NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 16

1965

Publl81uJd 611

THE NATIONAL INSTITUTE OF GENETICS
lIIWm4. Slmo,"""ke,,. JaptUI

1966

Annual Reportof theNational Institute of Genetics
No. 16, 1965

Published by

The National Institute of Genetics, Japan
1966

CONTENTS
General statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council............................................................
Projects of research for 1965
Foreign visitors in 1965 .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Researches carried out in 1965. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Genetics, cytology and biochemistry of animals
1. Distribution and persistence of lethal genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE
T. K.)....................................................
2. Pre-adult viability of heterozygotes for some persistent lethal
chromosomes (a persistence mechanism of lethal genes)(WATANABE T. K. and OSHIMA). . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Abnormal segregation in the offspring of persistent lethal
heterozygous male flies (a persistence mechanism of lethal
genes) (OSHIMA and WATANABE T. K.) . . . . . . . . . . . . . . . . . . . .
4. Chromosomal polymorphism in the Kofu and Katsunuma
natural populations (WATANABE T. and OSHIMA)..........
5. The effect of insecticide selection in experimental populationsof D. pseudoobscura (OSHIMA and WATANABE T.)..........
6. Functional differentiation of larval fatbody cells in Drosophilamelanogaster (TAIRA and RIZKI)
7. Further evidence supporting the optimum heterozygosity
hypothesis with respect to viability in a population of
DrosoPhila melanogaster (MUKAI);.....
8. Independence of optimum heterozygosity of the second chromosome on the heterozygosity of the third chromosome in
Drosophila melanogaster (MUKAI)
9. Suppressing system of SD in a natural population of Drosophilamelanogaster in Japan (HIRAIZUMI and KATAOKA)..........
10. Maternal age and non-disjunction in Drosophila melanogaster(NAKAZIMA and HIRAIZUMI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Further report on the heterotic effect due to linkage disequilibrium in Drosophila populations (IYAMA) . . . . . . . . . . . .
12. Effect of other species on the genetic constitution of Drosophilamelanogaster (NARISE) .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

13. Selection for bilateral asymmetry in wings of Drosophilamelanogaster (SAKAI and NARISE) . . . . . . . . . . . . . . . . . . . . . . . . .
14. Deoxyribonucleic acid from SR spirochetes in Drosophila(SAKAGUCHI, NAWA and POULSON)
15. Effect of DNA treatment on the wing scale in Ephestia(NAWA)
16. Genetic and biochemical studies of chromogranules in the
hypodermal cells of the silkworm (TSUJITA and SAKURAI)..
17. Location of silkworm rm gene on the 3rd chromosome (TSUJITA
and SAKURAI) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18. Experimental induction of androgenesis, gynogenesis andpolyploidy in Bombyx mori by treatment with C02 gas(TAZIMA and ONUMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19. Autoradiographic studies of silkworm spermatogenesis using
tritiated thymidine (SADO) . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . .
20. Inter-breed competition in growing chickens (FUJISHIMA).. ..
21. Variance and covariance analysis of egg production and relatedcharacters in the domestic fowl (KAWAHARA and INOUE) . .
22. Estimation of genetic parameters of the productive traits ofJapanese quail (KAWAHARA and INOUE)....................
23. Further study on chromosome of plasma cell tumors of
BALB/c mice (YOSIDA, IMAI and POTTER).. . . . . . . . . . . . . . . . .
24. Chromosomes of normal plasma cells of mouse (YOSIDA and
IMAI)
25. Further study on chromosomes of sensitive and drug resistant
sublines of mouse lymphocytic neoplasm, P388, growing invitro (YOSIDA, OHARA, ROOSA and FUKAYA)
26. Chromosome study of survived sublines of mouse fibroblastic
in vitro line L·5 after treatment with 8-azaguanine (OHARA,
FUKAYA and YOSIDA)
27. The effect of 8-azaguanine on DNA synthesis of Yoshidasarcoma cells (YOSIDA, KURITA and TSURUTA).............
28. Chromosome aberrations induced in mouse bone marrow cellsby 20-methylcholanthrene (TSURUTA, KURITA, YOSIDA andSAKAKIBARA)
29. Autoradiographic analysis of the effect of Mitomycin C on
chromosomes of Ehrlich ascites tumor cells (KURITA, YOSIDA
and TSURUTA) . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ..
30. Alteration of karyotype in a transplantable granulocytic
leukemia (chloroleukemia) in the rat (SAKAI, YOSIDA andSHIMIZU)

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CONTENTS
31.

32.

33.
34.
35.
36.
37.

Population survey of Japanese and Korean black rats withchromosomal polymorphism (YOSIDA, FUKAYA, KANG, MORIGUCHI, OHARA and NAKAMURA)
Crosses between black rats characterized by chromosomal
polymorphism (YOSIDA, MORIGUCHI, FUKAYA and NAKAMURA)....................................................
Chromosomal polymorphism of the rat, Rattus norvegicus, andits segregation (YOSIDA, TSURUTA and MORIGUCHI)........
Chromosomes of male ants Aphaenogaster osimensis producedby parthenogenesis of workers (IMAI and YOSIDA)
Polyploid cells observed in male and queen ants of Aphaenogaster osimensis (1MAI and YOSIDA). . . . . . . . . . . . . . . . . . . . . . . .
Nuptial flight and multiple mating observed in the formicine
ant, Lasius niger (1MAI)........................
Ovary development of workers and queens in the ant
Aphaenogaster osimensis (IMAI).

B. Genetics, cytology and biochemistry of plants
38. Hereditary characteristics of three cytoplasms revealed bylong-time substitution experiments (KIHARA and TSUNEWAKI)
39. Preliminary study on free amino acids in the anthers of
nucleus-substitution lines (KIHARA) ... . . . . . . . . . . . . . . . . . . . .
40. Comparative gene analysis of common wheat and its ancestral
species II. Waxiness (TSUNEWAKI)
41. Comparative gene analysis of common wheat and its ancestral
species III. Growth habit and awnedness (TSUNEWAKI)....
42. Comparative gene analysis of common wheat and its ancestral
species IV. Glume hairiness (TSUNEWAKI)
43. Awn inhibitor in Redman wheat (TSUNEWAKI)
44. Genome analysis of the genus Eremopyrum (SAKAMOTO)
45. An intergeneric hybrid between Eremopyrum orientale (Linn.)
Jaub. et Spach. and Aegilops squarrosa Linn. (SAKAMOTO)..
46. Intergeneric hybrids between two Eremopyrum and Agropyron
species (SAKAMOTO) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47. Photoperiodic response of various Oryza species, VIII (KATAYAMA)....................................................
48. Diallel crosses among Sikkimese rice types, II (KATAYAMA). .
49. Embryo transplantation in the genus Oryza (KATAyAMA)....
.50. Anatomical changes in the seminal root of Oryza sativa after
the removal of adventitious roots (KATAyAMA)............

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

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52.

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68.
69.

70.

71.

Variations in the breeding system of a wild rice, Oryza
perennis (aKA and MORISHIMA).... . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of indices showing genetic plant types and theircorrelations with yielding capacity in a segregating
population of rice (MORISHIMA, aKA and CHANG)..........
Seasonal change in genetic plant type of rice (MORISHIMA,aKA and CHANG).........................
Analysis of genes controlling heading time in Taichung 65
and other rice varieties (TSAI and aKA) . . . . . . . . . . . . . . . . . .
Weakness of F 1 hybrids in the Sativa rice group (CHU, aKA
and T AKENAKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deterioration of Fl embryos in hybrids between Oryza sativa
and O. perennis subsp. barthii (CHU and aKA)...... . . . . . .
Fl pollen and embryosac sterilities in the Sativa rice group(CHU and aKA)...........................................
Developmental instability in leaves, panicle and culm internodes of rice (SAKAI and BALAL)
Genetical studies on clustered spikelets in rice (MORIMURA andSAKAI)
Biometric-genetic study on rachis deficiency in rice (WASANO
and SAKAI)...............................................
Studies on genetic relationships among leaves, culm internodes and the panicle in rice (BALAL and SAKAI) .. . . . . . . .
Cytogenetic studies in the genus Nicotiana (TAKENAKA) ....
Karyotypes in the genus Nicotiana (CHU and T AKENAKA). . . .
Genetical studies on vein formation in Nicotiana tabacum(SAKAI and WASANO)
Inheritance of developmental instability in leaves of tabacco(SHIMAMOTO and SAKAI)
Competition III forests and an evidence that plants of thesame genotype do not compete (SAKAI, MUKAIDE andTOMITA)..................................................
Callus tissue culture in Japanese morning glory (TAKENAKA,
YONEDA and CHU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic analysis of a tumorous strain in Japanese morning
glory (YONEDA)
Auxin destruction and growth in Japanese morning glory
stems (STONIER and YONEDA) .. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Induction of maize seedling catechol oxidase by maleic
hydrazide (ENDO and SCHWARTZ)..........................
Tulip breaking virus and synthesis of water soluble proteins

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CONTENTS

in tulip petals (OGAWA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

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C. Mathematical genetics
72. A stochastic model concerning the maintenance of genetic
variability in quantitative characters (KIMURA)............
73. Quasi linkage equilibrium (KIMURA) .. . . . . . . . . . . . . . . . . . . . . . . .
74. Simulation studies on the number of self-sterility alleles
maintained in a small population (KIMURA). . . . . . . . . . . . . . . .

85
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D. Genetics and biochemistry of microorganisms
75. Leaky mutants of flagellation genes in Salmonella (IINO and
76.
77.
78.
79.

80.

81.
82.
83.

MITANI)
Heteromorphism of flagella in a Salmonella mutant (IINO andMITANI)
Dimorphic nature of bundled flagella in a small-amplitude
mutant of Salmonella abortus-equi (MITANI and IINO)......
An assay for newly-synthesized flagellin of Salmonella (SUZUKI
and IINO)
Composition of chromosome fragments participating in P22
mediated transduction of fla· Hi· mot loci in Salmonella
typhimurium (ENOMOTO)
Dependency of argos mutant of Salmonella typhimurium
not only on uracil but also on arginine during arginine
inhibition (ISHIDSU) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Subunit analysis of g-complex antigens of Salmonella (YAMAGUCHI)
Mutation of g-complex antigen in Salmonella (YAMAGUCHI
and IINO)
Lack of host cell reactivation of bacteriophage TI inactivatedby 32p decay (IKENAGA and KONDO). . . . . . . . . . . . . . . . . . . . . . . .

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E. Radiation genetics in animals
84. Mechanisms underlying the enhancing effect of inducedmutation frequency after fractionated r-irradiation ofsilkworm gonia (TAZIMA and ONIMARU) .. . . . . . . . . . . . . . . . . . 96
85. Time of reversal of radiation dose-rate effect on mutationinduction in silkworm gonia (TAZJMA and FUKASE)
99
86. Further data on post-irradiation treatment of silkworm
spermatogonia with various metabolic inhibitors (TAZIMA
and ONIMARU)
100
87. Further studies on proliferation kinetics of silkworm spermatogonia during chronic r-irradiations (SADO). . . . . . . . . . . . 102

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

88.

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90.
91.

92.

93.

F.

Change in distribution of primary spermatogonia of thesilkworm at various phases of the cell cycle after irradiation(SADO and OISHI)..........................................
Radiosensitivity of silkworm spermatogonia during the cell
cycle I. Fate of primary spermatogonia irradiated in S phase(SADO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Variation in radiosensitivity during the early developmental
stage of the silkworm egg (MURAKAMI)...................
Enhancing effect of fractionated irradiation with 14 MeV
neutrons on the induction of visible recessive mutations
in silkworm gonia (MURAKAMI, KONDO and T AZIMA)
Chromosome aberrations induced in mouse bone marrow cellsby gamma irradiation (KURITA, TSURUTA, YOSIDA andSAKAKIBARA)
Persistent chromosome aberrations in mouse bone marrowcells after gamma irradiation (YOSIDA, KURITA, TSURUTA
and SAKAKIBARA)..........................................

Radiation genetics in plants
Photoreactivation of ultraviolet-induced mutation in maizepollen (MATSUMURA and MABUCHI)........................
95. Effects of dose fractionation of r-rays on mutation in maizepollen (MABUCHI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96. Storage and oxygen effects on r-ray induced mutation in
einkorn wheat (MABUCHI and MATSUMURA)................
97. Genetic effects of X-irradiated pollen in wheats (MABUCHI and
MATSUMURA)
98. Relation between polyploidy and radiosensitivity under chronic
condition (MATSUMURA and FUJII)
99. Development of mutated cells in diploid wheat (FUJII) . . . . . .
100. Effects of ultraviolet-rays on Arabidopsis thaliana (FUJII)....
101. Mutagenic efficiency of heavy ionizing particles in Arabidopsis
thaliana (FUJII, IKENAGA and LYMAN)

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107

109,

110

111

94.

G. Human genetics
102. Genetic epidemiology of Down's syndrome in Japan (MATSUNAGA)
103. Parental age and sporadic retinoblastoma (MATSUNAGA)
104. Genetically controlled variations of red cell acid phosphatase(SHINODA) .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
105. Alkaline phosphatase in human serum (SHINODA). . . . . . . . . . . .
106. Studies on haptoglobins (SHINODA) . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS

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107.

Cytogenetic survey of 127 cases with Down's syndrome (TONOMURA, OISHI and KIKUCHI)................................ 126
108. Identification of human somatic chromosomes from measurements of their areas (OISHI and TONOMURA) ............•• 127
109. Clinical conditions of patients with apparently normal
chromosomes, II (OISHI, KIKUCHI and TONOMURA) ........• 129
110. DNA replication pattern of the human Y chromosome(KIKUCHI)
130

H.

Technical note
Separax, a new Japanese cellulose acetate strip for electrophoresis (OGAWA)......................................... 131

111.

Books and papers published in 1965 by staff members..............

