NATI.ONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 151964

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima. Sizuoka-ken. Japan1965

Annual Reportof theNational Institute of Genetics
No. 15, 1964

(A Perspective of the Institute)Published by

The National Institute of Genetics, Japan
1965

CONTENTS
General statement. . . . .. .... . .. . . . .. .. . . . . . . . . . . .. .. ..... . . . . . .... . .
Abstracts of diary for 1964............................
Staff...............................................................
Council............................................................
Projects of research for 1964
Foreign visitors in 1964............................................
Researches carried out in 1964.....................................

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A. Genetics, cytology and biochemistry of animals
1. Genetic variability of a large natural population of Drosophilamelanogaster (OSHIMA and WATANABE, T. K.).. . . . . . . . . . . . . .
2. Allelic rate between lethal genes concealed in natural populations of Kofu and Katsunuma (OSHIMA and WATANABE,

13

T. K.)....................................................

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3.
4.

5.
6.
)r

7.
8.
9.

10.
11.

12.

Location of lethal genes on the second chromosome (WATANABE, T. K. and OSHIMA).. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Frequencies of inversions in the Katsunuma natural population (WATANABE, T., WATANABE, T. K. and OSHIMA).... . .
Mechanism of maintenance of chromosomal polymorphism innatural populations (WATANABE, T. and OSHIMA)
The effect of insecticide selection on changing the frequencies
of four chromosomal types in experimental populations ofDrosophila pseudoobscura (OSHIMA and WATANABE, T.)......
Distribution pattern of tryptophan and pteridine metabolites
in Drosophila larval fatbody (TAIRA and RIZKI) .. . . . . . . . . .
Mapping of acid soluble nucleotides in insects (TAIRA andSUZUKI, S.) .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heterotic effect of linkage disequilibrium in Drosophila
populations (hAMA)
Position effect of spontaneous mutant polygenes controlling
viability in Drosophila melanogaster (MUKAI and YAMAZAKI).
The viabilities of heterozygotes for newly arising spontaneous
lethal genes in Drosophila melanogaster (YosHIKAWA andMUKAI)
Average degree of dominance of spontaneous mutant polygenes controlling viability (MUKAI and YAMAZAKI) .. . . . . . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

13. Homozygous loads and genetic structure of natural populations of Drosophila melanogaster (MUKAI). . . . . . . . . . . . . . . .
14. Synergistic interaction between spontaneous mutant polygenes controlling viability in Drosophila melanogaster(MUKAI)
15. Heterosis and Hardy-Weinberg equilibrium (MUKAI)
16. A condition for pseudo-overdominance (MUKAI) .. . . . . . . . . . . .
17. Examples of permanent linkage disequilibrium (MUKAI andyAMAZAKI)................................................
18. Persistence of the recessive gene ew in experimental populations in Drosophila melanogaster (YAMAZAKI and MUKAI). .
19. Partition of fitness into components concerning the recessive
mutant gene ew in two experimental populations ofDrosophila melanogaster (YAMAZAKI and MUKAI) .. . . . . . . . . .
20. Disappearance of "Sex-Ratio" condition by double infection
with two "Sex-Ratio" agents of different origin in
Drosophila (OISHI, K. and SAKAGUCHI)
21. Nature of the substance produced by nebulosa-SR agent
that kills willistoni-SR agent in Drosophila (SAKAGUCHI andOISHI, K.)................................................
22. Multiplication of SR agents in Drosophila (KOBAYASHI and
SAKAGUCHI)
23. Cellular studies of antibody formation, IV. Variation in the
kinetic indices of plasma cells in antibody response (SADO,
MAKINODAN, LEONARD and CHADWICK)
24. Chemical modification of DNA and its constituents (SHINODA)
25. Isolation and characterization of the deoxyribonucleic acidfrom Ephestia (NAWA)....................................
26. A new method for the study of chemical mutagenesis in thesilkworm (TAZIMA and ONIMARU)..........................
27. Purification of the three specific soluble chromoproteins fromchromogranules in hypodermal cells of the silkworm larva(TSUJITA and SAKURAI)....................................
28. Amino acid analysis of the three specific chromoproteins
purified from chromogranules in hypodermal cells of thesilkworm larva (TSUJITA and SAKURAI) . . . . . . . . . . . . . . . . .. . .
29. Amino acid analysis of the insoluble protein in chromogranulepellicles (TSUJITA and SAKURAI) .. . . . . . . . . . . . . . . . . . . . . . .. ..
30. Lipid contents in chromogranules in hypodermal cells of thesilkworm larva (SAKURAI and TSUJITA) . . . . . . . . . . . . . . . . . . ..
31. Multiple allelic genes for dilute lemon larval skin color of
the silkworm (TSUJITA and SAKURAI)......................

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CONTENTS

32.
33.
34.
35.
36.
37.

38.

39.

40.
41.
42.

43.

44.

45.
46.
47.
48.
49.

Variance and covariance analysis of egg weight, egg shapeand body weight in the domestic fowl (KAWAHARA)
Effect of heterosis and sex-linkage on sexual maturity inthe domestic fowl (KAWAHARA)............................
Bilateral asymmetry in the ribs of cervical vertebrae in thedomestic fowl (SAKAI and KAWAHARA).....................
Bilateral asymmetry in the legs of the domestic fowl (SAKAI
and FUJISHIMA). . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . .
Breed differences in competitive ability of growing chickens(FUJISHIMA) .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
Studies on chromosomal polymorphism, I. Autosomal polymorphism in Rattus rattus L. collected in Kusudomari and
Misima (YOSIDA, NAKAMURA and FUKAYA)..................
Studies on chromosomal polymorphism, II. Autosomal polymorphism in Norway rats, Rattus norvegicus (YOSIDA and
AMANO)
Further study on chromosomes of leukemias in the mouse
induced with Moloney leukemogenic virus (MLV) (YOSIDA
and LAW)
Karyotypes in drug resistant sublines of mouse leukemia L.
1210 (YOSIDA and OHARA)..................................
Chromosomes in drug sensitive and resistant lines of plasma
cell neoplasm 70429 of mice (YOSIDA) . . . . . . . . . . . . . . . . . . . . .
Karyotypic differences in mouse ascites hepatoma MH 134
and MH 129P maintained in different places (YOSIDA and
IMAI)
Autoradiographic analysis on the sensitive stage to 4-nitroquinoline I-oxide during division cycle of Yoshida sarcoma
cells (MORIWAKI, KURITA and YOSIDA)......................
Chromatid aberrations induced in Yoshida sarcoma cells by
the radiomimetic chemical, 4-nitroquinoline I-oxide (KURITA,YOSIDA and MORIWAKI)....................................
Autoradiographic analysis of the mitotic cycle in Yoshidasarcoma cells (KURITA, MORIWAKI and YOSIDA)
Strain difference of susceptibility to the teratogenic actionof ethylurethane in mice (TUTIKAWA and AKAHORI)........
Further studies on water soluble proteins in mouse skin following application of a tumor promoting agent (OGAWA).....
Chromosome observation in Japanese ants (IMAI and YOSIDA)..
Abnormal taster to phenyl-thio-carbamide (PTe) in Japanese
population (OGAWA)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

B. Genetics, cytology and biochemistry of plants
50. "Germless grain", a new effect of caudata cytoplasm on the
51.
52.

53.

54.

55.

56.
57.
58.
59.

60.
61.
62.
63.
64.
65.

66.
67.

68.
69.

manifestation of wheat genomes (KIHARA and TSUNEWAKI)..
An effective restorer to male-sterile ovata cytoplasm (KIHARA
and TSUNEWAKI)
Gene transfer between di-, tetra- and hexaploid wheats, I.
Transfer of v gene from common wheat to emmer wheat(TSUNEWAKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Geographical distribution of necrosis genes in common wheat,
I. Genetic basis of necrosis (TSUNEWAKI)... . . . . . . . . . . . . . .
Geographical distribution of necrosis genes in common wheat,
II. Distribution in Japanese local varieties (TSUNEWAKI and
NAKAI)
Geographical distribution of necrosis genes in common wheat,
III. Distribution in Pakistan, Afghanistan and Iran (TSUNEWAKI and NAKAI)
Taxonomical and ecological studies on Agropyron humidum
OHWI et SAKAMOTO (OHWI and SAKAMOTO)
Genome analysis of Japanese and Nepalese Agropyron(SAKAMOTO) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Natural interspecific hybrids in Japanese Agropyron (SAKAMOTO)
Morphological and cytological studies of two intergenerichybrids of Elymus sibiricus LINN. and El. dahuricus TURCZ.
with Agropyron tsukushiense var. transiens OHWI (SAKAMOTO)
Further observations on the flowering of Oryza species(KATAYAMA)
Diallel crosses in Sikkimese rice (KATAYAMA)
Preliminary studies on tillering in the genus Oryza (KATAYAMA)
Preliminary studies on embryo transplantation in the genus
Oryza (KATAyAMA)........................................
Photoperiodic responses of Oryze species, VII (KATAYAMA) ..
Genetic studies on developmental instability in Oryza sativa(SAKAI).
Survey of F 1 sterility relationships between strains of Oryza
perennis (MORISHIMA and OKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Variation in characters related to seed dispersion in Oryza
perennis (OKA and MORISHlMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deterioration of F 1 embryos in hybrids between subsp. barthii
and other forms of Oryza perennis (CHU and OKA)........
An observation of cultivation pressure on seed germination(OKA).....................................................

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76
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84
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CONTENTS
70.
71.

72.
73.
74.
75.
76.

C.

Pachytene analysis and an observation of chromosome association in haploid rice (CHU and TAKENAKA)
The genetic analysis of developmental instability in leavesof Nicotiana tabacum by diallel crosses (SHIMAMOTO andSAKAI)....................................................
Tumor formation in interspecific hybrids of Nico#ana,
especially in relation to injury (TAKENAKA and YONEDA)..
Hereditary tumor in Japanese morning glory (TAKENAKA and
YONEDA)..................................................
Chromosomes of some species of genus Paspalum (TERADA
and T AKENAKA)
The origin of Prunus yedoensis, II (TAKENAKA)..............
Method of estimating genetic parameters in a forest whereinter-tree competition is occurring (SAKAI and MUKAIDE)..

Mathematical genetics
The number of alleles that can be maintained in a finite
population (KIMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78. The mutational load with an epistatic interaction (KIMURA). .
79. Theory of the gene frequency cline in a habitat of finite
length (KIMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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87
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91

77.

D. Genetics and biochemistry of microorganisms
80. Hybridization between Salmonella abony and S. abortus-equi(IINO and SUZUKI, Y.) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
81. Mutation of flagellar antigen-1.2 in Salmonella (IINO andMITANI)
82. Flagellar mutants from a curly flagellar strain of Salmonella(MITANI and IINo).........................................
83. Bundled flagella of curly mutant cells in Salmonella(MITANI) . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84. A pauci-flagellated mutant in Salmonella typhimurium (ENoMOTO)
85. Sensitivity of g-group Salmonella to M8-phage, a host range
mutant of chi-phage (YAMAGUCHI)
86. Effect of arginine added at various growth stages to theculture of argos mutant of Salmonella tYPhimurium(ISHIDSU). . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
87. Effect of uracil together with arginine in the preculture ofargos mutant of Salmonella typhimurium (ISHIDSU) . . . . . . . .
88. Ribosomal RNA synthesis in the early stages of sporogenesis(SUZUKI, H.)..............................................

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100
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102

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

89. Changes in r-ray sensitivity with time after ultraviolet
irradiation of several strains of Escherichia coli (lKENAGA
and KONDO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

E.

Radiation genetics of animals
90. Effects of X-ray induced mutations on several components
of fitness (HIRAIZUMI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
91. Heterozygous effects of radiation-induced mutations on
viability in a synthetic homozygous genetic background(MUKAI, YOSHIKAWA and yAMAZAKI)........................
92. Radiation effect of the" Sex-Ratio" agents of D. willistaniand D. nebulasa (SAKAGUCHI, KOBAYASHI and OISHI, K.) . . . .
93. Mechanisms controlling two types of dose-rate dependenceof radiation-induced mutation frequencies in silkworm
gonia (TAZIMA)
94. Nature of post-irradiation metastasis as revealed by fractionated r-irradiation and application of high or low temperature
between the exposures (TAZIMA and ONIMARU)
95. Modification of radiation-induced mutation frequency bypost-treatment with KCN, chloramphenicol, high and low
temperatures and nitrogen gas (TAZIMA and ONIMARU) . . . .
96. Relative biological effectiveness of 14 MeV neutrons to
gamma-rays in the induction of mutations in germ cellsof hibernating silkworm embryos (MURAKAMI and TAZIMA)..
97. Induction of visible recessive mutations by X-radiation insilkworm germ cells at pre-embryonic stage (MURAKAMI
and TAZIMA)..............................................
98. Proliferation kinetics of spermatogonia of the silkworm
during chronic r-irradiations (SADO) . . .. . . . . . . . . . . . . . . . . ..
99. Correction factor (K) for estimating the number of spermatozoa bundles from serial sections of a testis of the silkworm(SADO)....................................................
100. Further studies of 32p induced mutations in the silkworm(IKENAGA and KONDO)......................................
101. Radiosensitivity of muscle tissue formation in regenerating
tissue of Triturus (OGAWA).

109

111
112

114

116

118

120

121
123

124
126
127

F. Radiation genetics of plants
102. Differences in effects of i-rays and fast neutrons from Po-Be
source on einkorn wheat and paddy rice (MATSUMURA).... 129
103. Differences in effects of r-rays and fission neutrons in thepolyploid wheat series (MATSUMURA) .... . . . . . . . . . . . . . . . . .. 130

CONTENTS

vii

104. Differences in effects of r-rays and 14 MeV neutrons on
einkorn wheat (MATSUMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 132
105. Effects of 14 MeV neutrons in T. monococcum (FuJII) . . . . .. 133
106. Lethal effects of heavy ionizing particles in Arabidopsis(FUJII). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 134
107. Effects of dose rate and fractionation of r-rays on mutationin maize pollen grains (MABUCHI) .. . . . . . . . . . . . . . . . . . . . . . .. 135
108. Photoreactivation of ultraviolet-induced mutation in maize
endosperm due to pollen irradiation (IKENAGA and MABUCHI). 137
109. Effects of temperature on post-irradiation storage in einkorn
wheat (MABUCHI). . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 138
110. Oxygen and storage effects on radiation damage in rice andeinkorn wheat seeds (MABUCHI and MATSUMURA)........... 139
111. Radio-sensitivity in pollen grains of Triticum and Aegilops(MABUCHI and MATSUMURA)
141
112. Radio-sensitivity of aged wheat seeds (MATSUMURA and FUJII). . 142
113. Killing effect of UV-rays in Arabidopsis (FuJII)............. 143
114. Effect of EMS on germination of einkorn wheat (FuJII) . . .. 144
115. Developmental-genetic study on culm internodes, leaf sheaths
and leaves in irradiated progeny of barley (TAKEDA andSAKAI). . . . . . . . . . . . . . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . . . . . . . . . . 145
116. Effect of gamma-ray irradiation and maleic hydrazide treatment on the pH 7.5 esterase of corn seedling (ENDo) ..... 146
117. Dose-rate distribution in the mcs gamma-greenhouse (KATO
and IKENAGA) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 147
118. Experimental study on depth dose curve on 60CO r-ray
irradiation (KATO and KONDo)
148
119. Fluorescence quenching and variation with solute concentration of fluorescence efficiency in 2,5-diphenyloxazoletoluene solutions (KATO).......... . . . . . . . . . . . . . . . . . . . . . . .. 149

G. Human genetics
120. Search for selection upon secretor status of ABO blood group
substances (MATSUNAGA, HIRAIZUMI, IZUMIYAMA and FURUSHO).
121. Effect of ABO-incompatibility on "waiting time" (HIRAIZUMI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
122. Studies on the possible genetic consequences of measures
affecting population growth (MATSUNAGA) .. . . . . . . . . . . . . . ..
123. Human red cell acid phosphatase variants (SHINODA)........
124. The nature of molecular heterogeneity between haptoglobin
2-1 and haptoglobin 2-2 (SHINODA) . . . . . . . . . . . . . . . . . . . . . . . .
125. Clinical conditions of patients with apparently normal chromo-

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viii ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

somes, I (OISHI, H. and TONOMURA)........................ 157
126. In vitro sensitivity of human lymphocytes to thalidomide
and its derivatives (TONOMURA and OISHI, H.)............. 157
Books and papers published in 1964 by staff members
Acknowledgment
Author index......................................................

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164
165

GENERAL STATEMENT
Already 15 years have passed since our Institute had been established.
The 15th anniversary was held on September 19,1964, in our roof garden.
Officials from the Government and many colleagues were present.
My address delivered on that occasion contained the statistics of ouractivities of the past 15 years. I wish to repeat them here.
Until now, two thirds of our main building (3,200 m 2) have been
constructed. We expect the remaining third part will be added in the
coming years to make the whole building complete. As additional
facilities, we have one radio-isotope laboratory, one insectarium, one
special silkworm laboratory, two mousaries, one rice laboratory with ashort day installation, one rabbit house, one glasshouse, one air-conditioned
greenhouse, one isolation greenhouse, one gamma-greenhouse, and 33
living quarters for research workers and administrators.
Our library is not rich but we are proud of having the collection ofgenetic reprints and books of the late Dr. GOLDSCHMIDT.
At first we have started with three departments of Morphological
Genetics, Cytogenetics and Physiological Genetics. However we havesucceeded to add until now six more departments of Biochemical Genetics,
Applied Genetics, Induced Mutation, Human Genetics, Microbial Genetics
and Population Genetics. As to our research staff, we have 40 research
workers, 38 full time technical assistants and about 50 part time
workers in addition to 18 administrative officials.
The number of our published papers reached to 539 until the end of
August, 1964. Most of them were read and corrected by Dr. F. A.
LILIENFELD, to whom we are most grateful.
In addition to research activities, we have organized several international
symposia and a genetic course for students of Southeastern Asia.
Our institute is governmental and is given every year by our government a budget. However we have been receiving grants from various
sources for special projects. For instance The Rockefeller Foundation
gave us a grant for the study of rice genetics and also funds forcollecting the rice species of the world. Also the National Science
Foundation and National Institutes of Health have contributed to thestudy of human as well as animal genetics. The Toyo Rayon Foundation
for Promotion of Science and Technology has also given us a grant forradiation work. To all these Foundations we want to express our hearty
thanks.

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ANNUAL REPORT OF NATIONAL INSTITUSE OF GENETICS NO. 15

ABSTRACT OF DIARY FOR 1964
Jan.
Feb.
Feb.
Mar.
Mar.
Mar.
May
May
May
June
July
July
July
Sep.
Oct.
Oct.
Nov.
Nov.
Nov.
Nov.
Nov.
Dec.

24
18
21
25
27
30
1
22
27
5

The 123rd Meeting of Misima Geneticists' Club.
The 56th Biological Symposia.
The 124th Meeting of Misima Geneticists' Club.
The 57th Biological Symposia.
The 125th Meeting of Misima Geneticists' Club.
The 58th Biological Symposia.
The 126th Meeting of Misima Geneticists' Club.
The 127th Meeting of Misima Geneticists' Club.
The 59th Biological Symposia.
The 60th Biological Symposia.

?} The 1st Seminar of the Institute.

11
24
19
2
30
2
6
13
20
28

The 128th Meeting of Misima Geneticists' Club.
Ceremony for the 15th Anniversary of the Institute.
The 129th Meeting of Misima Geneticists' Club.
The 61st Biological Symposia.
The 62nd Biological Symposia.
The 130th Meeting of Misima Geneticists' Club.
The 63rd Biological Symposia.
The 131st Meeting of Misima Geneticists' Club.
Public Lectures Memorializing the 15th Anniversary of theInstitute.
11 The 132nd Meeting of Misima Geneticists' Club.

STAFF(At the End of 1964)
Director

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

1. Department of Morphological Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
ONIMARU, Kimiharu; MURAKAMI*, Akio; Assistants (3)STAFF3

The 2nd Laboratory

2.

SAKAGUCHI, Bungo, D. Ag., Head of the Laboratory
SADO, Toshihiko, D. Ag.; KOBAYASHI*, Susumu; OISHI*, Kugao;
Assistant (1)Department of Cytogenetics
TAKENAKA, Yo, D. Sc., Head of the Department

The 1st Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
MORIWAKI, Kazuo, D. Sc. (in U. S. A.); NAKAMURA*, Akira;IMAI*, Hirotami; AMANO*, Kohachi; FUKAYA*, Takako; Assistants (4)The 2nd Laboratory

3.

TAKENAKA, Yo, D. Sc., Head of the Laboratory
YONEDA, Yoshiaki, D. Sc.; CHU*, Yaw-En; Assistants (2)Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department

The 1st Laboratory
OSHIMA, Chozo, D. Sc.. Head of the Laboratory
TAIRA, Toshifumi, D. Sc.; WATANABE*, Takao; Assistants (2)The 2nd Laboratory

4.

KIHARA, Hitoshi, D. Sc., Head of the Laboratory
TSUNEWAKI, Koichiro, Ph. D.; SAKAMOTO, Sadao;
KATAYAMA*, Tadao C., D. Ag.; NAKAI*, Yasuo; Assistants (3)Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ag., Head of the Department

The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory

The 2nd Laboratory
OGAWA, Yoshito, M. D., Head of the Laboratory
ENDo, Toru, D. Ag. (in U.S.A.)The 3rd Laboratory

5.

TSUJITA, Mitsuo, D. Ag., Head of the Laboratory
SAKURAI, Susumu; Assistant (1)Department of Applied Genetics
SAKAI, Kan-Ichi, D. Ag., Head of the Department

The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Labor';ltory
KAWAHARA, Takatada, D. Ag.; FUJISHIMA, Tohru; Assistants (4)The 2nd Laboratory* Research members under grant from the Toyo Rayon Foundation, the National
Institute of Health (U.S.A.), the Rockefeller Foundation, etc., and visiting
researchers.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
IYAMA, Shin-ya, D. Ag.; SHIMAMOTO*, Yoshiya; MUKAIDE*, Hiromasa;
EL-BALAL*, Mohamed S.; MARUYAMA*, Yukimitsu; Assistants (2)The 3rd Laboratory

aKA, Hiko-Ichi, D. Ag., Head of the Laboratory (in Philippines)
MORISHIMA-OKINO, Hiroko, D. Ag.; Assistant (1)6. Department of Induced Mutation
MATSUMURA, Seiji, D. Ag., Head of the Department

The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
MUKAI, Terumi, Ph. D., D. Sc.; YAMAZAKI*, Tsuneyuki; Assistants (4)The 2nd Laboratory
MATSUMURA, Seiji, D. Ag., Head of the Laboratory
FUJII, Taro, D. Ag.; MABUCHI*, Tomoo; Assistants (3)The 3rd Laboratory
MATSUMURA, Seiji, D. Ag., Head of the Laboratory
ISHIWA, Hiromi (in U.S.A.); IKENAGA, Mituo; KATO*, Takeshi;
Assistant (1)7.

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

The 1st Laboratory
MATSUNAGA, Ei, M. D., D. Sc., Head of the Laboratory
OISHI, Hidetsune; SHINODA, Tomotaka; Assistants (3)The 2nd Laboratory
TONOMURA, Akira, D. Sc., Head of the Laboratory
KIDA*, Mitsushiro; Assistant (1)8. Department of Microbial Genetics
KIHARA Hitoshi, D. Sc., Head of the Department

The 1st Laboratory
IINo, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi; MITANI*, Michiko; YAMAGUCHI*,
Assistants (2)Shigeru;

The 2nd Laboratory
IINo, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho; ISHIDSU, Jun-ichi; SUZUKI*, Yasuko

9.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department* Research members under grant from the Toyo Rayon Foundation, the National
Institute of Health (U.S.A.), the Rockefeller Foundation, etc., and visiting
researchers.

COUNCIL

5

The 1st Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
HIRAIZUMI, Yuichiro, D. Sc.; Assistants (3)10. Experimental Farm
MATSUMURA, Seiji, D. Ag., Head of the Farm
MIYAZAWA, Akira; Field Laborers (7)Honorary Members and Parl:-Time Sl:aff

KOMAI, Taku, D. Sc., Member of Japan Academy, Emeritus Professor of
Kyoto University
KUWADA, Yoshinari, D. Sc., Member of Japan Academy, Emeritus Professor of Kyoto University
LILIENFELD, Flora A., Ph. D.
OGUMA, Kan, D. Ag., Emeritus Professor of Hokkaido University
SHIRATO, Shiro, M. D.
TANAKA, Yoshimaro, D. Ag., D. Sc., Member of Japan Academy, Emeritus
Professor of Kyushu University
Deparl:menl: of Adminisl:ral:ion

MORINAGA, Tokuhiro, Head of the Department
MINAMIGUCHI, Toyotaka, Chief of the General Affairs Section
TANAKA, Mutsuo, Chief of the Finance Section
NAKANo, Hiroko; SEKINE, Akio; TSURUMI, Shigeru; MANo, Asakichi
Clerks (6), Librarians (2), Typist (1), Chauffeur (1), Janitors (4)Associal:ion for Propagal:ion of l:he Knowledge of Genel:ics

KIHARA, Hitoshi, President, Director of the Institute
TAKENAKA, Yo, Managing Director, Head of the Cytogenetics Department
TAZIMA, Yataro, Managing Director, Head of the Morphological Genetics
Department
MATSUMURA, Seiji, Manager, Head of the Induced Mutation Department
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Professor of Sizuoka University

COUNCILOKADA, YO, Chairman, Director of National Science Museum
MORIWAKI, Daigoro, Vice Chairman, Professor of Tokyo Metropolitan
University
FURUHATA, Tanemoto, Director of Scientific Research Institute of Police

6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

KAWADA, Akira, Director of National Institute of Agricultural Science
KAYA, Seiji, Emeritus Professor of Tokyo University
KIKKAWA, Hideo, Professor of Osaka University
KITAHARA, Kakuo, Director of Institute of Applied Microbiology, Tokyo
University
MAKINO, Sajiro, Professor of Hokkaido University
NAGAo, Seijin, Professor of Hokkaido University
OCHI, Yuichi, President of Azabu University for Veterinary Science
OGUMA, Kan, Emeritus Professor of Hokkaido University
SAKATA, Takeo, President of T. Sakata Company
T ACHI, Minoru, Director of Institute of Population Problems
TSUKAMOTO, Kempo, Director of National Institute of Radiological Sciences
WADA, Bungo, Professor of Sizuoka University

PROJECTS OF RESEARCH FOR 1964
Department of Morphological Genetics

Genetics of the silkworm (TAZIMA)
Chemical mutagenesis in the silkworm (TAZIMA, SADO, and MURAKAMI)
Studies on dose-rate dependence of radiation-induced mutation rates(TAZIMA and ONIMARU)
RBE of radiations for induced mutation frequency in the silkworm(MURAKAMI)
Hereditary infections in DrosoPhila (SAKAGUCHI, OISHI, K. and KOBAYASHI)
Genetical and embryological studies in insects (SAKAGUCHI and SADO)
Department of Cytogenetics

Study on chromosomal polymorphism of Muridae (YOSIDA, NAKAMURA and
FUKAYA)
Cytogenetical and biochemical studies on tumor cells (YOSIDA, MORIWAKI
and KURITA)
Mechanism of chromosomal abnormalities by treatment with chemicals(YOSIDA, KURITA and MORIWAKI)
Corelation between taxonomy and karyology of ants (IMAI and YOSIDA)
Experimental breeding and genetics of mice and rats CYOSIDA, MORIWAKI,
KURITA and NAKAMURA)
Determination and differentiation of sex in higher plants (TAKENAKA and
YONEDA)
Induction of abnormal mitosis and inhibition of growth by substances
extracted from certain plants (TAKENAKA)

PROJECTS OF RESEARCH FOR 1964

7

Interspecific hybridization in Nicotiana (TAKENAKA)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus Lannesiana (TAKENAKA)
Cytogenetics of Oryza species (TAKENAKA, YONEDA and CHU)
Morphological and genetical studies on some plant tumors (TAKENAKA
and YONEDA)
Depart:ment: of Physiological Genet:ics

Genetic studies on insecticide resistance in Drosophila pseudoobscura(OSHIMA)
Population genetics of deleterious genes in natural populations ofDrosophila melanogaster (OSHIMA and WATANABE, T. K.)
Studies on chromosomal aberrations of natural populations of Drosophilamelanogaster (OSHIMA and WATANABE, T.)
Eye-pigment formation in Drosophila (TAIRA)
Cytodifferentiation in Drosophila fat body (TAIRA)
Nucleotide-pool in insects (TAIRA)
Genetical isozyme in Drosophila (TAIRA)
Nucleus substitution in wheat and related species (KIHARA)
Comparative gene analysis with reference to the origin of wheat (KIHARA
and TSUNEWAKI)
Geographical distribution of necrosis genes in wheat (TSUNEWAKI and
NAKAI)
Genetic studies of wheat aneuploids (TSUNEWAKI)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Genetic bases of ecological differentiation in Agropyron (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Morphological study of Oryza (KIHARA and KATAYAMA)
Investigation of photoperiodic responses of Oryza species (KATAYAMA)
Depart:ment: of Biochemical Genet:ics

Studies on transformation in higher organisms (NAwA and TSUJITA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAwA and TSUJITA)
Studies on balanced lethality in the silkworm (TSUJITA)
Studies on the chromogranule formation in larval hypodermal cells of
the silkworm (TSUJITA and SAKURAI)
Analysis of genetic action on cell differentiation in higher organisms(TSUJITA and NAwA)
Studies on emanation of alkaloids from the leaves of tobacco plant(TSUJITA)8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA)
Genetical and biochemical studies of plant virus (OGAWA)
Biochemical research on the tissue specificity in plant (ENDO)
Studies on biosynthesis of protein and nucleic acid in plant seed (ENDO)
Comparative studies on seed proteins of rice plant by electrophoretic
analysis (SAKURAI)
Department of Applied Genetics

Studies on developmental instability in poultry (SAKAI, KAWAHARA andFUJISHIMA)
Quantitative genetic studies in poultry (KAWAHARA)
Competition studies in poultry (FUJISHlMA)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Studies on competition in plants (SAKAI and IYAMA)
Estimation of genetic parameters in forest trees (SAKAI and MUKAIDE)
Developmental genetics of quantitative characters in plants (SAKAI,SHIMAMOTO, EL·BALAL and TAKEDA)
Genetic studies on developmental instability in plants (SAKAI and SHIMAMOTO)
Studies on the effects of linkage disequilibrium in Drosophila population(IYAMA)
Genetic studies of Oryza sativaxglaberrima hybrids (OKA and MORISHIMA)
Survey of geographical variation in Oryza perennis (MORISHIMA and OKA)
Experiments on natural selection in wild and cultivated rice forms(MORISHIMA and OKA)
Analysis of sterility genes in Oryza (OKA and MORISHIMA)
Department of Induced Mutation

Radiation genetics of mice (TUTIKAWA)
Population genetics of Drosophila (MUKAI and YAMAZAKI)
Studies on the effects of irradiation on populations (MUKAI and YOSHIKAWA)
Radiation genetics of cereals and Arabidopsis (MATSUMURA, FUJII and
MABUCHI)Radiation genetics and its practical application (FuJII and MABUCHI)
Biophysical studies of radiation genetics (IKENAGA, ISHIWA, KATO and KONDO)
Radiation dosimetry (IKENAGA and KATO)
Genome analysis of Oryza species (MATSUMURA and MABUCHI)
Department of Human Genetics

Selection in ABO blood groups (MATSUNAGA and HIRAIZUMI)FOREIGN VISITORS IN 1964

9

Down's syndrome in Japan (MATSUNAGA and TONOMURA)
Cytogenetics in man (TONOMURA and OISHI, H.)
Sexual dimorphism in resting nuclei (TONOMURA)
Biochemical studies on plasma proteins, haemoglobins and G-6P-D(SHINODA)
Chemical modification of ribonucleic acid and their constituents (SHINODA)
Depar~men~of Microbial Gene~ics

Immunogenetics of Salmonella (IINo, ENOMOTO, YAMAGUCHI and MITANI)
Genetics of motility in bacteria (ENOMOTO)
Genetics of cellular regulatory mechanisms (SUZUKI, H., ISHlDSU andSUZUKI, Y.)
Genetic fine structure analysis on microorganisms (IsHlDsu and !INO)
Depar~men~of Popula~ion Gene~ics

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)FOREIGN VISITORS IN 1964
Feb. 18.-26 NAcE, G. W., Dept. of Zoology, Univ. of Michigan, Ann
Arbor, Mich., U.S.A.
Feb. 27
OETJEN, R. A., National Science Foundation, Tokyo
Feb. 27.-28 EMERSON, A. E., Dept. of Zoology, Univ. of Chicago, Chicago,
Ill., U.S.A.
Mar. 24.-25 SANCHEZ-MoNGE, E., Director, Centro de Mejora del Maiz,
Instituto Nacional de Investigaciones Agron6micas, Avenida
Puerta de Hierro, Madrid, Spain
Mar. 30
GRAY, L. H., British Empire Cancer Campaign, Research Unit
in Radiobiology, London, England
Apr. 6
NEUREITER, N. P., National Science Foundation, Washington,
D. C., U.S.A.
Apr. 6O'CONNELL, J. E., National Science Foundation, Tokyo
Apr. 11
VERLEUR, J. D., Dept. of Botany, Free Univ., Amsterdam,
The Netherlands
Apr. 20
FORREST, H. S., Genetics Foundation, Univ. of Texas, Austin,
Texas, U.S.A.
Apr. 30
KAMEMOTO, H., Univ. of Hawaii, Honolulu, Hawaii, U.S.A.

10

May

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

WHEELER, M. R., Genetics Foundation, Univ. of Texas,
Austin, Texas, U.S.A.
May 7
THROCKMORTON, L. H., Dept. of Zoology, Univ. of Chicago,
Chicago, Ill., U.S.A.
May 9
KWAcK, B. H., Hyosung Women's College, Taegu, Korea
May 25
SOON, I. K., Nam-do, Korea
May 25CHUNG, S. H., Nam-do, Korea
May 27
AUXIER, ]. A., Health Physics Division, Oak Ridge National
Laboratory, Oak Ridge, Tenn., U.S.A.
May 27
CHEKA, J. S., Health Physics Division, Oak Ridge National
Laboratory, Oak Ridge, Tenn., U.S.A.
HOWES, J. R., Auburn Univ., Auburn, Ala., U.S.A.
June 5
MEHRA, P. W., Head of Botany Dept., Panjab Univ., ChanJune 9
diarh, India
VENKATESWARLU, J., Head of Botany Dept., Andhra Univ.,
June 25
Waltair, India
RAo, C. B.]., Foreign Research Student, Faculty of AgriJune 25
culture, Univ. of Tokyo, Tokyo
PARK, Y. S., Section of Agricultural Production, Dept. ofJune 28
Agricultural Production, Ministry of Agriculture, Seoul,
Korea
LAMMERTS, W. G., Director of Research, Germain's HorticulJune 29
tural Research Division, Livermore, Calif., U.S.A.
July 16
BAE, S. H., Crop Experiment Station Office of Rural Development, Suwon, Korea
RAYCHAUDHURI, S. P., Indian Agricultural Research lnst., New
July 17
Delhi 12, India
TER-AvANERYAN, D. V., Dept. Director, All Union Inst. ofAug. 18
Plant Industry, Leningrad, U.S.S.R.
Aug. 18
KORsAKov, N. I., All Union Inst. of Plant Industry, Leningrad,
U.S.S.R.
Sep. 7,..,,8 DASGUPTA, K. P., Central Sericultural Research Sub-Station,
Kalimpong (West Bengal), India
Sep. 14,..,,15 TAN, W. Y., The Inst. of Botany, Academia Sinica, Nankang,
Taipei, Taiwan
Sep. 27,..,,28 CHANG, T. T., International Rice Research Inst., Los Baros,
Laguna, Philippines
Oct.
5
DOBZHANSKY, Th., The Rockefeller Inst., New York, N. Y.,
U.S.A.
Oct. 13,..,,14 DOBZHANSKY, Th., The Rockefeller Inst., New York, N. Y.,
U.S.A.
7

FOREIGN VISITORS IN 1964

1129-30 WOLFF, S., Oak Ridge National Laboratory, Oak Ridge, Tenn.,
U.S.A.
Oct. 29-30 DOBZHANSKY, Th., The Rockefeller Inst., New York, N. Y.,
U.S.A.
OAKBERG, E. F., Oak Ridge National Laboratory, Oak Ridge,
Oct. 30
Tenn., U.S.A.
RUSSELL, W. L., Oak Ridge National Laboratory, Oak Ridge'
Oct. 30
Tenn., U.S.A.
OAKBERG, E. F., Oak Ridge National Laboratory, Oak Ridge,
Nov. 2
Tenn., U.S.A.
RUSSELL, W. L., Oak Ridge National Laboratory, Oak Ridge,
Nov. 2
Tenn., U.S.A.
CHU, E. H. Y., Oak Ridge National Laboratory, Oak Ridge,
Nov. 2
Tenn., U.S.A.
HINDY, S. S., Rice Breeder, Cairo, Egypt, U.A.R.
Nov. 6
INTSIFOL, J. K., Crops Research Inst., Kumasi, Ghana
Nov. 6
CHU, E. H. Y., Oak Ridge National Laboratory, Oak Ridge,
Nov. 7
Tenn., U.S.A.
WOLFF, S., Oak Ridge National Laboratory, Oak Ridge, Tenn.,
Nov. 7
U.S.A.
KIMBALL, R. F., Oak Ridge National Laboratory, Oak Ridge,
Nov. 7
Tenn., U.S.A.
ELKIND, M. M., National Cancer Inst., Bethesda, Md., U.S.A.
Nov. 7KALLMAN, R. F., Stanford Univ., Palo Alto, Calif., U.S.A.
Nov. 7OSTER, I. I., The Inst. for Cancer Research, Philadelphia,
Nov. 7
Pa., U.S.A.
SINCLAIR, W. K., Argonne National Laboratory, Argonne, Ill.,
Nov. 7
U.S.A.
SPARROW, A. H., Brookhaven National Laboratory, Upton,
Nov. 7N. Y., U.S.A.
HOWARD-FLANDERS, P., Yale Univ., New Haven, Conn., U.S.A. ~Nov. 7
ARVEY, M. D., National Science Foundation, Tokyo
Nov. 7KIMBALL, R. F., Oak Ridge National Laboratory, Oak Ridge,
Nov. 9Tenn., U.S.A.
SPARROW, A. H., Brookhaven National Laboratory, Upton,
Nov. 9N. Y., U.S.A.
DOBZHANSKY, Th., The Rockefeller Inst., New York, N. Y.,
Nov. 12
U.S.A.
Nov. 12-13 GAUL, H., Max·Planck Inst. fUr Ziichtungsforschung, Deutschland
Nov. 13SONNEBORN, T. M., Dept. of Zoology, Indiana Univ., Bloomington, Ind., U.S.A.

Oct.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Nov. 23--24 OSTER, I. I., The Inst. for Cancer Research, Philadelphia,
Pa., U.S.A.
Nov. 23--24 DOBZHANSKY, Th., The Rockefeller Inst., New York, N. Y.,
U.S.A.
OsTER, I. I., The Inst. for Cancer Research, Philadelphia,
Dec. 1
Pa., U.S.A.
DAVE. M. J., Indian Cancer Research Center, Bombay, India
Dec. 10
RAo, C. B. J., Foreign Research Student, Faculty of AgriDec. 10
culture, Univ. of Tokyo, Tokyo
SUBBADAH, K. C., Biology Division, Atomic Energy EstablishDec. 12
ment, Torombay, Bombay, India
CHEN, C. L., The Inst. of Botany, Academia Sinica, Nankang,Dec. 12
Taipei, Taiwan
OMATA, R. R., N. I. H. Pacific Office, American Embassy,
Dec. 28
Tokyo

RESEARCHES CARRIED OUT IN 1964
A.

1.

GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF ANIMALS

Genetic variability of a large natural poPulation of
Drosophila melanogaster 1)
Chozo OSHIMA and Takao WATANABE

In late October, 1964, many male flies were collected from large natural
populations in five different graperies of Kofu and Katsunuma. The
second chromosome isolated from each male fly was duplicated, in orderto estimate the effect of genes on viability. Seven hundred and forty
2nd chromosomes were divided into four classes; lethal, semi-lethal,
subvital and normal. Their respective frequencies were 12.30, 22.84, 7.57
and 57.30 per cent. The frequency of lethal and semi-lethal chromosomes
was found to be a similar high level as that of samples in 1963. On theother hand, the mean viability of homozygous flies for 480 quasinormal
chromosomes was 90.5, when the viability of Curly and quasinormal
heterozygous flies was standardized as 100. This mean value was muchhigher than that of samples of 1963, because the observed frequency ofsubvital chromosomes was very low.
The two graperies in Kofu and the three graperies in Katsunuma,
where the flies were collected, are about one or two kilometers apartfrom each other, but the distance between both localities is about 13
kilometers. The distribution frequencies for the four classes of about
150 chromosomes isolated from each of the natural populations of those
graperies, were found to be very similar. From the results, their genetic
structure, in spite of their locations over a wide area, is assumed to be
fairly uniform.

2.

Allelic rate between lethal genes concealed in natural
populations of Kofu and Katsunuma 1 )Chozo OSHIMA and Takao WATANABEOne hundred and fourteen lethal chromosomes were isolated fromdifferent male flies collected at the two localities in the middle of
1l This work was supported by a grant from the Japan Society for the Promotionof Science as part of the Japan-U.S. Cooperative Science Program and also partly
supported by the United States Public Health Service Grant RG 7836.

13

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

October, 1963. A total of 6,441 crosses were performed between thelethal and the Curly balanced strains. The crosses were divided into
three groups; two of them represented crosses within lethal strains
extracted from the same population and the third was a cross between
Table 1.

The allelic rate between lethal genes isolated from natural
populations in two localities, Kofu and Katsunuma.

Year

Population

No. oflethalchromosome

No. ofcrossesNo. ofallelic
crossesAllelicrate (%)

1963

Within lethals of Kofu
Within lethals of Katsunuma
Between lethals of Kf. and Kn.

61
53
114

1830
1378
3233

60
30
80

3.28
2.18
2.47

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

Total
Table 2.

114

6441

-

170

Allelic groups of single, double and multi-locilethal genes.

