concentrations were prepared from the above
three chemicals, and living roots of A. scorodoprasum var. viviparum wereplaced in the solutions for 4 to 24 hours. The living root-tips weretreated according to the hydrochloric-acetic acid-orcein method and oberved under microscope. Using water solutions of swertiamarin andDays after transplantation
10

100

20

30

~

.....,al....al....>~=~~\ \\ \.---\

50

\.S~
~ .

t:

:>

\ ..........

U)

0

-

'\
C.

G.

C.

\ '\.
""-

A.

'----.----_.- - - - -s.
Control

S'.

Swert iamarinG.

Gentiopicrin

A.Aml,lrogenin

Fig. 1. The effect of three compounds extracted from Swertia japonica onlife-prolongation of the host rat.

gentiopicrin, no structural change was found in the living cells, but thatof amarogenin caused remarkable radiomimetic phenomena, even after
4 hour treatment with 4% solution.

47

RESEARCHES CARRIED OUT IN 1961

After transplantation of the ascites tumor to a Wister rat (two monthsold), the above three chemicals were immediately injected at the rate of
0.5 mg per 1 g body weight and the same injections were made again
after 24 hours. Their toxicity, systemc symptoms and prolongation oflife of the host rat were investigated.
Groups treated with swertiamarin and gentiopicrin showed no difference
from the non-treated control in systemic symptoms and in the duration of
host's life. But the group injected with amarogenin showed 4 days after
the last injection, a remarkable recovery in systemic symptoms and 60%of the treated animals remained alive for about 16 days (Fig. 1), though
the control group died out within 8 days after transplantation. These
three chemicals showed no toxicity at the above-given doses.

31.

Anti-cancer activity of extract from
Glycyrrhizae Radix*(By Yoshito OGAWA)

The effect of methanol and ethyl-ether extract of Glycyrrhizae Radix(Chinese Licorice) on Yoshida-sarcoma, transplanted into a strain of rat,
Wister (two months old) was examined. Glycyrrhizae Radix, which is
obtainable in Japanese drug stores, was first treated with the same volume
of methanol on the water bath. The methanol insoluble tissue was thenextracted five times with the same volume of ethyl-ether at room
temperature. The yields of methanol and ethyl-ether fractions were 7.8%
and 0.5% in weight of original plant tissue, respectively.
DayS after

tra~splantation

60

40

20
100

~
Q)

.
....

75

Treated with~ethanolextract

-;;;

'"

50

~

25

'""

0

Treated with ethyl-ether extract[;

Clinical stage
Non~treated control

Immediately after the transplantation of the ascites tumor into theabdomen of a rat, the obtained extracts were injected at the axilla sub-* This work was supported by a Grant-in-Aid for Fundamental Scientific Research(No. 0433) from the Ministry of Education in Japan.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

cutaneously two times with a 24 hour interval making up a total of 100
mg per 100 gr. body weight of the host rat, and its influence on thesarcoma was investigated in respect to the following three points: 1)
toxicity of the extracts; 2) systemic symptoms of the rat bearing thesarcoma (appetite, ascites, icterus, metastasis and body weight); 3) prolongation of the host's life.
The methanol fraction showed an inflammation at the site of injection
and some collapse of the regional tissue was found 7 days after treatment, though the ethyl-ether fraction showed no remarkable toxicity inthe injected region and no systemic symptom at the above mentioned
doses. As to the prolongation of the host's life, 40% of the animals
treated with the methanol fraction died in the first seven days. Thismay be due to the toxicity of the injected substances. But the remaining 60% of this group, shewed a remarkable recovery in systemic symptoms ten days after the injection and were alive for more than twomonths, though the non-treated control group died out between the 8th
and 12th day after the transplantation of sarcoma. But in the grouptreated with the ethyl-ether fraction, no animal died before the 12th day
after transplantation and about half of this group remained alive formore than 2 months after transplantation. The progress of their recovery in systemic symptoms is just the same as that found in thegroup treated with the methanol fraction.
These results seem to indicate that the effective substance of Glycyrrlzizae
Radix shows no toxicity to the animals and is easily separable from thetoxic component by treatment with ether. Further chemical and pharmacological researches of this effective substance are now being carried
out.

B.

GENETICS, CYTOLOGY AND BIOCHEMISTRYOF PLANTS
32. Cytoplasmic male sterility of common wheat(By Hitoshi KIHARA)

In order to study the effect of alien cytoplasm on male fertility ofcommon wheat, genomes of 3 hexaploid species, i.e., Triticum vulgare,T. spelta and T. compactum have been introduced by successive backcrosses into the cytoplasm of Aegilops caudata. Donor of the cytoplasmwas" SBa" (Kihara 1958), that possessed caudata cytoplasm and genomesof T. vulgare.

RESEARCHES CARRIED OUT IN 1961

49

Pollen and seed fertilities of the successive backcross generations are
summarized in Table 1.
Table 1. Pollen and seed fertilities in nucleus-substitution lines of
3 common wheat species

Strain
SB a
SB a xvulg. (SB 9)
If
xvulg. 2 (SB lO )If
X vulg. 3 (SB ll)
If
xvulg} (SB I2)
SB a xspelta
If
xspelta 2
xspelta 3
xspelta'
SB a xcomp.
If
xcomp.2
If
xcomp.3
If
xcomp.4
T. vulgare
T. spelta
T. compactum
f(

f(

Year

Pollenfertility (%)

Seed fertility (%)
Selfing

I

Crossing

1957
1958
'59
'60
'61

0.0
0.3( O.O~ 0.5)
0.2( O.O~ 0.5)
2.0( O.O~ 6.8)
0.1( O.O~ 0.9)

0.0
23.2(12.0~ 52.8)
0.0
41.2( 2.5~ 58.8)
0.0
56.3(42.5~ 75.0)
0.0
50.9( 5.0~100.0)
0.6( o.o~ 1.6) 66.5(50.0~ 85.0)

1958
'59

0.0
0.0
0.1( o.o~ 1.1)

0.0
27.9( 3.6~ 40.0)
1.3( O.O~ 6.3) 39.1( 5.6~ 62.5)
0.2 ( O.O~ 1.5) 7.5( O.O~ 25.0)
0.1 ( O.O~ 0.2) 52.7(40.0~ 68.2)
6.2( 2.4~11.4) 33.4(16.7~ 50.0)
51.6(19.2~90.7) 53.6(27.5~ 67.6)
23.6( 0.0~67.2) 48.3( O.O~ 95.0)
45.5 (28.1~62.9) 92.8 (86.4~ 100.0)
83.6(78.4~86.0) 78.3(65.0~ 95.0)
63.1 (47 .2~71.1) 89.4(85.0~ 91.7)
65.6 (63.6~77.5) 83.6(63.3~100.0)

'60
'61
1958
'59
'60
'61
1961
If
If

0.0
33.1
39.9( 6.7~52.6)
85.5(64.6~96.7)
90.1 (78.0~96.2)
98.8
97.0
93.8

Apparently, the cytoplasm of Ae. caudata causes almost complete malesterility of T. vulgare and T. spelta, while T. compactum possesses genesthat restore male fertility.
In order to investigate the number of the restoring genes in T. compactum, "SB Il " plants (~) were pollinated with the pollen of the F 1
hybrid, T. vulgare X T. compactum. In the subsequent generation completely male-sterile and partially male-fertile plants were segregated toa ratio of 37 to 12, suggesting 3: 1 segregation ratio. The average fertility of the latter group was 4.8% ranging from 0.7-20.8%.
This fact seems to indicate that T. compactum carries 2 complementary
genes for restoration of male fertility; based on this assumption, thegenotypes, Ms 1Ms 1Ms 2Ms 2 and mSlmSlmS2mS2 are tentatively assigned toT. compactum and T. vulgare, respectively. The partially fertile plants,
which were segregated in the above cross (mslmslms2ms2xMslmsIMs2ms2)
are assumed to be of a genotype MSlmslMs2ms2. The designation proposed is supported by a fact that "SBa" (mslmslms2ms2) X T. compactum

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12(Ms1Ms1MszMs z) produced partially fertile F 1 hybrids, whose fertility (6.2%
on the average ranging from 2.4 to 11.4%) is well comparable with thatof the partially fertile plants segregated in the cross, "SB l l " x(T. vulgare
X T. compactum F 1).
33. Pistillody of Triticum durum induced by an
Aegilops cytoplasm(By Hitoshi KIHARA and Koichiro TSUNEWAKI)

Nucleus of Triticum durum (2n=28) was placed into cytoplasm of Aegilops caudata (2n=14) by successive backcrosses. Since hybridization
between T. durum (0) and Ae. caudata (Q) was not easy, a strain thatpossessed caudata cytoplasm and genomes of T. vulgare (2n=42) was usedas the donor of caudata cytoplasm.
T. durum with caudata cytoplasm became completely male sterile because of the pistillody of all stamens. The pistilloid stamens had noovule and instead had only a cavity. In some pistilloids even such acavity was not formed. In order to test the function of pistilloid stamens
as female organs, a normal pistil was carefully removed from each floret
and the remaining pistilloid stamens were artificially pollinated. 204
floerts were treated in this way but did not produce any seed. These
results clearly indicate that the pistilloid stamens of T. durum inducedby caudata cytoplasm are not functioning as female organs.
Seed fertility of T. durum with caudata cytoplasm, when cross-pollinated,
was only slightly higher than 10%, being much lower than that of normal
T. durum. An anatomical study of apparently normal pistils indicatedthat a majority of ovaries had abortive ovules. This seems to be a causeof the depression of female fertility.
These results indicate that effect of caudata cytoplasm on the manifestation of the durum genomes is twofold, i.e., induction of pistillody
and depression of female fertility.

34. Progressive necrosis of seedlings in hexaploid wheat(By Koichiro TSUNEWAKI)

Tsunewaki (1960) and Tsunewaki and Kihara (1961) found that progressive necrosis of common wheat is controlled by 3 complementary
genes, Nelon chromosome 5B, Nez on chromosome 2A and Nes on chromosome 3D, and that T. aestivum var. Kharkov, T. aestivum var. Prelude
and T. macha var. subletschumicum are of genotype, ne1NezNes, NelnezNes

RESEARCHES CARRIED OUT IN 1961

51

and NetNe2ne3, respectively.
In order to investigate the distribution of the 3 necrosis genes in common wheat and its ancestral species, the author crossed the above-mentioned varieties as testers to 16 varieties of common wheat (including 5
species) and 12 strains of synthesized hexaploids (including 6 species of
Emmer wheat and 3 varieties of Ae. squarrosa).
Necrosis observed in the seedling stage of those F t hybrids is summarized in Table 1.
Table 1.

Seedling necrosis in the F t hybrids between 3 tester varieties
and 16 common wheats and 12 synthetic hexaploids
Testers

Tested varieties
Chinese Spring, Marquillo, Trumbull,
American Banner, Pawnee, S·615,
T. vulg. eryth., T. compo 44, T. spelta
Duha, T. sphaero.
Nowinka, T. macha paleo, ABD.l,
ABD-8, ABD.9, ABD-14, ABD-16,
ABD (Pentad), ABD-22
Riebesel
ABD-3, ABD-13
T. macha sublet.
Prelude, ABD-U, ABD-23, ABD (Golden
Ball)
Kharkov, Jones Fife, H l l , Fulcaster,Blachhull+ : necrotic,-:

Kharkov

I Prelude IT. (NetNe2ne3)
macha sublet.

(ne,Ne2Ne3) (Ne,ne2Ne3)
-

-

+

-

-

-

+

-

-

-

+

+
+

+

-

-

+

-

+

+

normalFrom this result, at least, one variety representing each one of the 7possible genotypes has been found. It is interesting to notice that all
three, Net, Ne2 and Ne3' are widely distributed in common wheat, while
Ne 2 is rarely found in Emmer wheat, in which Net widely occurs. Ne3
is also widely distributed in Ae. squarrosa.
35.

Relation between chlorophyll content and free aminoacids in mutants of Triticum monococcum(By Taro FUJII and Yukio ONO)

Free amino acids in chlorina, virido-albina and their double recessives
were examined with paper chromatographic method. On the two-dimensional development of extract in seedling stage, seven spots were observed

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

in normals and chlorina showing the presence of aspartic acid, glutamic
acid, glycine, glutamine, phenylalanine; in addition, two unidentified spots
a and b appeared. On the other hand, nine spots were observed in bothvirido-albina and double recessive plants. The two additional spots indicate alanine and leucine.
Eight spots of the same kinds were observed in normals and chlorina
in maturing stage, but at that time ten spots were observed in viridoalbina and double recessives. Alanine is absent only in chlorina but it ispresent in the other three strains as a diluted spot, and glutamine isabsent in normals and chlorina though they are present in them at seedlings stage. Leucine and spot a were absent in four strains, though spot
a was observed in all strains in seedlings and leucine was observed invirido-albina and double recessives at early seedling stage. Furthermore,
spots c, d and e newly appeared at this stage in all strains.
The chlorophyll content in double recessive plants was recovered to thechlorina level and no further increase occurred (d. Ann. Rep. No.8).
The chlorina gene, therefore, is epistatic to virido-albina gene as to theability to recover, nevertheless the same kinds of amino acids were observed in both virido-albina homozygotes and double recessive plants at
seedling stage or at ripening stage.

36.

On the occurrence of chlorophyll mutations in clusters
in einkorn wheat ll(By Taro FUJII)

Dormant seeds of Triticum monococcum were subjected to 10, 20 and
30 kr of gamma-rays. All spikes from all irradiated Xl individuals were
harvested and chlorophyll mutations were scored in the X 2 generation.
Number of spikes with chlorophyll mutants increased with increasing
dosage. In some cases, several spikes proved to carry the same kind of
mutation from the same Xl individual. Namely, 8 spikes among 16 from
one individual segregated albina, while in another case, only one spikeamong 60 spikes xantha was found. Thus distribution of cluster mutations showed a wide variation, and the average number of cluster mutants
per Xl individual was not related to dosage. Frequency distribution ofcluster mutants of albina type was calculated according to KONDO (d.
KONDO, S. 1961, Jap. Jour. Genet. 36: 6-17), as given in Table 1. According to the results, the number of original cells in embryonic stage was1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

RESEARCHES CARRIED OUT IN 1961

53

estimated as 35±3, and some of these differentiated cells seem to havefurnished the initials for the spikes.
Table 1.
No. of spikes
which segregatedalbina seedlingsin X 201237.

Frequency of albina clusters
No. ofX, plants

151
24
8

3

4

4
5
6
7

3

0
0
0

Calculation
According toObserved
KONDO

I

0.795
0.126
0.042
0.021
0.016
0
0
0

0.795
0.122
0.055
0.019
0.005
0.002
0.00050.0001

Crossing experiments with chlorophyll mutantsof einkorn wheat(By Taro FUJII)

Crossing experiments among several viable chlorophyll mutants of
Triticum monococcum were carried out and F , plants were obtained from28 combinations. Among them, some were between different types ofmutant such as basi-viridis or virido-albina and chlorina. And others were
between different strains within the same type of mutant such as basiviridis-5061x-5061 or virido-albina-5066x-5079, etc. All F , plants from 27
combinations showed normal green color and relatively high fertility andin the F 2 generation they segregated plants with both parental charactersand double recessives. Linkage relationships between these mutants are
not yet determined because of low germination rate and high mortalityof some mutant strains and double recessive segregants.
The F 1 seedlings between virido-albina-5066 and -5079 showed viridoalbina character in reciprocal crosses. There was a small difference between both strains concerning the recovery of chlorophyll pigment. Therefore, it is concluded that the genes for their characters are located on
the same locus, being multiple alleles. A large number of lethal mutants
such as albina and xantha are listed in our collection. From these facts,
many genes seem to have contributed to chlorophyll development and it
is suggested that the chlorophyll pigment is synthesized in several steps.

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

38.

Developmental anatomy of glume epidermis
in the genus Oryza ll(By Hitoshi KIHARA and Tadao C. KATAYAMA)The surface structure of the glume is an important taxonomic character in Oryza, as was recognized by Roschevicz. In order to examine
the details, the present authors studied the glume of 18 Oryza species by
SUMP (Suzuki's Universal Microprinting) method (1960). Histologicalinvestigations of epidermal cells of glumes were carried out this year.
The distinction between the glume characters of the four sections is very
conspicuous.
The cells of O. subulata (Section Rhynchoryza) are of equal size and arearranged like fish scales, and seldom short cells occur. In some casessuch short cells develop into hairs. In all other three sections (Sections
Sativa, Granulata, Coarctata) the epidermal cells are arranged more orless in regular longitudinal rows. The cells of species belonging to Section Sativa are arranged also in regular crosswise district, with the result of a checkerboard arrangement. In the case of species belonging to
the latter three sections, each cell divides into two kinds of cells at thelate developmental stage; one is strongly elongated and the other is shortand either remains undifferentiated or develops into a hair.
The aspect of cells in the maturing stage of each section were summarized as follows:
In Section Sativa, where the short cells and the long cells are immediately adjacent to one another, the tubercles result from uneven thickening at the points of contacts of six cells which consisted of four longand two short cells. The short cells are much reduced in size.
In Section Granulata, with short cells not usually adjacent in arrangement, the tubercles are formed over the area, where two short and twolong cells are in contact. They are every so often fused together andform large warts.
In Section Coarctata, species belonging to this section show a great
variation in the structure of epidermal cells. One of the species, O.
ridleyi has not tubercles. Otherwise the epidermal cells are very similar
to that of the species belonging to Section Sativa.
In Section Rhynchoryza, small cells, which occurred rarely in the epidermis, have either a short hair or a protrusion. The cells are diagonally arranged. Cell walls between two cells are thin and apparently
1) This work was supported by Grant RF 57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1961

fused, then the dual partition between its cells is not discernible.
epidermal cell has one tubercle on its surface.

55

Each

39. Genome analysis in the genus Oryza IJIll(By Hitoshi KIHARA, Tadao C. KATAYAMA,
Seiji MATSUMURA and Tomoo MABUCHI)

Genome analysis of Oryza species was continued this year. 10 interspecific hybrids were newly produced, among which one hybrid was morphologically and cytologically investigated.
The F 1 hybrid between O. grandiglumis and O. latifolia showed invariably 24 bivalents at meiosis. The genome constitution of O. grandiglumis is apparently the same to that of O. latifolia and should be CCDD.
However, the hybrid was highly sterile, i.e., its seed fertility was only
2.7%. Those two species, therefore, seem to belong to different intersterile groups with the same genomes.

40.

Variation in blast susceptibility in wild rices closely
and distantly related to cultivated rice ll(By Keizo KATSUYA)In several wild rices, which are closely or distantly related to the cultivated rice and were collected from various countries, variation was
estimated in susceptibility to P-2 strain of Piricularia oryzae. The susceptibility was tested by the leaf-sheath method using the leaf sheath of
the next below the youngest leaf. 30 individuals were used in each strain,
and each individual was tested twice.
In general, closely related species to cultivated rice, Oryza perennis,
O. sativa f. spontanea, O. barthii and O. breviligulata, were highly heterogeneous with respect to blast susceptibility, while distantly related
species, O. officinalis, O. eichingeri, O. minuta, O. latifolia, O. alta andO. malabarensis, were homogeneous compared with the former. In susceptibility of species closely and distantly related to cultivated rice highly significant differences occurred between and within strains. O. latifolia
collected from Cuba and Guatemala, O. minuta from Malaya andPhilippines and O. perennis from Cuba were highly susceptible.
1) This work was supported by Grant RF 57080 from the Rockefeller Foundation.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

41. Some considerations on rice cultivation in New Guinea 1)(By Tadao C. KATAYAMA)

In Territory of Papua and New Guinea and Netherlands New Guinea,
several experiments on rice were started a few years ago at some experiment stations and are extensively carried on.
Some rice cultivation by the natives was found in several districts, but
it was mainly limited to those where Indonesians are living. Recently
those people have been routed out from their places, so that rice cultivation markedly decreased. However, in some areas rice is cultivated bya primitive shifting method. The strains used include both of "japonica
type" and "indica type" from the view point of morphological and physiological characters. The former are mainly used in Territory of Papua
and New Guinea and the latter are mainly used in Netherlands New
Guinea.
Some diseases were found in several districts such as Ramularia oryzae
and Sphaerulina oryzina. These diseases covered a large area in some
district. Two insects were found in New Guinea. One is Cnaphalocrocis
medinalis and the other is Nephotettix bipunctatus cincticeps. These insects occupy a large area and seem to become a serious problem in near
future. The natives usually leave rice fields, which have been severely
infected by insects or diseases, lying idle for several years.
As to the future of rice cultivation in New Guinea, an extension ofrice cultivation at the sea-side seems to be promising, because of abundant rain fall and high temperature; guidance and efforts of the government, especially in employing more efficient methods for upland cultivation and irrigation systems for lowland rice fields seem to be successful.
However, popularization of rice cultivation among the natives plannedby the government seems to be very difficult, because the natives hate
hard work and the laboring force is small, although rice is their favoredfood.