135

GENERAL STATEMENTOn the 20th of April, our Institute was honored by the visit of His
Majesty the Emperor and Her Majesty the Empress. This was the second
visit of His Majesty since 1954. The Emperor and the Empress haveinspected our laboratories, listened to our recent progress and encouraged
our staff members.
On the 20th of November we had an augural ceremony for the completion of a two-storied building which comprises on the first floor rooms forseed storage, herbarium, exhibit and research and on the second floor
an auditorium with 200 seats. We are most grateful to the Rockefeller
Foundation, who has given us a grant for erecting the first floor.
This year (1965) the geneticists were busy in celebrating Mendel'scentennial all over the world. Our members attended various meetings
as given below.
Impact of Mendelism on Agriculture, Biology and Medicine. New
Delhi, India, February. Participant: H. KIHARA.
2. G. Mendel Memorial Symposium. Brno and Prague, Czechoslovakia,
August. Participant: C. OSHIMA.
3. Mendel Centennial. Fort Collins, U.S.A., September. Participant:
H. KIHARA.
4. Lectures and Exhibits in Commemoration of Mendel's Centennial.
Tokyo and Kyoto, Japan, October.
In this paragraph it was our custom to mention the honors and degrees
given to our members. However we will mention only the award given
this year to Dr. M. KIMURA. He has received Weldon Memorial Prize
from the University of Oxford for his contributions to population genetics.
The prize is given every three years and is a great honor to him as wellas to our Institute.
1.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

STAFF(At the End of 1965)
Director

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

1. Department of Morphological Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag.
The 2nd Laboratory
SAKAGUCHI, Bungo, D. Ag., Head of the Laboratory
SADO, Toshihiko, D. Ag.; KOBAYASHI*, Susumu
2. Department of Cytogenetics
TAKENAKA, Yo, D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
MORIWAKI, Kazuo, D. Sc. (in U.S.A.); OHARA*, Hiroshi, D. Sc.;
IMAI*, Hirotami; FUKAYA*, Takako; TSURUTA*, ReikoThe 2nd Laboratory
T AKENAKA, Yo, D. Sc., Head of the Laboratory
YONEDA, Yoshiaki, D. Sc. (in U.S.A.)
3. Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department
The 1st Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
WATANABE*, Takao, K.
The 2nd Laboratory
TAIRA, Toshifumi, D. Sc., Head of the Laboratory
SAKAMOTO, Sadao, D. Ag.; KATAYAMA*, Tadao C., D. Ag.;NAKAI*, Yasuo; HORI*, Tadaaki* Research members under grant from other organizations or visiting researchers.

STAFF3

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

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

The 2nd Laboratory
OGAWA, Yoshito, M. D., Head of the Laboratory
ENDO, Toru, D. Ag. (in U.S.A.)The 3rd Laboratory
TSUJITA, Mitsuo, D. Ag., Head of the Laboratory
SAKURAI, Susumu; NARISE*, Sumiko, M. D.

50 Department of Applied Genetics
SAKAI, Kan-Ichi, Do Ago, Head of the Department

The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, Do Ag.; FUJISHIMA, Tohru;
NARISE*, Takashi, D. SCo; INOUE*, Teruo

The 2nd Laboratory
SAKAI, Kan-Ichi, D. Ago, Head of the Laboratory
IYAMA, Shin-ya, Do Ago; SHIMAMOTO*, Yoshiya;
EL-BALAL*, Mohamed S.; HAYASHI*, Shigesuke;
MORIMURA*, Katsuyoshi; TOMITA*, Koji; TSUZAKI*, Kazuo;
WASANO*, Kikuwo

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

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

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

The 3rd Laboratory
MATSUMURA, Seiji, Do Ago, Head of the Laboratory
IKENAGA, Mituo; MABUCHI, Tomoo* Research members under grant from other organizations or visiting researchers.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

7. Department of Human Genetics
MATSUNAGA, Ei, M. D., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, M. D., D. Sc., Head of the Laboratory
OISHI, Hidetsune; SHINODA, Tomotaka (in U.S.A.)The 2nd Laboratory
TONOMURA, Akira, D. Sc., Head of the Laboratory
KIKUCHI, Yasumoto, D. Sc.; OHDO*, Shozo
8. Department of Microbial Genetics
IINO, Tetsuo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi; Ml'fANI*, Michiko; YAMAGUCHI*, Shigeru
The 2nd Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.; ISHIDSU, Jun-ichi; SUZUKI*, Yasuko
9. Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
HIRAIZUMI, Yuichiro, D. Sc. (in U.S.A.)
10. Experimental Farm
MATSUMURA, Seiji, D. Ag., Head of the Farm
MIYAZAWA, Akira
Honorary Members and Parl:-Time Sl:aff

KOMAI, Taku, D. Sc., Member of Japan Academy, Emeritus Professor of
Kyoto University
KUWADA, Yoshinari, D. Sc., Member of Japan Academy, Emeritus Professor of Kyoto University
LILIENFELD, Flora A., Ph. D.
OGUMA, Kan, D. Ag., Emeritus Professor of Hokkaido University
TANAKA, Yoshimaro, D. Ag., D. Sc., Member of Japan Academy, Emeritus
Professor of Kyushu University* Research members under grant from other organizations or visiting researchers.

COUNCIL

5

Depart:ment: of Administ:rat:ion

MORINAGA, Norihiro, Head of the Department
KANAMORI, Shigeru, Chief of the General Affairs Section
TANAKA, Mutsuo, Chief of the Finance Section
Associat:ion for Propagat:ion of t:he Knowledge of Genet:ics

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

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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

PROJECTS OF RESEARCH FOR 1965
Department of Morphological Genetics

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

Cytogenetical studies on tumor cells (YosmA, KURITA, OHARA, IMAI,FUKAYA, TSURUTA and MORIWAKI)
Mechanism of chromosomal abnormalities by treatment with chemicals(YosmA, KURITA, TSURUTA and MORIWAKI)
Study on chromosomal polymorphism of Muridae (YosmA, FUKAYA, MORIGUCHI and NAKAMURA)
Experimental breeding and genetics of mice and rats (YosmA, KURITA,SAKAKIBARA, MORIGUCHI and MORIWAKI)
Correlation between taxonomy and karyology of ants (IMAI and YosmA)
Interspecific hybridization in Nicotiana (TAKENAKA)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus Lannesiana (TAKENAKA)
Cytogenetics of Oryza species (TAKENAKA, YONEDA and CHU)
Morphological and genetical studies on some plant tumors (TAKENAKA,
YONEDA and CHU)
Department of Physiological Genetics

Genetic studies on insecticide resistance in Drosophila pseudoobscura(OSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE, T. K.)
Studies on chromosomal aberrations of natural populations of Drosophilamelanogaster (OSHIMA and WATANABE, T.)
Eye-pigment formation in Drosophila (TAIRA)PROJECTS OF RESEARCH FOR 1965

7

Cytodifferentiation in Drosophila fatbody (TAIRA)
Nucleotide-pool in insects (TAIRA)
Genetical studies on isozymes in Drosophila (TAIRA)
Nucleus substitution in wheat and related species (KIHARA)
Comparative gene analysis with reference to the origin of wheat (KIHARA
and TSUNEWAKI)
Geographical distribution of necrosis genes in wheat (TSUNEWAKI and
NAKAI)
Genetic studies of wheat aneuploids (TSUNEWAKI)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Genetic bases of ecological differentiation in Agropyron (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Morphological studies of Oryza (KIHARA and KATAYAMA)
Investigation of photoperiodic responses of Oryza species (KATAYAMA)
Depart:ment: of Biochemical Genet:ics

Studies on transformation in higher organisms (NAWA, YAMADA and
TSUJITA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA and TSUJITA)
Studies on a gene for retarded moulting (rm) in the silkworm (TSUJITA)
Studies on the chromogranule formation in larval hypodermal cells of thesilkworm (TSUJITA and SAKURAI)
Analysis of genetic action on cell differentiation in higher organisms(TSUJITA and NAWA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA)
Genetical and biochemical studies of plant virus (OGAWA)
Comparative studies on seed proteins of rice plant by electrophoretic
analysis (SAKURAI)
Genetics on isozymes in plants (ENDO)
Depart:ment: of Applied Genet:ics

Studies on developmental instability in poultry (SAKAI, KAWAHARA andFUJISHIMA)
Quantitative genetic studies in poultry (KAWAHARA, FUJISHIMA and INOUE)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Studies on competition in plants and animals (SAKAI, IYAMA, FUJISHIMA
and NARISE)

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

Estimation of genetic parameters in forest trees (SAKAI, HAYASHI andTOMITA)
Developmental genetics of quantitative characters in plants (SAKAI, SHIMAMOTO, El-BALAL, MORIMURA and W ASANO)
Genetic studies on developmental instability in plants (SAKAI and SHIMAMOTO)
Studies on the effects of linkage disequilibrium in Drosophila populations(IYAMA)
Genetic studies of isolating barriers in Oryza (OKA and CHU)
Survey of geographical variation in Oryza perennis (MORISHIMA and OKA)
Experiments on natural selection in wild and cultivated rice forms (MORISHIMA and OKA)
Analysis of sterility genes in Oryza (OKA and MORISHIMA)
Analysis of genetic plant types (MORISHIMA and OKA)
Department of Induced Mutation

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

Genetics of retinoblastoma (MATSUNAGA)
Down's syndrome in Japan (MATSUNAGA, TONOMURA and OISHI, H.)
Cytogenetics in man (TONOMURA, OISHI, H. and KIKUCHI)
DNA replication in human chromosomes (KIKUCHI)
Biochemical studies on plasma proteins and enzymes (SHINODA)
Chemical modification of ribonucleic acid and their constituents (SHINODA)
Department of Microbial Genetics

Immunogenetics of Salmonella (IINO, ENOMOTO, YAMAGUCHI and MITANI)
Genetics of motility in bacteria (ENOMOTO)
Genetics of cellular regulatory mechanisms (SUZUKI, H., ISHIDSU andSUZUKI, Y.)

FOREIGN VISITORS IN 1965

9

Genetic fine structure analysis on microorganisms (ISHIDSU and IINO)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA)
Uses of computers in the theoretical studies of population genetics(KIMURA)
Effects of radiation-induced mutation on fitness (HIRAIZUMI)
Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)FOREIGN VISITORS IN 1965
LI, S., Taiwan Sugar Experiment Station, Taiwan
CHENG, M. C., Taiwan Sugar Experiment Station, Taiwan
BANDURSKI, R. S., Dept. of Botany, Michigan State College,
Mich., U.S.A.
ROBINSON, H. F., Institute of Biological Sciences, North CaroFeb. 9lina State Univ., N. C., U.S.A.
Mar. 1-2 ZELLE M. R., Division of Biological and Medical Research,
Argonne National Laboratory, 111., U.S.A.
Mar. 5-6 GUSTAFSSON, A., Institute of Genetics, Royal College ofForestry, Stockholm, Sweden
Mar. 5-6 KONZAK, C. F., Agronomy Dept., Washington State Univ.,
Wash., U.S.A.
Fu, T. T., Ministry of Economic, Republic of China GovenApr. 3
ment, Taiwan
SAUNDERS, J. W., Dept. of Biology, Marquette Univ., Wis.,
Apr. 5
U.S.A.
RUBEN, 1. N., Dept. of Biology, The Reed Institute, Ore.,
Apr. 5
U.S.A.
STERN, H., Dept. of Botany, Univ. of Illinois, 111., U.S.A.
Apr. 5SCHNEIDERMAN, H. A., Dept. of Biology, Western Reserve
Apr. 5
Univ., Ohio, U.S.A.
Apr. 15SHEBESKI, 1. H., Dept. of Plant Science, Univ. of Manitoba,
Manitoba, Canada
Apr. 19-20 MICHAELIS, P., Max-Planck-Institut fur Zuchtungsforschung,
Abteilung fUr Plasmavererbung, 5 Kaln-Vogelsang, West
Germany
May 8-13 SAMPATH, S., Central Rice Research Institute, Cuttack, India
Jan.
Jan.
Jan.

8
8
30

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

May 10
May 25June

8-9June 16
June 21
June 25
July
July
July

8
8
9

July 19
July 31
Aug. 7
Aug.
Aug.
Sep.

7
7
1

Sep.

1

Sep

10

Sep. 11
Sep. 27
Sep. 29
Oct

23

Nov.

9

Nov.
Nov.

9
9

KOJIMA, K., North Carolina State Univ., N. C., U.S.A.
AFRIKIAN, E. G., General Microbiology, Antibiotics Instituteof Microbiology, Academy of Sciences of the Armenian
S.S.R. Charenty Street 19, Erevan, U.S.S.R.
MCCLUNG, A. C., International Rice Research Institute, Los
Banos, Philippines
SVED, J., Dept. of Genetics, Univ. of Adelaide, South Australia
OJIMA, M., Institute Agronomico, Secao Frutas de Clima
Temperado, Sao Paulo, Brasil
LAMBERS, H.B., Wageningen Agricultural Univ., Wageningen,
Netherlands
LEE, I. S., Univ. of Seoul, Korea
LEE, S. C., Institute of Plant Circumstances, Korea
VELTMAN, P. L., Vice President, W. R. Grace & Co., Clarksville, Md., U.S.A.
OLAH, L., Biology Dept., Southern Illinois Univ., 111., U.S.A.
NEEL, J. V., Univ. of Michigan, Medical School, Mich., U.S.A.
GLASS, B., State Univ. of New York at Stony Brook, N. Y.,
U.S.A.
GLASS, S. S., Baltimore, Md. and Stony Brook, N. Y., U.S.A.
WELCK, C. A., Michigan State Univ., Mich., U.S.A.
LUERS, H., Institut fUr Genetik, Freie Univ. Berlin, West
Germany
LUERS, T., Institut fUr Genetik, Freie Univ. Berlin, West
Germany
BEACHELL, H. M., International Rice Research Institute, Los
Banos, Philippines
SCHICK, R., Institut fUr Pflanzenzuchtung, Deutsche Akademiedel' Landwirtschaftswissenschaften, Berlin, East Germany
LI, H. W., Institute of Botany, Academia Sinica, Nangkang,
Taipei, Taiwan
LUGG, J. W. H., Dept. of Biochemistry, Univ. of Western
Australia, Nedland, Western Australia
MARAMOROSCH, K., Boyce Thompson Institute for Plant
Research, N. Y., U.S.A.
Kou, K. E., Taipei District Agricultural Improvement Station,
Taiwan
POUN, T. T., Taiwan Agricultural Research Institute, Taiwan
JAN, S. C., Chia-i Branch Station, Taiwan Agricultural
Research Institute, Taiwan

FOREIGN VISITORS IN 1965

Nov.

9

Nov.

9

Nov. 12
Nov. 13
Nov. 15
Nov. 15
Nov. 16
Nov. 18
Nov. 18
Nov. 19
Dec.

