NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 14

1963

Published by

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

1964

--of

Annual Report:the

National Inst:itut:e of Genet:ics-

No. 14, 1963

Published by

The National Institute of Genetics, Japan
1964

CONTENTS
General statement, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,Abstracts of diary for 1963 , , , , , , , , , , , , , , , , , , , . , , , , , , , , , , , , , , . , , , , ,Staff. , ,,,,,,,,.,,,,............
Council,,,.....
,,.,....... . . . . . . . . . . . .
Projects of research for 1963
Foreign visitors in 1963",.,.....................
Research carried out in 1963.......................................

1
2
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5
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11

A. Genetics, cytology and biochemistry of animals
1.

2.
3.
4.

5.
6.
7.

8.
9.

10.

11.

Genetic variability of deleterious genes in a large natural
population of Drosophila melanogaster (OSHIMA, WATANABE
and WATANABE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanism of persistence of a lethal gene in a small natural
population (OSHIMA, WATANABE and WATANABE)
Location of lethal genes on the second chromosome (WATANABE and OSHIMA),.........
Frequencies of inversions in a large and a small natural
populations (WATANABE and OSHIMA)......................
Cytodifferentiation in the fatbody of Drosophila melanogaster(TAIRA and RIZKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A drive element, Nomadic Hunter (NH), in a natural populationof Drosophila melanogaster (HIRAIZUMI and NAKAZIMA) . . . . .
Further evidence supporting a high spontaneous mutation
rate of polygenes controlling viability of Drosophila melanogaster (MUKAI, CHIGUSA and YOSHIKAWA)
Polygenic mutation rate and homozygous genetic load in
Drosophila populations (MUKAI)...........................
Overdominance of spontaneous mutant polygenes controlling
viability in homozygous genetic background in Drosophilamelanogaster (MUKAI, CHIGUSA and yOSHIKAWA)............
Deleterious heterozygous effect of spontaneous mutant polygenes controlling viability in heterozygous genetic backgrounds in Drosophila melanogaster (MUKAI, CHIGUSA andYOSHIKAWA)
Allelism frequency in spontaneous recessive lethal genes in
Drosophila melanogaster (YoSHIKAWA and MUKAI)..........

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

12.

13.
14.
15.

16.

17.
18.

19.
20.

21.

22.
23.

24.

25.
26.
27.

28.

Spontaneous mutation rate of polygenes controlling developmental time in Drosophila melanogaster (MUKAI and
YAMAZAKI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Correlation between viability and developmental time in
Drosophila melanogaster (YAMAZAKI and MUKAI) . . . . . . . . . . . 23
Persistence of two recessive genes in experimental populations in Drosophila melanogaster (III) (CHIGUSA and MUKAI). 24
Interruption of "sex-ratio" condition by double infection
with two SR agents of different origin (SAKAGUCHI andOISHI)
24
Nature of the interference phenomenon between the "sexratio" agents from D. willistoni and D. nebulosa (SAKAGUCHI, OISHI and KOBAYASHI). . . . . . 26
Low temperature effects on abnormal "sex-ratio" condition
in Drosophila (OISHI and SAKAGUCHI)...................... 27
Morphology of "sex-ratio" agents of D. willistoni and D.
nebulosa studied by electron microscope (KOBAYASHI and
SAKAGUCHI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Cellular studies of antibody formation I. Proliferative capacity of antibody-forming cells (SADO and LEONARD)....... 29
Cellular studies of antibody formation II. The cell cycle ofblast cells involved in secondary antibody response (SADO
and MAKINODAN). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Cellular studies of antibody formation III. Suppression of
appearance of antibody-synthesizing cells with supraoptimaldose of antigen (SADO and CHADWICK)
32
Change of a somatic character by DNA in Ephestia (NAWA
and CASPARI)" ..
...
. . .. .
. .. 34
Genetical and biochemical studies of chromogranules contained in the larval hypodermis of the silkworm (TSUJITA
and SAKURAI)............................................. 34
Chromogranules in the hypodermis of normal and several
oily skin mutant larvae of the silkworm (TSUJITA andSAKURAI)
1. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •36
Genetic analysis of growth of early chick-embryos (KAWAHARA)
Heterosis and asymmetry in testis weight in domestic fowl
(KAWAHARA)
38
Chromosomal study of mouse lymphocytic neoplasm, P388,
growing in vivo and in vitro (YOSIDA, LAW and ROOSA).... 39
Karyotypes of various drug-resistant sublines of P388 neoplasm growing in vitro (YOSIDA, LAW and ROOSA)......... 40

CONTENTS

Karyological analysis of the mouse lymphocytic neoplasm,
P388, growing in synthetic culture medium (YOSHIDA, EVANS
and LAW):30. Karyological study on mouse leukemias developed by virus
infection (YOSIDA, LAW and PRECERUTTI). . . . . . . . . . . . . . . . . . .
:31. Relation between chromosome constitution and protein specificity in mouse plasma cell leukemias (YOSIDA, POTTER,
KURITA and MORIWAKI):32. Chromosomes of normal and dwarf cattle (YOSIDA and LAMONTAIN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
:33. Karyological observations on a Misima subline of hyperdiploid Ehrlich tumor and its three clones (YOSIDA)........
:34. Induction of specific protein synthesis in tumor cells by theaddition of exogenous RNA (MORIWAKI):35. Electrophoretic analyses of esterase isozymes in various
strains of mice (OGITA, OGITA, KURITA and MORIWAKI) ....
:36. Cytological analysis of the mechanism of induction of chromosome aberrations by 4NQO (MORIWAKI, KURITA andYOSIDA)'37. An unusually Y chromosome found in a strain of the mouse(NAKAMURA and TONOMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
'38. Electrophoretic analysis of water-soluble proteins in Triturusembryos on cellulose acetate (OGAWA and KOBAyASHI).....
39. Changes in the soluble proteins of mouse skin following theapplication of Sweet Orange Oil, a tumor promoting agent(OGAWA and KOBAyASHI)....................................
40. Selection for larval growth in Tribolium under two nutritional levels (YAMADA and A. E. BELL) . . . . . . . . . . . . . . . . . . . .

iii

29.

. Genetics, cytology and biochemistry of plants
41. Discovery of three cytoplasms inducing male-sterility inwheat (KIHARA)
42. Effect of caudata cytoplasm on the development of 4 nucleus-substitution lines of common wheat (KIHARA)
43. Production of tetraploid rice by N 2 0-treatment (KIHARA and
TSUNEWAKI) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
·44. Frequency of aneuploids in the offspring of 7 synthesized
6x wheats (TSUNEWAKI)...................................
45. Genetic studies of a 6x-derivative from an 8x Triticale(TSUNEWAKI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46. Transmission of monosomes and trisomes in an Emmer
wheat, T. dicoccum var. Khapli (TSUNEWAKI)

41
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43
44
44
45

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51

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iv ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

47.
48.
49.
50.
51.
52.

53.
54.
55.
56.
57.
58.
59.
60.

61.
62.

63.
64.

65.
66.
67.
68.

An Emmer wheat with 15 chromosome pairs (TSUNEWAKI)..
Further proof that type 1 necrosis is controlled by the Negenetic system (NISHIKAWA)
Distribution of necrosis genes in Emmer wheat (NISHIKAWA).
Frequencies of Nel and Ne2 genes in Emmer and common
wheats (TUNEWAKI and NISHIKAWA)........................
A polyhaploid plant of Agropyron tsukushiense var. transiens
Ohwi found in a state of nature (SAKAMOTO)
Preliminary studies on the relationship between Eremopyrum
distans Nevski and E. triticeum Nevski (Gramineae) (SAKAMOTO and MURAMATSU)....................................
Spodogram analysis of leaf-blade in the genus Oryza (KIHARA
and KATAyAMA)...........................................
Occurrence of indica and japonica types among Sikkimeserice varieties (KIHARA and KATAYAMA) . . . . . . . . . . . . . . . . . . . .
Photoperiodic responses of Oryza species, VI (KATAYAMA) . .
Characteristics of Oryza officinalis Wall. found in Borneo(KATAYAMA) .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Studies on the intercellular spaces of an Assamese floating
rice (KATAyAMA)..........................................
A genetic study of developmental instability in tabacco leaf
(SAKAI and SHIMAMOTO) ... ... .. . . . .. . . . . . . . . . . . . . . . . .. . . . .
A developmental-genetic study of panicle characters in rice
(SAKAI and SHIMAMOTO)
Density response and competitive ability in barley (SAKAI
and IYAMA)...............................................
Survey of F 1 sterility relationships between strains of Oryza
perennis (MORISHIMA and OKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Characters relative to the Breeding System in Oryza species(MORISHIMA and OKA).....................................
Change of population genotype of rice hybrids in response
to different propagating methods (OKA and MORISHIMA) . . .
Observations of wild and cultivated rice species in West
and Central Africa (OKA)
Polyploidization and RNA and protein content of wheat seeds(ENDo)
Secondary association in Lillium auratum (TAKENAKA and
CHU)
Cytological studies on a Nicotiana tumorous hybrid and its
parents (YONEDA and TAKENAKA)..........................
Tumors in Japanese morning glory (TAKENAKA and YONEDA).

60
61
62

6465

66
66
67
68

69
70

71
72
73,

747475,
75

76
77
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79

CONTENTS

69.

Spring- and Winter-sown varieties of sugar beets, withspecial reference to photothermal induction (MATSUMURA).

C. Mathematical genetics
70. The mutational load in small populations (KIMURA).........
71. The stepping stone model of population structure (KIMURA)
72. Effect of maternal inheritance on the genetical variance ina segregating population (IYAMA).........................

v

80
81
82
83

D. Genetics and biochemistry of microorganisms
Allelic repression in transductional heterogenotes of Salmonella (lINO)...............................................
74. Peptides pattern of flagellin of normal and curly mutant
flagella in Salmonella abortus-equi (ENOMOTO and IINo)....
75. Inhibition and regeneration experiments of the flagellar
synthesizing system of Salmonella cells (MITANI).........
76. Mapping of the mutant sites in motility A-cistron of Salmonella typhimurium (ENOMOTO)..........................
77. Effect of phenol on the expression of multiple drug resistance of Shigella(R) (IINO and ENOMOTO) . . . . . . . . . . . . . . . . . .
78. Sensitization to phenol by infection of Salmonella with multiple drug resistance factors (R) (IINO and MITANI) . ; . . . . .
79. Preliminary studies on an arginine-sensitive mutant of Salmonella tYPhimurium (ISHIDSU)............................
80. Comparative studies on mutagenic efficiencies of incorporated
32p and r-rays with Salmonella (ISHIWA, YAN and KONDO).
81. Chemical modification of nucleic acids and their constituents(SHINODA)73.

E. Radiation genetics in animals
82. Heterozygous effects of radiation-induced mutations on viability in homozygous and heterozygous genetic backgrounds
in Drosopila melanogaster (II) (MUKAI, YOSHIKAWA and YAMA7.AKI)
83. Radiation-induced mutation rate and doubling dose in polygenes controlling viability in Drosophila melanogaster(MUKAI)..................................................
84. Radiation-induced mutation rates of polygenes controllingthe sternopleural bristle number in Drosophila melanogaster(MUKAI, YOSHIKAWA and CHIGUSA)
85. Further report on the increase in induced mutation frequency
after fractionated irradiation of gonial cells of the silkworm (TAZIMA and ONIMARU);..............

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87
88
89
90

91
92

93

95

96

97

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

86.

87.

88.

89.

90.
91.
92.
93.
94.

95.

Independence of induced mutation-rate from radiation doserate in the germ cells of hibernating silkworm embryo(TAZIMA and ONIMARU)....................... . . . . . . . . . . . . .
Enhancement of radiation induced mutation frequencey bypost-treatment of silkworm gonia with 5-bromodeoxyuridine(TAZIMA and MURAKAMI)
Relative biological effectiveness of fast neutrons in the induction of recessive visible mutations in the silkworm.
(I) Effect of 14.1MeV neutrons upon the gonia (MURAKAMI,. KONDO and T AZIMA)
Relative biological effectiveness of fast neutrons in the induction of recessive visible mutations in the silkworm.
(II) Effect of fission neutrons upon the gonia (MURAKAMI,KONDO and T AZIMA)
Relative biological effectiveness of 14.1 MeV neutrons inkilling dormant silkworm eggs (MURAKAMI and KONDO)...
Mutations induced by ingested 32p in silkworm (IKENAGA and
KONDO)...................................................
Mutagenic effects of ethyl methane sulfonate on silkworm(KONDO and IKENAGA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radiation response of actin synthesis in regenerating tissueof Triturus (OGAWA)......................................
Effects of L-gulonolactone and glucose on the synthesis of
the skeletal muscle protein, myosin, in X-irradiated Triturusembryos (OGAWA).........................................
Measurements of thymine dimers formed in ultraviolet irradiated bacteria and its related problem to photoreactivation (IKENAGA and KONDO)'"

F. Radiation genetics in plants
96. Induced mutation and pleiotropy of genes responsible forquantative characters in rice (SAKAI and SUZUKI) . . . . . . . ..
97. Relation of radiation effects to dose rates of gamma-rays in
diploid wheat (MATSUMURA and MABUCHI) . . . . . . . . . . . . . . . . .
98. Relation of radiation effects to dose rates of X- and gammarays in paddy rice (MATSUMURA and MABUCHI)
99. Effects of acute and chronic irradiations on growing wheat(MATSUMURA and FUJII)
100. Dose rate dependence of mutation rates of gamma-irradiatedpollen grains in maize (MABUCHI and MATSUMURA) . . . . . . ..
101. Relation between polyploidy and effects of gamma- andneutron-radiation on wheat (MATSUMURA)

98-

100

101

103·
106
107
108
110

111

114

116
117
117
118
119

121

CONTENTS

102.
103.
104.

vi

Relative biological effectiveness of 14 MeV neutrons to 60Co
gamma-rays in einkorn wheat (FuJII)..................... 12
RBE in Arabidopsis treated with fission and 14.IMeV neutrons
to gamma-rays (FUJII).................................... 12
Effects of gamma-irradiated pollen grains on pollen-tube
mitosis (IWANAMI and MATSUMURA)........................ 12

Human genetics
105. Studies on selection in ABO blood groups (HIRAIZUMI, MATSUNAGA, IZUMIYAMA and FuRusHo)............................
106. Down's syndrome in Japan (MATSUNAGA)....................
107. Clinical conditions of patients with apparently normal chromosomes (TONOMURA).....................................
108. Variation in length of the Y chromosome in man (TONOMURA
and ONO)109. Drumsticks of polymorphonuclear leucocytes in newborn
females (TONOMURA, ONO and SOGA)
110. Function of the amino group in the molecular complex consisting of hemoglobin and haptoglobin (SHINODA) . . . . . . . . .
111. Haptoglobin and G-6-PD deficiency in a Japanese population(SHINODA)

133

Books and papers published in 1963 by staff members.............

135

G.

12:
121
13(
13]
13~

132

GENERAL STATEMENT
According to the first construction plan for our main building,the erection of the remaining one third was started in August
1963. By the end of April 1964, the building will be completed.
In addition, the construction of a r-greenhouse was started at
the end of the year.
As to the research activities, the second laboratory of the
Department of Microbial Genetics was established in July. The
main object of this laboratory is the analysis of cellular regulation mechanisms of gene action.
Dr. M. Kimura finished his two year work at the Universityof Wisconsin. He attended the XI International Congress of
Genetics in Hague serving as vice president. He came back to
the institute in September. Other members who were working
in the U.S.A., Drs. S. Iyama, T. H. Yosida, Y. Yamada, T.
Taira and S. Nawa, came back to the institute, and are continuing
their researches.
At the XI International Congress of Genetics, Japan was given
the honor to be the host for the next Congress in 1968. In
order to organize and to operate the Congress, our members haveto work hard during the coming years.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

ABSTRACT OF DIARY FOR 1963
Jan.
Feb.
Mar.
Mar.
Apr.
May
May
June
July
July
July
Aug.
Aug.
Aug.
Sept.
Nov.
Nov.
Nov.
Nov.

25.
22.
15.
25.
26.
15.
24.
14.
5.
9.
29.

lllth meeting of Mishima Geneticists' Club.
112th meeting of Mishima Geneticists' Club.
113th meeting of Mishima Geneticists' Club.
114th meeting of Mishima Geneticists' Club.
115th meeting of Mishima Geneticists' Club.
49th Biological Symposia.
116th meeting of Mishima Geneticists' Club.
117th meeting of Mishima Geneticists' Club.
118th meeting of Mishima Geneticists' Club.
51st Biological Symposia.
summer Seminar of Genetics given by Junior Research
2. } Members.
6. 52nd Biological Symposia.
31. 119th meeting of Mishima Geneticists' Club.
20. 120th meeting of Mishima Geneticists' Club.
19. 53rd Biological Symposia.
20. 54th Biological Symposia.
21. 55th Biological Symposia.
23. 121st meeting of Mishima Geneticists' Club.

STAFF
Director
Hitoshi KIHARA, D. Sc., M. J. A., Emeritus Professor of Kyoto University

Members1.

Department of Morphological Genetics
Yataro TAZIMA, D. Ag., Head of Department
1st Laboratory: Yataro TAZIMA, D. Ag., Head of Lab.
Kimiharu ONIMARU, Akio MURAKAMI!)
2nd Laboratory: Bungo SAKAGUCHI, D. Ag., Head of Lab.
Toshihiko SADO, D. Ag. (in U. S. A.), Susumu KOBAYASHI 2 ), Kugao
OISHI 2 )2. Department of Cytogenetics
Yo TAKENAKA, D. Sc., Head of Department
1st Laboratory: Toshihide H. YOSIDA, D. Sc., Head of Lab.
Kazuo MORIWAKI, D. Sc., Akira NAKAMuRA3 )STAFF

2nd Laboratory: YO TAKENAKA, D. Sc., Head of Lab.
Yoshiaki YONEDA, D. Sc., Yao-Yen CHU 4 )3. Department of Physiological Genetics
Chozo OSHIMA, D. Sc., Head of Department
1st Laboratory: Chozo OSHIMA, D. Sc., Head of Lab.
Toshifumi TAIRA, D. Sc., Takao W ATANABE 2 ), Taishu W ATANABE 3 )2nd Laboratory: Hitoshi KIHARA, D. Sc., Head of Lab.
Koichiro TSUNEWAKI, Ph. D., Sadao SAKAMOTO, Tadao C. KATAYAMA 4 ).
Kozo NISHIKAWAsl, Yasuo NAKAI 4 )4. Department of Biochemical Genetics
Mitsuo TSUJITA, D. Ag., Head of Department
1st Laboratory: Saburo NAWA, D. Sc., Head of Lab.
2nd Laboratory: Yoshito OGAWA, M. D., Head of Lab.
Toru ENDO, D. Ag., Izumi KOBAYASHI 3 )3rd Laboratory; Mitsuo TSUJITA, D. Ag., Head of Lab.
Susumu SAKURAI
5. Department of Applied Genetics
Kan-Ichi SAKAI, D. Ag., Head of Department
1st Laboratoty: YAMADA, D. Ag., Head of Lab.
Takatada KAWAHARA, D. Ag.,
2nd Laboratory: Kan-Ichi SAKAI, D. Ag., Head of Lab.
Shinya lYAMA, D. Ag., Akira MIYAZAWA, Yoshiya SHIMAMOTOS )3rd Laboratory: Hiko-Ichi OKA, D. Ag., Head of Lab.
Hiroko MORISHIMA, D. Ag.
6. Department of Induced Mutation
Seiji MATSUMURA, D. Ag., Head of Department
1st Laboratory: Kiyosi TUTIKAWA, Acting Head of Lab.
Terumi MUKAI, Ph. D., Isao YOSHIKAWA 2 ), Tsuneyuki YAMAZAKI 2 ),Akira AKABORI S )2nd Laboratory: Seiji MATSUMURA, D. Ag., Head of Lab.
Taro FUJII, D. Ag., Tomoo MABUCHI 4 )3rd Laboratory: Seiji MATSUMURA, D. Ag., Head of Lab.
HIROMI ISHIWA, Mituo IKENAGA
7. Department of Human Genetics
Ei MATSUNAGA, M. D., D. Sc., Head of Department
1st Laboratory; Ei MATSUNAGA, M. D., D. Sc., Head of Lab.
Akira TONOMURA, D. Sc., Tomotaka SHINODA, Hiroshi ONO S )2nd Laboratory: Motoo KIMURA, Ph. D., D. Sc., Head of Lab.
Yuichiro HIRAIZUMI, D. Sc.
8. Department of Microbial Genetics
Hitoshi KIHARA, D. Sc., Head of Department
1st Laboratory: Tetsuo IINo, Ph. D., D. Sc., Head of Lab.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Masatoshi ENOMOTO, Michiko MITANI 2 )2nd Laboratory: Tetsuo lINO, Ph. D., D. Sc., Head of Lab.
Hideho SUZUKI, Jun-iche ISHIDsu
Research member under grant from the Toyo Rayon Foundation.
Research member under grant from the N. I. H. (U. S. A.).
3) Visiting researcher.
4) Research member under grant from the Rockefeller Foundation.
1)

2)

Part-time Staff and Research Associates
Taku KOMAI, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Sohei KONDO, D. Sc. Professor of Osaka University
Yoshinari KUWADA, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Flora A. LILIENFELD, Ph. D.
Kan OGUMA, D. Ag., Emeritus Professor of Hokkaido University
Shiro SHIRATO, M.D.,
Yoshimaro TANAKA, D. Ag., D. Sc., M.J.A., Emeritus Professor of Kyushu University
Tugub TATEOKA, D. Sc.
Kanko YOKOTA

Department of Administration
Tanryu OTOMO, Head of Department
Toyotake MINAMIGUCHI, Chief of General Affairs Section
Seiichi KOIZUMI, Chief of Finance Section
Hiroko NAKANO, Ry6ji OYAMA, Shigeru TSURUMI and Asakichi MANO
Clerks, Librarian, Typists, Chauffeur, Field Laborers and Janitors ..... 9

Misima Branch of Hatano Tabacco Experiment Station
Masao TANAKA, D. Ag., Head
Assistants .....................................•..................... 3

Association for Propagation of the Knowledge of Genetics
Hitoshi KIHARA, President
YO T AKENAKA, Managing Director
Yataro T AZIMA, Managing Director
Seiji MATSUMURA, Manager
Yosito SINOTO, Manager

COUNCIL

COUNCILyo OKADA, Director of National Science Museum, Chairman
Daigoro MORIWAKI, Professor of Tokyo Metropolitan University, Vice
ChairmanTanemoto FURUHATA, Director of Scientific Research Institute of Police
Akira KAwADA, Director of National Institute of Agricultural ScienceJ{akuo KITAHARA, Director of Institute of Applied Microbiology, University of Tokyo
Sajiro MAKINO, Professor of Hokkaido University
Seijin NAGAO, Professor of Hokkaido University
Yuichi OCHI, President of Azabu University for Veterinary ScienceJ{an OGUMA, Emeritus Professor of Hokkaido University
Toshio SAITO, Governor of Sizuoka Prefecture
TaW SAKATA, President of Japan Monopoly Corporation
Takeo SAKATA, President of T. Sakata Company
Minoru T ACHI, Director of Institute of Population Problems
Kempo TSUKAMOTO, Director of National Institute of Radiological Sciences,
Bungo WADA, Professor of Sizuoka University

PROJECTS OF RESEARCH FOR 1963
Department of l11orphological 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 and KOBAYASHI)
Genetical and embryological studies in insects (SAKAGUCHI)

Department of Cytogenetics
Cytogenetical and biochemical studies on tumor cells (YOSIDA, MORIWAKI
and KURITA)
Experimental breeding and genetics of mice and rats (YoSIDA, 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.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

extracted from certain plants (TAKENAKA and OGAWA)
Interspecific hybridization in Nicotiana (TAKENAKA)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus Lannesiana (TAKENAKA)
Cytogenetics of Oryza species (TAKENAKA, YONEDA and CHU)
Cytological studies on the yeast cell (YONEDA)

Department of Physiological Genetics'Genetical studies on insecticide-resistance in Drosophila (OSHIMA)
Physiological studies on eye-pigment formation in Drosophila (TAIRA andOSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila melanogaster (OSHIMA, WATANABE and WATANABE)
The persistence of some natural lethal genes in experimental populations(OSHIMA and WATANABE)'Studies on chromosomal aberrations of natural populations of DrosoPhila
melanogaster (WATANABE and OSHIMA)'Comparative gene analysis with reference to the origin of wheat (KIHARA,TSUNEWAKI and NISHIKAWA)
Nucleus substitution in wheat and related species (KIHARA)
Production of polyploids by N 30 treatment (KIHARA and TSUNEWAKI)'Genetic studies of wheat aneuploids (TSUNEWAKI)
Genetic basis 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)

Department of Biochemical Genetics
Biochemical genetics of insects (NAWA)
Embryological and biochemical studies in the silkworm (TSUJITA)'Genetical and biochemical studies of chromoprotein (SAKURAI)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA
and KOBAYASHI)
Chemical research in anti-tumor substances (OGAWA)
Biochemistry of the mechanism underlying variations in flower color(ENDo)

Department of Applied Genetics'Quantitative genetic studies in poultry (YAMADA and KAWAHARA)PROJECTS OF RESEARCH FOR 1963

7

Theoretical studies on plant breeding techniques (SAKAI and hAMA)Studies on competition and migration in plants (SAKAI and hAMA)
Biometrical study of cytoplasmic inheritance (SAKAI and hAMA)
Developmental genetics of quantitative characters in plants (SAKAI, lYAMA,SUZUKI and SHIMAMOTO)
Genetic studies on developmental stability in plants (SAKAI and SHIMAMOTO)
Studies on the effects of linkage disequilibrium in Drosophila population(IYAMA)
Comparison of evolutionary mode between series Sativa and series Glaberrima (MORISHIMA and aKA)
Survey of geographical variation in O. perennis (MORISHIMA and aKA)
Crossiong-experiments and observations of sterility of hybrids between
wild and cultivated rice strains (MORISHIMA and aKA)
Analysis of sterility genes in Oryza (aKA and MORISHIMA)

Department of Induced Mutation
Radiation genetics of mice (TUTIKAWA)
population genetics of Drosophila (MUKAI, CHIGUSA and YAMAZAKI)
Studies on the effects of irradiation on populations (MUKAI and YOSHIKAWA)
Estimation of polygenic mutation rates in Drosophila (MUKAI, YAMAZAKI
and YOSHIKAWA)
Radiation genetics of cereals (MATSUMURA, FUJII and MABUCHI)
Radiation genetics and its practical application (FuJII and MABUCHI)
Biophysical studies of radiation genetics (KONDO, ISHIWA, YAN and IKENAGA)
Mutagenic action of chemicals (KONDO, ISHIWA and IKENAGA)
Radiation dosimetry (KONDO and ISHIWA)
Male-sterility in sugar beets (MATSUMURA)
Genome analysis of Oryza species (MATSUMURA and MABUCHI)

Department of Human Genetics
Selection in ABO blood groups (MATSUNAGA and HIRAIZUMI)
Down's syndrome in Japan (MATSUNAGA and TONOMURA)
Cytogenetics in man (TONOMURA)
Sexual dimorphism in resting nuclei (TONOMURA)
Biochemical studies on plasma proteins, haemoglobins and G-6P-D (SHI"NODA)
Chemical modification of ribonucleic acid and their constituents (SHINODA)
Theoretical studies of population genetics (KIMURA)
Effects of radiation-induced mutations on fitness (HIRAIZUMI)8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)

Department of Microbial Genetics
Immunogenetics of Salmonella (IINO and ENOMOTO)
Genetics of multi-drug resistance in bacteria (lINO)
Genetics of motility in bacteria (ENOMOTO)
Genetics of cellular regulatory mechanisms (SUZUKI and ISHIDzu)
Genetic fine structure analysis on microorganisms (IsHIDzu and IINo)

FOREIGN VISITORS IN 1963
Feb.

23.

23.
Feb.
March 13.

April

29.

May

3.

May

14.

May

15.

May

15.

May
June

24.
3.

July

8.

July

20.

July

27.

Aug.

6.

Dr. N. E. JODON (Rice Experiment Station Crowley, Louisiana, U.S.A.)
Dr. S. BATRA (Bethune College Calcutta -12, India)
Dr. Y. TANADA (Professor, Department of Insect Pathology,
California U.S.A.)
Dr. S. WORTMAN (Associate Director, The International Rice
Research Institute, Los Banos, Laguna, The Philippines)
Dr. W. R. SINGLETON (Blaudy Experiment Farm, Universityof Virginia, Charlottesville Virginia, U.S.A.)
P. NARAHARI (Scientific Officer, Biology Division, Atomic
Energy Establishment, Tronbay Byculla, Bombay - 8, India)
Dr. E. MAYR (Comparative Zoology, Harvard University,
Cambridge, Mass., U.S.A.)
Dr. D. D. KECK (National Science Foundation, Washington
25, D. C., U.S.A.)
Prof. Dr. H. KAPPERT (Munster, i/Westf., Germany)
Dr. J. E. O'CONNELL (Deputy Chief, Tokyo office, National
Science Foundation)
Dr. R. C. PIGHETT (Professor, Department of Agronomy,
Purdue University Lafayette, Indiana, U.S.A.)
Dr. M. T. HENDERSON (Professor of Agronomy, Louisiana
State University, Louisiana, U.S.A.)
Dr. A. B. JOSHI (Dean, Post-Graduate School & Deputy
Director, Indian Agricultural Research Institute, New-Delhi12, India)
Dr. H. GRUNEBERG (Experimental Genetics Research Unit
(Medical Research Council) University College, London)FOREIGN VISITORS IN 1963

Sept.

17.

Sept.

19.

Sept.

27.

Sept.

28.

Oct.

13.

oct.
oct.

19.
21.

oct.

23.

Nov.

11.

Nov.
Nov.
Dec.

11.

Dec.

5.

Dec.

9.

Dec.

25.

18.
1.

9

Dr. H. W. LI (Director, Institute of Botany, Academi
Sinica)Dr E. M. HUTTON (Assistant Chief, C. S. L R. O. Division 0
Toropical Pastures. Cunningham Laboratry, Mill Road, St
Lucia, Brisbane, Australia)
Dr. A. SCHENK (Director of National Sericultural Experimen
Station, Ales, France)
Dr. A. W. NORDSKOG (Professor of Iowa State University
U.S.A.)T. J. LEE (Chungang University, College of Liberal Arh
and Science, Department of Biology)
Dr. D. HAMISSA (Plant Nutrition Section, Giza, Egypt)
T. S. PATTABIRAMAN (Member of Parliament, Secretary oj
Congress Party in Parliament, New-Delhi, India)
Dr. H. E. BREWBAKER (Sugar Beet Consultant, United StateE
Aid Mission to Iran, U. S. Embassy, Tehran, Iran)
Dr. C. STERN (Univerity of California, Berkeley, California,
U.S.A.)
M. S. EL-BALAL (National Research Center, Cairo, Egypt)
Dr. A. ABRAHAM (Professor of Kerala University, India)
Dr. A. G. STEINBERG (professor of Biology, Western Reseve
University. U.S.A.)
Prof. J.R.A. McMILLAN (Dean, Faculty of Agriculture, University of Sydney, Australia)
J. N. TEPORA (College of Agriculture, University of thePhilippines College. Laguna, Philippines)
Dr. D. ATSMON (The Weizman Institute of Science, Rebovoth,
Israel)RESEARCHES CARRIED OUT IN 1963
A. GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF ANIMALS
1.

Genetic variability of deleterious genes in a large natural
population of Drosophila melanogaster11{By Chozo OSHIMA, Takao WATANABE and Taishu WATANABE)

A great number of flies were collected on the lees of grapes in severa
graperies of Kofu and Katsul1uma cities in the middle of October 1963
More than seven hundred second chromosomes were isolated individuallyfrom collected male flies and duplicated by using the complete multiplE
inversion method. After four generations, 666 strains were obtained
which were heterozygous for Cy chromosome and each second chromosomEand their genetic backgrounds were substituted with isogenic chromosomes of the Samarkand strahl.
The viability of flies, having such genetic constitutions, was examined
and their second chromosomes were divided into four classes; lethal,semi-lethal, subvital and normal, by the degree of viability of all homozygous flies. The frequencies of the four classes were 17.06, 19.01, 27.40
and 36.53 per cent respectively.
The frequency of deleterious chromosomes in the large population(17.06+19.01 per cent) was remarkably higher than that in the small
Suyama-Juriki population (13.14+5.60 per cent). On the other hand, themean viability of homozygous flies for 415 quasi-normal chromosomes
was estimated to be 77.22, when the viability of Curly and quasi-normal
heterozygous flies was standardized as 100. This value was about ten
per cent lower than that (88.77) for 330 quasi-normal chromosomes inthe small population.
From the results, it may be concluded that the large population had alarger diversity in recessive deleterious genetic variability than the small
population. However, it is generally considered that the diversity could
have been reduced by increased chance of homozygosity in a small population, but the rates of reduction of the deleterious classes were notsimilar in our results. In the small population, the frequency of semilethal chromosomes was remarkably reduced, but that of lethal chromosomes has retained at a considerably high level.
II This work was supported by the United States Public Health Service Grant,
RG 7836.

12 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

2. Mechanism of persistence of a lethal gene in asmall natural population' )(By Chozo OSHIMA, Takao WATANABE and Taishu WATANABE)

Lethal second chromosomes had been isolated from a small population(Suyama and Juriki population) during the past four years (1959-1962)
and allelism tests were performed repeatedly between lethal genes isolated in two successive years. From the results, the evidence was obtainedthat a special lethal gene had been maintained in the same population
for a long period such as three years at least. The lethal strain has been
maintained in the laboratory by the Curly balanced system, and when
the salivary chromosome was observed, the lethal gene was found to bealways associated with a paracentric inversion (In(2L)A:26A-33E) on theleft arm of the second chromosome. On the other hand, its location on
the chromosome could be estimated to be 53.2 by the crossing overvalues from definite loci of several dominant marker genes. The locus
was very close to centromere on the left arm and it was presumed tolie between the centromere and the inversion.
The locations of other lethal genes isolated simulteneously from theSuyama-Juriki population in 1962 were determined. The majority were
distributed in the central region, with a few found in both terminal
regions. These findings may imply the existence, in the central and bothterminal regions of the chromosome, of an epistatic gene complex which
is made up of some closely linked loci, and behaves as a unit. If theparacentric inversion existing in both mid-arms of the second chromosome
were heterotic, it could contribute to the establishment and maintenanceof such a gene complex in a natural population.

3. Location of lethal genes on the second chromosome!)(By Takao WATANABE and Chozo OSHIMA)

About one hundred lethal chromosomes were isolated from many flies
caught in houses of Suyma and Juriki, and also those emerged fromseveral traps kept there for a week. These lethal chromosomes weredivided into 18 groups based on the results of allelism test. Six chromosomes belonging to three of these groups seemed to carry two lethal
genes each allelic to a different lethal. Thirty-eight lethal chromosomes
isolated from different flies were found to have one allelic gene in com1) This work was supported by the United States Public Health Service Grant,
RG 7836.