42. Some observations on the blooming of Oryza species ll(By Tadao C. KATAYAMA)

An investigation has been made on the mode of flowering of 21 Oryza
species, including date of flowering, order of blooming among the spikelets
1) This work was supported by Grant RF 57080 from the Rockefeller Founda·tion.

RESEARCHES CARRIED OUT IN 196157

of a panicle, number of flowers that bloom on successive days, time ofblooming, etc., because these characters are important for plant breeding
as well as ecological and phylogenetic studies of rice.
The blooming of spikelets in a panicle takes place in a regular sequence, that is, it is little influenced by environmental conditions suchas temperature and relative humidity. In all species blooming starts fromthe uppermost branch of a panicle and proceeds to the lower branches insuccession. However, some differences in the flowering order of spikelets
on a branch are found among several species. In many species, the blooming occurs first in the uppermost spikelet of a branch, followed by thelowest one and then proceeds toward the upper spikelets in consecutive
order. In 4 species, namely, O. glaberrima, O. breviligulata, O. stapfii
and O. coarctata, the blooming occurs first in the uppermost spikelet,
followed by a spikelet slightly nearer the base than the middle of the
branch and from there proceeds both upward and downward. In 2 otherspecies, O. brachyantha and O. subulata, blooming starts from the top ofa branch, proceeding straight downward.
In all species, but O. ridleyi and O. subulata, flowering of all spikelets
occurs continuously. In O. ridleyi, however, a male sterile spikelet on
the top of each branch flowers several days earlier than the other spikelets.
In O. subulata it takes several days between the flowering of a branch
and that of the following one.
The number of spikelets, which flower on each of successive days, isalmost constant for each species. The average number of spikelets,
which flower every day on a branch, is much smaller in the species ofSections Granulata, Coarctata and Rhynchoryza than in those of Section
Sativa.

43. Photoperiodic responses of Oryza species IVIl
(By Tadao C. KATAYAMA)

Using 23 Oryza species, intra-specific differentiation of photoperiodic
sensitivity and its relation to latitude were investigated.
Intra-specific differentiation. All species belonging to Section Sativa
contained both photoperiodically sensitive and insensitive strains, indicating intra-specific differentiation with respect to photoperiodic sensitivity. Percentages of sensitive strains were 79.6, 99.2 and 79.9%, respectively in the cultivated species (0. sativa and O. glaberrima), their
1) This work was supported by Grant RF 57080 from the Rockefeller Foundation.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

close wild relatives (0. sativa var. spontanea, O. perennis, O. barthii, O.
cubensis, O. breviligulata, and O. stapjii), and non-related species (0. australiensis, O. officinalis, O. minuta, O. malampuzhaensis, O. eichingeri, O.
punctata, O. latifolia, O. alta and O. grandiglumis). The heigher proportion of insensitive strains in the cultivated species than in the closely
related wild species seems to indicate that photoperiodic sensitivity isessential for the latter but its lack in the former is counterbalanced tosome extent by other economic characters.
The species belonging to the 3 other sections did not show any indication of intra-specific differentiation. All strains of O. granulata, O.
meyeriana and O. abromeitiana of the Section Granulata and O. subulataof the Section Rhynchoryza were photoperiodically insensitive, while thoseof O. coarctata, O. ridleyi and O. longiglumis of Coarctata section were allsensitive.

Relation between the latitude of habitats and the critical day length.
Among photoperiodically sensitive strains, a high negative correlation
was found between the latitude of their natural habitats and the criticalday length. The correlation coefficient obtained for all wild species was-0.6817 (d.f.=166). Strains collected in Assam showed some significant
deviation of their critical day length from that of the strains collected
at other localities in the same latitude, probably due to the high altitudeof Assam (ca. 5,000 ft.). The correlation coefficient became -0.8426(d.f.=111), when Assam's strains were omitted from the calculation.
The correlation coefficient for the cultivated rice, including both O.
sativa and O. glaberrima, was -0.4171 (d.f.=115), the correlation being
much smaller than that obtained for the wild rices. This fact also suggests that photoperiodic adaptation is more important for the wild rices
than for the cultivated ones.

44. Intra-plant variation in seed size in rice
(By Kan-Ichi SAKAI and Akio SUZUKI)

In the biometrical study of length and breadth of seeds in wild andcultivated rice strains, it was noticed that intra-plant variability wasless in the former than in the latter. This variability is the measureof the developmental stability in seed size. Among wild species, Oryza
perennis and O. rujipogon were much different from each other in respectof the intra-plant variability, though there seemed to be some difference
between geographical strains within the same species.
No evidence, supporting either less variability in the heterozygotes orintra-plant variability due to genic segregation, was obtained in a hy-

RESEARCHES CARRIED OUT IN 1961

59

bridization experiment of Japanese rice varieties. Thus, it seems rather
reasonable to assume that higher stability in seed size in wild riceforms than in the cultivated ones could be attributed to the effect oflong-term natural selections. (See also No. 90 on page 107)

45.

Variation in susceptibility to blast disease in wild
and cultivated rice
(By Iwasaburo GOTO and Kan-Ichi SAKAI)

The degree of susceptibility to blast disease in various strains of wild
and cultivated rice was determined by the leaf-sheath inoculation method
using strain 53-33 for Piricularia oryzae, kindly supplied by the National
Institute for Agricultural Sciences, Tokyo.
Among six geographical groups of cultivated varieties, a marked
difference was observed in the degree of susceptibility. The six groups canbe arranged in the order of their susceptibility as follows: Africa> Japan> South America> Java, Thailand and Ceylon. African varieties belong
to Oryza glaberrima, while those of Japan represent O. sativa japonica,
the remainders belonging to O. sativa indica.
The progeny of three wild populations formerly collected in Ceylon in
1959 and grown for one generation in Formosa in 1960, were tested fortheir within- and between-population variability. Partition of variance
was made into three components, i.e. variance due to environmental
causes, VI'), variance between plants within line within variety, VI. andvariance between varieties or lines, V:~. Variability between plants
within line is measured by VII( V B+ VI), and variability between lines orvarieties by VsI(Vs + V I + VB). There was no distinguishable differencein the degree of susceptibility between cultivated and wild rices. It
was found that the within-line variability was higher in wild strains
than in cultivated ones while the between lines within strain variability
was in the reverse order. This may mean that wild rice is more heterozygous with regard to blast susceptibility than cultivated rice. It is
widely accepted that the susceptibility of rice to blast disease considerably varies depending upon the level of nitrogenous fertilization, thehigher the dose of N-fertilizers the more susceptible become the plants.
Let the susceptibility of a given plant to a certain disease be expressedby the following model:s = c +b
where S is the susceptibility of the plant growing in a given condition
while c and b stand for the susceptibility of the plant determined

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

by resistant genes, if any, and that produced by environmental or cultivation conditions, respectively. Then, the inter-strain variance of theobserved susceptibility could be partitioned as follows:
V s = Ve + Vb + 2 Web.
Thus, we may be able to define
V e/ V s as degree of inherent susceptibility (De)V b/ V s as degree of environment-respondent susceptibility (Db)
2 W eb / V s as degree of covariation between the two (D be ).
On the basis of this principle of partition, it has been concluded thatin case of blast-disease, the degree of inherent susceptibility was verysmall while the degree of environment -respondent susceptibility waslarge, in other words, susceptibility of rice to Piricularia fungus would
be mostly dependent upon the pathological response of genotypes tofertilizer application. Wild rice seemed different from cultivated ricein that the former had a lower De but a higher Db than the latter.

46.

Rice varieties intermediate between wild and cultivatedforms and the origin of the Japonica type
(By Hiko-Ichi OKA and Wen-Tsai CHANG)

Investigations of rice varieties collected from the Jeypore Tract, India(partly reported by the writers in Ann. Rep. 9, 1958) were completed in
1961. Variations in the character complex of the strains were studied
with three discriminant formulas, for classifying perennis vs. spontanea
types of wild rice, wild vs. cultivated rice forms, and the Indica vs. Japonica
types of cultivated rice, respectively. A greater part of the strains wereintermediate between wild and cultivated forms, and those approachingwild forms were of the perennis type. This indicates that cultivated ricemight have evolved from the perennis type of wild rice. It was also
found that those strains would probably become differentiated into Indica
and Japonica types as they approach cultivated forms, indicating thatthe two types are monophyletic. Most of the Jeypore strains showed
high F 1 fertilities with test-strains of different types whose mutual F 1
hybrids were partly sterile, in the same manner as wild strains of O.
perennis. Their hybrid-sterility relationships did not show correlation
with variations in other characters. (Pub!. in Bot. Bull. Acad. Sinica 3,
in press).

RESEARCHES CARRIED OUT IN 1961

61

47. Responses to various growing conditions of wild
and cultivated rice forms(By Hiko-Ichi OKA and Wen-Tsai CHANG)

A set of ten rice varieties ranging from wild (spontanea type of O.
perennis) to improved cultivated forms CO. sativa), in which intermediate
wild-cultivated forms from the Jeypore Tract, India, and native varietiesof tropical Asian countries were included, were tested in different growing conditions in the experimental field of Taiwan Provincial Agricultural
College, Taichung. It was found in general that the nearer the genotype
approached improved cultivated varieties, the higher would become theresponse to fertilizer application, weeding and transplanting, which may
be regarded as elements of intensive rice culture in Asia. It was suggested that the evolution of genotype and that of environment mightproceed in parallel.

48. Intra-specific variations of Oryza breviligulata
in West Africa(By Hiroko MORISHIMA and Hiko-Ichi OKA)

Oryza breviligulata A. Cheval. et Roehr. is a wild species geneticallyin close relation with the cultivated rice species, O. glaberrima Steud.,and is distributed in West Africa. From the results of variation studies
in various characters of this species, the directions of intra-specific differentiation were investigated by using the technique of factor analysis.
Only the first factor had an important contribution, which represented
the variation of those wild forms in the direction of cultivated type.
Actually, some strains were in various characters approaching cultivatedvarieties of O. glaberrima (c.f. Morishima and Oka, 1960, Ann. Rep. 11).
It was pointed out that O. stapfii Roschev. was synonimous with O.
breviligulata. It was found further that O. breviligulata had a smaller
amount of genetic variability in its populations than O. perennis, butvaried in a wider range among populations. The results of analysis of
intra-populational variations suggest that this annual wild species might
have a relatively high amount of self-pollination.
49. Differences in breeding behavior between wild
and cultivated rice species(By Kokichi HINATA, Hiroko MORISHIMA
and Hiko-Ichi OKA)

It is known that O. perennis is partially allogamous, while O. sativa

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

is nearly autogamous. With regard to this difference in breeding behavior, the pollinating systems of the two species were compared. It
was found that O. perennis had 1) longer anthers (more pollen-grains),
2) longer duration of life of pollen-grains emitted from the anthers, 3)
longer time interval from flower openning to pollen emission, and 4)
more extruded stigmas, than O. sativa. The former two conditions
might be related to the capacity of plants to pollinate other plants, andthe latter two to the probability of being pollinated by others. Thus, inboth respects, O. perennis seems to be suited for outcrossing. Further,regarding the longevity of seeds, it was found that seeds of O. perennis
did not germinate when immersed in water, but could be kept alive inwater or in moist soil for more than two years. This behavior would
bring about overlapping of generations in the natural habitat.

50.

Tetraploid F 1 hybrid between Oryza sativa
and O. glaberrima(By Kokichi HINATA and Hiko-Ichi OKA)

Two tetraploid strains induced by colchicine treatment of seedlings,
one belonging to O. glaberrima and the other to O. sativa, were crossed.
The F 1 plants showed partial fertility, namely about 45% good pollengrains and 25% seeds set. The number of quadrivalent chromosomes
per P.M.C. was 4.37 on the average, ranging from 3 to 10; it was smaller
by about two than that found in the parental autotetraploid strains.
However, the same decrease in the number of quadrivalent chromosomes
is also found in tetraploid F 1 hybrids between varieties of O. sativa. It
then appears difficult to evaluate by this method how the two species
differ in chromosome structure. Observation of segregation ratios intetraploid F 2 hybrids is under way.
51.

Hybrid-sterility relationships among Asian strainsof Oryza perennis and O. sativa(By Kokichi HINATA and Hiko-Ichi OKA)

A number of strains of O. perennis Moench. (including those of O. sativa
f. spontanea Roschev.), collected from India, Thailand, Malaya and other
Asian countries, were each crossed with a set of test-strains belonging toO. sativa and O. perennis, and the F 1 fertility data were compared withthose formerly obtained by the second writer for sativa strains. As pointed
out by the writers before (Ann. Rep. 11, 1960), the perennis strains mostly

RESEARCHES CARRIED OUT IN 1961

63

showed high F I fertilities with the test - strains. It was suggested,from the results of multi-variate analysis of the data, that the F , sterility
found between sativa varieties might be on the increase with the approach
to cultivated forms. However, as aka and Chang (1959, Ann. Rep. 10)
showed, populations of O. perennis were found to contain variations ofsterility factors, mainly in the form heterozygotes. These features arewell explained by the hypothesis of gametic-development genes for the F I
sterility between sativa varieties.

52. Investigation on the measuring method of theintercellular spaces on several plants(By Tadao C. KATAYAMA)On the aerating system of roots and leaves and its physiological significance in several plants many works have been published. In the caseof rice plants, possibility of oxygen transfer from the top to the roots
has been discussed with a view to understanding root respiration underpaddy field conditions, which may be closely related to their resistance
to excessive soil moisture.
As a quantitative determination of the total intercellular air space inwhole part of plant is lacking, the author investigated the measuringmethod of its volume using several plants, as Oryza sativa, Ligustrum
japonicum, Daphne odora, etc.
The volumes of plant organs and their intercellular spaces can be
simply measured by means of measuring both gravity and buoyancy
methods. The latter method surpasses the former one in accuracy ofmeasurement. The buoyancy method is achieved by the following way;materials are first weighed in air (G I ), secondly weighed in the condition
submerged in fluid such as water or liquid paraffin (G 2 ), and once again
weighed in fluid after having been infiltrated with fluid using an electricvacuum pum) (G 3 ); in the case of gravity method, materials are once
more weighed in air (G.) in addition to the weighing procedure of threetimes mentioned above.
Computations were made by the following formulae:
VI
V2
Va

G,-G 2p-Pa

G.-G 3p-Pa

G3 -G 2p-Pa

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

C,-C I
p-Pa

VI and V2 :volume of organs in the case of buoyancy and gravity
methods,
Va and V g : volume of intercellular spaces in the case of buoyancy
and gravity methods,
P and Pa: density of fluid and air, respectively.
At the measurement of organ volume, it was confirmed that themeasuring error was less than 1%. By these measuring methods, it ispossible to measure not only in the case of turgescent organ but also
wilted one. Using vacuum pump, precaution should be taken that air
should not be remained in the organ. However, it was also confirmedthat the measuring error owing to the above cause did not exceed 2%
at 20°C. In addition, some conceivable theoretical errors caused byshrinking, deformation, infiltration of the organs were discussed. But
it was certified that they were quite negligible.

53.

Arisaema triphyllum, Jack-in-the-Pulpit, in
Minnesota, especially at the Cedar Creek
Natural History Area
(Sadao SAKAMOTO)

In Minnesota two species of Arisaema, A. triphyllum (L.) Schott. with
two subspecies: subsp. triphyllum Huttleston and subsp. Stewardsonii
Huttleston and A. Dracontium (L.) Schott., are found. The distribution
map of these species based upon the herbarium specimens was studied.
Arisaema triphyllum subsp. triphyllum is most common in Minnesota; A.
DracontiuJ11 occurs only in the southeast corner of the state. In specimens of subsp. triphyllum, three color phase variations of the spathe
were observed; pale green with no stripes (type A), pale green withlight purple stripes (type B) and pale green with deep purple stripes(type C). Type B is most common in Minnesota. Type A is distributedat random. Type C is only found in the northeast part of the state.
The following observations were carried out at three observation-stations in the Cedar Creek Natural History Area, Minnesota.
Color variations in inflorescence and flower parts were very characteristic. Generally, the plant having purple color in spathe, in spadix
and in anthers (male plant), and no purple color in stigmata (female
plant), are most common in this Area.
Flowers in the male inflorescence start to bloom from the middle of

RESEARCHES CARRIED OUT IN 1961

65

inflorescence toward both upper and lower extremities. Range andaverage number of flowers per inflorescence are greater in female thanin male. Three types of inflorescence, male, intermediate (monoecious)and female, were found. Proportions of these types differed among thestations according to the environmental conditions. In total, the proportion of male plants was 71.4%, of intermediate 5.9%, and female 22.7%
in the populations studied. The plants with a single foliage leaf wereusually males, and those with two foliage leaves usually females.
Of 12 quantitative characters measured, all are greater in female thanin male except length of the peduncle.
Fruit- and seed-fertility of each plant were decidedly variable and low,
and the average of 12 plants showed 52.5% and 17.6%, respectively. On
the contrary, average number of seeds per fruit of each plant was very
uniform, ranging from 1.0 to 2.2 and with 1.6 as the average. The ovary
contained 3 to 7 ovules and 4.7 ovules per ovary was the average.
Number of seeds per fruit was very different from the number of ovules
per ovary. It is assumed that one of the factors causing such prominent
differences would be something like developmental competition for foodamong the fertilized ovules during the young stage of fruit development.

54. Fertility and crossability of certain wild bramblesof Minnesota and Wisconsin(By Sadao SAKAMOTO and Arthur N. WILCOX*)

The identification of 48 strains of Rubus collected in Minnesota and in
Wisconsin was studied. In this collection, 7 species were identified: R.
strigosus, R. occidentalis, R. allegheniensis, R. flagellaris, R. minnesotanus,
R. Groutianus and R. vermontanus.
From their records of performance under cultivation at the Fruit Breeding Farm during 1958-1961, 9 strains were recognized as relatively winterhardy.
Seventeen strains of blackberries and dewberries were selected forfurther examination. Chromosome numbers of each strain were determined. In R. allegheniensis, diploid and triploid strains were found. In
R. minnesotanus, tetraploid (?) and pentaploid were observed. R. flagellaris was pentaploid. R. Groutianus and R. vermontanus were all tetraploid. The origin of different ploidy forms was discussed.
Several horticultural characters, such as tip-rooting, seed weight, etc.,
were observed.
* Department of Horticulture, Institute of Agriculture, University of Minnesota,
St. Paul, Minnesota, U.S.A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Pollen fertility and pollen germination on culture media were variablefrom strain to strain.
Fruit fertility was also very variable. The highest self- and openpollinated fruit fertility obtained were 28 and 38 per cent, respectively,
in a strain of R. minnesotanus. The ratio between self- and open-pollinated fertility was calculated. Three strains of R. allegheniensis andone of R. minnesotanus showed low values of the ratio.
Crosses among blackberries and dewberries, and between the blackberry
and dewberry were made and studied. Cross-pollinated fruit fertility
ranged from 0 to 12.4 per cent. Crossability was higher in the crosses
within sections of the genus than in those of intersectional combinations.
Crosses between the blackberry or the dewberry and the cultivated red
raspberry were made in an exploratory way, looking toward breeding for
new combinations of desirable characters. When crosses were made between the red raspberry as the female parent and the blackberry or thedewberry as the male parent, they were much more successful in the
production of seed than in the reverse combination.

55.