8

11

TSUAN, S. R., Tainan District Agricultural Improvement
Station, Taiwan
HON, C. T., Taichung District Agricultural Improvement
Station, Taiwan
KAIJSER, K., Paediatic Dept. of Central Hospital of Eskilstuna,
SwedenLENZ, W., Institut fUr Humangenetik der Univ., Munster,West Germany
WEICKER, H., Institut fUr Humangenetik der Univ., Bonn,
Germany
FLATZ, G., Univ.-Kinderklinik und Poliklinik, Bonn, West
Germany
DAY, R. I., Planned Parenthood-World Population, U.S.A.
JENNINGS, P. R., International Rice Research Institute, Los
Banos, Philippines
TANAKA, A., International Rice Research Institute, Los Banos,
Philippines
HULSE, F. S., Dept. of Anthropology, Univ. of Arizona,Ariz., U.S.A.
TOMES, M. 1., Dept. of Botany and Plant Pathology, Purdue
Univ., Ind., U.S.A.

RESEARCHES CARRIED OUT IN 1965
A.

1.

GENETICS, CYTOLOGY AND
BIOCI-IEMISTRY OF ANIMALSDistribution and persistence of lethal genes in natural
populations of Drosophila melanogaster1l
Chozo OSHIMA and Takao WATANABE

Many male flies were captured simultaneously in October of the past
two years from large natural populations in five different graperies of the
Kofu and Katsunuma territory. The frequencies of complete recessive
lethal chromosomes were 17.06% and 19.46% in isolated about 700 second
chromosomes from each different male fly sampled in 1963 and 1964
respectively.
A series of allelism tests between 188 lethal genes were performed by.a total of 16,126 crosses and the results are given in Table 1.
Table 1. Results of allelism test between lethal genes isolated
from Kofu and Katsunuma populations in 1963 and 1964.

Population
No. of lethal·chromosomes
Allelic rate
(%)

1964

1963

Year

within within between wholewithin within between wholeKF
KN KF&KN territoryKF
KN KF & KN territory
61

53

114

114

31

43

74

74

3.28

2.18

2.47

2.64

3.87

3.65

4.13

3.92

between old and new lethals
No. of lethal
chromosomes
Allelic rate

72

97

(%)

2.65

KF: Kofu population, KN: Katsunuma population

Six lethal genes were found to be distributed over a wide area (15 x 3 km 2 )of the Kufu and Katsunuma territory and their frequencies had been highly
stable during two years as given in Table 2.
1) These works (1~5) were supported by a grant from the Japan Society for the
Promotion of Science as part of the Japan-U.S. Cooperative Science Program.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
Table 2. Number of persistent lethal genes in Kofu andKatsunuma populations during two years.
Number of appearance andtheir linked inversion

Symbol

1963

l201
l202
l203
l204 *
1207 *
l208+ or In(2R)C+ or In(2R)C+++ or In(2L)B
+

13
8
6
6
4
3

Locus on thegenetic map

1964

I
6
6
3
7
2
1

+ or In(2R)C+ or In(2R)C+ or In(2R)C+ or In(2R)C
+
+

47.9
33.7
5.5
32.1
58.4
16.7

* allelic lethal genes were found among lethal genes isolated from the same
population in 1959.

The number of such persistent lethal genes reached to about one thirdof the total of lethal genes and their zygotic frequencey in the population
was estimated to be 0.12. One third of them were associated with inversion
C on the right arm, but they were located on the left arm except for gene1207.

2. Pre-adult viability of heterozygotes for some persistent lethal
chromosomes (a persistence mechanism of lethal genes)
Takao WATANABE and Chozo OSHIMA

One double-lethal chromosome, ll(2)301a and three single-lethal chromosome, 1(2)301d, 1(2)302c, 1(2)302d, were used as persistent lethal
chromosomes, because they contained the same lethal gene as 1(2)201 or
1(2)202, located at 47.9 or 33.7 on the genetic map. These persistentlethal chromosomes having no inversion were picked up for the analyticalexperiment. On the other hand, about ten chromosomes were picked up
randomly from each group of four homozygous viability classes: normal,
subvital, semi-lethal and lethal. Then, the viability of heterozygotes
having combination of the persistent and the various other chromosomes
was estimated by Cy-Pm technique. The results are given in Table 3.
The persistent lethal chromosomes had a tendency to produce better
heterozygous viabilities than normal heterozygotes, when they werepaired with various kinds of chromosomes. On the contrary, the effectof subvital chromosomes was significantly deteriorating the viabilityof heterozygotes by about 4% compared with that of normal chromosomes,

RESEARCHES CARRIED OUT IN 1965
Table 3.

Pre-adult viabilities of heterozygotes for persistent lethal
chromosomes and various kinds of chromosomes.
Mating

n
L
LL

n
sv
sl
I

15

n

sv

sl

nln
Lin
LL/n

nlsv
L/sv
LL/sv

nisi
L/sl
LL/sI

n

L

LL

nln
nlsv
nisI
nil

Lin
L/sv
L/sl
Lil

LL/n
LL/sv
LL/sl
LLII

Pooled basis

nil
Lil
LLII

No. of counted
flies

Relative viability

103,035
161,218
52,6251.0537 ±0.00929
1. 0585 ± 0.00748
1.0618 ± 0.01304

88,27883,19868,59976,803

1. 0752 ± 0.01027]
1.0321 ± 0.01014] **1,0686±0.01156 *1. 0554 ± 0.01074

The viability of Cyl Pm fly = 1.0000.
n: normal, sv: subvital, sl: semi-lethal, I: lethal chromosome.
L, LL: persistent lethal chromosome.
** significant at the 1% level, * significant at the 5% level.

but other semi-lethal and lethal chromosomes expressed slight and nonsignificant deleteriousness by 1 or 2%, when they were paired with normal
and persistent lethal chromosomes. The results suggest the existence of
epistatic gene complexes in the persistent lethal chromosomes. Thismight be one of persistence mechanisms of the particular lethal genes
in natural populations.

3. Abnormal segregation in the offspring of persistentlethal heterozygous male flies (a persistencemechanism of lethal genes)
Chozo OSHIMA and Takao WATANABE

When the viability of heterozygotes for each lethal chromosome:ll(2)301b, l(2)301b and l(2)301c, was examined by 'Cy-Pm technique, it
was found to be strikingly better than that of normal heterozygotes.
These chromosomes had the inversion, In(2R)C, on the right arm, whose
frequency has been maintained in about 30% of natural populations of
the Kofu and Katsunuma territory. The single-lethal chromosome has
one lethal gene located at 47.9 on the genetic map and the double-lethal
chromosome has in addition another lethal gene located at 33.7. In the

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

reciprocal matings, two kinds of offspring emerged, one having the lethal
and the other the Pm chromosome; they were scored in five successive
subcultures. The results are shown in Table 4.
Table 4.

Mating!i1

X(')

Cy/n-lxPm/LL
Cy/n-lxPm/L

Abnormal segregation in the offspring of persistentlethal heterozygous male flies.
no. ofNo. and
period of
subculture

No. of
F 1 flies
per day

non-

Pm

Pm

1. 3 days
II. 2 days
III. 2 days
IV. 2 days
V. 3 days

69.2
124.0
124.7
83.7
67.7

6090
6936
6934
5021
5890

3664
4723
4785
2846
3657

1. 66 ± 0.035J
1.47±0.028 **1.45±0.027}*
1.76±0.041
1.61 ±0.034}*

93.9

30871

19675

1.57

82.3
142.5
130.5
92.8
77.6

5223
5945
5436
3863
4780

4905
5737
5268
3744
4762

1.06±00021
1.04±0.019
1.03±0.021 *1.03±0.024
1.00±0.O21

105.1

25247

24416

1.03

12 days

Ratio of(mean)

Cy/LLxPm/n-l
Cy/LxPm/n-l

1. 3 days
II. 2 days
III. 2 days
IV. 2 days
V. 3 days

12 days(mean)

non-Pm/Pm

1

L, LL: persistent lethal chromosome.

n-l: normal, subvital, semi-lethal and lethal chromosome.
** significant at the 1% level, * significant at the 5% level.

When the rasults of the reciprocal matings were compared, a great
difference in the ratio of non-Pm and Pm flies in the offspring wasnoticed. The ratio of the mating in the upper part of Table 4 was on
the average 1.5 times higher than that in the lower part of the sametable and fluctuated significantly among the five successive subcultures.
Such abnormal segregations could be brought about by some complicatedfactors, but it could be simply assumed that the results may be ascribedto a difference in activity (certation effect) between two kinds of sperm,
one with Pm chromosome and the other with the persistent lethal
chromosome.
On the other hand, when male flies heterozygous for the persistent lethal
chromosome and one of en bw, bw, dp, en bw, el, ltd or vg marked chro-

RESEARCHES CARRIED OUT IN 1965

17

mosomes were mated with such mutant homozygous female flies, thegreat majority of emarged Fl flies (80-90%) were wild type and mutant
flies were very few. In these cases, the abnormal segregations werequite clearly detected like in the case of SD chromosome investigatedby HIRAIZUMI. The paracentric inversion (In(2R)C) was assumed to occur
in the persistent lethal chromosome as well as in SD or NH chromosomes,
but the lethal gene located at 47.9 in the former was not the same as any
lethal gene in the latter.

4.

Chromosomal polymorphism in the Kofu and Katsunuma
natural populations
Taishu WATANABEll and Chozo OSHIMA

In late October of the past three years, many female flies were captured
simultaneously from the same natural populations in the Kofu andKatsunuma territory. These female flies were allowed to lay eggs
individually in a vial and then the salivary chromosomes of 100 larvae
Table 5.

Frequencies of inversions in the Kofu and Katsunuma
natural populations during three years
Kofu pop.

Population
Year
Second chromosome
standard (left arm)
In(2L)AIn(2L)B

standard (right arm)
In(2R)C

Third chromosome
standard (left arm)
In(3L)E
In(3L)F

standard (right arm)
In(3R)GIn(3R)HIn(3R)IIn(3R)J
1l

Katsunuma pop.

1963

1964

1965

67.0
0.5
32.5
73.0
27.0

64.0
0.0
36.0
74.5
25.5

92.0
6.5
1.5
63.5
11.0
14.0
11.0
0.5

95.0
5.0
0.0
76.0
9.0
7.0
8.0
0.0

1963

1964

1965

57.5
0.0
42.5
70.0
30.0

68.0
0.0
32.0
79.0
21.0

73.0
0.0
27.0
74.5
25.5

65.0
0.0
35.0
74.5
25.5

88.0
11.5
0.0
61.0
20.5
12.5
6.0
0.0

89.5
8.5
2.0
61.0
18.0
10.0
11.0
0.0

89.5
10.5
0.0
71.0
12.0
8.5
8.5
0.0

89.5
10.0
0.5
58.5
26.5
8.0
7.0
0.0

(%)

(%)

Department of Biology, Faculty of Science, Kyushu University.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

sampled from different cultures were observed. The results of therepeated experiments during three years are given in Table 5.
Individual frequencies of the common inversion: In(2L)B, In(2R)C,
In(3L)E, In(3R)G, In(3R)H and In(3R)I, in both natural populations weresimilarly maintained, but In(3R)G only seemed to increase about two times
in 1965.
As to the mechanism for maintaining each frequency of these common
inversions in equilibrium, it was indicated in a previous report (No. 15)that total observed numbers of double inversion heterozygotes of second
and third chromosomes were higher than expected.
In the present experiment, about 600 male flies, captured from a
Katsunuma population simultaneously, were individually mated with several
virgin female flies of Samarkand strain. The salivary chromosomes of 8
or 10 Fl larvae of each culture were observed. Then, the chromosomal
types of 300 male flies could be determined and the frequency of eachkind of chromosomes was found. The expected numbers of all possible
homozygous and heterozygous combinations of the standard and various
inversions of second and third chromosomes were calculated, using theobserved frequencies of each chromosome, on the basis of Hardy-Weinberg's
Table 6. Comparison of observed and expected numbers of various
chromosomal types of second and third chromosomes.
Chromosomal typeI Observed Total Expected Total Difference
numbernumberSecond chromosome Third chromosome
homo (+and inv.) homo (+and inv.)

32

32.55

single iny. hetero homo (+and iny.)
homo (+and iny.) single iny. hetero
single iny. hetero single iny. hetero

46
46
65

double iny. hetero home (+and iny.)
double iny. hetero single iny. hetero
home (+and iny.) double iny. hetero
single iny. hetero double iny. hetero
double iny. hetero double iny. heterohomo (+and iny.) triple iny. hetero
single iny. hetero triple iny. hetero
double iny. hetero triple iny. hetero
Total

-0.55

157

46.74
48.85
70.14

165.73

-8.73

21
23
26
21
11

102

15.82
23.72
18.64
26.79
9.05

94.02

+7.98

1
7
1

9

2.24
3.18
1.07

6.49

+2.51

300

298.79RESEARCHES CARRIED OUT IN 1965

19

law and the observed numbers of all chromosomal types found in thesamples were compared with the expected numbers as shown in Table 6.
It was confirmed by the results that the equilibrium states of thesecommon inversions could be maintained by heterosis of double or triple
inversion heterozygotes.

5.