RESEARCHES CARRIED OUT IN 1963

13

mono Such a high frequency of appearance of a certain lethal gene coul<be caused by genetic drift.
The balanced strain with dominant marker gene (SP Bl L) and Curl.
was crossed with fifteen balanced strains involving each a lethal an(
Curly gene. F 1 female flies (lethal heterozygotes) carrying dominant marke
genes were back-crossed to the original Cy-lethal heterozygous males
Non-Cy and Cy flies, having eight different phenotypes each, were obtaine<in the F 2 generation. The crossing over values between these dominan
marker genes were examined by the rate of recombinants among C:
offspring. The location of inversion, if the lethal chromosome had one
was determined at the same time. The number of non-Cy offspring if
these cultures was reduced; especially the number of some phenotype:decreased due to the presence of the lethal gene. It could be inferrec
from this result, which one of the four regions included the lethal geneand the locus of the gene could be determined by the crossing over valUEfrom the marker gene. The results are shown in Fig. 1.

Fig. 1. The location of lethal genes in the second chromosome
extracted from the Suyama-Juriki natural population.
* associated with In(2L)A: 26A - E**
ff
In(2LR)D: 36F - 49B

The majority of lethal genes were distributed in the central region,
and a few were located in both terminal regions. These lethal genes
such as 1(2)62j 104, 106 and 113 were associated with an inversion.
However, no lethal genes seemed to be included within the inversion.
Two loci of lethal genes in the double lethal chromosome were separated
by more than 50 units in all cases.

14 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

4. Frequencies of inversions in a large and asmall natural population1 )(By Taishu WATANABE2) and Chozo OSHIMA)

By the observation of salivary gland chromosomes, the chromosomal
polymorphism was analized in a large population in Kofu and Katsunuma
cities, Yamanashi Prefecture, and also in a small one in Suyama andJuriki villages, Shizuoka Prefecture.
One hundred larvae were sampled from a cage population, originatedfrom the Suyama and Juriki population and kept in a population cagefor eight months, and on the other hand, one hundred larvae were employed, developed from different female flies caught in the Kofu andKatsunuma population.
Ten kinds of autosomal inversions were observed; their break points
are shown in Table 1 and their frequencies in the small Suyama and thelarge Kofu and Katsunuma population are given in Table 2.
Table 1.

Autosomal inversions found in Japanese natural populations
and their locations.

Chromosome

Inversion

Break points

2

In(2L)AIn(2L)BIn(2R)CIn(2LR)D

26A - 33E
22D - 34A
52A - 56F
36F - 49B

In(3L)E
In(3L)FIn(3R)GIn(3R)H

66C -71B
89D -96A
92D -lOOF

In(3R)IIn(3R)J

93D - 98F
96E - 98F

3

63A -74C

The large population carried more inversions on both arms of the two
autosomes. No inversion was found in the X chromosome. A pericentric
inversion (In(2LR)D) was only found in a lethal chromosome isolated
from the Suyama population. In(2L)A, In(3L)F and In(3R)J were rather
infrequent and seemed to be endemic. But In(2L)A was observed tohave been maintained for at least three years in the Suyama-Juriki
1) This work was supported by the Grant of the Japan Society for the Promotionof Sciences.
2) Visiting researcher from Department of Biology, Kyushu University.

15

RESEARCHES CARRIED OUT IN 1963
Table 2.

population

Frequencies of inversions in the small andthe large population.
SuyamaI Katsunuma ,-Inversion & standard----

2L (standard)
In(2L) A
In(2L) B

Kofu

Frequency (%)
84.5
0.5
15.0

68.0
0.0
32.0

67.0
0.5
32.5

100.0
0.0

79.0
21.0

73.0
27.0

97.5
2.5
0.0

89.5

92.0
6.5
1.0

82.0
2.5
15.5
0.0
0.0

61.0
18.0

~

2R (standard)-

In(2R) C
3L (standard)
In(3L) E
In(3L) F
3R (standard)
In(3R) GIn(3R) H
In(3R) IIn(3R) ]

8.5
2.0

I

10.0
11.0
0.0

63.5
11.0
14.0
11.0
0.5

population in association with the lethal gene (l(2)62j 106). Other con
man inversions, such as In(2L)B, In(2R)C, In(3L)E, In(3R)G, In(3R)H an
In(3R)I seem to be in the state of equilibrium in the large populatiorbecause the number of their hetero and homozygotes closely fitted thfrequencies expected from Hardy-Weinberg's law.

5. Cytodifferentiation in the latbody 01 Drosophila melanogaster(By Toshifumi TAIRA and T. M. RIZKI)In the wild type strain of D. melanogaster, the anterior region of th
fatbody at the prepupal stage is clearly distinguishable from the posterio
region due to a difference in autofiuorescent cytoplasmic inclusions. Th
inclusions in the cells of the anterior region were identified as kynurenine and those of the posterior region were confirmed to be isoxantho
pterin, on the basis of chromatographic and spectrophotometric examina
tions. In the vermilion mutant, the cells of the anterior region of thl
fatbody have no cytoplasmic kynurenine granules, but the posterior regior
has as many isoxanthopterin particles as the wild strain. The sepia (semutant remarkably differs from the wild type strain in that the anteriol
region containing kynurenine granules extends farther toward thE

16 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

front area of the posterior region. The distribution of isoxanthoPteri
particles in the cells of the posterior region appears to be similarboth strains, but the number of particles per cell in the se strain i~
considerably smaller than in the wild type strain. The rosy mutant has
many granules of 2-amino-4-hydroxypteridine instead of isoxanthopterinin the cells of the posterior region. In the Henna-recessive mutant, many
particles of sepiapterin, which never were detected in other strains, Werefound among a smaller number of isoxanthopterin particles in the cellsof the posterior region. These observations are in good agreement withthe results from qualitative and quantitative analyses of those fatbody
compounds.
Our observations show that specific enzymes releasing action of therelevant genes of the examined eye-color mutants appeared in the larval
fatbody as early as even before the formation of the compound eyes.

t·6.

A drive element, Nomadic Hunter (NH), in a natural
population of Drosophila melanogaster(By Yuichiro HIRAIZUMI and Ky6ko NAKAZIMA)

About 600 males of D. melanogaster were collected from a natural
population of Ohdate, Akita Prefecture. These males were then crossed
Table 1.

The number of tested and NH-carrying males, andthe time of fly collections in 1963.

No. of tested males

No. of NH males

Time of collection

120Aug. 23-Aug. 28

120

2

Sept.

63

1

Sept. 12-Sept. 15

230

2

Sept. 23-Sept.27

73

1

Oct.

57

1

Oct. 17-0ct. 20

48

1

Oct. 30-Nov.

0*0

Middle of Nov.

1

0

End of Nov.

Total 604

83-Sept. 7

4-0ct.

103* Fly collections were continued but no melanogaster was captured.

17

RESEARCHES CARRIED OUT IN 1963

'ngly to cn bw females.

From the F 1 progenies of each cross, 5 males
In the F 2 genera~on the ratio of pheno.typically. en bw to wild flies is expected to be
pproximately 1:1, and 1ll fact thIs was so for almost of all tested males.
~here were however 8 exceptional cases where some of the wild second
hromosomes behaved as if they carried segregation-distorter (SD) locus.
~hiS probably new drive element will be called, for the time being, as
Nomadic Hunter (Symbol NH). The numbers of tested males, NH-carrying males, and the time of fly collections are presented in Table L
Examination of salivary gland chromosomes revealed that all of the 8
Nfl-bearing chromosomes carried, in their right arms, a inversion complex which was apparently different from those in SD-5 and SD-72
chromosomes. Further studies on this NH element are in progress.
SI ere collected to back-cross singly to cn bw females.

7. Further evidence supporting a high spontaneous mutation
rate of polygenes controlling viability of
Drosophila melanogaster 1J(By Terumi MUKAI, Sadao CHIGUSA and Isao YOSHIKAWA

As previously reported (Mukai, Chigusa, and Yoshikawa, Annual Report13) spontaneous mutant polygenes controlling viability have been accumulated by the following method: A single male Pm!+ from the cross
Cy!PmX +!+ (an isogenic stock of Burdick: W160) was sampled andmultiplied by the cross Cy!Pm (~~)xPm!+(lo), and 104linesofCy!Pm
X Pm! + i (i = 1, 2, ..... , 104) were established. In each line, the second
chromosome has been maintained through a single male by the cross
Cy!Pm (5~ ~)xPm!+i(o). In several generations, homozygous and heterozygous viabilities of these chromosomes have been tested and theresults from Generation 10 through Generation 25 were previously reported.
Table 1.

Analysis of variance for homozygote viabilitiesof quasi-normal lines in Generation 32.

SourceSum of squaresd.£.

Mean squareF

Between lines
Error
Total

4026.30
3239.317265.61

79
560
639

50.96585.78458.81****** significant at a low level.
1) This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

18 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

In Generation 32, the homozygous viabilities of those chromosome lines
were estimated with the aid of Cy-method for SO quasi-normal lines. The
result of variance analysis is presented in Table 1.
The genetic variance among quasi-normal lines was estimated from
Table 1 to be &~=5.6477. The reduction of the mean from the control
was 4.76. This value was estimated by a similar method to that describ.
ed in the paper presented in Annual Report No. 13. The average numberof mutations was estimated by the following formula which was also
given in a previous paper (Annual Report No. 13):

K<p
B -

(IJ

where A is the reduction of mean viability and B stands for the genetic
variance. The result is 4.011S::;:p, which implies that polygenic viability
mutations occurred at the rate of 0.1254/second chromosome/generation.
If interaction existed among loci, this figure might be a slightly biased
estimate, but would not deviate much from the true value. The resultshows a remarkable consistence with the previous estimate (0.130l/second
chromosome/generation).
S.

Polygenic mutation rate and homozygous genetic
load in Drosophila populations 1 )(By Terumi MUKAI)

In an equilibrium random mating population where frequency of Gene
A is p and that of Gene a is q, q is approximately p/hs (p indicates.
mutation rate from "An to "an and h the degree of dominance of "a"gene). Thus, homozygous load becomes p/h per locus under the "Classical" model.
If there is no complicated interaction among loci, the expected survivalrate, when the population becomes suddenly homozygous without selection, can be expressed as follows:

S =e

-L,~
h

(l}

In the previous Annual Report (No. 13), we have estimated the total
mutation rate in the second chromosome of D. melanogaster to be ~tt=0.1301. Under the assumption of ii=0.04 (Stern, et al. 1952), calculated
S becomes 0.03868. According to Greenberg and Crow (1960), the relative
survival rate of homozygotes from equilibrium populations was estimated
1) This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

19'

RESEARCHES CARRIED OUT IN 1963

to be 0.632 in comparison with that of random heterozygotes. ThesetwO figures are significantly different. The difference must have beencaused by the simple assumption of the Classical Hypothesis. In concluion, it can be stated that the Classical Hypothesis in its simplest form~s inconsistent with the actual situation of random mating populations.

9. Overdominance of spontaneous mutant polygenes controlling
viability in homozygous genetic background in
Drosophila melanogaster1 )(By Terumi MUKAI, Sadao CHIGUSA and Isao YOSHIKAWA)

In Generation 32 of the experiment described in the foregoing article(7), dominance degrees of accumulated mutant polygenes were tested inheterozygous condition with a chromosome supposed, since it did not undergo any mutation, to be identical to the original chromosome. Line92 (abbreviated as +0/+0) was chosen for this purpose on the basis of theresult from the preliminary test in which its viability in homozygous condition was estimated as normal (viability index =32.08). The viabilities ofheterozygotes were estimated by counting the number of offspring in the
crosses +0/+0 (7"1 "I) x Cyl +i (51) 1)). The relative viability was expressedby the percentage of phenotypically wild-type flies in one culture.
For the sake of simple presentation, the 80 quasi-normal lines weredivided into 5 groups in the order of degree in homozygote viability.
Thus, each group consisted of 16 lines. The relationship between the
average of homozygote viabilities and that of heterozygote viabilities ispresented in Table 1.
Table 1.

Relationship between homozygote and heterogote viabilities.

Rank

1

2

Homozygote*
Heterozygote**

31.66
51.11

3

4

5

29.18

27.98

51.38

51.51

26.82
51.70

51.85

* Result of counting 427,886 flies.

** Result of counting 392,077 flies.

24.61

Expected viability of normal flies=33.3.
Expected viability of normal flies=50.0.

The genotypic correlation between homozygote and heterozygote viabilities was estimated to be YGG'= -0.45. These results clearly indicatethat newly arising mutant polygenes show overdominance with respect
to viability in homozygous genetic background at least when the mutant
1) This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

polygenes are located only in one chromosome.
For reference, the control viability of the heterozygotes was estimatedto be 49.91 by using viabilities of 5 heterozygotes whose corresponding
homozygotes had shown the highest viabilities in the 80 homozygoUsly
quasi-normal lines.

10. Deleterious heterozygous effect of spontaneous mutant polygenes
controlling viability in heterozygous genetic backgrounds
in Drosophila melanogaster11(By Terumi MUKAI, Sadao CHIGUSA and 1sao YOSHIKAWA)

In Generation 32 of the experiment for spontaneous mutations, similar
experiments to those described in the foregoing article (9) were conductedusing two normal second chromosomes; one (abbreviated +w!+w) fromthe population from where the original chromosome (+0) has been extracted, and the other (abbreviated -h!+B) from an entirely unrelated
population.
The viabilities of heterozygotes were estimated by the same method
as that for the heterozygotes with the original chromosome: Crosses+w/+w (799) X CY!+i (500) and +B!+B (79 \1) XCY!+i (500) were con.
ducted for each line. The percentage of wild-type flies in the offspring
was employed as viability index for heterozygotes.
For the sake of simple presentation, the 80 quasi-normal lines weregrouped into 5 in the order of degree in homozygote viability. Thus,
each group consisted of 16 lines. The relationship between the averageof homozygote viabilities and that of heterozygotes is presented in Table 1.
Table 1.

Relationship between homozygote and heterozygote viabilities.

Rank
Homozygotes*
Intra-populational
heterozygotes +w/ +i**Inter-populational
heterozygotes +B/ +i***

I

1

2

3

4

5

31.66

29.18

27.98

26.82

24.61

50.92

50.22

50.29

50.06

49.87

55.28

54.41

55.26

54.60

54.78

* Result of counting 427,886 flies.
** Result of counting 390,152 flies.
*** Result of counting 465,820 flies.

Expected viability of normal flies=33.3.
Expected viability of normal flies=50.0.
Expected viability of normal flies=50.0.

The genotypic correlation between the homozygote and heterozygote
1) This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

21

RESEARCHES CARRIED OUT IN 1963

'abilities was estimated to be rGG' = +0.56 (intra·populational) andrGG'~ +0.27 (inter·populational). These results clearly indicate that newly
rising mutant polygenes are heterozygously detrimental to their carriers~ heterozygous genetic background.

For reference, the control viability of the heterozygotes was estimatedto be 51.05 and 55.95 in intra· and inter·populational hybrids, respectively.

1)1

11.

Allelism frequency in spontaneous recessive lethal
genes in Drosophila melanogaster1 )(By Isao YOSHIKAWA and Terumi MUKAI)

The method for accumulating spontaneous mutations was described in
the Annual Report No. 13. In those experiments, the tests for the detection of recessive lethal mutations in the second chromosomes were conducted together with the estimation of the mutation rate of polygenes
controlling viability. Each lethal mutation has an almost equal probability to be established in each line. The twenty-five lethal chromosomes
have been collected through the forty·first generation.
The allelism frequency among all the collected recessive lethals wasestimated. For a series of lethal chromosomes all possible cross combinations were made, The experimental results are shown in Table 1.
Table 1.

The result of allelism tests.

No. of lethal
chromosomes

No. of crosses

25

300

Frequency of
appearance
1
2
3
21

2

o

Frequency ofallelism

0.0067It has been reported that the allelism frequency among recessive lethal
genes extracted from unrelated populations is 0.0025 (Prout 1954). The
test of significance for the difference between this figure and the present
estimate was carried out, but it was impossible to detect it.
On the basis of this finding and another estimate in the present experiment with respect to the recessive lethal mutation rate (O.0063/second
chromosome/generation), which is almost equal to that of Wallace (1956),
it may be concluded that spontaneous recessive lethal mutations occurredat random in the second chromosome loci which are mutable to therecessive lethal.
1) This work has been supported by Grants GM-7836 and RH-34 from the PublicHealth Service, U. S. A.

22 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

12. Spontaneous mutation rate of polygenes controllingdevelopmental time in Drosophila melanogaster '1(By Terumi MUKAI and Tsuneyuki YAMAZAKI)As a by-product of the experiment of Generation 32 for the estimationof mutation rate of polygenes controlling viability, we have estimated thedevelopmental time of 80 quasi-normal lines in homozygous condition.
Using Cy-method, two types of fiies, Le., phenotypically Cy flies andwild-type fiies, segregated in one vial. The difference between the
average developmental time of these two types [(+)-(Cy)] was employed,as index.
The analysis of variance was conducted for the trait in question, and theresult is given in Table 1.
Table 1.

Analysis of variance for developmental time in Generation 32.

SourceBetween lines
Error
Total

I

S.S.

d. f.

M.S.

F

41.9997
63.022279560

0.53160.12544.24**

105.0219

639** significant at the 1% level.
The genetic variance was estimated as &5=0.05078. The delay of theaverage developmental time in comparison with the control was 0.5785
days. The control was estimated as the average developmental time of
the five lines. The criterion of selecting these five lines was described in the foregoing article (9). The high negative genotypic correlation
between viability and developmental time described in the following
article (13) justifies this method.
Mutation rate of polygenes controlling the trait in question was estimated by using Formula (1) [Mukai et al. (7)]. The result is as follows:(0.5785)2 < P
0.05078 6.5892 :c::pwhere p indicates the average number of mutations per second chromosome. From this result, it can be said that the mutation rate of polygenes controlling developmental time is 0.2059/second chromosome/generation. This rate is of the same order as that of viability mutations.
II This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

RESEARCHES CARRIED OUT IN 1963

23

The reliability of this estimate might be lower than that of viability
mutation rates, because the estimated control value has larger variancein the former than in the latter.

13. Correlation between viability and developmental
time in Drosophila melanogaster11(By Tsuneyuki YAMAZAKI and Terumi MUKAI)

In the present experiment, correlation between viability and developmental time was examined. Study of this kind was first carried out by
Dobzhansky and Spassky (1942).
As a by-product of the experiment for the estimation of polygenic
viability mutations (7), we have estimated the developmental time ofhomozygotes and heterozygotes produced by random combination ofhomozygote lines in Generation 32. Using Cy-method, two types of flies,
i.e., phenotypically Cy and wild-type, are found in one vial. The difference between the average developmental time of these two types[(+)-(Cy)] was employed as index.
The result of the experiment is given in Table 1 which shows thatthe average developmental time is negatively correlated with viability,i.e., both phenotypic and genotypic correlations between viability anddevelopmental time are negative.
Thus, the mutant polygenes control both developmental time and viability pleiotropically, and it can be concluded that more viable lines. develop faster than less viable ones at least when these lines have notexperienced any natural selection.
Table 1.

Characteristics of the average developmental timeof each genotype in Generation 32.

Item
No. of lines tested
Average viability index
Genetic variancePhenotypic correlation with viability
Genotypic correlation with viability

Homozygote
80+0.56(day)0.05078-0.2565**-0.7358**

Ramdomly combined
heterozygote
77+0.49(day)0.07838-0.4238**-0.8947**** significantly different from zero at the 1% level.
1) This work has been supported by Grants GM-7836 and RH-34 from the Public
Health Service, U. S. A.

24 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

14.

Persistence of two recessive genes in experimental
populations in Drosophila melanogaster (III)1)(By Sadao CHIGUSA and Terumi MUKAI)

Two cases of heterosis associated with the recessive genes sepia andexpanded wing have been examined as to whether or not these phenomena
have been caused by overdominance.
Four artificial populations (Pop. A-I and Pop. A-2 for se, and Pop.
B-1 and Pop. B-2 for ew) were established, and the gene frequencies ofPop. (A or B)-l and Rop. (A or B)-2 in the starting generation were 0.5
and 0.1, respectively. The homozygosity of the genetic backgrounds in
A-populations is extremely low, while that of B-populations is relatively
high. Each population consisting of 8 sub-populations has been main.
tained by so-called Pearl's method, and the frequencies of recessive homo.
zygotes were estimated in some generations. Four populations seem tohave reached equilibria since Generation 20-25, which indicates thesuperiority of the heterozygote over either homozygote with respect tofitness. The gene frequencies that were estimated in the latest genera.
tions of these populations are given in Table 1.
Table 1.
Population
A-I (se)A-2 (se)B-1 (ew)
B-2 (ew)

I

Generation

Gene frequencies in populations.
count Progeny test* Gene freq.
IIPopulation(recessive+/+ +/x x/x
homo.)

54
44
50
46

4.87
7.45
1.01
3.90

(q)

(%)

(%)

184
98
122
73

76
46
68
40

13
12
2
5

18.7 ±1.7
22.4±2.418.8±2.0
21.2±2.7* x indicates se or ew.
From Table 1, it can be seen that the recessive genes in question havebeen maintained in high frequencies in populations. This might havebeen caused by overdominance with respect to fitness. Further analysis
is now in progress.

15. Interruption of "sex-ratio" condition by double infection
with two SR agents of different origin 2 )(By Bungo SAKAGUCHI and Kugao OISHI)As to the nature of the so-called SR agents, it was demonstrated by
1) This work has been supported by Grant GM-7836 from the Public Health Service, U. S. A.
2) This work was supported by U. S. Public Health Service Research Grant GM
10238 from the Division of General Medical Sciences.

25

RESEARCHES CARRIED OUT IN 1963

poulson and Sakaguchi (1961) that they are minute filamentous microorganisms belonging to the order of treponema-like spirochetes with 10f.!
long and O.lp wide filaments. They occur in high concentration in thehemolymph of adult females of D. willistoni as well D. nebulosa.
Recently, Sakaguchi and Poulson (1961) found that they can be easilytransferred from either of the two species, willistoni and nebulosa, to
the Oregon strain of D. melanogaster where they become stable andpersistent.
In order to find the difference, if any, between the willistoni and thenebulosa spirochetes, an attempt was made to combine by superinfection
both kinds in the same host. For this purpose two lines of the Oregon
strain were established by artificial transfer, one hosting the willistoni
and the other the nebulosa spirochetes. The females of these two preinfected Oregon lines were superinfected, so, as to bring together thewillistoni, SRB-3, and the nebulosa, NebSR, spirochetes.

0
0

to

OJ

'"

'T

o=@~~~@=o=o~@""""'@/ ~,@-=@

fA/&

~

l
... l
00

to

..,

..
"
OJ

.c

'"

.'0
0

..,

""
OJ

to

"

i>o

@

'1
m::

·1

I)

3

Ii

!_,

9

12

,..@

r
.

'_'",""",",",,!
15

HI

21

24

@/@~
0 OR I,,,,,).'R(M,'",)

fA OR.

(NehSR)<\1=S.Im:J(Ii OR(SP.lfl3)~Ne"SR

I
27

l,~

30

33

Days after injection

Fig. 1. Interruptions of SR condition in D. melanogaster by superinfection
with SR agents from D. willistoni and D. nebulosa.
OR(SRB-3): SR line of D. melanogaster hosting willistoni SR agent.
OR(NebSR): SR line of D. melanogaster hosting nebulosa SR agent.

In detail, 14 females of the Oregon strain, hosting willistoni SRB-3
were superinfected with the original nebulosa agent (NebSR) and thepercentage of females in the progeny was examined. Figure 1 shows astriking effect in that the percentage of females was reduced from 94%

26 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

in the first brood to 45% at the third brood (after 6 to 9 days from thetime of injection), but it increased again gradually and finally reachedupwards to 80% in the tenth or eleventh brood. In the control superin_
fected by the same SRB-3 spirochetes (to which it was the host), or left
without superinfection no males were produced (Fig. 1)
The reciprocal infection was carried out in similar fashion, namely 12
females of the Oregon line, preinfected with NebSR agent, were superin_
fected with willistoni SRB-3 spirochetes. The examination of the sex
relation revealed the same tendency, except that the percentage of females
was considerably less decreased than in the other direction (Fig. 1). In
the pertinent control no males at all were produced.
The interruption of the SR condition is assumed to be due to interference between two genetically different kinds of SR spirochetes. The
mechanism of this phenomenon was demonstrated by in vivo and in vitro
experiments described in another article of this Annual Report.

16. Nature of the interference phenomenon between the "sex-ratio"
agents from D. willistoni and D. nebulosa 1 )(By Bungo SAKAGUCHI, Kugao OISHI and Susumu KOBAY ASRI)

So-called maternally inherited "Sex-Ratio" (SR) condition in D. willistoni
and D. nebulosa is caused by the presence of Treponema-like spirochetes
in the female bodies of the species hosting them (Poulson and Sakaguchi1961).
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 (as described in an other article of this Annual Report).
In order to analyse the phenomenon of interference between the twokinds of SR spirochetes, phase- and electron-microscopic examination ofhemolymph taken from superinfected females hosting both was carriedout. Many clumps were found in the hemolymph taken from suchfemales. Their formation was observed from three to six days from thetime of superinfection, and the number of free spirochetes was distinctly
reduced. Furthermore, the clumps gradually disappeared from thehemolymph of the infected females after 9 to 12 days and the numberof free SR spirochetes gradually increased again. However, no clumps
were observed in the hemolymph taken from the control which wassuperinfected with the same kind of SR spirochetes to which it was thehost.
1) This work was supported by U. S. Public Health Service Research Grant GM
10238 from the Division of General Medical Sciences.

RESEARCHES CARRIED OUT IN 1963

27

ClumP formation between the two kinds of SR spirochetes was also exmined in vitro. Hemolymphs of females of willistoni SR and nebulosa~R strain were separately removed with micropipette and were thenixed on a hollow-slide. The mixed hemolymphs were allowed to stand%r some time and were examined by phase- and electron-microscope.
Formation of clumps of SR spirochetes began after one hour from the time
f mixture and the size of clumps gradually increased. The clumps were:suallY of irregular round shape and measured about 20 to 50p in diameter.
Electron microscopic observation revealed that the clumps after one hourfrom the time of mixture consisted of many filaments which could beclearly distinguished. However some filaments were partially obliterated>6 hours later. As a control experiment, hemolymphs were mixed, eachkind among themselves. It was found that no clumps were formed in
the preparation.
Extract freed from SR spirochetes ,vas prepared from nebulosa SR
females by ultra-high speed centrifugation and was added to willistoni SR
spirochetes, and the effect was examined by electron microscopy. No
dumps were observed in the preparation but partial obliteration of filaments was seen.
Another experiment was carried out to detect the effect of the extract
freed from SR spirochetes of nebulosa females on willistoni SR condition.
The extract was injected into SR females of willistoni and the sex proportion in the progeny from the injected females was examined in eachbrood. It was found that the proportion was 100 per cent females in an,early brood but gradually decreased and finally reached to about 50 per
cent, i.e. normal sex-ratio, in 9 to 12 day broods, and SR condition wasnever restored.
These results indicate that SR spirochetes of nebulosa may produce a
substance which kills those of willistoni. It is unknown at present
whether the latter also produce a substance which has an influence on
the spirochetes of nebulosa.

17. Low temperature effects on abnormal "sex-ratio"condition in Drosophila' )(By Kugao OISHI and Bungo SAKAGUCHI)

It has been demonstrated by Poulson and Sakaguchi (1961) that thephenomenon of almost exclusively female progeny in D. willistoni andD. nebulosa, referred to as maternally transmitted "Sex-Ratio" (SR)1) This work was supported by U. S. Public Health Service Research Grant GM
10238 from the Division of General Medical Sciences.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

condition, is caused by treponema-like spirochetes present in the bodyof the females. The spirochetes of D. willistoni and D. nebulosa injectedto the Oregon-R strain of D. melanogaster have proved to be stable andpersistent (Sakaguchi and Poulson, 1962). However, some males Usuallyappeared in the earlier broods (1 to 8 days after mating) in every generation.
To test the effect of low temperature on the SR condition, young flies
of OR (SRB-3) strain which was established in Oregon-R by infection
with willistoni spirochetes, were raised at first at 15°C for 46 days andthen maintained at 24°C. The sex proportion in the first generation
hatched from those cultures at low and high temperature were examined
as shown in Table 1. It was found from the results that the percentageof the females in both cultures was 100 showing complete SR condition.
(Table 1).
Table 1.

SR condition in first and second generation derived fromparents treated with low and high temperature.

Parents culture
kept at

24°C

15°C

---b
5-8

g
i
c
d
f
h
j
e
k
9-12 13-16 17-20 21-24 25-28 29-46 47-51 52-56 57-

26:0

9:0

10:0

3:0

9:0

5:0

5:0

11:0

25:0 11:0

0

25

6

8

3

9

5

4

i
111

24

11

0

10

4

2

1

3

1

1

11

18

6

0

2

0

0

0

0

0

0

11

18

6

Brood,adays after mating 1-4----No. of females
and males in first 3:0
generation
No. of first generation flies tested
No. of first generation flies that
produced progeny
No. of SR

The females of the first generation hatched from both cultures weremated with normal Oregon-R males. All were culture at 24°C and the
proportion of the sexes in the second generation was examined. The
SR condition in the second generation derived from the parents cultured
at low and high temperature showed a different behavior. The condition
almost disappeared in the second generation derived from low temperature culture, except for 2 flies in the early broods. But, when theculture was kept at 24°C after staying at 15°C for 46 days, stable SR
condition was transmitted through first to second generation (Table 1).
The cause for the disappearance in the the former case of SR conditionin the second but not in the first generation is now investigated.

RESEARCHES CARRIED OUT IN 1963

29

18 Morphology of "sex-ratio" agents of D. willistoni and D. nebulosa

studied by electron microscope!)(By Susumu KOBAYASHI and Bungo SAKAGUCHI)

poulson and Sagakuchi (1961) were able to show that "sex-ratio" (SR)agents are small spirochetes, presumably Treponema, present in each of
the two species studied, D. nebulosa and D. willistoni. In order to makeclear whether there is any morphological difference among the SR agentsof those species, electron microscopical observations have been undertaken.
The morphology of the spirochetes in adult D. willistoni is very similar
to that of D. nebulosa. The size of the spirochetes of the two species
can be estimated to be of the order of about 0.15 to 0.08,u in diameterand 8 to 16,u in length. In most instances the spirochetes showed irregular undulation running along the filaments as represented in the photographs (Fig. 1 in page 30). It seems that in D. nebulosa the undulation
is more pronounced that in D. willistoni. Occasionally, the filaments are
branched.
A striking morphological feature are knob-like bodies ranging from
0.8 to 1.2,u in diameter and showing a weak electron density. Thosebodies may occur at any point along the filament, i. e. they may be foundeither in an intercalary or a terminal position. Furthermore, they form
one or two minute granules (buds) which may develop into young
spirochetes.
In the spirochetes so-called "protoplasmic cylinder" and "cell envolope"can be distinguished. The cylinder is 8 to 12m,u in diameter and theenvelope is about 2m,u wide. In some spirochetes the protoplasmic cylinder appeared to be double coiled, each coil approximately 5m,u thick.
Occasionally the cell envelope could not be distinguished from the cylinder by negative contrast technique.

19. Cellular studies of antibody formation 1. Proliferative
capacity of antibody-forming cells(By Toshihiko SADO and Martha R. LEONARD2»

An understanding of the process of cellular differentiation is of fundamental importance in biology. We feel that the one approach in understanding this complex cellular process is to study the cellular changes
1)This work was supported by U. S. Public Health Service Research Grant GM

10238 from Division of General Medical Sciences.

2) Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

30 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Fig. 1. Electronmicrographs of SR agents of D. willistoni and D. nebulosa.
Negative contrast technique.
A and B: SR agent of D. nebulosa (A: X 28,000, B: X 18,000). C: SR
agent of D. willistoni (X 21,000). b: Bud, br: Branch, c: Cell envelope,k: Knob-like body, p: Protoplasmic cylinder.

RESEARCHES CARRIED OUT IN 196331

that take place in an immune response because past studies have shownthat lymphoidal cells of the spleens and lymph nodes undergo functional and1Uorphological changes when stimulated by a specific antigen. Moreover,this alteration may persist for many years, and, hence, raises the question of how the information to synthesize a specific protein is transmitted
through generations of cells. In view of this consideration, the cellular
aspects of antibody formation is, in a sense, a problem of somatic cell
genetics.
It is known that somatic cellular division is characteristic of the im1Uune response. However, little is known about the proliferation of cells
that are actually synthesizing antibodies. In order to obtain better insight
into this problem, the following experiments were carried out.
Spleen cells from rabbits preimmunized against bovine serum albumin(BSA) were cultured together with the test antigen in O.l,u-porosity diffusion chambers which were implanted into sublethally irradiated mice.
At various intervals after culture, the proliferative capacity of antibodyforming cells were studied by combined W-thymidine (W-Tdr) labelingand immunofluorescence techniques. Antibody-synthesizing cells wereclassified into two types on the basis of size and appearance: a large, or
immature, cell type and a small, or mature, type. The results show thatprior to the differentiation of antibody-synthesizing cells (day 3) there
was a very rapid proliferation of blast cells. During the log phase ofantibody production (day 5) large antibody-synthesizing cells were very
active in DNA synthesis. Very few such cells are capable of synthesizing
DNA on days 9 and 11. On day 5 some of the mature cells incorporated
W-Tdr but no such cells were labeled on day 6 or later. This indicatesthat these cells are non-dividing, differentiated, terminal cells. These
data suggest that antidody response is a reflection of proliferation of
antibody-synthesizing cells.

20 Cellular studies 0/ antibody formation II. The cell cycle 0/
blast cells involved in secondary antibody response(By Toshihiko SADO and Takashi MAKINODAN1)

Studies on antibody response to suboptimal, optimal and supraoptimaldoses of antigen clearly indicate that cytokinetically the blast cells are
one of the most important cell types involved in an immune response
in a lymphoidal organ. The previous observations have suggested thatblast cells are the precursor of antibody-synthesizing cells and they do
proliferate very rapidly during the early phase of antibody response.
1)

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

32 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

For this reason, we have focused our attention to the cell cycle of blast
cells involved in secondary antibody response. When spleen cells frolll
primed rabbits are cultured with the test antigen in in vivo diffusion
chambers which are implanted in the irradiated mice, the number of
blast cells reaches a state of dynamic equilibrium between three andfour days after culture. The cell cycle of blast cells was, therefore
analysed during this period by determining the frequency of labeled
mitoses as a function of time after a single pulse injection of H 3·Tdr
to the recipient mice. From this experiment each phase of the cell cYcle
was estimated as follows: G2 , 0.7hr; M, 0.5hr; G b 0-lhr; 5, 5-6.8hr; andthe generation time, 8-9hr. To our knowledge this is the shortest generation time observed among differentiating mammalian cells. In spite ofthis short cell cycle, the estimated time for the 5 phase of 7hr is COlllparable to that of other mammalian somatic cells with a longer generation time. Our data thus support the current view that the 5 phase isrelatively constant independent of the cell type and generation time
among mammalian cells (Defendi and Manson 1963, Cameron and Greulich1963). It is of interest to note that if we accept 7hr as the length of S
phase, then the estimated generation time with use of H 3 -Tdr index(percent of labeled cells after one hour exposure to H 3 -Tdr) is also "-'%r(G. T. =7hr/H 3 - Tdr).
Insight into the problem of DNA metabolism of blast cells during the
5 phase was gained by estimating the mean grain number per metaphaseas a function of time after pulse injection of H 3 ·Tdr. The results showedthat the relative uptake of H 3 -Tdr is maximal during the latter third of
the 5 phase. These data are comparable to those reported by Humphrey
et al. (1963) in their study with Chinese hamster cells cultured in vitro.
They concluded that data of this nature reflect the rate of DNA synthesis
by cells at different stages of the 5 phase. We, on the other hand, feelthat the availability of H 3 ·Tdr with respect to the target DNA syntheticsite is variable with time, and, therefore, consider data of this nature
as reflections of the diffusion rate of H 3 -Tdr, the relative pool size of
Tdr and the DNA synthetic rate of cells in the 5 phase.