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

Symbol of
single lethal
gene

No. ofallelic
strainNo. ofSymbol of double,
multi-loci and synthetic allelicstrainslethal genesl(2)6}j 201

12

202

8

20}
204

5
6

205

4

206
207

4
2

208

2

209

2

210

2

11 6}j 272

1

211

2

11 6~j 207

1

212

2

1

21}

2

214

1

11 6}j 27}

111 6}j 20~

1='P14~-11 6~j 269

1

215

2

11 6}j 225

217

111 6}j 227

1

218

1

11 6}j 2}}

1(219 - 265)(40)

11 6}j 259

1

266

1

25}

1

11 6}j 274

1

267

0

11 6}j 271

1

268

0

Total strains

100

11

2.64

RESEARCHES CARRIED OUT IN 196415

lethal strains extracted from different populations. The results are given
in Table l.
The lethal genes were classified into sixty·one non-allelic groups andthe frequencies of their appearances could be determined by the resultsof diallel crosses mentioned above. The results are given in Table 2.
The allelic rate between the populations was relatively high as comparedwith those within the populations, although the two populations are about
thirteen kilometers apart. This could be due to frequent migration offlies between the localities or to persistence of some common lethal genes
in both natural populations.
Eleven chromosomes seemed to have multi-loci lethal genes. Fivechromosomes among them were ascertained to have two lethal genes
which were allelic to two different lethal genes respectively. However,
the other six chromosomes wereassumed to have multi-loci lethal~ In't.B
I
genes, whose loci could not be: (21')
exactly determined.
I
I

I

I
I

3. Location of lethal genes on
the second chromosome l )Takao WATANABE and
Chozo OSHIMAIn the previous annual report(No. 14, 1963), loci of fifteen lethal
genes were determined by thecrossing over value between themand the dominant marker genes
such as Sp, BI and L. Their locations
on the genetic map seemed to be
non·random, but their number was
too small for evaluating our results
and comparing them with the dataof other investigators. However,recently loci of 55 single lethal genes
isolated from the natural popu·
lations in Kofu and Katsunuma were
determined by the same method.

I

-

'"

I

,
I

,
I

, ...eI
I
I UU): (1" Ii

1
Fig. 1. Loci of lethal genes extracted
from Kofu and Katsunuma popu·
lations.

I) This work was supported by a grant from the Japan Society for the Promotionof Science as part of the Japan-U.S. Cooperative Science Program and also partly
supported by the United States Public Health Service Grant RG 7836.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

The results are shown in Fig. 1. About half of them (51.8%) werelocated in the central region (40--70) of the genetic map and twelve
lethal genes were strikingly clustered within only five units, between 55
and 60.
The distribution of these lethal genes on the genetic map was comparedwith that of recessive visible mutant loci (N =i= 100) described in the book
"The Mutants of Drosophila melanogaster" by BRIDGES and BREHME (1944),
and no significant difference between them was detected. Their distribution was confirmed to be non-random. As it is well known that theoccurrence of recombination is not random on both arms of the large
V-shaped chromosome, it may be assumed that the lethal genes are locatedrandomly on the chromosome as well as the visible mutant genes. Twokinds of double lethal chromosomes had one lethal gene in the central
region and another in a terminal region. Three different lethal genes
were found to be surely included in inversion B or C, but their locicould not be determined.

4. Frequencies of inversions in the Katsunuma
natural population1 )Taishu WATANABE, Takao WATANABE
and Chozo OSHIMA
Frequencies of inversions which were found in small and large natural
populations were reported previously (WATANABE, T. and OSHIMA, Annual
Report No. 14, 1963). In late October, 1964, many female flies werecollected simultaneously from two natural populations at Katsunuma
locality. These females were allowed to lay eggs individually in a vial
and then the salivary gland chromosomes of 100 larvae sampled fromdifferent cultures were observed. The results were compared with thedata obtained in 1963.
These common inversions might have been maintained in the same
population, but an endemic inversion, In (3L) F, could not be found in
1964.
The viabilities of heterozygotes for B or C inversion and the normal
chromosome and of transformed heterozygotes for Band C inversion
chromosome were estimated by using Cy-Pm technique. The materials
used in the experiment were chromosomes extracted in 1963 from the
Kofu and Katsunuma populations. The results are shown in Table 2.
1> This work was supported by a grant from the Japan Society for the Promotionof Science as part of the Japan-U.S. Cooperative Science Program and also partly
supported by the United States Public Health Service Grant RG 7836.

RESEARCHES CARRIED OUT IN 1964

17

The inversion heterozygotes did not show heterosis in viability, but theviability of double inversion heterozygotes was proved to be significantly
higher than that of normal heterozygotes.
Table 1. Frequencies of inversions in natural populationsof Katsunuma.
Year

1963

1964

Standard and inversion

Frequency (%)

2L (standard)
In (2L) B

68.0
32.0

73.0
27.0

2R (standard)
In(2R) C

79.0
21.0

74.5
25.5

3L (standard)
In(3L) E
In(3L) F

89.5
8.5
2.0

89.5
10.5

3R (standard)
In(3R) GIn(3R) H
In(3R) I

61.0
18.0
10.0
11.0

71.0
12.0
8.5
8.5

Table 2.

Relative viability of inversion heterozygotes.
Pooled basis

Chromosomal
type
+!+
+/B
+/C
B/C

Line basis

No. ofR I'. bT
counted flies e ative VIa 1 Ity
98,631
53,97851,70755,086

1.0583+0.00941 ]1. 0587±0. 01277
1.0512±0.01295
1. 1253±0.01361**

Viability of Cy/Pm flies = 1.0000

No. of
lines
78
46
40
49

Relative viability

1.0699+0.01683 ]1. 0727±0. 01640
1.0539±0.01808
1. 1490±0.02678** Significant at the 5 % level** Significant at the 1 % level

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

5. Mechanism of maintenance of chromosomal
polymorphism in natural populations!)
Taishu WATANABE and Chozo OSHIMAOne of causes of maintained chromosomal polymorphism could be assumed
to be the situation described in the preceding report. This assumption
was doubly confirmed by the following experimental results. One hundred
male flies, collected from natural populations at Kofu and Katsunuma
in late October, 1964, were used in this experiment. Each male fly wascrossed with several virgin female flies of Samarkand strain, and thesalivary gland chromosomes of 8 or 10 F! larvae in each culture wereobserved. The chromosomal type of each male fly could be detected andthe frequencies of various inversions in second and third chromosomes
were also determined. The expected frequencies of all possible combinations of the second and the third chromosomes were calculated on thebasis of Hardy-Weinberg's law and were compared with the observed
Table 1. Comparison of observed and expected frequencies ofvarious inversion second chromosomes.
Observed
frequency

Chromosomal type

Inversion
homozygote

+!+
B/B
CfC
BCfBC
L*/L*

27
4
2
0
0

Inversion
heterozygote

+/B
+/C
+/L*
B/BC
CfBC

27
13
0
4
0

Double inversion
heterozygote

B/C
B/L*
CfL*
BC/+
BCfL*

10
0
1
12
0

Standard-

Total

Expected
frequency

Total

Difference

33

28.09
6.00251.96
0.64
0.002536.695-3.695

44

25.97
14.84
0.53
3.92
2.24

47.50

-3.50

23

6.86
0.245
0.14
8.48
0.08

15.805+7.195~'.'---

100
100
A new endemic inversion, which was found in 1964, is located on
the right arm (43F -49F) of the second chromosome.

* In (2R) L:

11 This work was supported by a grant from the Japan Society for the Promotionof Science as part of the Japan-U.S. Cooperative Science Program and also partly
supported by the United States Public Health Service Grant RG 7836.

RESEARCHES CARRIED OUT IN 1964
Table 2.

Comparison of observed and expected frequencies ofvarious inversion third chromosomes.
Observed
frequency

Chromosomal type

Inversion
homozygote

+1+
E/E
GIG
H/H
III
EG/EG
EH/EH
EI/EI

46
0
1
0
1
0
0
0

Inversion
heterozygote

+/E
+/G
+/H
+/1
E/EG
E/EH
E/EI
G/EG
H/EH
I/EI

7
9
13
9
0
0
0
0
0
0

Double inversion
heterozygote

E/G
E/H
Ell
G/H
Gil
H/I
EG/EH
EG/EI
EH/EI
+/EG
+/EH
+/EI
G/EH
G/EI
H/EI
H/EG
I/EG
I/EH

1
1
0
3
2
0
0
0
0
1
2
3
0
0
0
0
0
1

Standard

19

100

Total

Expected
frequency

Total

Difference

48

46.24
0.20250.72250.72250.49000.00250.02250.022548.425-0.425

38

6.12
11.56
11.56
9.52
0.045
0.135
0.135
0.085
0.255
0.210

39.625-1.625

14

0.765
0.765
0.630
1.445
1.190
1.190
0.015
0.015
0.045
0.680
2.040
2.040
0.255
0.255
0.255
0.085
0.070
0.210

11.950+2.050100

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

frequencies in Tables 1 and 2.
Total observed frequency of double inversion heterozygotes of both
chromosomes was higher than the expected one regardless of their chromosomal types in any other chromosome. From the results, heterosis of
double inversion heterozygotes could be a strong mechanism for maintaining
chromosomal polymorphism in natural populations.

6.

The effect of insecticide selection on changing the frequencies
of four chromosomal types in experimental populationsof Drosophila pseudoobscura 1l
Chozo OSHIMA and Taishu WATANABE

About forty homozygous strains for four kinds of chromosomes, ST,
AR, CH and PP, were used in the experiments. These strains, originatedfrom the mather population in California, had been established by
DOBZHANSKY (The Rockefeller Institute) in 1963. Heterozygous female
flies for ST and AR were crossed with heterozygous male flies for CH
and PP. Two initial populations were made with 250 female and 250
male offspring and, after two generations (one generation period lasts
about 20 days), the salivary gland chromosomes of 150 larvae hatchedfrom sampled eggs were observed. In the F2 generation of both populations, the frequencies of the four kinds of chromosomes should betheoretically equal, i.e. 25 per cent of each. However, the observed
frequencies were ST:33.3, AR:26.3, CH:22.7, PP:17.7 per cent in Population I and ST:26.3, AR:28.0, CH:26.3, PP:19.3 per cent in Population
II. Further, each population was divided into two populations A and B.
Flies in Population A were exposed to insecticide test paper (DDT 1 %,2 % or Dieldrin 0.1 %) for one hour and transferred into a new cage ineach new generation. Flies in Population B were transferred into a newcage without exposure. The test papers and test kits were prepared by
WHO in Geneva and sent to us. These populations are being maintained,
but the varying frequencies of chromosomes from F 2 to F 7 in each
population were observed as shown in the following table.
The frequency of ST chromosomes has increased in both selected andnon-selected populations, but after F s the increase in the former (lA,
IIA) was more remarkable than in the latter (lB, lIB). On the other
hand, AR chromosomes showed changes which were opposite to thoseof ST. The frequencies of both CH and PP chromosomes have decreased.
However, their relative frequencies in selected populations were differentI) This work was supported by a grant from the Japan Society for the PromotioDof Science as part of the Japan-U.S. Cooperative Science Program.

21

RESEARCHES CARRIED OUT IN 1964
Table 1. Changing frequencies of four kinds of chromosomes in DDTor Dieldrin selected and non-selected populations.
Frequency (%)

FaPop. I

Pop. II

ST
AR
CH
PP

ST
AR
CH
PP

33.3
26.3
22.7-

55.3

73.3

24.3
8.3
12.0

15.7
4.0
7.0

45.7
25.7
17.3
11.3

53.7
26.3
10.0
10.0

65.3
22.0
7.0
5.7

43.7
19.7
15.3
21.3

41.0
30.0
9.7
19.3

43.0
29.7
10.3
17.0

71.0
16.7
4.3
8.0

36.3
21.7
23.3
18.7

45.7
29.7
14.7
10.0

49.3
27.3
13.3
10.0

52.3
30.0
12.0
5.7

-IA (DDT selected)

ST
AR
CH
PP

40.3
18.3
22.0
19.3

55.0
23.0
11. 7
10.3

-IE (non-selected)

ST
AR
CH
PP

42.0
24.7
18.3
15.0

-IIA (DL selected)

ST
AR
CH
PP

-IIB (non-selected)

ST
AR
CH
PP

17.7

26.3
28.0
26.3
19.3

from those in non-selected populations; PP became more frequent thanCH after several selections, and, on the other hand, CH was found more
frequent than PP in non-selected populations. These results seem to·
be an interesting illustrations of the genetic changes which DOBZHANSKY
observed for about twenty years from 1940 in natural populations ofCalifornia locality.

7.

Distribution pattern of tryptophan and pteridine
metabolites in Drosophila larval fatbody
Toshifumi TAIRA and Tahir M. RIZKIll

Larval fatbodies are morphologically symmetrical and those of bothsides are connected by a narrow band of single cells at the top of theanterior region. The fatbody of each side is a lobed monolayered cellmass, which consists of about 900 cells at the 96 hour stage. Its cells are
distinguishable under the UV-microscope in the anterior and the posterior
II Professor of Department of Zoology, University of Michigan, Ann Arbor.
Michigan, U.S.A.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

region by cytoplasmic autotluorescent inclusions. Kynurenine granules
are characteristically found in the anterior region cells constituting thetryptophan metabolic zone, and pteridine granules are observed in theposterior region which is the pteridine metabolic zone. Such distribution
pattern is highly specific for eye color mutants. The pattern of both
metabolic zones of the fatbody, however, can be changed by feeding thelarvae with L-tryptophan and 2-amino-4-hydroxypteridine (AHP). Due totryptophan feeding, the distribution pattern of kynurenine cells in theanterior region extends to the posterior pteridine zone along the ventral
line of the fatbody, and kynurenine granules can be often observed in thepteridine cells of the posterior region. Such an extension of the kynurenine zone can not be suppressed by pre-treatment with chloramphenicolfeeding. In AHP feeding experiments, kynurenine granules in the anterior
region gradually disappear, changing their shape, in the direction fromthe rear to the front, along the dorsal line of the fatbody. Eventually,
the region is completely converted to cells containing pteridine granules
within one hour. Such a conversion is considerably suppressed by pretreatment with chloramphenicol feeding, but not all by the same treatment given simultaneously. The pre-treatment with AHP feeding given
even at the 93 hour stage causes kynurenine cells to convert to pteridine
cells in the anterior region. After 7 hours, the converted pteridine cells
revert only in the anterior region to kynurenine ones, even if tryptophan
is not fed to the same larvae.
These observations lead to following conclusions: (1) Distribution patternof tryptophan and pteridine metabolic zones found in the larval fat bodyis determined by the relative amount of both metabolites produced invivo. (2) The activity of tryptophan pyrrolase by which tryptophan isconverted to kynurenine, is induced by the additional feeding without a
new synthesis of the enzyme protein. However, the enzyme catalyzing
pteridine is probably synthesized de novo. (3) These phenomena art::
assumed to depend on the physiological potential at cellular level, suchas permeability and so on.

8. Mapping of acid soluble nucleotides in insects
Toshifumi TAIRA and Sakaru SUZUKI\)

A standard mapping of acid soluble nucleotides in insects has been
attempted using fertilized eggs, larvae, pupae and adults of Drosophila,
Bombyx and Ephestia, by means of the following procedures:\) Professor of Department of Chemistry, Faculty of Science, Nagoya University,
Nagoya.

RESEARCHES CARRIED OUT IN 1964

23A) Extraction and separation: Materials were homogenized in a Waring
Blender in cold 50 % ethanol, at the ratio of 1 g per 5 ml. The homogenate was boiled for 2 min in a water bath and, after cooling, wasquickly filtrated. This extraction was carried out at least twice. The
combined filtrate was concentrated to one third in volume with a rotary
evaporator. The concentrated solution was mixed with a given weightof charcoal (Nolite extra) in an ice bath under acidic condition (pH 5),
and left standing for 15 min under occasional stirring. The cold mixture
was again filtrated and the charcoal adsorbed the nudeotides on thefilter paper was quickly washed with cold 0.001 N Hel. The washedcharcoal was placed in a given volume of 50 % ethanol containing 0.1 %of ammonium water and left standing for 30 min at room temperature,under occasional stirring. After two elutions, the solution was concentrated again to a small volume for paper chromatographical fractionation of the nudeotides. In order to remove other compounds, a descending
paper chromatography was used with filter paper (Toyo, *f 527, 60x60 cm),by a solvent of n-butanol, ethanol and water (52: 32: 16), for 6 to 8 hours atroom temperature.
B) Indentijication and maPPing: Two-dimensional chromatography was

12
14
7
6

+J

C

(I)

11

>

(5
(/)

1

23
4 5

16
18

L

o

~

1
9 °13 15
I

k

22

21
1718 20--- Electrophoresis
Fig. 1. Map of acid soluble nucleotides.
1. p-NAD 2. CMP 3. AMP 4. GMP 5. IMP 6. UMP
7. TMP 8. NADP 9. CDP 10. ADP 11. ADP-ribose
12. UDP-N-acetyl-D-glucosamine 13. GDP 14. UDP-Dglucosamine 15. CoA 16. TDP 17. CTP 18. UDP
19. ATP 20. GTP 21. UTP 22. TTP24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

applied to identify and to map the nucleotides, i.e. first by paper electrophoresis and then by paper chromatography. Paper electrophoresis
was conducted on filter paper (Toyo, :If: 51, 20x60 em) using 0.05 M
ammonium acetate and acetic acid buffer (pH 5), a cooling system ofcarbon tetrachloride and an electric run with 30 volts per em for 90 min.
The separated electrograms were eluted from each fraction with water
after localization by UV-photograph, and every fraction was simultaneously
applied to second paper chromatography. A filter paper (Toyo , :If: 50,
60x60cm) was used for carrying out descending chromatography, andthe solvent "Leloir", i.e. mixed solution of 95 % ethanol and 1 M ammonium acetate (15:6), pH 7, containing 2% of EDTA, was applied for 48 to
80 hours at room temperature. The chromatograms were located on thefilter paper by UV-photograph. In each separated nucleotide, furthermore, the base component, sugar content and the amount of phosphorus
were determined. Thus, the results allowed to draw a map of nucleotides,
as shown in Fig. 1. Detailed analyses are now in progress.

9. Heterotic effect of linkage disequilibrium in
Drosophila populations
Shin-ya IYAMAThe segregating population of Drosophila melanogaster which wasderived from the cross between a natural population collected in St. Paul,
Minn., U.S.A., and the ebony mutant (e) stock, have been kept for morethan 70 generations under random mating so as to reach nearly linkage
equilibrium between the ebony locus and other loci concerning fitness.
Both kinds of the third chromosome, one carrying the wild and the otherthe mutant allele for ebony locus, were sampled from this population.
The third chromosomes of the isogenic line derived from one genome of
the Samarkand inbred stock were replaced by the sampled chromosomesin heterozygous condition, i.e., +/e.
Experimental populations were initiated by mating the above mentioned
flies with eight replications as follows:
Population I:
one pair of identical flies.
Population II: one pair of unrelated flies.
Population III: six pairs of unrelated flies and one pair of identicalflies.
Each replicate was kept in a 200 ml milk bottle containing 250 to 300
flies. Soon after the emergence, in every generation wild and ebony fly
frequencies were counted and ebony flies were discarded before mating
making the ebony gene artificially lethal. The change in average ebony

RESEARCHES CARRIED OUT IN 196425

fly frequencies in each population is shown in Table 1 up to the eleventh
generation together with the expected frequencies calculated on theassumption of equal fitness for +1+ and +Ie.
Table 1.
Population
I
II
III
Expected

Go

Ebony fly frequencies in per cent in three populations I~ III
in which ebony flies were made artificially lethal.

Ga

Gs

23.42 18.30 18.02 17.31 13.64 14.68 10.08 11.01 15.45 12.83 10.46 10.09
23.08 14.82 13.37 11.26 9.52 8.62 7.58 8.21 8.51 8.93 8.44 8.39
21.0213.18 10.81 6.54 4.66 3.58 3.60 2.59 3.78 4.19 3.72 3.36
25.00 11.11 6.25 4.00 2.78 2.04 1.56 1.23 1.00 0.83 0.69 0.59

The results reveal the fact that ebony fly frequencies were higher
than expected in all three populations, indicating a heterotic effect in+ Ie heterozygotes; its magnitude was largest in population I and smallest
in population III, depending on the number of pairs used for the initial
mating, that is, on the degree of linkage disequlibrium involved in the
population with regard to linkage between ebony and other loci concernedwith fitness.

10. Position effect of spontaneous mutant polygenes controlling
viability in Drosophila melanogaster
Terumi MUKAI and Tsuneyuki YAMAZAKI

As described in previous reports (MUKAI et al., Annual Report No. 12,
13 and 14, 1961--1963), spontaneous polygenic mutations affecting viability
had been accumulated under the minimum pressure of natural selectionin 104 chromosomes derived from a single chromosome. In Generations
25, 32, 52 and 60, homozygous viabilities of these chromosome lines were
tested. In addition, the viabilities of flies carrying random combinationsof these chromosomes were estimated in Generations 25, 32 and 52 andthe correlation coefficient between the sum of homozygote viabilities andthe corresponding heterozygote viability was calculated for each generation. The estimated values were positive and highly significant.
On the other hand, in Generations 32 and 60, the viabilities ofheterozygotes between these experimental chromosomes and a chromosome supposed to be identical to the original chromosome were
estimated and the correlation coefficient between homozygote and heterozygote viabilities was calculated for each generation. The results showedsignificantly negative values.
From these experimental results, it can be said that newly arising

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

mutant polygenes clearly show overdominance in homozygous genetic
background when the chromosomes carrying them are combined withthe original normal chromosome, but are heterozygously deleterious intrans-phase heterozygotes. These phenomena are significant in clarifying
the mechanism by which natural populations carry genetic variation.

11.

The viabilities of heterozygotes for newly arising spontaneous
lethal genes in Drosophila melanogaster
Isao YOSHIKAWA and Terumi MUKAIIn the experiment described above (This Report, 10), forty-one second
chromosomes carrying newly arising spontaneous recessive lethal genes
were accumulated by Generation 60. These chromosomes were derived
from a single normal second chromosome. Heterozygous effects ofthese lethal genes on viability were tested in three genetic backgrounds(almost homozygous, intra-population hybrid and inter-population hybrid).
The control experiment was conducted by employing 41 chromosomes
which were randomly picked up from lethal-free chromosomes. Thesechromosomes are of the same origin as the lethal-carrying chromosomes
and had been maintained simultaneously in the same way. Therefore,
the genetic backgrounds of lethal heterozygotes and their controls were,on the average, the same.
The following findings were obtained by statistical analysis: (1) All
these lethal genes were detrimental in heterozygous condition, i.e., any
lethal gene did not show overdominance. (2) Interaction between lethal
genes and genetic backgrounds could not be observed, namely, thatlethal genes that showed more deleterious effect in one genetic backgroundwere more detrimental in any other genetic background. (3) Overall
average degree of dominance (ii) was 0.0143.

12. Average degree of dominance of spontaneous mutant
polygenes controlling viability
Terumi MUKAI and Tsuneyuki YAMAZAKI

After the sums of the viabilities of homozygotes corresponding torandom heterozygotes (This Report, 10) were divided into four groupsin Generations 32 and 52 according to the order of their magnitudes, therelationship between the averages of the sums of homozygote viabilities
and those of corresponding heterozygote viabilities were examined, taking
all the data together into consideration. A clear linear relationship was

27

RESEARCHES CARRIED OUT IN 1964

observed between them. Thus, the average degree of mutant polygenes(ii) in the classical model, in which the viabilities of AA, Aa and aa are
assumed to be 1, 1-hs and 1-s (A = wild-type gene, a = mutant geneand s = selection coefficient against the mutant homozygote), was estimated. In the present analysis, only chromosome lines which had new
mutant polygenes and their heterozygotes were employed.
The average degree of dominance (ii) was estimated by the following
two formulae, under the assumption of no correlation between selection
coefficient against mutant homozygotes and the h in the range ofpolygenes:/'-.
COV(Vi+Vj and Vij)ii =••••••••••••••••••••• ( 1)

2a~

and
-

Vo-it

h = 2(vo-v)( 2)

where vo = viability index of the original homozygotes (ca. 33.3); iJ = average viability of homozygote lines; it = average viability of random heterozygotes; O'~ = genotypic variance among homozygote lines; cov (Vi + Vj
and Vi;) = covariance between the sum of the homozygote viabilities ofLine i and Line j and viability of their hybrid.
The estimated ii value is 0.403±0.017, which is remarkably large incomparison with that of lethal genes (ii = 0.04, STERN et al., 1952).
13.

Homozygous loads and genetic structure of natural
populations of Drosophila melanogaster
Terumi MUKAI

According to the results in Generation 60 reported in a precedingarticle (This Report, 10), the viabilities of heterozygotes with the original
chromosome increased with the number of mutant polygenes, but reacheda plateau when about eleven mutant polygenes, on the average, havebeen accumulated. This is one restriction to the manifestation of overdominance in natural populations. In addition, these mutant polygenes
cannot be overdominant in trans-phase.
GREENBERG and CROW (1960) have developed a method for the test of
the existence of overdominance in natural populations, using homozygous
loads. On the basis of our findings, homozygous loads were calculated
assuming no overdominance. Using the relationship derived by WRIGHT(1931) for an equilibrium random mating population,

q """

:s'

where p.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

is the mutation rate per locus; h stands for the degree of dominance;s for selective disadvantage of mutant homozygote; and q for thefrequency of the mutant gene, the homozygous loads were calculated tobe~ %s s= ( ~) ~ p.

In this derivation, it was assumed that~ and pare uncorrelated.
The magnitude of homozygous load due to lethal genes in the second
chromosomes (L) was approximately 0.0063 = 0.4406 where
0.0143(I)=_1_h0.0143was employed as an approximate value, because we could not estimate( ~ ).

h (= 0.0143) and p (= 0.0063) were estimated in our experiment.

The load due to detrimental genes (D) was approximately 0.1411 =0.3501.
0.403
p(=0.1411) and h(=0.403) were also estimated by us (MUKAI, 1964; This
Report, 10). Therefore, D:L = 0.79. The estimated D:L values fromrandom mating populations ranged from 0.502 to 1.012 (GREENBERG and
CROW, 1960).
On the other hand, D:L ratio was calculated for newly arisingmutations, by using the experimental result in Generation 32. The
results are: D:L = 0.98 (L = 0.1843 and D= 0.1797 in Generation 32).
Thus, the D:L ratio of equilibrium populations to that of new mutants
becomes 0.806. This ratio is very close to 1 which can be expected fromhs = constant (GREENBERG and CROW, 1960). Actually, our data almost
satisfy this relationship.
In conclusion, it might be said that the manifestation of overdominancein natural populations is rare. Accordingly, the classical hypothesis
might be generally accepted.

14. Synergistic interaction between spontaneous mutant polygenes
controlling viability in Drosophila melanogaster
Terumi MUAKIAs reported in a preceding article (This Report, 10), homozygous
viabilities of the chromosome lines derived from a single second chromosome were estimated in Generations 10, 15, 20, 25, 32, 52 and 60. Excluding
lethal and semi-lethal lines, seventy-two lines were examined out of 104
lines. However, since the result in Generation 15 was abnormal, it was
not employed in the present analysis. The probable reason for thisabnormal phenomenon was described in my report (MUKAI, 1964). The
average viability in each generation is given in Table 1.

RESEARCHES CARRIED OUT IN 1964

29

Table 1. Relationship between average homozygote viabilities
and generation number.
Generation

10

20

25

32

52

60

Average homozygote viability

31.57

30.85

28.53

28.14

21.32

16.42

The viability of the original homozygotes was ca. 33.0 (per cent of wild-type
flies by Wallace's Cy method (1956)).

From Table 1, it can be said that a linearity does not exist between
generation number and average viability, and especially, the decrementof viability per generation became large with increasing generation
number after Generation 32. This result implies that there is a synergistic interaction among mutant polygenes. A quadratic equation was
fitted to the experimental data by the least-squares method and thefollowing equation was obtained:
Y=-O.00469Ix 2 +O.03547x+31.57( 1)

where x and y stand for generation number and the average viability,
respectively. This formula shows a good accord with the experimental
data.
From this formula, it can be expected that the average viability
becomes zero in Generation 85.9 where the average number of polygenic
mutations per second chromosome is expected to be: O.I411x85.9=;:I2.I2.

15. Heterosis and Hardy- Weinberg equilibrium
Terumi MUKAI

Fitness of Drosophila may be separated into several components. In
this short report, the author will discuss the characteristic of equilibrium
populations where a mutant gene has been maintained by the superiorityof heterozygotes over either homozygote with respect to viability, female
fecundity or male mating ability.
In order to set up mathematical models, the following parameters
were defined: f(m, v) = selection coefficient in female fecundity (malemating ability, zygotic viability) against recessive homozygotes (aa),that of wild-type homozygotes (AA) being O. We assume that theseparameters are positive and less than L h = degreE; of dominanceof a gene over A gene in female fecundity, male mating ability
or zygotic viability. Thus, for example, female fecundities of AA, Aa
and aa individuals are 1, I-hf and I-f, respectively. x = frequencyof heterozygotes in an adult population; P = frequency of a wild-type

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

gene (A); and q = frequency of the mutant gene (a) allelic to A gene,and P+q = 1.
Zygotic viability: When the fitnesses of wild-type and heterozygotic
flies are different only in zygotic viability, the necessary and sufficientcondition for the maintenance of the recessive gene in stable state is
obtained to be h<O by the ordinary procedure. The equilibrium frequency of the recessive gene becomes -hh ,and the Hardy-Weinberg
2 -1
equilibrium can not be formed in adult populations.
Female fecundity: The zygotic array in an arbitrary population can be
represented as that in Table 1. In an equilibrium population, theTable 1.

Zygotic array and female fecundity of eachgenotype in an arbitrary population.

Genotype
Frequency
Female fecundity
Male mating ability

AA

Aa

aa

p--

x
2

x

x
q-2

1
1

I-hf

I-f

1

1

magnitudes of change in gene frequency and heterozygote frequency
should be zero. From these conditions, the following simultaneous equations (1) can be obtained:
x{P(1-h)+h(1-P)}-2P(1-P) = 0~{2f ( h- ~)}X2-{f(h-2+P)+2}X+2P(I-P)(2-f)=OJ •....... (1)Under the condition of O<p<1, x and p can be obtained as follows:1
p= :h__ 1(q=}(2)
2(h-l)hx = (2h-l)Z

2h~1)

A

The necessary and sufficient condition for these p and x being stableis h<O.
Exactly the same relationship as the above can be obtained when only
male mating abilities are different among the three genotypes.
The following important finding should be pointed out: When only
female fecundity or only male mating ability is concerned with themaintenance of mutant genes in populations, the Hardy-Weinberg
equilibrium can be formed, in contrast with the case of zygotic viability.

RESEARCHES CARRIED OUT IN 1964

31

16. A condition for pseudo-overdominanceTerumi MUKAI

It has been said that single mutant genes can be maintained in
populations with the help of adaptive genes linked with them, eventhough they are detrimental to their carriers both in homozygous andheterozygous conditions. In this article, the condition for such pseudooverdominance is derived.
Following parameters are defined for setting up a mathematical model:
A (or a) = wild-type (or mutant) gene in Locus 1; p = gene frequency of
A; 1-P = gene frequency of a; B(or b) = wild-type (or mutant) gene in
Locus 2, and Loci 1 and 2 are localized in the same chromosome. q = genefrequency of B; 1-q = gene frequency of b; D= magnitude of linkage
disequilibrium (LEWONTIN and KOJIMA, 1960), PS-QR in Table 1.
Table 1. Frequencies of gametes and zygotes and adaptive valuesof zygotes in an arbitrary generation.
~

--~

Male
gamete

P(AB)

Q(Ab)

R(aB)

SCab)

P(AB)

P2(AB)1 AB

PQ (Ab)1-hs ABPR (aB)
1-hs ABPS (ab)1-2hs ABQ(Ab)

QP (AB)
1-hs Ab

Q2 (Ab )l-s AbQR (aB
1-2hs Ab )1-hs-s Ab

R(aB)

RP (AB
RQ (Ab)1-hs aB I 1-2hs aB

R2 (aB)l-s\aB

RS (ab)1-hs-s aB

SCab)

SP (AB)
SQ (Ab)SR (aB)
1-2hs ab1-hs-s ab
1-hs-s ab

FemalegameteQS(ab)

S2 (ab)1-2s ab,With these parameters, the frequencies of four kinds of gametes canbe expressed as follows:

AB: pq+D=P
Ab: P(l-q)-D=Q
aB: (l-P)q-D= R
ab: (l-P)(l-q)+ D = S
The adaptive value of each genotype is given in Table 1, where hdenotes the degree of dominance of a or b gene over A or B gene,
respectively, and s indicates the selection coefficient of aa or bb homozygotes in comparison with AA or BB homozygotes (h and s are positive).
If we calculate the average adaptive values of AA homozygotes (WAA)
and Aa heterozygotes (W Aa) and the difference of these values, the32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

following formula can be obtained:

WAa- WAA = {D2-P(1-q)D}s-{2D2+P(2q-1)D+p 2(1-P)}hs·····( 1)If this value becomes positive, it implies that, on the average, Aa heterozygotes show adaptive superiority to the homozygotes (pseudo-overdomi·nance) even if a gene itself does not show overdominance.
The necessary and sufficient conditions for the above pseudo-overdominance can be obtained on the assumption that h is very small incomparison with 1. The results are:1) When P+q> 1 ,-lP(l-q) {12P )}<D
V/1+ !h(l-:(1-q)2(2-l)(1-q)2>hp2
2) When P+q<l ,-lP(l-q){l- 11 + 4h(1~P)}>D
2V(1-q)2q>h(l-P)(2-2)

Accordingly, it can be concluded that some magnitude of linkage
disequilibrium could cause pseudo-overdominance of a certain gene in a
population and that, if this D is not changed permanently by epistatic
effect of these genes, heterozygote superiority and the mutant gene awill be maintained permanently in the population.

17. Examples of permanent linkage disequilibrium
Terllmi MUKAI and TSlineYliki YAMAZAKICHIGUSA and MUKAI (1964) have established eight random mating populations using sePia-carrying and wild-type third chromosomes of Drosophilamelanogaster. The former were originated from ten chromosomes andthe latter from a single chromosome. Initial gene frequencies of sePia
were 0.5 in all populations. Thus, the expectation of sepia homozygote
frequencies was 25 per cent. The frequencies of sepia homozygotes
decreased to less than three per cent in six of the eight populations byGeneration 55. However, after the frequencies of sepia homozygotes
decreased to about seven per cent, they began to increase in Populations
6 and 7, and reached equilibria approximately in Generation 50, where
their frequencies were 15,...,20 per cent. Since then, the sepia genes
have been maintained at ,high frequencies. The results are shown in
Table 1.
These results can be best explained by the appearance of permanent

RESEARCHES CARRIED OUT IN 1964

33

linkage disequilibrium owing to the development of some gene complex
around the sepia genes.
Table 1. Frequencies of sepia homozygotes in experimental
populations of D. melanogaster.
Generation
Population(Average)
67
51-55 ....

81

82

83

84

(Average)
81-84

#6

0.15

0.20

0.24

0.24

0.23

0.22

0.23

#7

0.21

0.26

0.13

0.15

0.14

0.13

0.14

18. Persistence of the recessive gene ew in experimental
populations in Drosophila melanogaster
Tsuneyuki YAMAZAKI and Terumi MUKAIThis experiment was designed to obtain some information as towhether or not the third chromosome recessive mutant gene ew (expandedwing) shows overdominance. Two populations, A-I and A-2, were set
up in 1961 with the initial frequencies of ew 50 and 10 per cent, respecEach population consisting of 8 sub-populations has been
tively.
maintained by so-called Pearl's method. The frequency of ew gene ineach sub-population was examined in several generations after Generations
37 and 32 in Populations A-I and A-2, respectively.
The results are given in Table 1. Although each population had 8
sub-populations, the gene frequencies of the highest (Sub-pop. 3 in A-I
and Sub-pop. 8 in A-2) and the lowest (Sub-pop. 6 in A-I and Sub-pop.
1 in A-2) of all sub..populations between the 60th and the 70th generation,and the average in these 8 sub-populations are summarized in Table 1
for simple presentation. From Table 1, it can easily be seen that genefrequencies in both populations decreased not only on the average, butalso in the frequencies of the highest and the lowest sub-population
with increasing number of generations. All sub-populations had genefrequencies of more than 10 per cent in both populations (A-l and A-2)
until Generation 50. Between the 60th and the 70th generation, thehighest gene frequencies were still more than 10 per cent in the respective populations, but the lowest ones were less than 4 per cent; namely,in early generations the differences in gene frequencies were not observed,
but between the 60th and the 70th generation they became very clear.
This phenomenon indicated that the ew heterozygotes showed adaptive

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table 1. Gene frequencies of the recessive gene ew
in experimental populations.
Generation

Initialgenefrequency

Gen. 32~461 Gen. 37 ~50 I Gen. 62~66IGen. 66~70(38.2)(42.4)(64)(68)
Average
Sub-pop. 3
I (the highest)
Sub-pop. 6(the lowest)
I

A-I

0.5

-

0.1485-0.1099-0.1940-0.1917-0.1167-0.00190.2546-0.1347-0.2892-0.2129-0.2062-0.0383I
I

Average
Sub-pop. 8
I (the highest)
Sub-pop. 1! (the lowest)
I

A-2

0.1

I

The figures in parentheses stand for the average generation number.

superiority due to linked genes in early generations, but later recombination occurred between them and the superiority of heterozygotes
disappeared in some sub-populations (Sub-pop. 6 in A-I and Sub-pop. 1 in
A-2). On the other hand, in other sub-populations (Sub-pop. 3 in A-I
and Sub-pop. 8 in A-2) it has not disappeared because linkage has still
remained between the 60th and the 70th generation and/or a new adaptive
gene complex was developed around ew gene and this complex has been
maintained by epistatic interaction. Therefore, it seems that ew gene inthese populations has not been maintained on account of overdominance.

19. Partition of fitness into components concerning therecessive mutant gene ew in two experimental
populations of Drosophila melanogaster
Tsuneyuki YAMAZAKI and Terumi MUKAI

A recessive mutant gene (ew) has been maintained without eliminationin experimental populations almost for four years as described in aprevious report (This Report, 18). However, great differences in ew
gene frequencies have developed among the sub-populations. This work
was designed to find out the reason for those differences. Several ewcarrying and wild-type chromosomes were extracted from each of the
sub-populations showing the highest (Sub-pop. 8) and the lowest (Subpop. 1) gene frequency in Population A-2. Three components of fitness(viability, female fecundity and male mating ability) of their homo- andRESEARCHES CARRIED OUT IN 1964

35

Table 1. Relative values of viability, female fecundity, and male mating
ability in Population A-2. The value of each fitness component
for wild-type homozygotes (+ /+) is taken to be unity. In
male mating ability both genotypes, +/+ and +/ew,
are assumed to be unity.
-----_ _ - ..

Genotype
Subpopulation

+/ew

I

Female1- Viability fecundity

-----

ew/ew
Male
matingability

Viability

FemalefecundityMalematingabilit_y_

IP1.03040.95561

0.97060.81700.6116#8

1.04641.08661

0.9877

0.9462

1.0273

hetero-zygotes were examined using the above chromosomes.
The results of this experiment are summarized in Table 1. From thistable, the heterozygotes (+ lew) in both sub-populations proved to be more
viable than the corresponding homozygotes (+/+, ew/ew). On the other
hand, an extreme difference was found between Sub-pop. 1 and Sub-pop.
8 concerning female fecundity and male mating ability; in Sub-pop. 8,
female heterozygotes were more fecund than the corresponding homozygotes (+/ew>+/+>ew/ew), while in Sub-pop. 1, one of the homozygotes(+/+) was more fecund than the heterozygotes (+/+ > +/ew>ew/ew). As
to male mating ability, ew/ew homozygotes in Sub-pop. 8 were stronger
than the other genotypes (ew/ew>+/+, +/ew), but the opposite result
was obtained in Sub-pop. 1. Homozygous individuals with respect to ew
gene in Sub-pop. 1 were weaker than the other genotypes (ew/ew< +/+,+/ew). From these results, the difference in gene frequencies between
Sub-pop. 1 and Sub-pop. 8 seems to be mainly due to differences in malemating ability and female fecundity, but not in viability. As ew genes
in these sub-populations were derived from a single mutant gene, it
might be said that the differences in the magnitudes of fitness components between these two sub-populations are caused by the effects ofthe genetic background and/or interaction between the gene in question
and its genetic background.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

20. Disappearance of" Sex-Ratio" condition by double
infection with two" Sex-Ratio" agents ofdifferent origin in Drosophila 1)
Kugao OISHI and Bungo SAKAGUCHI
So-called maternally inherited "Sex-Ratio" (SR) condition is characterized by an extreme departure from the normal 1: 1 sex ratio to give
all or nearly all female offspring. The condition in D. willistoni and D.
nebulosa is caused by the presence of infectious Treponema-like spirochetes(SR spirochetes) in the female bodies of the species hosting them (POULSONand SAKAGUCHI, 1961). Furthermore, the SR condition is interrupted when
the two kinds of SR spirochetes, i.e., from D. willistoni and from D.
nebulosa, are combined in the same host (SAKAGUCHI, OISHI and KOBAYASHI,
1964). Such an experiment was carried out and it was tested whether the
SR condition persists into subsequent generations. For this purpose a lineof the Oregon-R strain of D. melanogaster was established by artificialtransfer of willistoni spirochetes. This line will be referred to as ORSRB-3.
The females of this line were superinfected, so as to bring together thewillistoni, SRB-3, and the nebulosa, NebSR, spirochetes and were matedsingly with males of normal Oregon-R strain.
In detail, 14 females of the ORSRB-3 were superinfected with nebulosa
spirochetes and the sex ratio in successive broods was examined. Table 1
shows a striking effect that the proportion of males in the first generation was gradually increasing (after 1- 3 days from the time of injection)and reached to about 50 per cent in the third to fifth broods, but it
decreased again gradually from the sixth to the following broods.
Furthermore, SR condition disappeared in the flies of the next generation
except in the progenies of the first and the second broods. The SR
condition was never restored in the subsequent generations.
In the control superinfected by the same SRB-3 spirochete (to which
it was the host), or left without superinfection, no males were produced
except in the first brood. The SR condition in this case was very stablytransmitted into progenies as shown in Table 1.

1) This work has been supported by U. S. Public Health Service Research Grant
GM 10238 from the Division of General Medical Sciences.