The effect of insecticide selection in experimental
populations of D. pseudoobscura
Chozo OSHIMA and Taishu WATANABE

About forty homozygous strains of four kinds of chromosomes, ST, AR,
CH and PP, were used in the experiments. These strains, originated from
the Mather population in California, had been established by Dobzhansky
in 1963.
Three initial populations were made with given frequencies of fourchromosomes represented in Table 1; further these populations weredivided into two A and B. Flies in population A were exposed toinsecticide (1 or 2% DDT or 0.1% Dieldrin) test paper for one hour andtransferred into a new cage in every new generation. Flies in population
B were transferred into a new cage without exposure. The salivary
chromosomes of 150 larvae hatched from sampled eggs were observed
for detecting the frequency of each chromosome in these populations.
The varying frequencies of chromosomes from F 2 to F 20 in the populations
IA and IB and from F 2 to F12 in the populations IIA and lIB, IlIA and
IIIB were observed as shown in Table 1.
The frequency of ST chromosomes has increased in both selected andnon-selected populations, but after F 7 the increase in selected populations
was greater than in non-selected populations. On the contrary, AR
chromosomes decreased after F 7 in selected populations, but did not change
so much in non-selected populations, and rather increased after F12 in
the population lB. The frequencies of both CH and PP chromosomes havedecreased gradually in all populations except the selected population lIlA.
The initial frequency of PP chromosome in the population lIlA has been
maintained or rather slightly increased. These results seem to suggestthat the striking evolutionary changes in natural populations of D.
pseudoobscura in California during about twenty years might be due toinsecticide selection: ST chromosomes increased and AR chromosomes
underwent changes in the opposite direction. CH chromosome decreased
and became rare and PP chromosome emerged with spectacular frequency.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
Table 1. Changing frequencies of four kinds of chromosomes in DDTor Dieldrin selected and non-selected populations.
F2

Fa
IA (DDT selected)

ST 33.3
AR 26.3
Pop. ICH 22.7
PP 17.7
IE (non-selected)

ST 26.3
AR 28.0
Pop. IICH 26.3
PP 19.3
IIB (non-selected)

ST 40.0
AR 40.0
Pop. IIICH 17.0
PP 3.0IlIB (non-selected)

F7

F9

F l2

F 15

F20

ST 42.045.753.765.372.367.065.052.7
AR 24.725.726.322.0 17.0 27.0 34.3 46.7
CH 18.3 17.3 10.0 7.0 6.3 4.7 0.7 0.0
PP 15.0 11.3 10.0 5.7 4.3 1.3 0.0 0.7
Fa

F,

F5

F7

F9

F l2

ST
AR
CH
PP

43.7
19.7
15.3
21.3

41.0
30.0
9.7
19.3

43.0
29.7
10.3
17.0

71.0
16.7
4.3
8.0

84.3
5.7
6.0
4.0

90.3
2.0
4.7
3.0

ST
AR
CH
PP

36.3
21.7
23.3
18.7

45.7
29.7
14.7
10.0

49.3
27.3
13.3
10.0

52.3
30.0
12.0
5.7

64.3
25.3
5.3
5.0

68.7
26.3
3.0
2.0

F2

Fa

F,

F5

F9

F l2

48.0
32.7
16.3
3.0

52.7
36.7
6.3
4.3

59.3
32.0
1.7
7.0

73.2
19.7
0.9
6.1

93.0
3.0
0.3
3.7

90.3
4.7
0.0
5.0

ST 41.3
AR 43.3
CH 14.0
PP
1.3

59.0
33.7
6.3
1.0

63.6
30.4
2.5
3.5

73.7
20.7
3.7
2.0

87.3
11.0
1.3
0.3

90.3
8.7
1.0
0.0

F1
IlIA (DL selected)

F5

ST 40.3 55.0 55.3 73.3 77.0 92.7 86.3 87.0
AR 18.3 23.0 24.3 15.7 18.7 6.3 7.7 9.7
CH 22.0 11. 7 8.3 4.0 0.7 0.0 0.0 0.0
PP 19.3 10.3 12.0 7.0 3.7 1.0 6.0 3.3

F2
IIA (DL selected)

F,

ST
AR
CH
PP

21

RESEARCHES CARRIED OUT IN 1965

6.

Functional differentiation of larval fatbody cells in
Drosophila melanogasterToshifumi TAIRA and Taheir M. RrzKrl)

Physiological analyses: As mentioned in our previous report, in normal
condition Drosophila larval fatbody consists of two kinds of functionallydifferentiated cells; about thousand cells in the anterior region are concerned with producing tryptophan pyrrolase and about 700 cell in theposterior region contribute to the production of pterine dehydrogenase.
Experimental induction to produce those enzymes by feeding the respective precursors to various larval stages gave the following results (Table 1).
Table 1. Relative amounts of kynurenine and isoxanthopterin extracted
from 96 hr old larvae, by feeding L-tryptophan or 2-amino4-hydroxypteridine (AHP) to 65 hr old larvae, at 25°C.
FeedingNutrients only
Tryptophan (0.03 M)
AHP (0.02M)(~)

Kynurenine(pg)

Isoxanthopterin
(pg)

45
45
45

1.62
3.42
0.82

0.58
0.35
1.39

No. of LarvaeA) L-tryptophan feeding (0.03 M, pH 8.0): Induced kynurenine cells in
the posterior region were revealed only in larvae treated before the 70 hrstage. The induction of tryptophan pyrrolase in the posterior region could
not be suppressed by pre-treatment with chloramphenicol (0.06 mM) fed
to the larvae. At the pupation stage (108-110 hrs), however, the induced
kynurenine cells in the posterior region changed into pteridine cells
normally produced in this region. This replacement of cellular inclusions in an early pupa was more rapid in individuals grown on a mediumrich in nutrients at the larval period.
B) 2-amino-4-hydroxypteridine feeding (0.02 M, pH 8.5): The inductionof pterine dehydrogenase was clearly observed within a half hour in theanterior region, which normally contained only kynurenine cells, when the
inducer was given to the larvae before the 93 hr stage. Such an induction
was effectively suppressed by feeding chloramphenicol (0.06 mM). The
induced pteridine cells in the anterior region changed into the normally
produced kynurenine cells, at the pupation stage (108-110 hrs). Rich
nutrients given through the whole larval life accelerated the replacementof cellular inclusions.
From these observations, the following conclusions may be drawn:1) Department of Zoology, University of Michigan, Ann Arbor, Michigan, U.S.A.

'22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16(1) The inductivityof tryptophan pyrrolase is recognized in every cell of

1arval fatbody before the 70 hr stage. Then, it is gradually lost from thecells of the posterior region, and it can not be detected there in any cell
after the 85 hr stage. This indicates that the gene which controls the
production of tryptophan pyrrolase becomes inactive in the cells of theposterior region. (2) Pterine dehydrogenase can be induced in larval
fatbody cells until the larvae reach nearly the pupalium formation time
(96 hrs). The gene controlling the formation of pterine dehydrogenase isalways active in the anterior as weIl as in the posterior region. This iscontradictory to the actual observation that normal cells in the anterior
region have no isoxanthopterin, which is converted from 2-amino-4hydroxypteridine by the action of pterine dehydrogenase. Such an
apparent loss of pterine dehydrogenase in the anterior region appears to
be caused by the relative concentration of those inducers which areproduced in vivo. (3) Larval fatbody is fully enclosed with surrounding
body fluid, which contains many soluble substances. If it is so, the induction of pterine dehydrogenase should take place in every fatbody ceIl·simultaneously. In fact, the induction occurs successively in the adjacentcells from tail to head. Along the direction from tail to head, particularly,
the appearance of induced cells is faster on the dorsal than on the vental
side. These observations remind of the "bipotential hypothesis" in'embryology. However, it remains to be elucidated how does the gene
control the bipotentiality in the development. (4) The present induction
can not fundamentaIly change any system of the gene action.
Fate of larval fatbody cells in pupal development: The morphologicalchange of larval fatbody through the whole pupal period were observedin the living condition under UV-microscope. In an early pupa (108 hrstage), the histolysis of larval fatbody occurred successively starting fromthe front of the anterior region. Most of kynurenine in the cellular
granules were converted into 3-hydroxykynurenine around the 110 hr stage.
The dorsal leaf-shaped cells of the posterior region were also released byhistolysis. Those detached ceIls migrated to the developing thorax andhead. Consequently, they were incorporated into every organ tissue ofthe adult fly. In the process of compound eye formation, two kinds of,cells, having 3-hydroxykynurenine or pteridine, were surely seen to beincorporated into eye tissue. These incorporated ceIls might be theinitiators of the pigmentary ceIls of the compound eye.
Most cells of the posterior region assembled to surround the gonads.
In the male fly, the number of pteridine granules in each cell of theposterior regions gradually increased, but not in the female fly. It is a,question, if the activation of the pteridine enzyme might be closely related

RESEARCHES CARRIED OUT IN 196523

to the sexual hormone.

7. Further evidence supporting the optimum heterozygosity
hypothesis with respect to viability in a populationof Drosophila melanogaster 11
Terumi MUKAIAs previously reported (MUKAI 1964 Genetics 50: 1-19), spontaneous
mutant polygenes controlling viability have been accumulated independently
in 104 second chromosome lines which originated from a single second
chromosome.
In Generation 32, the degree of dominance of accumulated mutant
polygenes controlling viability was tested in heterozygous condition witha chromosome thought to be identical to the original chromosome, adifferent chromosome from the population from which the original
chromosome was extracted, and a chromosome from an entirely unrelated
population. The results were given in Annual Report 14.
Based on the above experimental results, we proposed the optimum
Table 1. Relationship between the viabilities of homozygotes andthose of heterozygotes with the" original" chromosome (or achromosome carrying very few minor mutant genes)(Pooled result of Generations 32, 60, and 78)
Range ofhomozygote
viabilities

No. of lines*
testedHomozygote

Heterozygote

0> - .09***.10-.19.20- .29.30-.39.40- .49.50-.59.60-.69.70-.79.80-.89
.90-

14
17
17
13
15
11
7
23
46
33

0.06170.15010.25320.34900.44810.54770.65510.75670.8483
0.97341.0736
1.07831.07581.09211.0862
1.09171.0756
1.04161.03441.0205

Average viability*** Each line is represented two or three times (Generations 32, 60 and 78).
** The viability of the original homozygote = 1.00.
*** Lethal lines are not included.
1) This work was conducted in the Department of Medical Genetics, Universityof Wisconsin, Madison, U.S.A.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

heterozygosity hypothesis (MUKAI, CHIGUSA, and YOSHIKAWA 1965 Genetics
52: 493-501), Le., a hypothesis according to which there is overdominanceif the number of heterozygous loci is very small. But the viabilities ofheterozygotes decrease with an increase in the number of heterozygous
loci after the viability has reached as optimal level.
In Generation 78, the viabilities of 44 randomly chosen lines were
estimated in homozygotes and in heterozygotes with the "original"
chromosome (or a chromosome carrying very few minor mutant genes).
The results were standardized and pooled with those of Generations 32
and 60 which were given in Annual Reports 14 and 15, respectively.
The relationship between homozygote and heterozygote viabilities isshown in Table 1. The results presented in this table clearly support
the optimum heterozygosity hypothesis.

8.

Independence of optimum heterozygosity of the second
chromosome on the heterozygosity of the third
chromosome in Drosophila melanogaster 1)
Terumi MUKAI

On the basis of interaction of different loci within the second chromosome, we have proposed that an optimum level of heterozygosity exists
for manifestation of overdominance with respect to viability. In orderto obtain information regarding the effects of interaction of heterozygosityof different chromosomes, the following experiment was conducted. Basedon the Generation 78 test described in the previous article No.7, 15 lines
were selected, of which 10 lines (Group A) showed the highest amountof overdominance [magnitudes of heterozygosity were near-optimal, see
Article No.7] and the other 5 lines (Group B) exhibited very little, owing
to a very small number of newly arising polygenic mutations controlling
viability. Crosses were made between CylPm SblUbx (~~ )XCyl+i +1+(many;);)) where i indicates line number. From the offspring, Cyl+i
Sbl + males were chosen and mated to the "original" isogenic strain (+ 1++1+, normal viability). The third chromosomes of Cyl+i +1+ and +1++1+ are homozygous and idential to each other except for recently arisingmutations. In the following generation Cyl+ Sbl+, +I+i Sbl+, CYI+
+1+, and +I+i +1+ segregate. Thus, the viability of +I+i relative to
CYI+ for the second chromosome can be estimated in the homozygous(+ 1+) and heterozygous (Sbl +) third chromosome genetic backgrounds.
1) This work was conducted in the Department of Medical Genetics, Universityof Wisconsin, Madison, U.S.A.

RESEARCHES CARRIED OUT IN 1965

25

The pooled results are presented in Table 1 and the analysis of varianceof heterozygote viabilities is shown in Table 2.
From the results of Tables 1 and 2, the following conclusions can be
drawn: (1) The manifestation of overdominance in the second chromosome
is independent of the genetic background in the third chromosome. If
Table 1.

Relative average viabilities of four kinds of genotypes ll
Genotype

Cy/+ Sb/+

I+/+i Sb/+II

Cy/+ +/+

Group A21

1

1.1589***1Group B31

1

0.92211

I +/+i +1+
I 1.2325***I

0.9574liOn the basis of counting 34,055 flies.
21 10 lines carrying many polygenic mutations.
3) 5 lines carrying very few polygenic mutations.
*** Highly significant.
Table 2.

Analysis of variance for the viabilities of heterozygotes
with the" original" chromosome II

Source of variation

Degrees of
freedom

Mean square

Between lines
Between genetic
backgrounds
Lines Xbackground
Error

14

0.0261091

0.013612

4.86*

14
270

0.002798
0.003162

0.88

Total

F

9.33***

299* Significant at the 5 per cent level.
*** Highly significant.
the second and the third chromosomes were not independent, overdominance would not be manifested in the second chromosomes under
the optimum heterozygosity hypothesis, because the level of heterozygosityof the third chromosomes is probably much higher than the optimum
level for the second chromosome. (2) Genetic background (the third
chromosome) affects the relative viability of the second chromosomes.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

9.

Suppressing system of SD in a natural population of
Drosophila melanogaster in Japan
Yuichiro HIRAIZUMI and Yukiko KATAOKA

SD was first discovered in a natural population of D. melanogaster inMadison, Wisconsin (SANDLER, HIRAIZUMI and SANDLER, 1959), and recently
it was also discovered in a natural population (ODATE population) in Japan(HIRAIZUMI and NAKAZIMA, 1965). Since then, experiments have been
carried out to study the genetic systems modifying SD action in that
population. Mating scheme relevant to the present study is presented
in Figure 1. The circled chromosome in this figure indicates that it
originated from the ODATE population,
dnd SDbw is the SD-bearing chromosome (SD from the ODATE population)marked by recessive bw (brown eye
color) mutant.
If there is any suppressing factor
present in the ODATE population, someor all of the males in (2) and (3)matings will show much reduced
amountof distortion (for simplicity,
Fig. 1.
let k=1 and k=0.5 refer respectively
to the distorting and the non-distorting segregation ratio), and the comparison between (4) and (5) matings will show in what chromosome pairsthat suppressing factor locates; Le., A) if all of the males in (4) and (5)matings show k=l, the suppressor must locate on the second chromosome,B) if all of the males in (4) mating show k=1 and all of the males in (5)mating show k=0.5, the suppressor must locate on the X-chromosome, andC) if the suppressor locates on the third or the fourth chromosome, halfof the males in both of the (4) and the (5) matings will show k=0.5(remaining half, k=I).
In total 19 independent sample females from the ODATE population (38chromosomes for each pair) were tested. Of 38 X-chromosomes examined,
24 (0.63) were found to carry suppressor and of 38 second chromosomes
tested, only one was found either 1) to carry suppressor or 2) to carryinsensitive SD+ allele. None of the third and the fourth chromosomes
was found to carry suppressor. The effect of suppressor was complete
for all of the cases, resulting in a normal, 1: 1, segregation ratio.
Mappings of these suppressors are in progress.