21.

Cellular studies of antibody formation III. Suppresionof appearance of antibody-synthesizing cells withsupraoptimal dose of antigen(By Toshihiko SADO and Carol CHADWICKl)

It is known that competent cells appear in reduced numbers or do not
1

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

RESEARCHES CARRIED OUT IN 1963

33

appear at all when nonprimed lymphoid cells are stimulated with eithel
suboptimal or supraoptimal doses of antigen. Previous studies in thi~laboratory showed that suboptimal anti-RBC response can be induced ina similar manner even with use of lymphoid cells from primed donors.
I!owever, we do not know whether the suboptimal response is due tc(a) decrease in the efficiency of antibody synthesis per functional cell,(b) decrease in the number of functional cells or both (a) and (b). In an
attempt to have better insight into this problem, we have used soluble BSA
antigen in this study. This permits us to assess immunohistochemicallythe number of antibody-containing cells in relation to the amount ofantibodies synthesized.
Spleen cell donors were either primed adult mice or albino rabbits.
Generally 24x 10 6 primed spleen cells were cultured in O.I.a-porosity diffusion chambers with varying doses of BSA ranging from 5 X 10- 6 mg to30mg per chamber. Anti-BSA titers were determined by Stavitsky's
passive heamagglutination method. The number of antibody-containing
cells per chamber was estimated by multiplying the number of cells perchamber by the percentage of antibody-containing cells determined with
Coon's immunohistochemical method.
The results showed that the optimal dose of BSA is approximately
5 X 1O-2mg per 24 x 106 primed spleen cells. Furthermore, a response of
approximately 5% of the maximum was obtained with an antigen .dose
either lOG-fold more than or less than the optimal dose. As regards thenumber of antibody-containing cells produced, two points are noteworthy.
(i) The appearance of these cells was suppressed remarkably with supraoptimal doses of antigen, i.e., an approximately 75 and 97% decrease in thenumber of competent cells was obtained by increasing the antigen dose
IOO-fold and 600-fold, respectively. (ii) The efficiency of functional cells
to synthesize antibodies was reduced markedly when these cells were
cultured in presence of excess antigen. From these observations we
can conclude that suppressed secondary antibody response caused byexcess doses of antigen is due first to decrease in the number of functional cells and secondly to decrease in the efficiency of functional cells.
These results indicate that on the cellular level the problem of immunological responsiveness and unresponsiveness resides not only on thecells in the multipotent, primary compartment but also on the cells in
the unipotent, secondary compartment, both of which require antigenic
situmulation before they are transformed into functional cells (Makinodan
and Albright, 1963).

34 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

22. Change of a somatic character by DNA in Ephestia 11(By Saburo NAWA and Ernst CASPARI21)

This work has been designed to test the possibility of somatic trans_
formation by extracted DNA in a higher organism. The wild tYPe of
Ephestia has a pattern of black scales on its forewing, while a recessive
mutant ml has a uniform type of scales without pattern. There aresome advantages in using this gene for transformation work: it ispossible to observe a large number of cells, since a single wing contains
thousands of scales each corresponding to one cell. It is, moreovereasy to detect a black scale among a large number of light-colored
scales.
Fibrous DNA was prepared either by the sodium lauryl sulfate andchloroform method or by the phenol method from larvae, pupae or adults.
Experiments were mainly carried out by using larval DNA, since it hasless impurities than pupal or adult DNA and since it is easy to collectlarvae.
DNA from normal larvae was injected into ml/ml larvae on the eighth
day of the fifth instar. The wings of adults developed from the larvae
treated with DNA were scored for black scales. The frequency of theappearance of black scales was variable in different experiments, as was
the survival of treated animals. In the best case, three black scales
were found on the wings of twenty-five adults in an experiment in which
individual larvae were treated with 3.2 pg of DNA.
This phenomenon may correspond to transformation observed in somemicroorganisms, although further experiments, now in progress, arenecessary for drawing valid conclusions.

23. Genetical and biochemical studies of chromogranules containedin the larval hypodermis of the silkworm(By Mitsuo TSUJITA and Susumu SAKURAI)

In our previous paper it was reported that the presence of yellow
chromogranules which contained sepiapterin combining with a specificprotein in the hypodermal cells gives a yellow color to the larvae. It
has been shown by several investigators Oucci 1932, Hatamura 1943,
Shimizu 1943, Doira and Chikushi 1962) that the opaque skin of thesilkworm is due to the occurrence of a large amount of uric acid in the1) This work was carried out at
The University of Rochester, U.S.A., underContract AT (30-1)-2902 with the U.S. Atomic Energy Commission.
21 Department of Biology, The University of Rochester, Rochester, New York.

RESEARCHES CARRIED OUT IN 196335

h podermal cells. On the contrary, it was found from our investigation:at there is an intimate relation between larval color or oily skin~haracter and the shape, color or amount of chromogranules in theh podermal cells. Therefore, to confirm whether the chromogranules~ntain a large amount of uric acid or not, experiments were carried
Cut and the following results were obtained.
o The chromogranules contain soluble proteins enclosed in an outermembrane or pellicle, which is mainly composed of proteins and lipids.
By the purification procedure described in our previous paper (Tsujita
and Sakurai 1963) the chromogranules were isolated. After adding distiIled water, the chromogranules were homogenized and centrifuged.
The supernatant containing soluble proteins was loaded onto the DEAE
(diethyl-amino-ethyl) cellulose column, and was eluted by phosphatebuffer solution at pH 8.0. It could be fractionated into the following
three parts.
i) 1st fraction contained proteins combining with yellowish brown
pigment.
ii) In the 2nd fraction of the normal strain sample proteins, among
them the protein combining with sky-blue fluorescent substance,

I

I

I.

E-lo;:;o,I
I
II....... -

.-1>~

--

lPD S -

O.OO5-o.02sro ~~tf.l b]n:l'ieGe

1~8 x lOms

1

1

I

1.0

I

.I

I
I

I
I
I

I

I
I
I
I
I

\

\

~

~

~

"'"

Fig. 1. Absorbed in DEAE cellulose column, soluble proteins ofchromogranules were eluted by gradient elution method with 0.0050.025 M phosphahe buffer at pH 8.0 and were collected all in thesame amount of 4 ml in each of the test-tubes. Y band: the yellow
pigment-protein complex.

36 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14
were eluted, while in the same fraction of the lemon strain sample
the protein combining with sepiapterin was collected. The latter
protein was eluted before the former as shown in Fig. 1.
iii) 3rd fraction contained the protein combining with uric acid and thatcombining with isoxanthopterin.
A fairly large amount of uric acid and isoxanthopterin could be also
detected in the insoluble protein which is a major component of thepellicle of chromogranules.
0.1 M sodium citrate solution and 0.1 N hydrochloric acid were mixed
in proportions, 1:9, 2:8, 3:7,
The pH values of each of the
mixtures are given in Table 1.
Table 1.

preciPitatesj
pH value
Yppt:

Relationship between precipitates and pH of sodium cit rateshydrochloric acid solution.
Yppt

1

I

Wppt

I

l~~r53 2 . s+~ ~4~1-15-.1'115-.-35'i5-.7-0'16-.1'516-.-45'16-.7-s'17.-85.

yellow precipitates\i\Fppt:

white precipitates

A few drops of the soluble proteins squeezed out from the chromogranules was added into 2 ml of each of the above-mentioned mixtures.
Specific precipitates could be produced at definite pH intervals; in theinterval between pH 2-3, flattened crystals containg sky blue fluorescent
substance could be produced from a normal larval sample, and similar
crystals containing sepiapterin were produced from the lemon larval
sample, while in the interval between pH 4-5, white colored powdery
precipitates were produced from both samples, which contained isoxanthopterin and uric acid (Table 1). Therefore, it seems that theformer crystals were derived from the protein of the 2nd fraction andthe latter precipitates from that of the 3rd fraction among the threewhich were fractionated using DEAE cellulose.
It was concluded that a number of genes inducing oily skin characterof the silkworm larvae are concerned directly or indirectly with theformation of chromogranules.

24. Chromogranules in the hypodermis of normal and several
oily skin mutant larvae of the silkworm(By Mitsuo TSUJITA and Susumu SAKURAI)

Using normal and several oily skin mutants of the silkworm, theRESEARCHES CARRIED OUT IN 196337

chromOgranules in the cytoplasm of larval hypodermis were studied.
The results are as follows:i) E-Iem: The skin color is yellow and non-transparent. The chromogranules obtained by purification method from the cytoplasm either of
hypodermal cells of the lemon larvae or those of the dilute lemon larvae
had almost the same shape, that is, they were irregularly sphericalmeasuring 2-3,u in diameter. The granules obtained from the hypodermis of larvae with the genotype lem/lem;had muchdarker yellow color than those obtained from larvae with the genotype
lem/lem; d-Iem/d-lem.
ii) Normal larvae (C124): The larval skin is opaque. Similar spherical
granules were obtained from the hypodermal cells of normal larvae by
the same purification method as that used for the lemon larvae. The
diameter of these granules was lo5-2.5,u, being shorter than that of the
granules from E-Iem. They were white.
iii) Lemon semi-oily mutant: The skin color is yellow and semitransparent. In the hypodermal cells the amount of chromogranules issmaller than in the normal lemon larvae. The diameter of the granules
is l.5-2.0,u and they are often joined like beads in a string. It was difficult to isolate them from the cytoplasm.
iv) the ad oily mutant: The skin exhibits strong transparency. A
small amount of chromogranules can be observed in the cytoplasm.
Their shape is somewhat different from those in the hypodermal cellsof normal larvae. Since the chromogranules are so few, it is difficult
to isolate them from the cytoplasm.
v) The w'" oily mutant: This mutant larvae have the strongest skintransparency among the several mutant strains used in the present experiment. The chromogranules can be scarcely observed in the cytoplasm of the hypodermal cells.
vi) The w-3 semi-oily mutant: The larval skin is almost opaque; itis weakly translucent. In the cytoplasm very light-brown chromogranules are found. The size and amount of chromogranules are somewhat smaller than in the hypodermal cells of normal larvae. They canbe isolated from the cytoplasm.
It may be said from our observations that non-transparency or transparency of larval skin depends on the presence or absence of chromogranules contained in the hypodermal cells of the larvae.
When the larvae were fed on mulberry leaves painted with 1% neutral
red solution the skin all over the larval body became reddish purple.
It was found in our observations that this color change is due to staining of the hypodermal granules with neutral red. However, when thestained larvae fed on mulberry leaves painted with 1% melamine acetate

38 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

solution, the chromogranules gradually broke down and the pigment
neutral red, which stained the granules became released and was los~from the hypodermal cells.
Not only the chromogranules in the larval hypodermis but also vari.
eus types of granules in the cytoplasm of the epithelial cells of several
tissues such as mid-gut, Malpighian tubules, silk-glands and so On canbe stained with neutral red. It may be considered, therefore, that themechanism of staining with neutral red of the chromogranules in the
hypodermal cells is somewhat different from that of absorption andmaintenance of pteridine compounds as well as uric acid within the
granules.

25. Genetic analysis of growth of early chick-embryos(By Takatada KAWAHARA)

Three purebreds: White Leghorns (WL), Barred Plymouth ROcks(BPR), Nagoyas (NG), and the reciprocal Ft's, F/s, and back-cross hybrids between WL and the two others were investigated. The numberof somite pairs was examined in embryos at thirty-six and forty-eight
hours after incubation. As preliminary experiments conducted before
suggested that early embrynic growth in chickens showed seasonal vari·ation, special attention was paid at the same time to between-strains
comparison. Results of this investigation are summarized as follows:
Growth rate estimated from the number of somite pairs in early embryonic stages varied among strains, indicating that it is a genetic trait.
The number of somite pairs was 16.51, 17.24 and 17.90, in purebred WL,
EPR and NG respectively, while it was 15.96, 18.47, 14.30 and 18.08, in
WL Q xBPR 1), BPR Q xWL 1), WL Q xNG 1) andNG Q xWL 1), respectively. Marked differences were found between breeds in the number ofsomite pairs when examined 48 hours after incubation, NG and BPR
showing a larger numJer than WL. The number of somite pairs in
WL was 1.06, which was 1.04 less than that in NG or BPR. Significant
differences were found between reciprocal F 1 hybrids (48 hours embryo).
Namely, F 1 hybrids of WL dams with NG or EPR sires produced lesssomite pairs than the reciprocals (3.78 pairs in the WL-NG cross and2.51 pairs in the WL-BPR cross). The reciprocal differences were also
found in F 2 and back-crosses, as follows: WL-BPR F 2 : 0.84, WL-NG
F 2 : 1.31, F1(WL-BPR) Q X WL 11 : 0.98 and F1(WL-NG) Q NG 11 : 1.04 pairs.
The heritability values, h}, hi, h;+d and c 2 , for the number of somite
pairs estimated in the embryos 48 hours after incubation were 0.07,
1.11, 0.59 and 0.26, respectively. These results suggest that early

RESEARCHES CARRIED OUT IN 196339

wth of chick embryos is strongly influenced by maternal factors,andg~~t the differences between reciprocal F, hybrids may be partly due to~aternal cytoplasm-hybrid genotype interaction.
26. Heterosis and asymmetry in testis weight in domestic fowl
(By Takatada KAWAHARA)

A comparison between purebred and crossbred strains was made regarding testis weight at various developmental stages. The materials
used were cockerels of White Leghorns, Barred Plymouth Rocks, Nagoyas, and F, hybrids between WL and two other breeds. Testis
weights were examined 0, 5, 12 and 43 weeks after hatching. The
results obtained are summarized as follows:
1) Correlation coefficients between testis weights and body weights
were always positive ranging between 0.307 and 0.595 over all fourstages of observation.
2) Weight of testis, either absolute or expressed in percentage of
total body weight, was larger in the F, hybids than in their parents,
indicating heterosis, especially in the later stages of development.
3) Generally, the left testes of cockerels were heavier than the rightones. Asymmetry was shown by an index, namely ILeft testis weightRight testis weightl/Left testis weight+Right testis weight, in percent.
The asymmetry index of F, hybrids was 9.82%, while that of purebreds
was 8.55%. 0, 5, 12 and 43 weeks after hatching F, hybrids had 3.51%,
1.93%, 0.56% and 0.78% larger indices than the purebreds, respectively.
Crossbred cockerels were more asymmetrical in testis weight than the
purebreds, especially when young; as they continued to grow, theasymmetry of crossbreds was approaching that of the purebreds.

27.

Chromosomal study of mouse lymphocytic neoplasm,
P388, growing in vivo and in vitro')(By Tosihide H. YOSIDA, Lloyd W. LAW 2 ) and Robert A. ROOSA3»)

Karyotypes of several lines of mouse lymphocytic neoplasm, P388,
growing in vivo and in vitro were examined. Parental P388 ascites line
was characterized by stem-line cells with 41 telocentric chromosomes.
Modal chromosome number of another ascites subline, P388/L, was 44",') This study was carried out in the National Institutes of Health, Bethesda,
Md., U.S.A. in 1962-1963.
2) National Cancer Institute, Bethesda, Md., U.S.A..
3) Wistar Institute, Philadelphia, Penn., U.S.A ..

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

all of telocentric type. Although all chromosomes in these two line
were telocentric, their length was considerably different from that o~normal somatic chromosomes. On other hand, P388jP line, adapted toin vitro culture was remarkable owing to a drastic change of karyotype
which consisted of telocentric, medIan and submedian metacentric chromo_
somes. Majority of cells of this line had 34 telocentrics, 11 medIan
metacentrics and 4 submedian metacentrics. Four single-cell clones
derived from P388jP line had all similar karyotypes to that of theparental line, but number, length and arm index of metacentric chromo_
somes were slightly different. R-26 line which developed by reinocula_tion of culture adapted cells had also a diversified karyotype like theparental culture line, but the chromosome number, especially that of
metacentrics, was lower.
It may be concluded from the present study, that chromosome alteration may occur frequently in tumor cell populations growing in vivo
and in vitro. Thus, tumors are usually in heterogeneous condition. The
most vigorous cell type, however, may control the mode of a cell population under certain conditions.
28.

arYOitytles of various drug-resistant sublines of P388
neoplasm, growing in vitro l )(By Tosihide H. YOSIDA, Lloyd W. LAW and Robert A. ROOSA)The chromosome pattern was examined in a sensitive and several 8azaguanine (AZG)- and amethopterin (AMT)- resistant lines of lymphocytic neoplasm, P388, growing in vitro. Karyotype of sensitive P388jPline was characterized by the modal chromosome number 49 and included 15 metacentrics. However, the karyotypes of various drug resistant lines were quite different from that of the sensitive line. A remarkable increase in chromosome number was found in resistant lines
102E AZG, 102H AZG and 146D AMT, developed by treatment with fairly low drug concentrations. A secondary decrease in chromosome
number was observed in AMT- resistant lines 102J AZG and 146J AMT
treated with higher drug concentration. In AZG-resistant lines, the de,crease in the number of submedian metacentric chromosomes was striking and in the AMT-resistant line that of telocentric chromosomes was
remarkable.
From the observation of chromosome number distribution in thesensitive and resistant lines and in several single cell clones it wasI) This study was carried out
in the National Institutes of Health, Bethesda,
Md., U.S.A. in 1962-1963.

RESEARCHES CARRIED OUT IN 196341

sidered that the parental 1'388 and the resistant lines were a mosaic

c~n various karyotypes, although the type of the stemline cells was theo ost frequent in a cell population. A resistant cell or cells which haveT-fferent karyotypes may be selected from such a cell population byleatment with drugs as selective agents. Therefore, resistant cell lines
tray have different karyotypes from those of the sensitive or formerlyrnresistant l'meso
29. Karyological analysis of the mouse lymphocytic neoplasm, P388,
growing in synthetic culture medium l )(By Tosihide H. YOSIDA, Virginia J. EVANS2) and Lloyd W. LAW)

To examine whether different conditions of the culture medium produce different effects on the karyotypes of cultured cells, synthetic andserum media were used in in vitro cultivation of mouse lymphocytic
neoplasm, P388. P388 ascites and two in vitro lines, NCTC 4299 maintained in the serum medium and NCTC 4366 cultured in the synthetic
medium, showed 41 as the modal chromosome number. Distribution ofchromosome numbers, however, was different, NCTC 4366 showing thewidest variation. In in vitro adapted P388 sublines, however, variationof chromosome numbers in NCTC 3749, maintained in the synthetic
medium, was rather narrower than in P388jP line kept in the serum.
The number of metacentric chromosomes of NCTC 3749 was less thanin the P388jP line. P388jDEP, an in vitro-derived acites tumor cell line,
also showed a decrease by one metacentric chromosome as comparedwith the parent, NCTC 3749. Based on the above investigation, it isconsidered that under conditions in vivo as well as in vitro, the mostadaptive karyotype will control the mode of a cell population representing its stem-line.
30.

Karyological study on mouse leukemias developed byvirus infection 3 )(Tosihide H. YOSIDA, Lloyd W. LAW and A. PRECERUTTI4))

Chromosomes of 19 mouse leukemias induced by Moloney virus inThis study was carried out in the National Institutes of Health, Bethesda Md.,
U.S.A. in 1962-1963.
2) National Cancer Institute, Bethesda, Md., U.S.A..
S) This study was carried out in the National Institutes of Health, Bethesda, Md.,
U.S.A. in 1963.
4) National Institute of Health, Buenos Aeres, Argentina.
1)

42 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

fection were observed. They were examined in spleen, thymus, andmesenchymal lymph nodes. Among 19 leukemias, 15 (78.9%) had thmodal chromosome number 40, all characterized by telocentric centro~
meres. No difference in karyotype could be found from that of normal
somatic cells. Further, 3 leukemias were characterized by the mode at
41, and the remaining one had bimodal distribution of chromosome
numbers with modes at 40 and 41. Also in these last four leukemias, alI
chromosomes were characterized by telocentric centromeres. Relation
between the various leukemic mouse strains and their neoplasmic karyo.
types are shown in Table 1. Almost all neoplasms examined here Werecharacterized by lymphocytic leukemias.
The original leukemia developed by poLLY virus (Precerutti and Law1963)1) was characterized by the modal chromosome number of 41 andTable 1.

-Frequency of several mouse leukemias (Moloney)
with 40-41 chromosomes----~~

Strains C3HfjLW
Chromo No>_ _40
41
40, 41

I

Total No. ofmice

7

C3HfjGs 1 C3HfjFg

BALBjc

RFM

Total

-

0
0

2
1
1

2
0
0

2
1

2
1

0

0

15(78.0%)
3
1

7

4

2

3

3

19

by telocentric centromeres. The viruses obtained from this leukemia
were injected into C3Hf/Bi and BALB/c mice which developed typicallymphocytic leukemias. Their stemline had 40 chromosomes like mostof Moloney leukemias. Based on the above investigations it can besaid that the karyotypes of the great majority of mouse leukemias induced by virus infection had apparently normal karyotype.
31.

Relation between chromosome constitution and protein
specificity in mouse plasma cell leukemias 2 )(By Tosihide H. YOSIDA, Michael POTTERS), Yoshinori KURITA
and Kazuo MORIW AKI)

Chromosomes of 7 strains of mouse plasma cell tumors were observed
Precerutti, A. and Law, L. W. 1963. Nature 198: 801-803.
A part of this study was carried out in the National Institutes of Health,Bethesda, Md., U.S.A. in 1963.
3) National Cancer Institute, Bethesda, Md., U.S.A.
1)

2)

RESEARCHES CARRIED OUT IN 1963

ill r

elation to the production of specific myeloma protein. As Table 1'Table 1.

--~

StrainKaryotypes and protein specificity of plasma cell tumors in mouse.
Total no. of
Karyotype**
Protein specificity
chromo

RP C·6A
RP C-6C
RP C-20

MOPC-9MO PC-3IB
X5563 solid
X5 563 ascites-

43

51-78(77)*70-96 (95)
62-86(84)98-80 (80)
55-83 (81)62-84 (84)33-92(84)75T+1M+1m
90T+5M
82T+IM
79T+IM
8IT
82T +lSM +IST
80T +2SM+1ST +lm

f3-2A euglobulin
f3-2A urinary protein
Bence Jones unusual protein
Bence Jones unusual proteinr-2' myeloma globulin***r-2 myeloma globulin
r- 2 myeloma globulin

I* In parenthesis, modal chromosome number.
** Telocentric, metacentric, subtelocentric and minute chromosomes are denotedby T, M, ST and m.
*** position of electrophoresis band was slightly different from r-2 myeloma
globulin of X5563.

shows, all plasma cell neoplasms examined were characterized by neartetraploid chromosome constitution. The majority of the neoplasms had
marker chromosomes such as metacentrics, subtelocentrics or minutes.
An ascites developed from the solid type X5563 neoplasm. It produced
the same r-2 globuline, but its karyotype was different from that of thesolid type. No constant relationship between karyotype and protein
specificity could be found at the present time.

32. Chromosomes of normal and dwarf cattle 1 )(By Tosihide H. YOSIDA and Edward J. LAMONTAIN2»)

Chromosomes of dwarf female cattle were observed by skin tissue
culture in comparison with those of normal females and males. The
animals were 2 years old Herefords obtained from "Bay-Manor-Farm"
Delaweare, U.S.A.. Normal as well as dwarf female cattle had 60
chromosomes, namely 29 telocentric autosome pairs and one submedianmetacentric X pair. There was no difference in the karyotype between
normal and dwarf females. Male normal cattle had 29 telocentric autosome pairs and in addition a submedian X and a median metacentric Y
chromosome. From the above investigation, it was concluded that the1) A part of this study was carried out in the National Institutes of Health,Bethesda, Md., U.S.A. in 1963.
2) National Cancer Institute, Bentesda, Md., U.S.A ..

44 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

character of bovine dwarfism is not reflected in the change of karyotype
being due to a single gene mutation.

33. Karyological observations on a Misima subline of hyperdiploid
Ehrlich tumor and its three clones(By Tosihide H. YOSIDA)

The hyperdiploid Ehrlich tumor (ELD) used in the present study was
obtained from Children's Cancer Research Foundation, Boston, in December, 1958. Its origin goes back to Dr. Klein's stock at the KaroHnska Institute in Stockholm. Since that time, this tumor has been
maintained in Misima by serial animal passages using mice of a Swiss
strain.
Chromosome number and karyotypes of the Misima subline of ELD
tumor and three single cell clones derived from that subline were examined. The stemline cells of the Misima subline were characterized
by the modal chromosome number of 44 including one A-chromosome(telocentric with secondary constriction), two B-chromosomes (large metacentrics), one Sa-chromosome (short arm) and two or three minutes.
One small metacentric was remarkable, especially as it appeared at firstafter the 61st transplant generation. The three clones had almost thesame karyotype as that of the stemline of the parental cell population.
All ELD sublines reported until now by several investigators werecharacterized by 45 or 46 chromosomes including one A and one B
chromosome. Based on the above investigation, it may be concluded
that the Misima subline has a remarkablly different karyotype from theother sublines of ELD tumor.

34. Induction of specific protein synthesis in tumor cellsby the addition of exogenous RNA(By KaZliO MORIWAKI)

Genetical information is assumed to be transferred sequentially from
DNA to protein through RNA. To reveal the role of RNA in thissequential pathway, the inducing ability of exogenous RNA in a specificprotein synthesis of tumor cells was studied in the present experiment.
RNA fraction, containing soluble RNA, ribosomal RNA and probably
larger molecular RNA, was extracted from plasma cell tumor (X5563)
of C3H mouse by phenol method, slightly modified from that describedby Kirby (1956). The plasma cell tumor strain employed was kindly

RESEARCHES CARRIED OUT IN 1963

supplied by Dr. M. Potter of NIH, U.S.A.; its cells could produce w
high efficiency the specific protein gamma myeloma globulin. Ehrl
ascites tumor cells, which can not produce gamma globulin were tre.cd for 15 hours with higher concentration of RNA (O.D' 26om l"=50jml)
ODC. After contact with RNA, Ehrlich tumor cells were shaked forhours at 37°C with a suitable medium containing radioactive amino al(C4.1eucine). During this incubation, oxygen consumption of the ce
was determined by manometer. Then, the tumor cells were disrupt
by freezing and thawing. To the soluble protein extracted from drupted cells, anti-myeloma grobulin rabbit serum was added. RN
induced myeloma protein synthesis could be estimated by radioactiviof amino acid in the precipitate.
Although protein synthesis of Ehrlich tumor cells was apparent
inhibited on the whole following the treatment with higher concentratil,of plasma cell tumor RNA, induction of gamma globulin synthesis cou
be observed in the tumor cells, when the ratio of gamma globulin sy
thesis to total protein synthesis was compared. RNase digestion aI
boiling of RNA caused loss of this ability.
These results indicate that a foreign protein synthesis can be inducf
in intact tumor cells by incorpolation of exogenous RNA.

35. Electrophoretic analyses of esterase isozymes in various
strains of mice
(By Zenichi OGITA1), Sachio OGITA1), Yoshinori KURITA and Kazuo MORIWAKI)

Enzymes having multiple molecular forms but the same substrat
specificity were named "isozymes" by Markert and Moller (1959). Il,order to study whether or not any differences in isozyme patterns exis
among various strains of mice, the zymograms of liver esterases wen
compared using thin layer agar electrophoresis. The details of tht
technique have been already reported by Ogita (1962). Liver esterase~of various strains of mice were separated into 34 bands, and the positiomof the bands on the agar plate were designated as Et, E 2 , •• , E 24 if·order of faster migration towards the anodal side.
In the present analyses, 10 inbred strains, AKR, C57BL, C57L, CBA,
CFW, m03, DBAj2, dd, NH, SL, and 8 mutant strains, ap, A Y , C", Pc
hrr ", se, t, W were examined. Among them, C57BL and C57L lacked
E2 band and the rest lacked E 1 band. In the hybrids between C57BL(E 1 type) and CBA (E 2 type), enzyme activities appeared at both posiV,

1) Department of Genetics, Medical School, Osaka University.

46 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

tions, E 1 and E 2 , through they were appreciably weaker. Similar result
has been obtained in the case of hybrids between NH and se. NB:
mutant strain lacked E 13 enzyme and the activities at Ell and E 12 posi.
tions were apparently strong. On the other hand, se strain lacked Elland showed strong activities at E 12 and E 13 • In the hybrids between
them, the esterase activities could be observed at three positions, Ell,
E 12 and E 13 • Also in this case, the enzyme activities at Ell and E 13 in
the hybrids were weaker than in either parent.
These findings suggest that the protein specificities in mouse liver
esterases are controlled by genes, and that a gene dosage effect exists
in the synthesis of the enzyme proteins.

36. Cytological analysis of the mechanism of induction ofchromosome aberrations by 4NQO
(By Kazuo MORI\tVAKI, Yoshinori KURITA and Tosihide H. YOSIDA)

The present study was attempted to find out which phase is the mostsensitive to 4-Nitroquinoline N-oxide (4NQO) during the division cycleof mammalian cells with respect to chromosome aberrations, and toreveal the mechanism of its mutagenic action.
Five days after intraperitoneal transplantation of Yoshida sarcoma
cells to wistar rat, 0.8 ml of 10- 3 M 4NQO solution was injected into theascites fluid. Final concentration of 4NQO in the ascites fluid wasestimated as at the order of 10-5 M by determining the optical densityof the fluid at 365 mIt. The metaphasic chromosome figures were examined at 0, 4, 8, 12, 24 and 48 hours after the injection.
The first peak in the frequency curve of chromosomal aberrations,representing mostly chromosome elongation, appeared from 0 to 3 hours
after the treatment. The second peak represented chromatid breaks andcould be seen from 4 to 12 hours after injection. The third peak appeared from 24 to 48 hours after the injection, and represented predominant chromosomal translocations.
The chromosomal aberrations of the first peak were probably causedby the direct action of 4NQO on metaphase chromosomes. If it isassumed that one-generation time for those tumor cells is 19 hours as
already reported by Ferymann (1956) and the division delay of several
hours occurred following treatment with 4NQO, chromosomal aberrations
at the second and third peaks may be considered to be induced bystructural changes of chromatin threads caused by 4NQO in G2 phasecells.
These results probably indicate that the chromatin materials in G:

RESEARCHES CARRIED OUT IN 196347

and M phase cells are remarkably sensitive to 4NQO and that distortionsof chromosomal structure caused by this' drug induce secondarily certain
gene mutations, as suggested by Endo (1962). On the other hand, thedisappearance of chromosomal aberrations during 13 to 23 hours after
4NQO injection shows perhaps that S phase cells may be less sensitive
to this drug or that DNA synthesis itself might not be affected directly
by 4NQO.

37.

An unusually Y chromosome found in a strain of the mous?
(By Akira NAKAMURA and Akira TONOMURA)

For the past two years, a detailed analysis of the somatic metaphase,chromosomes of cultured embryonic cells has been carried out ingenetically different mouse strains and it was found that one of theinbred strains characterized by post-axial polydactyly (gene Po) has anusually long Y chromosome as compared with other strains, such as
Swiss, DM/Ms, C3H and A/HeMs. The presence of a similar long Y.chromosome has also been found in an inbred strain, C57Bl (Levan, Hsu
and Stick; 1962).
In the present paper the results are reported of a comparative analysisof the relative length of the Y between the polydactylous strain, C57Bl
and DlVI;Ms. Since the length of metaphase chromosomes depends on
the state of contraction of the individual cells analysed, the relative
length of the Y in each strain was expressed as the ratio between thelength of the Y and that of the smallest autosomal pair, No. 20. The
results obtained are summarized in Table 1.
Table 1. Comparison of the relative length of Y chromosome of thepolydactylous strain with that of C57Bl and DM/Ms strains.
Strains

Relati ve length of Y*

Mean of Y

No. of cells
examined

Polydactylous

1.2 1.5 1.3 1.3 1.4 1.2 1.1
1.1 1.1 1.1 1.3 1.2 1.2 1.1
1.1 1.1 1.2 1.3 1.1 1.1 1.5

Xl=1.21

21

C57Bl

1.2 1.2 1.4 1.3 1.3

X2=1.28

7

DM/Ms

1.0 0.9 1.0 0.9
0.8 1.0 1.0 0.9

X3=0.9011

1.5 1.1

0.9 0.7 0.9* Length of the Y/mean length of No. 20 pair.

48 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

There was no significant difference between Xl and X2 (t 26 =1.2, 0.20<:
P<0.30), but between Xl and X3 the difference was highly significant(t30=6.7, P<0.001). From these results it is clear that the Y chromo.
somes of Swiss, DM/Ms, C3H and A/HeMs mice are slightly shorter
than the smallest autosomal pair, No. 20, while in C57B1 and the poly.
actylous strain here examined the Y chromosomes are intermediate between Nos. 19 and 20. Since the Y chromosome of the mouse does notform a regular pair or chiasma during pachytene, the greater variationin the appearane of the Y may suggest its indepentent evolution.

38. Electrophoretic analysis of water-soluble proteinsin Triturus embryos on cellulose acetat/ I(By Yoshito OGAWA and Izumi KOBAYASHI)

Water-soluble proteins in Triturus embryos immediately after fertili.
zation were examined by means of electrophoretic analysis on cellulose
acetate.
Embryos from 5 to 35 hrs. after fertilization and unfertilized eggS
were mushed with the same volume of ice-cold distilled water in a glass
homogenizer. The homogenate was allowed to stand for one hour at
O°C. under constant stirring and then was centrifuged at 2,600 g for 10
minutes. The clear supernatant was analysed using veronal buffer (pH
8.6, 0.07 M) at room temperature, 0.6 rnA/em width of strip. The
quantitative analysis was carried out after staining with Ponceau 3R21
or Nigrosine3 ) solution.
The analytical results are given in Table 1. Eleven fractions weredivided into four groups according to their mobility by analysis andtheir changes in amount during the development of embryos. Group I
moved fastest to the anode side; it commanded 31.68% in total soluble
proteins of unfertilized eggs, and was composed of fractions A, B, C
and E. Fractions A and E were found only in the unfertized eggs.
This group decreased in amount immediately after fertilization and disappeared 35 hrs. after fertilization. Group II, fractions D, G and H,
account for 74.97-77.67% of the material. The total quantity of thisgroup was practically constant but the amount of the individual fractions
changed remarkably during the development of the embryos. Group III,
1) This work was supported by a Grant-in-Aid for
Fundamental Scientific Research (No. 710273) of the Ministry of Education in Japan.
2) Y. Ogawa:Med. and BioI. 66: 234, 1963.
3) M. Ortega:Nature 179: 1086, 1957.

RESEARCHES CARRIED OUT IN 1963
Table 1.

-B rs. afterfe rtilizatio nControl(V nfertilized

49

Results of electrophoretic analysis of water-soluble
proteins in Triturus embryos.