The origin of Prunus yedoensis(By YO TAKENAKA)

Prunus yedoensis Matsumura (Somei-Yoshino) is the most famous flowering cherry tree grown in Japan. Its origin has been unknown, however.
This species has abundant beautiful flowers, but sets only a few seeds.
Accordingly, it can be propagated only by grafting. However, it grows
more rapidly than any other cherry tree. From these facts, I haveassumed that it might be a hybrid. I gathered seeds from many treesof this species in 1952. The seeds were sown in 1953, and the seedlings
were observed from 1954 to 1962. From these observations, P. yedoensis
was considered to be a hybrid between P. lannesiana var. speciosa (Oshima-Zakura) and P. subhirtella var. pendula forma ascendens (Edo-Hig:m),whose characteristics are as follows: speciosa is distinguished fromascendens by underneath glabrous and larger leaves, and vigorously
growing stems. The seedlings of yedoensis showed in these respects
a series of intergrades ranging from speciosa to ascendens types. Someof the seedlings bloomed in the spring of 1959, and since then, 25 trees
had flowers. In many flower characters, viz., size, color, and hairinessat peduncle, receptacle, calyx, style and ovule, the trees showed a wide
range of variation from speciosa to ascendens.
In further experiments started in 1957, I made reciprocal crosses between speciosa and ascendens. In 1961, 8 hybrid plants bloomed. TheyRESEARCHES CARRIED OUT IN 196167

were intermediate between the parents in the characters of stem, leaf
and flower, and appeared as a whole to be similar to one another, though
they showed some minor differences. Two of them had hairs on thestyle and ovule like P. yedoensis, and the other two had a few hairs,
while the rest were hairless. In other respects, they were similar to
P. yedoensis, though the size of leaves and flowers was about 15% to
20% larger.
It seems that the parental trees used for this crossing experiment,
which were taken from populations of the respective species, have beenheterozygous. Accordingly, the hybrid plants might have segregated as
to hairiness and some other minor characters. It may be concluded that
P. yedoensis is a hybrid between speciosa and ascendens.
The question arises from where P. yedoensis was introduced. Tradition
says that it was brought over from Oshima, one of the Izu islands. But
on the island the only flowering cherry growing wild is P. lannesiana
var. speciosa.
Dr. E. Koehne (1912) named a flowering cherry tree collected by Taquat(a missionary) from Quelpart island of Korea P. yedonisis var. nudijiora.
Later, Dr. G. Koizumi believed that P. yedoensis was brought from
Quelpart island. On the other hand, Dr. E. H. Wilson (1916) stated: "Tome P. yedoensis Matsumura strongly suggests a hybrid between P. subhirtella var. ascendens Wilson and the wild form P. lannesiana Wilson.
It has many characters of the latter and in its venation, pubescens andshape of the cupula resembles the former." Dr. Koizumi went to Quelpart island in 1932 and found one tree which he identified with P.
yedoensis. Since his identification was questionable, I visited the island
in 1933 and observed the tree. The tree, which was growing wild,
showed difference from P. yedoensis; the amount of hairs on calyx lobes
and back side of leaves was less, and the peduncles were shorter thanthose of the latter. At any rate, it was not P. yedoensis. I assume thatit has been a hybrid between P. subhirtella var. pendula f. ascendens
and P. quelpartensis (Tanna-Yamazakura; perhaps a form of P. verecunda)or other cherry species.
In Japan, the putative parental species of P. yedoensis, P. speciosa andP. ascendens, were reported to grow together in Boso-peninsula and Izupeninsula. I surveyed wild cherry trees in these two districts. Thoughboth species were found in Izu-peninsula, only speciosa was found in
Boso-peninsula. In Izu, further, I found trees which were presumably
the offspring of P. yedoensis, and a tree which appeared to be a new
hybrid between speciosa and ascendens. That tree was identical with
one of my synthesized trees. It then seems most probable that P.
yedoensis has originated in Izu-peninsula.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

56. Cytogenetic studies in the genus Nicotiana XIV(By YO TAKENAKA)

The reduction division in PMC's was studied in 4 hybrids between N.
paniculata and 4 other species of section Alatae, namely N. paniculata X
N. alata, N. paniculataxN. langsdorffii, N. paniculataxN. plumbaginifolia
and N. paniculataxN. longiflora.
1) F 1 of N. paniculata (n=12)xN. alata (n=9).
At MI of PMC's of this hybrid, the number of bivalents ranged fromo to 6, with the mode at 3. The frequency of PMC's with 4 and 2
bivalents followed that of PMC's with 3 bivalents. PMC's with 1 and5 bivalents were occasionally found but those with 6 bivalents or without
bivalents were very rare.
In the same hybrid, Kostoff (1943) found that the number of bivalents
ranged from 2 to 7 and observed occasionally a few trivalents. His observation of trivalents is doubtful, because the trivalent-like chromosomes
in my preparations were heteromorphic bivalents. Otherwise Kostoff's
results generally agree with mine.
2) F 1 of N. paniculata (n=12)xN. langsdorffii (n=9).
At MI of PMC's of this hybrid, bivalent range was from 0 to 4 withthe mode at 1 and 2. The frequency of PMC's with 0 bivalents followedthat of PMC's with 1 or 2 bivalents. PMC's with 3 bivalents were occasionally found but those with 4 bivalents were very rare.
In the same hybrid, Goodspeed (1954) found that the number of bivalents ranged from 0 to 4, with the mode at 2. His results agree withmine. According to Kostoff's description (1943), he and also Dremlug
observed in the same hybrid that the number of bivalents ranged fromo to 7 and some trivalents were present. The difference between the
results observed by Goodspeed and also myself and those of Kostoff andDremlug is too large to be attributed to different strains used by theauthors.
3) F 1 of N. paniculata (n=12)xN. Plumbaginifolia (n=10).
So far as I know, no investigation of this hybrid has been published.
At the MI of PMC's, the bivalent range was from 0 to 3, with themode at O. Accordingly N. paniculata is assumed to be less related toN. plumbaginifolia than to N. alata and N. langsdorffii.
4) F 1 of N. paniculata (n=12)xN. longiflora (n=10)
No findings on this hybrid have been reported till now, so far as Iknow.
At MI of the PMC's of this hybrid, the number of bivalents ranged

RESEARCHES CARRIED OUT IN 1961

69

from 0 to 2, with the mode at O. N. longijlora is very closely related
to N. plumbaginifolia and the chromosome pairing in the hybrids N.
paniculata x N. longijlora and N. paniculata x N. plumbaginifolia wassimilar.
From the results of the chromosome pairing in the four hybrids mentioned above, it is assumed that N. paniculata is not related to the above
four species belonging to section Alatae.

57.

Alkaloid decomposition by tobacco extract(By Masao TANAKA)

The citric acid extract of pulverized tobacco leaves developed on achromatogram with 50% acetone contains a substance which decomposes
tobacco alkaloid.
On the paper strip on which the extract has been developed, a yellowish
orange spot of alkaloid appears at the position of Rf 0.85 when it is exposed to the vapour of cyanogen bromide after spraying with an ethanolic
solution of p-aminobenzoic acid. When the developed chromatogram istreated in the same way as that of the cited above after spraying witha dilute solution of alkaloid, two spots of lighter hue resulted fromthe lack of reaction appear adjacent to the anterior and posterior ends ofthe original spot of alkaloid on the yellowish orange background of thepaper. The posterior spot is larger than that of the anterior one.
Decomposition of alkaloid can be demonstrated numerically by nephelometric analysis-by measureing the density of precipitation caused byadding a solution of silicotungustic acid-of alkaloid solutions in which
divided chromatograms have been dipped and incubated for several hours.
Alkaloid decomposition by tobacco extract is detectable in majority of
tobacco varieties. It is activated by the exposure of ultraviolet light andprevented by the spray with a dilute solution of pepsin. Nornicotine
seems to be more easily decomposed than nicotine. This phenomenon
corresponds with the facts that leaves of the tobacco varieties which
possess nicotine-nornicotine converting factors have a lower level ofalkaloids in comparison with those of the varieties without it, and thatmajority of the low alkaloid species of Nicotiana are nornicotine types.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

58.

Inheritance of anthocyanin concentrations
in Torenia flowers(By Toru ENDO)

Torenia fournieri Lind. is known to occur in flower colors, deep bluepurple and white. Two plants with pale blue-purple flowers appearedin the sample of the deep blue-purple form. The flowers of deep andpale blue-purple forms and those of six F I hybrids among all three forms
were determined to contain the same three anthocyanins, namely, malvidin-, petunidin- and delphinidin-glycosides. The white form is slightly
purplish on the lower lip, where traces of the anthocyanins were detected.
Malvidin-glycoside is the major component in all materials; it was isolated
in crystalline form and identified with malvino The other two are minor
components.
It was concluded from F 2 segregations that the genotypes of the threeforms were AABB, AAbb and aaBB for deep blue-purple, pale blue-purple
and white, respectively, and that A and B are basic complementary genes
controlling anthocyanin formation. The genotype aabb may be ascribedto the acyanic segregants with pure-white flowers. Further, the effects
of genes A and B on anthocyanin formation were estimated for six different genotypes, as shown in Table 1. In the table, the relative concentrations of anthocyanins are optical density at absorption maximum,
524 mp, of 1% aqueous hydrochloric acid extracts from fresh flowers.
Thus the concentrations of the anthocyanins show the following reTable 1.

Parentage
BP
pP
W
BPxW
WxBP
BPxpP
pPxBP
pPxW
WxpP

Relative concentrations of anthocyanins in Torenia
flowers of different genotypes

I Flower color IGenotype INo of I Variation
I O.D./flower
in O.D.
pl~ntsdeep blue-purple
pale blue-purple
white
deep blue-purple
deep blue· purple
deep blue-purple
deep blue·purple
blue-purple
blue-purple

AABB
AAbb
aaBB
AaBB
AaBB
AABb
AABb
AaBb
AaBb

25
25
5
25
25
25
25
25
25

O. 737 ~1.140
0.037 ~0.242
0.013~0.032

o.464 ~ 1. 060
0.710~1.130
0.592~ 1.125

0.472~0.862
0.441~0. 760
O. 264~0. 772* sIgnificant at the 5% level as compared with AABB.

0.869±0.254
0.096±0.013
0.024±0.007
0.898±0.215
0.902±0.288
O. 824±0 .128
0.703±0.103*
0.596±0.091*
0.416±0.157*

RESEARCHES CARRIED OUT IN 196171

lations,
AABB= AaBB > AABb > AaBb > AAbb > aaBB > aabb=O.
It was found that dominance of A over a is complete and that of B is
incomplete under the present environmental conditions.

59.

Cytological studies on the nuclei of yeasts(By Yoshiaki YONEDA)

The nuclear structures and the division process of five species ofyeasts, namely a strain of Saccharomyces, Schizosaccharomyces pombe,
Lipomyces starkeyi, Torula utilis and Torula rubra were studied.
The nucleus of all species contained chromatin and a fairly large
nucleolus. The chromatin was Feulgen-positive and stained with acetoorcein and Giemsa. The chromatin of Saccharomyces, Schizo pombe andT. utilis also stained with methyl green. The nucleolus was Feulgennegative and stained densely with haematoxylin and aceto-carmine.
Giemsa staining revealed both the chromatin and the nucleolus, but thelatter took a more reddish color than the former. The nucleolus of
Saccharomyces also stained with pyronine. Besides the chromatin and thenucleolus, a nuclear membrane and karyoplasm were also recognized in
the nucleus.
The nuclear division of yeast proceeded in two stages, namely in thefirst stage the division of the chromatin (prophase to telophase) took
place (dividing stage), and in the second stage followed the separationof the divided chromatin (separation stage). The homogeneous chromatinat resting stage granulated at prophase and chromosomal bodies appearedat metaphase. Six or more such bodies were recognized in Saccharomyces,
about four in Lip. starkeyi and four in T. utilis. In T. rubra, such bodies
were also recognized, but their number was not determined. In Schizo
pombe, chromosomal bodies were not clearly distinguished. Anaphaseand telophase were carried out on one side of the nucleoli. In thesestages, the nucleoli of four species except Saccharomyces kept theirspherical shape. The nucleolus of Saccharomyces changed the shape inthese stages. The divided chromatin condensed at telophase.
In the separation stage, the nucleoli of Saccharomyces and Schizo pombe
elongated, became constricted at the midregion and separated into daughter nuclei, while those of Lip. starkeyi, T. utilis and T. rubra were left
behind in the mother cells and disintegrated there. In the latter species,
the daughter nucleoli seem to be synthesized de novo in the daughter
nuclei. Thus, two types of nucleoli were found with regard to their

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

behavior during nuclear division.
In T. utilis and Lip. starkeyi, a slender structure was found, whichconnected two masses of separating chromatin. The chromatin was
assumed to be separated by elongation of this structure. This findingof the spindle-like structure suggests a possibility that the spindle mechanism also operates during the dividiug stage of T. utilis and Lip.
starkeyi.
It was confirmed by staining with Feulgen-carmine method that thenucleus of Saccharomyces is a small body lying outside of the central
vacuole. A central granule was found at the center of metaphase chromatin ring in Saccharomyces. It seems to divide at anaphase.
The vegetative nuclei of yeasts are assumed to multiply by mitosis.
However, the behavior of the nucleoli during nuclear division seems to
be different from those of higher organisms.

C.
60.

MATHEMATICAL GENETICS

A general formula for the probability of fixationof mutant genes in a population(By Motoo KIMURA)

In this report I will consider the probability of fixation of genes in arandom mating population of effective size N. Let u(P, t) be the probability that a mutant gene becomes fixed in the population (Le. its
relative frequency becomes 1) by the tth generation, given that its frequency is p at t=O. If M 8p and V8p are respectively the mean and thevariance of the rate of change in p per generation, then u(P, t) satisfies
the following equation,1)
au(p,t) 1 V: a2u(p, t) + M Du(P,t)(
at
2 8p ap2
8p
ap ,with boundary conditions ofu(O,t)=o,u(l,t)=l.
The probability of ultimate fixation denoted by u(P), i.e.
u(P) = lim u(P, t)

(2)

t~<x>

may be obtained from (1) by putting au/at=o:~ V: d 2u(P)+M du(P)_O(3)
2 8p dp2
8p dp ,{
u(O)=O, u(l)=l.

RESEARCHES CARRIED OUT IN 1961

73

Thus we obtain
(4 )

~:

_
G(x)dxu(P)- ~l'G(x)dx
o

where

r

2MOX

G(x) = e-rvS;dX•

The above formula (4), which gives the probability of ultimate fixationof a mutant gene with initial frequency P, has simplicity and generality
comparable to Wright's well·known formula for the gene frequency distribution at equilibrium. It includes previous results as special casesand can be used to solve problems where there is random fluctuation in
selection intensity.

61. On circular mating systems(By Motoo KIMURA)

It is well known that in a random mating population of N monoecious
individuals, the frequency of heterozygotes decreases at the rate of
1/(2N) per generation, as first shown by Wright. It was also found by
him that in a population consisting of n males and n females (N=2n)the heterozygosity decreases asymptotically at the rate of 1/(2N) under randommating, while it decreases at the rateof 1/(4N) under what he calls maximum
avoidance of consanguineous mating.
The purpose of this note is to reportan unexpected finding that in matinglasystems which I would like to call circular mating systems, the asymptotic
rate of decrease of heterozygosity ismuch smaller.
In the simplest case of what may becalled the individual circular matinglb(Fig. 1a) n males and n females are
Figs. la and lb. Examples ofarranged alternately and each individual
circular mating systems involving
is mated to adjacent individuals to form, 8 individuals. ]a: circular indiviso to speak, a circle. For this mating dual mating. lb: circular pairsystem, the ratio A by which heterozy- mating.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

gosity decreases each generation satisfies the equationI(l)

A=.l+.lcos 022

sin O=cot nO,

from which we can obtain the ultimate rate of decrease in heterozygosity:
rr 2

(2 )

.o=l-:-A=----,..,----

16(n+l)2

2 54!(n+1)4

where A is the largest root of the equation (1).
rr

(3 )

Thus for a large N,52

e-- 4N2 = 2N2 .

This may be compared with the corresponding value in the case of"maximum avoidance" of consanguineous mating, which is
1(4)

s......., 4N-

For example, 100 individuals in (3) is approximately equivalent to 1,000
individuals in (4).
Circular mating may also be carried out between subpopulations insuch a way that they are arranged on a circle and males in each subpopulation are mated to females in a neighboring subpopulation. The
simplest case of this type of mating is circular pair mating illustrated
in Fig. lb. In this case, it can be shown that the ultimate rate ofdecrease in heterozygosity is asymptotically(5 )i.e. only 1/4 as slow as in the case of the circular individual mating,though it can still be much better than the "maximum avoidance" system in keeping heterozygosity if N is large.
Investigation on the properties of the circular mating systems is inprogress in collaboration with Dr. J. F. Crow.

62.

The maintenance of supernumerary chromosome
in wild populations of Lilium callosum
by preferential segregation(By Motoo KIMURA)

In Lilium callosum, a supernumerary chromosome called fl (meaning
long fragment) by Kayano shows meiotic drive and tends to increase

RESEARCHES CARRIED OUT IN 196175

its number through maternal line transmission. It markedly reduces
both pollen and seed fertility when more than one is present. The frequency distribution of fl in natural populations was investigated. The
analysis has shown that individuals with fl's are not only less fertile but
also less viable. A mathematical theory was developed to analyse themechanism of distribution of fl in the populations. The concept of distortional load (or load due to meiotic drive) was applied to the maintenance of fl in the natural populations and it was estimated that thisload amounts to about 17 percent. The detail of the present investigation has been published recently in GENETICS in collaboration with H.
Kayano.
Results of this investigation seem to suggest a general mechanism bywhich a relatively large but inert supernumerary chromosome is evolvedin the history of a species.
For any supernumerary chromosome to be permanently maintained ina population, probably it must be endowed with meiotic drive (i.e. segregation distortion) mechanism from the outset so that it is protected fromloss either by selection or random genetic drift. Such a supernumerarychromosome which is derived originally from the proper set of chromosomes must be deleterious for the possessor because of the trisomic condition that it creates and the segregation distortion has to be sufficiently strong to counterbalance the selection pressure. The spontaneous
origin of the meiotic drive mechanism might be due to mutation in thecentromeric region or by some other unknown cause, but recent discovery of SD factor in Drosophila melanogater by Hiraizumi seems tosuggest that such event is quite probable in the long history of at least
some species. Once infected by such mechanism, the genetic load will
immediately be imposed by such segregation distortion. Thus for the
species to survive, it must either get rid of the supernumerary chromosome by suppressing the distortion or to reduce the load by making
the effect of the supernumerary chromosome less deleterious. In thelatter case, which is of interest in the present context, both evolutionary
modification of the proper genotype by natural selection and loss infunction of the supernumerary chromosome due to the accumulation of
amorphic mutations in it must be effective. The relative importance of
these two factors may be evaluated by finding two completely isolated
but closely related races, one having a large supernumerary chromosome
at a very high frequency and the other entirely lacking of it and bycrossing them to see if introduction of the supernumerary chromosome
from the former race into the latter will greatly influence the fitnessof the latter or not. If the apparent inertness of the supernumerary chromosome is really due to accumulation of amorphic mutations, no exces-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

sively strong deleterious effect will be found upon introduction into theother race.
63.

Distorted segregation and substitutional load
(By Yuichiro HIRAIZUMI)Let Pt and qt be respectively the frequencies of a driven (A 2 ) and its
normal element (AI) in the gamates of t-th generation. The fitnesses ofthree genotypes and their frequencies are listed below. Here we assume
random mating.
Genotype
Fitness
Frequency

AlAI
l-s
qt 2

A IA 2l-s(l-h)2Ptqt

A 2A 21
Pt 2

The average fitness of this population is
w=1-s(1-Pt){Pt(1-2h)+1} ,and the load in the t-th generation is given by
L t =s(1-Pt){Pt(1-2h)+1} .

Let k be the segregation ratio of A 2 from a heterozygote A IA 2 in thenext generation (1~k>O.5), then we have the following equation for thefrequency change of A 2 •dPt =Pt(l-P,) {Pt s(1-2h)+2k(1-s+sh)-(1-s)}.
dt
w

The total substitutional load is then computed to beL.~lcoL,dt=.Jo

II s{Pt(1-2h)+1}[1-s(1-Pt){Pt(1-2h)+1}] #th o Pt{Pts(1-2h)+(1-s)(2k-l)+2ksh}.

If s~l, this is approximated by

L.=sllPt(1-2h)+1

h o Pt{Pts(1-2h)+(1-s)(2k-l)+2ksh}#t= -[s log Po+{2ks(1-h)-(2k-l)}s(l-2h)+(1-s)(2k-l)+2ksh
xlog{ Pos(1-2h)+(1-s)(2k-l)+2ksh}Jj{(1-s)(2k-l)+2ksh} ,where Po is the initial frequency of A 2 • When 2k-l=O (segregation
normal), the above solution can be reduced to a simple form obtainedby Kimura (1959). The numerical examples of L. are given in Table 1.

RESEARCHES CARRIED OUT IN 1961

77

When 2k-1 =0, L is independent of s but with drive it is no more
independent; a larger value of s involves a larger substitutional load.
It is worth noting that h=l, Le., complete dominance of A z over All
reduces L from 103.6 to 4.6 when k=0.50oo. On the other hand, k=
0.5100 (only 0.01 in excess) reduces the load to the same, or less extent.
This shows how effectively a slight drive works to accelerate the accumulation of a beneficial element in a population.
B

B

Table 1. Substitutional load, L B , under various values ofk, h, and 8. The initial frequency of p is
0.01 for all the casesk

LB

I 8=0.005 I 0.01 I 0.02

00.50000.50010.50100.51000.5500

103.6
47.0
9.7
1.3
0.3

103.6
57.1
16.2
2.5
0.6

103.6
71.4
25.5
4.6
1.1

0.001

0.50000.50010.50100.51000.5500

99.0
42.0
9.7
1.3
0.3

99.0
55.2
16.2
2.5
0.6

99.0
67.4
25.5
4.6
1.1

0.01

0.50000.50010.50100.51000.5500

73.2
37.9
9.6
1.3
0.3

73.2
48.9
15.7
2.5
0.6

73.2
58.5
23.8
4.6
1.1

0.1

0.50000.50010.50100.51000.5500

27.0
21.6
8.9
1.2
0.3

27.0
23.9
11.8
2.4
0.5

27.0
25.4
17.0
4.3
1.0

1

0.50000.50010.50100.51000.5500

4.6
4.3
2.9
0.7
0.2

4.6
4.3
3.2
1.3
0.3

4.6
4.5
4.2
2.0
0.9

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

64.