RESEARCHES CARRIED OUT IN 1965

27

10. Maternal age and non-disjunction in Drosophilamelanogaster
Kyoko NAKAZIMA and Yuichiro HIRAIZUMIMany years ago MOTTRAM (1930) and LAMY (1949) reported that thefrequency of non-disjunction in female Drosophila increased with theirage. ANDERSON (1927), on the other hand, reported a negative result.
Recently, the above papers were reviewed and critisized by KELSALL(1963), and he presented data showing no correlation between maternal
age and non-disjunction in Drosophila.
The experiment which will be described here was initially designed
as one of a series of studies on the segregation distorter system of
D. melanogaster, but during the course of experiments, we obtained
results showing some relation between the frequency of non-disjunction
and maternal age.
Three y!y females (y: yellow body color, sex-linked recessive) each lessthan one day old were crossed with two or three wild males from Tokyo
stock, and the parents in each culture vial were transfered to a fresh
food vial every three days (parents were kept for 7 days in the last,
7th, vial). Results are summarized in Table 1. Tests showed that each
Table 1.

The numbers and percentages of non-disjunctional flies

Age of female
in days

Total No.
of flies

No. of
XXY female

No. of
XO maleTotal No. of
non-disjunctions

2
5
8
11
14
17
22

24,716
17,66014,182
16,103
12,21011,812
18,9373(0.012%)2(0.011%)3(0.021%)7(0.043%)6(0.049%)3(0.025%)3(0.016%)0(0.000%)0(0.000%)0(0.000%)
1 (0.006%)0(0.000%)2(0.017%)1 (0.005%)

3(0.012%)2(0.011%)3(0.021%)8(0.049%)6(0.049%)5(0.042%)4(0.021%)

Total

115,62027(0.023%)

4(0.003%)31(0.027%)of the 27 exceptional, yellow body color females (XXY) actually carried
an extra-Y chromosome and all of the 4 exceptional, normal body color
males (XO) were found to be sterile. Table 1 clearly shows that thematernal ageing effect is not simply linear. The frequency of nondisjunction in the females increases with their age until they reach theage of about 2 weeks, but then, beyond this age, it starts to decrease.
Since the synaptic condition of chromosome pairs during meiosis may-affect the frequency of non-disjunction and of crossing over, we may

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

expect to have a negative correlation between them. Old studies by
Bridges (1927, 1929) are available to show the relation between thefrequency of crossing over and maternal age. Although Bridges's studies
were on the third chromosome, we can see a clear, negative correlation
between the frequency of non-disjunction and of crossing over (Fig. 1).
05

40

0.04

0.01

2

5

8

11

14

17

Age of femal. in dlYs

22

2

5

8

11

14

17

22

Age of femlll" in days

Fig. 1. Graph showing the relations between the age of female andthe frequencies of non-disjunction and crossing over.

The overall frequency of exceptional females in the present study,.
27/115,620, agrees quite well with that obtained by KELSALL (1963), namely,
27/123,000 but there was a big difference in the frequency of exceptional
XO males (4/115,620 in the present and 64/123,000 in the Kelsall's studies).
The reason for this is not yet understood, but a very reduced viabilityof XO male, X chromosome from the Tokyo stock, may account for this.
H is interesting to note that, instead of its reduced frequency, thefrequency of XO males also tends to increase with the maternal age.
Further studies are in progress. Any comments to the present report
should be addressed to: Y. HIRAIZUMI, Dept. of Genetics, Univ. of Hawaii...
Honolulu, Hawaii, U.S.A.

RESEARCHES CARRIED OUT IN 1965

29

11. Further report on the heterotic effect due to linkage
disequilibrium in Drosophila populations
Shin-ya IYAMA

Three kinds of populations described in the 1965 issue of the Annual
Report were further examined through the 30th generation (ExperimentI). A similar experiment for the third chromosome bearing the ebony
locus was again conducted (Experiment 11). The isogenic background of
those flies in this experiment was derived from the original population
instead of that from the Samarkand stock used in the previous experiment. Each population included eight replicates, each batch comprising250-300 flies in a 200 c.c. milk bottle. The number of wild and ebony flies
was counted in every generation and the ebony flies were discarded
before mating, artificially causing the ebony gene to be lethal. Numberof pairs of + Ie heterozygotes initiating the population was as follows:
Population 1
Population 2
Population 3

Experiment I
One pair of identical flies.
One pair of unrelated flies.
Six pairs of unrelated andone pair of identical flies.

Experiment II
One pair of unrelated flies.
Four pairs of unrelated flies.
Ten pairs of unrelated flies.

As shown in Figures 1 and 2, the results of the two experiments werein good accord with each other: (1) in all populations, the frequency ofebony flies was apparently higher than the theoretical expectation shownby the dotted line, which was calculated on the assumption of the same
fitness for +1+ and +Ie flies, (2) the smaller was the number of initialpairs used, the slower was the decrease in the ebony fly frequency, (3)a similar rate of the decrease was observed in populations initiated withcomparable number of pairs, i.e., between Population 2 of Experiment I
and Population 1 of Experiment II, and between Population 3 of Experiment I and Population 2 of Experiment II.
It is concluded from this study that the manifestation of heterotic
effect of +Ie heterozygotes depends upon the number of pairs of fliesat the initial mating. This fact may support the hypothesis that linkage
disequilibrium maintained between the ebony and other loci concernedwith fitness in the third chromosome could be responsible for the heterotic
effect. The following observation would also support the hypothesis. A
part of each replicate of Population 2 of Experiment I was mixed at the13th generation and the change in ebony fly frequency was examined.
As indicated by the broken line in Figure 1, the ebony fly frequency inthis population dropped rapidly, suggesting that it might have resulted
from the loss of linkage disequilibrium due to mixing.

~

25
'Yoh

~

I- ~ PArR
JJ: I PAIR
III· 6·5 PAIRS

~
~

t'l

.~..,

I-

~IO

u

"'w
a. 5

~

o~,
o

I

2

3

4

.... _-

_-

6

7

5

.

- -.... _-- OJ'
8

9

10

-•.• _If-t.:..=.:.=..:l=:-=..:.:..L~_=_=__=__:l_~.:..::..~l.:_::_~.L~~--·----:=~~~--:"?~~III

•
12

13

14

15

lG

17

18

19

20

21

22

23

21

GENERATION

25

26

27

20

29

30

Z
;p

g
z

Fig. 1.

F:

z

25

...,

(J)

<1/,,,,~...,c:

20

I' I PAIR
II, 4 PAms
III- 10 PAins

t'l

~
C)

~
~

(=)

~I

"''"a.

_ •.- - - - - - - -

o
(J

I~~

-

'\../'

(J)

pll

z

-

i~~·2--3-----;t- ~----~6--(~~·~~~-~~~-·-··-;;;~=-:IL~~~~lr:--~:-I-.~:~~~.. JIII
I: N E R 1\ T I 0 H J

IL

I:)

14

15

16

17

JO

19

9

......

Cl>

Fig. 2.

RESEARCHES CARRIED OUT IN 196531

12. Effect of other species on the genetic constitution of
Drosophila melanogasterTakashi NARISE

Frequency of genes in a closed population of an animal species is
expected, after a number of generations, to be in an equilibrium which
may be interrupted by genetic contributions from immigrants from otherpopulations of the same species. How about the situation when immigration by a different species occurs as is usually expected in nature?
The paper describes the result of a study conducted in order to answer
this question.
Animals used in the study were two mutant strains, ss and ssa, of
Drosophila melanogaster and wild type flies of a different species, D.
simulans. The experiment started with twelve half-point milk bottles
with yeast-sugar-agar medium, each containing 50 male and 50 female F 1
hybrids between the two strains of D. melanogaster. The experiment
was conducted at 25 0 ± 0.5°C. The transfer of flies into new bottles wasmade three times per week: Monday, Wednesday and Friday. The
practice followed the method of Buzzati-Traverso (1955).
Relative frequency of ssa gene was determined every two weeks bysampling the eggs. In 9th generation when the frequency of two genes
had attained equilibrium, various numbers of flies of D. simulans wereintroduced into the melanogaster bottles, namly per bottle 0, 100, 200 and400, each with three replications.
The frequency of ssa gene at equilibrium in a pure culture of D.
melanogaster was 0.45. The same also held in a mix-culture of 100
simulans and the melanogaster population. Mix-culture of 200 flies ofsimulans, however, brought about a decrease of relative frequency of ssa
gene, the equilibrium being at 0.39 for the three and later consecutive
generations. Further decrease in gene frequency took place in the
mixture of 400 flies of simulans, no equilibrium being attained even after
5 generations.
It is suggested from this experiment that the genetic equilibrium in a
population of an animal species is subjected to change not only by
immigration from other populations of the same species, but also bycoexistence with other species which are not able to form Mendelian
population with the former occupants.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

13. Selection for bilateral asymmetry in wings ofDrosophila melanogaster
Kan-Ichi SAKAI and Takashi NARISE

Wings of Diptera flies are usually highly symmetrical in respect oflength. The same also holds for wild flies of Drosophila melanogaster.
The wings of the vestigial strain, however, are found to be not always
symmetrical. This study aimed at establishing lines with highly asymmetrical vestigial wings on the one hand, and on the other hand at
finding to what extent the bilateral symmetry of wings of wild type flies
could be destroyed by the genotype of the selected vestigial line.
All experiments were conducted at 25° ±0.5°C. The results of a survey
on wing asymmetry in three wild strains and a vestigial strain showedthat irrespective of whether cultured for long or short duration in halfpint milk bottles, the wild flies were highly symmetrical in the length oftheir wings. Flies of the vestigial strain, however, were very variablewith respect to symmetry. Sixty per cent of them were more or lessasymmetrical in wing length, symmetrical flies being only 40 per cent.
There seemed to be little difference between males and females. A
selection experiment was conducted in those vestigial flies for and against
bilateral asymmetry, the intensity of selection being 10 for higher and
10 for lower degree of asymmetry among 100 flies. Table 1 gives the
Table 1.

The degree of asymmetry* in wing length of vestigial
flies in succesive selection generations.

Generation
High
Low

Male
FemaleMale
Female

0

1

2

3

4

0.61250.52500.61250.5250

1.195
1.570
0.620
0.785

1.295
1.845
0.785
0.955

1.440
1.940
0.920
1.160

1.470
1.905
0.725
0.650

The unit is 0.1 mm.

* The degree of asymmetry is expressed in terms of the absolute difference
between right and left wing length on an individual basis.

result of selection experiment, from which we find that the selection forhigher asymmetry was quite successful, whereas the selection for symmetry was ineffective. Females in the selected lines were higher inbilateral asymmetry than males.
It was observed in the course of the selection experiment that bilateralasymmetry in wings is likely to be accompanied by lower fecundity.
lower hatching ability and low viability of larvae and adult flies.

RESEARCHES CARRIED OUT IN 196533

14. Deoxyribonucleic acid from SR spirochetes in Drosophila 1)
Bungo SAKAGUCHI, Saburo NAWA and Donald F. POULSON21

It has been demonstrated by POULSON and SAKAGUCHI (1961) that theinfectious agents responsible for the maternally transmitted" Sex-ratio"condition (SR) in D. willistoni and D. nebulosa are treponema-like spirochetes. Furthermore, POULSON and SAKAGUCHI (1961, '63) found that they
can be easily transferred from either of the two species, D. willistoni
and D. nebulosa, to the Oregon strain of D. melanogaster where become
stable and persistent.
Female flies of a SR line of D. melanogaster, Oregon strain, with nebulosa
SR spirochetes were used for isolation of spirochete DNA. The SR
spirochetes from a large number of the host flies were partially purified
by differential centrifugation. The procedure of MARMUR (1961) for theisolation of DNA was followed, except that the alcohol precipitation steps
in the procedure were omitted. This was to avoid loss of the small
amount of DNA of the SR spirochetes. The chemicals used for the isolationof DNA were removed by dialysis against sodium chloride and sodium
citrate solution.
The DNA isolated was analysed by CsCl density gradient centrifugationin the Spinco model E analytical ultracentrifuge. The CsCl solution was
at a mean density of 1.70 g/cm 3 and contained about 2 pg of the test
DNA and 1 pg E. coli DNA used as the density reference. After 20 hrs
centrifugation at 44,770 rev./min. at 25°C, UV absorption photographs ofthe cell were taken.
Examination of DNA obtained from homogenates of normal whole flies
by the analytical density gradient centrifugation reveals two peaks in thedensitometer tracing of the UV photographs; one peak shows the major
DNA derived from the flies and the other shows the minor DNA (Figure1, A). The minor DNA does not constantly appear and its orgin is not
yet known. The major DNA has a density of 1.704 and the minor one
has 1.689. When the DNA obtained from whole cells of the homogenateof SR flies was examined, three peaks were shown in the densitometer
tracing of the density gradient UV photographs (Figure 1, B). Two peaks
out of the three were identical with the DNA from normal flies, but theother minor peak was shown specifically only in the DNA isolated fromthe SR flies. The minor peak has a density of 1.681. From this density
value, the guanine-cytosine content of the DNA is estimated to be 21
II This work has been supported by U.S. Public Health Service Research Grant
GM 10238 from the Division of General Medical Sciences.
2) Department of Biology, Yale University, U.S.A.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

per cent. The G-C content is rather low compared with that of mostother organisms. The major DNA peak from the partially purified SR

A
Normal fly whole cell DNA (Control
of B)

B
UI''-' 1'--

SR fly whole cell DNA

C
Partially purified SR spirochete DNA---t--+--+-+-1.710

D
DNase treated partially purified SR
spirochete DNA.1.689

1.704

1.681

Fig. 1. Densitometer tracings of UV photographs made in the Spinco model
E ultracentrifuge. DNA's obtained from the fractions indicated had been
centrifuged in CsCI for 20 hours at 44,770 rev./min. E. coli DNA ofdensity 1.710 was the marker. The densities of host fly DNA, host fly
minor DNA and partially purified SR spirochete DNA are 1.704, 1.689
and 1.681 respectively.

spirochete fraction (Figure 1, C) has a density of 1.681, identical with thepeak seen in whole cell DNA of the SR flies (Figure 1, B).
The UV absorption spectrum of the material of the SR specific peakisolated by CsCI gradient using SW 39 rotor of the Spinco centrifuge
was shown to be typically that of nucleic acid; the maximum absorption
was at 260 mf-!. This maximum absorption disappeared after DNase
treatment. However, after the treatment with DNase the peak (densityof 1.681) in the CsCI gradient by the analytical ultracentrifuge was stillpresent (Figure 1, D). The material remaining in this peak contained
abundant hexose, presumably glucose. These facts suggest the possibility
that the SR specific peak may represent an overlap between the SR DNA
and polysaccharide in the position corresponding to density of 1.681.
More detailed examination of the nature and properties of the SR spirochete DNA using the method of the CsCI density gradient is now underway.