I_GI_H

A

B

C

1.07

4.42

10.78 14.10 15.41 17.52 21.82

2.15
0.30

5.70 13.62
3.56 16.53
26.93_ DI _ E _ F

I _ II _ J

K

9.62 1.97

1.72

1.57

9.83 38.73 23.79 3.80
12.57 23.28 37.86 3.58
16.69 31.59 16.45 4.65

1.40
1.10
2.02

0.98
1.22
1.67

egg)
5
20
35

fraction I, J and K, migrated fatest to the cathode side and no fractionin this group showed any significant change in the amount. The lastgroup IV, fraction F, remained at the point of application on the cellulose acetate strip. This fraction showed a temporary decrease immediately after fertilization.
These results showed the same tendency as the results of electophoretic analysis of water-soluble proteins in the eggs of sea urchin ' ) or
medaka (Orysias lapites)2) when agar or filter paper was used. Group I
is equivalent to Nos. 3 and 6 in sea urchin and fraction A in medaka(phospho-protein), Group II to Nos. 1, 2 and 2a in sea urchin and fractions B, C and F in medaka (phopholipo-protein). Group III to Nos. 4
and 5 in sea urchin and fraction D in medaka (glyco-protein), and GroupIV to fraction E in medaka.
The analytical results on cellulose acetate seems to be more detailed
than that on agar or filter paper.

39. Changes in the soluble proteins of mouse skinfollowing the application of sweet Orange Oil,a tumor promoting agentS)(By Yoshito OGAWA and Izumi KOBAYASHI)

Sweet Orange Oil is a component of the peel of Citrus sinensis and isused as an additive to soft drinks, in confectionery, bakery and drugtrade in Europe and United States. The main constituents are limonene(about 90 percent) and decanal. Roe (1959)4) belives that this oil shows
A. Monroy: Exptl. Cell Research 1: 92, 1950.
Y. Ohi: Embryologia 7: 208, 1962.
3) This work was supported by aGrant-in-Aid for Fundamental Scientific Research (No. 95625) of the Ministry of Education in Japan.
4) Roe, F. J. C.:
Brit. J. Cancer 13: 92, 1959.
1)

2)

50 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

co-carcinogenic activity in the skin of mice by combined treatment .
9, 1O-dimethyl-1, 2-benzanthracene (DMBA), and Meek (1963)51 sugge:: ththe possibility of its acting as a promoting agent in experimental sk:dcarcinogenesis. In the present paper the changes in water-solUb;nproteins of mouse skin (dd strain) treated with Sweet Orange Oil (B. pe
C. grade) are described.
.
In the treated group, Sweet Orange Oil was applied to the dorsal skiof mice six times with two day intervals until a total per animal ~2.4 g was reached. Some skin inflammation was found at the site ~fapplication immediately after treatment, and hair loss and considerable
thickening of the epidermis without ulceration were recognized on the
7th day. 4 days after the last treatment, the treated part of the skin
was cut off and mushed with the same volume of distilled water in a
glass homogenizer. The homogenate was centrifuged at 1,500 g for 10
minutes. Electrophoresis of the clear supernatant was carried out atroan temperature on cellulose acetate membrane by using veronal buffer(pH 8.6, 0,07 M), 0.7 rnA/em width of strip for two hours. Nigrosine
staining was used for the estimation of the analysed proteins. The non.
treated group was prepared for control.
The results are shown in Table 1. The soluble proteins extracted
Table L

Results of electrophoretic analysis of water-soluble proteinsin mouse skin treated with Sweet Orange Oil.
(%)~_F_ G
Control(Nontreated)

17.54

Treated(Normal components only)

22.11(34.62)

,:J

B

46.61

I

14.50 10.30

I

18.40
(29.03)

H

D

23.41

7.72 11.43 14.89(12.16)(23.49)

I

E

0.7

-

I

0.45 0.20(0.70)

from the treated group were separated into nine fractions (A, B, .. ·1),
but that from the control group could be divided only in five fractions(A, B, .. ·E). Therefore, four more fractions (F, G, H. and I) resulted
from the treatment with Sweet Orange Oil. In the treated group, fraction A was increased and fraction B was considerably decreased as compared with the control. But no significant difference was found in fractions C, D and E.
The biochemical and cytochemical examination of the relation between
the above findings and the carcinogenic activity of Sweet Orange Oilmust be furtuer investigated.
5)

Meek E. S.:

Exptl. Cell Research 29:

389, 1963.

RESEARCHES CARRIED OUT IN 1963

51

40. Selection for larval growth in Tribolium under two
nutritional levels(By Yukio YAMADA and A. E. BELL)

Selection for large and small larval weight in Tribolium castaneUi
investigated for 16 generations to evaluate the effectiveness of var'Was selection methods and the importance of genotype-environment inte
OU:ion under two levels of nutrition. Chemical composition of the t'i'i~~tions is listed below.

--------Rat ion

Protein

Fat

Ash

Water

Others

Total

Goo d
poo r

18.3
13.2

8.5

2.8
2.2

10..4

4.2

60.0
70.5

100

9.9

100

The character for selection is the I3-day larval weight. Genetic par;meters of the base population for the character were O.25±O.03 i
heritability under both nutritional levels and the genetic correlation h
tween the character under the two rations was 0.62±0.12.
The experimental populations are listed below together with U
respective selection methods.
Symbol of population

Selection method

GL=selected for large under Good level every generation,
PL=selected for large under Poor level every generation,m=selected for large on average under both levels,
GPL=selected for large under Good and Poor in alternating generations,
GS=selected for small under Good level every generation,
PS=selected for small under Poor every gen.cration,
GPS=selected for small on average under both levels,
GPS=selected for small under Good and Poor in alternating generations,
C=unselected control consisted of 20 pair-matings, each contributing one rna]and one female to next generation.

The mating and selection were made in such a way that each pair (parents produced fertile eggs in the creamer which contained standar
wheat media for the first 48 hours after removal from the matin
creamer, in which they stayed for approximately a week after emej
gence, and then the parents were transferred to two Good rations, eac52 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

for 24 hours, two Poor rations, each for 24 hours.
Once the families for selection were chosen, the sibs raised in thestandard wheat media were picked up in their pupal stage and seXed
for mating to produce the next generation without weighing.
Each population consisted of 40 single-pair matings and best eightfamilies were selected out of all fertile matings. Five males and fivefemales in each selected family were drawn from the standard media
and mated at random with the restriction that full sib mating Was
avoided.
All populations were raised in the climatic chamber of 33°C and 70%
R. H. The whole experiment was run at thirty-five days interval per
generation throughout from generation 0 to 16.
Average gain, in each population, per generation for the entire periodof selection adjusted to the unselected control is as follows:
Population

Under Good

Under Poor

GL
PL
GPL
GPL
GS
PS
GPS
GPS

66.0± 3.853.6± 2.952.6± 3.459.2± 1.4
-106.0+ 8.0
87.8±10.585.0± 5.994.9± 8.759.1±9.4
98.7+9.672.5±7.4
83.4±8.4-53.2±3.3-75.3+4.7-61.0±4.0-60.3±4.1This shows very clearly that direct selection responses are generally
larger than indirect selection under both Good and Poor nutritional levels.
That is, GL and GS were both extremes in performance when they
were tested under Good ration, while PL and PS were extremes when
they were tested under Poor ration. This shows in turn that strainenvironment interactions are quite obvious in later generations of
selection. On the contrary, direct selection gains in the populations
selected for the average of the two environments -the gain in averagefor GPL and GPS- do not give the highest response, probably because
the amount of information for selection in those two populations was
relatively smaller than in the other populations.
It is quite interesting from the view point of animal breeding that
selection under Good environment has parallel response in the populationmeans under two nutritional levels when the population has been selected
for large (i.e. GL), while the population selected for large under Poor(i.e. PL) shows that the two response lines under Good and Poor levels

RESEARCHES CARRIED OUT IN 1963

53

approach gradually each other as the generations of selection proceedo
The response regression line under Poor is definitely steeper not only
than that on Good of the same population but also than those in GL
population on either environment. On the other hand, selection forsrnall size was exactly reverse in selection response in that PS had
parallelism but GS not. As an extreme, one of GS lines showed completely reverse ranking under two environments after generation 12
inclusively.
Asymmetrical selection responses in opposite direction were observedo
One of the interesting aspects of the asymmetry in this study is thedependency of the asymmetry on the tested environment. Downward
selection was favored under Good, while upward selection gave larger
response under Poor.
Average realised heritabilities for two directions of selection gave
0.329 for large and 0.444 for small size. This suggests that in theprocesses of selection toward these opposite directions, gens that responded to selection for larger body and those involved in selection forsmaller body may not belong to the same loci. (Supported by NSFG
15824)-B.

GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF PLANTS

41. Discovery of three cytoPlasms inducing male-sterility in wheat(By Hitoshi KIHARA)

I have started my work on nucleus substitution in wheat only for its
scientific interest. However, some of the results seem to have givenrise to considerable research activities among the American wheat
breeders, aiming at the development of a new breeding system for thisimportant crop. They consider the male-sterile lines to be a promisingmeans in establishing hybrid wheat. Therefore, a brief description will
be given here on 3 hitherto known cytoplasms which induce male-sterility in wheat.
(a) caudata cytoplasm: A male-sterile line with plasma of Aegilops
caudata and the genome of Triticum vulgare var. erythrospermum,
initiated in 1935 by the production of the F , hybrid, retained its cytoplasmic sterility until the present time (1963). Eighteen male-sterilelines (10 of hexaploid and 8 of tetraploid wheats) were derived fromthis male-sterile wheat. So far 2 fertility-restoring genes have become54 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

known, one in T. compactum var. No. 44 and the other in strain P 168(6x-wheat with a pair of caudata chromosomes).
( b) ovata cytoplasm: Nucleus-substitution lines of tetraploid whe twith the cytoplasm of Aegilops ovata were all male-sterile. No resto/ S
were discovered among 8 tetraploid wheat varieties which I have stUdi:~s
According to Fukasawa, however, T. dicoccoides var. kotschyanum .
stores pollen fertility lost in ovata cytoplasm.
reo(c) timopheevi cytoplasm: In my previous paper (Kihara 1959) I
mentioned briefly that T. dicoccum var. Hokudai with timoPheevi c;to.
plasm was fertile. Later it was revealed that T. durum var. reichen.
bachii with timopheevi cytoplasm produces highly sterile pollen. There.
fore we may say that the timopheevi cytoplasm induces male sterility
and that T. dicoccum var. Hokudai has the ability to produce functionalpollen grains in timopheevi cytoplasm.

42.

EjJeet of caudata cytoplasm on the development of 4
nucleus-substitution lines of common wheat(By Hitoshi KIHARA)

Using 4 advanced nucleus-substitution lines of 6x wheat, effects of analien cytoplasm introduced from Aegilops caudata have been studied in
1963 in respect to several important characters. Performance of thesubstitution lines on those characters was compared with that of therespective normal lines and their difference was statistically tested. The
results are summarized in Table 1. Pollen and seed fertilities are takenfrom the 1962 record, because of extraordinary wet ripening season in
1963. For estimating the frequencies of haploids and twin seedlings,
data of earlier substitution-backcross generations were combined withthose of 1963.
Substitution line of T. compactum var. No. 44 was the most male·fertile among 4 substitution lines and no haploid or twin seedlings havebeen found so far. Neither was its performance as to 3 other agronomic
characters different from that of the normal line. On the other hand,the substitution line of T. vulgare var. Salmon behaved quite different·ly from its normal line. It was completely male-sterile and thefrequencies of haploids and twins were extremely high. Also its performance concerning 3 other characters deviated significantly from that of
the normal line. Responses of 2 other varieties to the same alien cytoplasm were, in general, between those of the above two. From thoseresults it can be said that the degree of influence of an alien cytoplasm

Table 1.

Performance of normal and nucleus-substitution lines of 4 hexaploid wheats.

StrainPollenfertility(%)

Seed
fertility(self)(%)

Freq. ofhaploids
(%)

96.7
0.2**

60.5
0.0**

II Freq.
of Heading
twins
date
(%)

(day)

0.3

0.2

3.1**

3.9**

199.3
199.3

Plant
height(cm)

Drymatterweight(gm)

T. vulgare var. erythrospermum
Normal line
Substitution line

135,4
131.0

179
178

T. vulgare var. Salmon
Normal line
Substitution line

89.4

75.1

0.0

0.3

0.5**

0.0**

27.9**

10.7**

197.8
192.2*

112.2

95

120.2*

125*

::r:
t:r:1

UJ

;J>

::0
::0

~

tJ

88.2

79.4

0.0**

0.5**

0.0
2.0

0.0
2.0

203.0
204.4

120.0
114.3**

257
194*

o
c:

>4

Z

T. compactum var. No. 44
Normal line
Substitution line

;J>
;>;:I
(")

n

T. spelta var. duharnelianum
Normal line
Substitution line

::0
t:r:1
UJ
t:r:1

98.0

80.2

84.7**

35.3**

,~-

0.0
0_0

0.0
0.0

198.3
199.4

110.3

I 106.6

181
139

>-'

<!)

m

w

* and **: Substitution line is significantly different from the corresponding normal line at the 5% and 1% level, respectively.

<.n

\11

'56 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14<on the manifestation of a wheat nucleus is mostly determined by its
whole genotype.

43. Production of tetraploid rice by N 2 0-treatment*(By Hitoshi KIHARA and Koichiro TSUNEWAKI)

Treatment of pollinated florets with nitrous oxide (N 2 0) was demon_'strated in wheat to be a very effective means for production of polyploids (Kihara and Tsunewaki 1960). The same treatment was applied
last year to a cultivated rice variety in the following way. All florets,of potted plants, except those which flowered just one day before thetreatment, were removed from every panicle. The plants were placedin a steel tank and the air was sucked out by a vacuum pump, until
the pressure inside the tank became 1/3 atm. Then it was filled with
N 20 gas up to the pressure of 5 atms. Plants were kept in this condition for 12 hrs. After the treatment they were grown under natural,condition. The seeds were sown and the seedlings were examined fortheir chromosome number. The result of this experiment, as shown in
Table 1, indicates that N 2 0-treatment is useful for the production oftetraploid rice.

Table 1.

Frequency of tetraploids in the offspring of N 20-treated rice;treatment was given for 12 hrs. at 5 atms., from one
day after pollination.

PanicleNo.

No'
florets
treated

I

36
36
27
25
37
22
183

2
3
4
5
6
Total

No.
seeds
set

%

seeds
set

No. I
%
seeds
germi- germinationnated--- --- --34
35
26
25
35
21
176

94
97
96
100
95
96
96

23
24
20
23
17
14
121

68
69
77
92
49
67
69

No.
% tetraploids
tetraploids
per germi- per(including
natedtreatedaneuploids)seedfloret

7
7
3
3
9
3
32

30
29
15
13
53
21
26

19
19
11
12
24
14
18

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

'

RESEARCHES CARRIED OUT IN 1963

57

44. Frequency of aneuploids in the offspring of 7 synthesized
6x wheats(By Koichiro TSUNEW AKI)

More than 30 6x wheats have been synthesized from different varietiesof Emmer wheat and Aegilops squarrosa (MacFadden and Sears 1944,
J{ih ara et al. 1950, Tanaka 1961, Sears unpubl.). Investigation of their
genetic stability has a great significance in the considerations on theorigin and evolution of common wheat. Among those synthetics, one
strain, produced from Triticum persicum var. stramineumx Ae. squarrora
var. typica, was studied by Tabushi (1957). He reported high frequency (about 27%) of aneuploids in its offspring. In order to obtain ageneral picture of the genetic stability of synthesized 6x wheat, chromosome numbers in the offspring of 7 synthetics, produced by Kihara etat., Sears and Tanaka, were investigated. The result is summarizedin Table 1 in which the frequency of apparent aneuploids with moreor less chromosomes than 42 is indicated. However, plants with 42
chromosomes are not necessarily assumed to be euploid; they might be
deficient for one chromosome and have another one duplicated.
This result clearly indicates that all synthesized 6x wheats are
more or less genetically unstable. In total, 28.2% of the offspringwere aneuploids, the majority being hypo-aneuploids. No clear-cut
difference in aneuploid frequency was found among the 7 strains,though 2 of them, whose Emmer parent was T. dicoccum var. Vernal
or T. dicoccoides var. spontaneo-nigrum, seemed to be slightly stabler
than the others.

45. Genetic studies of a 6x-derivative from an 8x Triticale*(By Koichiro TSUNEWAKI)

A 6x strain named "Salmon" (2n=42) was obtained in the progeny ofa hybrid between two strains of 8x Triticale (2n=56). In order to elucidate the genetic make-up of this derivative, several genetic investigations were undertaken.
(1) Salmon was crossed to Triticum aestivum var. Chinese Spring.
The F 1 hybrid showed 21 n in a majority of PMC's. The extent of
meiotic irregularities was definitely comparable to that observed in interor intrasubspecific hybrids of T. aestivum.
(2) A detailed analysis of chromosome configurations in the F 1 hy-* This work was supported by Grant RF 62073 from the Rockefeller Foundation.

<:.n

00

~

Z
Z

c:::
~

r

;;>;i

Table 1.

trJ
>-0

o

Chromosome numbers in the offspring of 7 strains of synthesized 6x wheat.

?:I

I

Strains*

T. orientale var. insigne
T. persicum var. stramineum**
T. durum var. Golden Ball
T. durum var. Carleton
T. durum var. Pentad
T. dicoccum var. Vernal
T. dicoccoides var. spontaneo-nigrum
Total

No.
progenies

No. plants
examined

1
3
1

15
112
16

10
1
1
1

353

19
18
17
550

I

>-l

Somatic chromosome no.
44 or
more

43

42

41

40

39 or
less

0
1
0
3
0
0
0
4

1

10

8
0
20
1

76
11

3
22
4

253
14

58
4

1
3
1
16

0
0
30

15
16
395

3
1

0
2
0
3
0
0
0

* Strains of the synthesized 6x wheat were indicated by their Emmer parent.
typica was used as the squarrosa parent.
** Including the result of Tabushi (1957).

95

0
0
0
21

5

%
aneuploids

o

ITI

Z
33.3
32.1
31.3
28.3
26.4
16.7
5.9
28.2

In all cases, Aegilops squarrosa var.

~

>-l
....,

o

z~

r

Z

Ul

>-l

::J

c:::

>-l

t'.rJ

o

ITI
CJ
trJ

Z

trJ

>-l

o

Ul

z

?

.,.

......

RESEARCHES CARRIED OUT IN 196359

'd and a comparison of karyotypes of its parents in root-tip mitosis
bfl ealed that the satellited arm of one of the two wheat Sat-chromofe~es was replaced in Salmon by an arm of a rye chromosome.
so( 3) Monosomic analysis of Salmon on waxiness indieated that W
ne responsible for waxiness of common wheat is located on chromogerne XIII. Non-waxy character of Salmon is attributable to the de~~iency of a chromosome segment comprising the W locus.
(4 ) Nucleus substitution lines of Salmon and three other 6x wheats,
1l of whieh were produced by Dr. H. Kihara, were compared with the~he respective normal lines. The substitution line of Salmon was superior to Salmon itself, while those of the other wheats were in generalinferior to the corresponding normal lines.
From these results it is concluded that 8x Triticale was converted to
the 6x wheat by elimination of chromosome complement of rye. However, the remaining wheat chromosome complement was slightly modified by integrating some chromosome segments of rye and losing someof wheat. This modification seems to be the main factor responsible
for the peculiar developmental behavior of Salmon's nucleus substitution line.

46.

Transmission of monosomes and trisomes in an Emmer
wheat, T. dicoccum var. Khapli(By Koichiro TSUNEW AKI)

As reported earlier (Kihara and Tsunewaki 1960, 1962; Tsunewaki,1962), several aneuploids of an Emmer wheat, T. dicoccum var. Khapli
were produced among polyploids by N 20-treatment of artificially pollinated spikes. Three monosomics and 6 trisomies were obtained so far.
Transmission of the monosomes and trisomes to their offspring has beenstudied. The result is summarized in Table 1.
No monosomies were recovered in the offspring of all 3 monosomics,
although 2 trisomies were found. No transmission or a very low one ofmonosomes seems to be due to the low fertility of chromosome-deficient
female gametes, because seed fertility of the monosomies was very low(about 30%) even at artificial pollination with pollen grains from disomic
Khapli. This fact indicates that monosomics of, at least, this Emmer
wheat are not suitable for genetie investigation, except that they mightserve as a source of trisomies.
On the other hand, many trisomies (24% on the average) were obtainedin the offspring of trisomies. A single tetrasomic was also found.
Transmission rates of the trisome through female and male gametes

60 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14
Table 1. Fertilities of mono- and trisomies of T. dicoccum var.
Khapli and the frequency of aneuploids in their offspring.
No. of offspring% seedPlant

Monosomies
K-mono-l
2
II
II
3
Total
Trisomies
K-tri-la
II
Ib
Ie
II
II
2
II
3
II
4
Total

set (selfed)

Total
examined

Monosomic

Trisomic

Tetrasomic

20.0
35.0
39.3*
33.1

8
5
11
24

0
0
0
0

1

-

100.0
92.5
83.3
82.5
62.5
73.3
82.3

36
38
35
35
24
37
205

-

-

-

~

1
2
9
6
9
10
6
9
49

-

-

0
1
0
0
0
0
1

-

Haploid s

1

-

-

1

* Seed fertility by artificial pollination with normal pollen.
were estimated to be 23% and 2%, respectively. Transmission rate of
the trisome in this Emmer wheat is much lower than that reported
for common wheat but is almost the same as in barley. Trisomies ofthe employed Emmer wheat, therefore, may be convenient for genetic
study.

47.

An Emmer wheat with 15 chromosome pairs!)(By Koiehiro TSUNEW AKI)

Since the work of Sakamura (1918) and Sax (1918), the chromosome
numbers in Triticum are known to be 2n=14, 28 and 42 for 2x, 4x and6x species, respectively. No exception to this polyploid series has beenreported. During the course of a cytological investigation of the 4x
wheat, the present author found 15 instead of the expected 14 chromosome pairs in a strain of Triticum durum Desf. var. melanopus (Al.)
Korn. Thirteen pairs consisted of median or submedian chromosomes,
while those of the other two pairs were telocentries. Both telocentric
pairs showed some meiotic irregularities. In a great majority of PMC's,
1)This work was supported by Grant RF 62073 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 196361

however, they formed bivalents in diakinesis (Fig. 1) and metaphase(Fig. 2) and segregated in 1 : 1 fashion in anaphase. Karyotype analysis(ef. Fig. 3) indicated that the two telocentric pairs must have arisen byfragmentation of a submedian chromosome in the normal complementof Emmer wheat.

Figs. 1-3. Chromosomes of a strain of Triticum durum var. melanopus. x 700.
Fig. 1. Diakinesis with ISH. Fig. 2. Metaphase I with IS n . Fig. 3. Mitotic
metaphase in a root-tip cell of haploid spontaneously occurred in this durum.

48.

Further proof that Type 1 necrosis is controlled by theNe- genetic system(By K6zo NrsHIKAw A)

It was shown in an earlier paper (Nishikawa 1962) that Type 1 necrosis
found in hybrids from certain Emmer-squarrosa crosses could be attributed to Ne-genetic system. The new data confirm the earlier interpretation in showing that Neg, which causes necrosis when present together with Nei and Ne2' is responsible for the expression of Type 1
necrosis in the hybrids involving Khapli (T. dicoccum var. arras), andthat necrosis genes carried by Khapli, which interact with Ne3' are thesame as Nei and Ne2 of common wheat.
Chinese Spring monosomic series was crossed with Khapli. The F i 'swere scored for chromosome number and occurrence of necrosis (Table
1).
All F i plants were necrotic except those nullisomic for 3D. Comparable data were already presented by Tsunewaki and Kihara (1961) incrosses involving T. macha var. subletshchumicum (NeiNeiNe2Ne2ne3ne3)
instead of Khapli.
A hybrid between T. macha var. sublet. and Khapli was normal but
did not segregate any normal plants when crossed with Prelude(NeiNeine2ne2Ne3Ne3) or Jones Fife (neineiNe2Ne2Ne3Ne3)'
These results indicate that Khapli carries Nei and Ne2 on chromosome 5B and 2A, respectively. In other words Type 1 necrosis foundANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

62

in certain Emmer-squarrosa hybrids is controlled by the Ne-geneti
system established by Tsunewaki and Kihara (1961).
c
Table 1.

Strains

cm- lA X Khapli*
2A X

"
"

3A X
4A X

"

5A X
6A X

"
"
"

7A X

"
"
"
"
"
"
"
"

"

lEx

"

2B X

"

3B X

"

"

4B X

"

5B X

"
"
"
7B X
"
"
" lDx "
"

6B X

"

2D X

"

"

3D X

"

4D X

"

5D X

"
"
"

"

6D X

"

" 7Dx "

Occurrence of necrosis in F 1 between Chinese Springmonosomics and Khapli.
No. ofchromosomes
34
35
34
34
35
34
34
35
34
35
34
35
34
35
34
35
34
35
34
35
34
35
34
34
35
34
35
34
35
34
35
34
35
34
35
34
35
34

No. of plants--

normalnecrotic

total

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
0
0
0
0
0
0
0
0

5
4
6
10
2
8
7
3
7
2
6
1
9
2
6
2
8
2
8
4
6
5
5
8
2
8
2
8
2
0
2
8
1
9
3
7
2
7

5
10

-

10
10
7
10
8

10
8

10
10
10
10
8
10

10
10
10
10
10
9

* em: Chinese Spring monosomics.

49. Distribution of necrosis genes in Emmer wheat(By K6zo NISHIKAWA)

There are 2 genetic systems for necrosis in wheat hybrids, namely Ne

RESEARCHES CARRIED OUT IN 1963

63

d Net systems. In Emmer wheat, 4 genotypes, namely NelNelNezNez~eINel1Zez1Zez, nelnelNezNez and nelnelneZneZ are possible in Ne-system bU1
ly one gene and consequently only 2 genotypes occur in the Net-

~nstem, namely NetlNet l and netlnetl . In order to get an informationy the frequency of those genotypes, hybrids were produced between 4

op t varieties on one hand and 40 Emmer varieties on the other. Occur·. .m theF'.
tesn of necrOSIS
l s was 0 bserve d and t h e pertment
genotypece;er each Emmer variety was formulated. As previously shown by the~esent author (1962), Type 1 is epistatic over Type 2 necrosis. Khapli,
fha t carries Net l in addition to Nel and Nez, produced hybrids showing
Type 1 necrosis, when crossed with ABD No.1, the carrier of Nea andNetz' Therefore, it could not be determined from F l phenotype whether
J{hapli carried Net l or netl . The same applies to 4 varieties of T. dicoccoides, i.e. kotschyanum, straussianum, P. B. 1. and aaronsohni.
The result is summarized in Table 1 showing the relative frequencies
of all possible genotypes in 8 Emmer species. It is remarkable thatgenotype NelNez is confined to T. dicoccoides, with the exception of adicoccum variety. This suggests that the dominant alleles Nel and Nez
were the prototypes and in the evolutional course have mutated to therecessive alleles, nel and nez . The most widely spread genotype in Emmer is Nelnez, followed by nelneZ and NelNez. It is noteworthy thatnel Ne Z has never been found in Emmer varieties so far tested. As to
Net system both Net l and net I are almost equally distributed among the
8 Emmer species.

Table 1.

Relative frequencies of genotypes of Ne- and Net-genetic
systems in Emmer wheat.

Net-system

Ne-system
SpecieNelNez NelneZ nelNez nelneZT. dicoccoides
T. dicoccum
T. durumT. orientale
T. pyramidale
T. turgidum
T. polonicum
T. persicum

0
0
0
0
0
0

Total

6

-

5
1

5
0
13
1
1

0
0
0
0
0

0
2
3
25

0
0
0
0

3
2
2
0
0
2
1
0
10

Total

Netl

netl

Total

13
3
15
1
1
2

3
4

7
1

10

7
0
0
1
2
0
17

10
2
1
1
1

3
3
41

3
26

5
17
2
1
2
3
3
43

64 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

50. Frequencies of Nel and Ne2 genes in Emmer andcommon wheats(By Koichiro TSUNEWAKI and K6zo NISHIKAWA)

Three complementary genes, Nel' Ne2 and Ne3' cause in combinationa progressive necrosis in common wheat. Nel gene is located in B, Ne 2
in A and Ne3 in D genome. Among them, Nel and Ne2 were extensive.
ly investigated by Caldwell and Compton (1943), Heyne, Wiebe and Painter (1943), Hermsen (1957, '59, '62, '63), Schmalz (1959), Tsunewaki (1960}
and Tsunewaki and Kihara (1962). The number of examined varieties
amounts to 520. For 4 of them, a contradictory result was reported bydifferent authors. Those two genes are also found in Emmer wheat andtheir distribution among 41 varieties was investigated by Tsunewaki
and Kihara (1962) and Nishikawa (1962a, b). Excluding the contradicto_
ry cases, frequencies of 4 possible genotypes in two groups of wheat
are summarized in Table 1. From these results, frequencies of individual
alleles were calculated and are listed in the same table.
In Emmer wheat, frequencies of 4 genotypes are in good agreementwith those expected from random assortment between two pairs of alleles.
Concerning each allelic pair, the dominant allele predominates in the Nelpair, while the recessive is prevalent in the Ne2 pair. In common wheat,frequencies of 4 genotypes deviate from those expected from random
assortment. Frequencies NelNelne2ne2 and nelne 1Ne 2Ne2 are much higher
Table 1. Frequencies of 4 genotypes and alleles for necrosis
in Emmer and common wheats.
Emmer wheat
ActualI Expected*

Freq. of each genotype (No. of varieties)
6
N elNe 1Ne2Ne2
25
N elNelne2ne20nelnelNe 2Ne2
10nelnelne2ne2
Total
41
Freq. of each allele (Relative freq.)
0.756
Nel
0.244
nel
0.146
Ne2
0.854
ne2

4.53
26.47
1.46
8.54
41.00

Common wheat
ActualExpected*

1
87
206
222
516

35.38
52.85
171.53256.23515.99

0.171
0.829
0.401
0.599

* Expected from random assortment between two pairs of alleles.

RESEARCHES CARRIED OUT IN 1963

65

than the expected ones from randomness. In this group of wheat, therecessive allele is predominating in Ne1 allelic pair, while the frequencies of the 2 alleles of the other pair, were not markedly different fromeach other.
Those results clearly indicate that: (1) two genotypes, Ne1Ne1ne2ne2
and ne1ne1Ne2Ne2, are preferentially selected in common wheat against
the two others, and (2) a shift in the gene frequencies must have occurred at the hexaploid level in favor to Ne2 and, especially, ne1'
51.

A polyhaploid plant of Agropyron tsukushiense var. transiens
Ohwi found in a state of nature(By Sadao SAKAMOTO)

A. tsukushiense var. transiens, a common weed growing in fields alongroad-sides, is widely distributed in Japan, China, Manchuria and Korea.
This species is hexaploid (2n=42).
In a valley of the hilly neighborhood of Misima, spontaneous polyhaploid plant (2n=21) of the species was found. It was smaller thanthe hexaploid specimens and had slender spikes, but its tillering wasvery vigorous. In natural condition it yielded two seeds which wereTable 1. Chromosome pairing at MI of PMCs of the polyhaploidand a monosomic of A. tsukushiense var. transiens.
polyhaploid:

2 11

1m
lIV

1n +191+181
2n+ 17I+17r
3n+ 15I

Total

No. of cells
observed

%

369
61
2

83.5
13.8
0.5

8
1
1

1.8
0.2
0.2

442

100.0

164
5

97.0
3.0

169

100.0

Monosomic:
20n + 1119n + 31Total

66 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

assumed to have been produced by pollination with the pollen fhexaploid plants growing in its proximity. The two seeds gave t:plants, one a disomic (2n=42) and the other a monosomic (2n=41).
0Pollen fertility of the polyhaploid, the monosomic and the diso rn '.
was 0.001, 28.5 and 78.5% and seed fertility was 0, 53.3 and 76.6~crespectively. The polyhaploid was crossed by the disomic and by t':~A. tsukushiense vax. transiens strains, and the crossing success amounted
to 1.0.......,1.6%.
Chromosome pairing at MI of PMCs of the polyhaploid and the monoso_
mic is given in Table 1. Of 442 cells of the polyhaploid 83% showed
211 and 14% showed In (terminal pairing)+19I · In a few cells lUI Or
2n were observed. In the majority of cells of the monosomic, chromosorne
pairing was 20 n + h. This result indicates that A. tsukushiense var.
transiens is an allohexaploid whose genome constitution comprises threedifferent genomes.

52. Preliminary studies on the relationship between diPloid Eremopyrum
distans Nevski and E. triticeum Nevski CGramineae)(By Sadao SAKAMOTO and Mikio MURAMATSU*)

E. distans was crossed as the female parent with E. buonapartis Nevski
var. buonapartis, E. triticeum and Heteranthelium Piliferum Hochst. Only
seeds from the cross E. distans X E. triticeum germinated. The F 1 hybrid
showed vigorous and uniform growth. Most of the characters of the F1
hybrid were intermediate between the parents. The F 1 offspring set noseeds. Chromosome pairing at MI of the F 1 was 0-3n with On as themode. All bivalents were loosely associated by a terminal chiasma. It
is concluded that the genomes of E. distans and E. triticeum are
different.
53. Spodogram analysis of leaf-blade in the genus Oryza**(By Hitoshi KIHARA and Tadao C. KATAYAMA)The surface structure of the glume is an important taxonomic characterin the genus Oryza, as was recognized by Roschevicz. In order to examine
the details, the present authors have studied the glume of 18 Oryza species
by SUMP method (1960) and by histological observations (1962). Exploringother taxonomic characters similar to glume characteristics, ash figures.
* Kihara Institute for Biological Research, Yokohama.
** This work was supported by Grant RF 62027 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 196367

f leaves, with special consideration of the silica cells, were examined°h's year, using 30 strains of 21 Oryza species.
t ~n both ends concave silica cells were found in the species of section
S tiva, while those with fiat ends were observed in the sections Granulata,aaretata and Rhynchoryza. The silica cells were classified into 5Co. I types.
orpholog1ca
t1l (1) Officinalis type: Peanut-shaped (or dumbbell-shaped) silica cells
concave at both ends were found in O. officinalis, O. sativa, O. sativa
var. spontanea, O. perennis, O. barthii, O. glaberrima and O. breviligulata.
(2) Minuta type: Cross-shaped silica cells were observed in O. minuta,
O. lati/olia, O. alta and O. grandiglumis.
(3) Australiensis type: Silica cells are of slender peanut-shape, concave
at both ends as observed in Officinalis type. This type was found onlyin O. australiensis.
(4) Granulata type: Silica cells of many different shapes, elliptic-,...,
peanut-shaped, were found in this type. Their arrangement and size
were also irregular. This type was detected in O. granulata, O.
meyeriana, O. abromeitiana, O. coarctata, O. ridleyi, O. longiglumis andO. subulata.
.
(5) Brachyantha type: The silica cells of this type were a mixture of
two kinds, namely Granulata type and modified wider Granulata type.
Found in two species, O. brachyantha and O. tisseranti.
It is concluded that the morphology of silica cells could be used asa good taxonomic character in the genus Oryza.

54.

Occurrence 0/ indica and japonica types among Sikkimeserice varieties*(By Hitoshi KIHARA and Tadao C. KATAYAMA)

Kihara and Nakao (1960) reported indica and japonica types in Sikkimeserice based on the examination of morphological characters, phenol reaction
and seed fertility. The most important key for the classification ofcultivated rice into indica and japonica types is hybrid sterility between
strains. Therefore, in order to confirm our classification of Sikkimesevarieties, pollen fertility of F 1 hybrids with indica and japonica tester
strains was studied.
Fourteen strains of Sikkimese rice were selected and crossed with twotesters, namely an indica strain from Formosa, and a japonica strain
from Japan. Phenol reaction of those Sikkimese strains was reexamined.
In confirmation of our classification, pollen fertility of F 1 hybrids* This work was supported by Grant RF 62027 from the Rockefeller Foundation.