Population dynamics of sex-linked recessive lethals(By Terumi MUKAI)

In general, the heterogametic sex has a mechanism working towardelimination of detrimental genes from the sex chromosomes, contributing a great deal to the formation of their specific genic systems (MUKAI
1961). A study was carried out with respect to a single locus in orderto understand the process of elimination from the population of the recessive lethals, the most detrimental genes of the sex chromosomes.
In order to set up mathematical models in the homogametic sex, thefollowing parameters are defined:
Qn = relative frequency of lethal heterozygotes (which are describedas Aa) in the n-th generation.
l-Qn = relative frequency of AA individuals in the n-th generation.
m = relative mating ability of Aa males, that of AA being 1.
f = relative fecundity of Aa females, that of AA being 1.
v = relative zygotic viability of Aa individuals, that of AA being 1.
s = relative competitive ability of a sperm, that of A being 1.
n = the number of generations.
In the case of Drosophila-type sex-linked recessive lethal genes (females
are homogametic), the following general formula can be obtained:_
Q,(2-fv)(fv)n-l
Qn - 2n-'[(I-Q,)(2-fv)+ fQd+Q,[2- f(l +v)](fv)n-l(1)
Although the lethal gene can be maintained in a population under thecondition of fv > 2 (the equilibrium frequency Q is given as formula (2»,
it is practically impossible to satisfy this condition.
2-fv
(2)
Q
= 2-f(1+v)
In the case of Bombyx- or chicken-type sex-linked recessive lethals,
which might be assumed to be completely lethal in the heterogameticsex (female), the following general formula can be obtained:
Q,(1 +s-smv)(smv)'d(3)
Qn =-----.:::..-'C"---'----'-'LJ-'-'--'--'-'-'--'-'--'--'------where ~=(1 +s-smv)[(I-Q,)(1 +s)n-l +Q,(smv)n-,] +Q,m[(l +s)n-l_(smv)'d].
Under the condition of l+s<smv, the lethal gene is maintained in the
population. The equilibrium frequency is as follows:
Q= l+s-smv(4)l+s-m(l+sv)
However, it is too difficult to satisfy this condition, and all lethal genes

RESEARCHES CARRIED OUT IN 196179

will be eliminated from the population.
Details of this work will be published elsewhere.
65.

Equation for polygenic mutation rate
per locus per unit dose
(By Sohei KONDO)Let us assume that an individual of an arbitrary isogenic line has npolygene loci with gene gi at locus i (i=l, "', n), that gi mutates fromthe wild type gio to an allele gir with the probability Pir (r=l, "', lJi; lJibeing the total number of the polygene alleles at locus i) and with acontribution of amount Xir to change the quantitative character X underconsideration and finally that Xir is small enough to assume the validityof additivity law for change in X from the contribution of simultaneous
mutations in g;'s in an individual. Then, we obtain the probability f(x),that an irradiated organism gives rise to an overall genetic change x in
X, as the coefficient of ZZ in the expansion of the generating function:
G(z) ~ exp [- 'Z Pir+ 2: Pirz Zir ]= 'Zf(x)zZ,
(l)
i.r

i,r

x

where we have neglected higher order terms of Pir' Assuming Pir=cirDk(D: radiation dose, k: a contant, Cir: proportionality constant), from eqution (1) we have equations for j-th moment of x as follows:
pj=AjDk; A j = 'Z CirCXir)i(j=1, 2, 3,).
(2)
i.rThis means that the mean, the variance and the third moment of x havethe same relationship with dose except for the difference in proportionality constant Aj. If k=l as the case of major genes, then variance P2
must increase linearly with dose.
To obtain an explicite formula for average mutation rate per locus perunit dose, we assume that k=l, Cir=C, lJi=lJ, Xir=Xi and that Xi is distributed about mean a in a normal distribution with standard deviation(la.
Then, from equation (2) we obtain
PI = nlJcDa,)

P2 = nlJcD(a 2 +aa2) ,P3 = nlJcD(3aa a +a ) .
That is, mutation rate per locus per unit dose lJC is given by
lJC = pdnDa,
where
2

(3)

3

(4)
(5)

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

D.

66.

GENETICS AND BIOCHEMISTRYOF MICROORGANISMS

A temperature independent flagellation mutant
in Salmonella*(By Tetsuo IINO)

A strain of Salmonella typhimurium, TM2, produces peritrichate flagella(six to ten flagella per bacterium) when it is cultivated in the nutrient
broth at temperatures below 40 DC. The optimal temperature for flagellar
production is for this strain around 30 D C. At 44°C the cell can divide
but they lose the ability to produce flagella whose number per bacterium
decreases as the cells multiply though the preformed flagella are not destroyed at that temperature. In ten to fifteen cell generations after theculture of flagellated cells was transferred to 44 DC, more than 9996 of
the cells become non-flagellated and are therefore non-motile. The remaining cells carry only a few flagella and rotate in the broth culture.
A mutant which produces flagella at 44°C was isolated from the strain
TM2 cultivated on a semisolid nutrient agar plate at 44Co. The mutant
cells can produce flagella at 44 DC as well as at 37 DC, 30°C and 20°C, ineither antigenic phase-1 or phase-2. No differences in the specificity of
H-antigen between the flagella of TM2 and the mutant or between those
produced at 30°C and 44 DC by the mutant could be detected by a crossabsorption experiment. The mutant marker is transduced independentlyof any known genes which control flagellation or motility in Salmonella.
The physico-chemical comparative study of the flagella produced by thewild type and the mutant is on the way.

67.

A modified selective medium for the isolationof motility mutants of Salmonella**(By Masatoshi ENOMOTO and Tetsuo IINO)

With the exception of some specific serotypes (Sasaki, Annual Report,
No. 11, 1960), chi-phage infects motile cells of Salmonella. The non-motile
cells are resistant against chi-phage, regardless of the presence of* This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.
** This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1961

81

paralyzed flagella. Therefore, a nutrient agar plate spread with chi-phage(X-NA) has been used as a selective medium for the isolation of nonmotile mutants. On X-NA, however, the cells of many motile strains
tend to lose temporarily their motilty, and grow to minute colonies.
Moreover, some motile clones, which may be temporary carriers of chiphage, as well as the non-motile mutants can form colonies. A modified
selective medium, X-NGA, was devised to remove these defects of X-NA
and to improve the procedure of isolation of non-motile mutants. In
X-NGA, the nutrient agar of X-NA was replaced by semisolid nutrient
gelatine agar which has been used for the isolation of motile mutantsfrom non-motile bacterial cultures (Iino, Annual Report, No.9, 1958).
When the motile cells of Salmonella are plated on X-NGA and incubated
4 hours at 37°C and then the colonies are allowed to develop at 24°C
for 18 hours, a non-motile mutant clone will form a compact colony,
while the clone of a temporary phage carrier spreads as a swarm and thedevelopment of non-heritable chi-resistant minute colonies is suppressed
remarkably. In certain salmonella strains, e.g. S. typhimurium TM2,
S. abony SW 1391 and S. abortus-equi SL 23, it was also found that thecolonies formed on the plate by paralyzed and non-flagellated mutants
are different, the former being large, opaque and yellowish-white, while
the latter are small, translucent and white. By the use of NGA and
X-NGA as selective media, it is now possible to select with high efficiency
back and forth occurring motility mutations in Salmonella.

68. H-antigen of heterozygous hybrids between Salmonella
abony and Salmonella typhimurium*(By Hideo HIROKAWA** and Tetsuo IINO)

An Hfr strain of S. abony, SW1391 (M-Sh-H,bHzen z ), was crossed with
S. tYPhimurium SW1292 (p-L-Ara-Gal-Xyl-Rha-H1iHzl,Z). The auxotrophic markers of the parental strains were adopted as the selective
markers. Each prototrophic clone isolated from the minimal selective
media was transferred successively to nutrient agar plates (NA). Hantigens of the colonies grown on NA were typed by slide agglutination
test. The nutritional requirements and sugar fermentation of the colonies
were examined by the replica plating method.
* This work was supported by a research grant from the National Institute ofAllergy and Infectious Disease (E.2872), Public Health Service, U.S.A.
** Present address: Biochemical Institute, Faculty of Science, University ofTokyo, Tokyo.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Among the hybrids isolated, considerable numbers of persistent heterozygotes were obtained in respect to the selective and the unselective
markers. The frequency of segregation of these persistent heterozygotes
was less than 1%. When a suspension of the heterozygous cells, whichcontains 0.7% segregants, was irradiated by ultra-violet germicidal lamp
(340 erg/mm 2 ), and plated on the indicator media, 20% of the colonies
grown on them segregated.
The heterozygous cells carried the same H-antigens, i or 1.2, as thefemale parental strain when isolated from the selective medium, andmost of them continued to express i and 1.2 antigens alternatively insuccessive subcultures. By selection with anti-i or anti-i.1.2 serum, thesubclones which express alternatively either band 1.2 or band e.n.x
were isolated. Two antigen types of the same H-phase had never been
expressed simultaneously by a heterozygous clone. These subclones
were usually still heterozygotic and dissociated cells expressing thehidden antigen i at a low frequency.

69. Chi-phage resistance of the salmonella
serotyPes having g-antigen*(By Itiro SASAKI)

It has been known that the salmonella serotypes having g-antlgen areresistant against chi-phage, and that the essential genetic factor of theresistance is associated with the determinant of flagellar antigen g. So
far, the attempt at isolating host range mutants of chi-phage against
these serotypes has been unsuccessful by the standard method of selection, namely plating resistant bacterial cells with an excess of phage
particles. However, some host range mutants (h G ) have been isolated
from chi-page by serial mixed culture method, multiplying the phagein a mixed broth culture of both sensitive and resistant strains. A chiphage resistant mutant SJ 100 (i: 1, 2) was isolated from a sensitive
strain, of S. typhimurium, TM 2. The strain forms smaller and thinnercolonies than TM 2, and in antigenic 1, 2 phase it is resistant to chi-phage.
In contrast to hG, the host range mutants (h T ) against SJ 100 is easily
isolated by the standard method of selection. Among 105 h T mutants,
only two can attack g-type Salmonella, while all of the h G mutants, so
far tested, can attack SJ 100 in 1,2 phase as well as in i phase.
From these results and the result reported last year (Annual Report,* This work was supported by a research grant from the National Institute ofAllergy and Infectious Disease (E-2872), Public Health Service, U.S.A. to T. lino

RESEARCHES CARRIED OUT IN 196183

No. 11, 1960) it is inferred that chi-phase resistant factors in g-type
Salmonella consist of more than two blocking steps of phage multiplication, and that at least one factor is involved with the resistance factor
of SJ 100. The following result further supports the above conclusionand suggests that in TM 2 there is a factor which blocks the infectionof hG 12 in cooperation with a resistance factor associated with the determinant of g-antigen. A clone of hG mutant, hG 12, can attack both
TM 2 and a strain of S. dublin (gp:-), NTCT 4197, while the gp: 1, 2
type recombinant, SJ 26, obtained by transduction of the gp-determinantfrom NTCT 4197 to TM 2, is resistant against hG 12.
The salmonella strains tested fall into four groups in regard to sensitivity to the host range mutants hG and hT.
(1) A type from which a higher yield of phage is obtained.
(2) A type from which a lower yield of phage is obtained.
(3) A type from which 'lysis from without' occurs only by the phages.
(4) A type which is completely resistant to the phages.

E.

RADIATION GENETICS IN ANIMALS

70. Radiation-induced mutation rate of polygenes controllingthe number of sternopleural bristles in Drosophila melanogaster(Terumi MUKAI and Sadao CHIGUSA)In order to obtain a fundamental information on the influence ofradiation on human populations, the mutation rates of polygenes controlling the number of sternopleural bristle numbers have been estimated.
The males of an isogenic line extracted from a wild population ofErie, Pa. (U. S. A.) were irradiated with X-rays at 250r and 500r. Immediately after irradiation, the irradiated males were mated to thefemales of the same line. The numbers of sternopleural bristles infemales and males which had hatched on or before the 13th day
after the mating were scored to test the heterozygous effects of radiationinduced mutations. The experiments are still in progress, but the results
at hand are presented in Table l.
One of the most important results shown in Table 1 is that thevariances of irradiated groups have increased in females but decreasedin males as compared with the controls. We may attribute this difference to mutations (some radiation-induced changes) in hetero-chromatic
parts of Y chromosomes, although this hypothesis should be re-examined.
Thus, the mutation rates were estimated by using the data for females
only.

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12
Table 1. Means, variances, and third moments about the means of thesternopleural bristle numbers
500r Experiment250r Experiment
TreatedControl
Ii!

0

Ii!

TreatedControl

Ii!

0

Ii!

0

0

Mean

* 16.1881
16.6244 16.0355 16.6778 15.9579 16.7538 16.1350 16.8980

Variance

1.44081.5872**
1.64791.48141.59071.7173*
1.7340**1.2629

Third moment
about the mean

0.4406-

0.5228-0.5989-0.6620-No. of genomes
tested

1592

1156

1704

1307

975

674

1088

914

* significant at the 5% level.

** significant at the 1% level.

1) The differences of the third moments about the means between the control
and treated groups were not tested statistically.
Table 2.

Estimation of radiation-induced mutation rates of polygenes
controlling the number of sternopleural bristlesI

250r Experiment 1500r Experiment

Heterozygous effect of each mutation
Number of positive mutations per genome
Number of negative mutations per genome
Mutation rate

1.240
0.710
0.089
0.243
0.046
0.041
1.08x 1O-6/locus/r 1.14x 1O-6/locus/rA method for the estimation of polygenic mutation rates developed byIn
this method, the means, variances, and the third moments about themeans of the distribution pattern have been used. The results are shownin Table 2.
The mutation rates were estimated on the assumption of 500 loci which
control the number of sternopleural bristles. The estimated polygenic
mutation rates were of the order of 10-6 jlocusjr, and this is very close
to the estimate of the mutation rates of polygenes controlling theviability (ca. 1.26xlO-6 /locusjr) (MUKAI 1961).
Indeed, the assumption of 500 loci may be not an underestimate when
FALCONER'S (1960) estimation that the number of loci controlling the

MUKAI (1961) with reference to BATEMAN'S work (1959) was employed.

RESEARCHES CARRIED OUT IN 196185

abdominal bristles is 100 is considered. BATEMAN (1959) and KONDO(unpublished) have mentioned that the mutation rates estimated by thiskind of method are underestimates, while the effect of each mutation,
which is assumed to be a constant, is an overestimate. Thus, it may
be concluded that the radiation-induced polygenic mutation rates aresubstantially higher than those for major genes, and are at least of theorder of 1O-6jlocusjr.

71.

Further radiation-genetical consideration concerning
the genetic structure of natural populations(Terumi MUKAI)

Following the balance hypothesis (DOBZHANSKY 1955), we can say thatheterosis (overdominance with respect to fitness) is a product of natural
selection within populations.
Suppose that there are two isogenic lines, AA and BB extracted fromdifferent natural populations. A and B indicate the genomes. After
irradiation (indicated by superscript [' D, heterozygotes AA' and AB'
are produced. In expectation of radiation-induced mutations in A' andB', we compare the viability of AA' and AB' with AA and AB,
respectively.
On the basis of the classical hypothesis, a reduction of viability inAA' and AB' can be expected in comparison with AA and AB, respectively.
On the contrary, according to the balance hypothesis, the results
should be different, i. e., the viability of AA' would increase as comparedwith AA, because a genic system, which will manifest heterosis when
it becomes heterozygous by mutation, has been produced by natural
selection. However, we can not expect increased viability in AB' as
compared with AB, because A and B have independently experienced
natural selection in different populations. Consequently, the genotype
AB would not be a genic system that could reveal heterosis.
Thus, if AA' and AB' would manifest lower viabilities than AA andAB respectively, this might be one evidence in the favor of the classicalhypothesis. However, if AA' would reveal better viability than AA,
and AB' would show lower viability than AB, this would support thebalance hypothesis.
BURDICK and MUKAI (unpublished) irradiated the males of the two
isogenic lines with X-rays at doses of 30, 60, 90, 120, 150, and 180r, andtested the heterozygous viabilities in F 2 with respect to the radiationinduced mutations. As a standard of the viability estimation, Xa

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

heterozygotes with one of the isogenic lines (AA), ~ were employed.
+.+.1
In every radiation treatment, the viability of AA' increased in comparison
with AA, while that of ABt was decreased as compared with that of
AB. These experimental results indicate that the balance hypothesis
might be valid on the polygene level.
To follow up this argument, an experiment on a larger scale is now
in progress.

72.

further studies on two types of dose·rate dependence oj
radiation·induced mutation rates in spermatogonia
and oogonia of the silkworm(by Yataro TAZIMA and Sohei KONDO)

Two types of dose-rate dependence of radiation-induced mutation
frequency have been found in early gonial cells of the silkworm. In
10- 5
100

e6/1

0.1

Ol:Sgon1a

1
Dose

10
rate

100r/m1n.

Fig. 1. Dose-rate dependence of radiation· induced mutation rate forlate gonia. Solid line represents the changes in mutation frequency
for +pe locus and broken line those for +re locus.

RESEARCHES CARRIED OUT IN 196187

one type mutagenic effectiveness of chronic irradiation is lower thanthat of acute irradiation, and in the other the relation is reversed. The
occurrence of either type depends upon the stage of germ cells. Thesefindings have been interpreted by assuming, 1) differential repair for theformer and 2) selective killing for the latter type (Genetics, 1961).
Further evidences compatible with the hypothesis of selective killing
have been obtained. Variation in induced mutation frequency wasstudied along with the development of germ cells both for acute andchronic irradiation. It varied drastically after acute exposure, being
highest at the time of hatching and decreasing rapidly toward laterstages, while it was almost constant after the chronic treatment.
Irradiation with various dose-rates in later stages revealed that mutagenic
effectiveness of radiation decreases gradually with the increase in doserate within the range from 0.14r/min to 320r/min (Fig 1). In thisstage mutation frequency for the acute treatment did not deviate, evenat 150r, from the linear relation extapolated from higher doses, suggesting that the delivered dose was too high to realize the expectation thata small dose of acute irradiation might give a higher frequency than expected from the linear relation.
Although directly supporting evidences are still meagre, beyond doubt
two distinct types of dose-rate dependence occur, one of which can not
be interpreted by the repair hypothesis.

73.

Comparison of acute and chronic irradiations in respect toradiation-induced lethal mutation rates in the silkworm(by Yataro TAZIMA and Takao KOBAYASHI)

By using the lethal-free strain described in the preceding issue of thisAnnual Report (No. 11, 1961), experiments have been continued tocompare radiation-induced lethal mutation rates between acute andchronic irradiations. Egg layings from a lethal-free strain, which had
been checked again in P, generation, were divided into three groups,
two for irradiation and one for control. The experiment was run intwo series, with two parental lines. Both acute (320/min) and chronic(0.139r/min) r-irradiations were administered to very young larvae withthe same doses, 1000r. The dose-rates ratio was ca. 2300: 1. Acuteirradiation was given in one exposure of 3.12 minutes just after hatching,while the chronically irradiated group was exposed for 120 hours, starting two days before and continuing three days after hatching. During
this period the germ cells are known to be at the primordial stage, or
the most advanced ones at the stage of primary gonia. After the emer-

88

ANNUAL REPORT OF NATIONAL INSTTUTE OF GENETICS NO. 12gence of irradiated individuals the females were crossed to males of thelethal-free strain and about 600 eggs were extracted at random from 30
layings for each group and were treated with hot hydrochloric acid toobtain the next generation, Gg •By backcrossing Gg females to males of the lethal-free strain about
200 Gs layings were produced. Among them 100 Gs layings were raised
separately after the application of artificial hatching treatment. Bysibmating Gs moths within each batch, more than 100 layings of G4 eggs
were produced for each batch, to determine the frequency of radiationinduced lethaIs in G I • The results are given in Table 1.
Table 1. Data obtained after the test for lethaIs in G4 of theirradiated groups and scored on Gs line basis.
Number of G a lines
Expt.