Table 1. SR condition in the progenies of ORSRB-3+ females injectedwith the nebulosa and wiUistoni spirochetes.
Brood. days after injection

Direction of injection
0~3

0

'i1

Host
Donor

Host
Donor

Host
Donor

A:
B:
C:
+
*
**

ORSRB-3+
NebSR*ORSRB-3
SRB-3**ORSRB-3

4~6

'i1

0

7~9

0

'i1

1O~12

0

'i1

13~15

0

'i1

16~18

0

'i1

19~21

'i1

0

22~24

'i1

0

25~27

0

'i1

A:

101:8

138:50

B'
.

37

49

29

55

57

33

29

c:

12

15

o

o

o

o

o

106:125 152:148 108:104

70:53

67:52

42:14

61:22

::0
tTl

en

tTl

A:

83:13

132:0

108:0

134:0

118:0

90:0

B:

46

33

37

28

33

c:

21

24

37

28

30

A:

81:0

53:0

31:0

39:0

39:0

26:0

B:

25

24

15

24

15

c:

11

23

15

24

15

64:1

36:0

;p

::0

g
tTl

en
(')

21:0

;p

28

9

::0
::0

27

9

til

t:l

o

20:0

C
>-l

17

17

Z

17

17

~

15:0

9:0

......

Sex-ratio of the 1st generation flies.
No. of the 1st generation flies tested.
No. of SR.
SR strain of D. melanogaster. Oregon-R, derived by transfer of SR from D. wiUistoni SRB-3 strain.
SR strain of D. nebulosa.
SR strain of D. wiUistoni.
~

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

21.

Nature of the substance produced by nebulosa-SR agent
that kills willistoni-SR agent in Drosophila l )Bungo SAKAGUCHI and Kugao OISHI

So-called maternally inherited" Sex-Ratio" (SR) condition in Drosophila
willistoni and D. nebulosa are caused by the presence of a Treponemalike spirochetes in the female bodies of the species hosting them
(POULSON and SAKAGUCHI, 1961). When the two kinds of SR agents (SRspirochetes), i.e., from D. willistoni and from D. nebulosa, were combinedin females of Oregon-R (OR) strain of D. melanogaster, the expressionof SR condition was temporarily interrupted. The phenomenon of interruption is the consequence of interaction between the two kinds of SR
spirochetes, and finally SR spirochetes of nebulosa replace those ofwillstoni in the host's females. Furthermore, SR spirochetes of willistoni
are always eliminated in the host in which the two kinds are combined(SAKAGUCHI, OISHI and KOBAYASHI, 1964).
In an attempt to gain a better understanding of the mechanism of
the interaction between the two SR spirochetes, about one gram of
OR females hosting nebulosa spirochetes was homogenized in 3 ml of
0.25 M sucrose or Drosophila Ringer solution and the homogenate wascentrifuged at 5,000 rev/min for 15 minutes. The supernatant was again
centrifuged at 30,000 rev/min (80,000 g) for one hour. A part of thesupernatant which was almost free of spirochetes was injected into thetest flies of OR females hosting willistoni spirochetes and the proportionof males and females in the successive broods from the injected flies
was determined (control in Fig. 1). Another part of the supernatant
was divided into three parts and each was then subjected to the following
procedures. (1) The supernatant was treated by heating for 10 minutes
at 60°C. After the treatment it was quickly chilled and centrifuged at
10,000 rev/min for 15 minutes. The supernatant was injected into thetest flies and the sex ratios in the progenies of the injected flies were
examined. (2) Another part of the supernatant was digested overnightwith 0.3 percent trypsin at 37°C. The trypsin was then removed bysaturation at 40 percent of ammonium sulfate. The digested fluid was
also used for injection. (3) A third part was dialysed against distilledwater with collodion membrane and the dialyzate solution was then used
for injection. These results are shown in Fig. 1.
The proportions of females in the progeny of test flies injected with
two fractions of nontreated supernatant (control) and those that wereI) This work has been supported by U. S. Public Health Service Research Grant
GM 10238 from the Division of General Medical Sciences.

RESEARCHES CARRIED OUT IN 1964

39

heat treated were more than 90 percent in the first brood (1 to 3 days
after injection), but suddenly decreased to about 50 percent in the third(6 to 9 days) and subsequent broods: SR condition was never restored.
The hemolymph of adults was examined by phase-contrast microscopy.
100:\~-'-"'_.l(_.l(_.l(-l(

.~

4

e

control••

60'C 10 min.
dialyzateXtrypsindigestion

4:__:_-:-_.-i-e-

e

9

24

50

3

6

12
15
18
21
Days after injection

Fig. 1. Effects of various treatments of the substanceproduced by nebulosa spirochetes on willistoni SR
condition.

SR spirochetes were observed in the first but not in the third andfollowing broods. While no males were produced after injection oftrypsin digested and dialyzate fluids. But, when nondialyzable solution
was injected into test flies, the proportion of females was decreased insuccessive broods as in the control, though the result is not presented
here.
These results indicate that the supernatant from homogenate of the
SR females hosting nebulosa spirochetes contains some substance which
is produced by nebulosa spirochetes and the substance may kill orincapacitate willistoni spirochetes. Furthermore, it is suggested thatthe nature of the substance is some kind of protein which might becalled Spirocin.
An experiment to purify spirocin by means of column chromatography
and electrophoresis is now under way.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

22. Multiplication of SR agents in Drosophila l )Susumu KOBAYASHI and Bungo SAKAGUCHIThe SR agents in D. willistoni and D. nebulosa are Treponema-likespirochetes, about 4-16 microns in length and 0.1 microns in width(POULSON and SAKAGUCHI, 1961). A striking morphological feature revealed
by electron microscope are vesicles which may occur at any point along
the filament; they may form at least one to four granules (buds) which
may develop into young spirochetes (A in Fig. 1). Another feature isbranching of filaments at any point (A in Fig. 1). Free vesicles, about
0.3 microns in diameter, are also present in the hemolymph of host flies.
They also form minute granules which may develop into young spirochetes(B, C and D in Fig. 1).

Fig. 1. Electron micrographs of SR spirochetes of Drosophila nebulosa.
Negative contrast technique.
A. Mature SR spirochete. Large vesicle which occurs along the filament
and two minute granucles (buds) which may have developed from the vesicle
can be seen. A branch of filament can also be observed. x 24,000.
B, C and D. Free vesicle which may represent a stage in the life cycleof SR spirochetes. Note two granucles (buds) or a long tail attached to thevesicle. x 27,000.
B: bud; BR: branch; FV: free vesicle; V: vesicle.
I) This work has been supported by U. S. Public Health Service Research Grant
GM 10238 from the Division of General Medical Sciences.

RESEARCHES CARRIED OUT IN 1964

41

These facts indicate the possibilities that the SR spirochetes may
multiply by forming free vesicles which may represent different stages
in the life cycle, by budding, or by separation of filament branches.
Experiments to determine the multiplication unit by a breakdown of
the agent with sonic treatment and separation into elements of various
size by centrifugation are now in progress. An autoradiographicalattempt will be also made after the incorporation of isotopes into the
SR agents. It is hoped that these studies will give the answer to themethod of multiplication.

23. Cellular studies of antibody formation, IV. Variation in the
kinetic indices of plasma cells in antibody response
Toshihiko SADO, Takashi MAKINODAN,lI Martha R. LEONARDlI
and Carol J. CHADWICKlI

Two of the major problems in the field of cytodynamics of antibody
formation are (a) the fate of terminal plasma cells, and (b) the intercompartmental transit time (ITT), or the time it takes for a cell totransform from one metabolically unique state into another. The present
study was carried out in an attempt to resolve these problems by using
H3-thymidine as a cell marker.
A fixed number of primed spleen cells (2.4 x 107 ) were cultured in 0.1 p.
porosity diffusion chambers together with an optimum dose of antigen(- 50 p.g of bovine serum albumin). Autoradiographic and immunofluorescent methods were employed in detecting antibody-forming plasma
cells. The results showed that mature antibody-forming cells in thestationary phase are derived from proliferating immature plasma cells
in the late latent and log phases of response and that 70 % of all plasma
cells at this time are immunofluorescent positive, antibody containing
cells. This indicates that with use of this culture method it is possible
to assess the kinetics of antibody-forming cells by direct plasma cell
analysis. The evidence indicated that the life cycle of immature plasma
cells was changing during the log-stationary phase of response, possibly
due to a change in the G 1 phase of immature plasma cells, and that the
ITT between immature and mature plasma cells was varying from 3 to
12 hrs. Finally, most of the mature plasma cells was shown to be dying
exponentially with a Ti of approximately 32 hrs. The number ofdegenerating plasma cells increased abruptly during the late log phaseand remained relatively constant during the stationary phase.
These results indicate that because the ITT can vary from 3 to 12 hrs
1l

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn., U.S.A.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

there must exist many" transitional" plasma cells that possess properties
between a typical immature and mature plasma cells, and such cells havebeen observed by morphologists. The data also show that at any given
time a newly formed mature plasma cell has an equal probability ofdying as one that was formed much earlier. It follows then thatsenescence in the classical sense cannot be the primary cause for deathof mature plasma cells. It is of interest to correlate these changes inkinetic indices of plasma cells with the drastic changes in cellularenvironment, e.g., by increasing the concentration of antibody passively.
Thus, in this manner we may be able to assess the role of specificantibody as a feedback regulator on the progenitor cells.

24. Chemical modification of DNA and its constituents
Tomotaka SHINODA

Attempts have been made to modify DNA and its individual constituentsby using sodium trinitrobenzene sulfonate (TNBS). Under the several
conditions employed, reaction with free bases and with nucleosides occurred
only with guanine and its deoxyriboside, and none of the others werereactive towards the reagent. When purified calf thymus DNA was usedin the reaction under a pH above 9 and the reaction was followed
spectrophotometrically, the curve shown in Fig. 1 was obtained. The
absorption maximum of modified DNA appearing in Fig. 1 disappeared
in a medium whose pH was below 3. Trinitrophenylated guanine wasobtained from an acid hydrolysate of modified DNA followed by paper
chromatography by which it could be easily detected as a distinct yellow
spot. The molar extinction coefficient of TNP-guanine was calculated tobe 1.45 x 10· (A max 430 mil at pH 10). This substance was rather stable inacid medium but it appeared to be unstable under alkaline condition.
When the material was treated with N KOH at 100°C, most of it
decomposed into picric acid and other brownish degradation products.
A recovery of free guanine from the hydrolysate was somewhat poor,
which might suggest that a considerable disintegration of the base ring
was caused by the treatment. In contrast with the result obtained in
the intact material, the modified substance was not affected by nitrousacid treatment. When the material was treated by usual alkylating
agents such as dimethyl sulfate and ethylmethane sulfonate, similar
results were obtained suggesting alkylation of guanine at position 7.
This finding strongly suggests that a trinitrophenyl residue was locatedin the amino group of guanine at position 2.
Two interesting problems concerned with the investigation of the

RESEARCHES CARRIED OUT IN 1964

43

...
I

~
X

290

390

470

550 (ml')Fig. 1. Absorption spectra of TNP-DNA.
Curve I, at pH 10; Curve II, at pH 3.

chemical structure of DNA and of mutagenic effect induced by visible
light other than UV have arisen from the utilization of the employedtechnique. One promising related problem is that substrate specificity
of deoxyribonuclease was found to be limited by modification of DNA
with TNBS. The other advantage is that in the analytical proceduresof enzymatic digest of DNA oligonucleotides with TNP-guanine can bedirectly detectable by their intense yellow color, which no doubt is very
convenient in the following analytical procedures.

25.

Isolation and characterization of the deoxyribonucleic
acid from Ephestia
Saburo NAWA

As previously reported (NAWA and CASPARI, Annual Report No. 14,
1963), a method to demonstrate transformation in Ephestia was developed.
In order to achieve transformation, it was necessary to prepare pure,high molecular DNA from Ephestia. While methods for microorganisms
and mammalian tissues are available and fairly routine, their application
to insect has been accomplished only rarely. Whole animals were used
for extraction of DNA, since it was difficult to separate a specific tissueof Ephestia in great quantities. Care was taken to avoid the degradationof DNA when homogenized. Larvae were gently homogenized withsaline-citrate solution in a glass homogenizer by hand and then centrifuged.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

DNA was extracted from the pellet with sodium dodecylsulfate according
to the method of MARMUR. Protein denaturation was carried out byshaking with chloroform repeatedly. Ribonuclease was used to depolymerize RNA. The resulting preparaton was viscous high molecular DNA
contaminated with not more than 2 % protein and 1 % RNA by weight.
The slightly low phosphorus content (7 %) seems to be due to the presenceof polysaccharides (probably mainly glycogen, since only glucose wasfound by hydrolysis with HCl and separation by paper chromatography).
The molecular extinction coefficient at 260 m,u with respect to phosphorus
was 6,800. The preparation showed a 30 % absorption increment at 260 m,uwhen treated with deoxyribonuclease or alkali. DNA was prepared by
the same procedure from pupae and adults. It was, however, difficult toremove a trace of pigments (ommochromes) from adult DNA preparation.
Phenol method was also available to get fibrous DNA's. Base compositions
were determined by formic acid decomposition of DNA, following
paper chromatography. There seems to be no difference of the basecomponents among larval, pupal and adult DNA: Larval DNA; G, 16.4,
A, 33.3, C, 18.2, T, 32.1. Pupal DNA; G, 17.4, A, 33.3, C, 18.2, T, 31.3.
Adult DNA; G, 17.1, A, 32.8, G,17.5, T,32.5.

26.

A new method for the study of chemical mutagenesis
in the silkworm
Yataro TAZIMA and Kimiharu ONIMARU

With a purpose of exploiting a new technique for the study of chemicalmutagenesis, a simple method of treating eggs of the silkworm has beenstudied. The egg shell of this insect is very porous having many spiracles
on the surface. It is, however, uncertain whether or not a given chemical
can pass through the spiracles and penetrate into the egg plasm. If thepenetration of such chemical is confirmed, the experimenter could performthe experiment with comparative ease in the study of chemical mutagenesis.
In this regard a series of experiments have been carried out with somechemicals having lower molecular weight. The experiment to be reported
here concerns mostly KNO z, a known agent that reacts to purine andpyrimidine bases of DNA. Newly laid eggs were immersed in an aqueous
solution of KNO z at 30°C. The effectiveness of the treatment wasdetermined by the killing effect and/or mutagenicity. The eggs wereapproximately at the age of 30 minutes when the treatment was started.
By this time egg- and sperm-nuclei are not fused yet. They are presumed
to undergo syngamy around two hours after deposition. There would be

RESEARCHES CARRIED OUT IN 1964

45

expected, therefore, an incidence of mosaic patches if the penetratingchemical causes mutation. The experimental conditions are given indetail in Table 1 together with the results.
Table 1.
Group

Experimental conditions of KN0 2 treatment and survival.

. I Cration
oncen t - p H S ugar
C h emlca
Mol

Cont.

1
2
K-2

- - - _ . _ . _ - ~34--------_.-

5
3

Dist. Water
Dist. Water
S.A. BufferMol-_._---~,

min
60
120
60

4,897
7,585
9,575

98.16
98.26
98.25

30
300

30

16,224
12,364

1.12
6.23

30

30

13,155
16,224

1.00
1.12

30

30

13,155
15,134

1.00
97.87

30

30

15,41014,040
4,285

14.18
39.84
54.03

30

60

5,496
11 ,199
5,920

0.29
3.96
14.39

30

30
60
90

9,966
10,105
8,261

83.30
14.20
0.98

min
30

4.2

----.-_.--------------- - - - - -KN0 2

Age Duration Number Survl·val
ff eggsa ft er
00
in %oviposit treatment observed

0.2

4.2

-----_._._----._--------

KN0 2

0.01
0.2

4.2

-----------------------

5
6

KN02

OP-l
2
3

KN02

0.01

-----------_.

0.2

4.2
5.6
4.2

0.0
0.25
1.0

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

OP-4
5
6

KN0 2

0.2
---.

C-l
2
3
S.A. :

NK0 2

4.2

0.0
0.25
1.0

- - - - - ~ - - _ .

0.2

4.2

._--

sodium acetate

It has been known for T 2 phage that KN0 2 is most effective in its
killing action at pH 4.2. Therefore, a similar pH condition was preparedby regulating the pH value with sodium acetate buffer. As clearly seenfrom the table the killing effect of KN0 2 treatment was remarkable at
pH 4.2 but almost negligible at pH 5.6. This suggests that the chemical
did act upon the egg cell after passing through the shell. However, it
is not certain whether this reaction occurred due to the permeation of
the chemical via aqueous solution.
This question could be answered by an experiment in which theosmotic pressure of the KN0 2 solution were regulated by adding canesugar at various concentrations. The results of such experiment are
given in the same table; they show that reaction to the chemical did
actually occur due to its permeation.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

From these results it can be inferred that any chemical, as far as its
molecular weight is small, can be brought into the egg plasm by simply
immersing them into an aqueous solution. Preliminary experiments, inwhich BU and BUdR were utilized, indicated that this method could
conveniently be used in the study of chemical mutagenesis.

27. Purification of the three specific soluble chromoproteinsfrom chromogranules in hyPodermal cells of
the silkworm larva
Mitsuo TSUJITA and Susumu SAKURAI
It was reported in our previous paper (TSUJITA and SAKURAI, 1963, 1964)
that the chromogranules in hypodermal cells of silkworm larvae are an
important factor participating in the manifestation of larval skin color ortransparency. These chromogranules are composed of proteins, lipids,
RNA and sugar. Two kinds of proteins exist in the chromogranules,
that is, soluble protein of the inner part of the granules, and insoluble
protein of granule pellic1es. It was shown in our previous paper thatthe soluble protein combines with sepiapterin and can be separated fromother proteins, such as those which combines with uric acid, or withisoxanthopterin. Therefore, we continued our experiments in order toremove some impurities from the protein that combines with sepiapterin.
Moreover, experiments to separate the protein combining with uric acidfrom that combining with isoxanthopterin were carried out. In ourreport (TSUJITA and SAKURAI, 1964) these purification procedures were
described.

28. Amino acid analysis of the three specific chromoproteins
purified from chromogranules in hypodermal
cells of the silkworm larva
Mitsuo TSUJITA and Susumu SAKURAI
It was reported in our previous paper that the three chromoproteins
were separated and purified from the soluble proteins of chromogranules
in hypodermal cells of silkworm larvae. In our report (TSUJITA andSAKURAI, 1964) absorption spectra of the purified chromoproteins and theexperimental results of an amino acid analysis of those chromoproteinsare described.

RESEARCHES CARRIED OUT IN 1964

29.

47

Amino acid analysis of the insoluble protein
in chromogranule pellicles
Mitsuo TSUJITA and Susumu SAKURAI

Chromogranules collected from larval hypodermal cells of the normal
strain (Cl24) was purified. Chromogranule pellicle samples prepared byremoving their soluble proteins were analysed by Shibata's auto-aminoacid analyser. Seventeen amino acids, lysine, arginine, histidine, aspartic
acid, glutamic acid, threonine, serine, proline, glycine, alanine, cystine,
valine, methionine, leucine, isoleucine, tyrosine and phenylalanine, weredetected and quantitative analysis of each amino acid was carried out.

30. LiPid contents in chromogranules in hypodermal cellsof the silkworm larva
Susumu SAKURAI and Mitsuo TSUJITA

The. present experiments were undertaken in order to detect the lipid
contents in the chromogranules of silkworm larval hypodermis. The
chromogranules which were collected from the larval hypodermis ofnormal (C124 and Daizo) and mutant strains (lem and d-lem) were usedas materials. Using thin layer chromatography, containing corn starchor calcium sulfate as the binder, a qualitative analysis of the lipid contents
extracted from those granules by benzene-chloroform mixture or bybenzene-petroleum ether-ethanol mixture was carried out.
When benzene-chloroform mixture was used as solvent, the normal
strain samples, unlike the mutant strain samples, produced a supernumerary spot in addition to 5 common spots. On the contrary, whenbenzene-petroleum ether-ethanol mixture was used as solvent, the normal
strain samples also produced a supernumerary spot in addition to 6
common spots. It is considered that 3 spots among the 6 common spots
are palmitic, stealic and oleic acids. Judging from coloring reactions, thesupernumerary spot observed in the chromatograph of the normal strain
samples may be an unsaturated fatty acid.
The lipid composition may play an important role in the formation of
the granules, especially in the binding of polypeptide chains in the
granular pellicles.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

31. MultiPle allelic genes for dilute lemon larval skincolor of the silkworm
Mitsuo TSUJITA and Susumu SAKURAI

Further studies on amino acid composition of the three specific proteinscombining with pigments, i.e., sepiapterin-protein, isoxanthopterin-protein
and uric acid-protein complexes. are now under way.
For the purpose of studying the protein combining with sepiapterin
several dilute lemon strains in addition to the normal lemon strain havebeen prepared. One of the dilute lemon strains is d-Iem strain (TSUJITA,
1963) and the others are mutant strains artificially induced by X-rays.
The method used for induction of the mutants with dilute lemon larvalskin color was as follows. Female pupae of the normal lemon strain
about 7 days after pupation were irradiated with X-rays at doses of 5,0008,000 r and were mated with d-Iem males. Since mutants with dilutelemon larval skin color appeared in relatively high frequency among lemon
larvae in the next generation, they were reared to imaginal stage andback-crossed to d-Iem individuals. When new type dilute lemon larvae
segregated in the next generation, they were reared. The characteristics of three new strains, A, Band C obtained in this manner, are as
follows:
A. Mutant with remarkably dilute lemon larval skin. Although mostof the larvae in 5th instar exhibit almost normal opaque skin color, apart show remarkably dilute lemon skin color. The activity of pterine
reductase in this mutant individuals is weak just as in the lem and d-Iem
mutants. Besides, the content of isoxanthopterin in their hypodermal
cells is smaller than in the normal lemon larvae.
B. These mutant larvae resemble A. In addition, the content of isoxanthopterin in larval hypodermal cells is rather smaller than in A
mutant larvae, but we could not find any difference in the activityof pterine reductase.
C. These mutant larvae show dilute lemon skin color. Just like A and
B the activity of pterine reductase is weak. However, the content of
isoxanthopterin in larval hypodermal cells is to some extent larger thanin the lemon larvae.
Hybrid larvae between 2 strains of the three dilute lemon mutants,
A, Band C, show dilute lemon skin color. Moreover, hybrid larvae
between d-Iem individuals and those of one of the three dilute lemonmutants exhibit also dilute lemon skin color. Normal larvae, larvae withlemon skin color and those with dilute lemon skin color segregated in theF 2 generation of the crosses between individuals of the normal strain andthose of one of the new dilute lemon mutants (Table 1).

49

RESEARCHES CARRIED OUT IN 1964
Table 1. Segregation in the F 2 generation of the crosses between
individuals of normal strain and those of one of the new
dilute lemon mutants.