68 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

dearly indicated the existence of both indica and japonica tYPes in
Sikkimese rice. Of 14 strains studied, C7716, C7725 and C7729 are of
typical indica type, while C7707, C7719 and C7735 represent the typicaljaponica type.
Considering the present results obtained in pollen fertility, it is assumed
that the differentiation of indica and japonica types in cultivated Orvza
sativa might have occurred in the Indian sub-continent, particularly in
Sikkim.

55.

Photoperiodic responses of Oryza species, VI*(By Tadao C. KATAYAMA)

Several factors are responsible for different photoperiodic sensitivities
among various Oryza strains. The acceleration degree, which was
expressed by the differential heading acceleration under short day
condition, was considered in a previous paper. In this report, the acceleration degrees of several strains distributed in high altitudes isreported. Nineteen strains belonging to Oryza sativa var. spontanea
Roschev. and O. perennis Moench collected at Sikkim and Assam wereused in this study. Two cultivated strains, Le., No. 124 from India andKA from Japan were also used for the sake of comparison.
The acceleration degree was expressed by (1) the coefficient of linear
regression of growth period on sowing time (denoted by LRC-l), (2) the
coefficient of linear regression of growth period on day length at the timeof flower bud formation (LRC-2), and (3) the angle of the slope of theregression line used in (2) (Angle). The results are summarized in Table 1.
All strains of high altitudes were highly sensitive having 0.85 to 1.00
in LRC-l, 2.55 to 29.50 in LRC-2 and 68.6 to 88.1 in Angle, respectively.
Their heading dates strongly depended upon day length. They showeda rather unusual deviation of their critical day length from the generaltendency, which was found in the correlation between critical day lengthand latitude. This phenomenon was explained as follows: Since theirnative places are very high (ca. 5,000 ft.), temperature rather than day
length seems to have played the essential role in their adaptation.
It is assumed that rice plants growing in such high altitudes as in
Sikkim and Assam might complete their life cycle during a short period
when the natural temperature is suitable for their growth. This resultclearly indicates that at high altitudes the predominating strains respondto a small change in day length which gives them a selective advantage
in their natural conditions.

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

RESEARCHES CARRIED OUT IN 1963-

69

Table 1. Acceleration rates of heading dates.

-Strain

Heading date when sown on
LRC-l

LRC-2

Angle

10-29
10-26
10-26
10-26
10-26
10-26
10-26
10-26
10-17
10-17
10-17
10-25
10-26
10-24
10-25
10-24
10-24
10-26
10-22
10-22
10-21
10-21
10-21
10-22
10-10

0.85
0.93
0.98
0.97
1.00
0.97
1.00
1.00
0.98
0.97
0.98
1.00
0.92
0.97
0.90
0.98
1.00
0.97
0.95
0.95
0.97
0.98
0.98
1.00
0.98

2.55
6.22
19.89
11.60
11.60

68.6
80.9
87.1
85.1
85.1

-

-

-

-

29.50

88.1

10- 7
9-26

0.80
0.65

1.92
0.96

62.5
43.8

Apr. 17

June 16

W0 157
W0635
WI079
WI081
WI082
WI083
WI084
WI085
WI090
WI091
WI092
WI095
WI098
W1099
WllOO
Wll02
Wll04
Wll05
Wll07
Wll08
Wl115
WI116
Wl117
Wll21
Wll34

10-20*
10-22
10-25
10-24
10-26
10-24
10-26
10-26
10-16
10-15
10-16
10-25
10-21
10-22
10-19
10-23
10-24
10-24
10-19
10-19
10-19
10-20
10-20
10-22
10- 9

124
KA

9-25
9- 7

-

-

-

19.89
11.60
19.89

87.1
85.1
87.1

-

-

4.58
14.50
4.15
29.50
11.60
8.14
8.14
14.50
29.50
29.50

77.7
86.1
76.5
88.1
-

85.1
83.0
83.0
86.1
88.1
88.1

* Headed on October 20.
56. Characteristics of Oryza officinalis Wall. found in Borneo**(By Tadao C. KATAYAMA)

During the collection tour of Oryza species in Borneo made in 1963,** This work was supported by Grant RF 62027 from the Rockefeller Foundation.

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

46 strains of O. officinalis were found at different localities in North
Borneo, Brunei, Sarawak and Kalimantan. These strains were Conspicuously different from those collected in other tropical Asiatic regionsin the following characteristics.
1) Native localities of 46 strains of O. officinalis were widely scatteredover the whole island of Borneo, whereas those of strains found inother regions were rather limited to narrow areas. Population size ofeach strain found in Borneo was much larger than that of the strains
found in other regions of tropical Asia.
2) In Borneo O. officinalis was found not only in shady places under
the trees but also in open fields. Sometimes it was growing sympatricallywith O. sativa in the paddy fields. On the contrary, the natural habitatsof most strains of this species collected in other regions were shadyspots in the forest.
3) High variation was found in size and shape of grains, leaf lengthand plant height of O. officinalis collected in Borneo.
4) The ratio of photoperiodically sensitive vs. insensitive plants among
46 strains of O. officinalis collected in Borneo was 7 to 5. The value
indicates a considerable difference from the ratio, 1 sensitive to 3
insensitive, obtained for the strains of this species found in other regions.
It is noteworthy that many sensitive strains were detected even in Borneo,
an equatorial island, situated between 7°N to 4°S latitude.
Judging from those characteristics of O. officinalis found in Borneo,
it is assumed that O. officinalis might have originated in Borneo or its
adjacent islands, and that photoperiodically sensitive strains of thisspecies might be the original form from which insensitive strains would
have derived.

57.

Studies on the intercellular spaces of an Assamese floating rice*(By Tadao C. KATAYAMA)Intercellular spaces in leaves of Oryza sativa, Zea mays and Vicia faba
were examined by the present author in the 1961. This year the leavesof an Assamese floating rice (strain No. C7795) were investigated. The
seeds were sown and seedlings were grown both in paddy and uplandconditions. The leaves were cut off with sharp scissors and divided
into pieces each 3 em long. The intercellular spaces were measured at
different growth stages. Table 1 shows an example of total intercellularspaces in leaf blade and leaf sheath of 3rd, 4th and 5th leaf in a given
stage.

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

RESEARCHES CARRIED OUT IN 1963

The sheath of a fully expanded leaf had always a wider total intercellular space than the blade. The total intercellular space in leaves ofplants grown in paddy condition was larger than that of plants in uplandcondition. This difference was recognized in any growth stage of theplant. The most interesting result was that the development of air
spaces in the leaf was markedly influenced by cultivation conditions,especially by the water content of the soil. The present result is anexperimental evidence for the well-known opinion that the floating rice
is very well adapted to deep water condition.
Table 1.

Percentage and standard deviation of total intercellular spacesin leaves of an Assamese floating rice strain (C7795).

3rd leaf
4th leaf
5th leaf

58.

blade
sheathblade
sheathblade
sheathPaddy

Upland12.80+0.02
13.22±8.79
18.83+3.78
31.75±1.96
14.07±4.36
20.67±O.19

8.73±4.16
9.07±6.62
5.18±1. 71
9.07±6.62
3.40±O.41
6.32±3.18

A. genetic study of developmental instability in tobacco leaf
(By Kan-Ichi SAKAI and Yoshiya SHIMAMOTO)

Diallel crosses were conducted with four varieties of tobacco. Twoof the four were unstable in leaf formation while the remaining two
were stable. Instability in leaf formation was observed in three ways:
bilateral asymmetry, within-leaf variation in the distance between
adjoining veins on the mid-rib and vein-deviation index measured by
the degree of non-parallelity between two adjoining veins. The threemeasures of instability were in close association.
Analysis of variance of the data shows that inter-line variation was
statistically significant for each of the three measures of instability.
Dominance deviation associated with specific parents was significant forvein distance and asymmetry but not for vein-deviation index. Reciprocal
differences were not detected. It is thus concluded that the instability
is due mainly to the additive effect of genes, but in vein distance andin asymmetry, partial dominance effect seems also to be operative in
Some parental strains.
The summary of our data is presented in the following table.

'72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14
Table l.

Genetic behavior of developmental instabilities in leaves in P 1
hybrids of Nicotiana tabacum.
Vein distance

Asymmetry

Midparent

177
165
207
125
167
155

159
9.7
7.3
119
193 -20.3
113
0.3
187
2.2
129
7.9

4.97
5.32
6.38
4.04
5.10
5.45

996

900

F1

Midparent

6.90
8.12
9.88
4.93
5.77
7.37

0.45
0.12
0.23
-0.16
0.05
0.52

AxC
AxS
AxT
CxS
,CxT
SxT

6.75
7.63
8.92
5.13
6.42
7.30

Total

42.15 42.97

F1

----

Vein-deviation ~Recip.
diff.

Recip.
diff.

Midparent

F1

Recip
diff..

5.09
0.72
5.02
0.60
6.27
0.60
3.95 -0.03
4.71 -1.29
5.63
0.32
31.26 30.67---

59. A developmental-gentic study of panicle characters in rice
(By Kan-Ichi SAKAI and Yoshiya SHIMAMOTO)

Forty-six X 4 lines and the same number of control lines, each derived
from the same rice variety, Norin No.8, were examined for fivepanicle characters. The five characters were lengths of the main rachis
basal internode, and second, third and fourth rachillas from the base:Measurements were taken on a single panicle basis for all panicle-bearing
tillers of a plant. Number of plants examined per line was 12.
The present study consists of two parts, one being the estimation ofpleiotropic effect of main rachis genes over the other four characters,
and the other, the inference of developmental relationships between themon the basis of their developmental instability. The estimation ofpleiotropic parameters was made by the method of Sakai and Suzuki
described in our companion contribution to this Annual Report. The
inference of developmental relationships among different characters on
the basis of their developmental instability is based on the following
principle: If two given characters are found to be pleiotropicallycontrolled by the same gene, they may either take their own develop·
mental path separately at an early stage of development or they rriay
start developing together in a single process until differentiation sets
in at a later stage.
If developmental instability of a full-grown character is the reflectionof accumulated effects of developmental noise occurring in the process,then it is expected that late differentiated characters may have a goodshare in instability, while early differentiation may make their instabilities
independent from each other. Conversely speaking, genetic correlation

RESEARCHES CARRIED OUT IN 196373

oefficients of developmental instability between two characters may~eveal their developmental relationship which is genetically controlled.

Results of the present study have led us to conclude that: (1) Fortyis: lines in the X 4 as well as in the control group involved genetic~ariation in all five characters investigated. (2) Genes responsible forlength of the main rachis are pleiotropically effective on the length ofthe other four characters, though the intensity of pleiotropy varies among
the latter. (3) It is found that the pleiotropic influence of the mainrachis genes is less pronounced on the lower than on the higher rachilla.
Basal internode is also partly affected by the same genes. (4) Forty-six
Jines of the control group were found to involve genetic variation indevelopmental instability of the five characters. (5) Computation oj
genetic correlations among instabilities of the five characters have showrthat the main rachis and the fourth rachilla are developmentally very
closely related, while the second rachilla is remotely related to it.
Details of the present study will be published before long.

60. Density response and competitive ability in barley(By Kan-Ichi SAKAI and Shin-ya lYAMA)In 1962, the density response was examined with twelve varieties 0:barley. Spaces assigned to each plant were 2 x 2, 4 x 4, 8 X 8, 16 x 16 an<
32x32cm 2 • Examined were on an individual plant basis plant heightnumber of tillers, number of panicles, length of the longest panicle, dn
plant weight and seed weight. Analysis of variance showed that th(effect of density was statistically highly significant for all character:
except plant height.
It was found that the number of tillers increased linearly in proportioI
to widening the interplant spacing, while seed yield per plant behave<somewhat differently. The increase in seed yield was linear up t<
l6x 16 cm 2 , but at wider spacing, it appeared to attain a plateau in som(varieties. Plant weight was intermediate between tiller number an<
seed yield so far as density response was concerned. This is natura
because dry plant weight consists of seed and straw weights, the forme]
representing a good part of it. It was concluded from this experimen(1) that the spacing at which the increase in seed yield attained a platem
was different among genotypes, (2) that the overall response to plan
density was different among genotypes, and (3) that attaining a platem
in seed yield at wider spacing was not necessarily associated with th(vegetative growth of the given plant. It was considered the non-parallelisn: between reproductive and vegetative growth could be due to intraplan

74 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

competition in seed setting for the limited supply of available assimila.
tion products.
In 1963, competitive ability of the same 12 varieties was examinedwith the aid of one variety as a tester. The experiment was conductedwith four replications. The results proved that competitive ability wassignificantly variable among the 12 varieties.
Combining the two years' data mentioned above, it was examined
whether or not competitive ability and density response were related:
The correlation coefficients between density response and comptitive
ability were 0.548 for plant weight, 0.410 for number of tillers and 0.145
for seed yield, all falling short of the 5% level of significance.
There is a general tendency among ecologists and agronomists toconfound the effect of density increase with the effect of competition.
The present experiment, however, indicates that the two should not
be mixed, but should be considered separately, one as density response
and the other as competitive reaction.

61.

Survey 0/ F 1 sterility relationships between strains 0/
Oryza perennis(By Hiroko MORISHIMA and Hiko-Ichi OKA)

Oryza perennis is distributed throughout the tropical countries. of theworld. It comprises various geographical races. A survey of F 1 sterility
relationships between Asian strains was reported previously (Jap. ].
'Genet. 37: 314-328). The same work was extended to South Americanand African (barthii) materials. The American strains, forming an
intra-fertile group, generally showed a high F 1 sterility in crosses with
Asian and African strains. The F 1 hybrids between African and Asianstrains showed a wide range of pollen fertility. In some of those crosses,
crossability was low, or the F 1 seeds did not germinate well because of
the poor development of the embryos. These phenomena are still under,observation.
62.

Characters relative to the breeding system in Oryza species(By Hiroko MORISHIMA and Hiko-Ichi OKA)

Wild rice species, O. perennis as well as O. breviligulata, are partly,cross-pollinated. With the view to looking into their breeding system,
a number of strains belonging to these species were investigated regarding 1) time interval from spikelet openning to pollen emission, 2)

RESEARCHES CARRIED OUT IN 196375

I ngevity of pollen grains emitted from the anther, 3) number of pollen
aainS per anther, 4) anther length, and 5) the size of stigmas. In
g~dition, regenerating or ratooning ability was estimated by measuring:he development of adventitious roots from stem-cuts with the first or
eco nd node placed in moist sand for three days. In each character, as ide variation was found among the strains. The characters supposedly~omoting cross-pollination (1-5) were correlated with one another, and~ISO with regenerating ability.

63.

Change of population genotype of rice hybrids in response
to different propagating methods(By Hiko-Ichi OKA and Hiroko MORISHIMA)

Hybrid populations between wild (Oryza perennis) and cultivated (0.
sativa) strains were propagated until F 7 by three different methods,
selfing in pedigrees, open-pollination (seedlings raised in pedigrees weremix-planted in bulk), and mix-harvesting in bulk. The plants propagatedin mix-harvesting bulk rapidly approached cultivated forms and had arelatively small heterozygosity, possibly because of increasing selfing
rate. Those selfed in pedigrees did not approach cultivated forms so
rapidly. These facts suggest that a wild population containing a sufficient amount of genetic variability can change toward cultivatedforms in response to selection pressure under cultivation. This work
is still under way.

64. Observations of wild and cultivated rice species in West andCentral Africa(By Hiko-Ichi Oka)

From October 1963 to January 1964, together with Dr. Wen-Tsai Changof the Academia Sinica, Taiwan, I visited Sierra Leone, Guinea, Mali,
Niger, Dahomy, Nigeria, Cameroun and Tchad for studies of wild andcultivated rice species. Our main tasks were to investigate variations in
Oryza perennis subsp. barthii, hybrid swarms between O. breviligulata andO. glaberrima, and other Oryza species, and to study basic problems
underlying rice breeding in Africa. The results of observations havebeen described in a mimeographed report submitted to the Institute.
To our great sorrow, almost at the end of the expedition Dr. Changlost his life in a car accident.

~

Cl'l

Table 1.
Species and Genome

Protein and RNA content of wheat seeds

Prolamin(60% EtOH)

Albumin(H 2O)

Gloublin(2% NaCI)

Glutelin(.2% NaOH)

Total

0'>

?1

>
Z

~

c:

~

RNA-P

'<::l-~...
~

0.54 mgNjg

0.32 mgNjg

4.72 mgNjg

(37%)

( 6%)

( 4%)

(53%)

1.93
(31%)

0.58
( 9%)

0.56
( 9%)

3.20
(51%)

6.27

2.21
(36%)

0.48
( 8%)

0.29
( 5%)

3.16
(52%)

6.14

3.32 mgNjg

T. spelta

AABBDDT. durum X Ae. squarrosa

AABBDDT. durum

AABB
T. dicoccoides

AABB
T. monococcumxAe. squarrosa

AADD
Ae. squarrosa 4x

DDDD

1.98
(33%)

0.71
(12%)

0.39
( 7%)

2.91
(49%)

8.92 mgNjg

N

229f1gjg

5.98

241

~

0

~

~

;:s

tz:J

>:>...

..,

ztj

<::l

'<::l-

0.34
( 4%)

0.44
( 5%)

5.34
(60%)

8.92

311

(32%)
2.38
(30%)

0.71
( 9%)

0.61
( 8%)

4.16
(53%)

7.86

190

;:s

2.84

2l
-S ""-

...

'"

2.76
(34%)

0.65
( 8%)

0.54
(7%)

4.15
(51%)

8.10

176

'"'
;:s
'"

3.34
(37%)

0.92
(10%)

0.28
( 3%)

4.46
(50%)

9.00

178

~

0

;:s

T. monococcumAA
Ae. squarrosa

DD
Abbr.:

""""-

~

~

'"!j

Z

>
,..,
0
Z
>
t""
>-<

Z

,..,
,..,
c:
,..,

Ul
>-<

tz:J
0

'"!j

C)

tz:J
Z
tz:J
,..,
>-<
(")

Ul

'"""-

Z

~

,p.

<::l

T.; Triticum and Ae.; Aegilops

,..,
0

;:s

,..,

0

c'
;:s
<::l

til
'<

::>;i

tz:J

'1:1
::>;i

>:>...

212

>
t""

<::l

""-

208

Z

~

9

,...

RESEARCHES CARRIED OUT IN 1963

77

Ribosomal RNA (rRNA), which represents 80 to 90 per cent of total
RNA, is synthesized in conjuction with the nucleolar-associated DNA
and then probably moves into the cytoplasm. The purpose of this work
was to test whether an increase in DNA content due to auto-and allopolyploidization has any effect on the rRNA and protein content of wheat
seeds.
Materials used were selfed seeds of Triticum and related naturally
ocurring polyploids as well as artificial amphidiploids. In each experiment, 400 to 800 mg of seeds was used. For the estimation of protein
contents, seeds were macerated in cold acetone before extraction. Differential extraction of the proteins were made with the solvents listed
in Table 1. Protein content was estimated by the phenol method andcalculated in terms of serum albumin. Determination of RNA content
was carried out by the Ogur-Rosen method.
There is positively a similarity in the relative concentration of eachspecific protein (Table 1). Also, no significant correlation was observed
between the RNA content and polyploidization.
The present results seem to show that the increase of DNA content
by polyploidization has in general no appreciable influence on the controlling mechanism of rRNA synthesis except for AADD type. Considerablylarger amount of RNA in AADD than in the other employed tetraploidwas observed.

66. Secondary association in Lillium auratum(By Yo TAKENAKA and Y. E. CHU)

Secondary association in PMCs and karyomorphology of somatic chromosomes of Lillium auratum were studied. For karyological analysis
the somatic chromosomes were divided into four groups (A, B, C, D)
by their length, place of constriction and other chracters, and the occurrence of each type was expressed; AIA2BIB2CIC2C3C4CsDID2D3'
The investigation of secondary association at diakinesis showed 12(1)*,.
1(2)+10(1)** and 2(2)+8(1)*** figures with frequencies of 16.3% 30.4%
and 27.5% respectively. At MI 12(1), 1(2)+10(1) and 2(2)+8(1) figures
Were observed with frequencies of 50.1% 30.6% and 11.7% respectively.
According to the above data, the frequencis of secondry association at
MI were less than those at diakinesis. This is assumed to be due to* 12(1) indicates 12 bivalents.
** 1(2)+10(1) indicates 1 secondary association of 2 and 10 bivalents.
*** 2(2)+8(1) indicates 2 secondary association of 2 and 8 bivalents.

78 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

the decrease of attraction between the chromosomes, during the progre..
Sso f melOtlc stages.
If the phenomenon of secondary association is due to specific affinity
between homologous chromosome segments, secondary association should
occur always between the same chromosomes. At diplotene, it was foundthat there was high frequency of association between A1A l and D1D l andalso between AzA z and BzB z or B1B l (it was very difficult to distinguish
Bl from Bz chromosome). From this finding, it could be assumed that
there were some homologous segments between non-homologous chro.
mosomes. At pachytene, it was found that D l always touched the shortarm of A l and the short arm of B l orB z always touched the short armof A z•From the above results, it is assumed that this species could havederived by hybridization between two closely related varieties.

67.

Cytological studies on a Nicotiana tumorous hybrid and its parents(By Yoshiaki YONEDA and YO TAKENAKA)

Cytological and growth characters of a tumorous hybrid, N. glaucax
N. plumbaginifolia, and its parents were studied in vitro. The materials
used were as follows: "Normal" and "tumor" tissues were isolated
from normal stems and tumor tissues of the hybrid respectively, andcallus tissues were obtained from parental hypocotyle.
As control, we first observed mitotic cells of small, young in vivo
tumors produced on the stem of hybrid. They had almost all 22 normal
somatic chromosome. Polyploid and aneuploid cells were rarely found.
Chromosome number of the above four tissues cultured on White+yeast extract+2,4-D agar medium were counted. Though the frequency
of normal diploid cells was high, polyploid and aneuploid cells were alsofrequently found, especially in the callus of N. glauca.
In order to reveal the relation of the variation in chromosome number
to medium components, the tissues were cultured on basic media con·taining auxins, kinetin, single orjn combination. In the "normal" tissueof the hybrid cultured on media containing both auxin and kinetin, thefrequency of diploid cells was higher than that of polyploid ones, whilein the "tumor" tissue polyploid cells became more frequent on the same
media. In kinetin (2 ppm) medium, mitotic cells of the "tumor" tissue
were predominently polyploid. Thus "normal" and "tumor" tissues
were assumed to respond differently to kinetin concerning the occurrenceof polyploid cells. The cultured tissue of N. plumbaginifolia had

RESEARCHES CARRIED OUT IN 196379

osistently a very high frequency of diploid cells. On the other hand,
polyploid
cells were found in the callus of N. glauca at the timeo..
f this experIment.
,a In "normal" and "tumor" tissues of the hybrid and in the callus of
No glauca, anaphase bridges were found in high frequency on the various
media used. A few such bridges were also found in the callus of N.
plurnbaginifolia. As most of the bridges were not accompanied by<chromosome fragments, these might have been sticky bridges. Suchbridges were demonstrated in the "tumor" tissue of the hybrid and theparental calli cultured on White's basic medium. As the mitotic cells
were very few, such sticky bridges were not yet recognized in "normal"
tissue cultured on the basic medium.
The "normal" and "tumor" tissues of the hybrid grew slowly on
White's basic medium. The calli of parental species increased in fresh
weight and some mitotic cells were also recognized when they were
cultured on the basic medium. This hormone-independent growth ofthe callus tissues is considered to represent the so-called habituation
under cultured conditions. The growth of N. glauca callus was stimulatedby the addition of kinetin, auxin or both to the basic medium, whereas
the callus of N. plumbaginifolia showed the stablest growth on White+
2,4-D+kinetin+yeast extract.
C nly

68.

Tumors in Japanese morning glory(By Y6 TAKENAKA and Yoshiaki YONEDA)

We found tumorous outgrowths in a strain, 056, of Japanese morning
glory, which was supplied to the senior author by Mr. Omori in 1960.
Since then many tumor-bearing plants have been cultivated in thisinstitute. We succeeded in obtainin\S in vitro cultures of the tumor tissue
in 1962. The present report is concerned with morphological and cytological characters of both in vivo and in vitro tumor tissues.
The tumor-producing plants retained the cotyledons throughout theirwhole life. Their leaf development was very variable. Some plants
had curled and crooked leaves with sharp-pointed lobes. These phenotypiccharacters are assumed to be due to the genes "Blown", "delicate" and"feathered" . They often bloomed, but the flowers were completely
sterile. In other plants after the cotyledons no leaves appeared, only
clumps of abnormal shoots. Such a plant had the appearance of teratoma.
The small, swollen shoots had many leaflet-like protuberances below theapex. We often found a spheroidal lump of tumor tissue on such an

80 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

abnormal shoot. It is unknown at present how to explain these various
phenotypes. One of the causes might be the complexity of the genetic
background system in the original horticultural stock.
Histologically the tumor showed much disturbances in tissue organiza_tion. Meristematic tissues were distributed at the surface of the tumor
and procambium strands ran through the parenchyma. In the interiorvarious types of vascular nodules were found. In general, the vascula;tissues were developed strongly, but their distribution was irregular.
In cultured condition, the tumor tissue could grow on White's basic
medium, while normal callus isolated from the wild type of morning
glory showed no growth on this medium. This result indicated that thetumor growth was hormone-independent; it was promoted by yeast
extract. The tumor tissue was yellow-brown and had very hard texture.
Histologically a great amount of vascular tissues developed in its interior
and the meristematic tissues were found only on the surface. For
constant growth of normal callus, addition of auxin, kinetin and caseinhydrolyzate or yeast extract to the basic medium was necessary; it was
yellow and very soft in texture and mostly consisted of meristematicand parenchymatous tissues. Thus the vascular development in thetumor tissue was very different from that of the normal callus.
In cytological studies a difference was found; namely, polyploid cells
were recognized in the tumor tissue cultured on White+2,4-D+yeast
extract medium, while normal callus contained only diploid cellss on
the same medium.
Tumor formations in Japanese morning glory are considered to berelated to some genic systems (or genic combination). In the present
case, at least three genes were identified in the tumorous plants. From
the result, we have assumed that the tumor production in this plant
may be included in the category of genetic tumors.

69. Spring- and Winter-sown varieties of sugar beets, withspecial reference to photothermal induction(By Seiji MATSUMURA)

Induction of flowering in sugar beets (Beta vulgaris L.) has two'
important practical aspects. Development of seedstalks, i.e. bolting, isobjectionable when the crop is grown for sugar production, but when
the crop is grown for seed production initiation and complete development of seedstalks is desirable. All investigated varieties are longday
and biennial plants.
Experiments with Spring-sown (Hon-iku 192, GW 359, etc.) and Winter-

RESEARCHES CARRIED OUT IN 196381

sown varieties (Kleinwanzleben-AA, Cesena-P, etc.) confirmed that treatment of 5,...,6 month old beets with the temperature of 1,...,4°C for 1,...,2months (July and August) increased the tendency to bolting; one monthtreatment was not effective in inducing bolting in Winter-sown varieties,
especially in polyploids, but was effective in Spring-sown ones. Thevernalized beets could be devernalized by subsequent treatment even at
Zooe day and 19°e night temperature in an air-conditioned greenhousewith continuous lighting. Subsequent treatment at about lOoe in a
growth cabinet at natural day length was markedly more effective thanthat in field cultivation. It is assumed, therefore, that the climaticconditions in Hokkaido district are suitable only for seed production of
Winter-sown varieties, which are promising for sugar production inSouthwestern districts of Japan, where (for instance, islands of theInland Sea, Kagawa-ken) even seeds of Spring-sown varieties, mostly
cultivated in Hokkaido for sugar production can be produced.

C.
70.

MATHEMATICAL GENETICS

The mutational load in small populations(By Motoo KIMURA)

It is customary, in the calculation of the mutational load, to assume
that the population is infinitely large and the gene frequency is exactly
determined by the balance between mutation and selection. However,in a finite population, the factor of random drift becomes involved andboth the gene frequency and the load become random variables.
Applying Wright's formula for the gene frequency distribution in
finite populations, the mean value of the mutational load was calculated
numerically by the m:e of an electronic computer.
The study showed that in a small population the load is considerablylarger than in a large population. Futhermore, a rather unexpected
result emerged: Namely, for a wide range of population sizes, amutant that is slightly harmful is more damaging to the fitness of the
population than a mutant with a much greater harmful effect.
The variance of the mutational load has also been computed. The
results show that intergroup selection is ineffective in reducing thisload.
The present study was done in collaboration with Dr.]. F. Crow and
Mr. T. Maruyama, while the author was visiting the University of

82 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Wisconsin. For details, see Kimura, M., J. F. Crow and T. Maruyama(1963) Genetics 48: 1303-1312.

71.

The stepping stone model of population structure(By Motoo KIMURA)

In Number 3 (1953) of these annual reports, the author proposed a newmodel for the breeding structure of populations which he called stepping_stone model. In this model it is assumed that the entire population is
subdivided into colonies and the migration of individuals in each generation is restricted to nearby colonies. At that time, no satisfactorymathematical treatment of the model was obtained.
Recently, while the author was visiting the University of Wisconsin,
the complete solution of the model concerning the decrease of genetic
correlation with distance was obtained in collaboration with Dr. G. H.
Weiss of the University of Maryland.
It has been shown that the decrease of genetic correlation with distancedepends very much on the number of dimensions:
In one dimension, the correlation decreases approximately exponentiallywith distance:r(p)cce- Ap

where p is the distance and A is a positive constant. In two dimensions,it falls off more rapidly and if the migration is isotropic in X and Y
directions, we obtain for a large value of p, the relationr(p)cce-Bph/P;

where B is a positive constant. Finally in three dimensions, it falls off
still more rapidly and asymptotically,
1'(p)cce-cP/p,

where C is again a positive constant.
The quantity 1-1'" where 1'1 is the correlation coefficient between two
adjacent colonies, is also relevant when the tendency toward random
local differentiation is discussed. It has been shown that the relationof this quantity to mutation and migration rates depends very much on
the number of dimensions. This, together with the above results on
the decrease of correlation at a large distance, clearly shows that thetendency toward random local differentiation is strongest in one dimension
and becomes weaker as the number of dimensions increases. The details
will be published in the April 1964 issue of Genetics.

RESEARCHES CARRIED OUT IN 1963

72.

83

Effect of maternal inheritance on the genetical variance in asegreating population(By Shin-ya IYAMA)In the Annual Report No. 10, the author reported on the effect ofmaternal inheritance on the variance in a hybrid population of autog31l10US plants.
More general formula is now presented and maternal
effect in a random mating population is discussed.
According to Sakai's model (1960),
P n=(1-m)Gn+mPn- 1(1)

where P n and Gn stand for the genotypic values with and withoutmaternal effect in the n-th generation respectively, and m represents
the proportion of maternal effect in the total hereditary effect. In caseof allogamous organisms, the maternal parents are to be traced back.
Formula (1) may be written by repeated substitution as follows:
P n=(1-m){Gn+mGn- 1 +··· +mn- 1 Gd+m np o(2)Then, the mean and the variance in the n-th generation are,Pn=(l-m)i mn-iGi+mnpO

(3)

i=l

+2mn(1-m)

i mn-iWpOGi+m2nvpo(4)'/..=1

where VGi and V po stand for the variances of Gi and Po respectively,
and WGiGj and WPOGi stand for the covariances between Gi and Gj andbetween Po and Gi respectively.
Assuming random mating and no change of gene frequency, we may
obtain the following relationships:
Gi=Gj=G, VGi= VGj= V G, W GiGj = W GkGl

if i-j=k-l,

WGiGj+t=(!)t WGiGj and WPOGi+t=(!)t WPOGi'

Then,

G (n~oo)
n-l

n-2

n-j-2

,[.=0

J'=O

j=O

=(1-m)2VG l m 2i +(1-m)2Wl (~)j
n-l

1

2m 2i

+2mn(1-m)WpoGl(~t-l1 (2m)i+m2nvpo
i=O84 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

where W stands for convariance between genotypic values of the maternal
parent and its offspring. Since V G =a/+ad 2 and W=~a/, total geneticalvariance in a random mating population will approach toV- (1-m) (2+m) 2+ (1-m) 2(l+m) (2-m) ag(1+m) ad .

P noo -

After sufficiently large number of generations, the maternal effect On
the population mean will be lost and the genetical variances will reacha constant value, both the additive genetic variance and the dominancevariance being smaller than when no maternal effect exists.

D.

73.

GENETICS AND BIOCHEMISTRYOF MICROORGANISMS

Allelic repression in transductional heterogenotes of Salmonella*(By Tetsuo IINO)

In Salmonella-phage P22 system, a heterogenotic line occurring bytransduction of an Hl-fla segment from a flagellated cell to a nonflagellated fla-A- mutant cell produces a trail of minute colonies on semisolid
nutrient medium (lino, Annual Report No.9, 1958). Trail formation
by fla+jfla- heterogenotes is suppressed when specific antisera against
H (flagella) antigens of their component cells are added to the semisolid
media (lino, Annual Report No. 13, 1962). This phenomenon was applied
to identify H-antigen type expressed by H 1i jH/ heterogenotes.
Transductions of Hrfla segments were carried out from S. tYPhimurium(i:J,2) to a phase-1 culture of a fla-A- mutant of S. abony (b:e,n,x).
Genotype of the resulting heterogenotes is expected to be H 1"fla-jexH 1'fla+.
In this combination, trail production on semisolid media is suppressedby anti-b,e,n,x but not by anti-i,e,n,x sera. This result ind"cates thatthe Hl"fla-jexHlifla+ heterogenotes produce flagella carrying b antig-en.
In transductions from S. abony to a fla-A- mutant of S. typhimurium,
trail production by the resulting heterogenotes, H 1ifla-jexH/fla+, is suppressed by anti-i,J,2 but not by anti-b,1,2 sera.
A general rule deduced from these results is that heterogenotes of
H 1 gene, resulting from the P22-phage mediated transduction between
two Salmonella serotypes, produce flagella determined in their specificity* 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 196385

bY the endogenote, and the function of exogenotic Hi is repressed byendogenotic Hi'
Allelic repression of endogenotic Hi to exogenotic H l was also observedin transductions between S. tYPhimurium (i:l,2) and S. dublin (g,p:-).
When phase-2 cultures are used as recipients in the transductions
described above, expression of both exo- and endo-genotic Hi is repressedby the phase-2 antigen type determinant, Hz·

74.

Peptides pattern 0/ flagellin 0/ normal and curlymutant flagella in Salmonella abortus-equi*(By Masatoshi ENOMOTO and Tetsuo lINO)

A curly flagellar mutant, SJ30, originated from a wild type strain of
S. abortus-equi, SL23, has flagella of half wavelength compared with thewild type. It is attributed to a mutation in the structure gene of phase-2
flagellin, Hz. H-antigen type of the curly flagella is the same as that of
the wild type, enx. Flagellins of these two strains, SL23, and SJ30, were
crystalyzed (Enomoto and lino, Annual Report No. 13, 1962), and two
dimensional patterns of peptides from their tryptic digests were comparedwith each other. For the tryptic digestions of heat denatured flagellin,
2% flagellin and 0.04% trypsin in 0.05M-phosphate buffer (pH 8.0) wereincubated for 20 hr. at 25°C with a drop of toluene. Two-dimensional
separation of the produced peptides was performed by electrophoresis
followed by chromatography. By electrophoresis at pH 6.5 in pyridirre
acetate buffer, peptides from both, SL23 and SJ30 flagellins, were divided
into eleven groups based on charge differences. Distributions of thesegroups on the electrophoreograms were identical for the two strains.
By subsequent chromatography with n-butanol-acetic acid-water system,
34-35 spots were separated further from eleven groups of peptides. Amongthem, the patterns of 32-33 peptides, among which 4 were histidine- and8 were arginine-containing, were indistinguishable between SL23 andS130. One peptide spot involved in group 4 of the electrophoreogramof SL23 was absent in SJ30. Instead, a new peptide spot appeared ata more distal point from the original line. The remaining peptide
appeared at the same position in both strains but its amount was larger
in 8J30 than in SL23. Together with the genetic data (lino, Annual
Report No.9, 1958), these results support the previously proposed hypothesis that the morphological change of flagella by curly mutation is
primarily caused by an alteration of an amino acid code in the structure
gene of fiagellin.