No. 211
No. 221

Detcted lethals
per line

Irradiation

Acute
I Chronic

I

0

1

2

Total
observed

63
78

33
19

4
3

100
100

I

Total No.
of lethals
detected

Freq. of.
lethals
per Gs line

41
25

0.41000.2500I

The detected lethals in Table 1 comprise radiation-induced and spontaneous lethals contained in the gametes which were transmitted fromGg females of the irradiated strain and spontaneous lethals contained in
the gametes which were transmitted from G 2 males of the lethal-freestrain (marked by D in Fig. 1). Therefore, to calculate radiation-induced
mutation rates we have to subtract all those background spontaneous
lethals from the observed data.
With regard to spontaneous lethals, their accumulation during three
generations, P g , PI and Gl> in the irradiated line and in P g and PI of
the lethal-free strain (D), should be taken into account, because all lethals
accumulated down to P a had already been eliminated and those newly
arisen in G 2 and G a cOcl1d not be detected in the present test. In theirradiated line, two kinds of gametes are transmitted in each batchfrom G I to G2 , one coming from the irradiated mother and the otherfrom the non-irradiated lethal-free father (C).
Since we have found that the probability of the occurrence of spontaneous lethaIs is 0.004046 per gamete per generation (Annual Report
No. 11), the number of accumulated lethaIs in each gamete transmitted
to G2 could be calculated as follows:in the lethal-free strain (D)0.016184 (for two gametes, P 2 and PI)

RESEARCHES CARRIED OUT IN 1961

89

(c)

lethal test

lethal test

lethal test

Fig. 1. Breeding scheme employed in the experiment. Single-pair culture isrepresented by a circle. Mass culture of s ~veral layings is indicated by arectangle. Irradiation was given in G, generation.

in the irradiated strain one gamete from non-irradiated (C)......0.008042 (in P z and G,),
the other one gamete from irradiated G, . ... 0.012138 (in P z , P, and G 1).
The total number of spontaneous lethals in the genetic background of
this experiment, which can be detected in G•• should therefore be 0.036418.
By subtracting the above figure from the observed values for both
irradiated groups, the induced mutation rates were obtained (Table 2).
As may be clearly seen from the table, the calculated induced mutation-rate is higher in the acute group than in th~ chronic group, showing

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12
Table 2.

Calculated induced lethal mutation rates for both irradiated groups.

Expt.

Irradiation

Freq. oflethals
detected

Background
lethals

Inducedlethals

Induced rate
per gamete
per 1000 r

AcuteChronic

0.41000.25000.03640.03640.3736
0.2136

0.3736
0.21362111

No.
No. 221a good agreement with the results obtained by specific loci method(T AZIMA et al. 1961).
This work has been originally carried out in 1960 under the contract
between 1. A. E. A. and this Department. The results reported now
were obtained in 1961 after the discontinuation of the contract.

74.

Differences in response of silkworm gonia to acuteand chronic r-irradiation(by Toshihiko SADO)

Two types of dose-rate dependence of radiation-induced mutation rates
have been revealed both for the spermatogonia and the oogonia of thesilkworm. In one type, mutagenic effectiveness is lower at chronic thanat acute irradiation, and in the other type it is higher at chronic thanat acute irradiation. The second type is found in the later stages ofgonial development (Tazima et al. 1960, 1961). As a possible explanation
it was assumed that germ cells at highly sensitive stages are selectively
killed by acute but not by chronic irradiation, under the same dose. In
order to obtain cytological evidence supporting this assump.ion, thepresent experiment was undertaken.
Both sexes of a wild type silkworm strain, C108, were exposed to
Cs-137 r-rays of high dose-rate (320r/min.) or to Co-60 r-rays of low
dose-rate (0.128r/min.) in early larval stages. Total doses given were
1000r in both groups. In the acute group, the insects were irradiated atearly third instar, while in the chronic group, the irradiation time wasso adjusted that it was terminated at the same time as for the acutegroup.
Acutely treated cells. Under acute irradiation the results obtained formale germ cell were quite similar to those of the earlier experiments(Sado 1958, 1961). High incidence of necrosis of the secondary spermatogonia was observed 24 and 48 hours after the exposure and thenecrotic cells rapidly disappeared from the testes. Five days after the

RESEARCHES CARRIED OUT IN 1961

91

exposure, regeneration of spermatogonia was observed to set in. In thefemale, no corresponding stages are found with cells mitotically as active
as the secondary spermatogonia. No necrotic cells were observed in theovary 24 and 48 hours after the treatment. But on the third day after
the exposure, a few degenerating cells were observed in the proximal
part of the ovarioles where the oocytes and nurse cells were differentiat·
ing. However, the damage in the cells was much less severe in thefemale than in the male. Acutely irradiated oogonia, if not all, appear
to degenerate around the critical stage when differentiation into oOcytes
and nurse cells would occur if they had not been exposed to radiation.
Chronically treated cells. In the case of chronic irradiation, the insects
were examined every 24 hours after the onset of the treatment. At 24 and48 hours a few degenerating secondary spermatogonia were observed in
the irradiated testes. Mitoses of spermatogonia, but not many, wereobserved at this time. 24 and 48 hours after the treatment was started
the total doses given reached 200 rand 400 r, respectively. During thefirst 3 to 5 days, a reduction in the number of spermatogonia wasremarkable. An increase in the number of secondary spermatogonia
was not observed, mainly due to mitotic inhibition of the primary andperhaps of very early secondary spermatogonia. At the time of termi·
nation of the exposure the testes contained a few primary spermatocytes
but very few, or none, secondary spermatogonia. Thus, it seems thata part of the secondary spermatogonia present at the time of the onsetof the treatment were killed but others differentiated into primary
spermatocytes although they were less numerous than in the non·
irradiated testes. In the chronically irradiated females, degeneration ofirradiated oogonia was not observed even at the termination of thetreatment. But the development of the germ cells was retarded due tomitotic inhibition of the oogonia.
Thus, the experiment failed to furnish a conclusive evidence supporting the validity of the selective killing hypothesis which has been
proposed by Tazima et al. (1961) for the explanation of the second typeof dose-rate dependence.

75.

Number of gonial cells of the silkworm at earlystages around hatching(by Takao KOBAYASHI)

Tazima, Kondo and Sado (1960) obtained many cluster mutations intheir experiments on dose-rate dependence of radiation-induced mutations.
In these experiments, the earlier was the irradiated stage, the larger

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

became the cluster size. This finding suggests that the cluster appearanceof mutation is intimately correlated to the number of gonial cells present
at the irradiated stage. That is to say, the mutation-bearing gonial
cells multiply with the development of the insect.
In order to provide a cytological evidence for the theoretical considerations of the above authors, a cytological investigation has been carriedout before and after hatching, in which special attention was paid to
the number of germ cells in the gonads.
There are scarcely any follicular septa in the gonads before hatching.
They appear first on the hatching day, and are completed on the nextday. When septa are formed, the gonial cells seem to gather on the
periphery of each follicle. From cytological observation, it was difficult
to determine the sex of the gonads at those stages.
The number of gonial cells was counted carefully in each gonad fromtwo days before to one day after hatching. The results are shown in
Table 1.
Table 1.

Number of gonial cells before and after hatchingStage
Two days before hatching
One day before hatching
Hatching day
One day after hatching

Number of
gonadsexamined

Number of germ cells per gonad
Minimum Maximum

17
28
11
15

29
37
82
102

57
79
162
236

Mean
47.5
59.1
119.5
171.4

From these results, it is evident that the number of gonial cells
increases rapidly after hatching.
The observed numbers agree in general with the theoretical calculationof Kondo (1961).

76. Induction of a presumably somatic mutuation
with 5-bromouracil in the silkworm(by Makoto NAKAJIMA)

It has been known in bacteria that certain base analogues incorporate
into deoxyribonucleic acid resulting in a high incidence of mutations.
So far, in higher organisms such investigations have been scarcely
attempted.
In order to examine whether or not a similar situation may be foundRESEARCHES CARRIED OUT IN 196193

in higher organisms, the present study has been undertaken with thesilkworm. We confined ourselves to the detection of the mutagenic
action of two base analogues in producing spots due to somatic mutationof the larval skin. For this purpose, heterozygotes for opaque (+) andtranslucent (od) served as a convenient material. The detection of
mutation was carried out on the epidermis of the fifth instar larvae
which had been raised during the first instar by feeding mulberry leaves
supplemented with a solution of either of the two base analogues. The
results obtained are as follows:(1) With the administered doses from 4 to 38 p.g. per individual, 5·bromodoexyuridine induced a large number of translucent spots, eachcomprising a number of cells. The supplementation of aminopterin,
known as an inhibitor of thymidine synthesis, showed no significant
effect on the production of the spots. The effect of 5-bromodeoxyuridine
on producing mutations was about 8 times as high as that of 5-bromouracil for equimolar administration.
(2) The strongest effect of 5-bromodeoxyuridine was observed when
it was administered on the second day of the first instar, showing thelowest viability among those treated on the first or third day.
(3) The translucent spots induced by 5-bromouracil as well as by
5-bromodeoxyuridine were similar to those induced by r-irradiation at
the same stage. But the mean size of the spots produced by the baseanalogues was about half as large as of those produced by r-rays.
Although the mechanism of production of such spots is at present notclear, at least two possibilities may be considered. First a mutation
could have occurred in some of the epidermal cells during development
as a result of the replacement of the normal base in DNA with 5-bromouracil. Second, it may be that somatic crossing over was induced insome cells by the stimulating action of the base analogues.

77.

Theoretical estimation of " survival cell number versus dose"
curve from experimental frequency distributionof the number of mutants(by Sohei KONDO)

Assuming that the initial mutative events in germ cells are distributed
in a Poisson distribution, the probability distribution function f(m) of mmutants to be recovered later in the offspring from each mating pairhas been theoretically derived in terms of the number of the offspring
per mating pair, the number n of germ cells at the time of occurrenceof the initial mutative events and the frequency of the initial mutation

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

per germ cell (Kondo: 1961). It has been previously found that byadjusting n, the theoretical frequency distribution f(m) can be fitted
very well the observed f(m) for the case of silkworm exposed to acuter-rays at the gonial stage.
Conversely, from experimental f(m) distribution curves for various
values of radiation dose D we can construct the curve of the number of
viable germ cells, n, versus dose. Such a curve is useful for evaluating
the contribution of killing effects on germ cells to mutation mechanism,
especially, the dose-rate dependence of radiation-induced mutation.

700
600

lotus
pe re

,00
0+>

..,
u

(l;-

'" 400
.:i

~

.....

• •
~

0

<lJ

P.

3
4;

<..>

c:s
'",

250

0

C»

15

200

...

<lJ

..0

's="

: s ISo

•

g

.~

::s

V)

100

90

ao0

~Jo

/o~o
,doo
,Exposure dose

2000 rFig. 1. Theoretical estimation of "survival cell number versus dose" curvefrom experimental frequency distribution of the number of mutants.

Fig. 1 shows two n-versus-dose curves constructed in the abovementioned way from the experimental datal) of silkworm exposed toacute r rays at the early gonial stage (time of hatching). The LD50 isabout 1000 and 2000 r for spermatogonia and oogonia, respectively.
1)

Y. Tazima (unpublished).

95

RESEARCHES CARRIED OUT IN 1961

78. Deformities in chick embryos caused by r-irradiation(by Takatada KAWAHARA)

After a few preliminary experiments, it has been concluded that induction of deformities by r-irradiation in chicks was most effective when
48 hours old embryos were treated after incubation. It was also foundthat in that stage embryonic lethality was rather low in comparison withtreatments started earlier. On the basis of these findings, chick embryosof 48 hours were irradiated by r-rays, the does applied being 600r by
l37es. The materials used were chick embryos of White Leghorns (WL),
Barred Plymouth Rocks (BPR), Nagoyas (NG) and hybrids between WL
and the two others.
Deformities noticed were mainly toe defects, though a few individuals
having slightly or strongly defective legs or wings were found. The
frequency of deformities in purebred and crossbred chick embryos ispresented in Table 1.
The results of statistical analysis of the data obtained are summarized
as follows; The difference between the races in respect of the incidence
of deformities was remarkable, BPR showing a very low frequency incomparison with WL or NG. The frequency in White Leghorns was
5.25% higher than that in Nagoyas though it was statistically not significant. A considerable difference was observed between the reciprocal
hybrids. Hybrid chicks born from White Leghorn dams showed a muchhigher frequency of deformities than the reciprocals. There was no observable relation between sex and frequency of deformities.
Those results suggest that the frequency of deformities due to rirradiation in chick embryos is largely controlled by maternal factors.
Table 1.

Breed or crossDeformities due to r-irradiation of chick embryos
Treated (600r)
of
No. of tested , Incidence
deformities
embryos

(%)

WL
BPR
NG
WL\2 xBPR o
BPR\2 xWL o
WL\2 xNG o
NG\2 xWL o
Total

799
296
138
317
269
261
213
2293

32.79
5.07
27.54
19.56
9.29
38.38
22.53

Non-treated (control)
Percent of
No. of tested
abnormalembryosembryos

I

421
78
12
618
150
158
47
1484

0.48
0
0
0
0.61'
0
0

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

79. Genetic effects of chronic tow dose irradiation on mice
(Susumu MURAMATSU, Tsutomu SUGAHARA and Yoshiko OKAZAWA)

The genetic effects of radiation on mice were studied by the treatment of gamma-irradiation from conception to reproduction for threesuccessive generations. Progenies of mice of a multiple recessive stock," wavy" stock,.,irradiated chronically with 34.4 r (the dose rate of 0.43r/
22 hr.-day) of 60Co-gamma-rays for three successive generations, as
described in the previous paper (Sugahara et at. 1961), were tested forrecessive lethal mutations on the basis of four different criteria (Haldane's
method (1956), litter size, rate of early death and sex ratio) by matingthem with the wild CBA strain and studying their F I and F 2 progenies.
Table 1.

Number of animals used for the analysis
Male line

Female line

Total

100
1,170
8,337

100
1,362
8,450

200
2,532
16,787

100
1,158
8,011

100
1,071
7,739

200
2,229
15,750

Irradiated series
PI

FI
F2

Non-irradiated series
PI

FI
F2

The numbers of animals used for analysis are given in Table 1 withregard to generations and lines. Male line or female line means F 1 andF 2 of recessive PI males or females, respectively.
Table 2.

MethodHaldane's method
Litter size in F z
Early death rate
Sex ratio

Mutation rates calculated from four different data on
the same materials
Lethal effective in the period

Mutation ratefrom conception to weaningfrom conception to birthfrom birth to weaningfrom conception to birth

4.8x lO- s/r/total autosomes
0.5xlO- s
2.1XlO- s
2 X lO-s/r/X-chromosomes

Results obtained are summarized in Table 2.
The calculated values seem to be practically in good accordance withRESEARCHES CARRIED OUT IN 1961

97

each other :CO.5-4.Sx 10-3/r/total autosomes) except for that from sex
ratio (2x 1O-3/r/X-chromosome). But, unfortunately, neither linearlity
nor dose rate dependency can be tested from the present data. Thus,
it can be said that the genetic effects of radiation on mice were demonstrated at a low dose with a low dose rate. The results may be verysignificant for the assessment of human radiation hazards. Detailed
analysis is now in progress and the results will be published elsewhere.

F. RADIATION GENETICS IN PLANTS
Boron effects upon gamma-ray and thermal neutron irradiationsof einkorn wheat; RBE of heavy particles
from lOBCn, a)7Li reaction ll

so. I. Effects on seedling growth, fertility andchromosome aberrations(By Seiji MATSUMURA and Tomoo MABUCHI)

As an approach to the investigation of boron effects, seeds of Triticum
monococcum fiavescens soaked in water and 0.1, 0.5 and 1.0% aqueous
solutions of borax for 2 days were exposed to gamma-rays in our Instituteand to thermal neutrons in the Japan Atomic Energy Research Institute's
Nuclear Reactor, JRR-1. Gamma-irradiation with 137es was applied at
the dosages of 0.5, 1, 2 and 3 kr. The thermal neutron flux was calculated to be 5.2x109 nth/cm 2 ·sec when the reactor was operated at 500
watts. The thermal neutron integrated flux ranged from 1.3 to 10.4 X
10 11 nth/cm 2 for 25......200 seconds. Gamma contamination dose rate was
117 r/min. The measurements of thermal neutron integrated fluxes andgamma contamination dosages were made with glass plates of different
AgP0 3 concentrations.
There was no marked difference in the germination rate among gammairradiations with various dosages and various borax concentrations.
Only a slight decrease in the germination rate was found in thermal
neutron treatment at higher dosages, especially with higher concentrations of borax. The higher were the dosage of gamma-rays and thermal
neutrons and the concentration of borax in the thermal neutron treatments, the more reduced was the growth of seedlings and seed fertility
and the more increased were mitotic disturbances in the root tips
immediately after germination.
1) This work was done partly under Research Contract No. 27 with the International Atomic Energy Agency.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

The frequency of chromosome aberrations in PMC'sincreased markedly
with the increase of the dosage of gamma-rays and thermal neutrons,
and especially with increasing borax concentration for the neutron
treatments. Assuming that the chromosome aberration frequency versus
gamma-ray dose curve is linear and independent of dose rate as well asof borax concentrations, the overall effective doses of thermal neutronsof 1.3 x 1011 and 5.2 x 10 ll nth/cm 2 can be converted to equivalent gammaray doses for the various borax concentrations as given in line 4 of
Table 1. Subtraction of the equivalent gamma-ray dose for 0% borax
concentration from that for 0.1 or 1.0% borax concentration at the same
integrated neutron flux gives the biological response to thermal neutron
in units of gamma-ray dose due to the added borax (see line 5 ofTable 1).
Table 1.

1.

Estimated RBE values of the heavy particles fromlOB (n, a) 'Li and the relevant data

2. Content of borax aqueous solution (weight% relative to dry seed)

3. Integrated thermal neutron flux (101l/cm 2 )

1.3

4.

Biological dose of thermal neutron equivalent to gamma-ray dose (krad)

0.25

5.

Biological response to thermal neutrons
due to added borax in units of gammaray dose (krad)-

6. Dose calculated from 10B(n, a)'Li reaction(krad)

7.

0

0.1

1.0

91

85

79

Borax concentration (%)

RBE of (a+'Li) (5-7-6)

5.2

1.3

5.2

1.3

5.2

- - - -- - - - - 2
53
1.6 0.9- - - - - -- -- -

-

0.65

1.4

1.75

3.4

- - - - - -- - - ---0.011 0.044 0.105 0.42
60

- -- -- 32

17

8

The physical absorbed doses in rads due to heavy particles from
10B(n, a)'Li reaction were calculated by Dr. KONDO, as given in line 6 ofTable 1. The RBE (relative biological effectiveness) of (a+ 'Li) relative
to gamma-rays is shown in line 7 of Table 1. That the higher borax
concentration results in a lower RBE value will mean that the boron
atoms cannot penetrate the surface covering of the seeds as freely aswater molecules. The lower RBE values for higher neutron integrated
flux means that the linear assumption of chromosome aberration versus
gamma-ray dose curve does not hold true.

RESEARCHES CARRIED OUT IN 1961

81.

II.

99

Effects on chlorophyll mutations(By Tomoo MABUCHI and Seiji MATSUMURA)

In order to study RBE for gene mutations of (a+ 'Li) from the thermal
neutron capture reaction lOB(n, a)'Li, compared with gamma-rays, chlorophyll mutations in the X 2 seedlings were investigated. Their frequency
increased roughly in a linear relation to th~ dose of gamma-rays andthermal neutrons and also markedly with increasing boron concentration
only for thermal neutron treatments, as
could have been expected. From these data,ochlorophyll mutation rates per 1011 nth/cm 2of thermal neutrons are plotted in Figure
1, for the various borax concentrations. The £mutation rate per kr of gamma-rays is i! 3
1.3%. An absorbed dose due to lOB(n, a)'Li ~reaction is calculated as 82.6 rad per 1011 .g 2
Enth/cm 2 in 1.0% borax solution by Dr. KONDO :1o(see Part III of this series). The mutation rate per rad due to lOB(n, a)'Li reaction is about 4 x 10- 4 • Therefore, we mayo LO~Q-I-_~O.5~---I""'.oconclude that the RBE value of the heavy
Borax concentration (%)
particles from lOB(n, a)'Li relative to
Fig. 1. Relationship between
gamma-rays is about 30 for gene mutations.
mutation rate and borax
concentration.

82.

III.