RESEARCHES CARRIED OUT IN 196535

15. Effect of DNA treatment on the wing scale in Ephestia
Saburo NAWA

The wild type of Ephestia (NCR) has a pattern of black scales (about
20 per cent) on its fore wing, while a recessive multant ml has a uniform
type of scales without pattern. As previously reported, the appearanceof black scales on the fore wings of ml/ml was observed, when larvae
were treated with DNA prepared from wild animals. The black patternscales constitute nearly 100 per cent of the total number of scales on thefore wing of a mutant bib. Thus, it might be expected that treatmentof ml/ml; bib with NCR-DNA produces about five times as many blackscales as does treatment of ml/ml; +b/+b with the same DNA. Three
black scales were found in 58 ml/ml; bib animals treated (1 black scale/39
wings). In the case of ml/ml; +b/+b, the frequency was 1/37 wings(19/712). It appears that the frequencies are the same in both cases,
although the number of ml/ml; bib animals treated was small. This may
mean that any scale, wherever it is located on the wing, is able to beaffected with DNA. Effect of DNA from various sources was also examined.
So far, no black pattern scales have been found in the experiments using
calf thymus DNA or E. Coli DNA. The appearance of black scales wasobserved on the wings of ml/ml animals treated with Bombyx DNA,
although the frequency was low (2/226). This may represent a case ofinterspecies-transformation.

16. Genetic and biochemical studies of chromogranules
in the hypodermal cells of the silkworm
Mitsuo TSUJITA and Susumu SAKURAI

A large amount of spherical chromogranules that contain pteridine
compounds such as isoxanthopterin and sepiapterin, and uric acid arefound in the larval hypodermis of the silkworm. Owing to the presenceof those chromogranules, normal larvae exhibit a non-transparent hypodermis. Since the chromogranules of the hypodermal cells of normal
larvae contain mainly the pteridine compound isoxanthopterin, and uricacid, their presence gives white color to the larval skin, but the yellow
chromogranules of the lem mutant which contain mainly the pteridine
compound sepiapterin, and uric acid give yellow color to the larval skin.
The present experiments were undertaken to examine the RNA contents of chromogranules.In order to gather the granules, larvae at 4-5th days of 5th instar

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

were cut open at their dorsal side, all their inner organs were removed
and the cytoplasm of the hypodermal layer which contained the granules
was scraped and gathered. The raw fresh material of cytoplasm thus
obtained was mixed with 2.2 M sucrose solution (volume ratio of sample
to sucrose solution 1: 10) and then centrifuged at 14,000 g for 60 minutes.
The supernatant and floating cell components were discarded and theprecipitate that contained the chromogranules and nucleus was placed in
20 ml of 3 M sucrose solution in a test tube and centrifuged at 40,000 g for
60 minutes. Four layers could be discriminated in the test tube, namely,
1st upper layer containing floating cell components, 2nd transparent layer,
3rd layer containing a suspension of chromogranules and 4th layer ofprecipitated chromogranules. In order to precipitate all of the chromogranules the 3rd layer of the chromogranule suspension was centrifuged
at 54,000 g for 90 minutes.
Thus separated chromogranules were lyophilized. On the other hand,
the larval hypodermis of each of the strains was dried and ground to
powder. The isolated chromogranules and powdered, dry hypodermis wereused for the determination of the RNA amount. RNA, protein and lipid
fractions were prepared from purified chromogranules according to theprocedure of Schmidt-Thannhauser and Schneider (1946), and RNA wasdetermined by the orcinol method of Mejbaum (1939).
Protein and lipid fractions were dried to constant weight and estimatedquantitatively by weighing. Experimental results are given in Table. 1.
Table 1.

Biochemical composition of chromogranules~ IE-lem Id-lem IC-12411~IE'leml d-lem IC-124) Daizo I w
RNA

170.4174.7

87.2--jL-i-Pi-dp-G--'-1-0-.9-113.4
pg mg . .

8.8

pgjmg P.G.

Proteinpgjmg G.

Abbreviation,

364

oz

pgj~~~.m.

51.9

49.4165.5156.9179.6

jLipid--pg mg d .m.

~I~ ~I~ -27-4-IiI-d-r-y-m-at-t-er--1-2-9-7-1--;: -;;l~ ~pgjmg H.

d.m.:
G.:
P.G.:
H.;

dry matter
granuleprotein in granules
hypodermis

In the table the amount of RNA is shown by flg RNA/mg protein ofchromogranules, the amount of lipids is shown by flg lipid/mg proteinof chromogranules and the amount of protein is given by flg protein/mg

RESEARCHES CARRIED OUT IN 196537

chromogranules.
For the study of the effect of ribonuclease on RNA contained in thechromogranules and in the hypodermal cells, 50 mg of chromogranulesof the [em larvae or 100 mg of powdered dry hypodermis of C-124 andwO~ were treated with 300 fig RNase at 37°C for 1.5 hrs. and then theamount of remaining RNA in each of three sample was determined.
Experimental results are given in Table 2.
Table 2.

The loss of RNA in the granules and hypodermis
treated with RNase.

E-lem granules
RNase treated E-lem granules

70.4 p.g/mg P.G.
28.3 p.g/mg P.G.

C-124 hypodermis
RNase treated C-124 hypodermis

65.5 p.g/mg d.m.
23.5 p.g/mg d.m.

WO~ hypodermis
RNase treated WO~ hypodermis

79.6 p.g/mg d.m.
16.5 p.g/mg d.m.

Abbreviation,

P.G.:
d.m.;

protein in granule
dry matter

17. Location of silkworm rm gene on the 3rd chromosome
Mitsuo TSUJITA and Susumu SAKURAIYD 4 is a silkworm in which strain the [em l gene is located at 22.3'
units on one of the 3rd chromosomes. However, a gene for retarded
moulting (gene symbol: rm) arose by spontaneous mutation at a locus
near +lem gene on the other of the 3rd chromosomes. Consequently, ineach batch of the next generation produced by sib-mating of individuals
with normal larval phenotype segregated from YD4 strain, the following
segregation was found; about one fourth of the larvae became lethal
yellow immediately after the 1st moulting, about another fourth had
retarded moulting owing to slow growth, and the remaining two fourth
showed the normal phenotype. It may be safely said that larvae having
the normal phenotype had the genotype rm + 1+ [em l •From the results of linkage experiments between rm and Ze gene, it
was concluded that the rm gene locates relatively near the +lem gene,but an accurate crossing-over value could not be determined.
It is assumed from various cross experiments that the penetrance ofthe rm gene is considerably changed by the presence of suppressors
affecting its manifestation, although the suppressing genes have not yet

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

been sufficiently analysed. It seems that there is some relation between
the rm gene and the moulting hormone. As a means to explore its
biochemical action, an electrophoretic study of soluble proteins containedin the body fluid is now under way with polyacryl amide as supporter.
18.

Experimental induction of androgenesis, gynogenesis andpolyploidy, in Bombyx mori by treatment with C02 gas
Yatar3 TAZIMA and Akio ONUMA

Experimental induction of parthenogenesis and dispermic merogony
has been investigated in the silkworm by many workers. The mostpowerful agents used so far were hot hydrochloric acid, thermal stimuli
and X- or r-rays. Quite recently, it was found in this laboratory that
CO 2 gas is also a very effective agent for the induction of androgenesis,
gynogenesis as well as polyploidy. The immediate effect of this gas
permitted us the estimation of the progress in time of the maturation
division, syngamy and cleavage.
When eggs from wild type females, mated with double recessive pe remales, were subjected to C02 gas soon after deposition, a surprisingly
high incidence of pe eggs as well as mosaics for pe and + was observed.
Although most of pe eggs died during embryonic development, somehatched out and attained maturity, all giving males with pink eyes. The
genetic test revealed that they were homozygous for pe and reo Taking
these results into consideration, together with those of HASIMOTO (1934)
and ASTAUROV et al. (1956), those pe eggs were assumed to be the productof dispermic merogony. Following this experiment, it was soon confirmedthat C02 gas is also effective for the induction of parthenogenesis as wellas polyploidy (detected by non-disjunction of two genomes). Nitrogen gas
was completely ineffective.
By marking each parental genome by a gene for a different egg colour,
detailed analyses have been made of the changes in frequency in theexperimental induction of those exceptional cases, which escaped
fertilization, in relation to the time that elapsed since deposition of theeggs. Female moths were allowed to deposite eggs for 20 minutes.
After collection those eggs were divided into several batches and each
was placed according to the experimental design in a glass vial, throughwhich a stream of CO 2 gas was passed for a period of definite duration
at various ages of the eggs. Except time of the treatment, the eggs
were kept at 25°C. The cross combinations used were; +1+ ~ xrelreo
for the detection of androgenesis, relre ~ X +1+ 0 for gynogenesis andpe+l+re ~ xpe relpe re 0 for non-disjunction of two genomes.

RESEARCHES CARRIED OUT IN 1965

39

The incidence of non-disjunction of homologous genomes declined soonafter deposition and disappeared after 50 minute egg age. Then a brisk
incidence of androgenesis came into appearance representing two cyclic
peaks, a major one at egg age of 70 minutes followed by a minor peak

,.

100

0

90

10

unpigmented

eo

20

70

)0

60

40

50

50

40

60

)0

surviving(blaok)

20
10

70

eo
90
100
f,

Fig. 1. Changes in the incidence of unpigmented (unfertilized) and earlydying eggs and androgenic re eggs with the progression of egg age.
CO2 gas treatment for 180 minutes. (Expt. 653) Abscissa: egg age inminutes, Ordinate: frequency, Broken line with black circle: androgenic
re eggs, Solid line with black circle: mosaics for re and +, Heavy solidline with black circle: non-disjunction, obtained from another experiment.

at egg age of 150 minutes. An almost similar type of relation was obtainedfor gynogenesis, although the frequency was very low. From the observed
survival of the treated eggs the net survival for the egg nucleus has been
calculated by subtracting the frequency of androgenic eggs. The net
survival curve declined immediately after oviposition and reached the firstminimum at 90 minutes, then went up again to 150-170 minutes. Thereafter, it showed cyclic changes in 50 minute periods.
These results furnish a very good clue for the estimation of the progressin time of the maturation and further developments. For instance, thenet survival decline during 10 to 90 minutes seems to indicate the durationof maturation division and the cyclic changes after 170 minutes appear tocorrespond with cleavage cycles. Furthermore, the finding that nondisjunction ceased to occur by the egg age of 50 minutes suggests thatthe reduction division takes place in this insect at meiosis I.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

19. Autoradiographic studies of silkworm spermatogenesis
using tritiated thymidine
Toshihiko SADO

Informations on the timing of spermatognesis are essential for studying
the effects of physical or chemical agents on various phases of the
spermatogenic cycle. In the silkworm, a rough estimate of this cycle
has been made previously by myself (SADO 1959). This estimation was
based on a time table recording the first appearance of cells of one stage,then those of the next and so forth in both normal and irradiated testes.
In order to furnish a more complete time table of the spermatogenic cycleof this insect, further experiments have been carried out with the use of
H3-thymidine (H3-Tdr) as cell marker.
In one experiment, 10 day old male larvae were injected with 0.6 f-IC
H3-Tdr into the dorsal vessel, using a microinjection apparatus devised
by RITZKI (1953). At intervals, testes were fixed with Carnoy's fluid,
sectioned at 6 f-I and autoradiographed using SAKURA NR-M2 emulsion andexposed for 4 weeks. As expected, the last stage at which H3-Tdr wasincorporated was that of the youngest spermatocytes (64 cell stages) justfollowing the last spermatogonial mitosis. 24 hours later many of the
spermatocytes at synapsis (or zygotene) were found to be labeled. The
labeled spermatocytes in metaphase I-anaphase II were for the first timeobserved nine days after injection of the isotope. A few young spermatids
were also found to be labeled at this time. On the 17th day there weremany labeled spermatids but no labeled spermatozoa. On the 18th day,
30 to 40% of the spermatozoa cysts were found to be labeled. Thus it isclear that both meiotic prophase I and spermatid stage (spermiogenesis)
last for nine days. This means that it takes 18 days from the last DNA
synthesis to the completion of spermatozoa.
Spermatogonia of this insect can be classified into two categories; primaryand secondary spermatogonia. Using (a) the time in which the numberof primary spermatogonia doubled during early larval stages, (b) half-timeof the mean grain number/cell as a function of time after a single pulse
injection of H3-Tdr and (c) colchicine method (Leblond 1959), the generation time of primary spermatogonia was estimated to be approximately
34 hours. The secondary spermatogonia comprise 6 generations and thetotal duration of this stage (sum of 6 generations) amounts to 5 days as
seen from the fact that the testis of a newly hatched larva contains nosecondary spermatogonia whereas it contains early spermatocytes 6 days
later. The time table of spermatogenesis of this insect is summarized inTable 1, in which the earlier estimates are also given for comparison.

RESEARCHES CARRIED OUT IN 1965

41

Table 1.
Spermatogenic stages

Present result

Sado (1959)

Primary spermatogonia
Secondary spermatogonia(Sum of 6 generations)
Spermatocytes
Resting stage
Meiotic prophase I(Leptotene to diakinesis)
Metaphase I-Anaphase II
Spermatid stage
Spermatozoa
Total spermatogenesis

34 hours
5-6 days

A few days

«1

9

«1
9

/I
/I

/I
/I

(1-14 days)*
Approx. 26 days

10-11 days
Within 1 day
5-6 /I
Approx. 20 days

* Storage time in the testes.