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

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

75. Inhibition and regeneration experiments of theflagellar synthesizing system of Salmonella cells*(By Michiko MITANI)

Phenol, lithium chloride and 44°C have been known as the specificagents which inhibit flagellar formation of growing bacteria (lino &Mitani, Annual Report No. 13, 74, 1962). In an attempt to disclose aflagellar synthesizing system in bacterial cells, the search for specificinhibitors of flagellar synthesis of S. typhimurium was continued, and theireffects were compared.
In addition to the above three agents, lithium carbonate and cresol
were found to inhibit flagellar synthesis. The concentrations which
inhibit flagellation of bacterial cells and yet minimize the growth inhibition are 0.12% and 0.20%, respectively, for phenol and lithium carbonate.
Although final growth is diminished at those concentrations approximately
by one tenth in contrast with the control, the number of flagella perbacterium is progressively decreased following cell divisions. But if
the resulting deflagellated bacteria are transferred to synthetic regeneration medium, de novo flagellar synthesis is performed after a lag Corresponding to the time requiring at least one bacterial division.
In the course of the regeneration experiment of flagella, the bacteria
deflagellated previously by an inhibitory agent are found to regenerate
their flagella in the regeneration medium containing certain otherinhibitory agents. Namely, after preincubation in the medium withadded 0.12% phenol, the deflagellated bacteria were transferred intobroth containing 0.20% lithium carbonate or to a synthetic regeneration
medium at Moe. Then, flagellar regeneration of the cells was observedin the medium incubated at 44°C but not in the broth containing 0.20%
lithium carbonate. When the cells were deflagellated by lithium carbonate
treatment, flagellar regeneration was well observed both in the regeneration medium containing 0.12% phenol and in the medium incubated at 44°C.
On the contrary, if the deflagellated cells were obtained by cultivation
at 44°C, the cells did not regenerate their flagella either in the medium
with added 0.12% phenol or with 0.20% lithium carbonate.
From these results it is assumed that each of the inhibitors affects adifferent step of flagellar synthesis; the most plausible order of sites
inhibited by each agent is considered to be: 44°e~phenol-,lithium
carbonate.

* This work was partly supported by a research grant from the National Instituteof Allergy and infectious Diseases (AI-02872), Public Health Service, U.S.A., toT. lino.

87

RESEARCHES CARRIED OUT IN 1963

76. Mapping of the mutant sites in motility A-cisronof Salmonella tYPhimuriuln*(By Masatoshi ENOMOTO)

With a few exceptions, mutant sites causing paralysis of flagella inS. typhimurium, TM2, are classified into two functional units, either
ffl ot -A or mot-B cistron, by means of the cis-trans test (Enomoto, Annual

Report No. 13, 1962). From 48 mutants in A-cistron, 12 mutants showing
nO tendency to revert were chosen and transductions of the motility
genes were carried out with phage P22 in all pairwise combinations. It
waS found that 10 out of 12 mutants failed to give motile recombinantswith one or more mutants, and the remaining two recombined with allother mutants (Fig. 1). In order to map the reverting mutants, that is,
those containing small alterations, further qualitative (yes-or-no) tests
for recombinations were carried out between 12 stable mutants and allother members of A-cistron. To avoid confusions between recombination
and reverse mutation, 12 stable mutants were used as recipients andthe reciprocal tests were carried out only in crosses with relatively
stable mutants. Out of 36 mutations tested, 15 mutations overlapped
at least with one of the stable mutations including both SJ631 and SJ634..... - ~.. l

SJ444

SJ597
SJ442---

8J472

--

<f

• ~ --.

I

I

I

I

I

I

I
:

I

I

i
I

I

;

:

-:

""Q~GlB~ ... _ ..
I
I
I

I

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

: : : : ~-!-~-~--~--: --:---t-

~

SJ428

:

SJ605

"I :

SJ615

I

I
I

:----H1

I:--:---:--:--:-:--:----~-

::

~

:

~.~,-

;
- - - - :-I~~-~~... ~---_ . ---:-,~-;-

I::: _ :__~--_- _:
I

I
I

I
I

I

I

, 0------"--I

I

I

:~
I

I

'
I

I

I

SJ60
I
;
:--:-;-SJ630----,.L
I ---:--:----:
SJ611---···--- - - . - - . - - - - - - . - - --~----:-.---:-;-.
s . 1 e ' l - - - - - · - - - - - - - · - - - - - · · - - · · · · - - ------ ~-:-:-­S J 6 3 4 - - - - - - - - - - ·- - - - - - - - - - - - -.. ---------~!

Fig. 1. Genetic map of motility A-cistron. Strains described verticallyare extended (deletion) mutations. Horizontals represent the tentativealignment of mutation sites which overlap with extended mutations.

* This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (AI-02872), PUblic Health Service, U.S.A. to T.
lino.

88 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

which gave recombinants with all other stable mutants (Fig. 1). None·of 36 mutations overlapped with the stable mutations, SJ444 and SJ597
which gave no recombinant with each other. These two mutants wer~found to revert at a very low frequency in repeated experiments, Sothat they are likely point mutants in closely adjacent sites. Except
these two, stable mutants included relatively large alterations, so-called
deletions, and their sites could be arranged in a linear sequence.

77.

Effect of phenol on the expression of multiple drug resistanceof Shigella(R)(by Tetsuo lINO and Masatoshi ENOMOTO)

Shigella flexneri 2b-103 carries an episome, R, which confers on theinfected cells resistance to four antibiotics, namely sulfonamide (Sa),streptomycin (Sm), tetracyclin (Tc) and chloramphenicol (Cm).
From growth experiments in media containing both 0.06% phenol andvarious concentrations of the antibiotics, it was found that the additionof phenol increased remarkably the degree of resistance of the 2b-103
cells to Cm and to less extent to Sm, and reduced the resistance to Tc(Fig. 1).
Preincubation of the cells in phenol containing media increases thedegree of resistance more than 100 times to Cm as compared with theculture without preincubation in phenol broth (Table 1).
The modification of resistance is maintained only in the presence ofphenol. When the cells are transferred from a phenol containing to aphenol free medium, the degree of resistance reverts to the originallevel.
Table 1.

Effect of phenol on the resistance of Shigella 2b-103(R) to Cm.
no. of surviving cells per ml of broth culture*containing

pre-incubation.________ Cm Cr/ml)

Pheno~
0
broth
0.06

100

0

I
I

2.6x10 9
2.8x10 8

I 2.4x10
0.06
I 4'7x10* 20 hr. incubation at 37°C; initial inocula: 10 cells/m!.

broth + 0 .06 %phenol

0

9

8

4

I
I
I
I

<10 3
3.4x104<10 3
5.8x10 6

RESEARCHES CARRIED OUT IN 193689

It has been inferred that infection with R-factors causes an alterationof the surface structure of the infected cells leading to reduction of
permeability of the cell membrane for antibiotics. Thus, phenol may
modify the surface structure of R-infected cells so as to favor the permeation of Tc and prohibit that of em and Sm.

COl"!centra t:ion

Fig. 1.

78.

of To rnicrogram/ml

ern microgJ.""@,ID/ml

Effect of phenol on drug resistance of Shigella flexneri.
Colony number on nutrient agar (drug free) is taken as 100.

Sensitization to phenol by infection of Salmonella with multiple
drug resistance factors (R)(By Tetsuo IINO and Michiko MITANI)

It was found that the infection of R-factors to a strain of S.
fyphimurium TM2 results in sensitization of the infected cells to phenol
as well as conferring resistance to four antibiotics, namely sulfonamide(Sa), streptomycin (Sm), tetracyclin (Tc) and chloramphenicol (Cm).
TM2 cells are sensitive to all four at the concentrations of 10,ug/ml of
Sm, 10,ug/ml of Cm, 5,ug/ml of Tc and 50,ug/ml of Sa. In the growthof broth culture this strain is reduced to 50% by adding 0.05% phenol
to the media and is completely inhibited by 0.25% phenol.
An R-factor originally detected from Shigella flexneri 2b-103 was transferred via Escherichia coil W3102 to TM2. Resistance of the R-infected
TM2, designated TM2(R), to antibiotics Sm, Tc, Cm and Sa, increased
up to the level of 50 to 100,ug/ml against the first three and to lOOO,ug/ml
against the last one. While its resistance to phenol decreased afterR-infection to about 50% in comparison with TM2. Complete growth
inhibition occurred already in 0.12% phenol broth. Subclones of TM2(R)which spontaneously lost resistance to Tc andlor Sm still maintained
the same level of sensitivity to phenol as the four drug resistant clones.
Occurrence of this phenomenon in other bacterial strains is underinvestigation.

'90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

79.

Preliminary studies on an arginine-sensitive
mutant of Salmonella typhimurium(By Jun-ichi ISHIDSU)

A new arginine-sensitive mutant (arg-s-l) was isolated from a wildtype strain of Salmonella typhimurium LT-2 by 2-aminopurine treatment.
This mutant grows normally in synthetic minimal medium (K2HP04,
10.5 g; KH 2 P0 4 , 4.5 g; MgS0 4, 0.1 g; (NH 4hS04' 1 g; sodium citrate, 0.47 g;
glucose, 4 g; water, 1,000 ml) and can also grow almost normally innutrient broth. But when the synthetic medium is supplemented witharginine (either hydrochloride or carbonate), its g-rowth is specificallyinhibited. The critical point of arginine concentration effective in growth
inhibition of arg-s-l in liquid medium lies between 10- 5 M and 10- 4 M(Fig. 1). If the concentration of arginine is not too high but sufficient toinhibit at least the initial growth, the mutant suddenly becomes freefrom inhibition after a lag period, and starts growing at the same rate
as it does in the absence of arginine. The lower is the original arginine,concentration, the shorter is the lag period, and once growth starts, it
continues until a similar level of stationary phase is attained having no
relation to the original arginine concentration. This phenomenon
suggests that arginine in the medium is being exhausted or inactivatedin some way during the lag period even without significant growth of
the bacteria. In addition, it was found that, if the glucose concentration is brought up to 1%, or higher, the inhibition period is shortenedby the hour.
Among various common amino acids, vitamins, purines, or pyrimidines,
so far as tested, only arginine can affect the growth of arg-s-1Most interestingly, this growth inhibition by arginine is removed immediately by adding a sufficient amount of uracil or some of its precursors or related substances, such as orotic acid, uridine, cytosine,
cytidine, isocytosine, or uridylic acid. No other chemicals so far as
tested could readily rescue arg-s-l from arginine inhibition. Tests withuracil indicate, as shown in Fig. 2, two facts: (1) When an equivalentamount of uracil to arginine, or more, is added to the medium, thegrowth inhibition is removed at once and the bacteria grow at thenormal rate and attain the normal stationary level, and (2) when an
insufficient amount of uracil, but higher than 10- 5 M in concentration,is supplied, the bacteria start growing at the normal rate but the growthsuddenly stops when a certain level, far below the normal stationary
level, is attained. This stop is not brought about by arginine concentration but by uracil concentration only.
Since this mutant has no auxotrophic requirements, the damage of

RESEARCHES CARRIED OUT IN 196391

its DNA is, most probably, not concerned with the parts controlling
protein structure (structural genes), but those that are responsible forsome kind of cellular regulation systems (regulator or operator genes).
Fig. I. Grow'rn WIVm< of Ol"lZl-\l-! under\'ariau~ OIr~inmil:

3.0

x

o
o

.E

1O-5 M
IO-oe M

D.

10-3~

"

of

\lOrlOU5

concc;ntrations

x NO URA a 10-6 M UAAolo-5M URAoD

IO-'lM URA

IO-3M URA
fA 10-2 M URA

D.

IO~2M

ARG conc.lo~3M-- --- ARG cone. la-2 1M....... ORA cone. WQ~

Eo

o

...

~rQcil3.0

NO &'RG
IO-G MebFig. 2.. Rec.overy 01 atlilinine jnhibilion by

conc.l2I'Itrntionl>"

~
~

2.0

...
>...
;;;

2Or

4

4

z

~

0
~

4
U

ii: 1.0

0

•
TIME

80.

;N

HRS.

TIME

G
IN

HRS

Comparative studies on mutagenic efficiencies of incorporated
1
32p and r-rays with Salmonella )(By Hiromi ISHIWA, Yonhon YAN21 and Sohei KONDO)

The mutation inducibility due to the decay of 32p incorporated in
Salmonella TL2 was compared with non-radioactive control Salmonella
irradiated by gamma-rays. The forward mutation was observed fromgal+ to gar colony plated on EMG-galactose complete agar plates(Lederberg), when the mutated colony showed pink or red color among
many green wild type colonies.
The mutation frequencies (y) for two different kinds of mutagens,
32p and gamm-rays, were plotted in Fig. 1 against survival fraction andlethal hits (x), respectively. As the figure shows, 32p induced mutation
11 This work was supported by Grant from the Toyo Rayon Foundation for the
Promotion of Science and Technology.
21 Department of Nuclear Science, Faculty of Science, Kyoto University, Kyoto.

92 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

frequency was rather lower than that caused by gamina-rays at thesame survival fraction. From our experimental results, transmutationof 32p atoms incorporated into the genetic substance seems to be little
effective in inducing non-lethal mutations. Hence the mutations detectedin this case may be mainly due to the internal as well as external effects
of 32p .a-rays. Many contradictory results against this conclusion werereported by Dr. KAUDEWITZ et al (1958).

F
'"' 0

o

x

o

o

5

4
/0- 1

/0-2

6

7

8

9

10-'

IO-~

.surviving fraction
Comparison of mutation inducibility by disintergation of 32p incorporated in
Salmonella with that of r-ray irradiation.

81. Chemical modification of nucleic acids and their constituents(By Tomotaka SHINODA)

In order to investigate the aromatization of ribonucleic acid by TNBS
and the specificity limitation of ribonuclease action towards the substrate,
the effect of this reagent upon the individual constituents of RNA wasdetermined. Under several conditions employed, the reaction with freebase occurred only with guanine, and the others were not reactive towards
the reagent. Up to the present, the following 12 derivatives of the constituents of RNA were obtained in monotrinitrophenyl form; adenosine,
methyl adenosine, O-acetyl adenosine, guanine, guanosine, O-acetyl
guanosine, cytidine, O-acetyl cytidine, adenylic acid and cytidylic acid,and in addition to these inosine derivative was also prepared. Funda·

RESEARCHES CARRIED OUT IN 196393

mental physicochemical properties of these materials were observed inultraviolet and infrared spectra and for electrophoretic and chromatographic behavior. A characteristic absorption of these derivatives in alkali
medium was seen at 410 to 430 mp, and in addition to that the absorption
at 260 mp which corresponds to that of bases. On the other hand absorption at 410 to 430 mp seen in the alkali disappeared in acid medium
while at 260 mp it was unchanged or several of the derivatives showed
an increase in absorption.
Preliminary studies of the effects of UV- and ionizing radiation on
the derivatives were carried out by ultraviolet spectrophotometry andpaper chromatography. In some cases liberation of picric acid was observed both in spectrophotometry and paper chromatography. Effects
of chemical agents such as iodoacetate, dimethylsulfate, nitrous acid andperiodate upon the trinitrophenylated derivatives of base were alsostudied.
purified yeast RNA was used in the reaction, and the base composition of the material followed by the treatment with alkali was analyzed
by paper chromatography. Based on the results, it has been determinedthat the reaction of RNA with the reagent occurred mainly in the sitesof purine moieties of RNA. Ultraviolet absorption spectrum of modified
RNA was very similar to that of modified guanosine, this observation
being in good agreement with the result from base composition of modified RNA. Relative velocities of the action of ribonuclease upon intact
and modified RNA were compared in the usual fashion, and a decreased
velocity was observed in the latter. This is assumed to be due to theresult of the reaction upon purine moiety of RNA.

E.

RADIATION GENETICS IN ANIMALS

82. Heterozygous effects of radiation-induced mutations on
viability in homozygous and heterozyous genetic
backgrounds in Drosophila melanogaster (II)*(By Terumi MUKAI, Isao YOSHIKAWA and Tsuneyuki YAMAZAKI)To confirm the experimental results concerning the heterozygous effectof radiation-induced mutations on viability presented in the Annual
Report No. 13, experiments were conducted in one homozygous and two* This work has been supported by Grant RH-34 from the Public Health Service, . U.S.A..

94 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

heterozygous (intra- and inter-populational hybrids) genetic backgroUnds
Two isogenic lines, A,A, (W160S) and A zA 2 , extracted from the sam'
population (W-1), and another isogenic line (BB) obtained from aeentirely unrelated population were prepared for the irradiation wit~
respect to the second chromosome. The X, the third and the fourthchromosomes of AzA z and BB were substituted by those of A,A,.
After X-ray irradiation at the dose of 500 r, (irradiation is indicatedby superscript (')), heterozygotes A,A/, A/ A z and A/ B were producedby the aid of Cy/Pm Ubx/Sb strains. The relative viabilities of irradiated heterozygotes with respect to the second chromosome were
estimated in comparison with CY/+L1 , (in A,A,'), CY/+A 2 (in A,'A z) andCY/+B (in A/B). The irradiated X and third chromosomes were Completely substituted by the unirradiated ones of A,A, before the estima_tion of viability. The control experiments were conducted in the same
way without irradiation. The results are presented in Table 1.
From Table 1, it can be seen that radiation-induced mutations which
occurred in homozygous individuals were heterozygously beneficial totheir carriers, i.e., the average viability of A,A,' was larger than thatof A,A, by 0.0264 and this value is significantly different from zero andconsistent with the previous result, while those that occurred in heterozygous genetic backgrounds, both in intra- and inter-populational hybrids
Table 1.

Heterozygous effects of radiation-induced mutations on
viability in Drosophila melanogaster.
(2nd chromosome, sperm, acute 500r)

No. of chromosome lines tested

Total no. offlies counted

Averageviability(irradiated)-(control)

292
291

222,446
227,788

1.01501.04040.0264**308
305

283,633
271,479

1.0322
1.04380.0116

A,'Az
A,B
A,'B

324
312

391,479
368,877

1.1474
1.1423-0.0051

Genotype

A,A,
A,A,'

A,Az

In A,A, and A,A,', viability of.!!.JL +L11= 1.0000.

In A,A z and A,' Az, viability ofCy+A, +A,+ A, = 1.0000.

+A 2 +A,
In A,B and A,'B, viability of .!!.JL +A' =1.0000.
+B +A,** significant at the 1 per cent level.

RESEARCHES CARRIED OUT IN 1963

id not rnanif~st thei~ ~ffects sign~ficantly.

95

.

.

<i After obtainIng addltlOnal expenmental results, the effect of lrradla. on populations and the relation between heterozygous manifestationn
tlO the radiatlOn-l~.' dta t'mns an d evalutlOnary'
h'Istory af t he m
. d'1,ofuced
mu"idual s will be dIscussed.

83. Radiation-induced mutation rate and doubling dose in polygenes
controlling viability in Drosophila melanogaster*(By Terumi MUKAI)

As described in another paper of the present report, we irradiatedthe sperms of D. melanogaster with X-rays at the dose pf 500r andtested the heterozygous effect of radiation-induced mutations in homozygous genetic background.
With the aid of variance analysis we have estimated the genetic variance caused by radiation-induced mutations as a~ (x) =0.001161. The increment of the mean viability was 0.03020 viability index.
Under the assumption that mutations are distributed on chromosomes:according to Poisson distribution, the following formula can be obtained:
22
B
0:<=:17 a :<=:4A 2(1)

ii:<=: ~ •.•..••••.•.••••••••.••••••••••.••••••••••. (2)2A
B:<=:P •..•••.••••••..••.•.•••.•••••.•••••••••...• (3)where A is the increment of viability mean, B is the genetic variancecaused by radiation-induced mutations, a is the heterozygous effect of asingle mutation whose mean and variance are ii and 17 a 2 , respectively,
and p indicates average number of radiation-induced mutations in thesecond chromosome.
Using Formulae (1), (2) and (3), the following estimates are obtained:
0:<=:17/:<=:0.000369
ii:<=:0.0384
0.7860:<=:pThese results indicate that by 500\ irradiation, at least 0.7860 mutations, on the average, occurred in the second chromosome. This result* This work has been supported by Grant RH-34 from the Public Health Service,
U.S.A..

96 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

is consistent with that obtained previously (Mukai 1961).
Doubling dose was estimated by employing the result of spontaneous
mutation rate estimated for the same experimental material as that for
the radiation experiment (O.1301/second chromosome/generation). The
result thus obtained is ;')2.8r.

84. Radiation-induced mutation rates of polygenes controllingthe sternopleural bristle number in Drosophilamelanogaster 1 )(By Terumi MUKAI, Isao YOSHIKAWA and Sadao CHIGUSA)

To obtain fundamental information on the genetic influence of radiation on human populations and on the estimation of spontaneous mutation rate, radiation-induced mutation rates of polygenes controlling thesternopleural bristle number in Drosophila melanogaster have been estimated.
An isogenic line extracted from a wild population of Erie, Pa. (U.S.A.)
was employed. The males of the isogenic line were irradiated with Xrays at 250r and 500r. Immediately after irradiation, the irradiatedmales were crossed to the females of the same line in half pint milkbottles. The number of sternopleural bristles in females and males.
which had hatched on or before the 13th day after the mating werescored and the means and the variances were calculated to test theheterozygous effects of radiation-induced mutations. The results are
summarized in Table 1.
Table 1.

Means and variances of sternopleural bristle numbers.
Control
\?

250r
1)

\?

500r
1)

I

?-

1)
---

--

Mean

17.0389

16.4057

17.0204

16.3750

17.0834

16.4252

Variance

1.42701.41061.47901.3253*1.5548**1.3309*

6497

5411

5983

5313

4306

----

No. of irradiated genomes

4827
1

I

* significantly different from the control at the 5% level.
** significantly different from the control at the 1% level.
1} This work has been
supported by Grant GM-7836 from the Public Healthl
Service, U.S.A..

RESEARCHES CARRIED OUT IN 1963

97

The variances of the irradiated groups have approximately linearly inrea sed with the dose in females (the rate of variance increase: 2.32x~O-4/r), but have decreased significantly in males as compared with that of
the controls. This difference might be attributed to some change in theheterochromatic parts in Y-chromosomes. The means of sternopleuralbristle numbers did not change, either in males or females. Thus, it
way be suggested that heterozygously positive and negative mutations,occur in equal frequencies and amounts. This might indicate that thetrait in question is not strongly correlated with fitness.

85.

Further report on the increase in induced J1'zutation
frequency after fractionated irradiation of gonial
cells of the silkworm(By Yataro TAZIMA and Kimiharu ONIMARU)

A marked increase in induced mutation frequency was revealed forboth spermatogonia and oogonia when an X -ray dose of lOOOr was given
in two or three fractions separated by 24 or 48 hour interval (Tazima
and Murakami, 1962, Ann. Rept. No. 13). This finding led us to examination, in a new experiment, of the most effective period for thesecond exposure and the length of duration during which the enhancing
effect is observed.
Total dose of 1000r r-rays was divided into two halves. 500r wasadministered to the larvae at the 7th day after hatching and the remaining 500r was given at several intervals, i.e., 2, 12, 24, 36 and 48
hours after the first exposure. The radiation dose-rate was 60r/min.
For the estimation of mutation frequency the specific locus method forpe and re loci was used as usual.
The results are shown in Fig. 1.
It can be seen from Fig. 1 that fractionated irradiation enhances
mutation frequency throughout the whole period examined, Le., from 2
to 48 hours after the first exposure. The mutation frequencies showed
their peak at 12 hours for spermatogonia and at 24 hours for oogonia,
They were 4-8 times as high as those from single exposure, surpassing
the level of average mutation frequencies obtained from chronic irradiation.
That mutation frequencies never fell below a single acute exposurelevel does not support the view that enhanced mutation frequencies
were due to synchronization of the cell cycle,but indicates that sensitive cells were not killed but survived in a highly mutable state during
a definite period of post-irradiation.

98 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

level.......

,o.~'~~~~.~"~~<f?"'~..0...... '-..

<:>-....~----() ohronic level...

~~ •• ~ ••••

chronic level~ _------------::--:.~-~~,•,1"ohronic level ----0---------------lJo-------------0--------~

o 2

12

24
intervals in hr.

36

48

Fig. 1. Variation in induced mutation frequency with time interval between
two exposures. Solid line represents the changes in mutation frequency
for +pe locus and broken line those for +re locus.

This argument, however, does not necessarily rule out the selective
cell-killing hypothesis. Because, even when sensitive cells are killed
off, surviving resistant cells could react to fractionated irradiation in asimilar manner as do the sensitive cells and give rise to analogous
results.

86. Independence of induced mutation-rate from radiation
dose-rate in the germ cells of hibernating
silkworm embryo(By Yataro T AZIMA and Kimiharu ONIMARU)

As a possible interpretation for the reverse type of dose-rate dependence of radiation-induced mutation rate in the silkworm gonia, thehypothesis of selective cell-killing was proposed by Tazima, Kondo andSado (1961); namely, they assumed that sensitive cells at an advanced
stage are killed by acute irradiation, whereas cells in dormant state
can survive the irradiation and achieve repopulation afterwards, resulting in lower mutation frequencies of resistant cells.
According to this hypothesis, irradiation of gonads consisting of homo-

RESEARCHES CARRIED OUT IN 196399

geneOUS dormant germ cells, if existed, should give rise to low mutation frequencies or at least equal to those for acutely irradiated gonads
at the reverse type (type II) stage, regardless of dose-rate. Germ cellsof hibernating embryos could be regarded as an nearly ideal model ofsuch a homogeneous cell population.
Therefore, silkworm eggs, which had passed through hibernation but
were still preserved in a refrigerator at 2.5°e, were chosen as thematerial for this experiment. Twenty batches of a wild type strain,
Cl08, were divided each into two halves. One group was exposed tc
1000r r-rays for one minute (acute), while the other group was irradiated chronically with 1000r in total during 10 days (chronic). In bottcases irradiation was carried out at 2°e. After irradiation, eggs of bottgroupS were stored again in the refrigerator at 2.5°e for nearly ommonth until incubation. For the estimation of induced mutation frequency, specific loci method was used.
The results are given in Table 1.
Table. 1.

Induced mutation frequencies obtained after r-irradiation ofhibernating eggs at different dose-rates.
female germ cells

male germ cells
Treatment

Dose-rate

Acute*
Chronic*** 137Cs-r-rays.

1000r/min
0.0695r/min

Mut. freq.

Mut. freq.
No. obsd.

128,180
139,340

No. obsd.

perex10- 462.6
64.2

x10- 4
21.2
25.8

pe
32,40048,600rex10- 4 xlO31.3
4.
21.5 12.

** 60Co-r·rays.

The results were in complete contradiction to our expectation; nam(ly, induced mutation frequencies were surprisingly high in both acutand chronic groups. In some F 1 batches mutation occurred in fair!
large clusters. Furthermore, no significant difference in mutation fn
quency was observed between the two groups.
These results seem to indicate that germ cells of hibernating embryc
are not similar to suposedly dormant germ cells in growing gonads ,young larval stage, being far more mutable in the embryos. Howeve
before drawing a definite conclusion, it must be taken into consideraticthat the observed high frequencies might not be genuine, but could hal
been exaggerated to a considerable extent by cold storage as a resulof disturbances in the repair process of premutational damages. 1

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

answer this question, further experiments are in progress.

87. Enhancement of radiation induced mutation frequencv bypost-treatment of silkworm gonia with
5-bromodeoxyuridine(By Yataro TAZIMA and Akio MURAKAMI)

As reported previously (Ann. Rept. Nat. lnst. Genet. No. 13), 5- bromo.
uracil (BD) or 5-bromodeoxyuridine (BDdR), analogues to the normalbase thymine or normal nucleoside thymidine of DNA, enhance radiation.
induced mutation frequency to an appreciable extent, when administeredafter X-irradiation. As to the cause of this phenomenon, it was specul.
ated that BD is incorporated into X-rayed DNA in the place of thymine
and gives rise to a copying error during its replication. Whether the
speculation holds true or not, has been examined as follows.
1) Eexperiments to test whether post-treatment with thymidine increases
radiation induced mutation frequency.
Following a similar procedure to that used for BUdR, the effect ofthymidine supplementation was studied. The chemical was administer.
ed to young larvae of a wild type strain by feeding them on leaves
supplemented with thymidine (16 mg per 1500 larvae) for about one day
after r-irradiation.
Thus far, the experiment has been repeated four times at different
stages, from hatching day to the second instar and more than four mil·lion F 1 eggs have been examined. No significant increase, however, hasbeen confirmed. Hence, it may be concluded that thymidine, even inexcess, does not increase radiation induced mutation frequency.
2) Simultaneous feeding of BUdR and thymidine immediately after rray irradiation.
If an enhancement were due to replacement of thymine by BU, thenit could be anticipated that the chance of its replacement is decreased
by simultaneous administration of BUdR and thymidine. This would
give rise to the decrease or disappearance of enhancing effect. In thisrespect, experiments have been conducted with silkworm gonia. Bothchemicals were administered to young silkworm larvae in the moleular
ratio of 1 : 1 (20 mg BUdR: 16 mg thymidine) and 1: 2 (20 mg BUdR:
32 mg thymidine). Two series of experiments, one at first instar day 2
and the other at second instar day 1, have been performed. The r-ray
doses were 500r for the former and 1000r for the latter. The method
used for the estimation of mutation frequency was similar to that previously used.

RESEARCHES CARRIED OUT IN 1963

101

The results are, however, not conclusive, showing no definite increase
decrease in mutation frequencies among similarly treated groups.
orThus, it is doubtful in the silkworm whether the replacement of thymine by BU induces mutation or not.
88. Relative biological effectiveness of fast neutrons in the
induction of recessive visible mutations in the silkworm(I)

Effect of 14.1 MeV neutrons upon the J5onia*(By Akio MURAKAMI, Sohei KONDO and Yatara TAZIMA)

Effectiveness (RBE) of 14.1 MeV neutrons from T(d, n) He reaction has
been compaired with that of mCs-gamma rays in the induction of visible
recessive mutations upon gonia.
It has been reported by Tazima et al. (1961) that goniaI cells of thesilkworm respond quite differently to low LET radiations of high or low
dose-rate at early and late developmental stages. In order to examine
whether the same manner of response is observed for high LET radiations, this experiment has been undertaken
The exposure to 14.1 MeV neutrons was carried out at the ResearchInstitute of Nuclear Medicine and Biology, Hiroshima University, bycourtesy of Professor Haruma Yoshinaga. Young larvae of a wild typestrain, C10S, were exposed to fast neutron beams for 114 minutes byplacing them in aery 1 tubes at several distances from 3.5 cm to 6.0
after hatching (abbreviated to early) and on the 7th day after hatching(abbreviated to late). Radiation dose-rate ranged from 6.7 rad to 19.6
rad per minute. The dose calculation was made by following the firstcollision dose method developed by Randolph, based on the analytical
data of elemental composition. For the estimation of mutation frequency we applied the specific loci method with egg colour markers.
Mutation frequencies obtained for each locus were plotted against thedose of 14.1 MeV neutrons for spermatogonia and oogonia irradiated atearly or late stages.
For the comparison of effectiveness, dose-response curves for mutation
by l37Cs-gamma rays were drawn from old (Tazima and Kondo, 1963)
and new data.
Those curves are shown in Figure 1 for spermatogonia and 06gonia. In
oogonia mutation frequencies are distinctly higher in groups irradiated at
late stage than in those treated at early stage, throughout the whole dose

* This work was supported by Grant from Toyo Rayon Foundation for the
Promotion of Science and Technology.

102 ANNUAL RERORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

range examined. The results are in sharp contrast to those obtained
after acute gamma-ray exposure, where induced mutation frequencies
were higher at early stage. The phenomenon may presumably be due
to dose-rate effect of neutrons. It may be noted that mutation frequencies from fast neutrons in the present experiment are clearly higher
than those from chronic gamma irradiation, irrespective of the stage at
irradiation.
x10- 5
1500

pe

Laie

2500

o
Dose ill rad

Spermatogoniarex 10- 5

1000

I

r

I
I

I
I

u

I

""g.

I
I
I

...."

I

~

"o

~

pe

I

>,

I

I
I

500

~

."

"

::I

o

200

400

GOO

800

1000

Dose in rad

Oiigonia
Fig. 1. Dose-mutation frequency curves obtained after 14.1 MeV neutron
irradiation at early and late gonial stages.

RESEARCHES CARRIED OUT IN 1963

103

Since the dose-irequency curves do not follow linear relationship, it ishardly possible to make exact comparison of effectiveness between thetWO kinds of radiation for the whole dose-range. However, at an arbitrary level of mutation frequency, estimation is possible. The values
estimated, for example, at the level of 100x 10- 5 mutation frequency
are as follows.
At early stage RBE's of 14.1 MeV neutrons are 1.0 and 0.8 for spermatogonia and 1.2 and 1.2 for oogonia, while at late stage they are
1.8 and 2.1 for spermatogonia and 1.1 and 1.3 for oogonia at pe and relocus, respectively. Thus, RBE values of 14.1 MeV fast neutrons arehigher in late than in early stage at the level of 100 x 10- 5 mutation
frequency. Though the values are fairly small at the lower level ofmutation frequency, they become larger at higher mutation frequency
levels. At still higher dose levels, however, RBE values decrease again.

89. Relative biological effectiveness of fast neutrons in the
induction of recessive visible mutationsin the silkworm(II)

Effect of fission neutrons upon the gonia I )(By Akio MURAKAMI, Sohei KONDO and Yataro TAZIMA)

By the courtesy of AEC of United States and Dr. l.A. Auxier of
Oak Ridge National Laboratory, an irradiation experiment of silkworm
gonia with 1.5 MeV 235U-fission spectrum fast neutrons has been conducted at Oak Ridge National Laboratory. The health physics research
reactor (HPRR) was used. The neutrons generated from the reactor
comprise 55 per cent neutrons within energy range from 0.1 to 1.5 MeV,
38 per cent within energy range from 1.5 to 2.5 MeV and 7.0 per centwithin energy range from 2.5 to 6 MeV, the representative energy level
being 1.5 MeV. The mean linear energy transfer (LET) is estimatedas ca. 50 keV/p.
The biological material and method are similar to those used for 14.1
MeV fast neutron experiments. The young larvae were placed at 100cm
distance from the core of the reactor. Neutron dose-rates ranged from
229 to 259 rad per minute.
The results are given in Figure 1a and b. The mutation-response
Curves for 1.5 MeV fast neutrons rose more rapidly with the dose than1) This work was supported by Grant
from Toyo Rayon Foundation for the
Promotion of Science and Technology.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Jar 14.1 MeV neutrons. The relation, however, can be regarded approximately as linear under 100x 10- 5 mutation frequency level. At thislevel REE values of 1.5 MeV neutrons at early stage are 1.7 and 1.9 forspermatogonia and 2.1 and 2.4 for oogonia, while at late stage they
are 2.8 and 3.0 for spermatogonia and 2.5 and 3.0 for oogonia at pe andre locus, respectively. Namely, the REE values of 1.5 MeV neutrons ingonial cells are higher at late than at early stage at the 100x 10- 5 mutation frequency level. A similar tendency has also been observed for14.1 MeV neutrons although the values were smaller (first report of thisseries). Combining both data, it may be pointed out that REE frominduction of visible recessive mutation increases with increasing LET
within the energy range examined (Figure 2).
It has been known that in Drosophila the REE values for fast neutrons
in the induction of sex-linked recessive lethal mutations are about 1 but
they are 4-6 times as high in the induction of dominant lethals.
The REE values obtained in the present experiment are clearly higher
than those expected from visible recessive mutation on Drosophila, indicating that the recovered mutations comprise not only recessive visible
mutations, but also small deficiencies and some kinds of gross structualchanges.
This conclusion is also supported by the finding that, in most cases,
dose-response curves do not follow linear relationship, but are rather,exponential.