Calculation of absorbed dose due to lOB(n, a)'Li reaction(By Sohei KONDO)

The physical absorbed dose in rad units due to heavy particles fromlOB(n, a)'Li reaction has been calculated by the first collision dose: dB=1.602 x 1O- 8 aEN1>(rad) where 1.602 x 10- 3 is the conversion factor from
MeV/gram to rad, a(=755xlO- 24 cm 2 ) the thermal neutron capture crosssection for boron, E(2.34 MeV) the sum of energies of a and 'Li particles
emitted by the capture reaction, N the number of boron atoms per gramof seeds and 1> the integrated flux of thermal neutrons. The values of
N have been estimated by assuming that boron atoms can enter seeds
as freely as water molecules. That is

N-

4NoxY

100Aborax(1 + }') ,where No is AVOGADRO'S number, Aborax the molecular weight of borax,

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

factor 4 the number of boron atoms per borax molecule, x(%) the concentration of the borax solution used and Y the increase in weight ofthe soaked seeds relative to dry weight.
Table 1 shows the calculated absorbed dose per 1011 thermal neutrons
per cm 2 •Table 1.

Calculated absorbed dose dB due to 10B(n, a)'Li reaction
and relevant experimental data

x (%)

y

N (B atoms/g)dB (rad per 1O l1 nth cm- 2 )

0.1
0.5
1.0

0.85
0.83
0.79

3.05x10 18
1.50x 1019
2.93x10 19

8.6
42.5
82.6

Let us plot a biological response of treated seeds at a given dose ofthermal neutrons fb(nth/cm 2) mixed with r rays against the values ofborax concentration x together with the response of pure r ray treatment against the dose. Then, the overall effective dose of the thermal
neutron field at the integrated flux of fb can be converted to equivalent
gamma-ray dose for various borax concentration'S. Subtraction of theequivalent gamma-ray dose for 0% borax concentration from that for
0.1, 0.5 or 1.0% borax concentration gives the biological response to thereaction of thermal neutrons with the added borax in rad units of gammaray dose. Division of this value by the absorded dose due to heavy
particles from l°B(n, a)'Li reaction calculable from Table 1 gives theRBE of the heavy particles relative to r rays. This method does not
require the knowledge of the r ray and fast neutron contamination in
the thermal neutron field nor the contribution of the thermal neutron
reaction with other elements contained in the seeds.

83. Radiological study of wheat monosomics(By Koichiro TSUNEWAKI)

The experiment reported here was designed to study the effects onradiosensitivity of wheat produced by elimination of a specific chromosome
in each of 21 monosomic lines of Triticum aestivum var. Chinese Spring.
Dormant seeds were treated with r-rays from COBO. The r-ray dosage
per hour was 250r. The experime~al design used was a split plot in arandomized block design with 3 r~lications and 10 r-ray dosages, i.e.,0, 10, 20, 25, 30, 35, 40, 45, 50 and 60 Kr, for units, and 22 genetic

RESEARCHES CARRIED OUT IN 1961

101

strains, i.e., the disomic and 21 monosomics for subunits. About 50
seeds from each treatment were planted per strain per replication.
At the moment data on survival rate and plant height at the 5th leaf
stage of the R, generation have been analyzed. There is a differential
radiosensitivity between the disomic and the monosomics, the former
being more resistant than any of the latter. A differential radiosensitivity also exists among the monosomics. Comparing with the survival
rate of the disomic, Mono-3B showed a distinctive drop in survival ata dosage of 30 Kr, Mono-5B at 35 Kr, Mono-2A, 2B, 2D, 4A, 6A and 6D
at 40 Kr, and Mono-lA, lB, ID, 4D, 5D and 6B at 45 Kr. Mono-3A,3D,
4B, 5A, 7A, 7B and 7D did not show any significant drop.
Three monosomic lines belonging to each of the homoeologous groups
2 and 7 showed a very similar pattern of radiological response. In the homoeologous groups 1, 3 and 6, Mono-IA and ID, 3A and 3D, and 6A and 6D,
respectively, showed a similar radiological response but the third member of each group differed distinctly from the others. Among all 3
monosomics of the homoeologous groups 4 and 5 the responses weredistinctly different one from another.
These facts seem to indicate that functional differentiation of chromosomes has taken place to a different extent in the 7 homoeologous
groups. It is also interesting to notice that monosomics of homoeologous
chromosomes in A and D genomes showed, in general, a similar response
to irradiation, while those of B genomes differed from the others.

84. Changes in rust susceptibility due to partial irradiation ofwheat seedlings and its dose dependence(By Keizo KATSUYA)

The stems and leaves of seedlings at leaf stage of Triticum vulgare(Norin No. 16) were irradiated by X-rays at 5 kr. The seedlings were
inoculated 10 days after irradiation by spraying with an aqueous uredospore suspension of Puccinia triticina. The first leaves of the seedlings
were removed 10 days after inoculation and the size of uredosori wasmeasured. They were on plants whose whole bodies or stems were
irradiated about 1.7 times larger than on the control and plants whose
leaves only were irradiated.
Seedlings at 2 leaf stage of T. vulgare (Norin No. 50) were irradiatedby gamma-rays, at 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 kr. The seedlings were
inoculated 6 days after irradiation by spraying with the spore suspension.
Seedling length was measured 7 days after irradiation, and then the102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

size of the sori was measured 12 days after inoculation. Inhibition of
seedling growth increased with the increase of dosage and the seedlinglength at dosage above 1 kr remained unchanged. Also, the size of thesori increased with the increase of the dosage, however it did notfurther increase at dosages above 5 kr.

85.

A study on the relation between dose rates of gamma-rays
and chromosomal aberrations!)(By Noriko SAKURAI* and Seiji MATSUMURA)

The frequency of root tip cells showing the first mitotic division andthe time of its occurrence after germination in various temperatures
were preliminarily investigated in non-irradiated seeds of two-rowedbarley (Hordeum sativum JESS. var. distichum HOCK.). In relatively
high temperature (l2°~16°C), the first mitosis began 20~24 hours aftersowing, and then the root elongated to 1.2~1.6 mm.
Further, a study on the relation between chromosomal aberrations inroot tip cells and various dosages was carried out. Dormant seeds were
subjected to gamma-rays (137CS) of 2, 5, 10, 20 and 50 kr. After irradiation the seeds were kept in a dark room at 16°C and their root tips
were fixed 34, 42, 58 and 60 hours later. The observation of chromosomal aberrations was arbitrarily limited to cells in early telophase I justbefore the formation of nuclear membrane. The frequency of chromosomal aberrations involving fragments and chromatin bridges increased
with the increase of dosage, but it decreased in the later stages. These
results indicate that some of the cells which showed abnormal division
recover their normal behavior in stages later than 1st division.
In addition a study was carried out for a detailed comparative studyof dose rate and storage effects on growth and chromosomal aberrations.
Two lots, each of 100 dormant s~eds, whose water content was 12.3%,
were subjected to gamma-rays of 5 and 10 kr. For acute and chronic
exposure to gamma-radiation two dose rates, namely 312.5 r/min with
137CS and 20 r/hr with 60CO were adopted respectively. The acute treatments
were given in three ways; namely, at the beginning or at the endof the chronic irradiation and in two fractionations at both.
Irradiated seeds were sown in the greenhouse and the seedlings were
* Kihara Institute for Biological Research, Yokohama.
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

RESEARCHES CARRIED OUT IN 1961

103

measured 14 days after sowing. There was no striking difference between 5 and 10 kr, in so far as the germination is concerned, but growthof seedlings was slightly delayed at 10 kr as compared with 5 kr. At
the two above dosages, chronic irradiation and the acute one which wasapplied at the start of the former were more effective in inhibiting
growth than the others. This may indicate that storage of dormantbarley seeds after irradiation increases radiation damage.
For the observation of chromosomal aberrations, root tips were fixed
58 hours after sowing. At 10 kr the frequency of abnormal cells andbreakages per cell at high dose rate was higher than at low dose rate,
especially when acute irradiation was applied at the start of the chronic
one. It is assumed that the frequency of "two-hit" aberrations was
relatively high and dependent upon the intensity of radiation. Unexpectedly, chronic irradiation with 5 kr was more effective than acuteirradiation given in fractions. It is supposed that storage effect wereinvolved.

86.

PolYPloids and aneuploids of Triticum dicoccum
produced by N 20-treatment(By Hitoshi KIHARA and Koichiro TSUNEWAKI)

As previously reported, the present authors treated an Emmer wheat,
T. dicoccum var. Khapli (2n=28) with nitrous oxide and obtained polyploids and aneuploids from almost all florets treated under the optimum
condition. In this report a result of progeny test of those plants will
be described with a consideration on the prospect of aneuploid studies
in Emmer wheat.
All tetra- and hypotetraploids (2n=54-56) produced offspring whichwere tetra-, hypertetra- or hypotetraploids (2n=51-61). In those groups,
there was on the average a strict correlation between the chromosome
number of the parents and that of the offspring.
Progenies of 2 monosomics consisted of 12 disomics and 1 trisomic
but no monosomics were recovered. The trisomic seemed to be produced
from an aberrant meiosis in the monosomic parent as is known in mono·
somics of common wheat. A plant, whose roots were a mosaic of monoand trisomic cells, gave in its offspring 4 plants, one of which wasmonosomic. Since the seed-fertility of those monosomics including themosaic plant was about 25%, the very low transmission rate of themonosomic condition in this Emmer variety was seemingly due to strong
selection against chromosome-deficient gametes and/or zygotes. A

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

trisomic plant produced 17 descendants among which 2 trisomics wererecovered. The transmission rate of the trisomic condition is also significantly lower than that known in common wheat.
These facts suggest that it will be very difficult to establish an aneu'
ploid series in Emmer wheat and that both monosomic and trisomic
analyses will mean hard work.

87.

Comparison of 32p f3 ray and 137Cs r ray effects
on seedling height of wheat ll(By Sohei KONDO and Hiromi ISHIWA)

The previous expriments turned out that 32p f3 rays were less effectiveon wheat seeds than expected from the calculated dose by assuming that
seeds are in an infinite 32p homogeneous solution. This discrepancy may
be attributable to the partial shielding of f3 rays by the proximity ofseeds touching each other when they were soaked in the 32p solution inbatches of 20 seeds per a gauze pouch.
The present experiment has been improved by minimizing such apartial shielding. Seeds of Triticum monococcum jlavescens have been
soaked in 2 inch petri dishes (100 seeds/dish), with blotting papers elevated by thin glass fibers about 2 mm from the glass bottoms so that
Table 1. Comparison of radiation effects on seedling height between seeds
soaked in S2p solution of 0.154 mcJml and those exposed to 137Cs r rays.
Time after
treatment(days)
16

33
1)

r ray dose
equivalent
to S2ptreatment(kr)

Assumedcontributionof 32p inside
embryosl)(kr-equiv.)

Calculated doseto soaked embryos
due to external
32p f3 rays
(kr-equiv.)

Total embryodose due
to 32p(kr-equiv.)

4.1
4.8

1.15
1.85

2.0
2.0

3.2
3.9

The increase (0.7 kr-equivalent per 17 days) in effectiveness of 32p treatment
shown in column 2 has been assumed to be due to disintegration (34%) of 32p
inside the soaked seeds during the 17 day period, and the effect of suchdisintegration during the first 16 days has been calculated by multiplying 0.7
kr by the ratio of disintegration during the first 16 days to that during thelatter 17 days.

1) This work was done under Contract No. 27 with the International Atomic
Energy Agency.

RESEARCHES CARRIED OUT IN 1961

105

seeds can be assumed to have been approximately in homogeneous infinite solution. The seeds have been soaked for 48 hrs but exposure to
32p solution or to Cs-137 r rays has been limited within the intervalfrom 12 to 36 hour after the initiation of the soaking.
Table 1 shows the comparison of effectiveness of 82p f3 rays with mcs
r rays on seedling height. The higher effectiveness of 82p f3 rays than
expected from calculation is partly attributable to the 82p which penetrated into the embryos, as suggested by the observed increase in therelative effectiveness of 82p f3 rays to r rays with increase in time after
the soaking period (see column 2, Table 1). Furthermore in our previous
calculation of the effective f3 ray dose of embryo the curvature of seeds
was not taken into account. Improvement of the dose calculation is now
under way.
88.

Effect of surface dose change on small seeds exposed
to Co-60 r rays
(By Sohei KONDO and Hiromi ISHIWA)

It is often the case that when small seeds are exposed to Co-60 r
rays, seeds placed in the surface layer show a difference in biologicalresponses compared with those in the interior. This is mainly due tothe well-known fact that the absorbed dose varies appreciably withdepth in the surface region of the materials exposed to high energy
photons.
(,..)

'<;1

7·1-=:::::::----_ _.:.

4:u~",arcl'<;1:

M"",,,,,,rd.

°n~----------,l:_---....J,.----.LExposure

cios.eFig. 1. Effect of surface:dose change on small seeds exposed toCo-60 r rays.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

Since the genetic system of a seed is confined to the embryo the effective dose of an irradiated seed will be equivalent to that of the embryo.
From this reasoning, we have irradiated rice seeds arranged in a monolayer, one group of seeds with the embryo facing upward toward the 60CO
source and the other downward.
The irradiation has been carried out by using our Co-60 7 ray facility
whose depth dose curve is known; the surface dose is higher about 50%
than the interior dose because of the high contamination wIth external
secondary electrons. The experimental results are shown in Fig. 1.
Radiation effects on seedling heights for rice seeds with embryos facing
upward have been about 50% more effective than those for the seeds
with embryos facing downward as expected from the physical measurement of the depth dose curve.

89. Relation between chlorophyll mutations and dose
rates of gamma-radiation in rice
(By Seiji MATSUMURA and Tomoo MABUCHI)

In order to investigate the relation of radiation effects to dose rates,
dry seeds of upland rice were irradiated by gamma-rays from a 60CO or
137CS source at the doses of 5, 10, 20 and 30 kr and three different dose
rates of 10 r/hr, 300 r/hr and 10,000 r/hr, for the first 3 doses. These
chronic, moderate and acute irradiations were terminated almost simultaneously before sowing. The germination rate, seedling height, seedfertility and chlorophyll mutations were investigated. Chronic irradiation, which was applied from the start, was the most effective in inhibiting the growth of seedlings and decreasing the fertility, especially at
20 kr, indicating an intensification of radiation damage due to storage(d. Ann. Rep. No. 11: 100).
The spectrum and frequency of chlorophyll mutations in the X 2 generation are shown in Table 1. In the untreated lot were found 36 panicle
progenies which segregated albina. They originated from the 4 panicles
of each of 9 Xi-plants, which must have been heterozygous for albina.
This was due to have obtained a wrong sample for the control seeds.
Therefore, similar panicle progenies from treated lots, which segregatedalbina, shown in brackets in Table 1, and also those segregating striata,were excluded from the calculation of mutation rates. As expected,
the frequency of chlorophyll mutations increased roughly in a linear
relation to dose, except at chronic 10 kr. There was no marked difference between chronic and moderate irradiation, while the acute one

RESEARCHES CARRIED OUT IN 1961
Table 1.

Spectrum and frequency of chlorophyll mutations in the X 2 -generation

Dosage Dose
rate
(kr)
(rjhr)
0107-

No. of~

X,- X2 panicle Albina*
plants progenies

V"~n-. X an- T'~g. Albo..
Stri- Chlorophyllmutationdis
tha rina m,,:~- ata
d~srate (%)

307

1,085

36 (36)

0

995
950

26 (19)46 (34)28 (15)

0

0

0

0

0.00

4

3
0
1

0
0
4

3

3
8

0
1

9
9
9

1.71
1.58
2.70

5
5
5

10
300
10,000

288
279
287

10

297
236
292

1,039

10

10
300
10,000871
1,034

63 (43)16 (11)27 (6)

16
14
7

0
0
4

0
0
2

5
0
3

8
10
9

3.95
2.18
3.58

20
20
20

10
300
10,000

219
321
268

686
1,016
819

25 (18)52 (32)44 (12)

17
6
25

2
7
3

4
1
2

1
12
0

4
5
9

4.81
4.53

30
30

300
10,000

300
230

988
644

64 (28)43 (10)

20
20

5
1

2
1

2
1

7
12

10

999

7.57
6.58
8.70

* See text.
showed clearly a higher mutation rate than the other two indicating adose rate dependency in the frequency of chlorophyll mutations.

90. Occurrence of variants in developmental stability
in seed size in the X-rayed progeny of rice
(By Kan-Ichi SAKAI and Akio SUZUKI)

Two groups of plants derived from the same variety of Japanese rice,
but different from each other in that one group was X-irradiated (20 kr)
in 1959 while the other remained untreated, were propagated year after
year by one plant-one offspring method in order to minimize the effectof natural selection.
Each group included approximately 1500 plants each year. In the X 3
generation, 400 plants selected at random among the X-irradiated progeny were compared with the same number of plants taken from thecontrol plot. It was then found that the former X-treatment has significantly increased intra-plant variability in seed size.
The intra-plant variabilities for seed length and for seed breadthcaused by X-rays were positively correlated, r= +0.4345, suggesting that

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

a common genic system is, to an extent, responsible for both variabilities. Intra-plant variability was not correlated with the absolute valuesof length or breadth of seed.

91.

Mutations in Pharbitis Nil induced by gamma rays
(By Hitoshi KIHARA and F. A. LILIENFELD)

Further observations on the progeny of r-irradiated wild and cultivatedMorning Glory are reported.
Five new mutants, all of them recessive, could be added to thosepreviously reported (Annual Report, No.9, 1958). All hitherto observed
are given in Table 1.
The great majority of deviating forms are found in the cultivatedstrain P7. They all represent known types of cultivated Morning Glory
which have arisen by spontaneous mutation in the course of many yearsof cultivation.
It is noteworthy that no albinotics were found which occur very often
in the offspring of irradiated cereals.
Table 1.

Mutants found in 3 strains of Pharbitis Nil

Material

MutationsWild strain from Nepal
with standard leaves andblue flowers

1) maple-like leaves
2) chlorina

Wild strain Tendan (North
China) with standard
leaves and blue flowers

3)

Japanese cultivated strain(P7) with standard leaves
and blue flowers

4)
5)
6)

• Remarks
viability good
weak, highly sterile

deep purple flowers*(lost in a typhoon)

viability gooddelicate leaf (Sasa)*
maple leaves (Tatsuta)*
dragonfly leaves (Tombo)
7) white flowers, black seeds
8) white flowers, white seeds

dwarf, sterileviability good* mutants reported in 1958.

92. Irradiation experiments with cultivated chrysanthemum ll(By Taro FUJII and Tomoo MABUCHI)

Rooted cuttings of two varieties of chrysanthemum were irradiated
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

RESEARCHES CARRIED OUT IN 1961

109

with 2, 4 and 8 kr of gamma-rays. Per cent of survivals was slightly
decreased at 2 kr irradiation and at 4 and 8 kr it was very low inboth varieties. About 25 per cent of planted cuttings died at 8 kr andthe plant height of survivals was depressed to about one half of that of
the non-irradiated control. 8 kr irradiation seems to be too high formutation experiments with chrysanthemum cuttings. All flowers from
irradiated cuttings of the variety Kinrei showed the original yellow
color like those of non-irradiated ones. Bud mutation occurred in thevariety Hakuei; flower color changed, sometimes sectorially, fromwhite to yellow. In some cases, all heads on the same branch were
yellow, while in other cases a part of a head was yellow and the remaining part was white. In the calculation of mutation rate, these brancheswith mutated flower heads were scored as one mutation and the linear
relationship between mutation rate and dosage was observed. At 2, 4
and 8 kr the frequency of branches with somatic mutation was 0.24, 0.61
and 4.95 per cent of all branches, respectively.
Cuttings and rooted shoots from the mutated plants of Hakuei wereplanted. The plants from the mutated branch showed the same yellow
color and those from non-mutated branches within the same individual
had white heads. This case therefore represents a typical bud mutation.

G.