In another experiment where H3-Tdr was injected into larvae 19 days
after hatching, or 17 days prior to eclosion of the moths, no spermatozoa
were found to be labeled at the time of emergence. However, when theisotope was injected 16 and 17 days after hatching, approximately 6 and
0.4%, respectively, of the spermatozoa bundles in the seminal vesicles of
newly emerged moths were found to be labeled. Maximum labeling of
spermatozoa bundles was obtained when H3-Tdr was injected between 8
and 12 days after hatching. In these instances, as many as 25% of the
spermatozoa bundles were found to be labeled. This indicates that inany attempt to test a transmutation effect of an isotope incorporated into
DNA or to induce transformation by external DNA using spermatogenic
cells the administration of these agents must be done in this insect during
this period.

20.

Inter-breed competition in growing chickens
Tohru FUJISHIMA

Three breeds of demestic fowl, White Leghorn, Barred Plymouth Rockand Rhode Island Red, were crossed by each other following the schemeof 3 X 3 diallel crosses, and the progeny were investigated with regard tointergenotypic competition.
At one week of age, the hybrid males and females obtained from thosecrosses were divided into three paternal strain groups and two intermingled
groups involving three different paternal strains. Each paternal straingroup contained twenty seven birds, comprising 9 birds of each of threedifferent dams in a paternal strain, while each intermingled group con-

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

tained a total of 27 birds consisting of 3 birds of each of three different
dams in every parternal strain. The experiments were repeated threetimes. Competitive ability for each paternal strain was estimated on thebasis of mean superiority or inferiority of body weight in intermingled
groups as compared with body weight in the paternal strain groups. The
experiments were conducted under restricted and full feeding conditionsin the intermingled groups, but only under restricted conditions in thepaternal strain groups. The same amount of feed consumed by the full· fedgroups in the previous week on a per bird basis was fed to the restrictedgroup every week during the experimental period. The restricted groupalways received the same quality of food as the full-fed group, the only
difference being in the quantity of feed received.
The results of the present experiment, as shown in Table 1, indicate
Table 1.

~

Inter-breed competitive abilities in body weightat 5 weeks of age.

White Leghorn

Rhode Island Red

Dam

C1l

C;;

::E

<I)

C;;

E
C1l

""

Barred Plymouth
RockInter- Paternal Com·Inter- Paternal Com·Inter- Paternal Commingled strain petitive mingled strain petitive mingled strain petitive
group group ability group group ability group group ability
Mean Mean Mean Mean Mean Mean Mean Mean Mean
gr.
gr.
gr.
gr.
gr.
gr.
gr.
gr.
gr.

W.L.
R.I.R.
B.P.R.

233.4 244.7 -11.3
217.8 231.5 -13.7
0.2
245.6 245.4

233.8 248.8 -15.0
180.0 200.9 -20.9
246.2 250.9 -4.7

248.3 231.7
206.8 204.1
202.6 191.1

16.6
2.7
11.5

Mean

232.3 240.5-8.2

220.0 233.5 -13.5

219.2 209.0

10.2

W.L.
R.I.R.
B.P.R.

229.2 232.6
239.4 234.8
256.2 244.4

-3.4
4.6
11.8

219.4 238.8 -19.4
200.9 207.9 -7.0
244.2 264.4 -20.2

225.9 230.7 -4.8
204.8 218.8 -14.0
190.1 211.5 -21.4

Mean

241.6 237.3

4.3

-15.5

206.9 220.3 -13.4

221.5 237.0

that there are general combining abilities for competitive ability in thedomestic fowl, because the male progeny from B. P. R. used as parental
breed have generally statistically significant superiority in competitive
ability to those from the other breeds (P<O.01), while in female progeny,
those from W. L. have significant superiority to those from the other breeds(P<O.OI), and there are statistically significant sire X dam interactions incompetitive ability of female progeny (P<O.05), i.e., non-additive genetic
effects in inter-breed competitive ability.

RESEARCHES CARRIED OUT IN 1965

21.

43

Variance and covariance analysis of egg production
and related characters in the domestic fowl.
Takatada KAWAHARA and Teruo INOUE

Heritability of and genetic and phenotypic correlations among egg
production (up to 120 days after first egg), egg weight, egg width, egg
length, body weight and sexual maturity were investigated in a closed
flock of White Leghorns. Data were collected from 907 pullets originatedfrom 65 sires and 284 dams. The estimates of heritability from ananalysis of variance are given in Table 1.
Table 1. Heritability estimates of egg production, egg weight,
egg width, egg length, body weight and sexual maturity.
Heritability

h2•
h2 d
h~+d

Egg
I Egg
production weight

I

0.309
0.189
0.249
Table 2.

0.386
0.465
0.426

I widthEgg

Egg
lengthI weight
Body

I maturity
Sexual

0.392
0.292
0.342

0.506
0.339
0.396

0.895
0.346
0.620

0.407
0.483
0.445

Genetic and phenotypic correlations between egg
production and related traits.

TraitsGenetic
correlation

Phenotypiccorrelation

Egg production-Egg weight
Egg production-Egg width
Egg production-Egg length
Egg production-Body weight
Egg production--Sexual maturity-0.217-0.090-0.232-0.132-0.113-0.140**-0.064*-0.216**-0.023-0.048* Significant at the 1% level.
** Significant at the 5% level.
Table 2 represents the estimates of coefficients of genetic and phenotypiccorrelations between egg production and related. traits.
Both genetic and phenotypic correlation coefficients between egg production and the related characters are all negative. Interesting is the findingthat the genetic correlation between egg production and egg length was-0.232 and that between egg production and egg width was -0.090.
This indicates that the generally accepted negative relationship between
egg number and egg weight could be rather due to a change in egg lengththan in egg width.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

22. Estimation of genetic parameters of the productive
traits of Japanese quail.
Takatada KAWAHARA and Teruo INOUE

Heritability of and genetic and phenotypic correlations among egg
production index (up to six months after hatching), age at sexual maturity,
egg weight (average first three eggs) and body weight (at sexual maturity)
were investigated in a closed population of the Japanese quail. Data werecollected from 235 female offspring originated from 47 pair matings of one
male and one female each. The estimates of heritability from an analysis
of variance are given in Table 1.
Table 1.

Heritability estimates of various productive traits
in Japanese quail.
TraitsHeritability ± S.E.

Egg production index(P)
Age at sexual maturity (S)Egg weight(E)
Body weight(B)

0.203± 0.093
0.329 ±0.104
0.542 ± 0 .124
0.422 ± 0 .113

Table 2 represents the estimates of coefficients of genetic and phenotypiccorrelations between various characters.
Table 2.

Genetic and phenotypic correlations between
various traits in Japanese quail.

TraitsGenetic correlation

I Phenotypic correlation

P-S
P-E
P-B
S-E
S-B
E-B

-0.514±0.198-O.387±0.195-O.118±0.123+0.390±0.163+0.542±0.149+0.872±0.042-0.109±0.065-0.261±0.061+0.153±0.064+0.393±0.055+0.220±0.062+0.394± 0.055

RESEARCHES CARRIED OUT IN 196545

23. Further study on chromosomes of plasma celltumors of BALB/c mice 1 )Tosihide H. YOSIDA, Hirotami T. IMAI
and Michael POTTER2)

Chromosomes of 7 mouse plasma cell tumors had been already observedby us CYasIDA et al. 1964). All of them were characterized by a neartetraploid chromosome distribution. These tumors were remarkable byproducing several kinds of specific r-globulins. Two important question
Table 1.

Karyotypes of 9 plasma cell tumors in BALB/c mice

I Variation

No. of marker
chroms.
(mode)

No. ofcells
observed

Protein
specificity

0-5 (1)0-1 (0)50

rA serum
protein0-2(2)
0-2(2)

50

Tumor linesof chromo
number(mode)

RPC-6A
positive164-176 (68) IMll
SM2)

RPC-6A
negative153-162 (73)

M
SM

RPC-9

0-1 (1) i
153-I48(~~) MSM S1'31 0-2
(2)

50

SM
M
m')

0-2 (1)

50

SM
M
m

0-1 (0)1-3 (2)0-1 (0)20

48 69 (67)-M

0-2 (1)53

40-90 (44)MSM

0-2 (1)0-1 (0)50M

0-1 (0)50

RCP-20positive
RPC-20negative67-154 (79)169-142 (72)III
0-2(1) I
1-3 (1) I

I, generation.
Transpl.
I

56

I

-

I

I

I

Kappa
chain

I

Lambda
chain

70
13

69

I
I

-

I

70

I

I

MOPC-88
1

MOPC-3IC

MOPC-70A

37-97 (73)66

IrA urinary!
proteinI
rF serum !
protein only I

48

I

rFprotein"rom I+ excess I

12I

Kappa chain !
MOPC70A·10A

57-68 (66)SMm1) M=median metacentrics.
3) ST = subtelocentrics.

0-1 (0)0-1 (0)

50

I

I

"

iI

I

69

:

2) SM=submedian metacentrics.
4) m = minutes.

1) This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.
2) National Cancer Institute, Bethesda, Md., U.S.A.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

arose; (1) Why did they have tetraploid karyotypes? Was our investigation
carried out in some exceptional material? (2) Is there a correlation between
karyotype and protein specificity of tumors?
To answer those questions, the chromosomes of 9 plasma cell tumors(RPC-6A positive, RPC-6A negative, RPC-9, RPC-20 positive, RPC-20
negative, RPC-88, MOPC-31C, MOPC-70A and MOPC-70·lOA) were
examined again. They had been induced in BALB/c mice by one of thepresent authors, M. Potter, in National Cancer Institute, Bethesda, byinjection of mineral oil, and the materials were sent to this institute.
The karyotypes and protein specificity of these tumor cells are given in
Table 1. As may be seen from the table, only MOPC-31C showed a
hyperdiploid stemline. The other 8 tumors were remarkable by having
hypotetraploid karyotypes. In general, the range of distribution of chromosome numbers was narrower in the present materials than in the previous
examinations of the same strains. Two sublines of protein positive(protein producing) and protein negative (not producing protein) werecompared using two strains (RPC-6A and RPC-20). The chromosomes of
the negative sublines were markedly different from those of the respective
positive lines. No constant relationship between karyotype and protein
specificity could be found until now.

24. Chromosomes of normal plasma cells of mouse 1)
Tosihide H. YOSIDA and Hirotami T. 1M AI

Almost all plasma cell tumors of BALB/c mice induced by injection ofmineral oil or mineral oil adjuvant and those of C3H mice developed
following ovariectomy were characterized by near-tetraploid karyotypes
including a few marker chromosomes (YasIDA et al. 1964). What are thekaryotypes of normal plasma cells of mice was an important question. On
this point, however, no information had been yet obtained. In order toobserve chromosomes of normal plasma cells, an adjuvant-vaccine emulsion
was injected into the sole of the feet of the animals. One to two weeks after
the injection hypertrophic lymphnodes were observed in the inguinal
region. A remarkable increase followed of macrophages, lymphocytes andplasma cells characterized by endoplasmic reticulum in the cytoplasm andmitotic cells in the lymphnodes. In metaphase cells, however, recognitionof plasma cells among other cells was difficult, because the characteristic
figures observed in their cytoplasm had disappeared. Under such con11 This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 196547

ditions, it was very hard to identify the karyotype oi plasma cells.
About 5 percent of all interphase cells in the lymphnodes were examined
16 days after injection. From that information at least 5 percent of
metaphase cells in the lymphnodes should be plasma cells. However, 579
mataphase cells obtained from hypertrophic lymphnodes were all characterized by a strictly diploid karyotype. Tetraploid cells were neverobserved in this cell population. The above investigation strongly suggeststhat normal plasma cells are characterized by a normal diploid chromosome complement.

25. Further study on chromosomes of sensitive and drug
resistant sublines of mouse lymphocytic neoplasm,
P388, growing in vitro!)
Tosihide H. YOSIDA, Hiroshi OHARA, Robert A. ROOSA21
and Takako FUKAYA

Chromosomes of sensitive sublines and 8-azaguanine and amethopterin
resistant sublines of mouse lymphocytic leukemia, P388, growing in vitro
had been previously observed. Those materials were fixed in National
Cancer Institute, Bethesda, U.S.A. in 1962. We found at that time thatthe chromosome pattern of sensitive lines differed markedly from those ofresistant sublines. In order to reinvestigate this problem, we have newly
obtained in November 1965, sensitive and drug resistant sublines of P388
growing in vitro from Wistar Institute, Philadelphia, namely, 3 sensitivelines, 5 8-azaguanine resistant, one FUDR resistant and one amethopterin
resistant lines. Although the total chromosome number (44) in the sensitiveparental line growing in a suspension culture was less than that (47 to 49)of the attached culture lines, the number of maker chromosomes was quite
similar to that of the three sensitive lines. They were characterized by
8 to 9 metacentric chromosomes and 4 to 5 submetacentric markers. Thesefindings were similar to those obtained by us previously.
On the other hand, several drug resistant lines showed alterations in theconstitution of their biarmed chromosomes as well as in the total chromosome number. In an 8-azaguanine resistant line, AZGr-3 (1 x 10-4 M AZG),
the number of metacentric chromosome was decreased. A FUDRr-3
resistant line (5 X 10-8 M FUDR) was characterized by a marked decreaseof the total chromosome number and a marked increase of biarmed
1) This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.
21 Wistar Institute, Philadelphia, Penn. U.S.A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

chromosomes. In the amethopterin resistant line, AMTr-5 (8X 1O- 6 M AMT),
a slight decrease of metacentric chromosomes was noted. From the above
investigations, it can be said that the chromosome patterns of cultured
lines varied according to the culture method employed, and those of
sensitive lines were different from those of drug resistant lines.