.

~~~y;--- -=--NO~--- +----. lemo~_ _1

Ax+CDaizo)2, __138
280

3

271

--I dilute k~onl- Total-'=~3~~~ '1-=~i2-! -.-

78

20

46-

16---

-

4

240

----

48

Total
----

-------

-----

378

I

----

333

1 - -

16

304
--------1

203

45

15

1,132

253

79

1,464

159

35

14

208

137

39

14

190

275

30

11

316

263

--

1

+(Daizo)xA

186._- - - - - - ---!------

5-=r--! ----

2
3

I

I

= -; ~~==_:;-.--!--:-I--~-~--,--:-:-:i

955

1
1---2

150

Total

+(Daizo)xB

i

174

1

61

35

1,190

-_._-

11

196

- - - _ . _ -

1

152

1

10

32

-----1----

3

194

-----

201

44

--~.

11

256

10

214

10

143

-----

4

172

5

103

1----1

i

32
30

- - -

778

'Total

I
I

I

1-

-

173

------c-----';------------I-(Daizo) x C

1
2

1

I

1

-

--3 -

52

I

208
I
46
1-- 240- - --6 - 5

I

1,003

----~_._-

15---16--

11

269
_ __
307
---3-43--I

269----- ------

58 - -

__ 1
1

- -

4

197

50

14

261

5

183

52

17

252

Total

1,097

262

73

1,432

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

It was confirmed that d-lem belongs to the 2nd chromosome, therecombination value between d-lem and Y being 6.0 and between d-lem
and p 30.2. It seems from the results of the crosses mentioned abovethat d-lem and the genes for A, Band C strains form a mutiple allelic series.

32.

Variance and covariance analysis of egg weight, egg shapeand body weight in the domestic fowl
Takatada KAWAHARA

Heritability of and genetic and phenotypic correlations among egg weight,
egg width, egg length and body weight were investigated in a closed
flock of White Leghorns. Data were collected from 479 pullets originatedfrom 32 sires and 153 dams. Experimental records were taken for aperiod of approximately 11,..., 12 months after hatching. The estimatesof heritability from analysis of variance are shown in Table l.
Table 2 presents the estimates of coefficients of genetic and phenotypiccorrelations between various traits.
It is found from Table 1 that the body weight and egg length aremoderately heritable whereas the heritability of egg width is low.
Table 1.

Heritability estimates of body weight, egg weight,
egg width and egg length.

Heritability

Egg weight

Egg width

Egg length

Body weight

h28
h2d
h;+d

0.336
0.324
0.330
-0.003

0.303
0.177
0.240
-0.031

0.447
0.455
0.466
0.005

0.791
0.526
0.658
-0.066c2.'--,----

Table 2.

Genetic and phenotypic correlations between body weight,
egg weight, egg width and egg length.

TraitsGenetic correlation

Phenotypic correlation

Body weight-Egg weight
Body weight-Egg width
Body weight-Egg length
Egg weight-Egg width
Egg weight-Egg length
Egg width-Egg length

0.543
0.476
0.287
0.829
0.820
0.309

0.298**
0.263**
0.180**
0.718**
0.681**
0.271**- - - - --------~-------------------** Significant at the 1 % level.

RESEARCHES CARRIED OUT IN 1964

51

Notwithstanding the higher value of heritability of egg length than egg
width, Table 2 shows that genetic correlation between egg length andbody weight was as low as 0.287, while that between egg width and bodyweight was moderately high, i.e., 0.476. It suggests that genes governing
egg length are rather independent from those governing body weight
and/or egg width.

33. Effect of heterosis and sex-linkage on sexual maturity
in the domestic fowl
Takatada KAWAHARA

Three breeds, i.e., White Leghorns (WL), Barred Plymouth Rocks (BPR)
and Nagoyas (NG), and reciprocal Fl'S and backcross hybrids between
WL and the other two breeds were investigated for sexual maturity.
Method of mating was such that a mixture of spermatozoa from two
breeds was inseminated to the dams to produce purebreds and F l hybridsof the two breeds in paternal or maternal half-sib combinations. Reciprocal
backcrosses were carried out between purebreds and F1 hybrids in orderto analyze sex-linked and maternal effects on sexual maturity. Several
marker genes were used, namely for rapid (k) and slow (K) feathering,for dominant white (f) and barred (B) plumage, for melanin formationin the dermis (id) and its inhibitor (fd).
Results of this investigation are summarized as follows: The average
number of days to attain sexual maturity was 189.05, 219.86, 192.55, for
WL, BPR and NG, and 192.45,178.34,177.35 and 171.09 for WL ~ xBPR (;,BPR~ xWLo, WL~ xNGo and NG~ xWLo, respectively. Fl hybridsfrom the WL-BPR combination attained sexual maturity 19.06 days
earlier than the corresponding mid-parents, and those from WL-NG
combination 16.58 days earlier than the mid-parents, possibly due toheterosis. The BPR ~ x WL 0 F l hybrids showed sexual maturity 14.11
days earlier than the reciprocal cross, the difference being statisticallysignificant. This finding was confirmed by the backcross experiment.
The average number of days to attain sexual maturity was 188.36, 192.62,
189.04 and 194.78 for WL ~ XF1(WL-BPR, k) 0, WL ~ XF1(WL-BPR, K) 0,
BPR ~ xF l (WL-BPR, k) 0 and BPR ~ (WL-BPR, K) 0, respectively. Ageat sexual maturity in backcross progenies carrying WL sex-chromosome
was 6.27 days earlier in WL~ xFlo and in BPR~ xF l (; 5.74 days earlier
than in those carrying BPR sex-chromosome, the differences being
statistically significant.
Thus, it was concluded that the gene(s) located on the sex-chromosome,possibly the k allele, could be resposible for early sexual maturity.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

34. Bilateral asymmetry in the ribs of cervical vertebrae
in the domestic fowl
Kan-Ichi SAKAI and Takatada KAWAHARA

Bilateral asymmetry in the ribs of cervicae vertebrae in the domestic
fowls was investigated. Carcasses of birds from inbred or non-inbred
strains of three breeds, White Leghorns, Rhode Island Reds and Nagoyas,
were treated with papain and the cervical vertebrae were carefully
separated in boiling water. Measured were lengths of right and left ribsof five central of fourteen consecutive vertebrae. The degree of bilateralasymmetry was measured by the absolute difference between left andright rib of a vertebra. Analysis of variance revealed that strain differences were statistically significant. Table 1 presents mean values ofbilateral asymmetry of six strains. Of interest is to find in Table 1 that
Table 1. Bilateral asymmetry of the ribs of cervical vertebrae in 5 inbredand one closed strains of three breeds of the domestic fowl.

Breed

StrainAsymmetry (ILeft side-Right sidell ofcervical vertebrae rib length
5

6

7

8

9

Mean

0.300
0.413
0.300
0.265
0.333
0.318

0.288
0.428
0.277
0.278
0.380
0.323

_._---White Leghorn
White Leghorn
White Leghorn
White Leghorn
Rhode Island Red
NagoyaKO-l, inbred
OW-2, inbred
OW-4, inbred
Closed flock
KR-2, inbred
KN-l, inbred

0.332
0.463
0.214
0.368
0.350
0.391

0.291
0.438
0.257
0.221
0.258
0.182

0.236
0.300
0.329
0.259
0.367
0.364

0.282
0.650
0.286
0.309
0.550
0.355

among three inbred strains of White Leghorns, the OW-2 strain showed
an unusually high asymmetry in comparison with others. These three
inbred strains had approximately the same inbreeding coefficient, about
60 percent, whereas they differed with respect to the degree of inbreeding
degeneration, the OW-2 strain being more vigorous and productive thanthe other two.
35.

Bilateral asymmetry in the legs of the domestic fowl
Kan-Ichi SAKAI and Tohru FUJISHlMA

Bilateral asymmetry in the legs of chickens was investigated in 714
birds of two breeds, i.e., 436 of White Leghorns and 278 of Rhode Island
Reds. Lengths of shank and four toes were measured at 0, 3 and 6 weeks

RESEARCHES CARRIED OUT IN 1964

53

of age with slide calipers, and the value, left minus right, was used as
the index of bilateral asymmetry for each part.
It was found from this study that in one day old chickens the left side
was generally longer than the right, except for the fourth toe, suggesting
the presence of directional asymmetry (Table 1). Its degree was significantly different between breeds, suggesting presence of a genetic control.
Table 1.

Bilateral asymmetries in legs of the domestic fowl.

White Leghorn
Wks
of
No. Left Asymage i of mean metry (J. birds mm
mm

Part

I

Rhode Island Red
No. Leftof meanbirds mm

Asym.
metry
mm

I Breed diff.
I(J---------------

1 P<O.Ol

Shank

0
3
6

**
**
I 278 28.30 0.31±0.47
436 28.78 0.51±0.41
319 42.95 -0.05±0.41 i 219 44.43 -0.01±0.38I**. **I, 220 61.63 -0.17±0.45I334 61. 72 -0.13±0.47
436 7.24
319 11.57
334 16.35

0.47±0.52

1st toe0
3
6

2nd toe03
6436 13.37
319 19.02
334 26.07

0.26±0.44I 278 13.62**
0.13±0.54
219 20.17**
0.14±0.65 I 220 27.02

I----------**----1
1

278 7.33**
0.25±0.70 I 219 12.25**
220 17.01
0.38±0.70****

0.27±0.49**
0.26±0.71**
0.33±0.69

**

N.S.
N.S.

1----P<O.Ol
1

N. S.
N. S.

1

0.4~±0.441

03
6

P<O.Ol
N.S.
0.13±0.50 I**
0.05±0.56 I
N. S.
1--436 19.19 0.14±0.451 278 18.88 0.03±0.50 i P<O.Ol****
319 27.65 -0.18±0.54I
219 28.85 -0.31±0.50
P<O.Ol****334 37.47 -0.29±0.60
220 38.45 -0.41±0.58
P<O.Ol

0
3
6

**
**
I
436 14. 75 -0.1~t°.40
278 14.94 -0.23±0.42I319 21.73 0.17±0.47
219 22.98 0.04±0.59***
I
334 29.45 0.34±0.59
O. 18±0.51
I 220 30.88---------

3rd toein

I asymmetry!

4th toe

1

N.S.
P<O.OlP<O.Ol* Significant at the 5 % level.
** Significant at the 1 % level.
N. S. Non-significant.

The heritabilities of these asymmetries estimated were 0.36 for the shank,
0.00 for the first toe, 0.12 for the second toe, 0.09 for the third toe, and0.19 for the fourth toe.
This directional asymmetry, however, seems to be interfered by later
development. Thus, in 6 weeks old chickens, the shank and the third toe
became longer in the right side, but the first, second and fourth toes werelonger on the left side. Particularly, the asymmetries in the shank andthe third toe changed in the negative direction, while that in the fourth toe

54

ANNUTL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

changed in the positive direction. Thus, the asymmetries in the third andfourth toes changed in the opposite direction with the growth of chickens.

36. Breed differences in competitive ability of growing chickens
Tohru FUJISHIMA

Experiments concerned with the competition during chick growth arevery few, in spite of their importance in poultry husbandry, particularly
in growing breeding birds or broilers. The present experiment was
attempted to investigate differences, if any, between breeds in competitive
ability of chickens, growth stage at which competition sets in and relationship between initial body weight and competitive capacity of domestic
fowl.
Male and female chickens of three breeds, i.e., White Leghron, Barred
Plymouth Rock and Rhode Island Red, were divided into three single
Table 1. Body weights in single and mixed groups at 4, 5 and 6 weeks
of age, and regression coefficients of gains in body weight (4-6 wks)
on 4 wks body weight estimated within and among breeds.
Male
Weeks
of ageBreed

I Single group I Mixed group

I
W

4

I

L

B P R
R I

R

(g)

(g)

191.1
199 5
208.9

I

I

191. 8
201 4
200.8

I
191.6

191.1

------, - - - - -

- -

5

W
L
B P R
R I R

244.0
245.7
250.9

241.4
256.0
235.5

236.1
239.4
246.6

6

W
L
B P R
R I R

300.8
286.4
311.2

309.2
318.2
293.9

291.8
282.5
298.3

Regres.
coef.
within
breed

W
L
B P R
R I R

0.398**
0.430*
0.463**

0.415*
0.953**
0.575

0.417**
0.212
0.619**

0.977

1.229

------,--

230.5
250.7
236.1
-----

Regres. coef.
among breeds

I
I

I

I

287.3
311.3
294.4
-

* Significant at the 5 % level.
** Significant at the 1 % level.

-~

0.435*
0.413
0.515*
0.605-0.260I

RESEARCHES CARRIED OUT IN 196455

breed groups and a mixed group. Each single breed group contained 30
birds of the same sex and breed, while each mixed breed group contained
30 birds derived from all three breeds, each contributing 10 birds of thesame sex. They were reared until the age of six weeks and were weighed
every week. The experiment was repeated twice, and both replicateswere kept under restricted feeding condition, except for drinking water.
The results of this experiment, as shown in Table 1, indicate thatafter the age of 5 weeks, difference in competitive ability becomes recognizable especially in males among these breeds, Barred Plymouth Rock
being strongest competitor, White Leghorn intermediate and Rhode Island
Red weakest. The regression coefficients estimated within and among
breeds of gains in body weight on the body weight at 4 weeks of age,
after which the competition set in, were not significantly different between
mixed and single groups respectively, though they tended to be larger inthe mixed groups. Therefore, it is concluded that body weight may bean essentially different character from competitive ability.

37. Studies on chromosomal polymorphism, I. Autosomal
polymorphism in Rattus rattus L. collectedin Kusudomari and Misima
Tosihide H. YOSIDA, Akira NAKAMURA and Takako FUKAYA

Chromosomes of Rattus rattus L., collected in Kusudomari (Nagasaki)
and Misima (Sizuoka) were examined. The karyotype revealed a remark·able heteromorphism in chromosome No. 1. The homozygotic, i.e.,
standard type, was characterized by 13 pairs of telocentric and 7 pairs ofmetacentric chromosomes. Chromosome pair No.1 was telocentric. X
2,

4

1 VU.UI

5••

II" ",.. .. ,... -Figs. 1 and 2.

I'

I. 15

....

"17

11 \I

21 ,

•

A

. v _ v • •••••
,. -.
•• •

_

II

12

•

-

,

Serial alignments of chromosomes of bone marrow cells in
No. 1 chromosome pair in Fig. 1 is telocentric, while thatin Fig. 2 is heteromorphic concerning telocentric and subtelocentric chromosomes.

Rattus rattus.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

and Y chromosomes were also telocentric (Fig. 1). The heteromorphic
chromosome pair No. 1 was seen in 18.4 per cent of rats from Kusudomari and in 40 per cent from Misima. One chromosome of theheteromorphic pair was conspicuous by a subtelocentric centromere, while
the other was telocentric (Fig. 2). The total length of the telocentric
was almost the same as that of its subtelocentric partner. These facts
indicate that the subtelocentric No.1 chromosome might have arisen bya centromeric inversion of the telocentric partner. Individuals homozygous
for subtelocentric No.1 chromosome could not be found in either population. The difference in the frequency of the dimorphics collected in
Kusudomari and Misima was statistically significant.

38. Studies on chromosomal polymorphism, II. Autosomal
polymorphism in Norway rats, Rattus norvegicus
Tosihide H. YOSIDA and Kohachi AMANOPolymorphism in chromosome pair No.3 of Rattus norvegicus was foundin laboratory strains and in wild rats. Some of the animals had asubtelocentric pair No.3, while others had the same pair composed of
two telocentrics. Fourteen inbred strains were classified into two types
concerning the pair No.3.
1. WIS-type (characterized by a telocentric pair No.3): Donryu-,
NIG-III-, W/T- and WIS-strains are included in this type (Fig. 1).
2. YOS-type (characterized by a subtelocentric pair No.3): ACI-,
Albany Buffalo, CWo, Fischer-, Long-Evans-, NIG-IV-, Wayne Pink-Eyed

3
Figs.!, 2 and 3. Metaphase chromosomes of bone marrow cells in inbred rats
(Rattus norvegicus). 1. WIS-strain rat with a telocentric No.3 chromosome
pair. 2. YOS-strain rat with a subtelocentric No. 3 pair. 3. F 1 hybrid
between WIS- and YOS-strains, characterizing by telo- and subtelocentric
No. 3 chromosomes. Arrows indicate the No. 3 autosome pair.

RESEARCHES CARRIED OUT IN 1964

57

Hooded-, WKA- and YOS-strains belong to this type (Fig. 2).
F 1 hybrids between WIS- and YOS-strain rats had a heteromorphic
pair No.3 consisting of a telocentric and a subtelocentric chromosomes(Fig. 3).
Polymorphism in pair No.3 was also found in 43 wild rats collectedin Misima. They were classified into three types concerning pair No.3.
Among 43 rats, 17 were of WIS-type, 3 were of YOS-type and theremaining 23 were of hybrid-type.

39. Further study on chromosomes of leukemias in the mouse
induced with Moloney leukemogenic virus (ML V)1)
Tosihide H. YOSIDA and Lloyd W. LAw2)

The present authors have already reported on the chromosome numberof 19 Moloney mouse leukemias (YOSIDA and LAW, Annual Report No. 14,
1963). At that time 15 among them had 40 chromosomes as the modal
number, 3 had 41 chromosomes and the remaining one showed bimodal
distribution of chromosome numbers with the modes at 40 and 41. Last
year, chromosomes of 10 Moloney mouse leukemias were newly observed.
Among 10 leukemias 9 showed 40 chromosomes as the modal number,and only one had 41 chromosomes. Two leukemias have developed after
thymectomy from C3Hf/Lw strain mice. Both had also 40 chromosomesin their stemline cells.
Chromosomes of 29 Moloney mouse leukemias in total were hitherto
observed by us including the present study. Among them 23 leukemias.
(72.4 %) had 40 chromosomes as the modal number. On the other hand,about 50 % of spontaneous leukemias had 40 chromosomes and theremaining 50 % showed aneuploidy (STICH, 1960; KURITA and YOSIDA, 1961).
The majority of mouse leukemias developed by chemical treatment had
41 chromosomes (STICH, 1961), and those developed by X-irradiation hadvarious unusual karyotypes (FORD and MOLE, 1959; KURITA and YOSIDA,
1961; NODLER, 1962). From the above considerations it is assumed thatmost of virus leukemias develop without chromosomal alteration, and they
may be due to the transformation of cells at molecular level by virus
infection. Perhaps it would be well worthwhile to mention that theleukemias were from the artificially created high-leukemia lines G4 to·GI0 of several strains and all were lymphocytic except the 2 granulocytic
leukemias.
1) This work was supported by a research grant from the National Cancer Institute(CA 07798-01), Public Health Service, U.S.A.
2) National Cancer Institute, Bethesda, Md., U.S.A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

40. Karyotypes in drug resistant sublines of mouse
leukemia L. 1210 1 )Tosihide H. YOSIDA and Kyoko OHARA

Chromosomes in drug sensitive and 5 drug resistant sublines in mouse
leukemia L. 1210 were observed. They were established by Dr. LAW in
the National Cancer Institute, Bethesda, U.S.A. The 5 sublines used in
the present study were resistant to amethopterin (AM), 6-mercaptopurine
and fluorouracil (6-MP/FU), 6-mercaptopurine, fluorouracil and amethopterin (6-MP/FU/AM), anti-leukemic agent NSC No. 38280 (38280) andthioguanine (TH).
1. Variation in chromosome number: Chromosome number in sensitive L. 1210 varied from 34 to 46 with the mode at 40. Resistant sublines
to AM, 6-MP/FU, 6-MP/FU/AM and 38280 have also 40 chromosomes as
the mode of variation fluctuating from 36 to 45. TH resistant line only
had 41 chromosomes as modal number.
2. Marker chromosomes: A large metacentric chromosome was usuallyfound to characterize the sensitive line. In none of resistant lines,
however, this marker chromosome could be observed, while minute, small
metacentric and subtelocentric chromosomes were often occurring in
them.

41.

Chromosomes in drug sensitive and resistant lines ofplasma cell neoPlasm 70429 of mice l )Tosihide H. YOSIDA
Table 1. Chromosome number in sensitive and resistantlines of 70429 tumor.
Resistant to

38
39
40

41
42
Total number ofcells observed11
28 (84.9 %)
2
1

1
2

24 (75.0 %)4oAzauracil

Azaserine

o
o
o

o

42 (95.5 %)2

1
37 (90.2 %)o
3- - - - - - - - - - -- - - - - - - - - - - -------

33

32

44

41

Il This work was supported by a research grant from the National Cancer Institute(CA 07798-01), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1964

59

Chromosomes in sensitive and resistant lines of plasma cell neoplasm
70429 of mice were examined. All these sensitive and resistant lines
were obtained from Dr. LAW'S laboratory of the National Cancer Institute,
Bethesda, U.S.A. The distribution of chromosome numbers is shown in
Table 1.
As the table shows, sensitive and azauridine and azaserine resistantlines had 40 chromosomes in their stemline cells. Only the azauracil
resistant line had 41 stemline chromosomes. All chromosomes were rod
shaped in sensitive as well as in resistant lines.
42.

KaryotYPic differences in mouse ascites hepatoma MH
134 and MH 129P maintained in different Places!)
Tosihide H. YOSIDA and Hirotami IMAI

Mouse ascites hepatomas, MH 134 and MH 129P, used in the present
study were established in the National Institute of Health, Bethesda,
U.S.A. in 1952 (named tentatively MH 134A and MR 129A), and weretransferred to Tohoku University by Dr. SATO in 1956 (named MH 134J
and MH 129J). MH 134 was transferred to Misima in 1961 (named MH
134M). The two strains were maintained in these different places byserial intraperitoneal transplantation to C3H mice. Their chromosome
numbers were compared with the following result:
1. Frequency of near-diploid and near-tetraploid cells: Both MH 134A
and J and MH 129A and J lines were characterized by higher occurrenceof cells with near-diploid chromosomes. About 16 % cells in MH 134A
and J lines showed near-tetraploid chromosomes in their cell population
and about 50 % showed tetraploidy in MH 134M line. The frequencies
of tetraploid cells in MH 129A and J lines were about 10 %.
2. Karyotypes: MH 134A; The frequency of cells with 42 chromosomes was the highest showing fluctuation from 38 to 47. As markers,a large submedian metacentric chromosome and a minute one wereobserved. MH 134J; Cells with 40 chromosomes were of highest occurrence. A metacentric, a submetacentric and a minute chromosome wereobserved as markers. These karyotypes were also found in MH 134M
line. MH 129A; Cells with 46 chromosomes occurred at highest frequency.
As a marker, a large submetacentric chromosome with secondary constriction was observed. MH 129J; Cells with 45 chromosomes were ofhighest occurrence. A submedian metacentric, a submedian metacentricwith secondary constriction and a median metacentric chromosome were
1l This work was supported by a research grant from the National Cancer Institute(CA 07798-01), Public Health Service, U.S.A.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

observed as markers.
From the above investigations, it can be said that both lines, MH 134
and MH 129P kept in N. I. H., U.S.A., have considerably different karyotypes from those kept in Tohoku University.

43.

Autoradiographic analysis on the sensitive stage to
4-nitroquinoline i-oxide during division cycle of
Yoshida sarcoma cells
Kazuo MORIWAKI, Yoshinori KURITA
and Tosihide H. YOSIDA

It has already been shown that 4-nitroquinoline I-oxide (4NQO) exhibits
a mutagenic activity in several kinds of micro-organisms (OKABAYASHI,
1955, etc.), in addition to its well-known carcinogenic and carcinostatic
actions. The present study was undertaken to determine which stageof the division cycle of the Yoshida rat sarcoma cells is responsible forthe occurrence of the chromatid breaks in the metaphase following thein vivo treatment with 4NQO. The findings in the micro-organisms
mentioned above allow us to postulate that this drug might react
preferentially with DNA or DNA-synthetic processes in mammalian cells as
well. The present experiment, however, demonstrated that postsynthetic(G2) stage is more sensitive to 4NQO than the DNA synthetic (S) stage.
This can be shown as follows:
Chromatid breaks were observed about 4 hours after administrationof 0.8 ml of 10- 3M 4NQO into the ascites tumor which cells seem toproliferate at random with an average mitotic index of 3 %. The final
concentration of 4NQO in the ascites fluid was estimated as approximately
10- 5 M. On the other hand, retardation of G2 stage by 4NQO treatment
was indicated by the autoradiographic analysis of the incorporation oftritiated thymidine into the chromatids. One hundred and sixty pc
H3-thymidine in 0.2 ml saline solution was injected into the intraperitoneal
cavity of the tumor bearing rat. Grains on the autoradiographs ofchromosomes appeared about 2.5 hours after this injection. The simultaneous 4NQO treatment delayed the appearance of the grains until about
6 hours after the injection.
These results suggest that this drug is able to give some structuralchange on the chromosomal materials during G2 stage, and not during
S stage. The precise mechanism of this action is yet to be studied.

RESEARCHES CARRIED OUT IN 1964

61

44. Chromatid aberrations induced in Yoshida sarcoma cellsby the radiomimetic chemical, 4-nitroquinoline l-oxide
Yoshinori KURITA, Tosihide H. YOSIDA and Kazuo MORIWAKI

Chromosome aberrations induced in Yoshida sarcoma cells by theradiomimetic chemical, 4-nitroquinoline I-oxide (4NQO), are only of thechromatid type.
Chromatid intrachanges (breakage) occurred withmaximum frequency 8 hours after the treatment, and their distribution
among cells was nonrandom. The frequency of intrachanges was notproportional to the length of metaphase chromosomes. Chromatid interchanges (translocations) usually occurred most frequently 24 hours after
treatment, and their distribution among cells was also nonrandom. The
aberrations were mostly localized in the neighbourhood of the secondary
constriction. Frequency of both, intrachanges and interchanges, increased
proportionally with the doses 8 hours after treatment; the former
increased with the square of the dose. However, the frequency ofinterchanges which occurred 24 hours after treatment showed no
proportional relation to the dose.
The mechanism of aberration induction is supposed to be the resultof cross-linking of the twin molecular chain of the chromonema, sincecomplete lack of chromosome type aberration suggests that direct
chromatid breakage in the cells of the Gl phase at the time of treatment
is very rare. Furthermore there is no evidence to support the possibility
that the main function of the alkylating agents is hydrolysis of chromosomal macromolecules. The localized aberrations near the secondary
constriction are supposed to be closely related to the location of nucleoli
and of nucleoli organizers in the constrictions; the nucleoli are the sitesof r-RNA synthesis which seems to be completely inhibited by thechemical treatment.

45. Autoradiographic analysis of the mitotic cycle in Yoshidasarcoma cells
Yoshinori KURITA, Kazuo MORIWAKI and Tosihide H. YOSIDA

The mitotic cycle of Yoshida sarcoma cells was studied by means oftritiated thymidine and autoradiography.
The duration of each phase of the mitotic cycle was estimated asfollows:(1) Early interphase (Gl) lasted about 5.5,..., 6.0 hours.
(2) Phase of DNA synthesis (S) lasted about 9.0,..., 9.5 hours.
(3) Antephase (G z) was about 2.5 hours long.

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

The generation time of the sarcoma cells was determined to be about
18.5 hours, which is almost in complete agreement with FREYMANN'S
result obtained by X-ray experiment.
46.

Strain difference of susceptibility to the teratogenic
action of ethyturethane in mice
Kiyosi TUTIKAwA and Akira AKAHORI

It has been well recognized that the frequency of cleft palate following
treatment of mice with cortisone varies with the genetic constitution of
the mother and embryo. Recently, GOLDSTEIN et at. (1963), who used
6-aminonicotinamide as a teratogen, pointed out that cytoplasmic factors
influencing the embryo's response to the teratogen may be active in thecase of cleft palate, and may also exist for vertebral fusions.
The purpose of the study was to provide further information on thegenetic basis for a strain difference in frequency of induced malformations
following treatment of mice with ethylurethane. Mice of two inbred
strains, CBA and C3H eB/Fe, subsequently referred to as C and T, wereTable 1. Frequencies of late death (LD) and exencephaly following maternal
treatment with ethylurethane on day 8.5 of gestation.
---._---Cross

No. offemales CL

IMP PIL---------_.-CxC
Control
TxTControl
CxT
TxC
CTxCT
TCxTC
CxCT
CxTC
CTxC
TCxC
TxCT
TxTC
CTxT
TCxT

12
6
10

10
11
10
10
10
13
7
3
20
12
6
3
18

ED

LD

LE

Percent
LDjIMP of embryos
withexencephaly- - - - ~ . _ . _ - . _ - -

126
59
107
101
111
114
119
120
137
71

35
231
137
65

36
202

99
44

100
90
78
105
110
103
127
64
31
207
118
59
32
183

27
15
7
11
33
9
9
17
10
7
4
24
19
6
4
19

5
1
4
8
9
7
0
3
15
6
1
5
4
7
4
6

16
2
40
6
10
16
7
17
25
6
6
32
21
11
7
25

78
41
56
76
59
82
103
83
87
52
24
170
93
41
21
152

16.2
4.6
40.0
6.7
12.8
15.2
6.4
16.5
19.7
9.4
19.4
15.5
17.8
18.6
21.9
13.7

0
0
21.9
0
0
11.2
10.0
11.0
0
0
0
4.0
12.3
14.7
10.7
13.0
-------_."--RESEARCHES CARRIED OUT IN 1964

63

used in the present experiment. Pregnant females were injected with
1.5 mg/g of 10 % ethylurethane aqueous solution once on the 8.5 day ofpregnancy and killed on day 17.5.
In treated mice, exencephaly was induced in 21.9 % of the susceptible
T embryos and 0 % in the resistant C embryos. In reciprocal crosses
between these two strains, the TxC embryos had a higher frequencyof the defect than the CxT embryos. While, similar frequencies of
exencephaly resulted from matings among the reciprocal Fl themselves
and from matings of the reciprocal Fl females to susceptible T males(Table 1).
Similar result was obtained from the search for induction of posterior
shift of thoracolumbar border following same maternal treatment. Thus
the factors controlling the susceptibility of an embryo to ethylurethane
are not cytoplasmically transmitted, and involve the interaction of fetal
and maternal genotypes.

47. Further studies on water soluble proteins in mouse skinfollowing the aPPlication of a tumor promoting agent
Yoshito OGAWA

In a previous preliminary note (OGAWA and KOBAYASHI, Annual Report
No. 14, 1963), the effect was described of Sweet Orange Oil on the watersoluble proteins in the skin of the mouse. Sweet Orange Oil which is
a component of the peal of Citurus sinensis aroused the interest of cancer
researchers as a promoting agent in experimental carcinogenesis. The
present communication corrects some errors that occurred in our previous
note resulting from the use of denaturalized materials due to refrigerationand describes the changes in those soluble proteins following the application of Sweet Orange Oil (B.P.C. grade).
0.4 g of Sweet Orange Oil was applied individually to the dorsal skinof mice with two day intervals, and the treated part of the skin was
cut off with three day intervals after treatment. The cut skin tissue
was mashed with the same volume of distilled water in a glass homogenizer at ooe. Immediately after centrifuging the homogenate at 1,500 g
for 10 minutes, electrophoretic analysis of the obtained clear supernatant
was made at room temperature on cellulose acetate strips (OXOID) of
2.5 X 9.0 cm size by using veronaI buffer (pH 8.6, p. 0= 0.06) at 0.7 mA/cm fortwo hours. Nigrosin staining was used for the estimation of the analyzed
proteins. A non-treated group was prepared for control.
The analytical findings are given in Table 1. The total protein
concentration increases immediately after the application of Sweet Orange

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table 1. Effect of Sweet Orange Oil on the water soluble proteinsin mice skin tissue (mg/g skin tissue weight).

Day after
treatment
Normal(Control)
3
6
9
12
15

Total
I protein
12.52
20.66
23.21
31.30
19.07
16.58

I(Alb~min) F

G

B

H

D

E

3.25

1.40

1.06

1.90

0.00

2.28

2.16

0.47

5.23
4.24
8.21
4.46
3.85

1.44
2.25
0.00
0.00
2.02

1.18 5.10
2.25 4.74
0.00 11.20
0.00 3.25
1.45 4.34

0.00
0.00
0.00
5.41
0.00

4.18
8.98
7.31
3.11
2.22

3.01
0.00
2.06
1.90
1.93

0.52
0.75
2.52
0.94
0.77

Oil. It was 250 % higher than in the control on the 9th day after thetreatment. This finding was closely related to the progress of themitotic index in the skin tissue. The most remarkable increase of themitotic index was recognized on the 9th day and then it decreased
gradually with the lapse of time. The soluble proteins extracted fromnormal skin tissue were separated into seven fractions (A, F, G, B, D, E
and I). Fraction C of our previous note was not recognized in freshmaterials either in normal or in treated skin. No remarkable change
was found in the components of soluble proteins during the first threedays after treatment. But on the 6th day, fraction E disappeared
temporarily, and fractions F and G disappeared from 9th to 12th day
after treatment. On the contrary, a new fraction H was recognized on
the 12th day. This transient fraction resulted from the treatment with
Sweet Orange Oil.
The physiological and chemical nature of this new fraction is now studied.

48. Chromosome observation in Japanese ants
Hirotami 1M AI and Tosihide H. YOSIDA

Since there have been made only a few studies on the chromosomesof ants, taxonomists could not utilize cytological data in evolutional
considerations of this insect. The present authors made an attempt tocoordinate karyological data with problems of ant phylogeny. As thefirst step, the authors made a detailed analysis of their chromosomes byusing the hyaluronidase-aceto-orcein squash method in combination withthe drying-method, and obtained suitable preparations. The present
paper reports the chromosome numbers of 22 Japanese species belonging
to 4 subfamilies (Table 1).
From these results, it became clear that the Ivariation of the chromo-

65

RESEARCHES CARRIED OUT IN 1964
Table 1.

Chromosome numbers of Japanese ants.
Subfamily PONERINAE

Brachyponera luteipes SMITH
Cryptopone sauteri WHEELER

n

2n

. 11
.

22

. 14

28

. 10
. 11
. 12
.
.
. 14
. 16
. 17
.
. 22

20

.

18
28

28

Subfamily DOLICHODERINAE

Iridomyrmex itoi FOREL
Subfamily MYRMICINAE

Pheidole fervida SMITH
Monomorium pharaonis LINNE
Pristomyrmex pungens MAYR
Leptothorax spinosior FOREL
Crematogaster laboriosa SMITH
Tetramorium caespitum jacoti WHEELER
Aphaenogaster sp. .
Aphaenogaster famelica SMITH
Vollenhovia emeryi WHEELER
Messor aciculatum SMITH

24
24
26
28
34
36
44

Subfamily FORMICINAE

Camponotus sp
Camponotus kiusiuensis SANTSCHICamponotus japonicus MAYR
Lasius niger LINNE
Lasius talpa WILSON
Formica sanguinea LATREILLE
Formica truncorum yessensis FOREL
Formica japonica MOTSCHULSKY
Polyergus samurai Y ANO

9

.
. 14
.
.
. 26
. 26
. 27
. 27

30
30
52
52
54
54

some numbers is very wide among the genera of the same subfamily,
namely, from n = 4 to n = 27 (including the data of PEACOCK et at. (1954)
and HAUSCHTECK (1961, 1962)). Striking heteroploidy has been developedin two subfamilies, Formicinae (n = 8 ,..., 27) and Myrmicinae (n = 4,..., 22).
Based on this fact, it is presumed that both subfamilies evolved independently from a very early evolutional stage in the direction toward
increasing the chromosome number and developing heteroploidy. This
assumption agrees with the opinion of BROWN (1954) who divided thefamily Formicidae into two large groups based on morphological traits, i.e.,
Poneroid-complex including Myrmicinae and Myrmecioid-complex including
Formicinae. The assumption is also supported by their wide distribution
and mode of reproduction. WHEELER (1928) thought that the origin ofants is tropical, but both those subfamilies became distributed all over

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

the world. Therefore it may be assumed that the heteroploidy occurredin various genera, one after another, in the course of their migration to
new environments. At the same time, their complicated system ofparthenogenetical reproduction (haploid or diploid parthenogenesis of eggs
laid by the queen or especially by the workers) may have also contributed
to the development of heteroploidy as know in many other parthenogenetical animals.

49.

Abnormal taster to phenyl-thio-carbamide (PTe)in Japanese population
Yoshito OGAWA

This report furnishes a few data on abnormal tasters for PTC among
the Japanese population, namely those with sweet, puckery, salty,
pungent and other taste responses.
Within the age range from 15 to 18 years, 2,304 persons residing in
Matsumura (Niigata Pre£. : 354 persons), Chiba (Chiba Pref. : 109 persons),
Okazaki (Aichi Pref. : 141 persons), Katsuyama (Fukui Pre£. : 166 persons),
Hamada (Shimane Pre£. :246 persons), Kure (Hiroshima Pref. : 429 persons),
Nakamura (Kochi Pre£. : 310 persons), Miyazaki (Miyazaki Pref. : 176 persons) and Tokyo (Tokyo: 373 persons) were examined for their taste
reaction to PTC solutions prepared in accordance with Hartman's report(1939).
In distilled water diluted solutions (1:2, 1:4, 1:8, etc.) were preparedfrom a stock solution (0.16 %) of PTC. According to their strength,
the solutions were numbered from 1 to 12, 1 being the stock solution
and 12 the weakest solution (0.78125- 4 %). The threshold value of taste
sensitivity, irrespective of the kind of taste, was investigated twice ineach individual. When different results were obtained between first andsecond test, the examination was made again next day and only sure
results were used in this report.
According to the taste response to PTC, the subjects were divided
into two groups. One represented the normal tasters (simple tasters)
and the other the abnormal tasters (complicated tasters). The normalgroup showed only bitter response to every concentration of PTC, also
in respect of their taste threshold, and non-tasters even when the stock
solution of PTC was used. This group consisted of 1,987 persons (86.24 %).
The abnormal group, comprising 317 persons (13.76 %), showed someother taste response, e.g. sweet, puckery, etc., on the taste thresholdpoint. And some of the people in this group showed often bitter responseto stronger PTC solutions.

RESEARCHES CARRIED OUT IN 1964

67

In the group of abnormal tasters, 25.24 percent (80 persons) showedno bitter taste response even to No.2 solution of PTe. But in the groupof normal tasters, only 8.91 percent (177 persons) were non-tasters.
The distribution of bitter taste threshold values between these two groups
is clearly different (X 2 = 38.52, DF = 7, P < 0.01) (Table 1).
Table I.

Group
Normaltasters
Abnormaltasters

Distribution of threshold values of bitter taste response
in normal and abnormal taster groups.

Non.
tasters<1,1,2

3

4

5

6

7

8

177

56

66

127

224

415

393

325

204

1,987

80

21

19

25

26

47

49

34

16

317

Tasters

Total
9 10-12Moreover the distribution of threshold values of taste response in the groupof abnormal tasters, irrespective of the kind of taste, was also significantly
different from that of the threshold value of bitter taste response of thenormal group as showed in Table 2 (X2 = 267.89, DF = 9, P < 0.01).
Table 2.

I

Group
Normaltasters
Abnormaltasters

Non.

I tastersiDistribution of threshold values of taste response
in normal and abnormal tasters.*
Tasters<1,1,2

3

4

177

56

3

4

Total

10

11

12

66 127 224 415

393 325 108

68

28

1,987

11

44

49

55

317

5

12

6

18

7

23

8

9

47

51

"-_._"---"------*

Irrespective of the kind of taste response to PTC solution.

These results indicate that the threshold value of taste response in
the abnormal group does not correspond to that of the normal group.
The abnormal taster group must be regarded as a different populationfrom that of normal tasters.

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

B. GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF PLANTS

50. "Germless grain ", a new effect of caudata cytoplasm
on the manifestation of wheat genomes
Hitoshi KIHARA and Koichiro TSUNEWAKI

It is already known that caudata cytoplasm exerts various important
effects on the manifestation of wheat genomes, such as induction ofmale sterility or pistillody of stamens, reduction of plant vigor, andincreased occurrence of haploids and twin seedlings (KIHARA, 1949-1964;
KIHARA and TSUNEWAKI, 1961-1964). Recently it has been found to increase
the occurrence of germless grains which is here reported.
Male-sterile plants of Triticum aestivum ssp. vulgare var. erythrospermum(Abbrev. T. v. e.) with caudata cytoplasm were interplanted with normal
plants of the same variety. About 40 spikes of both male-sterile andnormal lines were emasculated before flowering and then artificiallypollinated with normal pollen grains. The hand-pollinated spikes were
harvested together with some open pollinated spikes, and the caryopsis
was examined for the presence of an embryo. The result is summarizedin Table 1.
Table 1. Frequency of germless grains among seeds produced by malesterile T. v. e. with caudata cytoplasm and the normal line.
Lines

Type ofpollination

No. of seeds
examined

No. of germless grains

NormalArtificial
Open
Artificial
Open

593
15,520
506
1,607

1
17

Male-sterile

48

276

%

0.2
0.1
9.5
17.2

The frequency of germless grains in normal T. v. e. was, on the average,
0.1 %, and no difference was found between artificial and open pollination.
On the contrary, germless grains were found in abundance among seeds
set on male-sterile plants. Their frequency was higher when the seeds
were produced by open pollination than when they were obtained fromhand pollination. From those results, it can be said that caudata
cytoplasm increases the frequency of germless grains and that open
pollination of male-sterile plants further increases their frequency
probably due to inadequate timing of pollen transfer. In order to test

RESEARCHES CARRIED OUT IN 196469

the germination ability of the germless grains, they were seeded onmoist filter paper and their germination was compared to that of normal
grains. In this test all normal grains germinated but none of the germless grains, as shown in Fig. 1.

Fig. 1. Germination test of normal and germless grains obtained from malesterile T. v. e.
Left, normal; right, germless.

51.

An effective restorer to male-sterile ovata cytoplasm
Hitoshi KIHARA and Koichiro TSUNEWAKI

Since the work of FUKASAWA (1953) the cytoplasm of Aegilops ovata
is known to cause male sterility in wheat. In order to find restorers forthis cytoplasm, WILSON and Ross (1961) crossed 112 varieties or strainsof common wheat to FUKASAWA'S male-sterile Norin 26 and tested thefertility of the Fl hybrids. They could not find any restorer that would
recover high fertility in heterozygotes.
The authors crossed FUKASAWA'S male-sterile Norin 26 as the female
parent to three known restorers of caudata cytoplasm, i.e., P 168, compactum 44 and ABD-13. Restoring genes of the last two did not function
effectively in ovata cytoplasm. In contrast, Fl hybrids with P 168
restored fertility almost completely, i.e., their selfed seed fertility, on
the average, was 91.3 %, which is well comparable to that of normal
Norin 26 (97.5 %) or P 168 (85.2 %). Spikes of the F 1 and its parents are

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS No. 15

Fig. 1. Spikes of male-fertile F 1 from the cross, male-sterile Norin 26 x P 168.
From left to right: normal Norin 26, male-sterile Norin 26, (male-sterile
Norin 26 x P 168) F 1 and P 168.

RESEARCHES CARRIED OUT IN 196471

shown in Fig. 1. This restorer in combination with ovata cytoplasm
seems to be promising as a new material for hybrid wheat breeding.

52. Gene transfer between di-, tetra- andhexaploid wheats, I.
Transfer of v gene from common
wheat to emmer wheat
Koichiro TSUNEW AKI

A virescent gene of common wheat that does not affect germination
in homozygous condition was found by NEATBY (1933). It was later
designated by v and located on chromosome 3B by SEARS (1954). At atemperature of 13 - 16°C, seedlings of common wheat homozygous for v
are devoid of chlorophyll in the first and second leaves, and die at thethird or fourth leaf stage. However, if they are grown at 21 _24°C,
the extreme tip of the first leaf shows a trace of chlorophyll. All later
leaves are pale at first but become gradually green.
Since v is located on chromosome 3B, transfer of this gene to tetraploidemmer wheat should be possible. For this purpose, heterozygous plants(Vv) of common wheat were crossed to an emmer wheat, Triticum carthlicum
var. stramineum. The pentaploid F 1 hybrids were backcrossed to theemmer parent. In the B1 generation plants having 14 bivalents wereselected and again backcrossed to the same emmer. At the same time,
the B 1 plants were self-pollinated and their offspring were investigated
Table l. Germination and segregation of virescent plants in the selfed
progeny of B1> T. aestivum (Vv) X T. carthlicum (VV)2.
---------------

B1 lines

Genotypeof B 1

No. seedssown

No. seeds
germinated

Per cent
germinationNo.
normalsNo.
virescents

1145-31149-11149-3

Vv

20
20
20
60
20
20
20
20
80

14
14
14
42
19
18
19
17

70
70
70
70
95
90
95
85
91

13*
14*
13*
40
19
18
19
17
73

1
0
1
2
0
0
0
0
0

q
q

Total

1147-1
1147-21149-21151-3

VV

Total

q
q
q

73

* Some showed a tendency to virescence but distinction between Vvand VV
was difficult.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

for the segregation of virescent plants. The result is summarized inTable 1.
Only two virescent seedlings were obtained among 42 offspring of the
Vv plants. The frequency (less than 5 %) was much lower than the
expected 25 %. In addition, germination rate of the selfed progeny (Blline) of Vv plants was only 70 %, whereas that of VV was higher than
90 %. These facts clearly indicate that vv homozygotes of emmer wheat
are mostly seed-lethal. Since the v gene, in common wheat, causes
virescence without affecting germination, it can be said that the gene
upsets the physiology of embryos more drastically on the tetraploid thanon the hexaploid level.
53.

Geographical distribution of necrosis genes incommon wheat, 1.
Genetic basis of necrosis
Koichiro TSUNEWAKI

Based on F 2 and Bl data of a cross, Chinese SpringxMacha sub.,necrosis (NISHIKAWA'S type 1) has been assumed by TSUNEWAKI and KIHARA(1961) to be controlled by three complementary genes, Nel, Ne2 and Nea.
Furthermore, two distinct types of expression have been noticed, whichwere thought to be due to different alleles of Nel, namely, Nel 8Y forsystemic type and Nele p for epidemic type (NISHIKAWA, 1963).
However, recent results of the author obtained by test crosses of F 2
and Fa of the same hybrid, Chinese SpringxMacha sub., indicated thatthe two types of necrosis are under control of two independent complementary gene systems. For simplicity the systemic type of necrosis
will be here called" chlorosis ", because of prominent chlorosis of leaves
prior to necrosis, and the term "necrosis" will be retained for the
epidemic type.
Chlorosis is caused by two complementary genes, Chi located in A or
B genome (chromosome is yet undetermined) and Ch 2 (former Nea) on
chromosome 3D, while necrosis is due to Nelon chromosome 5B and Ne2
on chromosome 2A. The results of a detailed gene analysis revealed
the following genotypes in haploid phase for the three testers previously
offered for necrosis:
Jones Fife
ne,Ne2chlCh 2
PreludeNelne2ch£h 2Macha sub.
Nelne2Chlch 2
For the two complementary gene systems, nine genotypes are expected as Table 1 shows. The phenotypes of F I hybrids between threeRESEARCHES CARRIED OUT IN 1964

73

Table 1. Genotype of three testers and phenotypes of F l hybrids
between them and unknown varieties with each of ninepossible genotypes.
------~---

Nine possible
genotypes
1.

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

Nelne 2Ch lch 2
Nelne2ch lCh2
Nelne2ch,ch 2
ne l Ne 2Ch lch 2
ne l Ne2ch l Ch 2
ne l Ne2ch lch 2
nelne 2Ch lch 2
nelne2ch l Ch2
nelne2chlch2------_.-----_._---

Ii-

Testers
Jones Fife
(ne l Ne2ch l Ch 2)

Prelude(Nelne2ch l Ch 2)

Macha sub.
(Nelne 2Ch lch 2)

chlorotic
necrotic
necrotic
chlorotic
normalnormalchlorotic
normalnormalchlorotic
normalnormalchlorotic
necrotic
necrotic
chlorotic
normalnormalnormalchlorotic
normalnecrotic
chlorotic
necrotic
normalchlorotic
normal- - - -_

. . .-----

testers and an unknown variety with each of the nine genotypes arealso shown in Table 1. It must be noted that chlorosis is epistatic overnecrosis.
From this table, it is evident that genotypes 1 and 7 can not be
distinguished by the use of the present tester series, while all the otherseven are distinguishable from each other.

54. Geographical distribution of necrosis genes incommon wheat, II.
Distribution in Japanese local varieties
Koichiro TSUNEWAKI and Yasuo NAKAI

There are about 250 varieties in Japan which can be called local. Of
those 194 varieties were crossed to the testers for necrosis and their
genotypes were determined. The result is summarized in Table 1.