HUMAN GENETICS

93. Prezygotic selection in ABO blood groups(By Ei MATSUNAGA and Yuichiro HIRAIZUMI)There are three possible selective mechanisms at prezygotic stages:
1. meiotic drive or unequal production of gametes carrying different
alleles in heteorozygouS parents: 2. sperm competition which may occurt independently of the female genotype: 3. sperm competition which may
occur as a result of serological incompatibility between the A or B
antigen of the sperm and the anti-A or anti-B antibody in the secretionof female genital organs.
Although by statistical methods a distinction cannot be made at present
between the first two possibilities, it is possible to test whether or not
prezygotic selection is operating to an appreciable extent. In order tocompare different family sizes of various matings in terms of the numberof children of a specific genotype and thus to test the possibility of
prezygotic selection in males, a concept of "family size equivalent" forchildren of a given genotype has been introduced. The family size110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

equivalent for 0 children of 0 X0 matings is of course the mean numberof observed children for such matings. The corresponding value for 'f 0
X (') A matings may be given by the following formula:11oeq('fOX (')A)=mx-Xn
Po

where n is the observed number of 'f 0 X (') A matings, m is the observed
number of 0 children from such matings, and Po is the probability thatan 0 child will be produced in such matings. This value represents themean number of children produced if sperm carrying A gene in AO
fathers were all eliminated and only sperm carrying 0 gene participatedin the fertilization. If there were no prezygotic selection, then no difference would be seen between Oei 'f 0 X (') A) and Oei 'f 0 X (') 0). But if
there were such selection, say, for O-sperm and against A-sperm, thenthe value for Oeq( 'f 0 X (') A) would be larger than Oei 'f 0 X (') 0). It is
important to realize that zygotic selection would not affect the comparison between the two equivalent values, since they were calculated
respectively for 0 children in 0 mothers. Prezygotic selection in the BO
father can be tested by comparing Oeq( 'f 0 X (') 0) with Oei 'f 0 X (') B).
Many other comparisons between two mating types in which the genotypes of mothers and children are constant but only fathers' genotypes
differ may be used to check the above hypothesis.
Using Japanese family data, it has been demonstrated that the sperm
carrying 0 gene are at an advantage over those carrying A and B genes.
The ratio of 0 sperm in heterozygous male has been estimated at about
0.55. Moreover, it seems that this selection is due either to meiotic
drive or to sperm competition without interaction with the maternal
genotypes.
A preliminary report of this work together with a table will be published in Science 135: 432-433, 1962.

94. Selection in ABO blood groups: Viability and its relatiOit
to the proportion of prenatal deaths ll(By Yuichiro HIRAIZUMI and Ei MATSUNAGA)

Based on a total of approximately 2,200 Japanese family data, Matsunaga and Hiraizumi (in press) found an evidence of pre-zygotic selectionin ABO blood groups; namely, about 55% (=k), instead of the expected
50%, O-bearing sperm from AO and BO fathers are transmitted to theirchildren.
1.

This work was supported by Grant RF 61113 from The Rockefeller Foundation.

111

RESEARCHES CARRIED OUT IN 1961

We now study the post-zygotic selection (viability) operating on thissystem.
1. Estimation of viability.

The relative viability of each genotype can be estimated by comparing
the family size equivalent value for the children of the genotype inquestion with that of a standard genotype (=00)2 in the same mating.
The equivalent values must be corrected in an appropriate way (substitution of k value) when the mating involves either AO or BO fathers.
For convenience we let the relative viability of the standard 0 children
be unity. The viabilities thus estimated are listed in Table 1.
Table 1. Viabilities of genotypes estimated on the basis of k;cO.55
Viability00
Compatible
Incompatible

1.0000-I AA I BB I AB I BO I
1.0674-

1.4466-1.38451.04750.85980.9413AO

1.06720.9792 (in 09)
1.4319 (in B 9)** A surprisingly high viability of incompatible AO children in B mothers is observedin both matings B9 xA o and B9 xAB o . The incompatible AO children also
show very high sex-ratio (c.£. paragraph 3 in this report). Perhaps this highviability is real but the reason for it is not yet known.

It is interesting to examine whether there is any evidence of heterosis
in viability. The figures in Table 1 were rearranged for this purpose
and are presented in Table 2.
In Table 2 we see no sign of heterosis. However, if we do not takeaccount of pre-zygotic selection, we shall be led to quite an opposite
conclusion. Table 3 represents the same comparisons as Table 2, but
this time the viabilities are computed assuming that the proportion of
O-bearing sperm from the AO and BO fathers is 50%.
In Table 3 heterosis is clearly seen in all the three pairs. This shows
2. Even a mating which does not produce standard 0 children can be used for
estimating viability. For example, no 0 children will be produced from the AB 9xAO 0 mating, but we may compute Ve AR by comparing the equivalent value ofAB children with that of BO children whose viability is estimated from otherindependent matings (AB 9 xO 0, B 9 xO 0). However, the reliability of Ve AB thus
estimated will be much lower than that Ve RO • The reliability of Ve AA (and Ve BB )may be also very low, because for this estimation we must substitute the estimate()f VeAO (Ve BO ets.).

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12
Table 2. Comparison in viability of homozygote with heterozygote. Vi}
represents the viability of the compatiable genotype ij. k~O.55
Pair No.
1
2
3

Viability
VOO=l.OOOO=1. ‫סס‬oo
V AA =1.0674

VO O

V Ao =I.0672
VBo=l.0475
V AB =I.3845

V AA =l.0674
V BB =I.4466
V BB =1.4466

how important it is to examine first pre-zygotic selection when we
discuss the phenomenon of heterosis.
Table 3.

Comparison in viability of homozygote with heterozygote. k=O.50

Pair No.

Viability

1

VOO=l.OOOO

2
3

Voo=l.OOOO
V AA =O.7420

VAo=I.0672
VBo=l.0475
VAB=I.0334

V AA =O.7420
V BB =l.0269
V BB =l.0269

2. Relation between viability and proportion of prenatal deaths.
Substituting the estimates of viabilities and the value of k, the averageviability of children can be computed for each mating. This makes it
possible to study the relation between the average viabilities and the
proportions of prenatal deaths (=r). Two reports by Matsunaga and Itoh(1958) and Haga (1959) are available for estimating r in each mating. Fromtheir data the average of r's, r, in the compatible matings is computed
to be 0.077 while it is 0.116 in the incompatible matings. On the other
hand the expected value of r (this can be computed by substituting theestimates of viabilities and of k) in the incompatible matings is com·puted to be 0.158. Thus the two values, observed (=0.116) and expected(=0.158), agree reasonably well suggesting that our viability estimates
are fairly reliable.
If we compute the expected value of r in the incompatible matings
assuming k=0.50, we get r=0.209. Thus the value of r on the basis of
k=0.55 agrees with the observed value better than the one which iscomputed by assuming k=0.50.
3. Relation between viability and sex-ratio.
It is generally accepted that the viability of males is, on the average,
lower than that of females (r is larger in male than in female) andtherefore males will suffer more than females from reduced viabilities.
Admitting this is true, we may expect a positive correlation between

RESEARCHES CARRIED OUT IN 1961

113

sex-ratios (proportion of males) and viabilities ofgenotypes.
Hence we can make one more check of our viability estimates forreliability.
For simplicity we classify the genotypes of children into two groups
according to their viabilities; group 1 includes genotypes with therelative viabilities larger than, or equal to, unity, and group 2 includes
those with viabilities less than unity. In group 1 the average sex-ratio
is 0.5324 while it is 0.5196 in group 2. Thus there is an indication thatthe difference in viability is reflected reasonably in the sex-ratio, although
the difference in sex-ratio between the two groups is statistically insignificant 3 ) • Further accumulations of data are required to confirm this point.

95.

Selection in P blood groups in man: A probable case of
pre-zygotic selection operating in both sexes!)(By Yuichiro HIRAIZUMI)A number of instances of pre-zygotic selection have been reported forvarious organisms, but in cases so far investigated it operates only ineither one of the two sexes. Recently Matsunaga and Hiraizumi foundfor ABO blood groups that more than 50 % (approximately 55 %) a-bearing sperm from heterozygous AO and BO fathers were transmitted to their children, while the segregation ratio from heterozygous
mothers was normal.
The purpose of the present investigation was to find out whether prezygotic selection is operating on the P blood groups in man. P locus
consists of two alleles, P and its recessive allele p. The Japanese family
data used in this study are those published during the period from 1937
to 1959. They consist of 7 publications, dealing with 401 families with
997 children in total. The frequency of p allele (=0.8142) is estimatedfrom these data (frequency of p~.663). In addition, there are several
published data available but they are omitted from this analysis because
they seem to be biased as to gene frequency distributions, perhaps due
to biased sampling procedures. Since a complete analysis will be presented in detail elsewhere, only the perliminary results are reported
here.
3. The sex of children was not indicated in many of the family data used inthis study so that the number of families available for analizing sex-ratio wasrelatively small. This may be the reason why the clear difference between thetwo groups is not statistically significant.
1) This work was supported by Grant RF 61113 from the Rockefeller Foundation.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

1. Pre-zygotic selection in the father.

A method of comparison in terms of the " family size equivalent forchildren of a given genotype" was a powerful tool in studying thepre-zygotic selection in ABO system, so this method was again used inthis study. For family size equivalent, see Matsunaga and Hiraizumi,
1962, Science.
The pairs of equivalent values are listed in Table 1.
Table 1. Comparisons of family size equivalents for p-children
Pair No.

1.
2.
Total

Family size equivalents for p-children(P5i!xpo):2.119(P5i! xPo): 2.240: 2.144(p 5i! X Po) : 2.405(P 5i! xP 0): 3.964: 2.689

Difference+0.286+1. 724+0.544±0.36

The value of k 1 (segregation ratio of p from heterozygous pp fathers)
is estimated to be 0.6269 (0.10>Pr.>0.05). Thus, instead of the expected
50%, about 60% p-bearing sperms from Pp fathers are transmitted totheir children, although the difference between the estimated k 1 and the
expected 0.50 is not statistically significant.
2. Pre-zygotic selection in the mother.
Principally the same method may be applied to examine the segregation
ratio from heterozygous mothers. In this case, however, we must firstcheck the female fertility difference between the two genotypes, P- andpp. The average number of children from P- and pp mothers is 2.2027
and 2.1211, respectively. This suggests that there is no, or little if any,difference in female fertility between P- and pp mothers. This view isalso supported by Matsunaga and Itoh's observation (1958). We therefore proceed assuming that there is no female fertility difference. Table
2 represents similar comparisons as those in Table 1, but this time thegenotype of the children and of the fathers is the same in each pair.
Table 2 suggests that pre-zygotic selection is also operating in theheterozygous mothers, and that it does so in the same manner as in thefathers (k 2 , the segregation ratio of p from Pp mothers, is 0.5870),
although again the value of k 2 does not differ significantly from theexpected value of 0.50.
However, if we combine k 1 and k 2 together, then the deviation from0.50 becomes significant. From those observations it is suggested thatthe pre-zygotic selection favouring p allele is operating on the two sexes
in the same way.

RESEARCHES CARRIED OUT IN 1961
Table 2.
Pair No.
1.

2.
Total

115

Comparisons of family size equivalents for p-children

Family size equivalents for p-children(p9XPi'i):2.119(p 9 x Po) : 2.405: 2.179(P \! XP i'i ): 2.240(P1l XPi'i): 3.964: 2.558

Difference+0.121+1.559+0.379±0.42

3. Viabilities of three genotypes
Viabilities of the three genotypes, Vpp, V-;'p (compatible), V~p (incompatible) and Vpp , can be easily estimated from the present data, i. e.,
Vpp =0.6940, V-;'p=0.8204, Vj,p=0.8223, and Vpp=l.OOOO. Matsunaga andItoh (1958) reported that there was no demonstrable difference in themean number of children, proportion of natural abortions, etc., between
P-compatible and P-incompatible matings. In the present study, again
there is no sign of difference between V-;'p and V~p. We therefore use
the average of the two estimates, 0.8214, as a measure of viability of
pp heterozygotes.
It is worth noting that the estimates of viabilities of three genotypes
are in favour of the so-called classical hypothesis founded on the superiority of homozygotes in fitness.
4. Conditions for the co-existence of two alleles
The fitness of an organism is the resultant of many components, andtherefore the informations on viability alone are not always sufficient tounderstand the total fitness. But since viability is one of the mostimportant components of fitness, we assume, for the time being, that
viability is a good measure of the total fitness.
Let the viability of aa, Aa (compatible and incompatible) and AA be
V aa , V~a, V~a and 1 respectively, and let k, and k 2 be the segregation
ratio for a from Aa heterozygous males and females, respectively. Aftersome calculations we get the following sufficient conditions for the coexistence 2} of the two alleles, although it is extremely difficult to obtainthe necessary conditions.

The viabilities of the three genotypes and the value of k estimated inparagraph 3 may be substituted into these conditions: clearly the2) Conditions for co-existence are not always the same as those for the stableequilibrium; two alleles can co-exist without maintaining stable equilibrium if there
are regular periodical changes in frequencies. For this see, for example, Kimura
1958, Wright 1955, etc.

116 ANNUAL REPORT OF NATIONAL INSTITUTS OF GENETICS NO. 12

estimates do not satisfy the two conditions, or the p allele would replace
P when the frequency of p is sufficiently close to 1. This, of course,
does not always imply that P and p cannot co-exist, because the above
two are sufficient conditions, but may not always be the necessary ones.
It is, perhaps, worth noting that by trial and error procedure, we canfind the following set of coefficients which meets the above two conditions: Vpp = 1.0000, V pp =0.8387, V pp =0.6498 and k=0.6100. With thesecoefficients P and p can maintain an equilibrium state with the zygotic
frequency of 0.63 for pp and 0.37 for P-. Note that these coefficients andfrequencies are very close to the estimated and actually observed ones.
However, further studies must be made on this point because the equilibrium frequencies, and their stabilities, are very sensitive to even aslight change in anyone of the coefficients.
Finally, it is very interesting that there are some similarities in selection mechanisms between ABO and P blood groups, i. e., in both systems
the recessive alleles (0 in ABO and p in P systems), which are lessviable when homozygous, are favoured by pre-zygotic selection.

96. Genetic study on sporadic retinoblastoma in Japan*(By Ei MATSUNAGA)

In our previous endeavor (1959) to establish as complete a roster as
possible of retinoblastoma cases occurring in Hokkaido in the period
from 1945 to 1957, 69 cases were located, of which 68 were sporadic
while there was only one familial case. The incidence of the disease
was calculated to be approximately 1 for each 23,829 live births or 4.2
x 10-5 for one live birth. This figure is rather close to that found forEuropean populations. However, the question remained open what fraction of sporadic cases was actually due to fresh mutations.
In order to answer the above question, a follow-up study of patientswith retinoblastoma treated at three University Hospitals in the period
from 1900 to 1935 has been carried out. In parallel with this investigation, questionaires were sent to 76 institutions for blind persons asking
about the outcome of marriages of persons whose eye balls had been
enucleated because of retinoblastoma in childhood.
Up to present, altogether 43 cases of sporadic retinoblastoma that had
survived the disease could have been followed up, of which 37 were ofunilateral and 6 were of bilateral retinoblastoma. Among the 37 unilateralcases, 28 have" married, of which 22 have got one or more children. Out* This study was supported by a Grant·in-Aid from the Ministry of Education.

RESEARCHES CARRIED OUT IN 1961

117

of these 22, however, there was only one single case, a man who transmitted the disease to one of his two children. Assuming that thepenetrance of retinoblastoma gene is approximately 0.75, as was calculated
in the previous study, and also considering the distribution of the numbersof children born to the affected parents as well as the mean age at theonset of the disease, the fraction of sporadic unilateral retinoblastoma
actually due to germinal mutation was estimated to be only about 8%,
the remaining 92% being regarded as phenocopies.
As for cases of bilateral sporadic retinoblastoma, it was found that
there was only one case married out of the 6 whose ages were over 20
years. Surprisingly enough, however, this man transmitted the disease
to his single child, a son who was affected with bilateral retinoblastoma
like his father.
Although the number of available cases is still very scarce, the discrepancy between unilateral and bilateral retinoblastoma with regard to
the probability of transmission of the disease to children seems to bevery striking. Thus, it appears that the theory advanced by Vogel(1957) that about 17-18% of unilateral sporadic cases and 50-100% ofbilateral cases are due to fresh mutation, may also be applied to Japanesematerial. The rate of mutation of the gene for retinoblastoma in Japanis calculated to be about 8xlO- 6 , which is very close to the figure of
6-7 x 10-6 obtained by Vogel for Caucasians.
Reference
1. Matsunaga, E. and Ogyu, H.: Genetic study of retinoblastoma in a
Japanese population. Jap. J. Human Genet. 4: 156, 1959.
2. Vogel, F.: Neue Untersuchungen zur Genetik des Retinoblastoms(Glioma retinae). Z. menschl. Vererb.- u. Konst.-Lehre 34: 205-236,
1957.

97. Dimorphism in human normal cerumen(By Ei MATSUNAGA)

Among a number of polymorphic traits which have been dIscoveredin man, the dimorphism in human normal cerumen or ear-wax has beenstudied almost exclusively in Japan, while European and American
workers have hitherto paid very little attention to this phenomenon.
This may perhaps be due to the fact that the variation in cerumen is
not so marked among Caucasians and Negroes as in Mongolian race.
In a paper by the auther which will be published in the Annals of118 ANNUAL REPORT OF NATIONAL INSTITUTS OF GENETICS NO. 12

Human Genetics, Vol. 26: 273-286, 1962, various aspects of this dimorphism
have been reviewed on the basis of the author's own observations inaddition to the results collected by other investigators. It was shownthat the ear-wax types are apparently determined by a simple genetic
mechanism, and can be used as a polymorphic system for both genetics
and anthropology. Frequencies of the alleles for the ear-wax types havebeen estimated for different ethnic groups. It was pointed out that themarked difference in the distribution of ear-wax types among the major
races is probably a consequence of natural selection having acted in along course of evolution, and there may be some differences in theselective value between individuals with wet cerumen and those withdry. In view of the apparent association with axillary odor, it wassuggested that the alleles for ear-wax types may control certain metabolic
processes, through which individuals with different types of cerumen
may display different reactions to environmental changes. The need for
future studies along this line is stressed.

98.

The frequency distribution of the so-called "drumsticks"
in the polymorphonuclear neutrophil leucocytes
in Japanese female blood films ll(By Yasuko TONOMURA (ToYOFuKu) and Akira TONOMURA)

The neutrophil method proposed in 1954 by Davidson and Smith is acytological test for the detection of the genetic sex in man. The basisof this test is that 2 to 3 per cent of polymorphonuclear neutrophilleucocytes of females have a solitary chromatin mass- generally referred toas "drumstick" -- attached to a segment of the nucleus by a thinstrand of chromatin, while this characteristic nuclear appendage does notoccur in neutrophil leucocytes of males. The fact of nuclear sexual dimorphism in neutrophils has firmly been confirmed by many independentstudies. However, some investigators have questioned the accuracy ofthis method in detecting the genetic sex in certain patients (Briggs andKupperman 1956, Ashley 1958).
In order to scrutinize the validity of this neutrophil method, the authors
examined the frequency distribution of the neutrophils showing thetypical drumstick in Japanese females, aged 1 to 79 years. For thepresent investigation five hundred neutrophil leucocytes were examinedin each of 250 female blood films. The first cell having one drumstick
was found on the average in 42.74 neutrophils. Six drumsticks wereobserved on the average in 255.37±8.17 neutrophils. The correctness of
1.

This work was supported by Grant RF 61113 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1961

119

these average figures was readily corroborated by the results of Davidson
and Smith (1954), Tenczar and Streitmatter (1956) and others. The
number of drumsticks found in 500 neutrophils was, on the average,
1l.84±0.38 in 250 females, aged 1 to 79 years. However, the negative
correlation between age and the frequency of drumsticks indicate indicatesthat the incidence of drumsticks decreases significantly with age (r=-0.258, P<0.01), although the presence of drumsticks was demonstrated
for all ages here examined.
On the other hand, five hundred neutrophils were examined in each of
50 male blood films, and no characteristic drumstick could be observed.
At the present, the origin of the drumsticks still remains an unsolved
problem, but it is suggested that the determination of the genetic sex
in neutrophil leucocytes is highly accurate and much easier than skinbiopsy or oral smear methods.

99.

Chromosome conditions zn twenty patients(By Akira TONOMURA)~no.

Conditions

1
2

Turner's syndrome
Sister of a mongol boy
Mother of two children, one with cleft palate
and the other with cerebral palsy
Husband of case 3
Down's syndrome
Bourneville-Pringle's dis~ase

3
4
5
6
7
8
9
10
11

12
13
14
15
16
17
18
19
20

f/

Microcephaly(sister of case 8)
Adrenogenital syndrome
Klinefelter's syndrome
Congenital anhidrosis
Testicular feminization
Boy with cleft palate
Girl with cleft palate, hair lip and heart disease
Boy with hypoplastic penis
UHrich syndrome
Down's syndrome
f/

'1
'1

Chrom.

I no.
F
F

27
23

45
46

F

36

46

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

37
10 months

46
47
46
46
46
46
46
47
46
46

11

10

27
20
6
19
4 months
20
-

46

-

46
46
46
47
47
47

8
5
7

1

120 ANNUAL REPORT OF NATIONAL INSTITUTS OF GENETICS NO. 12

The chromosome complement of twenty selected patients was examinedusing short term culture of either bone marrow or peripheral blood. In
all cases a minimum of three cells were analysed in detail. The chromosome conditions of each case are summarized in the above table,
together with brief clinical conditions.