1000oSpermatogonia

RESEARCHES CARRIED OUT IN 1963

xlO- 5
20UO

""

1500

~

ID

':"r

"
~

'"~ 1000

.-0

."
~

."

'"

::II

500·

o
Dose in rmd

06goniaFig. 1. Dose··mutation frequency curves obtained after fission neutron
irradiation at early and late gonial stages.

3o'------'--~---'"----d-_--...L.

o_ __.:.I_ _ _ l _

20
30
40
50
60
LET
keV/flFig. 2. Relation between RBE and LET in the induction of recessive·
mutation in silkworm gonia.
@ indicates the early stage gonia.
indicates the late stage gonia.

o

10

10&

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO, 14

90.

Relative biological effectiveness of 14.1 Me V neutrons inkilling dormant silkworm eggs l )(By Akio MURAKAMI and Sohei KONDO)

Although killing effects of low ion density radiations, such as X- or
gamma-rays, upon silkworm eggs have been studied by many investigators, scarcely anything is known about the effects of high ion density
radiations. Our study of RBE in mutation induction has yielded muchinformation in this respect. The need of examination of RBE of highenergy neutrons for dormant silkworm eggs came from another COnsideration.
In this experiment biological effectiveness of 14.1 MeV neutrons has
been compared to that of gamma-radiations concerning their killing effects
on silkworm eggs at dormancy.

100

50
~

"
Cl)

'"

;.;

Cl)

P<

'"'">,

10

+'
'''';

,...;
'''';

,0

'"

~

'"

~

'"

IJ:I

1

0

1000

2000

3000

4000

5000

Dose in rad
Fig. 1. Hatchability curves obtained after irradiation of dormant silkworm
eggs with gamma-rays and 14.1 MeV neutrons.
I) This work was supported
by Grant from Toyo Rayon Foundation for the
Promotion of Science and Technology.

RESEARCHES CARRIED OUT IN 1963

107

The exposure to 14.1 MeV neutrons was applied at the Biology
Division, Oak Ridge National Laboratory, with use of Cockcroft-Walton
accelerator. Dormant silkworm eggs were exposed to neutron beams at
a distance of 6.3 em from the tritium target for a variable length ofduration, from 60 to 180 minutes. The neutron dose-rate was 5 rad per
minute and total doses given were 500, 1000 and 1500 rad. In parallelto this, l31Cs-gamma ray treatment was carried out at our Institute withthe same material. Gamma-ray doses were 500, 1000, 3000 and 5000r at adose-rate of 100r per minute.
The percentages of hatchability were plotted against doses given on
semilogarithmic scale both for fast neutrons and gamma-rays (Figure 1).
The hatchability curve for 14.1 MeV neutrons declines more rapidly
than that for l37Cs-gamma rays. Calculated doses that allow 50 per centhatchability (LD 50 ) are 600 rads and 2000 rads for 14.1 MeV neutrons and
137Cs-gamma rays, respectively. That is to say 14.1 MeV neutrons are
more effective than l37Cs-gamma radiations with regard to their killing
action. RBE thus calculated is 3.3.

91.

Mutations induced by ingested 32p in silkworm l )(By Mituo IKENAGA and Sohei KONDO)

The mutagenic effect of decay of 32p incorporated into the genetic
substance could be due either to the effects of radiation accompanying
radioactive decay or to some noxious events caused by nuclear transmutation of 32p atom, such as chemical valence change (STRAUSS: 1958,
STENT and FUERST: 1960). In order to elucidate the relative contributionof p-electron and transmutation effect to induced mutation, the mutagenic effectiveness of 32p ingested by the silkworm on the third dayof fourth instar was compared with that of corresponding doses of rirradiation from l37Cs, using egg color mutants, pe and reo Since 32p
p-ray dose absorbed by the gonads is strictly proportional to the specificradioactivity of the silkworm, it was calculated in the present experiments by measuring the activity of all the pupae at middle pupal stage.
The results obtained in both series, 32P-treatment and r-irradiation, areshown in Fig. 1. The mutants scored for mutation frequency are thetotal of pe, re and mosaic-reo As clearly seen from the graph, themutation frequency induced by 32p in males is quite the same as thatinduced by the corresponding dose of r-rays, while 32P-treatment inI) This work was supported by Grant from the Toyo Rayon Foundation for the
Promortion of Science and Technolegy.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14(a) Maleo 32 p _ trea tmen t~

r-rays irradiation....+'§ 50
oJ

+'

~

F

.oP .........

..-....-

O'--_ _....!.I_=_~=~~bl=~_ _=,""'1=

o

500

1000

1500

_ _.J.I~_~..l

2000

2500

Radioactivity of pupa (cpm)Fig. 1. Dose response curve for 32P-treatment and r-irradiation in silkworm:
irradiation with 20r(day r-rays corresponds to 500 cpm for male and 590 cpm
for female pupae.

females induces mutations more efficiently than r-irradiation. In otherwords, there seems to be observed a transmutation effect on induced
mutation for female silkworm, but not for males. The observed results
and the methods of calculating absorbed dose of 32p will be publishedin detail elsewhere together with a discussion.

92. Mutagenic effects of ethyl methane sulfonate on silkworm(By Sohei KONDO and Mituo IKENAGA)

It has been shown that ethyl methane sulfonate (EMS) induces mutations in lower organisms (LOVELESS: 1958) and in maize (NEUFFER andFICSOR: 1963). To interpret the mechanism of its mutagenic action it is
supposed that this alkylating agent can ethylate the guanine moity in

RESEARCHES CARRIED OUT IN 1963

109

position 7, thereby causing guanine to pair with thymine rather thanCytosine during DNA duplications. However, EMS mutagenicity inhigher animals was not yet reported. It was examined in the present
ex:periment using the egg color mutant pe, re of the silkworm. For
the estimation of mutation frequency the abdomen of both sexes of awild strain was injected with 0.2 ml of EMS solution in late pupal
stages (4 days before emergence) and then the insects were mated withthe marker strain. The results obtained are given in Table 1. The
Table L-a.

pe re '? X ++ 13 (treated)

Concentrationof EMS(Mol.)

Total
no. ofeggs
observed-

34,1008.0X10- 5 28,657
8.0X10- 4 16,806
8.0X 10- 3 15,507
Control

b.

pe
Whole

re
Fractional

Whole

FractionalI

No. of Freq. No. of Freq. No. of Freq. No. of Freq.
detd. 11 (X 10- 5) detd. (X 10- 5) detd. (xlO-5) detd. (xlO-5)

3
4
3
1

8.80
14.0
17.9
6.45

1
0
32
83

2.93
190,
535

5
6
4
21

16.0
44.3
30.9
397

-

I

0
0
1
0

5.95

-

0
0
66
206

393
13"28

0
1
2
19

7.39
15.5
539

-

++ Q (treated) X pe re 13

Control

8.0X10- 5
8.0X10- 4
8.0X10- s
1)

The frequency of egg color mutants induced byinjection with EMS at pupal stage.

31,20013,54012,939
5,287

3
1
3
1

9.62
7.39
23.2
18.9

0
3
2
1

22.2
15.5
18.9

-

detected.

table clearly shows that EMS was effective. A remarkable difference
between the relative frequencies of whole- and fractional-mutations wasobserved at high concentration of the chemicals (bottom rows for both
sexes), and in most of the fractional-mutants a quarter of the egg orless is involved.
This abnormality of mutagenic action of EMS may be interpreted asfollows; (1) EMS acts on dominant genes only during the developmental
stages after fertilization. (2) The appearance of the mutation is somewhat delayed, since its stabilization requires one or two cycles of DNA
duplication. (3) Most of the mutations are changes induced in one of
the two strands of DNA molecule.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

93. Radiation response of actin synthesis in regenerating
tissue of Triturus*(By Yoshito OGAWA)

Synthesis of muscle proteins with respect to X-irradiation at various
points of embryonal development of Triturus pyrrhogaster has beenreported beforel). From the same view point, the effect of X-ray irradiation on the synthesis of the contractile protein, actin, in the regenerating hind limb tissue of adult Triturus amputated at the knee
was examined in detail and the findings are here reported. They may
be helpful in the approach to the mechanism of chemo-differentiation ofmuscle tissue.
About 1,500 animals at different stages in the recovery progress
after operation were exposed to single doses of X-rays with 20r., 50r.,
200r., 500r., 1,000r. and 2,500r., and the examination for the first detectable trace of actin in regenerating limb tissue was carried out 3, 9, 15,
18, 19, 20, and 22 days after operation. The technique employed wasessentially the same as in the experiments with early embryonal stages2 ).
Pure G-actin was isolated from the skeletal muscle of adult Triturus,
and the anti-serum was prepared by injecting it into rabbits. Before
carrying out the precipitin reaction with saline extracts of granulated
tissue of the amputated limb, the titer of anti-serum was adjusted to
1:512. In non-irradiated group, actin first became detectable in theregenerating tissue 20 days after operation21 • The time of actin formation after operation in the X-irradiated group is represented in Fig. 1.
When a dose of 20r. is given 3 days after amputation, the time of actin
formation changes to the 18th day after operation. In the case of irradiation 19 days after operation with a heavy dose of 2,500r., thesynthesis of actin was remarkably inhibited and the time of actin
formation changed to the 30th day after amputation.
The curves in. Fig. 1 show a similar response of actin formation. to
X-ray as in. embryonal stages. These results may suggest the presenceof two factors sensitive to X-rays in actin synthesis during tissue regeneration as well as in early development. One (R-Aa) suppresses thesynthesis of actin and the other (R-Ba) markedly promotes it after Xirradiation. The inhibiting factor R-Aa is represented by the points
found on the right of the vertical control line (Fig. 1, 0) and the promoting factor R-Ba by the points on the left (Fig. 1, ~). Factor R-Aa
does not react to X-rays until doses over 500r. are applied, but factor* This work was supported by a Grant-in-Aid for Fundamental Scientific Research (No. 710273) of the Ministry of Education in Japan.

RESEARCHES CARRIED OUT IN 1963

22

111

c

0

<-'

,..ro

20
19
J3

~J'OOor

~

:;~

<-'

~

ro

~

15

"

2,500r

/

,..

:;

'"

0

o Resl.i.l ts of
R-·Aa ac t i on

<-'

.;:;
-0

~

"

e Results of
R-Ba action

<-'

~

'"
~

'"

3

l,OOOr

L-

200r

500r
2,500r~4

8

12~ _ . l_ . _

20
Z4
16
Days after action formation

(Days
____=
28

after operation)
32

Fig. 1. Synthesis of actin in regenerating limb tissue of Triturus irradiated by
X-rays with doses from 20 r to 2,500 r.

R-Ba is very sensitive to X-rays and is easily detectable with only 20r.
dose. The factors R-Aa and R-Ba are detectable, respectively, at irradiations between 18th and 19th day and between 3rd and 15th day
after operation. The most effective time of irradiation is the 19th day
after amputation for factor R-Aa and the 3rd day for factor R-Ba under
the above experimental condirtons.
1)
2)

Y. Ogawa:
Y. Ogawa:

94.

B. B. A. 54: 397, 1961.
Exptl Cell Research 26: 269, 1962.

Effects of L-gulonolactone and glucose on the synthestsof the skeletal muscle protein, myosin, in
X-irradiated Triturus embryos*(By Yoshito OGAWA)

Previously, the effect was reported of sodium-glucuronate on abnormal
differentiation caused by X-rays of muscle proteins during early em-* This work was supported by a Grant-in-Aid from Tokyo Biochemical Research
Fundation.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

bryonal development of Triturus pyrrhogaster 11 • This paper deals withthe effects of L-gulonolactone and glucose on the synthesis of myosinin X-irradiated material of the same animal. These experiments ate
partly carried out in order to investigate the protective action of
glucuronic acid and its derivatives on abnormal differentiation in early
embryos induced by X-rays.
Pure myosin was isolated from skeletal muscle tissue of adult Triturusand the anti-serum was prepared by injecting it intravenously into
rabbits. Immediately after fertilization, the embryos were raised in
0.01% solution of L-gulonolactone (most effective concentration for promoting the synthesis of myosin zl ) or glucose, at 18°C., and total body
X-irradiation of embryos with 50r., 200r. and 500r. was performed 108
hrs. after fertilization. After irradiation, the treatment with L-gulonolactone or glucose was continued and the analysis of myosin was immediately carried out. Embryos from 108 to 204 hrs. after fertilization
were mashed in ice cold potassium chlorid solution (0.6 M). The homogenate was allowed to stand for one hour at O°C. under constant stirring and then was centrifuged at 2,OOOg. for 30 minutes. Before carrying out the precipitin reaction with the anti-serum, the clear supernatant
was placed in a collodium membrane tube and treated with large amountof distilled water for 48 hrs. at O°C. to eliminate the potassium chloride.
Constant volume of anti-serum adjusting the titer to 1:512 and progressively decreasing amounts of antigen, ranging from 1:10 to 1:160 inembryo weight, were used for preventing a possible failure due to excess
of antigen 1 • 31 • Non-treated and irradiated, treated and non-irradiatedand normal (non-treated and non-irradiated) control groups were prepared.
In normal embryos, myosin became at first detectable 176 hrs. after
fertilization 31 • In the present experiment, myosin formation in thenormal group was first recognized 180 hrs. after fertilization, since theexamination was carried out in periods of 12 hrs. In the case of nontreated and irradiated material, the time of myosin formation after
fertilization was changed to 156 hrs at 50r., to 144 hrs. at 200r. and 132
hrs. at 500r. 41 • Moreover, the production of synthesized myosin was·markedly promoted, and the acceleration was propotional to X-ray
dosage (Figs. 1 and 2).
In the non-irradiated but treated with L-gulonolactone embryos,
myosin appears 132 hrs. after fertilization. The development of this.
protein, therefore, was acclerated by 48 hrs. as compared with thecontrol. But no significant difference was found in the amount of thesynthesized protein (Fig. 1). In the irradiated and treated with Lgulonocatone group, the tendency to increasing the rate of formation as

RESEARCHES CARRIED OUT IN 1963

113

Lo.... est nt igenconcent ation
giving r sitive
resu ts

192

8 fterfert il iza.t ion

lrrediated08'Non-irradiated .con.tr.ol

Fig. 1.

Effect of L-gulonolactone on the synthesis of myosin in X-irradiatedTriturus embryos.

SCOr

Non-irradiated control

Fig. 2. Effect of glucose on the synthesis of myosin in X-irradiated Trituru8
embryos.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

well as amount of myosin was remarkably strong when irradiated with50r. and 200r. However, a heavy dose of 500r. showed no influence on
the rate of myosin formation and suppressed its amount (Fig. 1). These
results show the same tendency as found in the experiment with sodium_
glucuronate 1l •In the non-irradiated but treated with glucose group, the rate ofmyosin formation was not influenced as compared with the control, butsome reduction of the amount was recognized (Fig. 2). In the case ofirradiated and treated with glucose material, the promotion of the rateof myosin formation by X-rays was accelerated by about 24 hrs. irrespective of the dose of X-ray, though no remarkable effect was foundon the amount (Fig. 2).
Thus, it was found that L-gulonolactone as well as sodium-glucuronateshows a significant protective effect on the development of myosin inembryos irradiated with light X-ray doses (under 200L) and the effects
of both on myosin synthesis are clearly different from that caused by
glucose.
1) Y. Ogawa:2) Y. Ogawa:3) Y. Ogawa:4) Y. Ogawa:

Reports of 7th. Symp. on Glucuronic Acid 128, 1961.
Med. and BioI. 66: 216, 1963.
Exptl. Cell Research 26: 269, 1962.
Nature 186: 77, 1960.

95. Measurements of thymine dimers formed in ultraviolet
irradiated bacteria and its related problem
to photoreactivation(By Mituo IKENAGA and Sohei KONDO)

It is well known that ultraviolet radiation prevents colony formationin microorganisms. A reasonable explanation is that a large part ofthe inactivation of cells, after ultraviolet irradiation, results from theformation of thymine dimers in DNA polynucleotide chains. It is also
known that the effects of ultraviolet irradiation on colony formation may
be reversed by the action of visible light which splits thymine dimer
into monomers by some enzymatic process (see recent reviews by
RUPERT: 1963, WACKER: 1963).
This phenomenon, photoreactivation, would
be expected to occur when the visible light is administered at the sametime as the ultraviolet treatment, unless long lag periods were involved.
For instance in the preliminary experiment with E. coli 15T-A-U-,
complete loss of photoreactivability was observed when the cells were
held for 60 min after inactivation in a half-synthetic medium at 37°C.

RESEARCHES CARRIED OUT IN 1963

115

The purpose of the present study was to obtain data accounting for the1055 of photoreactivability.

E. coli 15 T - A - U - was grown in a half-synthetic medium in presenceof 2 p.g thymine-2-C 4 /ml. Ultraviolet irradiation with 160 ergs/mm 2 wascarried out at 254 mp. during the logarithmic growth of bacteria insynchrony. After a few different kinds of treatment (Table 1) bacterial
suspensions were sedimented, dried and hydrolysed in 70% perchloric
acid for 60 min at ::l0°e. The thymines and thymine dimers of the digest were separated by paper chromatography (86:14 n-butanol: H 2 0).
The radioactive regions were cut out and counted by a gas flow counter.
The experimental results are shown in Table 1. Thymine dimers wereTable 1.

The radioactivity of C1Lthymine dimers in irradiated E. coli
15 T- A - U- with various post-treatments.
C14 activity indimer region (cpm)*

Surviving
fraction (%)

No ultraviolet irradiation

-**

100

Irradiated only
Irradiated and photoreactivated***
Irradiated and grown 60 min at 37°C in
the darkness

64.2
28.2

54

10.2

9.1

8.1

* Sections of chromatograms containing dimers were cut into 1 cm strips,
counted by windowless 2", gas flow counter.
** Apparent thymine dimer activity was observed with unirradiated bacteria.
The values given in the middle column were subtracted according to thisapparent activity.
*** Photoreactivation was carried out for 15 min at 37°C, with illumination froma 500 watt flood lamp at distance of 3 cm from the sample tube.

most frequent in irradiated cells without further treatments. Based on
the obtained activity, 64.2 cpm, we were able to calculate the numberof thymine dimers per lethal hit to be about 250 dimers/cell/hit. For
the photoreactivation exposure the number of thymine dimers waschanged by a factor of about 0.5, which is reflected in the increasing
number of surviving cells. A considerable decrease in thymine dimers
was observed when irradiated cells were allowed to grow for 60 min,when the complete loss of photoreactivality could be seen. Thus theloss of photoreactivability may be due to the decrease in photoreactivable damages, i.e., in the number of thymine dimers, in spite of thefact that the surviving fraction at the time remains unchanged. A

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

possible molecular mechanism for the observed results is under Consideration.

F.

RADIATION GENETICS IN PLANTS

96. Induced mutation and pleiotropy of genes responsiblefor quantitative characters in rice
(By Kan-Ichi SAKAI and Akio SUZUKI)

Two groups of lines derived from the same variety of cultivated rice,
but different in one having been X-rayed in 1959 while the other wasleft untreated, were comparatively investigated on an individual plantbasis in respect of heading date, plant height, plant weight, tiller
number, panicle number, length of the biggest panicle and grain yield.
Comparison of genetic variance between the two has indicated that Xray treatment succeeded in inducing mutations in those quantitative
characters. The X-induced mutations were unidirectional being mostly in minus direction. Irrespective of the increase in genetic variationin X 4 lines, the error variance measured by block x line interaction
appeared to be not different between X 4 and control lines.
By subtracting genetic covariance of the control population fromthat of the X-rayed group, genetic correlation due to pleiotropic effectof genes was estimated. The underlying principle for this estimation
is as follows: If we assume (1) that mutation at one locus does notaffect mutation in other loci which are located in the same or otherchromosomes, (2) that different polygenes are evenly scattered over all
chromosomes rather than forming assortative groups per chromosome,and (3) that X-ray induced deficiency of chromosome segments, if any,
is deleterious for survival in rice and accordingly non-existent in the X.
lines, then the genetic correlation in the X. population will involve
extra correlation due to pleiotropic effect of newly mutated genes in
addition to the effects of selection, gene linkage and pleiotropy involvedin the genetic correlation of the non-irradiated population.
It was found that panicle length and number of panicles per plant
were for the most part controlled by inversely operating pleiotropy of
the genes, the pG, that is the parameter of genetic correlation due
solely to pleiotropic effect of genes being -0.91. Details of this study
will be published in due time in Radiation Botany.

RESEARCHES CARRIED OUT IN 1963

117

97. Relation of radiation effects to dose rates ofgamma-rays in diploid wheat(By Seiji MATSUMURA and Tomoo MABUCHI)

In order to investigate the relation of radiation effects to dose rates,
dry seeds of Triticum monococcum flavescens were irradiated by gamma. rays at 4,....,15 kr. For acute and chronic irradiation the dose rates,
10,000 r/hr with 137Cs and 35.6 r/hr with 60Co, were used, respectively.
Chronic irradiation was slightly more effective in inhibiting seedling
growth than the acute treatment applied at the beginning of the chronic,
and was clearly less effective than that applied at the end of the latter.
Also, acute irradiation showed clearly a higher chlorophyll mutation
rate than the chronic did, which were terminated just before sowing.
The later was the irradiation at the same dose, the more pronounced
was the inhibition of seedling growth and the increase in chlorophyllmutations, especially at acute irradiation. Thus, almost no intensification of radiation damage due to storage effects was found, as alreadyreported in Ann. Rep. No. 13: 97-99.
A series of similar experiments with the same material were initiatedto verify the earlier studies. Dry seeds were exposed to gamma-rays
at the dose rates, 10,000 r/hr with 137Cs and 26 r/hr with 60Co, respectively. In general chronic irradiation was more effective inhibiting
seedling growth and increasing chlorophyll mutations than the acutetreatment, applied at the end of the chronic, especially at high dose (15kr). It is supposed, against the earlier studies, that a slight intensification of radiation damage due to storage effects was involved.

98.

Relation of radiation effects to dose rates of
X- and gamma-rays in paddy rice
(By Seiji MATSUMURA and Tomoo MABUCHI)

Dry seeds of Oryza sativa (Norin 8) were exposed to X- and gammarays at 5,....,20 kr. For acute and chronic irradiation the dose rates,
10,000 r/hr with X-rays (173 kVp, 25mA, 0.5mm Al+0.5mm Cu filter)or 137Cs and 20 r/hr with 60Co, were used, respectively, as shown in the
diagram of Figure 1. The germination rate, seedling height, seed fertility in Xl and chlorophyll mutations in X 2 were investigated (Tab. 1).
Chronic irradiation was more effective as to radiation damage of theabove properties at the highest dose (20 kr) than the acute treatment
applied at the end of the chronic radiation. Also, the earlier was the irradiation applied at the same dose in both treatments, the more reduced

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14_".,d j((20r/hr)

5 kr I

...,

,

~

-j

10

Y,oysI6Oco)

20

r

_(X)

Acute IHad,lIO,OOO IjIhr) . 10m(X}illl 0<1

m

mIX)

~

X o,y roys(""cs)20

Control0storage (hn)Fig. 1.

150
,

.

.

250

500

0,

Diagram of acute and chronic irradiations in rice (1962).

Table 1.
Dose rate(r/hr)

Dose
(kr)

,

1000

Effect of acute and chronic irradiations on rice.
Storage
(hrs)

0
5

20

1/

1/

1/

1/

750
500
250

Germination
rate (%)

Seedlingheight(cm)

Seedfertility(%)

ChlorophYll
mutationrate (%)

96.2

9.2

78.3

0.64

8.8
8.5
8.7
8.3
8.5
8.4

76.9
72.4
75.8
77.4
74.8
74.3

2.55
2.70
2.08
2.48
2.93
2.17

7.0
8.3
7.2
7.6
8.2

56.5
67.6
61.4
62.3

5.44
6.05
6.54
4.50
3.44

4.2
5.5
6.9

36.0
45.6

0

96.6
97.0
95.8
96.4

1/

500
0

97.8
95.8

10

20

500

1/

1/

96.8
96.0

1/

10,000

1/

1/

1/

20

1/

1/

1/

10,000

1/

0
750

11

500
0

97.4
96.4
96.0

1/

20
10,000

0
500

94.0
92.4

1/

1/

0

95.6

68.7

48.5

8.90
7.06
8.19

was seedling growth and seed fertility especially at higher doses. Thus
an intensification of radiation damage due to storage was taking place.

99.

Effects of acute and chronic irradiation on growing wheat(By Seiji MATSUMURA and Taro FUJII)

Seedling of di-, tetra- and hexaploid wheats were transplanted into

RESEARCHES CARRIED OUT IN 1963119'

the gamma-field. Six lots were placed in distances from 59 to 17 metersfrom the source. The total dosage was approximately 721.5 r at theminimum and 18,259r at the maximum intensity being 4.9......124r/20hr.
Growing seedlings or plants of the same materials were also irradiatedby acute gamma-rays in three different growing stages.
In the acute irradiation of the diploid wheat at an early seedlingstage and of young plants in spring, LD,o seemed to be around 0.5......0.8
kr. But in chronic condition LD 50 was around 2 kr. On the other hand,
LD50 of tetra- and hexaploid wheats in spring was about 1.5 kr in acutecondition, while that of the chronic condition was around 3......5 kr.
Young plants in early spring were more sensitive than in seedlingstage during winter and this phenomenon was common to all threeploids when acute radiation was applied.
A similar behavior was observed in tetra- and hexaploid wheats whenradiation was given to dormant seeds. But when growing plants weretreated, the hexaploid seemed to be slightly more resistant than thetetraploid.

100. Dose rate dependence of mutation rates of gamma-irradiatedpollen grains in maize(By Tomoo MABUCHI and Seiji MATSUMURA)

In order to investigate the relation of mutation rates to dose rates,.
pollen grains of maize, which had the dominant gene Su against sugary
endosperm, were collected from naturally dehisced anthers, or before
dehiscence, and were irradiated by gamma-rays from a 137Cs source withthe doses of 500......1,650r at two different dose rates of gl7r/min and1.25r/min. These acute and chronic irradiations were terminated almost
simultaneously before dusting the female ears having the recessive genesu with the treated pollen.
The endosperm alterations from normal to sugary resulting fromgamma-ray treatment of the pollen affected a large proportion of wholeendosperms and, in addition, only their parts producing chimeras (Tab. 1).
Acute irradiation was more effective in increasing the mutation rate
than the chronic one, especially at higher doses. Namely, the mutation
rates increased almost linearly with increasing doses in the latter, while
increasing exponentially in the former (Fig. 1). The mutation rate perroentgen was 5.5,.,.,8.1 X 10- 6 (average 6.6 X 10- 6 ) in chronic irradiation and12",23 X 10- 6 in the acute one.

....
~

~

Table 1.

ZZ

Pollination with gamma-irradiated pollen grains and mutation rate of Stt-gene in maize.

c:::No. of endosperms mutated (rate)
Dose
rate
(r/min)Dose(r)

No. ofDuration
female earsof
exposure
pollinated

917

917

917

Whole
Obs. (%)

ChimeraObs. (%)

Total
Obs. (%)% ofendosperms
whollymutatedMutationrate/r/locus

t""
"0
trJ
"0

o

;'Ii

~

o":rj

16

825

0

(0.00)

6hr40min

49

4,328

5

(0.12)

7(0.16)

12(0.28)

41.5

5.5x10- s

~
.....

1,000

13 20

69

5,414

33

(0.61)

11(0.20)

44(0.81)

75.0

8.1x10- S

z

1,500

20 00

69

3,735

24

(0.64)

10(0.27)

34(0.91)

70.0

6.1x10- S

t""

a

34

2,716

12

(0.44)

3(0.11)

15(0.55)

80.0}

b

37

4,142

28

(0.68)

3(0.07)

31(0.75)

90.3

a

49

5,418

95

(1.75)

21(0.39) 116(2.14)

81.9}

0
1.25

No. ofseeds set

~

500

550

1,100

1,650

36sec

72

108

z

19

1,732

28

(1.62)

7(0.40)

35(2.20)

80.0

a

29

3,337

101

(3.03)

19(0.57) 120(3.60)

84.2}

32

3,180

113

(3.55)

17(0.53) 130(4.09)

o

~

Z

(fJ

12 X10-s

~
.....
~

c:::
~

b

b

~

trJ

19 X 10-S

o":rj
C;;

86.9

trJ

23 X10- 6

Z

trJ

~

n
(fJ

a:

Anther irradiated before dehiscence,b:

Pollen irradiated.

z

9

J-'

"""

RESEARCHES CARRIED OUT IN 1963

121

The endosperm alterations are due usually, if not always, to deficiencies
rather than to gene mutations, as already STADLER and others reported.
It is assumed that the frequencey of "two-hit" aberrations was relatively
high, especially at acute irradiation, and it was dependent upon theintensity of radiation. The chronic irradiation was liable to give a highproportion of restitution from chromosome breakages effecting a linear
dose-response curve of the mutation rate, as in "one-hit" aberrations.

101.

Relation between polyploidy and effects of gamma- andneutron-radiation on wheat(By Seiji MATSUMURA)

Exposure to 14 MeV neutrons obtained from (D, T) reaction wa~
carried out in the Biology Division of Oak Ridge National Laboratory.
For the calculation of given doses, based on RANDOLPH'S method, theanalysed elemental compositions and the measured fast-neutron fluxe~
were used. The dose, applied to dormant seeds, ranged from 0.48 tc
1.80 krad for Triticum, monococcum and from 0.95 to 2.24 krad for T.
durum and T. vulgare. At the same time gamma-radiations were used
for comparison at 4.3,.....,17.2 krad in ORNL.
After exposure to 1.80 krad of fast neutrons, the seeds of T. monoco·
ccum germinated but most of the seedlings died in an early stage, whilE
in T. durum and T. vulgare slow growth of the seedlings continuec
even at 2.24 krad of fast neutrons. The higher was the dose of fas1
neutrons and gamma-rays, the more delayed were germination ane
growth of seedlings, the more reduced were survival rate and seec
fertility, and the more increased were chlorophyll mutations, with thE
exceptions of mutations in T. vulgare (Tab. 1). In general, T. monoco
ccum is the most sensitive to fast neutrons and gamma-rays. There i~
no significant difference between T. durum and T. vulgare. Also thE
relative biological effectiveness (RBE) of fast neutrons to gamma-ray~
was found to be 10",15 for those properties in T. monococcum. The RBE
values for the these characters in polyploids were higher than in T
monococcum, respectively.

122

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14
Table 1.

Effects of r-rays and fast neutrons (14 MeV)

Germina tionl
(%)

I

Dose

Length ofseedlings(em)Survial(%)

Fertility
in Xl
(%)

75.71

55.00

T. monococcum fiavescens (2n=14)
I

Controlr-4.3 kradI

r- 8 . 6
r- 12 . 9I----------

Chlor ophYl!
mutatio n ratein X2 (%)

70

11.07

75

10.03

56.00

41.57

68

8.09

58.82

24.96

4.47
5.93

57

5.96

54.39

24.24

4.59

0.0 o

N-0.48N-0.95

72

9.45

52.11

38.34

61

5.96

50.82

25.53

1.85
16.1 6N-1.43

50

3.85

32.00

10.45

6.0 6

N-1.80

38

2.04

0.00

-

-

I

T. durum Reichenbachii (2n=28)--

Control

72

10.21

72.22

38.37

0.00

r-8.6 In-ad

86

11.00

83.72

44.07

0.00

r- 12 . 9
r- 17 . 2

84

10.16

64.29

43.16

0.00

76

9.92

81.58

44.64

4.10

N-O.95

74

9.37

54.05

32.62

N-1.43

84

8.58

40.47

14.62

8.16
4.76

N-1.80

74

8.01

29.73

24.45

10.00

N-2.24

72

5.41

8.83

14.03

0.00

15.90

93.94

22.36

0.00

I

T. vulgare erythrospermum (2n=42)
66

Controlr-8.6 krad

58

14.82

89.66

13.11

0.00

r- 12 . 9
r- 17 . 2

68

14.36

89.66

7.92

0.00

68

12.31

70.59

5.62

0.00

N-O.95

70
82

14.10

71.43

9.22

N-1.43

13.15

53.66

9.25

0.00
0.00

N-2.24

71

9.05

19.35

4.18

0.00

102. Relative biological effectiveness of 14 Me V neutrons
to 60Co gamma-rays in einkorn wheat(By Taro FUJII)The RBE of 14 MeV neutrons to 60Co gamma-rays by using the

RESEARCHES CARRIED OUT IN 1963

ecific loci method in einkorn wheat was investigated.

123

F 1 seeds fromS~e croSS between the original normal strain and a chlorina mutant were~sed in this experiment.

Dormant F 1 seeds were irradiated at 4.3, 8.6
nd 12.9 krad of gamma-rays and 0.45, 0.95 and 1.43 krad of neutrons.
Mutations from dominant normal green to chlorina occurred by both'rradiations and appeared in the leaves and stems of the heterozygotic~1 plants as longitudinal stripes.
Around 80 per cent of seeds germinated in the control lot and in thelowest dosage lots from both neutron and gamma-ray irradiation, andgermination percentages were gradually decreasing with increasing dosageof both kinds of radiation. Moreover, a similar tendency was observedin early stages as to seedling growth which was gradually inhibitedwith increasing dosage. According to these results, neutron irradiation
was about 13 times as effective as that of gamma-rays. Survival ratein the non-irradiated control was about 90 per cent and about 50,....,,80
per cent of germinated seedlings survived in 0.45 and 0.95 krad lots
from neutron irradiation and from all lots irradiated by gamma-rays.
On the other hand, only about 4 per cent of germinated seedlings survived
in the highest neutron lot.
No mutation was observed in the control lot, and the number ofplants which contained striped tillers increased with increasing dosageof both kinds of radiation. The rates for striping were calculated per
spike and 0.19, 0.35 and 0.48 x 10-6 per rad were obtained from 4.3, 8.6
and 12.9 krad gamma-ray irradiation, respectively. 9.65 and 7.12xlO- 6
per rad were obtained from 0.45 and 0.95 krad neutron irradiation. 1.43
krad neutron irradiation is too high for einkorn wheat. From the results,
RBE value of 14 MeV neutrons to 60Co gamma-rays was about 13.