About one-third of Japanese varieties carry NCl gene, while NC2 isfound in less than one-tenth of them. The frequency of the former is muchhigher in South Kanto-Sansei-Shin-etsu district than in all other districts.
A great majority of NC2-carriers are found in northern Japan, mostly in
Hokkaido. This fact seems to suggest that they have been originatedfrom American or European varieties.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table 1.

Frequencies of various genotypes and alleles for necrosis
in Japanese local wheat varieties.
District

Chugoku Kinki,
S. Kanto, N. Kanto,
Kyushu Shikoku' Tokai,
Sansei,Tohoku, Unknown
Hokuriku Shin-etsu Hokkaido
Freq. of each genotype (No. of varieties)
Nelne2ch 1Ch 2
13
5
6
25
8
ne 1 Ne2ch 1Ch 2
0
3
0
1
12
nelne2ch 1Ch 2
35
15
18
18
25
nelne2ch 1ch 2
0
0
0
0
1
Total
48
23
24
44
46
Freq. of each dominant allele (Relative freq. in per cent)
Nel
27
22
25
57
17
N e2
0
13
0
2
26
Ch 1
0
0
0
0
0
Ch 2
100
100
100
100
98

Total
I

3
1
5
0
9

60
17
116
1
194

33
11
0
100

31
9
0
99

55. Geographical distribution of necrosis genes incommon wheat, III.
Distribution in Pakistan, Afghanistan and Iran
Koichiro TSUNEWAKI and Yasuo NAKAI

More than 240 strains of common wheat were collected in Pakistan,
Afghanistan and Iran by two members (Dr. H. KIHARA and Dr. K. YAMASRITA) of the Kyoto University Scientific Expedition (KUSE) to the
Karakoram and Hindukush, 1955. Of those 36 strains from Pakistan, 40
from Afghanistan and 143 from Iran, making 219 in total, were crossed
to the testers of necrosis genes and their genotypes were determined.
In some strains one of the three test crosses was unsuccessful and,
consequently, the genotype was only partially determined. The result
is summarized in Table 1.
Nel gene was very common in northern Iran, i.e., from Gorgan to
Ardabil and Tabriz, while its occurrence was very rare in all other
districts including Pakistan and Afghanistan. None of the Ne2-carriers
could be found. Chl was also rare. Ch2 occurred rather commonly.
From this result it can be safely concluded that the wheat populationof northern Iran is distinctly different from the other populations
regarding the very high frequency of Nel gene.

RESEARCHES CARRIED OUT IN 1964

75

Table 1. Frequencies of various genotypes and alleles fornecrosis in KUSE wheat.
Country and district
Iran
Mgh'"-I Subzuwar Isfahan Tehran
Gorgan ~~~~~' Total

Paki·stan I Istan
I

Freq. of each genotype (No. of varieties)
1
1
1
2
14
3
NelnezchlChz
0
0
1
0
0
Nel? chlChz
0
0
0
0
0
0
Nelnezch l ?
0000
nelNezchlCh z
0
0
0
13
14
16
15
0
nemezch l Ch z
9
2
2
1
0
nel ? chlCh z
0
0
0
0
1
0
0
0
? nezchlChz
0
1
0
0
0
0
? neZCh1ch z
19
8
4
26
3
7
nelnezch1chz
3
2
1
0
1
2
nelnezch l ?
36
19
Total
40
21
29
28
Freq. of each dominant allele (Relative freq. in per cent)
3
74
11
7
5
5
Nel
0
0
0
0
0
0
Nez
3
0
0
0
0
0
Chi
42
35
70
69
78
85
Ch z

35
1
1
0
0
1
0
0
8
0
46

55
1
1
0
45
4
0
1
30
6
143

80
0
0
82

40
0
1
77

56. Taxonomical and ecological studies on Agropyron
humidum OHWI et SAKAMOTO
Jisaburo OHWI and Sadao SAKAMOTO
Extensive studies of Agropyron species in Japan were first undertaken
by Dr. M. HONDA. In 1927 he described a new species, Ag. mayebaranum
HONDA, based on the herbarium specimens collected in Kyushu by Mr.
K. MAYEBARA.
Recently the present junior author reexamined the morphology andpollen fertility of the specimens collected by Mr. MAYEBARA. He foundthat the specimens collected by him are natural interspecific hybrids
between Ag. tsukushiense var. transiens OHWI and another hitherto
unknown species to which a new name was given; namely Ag. humidum
OHWI et SAKAMOTO. The hybrids have intermediate characters between
parental species.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Adaptation to moist environment is one of the most pronounced
characteristics of Ag. humidum. Two distinct characters of this new
species imply high adaptiveness to the habitat of fallowing paddy fields.
One is the formation of an abscission layer at maturity in the node
below the flag leaf. Thus, from this node on upward the spike is easily
separated from the rest of the plant by wind or other physical forces.
The other is the perennialization of the culm with the exception of theuppermost internode and spike. New shoots and roots are produced very
easily from the nodes when the conditions are favorable.
The two characteristics mentioned above have not been found in any
species belonging to tribe Triticeae which are mostly adapted to rather
dry habitats. With these two adaptive traits seeds and clones of thisspecies are dispersed uniformly in the paddy field during the preparation
for rice planting in June. This seems to be the reason why it occurs
in swarms and uniform populations in fallowing paddy fields as observedin the suburbs of Misima.

57. Genome analysis of Japanese and Nepalese Agropyron
Sadao SAKAMOTO
Morphological, physiological and cytogenetical studies were carriedout in 14 different interspecific Agropyron hybrid combinations among
five Japanese species, i.e., three tetraploids, Ag. ciliare (TRIN.) FRANCH.,
Ag. gmelini (LEDEB.) SCRIB. et SMITH var. tenuisetum OHWI and Ag. yezoense
HONDA, and two hexaploids, Ag. humidum OHWI et SAKAMOTO and Ag.
tsukushiense (HONDA) OHWI, two Nepalese tetraploids, i.e., Ag. semicostatum
NEEs and Ag. gmelini, and one American tetraploid, Ag. trachycaulum(LINK) MALTE.
The main characteristics of the hybrids were as follows: (1) Growthof hybrids was vigorous, and tillering, heading and flowering werenormal. (2) The general appearance of many characters was intermediate
between the parents or superior to both or similar to that of one of theparents, particularly in so far as quantitative characters are concerned.
(3) Complete pollen sterility and high seed sterility were the rule in all
hybrid combinations. The anthers were non-dehiscent at the flowering
time and the pollen grains were completely abortive.
Chromosome pairing in hybrids is summarized in Table 1. From theseresults, the following conclusions are drawn: (1) The Japanese species
are cytologically closely related with each other; two homologous genomes
are shared by three tetraploid and two hexaploid species with an added
third genome to the latter. (2) The genomes of Japanese and NepaleseTable 1. Chromosome pairing in interspecific hybrids of Japanese, Nepalese and American Agropyron.
Cross combination (~ X (')

No. ofcells
examined

No. of bivalents
Range

45

10~14

14

408
69

5~14

1O~14

12
12

325

2~

9

107
87
50
53

9~15

I

Average chromosome pairing
IV

III

II

I

0.36

0.20

11.29

2.29

:::0

0.14
0.04

0.15
0.04

11.34
12.26

4.20
3.17

:::0

5

0.02

0.07

5.33

17.10

14
14
13
14

0.06
0.15
0.18
0.09

0.08
0.33
0.16
0.02

13.06
12.24
12.28
13.17

8.44
8.81
8.84
6.45

11
10
12
11

0.11

0.16
0.02
0.11
0.08

9.80
10.66
11.30
11.92

14.05
13.62
11.57
10.76

~?02

20.74

Mode

V

Japanese 4xx4x:

Ag. ciliarexAg. yezoense
Japanese 4xxNepalese 4x:

Ag. ciliarexAg. semicostatum
Ag. semicostatum X Ag. yezoense

0.002

Japanese 4xxAmerican 4x:

Ag. ciliare X Ag. trachycaulum
Japanese 4xx6x:

Ag. tsukushiense X Ag. ciliare
Ag. tsukushiense X Ag. yezoense
Ag. gmelini (Japan) xAg. tsukushiense
Ag. humidumxAg. ciliare

7~15
11~15

12~16

Nepalese 4xxJapanese 6x:

Ag. tsukushiense X Ag. semicostatum
Ag. humidumxAg. semicostatum
Ag. gmelini (Nepal)xAg. tsukushiense
Ag. gmelini (Nepal) xAg. humidum
Japanese 6x X 6x:

Ag. humidumxAg. tsukushiense-~._-- - - - -

155
47
97
25

6~14
8~14
3~15
9~15

0.11
0.04

i

_~_L20~=-_ _21_ _1____.. _ _

~

>

(')

::t:

Bl
&:
:::0
:::0

til
t:I
o

c::

t-l

Z
....
~

0.46
.~-----

-1
-1

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Ag. gmelini are truly homologous except for a slight but definite reduction in pairing; this result was obtained indirectly through crosses of
both, Japanese and Nepalese Ag. gmelini, with Ag. tsukushiense. (3) The
genomes of the Nepalese species, Ag. semicostatum, are basically homologous to those found in Japanese tetraploid species, but some degreeof genomic differentiation which reduces the number of bivalents andincreases that of univalents in Japanese 4x x Nepalese 4x and Nepalese
4x x Japanese 6x hybrids has taken place. (4) The genomes of a tetraploid American species, Ag. trachycaulum, are partially homologous tothose found in a Japanese tetraploid species, Ag. ciliare.
58. Natural interspecific hybrids in Japanese Agropyron
Sadao SAKAMOTO
Two cases of natural interspecific hybridization in Japanese Agropyron
were studied in Misima. One is the pentaploid hybrid between Ag.
ciliare (TRIN.) FRANCH. (2n = 28) and Ag. tsukushiense (HONDA) OHWI
(2n = 42), and the other is the hexaploid hybrid between Ag. humidum
OHWI et SAKAMOTO (2n = 42) and Ag. tsukushiense. Clones of the former,
Ag. ciliare x Ag. tsukushiense, were found mainly in the hills and in theriver dikes in the suburbs of Misima.
No seed setting was observed in this hybrid due to extremely highsterility of both male and female organs. It is assumed that in thishybrid combination introgressive hybridization would not occur in natural
conditions.
As to hexaploid natural hybrids, two cases were observed; i.e., one
allopatric and the other sympatric. In the allopatric case Ag. humidum
and the common type of Ag. tsukushiense were involved, while, in thesympatric case Ag. humidum and the early ecotype of Ag. tsukushienseparticipated (SAKAMOTO, 1961). In both cases low seed setting wasobserved from examination of a considerable number of clones growing
on foot-paths of paddy fields of the former hybrid and in fallowing paddy
fields of the latter. From progeny tests of backcrossed plants obtained
from the sympatric natural hybrids between Ag. humidum and the early
ecotype of Ag. tsukushiense, the following conclusions are drawn: It isquite possible that introgression of characters from one species to theother, such as waxiness, might have occurred in the natural populations
through backcrossing to the parental species of natural F 1 hybrids followed
by segregation of the characters concerned. Rather quick restorationof fertility in the hybrid progenies makes the establishment of hybrid
swarms in a state of nature possible, having intermingled characteristics

RESEARCHES CARRIED OUT IN 1964

79

of the parental species. However, so far no introgressants have beenfound yet in the natural sympatric populations of these two species.
High sterility of FI bybrids might be a major cause of the restrictionof introgression.

59. Morphological and cytological studies of two intergenerichybrids of Elymus sibiricus LINN. and El. dahuricus TURCZ.
with Agropyron tsukushiense var. transiens OHWI
Sadao SAKAMOTO
Among the genera of the tribe Triticeae, the phylogenetic relationships
between Agropyron and Elymus are very complicated, because bothgenera are quite large comprising various and complex polyploid species
which distribute widely in the temperate regions of the world where
many natural hybrids between them have been found.
Exploring cytogenetical relationships of these two genera, two speciesof Asiatic Elymus, El. sibiricus (2n = 28) and El. dahuricus (2n = 42)
were crossed with a Japanese Agropyron, Ag. tsukushiense (2n = 42).
Pentaploid as well as hexaploid intergeneric F I offspring were morevigorous than the parental species. Shape of the spikes of F I was of
Elymus type. One of characteristics distinguishing Elymus from Agropyronis the production of two spikelets at most nodes of the rachis. This
character was in FI expressed only in 1/3--1/4 of all nodes of a rachis.
All pollen grains of both hybrids were completely abortive.
Average chromosome pairing at MI of PMCs in Fl was O.Olxv (0--1)+0.25m (0--1)+8.93u (4--13)+ 16.381 (9--27) in El. sibiricusx Ag. tsukushienseand 0.04v(0--1) + 0.281v (0--2)+0.32m (0--2) + 17.67n (13--21) + 4.4lx (0-:16)
in El. dahuricusx Ag. tsukushiense. (The parentheses indicate the rangeof respective pairing.) Ag. tsukushiense is an allohexaploid whose genome
constitution comprises three different genomes (SAKAMOTO, 1964). Fromthe present results, it is assumed that El. sibiricus and El. dahuricus
used in this experiment contain at least one genome in the former andtwo in the latter that are homologous to those of Ag. tsukushiense, andthat the third genome of El. dahuricus might be partially homologous
to the remaining genome of Ag. tsukushiense

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

60. Further observations on the flowering of Oryza species!)
Tadao C. KATAYAMA

Investigated were date of flowering, order of flowering among the
spikelets of a panicle, number of spikelets which open on successive
days and time of the day when they open. All characters areimportant in practical breeding as well as in ecological and phylogenetic
studies of rice. Some data have already been reported in 1960 and
1962. This year, further observations were carried out using four
wild species, O. australiensis, O. brachyantha, O. tisseranti and O. longiglumis, which were newly added to our collection.
Several strains of these species were grown in the greenhouse.
Observations were made from September 15 to November 16. Time offlowering was determined by the time of opening of the lemma andpalea of the first spikelet in a panicle. The duration of flowering was
indicated by the interval between opening and closing of a given spikelet.
Results of observations concerning the time of the day were as
follows: O. australiensis flowered in the evening. Its behavior wasvery different from that of cultivated species and their close relatives,
the wild Perennis group. On the other hand, the behavior of O.
brachyantha and O. tisseranti was quite similar; they started to flower
from 10 to 11 a.m. The spikelets of O. longiglumis opened very earlyin the morning as observed also in O. ridleyi. The results obtained infour years indicated that time and duration of flowering of taxonomicallyrelated species are similar. Generally the time was in any species
gradually delayed from September to November.
The opening of the spikelets in a panicle takes place in all species ina regular sequence; they start to open on the uppermost branch of apanicle continuing to the lower branches in succession. However, somedifferences in the order of opening spikelets in a branch are foundamong the species. In O. australiensis and O. longiglumis, flowering
occurs first in the uppermost spikelet of a branch, followed by the lowest
one and then proceeds again toward the upper spikelets in successiveorder. Consequently, the opening of the second spikelet from the top
is the latest in a branch. In other two species, O. brachyantha and O.
tisseranti, flowering starts at the top of a branch, proceeding straight
downward.
In O. australiensis, O. brachyantha and O. tisseranti, flowering iscontinuous. In O. longiglumis and O. ridleyi, a several days interval
was observed between the opening of the uppermost spikelet (always
11

This work was supported by the Grant RF 62027 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1964

81

male-sterile) and that of the second one in a branch. However, thisbehavior was not detected in the remaining three species, O. australiensis, O. brachyantha and O. tisseranti.

61. Diallel crosses in Sikkimese rice l )Tadao C. KATAYAMAKIHARA et al. (1960 and 1964) reported the occurrence of indica andjaponica types in Sikkimese rice based on the examination of morpho-

logical characters, phenol reaction, pollen and seed fertility and hybridization. Most important for the classification of cultivated rice into
indica and japonica types is the hybrid sterility between the two types.
Therefore, in order to reconfirm our classification of Sikkimese rice andto clarify the relationships between its strains, diallel crosses werecarried out using sixteen strains, namely, fourteen from Sikkimese rice,
one indica type strain and one of japonica type.
Most of crosses were successful and 1,583 hybrid seeds wereobtained. These seeds will be sown next year for the analysis ofpollen and seed fertility. The observed crossability fairly well indicateda considerable degree of intervarietal differentiation among Sikkimesestrains.

62. Preliminary studies on tiltering in the genus Oryza
Tadao C. KATAYAMA

Investigated were date of initiation and shooting of tillers, order oftillering, developmental state of tillers, whether vegetative or reproductive, and order of shooting of tillers after the main stalk had changed
to reproductive state. The study was undertaken in order to provide
information on the factors governing the type of growth habit, whether
annual or perennial, a character of great importance in rice breeding
and cultivation. This year 15 strains covering 8 species were used andthe plants were grown in wooden boxes. Observations of tillering weremade several times between June 15 and December 7.
In O. glaberrima, O. breviligulata and O. stapfii, tillering was detected
only from the first tiller of the main stalk. In the remaining fivespecies, O. sativa, O. perennis, O. barthii, O. officinalis and O. subulata,
however, tillering was observed in the first, second and higher tillersof the main stalk. The primordia of the tillers were generally initiatedI)

This work was supported by the Grant RF 62027 from the Rockefeller Foundation.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

in regular sequence during the life history of a plant.
As soon as the main stalk after forming the panicle was cut off,
primordia at the second or third node counted from the top developed
at once but none at the first node. Later, the lower primordia developed
gradually. An exceptional case was observed in O. subulata; in thisspecies only the first and second primordia from the top developed into
shoots, but not the lower ones.
Most of the tiller primordia were fixed after the main stalk hadchanged to reproductive state. Fixed materials were studied anatomicallyunder dissecting microscope. In perennial species, such as O. sativa,
O. perennis or O. officinalis, many primordia in relatively lower nodes
were found to remain in vegetative state. Such primordia could play
an important role in maintaining the plants as perennials. On thecontrary, in annual species, such as O. glaberrima or O. breviligulata,
all primordia observed were reproductive and no vegetative primordia
were found. It is concluded that the anatomical difference between
annual and perennial species in Oryza depends on the developmental
state of tiller primordia, whether a change to reproduction occurs or
they remain vegetative, when the primordium of the main stalkchanged from the vegetative into the reproductive state.

63. Preliminary studies on embryo transplantationin the genus Oryza
Tadao C. KATAYAMA

Embryo transplantation between several rice species was attempted.
This year, the technical possibility of embryo transplantation was
explored using 10 strains of five species of Oryza, O. sativa, O. sativa
var. spontanea, O. perennis, O. glaberrima and O. breviligulata. The
dormant seeds were sterilized for 30 minutes in 10 % chlorinated lime
solution and washed twice in sterilized water. The seeds were divided
by a sharp knife into embryo and endosperm. Four kinds of experimental
materials were used, namely, intact seeds, embryos alone, embryos
transplanted to their own endosperm (homogeneous transplantation) andembryos transplanted to alien endosperm (heterogeneous transplantation)
I of other strains or species. The materials were placed on aseptic agar
and kept under continuous dark condition at 25°C. Observations weremade from 30 to 60 days after incubation.
In general, the transplantation was successful not only in intraspecific,
but also in some interspecific combinations. Furthermore, fusion between
tissue of embryo and that of endosperm was ascertained in some intra-

RESEARCHES CARRIED OUT IN 196483

specific combinations. The growth of seedlings obtained from homogeneous transplantations was more vigorous than from heterogeneous
ones.

64.

Photoperiodic responses of Oryza species, VIP>
Tadao C. KAT AYAMA

Several factors are responsible for different photoperiodic sensitivities
among various Oryza strains. The acceleration degree, which was
expressed by differential heading acceleration under short day condition,
was dealt with in a previous paper. This year, the acceleration degree
is reported for several low latitude strains of O. sativa, O. sativa var.
spontanea, O. perennis and O. officinalis, which were recently collectedin Borneo and Java. Several cultivated and wild strains from India andJapan were also used for comparison. The acceleration degree of heading
date was expressed by three different methods as previously reported(KATAYAMA, 1963).
Heading date of the strains depended strongly upon day length.
According to the data reported in 1963, a high correlation was foundbetween geographical distribution and sensitivity, namely, strains
distributed in low latitudes showed in general greater sensitivity thanthose of high latitudes. The data obtained confirmed the validity ofthis correlation. This result answers the question, why photoperiodicallysensitive strains predominate even in low latitudes. It is assumed thatthe response of these strains to a small change of day length gives thema selective advantage in natural condition.
One of the factors influencing photoperiodic sensitivity is the so-called
accumulation effect. The accumulation effect is shown by the photoperiodic effectiveness of short day treatment interrupted by long day
condition. Accumulation effect of 20 strains of O. sativa, O. sativa var.
spontanea, O. perennis and O. glaberrima were analyzed this year. A
combination of 12h 30 m light + IP30 m dark was selected as short day condition, whereas natural day length from June to August was used aslong day treatment.
The effectiveness was measured by the degree of flowering induction
in relation to the frequency of short day plus long day treatment.
Generally, 10 day interruption of long day condition in short day treatment cancelled out thoroughly the effect of short day treatment. It was
clear that the shorter was the long day condition inserted between two
short day treatments, the more prominent was the accumulation effect.
1lThis work was supported by the Grant RF 62027 from the Rockefeller Foundation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Also, it was found that the lower was the latitude from where the strains
were originated, the smaller was the accumulation effect.

65. Genetic studies on developmental instability in Oryza sativa
Kan-Ichi SAKAI

Investigations have been carried out cooperatively by the staff membersof the writer's laboratory. They are still under way, but a brief accountis given.
By comparing control and X-rayed progeny lines of 6th generation
after irradiation, the genetic effect of X-rays was investigated on
developmental instability in leaves. It was found that the flag leaf andthe third leaf counted from it downward showed an increase in genetic
variation for intraplant variability. The mutation was observed to occur
toward higher instability. The next leaf to the flag, however, showed
at least no increase in genetic variation by X-rays. The mutation inlength of those three leaves was found to behave in a similar way.
In parallel with the above experiment, inheritance of intraplot variability or developmental instability has been investigated on plant height,
plant weight, muber of culms per plant, seed yield and panicle length.
It was found that correlation between two consecutive generations was
statistically significant and as high as 0.57 to 0.34 for the charactersgiven above except for the culm number per plant. Further study isrequired in order to draw a definite conclusion.

66. Survey of F, sterility relationships between strains of
Oryza perennis
Hiroko MORISHIMA and Hiko-Ichi OKA

We have been engaged in this work since 1958. This year, several
strains from New Guinea were observed in addition to other materials.
Some of them are of spontanea type (small annual plants), while the others
are of perennis type. In their natural habitats, they are completely
isolated from cultivated rice. This may prove that the spontanea type
is not necessarily a hybrid derivative from O. perennis and O. sativa.
The New Guinean spontanea strains showed a high F, sterility whencrossed with strains of Asian, American and African origins, but theperennis strains generally produced fertile F, hybrids. The survey is
still under way.

RESEARCHES CARRIED OUT IN 1964

67.

85

Variation in characters related to seed dispersion in
Oryza perennis
Hiko-Ichi OKA and Hiroko MORISHIMA

In wild grasses, the awn might be the organ responsible for seeddispersion. Its length, diameter (at the 1 em point from the apiculus)
and the length of bristles on its surface (at the same place as above)were measured in a number of strains of Oryza perennis. The threemeasurements, each varying within a range among strains, were foundto be inter-correlated. Their standardized values were then summed up
to obtain an index of seed dispersability. Among the strains used, theindex was positively correlated with grain shedding and seed dormancy,and negatively with the regenerating ability of stem cuttings.

68.

Deterioration of F 1 embryos in hybrids between subsp.
barthii and other forms of Oryza perennis
Yaw-En CHU and Hiko-Ichi OKA

Oryza perennis subsp. barthii is a wild form distributed in Africa.
When cross-pollination was made between it and other forms of O.
perennis in either direction, a part of the F 1 embryos were found to
deteriorate five to six days after fertilization. The frequency of deterIorated embryos differs according to cross-combination. It was found.:hat when deterioration occurs, the differentiation of embryonic cells'nto plumula and seminal root primordia could be blocked. Thisphenomenon may be regarded as a type of hybrid inviability taking"Jlace in young embryos.
69.

An observation of cultivation pressure on seed germination
Hiko-Ichi OKA

When a population of wild plants is cultivated, genes expressing
characters of cultivated forms will have an advantage under the pressureof selection due to cultivation, which we call "cultivation pressure".
Five hybrid populations between Oryza perennis and O. sativa were eachdivided into two plots; in one of them, seeds were harvested at maturity
and were sown next spring on puddled soil, while in the other plot
seeds were not harvested and the naturally shed seeds germinated after
the remaining plants of the previous generation were removed. Froma comparison between them, it was found that plants in the seededplots tended to resemble cultivated rice, while those of the naturally

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

germinating plots had many traits of wild rice, e.g., spreading paniclebranches, a high percentage of grain shedding, etc. This indicates thatwhen seeds are sown, certain cultivated types are selected, whilecharacters discriminating between wild and cultivated types are geneticallyinter-correlated. This experiment is continued in Chung-Hsing University,
Taichung, Taiwan.

70. Pachytene analysis and an observation of chromosome
associaion in haploid rice
Yaw·En CHU and YO TAKENAKA

The pachytene chromosomes of haploid plants of cultivated rice (0.
sativa) could be divided into 3 types according to their length and arm
ratio. When they were numbered in the order of length, chromosomes
3,4 and 7 were of median type, and chromosome 11 of sub-terminal type,while the rest were of sub-median type. In most cells, cromosomes 10
and 12 were attached to the nucleolus, the former with a higher frequency
than the latter. Chromosome 1 was 2.8 times as long as chromosome 12.
At diakinesis and metaphase I, 30 different association types were found,their number ranging from 0 to 8. The average number of associations
per cell was 3.23, 2.90 and 2.48 at pachytene, diakinesis and metaphase I,
respectively. The maximum configuration of associating haploid chromosomes has been regarded to be 2(3) + 3(2), namely association of 7, by
many researchers. But such configurations as including 3(3) and 1(4)were found, with the frequency of 36/680. It seems that 2(3) + 3(2) is
not maximum.
If the association of chromosomes in a haploid cell is due to a specificaffinity between partially homologous segments, it should occur between
definite chromosomes. Distinction of individual pachytene chromosomes
was attempted on the basis of their length and arm ratio, and theposition of association in each chromosome was recorded. Distinguishing
between chromosomes 5 and 6, and 9 and 10 was difficult, but chromosome
1 could be easily identified. It showed association with 3 different
chromosomes at different positions. This suggests that chromosome 1
can simultaneously associate with three others, forming a group of four.
In general, the distal part of chromosomes had more associations thanthe proximal part. Some association configurations appeared as if achiasma was formed. For estimating the residual homology between
chromosomes, it might be more useful to study their distribution frequency
in the associations than determining the maximum association which
represents the extreme case of variation.

RESEARCHES CARRIED OUT IN 1964

87

We may take it for granted that the association in haploid cells indicatesthe presence of homologous segments in chromosomes, but the pattern ofassociation actually observed might be influenced somewhat by the otherconditions.

71.

The genetic analysis of develoPmental instability inleaves of Nicotiana tabacum by diallel crosses
Yoshiya SHIMAMOTO and Kan-Ichi SAKAI

Developmental instability in leaves of N. tabacum was investigated by
the method of diallel crosses. Six varieties, Ambalema, T. I. 448A,
Bright Yellow, Daruma, Sumatra and Connecticut Broad Leaf and theirF , hybrids were grown in randomized blocks with two replications. Eightplants were selected at random from each strain and bilateral asymmetryof leaf blade and vein distance variability were measured in five leavesof eace plant.
Bilateral asymmetry was measured by the absolute difference between
left and right sides of blade, divided by the total width. The vein distance variability was measured by intraleaf standard deviation of veindistance from the mid-rib divided by average vein distance. The figures
thus obtained are understood to be the indices of developmental instability of tobacco leaves.
Experimental results generally showed no sign of heterosis in bothinstabilities, whereas the degree of dominance of genes governingbilateral asymmetry and vein distance variability was 0.6278 and 0.6347,
respectively. The dominance was toward instability for bilateral asymmetry, but toward stability for vein distance variability.
Analysis of variance proved that additive gene effect, dominance effectin specific combinations, maternal effect and reciprocal differences in
specific crosses were statistically significant.

72.

Tumor formation in interspecific hybrids of Nicotiana,
especially in relation to injury
Y6 TAKENAKA and Yoshiaki YONEDA

Among many specific hybrids in Nicotiana which were made forcytotaxonomical researches, we observed 11 tumorous ones, namely, F ,N. glauca X N. langsdorffii, F,N. glauca X N. longiflora, F,N. glauca X N.
plumbaginifolia, F , N. paniculataxN. langsdorffii, F, N. rusticaxN.
cavanillesii, F, N. suaveolensxN. langsdorffii, F, N. suaveolensxN. plumbaginifolia, F,N. suaveolens X N. longiflora, F,N. gossei X N. alata, F,N.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

gosseixN. longifiora and Fl 4x N. tabacumxN. alata. Among them, 3hybrids are newly found by us, namely, F 1 N. gosseixN. alata, F 1 N.
gossei X N. longifiora and F 1 N. suaveolens X N. plumbaginifolia.
The tumor shapes ranged from teratoma-type to massive-type. In F 1
N. glauca X N. langsdorffii whose tumor formation is most striking,spontaneous stem tumors begin to grow at the end of flowering. Theirlocation was: a) cicatriculae of flowers and leaves, b) apexes of lateralshoots, c) internodes and d) various parts of roots.
Injury of leaf veins and stems of F 1 N. glauca X N. langsdorffii wasobserved to stimulate tumor formation. Pricking experiments showedthat tumors were more frequently induced in the upper part of the stem
than in the lower one.
73.

Hereditary tumor in Japanese morning glory
Yo TAKENAKA and Yoshiaki YONEDAFor fourteen years we have cultured many strains and hybrids ofJapanese morning glory (Pharbitis Nil). Among the offspring of strain
056 which is assumed to have the dominant gene Blown (B) and recessive
genes delicate (dl) and feathered (fe) and also those of strain 058 assumed
to have the genes B and fe, neither producing seeds in homozygous
condition, we found many individuals showing tumor-like shoot development at cotyledonary stage. As the result of our observation of theexternal morphology of cotyledons and normal parts of those individuals,
the co-existence of Band fe genes in homozygous condition is considered
to be connected with the occurrence of the tumor.
In in vivo tumor tissues, we found various irregular configurations ofvascular elements.
In vitro tumor tissues grew slowly on modified Whites' agar medium
but the addition of yeast extract stimulated the growth considerably.
Those cultured tissues showed frequently remarkable high polyploidy.

74.

Chromosomes of some species of genus Paspalum
Yasumichi TERADA and YO T AKENAKA

As the first step for finding good pasture species of genus Paspalum,
chromosome numbers and meiotic behavior of those grasses were ob·served. The results are summarized in Table 1, together with otherresearchers' results.
The somatic chromosome number of P. notatum was reported to be 20
or 40 by SAURA (1948). The authors counted 20 somatic chromosomes

RESEARCHES CARRIED OUT IN 1964
Table l.
Species

Somatic and meiotic chromosome number of genus Paspalum.
Strain or origin

Chromosome number Meiotic
2nbehaviorn

SAURA, 1948"

P ensaco Ia 33195
F.C

20

10

10 II

"

BN
Pensacola 1309~57

20

10

10 II

Tifhi·1
Argentina
Paraguay
Common

20

10
10 II
10***
10***
20
20 II

"
"
"

P. dilatatum

40
50

KRISHNASWAMY,1940
BROWN, 1948

40

"

"
"
"
"
"

Researcher

20,40

P. notatum"89

L·B 230 ~2~050

30

2011+101

L·B 230 Pox46777
CommonYellow anther A & B*
Local A & B**

50
50
50
50

30
30
30
30

20II + 101
20II+10I
20II + 101
20II + 101

P. dilatatum
var. paucicilatum
Prostrate

40
40

P. thunbergii

20"

MORIYA and
KONDO, 1950
ONO and
TATEOKA, 1953

40

"
"

Kumamoto"

Okinawa

P. malacophyllum
*
**
***
****

PORODl, 1946
not
1011+101+
defined 101****

40
40
40

BURTON, 1940
20

20 II

Found in the collection of Kyushu Agri. Ex. Sta.
Collected in Kumamoto, Kyushu, Japan.
20 chromosomes are rarely found at diakinesis and MI.
Not yet confirmed.

and 10 bivalents in 3 strains, and 40 somatic chromosomes and 20 bivalents in one strain.
The somatic chromosome number in P. dilatatum was observed to be
50 by KRISHNASWAMY (1940) but 40 by BROWN (1948). The authors counted
50 somatic and about 30 meiotic chromosomes at diakinesis and MI,
being generally the configuration of 20 bivalents and 10 univalents.
PORODI (1946) reported 40 somatic chromosomes in P. dilatatum var.
prucicilatum. The authors also found 40 somatic chromosomes, but they

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

could not define the meiotic chromosome number, although they foundrarely 10 bivalents and 20 univalents at MI of PMC.
In P. thunbergii, MORIYA and KONDO (1950) counted 20 somatic chromosomes but ONO and T AKEOKA (1953) counted 40. The authors also
found 40 somatic chromosomes in a strain collected in Kumamoto.
BURTON (1940) reported 40 somatic chromosomes in P. malacophyllum andthe authors found also 40 somatic chromosomes and 20 bivalents at MIof PMC in a strain brought from Okinawa.
At present genome analyses of the genus are underway.

75.

The origin of Prunus yedoensis, II
Y6 TAKENAKA

The external morphology of about 130 seedlings of P. yedoensis wasobserved. A few typical P. lannesiana var. speciosa and P. subhirtella
var. pendula form ascendens were found among them and many others
ranged from speciosa to ascendens with wide distribution of various
character combinations. Needless to say a few P. yedoensis-like seedlings
were also found.
Fourteen hybrids between speciosa and ascendens grew well. Although
they showed minor differences from each other, they belonged all to theyedoensis group from the morphological point of view. But they hadlarger leaves and flowers and more stamens than P. yedoensis and theflower color of all plants except one was pink fainter than that ofP. yedoensis.
Hybrids between P. lannesiana var. speciosa and P. subhirtella var.
pendula had more delicate branches and a little smaller leaves and flowers
than P. yedoensis. In general, P. yedoensis may be assumed to be a
hybrid between speciosa and ascendens rather than between speciosa andpendula.
Nineteen hybrids between speciosa and yedoensis were observed. In
the hybrids between speciosa and ascendens, hairiness of leaves, peduncles
and calyxes and umbel inflorescence were assumed to be dominant tohairless and corymb respectively. In all mentioned hybrids, these
dominancies were also recognized.
Funabara-yoshino, which was found on Funabara-pass in Izu, had somewhat larger flowers and leaves and a few more stamens than P. yedoensis,
but was very similar in the other characters to P. yedoensis and showed
heterosis like P. yedoensis. Therefore this flowering cherry tree is also
assumed to be a new natural hybrid between speciosa and ascendens.
Kurama-zakura, which has been cultivated in Kumamoto since long

RESEARCHES CARRIED OUT IN 1964

91

ago, has leaves and flowers somewhat larger than those of yedoensis andits branches are slightly bent. But the other characters and appearanceof heterosis are the same as in yedoensis. Accordingly this tree may be
another hybrid between speciosa and ascensens.
Midori-yoshino (P. yedoensis var. Nikaii HONDA) has been cultivated in
Hagi from Edo-period but is not encountered there now. By identification from specimens, all characters are identical with those of yedoensis,
except one character, namely white petal color. This plant is also assumed to be a hybrid between speciosa and ascendens and might haveoriginated in Izu and have spread as a garden plant from there.
I found some trees which could presumably be the offspring of yedoensis in Izu, Boso and Noto. Similar plants were found by some taxonomists. Some of them may be the offspring of yedoensis itself and theothers may be that of the other hybrids of the same origin.

76.

Method of estimating genetic parameters in a forest whereinter-tree competition is occurring
Kan-Ichi SAKAI and Hiromasa MUKAIDESAKAI and HATAKEYAMA have published in 1963 a paper dealing witha new method of estimating the genetic parameters in a standing
forest of Abies. The method was based upon the principle of random
distribution of genotypes in a forest, in contrast to the heterogeneous
pattern of distribution due to environment. In a further study on
Cryptomeria, however, the method was found not to be appropriate
because of inter-tree competition in a forest, on the one hand, and of
the necessity of estimating the genetic parameters in face of competitive
pressure, on the other.
Inter-tree competition is concluded to occur: (1) if correlation between
two adjoining trees is zero or negative, (2) if variance of difference between two adjoining trees is equal to or larger than the two-fold varianceof single trees, (3) if correlation between the difference between adjoining
i and j trees and the difference between adjoining j and k trees, is equal
to or smaller than -0.5 or (4) if variance of difference between (i-j) and(j-k) trees is equal to or larger than the six-fold variance of single trees.
When we detect the occurrence of inter-tree competition in a forest,
estimation of genetic parameters can be made in the following way:
Variance of average value of x trees in each cluster is expected to
involve genotypic, environmental and competitional variances as follows:
VCil=Q+ Eb + T.,C ,
x

x

x

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

where G, E and C stand for genotypic, environmental and competitionalvariances, respectively, while b is a constant lying between 0 and 1
which relates to soil heterogeneity, and T z is a coefficient of C which
varies in accord with x. If we take various sizes of clusters in a forestand compute variances, then we can solve the following set of simultaneous equations for B, G, E and C, which show the best fit between
observed and expected variances.
VI!)
G + E +C,
2V I21
G + 2B E + 0.600C,
3V(ii)
G + 3B E + 0.535C,
4V(4)G + 4 B E + 0.300C,
5V(r;)
G + 5 B E + 0.340C,
G + 6B E + 0.235C,
6V(61
7V(7)
G + TB E + 0.270C,
8 VIS)
G + 8 B E + 0.215C,
9V(91
G + 9 B E + 0.173C,
where B=I-b. Finding those G, E and C values allows us to compute
heritability. Genetic correlations between two traits are also computedby finding covariances in place of variances.
Investigation in actual forests of Cryptomeria has proved this method
to be useful. Heritability values for stem diameter and tree height of
Cryptomeria japonica estimated by this study are so low as 0.1 to 0.4,
while genetic correlation between them is 0.6 to 0.9. An important
finding in the course of this study was that trees in an isogenic forest(a clone) do not compete against each other, which deserves a further
study. Details will be published before long.

C.

MATHEMATICAL GENETICS

77. The number of alleles that can be maintained in a finite population
Motoo KIMURA

It is not known for certain whether the so-called wild type gene in anatural population is usually a single entity or a complex consisting of
numerous isoalleles that are indistinguishable by any ordinary means.
Since each gene is made of a large number of nucleotide pairs, the latter
possibility can not be excluded. In the present work the number ofalleles that may be maintained in a finite population was calculated
assuming three models: (1) A system of selective neutral isoalleles whose

RESEARCHES CARRIED OUT IN 196493

frequency in the population is determined by the mutation rate and byrandom drift. (2) A system of mutually heterotic alleles. (3) A mixtureof heterotic and harmful mutants.
The work was carried out in collaboration with Dr. J. F. CROW of the
University of Wisconsin.
In the present calculation it is assumed that all homozygotes areequally deleterious and that each new mutant is an allele that does notalready exist in the population. Also random mating is assumed.
When there is no selection at all, the effective number of isoalleles
maintained in the population is approximately 4N e u + 1, where N c is theeffective population number and u is the mutation rate. The effective
number of alleles is defined as the reciprocal of the sum of squares ofallelic frequencies in a population. Thus, if 4N e is much less than thereciprocal of the mutation rate, most individuals in the population will
be homozygous for this locus.
When there is overdominance, the result is more complicated, but,the relationship between the effective number of alleles and the population number is given graphically for various combinations of mutation
rate and selection coefficient.
For details, see KIMURA and CROW (1964).

78.

The mutational load with an ePistatic interaction
Motoo KIMURAIn this report we will consider the following situation: Mutations aredeleterious in heterozygous as well as homozygous condition but theharmful effect of each mutant gene is small. The mutation rate perlocus, however, is much smaller than the selection coefficient against
each mutant gene, with the result that its equilibrium frequency is very
low. Because of a large number of loci involved, the total mutation
rate per individual may nevertheless be appreciable and the number ofmutant genes accumulated in each individual may be much larger thanunity. The population is very large and random mating is practiced.
Under such conditions, most of the mutant genes may be held inheterozygous condition, while in each locus the homozygous condition isso rare that its effect may be neglected in the following calculation.
The main purpose of the present calculation is to investigate themutational load when the deleterious effect of mutant genes against an
individual is proportional to the second order polynomial of the numberof mutant genes (x) contained in the individual.
Let Wz be the selective value of an individual having x mutant genes

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

Cx=O, 1, 2, .... ), assuming Wz=e-hlZ-hzZZ.
It is difficult to work out the exact probability distribution of x amongindividuals at equilibrium, but under free recombination and slow selection, it may be approximated with the normal distribution, unless themean number of mutant genes per individual is very small: Let 1 anda Z be the mean and the variance of such distribution, then e=Cx-l)ja
is distributed normally with mean 0 and variance unity, where a=vl:
1fce) = V2rr e-~z/z .

Here fCe) represents the distribution of mutant genes at fertilization, i.e.
after recombination but before selection. The selection coefficient of amutant gene may be obtained from-h= ~ CWz+l-wz)fce)de/~ wzfce)de .
Thus

h=l-

exp

{hl+hz+2h zl}
1+2hzl.

Let P be the frequency of the mutant gene in a locus, then 1=2:,2P,
where the summation is over all relevant loci.
At equilibrium in which mutation and selection balance each other,hP=f-l,where f-l is the mutation rate in the locus. This leads approximately to(1) 2hzl z+Chl+hz-2hzM)1-M=O,
where M= 2:,2f-l is the total mutation rate per individual and 1 is obtained
as a solution of equation (1). With this 1, the mutational load is(2) L=lV1:2hzl ex p {C~~~~:~)}.

If h zl<l, (1) and (2) may be substituted by much simpler relations:C1') 2h zl z+h 1l-M=O
C2') L=1-e-(h1+hz>.l>..

In the special case of hl=O, i. e., if the decrease of fitness of an individual is proportional to the square of the number of mutant genes
within the individual,
L=l-e- M / z .
Namely, the mutational load is roughly equal to half the total mutationrate.
On the other hand, if hz=O, i. e., if the decrease of fitness is proportional to the number of mutant genes,RESEARCHES CARRIED OUT IN 1964

95

L=l-e- M •This is the well-known case of mutational load being roughly equal to
the total mutation rate.
The above calculation is based on the normal approximation of distribution of x. It is probable that, because of the epistasis and linkage,u of the actual distribution is slightly smaller than -vT. Therefore, it
is interesting to note that at the limit of u=O the load is exactly givenby (1') and (2'). This means that these two relations (1') and (2') may
be used to calculate the mutational load with good approximation for
most of the cases.

79.

Theory of the gene frequency cline in a habitat of finite length
Motoo KIMURAIn the mathematical theories of cline developed by HALDANE and FISHER,
the length of habitat is assumed to be infinite. The purpose of thepresent study is to investigate the gene frequency cline assuming afinite length of habitat.
Consider a linear habitat represented by a finite interval [LI, L2] on one
dimensional coordinate axis and designate by X a position on the habitat.
Let y be the distance between the point of birth of an individual andthat of its parent. We assume that y is distributed with mean 0 andvariance u m 2. Let P(X) be the frequency of a gene Al at point X, andassume that it may be changed by migration and selection. The rateof change by selection is assumed to be represented by sP(l-P)f(P, X)
where s is a constant and f(P, X) is a function of P and X. Whenequilibrium is reached in the gene frequency cline, P(X) may be givenby the solution ofdp
2u m . dX2+sP(l-P)f(P, X)=O,
2

(1)

2

(LI<X<L2)

that satisfies the boundary conditions
(2)

dP =0
dX

both at X=LI and X=L2.
Thus
L2~ P(l-P)f(P, X)dX=O .
LI

If the cline is mild, P(X) may be expressed by the power series of X.