H. TECHNICAL NOTE
100. Measurements of r ray contamination in the reactorof ]RR-1 and response of silver-activated phosphateglass containing lithium and boron(By Sohei KONDO and Hiromi ISHlw A)

Two kinds of silver-activated glass dosimeters 6 mm long and 1 mm
in diameter are commercially available from Toshiba Electric Company(see Table 1).
Table 1.
Name of glass (usedonly for this paper)
K glass
Li glass

Compositions of glass dosimeters used
Base composition(weight %)KP0 3(25), Ba(P03M25)
Al(P0 3)3(50)LiPO(50), Al(P0 3)s(50)Added component(weight %)AgP0 3(8)Ag(P03)(8), B20 3(3)Let us expose these two kinds of glass rods encased in aluminum
container 1mm thick to <1J thermal neutrons per cm 2 mixed with r raysof r r and denote the readings of K and L glass by K and L r-equivalent,
respectively (1 r-equivalent means the output of fluorescence of glass
equal to that induced by 60CO r rays of 1 r). Then we have

k<1J+I'=K,
1<1J+r=L,(1)(2)

where k and I are, respectively, readings (in units of r-equivalent) of K
and L glass rods per one thermal neutron per cm'. Using the valuek = 1/(8.55 X 109 ) (r-equiv.ln.cm- 2 ) previously determined, we have thefollowing

r=K-

<1J

8.55x 10" 'I = ~[L-rl

(3 )
(4)

RESEARCHES CARRIED OUT IN 1961

121

Experimental measurements of (J)/t and FIt are given in Table 2, wheret is the exposure time. The values of (J)/t have been determined by gold
foils (Ichikawa and Amano, 1961).
Table 2.

Thermal neutron flux (/ilt and r contamination dose-rate fit
in thermal column of JRR-l

Position and power levelWlt(n/cm 2 • sec)f It(rlsec)
f/!/J(r/n. cm- 2)

excess (/J(n/cm 2 )excess fer)lll(n. cm- 2/r)

No. 16 Hole; 0.5 kw

No.7 Hole; 40 kw

5.6(5.2*) X 107
1.4(1.5*)
2.5(2.8*) X 10- 10
6.6 X 10 '0
113.6(2.5*) X 108

1. 33 X 107** 1. 7(2.0*) X 108
0.02**
0.03(0.18*)
1.2xlO- 9** 1.8(6.2*)x I O-10

2.3x10 8* Values obtained by glass plate dosimeters of different Ag concentration in 1960.
** The dosimeters presumably did not reach the ordinary position (11.8 em fromgraphite surface of the core of JRR-l).
Table 2 shows that the r contamination is in the same order both in
No. 16 Hole, which passes through the center of the reactor, and No.7
Hole which has been designed as the biological research hole.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12

101. Dose-rate distribution in the Cs-137 gamma ray room
(By Sohei KONDO and Hiromi ISHIWA)

The facility for the 2 kc 137CS r ray irradiation was built in March,
1960. The dosimetry of the r field was carried out by Victoreen condenser
chambers and the Fricke dosimeter (FeSO.(NH.)2S0•.6H 20 2 g,
NaCl 0.3 g, H 2S0.c95-98%) 110 cc, total 5000 cc). The measured
values of the effective r-ray intensity of the source were 708.7 rhm,
747.5 rhm and 754.8 rhm for the Fricke, Victoreen 100 r (r) and Victoreen
250 r (X) dosimeters, respectively. The curve of the dose-rate-versusdistance from the source along the vertical line passing through thecenter of source is given in Fig. 1 by using the Fricke dosimeter readings.

./

/
~

1/

,
~
-' 10
7
/

/

Sou.rce....

8...
I--.'U+.L--

~
~

II

12,11'1

U 20

.J.

~

.j

~

10

r= 40
~

T.•.•.••• ,.
100

8<: 6.

u r:r sCAle

-t!

P

200

~

float:..!jzt;cm
300 200

80

100'<l 40 30

20

10vertical distance from effective source center(em)

Fig. 1. Dose-rate distribution in the 137C8 r
field with 2 kc (Oct. 1961).

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

123

BOOKS AND PAPERS PUBLISHED IN 1961
BY STAFF MEMBERS
FUJII, T. and T. MABUCHI 1961 Irradiation experiments with chrysanthemum.
Seiken Ziho 12: 40-44
and Y. ONO 1961 Relation between chlorophyll content and freeamino acids in einkorn wheats. Proc. Jap. Acad. 37: 633-637
HIRAIZUMI, Y. 1961 Negative correlation between rate of development and female
fertility in Drosophila melanogaster. Genetics 46:615-624- - - , T. NARISE, and I. FUKUDA 1961 Heterotic viability in natural populations of Trillium kamtschaticum Pall. Jap. Jour. Genet. 36: 413-418
SANDLER, L., and Y. HIRAIZUMI 1961 Meiotic drive in natural populations ofDrosophila melanogaster. VII. Conditional segregation-distortion: A possible
non-allelic conversion. Genetics 46: 585-604- - - and
1961 Meitotic drive in natural populations of Drosophilamelanogaster. VIII. A heritable aging effect on the phenomenon of segregationdistortion. Canadian Jour. Genet. & Cytology 3: 34-46
IINO, T. 1961 Genetic analysis of O-H variation in Salmonella. Jap. Jour. Genet.
36: 268-275
1961 A stabilizer of antigenic phases in Salmonella abortus-equi. Genetics
46: 1465-1469
1961 Anomalous homology of flagellar phases in Salmonella. Genetics
46: 1471-1474
KADOTA, H., R. IMADA, A. KASAHARA and H. ISHIWA 1961. Measurement of
exposure doses with silver metaphosphate glass rods at the fingers of physicianson fluoroscopic examination of digestic tract (in Japanese). Hiroshima Med.
Jour. 15: 100-103
KATAYAMA, T. C. 1961 Investigations on the measuring method of the intercellular spaces on several plants. Proc. Crop. Sci. Soc. Jap. 30: 150-154
KATSUYA, K. 1961 Studies on the susceptibility of Oryzae to the blast fungus,
Piricularia oryzae (in Jap. with Eng. summ.). Ann. Phytopath. Soc. Japan 26:
153-159
and Y. ONO 1961 Effects of radiation on the susceptibility of wheat
seedlings to leaf rust (in Jap. with Eng. summ.). Ann. Phytopath. Soc. Japan
26: 147-152
KAWAHARA, T. 1961 Studies on heterosis in the domestic fowl IV. Hachabilityof F 1 hybrids and their parental purebred chick embryos (in Japanese withEng. resume). Jap. Jour. Zootech. Sci. 31: 309-313
KIHARA, H. 1961 Viruses and phenocopy (in Japanese). Nucleus and Cytoplasm
2: 1
and K. TSUNEWAKI 1961 Pistillody of Triticum durum induced byan alien cytoplasm. Seiken Ziho 12:
KIMURA, M. 1961 Genetical problems discussed at the 8th United Nations Assemblyof the Scientific Committee on the Effects of Atomic Radiation (in Japanese).
Kagaku-Asahi 2: 28-31

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12
1961 Radiation genetics in USSR, with special reference to the reports
presented at the 8th United Nations Assembly of the Scientific Committee on
the Effects of Atomic Radiation (in Japanese). Iden 15 (4): 4-8
1961 Natural selection as the process of accumulating genetic informationin adaptive evolution. Genetical Research 2: 127-140
and H. KA.YANO 1961 The maintenance of supernumerary chromosomes
in wild populations of Lillium callosum by preferential segregation. Genetics
12: 1699-1712
KONDO, S. 1961 A theory for the frequency distribution of cluster mutants. Jap.
Jour. Genet. 36: 6-17
1961 A biophysical consideration on radiation mutagenesis. Progr.
Theor. Phys. Suppl. 17: 129-142
1961 Simultaneous measurement of thermal neutron fluxes and gamma
contamination dose by silver-activated phosphate glass. In," Selected Topics
in Radiation Dosimetry." Intern. Atomic Energy Agency, Vienna, 491-496
1961 Radiation- physical basis for radiation biology - (in Japanese).
Biophysics (Japan) 1: 91-99
KURITA, Y. and T. H. YOSIDA 1961 Chromosomal alteration and the developmentof tumors, VII. Karyological analysis of spontaneous and induced leukemias
in mice. Gann 52: 257-264
MATSUMURA, S. 1961 Radiation genetics in wheat, VI. Biological effects ofthermal and fast neutrons on diploid wheat. Jap. Jour. Genet. 36: 84-96
1961 Ibid., VII. Comparison of radiation effects of beta-and gamma-rays
on diploid wheat. Rad. Bot. 1: 155-165
1961 Studies on artificial induction of mutations (in Japanese).
Jap. Jour. Breed. 11: 124-130
and K. KATSUYA 1961 Susceptibility of nullisomics wheat dwarfs andtheir respective gigas-plants to leaf-rust, Puccinia triticina. W. I. S. 12: 10
and M. NEZU 1961 Radiation effects of fast and thermal neutrons on
wheat, III. Relation between ploidy and radiation effects. W. I. S. 12: 9-10
and
1961 Relation between polyploidy and effects of neutronradiation on wheat. Effect of Ionizing Radiations on Seeds, I. A. E. A.,
543-552
OHTA, T. and S. MATSUMURA 1961 Mechanism of appearance of gigas-plantsfrom nullisomic dwarf wheat. Cytologia 26: 226-235
SAKURAI, T. and S. MATSUMURA 1961 On the relation of chromosomal aberrations
to gamma-ray dosage and dose rate in barley (in Jap. with Eng. summ.).
Seiken Ziho 12:
MATSUNAGA, E. 1961 Can the onset of hereditary disease be prevented? (inJapanese). Iden 15 (6): 17-18
CHUNG, C. S., E. MATSUNAGA and N. E. MORTON 1961 The MN polymorphismin Japan. Jap. Jour. Human Genet. 6: 1-11
TANAKA, K., E. MATSUNAGA, N. HANDA, T. MURATA and K. TAKEHARA 1961
Phenylketonuria in Japan. Jap. Jour. Human Genet. 6: 65-77
MATSUNAGA, E. 1961 Sex chromosome and sex chromatin in man (in Japanese).

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

••

125

Nihon Rinsho 19: 2243-2250
E. 1961 Human genetics, I (in Japanese). Iden 15 (12): 55-59
MORISHlMA, H., H. I. OKA and W. T. CHANG 1961 Directions of differentiationin populations of wild rice, Oryza perennis and O. sativa f. spontanea. Evolution 15: 326-339
MUKAI, T. 1961 A radiation-genetical consideration concerning the structure ofnatural populations. Jap. Jour. Genet. Suppi. 36: 139-150
1961 Recent topics concerning the maintenance of genetic variability innatural populations (in Japanese). Iden 15 (12): 29-32
and A. B. BURDICK 1960 Heterozygous effects of X-irradiation on theasymmetry of sternopleural bristle numbers in D. melanogaster. D. I. S. 34: 94
1961 Examination of the closely linked dominant adaptive gene hypothesis
as an alternative to single gene heterosis associated with 1 (2) 55 i in Drosophila melanogaster. Jap. Jour. Genet. 36: 97-104- - - . and S. CHIGUSA 1961 X-ray induced polygenic mutation rate in
Drosophila melanogaster (in Japanese). Jap. Jour. Genet. 36: 388
OGAWA, Y. 1961 Growth, differentiation and regeneration XXII. Biochemical
relation between the synthesis of actin and myosin in the regenerating limb
tissue of Triturus (in Japanese). Med. and BioI. 58: 8-11
1961 Examination in the taste ability (in Japanese). J. of Nutrition 18:
34-38
1961 Two factors sensitive to X-ray irradiation influencing the synthesisof skeletal muscle protein in early embryo (in Japanese). Kagaku 31: 92-93
1961 Growth differentiation and regeneration XXIII. Antagonistic effectbetween kinetin and Na-glucuronate in growth promoting activity on Triturusembryos (in Japanese). Med. and BioI. 58: 90-93
1961 Abnormal taster in Japanese to phenyl-thio-carbamide (in Japanese).
Med. and BioI. 58: 122-126
and N. KARUBE 1961 Growth, differentiation and regeneration XXIV.
Effects of glucuronic acid and related compounds on the development ofskeletal muscle proteins in early Triturus embryo (in Japanese). Med. and
BioI. 58: 185-187
1961 Growth, differentiation and regeneration XXV. Effect of Natriumglucuronate on the synthesis of actin in X-irradiated Triturus embryo (inJapanese). Med. and BioI. 59: 50-52
H. WATANABE and A. IDE 1961 The chemical structure of a componentof Citrullus colocynthis, SCHRAD. Agr. BioI. Chern. 25: 269-271
and N. KARUBE 1961 Growth, differentiation and regeneration XXVI.
Effects of sodium glucuronate and related compounds on the synthesis ofmuscle protein in the regenerating limb tissue of Triturus (in Japanese). Med.
and BioI. 59: 89-92
1961 Growth, differentiation and regeneration XXVII. Similarity of the
response to X-ray of actin formation in early embryonal stage and regenerating
limb tissue of Triturus (in Japanese). Med. and BioI. 59: 168-166
1961 On the experimental method of the examination in bitter taste-126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 12
blindness to phenyl-thio-carbamide (in Japanese). Iden 15(10): 45-49
1961 Protective function of sodium glucuronate on the abnormal differentiation to muscle tissue in X-irradiated embryo (in Japanese). Proc. of 7th
Symp. of Glucuronic Acid. 35-36
1961 Effects of X-ray irradiation on the syntheses of contractile proteinsin Triturus embryos. Biochim. Biophys. Acta 54: 397-399
OKA, H. I. and W. T. CHANG 1961 Hybrid swarms between wild and cultivatedrice species, Oryza perennis and O. sativa. Evolution 15: 418-430
1961 Horai varieties of rice and their adaptability in Taiwan (In Jap. withEng. resume). Jap. Jour. Trop. Agr. 5: 6-12
OSHIMA, C. 1961 Population genetical studies on insecticide resistance of insects.
Jap. Jour. Genet. 36: 359-376
OSHIMA, C. and O. KITAGAWA 1961 The persistence of deleterious genes innatural populations of Drosophila melanogaster. Proc. Jap. Acad. 37: 158-162
1961 Heterozygous effects of induced lethal genes on pre-adult viability
in Drosophila melanogaster and their persistence in experimental populations.
Suppl. Jap. Jour. Genet. 151-162
SADO, T. 1961 Spermatogenesis of the silkworm and its bearing on the radiationinduced sterility. Jap. Jour. Genet. (Suppl.): 136-151
SAKAGUCHI, B. and D. F. POULSON 1961 Transfer of the "sex-ratio" condition
from D. willistoni to D. melanogaster. (Abstr.) An'lt. Rec. 138: 381
and-1961
Evidence concerning the nlture of the "sex-ratio"
agent in Drosophila. (Abstr.) Anat. Rec. 138: 376-377
and-1961 Nature of "sex-ratio" agent in Drosophila. Science
133: 1489-1490
SAKAMOTO, S. 1961 An ecotype of Agropyron, Early ecotype (in Japanese withEnglish summary). Seiken Ziho 12:
LIYANAGA, D. V. and K. I. SAKAI 1960 Heritabilities of certain yield charactersof the coconut palm. Jour. Genet. 57: 245-252
SAKAI, K. I. and M. RODORIGO 1960 Studies on a laboratory method of testingsalinity resistance in rice varieties. Trop. Agr. 116: 179-184
1960 A comment on "The improvement of coconut palm by breeding
and selection" of Dr. S. C. HARLAND. Trop. Agr. 116: 185-190- - - , S. IYAMA and T. NARISE 1961 Biometrical approach to the cytoplasmic
inheritance in autogamous plants. Bull. Internat. Stat. 38: 249-257
1961 Competitive ability in plants: its inheritance and some related
problems. Symp. for Exp. BioI. 15: 245-263
TAKENAKA, Y., Y. OGAWA and T. ODASHIRO 1961 Induction of abnormal mitosis
and inhibition of growth by substances extracted from certain plants in relation
to tumor growth control I. Radiomimetic effects of extract from Gentiana
upon living plant tissue (in Japanese with English resume). Wakayama-Igaku
17: 1007-1016- - - , Y. OGAWA, T. ODASHIRO and N. KARUBE 1961 Induction of abnormal
mitosis and inhibition of growth by substances extracted from certain plants
in relation to tumor growth control II. Anti-cancer activity of Gentiana,.

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

127

extract (in Japanese with English resume). Wakayama-Igaku 12: 1325-1329
TAZIMA, Y. 1961 Theoretical basis of rediation breeding (in Japanese). Jap. Jour.
Breeding 11: 119-124
1961 Considerations on the changes in observed mutation rates in thesilkworm after irradiation of various stages of gametogenesis. Jap. Jour.
Genet. Suppl. 36: 50-64
S. KONDO ahd T. SADO 1961 Two types of dose·rate dependence ofradiation induced mutation rates in spermatogonia and oogonia of the silkworm.
Genetics 46: 1335-1345
TOYOFUKU. Y. 1961 Chromosomal polymorphism found in natural populations ofDrosophila immigrans (in Japanese). Jap. Jour. Genet. 36: 32-37
TOYOFUKU, Y. and Y. KIMURA 1961 Drosophila survey of Hokkaido, XIV. Distribution of drosophilid flies in Taisei-Mura, in the southwestern part of
Hokkaido, in relation to the flora. Jap. Jour. Zoo1. 13: 7-14
TSUJITA, M. 1961 Maternal inheritance of yellow lethal in the silkworm, Bomby:cmori. Nucleus and Cytoplasm 1: 9-14
1961 Maternal effect of +lem gene on pterine reductase. Jour. Seric.
Sci. Japan 30: 254
1961 Yellow pigments in the yellow lethal larvae and pterine reductase
acting on this pigments. Proc. Ann. Meet. Tokai Branch Seric. Soc. Jap.
9: 23-24
1961 Investigations in Nl type lethal mutants of the silkworm withspecial reference to Nl, Nl1 and Nl2 embryos. Jap. Jour. Genet. 7-8: 235-243
1961 Maternal effects of +lem on pterine reductase in Bomby:c mori.
Jap. Jour. Genet. 36: 337-346
M. and S. NAWA 1961 Functional relationship between lem and d-lem
genes in the silkworm. Jap. Jour. Genet. 36: 397
TSUNEWAKI, K. 1961 Gene evolution (in Japanese). Iden 15(2): 18-23
1961 Monosomic and conventional gene analyses in common wheat. IV.
Glume hairiness and ear density. Jap. Jour. Genet. 36: 55-62
and B. C. JENKINS 1961 Monosomic and conventional gene analyses incommon wheat. II. Growth habit and awnedness. Jap. Jour. Genet. 36:
and H. KIHARA 1961 F 1 monosomic analysis of Triticim macha. W. I.
S. 12: 1-3
YAMADA, Y. and O. KITAGAWA 1961 Doubling dose for polygenic mutations in
Drosophila melanogaster. Jap. Jour. Genet. 36: 76-83
1961 Population genetical approaches in animal breeding (in Japanese).
Jap. Jour. Breeding 11: 85-90
BOHREN, B. B., H. E. McKEAN and Y. YAMADA 1961 Relative efficiencies ofheritability estimates based on regression of offspring on parent. Biometrics
17: 481-491
MITSUMOTO, T. and Y. YAMADA 1961 Determination of parentage by the use of
polyvalent immune sera in chickens. Poultry Sci. 40: 1701-1704
YOSIDA, T. H. 1961 Chromosome and development of tumor (in Japanese). Nihon
Rinsho 19: 2273-2284

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETl'CS NO. 12
1961 Chromosomal alteration and the development of tumors, VIII.
Affinity of diploid and polyploid tumor cells of common Yoshida sarcoma strains
to certain organs. Gann 52: 313-320
UTSUMI, K. and T.H. YOSIDA 1961 On the behavior of tumor cells of the Ehrlich
ascites carcinoma growing in vitro. Annot. Zool. Jap. 34: 132-138
TANAKA, K. and T.H. YOSIDA 1961 Chromosomal condition of hypertriploid Hiro·saki sarcoma (in Japanise with Eng. resume). Zool. Mag. 70: 142-146- - - - , H. SAKAMOTO and T. ODASHIRO 1961 Genetical study on the new mutant.. falter" found in the house mouse. (Preliminary notes on experimental breed.
ing and genetics of small animals, No. 1 (in Japanese with Eng. resume).
Bull. Exp. Animals 9: 179-182- - - , Y. KURITA and S. T ANEDA 1961 Genetic study on two wild mutants ofrats (Preliminary note on ~xperimental breeding and genetics of small animals,
No. 2 (in Japanese with Eng. resume). Ibid 10: 20-22

ACKNOWLEDGMENTS
The editor wishes to acknowledge Dr. F. A. LILIENFELD for reading the manu·
scripts (Ei MATSUNAGA).

mft3l~*lilf~FJfif~

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IlBfll37 ~ 6 Ji 25 B ~1T~;fT1f

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