103. RBE in Arabidopsis treated with fission and14.1 Me V neutrons to gamma-rays(By Taro FUJII)RBE values of fission and 14.1 MeV neutrons to gamma-rays in
Arabidopsis were studied by specific loci method. The flower of thisplant is small and it is somewhat difficult to obtain many F 1 seeds.
Therefore, F 2 seeds from the cross between a single recessive hairless
and a wild strain (Landsberg) were used.
Dry seeds were subjected to 1.1, 2.1 and 2.9 krad of fission
neutrons in Oak Ridge National Laboratory and also to 0.7, 1.6, 2.3 and4.0 krad of monoenergetic 14.1 MeV neutrons in Hiroshima University. 24 to 47 krad of gamma-irradiations from 60Co and l37Cs were124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

also carried out in each neutron experiment in Oak Ridge National
Laboratory and National Institute of Genetics, respectively. About 1,000
seeds in each lot were sown in sterilized sand and experiments werecarried out under continuous artifical light in an air-conditioned room at
25±2°C. Around 90 per cent of seeds germinated in the controls andall irradiated lots and almost all of seedlings survived. Irradiations up
to about 50 krad of gamma-rays and 5 krad of neutrons had no effect On
germination or survival rate.
During the growth of seedlings, segregation ratio of hairless homozygotes was examined. Hairless homozygotes segregated according to
the ratio of 3:1 in all lots, as expected, and segregation fitted, according
to chi-square test, the simple Mendelian ratio. Therefore, hairy plants
must have consisted of homo- and heterozygotes, namely two thirds could
be assumed to be heterozygotes. Should somatic mutation from dominant hairy to recessive hairless occur in heterozygotic seeds it had toappear as hairless sectors for which they were examined. Number ofsuch plants with such sectors were counted. Mutations rates increased
with increasing dosage in each kind of radiation and the relation was
non-linear. Mutation rates were almost the same at24 krad of 60Co gammarays and 1.2 krad of fission neutrons and 0.70 per cent were obtained inboth lots. From the results, RBE value of fission neutrons to gammarays was about 16. On the other hand, mutation rate in the lowest
dosage lot of 14.1 MeV neutrons was somewhat high when it is compared with the other lots. But, RBE value of 14.1 MeV neutrons may be
estimated as about 15 from the curve of mutation rates and dosages.

104. Effects of gamma-irradiated pollen grains onpollen-tube mitosis(By Yoz6 IWANAMI* and Seiji MATSUMURA)

The effects of pollen grain irradiation on pollen-tube mitosis were investigated in Lilium auratum LINDL., Impatiens Balsamina L., Camellia
japonica L. and Camellia sazanqua THUNE. The data were obtained from
artifical culture media, sugar agar plates with 80% sucrose and 1.5%
agar. For irradiation gamma-rays were applied with 137Cs 6,000c at
dose rate 55 kr/hr. The doses ranged from 0.5 to 100 kr.
Inhibition of mitosis increased with increasing dose; namely the higher
the dose, the more delayed was the time of its occurrence and the more
reduced was the frequency of nuclear division. Division of the generative nucleus was delayed at small doses, namely at 0.5 kr in Lilium and* Biological Institute, Yokohama Municipal University, Yokohama.

125

RESEARCHES CARRIED OUT IN 1963

1 kr in Camellia and Impatiens.
In Lilium auratum the generative nucleus remained mostly undivided
in showing chromosome elements even at 5 kr, while at 20 kr a few
nuclei consisted of chromatin masses or chromosomes. The pollen grainsof Lilium did not germinate smoothly on the media. In natural condition, therefore, their pollen-tube mitosis may be more radio-resistant.
At 80 kr irradiation of Camellia japonica pollen the mitotic division of
the generative nucleus was almost completely inhibited. In Camellia
sazanqua the generative nucleus did not divide into 2 sperm nuclei above
25 kr, while relatively many generative nuclei with chromosomes wereobserved at 50 kr. The pollen grains of Impatiens Balsam-ina had thehighest radio-resistance. At 50~80 kr the generative nucleus underwent complete mitotic division.
Radio-sensitivity of pollen grains varied widely according to thespecies. Doses estimated as inhibiting pollen-tube mitosis by 50 per centare presented in Table 1 for the four species. In general, their pollentube mitosis was much more radio-resistant than the meiosis in PMC'sand mitosis in root tips.
Table 1.

LD 50 's doses (kr) for pollen-tube mitosis from gammairradiated pollen grains of 4 plant species.
Formation ofchromosomes

Species

Impatiens Balsamina
Camellia japonica
Camellia sazanqua
Lili~tm auratum

Prophasic condensationof chromatin threads

60~70

60~70

50

26~60

11~24
6~

G.

9

5
O.5~1

HUMAN GENETICS

105. Studies on selection in ABO blood groupsl)(By Yuichiro HIRAIZUMI, Ei MATSUNAGA, Hisajiro
IZUMIYAMA, Toshiyuki FURUSHO)

An extensive survey was carried out in the City of Ohdate in 1962 in~order to investigate the selective mechanisms operating on ABO bloodgroups. The data available covered approximately 2,500 couples withwife's age ranging from 30 to 40 years and more than 6,000 children.
I)

This work was supported by a grant (RF 61113) from the Rockefeller Foundation.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

Not only the ABO blood groups of parents and children, but also fertility of the mothers and mortality ot the children were determined.
The data have been subjected to statistical analyses. Among the results
obtained up to date, three points may be summarized as follows.
( 1) There were no differences in the proportion of infertile as well
as childless couples, in the mean number of living children, or in the
proportion of prenatal deaths between compatible and incompatible
mating groups (see Table 1).
Table 1.
No. ofmatings

Proportion
Proportionof couples Mean no. of prenatal
pregnancies deanths per
as yetinfertile
pregnancy

Proportionof couples Mean no.
ofas yetchildrenchildlessCompatible
matings

1389

0.022

3.189

0.108

0.030

2.590

Incompati·ablematings

1056

0.027

3.139

0.101

0.032

2.572

-

(2) The viabilities of children of various genotypes and thesegregation ratio of 0 genes in the AO and BO fathers were estimatedby means of maximum likelihood scoring method. Since a high speed
computer is not available in our Institute we had to use hand computers.
Because of this difficulty it was necessary to simplify somewhat theTable 2.
Definitions of parameters
Viability of compatible AD, BO, AB=1.00
Viability of incompatible AD, BO, AB=x
Viability of AA, BB=yViability of 00 =z
kA(k B ): segregation ratio ofgene from AD (BO) fathers.
hA (h B ): probability that a parent of type A (B) be AA(BB)°Group A
x=1.79±0.28
k A =0.65±0.07

y=2.51±0.69
z=1.02±0.08
h A =0.166±0.021 (Expected value of h A is 0.166)
Group B

x=1.74±0.50
k B =0.66±0.08

x=3.41±2.49
z=1.00±0.08
h B =0.135±0.034 (Expected value of h B is 0.135)

RESEARCHES CARRIED OUT IN 1963127

the procedures in numerical calculations. Hence, the mating types weredivided into two groups; (A) matings involving hA and/or kA and (B)those involving hE and/or k E (for h, k and other parameters, see Table 2).
The estimated viabilities are presented in Table 2.
All figures in this Table are those of the first approximation andsince the deviations from the null hypothesis of x=y=z=1, and k=0.5
are very large, it is necessary to continue the calculations to obtainmuch better estimates. However it is interesting to note that, in bothgroups, there was no indication of reduced viability of incomptible
heterozygous children. This result is consistent with (1). The 0 genes
from AO and EO fathers are transmitted to their children in more than50%.
(3) Based on the viabilities estimated in (2), the average viability ofchildren from each mating type was computed. These were then compared with the actually observed proportions of prenatal deaths as shownin Table 3.
Table 3.
Group 1
Prop. ofQ X 0 prenatal
deathsGroup 2
Prop. ofQ X 0 prenatal
deathsGroup 3
Prop. ofQ X 0 prenatal
deathsGroup 4
Prop. ofprenatal
QX 0
deaths

OxO

0.118

AxO

0.120

BxO

0.110

ABxO

0.136

Ox {~

0.102

AX{~

0.101

BX{~

0.113

0.089

OxAB

0.100

AxAB

0.100

BxAB

0.078

ABx {~
ABxAB

0.055

In order to eliminate the possible effect of mother's genotype on
prenatal death, 16 mating types were divided into 4 groups, each consisting of 4 mating types with the same genotype of the mothers. In eachgroup, the 4 mating types were placed in the order of the estimatedviability of children, i.e., in each group the first mating type has thelowest estimated viability of children. There is a clear correlation between them, i.e., a mating type with higher average viability of children
showed lower proportion of prenatal deaths. This agreement indicatesthat the survey made at Ohdate City was technically trustworthy.
As described above, the data obtained in Ohdate City so far indicatedno evidence of elimination of children due to ABO incompatibility,
which is in a remarkable contradiction to the previous results obtainedby others. The reason for this is yet unknown. The distorted segregation ratio of 0 genes in the AO and EO fathers, however, agreed wellwith that found in the previous study.

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

106.

Down's syndrome in Japan!)(By Ei MATSUNAGA)

Down's syndrome or "mongolism" is a well-known abnormality due
to chromosomal aberrations. A superficial resemblance in appearance inEuropean patients with this abnormality to normal Asiatic people hasoften been alleged, as is evident from the designation itself, but theclinical features found in typical cases in Japan are as remarkable as
those described for the European patients. As to chromosomal conditions, it seems that more than 150 cases, including unpublished data,.
have been examined in different laboratories of this country since thefirst report made by Makino, Tonomura and Matsunaga in 1960. In the
majority of cases standard 21-trisomy was revealed, while several cases
have been reported to be due to translocation or mosaicisms involving
the 21st autosome.
The incidence of this abnormality is about 1 per 500 total births inEurope and North America, but comparative figures from other parts of
the world have so far been very limited. Regarding the figures forJapan, Neel (1958) found, from an extensive survey made by ABCC, 6cases among 63,796 newborns in three cities of Hiroshima, Nagasaki andKure, but he added further 12 cases among 16,144 infants who were reexamined at the age of 8-10 months, so that the overall frequency may
be estimated to be about 1 per 1,200 infants. However, this figure appears to be an underestimate, since the clinical signs are not always.
wholly developed when the patients are under the age of one year.
As an indirect approach to estimate the frequency of Down's syndromein this country, a nation-wide survey has been conducted by correspondence with 218 institutions for mental defectives belonging to the 6-18
age group. The number of institutions that have returned the completed
questionaires is 77 (35%) up to the present time, providing various informations on 4,483 persons in all. Although the response rate is increasing, a preliminary result based on these data may be reported here.
There were 340 individuals with Down's syndrome among 4,483 inmates,.
the frequency being 7.6%. Examination of the distribution of LQ.
revealed the striking finding that almost all (96%) patients with Down's
syndrome showed LQ. less than 50, whereas the portion of those having
LQ. less than 50, regardless of whether they were affected with Down's
syndrome or not, amounted to 68% of all inmates. Thus, the frequency
of Down's syndrome among the inmates with LQ less than 50 may be
1)

tion.

This work was supported by a grant (RF 61113) from the Rockefeller Founda-

RESEARCHES CARRIED OUT IN 1963

129

calculated as about 10%. If one assumes that our subjects were representatives of mental defectives in a general population with respect
to LQ., and that, as the extensive survey carried out by Miki et at.
suggested, approximately 0.7% of the general population belonging to
the age group between 6 and 18 have LQ. less than 50, then the fre,quency of Down's syndrome among Japanese in the corresponding age
group is 1 per 1,400. This is of course a rough estimate, but, taking
into account the high mortality rate of the patients in early childhood,the incidence of this abnormality per total births in Japan seems to be
not so far from that reported for Europe and North America.
Table 1.

Maternal age inyears at birthof children
15-19
20-24
25-29
30-34

Distribution of births of children with Down's
syndrome according to maternal age
Percentage distribution
321 cases of
Down's syndromeI

All births inpopulation

Relativeincidence

0.9
13.7

2.0
26.1

35-39
40-44

18.4
18.7
27.7
16.8

35.5
21.9
11.2
3.2

45-49

3.7

0.2

0.85
2.48
5.31
18.73

All ages

99.9

100.1

1.00

Mean age

33.2

28.2

-

0.46
0.53
0.52

It has been repeatedly demonstrated that the incidence of Down's
syndrome increases with advancing maternal age. Results obtained inEurope and North America are generally consistent in that the increaseof this abnormality with maternal age is at first rather slow until themother reaches the age of 35, and then it becomes very rapid as themother approaches the age of menopause. Compared with the overallfrequency, a 2- to 4-fold increase for the age group of 35 to 39, a 5- to
10-fold increase for the age group of 40 to 44, and after the age of 45
a 10- to 20-fold increase was found (Lenz, 1959; Penrose, 1961). The
question remains, however, whether there is any geographical or racialdifference concerning the increase rate. The results of our study of 321
cases are summarized in Table 1. The control population is calculatedon the basis of the distribution of births in census reports of the years

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

corresponding to the birth dates of cases with Down's syndrome. As isapparent from the Table, there is for this abnormality no indication forany difference between Japanese and Caucasians as to suppression or acceleration of the increase rate with advancing maternal age.

107. Clinical conditions of patients with apparently
normal chromosomes(By Akira TONOMURA)

Based on cultures of peripheral blood and/or skin biopsy specimens
patients with the following clinical conditions were found to have 46chromosomes of apparently normal karyotype.

Age

Sex

Clinical conditions

1) 6 months2) 3 monthsMM3) 5 months
4) 2 months

M

Polydactyly; reduplication of left thumb
Multiple congenital abnormalities; heart
defect and low set ears
Oligodactyly; split hands
Multiple congenital abnormalities:
Megalocytosis5) 13 years6) 8 years7) 12 years3) 8 years9) 21 years10) 31 years11) 21 years12) 18 years13) 25 years14) 20 years15) 25 years16) 18 years17) 22 years18) 4 years

M

M
M

Laurence-Moon-Biedl's syndrome
Fanconi's syndrome

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

Gargoylism (Hurler's syndrome)
Pterygium coli
Primary amenorrhoea

Hypoplastic ovary
Testicular feminization
Female pseudohermaphroditism
Male pseudohermaphroditism

RESEARCHES CARRIED OUT IN 1963

108.

131

Variation in length of the Y chromosome in man(By Akira TONOMURA and Hiroshi ONO)

During our current survey of chromosomes in human subjects associated with various types of congenital disorders, we have found an unusually long Y chromosome in a 12-year-old mentally retarded boy and in his
apparently normal father. Variations in the length of the Y chromosome
in this family were investigated in comparison with those of two control
series obtained from normal males. The well-spread metaphase cells
were photographed and the chromosomes were measured. The relative
length of the Y chromosome in each cell was expressed as the ratio between the length of the Y and the mean length of nos. 21 and 22. The
results are summarized in Table 1.
Table 1. Comparison of the relative length of Y chromosome ofpatient and his father with that of normal males.
Mean of Y (X)

No. of cells examined

Propositus

Xl = 1.46

20

FatherX 2 =1.45

20

X 3=1.185

21

PersonControl series 1. (a male)Control senes 2. (1 cell fromeach of 53 males)II

53

The "t" test was applied comparing the means:
Xl with X2 ; t38=0.18 0.80<P<0.90
Xl with Z; ta9=5.71
P<O.OO1.
Xa with X';; tn=0.05 P>0.90..

The results of statistical analysis support the view that the Y chromosome in this family is significantly longer than in the control series andmay be a heritable characteristic. The presence of a significantly long
Y chromosome has been reported in several cases with phenotypic abnormalities as well as in their normal relatives. In general, variationin the relative length of a chromosome may reflect differences in chromatin content (duplication, deletion or translocation) or contraction statesof heterochromatin (differential spiralization). Since the Y chromosome
in man is highly heterochromatic, the increased length of the Y in thepresent family seems to be due to a difference in the degree of spiralization of the whole or a part of the Y chromosome rather than that inchromatin content.

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

109.

Drumsticks of polymorphonuclear leucocytes in newborn females(By Akira TONOMURA, Hiroshi ONO and Keiichi SOGA)

In has been generally accepted that the drumsticks found in poly.
morphonuclear leucocytes of female blood films are the counterpart ofthe sex chromatin in tissue cells, although the quantitative correlation
between drumstick and sex chromatin is still complicated. In a previous
paper (Tonomura, Toyofuku and Matsunaga; 1962) the authors demon.
strated that the number of drumsticks found in 500 neutrophils de.
creases slightly, but significantly with age. On the other hand, Smithet al. (1962) reported a lowered incidence of sex chromatin in normal
newborn females. Furthermore, it has been suggested by Taylor (1963)
that the reduction of the amount of sex chromatin is a reflection ofaltered metabolic processes in the neonatal period, possibly due to ex.
tremely high levels of oestrogens during the first few days after birth.
If this is so, it is very interesting to know whether the frequency ofdrumsticks in the newborn females is influenced by the metabolicchanges of this period.
In the present investigation blood smears were obtained from 10
females from 1 to 12 days after birth. Five hundred neutrophil leucocytes were examined in each smear, and the demenstrable drumsticks
were counted. All of the newborn females examined showed considerably high frequencies of drumsticks at the average of 32.0; thelowest number was 23 and the highest was 40 in the examination of 500
neutrophils. In the previous study the incidence of drumsticks in 250
females whose ages varied from 1 to 79 showed an average of 12.0±OA.
Though the numbers here examined are too small to be conclusive, theresults of our preliminary study may indicate that the drumstick situation of newborn females is somewhat different from that of sex
chromatin.

110. Function of the amino group in the molecular complex
consisting of hemoglobin and haptoglobin(By Tomotaka SHINODA)

In the endeavor to elucidate the mechanism of haptoglobin action
and the role of the amino group in its specificity and catalytic function,
an attempt has been made to convert a or e-amino groups of lysine into
the corresponding trinitrophenyl-amino residues. Human haptoglobin,
which was previously typed in starch gel electrophoresis and purified
with column chromatography, was dissolved in physiological saline solu-

RESEARCHES CARRIED OUT IN 1963133

don and treated with TNBS at 37°. The hemoglobin binding capacity(flBC) and electrophoretic behavior of the resulting materials were compared in the usual way with those of the control preparation. As to
the HBC of three types of haptoglobin 1-1, 2-1 and 2-2 at the stage ofintroduction of 1.8 moles of trinitrophenyl residues into the protein
molecule, no significant changes were observed in the first two while in
the remaining the decrease in HBC amounted to 15%. On the whole,the HBC of haptoglobin showed the tendency to be inversely proportional to the amount of trinitrophenyl residues introduced into protein
molecules. Using electrophoresis, a fine separation of a complex ofmodified haptoglobin with nemoglobin could be achieved from the unmodified one. The formation of a complex consisting of hemoglobin
and modified haptoglobin, has been observed on filter paper by crossinteraction technique during the course of electrophoresis. These findings are in good agreement with the result expected from tne loss ofamino residues of haptoglobin through the reaction with TNBS. On
the other hand, when hemoglobin was in the same way previously
treated with TNBS the formation of the complex consisting of modified
hemoglobin and haptoglobin was also observed but with less peroxidase
activity. So far about 70% of amino residues of hemoglobin molecule
have been altered by the reagent, and there was no more ability to form
a molecular complex with haptoglobin. These results may be considered
to be the consequence of the loss of functional amino groups which play
an essential role in the formation of the molecular complex between
the two components as well as conformation of the protein molecule atthe time of the formation of the complex.
Analysis of the results is now in progress by means of more rigorous
procedures.
111. Haptoglobin and G-6-PD deficiency in a Japanene population(By Tomotaka SHINODA)

During the summer season of this year, a field survey on glucose-6phosphate dehydrogenase (G-6-PD) deficiency was carried out in the
Shichikawa area of Wakayama prefecture in collaboration with a group{)f Wakayama Medical College. Blood samples were taken from 1,096
persons, and the activity of the enzyme was measured by means of"glutathione stability test". Throughout the survey, one male subject
was found to be deficient (the amount of erythrocyte glutathione wasless than 20 mg per 100 ml of packed erythrocyte after treatment ofblood specimen with oxidating agent), thus the frequency of the trait

134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14

in Japan being roughly 0.1%.
In addition to the screening of G-6-PD deficiency, sera were separated
from the blood samples and haptoglobin types were determined by starchgel electrophoresis. So far 171 specimens have been analyzed, and thefrequencies of the types 1-1,2-1 and 2-2 were 7,77 and 87, respectively,
Based on this result, the frequency of gene Hpl may be estimated asn.266.
Further anslysis of the samples is now in progress.

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

135

BOOKS AND PAPERS PUBLISHED IN 1963
BY STAFF MEMBERS
CHIGUSA, S. and T. MUKAI 1963 Two cases of heterosis associated with the receE
sive genes sepia (se) and expanded wing (ew) in D. melanogaster. Jap. ]Genet. 38: 177
ENOMOTO, M. and T. IINO 1963 Colonial dimorphism in nonmotile Salmonella. J
Bacteriol. 86: 473-477
FUJII, T. 1963 Effects of nitrous oxide on germinating seeds of Triticum mon(coccum (negative results). W. 1. S. 15-16: 5-6_
1963 Mutations induced by radiation in vegetatively propagated plantwith special references to flower color. (Collective review). Gamma Field SymI
1: 50-60_
1963 Relation between acute and chronic irradiations (Collective revie'
in Jap.). Recent Advances in Breeding 4: 60-69
HIRAIZUMI, Y. and T. E. REED 1963 Assumptions in tests for meiotic driVI
Science 139: 406-407
IMAMURA, S. and T. C. KATAYAMA 1963 Zur Bestimmung des Interzellularvolumer
der pflanzlichen Organe. Sci. Rep. Tohoku Univ. Ser. IV. 29: 131-142
KATAYAMA, T. C. 1963 Wild Oryza species of the Philippines, New Guinea, BornEand Java. Seiken Zih6 15: 35-46_ - - 1963 A survey of botanical studies on the genus Oryza, especially (photoperiodic studies. Seiken ZiM 15: 98-109
KIHARA, H. 1963 Classification of species and genome symbols in Oryza. Ja]
Jour. Breed. 13: 181-182_ _ _ 1963 Interspecific relationship in Triticum and Aegilops. Seiken Zih6 I!
1-12_ _ _ 1963 Nucleus substitution in wheat and Aegilops. II. Chromosome sui
stitution. Seiken ZiM 15: 13-23- - - - 1963 Genome-analysis in Oryza. Symp. on Rice Problems, Tenth Pacif
Science Cong.: 57-66- - - and K. TSUNEWAKI 1963 Increased occurrence of haploids and tw
seedlings due to an alien cytoplasm. W. 1. S. 15-16: 32-34- - - and
1963 Comparative genetics in wheat. Jour. Ind. Bot. SO(
Maheshwari. Festschrift: 118-136
KIMURA, M. 1963 A probability method for treating inbreeding systems, especial
with linked genes. Biometrics 19: 1-17
- - - and ]. F. CROW 1963 The measurement of effective population numbe
Evolution 17: 279-288- - - - T. MARUYAMA and J. F. CROW 1963 The mutation load in small popul
tions. Genetics 48: 1303-1312- - - and J. F. Crow 1963 On the maximum avoidance of inbreeding. Genetic
Research 4: 399-415
KONDO, S. 1963 A biophysical theory for radiation- induced polygenic mutatior
J. Genet. 58: 216-224

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 14- - - 1963 Fundamental aspects of relative biological effectiveness. Proceed.
ings of the 5th Japan Conference of Radioisotopes 2: 14-21____ 1963 Fundamental biological effects of ultraviolet light (reviews inJapanese). Seibutsu-butsuri 3: 9-24
MABUCHI, T. and S. MATSUMURA 1963 Dose rate dependency on mutation rates
of r-irradiated pollen grains in maize. Seiken ZiM 15: 64-74
MATSUMURA, S. 1963 Significance of the experimental control of environment (inJapanese). The Society of Heating, Air-Conditioning and Sanitary Engineersof Japan 37: 27-29____ 1963 Environment-controlled growth rooms for plants in Japan (in Japanese). The Society of Heating, Air-Conditioning and Sanitary Engineers ofJapan 37: 30-37____ and T. FUJII 1963 Effects of chronic irradiation on einkorn wheat. Proc.
5th Conf. Radioisotopes, Tokyo, 4: 243-245____ and
1963 Effects of chronic irradiations on growing wheat.
Seiken ZiM 15: 59-66_ _ _, S. KONDO and T. MABUCHI 1963 Radiation genetics in wheat. VIII. The
RBE of heavy particles from BlO (n, a)Li7 reaction for cytogenetic effects inEinkorn wheat. Rad. Bot. 3: 29-40
IWANAMI, Y. and S. MATSUMURA 1963 Effects of irradiation on pollen, I. Irradiation effects of 1"-rays on pollen tube mitosis' Bot. Mag. 76 (901): 246-255
MATSUNAGA, E. A. TONOMURA, INUI. N. and T. HONDA 1963 Embryonal sex:
ratio in Japanese determined by the sex-chromatin test.-A preliminary report
Jap. J. Human Genet. 8: 89- - - A. TONOMURA, B. YAMAGUCHI, and E. MATSUDA, 1963 A case of Down's
syndrome with 46 chromosomes. Jap. J. Human Genet. 8: 112-119
MORISHIMA R., K. HINATA and H. I. OKA, 1963. Comparison of modes of evolutionof cultivated forms from two wild rice species, Oryza breviligulata and O.
perennis. Evolution 17: 170-181
MORIWAKI, K. 1963 Activity change of acid phosphatase in the skin of the rhino
mouse preceding the hereditary hair loss (in Japanese). Zool. Mag. 72: 267-268
MURAKAMI A., Y. FUKASE, S. KONDO and Y. TAZIMA. 1963 Mutation frequenciesof silkworm gonia obtained after 14 MeV neutron irradiation. Jap. Jour. Genet.
38: 195-196 (Abstract in Japanese)
MUKAI, T., S. CHIGUSA and I. YOSHIKAWA 1963 Spontaneous mutation rate ofpolygenes controlling viability, and the genetic structure of natural populations
in D. melanogaster. Jap. J. Genet. 38: 195____ ----and
1963 Comparison of X-ray induced polygenic viability
mutations with those of spontaneous origin in Dro8ophila melanogaster. Ab·
stracts of the papers presented in the 5th Meeting of the Japan Radiation
Research Society: 114-115____ and I. YOSHIKAWA 1963 Heterozygous effects of radiation-induced mutations on viability in homozygous and heterozygous genetic backgrounds in
Drosophila melanogaster (Preliminary report). Jap. J. Genet. 38: 282-287'OGAWA, Y. 1963 Growth, differentiation and regeneration XXXII. Influence ofBOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

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temperature on the actin formation in Triturus embryo (in Japanese). Med.
and BioI. 66: 50-52______ 1963 Taste-ability for Citbittol A of Japanese I. Bitter taste thresholds(in Japanese). Med. and BioI. 66: 76-78______ 1963 Taste-ability for Citbittol A of Japanese II. Abnormal taste response(in Japanese). Med. and BioI. 66: 153-156_
1963 Cellulose acetate as a medium for ele'ctrophoresis and immunodiffusion technique (in Japanese). Igaku no Ayumi 45: 128-131_
1963 Effect of sodium-glucuronate on the survival of rats irradiated with
Cesium-137 i-rays. Reports of the 8th. Symp. of Glucuronic Acid. 72-76_
1963 Growth, differentiation and regeneration XXXIII. Effects of Lgulonolactone on the growth and differentiation of early embryos of Trituru8(in Japanese). Med. and Bio!. 66: 216-218_
1963 Use of "Ponceau 3R" for staining proteins on cellulose acetate strip
after electrophoresis and immuno-diffusion (in Japanese). Med. and BioI. 66:
234-237_
1963 Growth, differentiation and regeneration XXXIV. Effects of glucuronolactone on the syntheses of muscle proteins in the x-irradiated embryo (inJapanese). Med. and BioI. 66: 319-322
and I. KOBAYASHI 1963 Growth, differentiation and regeneration XXXV.
_Electrophoretic analysis of water-soluble proteins in Triturus embryos on
cellulose acetate (in Japanese). Med. and bio!' 67: 84-90_ _ _ and T. KAWAHARA 1963 Growth, differentiation and regeneration XXXVI.
Use of rabbit anti-serum against muscle proteins of fowl for serological determination of homologous tissue proteins of Triturus (in Japanese). Med. and
BioI. 67: 180-183_ _ _ 1963 An improved apparatus for cellulose acetate and filter paper electrophoresis (in Japanese). Med. and BioI. 67: 251-255_ _ _ 1963 Electrophoretic technique of serum proteins on cellulose acetate
strip (in Japanese). Japanese J. of Clinical Med. 21: 2419-2427.
_ _ _ 1963 Development of skeletal muscle proteins in embryonal stage andregenerating tissue of chick and Triturus (in Japanese). Medicine 20: 893-898
OKA, H. I. 1963 An induced mutant of rice showing abnormal development of innerglumes. Bot. Bull. Acad. Sinica, 4: 47-50- - - and W. T. Chang. 1963 A note on rice varieties of Japonica type foundin Northern Thailand. Bot. Bull. Acad. Sinica. 4: 163-168
OSHIMA, C. 1963 The persistence of some recessive lethal genes in natural populations of Drosophila melanogaster, III Proc. Jap. Acad. 39: 125-130- - - , 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---,
1963 Study on deleterious genes in a natural population of D. melanogaster. Zoological Magazine 72: 358
OSHIMA, C. 1963 Study on deleterious genes in a natural population of D. melano-·
gaster. (3) Jap. J. Genent. 38: 202

138 ANNUAL REPORT OF NANIONAL INSTITUTE OF GENETICS NO. 14
SADO, T. and M. R. LEONARD 1963 Autoradiographic studies on the proliferative
capacity of antibody producing cells. Radiation Research 19: 227-228- - - - 1963 Spermatogenesis of the silkworm and its bearing on radiation in.
duced sterility, I. J. Fac. Agr. Kyushu Univ. 12:359-386- - - - 1963 Spermatogenesis of the silkworm and its bearing on radiation in.
duced sterility, II. Ibid. 12: 387-404
SAKAGUCHI, B. and D. F. POULSON 1963 Interspecific transfer of the "sex-ratio"condition from Drosophila willistoni to D. melanogaster.
Genetics 48:
841-861
POULSON, D. F. and B. SAKAGUCHI 1963 Hereditary infections' and plasmic inheri_tance in Drosophila (Abst). Proc. 11th Inter. Congr. Genetic 206-207
SAKAI, K. I. 1963 Problems in selection in rice. Symp. on Rice Problems, Xth
Pacific Science Congress, 7-16
- - - - and I. GoTO 1963 Inherent and environment respondent susceptibility to
Piricularia oryzae in rice-plant. Ann. Phytopath. Soc. Japan 28: 124-130- - - - and S. HATAKEYAMA 1963 Estimation of genetic paramenters in forest
trees without rasising progeny. Silvae Genetica (West Germany) 12: 152-157
SAKAMOTO, S. 1963 On the well-designed small growth cabinets (A review). En.
vironment Control in BioI. 1: 35-40
and M. MURAMATSU 1962 Chromosome numbers of Gramineae species
collected in Pakistan, Afghanistan and Iran. C. I. S. 3: 32-33
and
1963 Preliminary studies on the relationship of diploid
Eremopyrum distans and E. triticeum (Gramineae). Can. Jour. Genet. Cytol.
5: 433-436
TAKENAKA, Y. 1963 The origin of "the Yoshino cherry tree". Jour. of Heredity
54: 207-211
1963 Cytogenetic studies in Nicotiana XX. Reduction division in threeinterspecific hybrids and one amphidiploid (in Japanese). Japanese Jour. Gene·
tics 38: 135-140
1963 Alpine plants (in Japanese). Alps Book Company, Tokyo.
and Y. YONEDA 1963 Chromosome studies in Nicotiana hybrid tumor(in Japanese). Japanese Jour. Genetics 38: 208
ISHIKAWA, S., Y. TAZIMA and T. HIRAO 1963 Response of the chemoreceptors ofmaxillary sensory hairs in a "Non-preference" mutant of the silkworm. J.
Sericult. Sci. Japan 32: 125-129
TAZIMA, Y. and S. Kondo 1963 Differential radiation-sensitivity of germ cells as apossible interpretation of sex difference in dose-rate dependence of induced
mutation rates in the silkworm. pp. 237-252 "Repair from Genetic Radiation
Damage" ed. by Sobels, Pergamon Pressand A. MURAKAMI 1963 The increase in induced mutation frequency
after fractionated irradiation of gonial cells of silkworm. Abstracts of thepapers presented in the 5th meeting of The Japan Radiation Research Society:112-113 (Abstract in Japanese)
1963 A new interpretation of a complexity of dose-rate dependence ofradiation induced mutation rates in the silkworm and its bearing on the

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

139

mechanism of radiation mutagenesis. Jap. Jour. Genet. 38: 207 (Abstract inJapanese)
TsUJITA, M. 1963 Manifestation mechanism of yellow larval color in the silkworm, with special regard to d-lem gene. Jap. J. Genet. 38: 48-60
and S. SAKURAI 1963 Specific protein combining with yellow pigments(dihydropteridine) in the silkworm, Bombyx mori L. Proc. Jap. Acad. 39:
247-252
and
1963 The association of a specific protein with yellow pigments (dihydropterin) in the silkworm, Bombyx mori. Jap. J. Genet. 38: 97105_
and1963 Melamine action in the phenocopy of pigment elimination from hypodermal cells of silkworm larva. Proc. Jap. Acad. 39: 513518- , S. NAWA, B. SAKAGUCHI and T. TAIRA 1963 Genetical and biochemical
studies of pteridine metabolism in insects. Jap. Jour. Genet. 38: 159-174
TSUNEWAKI, K. 1963 An Emmer wheat with 15 chromosome pairs. Can. Jour.
Genet. Cytol. 5: 462-466
1963 Analysis of the fertility-restoring gene in Triticum aestivum ssp.
compactum. Seiken Ziha 15: 47-53
1963 The transmission of the monosomic condition in a wheat variety,
Chinese Spring, II. A critical analysis of nine year records. Jap. Jour. Genet.
38: 270-281
WATANABE, T. K. 1963 Study on deleterious genes in a natural population of D.
melanogaster. (1) Jap. J. Genet. 38: 212- - - 1963 Study on deleterious genes in a natural population of D. melanogaster. (2) Jap. J. Genet. 38: 212
YAMADA, Y. 1962 Genotype by environment interaction and genetic correlationof the same trait under different environments. Jap. Jour. Genetict 37: 498509
YAMAZAKI, T. and T. MUKAI 1963 Correlation between viability and developmental rate in D. melanogaster. Jap. J. Genet. 38: 213-214
YONEDA, Y. 1963 Karyological studies on yeasts. Cytologia 28: 131-145
YOSIDA, T. H. 1963 The karyotypic analysis of various in vivo and in vitro linesof the lymphatic neoplasm, P388. Mam. Chrom.News Letter 9: 74
Evans, V. J., LARoCH, J. F., YOSIDA, T. H. and M. POTTER 1963 A new tissue
culture isolation and explanation of the P388 lymphocytic neoplasm in a chemically characterized medium. Exp. Cell Res. 32: 212-217
YOSHIKAWA, 1. and T. MUKAI 1963 Effects of radiation-induced mutations on
viability in D. melanogaster. Jap. J. Genet. 38: 214-215
ACKNOWLEDG MENTThe editor wishes to acknowledge Dr. F. A. LILIENFELD for reading the manuscripts (Motoo KIMURA)

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