For example, in the case of no dominance between a pair of alleles, Al

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

and A2, if Al is advantageous on the left side of point X o and disadvantageous on the right side of X o, and if the selection coefficient is proportional to the distance from Xo, thenf(P, X)=-(X-Xo) .
In this case,X o=)L2 XP(l-P)dX/~L2 P(l-P)dX.
L1

LI

Let x=(X- Xo)/a, where a 3 =um 2 /(2s), then (1) and (2) becomes respectively(1') P" =xP(l-P)
and.................
1--o.oL------'-------'-------'----0.0

0.5

x:

1.0

Fig. 1. Relation between x and p.

1.5

RESEARCHES CARRIED OUT IN 196497

where 11=(L 1-XO)/a and 12=(L2 -Xo)/a.
The solution is given by

P=A-Bx+ A(l-A) x 3 + B(2A-l) x 4 - ~X5+ ... ,
2·3
3·4
4·5
where constants A and B are determined by (2'). For the symmetrical(3)

case of -11=12=1 we have A=1/2 B=-l[2-~16+....
,
'8
4608
The power !,eries expansion (3) seems to be quite accurate within therange of -1.5<x<1.5. Relation between x and P is graphically given
in Fig. 1 for several values of I. Treatments for more general cases areunder investigation.

D.

GENETICS AND BIOCHEMISTRY OF MICROORGANISMS

80.

Hybridization between Salmonella abony and S. abortus-equi ll
Tetsuo IINO and Yasuko SUZUKI

S]457 is a non-flagellated (fla-), consequently non-motile, mutant onglnated from a slow motile Hfr strain SW1391 (gal-, sm r ) of S. abony.
Motility of SW1391 is characterized by the formation of 'flare' on
semisolid plates, in contrast with 'swarm' which is formed by motile
dones.
Mating experiments were carried out between S]457 and a strain of
S. abortus-equi S]152 (fla-F-, gal+, sm B ). When semisolid plates wereused as selective media, flares as well as swarms appeared from mixtureof the cells of both strains. Each of both, flares and swarms, was isolated, and genetic traits of the component cells were examined growing
them on indicator plates. As a result, swarms were found to be fla+recombinant clones between S]457 and S]152. The flares were grouped
into two types, namely stable and segregational. When cells of thestable type flares were dispersed in saline and spread on a semisolid
plate, the colonies grown on the plate were all flares; while those fromthe segregational type dissociated into compact colonies, swarms andflares. The compact colonies were composed of non-flagellated cells andtheir genetic markers were mostly those of S]152. The swarms werecomposed of the stable fla+ hybrid cells. The flares were of segregational type or occasionally stable.
1) This work was supported by aresearch grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), Public Health Service, U. S. A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

From those observations, it is assumed that a flare of stable type isa recombinant clone which received a slow motility gene from an Rfrcell, and that of the segregational type is a hemizygous clone currently
segregating recipient type cells and recombinants in its offspring.

81. Mutation of flagellar antigen-1.2 in Salmonella ll
Tetsuo IINO and Michiko MITANI

SW577 is a curly mutant strain derived from S. typhimurium TM2.
Its phase-l flagella are curly-i type and those of phase-2 are normal-1.2(IINO, Annual Report No.9, 1958). Cells of SW577 grown on semisolid
plates containing anti-1.2 serum are inhibited in their motility by thecontained antiserum, and cannot form swarms on the plates. Amongthe bacterial cultures, a mutant which can spread and form a swarm
appears on the plates at the frequency of 10- 6 to 10- 7 per cell. Eighteen
such mutant clones were isolated and their antigenicities were examinedby slide agglutination. They were all found to belong to 1.2-type. The
agglutination titers of anti-1.2 serum, which was prepared against
SW577, given to each of these mutant clones were measured before andafter absorption with each of the mutant cells in all pairwise combinations. From the absorption-agglutination test, it was found that eachof the mutants had lost a fraction of 1.2-antigen. Antigenic subunits
lost by the mutants are schematically represented in Fig. 1.
Agglutination test with anti-1 and anti-2 sera indicated that the antigenic loss of clone-1 covers both antigen-1 and -2, while others are declone

SW577

antigen
2

1

1
2

3

4
5,6,7,6
9,10,11,12

13,14
15,16,17,16
Fig. 1. Antigenic fractions lost in the mutant clones of SW577.
1> This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1964

99

ficient only in antigen-I. It was further indicated that antigen-I iscomposed of at least eight subunits.

82.

Flagellar mutants from a curly flagellar strain of Salmonella llMichiko MITANI and Tetsuo IINO

Curly mutant cells have flagella with wavelength half of that ofnormal type (IINO, Annual Report No.9, 1958). They cannot spread on
semisolid plates and their growth is confined to the region of inoculation.
After prolonged incubation on the plate, subclones which can spread andform swarms appear among the cultures. The edges of seven such
swarms of independent origin were isolated from the culture of a curlystrain of S. abortus-equi, SJ30 (phase-2 monophasic, (a): enx), and thecharacteristics of those subclones were compared with each other andwith the curly type. From the observation of motility by hanging drop
method under optical microscope, of swarm types spreading on semisolid
agar plates and of flagellar morphology under electron microscope, it
was found that they were divided into five groups as shown in Table 1.
Table 1.

Five type3 of flagellar mutants originated from acurly strain, SJ30.

Type

Rate ofspreading*Movementin broth

Wild

1.00

translation

2.45

translation
wigglenormal and curly

0.53translation

small amplitude

III

0.44

circulartranslation
wiggle

curly

IV

0.15

wigglerotation

Flagellar shapenormal_.

II__. _ - -short-

- -------

V

0.08

translation
wigglehooked curly

Curly

0.00

rotation

curly* Speed of spreading of the wild clone on a semisolid plate at 37°C was taken
as 1.00.
1) This work was
supported by a research grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), Public Health Service, U.S.A.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

The structure gene of phase-2 flagellin, Hz, was transduced with
P22-phage lysate from the five representative clones to the phase-I
monophasic strain of S. typhimurium SW1166 ((i):1.2). Motility andflagellar shape of the transductional clones in phase-2 showed the property of each donor. These results indicate that each of the motile
subclones appeared by a mutation presumably in Hz; the mutation may
have caused a structural alteration of flagellar protein resulting in thechange of flagellar shape and consequently of cellular motility.

83.

Bundled flagella of curly mutant cells in Salmonella Il
Michiko MIT ANI

Flagellar morphology and motility of a moving bacterium were alreadyobserved by microcinematography under dark field microscope by A.
PIJPER (1946). A swimming bacterium of a normal and a curly flagellar
strain appears as a bright particle trailing the helically bundled flagellaFig. 1. Bundled flagella of a curly mutant cell observed by chromium shadowing.
1) This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), Public Health Service, U.S.A., bestowed
on Dr. T. IINO.

RESEARCHES CARRIED OUT IN 1964

101

and moving forward by their screwing motion in the aqueous solutionof 0.5 % methylcellulose. Flagella of the normal type observed byelectron microscope dissociated from each other distributing around thebacterial surface, and no image of bundled flagella was demonstrated
under dark field microscope. During an investigation of flagellar shape,
bundled, not dissociated, flagella have been observed under electron
microscope on cells having curly flagella. Observed in a chromium
shadowed sample, they were regularly wound about each other helically(Fig. 1). By the extensive observation, bundled flagella have also beenfound on normal flagellar cells, but at far less frequency than on thosewith curly flagella. The difference between the two might be ascribedto the difference in the flagellar shape itself. This state of flagella was
expected to reveal their details in a moving bacterium. The connection
between bundle formation of flagella and bacterial movement and thegenetics of flagellar shape are now investigated.

84. A pauci-flagellated mutant in Salmonella typhimurium 'lMasatoshi ENOMOTO

A slow motile mutant, SJ399, was isolated from a strain of S. tyPhiIts cells were motile in brothculture at 37°C and their speed of movement and mode of locomotion
were indistinguishable from those of the wild type, TM2, while its cells
grown on semisolid medium did not give rise to any normal swarm
even after 40 hour incubation at 37°C, and produced a narrow band ofswarms at the margin of the inoculation site. From experiments on theswarm's progression on media containing a low agar concentration, it
was found that the motility of this strain was hindered by the viscosityof the semisolid medium. H-antigenicity and morphological charactersof this strain were the same as those of the wild type. The response
to motility phage, chi, was somewhat different between SJ399 and thewild type. Plaques on cells of SJ399 were small and smeary, while thoseon the wild type cells were clear and large, and the efficiency of plating
was 0.36 for SJ399. Moreover, it was found from the observations ofstained preparations of flagella that their number per bacterium decreased
to about one third of that of the wild type. The reduction in their
number was also ascertained by biochemical measurement of flagellar
protein which was purified after deflagellation of the cells. The content

murium, TM2, on semisolid medium.

II This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), Public Health Service, U.S.A., bestowed
on Dr. T. IINO.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

of flagellin in SJ399 was about 32 % of that of the wild type. The
mutant SJ399 was designated as "pauci-flagellated" mutant.
Genetic analyses by means of P22 phage mediated transductions from
SJ399 to non-flagellated (fia-) and paralysed (mot-) mutants showed thatthe mutant, SJ399, complemented 7 fia-s and 3 mot-s which wererepresentative mutants of the flagellation- and motility-cistrons,
respectively. By recombination test it was found that the site of SJ399
was co-transduced with both A and B motility cistrons. Further, by
experiments of two point crosses with mutants in motility A and B cistrons
and a cross with a deletion type of B cistron, the site of SJ399 was foundto be located in the latter.

85.

Sensitivity of g-group Salmonella to M8-phage, a host range
mutant of chi-phage ll
Shigeru YAMAGUCHIA Salmonella phage, chi, has been known to be unable to attack
Salmonella serotypes which have the g-group flagellar antigens, butseveral host range mutants of chi which can attack some of them havebeen isolated. Among the mutants, M8 has the widest host range on
the g-group Salmonella serotypes, but the degree of their sensitivity to
M8 varies with strains.
In regard to the response to M8 in a spot test, the g-group Salmonella
serotypes tested were classified into three types; that is, type-I which
produces clear plaques with high efficiency of plating (e.o.p.), to which
S. hato (gms:-), S. banana (mt:-) and S. dublin (gp:-) belong, type-II
which produces turbid plaques with the e.o.p. of 10- 1 to 10- 8 of that of
the type-I, to which S. kingston (fg:-), S. enteritidis (gm:-) and S. blegdam(gmq:-) belong and type-III which produces a vague patch when hightiter lysate is spotted and does not produce any plaque with low titerlysate, to which S. budapest (gt:-), and S. rostock (gpu:-) belong. In typeIII cells the phage cannot multiply.
From each of these g-group Salmonella serotypes, the antigentypedetermining gene was transduced to S. tYPhimurium TM2 (i: 1.2) and
S. abortus-equi SJ241 (a: (enx)), which are sensitive to both chi and M8.
Regardless of the type of the donor strains, all the transductants obtained
were sensitive to M8 and produced clear plaques with high e.o.p., butwere fully resistant to chi. Conversely, when the i-antigen-determining
1) This work was supported by a research grant from the National Institute of
Al1ergy and Infectious Diseases (AI-02872), Public Health Service, U.S.A., bestowed
on Dr. T. IINO.

RESEARCHES CARRIED OUT IN 1964

103

gene was transduced to the g-group Salmonella, each transductant showedalmost the same response to both M8 and chi as that of the recipient
to M8.
These results indicate that M8 can attach to all g-group antigenic
flagella, and the differences in the response to M8 among the g-group
Salmonella serotypes depend on some factors not associated with thedeterminant of the flagellar antigen, and also indicate that the hostrange mutant, M8, differs from the original phage, chi, only in theability to attach to the flagella having g-group antigens.

86.

Effect of arginine added at various growth stages to thecultures of argos mutant of Salmonella typhimurium
Jun-ichi ISHIDSU

The growth inhibition of an arginine sensitive mutant, argos-I, of S.
typhimurium caused by arginine is promptly removed by the addition of
uracil (IsHIDsu, Annual Report No. 14, 1963). On the contrary, the addition of arginine to a normally growing culture of this mutant did notcause immediate growth inhibition.
For example, when arginine was added to a culture growing normallyin synthetic minimal medium at early log phase (O.D. 0.07 at 660 mil, 2hours after the cultivation was started) so as to get the final concentrationof 10- 3 M, the optical density of the culture continued to increase to
0.62 in 6 hours, being followed by a sudden drop (Fig. 1). Viable cellcount and observation under a microscope showed that the residual O.D.
increase was brought about first by cell division and then by cell elongation (Fig. 2). Elongation of cells starts about two hours after the
addition of arginine and, at its maximum (in about six hours), they become as long as 6 to 7 times of the normal cell length. At the sametime cell agglutination proceeds which causes, at its maximum, a sudden
decrease in optical density.
When arginine was added at middle log phase (O.D. 0.5, 4.5 hours after
the start of the cultivation), the inhibitory effect was far less pronounced
and resulted in a slight delay only in O.D. increase and lowering of astationary level (Fig. 1).
These findings suggest that during the normal growth period uracil
itself or uracil synthesizing enzymes, or systems for synthesizing those
enzymes are established and accumulated to a level high enough topermit cells to grow residually upon arginine addition. Also it is clear
from Fig. 2 that arginine's action inhibits the growth of this mutant
but does not kill it at least for the first 10 hours.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

2.0

it

..
0

~
~
~

!
~

1.0

~

TIllE in HOURS,.-.:

Fig. 1. Effect of arginine added at various growth stages.
Arginine addition.
,\.
Growth in minimal medium.
x-x: Growth under the presence of 10- 3 M arginine.
0-0: Arginine was added at early log phase (at 2 hours).
0-0: Arginine was added at middle log phase (at 4.5 hours).
1 ~ 10 9

1.00

0.50

O.D. increased
mainly by cell
division.

~-----)I

O.D. increased mainly
by cell elongation.

5 x lOB+(---------)

if
0

...

:g 0.202 x 10 8

~

0
0

~

I x 10 8~ 0.10..
~'"'"--''
"""
""-'
~

;:

'"

~

..
"
";

5 x

0.05

10;

"'"'
"''"='z

0

2 x 10 7

0.02

1 x 10 7

0.01
10
THIE in HOUR:;.

Fig. 2.

Comparison of increases in O.D. and in number of viable cells.
• - . : Optical density.
0-0: Number of viable cells.

RESEARCHES CARRIED OUT IN 1964

105

87. Effect of uracil together with arginine in the precultureof argos mutant of Salmonella typhimurium
Jun-ichi ISHIDSU
Addition of argmme to a normally growing culture of arg-s-l of S.
tYPhimurium failed to inhibit immediately the growth of this arginine
sensitive mutant (This Report, 86). This made it very troublesome toinvestigate direct effect of arginine on the metabolic systems of themutant from the biochemical point of view.
Conditions under which the growth of arg-s-l is immediately andabsolutely inhibited upon arginine addition were searched for. Washingcells by saline, or starvation in minimal medium without carbon source
but supplemented with 10- 3 M arginine for 1 hour at 37°C was noteffective enough. Even after these treatments, cells showed considerable
residual growth when transferred to arginine medium.
Treatment with chloramphenicol (200 rlml) for 2 hours cut out thisresidual growth upon transfer to arginine medium that did not containchloramphenicol. Successive dilution and cultivation method was also
tried. Arginine was added to a normally growing culture at middle log
phase (O.D. 0.55) and the culture was diluted into fresh arginine medium
to the O.D. of 0.18 when the O.D. reached 0.70. Incubation was continued
under shaking, and similar dilutions and cultivations were repeatedseveral times successively. Step by step with repeated dilution, theslopes of growth curves became gentler suggesting that the accumulationof whatever it may be supporting the growth of the mutant even in
the presence of arginine was being exhausted without being synthesized
after the addition of arginine.
Absolute and immediate inhibition was observed when the mutant was
first grown in a synthetic medium containing both arginine (10- 3 M) anduracil (10- 3 M) and then transferred to the arginine medium. Fig. 1
shows that, even when this transfer is made at middle log phase (O.D.
0.56), the residual growth can be neglected. In the case of transfer fromthe culture grown in minimal medium at a similar phase resulted incontinuation of almost normal growth (This Report, 86). As has beenreported before (IsHIDsu, Annual Report No. 14, 1963), this mutant cangrow quite normally in the medium supplemented with both arginine
and uracil just as in the minimal medium. But the metabolic situationof cells under these two conditions is quite different. The following
conclusions can be drawn from these experiments. (1) Arginine inhibitionis caused by its inhibition of uracil synthesizing systems and resulting
uracil starvation. (2) Uracil, when supplied together with arginine, can106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

TIllE in HOURS.

Fig. 1. Growth curves of arg-s-l first grown in a synthetic medium containing both arginine (lO- a M) and uracil (10-a M) (x - x) and then transferred
to minimal medium (e-e) or to the same medium containing only arginine(10-a M) (0-0).

[I]:- - ...... :First culture.
Washing procedure.

[II]:
,/:

Second culture.
Uracil addition (10-a~M).

support the growth of arg-s-l but represses at least some part of uracil
synthesizing systems. (3) Cells first grown in the medium containing
both arginine and uracil suffer immediate growth inhibition when transferred to arginine medium since they lack established uracil synthesizing
systems. (4) Cells first grown in the minimal medium do not suffer
immediate growth inhibition upon transfer to arginine medium becauseof their established uracil synthesizing systems rather than because ofaccumulation of uracil itself.

RESEARCHES CARRIED OUT IN 1964

88.

107

Ribosomal RNA synthesis in the early stages of sporogenesis
Hideho SUZUKI

In a glucose-containing culture of Bacillus cereus strain T, pH of themedium decreases as the exponential growth proceeds and then rapidly
increases accompanied by the initiation of the sporulation cycle (NAKATA
and HALVORSON, 1960). A similar phenomenon was observed in the cultureof B. cereus NTCT 5691) grown in media containing glucose. Moreover,the growth curve of the culture, as depicted by intermittent logarithmic
increases in absorbancy at 660 mp (E660), revealed the existence of threephases prior to the stationary phase. The first phase of growth (phaseI) corresponds to exponential growth accompanied by dissimilation of
glucose and pH decrease in the medium. In the second phase (phaseII), E660 increased again logarithmically accompanied by pH rise andmorphological changes characteristic of the early sporulating process.
This :was followed by an additional increase of E660 in the third phase(phase III).
The cells in the late stages of each phase were fed with 14C-uracil(1 pc/100 ml medium) for 20 min. The nucleic acids were extracted withphenol from these cells after grinding them with quartz sand in the cold
and were analyzed by sucrose density gradient centrifugation. The
profiles of the radio activity of phase II in the centrifuge pattern were
scarcely different from those of phase I, the exponential growth phase,that is, the cells that had started the sporulation cycle synthesized 16s
and 23s RNA as well as soluble RNA; whereas in the nucleic acids fromthe cells at phase III there was no incorporation of radio-activity, suggesting the absence of RNA synthesis. This is in accordance with theobservation of the ability of Clostridium botulinum to sporulate without
net synthesis of RNA in the stationary phase (DAY and COSTILOW, 1964),
though the possibility cannot be eliminated that the cell membrane atphase III might be impermeable to uracil. From these observations it
is suggested that in B. cereus NTCT 569 the synthesis of ribosomal
RNA is not turned off by the initiation of the sporulation cycle but itceases at the transitional stage from phase III to the stationary phase.
It is in contrast to B. subtilis which appears to synthesize ribosomal
RNA during the stationary phase (DOl and IGARASHI, 1964; BALASSA, 1964).

1) Derived from a stock culture of Prof. H. CHANTRENNE at Universite libre deBruxelles, Belgique, and obtained through Dr. S. OSAWA of Hiroshima University.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

89. Changes in r-ray sensitivity with time after ultraviolet
irradiation of several strains of Escherichia coli
Mituo IKENAGA and Sohei KONDO

Ultraviolet light (UV) prevents in bacteria colony formation by forming
thymine dimers in their DNA. But UV irradiated bacteria regain theircolony forming ability under some appropriate post-irradiation conditions.
Among these post-treatment recovery phenomena, dark recovery is quite
interesting. To interpret the mechanism of repair of DNA in bacteria
during incubation in the dark after UV irradiation it was proposed thatthe cutting of thymine dimers together with DNA backbone leaves agap, i.e., a partially single stranded part in DNA, which is followed by~- ray

10°

10- 1
c:
.~

10

5

0

Dose ( K r)

:~~

0

Cl

c:
';;:
';;:
L.

20

,~
~,

tt
::J
C/)

15

~~
()

time

10- 2

treatments

r onlyafter UV

o
6

UV+

r

~~

......
:

6.

0
2
4
6

•

8

0

"'"

"-

.~

"-

()
~

0

Fig. 1. Changes in time of sensitivity to r-rays of Escherichia coli B phrafter UV irradiation.

RESEARCHES CARRIED OUT IN 1964

109

reconstruction of twin helical DNA due to partial DNA synthesis (SETLOW
and CARRIER, 1964; BOYCE and HOWARD·FLANDERS, 1964; PETTIJOHN and
HANAWALT, 1964). If this is true, bacteria some time after UV exposure
should have some single stranded parts in DNA and may be expected
to be more sensitive to additional agents, such as ionizing radiation,
than fully recovered bacteria long time after UV irradiation. With thispossibility in mind, we studied r-ray sensitivity of UV irradiated E. coli
at two hour intervals after irradiation. The used strains were E. coli
B, B phr- (mutant of B, lacking in ability of photoreactivation), B/r(radiation resistant mutant of B), B/r phr- (H/r) and BS-l (extremely UVsensitive mutant of B, which lacks the ability to excise thymine dimerfrom its DNA). After 254 mp. UV irradiation to the extent of a small
survival percentage, the bacteria were diluted in a 1/15 M phosphatebuffer and held in the dark at 21°C. After several two hour intervals,
a part of them was irradiated with 137Cs r-rays and then the survivors
were estimated by colony counting. Fig. 1 is a typical example for E.
coli B phr- which shows a depression in the resistance to r·rays 2,..., 4hours after UV irradiation. This may partly fit the UV recovery model
mentioned above, though it can not explain why UV irradiated B phrwas more resistant to r·rays than the unirradiated one, as judged fromthe slopes of the curves. On the contrary, the UV irradiated B/r and
Hlr showed soon after UV exposure a considerable depression in theresistance to r·rays and then gradually became resistant to r·rays andthe slopes of their survival curves finally reached to control level. The
B.-l showed no change in r·ray sensitivity at any time interval after
UV irradiation. So far the obtained data are rather complicated, but it
may be at least concluded that there are correlations between the dark
recovering ability and r-ray sensitivity changes after UV irradiation.

(:.

RADIATION G(:N(:TICS OF ANIMALS

90. Effects of X·ray induced mutations on several components of fitness
Yuichiro HIRAIZUMISecond chromosomes of D. melanogaster were X-rayed (sperm: 100 rlmin
for 5 min) and the effects of induced mutations on female fecundity,
rate of development and larval stage viability were examined. In order
to eliminate the effects of possible heterogeneity among the genetic
backgrounds of the stocks, the whole matings were repeated without

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

X-irradiation (=control set).
are summarized as follows.

Results obtained during the past three years

Female fecundity
One hundred and sixty-nine X-rayed (77 in the control set) chromosome
lines (four, sometimes three or two females per line) were tested.
Fecundity was measured by the total number of progenies produced
during the first 9 days. Results are given in Table 1, where E9 represents X-rayed second chromosome. In order to minimize the effects ofenvironmental fluctuations, experiments were designed such that comparisons among genotypes could be made pairwise. For each pair, £1 1 =(+/+ )-(E9/+) and £12=(+/+ )-(E9/E9).
1.

Table l.

Female fecundity.
.:1 1

Set

+/+

ffi/+

ffi/ffi

X-rayedControl

143.43139.88139.25142.16131.70140.14.:1 2

4.17±2.30 11.72±2.90-2.27±4.00 -0.25±4.00

In the X-rayed set, L1 2 was significantly and :3 1 nearly significantly
larger than zero indicating that female fecundities of E9/+ and E9/E9
were reduced. In the control set, the sign of L1 1 was negative, suggesting that the genetic backgrounds of the stocks used in this experimentwere not completely homogeneous.
2. Developmental rate
Two (sometimes more) fertilized females of a genotype in question,and two fertilized, standard, en bw females were put together into afood vial and allowed to lay eggs for 24 hours, and their progeny flies
were counted once a day until all the flies had eclosed. The averagetime from egg laying to eclosion was then computed for each genotype
separately in the two sexes. Let x and y be the eclosion time (in day)of en bw strain and of the genotype in question, taking the unweighted
average for the two sexes. Z= y- x was then computed for each culture,and the results are summarized in Table 2 (190 lines in X-rayed, and 81
in the control set. Larger value of Z represents slower developmentalrate). In Table 2, £1 1 (or £12)=Z of +/+ minus Z of E9/+ (or E9/E9).
Table 2. Developmental rate.
~
X-rayedControl~
0.0499±0.017
0.0088±0.025~-0.0312±0.020
0.0190±0.040

RESEARCHES CARRIED OUT IN 1964

111

In the X-rayed set, J1 was significantly larger than zero, or the developmental rate of E8/+ was significantly faster than +/+. Since there
is a positive correlation between rate of development and viability, thissuggests that the viability of E8/ + increased. Although not statisticallysignificant, E8/E8 revealed slower developmental rate than +/+. No:lear deviation was seen in the control set.
3. Larval stage viability
Two hundred and seven chromosomes (148 in the control set) were
tested. Matings were: +/CY!i2 x+/Vo, +/CY!i2 xE8/Vo and E8/CY!i2 X
E8/V o. In the F 1 of these matings, the number of wild flies was divided
by the number of +/V flies plus one (for .1 1) and of +/Cy flies plus one(for Ja) to estimate the relative viabilities of the three genotypes. Results
are summarized in Table 3, where Ja=(E8/+ )-(E8/E8).
Table 3. Larval stage viability.
Set

Standard genotype=+/Lz

X-rayed

L1 1 = -O.OI68±O.030

Control

J 1 = -O.OI05±O.035

Standard genotype=E8/Cy

In the X-rayed set, J a was significantly larger than zero. This suggeststhat either the viability of E8/+ increased or that of ffi/8 decreased (orboth). Further statistical analyses are in progress.

91.

Heterozygous effects of radiation-induced mutations on viability
in a synthetic homozygous genetic background
Terumi MUKAI, Isao YOSHIKAWA and Tsuneyuki YAMAZAKI

It has been generally accepted that specific co-adapted gene complexes
have been established within natural populations as a result of natural
selection (e.g., see WALLACE and VETUKHIV, 1955). In order to test whether
or not the manifestation of overdominance of radiation-induced mutations
previously reported (MUKAI, YOSHIKAWA and YAMAZAKI, Annual Report
No. 14, 1964) is a characteristic of co-adapted gene complexes at a chromosomal level, namely, if the nature of overdominance expression is the
product of natural selection (DOBZHANSKY, 1950), this experiment was
conducted.
An isogenic line (WI80) was synthesized (abbreviated as MM) using
entirely unrelated 64 wild-type stocks collected from almost all over theworld. This synthesis was conducted so as to bring about their equal
contribution to the isogenic line. After irradiating W180 males by X-

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
rays at the dose of 500 r, heterozygotes with respect to radiation induced
mutations were produced by the aid of Cy/Pm Sb/Ubx strain. The relative
viabilities of irradiated heterozygotes with respect to the second chromosome (MM') were estimated in comparison with CY/+M. The irradiated X
and third chromosomes were completely substituted by the unirradiatedones of W180 before the estimation of viability. The control experiments
were conducted in the same manner without irradiation. The results arepresented in Table 1, together with the results in homozygous genetic
background (AlAI) which was directly extracted from a random mating
population.
From Table 1, it can be seen that radiation-induced mutations which
occurred in synthetic homozygous individuals were not overdominant incontrast with the mutations induced in homozygous individuals extracted
from a random mating population. In conclusion, it might be said thatthe overdominance manifested by mutations induced in otherwise homozygous genetic background of the origin of an equilibrium random mating population is caused by interaction with their genetic background.
This might be a product of natural selection.
Table 1. Heterozygous effects of radiation-induced mutations on viability
in Drosophila melanogaster (500 r, 2nd chromosome).
--"----Average number of flies
No. ofAverage
Genotype
chromosomethat emerged in eachviability
chromosome line
lines tested----------._-

MM
MM'

315
306

1,694.76
1,683.74(-11.02±12.47)

1.00991.0096(-0.OOO3±0.0053)

287
286

766.63789.74(+23. 11*±12.89)

1.0141
1.0412(+0.0271**±0.0101)

Difference
AlAI
AlAI'

Difference, Indicates irradiated chromosomes.
* Significant at the 5% level.
** Significant at the 1% level.

92.

Radiation effect of the" Sex-Ratio" agents of
D. willistoni and D. nebulosa l )Bungo SAKAGUCHI, Susumu KOBAYASHI and Kugao OISHI
It was demonstrated by POULSON and SAKAGUCHI (1961) that the" SexI) This work has been supported by U. S. Public Health Service Research Grant
GM 10238 from the Division of General Medical Sciences, and Grant in Aid Scientific
Research of Ministry of Education (95212).

113

RESEARCHES CARRIED OUT IN 1964

Ratio" (SR) agents are the etiologic factor in the production of maternallytransmitted SR condition in Drosophila and that they are Treponema-likespirochetes (SR spirochetes). They were found in high concentration in
the hemolymph of adult females of SR strains in D. nebulosa, in D. willistoni and in D. melanogaster to which the SR condition has been
artificially transferred.
In order to make clear the inactivation and recovery of the functionof SR spirochetes by r-ray irradiation, the present experiments werecarried out.
Two SR lines of the Oregon-R (OR) strain of D. melanogaster were
established by artificial transfer, one hosting willistoni and the othernebulosa spirochetes. Females of these two preinfected OR lines were
exposed to Cs-137 r-ray with 30 kr dose. After one week from the timeof irradiation, the hemolymph containing a large amount of irradiatedSR spirochetes was injected into females of normal OR strain. Directionof the injection was always from one donor to one host. The injected
females were then mated singly with males of the same OR strain andthe sex ratios in the progenies were examined. Injection and examination
were repeated several times as shown in Fig. 1.
30 kr (r-rays)

i/i/

SR

'The<" (i~:: ::lm::,:::y :~:::::=

injection after
7 days from thetime of irradiation

fm SR ,ondition

Exam. SR)

I

I---->OR (II)-->progeny (Exam. SR)
injection after
7 days from thetime of injection---->OR (lII)-->progeny (Exam. SR)
injection
I

I_:---->OR (IV)-->progeny (Exam. SR)
injection
Fig. 1. Diagram representing the method for testing inactivation and recoveryof irradiated SR spirochetes.

The results are summarised in Table 1.
In the series of injections with irradiated willistoni spirochetes, only
one in each of II and III injections produced SR progenies. While in
the injection with irradiated nebulosa spirochetes, several flies produced
SR progenies from two to eight in first and second injection and finally114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

all injected flies produced SR progenies in the following injection steps.
These results indicate that the sensitivity against r-ray irradiation ofwillistoni spirochetes is considerably higher than that of nebulosa spirochetes, while multiplication after irradiation of nebulosa spirochetes isfaster than that of willistoni.
It is of interest that SR spirochetes were observed in the hemolymph
of a part of normal sex ratio progenies from the injected flies (Table 1).
Table 1. The manifestation of SR condition in successive injectionsof irradiated SR spirochetes.
IrradiatedSR spirochetes

willistoniI,-nebulosa-_ ..

Successive**
injection

.,

I

II

III

I

II

III

IV

11
0
8
3

11
1
8*
2

10
1
8*
1

12
2
3*
7

11
8
2*
1

8
8
0
0

6
6
0
0

..

No. of injected flies
No. of SR condition
No. of normal sex ratio
No. of sterile* SR spirochetes were observed in the hemolymph of a part of males
and females.
** See in Fig. 1.

93. Mechanisms controlling two types of dose-rate dependence ofradiation-induced mutation frequencies in silkworm gonia ll
Yataro TAZIMAThe dose-rate effect of radiation in the induction of mutation has beenstudied extensively for the past five years with silkworm gonia. The
results so far obtained are summarized and a new explanation is proposed.
As far as X- and r-rays are concerned, the mutational response ofyoung germ cells of the silkworm, mostly at gonial stage, is not simple.
At least two types of dose-rate dependence, one being the reverse of
the other, have been found in this insect. In one type, the mutagenic
effectiveness of chronic irradiation (0.1 r/minute) is lower than that ofacute irradiation (100-300 r/minute) (Type I), and in the other, mutagenic
effectiveness is higher for chronic than for acute irradiation (Type II).
Which type appears solely depends upon the developmental stage of theirradiated larvae. Irradiation of newly hatched larvae results in Type I
dose-rate dependence, while irradiation at a later stage, around the time1) Abstract of a paper presented before the conference on Dose-Rate Effect ofRadiation at Genetic and Cellular Levels, Oiso, 1964.

RESEARCHES CARRIED OUT IN 1964

115

when the larvae are eight days old, gives completely reversed results.
The complexity of the dose-rate effect is entirely due to a drasticchange in mutagenic effectiveness of acute irradiation according to thedevelopmental stage. The dose-mutation frequency curve shows thehighest peak at hatching time but decreases rapidly thereafter. In contrast, the mutagenic effectiveness of chronic irradiation remains almost
the same.
In order to interpret the reverse type of dose-rate effect, a hypothesis
involving selective cell killing was proposed. The hypothesis was basedon the finding that the homogeneous germ cell population which exists
at hatching time becomes gradually heterogeneous with the developmentof the larvae and the gonial cells at an advanced stage are highly
sensitive to the killing action of radiation. It was also assumed that cellkilling is more drastic in acute than in chronic exposure and that cells
sensitive to killing are also sensitive to having mutations induced inthem. However, we failed to obtain cytological evidence to support
this hypothesis.
Later a differential repopulation hypothesis was proposed but this wasagain found to be not satisfactory.
Since then, efforts have been made to obtain some decisive evidences
which may provide a clue to the solution of the problem. Extensive
experiments with fractionated doses and with neutrons were carried out.
The dose-fractionation experiments yielded very suggestive information
for the interpretation of the dose-rate effect. When 1,000 r acute irradiation was delivered in two fractions, 500 r+500 r, separated by various
time intervals from 2 to 48 hours, a remarkable increase in mutation
frequency was observed throughout the period examined, showing thepeak if the second dose was given 18 or 24 hours after the initial exposure.
Several possibilities have been considered for interpreting this effect.
Among them the most promising was the idea that metastasis producedby the exposure depresses for some while the repair of pre-mutational
damage, whose duration depends on the metabolic activity of the cell.
The same idea reasonably explained the drastic change in the mutational
response to acute irradiation during the progressing development of
the larvae.
A new hypothesis, based on this idea, has been proposed for the interpretation of the complicated features of the dose-rate effect as follows:(a) a large part of the radiation-induced pre-mutational damage is repaired during the post-irradiation recovery process,(b) the degree and extent of repair primarily depend upon the total
dose and dose-rate,(c) the occurrence of repair also depends upon the cellular conditions,

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
presumably metabolic activity, of the irradiated cell,(d) the final magnitude of the observed mutation frequency is determined by both factors combined.
With these assumptions, the two types of dose-rate dependence, one
being the reverse of the other, have been interpreted consistently.

94. Nature of post-irradiation metastasis as revealed by fractionatedr-irradiation and application of high or low temperature
between the exposures
Yataro TAZIMA and Kimiharu ONIMARU
As a possible interpretation of the enhancing effect of mutation frequency after two-fraction r-irradiation was administered from 2 to 48:
hours apart, the following assumption has been proposed (TAZIMA, 1964):
namely, a kind of temporary metastatic state, presumably a disturbanceof metabolism, was assumed to be produced in the cell that hinders therepair of pre-mutational damage until it disappears after a certain while.
If ~he second irradiation is given before the completion of repair, mostpre-mutational damages cannot be repaired so that higher mutation freq uencies are the result.
In order to look into the nature of post-irradiation metastasis, an experiment was carried out by subjecting irradiated larvae to different
temperatures, 15°C, 25°C and 30°C, between the two exposures. Total
1,000 r was divided into two halves and each 500 r was given to theTable l. Results of temperature treatment, performed in order to elucidate
the duration of the post-irradiation metastasis.* (643)
Oogonia

Treatment
Ohr
F 1
F 2

500 r
500 r

F 3 500 r
F4 500 r
F 5 1,000 r
F6 500 r
F7 500 r

24hr 48hr Temp. Observed
500 r
500 r

15°C

500 r
500 r

25°C
500 r

500 r

30°C

* Data for Type I stage.

Spermatogonia
Mut. freq.
(x 10- 5)

Observed

Mut. freq.
(x 10- 5)

pe

re

pe

160,146
159,278

133.6
148.8

82.4
89.2

133,420 152.2 72.0
120,594 141.0 53.1

150,598
165,788
179,676
167,958
183,148150.7 72.4
176.7 119.4
67.9 50.1
62.5 31.6
139.8 92.8

116,714
115,520
115,520
112,535
114,326

142.2
190.4
111.7
78.2
105.0

re

50.6
84.8
39.8
50.7
66.5

RESEARCHES CARRIED OUT IN 1964

117

larvae 24 or 48 hours apart. The first dose was delivered immediately
after hatching. Except for experimental treatments, all larvae wereraised at 25°C throughout their life cycle. The material used was wild
type C108 strain. For the estimation of mutation frequency we used
specific loci method with egg color marker genes, pe and reo
The results are shown in Table l.
It has been known from the previous experiments that under the
standard rearing temperature, 25°C, the maximum increase in the mutation frequency due to two-fraction irradiation appears in both sexes about
24 hours after initial irradiation. The present data for 25°C are consistent with the previous ones as far as the stage of appearance of thepeak is concerned.
It may be seen from the table that an enhancing effect of mutation
frequency disappears rapidly in 30°C group but persists fairly long whenirradiated larvae are kept at 15°C. Even 48 hours after initial exposure
mutation frequency was still high in 15°C group. These results suggestthat the length of the period during which the metastatic state persists
is temperature dependent.
The observed phenomenon can also be interpreted by assuming simply
a repair of pre-mutational damage. According to this assumption thelength of the period during which damage persists is taken to betemperature dependent. The assumption, however, can not explain thefollowing finding. It has been known for acutely r-irradiated gonia atlate stage that the dose-mutation frequency relation is linear up to 1,000 r,
increasing very slowly with increasing doses (TAZIMA and KONDO, 1962), but
turns into rapid non-linear increase at higher doses than 1,500 r (MURAKAMI and KONDO, 1964). The discrepancy between the two findings canonly be interpreted by introducing another factor in addition to premutational damage produced in genetic material. This factor is supposed
to be a disturbance of cellular metabolism produced by radiation.
It can be inferred from these findings that radiation produces in thecell at least two kinds of disturbance, a pre-mutational damage in genetic
material on one hand and a disturbance in metabolic system on the otherand that both factors in combination determine the final manifestationof a mutation.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

95. Modification of radiation-induced mutation frequency bypost-treatment with KeN, chloramPhenicol, high andlow temperatures and nitrogen gas
Yataro TAZIMA and Kimiharu ONIMARU

As described in the preceding article (This Report, 94), a post-irradiation
metastasis of the cell has been assumed. If this is actually the case, it
is foreseen that post-treatment of irradiated cells with agents that affectcellular metabolism would result either in an increase or in a decreaseof mutation frequency.
Experiments have, therefore, been carried out to confirm this assumption by applying, immediately after r-irradiation, various agents: namely
a known respiratory poison KCN, chloramphenicol, high and low temperatures and oxygen and nitrogen gases.
Prior to those treatments larvae were irradiated with 1,000 r r-rays
from a Cs-137 source at a dose-rate of 100 r per minute. Both females
and males of wild type C108 strain were exposed to radiation. For thedetermination of mutation frequency the specific loci method was utilizedwith use of egg color genes, pe and re, as markers.
Treatment with KCN and chloramphenicol was performed by feeding
the larvae on leaves supplemented with these chemicals. The administereddose of the chemical was determined by taking into account the results ofa preliminary experiment on the adverse effect on viability of the larvae;
it happened to be the same for both chemicals; i. e., 30 mg and 50 mg
per 1,000 larvae for newly hatched and 7 days old larvae, respectively.
The temperature treatment was carried out by exposing irradiatedlarvae to three different temperatures, 15°C for 48 hours, 25°C for 24
Table l.

Results of post-irradiation treatment of 7 days old larvae.
Oogonia(641)

Spermatogonia-------_._-------

Treatment

Observ.
numberMut. freq.
(x 10- 5)~---------"---

pe
1,000 r1,000 r+KCN (50 mg,
1 %, 6 hrs)
1,000r+CMP (50mg,
1 %, 6 hrs)
1,000 r+15°C (48 hrs)
1,000 r+30°C (24 hrs)
Or
137Cs-r-rays, 100 r/min

Observ.
numberMut. freq.
(x 10- 5)

pe

re

9.9
11.2

191,079
187,985

36.1
43.1

12.0
12.2

19.0

12.1

184,890

46.0

12.4

16.2
14.9
7.9

13.2
5.8
6.0

184,890
187,598
182,570

61.1
33.0
10.4

12.4
7.5
0.0

273,178
241,773

9.9
14.1

263,707265,701
275,671
266,698

CMP:

re

Chloramphenicol

RESEARCHES CARRIED OUT IN 1964
Table 2.

119

Results of post-irradiation treatment of newly hatched larvae.

(642)

Spermatogonia

Oogonia
Mut. freq.
(x 10- 5)

Mut. freq.
(X 10- 5)

Treatment

Observ.
number

pe

re

pe

re

1,ooor1,000 r+ KCN (30 mg,
1 %, 6 hrs)l,OOOr+CMP (30mg,
1%,6hrs)l,OOOr+15°C
1,000 r+30°C
Or

187,200
144,000

57.2
54.2

52.4
35.2

158,900
162,050

62.9
66.6

41.5
51.8

158,400

51.8

44.8

148,400

69.4

52.6

129,600
143,550
158,850

42.4
50.9
15.1

32.4
26.5
5.7

142,100
157,850
154,000

114.7
51.9
8.4

66.2
33.6
0.6

Observ.
number137Cs-r-rays, 100 r/min
Table 3.

Results of post-irradiation treatment of newly hatched larvae
with oxygen and nitrogen gases. (643)
Oogonia

Treatment

Mut. freq.
(x 10- 5)

Observ.
numberpe

re189,900
191,250

57.9
57.0

45.3
44.4

135,450
133,000

j ~~ ~~ ~ (24 hrs) 182,700

52.5

43.2

131,850

73.6

97.1

I,OOOrl,ooo~r+Oz (24 hrs)

1,000 r+Observ.
numberSpermatogonial,ooor+Nz (24hrs)

137Cs-r-rays, 100 r/min(Added at proof reading:Mut. freq.
(x 10- 5)

pe

re

70.1
64.7

36.2
48.1

136,850

46.8

27.8

152,950

94.8

27.5

N z gas was later on found not pure.)

hours or 30°C for 24 hours. All three groups were incubated and raisedin the same room at 25°C except during a treatment.
Gas treatment was performed in a glass cylinder. Irradiated larvae
were transferred immediately after irradiation into cylinders where either
oxygen or nitrogen gas was kept running for 24 hours. The gases usedwere obtained commercially.
The results are given in Table 1--3.
Throughout three different experiments, treatment with agents thatare known to depress metabolic activity of the cell resulted in an increasein mutation frequency. Namely, treatment with KCN, chloramphenicol,
low temperature and nitrogen gas gave higher mutation frequencies thanr-irradiation only, high temperature or oxygen gas treatment. These
results are consistent with the expectation that radiation produced

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

metastasis is intensified or prolonged by agents which depress or slow
down the metabolic activity of the cell. However, this does not necessarily imply that this is the only mechanism that underlies the present
findings.

96. Relative biological effectiveness of 14 MeV neutrons to
gamma-rays in the induction of mutations in germ
cells of hibernating silkworm embryos
Akio MURAKAMI and Yataro TAZIMA

It was reported in our previous paper that mutational RBE values ofboth 14 MeV neutrons and fission neutrons for goniaI cells of the silkworm differed between two early larval stages. This finding was explained by assuming a difference in metabolic condition of germ cells at
the two stages. It is, therefore, necessary, in order to obtain the
intrinsic mutational RBE, to utilize an ideal cell system where theinteraction of metabolic condition can be ignored. In this regard an
experiment has been carried out by using germ cells of hibernating
silkworm embryos as material. This is because in these cells metabolic
activity is regarded to be very low, if not negligible.
The method of testing mutation frequency is similar to that used in
our previous work. Hibernating embryos were treated with 14 MeV
neutrons from T(d, n)He reaction with the average dose rate, 8.7 rad per
minute, in four dosage groups: 320, 660, 900 and 1,300 rad. For thesake of comparison of effectiveness, 137CS gamma-irradiation was performedin parallel, in which irradiated larvae were treated similarly except forirradiation. In r-ray experiment seven different doses were administeredwith dose-rate 100 r per minute; i. e. 250, 500, 1,000, 1,500, 2,000, 2,500
and 3,000 r.
The results are shown in Fig. 1 only for the male sex. It can beseen from this figure that the dose-mutation frequency curve for 14 MeV
neutrons increases exponentially up to 600 rad and then turns into increasing gradually up to about 1,300 rad, whereas, the gamma-ray curve
increased approximately linearly below about 2,400 rad and then decreased
against the dose. The mutation frequencies for 14 MeV neutrons weresignificantly higher than those for gamma-rays throughout the whole
dose-range examined.
It was, however, difficult to obtain definite RBE value, because of the
non-linear relation of the obtained curves for both radiations. Consequently, RBE value was obtained tentatively by comparing the estimateddoses for each radiation at an arbitrary mutation frequency level of 10-8 •121

RESEARCHES CARRIED OUT IN 1964

Mal"

I.".r

I

,'vt~-"r--

c:
~200

.

,

~

I
I

,
I

I

100
I

I

I

"- rA1 s . NS

-1oFig. 1.
187CS

500

1.000

1.500

2.000

2._

3.000

Dose In Nld

Dose-mutation frequency curves obtained after 14 MeV neutron andr-ray irradiation of male hibernating embryo of the silkworm.

At this level RBE values of 14 MeV neutrons are 1.8 and 2.9 for pe andre locus, respectively. But the value decreases to a considerable extent
at higher dose levels.
The RBE values obtained in this manner are higher than those for
newly hatched larvae, but almost similar to those for 7 days old larvae,
in which the metabolic activity is presumed to be high. From thesecomparisons, it can be inferred that germ cells of hibernating embryos
cannot be regarded as an ideal model for getting intrinsic RBE. Perhaps
there are some unknown biological factors affecting mutation frequency.

97. Induction of visible recessive mutations by X-radiation insilkworm germ cells at pre-embryonic stage
Akio MURAKAMI and Yataro TAZIMA

It has been known in Drosophila that germ cells are mutagenicallymost sensitive to radiation around the time of fertilization (OSTER, 1958).
A similar phenomenon has been observed by NAKAO in the silkworm.
In order to study the relation more precisely and to elucidate the relevantmechanisms, the present experiment has been undertaken.
The egg material was obtained from actively laying wild type mothsof C108 strain. After allowing oviposition for one hour at 25°C eggs

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

were collected and subjected to X-ray treatment. The conditions for
X-irradiation were 20 rnA, 175 kVp and 50 cm in target distance. The
doses given were 500, 1,000 and 1,500 r with a dose-rate of 60 r per minute.
Irradiated insects were crossed, after emergence, to partners of themarker strain, pe and re, in order to examine the mutation frequency.
It is generally accepted from cytological evidences that syngamy offemale- and male-pronuclei occurs in this insect about two hours after
oviposition at 25°C. In the present experiment irradiation was administered at three different stages; 0.-1 hour-old eggs (before fertilization),
3.5.-4.5 hour-old eggs (soon after fertilization) and 48.-49 hour-old eggs
(early embryonic stage).
_ _-

fO"~

Male

---- -,.

--<>-~-~-

I- .... -rs- ...- -"'--r~

.

/f
I

r

~

!w

\ :

"

,~

\
\

,,1'
,

/7:-;~/

I ,

:1

~~

I

J
J
I

...,

Iff

10'

10.

10-1

5

5

,

,wi

/

\

/.

,/

I

/

/

0.5'.0 '.5 DosI in Kr

0.5 /.0 1.5

0.5 /.0 1.5

Ohr-olden

3.5",-o/d"f'J .....""'"

TJ:;..."'l::.

CIS 1I IS

0.5 1.0 IS

OtMM."

35vold."AS 1.0 /.5" - .. Kr'"""""-II ~Figs. 1-2. Variation in X-ray induced mutation frequencies at the earliest
developmental stages of silkworm eggs.

The results are shown in Figs. 1 and 2 for males and females, separately. As seen from the figures, dose-response curves for mutation
frequencies are essentially similar for both sexes, but differ dependingon the irradiated stage: Dose-response curves for 3.5 hour-old eggs and48 hour-old eggs represent a saturation effect, while no such effect isobserved for 0 hour-old eggs. The discrepancy seems to be correlatedwith the extent of the killing effect of radiation. Indeed it has been
observed that the hatchability of 3.5 and 48 hour-old groups was markedly
lower than that of 0 hour-old group.

RESEARCHES CARRIED OUT IN 1964

123

The mutation frequency obtained for 0 hour-old and 3.5 hour-old eggs
is approximately seven times as high as the maximum frequencies obtained for spermatids and mature oocytes. However, it decreased very
rapidly with the progress of embryogenesis and fell to about the samelevel as that of the maximum frequencies obtained for spermatids andmature oocytes at 48-hour age.
However, when the mutation frequency was calculated per batch basea very interesting information was obtained regarding the sensitivity tomutation. Mutation frequencies given in Figs. 1 and 2 were obtainedin accordance with the ordinary method from pooled data, although theoriginal data were recorded for each batch. From these records mutation frequency per batch base was calculated by dividing the number ofmutation yielding batches by their total number. There was no significant difference in the frequency among the three egg stages.
During the course of the analysis, however, it was found that a difference existed among those groups in the number of mutants per batch.
The size of mutant clusters recovered in the same batch was markedly
larger in 0 hour-old and 3.5 hour-old groups than in 48 hour-old group.

98.

Proliferation kinetics of spermatogonia of the silkworm
during chronic r-irradiations
Toshihiko SADO

During the past few years, attention has been directed in this laboratory to study the mechanism involved in the dose-rate effects on thefrequency of radiation-induced mutations. The type II dose-rate dependence (i. e., a higher mutation rate after chronic than after acute irradiation) is the characteristic of the dose-rate effect observed in the silkworm
gonia and was interpreted initially on the basis of a selective cell killing
hypothesis (TAZIMA, KONDO and SADO, 1961). This hypothesis, however,
was not favored by the subsequent cytological observations; e. g., asubstantial difference was not detected between acute and chronic groupin the number of gonial cells which survived the exposure. Secondary
spermatogonia, which are extremely sensitive to the killing action of
X-rays, were also found to be killed after chronic irradiations (SADO, 1962).
This observation led the author to postulate that synchronization of cells
at some mutagenically sensitive phase of the cell cycle during the chronic
exposure may be the primary factor involved in this dose-rate dependence.
This implies that the difference in mutation frequency between acute124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15:and chronic exposure is primarily a reflection of mutation rates at random
versus selected-sensitive phase(s) of the cell cycle.
In an attempt to gain a better insight into this problem, kinetics ofproliferation and killing of spermatogonia of the young larvae of thisinsect (3--8 days after the hatching) during chronic exposure (0.142 r/min,total doses 1,000 r in 5 days) were studied. The following information
was obtained. 1) The number of spermatogonia at the beginning and.at the end of the chronic exposure was essentially the same. 2) The
mitotic index of spermatogonia determined every 24 hours after the onset-of exposure was approximately the same as that of the control, and atsome intervals it was even higher in chronic than in the control group.
.3) Degeneration of spermatogonia was observed during the exposure.
There was a gradual rise in the frequency of pycnotic spermatogonia of
1--2 cell stages during the first 48 hours, followed by a sharp drop during
the subsequent 24 hours. Among the more advanced spermatogonia (4--32-cell stages) which began to appear 72 hours after the onset of the exposure,
13 percent were pycnotic.
These data emphasize the following. Under this experimental condition
mitotic inhibition of spermatogonia is not taking place, suggesting that'spermatogonia are proliferating apparently at a normal rate during the-chronic exposure. A considerable fraction of spermatogonia are dying,
however, during the exposure. Thus, the dynamic equilibrium is established between the newly occurring and dying cells. The experiments
to reconfirm and extend these findings are now in progress.

99.

Correction factor (K) for estimating the number of spermatozoa
bundles from serial sections of a testis of the silkworm
Toshihiko SADO

For the evaluation of radiation-induced mutation frequencies in relation
to the irradiated stages of the silkworm, or the stages of germ cells at
which irradiations were given, it is often desired to know the number·of spermatozoa produced by the irradiated insects in relation to thenumber of germ cells which survived the exposure. This is particularly
important when the cluster of mutations is taken into consideration(KONDO, 1961).
The number of spermatozoa produced per testis can be-obtained by multiplying the number of spermatozoa bundles produced
by 256 because a single spermatozoa bundle contains 256 spermatozoa.
For the estimation of number of spermatozoa bundles per testis, Machida's
method (MACHIDA, 1929) has been used in which the total number of:spermatozoa bundles in each testis was scored on the serial sections (20,u)RESEARCHES CARRIED OUT IN 1964

125,

only at the portion where the mass of nuclei were observed. A possible
source of error in this method was that a single mass of nuclei might
be scored two or more times. Therefore, the total number scored bythis method was corrected by the equation, N =KA, where N and A
represent scored and actual number, respectively, while K is a proportionality constant dependent on the size and form of the mass of nuclei
of a spermatozoa bundle and the thickness of the sections which was;calculated theoretically to be 2.0 for eupyrene, or typical, spermatozoa(MACHIDA, 1929). This indicates that, in general, a single spermatozoa
bundle may be scored twice on the serial sections of 20 p. thick. This.
value has been used in the past to assess the number of spermatozoa
bundles produced (KOGURE and NAKAJIMA, 1958; SADO, 1961, 1963). However, it was felt that this number was overestimated. An attempt was
made, therefore, to reexamine the validity of this value.
The number of mass of spermatozoa nuclei, regardless of the size, was·scored on the 20'" 30 successive sections of a serial paraffin sections(20 p.) of a testis of middle and late pupal stages on the one hand, andits actual number was determined on the other. This was done byeliminating the doubly scored spermatozoa bundles with the use of serial
microphotographs taken from the corresponding sections. K was thendetermined, using N=KA, or K=N/A. The result indicated that K was.
1.22 and 1.28, respectively, for testes from middle and late pupal stages,
the mean being 1.25. This supports the notion of MACHIDA (1929) that.
K might be smaller than 2.0.
Using this value, the number of spermatozoa bundles produced in a
testis was estimated to be approximately 4,000 '" 5,000 (recalculated fromMACHIDA, 1929; SADO, 1961, 1963). This indicates that one individual maleof this insect produces approximately 2.0'" 2.5 X 106 spermatozoa per
generation. Available data at hands suggest that 1,000 r of X- or r-rays.
given to the larvae before the third instar, independent of the dose-rate,
apparently does not affect the number of spermatozoa produced, eventhough almost complete loss of the secondary spermatogonia was observed
within a few days after the irradiation. However, it has been shown
previously that irradiation at later stages, e. g., early fifth inster, signifi-cantly reduced the number of spermatozoa produced (SADO, 1961, 1963).

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15.100. Further studies of 32p induced mutations in the silkworm 1)
Mituo IKENAGA and Sohei KONDO

In the previous paper (IKENAGA and KONDO, Annual Report No. 14, 1963)
the relative mutagenic efficiencies of r-rays and disintegrating 32p ingested to larvae were compared, and the mutation frequency of egg
color at an equivalent dose of r-rays and 32p {'-rays was found to be thesame for male groups but different for female groups. The latter discrepancy, i. e., existence of transmutation effects of 32p, could be accounted for a dosimetric error involved in the female group, since meiosis
occurs in females two weeks after 32p ingestion.
To prove the above assumption, we carried out similar experimentswith 32p injection given to the silkworm at the middle pupal stage, about
one week before meiosis in females. 32p induced mutation frequencies
were compared with those induced by daily irradiation with r-rays so
as to fit the daily 32p dose in the silkworm gonads. The gonad dose of
32p {'-rays was rather easily obtained, because a pupa is a closed system

Fig. 1. Mutation frequency versus dose curve for 32p treatment and,-irradiation. The 32p dose in rad units was determined experimentally.
1) This work was supported by grant No. 93001 for Intramural Cooperative Scientific Research from the Ministry of Education.

127

RESEARCHES CARRIED OUT IN 1964

showing no excretion of 32P. The results are shown in Fig. 1. The graphclearly shows that there is excellent agreement in induced-mutation
frequency at the same absorbed dose between the 32P-injected and the
r-rayed groups for both sexes.
From these experimental data and those of the previous experiment
for the male sex, our conclusion is that mutations induced by 32P-transmutation could not be detected in the silkworm under conditions suchas 32p ingestion or injection.

101. Radiosensitivity of muscle tissue formation inregenerating tissue of Triturus
Yoshito OGAWA

Actin formation in regenerating tissue of adult Triturus with respect
to X-irradiation at various point of the regenerating progress was dealt
with in a previous report (OGAWA, Annual Report No. 11, 1961). The
effect of X-ray irradiation on myosin formation in the regenerating hindlimb tissue of Triturus was examined and it was concluded that it isdue to radiosensitivity of chemo-differentiation of muscle tissue in theregenerating granula.

o Results of
R-Am action• Hesul ts of
H-Bm action;\on-irr!diatedcontrol

I

I
20r

4

6

16

20

24

I
28

32

Days

Myosin formation 4 - 34 days

Fig. 1. Synthesis of myosin in regenerating hind limb tissue of Triturusirradiated by X-rays with doses from 20 r to 2,500 r.

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

About 3,500 animals, at various stages of the recovery progress in thelimb tissue after amputation at the knee, were exposed to single doses of
X-rays of 20 r, 50 r, 200 r, 500 r, 1,000 rand 2,500 r and the examination for
the first detectable trace of myosin in the regenerating tissue was carriedout 3, 9, 15, 18, 21, 24 and 27 days after the operation. The antigen forthe preparation of anti-myosin serum by injecting into rabbits was isolated
from the skeletal muscle of adult Triturus. The titer of anti-serum wasadjusted to 1: 512 before using the precipitation with saline extracts ofthe granulated tissue.
In the non-irradiated control group, myosin first becomes detectable in
the regenerating tissue 27 days after operation (OGAWA, 1958). The timeof myosin formation in the irradiated group is shown in Fig. 1. A doseof 20 r given 3 days after amputation results in one day earlier myosinformation. When a heavy dose of 2,500 r was given 24 days after operation, the synthesis of myosin was remarkably inhibited and its formation

27
2500r.
24

21

IB.,on-i~radiated

9

3
1000r

4

500r
2500r

6

~Or

200r

12

16

20

24

26

32

Days

Days for muscle formation

Fig. 2. Chemo-differentiation of muscle tissue in regenerating limb tissue of
Trituru8 when X-irradiated with 20 r to 2,500 r.

RESEARCHES CARRIED OUT IN 1964

129

was first recognized on the 34th day after amputation.
The curves in Fig. 1 show a similar tendency as was found for myosinformation in early embryonal stages. These results suggest the presenceof two factors sensitive to X-rays in myosin formation during tissue
regeneration as well as in early development. One (R-Am) suppresses
the synthesis of myosin and the other (R-Bm) markedly promotes it after
X-irradiation. The inhibiting factor R-Am does not react to X-rays until
doses over 1,000 r are applied, but factor R-Bm is sensitive to X-rays andis detectable at the dose of only 20 r. The most effectful irradiation
time is the 24th day after amputation for factor R-Am and the 9th day
for factor R-Bm.
The time of complete actin and myosin synthesis in regenerating muscle
tissue of X-irradiated Triturus is graphically represented in Fig. 2.

F.
102.

RADIATiON GENETICS OF PLANTS

Differences in effects of r-rays and fast neutrons from
Po-Be source on einkorn wheat and paddy rice
Seiji MATSUMURA

Fp.st neutrons were obtained from a Po-Be source. The nuclear
reaettion is ~Be(a, n)l~C, and 2~~PO, whose half life is 138 days, is used as
an a-emitter. The resultant neutrons are emitted as discrete spectrum withthe maximum energy of 11 MeV and a mean energy of approximately
7.2 MeV. Dry seeds were attached to the surface of a stainless steel
cylinder of 10 cm diameter, which contained the Po-Be neutron source(210po 10 c) in the center.
The irradiation dose, preliminarily applied to einkorn wheat seeds,
ranged from 24 to 125 rad for 3--15 days. At the same time r-radiations
with I37CS 6,000c were used for comparison at 5--15kr and 20kr/hr.
Fast neutrons at 24--125 rad were not markedly effective in inhibiting
seed germination and seedling growth and decreasing survival in thefield or seed fertility in XI, but were for chlorophyll mutation effective
eve'11 at 75 --125 rad. It is assumed that these neutrons are more effective
than 14 MeV neutrons, and the RBE value for chlorophyll mutation
frequency is about 40 against r-rays.
Further, dry seeds of paddy rice were subjected to fast neutrons
from the same Po-Be source at 158,....,440 rad for 2--10 weeks. For
comparison r-rays were used at 5 -- 25 kr and 10 kr/hr. In this experiment also no clear inhibiting effects were noticed on seed germination,

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15- - r'rays - - neufrons

9

.,

0

90

Se~d ferTIlity~_i :~:;:/. ::
6

•

!I 5

60~
~

soi

2

•
001,00=-~5-.L-_---l+'15~--L--~25
158
282
364
440
'( ray or neutron (po) doseJ(kr)
( rod)

Fig. 1. Effects of neutrons (Po-Be) and r-rays on seed fertility (1963)
and chlorophyll mutation rate (1964) in rice.

seedling growth and survival in the field in the neutron-treated lots.
Fig. 1 shows the relation between doses of r-rays (kr) and fast neutrons(rad) and seed fertility or chlorophyll mutation frequency. From thefigure RBE of the fast neutron from Po-Be source against r-rays becomesnear 20 for seed fertility and mutation frequency. This value is also
higher than that of 14MeV neutrons.

103. Differences in effects of r-rays and fission neutrons
in the polYPloid wheat series
Seiji MATSUMURA

Dry seeds of Triticum monococcum flavescens, T. durum Reichenbachii
and T. vulgare erythrospermum were subjected on Oct. 21, 1963, tofission neutrons from the ORNL Health Physics Research Reactor, andr-irradiation from 60CO source was also carried out simultaneously in
Oak Ridge National Laboratory by courtesy of Dr. J. A. AUXER. Intensityof r-rays in this series was 175 kr/hr. Neutrons were given for 804
seconds to the seeds at various distances from the center of the neutron
reactor. The seeds of T. monococcum received doses of 103--1,027 rad
and those of T. durum and T. vulgare doses of 257,..., 2,057 rad. There wasapproximately 10 per cent additional exposure due to r-rays associatedwith the neutron field.

131

RESEARCHES CARRIED OUT IN 1964

The data for germination, seedling growth and seed fertility are given
for fission neutrons and r-radiation in Table 1. In June (harvest season)of 1963 we had unusually abundant rainfall. Therefore the seeds were
not good and germination was generally low. The higher was the doseof fission neutrons and r-rays, the more delayed were germination andgrowth of seedlings, and the more reduced was seed fertility. In general,
T. monococcum was the most sensitive to fission neutrons and r-rays.
There was as expected no significant difference between T. durum andTable 1. Effects of fission neutrons and r-rays on germination,
seedling growth, fertility and mutation in wheat.
Species

T. monococcum
jlavescens(n = 7)

Dose(%)

Chlorophyllmutationrate (%)

Control63

0.00

N-103 rad
N-257
N-514
N-770
N--1.027

58
59
39
40

3.04
6.52
11.07
9.60
17.19

r-5 krr-- 10
r- 15

59
52
42

5.22
8.38
7.58

ControlT. durum
Reichenbachii
N-257
rad
(n = 14)N-514
N-1,027
N-1.543N-2,057

63

72

0.00

54
70
24
30
32

2.54
3.25
10.87
7.14
23.81

36
28
24

0.00
0.00
14.29

Control

96

0.00

N-257 radN-514
N-1,027
N-1,543N-2,057

88
88
86
92
80

0.00
0.00
0.24
0.00
0.00

r- 10kr

86
88
86

0.00
0.00
0.00

r-10 kr
r- 17 . 5r- 25
T. vulgare
erythrospermum (n=21)

Germinationrate

r- 17 . 5
r-25

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

T. vulgare. The frequency of spike progenies with chlorophyll mutationsin X 2 was also investigated, as shown in the last column of Table 1.
As expected, the higher was ploidy, the lower was mutation frequency.
Only one albina mutation was found in hexaploids.
The RBE values of different characters were calculated roughly from
Table 1. For seedling height the RBE value of fission neutrons to thator r -rays was found to be 10 --15. It was calculated for seed fertility
as about 10 for diploids and as about 50 for tetra- and hexaploids andalso for chlorophyll mutations as 25 or more in di- and tetraploids. In
general, the RBE value was lower for the characters observed in earlier
stages than at maturity, especially in polyploids.
104. Differences in effects of r-rays and 14 MeV
neutrons on einkorn wheat
Seiji MATSUMURA
Dry seeds of Triticum monococcum were exposed in August, 1963, tomonoenergetic 14 MeV neutrons from T(d, n) reaction neutron generator
in Hiroshima University by courtesy of Dr. H. YOSHINAGA. The neutron
intensity was 9 X 109 neutrons/sec on average for 94 -- 281 min, excluding
r-ray contamination of less than 10 per cent. For comparison r-ray
exposure was carried out simultaneously with 3.1 kr/hr for 102 -- 510 min
with the 6,000 c mcs source in our institute.
Germination, survival in the field, seed fertility and chlorophyllmutation were investigated. The results of the present experiment are
roughly in good accord with those of our earlier works (MATSUMURA, 1961,1964) with 14MeV neutrons obtained from 3H(d, n)4He reaction producedin the 250 kV Cockcroft-Walton accelerator in the Biology Division, Oak
Ridge National Laboratory. The data summed up from both experiments
are given in Table 1. The RBE of 14MeV neutrons to that of r- andX-rays was calculated from the table. It was found to be about 10 for
germination rate showing a 10 % depression as compared with thecontrol and about 13 for seedling height showing a 25 % depression.
Also the RBE value was calculated as about 15 for seed fertility showing
a 30 % depression and for chlorophyll mutation rate amounting to 5 %.
It was generally lower for characters observed in earlier than in laterstages, at maturity.
According to the above mentioned experiments with fission and fastneutrons from Po-Be, it is assumed that they are more effective than
14 MeV neutrons, and also that fast neutrons (Po-Be) are clearly themost effective. In general, the RBE of fast neutrons is clearly larger

RESEARCHES CARRIED OUT IN 1964

133

Table 1. Effects of 14 MeV neutrons and r- or X-rays on germination,
seedling growth, fertility and mutation in einkorn wheat.
Dose

Germination(Index)Seedling
length(Index)

Survival(Index)Seed
fertility(Index)Chlorophyllmutationrate (%)

Control

100.0

100.0

100.0

100.0

0.00

N-0.5 krad
N-1.0
N-1.5
N-2.0
N-2.5

99.1
95.0
87.3
74.2
74.0

87.6
63.1
42.7
24.7
6.6

87.3
80.8
61.3
2.1
0.0

63.0
56.0
26.4
8.6

4.68
14.36
5.48

r~5 kr

108.1
92.3
83.8
64.8

90.6
76.6
53.9
28.4

89.1
82.4
86.7
34.9

76.2
61.8
56.8
64.2

4.00
6.98
4.81
9.30

r- or X-I0
r- 15
r- or X-20

for higher plants than for animals and microorganisms, and it increases
with increasing LET (linear energy transfer). The extraordinary large
RBE values for mutations in higher plants strongly indicate, as also
other evidences do (STADLER, 1932, 1954; MATSUMURA et al., 1963; KONDO,
1964), that the majority of ionizing-radiation induced mutations are due
to chromosome aberrations or gross deletions.

105. Effects of 14MeV neutrons in T. monococcum
Taro FUJII

F 1 seeds from the cross between the normal type and a chlorina mutant
were subjected to r-rays and 14MeV neutrons, as in the previous
experiment (FuJII, Annual Report No. 14, 1963). 8,600 rad of r-rays andup to 900 rad of neutrons had no apparent effect on germination rate.
Survival rate in the r-ray lot was slightly lower and was in the threeneutron lots slowly decreasing with increasing dosage. A similar tendency
was also observed in the average number of tillers, but it was not marked.
Somatic mutation rate was calculated on spike basis. As shown in
Table 1, the frequency of tillers with chlorina stripes increased withincreasing neutron dosage. The frequency of mutated tillers in thehighest dosage lot was 0.79 %; it was twice as large as that of thelowest dosage lot, and that of 8,600 rad of r-rays was about one half aslarge as that of the 324 rad neutron lot. An almost linear relation wasobserved between mutation frequency and neutron dosage, but the
mutation rate at higher dosages was too high for linear relation in the134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table 1.

Frequency of somatic mutations.
..

Survival
Treatment No. of Germination rate
rate
seeds
(krad)(%)(%)
Controlr- 8 ,600
n-324
n-661
n-900

239
400
399
399
399

44.8
50.2
50.6
50.1
43.6

87.9
83.2
77.2
76.2
73.0

~---_._-~.

Total A vera~e no. I Chlorina stripes
no. ofof spikes '--NO:-- of
No. ofspikes
per plant I plants~~p!k_~%)

r

3,228
6,101
5,617
4,806
3,902

34.3
35.3
36.0
31.6
30.7

i
I

0
6
13
11
11

11 (0.18). 22(0.39)~ 25(0.52)
31 (0.79)
1~---_._---

r-irradiation experiments. From the result, we may say that somatic
mutation is caused, for the most part, by chromosome aberrations.
Decrease of fertility was marked with increasing dosage. When thefertility was compared between mutated and non-mutated tillers withineach individual, average fertility of the former was lower than that of
the latter. These facts also support the view that most of somatic
mutation arises from chromosome aberrations.
Average number of mutated tillers per individual roughly increased
with increasing dosage, as the result of repeated occurrence of the samekind of mutation within the same individual.

106. Lethal effects of heavy ionizing particles in Arabidopsis
Taro FUJII

Heavy-ion linear accelerator (HILAC) is especially important in radiation
biology because accelerating beams of He-, Co, N- or Ar-ions, etc., canbe obtained from this machine. As the space age rapidly approaches,
information on the application of these beams to plants could be interesting to space biologists, such ions being abundant in the cosmos. Dry
seeds were subjected to 1--5 krad of He- and C-ions respectively, and
3 and 5 krad of Ar-ions from the HILAC machine with the energies of
10.4±O.2MeV per nucleon by courtesy of Dr. J. LYMAN, University ofCalifornia. Survival rate decreased markedly with the increase of dosage,
especially in the treatment with He-ions; in 5 krad lot it was reduced
to about one half of that of the control lot. Decrease of survival rates
in C-ion lots was not so severe, amounting to 66 per cent in the 5 kradlot. On the other hand, 3 and 5 krad treatments with Ar-ions showeda slight decrease of survival rate while their LET was strikingly higher
than with the other ions. RBE values increased with the increase of
LET and the peak of RBE was observed at about 70--100keV/.u of LET.

RESEARCHES CARRIED OUT IN 1964

135

The RBE decrease with increasing LET may be interpreted as follows.
The number of ions in the tissue increases with increasing LET. Buta certain number of ions in the given tissue is sufficient for producing
biological damage, and those produced in excess at a very high LET
range become ineffective and therefore the RBE values slow down. Thismay have caused a low effect in the Ar-ion lot. From the results, RBE
values for the killing effect seemed to be about 20 for He-ions, about 15
for C-ions and less than 10 for Ar-ions, when r-rays were used as
standard. These results are in good accord with those reported concerning RBE versus LET relationship.
Growth in the lots irradiated by those heavy ions was very bad, many
plants became dwarf and often had deformed leaves and branching which
occur seldom in r-irradiations. The facts suggest that the accelerated
heavy ions act not only on the nucleus or chromosomes but also on thetissues. It is known that the RBE values of neutrons were around 15
when they were examined for chromosome aberrations or somatic mutations, and their LET's were almost similar to those of He- and C-ions.
In my previous experiment, dosage up to about 5 krad of neutrons
had no effect on survival rate and caused no severe alteration of growthhabit (FuJII, Annual Report No. 14, 1963). A decrease in survival rateshould have been expected in the neutron irradiations because similar
dosages were applied both in experiments with neutrons and heavy ions.
This discrepancy awaits further investigation.

107. Effects of dose rate and fractionation of r-rays
on mutation in maize pollen grains
Tomoo MABUCHI

Mature pollen grains of a dominant line (Su) of maize were irradiatedby 137CS r-rays and dusted on the ears of a recessive line (su). Mutations(Su~su) were detected in the kernels developed from the fertilization by
the treated pollen. The results are shown in Tables 1 and 2.
The same dose was given to pollen grains at 6 different dose rates of
4-917 r/min for 1.2-275 min (Table 1). There was no significant
difference in the frequency of partial endosperm mutation between
different dose rates. But whole mutation was considerably higher athigher dose rate (more than 36 r/min) than at the lower rate (less than
15 r/min). A dose of 1,100 r at 917 r/min produced twice as many wholemutations as the same dose at 15 r/min. Total mutation frequency
decreased exponentially with decreasing dose rate and reached constantlevel. The data clearly show that there must be a threshold for a dose

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table l.

Effects of dose rate on mutation. (dose: 1,100 r)

Dose rate(rjmin)Duration of
exposure(min)

No. of
seeds
set

Whole

Partial

Total

917
250
125
36
15
4

1.2
4.06
8.12
30.66
73.31
275.0

2,869
1,502
1,655
2,454
1,861
2,010
2,314

0.03
2.26
2.48
2.36
1.67
1.19
1.30

0.06
0.73
0.48
0.81
0.70
0.65
0.52

O.10±0.06
3.00±0.44
2.96±0.42
3.18±0.35
2.36±0.35
1.84±0.30
1.82±0.28

Table 2.

Effects of storage and radiation fractionation on mutation.
(dose: 1,I00r, dose rate: 917rjmin)
Duration ofstorage orinterval (min)Single irrad.

Fractionation

Mutation frequency (%)

Mutation frequency (%)

No. of
seeds
set

Whole

Partial

Total

2,869

0.03

0.06

0.10±0.06

10
30
60

4,464
4,254
4,170

2.37
2.33
2.45

0.58
0.63
0.50

2.96±0.25
2.96±0.26
2.95±0.26

10
30
60

3,782
4,944
3,133

1.82
1. 76
1.56

0.42
0.40
0.41

2.25±0.24
2.16±0.21
1.98±0.25

11

required to raise the mutation rate.
A dose of 1,100 r at acute irradiation was applied to the pollen grains.
After irradiation they were stored in the r-room (23°C) for 10, 30 and 60
minutes and then dusted on the ears. A mutation rate of nearly 3 %was found in these 3 lots (Table 2). It is clear that there was no storage
effect on mature pollen grains.
Fractionated doses (each 550 r) were given twice with intervals of 10,
30 and 60 minutes. Partial mutation frequency was not significantly
different among 3 irradiated lots, but whole mutation slightly decreased
with increasing interval (Table 2). Therefore, the decrease in mutation
frequency was not due to storage effect but to restitution of chromosome
breakages.

137

RESEARCHES CARRIED OUT IN 1964

108. Photoreactivation of ultraviolet-induced mutation in maize
endosperm due to pollen irradiation
Mituo IKENAGA and Tomoo MABUCHI

The reversal of ultraviolet-induced damages by subsequent exposure
to visible light has been observed in various organisms over a numberof years. The phenomenon of photoreactivation is of great importanceto radiobiology, for it might be a specific process revealing the mechanismof ultraviolet (UV) effects. So far as photoreactivation of UV-induced
mutation is concerned, investigations have been scarecely carried out inhigher plants. In the present study an endosperm maize mutant, su,
was used. The pollen was irradiated in a single layer in a Petri dishwith UV of 254 m,u at a dose rate of 90.3 ergs/mm 2 /sec. Immediately after
UV exposure, the irradiated pollen
1.0
was divided into two halves, one
Dark
PR
o
whole ;
was re-spread on a Petri dish and0.9
partial : () , <D
exposed to visible light from 20 W
0.8
Toshiba fluorescent lamp for 30 min,,_<D---_
at the distance of 10 cm (PR treat0.7"'~ .....
ment), and the other half was held
"
/'
~
0.6
for 30 min in darkness (dark treatment). After PR or dark treatment,"6 0.5•the pollen was dusted on the silks,~of the recessive line, and the polli•\
nated silks of the dark treatment
03
i~()group were wrapped in aluminumr/foil as soon as possible in order too.2() "I,' ()
shut out the sun light.
OJ ~0/
Induced whole and partial mutants"o L .-o''----'---'--"'--~----'"-.........~(chimeras) were scored separately,oI2and the obtained mutation rates are
UV Dose ( 10 ergs;mm
given in Fig. 1. As seen from theFig. 1. Curves of whole and partial
graph, mutation rates of wholemutation rates versus UV dose withmutants were considerably decreased
or without photoreactivation.
by exposure to visible light. On theother hand, partial mutation rates were slightly increased by PR treatment. In a parallel experiment with pollen irradiated with 1,000 r of
r-rays, the same PR treatment was completely ineffective. From theobtained data and considering Ito's case with yeast (ITO et al., 1964) it
may be assumed that partial mutation may be caused by an UV-induced
alteration in one chromatid at Su locus and whole mutation by the~Ueration in two chromatids. since meiosis Occurs about ten days before.

" ,c;.

."

0,4

.

3

2

)

138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

UV irradiation. The fact that whole mutation is reversible by visible
light more frequently than the partial one suggests that photoreactivation
may proceed in some way from whole to partial mutation and from there
to normal endosperm. Finally, we may at least conclude that photoreactivation of mutation in higher plants really exists, though it is notcertain which source of visible light, fluorescent or natural daylight, areeffective for photoreactivation.

109. Effects of temperature on post-irradiation
storage in einkorn wheat
Tomoo MABUCHI

It is well known that post-irradiation storage increases radiation damagein X- or r-irradiated seeds, especially in super dry seeds (moisture: 4-8%).
Recently, MATSUMURA et at. (1961) reported that chronic irradiation was
more effective in producing radiation damage than acute treatment indry seeds of upland rice. However, they could not find such a relationship in seeds of einkorn wheat. An experiment was carried out to test
whether or not a storage effect exists during chronic irradiation of seedsof einkorn wheat.
Dry seeds of einkorn wheat were irradiated with 10 and 15 kr r-rays
at acute dose rate of 10 kr/hr and stored in constant temperatures ofooe, 13°e and 30 e for 436 hours. Furthermore, some of the treatedseeds with 15 kr were also preserved at 13°e for 138 and 700 hours. All
treated seeds were sown almost simultaneously, just after storage. The
0

Relation between post-irradiation temperatureand radiation damage.

Table l.

- ~ - _ .

Dose
(kr)

Storage
(hrs)

0

10

436

H

H

H

H

H

H

15
H

700
436

H

H

H
H
H

H

138
0

Temperature(0C)

Seedling height(em) (Index)

Seed fertility(%) (Index)

13

9.4 (100.0)

72.1 (100.0)

0
13
30
13

9.3 ( 98.9)
8.8 ( 93.6)
8.6 ( 91. 4)
9.2 ( 97.8)

66.5 ( 92.2)
65.6 ( 90.9)
60.1 ( 83.3)
66.3 ( 91.9)

13
0
13
30
13
13

6.7 ( 71. 2)
8.4 ( 89.3)
7.1 ( 75.5)
7.0 ( 74.4)
7.7 ( 81. 9)
7.8 ( 82.9)

51.5 ( 71. 4)
58.5 ( 81.1)
49.1 ( 68.0)
48.6 ( 67.4)
57.9 ( 80.3)
63.1 ( 87.5)

RESEARCHES CARRIED OUT IN 1964

139

data for seedling height and seed fertility in the Xl-generation are shownin Table 1.
There was found a slight radiation damage in acute 10 kr irradiation.
The seedling height and seed fertility were significantly reduced withprolongation of storage, when the irradiated seeds were stored at 13°C
after 15 kr irradiation. The results show that there was a clear storage
effect.
There was also a clear storage effect on seedling growth and seedfertility of r-irradiated seeds, stored at 13°C and 30°C for 436 hours
after 15 kr irradiation but no clear storage effect was found in seeds,
stored at O°C for 436 hours.
The results of this experiment show that storage effect was greatly
influenced by irradiation dose, temperature and storage duration. It istherefore considered that storage effect is noticeable if seeds are irradiatedchronically, but the effect may be often masked due to modifying factors,
i.e. temperature or water content.

110. Oxygen and storage effects on radiation damage
in rice and einkorn wheat seeds
Tomoo MABUCHI and Seiji MATSUMURA

1. RiceDry seeds of paddy rice with 12.0 % moisture were sealed in ampules
filled with oxygen, nitrogen and air, and were 120 hours after sealing
exposed to r-rays from mcs 6,000 c at 10 and 20 kr with dose rate 10 kr/hr.
Irradiated and unirradiated seeds were sown immediately after irradiation,and growth of seedling was investigated. Seeds in oxygen showedslightly lower growth of seedlings at 0, 10 and 20 kr than those in air.
On the other hand, seeds in nitrogen had similar growth of seedlings
at 10 kr and showed a slight protection effect for inhibition of seedling
growth at 20 kr, as compared to those in air.
Further, seeds similarly sealed and irradiated were stored in underground-room temperature (19-22°C) for 576 and 1,056 hours. Other
pretreated seeds were stored after similar sealing and irradiated in similar
conditions. The data are shown in Fig. 1. Pre-irradiation storage showeda slightly lower effect in inhibiting seedling growth in oxygen and air
treatments than post-irradiation storage, especially at 20 kr. On the other
hand, nitrogen treatments were generally more protective for radiation
damage as expected, than air and oxygen ones. Further, the relation
between pre- and post-irradiation storage in nitrogen seemed to be in
the reverse, compared to the results of oxygen and air treatments.

140 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

c:::J

14

_

IOkr or 20kr + no storooe

10k, or 20kr'" I056h.... storage~ 10k'''' 576 houn storoQO

12
~

10

E

u

~

8

f... 6
.5
~

~ 4
2

Air

AirO.N.

Fig. 1.

O.

Air

N.

O.

N.

20kr

10kr

Cont.

Oxygen!and storage effects on radiation damage in rice seeds (1964).

2. Einkorn wheat
Dry seeds of Triticum monococcum fiavescens containing 13 % water
were sealed in ampules filled with oxygen, nitrogen and air, and exposed
to 10 and 15 kr r-rays. For acute and chronic irradiation dose rates of
10,000 r/hr with l31Cs and 19.6 r/hr with 60CO were used, respectively.
~

Acute irrad. +504 hours storage
Acute irrad.+762 hours storage
c:::J Acute irrad. + storage
IT%i'.&J Chronic irrad. +no storage_(Ac. irrad.
(Ch. irrad.

10,000r/hr)
19.6r/hr)

10

E

8 f-

u

-

"'
0- 6

'0;

.c

~

4

'0

'"'"

(f)

o '-..1.-1'-'---'-_
Air 02 Nz

ConI

Air

Air

15 kr

Fig. 2. Oxygen and storage effects on radiation damage in einkorn wheat
seeds (1964).

RESEARCHES CARRIED OUT IN 1964

141

Two chronic irradiations (10 and 15 kr) were terminated simultaneously
before sowing. Acute irradiations were given at the time coinciding
with the beginning and termination of the chronic ones. The date forinhibition of seedling growth are shown in Fig. 2. In general, chronic
irradiations were found to be more effective than acute ones, especiallyat higher irradiation with 15 kr. There were no clear differences between
air and oxygen treatment either in acute or chronic irradiation. On theother hand, nitrogen treatments clearly showed a protective effect on
the inhibition of seedling growth, especially in acute irradiations.
Unexpectedly, post-irradiation storage had no effect on the inhibition,
also in acute irradiations.
These results might be explained by the relationship between the
production and decay of free radicals and radiation damage. It is assumed
that nitrogen treatment decreases the radical yield, and the production
and decay of the radicals are more gradual and slower in chronic thanin acute irradiation.

111. Radio-sensitivity in pollen grains of Triticum and Aegilops
Tomoo MABUCHI and Seiji MATSUMURA

Relation between radio-sensitivity and ploidy or genome constitution
was investigated in several species of Triticum and Aegilops. Anthers
before dehiscence were irradiated by X-rays (173 kVp, 25 mA, 1 mm Al
filter) at 1, 2 and 3 kr with dose rate of 200 r/min. Pollen grains were
dusted on the emasculated florets of the same species. Seed set, mean
grain weight and germination were recorded.
Seed setting was noticeably impaired even at 1 kr and reduced to·24---75 % and 3.1---25 % of control at 2 and 3 kr, respectively. Germination rate also decreased to 35---88 % of control at 1 kr and was markedly
affected (3.3---22.5 %) at 2 kr. Percentage of viable seedlings (per centsuccess of crossing) was calculated from seed settingxgermination rate.
It was for diploid Triticum and Aegilops species about 25 % of control
at 1 kr, and for several species of tetra- and hexaploids it was ranging
from 30 to 55 %. It is, therefore, concluded that diploid species aremost sensitive to radiation, T. monococcum as well as Ae. squarrosa, butthere is no clear difference between tetra- and hexaploid Triticum species.

142 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

112. Radio-sensitivity of aged wheat seeds
Seiji MATSUMURA and Taro FUJII

Difference in radiation susceptibility between aged and fresh seeds
was examined. 7.5 and 15 kr were applied to seeds of diploid wheat.
No germination was observed in the control lot of 2 year old seeds, andwe had no seedlings for irradiation. Non-irradiated 1 year old seeds
showed very low germination rate; a few seeds germinated in the 7.5
kr lot and no germination was observed in the 15 kr lot. Unfortunately
germination rate of fresh seeds was as low as 60 %; 7.5 kr of r-rays
did not much reduce it (54 per cent), and a few seeds germinated in
the 15 kr lot. Thus, the decrease in germinability by irradiation of 1
year old seeds was in the whole series markedly lower than that of fresh
seeds. A similar tendency was also observed in seedling length which
was measured 14 days after sowing, and the decrease of survival ratein April was also marked in the 1 year old series as compared with thatof fresh seeds.
10 and 20 kr of r-rays were given to tetra- and hexaploid seeds taking
into consideration their higher resistance to radiation than that of diploidseeds. Germinability of 2 year old seeds was very low in both, tetraand hexaploid lots, and no germination was observed at 20 kr irradiation.
But germination rate of control lots was better for younger than for
older seeds. This trend was marked in irradiated lots. For instance,
germination rate in 20 kr lots of 1 year old tetraploid seeds was 11.4 %amounting to index 55 when the index of 100 is given to the germination
rate in the control lot. On the other hand, 68 % germinated in the 20
kr lot of fresh seeds, the index being 73 against that of the control.
Seedling length and survival rates also showed a similar tendency. In
the hexaploid species, a similar decrease of germinability was observed
both in irradiated fresh and in 1 year old seeds. Namely, sensitivity
was almost the same in fresh and in 1 year old seeds of this species
whereas 2 year old seeds had higher sensitivity. From the results, it
can be said in general that older seeds have higher radiation susceptibility
than younger ones.
The decrease in germinability due to age was largest in diploid wheat
and smallest in hexaploid wheat. We may assume from the resultsthat at a higher ploidy level, we may expect a higher stability not only
in respect to radiations but also to the physiological changes caused byaging.

RESEARCHES CARRIED OUT IN 1964

143

113. Killing effect of UV-rays in Arabidopsis
Taro FUJII

Effects of ultraviolet-rays of 253.7 m,u on Arabidopsis seedling were
examined. Seeds were sown on wet filter paper in Petri dishes. 200
seeds were used for each lot. Experiments were carried out undercontinuous artificial light of about 1,000 lux in an air-conditioned roomat 25±2°C. About 5 mm long seedlings were exposed to 1-100 min of
90.3 ergs/mm 2/sec intensity and were kept in a dark room for 24 hours
for the elimination of photoreactivation effect or UV effect fixation.
Killing effectiveness of UV-rays was very high; about half of the treatedseedlings died at 10 min treatment and almost all died out at 100 min
treatment as shown in Table 1. Later, irradiated seedlings were placed
Table 1.

Killing effectiveness of UV-rays.

Per cent of survivals
Exposure time in min - - - - - - - - - - ; - - - - - - - - - - I With dark treatment !Without dark treatment

I

o

I

94.5
42.1

100.0

1

3

17.8

5

13.6
25.1
11.1
15.5
23.5

40.8
15.9

7

10
20
30
50
100

8.7
5.5

98.4

30.8
25.7
38.1
34.3
33.2

in the experimental room in light just after UV irradiation for examination of the photoreactivation effect. Survival rate became in all lots
better than in dark treatment; about 30 % of seedlings survived even in
the highest dosage lot. By this procedure a photoreactivation effect wasrecognized.
I do not know whether or not the severe killing effect coincides with
the induction of gene mutation, because the stem of young seedlings isvery soft and transparent and it seems that UV-rays act on the stem
rather than on the growing point. These questions are now examinedin experiments with heterozygotic material.

144 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

114. Effect of EMS on germination of einkorn wheat
Taro FUJII

Very high mutagenic effects of ethyl methane sulphonate (EMS) werealready reported by many researchers. Normal seeds, and heterozygotic
seeds from the cross between chlorina and normal green in Triticum
monococcum fiavescens were used in this experiment. The aim of thisexperiment was to determine the differences in mutagenic effect for a
specific gene and the difference in the appearance of mass mutation
between r-ray and EMS treatments. For two hours steeped seeds weretreated with 0.1, 0.3 and 0.5 % EMS solutions for 22 hours. For comparison, for 24 hours steeped seeds were subjected to 0.5, 1.0 and 1.5 kr
of r-rays. Moreover, EMS and r-ray treatments were combined to examine
the synergistic effect of both treatments. The treatments were so combined that 2 hours steeped seeds were placed in EMS solution for 22hours and r-rayexposure was done after 5 min washing with tap water.
Steeping and EMS treatments were done under room condition at 20°C.
r-rays had almost no effect on germination rate of the normal strain,
but the F 1 hybrids were affected. In EMS treatments, germination rate
in both strains decreased with increasing concentration of EMS, moremarkedly in the F 1 lot, as shown in Table 1. (Of course, germination
Table 1. Germination rates of r-ray and EMS
treatments of normal and F 1 seeds.
Treatment
r-ray 0.5 kr
1.0
1.5
EMS 0.1%
0.3
0.5
EMS 0.3 % +r-ray 1.0 kr
EMS 0.5 % +r-ray 1.0 kr

Normal

F1

86
86
88
91
80
70
78
52
90

54
50
46
50
48
32
38
14
78

rate of F 1 seeds was a little inferior to that of the normals because
they are usually smaller than normal seeds.) Moreover, combination of
EMS and r-ray treatments showed severer killing effect than each single
treatment; about 50 % and only 14 % seeds germinated in the normal
and the F 1 batch, respectively in the EMS 0.5 % and r-ray 1 kr treatment
lot. Almost all seedlings died out in these lots. Additive or synergistic
effects of both treatments are assumed. The appearance of chlorina
stripes due to somatic mutation is now under examination.

RESEARCHES CARRIED OUT IN 1964

145

115. Developmental-genetic study on culm internodes, leaf sheaths
and leaves in irradiated progeny of barley
Genkichi TAKEDA and Kan-Ichi SAKAI

Three groups of short-culm mutant lines derived from X- and r-rayedseeds of three barley varieties were investigated for six internodes, twoupper leaves and their leaf sheaths. Examined was the main culm ofeach plant. The lines were in generation X s or X 6 and the number oflines was 10 to 16 in each group.
Analysis of variance showed that variation among lines were found to
be statistically significant in all characters. It was also found in caseof internode length that interactions between internode position and line
were statistically significant.
Genetic correlations among internodes inter se, internodes and leaf
sheaths, and also leaf blades and leaf sheaths were investigated. Although internodes were more or less mutually correlated genetically,
genetic correlations between two adjoining internodes tended to be higher
than those between two distant ones, and particularly genetic correlations
between second and third, and third and fourth internode length werehighest in all line groups. Genetic correlation between leaf sheathlength and the corresponding internode length was high, about 0.7""'0.9,
whereas leaf blade was found to behave in another way: the genetic
correlation between leaf blade length and either leaf sheath or internode
length was so low as 0.4,...,-0.4. It is concluded that genes responsiblefor internode and leaf sheath formation should be to a certain extent
the same, whereas those responsible for leaf blade formation are different
from them.
Developmental instability in the length of internodes was investigatedon the basis of intraline interplant variation. It was observed that the
instability was different among lines in every group, and that the interactions between internode position and line were also statisticallysignificant in two groups. Of special interest was that the third andfourth internodes were highly stable while the upper as well as thelower ones were unstable.
Findings from this study are summarized as follows:(1) The same genes are likely to govern formation of culm and leaf
sheath, but they are different from those controlling leaf blade formation.
(2) Internodes in central position of a culm are developmentally highly
stable while upper and lower ones are unstable. It means that, in somesense or other, the central parts of a culm are developmentally highly
buffered, which deserves a further investigation.

146 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

116. Effect of gamma-ray irradiation and maleic hydrazide treatment
on the PH 7.5 esterase of corn seedling
Toru ENDO

One of the first detectable biochemical changes in germinating seedlings
as a result of irradiation of dormant seeds is an increase in activity ofa number of enzymes. This condition holds for the acidic esterases inmaize but not for the pH 7.5 esterase. In this study dry corn seeds
were irradiated with 50 kr gamma-ray using a cobalt-60 source, germinatedin the dark at 30°C, and changes in activity of the various esterases incrude seedling extracts during growth were determined by comparisonof zymograms developed following starch gel electrophoresis. Analysis
was made of extracts from the first internode and the coleoptilar region(which included the young leaves) of 4 through 9 day-old seedlings.
Growth inhibition in irradiated material became strikingly apparent 5
days after germination. The esterase levels in 4 day-old irradiatedseedlings were not different from the control. By 5 days the acidic
esterases increased greatly in activity in the first internode and theincrease presented through the 9 day material. The pH 7.5 esterase
did not show this increase and its level fell off during growth parallelingthe decrease in the control seedlings. On the other hand, in the coleoptilar region, the acidic esterases of the irradiated material remained
at about the same level as the control but the pH 7.5 esterase showeda marked decrease in activity with age relative to the control. Thus
the levels of the acidic and the pH 7.5 esterases are effected differently
by irradiation in the two plant parts and in no case did the irradiation
cause an increase in the pH 7.5 esterase. This is an interesting finding
since the pH 7.5 esterase in maize is associated with active growth.
Soaking of dry seeds in a 0.01 M solution of maleic hydrazide for 24hours decreases growth of the seedlings to about the same extent as 50 kr
gamma-rays. However, the esterase levels in the seedlings from thetwo treatments were very different. The acidic esterases were not increased in the first internode in maleic hydrazide treated material andthe pH 7.5 esterase level remained high in both tissues even in theolder seedlings

RESEARCHES CARRIED OUT IN 1964

117.

147

Dose-rate distribution in the 137CS gamma-greenhouse
Takesi KATOll and Mituo IKENAGA

The facility for continuous and extremely low dose rate r-ray irradiation
with 137es 40 c, the so-called gamma-greenhouse, roughly air conditioned,was built in March, 1964. The dosimetry of the r-room was carriedout by Victoreen condenser chambers, air saturated Fricke dosimeterand glass dosimeter. The measured value of the effective source intensity was 34.6 c (Sept., 1964) for the Victoreen 2.5 r (r) condenser chambers.
The relation between the dose rate and distance from the source isshown in Fig. 1 for the Victoreen condenser chamber readings.

Fig. 1.

Dose rate distribution in r-greenhouse.
(Measured in Sept., 1964)

1) Visiting researcher from Department of Fundamental Radiology, Faculty of
Medicine, Osaka University.

148 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

118. Experimental study on depth dose curveon 60Co r-ray irradiation
Takesi KATO and Sohei KONDO
To confirm the theoretical estimation of the absorbed dose in thesurface region of materials exposed to high energy photons presented
in The Annual Report No. 11, experimental studies of the depth dose
curves for 60Co r-ray of various materials have been carried out. In
order to measure the true depth dose curve it is necessary to eliminate
the external secondary electrons originating from the material outside
the dosimeter, since they affect depth dose curve as mentioned in theprevious report (KONDO et al., Annual Report No. 11, 1960). The parallel
plate ionization chamber used in the measurements has a very thin front
wall and the back wall is thick enough to give an electronic equilibriumfor 60CO r-rays. The relative depth dose curves were determined bymeasuring ionization currents for varying front wall thickness while
keeping the back wall thick enough to give a maximum reading. The
front wall thickness was varied by adding thin disks of appropriatematerial to the thin front wall. Materials used for the wall were polystyrene, aluminum, copper, silver and lead which have atomic numbersof 5.8, 13, 29, 47 and 82, respectively.
The results obtained for the five wall materials used are comparedwith the theoretical curves for which a correction was applied to take
into account the contribution of the backward emission of secondary
electrons produced by r-rays. It was found that the agreement between
the experimental and theoretical curves was satisfactory when Nakai's
approximation formula (NAKAI, personal communication) for energy dissipation of electron in matter was used in the calculation of depth dose
curve, while when Spencer's theory (SPENCER, 1958) was used a discrepancy
1.0
Q)'" : Experiment

'"o

-0

-

Q)

!:c 0.5: Nakai'S approxImation.. -- : Spencer's theory

0;
0::

o.

o

0.1

0.2

0.3

0.4

0.5

De pth ( g/cm')

Fig. 1. Comparison of the depth dose curve of polystyrene for
60CO r-ray with the theoretical curve.

RESEARCHES CARRIED OUT IN 1964

149

was found for the material of low atomic number. Fig. 1 compares theexperimental depth dose curve of polystyrene (circles) with the theoreticalcurves calculated by using Nakai's formula (solid curve) and Spencer's
theory. The discrepancies between experiment and theory may be attributable to neglecting the effects of electron straggling when thetheoretical formula is used.

119. Fluorescence quenching and variation with soluteconcentration of fluorescence efficiency in
2,5-diPhenyloxazole-toluene solutions
Takesi KAT01l

The variation of fluorescence efficiency with concentration for the 2,
5-diphenyloxazole CDPO) solute and for carbon tetrachloride in the toluene
solvent was studied under the aerated condition. The ratio of light yieldin absence of the quencher, 10. to that in its presence, I Q , at fixed soluteconcentration and at fixed irradiation intensity for direct excitation of
the solute by 302 mil UV was found to follow the relationship 10 /I Q =(1 +K2[Q]), where [Q] is the quencher concentration and K 2 is the quenching
constant by CCl4 relating to the solute DPO; the Kz value obtained was
6 liters/mole. Under indirect excitation of DPO via primary excitationof toluene solvent by 260 mil UV, the 10 /IQ vs. [Q] curve was found to
be described by the Stern-Volmer relationship lo/I Q = (I+K,[Q])(I+K 2·[Q]), where K , is the quenching constant relating to the solvent. We
obtained the following values for zero concentration of DPO; K I =640
liters/mole and kq=2.5xlO '0 liters/mole' sec, where kq is the specific rateof quenching calculated from the relation kq=K,/,', " being the decay
time of the fluorescence in the quencher-free pure toluene solution. The
specific rate, k t of excitation transfer from toluene molecules to DPO
molecules was determined from the observed relationship between fluorescence efficiency and DPO concentration and combined with otherrelevant data by other authors; k t =8.56x 10 10 liters/mole' sec.
The theoretical curves of fluorescence efficiency vs. DPO concentration
based on the energy-exchange processes and the experimental values ofrate constants mentioned above were found to fit satisfactorily the experimental values of fluorescence except for the low concentration range(Fig. 1). The disagreement may be explained by the assumption thatthe solvent emission is absorbed by the solute molecules, giving solute
fluorescence with quantum efficiency of about unity. The validity of1) Visiting researcher from Department of Fundamental Radiology,
Faculty of
Medicine, Osaka University.

150 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

.~

"

w
~

...
. ..
. .'
. 0'

I',

/

0.1

concenfrdtion of OPO

10

IOIMIJ

Fig. 1. Comparison of theoretical curves of fluorescenceintensity vs. solute concentration with experimental
values for the aerated DPO-toluene solutions excited by
260 mp UV. Curves A and B are calculated ones, (b)and (.....) are the observed values for CCl 4 concentrationof zero and 1.7 X 10- 3 Mil, respectively.

the assumption should, however, be checked experimentally. The quenching constant K I under 60Co r-ray excitation was larger by the factor of
2.2 than that under 260 m,u UV excitation.

G.

HUMAN GENETICS

120. Search for selection upon secretor status of ABO blood group
substances!)
Ei MATSUNAGA, Yuichiro HIRAIZUMI, Hisajiro IZUMIYAMA andToshiyuki FURUSH021

It has been established that the secretion of A, Band H substances
into the body fluids is controlled by the autosomal genes Se and its
recessive allele se. From the view-point of population genetics, perhaps
the most striking feature of this genetic system is that the relative
frequencies of secretor and non-secretor are almost the same among
different ethnic groups so far examined, except for small samples ofAmerican Indians and Australian aborigines. This fact suggests thatthe secretion phenomenon might have been subjected to a rather strong
natural selection.
II This work was supported by a grant (RF 61113) from the Rockefeller Foundation.
21 Tokyo Medical and Dental College.

RESEARCHES CARRIED OUT IN 1964

151

In order to investigate the selective mechanism, if any, operating
upon the secretor status, an extensive field survey was conducted in thesummer of 1962 in Ohdate City, located in Akita Prefecture, northernpart of Honshu. Approximately 2,500 couples with wife's age ranging
from 30 to 40 years and about 6,000 children were tested for ABO bloodgroups, and saliva samples were taken from each subject, absorbed intoa thin paper and kept as a dried stain. Marital histories were taken
carefully with the aid of trained midwives concerning the number ofpregnancies including induced abortions, pre-natal and post-natal mortalityof the children. As to the determination of secretor status, an appropriate serological method was devised by means of absorption experimentswith anti-A, anti-B and anti-H using the extracts of Ulex europaeus, sothat satisfactory results could be obtained with the dried stains ascompared with the results given by the raw material of saliva.
Among 11,978 persons tested in all, only four cases of "aberrant excretor" were encountered which showed dissociated secretion of A, B
or H substance. The overall frequency of non-secretors was 21.11 per
cent, and there were no differences in their distributions among A, B,
o and AB groups both in the parental and children's populations. Table
1 summarizes the pooled family data. Among 231 children born to 99
Table 1.

Summary of family data.

- - - - - - - - - - -------c---------cio

1 No. of couples'f'

I ChildlessI

1--------- --- I,

couples

Ex p . : No.

Obs.

SexSe I 1,461 1,459.4 ! 52
Sexse I
407
384.3
8
sexSe
362
384.3
15
sexse
99
101.1 I 1
1--- --- -Tot~i 2,329

-

-

-

1

No. of children,Sese% lObs.
3.6
2.0
4.1
1.0

--

I

UnexI
---------- - - - - - - aminedI_7~~ ..:.,..;

Exp.

3,274 3,186.1 !
699
699.6 i591
612.4'
1
0 i

Obs.

Exp.

370
320
301
230

357.9
319.4
279.6
231.0

152
36
41
6

--.------ ---------c----

4,565- - - -

J!.02_1---=3_5_

matings of non -secretor x non -secretor, one was found to be secretor.
Based on this result, the proportion of illegitimacy in our samples wasestimated to be O.0080±O.0079, which agrees well with the figure of
O.OI06±0.0029 estimated from ABO data.
Data concerning reproductive histories were summarized according tomating types in Table 2. Comparisons of the data between secretor
and non-secretor wives did not show any significant difference. There
were no differences in the proportion of sterile as well as childless152 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
Table 2.
I

IProportion0

Summary of fertility data according to mating types.
Mean no. of
pregnancies!M

I ean no.

ofof sterileIEId'living
I nc Iud'. mg xc u. lngl children
I couples
abortIOns abortIOns

!j1

I

SexSe
Sexse
sexSe
sexse

0.027
0.017
0.030
0.010

4.231
4.150
4.199
3.939

3.181 I
3.202 I
3.152 I
3.030 I
I

2.598
2.592
2.577
2.394*

Mean intervalfrom the beginning of cohabitation to 1st livebirth

Zygotic
loss
perpregnany(months)
22.91
22.75
21.87
21.75

0.183
0.190
0.182
0.210

* t=2.165, 0.05>P>0.02

couples, or in the proportion of pre-natal and post-natal deaths of thechildren among the four mating types. The mean number of living
children, however, was 2.394±O.089 for non-secretor X non-secretor matings, while it was 2.594±O.023 for the other matings, the difference
being significant at the 5 per cent level. This difference is presumably
due to a lowered number of pregnancies of non-secretor wives married
to non-secretor husbands.
The viabilities of children of secretor and non-secretor types as wellas the segregation ratio of the recessive gene se from the Sese parents
were estimated by the maximum likelihood scoring method. Preliminary
results obtained by hand computers are represented in Table 3. It is
Table 3.

Estimation of some parameters by means of the maximum
likelihood scoring method.

Definitions of parameters:
Viability of Se children.,.,.,., .. .,.,lViability of se childrenySegregation ratio of se gametes in Sese parentskProbability that an individual of Se phenotype is SeSeh

Estimated values:Ykh

InitialSecondThird

Standard deviation

1.00000.50000.37001.09650.49400.37711.10380.49330.37670.34190.06710.0173

evident from the table that there was no difference in the estimates of
viabilities between the two phenotypes, and no distortion in the segregation ratio.
To sum up, our data suggest so far that the gene for the secretor
status is apparently neutral with respect to prezygotic and postzygotic

RESEARCHES CARRIED OUT IN 1964

153

selection upon viability, but there might be some decrease in fertility of
non-secretor wives married to non-secretor husbands. The problem how
these findings are related to the mechanism by which this polymorphism
is maintained, remains to be solved.

121. Effect of ABO-incompatibility on "waiting time"Yuichiro HIRAIZUMIRecently, attempts have been made to associate "waiting time" toearly zygotic wastage due to some genetic mechanisms (R. KRIEGER,
personal communication). Accordingly, let the waiting time, Xi be theperiod between the date when the (i-1)·th pregnancy of a couple termi·
nated and the date when the next, i-th pregnancy started. For the firstpregnancy, Xi is the period between the date of marriage and the datewhen the first pregnancy started. If there are frequent occurrences of
unrecognized abortions at the very early stage of pregnancy, they willcontribute to increase the waiting time.
A preliminary analysis based upon the data collected at the City of
Ohdate, Akita, Japan, showed the following results. The values of Xl
and X2 were computed in month for each couple, and their average values
and proportions are presented in Tables 1 and 2.
Table 1.

Average values of Xl and X2 (in month).
- ~ _ .

A·incomp.

Mating Group
B-incomp.
AB-incomp.

__._-- "._--.-_._----

Incomp..
pooled

Compo

Xl

10.89

11.05

12.40

11.07

10.63

X2

21.36

20.65

20.74

20.99

20.31

Table 2.

Pregnancy

Proportions of couples with XI~ 1 for the firstand x2~10 for the second pregnancy.

A·incomp.

Mating Group
B·incomp.
AB-incomp.

Incomp.pooled

Compo

First

0.207

0.209

0.171

0.205

0.231

Second

0.195

0.209

0.143

0.198

0.236

- - - - - - - - - - - - - - - - - - - - - - - - - - -------The distributions of Xl and X2 (especially of Xl) among families weremuch skewed so that it was necessary to find out an adequate trans·
formation for statistical analyses. This transformation has not yet been

154 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

found, but taken at face values, x, and X2 were consistently larger in theincompatible mating group. This strongly suggests that unrecognizedabortions were occurring more frequently in this group than in the compatible group. This is also supported by the results presented in Table 2.
The difference between compatible and pooled incompatible groups was
not significant for x, (Xl 2 =2.24, O.10<P<O.20), but it was significant for
X2 (Xl 2 =4.45, O.02<P<O.05). Further analyses are in progress.

122. Studies on the possible genetic consequences ofmeasures affecting population growth
Ei MATSUNAGA

The problem of over-population is growing increasingly serious in many
parts of the world, and various methods of family planning are becoming
more and more widely used. Although the problems of population numbers
and of population quality are separate ones, it has been felt necessary toinquire what genetic consequences may result from measures taken to
limit the population growth.
It is well known that Japan was strongly motivated to limit family size
after the War. Based on the Eugenic Protection Law passed in 1948,
various measures have been actively pursued to slow down a too rapid
population increase. These measures include voluntary sterilization andinduced abortion for economic reasons as well as from physical consider·ations for the mother's health, putting emphasis on the encouragementof family planning by means of contraception. As a result, an unprecedented drop in births has been achieved during a short period in thiscountry.
From demographic data showing secular changes in Japan, some aspects
can be pointed out which could affect our genetic burden. Reduction in
the proportion of births to older mothers and those of high parity should
lead to a reduction in the frequencies of certain diseases such as Down's
syndrome and Rh-erythroblastosis. Decrease in family size should resultin a reduction in the frequencies of consanguineous marriages, and hence
reduce the expressed genetic burden. However, these two points arerather of minor importance. Perhaps the major effect of birth control
would result from the opportunity to use this measure for eugenic
purpose; if the birth of a child with some hereditary disorder could deter
the parents from further reproduction, a significant reduction in thefrequencies of the disease should be expected. The differential fertility
introduced by an uneven distribution of family planning practices among
different social strata may bring about some dysgenic effect, but it would

RESEARCHES CARRIED OUT IN 1964

155

most probably be a transient one. On the other hand, trends towards
lower mortality and smaller family size would result in relaxation ofnatural selection, but the nature and extent of the possible dysgenic
effect cannot be predicted at present.
A detailed account on this subject will be presented at the Second
World Population Conference to be held in Belgrade in 1965.

123.

Human red cell acid phosphatase variants
Tomotaka SHINODA

Variations of red cell acid phosphatase appear to have created a new
genetically determined polymorphism in man presumably controlled bythree autosomal allelic genes. When hemolysates are examined by starchgel electrophoresis, four zones can be observed of acid phosphataseactivity; a faintly active very fast migrating component, a fast one withintermediate activity, a strongly active intermediate component common
to all the patterns, and the slowest component with intermediate activity.
So far we have been able to distinguish at least four distinct acid phosphatase patterns characteristic of the given human individual. Theseelectrophoretic patterns may be detected as pink zones by incubation of
the gel with a solution of phenolphthalein diphosphate in citrate buffer,
pH 5.5, followed by addition of ammonium hydroxide to the gel. The
zones of enzyme activity can also be observed using p-nitrophenyl phosphate as substrate instead of phenolphthalein phosphate. In this case,however, p-nitrophenol liberated during incubation diffuses much more
rapidly than does the phenolphthalein and the zones are therefore muchless clearly defined. The attempt was unsuccessful to demonstrate thesezones using a- or .a-naphthyl phosphate as substrate with an appropriate
diazo coupling reagent. This result shows that it is easy to distinguish
red cell acid phosphatase from certain other human acid phosphatases
which hydrolyze a- or .a-naphthyl phosphate at a significant rate. It isof great interest to compare the substrate specificity of acid phosphatases
from different origins which may supply informations concerning both
structure and function and the genetic factor controlling them.
At the present stage, we have not yet sufficient data for the study ofthe frequency of the patterns, but the above finding may no doubt add
a new problem of polymorphism to human genetics.

156 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

124.

The nature of molecular heterogeneity between
haptoglobin 2-1 and haptoglobin 2-2
Tomotaka SHINODA

The molecular aspect of human serum haptoglobins was investigatedwith regard to the difference between Hp 2-1 and Hp 2-2. The haptoglobins were prepared from unhemolyzed fresh human plasma by DEAEcellulose column chromatography and ammonium sulfate precipitation(SHINODA, 1964). The polymers of Hp 2-1 and Hp 2-2 were fractionatedaccording to molecular size by gel filtration on Sephadex G-200. Haptoglobin fraction obtained by gel filtration was analyzed by starch- andurea-gel electrophoresis. As to Hp 2-1 the haptoglobin components eluted
first corresponded to those which migrated slowest in the starch gel,whereas those which were eluted later migrated faster in the gel. Whengel filtration was made of a sample to which an excess of hemoglobin wasadded prior to the experiment, the haptoglobin-hemoglobin complex was
eluted earlier than free haptoglobin alone. In Hp 2-2, similar results as
those with Hp 2-1 were obtained in both gel filtration and starch gelelectrophoresis. However, resolution between Hp 2-2-Hb complex andfree Hp 2-2 was not sufficiently successful in gel filtration under thecondition employed. By using an unfractionated Hp 2-1 it has been
demonstrated that Hp 2-1 contains both a l and a 2 polypeptide chains in
the molecule, as might be expected in the heterozygote. However, thisfinding does not make it certain whether these two polypeptide chains arederived independently from the respective components of Hp 2-1. The
present experiments demonstrated that the early fractions in the gel
filtration of Hp 2-1 consist entirely of polymers of slower migrating
components and lack the component corresponding to Hp 1-1. However,this fraction is shown, after reductive cleavage followed by urea-starchgel electrophoresis, to contain small amounts of a l chain which, therefore,should have been derived from the integral part of the polymers. It
may be concluded from these results that the three common haptoglobin
types possess in their structure disulfide bonds which are necessary for
maintaining the integrity of protein structure and perhaps also for polymer
formation, although at present it is not yet certain that the monomericunits are actually linked together by disulfide bonds. Since not allchanges in amino acid composition of polypeptides resulted in an alteredmobility in electrophoresis as was being demonstrated for some abnormal
hemoglobins, it might be premature to conclude that Hp 2-1 contained
both the a l and a 2 polypeptide chains which are identical to those of
Hp 1-1 and Hp 2-2, but it seems that Hp 2-1 contained both chains in
the molecule.

RESEARCHES CARRIED OUT IN 1964

157

125. Clinical conditions of patients with apparently
normal chromosomes, I
Hidetsune OISHI and Akira TONOMURA

Chromosome analyses performed on cultured leucocytes of patients
having various pathological conditions listed below showed that they all
had normal karyotypes of 46 chromosomes.
Name

1 YS
2

TK

3 SK
4 EE
5 HY
6 KY
7 MS
8 HM
9 YH
10 TN
11 KO
12 NS
13 MA
14 TS
15 KM
16 KY
17 AS
18 YI
19 SS
20 MK
21 SM
22 HT
23

24
25

KK
TY

MN

126.

Age

Legal sex

Clinical conditions

15 years
13 years
8 years
4 months
2 months
2 months
4 months
25 years
2 months
11 months
28 years
5 months
14 years
14 years
11 years
37 years
25 years
35 years
21 years
3 years
10 years
4 years
17 years
13 years
8 years

M

Laurence-Moon-Biedl's syndrome

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

M
M
M

M
F

M

M

H
H

Harelip; cleft palate
Microcephaly; polydactyly
Palatorrhaphy
Pierre Robin's disease
Hypopituitalismus
Ehlers Danlos syndrome
Chondrodystrophia
Madan's syndrome
Pterygium coli
Bonne-Viele Ullich syndrome
Testicular feminization
Adrenogenital syndrome
Gonadal disorder
Primary amenorrhoea
Eunuchoidism
H

Male intersex
Male pseudohermaphroditism"
H

True hermaphroditism
H

In vitro sensitivity of human lymPhocytes tothalidomide and its derivatives l )Akira TONOMURA and Hidetsune OISHI

From our present knowledge there is every reason to believe thatthalidomide exerts a teratogenic effect on foetal tissues of man and otherI) This work was supported by a grant from Scientific Research Fund of the
Ministry of Education.

158 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

animals. In the present study we have tried 1) to examine the effect ofthalidomide on human lymphocytes cultured in vitro and 2) to evaluate
the usefulness of human leucocyte culture for rapid and sensitive screen·
ing of potential drug toxicity.
Human leucocytes obtained from 10 ml of venous blood from healthy
donors were cultured according to a modified method of MOORHEAD et al.
(1960). The drugs used were commercial thalidomide (Isomin), phthalyl
glutamic acid, phthalyl glutamine, trinitrophenyl glutamic acid andtrinitrophenyl glutamine. The last four compounds were chosen becauseof their chemical resemblance to thalidomide and occasional questioningof their toxicity under similar experimental circumstances. The drugs
were dissolved in 0.02 M phosphate buffer solution (pH 7.4) and added to
culture media at various concentrations. The cultures were incubatedat 37°C for 72 hours. The preparations were made by the use of air·drying technique, and then stained with Giemsa solution. The lymphocytes
present were divided into 'small', 'intermediate' and 'blast' types as
described in 1962 by ELVES and WILKINSON (Fig. 1). A differential countFig. 1. Photomicrographs of three types of human lymphocytes.
1, small cells; 2, intermediate cells; 3, primitive' blast' cell.

of 500 cells was made on each slide, and percentages of lymphocytes
transformed into intermediate and blast types were determined.
The results obtained for the various groups are summarized briefly in
Table 1. In this table, the depression of intermediate and blast types
was expressed as a percentage; e.g., if the control culture had 50 % of
these cells and the treated 25 %, this would be expressed as a 50 %decrease. The control cultures usually showed 60.....70 % of intermediate
and blast transformations. Since the appearance of mitotic cells is

159

RESEARCHES CARRIED OUT IN 1964

coincidental with the appearance of these two types of cells and theincrease in DNA synthesis is probably due to the metabolism of thesecells, the depression of the two types of lymphocytes may indicate an
inhibition of some kind of cellular activity. When the lymphocytes were
exposed to thalidomide, a significant effect was obtained at a high degreeof confidence (P<O.01). Probably significant results (P<O.05) were also
obtained with phthalyl glutamic acid and trinitrophenyl glutamic acid.
Table 1. The depression of 'intermediate' and 'blast' lymphocyte
types in cultures with thalidomide and its derivatives.
Drugs used

Concentration
flg per ml

Depression(%)

dl·Thalidomide (Isomin)
Phthalyl glutamic acid
Phthalyl glutamine
Trinitrophenyl glutamic acid
Trinitrophenyl glutamine

15
28
28
17
17

38.3
27.4
19.5
31.8
16.6

Significance (P)*0.01~0.001
0.05~0.02

0.1 ~0.05
0.05~0.02

0.3 ~0.2* Statistically significant.
The results expressed by these figures may indicate that there aresome difficulties in proving the usefulness of human leucocyte cultures
for studying the potential side action of drugs. Recently, DIPAOLO andWENNER (1964), however, demonstrated that thalidomide did not show any
effect on the growth of Ehrlich ascites tumor cells in vitro. If this istrue, it is certain that the phenomenon of the significant sensitivity ofhuman lymphocytes against thalidomide and its derivatives is of greatinterest, and that much work remains to be carried out to solve thisproblem.

160 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15

BOOKS AND PAPERS PUBLISHED IN 1964
BY STAFF MEMBERS
ASAKURA, S., G. EGUCHI and T. IINO, 1964. Reconstitution of bacterial flagella invitro. J. Mol. BioI. 10: 42-56.
CHIGUSA, S. and T. MUKAI, 1964. Linkage disequilibrium and heterosis in experi·mental populations of Drosophila melanogaster with particular reference to thesepia gene. Jap. J. Genet. 39: 289-305.
CROW, J. F. and M. KIMURA, 1964. The theory of genetic loads. Genetics Today,
Proc. XIth Int. Congr. Genet. 3: 495-506.
FUJII, T., 1964. Relative biological effectiveness of 14 MeV fast neutrons to 60CO
gamma· rays in einkorn wheat. BioI. Effects of Neutron and Proton Irradiations
2: 217-231FUJII, T., 1964. Radiation effects on Arabidopsis thaliana I. Comparative efficiencies of r-rays, 14 MeV and fission neutrons in somatic mutation. Jap. J. Genet.
39: 91-101FUJII, T. and S. MATSUMURA, 1964. Radiosensitivity in plants, VI. Experimentswith aged wheat and rice seeds. Seiken Zih6 16: 21-27.
HIRAIZUMI, Y., 1964. Are the MN blood groups maintained by heterosis? Amer.
J. Human Genet. 16: 375-379.
HIRAIZUMI, Y., 1964. Prezygotic selection as a factor in the maintenance of variability. Cold Spring Harbor Symp. Quant. BioI. XXIX: 51--60.
IINO, T., 1964. Bacterial flagella (in Japanese). Seibutsu-Butsuri 4: 17-31IINO, T., 1964. Fine structure of gene (in Japanese). Seibutsu-Butsuri 4: 122-132.
IINO, T., 1964. Genetical studies of Salmonella flagella (in Japanese). Jap.]. Genet.
39: 313-335.
IINO, T., 1964. Genetics of flagellar antigen in Salmonella. Genetics Today, Proc.
XIth Int. Congr. Genet. 3: 731-740.
IINO, T. and J. LEDERBERG, 1964. Genetics of Salmonella. The World Problem of
Salmonellosis. Dr. W. Junk Publ. Co., The Hague, pp. 111-142.
INUI, N. and T. H. YOSIDA, 1964. Chromosomal alteration and the development oftumors, X. Karyological observations of ascites and metastatic tumors of
MH-134 mouse hepatoma. Gann 55: 49-55.
ISHIWA, H., Y. YAN and S. KONDO, 1964. Temperature-dependence of lethal andmutagenic actions of HN02 on phage T4. Biochim. Biophys. Acta 91: 160-162.
KATAYAMA, T. C., 1964. Photoperiodism in the genus Oryza, I. Jap. J. Bot. 18:
309-348.
KATAYAMA, T. C., 1964. Photoperiodism in the genus Oryza, II. Jap. J. Bot. 18:
349-383.
KATAYAMA, T. C., 1964. Short day field of National Institute of Genetics (inJapanese). Environmental Control in BioI. 2: 32-36.
KIHARA, H., 1964. Nucleus and chromosome substitution in wheat and Aegilops,
I. Nucleus substitution. Proc. 2nd Int. Wheat Genet. Symp.: 23-37.
KIHARA, H., 1964. Some notes on the XIth International Congress of Genetics (inJapanese). Iden 18: 3.

BOOK AND PAPERS WRITTEN BY STAFF MEMBERS

161

KIHARA, H., 1964. Records in sports (in Japanese). Iden 18: 10-12.
KIHARA, H., 1964. Pesticides and organisms (in Japanese). Chern. and BioI. 2: 225.
KIHARA, H. and K. TSUNEWAKI, 1964. Some fundamental problems underlying the
program for hybrid wheat breeding. Seiken Zihi'> 16: 1-14.
KIMURA, M., 1964. "The Statistical Processes of Evolutionary Theory" by P. A. P.
Moran (book review). The Quarterly Rev. BioI. 39: 86.
KIMURA, M., 1964. Diffusion models in population genetics. J. Applied Probability
1: 177-232.
KIMURA, M. and J. F. CROW, 1964. The number of alleles that can be maintainedin a finite population. Genetics 49: 725-738.
KIMURA, M. and G. H. WEISS, 1964. The stepping stone model of population structure and the decrease of genetic correlation with distance. Genetics 49: 516-576.
KONDO, S., 1964. Variation in mutagenicity and radiation resistance with genome
complexity and evolution. Jap. J. Genet. 39: 176-198.
KURITA, Y., K. MORIWAKI and T. H. YOSIDA, 1964. Autoradiographic analysis of
the mitotic cycle in Yoshida sarcoma cells. Gann 55: 397-402.
MABUCHI, T. and S. MATSUMURA, 1964. Dose rate dependence of mutation rates
from r-irradiated pollen grains of maize. Jap. J. Genet. 39: 131-135.
MAKINO, S., M. SASAKI and A. TONOMURA, 1964. Cytological studies of tumors,
XL. Chromosome studies in fifty-two human tumors. J. Nat. Cancer Inst. 32:
741-777.
MATSUMURA, S., 1964. Differences in RBE of gamma-rays and fast neutrons in thepolyploid wheat series. Jap. J. Genet. 39: 86-90.
MATSUMURA, S. and T. MABUCHI, 1964. Relation of radiation effects to dose rates
of gamma- or X-radiation in rice and wheat. Jap. J. Genet. 39: 120-130.
MATSUMURA, S. and Y. TAZIMA (Ed.), 1964. Radiation Genetics (in Japanese).
Syokabo, Tokyo, pp. 1-739.
Chapter I. Changes in genetic material caused by radiation (TAZIMA, Y.).
Chapter III. Problems in relation to radiation mutagenesis (TAZIMA, Y. and
S. KONDO).
Chapter V. Radiation effect for genetic material (NAWA, S.).
Chapter VIII. The effect of radiation on populations (KIMURA, M.).
Chapter XI. Radiation and quantitative characters (MUKAI, T.).
Chapter XII. Induction and application of useful mutations (MATSUMURA, S.).
MATSUNAGA, E., 1964. Genetic epidemiology of retinoblastoma (in Japanese).
Saishin-Igaku 19: 1284-1289.
MATSUNAGA, E., 1964. Dber die allgemeinen Grundsaze del' Vaterschaftsbestimmungen in Lichte del' Humangenetik (in Japanese with German summary). Jap.
J. Legal Med. 18: 268-294.
MATSUNAGA, E. and A. TONOMURA, 1964. Cytogenetic of Down's syndrome (inJapanese). Sogo-Igaku 21: 95-99.
MUKAI, T., 1964. Recent topics in the studies on the maintenance of genetic
variability in natural populations (review in Japanese). Seibutsu-Butsuri 4: 1-12.
MUKAI, T., 1964. The genetic structure of natural populations of Drosophilamelanogaster, I. Spontaneous mutation rate of polygenes controlling viability.
Genetics 50: 1-19.

162 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
MUKAI, T., 1964. Cis-trans effect between cistron in Drosophila melanogaster?
(in Japanese). Jap. J. Genet. 39: 357.
MUKAI, T., 1964. Polygenic mutations and the genetic structure of natural populations (in Japanese). Jap. J. Genet. 39: 380.
MUKAI, T., S. CHIGUSA and 1. YOSHIKAWA, 1964. The genetic structure of natural
populations of Drosophila melanogaster, II. Overdominance of spontaneous
mutant polygenes controlling viability in homozygous genetic background.
Genetics 50: 711-715.
MUKAI, T. and T. YAMAZAKI, 1964. Position effect of spontaneous mutant polygenes
controlling viability in Drosophila melanogaster. Proc. Jap. Acad. 40: 840-845.
MURAKAMI, A. and S. KONDO, 1964. Relative biological effectiveness of 14 MeV
neutrons to r-rays for inducing mutations in silkworm gonia. Jap. J. Genet.
39: 102-114.
OGAWA, Y., 1964. Growth, differentiation and regeneration, XXXVIII. Effect of
glucose on the abnormal differentiation of muscle tissue of X-irradiated Triturusembryos (in Japanese). Med. and BioI. 68: 271··273.
OGAWA, Y., 1964. Growth, differentiation and regeneration, XXXIX. Sensitivity ofthe synthesis of actin to X-ray during the early developmental stages (inJapanese). Med. and BioI. 69: 60-62.
OGAWA, Y., 1964. Growth, differentiation and regeneration, XL. Radiosensitivityof myosin formation in Trit'urus embryos treated with L-gulonolactone (inJapanese). Med. and BioI. 69: 122-125.
OGAWA, Y., 1964. Effects of L-gulonolactone and glucose on the synthesis ofskeletal muscle protein, myosin, in X-irradiated Triturus embryos. Reports of
the 9th Symp. on Glucuronic Acid, pp. 269-278.
OGAWA, Y., 1964. An apparatus for cellulose acetate electrophoresis (in Japanese).
The Phisico-Chemical BioI. 10: 120-121.
OGAWA, Y., 1964. Separax, a cellulose acetate strip for electrophoretic analysis
newly manufactured in Japan (in Japanese). Igaku no Ayumi 51: 467-471.
OGAWA, Y. and 1. KOBAYASHI, 1964. Growth, differentiation and regeneration,XXXVII. Zonal electrophoresis of water-soluble proteins in mouse skin treatedwith sweet orange oil, a tumor promoting agent (in Japanese). Med. and BioI.
68: 51-54.
OHWI, J. and S. SAKAMOTO, 1964. Taxonomy, ecology and distribution of Agropyron
humidum OHWI et SAKAMOTO. J. Jap. Bot. 39: 109-114.
OKA, H.1., 1964. Pattern of interspecific relationships and evolutionary dynamics
in Oryza. Rice Genet. and Cytogenet., 1. R. R. 1., pp. 71-90.
OKA, H.1., 1964. Considerations 011 the genetic basis of intervarietal sterility in
Oryza sativa. Rice Genet. and Cytogenet., 1. R. R. 1., pp. 158-174.
OKA, H.1. and W. T. CHANG, 1964. Evolution of responses to growing conditions
in wild and cultivated rice forms. Bot. Bull. Acad. Sinica 5: 120-138.
OSHIMA, C., 1964. Studies on deleterious genes in natural populations of D.
melanogaster, 1. Large and small populations (in Japanese). Jap. J. Genet.
39: 364.
OSHIMA, C., 1964. Genetics of resistance to insecticides in Drosophila. Proc.
XIIth Int. Congr. Entomol.: 243.

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

163

OSHIMA, C., T. K. WATANABE and T. WATANABE, 1964. The persistence of somerecessive lethal genes in natural populations of Drosophila melanogaster, IV.
Proc. Jap. Acad. 40: 125-130.
SADO, T., I. HOPPE and P. NETTSHEIM, 1964. Further cytokinetic studies on antibody
formation. Federation Proc. 23: 345.
SADO, T. and T. MAKINODAN, 1964. The cell cycle of blast cells involved insecondary antibody response. J. Immunol. 93: 696-700.
SAKAI, K. I. and A. SUZUKI, 1964. Induced mutation and pleiotropy of genes
responsible for quantitative characters in rice. Rad. Bot. 4: 141-151.
SAKAMOTO, S., 1964. A polyhaploid plant of Agropyron tsukushiense var. transiens
OHWI found in a state of nature. W. I. S. 17-18: 19-20.
SAKAMOTO, S., 1964. Cytogenetic studies in the tribe Triticeae, I. A polyhaploid
plant of Agropyron tsukushiense var. transiens OHWI found in a state of nature.
Jap. J. Genet. 39: 393-400.
SAKAMOTO, S., 1964. Cytogenetic problems in Agropyron hybrids. Seiken Ziho
16: 38-47.
SAKAMOTO, S., 1964. On the climatic chambers at the Swedish Seed Association (inJapanese). Environmental Control in BioI. 2: 48-50.
SHINODA, T., 1964. Biochemical studies on Drosophila melanogaster, I. Free aminoacids and pteridines (in Japanese). J. Jap. Biochem. Soc. 36: 816-820.
SUZUKI, H. and Y. HAYASHI, 1964. The formation of "ribosomal RNA" in
Escherichia coli during recovery from magnesium starvation. Biochim. Biophys.
Acta 87: 610-620.
TAZIMA, Y., 1964. The Genetics of the Silkworm. Logos Press Limited, London,
pp. 1··253.
TAZIMA, Y., 1964. Contribution from 14 MeV fast neutron experiments to theinterpretation of the mechanisms of dose-rate effects on mutation frequency insilkworm gonia. Jap. J. Genet. 39: 115-119.
TAZIMA, Y. and E. MATSUNAGA, 1964. The Genetics of Man (in Japanese). NHKBooks Series 14, pp. 1-262, Nippon Hoso Kyokai, Tokyo.
TONOMURA, A., 1964. A Technique for Tissue Culture (in Japanese). AsakuraShoten, Tokyo, pp.534-540.
TONOMURA, A. and T. KURITA, 1964. Triple chromosomal mosaicism in a Japanesechild with Down's syndrome. Acta Genet, 14: 67-75.
TSUJITA, M. and S. SAKURAI, 1964. Genetical and biochemical studies of thechromogranules in the larval hypodermis of the silkworm, Bombyx mori, I.
Rela,tion of uric acid to chromogranules (in Japanese). J. Seric. Sci. Jap. 33:
389-393.
TSUJITA, M. and S. SAKURAI, 1964. Genetical and biochemical studies of thechromogranules in the larval hypodermis of the silkworm, Bombyx mori, II.
Protein analysis of the chromogranules (in Japanese). J. Seric. Sci. Jap. 33:
447-459.
TSUJITA, M. and S. SAKURAI, 1964. Relationship between skin color or skint '~lJsparency of silkworm larvae and chromogranules in hypodermal cells.
. c. Jap. Acad. 40: 558-560.

164 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 15
TSUJITA, M. and S. SAKURAI, 1964. Relationship between chromogranules and uricacid in hypodermal cells of silkworm larvae. Proc. Jap. Acad. 40: 561-565.
TSUNEWAKI, K., 1964. Genetic studies of a 6x-derivative from an 8x Triticale.
Can. J. Genet. CytoI. 6: 1-11.
TSUNEWAKI, K., 1964,. Transmission of monosomes and trisomes in an Emmer
wheat, T. dicoccum var. Khapli. W. I. S. 17-18: 34-35.
TSUNEWAKI, K., 1964. Evolution of plants by polyploidy (in Japanese). Iden 18:
34-38.
TSUNEWAKI, K. and K. NISHIKAWA, 1964. Frequencies of Net and Nez genes in
Emmer and common wheats. W. I. S. 17-18: 4,0-41.
WATANABE, T., 1964. Studies on deleterious genes in natural populations of D.
melanogaster, III. Chromosomal polymorphism (in Japanese). Jap. J. Genet.
39: 376.
WATANABE, T. K., 1964. Studies on deleterious genes in natural populations of
D. melanogaster, II. Distribution of lethal genes on the second chromosome(in Japanese). Jap. J. Genet. 39: 376.
YAMAZAKI, T. and T. MUKAI, 1964. Overdominance of spontaneous mutant polygenes
in Drosophila melanogaster (in Japanese). Jap. J. Genet. 39: 377.
YOSHIKAWA, I. and T. MUKAI, 1964. The effects of radiation-induced mutations on
viability in Drosophila melanogaster (in Japanese). Jap. J. Genet. 39: 378.
YOSlDA, T. R., 1964. Chromosomal alteration and the development of tumors, IX.
Increase in the frequency of polyploid cells in metastatic tumors of the Ehrlich
hyperdiploid tumor of mice. Jap. J. Genet. 38: 342-350.
YOSlDA, T. R. and E. J. LAMONTAIN, 1964. Chromosomes of normal and dwarf
cattle. Jap. J. Genet. 38: 351-355.

ACKNOWLEDGMENT
The editor wishes to express his sincere gratitute to Dr. F. A. LILIENFELD forher kindness in reading the original manuscripts. (Tetsuo IINO)

I

1

165

AUTHOR INDEXAUTHOR INDEXArticle Number

Article Number

AKAHORI, A .•.......•..........•..... 46

NAKAI, Y •......•......••......... 54,55

AMANO, K .•.......................... 38

NAKAMURA, A •.....•..•............. 37

CHADWICK, C. J. oo
23
CHU, Y. E
68,70
ENDO, T ..................•........ 116
ENOMOTO, M .........•..•............ 84

NAWA, S. oo ............•..•......... 25

FUJII, T .•.•. . 105,106,112,113,114

OISHI, Hoo

FUJISHIMA, T ••...•••.•.......... 35, 36

OISHI, K
OKA, H

FUKAYA, T .•.........•..•............ 37.,

ONIMARU, K.
OSHIMA, C

IINO, T. oo"""
80,81,82
IKENAGA, M. oo ..•... . 89,100,108, 117

SADO, T

IMAI, H .......•............•..... 42, 48

SAKAGUCHI, B.

117,118,119
60, 61, 62, 63, 64
KAWAHARA, T
32,33,34
KIHARA, H. oo .................. • 50,51
KIMURA, M. '"77, 78, 79
KOBAYASHI, S"""" .. " .22, 92
KONDO, S ••.................. 89, 100, 118
KURITA, Y
43,44,45
LAW, L. W ..............•...••....... 39
LEONARD, M. R.,.,23
MABUCHI, T .. . 107,108,109,110,111
MAKINODAN, T.,.,
23
KATO, Too
KATAYAMA, T.C

MATSUMURA, S .•..•........•........

........ 102, 103, 104, 110, 111, 112
MATSUNAGA, E •............. . 120,122
MIT ANI, M., .,81, 82, 83

"

66,67
43, 44, 45
MUKAI, T ••..•••..•............... . 10,
11, 12, 13, 14, 15, 16, 17,18, 19, 91
MUKAIDE, H
76
MURAKAMI, A
96, 97MORISHIMA, H
MORIWAKI, K

.,

.,

""

FURUSHO, T •.............•......... . 120

IZUMIYAMA, H ....•...•.........•... . 120

II

OHWI, JHIRAIZUMI, Y ............. • 90, 120, 121

ISHIDSU, J
86,87
IYAMA, S ••....•.••......••..••...•... 9,

OGAWA, Y . . . ., ..... " ... .,47,49,101
OHARA, K . .............•............. 40

56
125,126
20,21,93
66,67,68,69
26,94, 95
1,2,3,4,5,6

RIZKI, T. M ..............•........... 7

23,98, 99
20,21,22,92
SAKAI, K
34,35, 65,71, 76, 115
SAKAMOTO, S .........•.•• 56,57,58,59
SAKURAI, S ........•...• 27, 28, 29, 30, 31
SHIMAMOTO, Y.,.,., . " ..... 71
SHINODA, T
24, 123, 124
SUZUKI, H •..............•..•••.••••• 88.,

SUZUKI, S ••...••.......•............. 8
SUZUKI, Y .•......•.•..•..•.••....... . 80
TAIRA, T.,
7,8
TAKEDA, G

115
70,72,73,74,75
TAZIMA, Y
26,93,94,95,96,97
TERADA, Y.,,, . .,74
TONOMURA, A. oo .. " " " ... " ,,125,126
TSUJITA, M
27,28,29,30,31
TSUNEWAKI, K • . . 50,51,52,53,54,55
TUTIKAWA, K ...........•........... 46
WATANABE, T
4,5,6
WATANABE, T. K
1, 2, 3,4
YAMAGUCHI, S ............••.•....... 85
YAMAZAKI, T ..... . 10,12,17,18,19,91
YONEDA, Y ............•....•.•••. 72,73
YOSHIKAWA, I. ........ " " " ..... 11,91
TAKENAKA, Y"

YOSIDA, T. H .............•..•.......•

37,38,39,40,41,42,43,44,45,48

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