26. Chromosome study of survived sublines of mouse
fibroblastic in vitro line L-5 after treatment
with 8-azaguaninel)
Hiroshi OHARA, Takako FUKAYA and Tosihide H. YOSIDA

The chromosome pattern of a parental mouse fibroblastic in vitro line L-5
and of the survived sublines developed by treatment with various concentrations of 8-azaguanine was examined in order to find the relationshipbetween the development of 8-azaguanine resistance and the change ofkaryotype. The cells were propagated in static closed bottles and in petri
dishes in a C02 incubator in MEM medium supplemented with 10 to 15%
of whole calf serum. For the experiment, 8-azaguanine was dissolved in
the above medium, in which the cells were kept for 7 days; then it waschanged to drug free medium in order to permit a rapid and abundant
growth of the survived cells. From the treatment with the chemical thefollowing sublines were obtained; 5 clonal lines (treated with 5,ug/ml),
5 lines (treated in a bottle with 5,ug/ml), 3 lines (treated with 0.1, 1 and10,ug/ml, respectively) and 10 lines (treated 5 times with 5,um/mI). !D soof parental L-5 line to 8-azaguanine was approximately 0.75 ,ug/ml.
However, some of the lines developed by the drug treatments, showed noperceptible growth when the medium contained the above concentrations.
Only one repeatedly treated line showed a slow growth at the drugconcentration of 2,ug/ml. Therefore, they were not true resistant lines,
and have probably developed a false resistance by physiological adaptation.
The majority of the parental line cells were characterized by 53 to 55
chromosomes including 10 metacentrics and 5 submetacentrics, among thelatter a strikingly large submetacentric D-chromosome. At the end of
1965, however, the karyotype of the parental line was slightly changed.
The chromosome pattern of the cells of the survived lines developed bytreatment with various concentration of 8-azaguanine was almost similar
to that of the parental line concerning the chromosome number andkaryotype. It may be reasonably assumed that these survived lines are1) This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 196549

not yet established true resistant lines which would be eventually developedby further succession of mutation and selection.

27.

The effect of 8-azaguanine on DNA synthesisof Yoshida sarcoma cells1 )Tosihide H. YOSIDA, Yoshinori KURITA
and Reiko TSURUTA

The anti-cancer agent, 8-azaguanine, inhibits the growth of a varietyof living cells. The present studies deal with the effect of the chemicalon the chromosomes and DNA synthesis of YOSHIDA sarcoma cells. Afterincubation of the cells with 10 p.c of IP-thymidine and 70 mg 8-azaguanine
in vitro, they were injected intraperitoneously into rats. Sampling was
made every 2 hours thereafter up to 24 hours after incubation. The results
were as follows:
In a total of 1,000 cells treated with the chemical DNA synthesis was
not affected. The generation time of a YOSHIDA sarcoma cell observed in
the present study was about 14 hours, and the duration of each phase of
the mitotic cycle was G1=3 hrs., S=8 hrs., G2 =2 hrs. and M=1 hr. The
results here obtained differ from those of the preceding non-treatedexperiment (KURITA, et al. 1964), in which one generation time of the cell
cycle was 18.5 hours.

28. Chromosome aberrations induced in mouse bone marrowcells by 20-methylcholanthrene ll
Reiko TSURUTA, Yoshinori KURITA, Tosihide H. YOSIDA
and Katsumi SAKAKIBARA

Since the chromosomes of chemically induced mouse leukemias generally
deviate from a normal complement, it is of particular importance to detect
whether carcinogens may affect chromosome rearrangements. In orderto throw a light on this problem, the types and frequency of chromosome
abnormalities induced in bone marrow cells of RF strain were examined
after treatment with 20·methylcholanthrene.
No chromosome aberrations were observed 8 hours after treatment with
10-3 M of the chemical. 24 hours after treatment, however, structural
chromosome abnormalities became evident. The aberrations observed1) This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

were only of chromosome type, namely, interstitial deletions and minutechromosomes which were never found in the control specimens. The
frequency of those aberrations was 7 percent. This seems to indicate that20-methylcholanthrene may weakly affect the chromosomes of bone marrowcells of mice.

29. Autoradiographic analysis of the effect of Mitomycin C
on chromosomes of Ehrlich ascites tumor cells!)
Yoshinori KURITA, Tosihide H. YOSIDA
and Reiko TSURUTA

Mitomycin C, a chemically reactive antibiotic derived from Streptomyces
caespitosus, is known as a selective inhibitor of DNA synthesis in E. coli.
The effect of the chemical on plant and human chromosomes has been
noted by several investigators. The present study was undertaken inorder to carry out an analysis of the types and frequencies of chromosome
aberrations under the influence of the chemical. Ehrlich mouse tumor
cells served as the material under particular consideration of the relation
between the type of induced aberration and the mitotic cycle.
Ehrlich ascites tumor cells transplanted into SL strain mice were treatedwith 10-3M and 10- 4 M Mitomycin C, and simultaneously with 10 pc of
H3-thymidine in vivo. Treatment with 10- 3 M Mitomycin C resulted in
an inhibition of DNA synthesis and mitosis. On the other hand, with
10-4 M treated cells showed a one hour lag of Ga phase. It was also observedthat the duration of S phase was greatly prolonged following the treatment. Although it is not clear whether this period represents the firstor the second post-treatment mitosis, almost all cells with chromosome
aberrations observed within 36 hours after treatment were labeld with H3
thymidine. Moreover, it was noted that chromatid breaks appeared
mostly 8 hours after treatment (0.35/cell), but the first chromatid exchanges
were seen 14 hours after treatment and their frequency gradually increased
from 14 to 22 hours. The frequency of chromatid exchanges observed
22 hours after treatment was 6.5/cell. These results may indicate that
Mitomycin C induces a cross-link in twin molecular chains of double
stranded DNA and then inhibits their separation which is essential for cell
division.

1) This work was supported by a research grant from the National Cancer Institute(CA 07798-02), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1965

30.

51

Alteration of karyotype in a transplantable granulocytic
leukemia (chloroleukemia) in the rat
Takao SAKAI, Tosihide H. YOSIDA and Mitsuyuki SHIMIZUIl

A transplantable granulocytic leukemia (chloroleukemia) used in thepresent study was transferred to this institute from Showa University,
Medical College, Tokyo, where the material was obtained from Dr. Harry
Shay, Fels Institute, Philadelphia, Penn. U.S.A. This tumor was originallyinduced in a male Wistar rat by gastric instillation of methylcholanthrene
in 1951. In 1961 the chromosomes of this tumor were studied by Norwell
et al. (Norwell et al. 1963). According to them, the tumor showed apersistent chromosome change, namely in the stemline cells with 43
chromosomes an extra chromosome was added to the group of mediansize acrocentrics.
The present observations revealed that the chromosome numbers varied
from 37 to 44 with the mode at 43 (809'6'). Karyotypes of the tumor
stemline cells were slightly different from those observed by Norwell et al.
In the present material, an extra-long telocentric and one small subtelocentric chromosomes were usually observed in the stemline karyotypes.
An extra chromosome in the group of medium size acrocentrics wasnoticed by Norwell et al. From the above observations it was assumed
that a distal part of an acrocentric element was translocated to one of
No.2 acrocentrics resulting in the extra-long element, while its other partwith the centromere remained as the small subtelocentric. Cells with 43
and 45 chromosomes were sometimes observed. From the idiogram analysis
it is assumed that those karyotypes were developed by nondisjunction of
one of No. 14 submetacentric chromosomes.

31.

Population survey of Japanese and Korean black rats
with chromosomal polymorphism
Tosihide H. YOSIDA, Takako FUKAYA, Yung Sun KANG,
Yukuo MORIGUCHI, Kyoko OHARA and Akira NAKAMURA

The black rat, Rattus rattus L., is characterized by polymorphism of
the largest No. 1 chromosome pair (YasIDA et al. 1965). According totheir morphology, three types were distinguished: telocentric homomorphic
pair (TIT), subtelocentric homomorphic pair (SIS) and telo- and subtelocentric heteromorphic pair (TIS). The animal occurs almost everywherein Japan, Korea and other countries, especially in the southern areas.
1)

Showa University, Medical College, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

We collected 124 animals from 7 places of Japan and 9 animals from Seoul,
Korea. Among 124 animals, 94 (75.8%) wers TIT type, 29 (23.3%) were
SIT type and the remaining only one animal (0.8.%) was SIS type. All
animals collected in Sugadaira, Nagano-ken, and Obama, Fukui-ken, were
TIT. The SIS animal was collected in Hamamatsu population. The
frequency of the three types varies considerably in Japan. Among 9
animals collected in Seoul 3 were TIT type, 5 were TIS type and theremaining one was SIS type. In Japan, SIS and TIS animals are scarce,
while in the Korean population their frequency seems to be higher. These
differences between Japan and Korea populations will be further investigated with more material.

32. Crosses between black rats characterized bychromosomal polymorphism
Tosihide H. YOSIDA, Yukuo MORIGUCHI, Takako
FUKAYA and Akira NAKAMURA

The black rat Rattus rattus L., is characterized by polymorphism of
the largest No. 1 chromosomes (YasIDA et al. 1965). As stated in theforegoing report, animals with subtelocentric homomorphic pair (SIS)
were very few in the natural population of Japan. In order to find out
why this particular type is so scarce in Japan, animals with different
karyotypes were crossed by each other in our animal rooms, and then thechromosomes of the F 1 hybrids were observed with the following results:
segregation from hybrids between TIT and TIS was into 3 TIT and 4 TIS
animals. The ratio of segregation closely approaches the theoretical ratio
1: 1. 28 Fl hybrids between TIS parents which were collected in Hamamatsu segregated into 9T/T: 15T/S:4S/S. The theoretical ratio (1:2:1)
would be 7 TIT: 14 TIS: 7 SIS. 7 F 1 animals between the Korean TIS
animals segregated into 1 TIT: 4 TIS: 1 SIS: I? 10 animals obtained from
TIT parents showed all the TIT homomorphic chromosome pair. From
this study, it seems that animals with the subtelocentric homomorphic
pair (SIS) were born and could survive in the animal room. A further
problem is to find out, why SIS animals are so few in the natural population though they are expected to be born in numbers comparable tothose of TIT animals.

RESEARCHES CARRIED OUT IN 1965

53

33. Chromosomal polymorphism of the rat, Rattus norvegicus,
and its segregation
Tosihide H. YOSIDA, Reiko TSURUTA
and Yukuo MORIGUCHIThe third chromosome pair of laboratory bred and wild rat, Rattus
norvegicus, is characterized by polymorphism (YOSIDA and AMANO 1965).
The differences in the character of the polymorphic chromosomes are very
striking especially in the segregating offspring of hybrids between animals
with different karyotypes. YOS-strain rats were characterized by No. 3
subtelocentric homomorphic pair (TIT), while WIS-strain had a telocentric
homomorphic pair (SIS). 20 Fl hybrids between YOS- and WIS-strain rats
showed all a heteromorphic telo- and subtelocentric No.3 pair (TIS). 16
back-cross hybrids between F 1 and WIS-strain segregated into 7 TIT,
S SIT and 1 (?) according to the ratio of 1: 1, as expected.
The karyotype in a new strain of rats, Toma, obtained from Toma
Institute, Tokyo, was examined. It was of WIS-type (TIT type) concerning
the No.3 chromosome. This strain may have originated from WIS-strain
rats which are widely spread in this country.

34.

Chromosomes of male ants Aphaenogaster osimensis
produced by parthenogenesis of workers
Hirotami T. IMAI and Tosihide H. YOSIDA

It is well known that normal males develop from unfertilized eggs
produced by the queen, and male haploidy was cytologically proved in
Monomorium by Smith and Peacock (1957). It is believed that unfertilized
eggs laid by the workers develop also into males, but very few cytologicalobservations are available.
Recently it was found that the vernal workers of Aphaenogaster osimensis
could lay eggs when they were isolated from the inhibition exercised by
the queen (IMAI 1966). Such eggs produced by parthenogenesis of vernal
workers were cultured and all developed into morphologically normal
males. The chromosomes of both their germ and somatic cells werestudied.
The chromosome number in spermatogonia and spermatocytes was
haploid (n=16), i. e. the same as in normal males (IMAI 1965). No reduction
division was observed. The brain cells had also the haploid chromosome
number. Those males have developed from haploid eggs laid by theworkers. The eggs are assumed to have undergone two normal matura-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

tion divisions reducing the chromosome number. Therefore those males
have a haploid chromosome number both in somatic and germ cells likenormal males.

35. Polyploid cells observed in male and queen antsof Aphaenogaster osimensis
Hirotami T. IMAI and Tosihide H. YOSIDAPolyploid cells in somatic tissues of worker ants have been alreadyreported by Hauschteck (1961). The present authors report polyploidy inboth somatic and germ cells of Aphaenogaster osimensis. The normal
chromosome number of this species is n=16 and 2n=32 (IMAI 1965). This
species is very convenient for the observation of ploidy, because large
submetacentric and subtelocentric chromosomes stand out in their karyotype and therefore ploidy can easily be determined by counting the numberof those marker chromosomes.
One example of polyploidy was found in a male prepupa, in which the
prepupal stage was considerably delayed but sperms had been alreadyformed. In this stage a few spermatogonial cell divisions are usuallyobserved and spermatogenesis begins only after an early pupal stage (IMAI1966). Among 55 somatic cells observed in the brain 39 were haploid, 14
were diploid, and 2 cells were tetraploid. The chromosomes of a testis in thesame individual were observed in 285 cells, of which 280 had the normal
haploid number, but three were diploid and two were tetraploid. In
addition ploidy was also found in the ovary of a queen pupa. Among 56
oogonial cells 8 were found to be tetraploid. It is interesting that ant
gonad contain polyploid cells, because ant eggs can develop by parthenogenesis, and the occurrence of polyploid individuals is possible in thisanimal.

36.

Nuptial flight and multiple mating observedin the formicine ant, Lasius niger
Hirotami T. IMAIThe author had the opportunity to observe the nuptial flight of Lasius
niger and to take a photograph of the copulation. This observation wasmade in August of 1964 at Kinugawa. It was a muggy and windlessevening. More than 10 thousand winged queens and males suddenly
appeared in a swarm around a street lamp. The flight was observed
from p.m. 7 to p.m. 9. Although the pairing flight could not be observed,

RESEARCHES CARRIED OUT IN 1965

some interesting events were seen.
Most ants soon landed and were creeping
around. The size of the male in this species
is about one half of the queen, but the numberof males seemed to be larger than that of
the queens. They were lively and followed
eagerly the queens. The photograph (Fig. 1)
represents one event. It was found that mostof the queens copulated with many males one
after another. This multiple mating was
already reported by Kerr (1961) in attine ants,
Mycocepurus. It is strange that a few queens
copulated with 3 males at the same time. This
multiple mating suggests that the fertilized
eggs of the queens might be genetically very
heterogeneous.

55

Fig. 1. Copulation of
L. niger.

37. Ovary development of workers and queens in
the ant Aphaenogaster osimensis
Hirotami T. IMAI

It is generally recognized that the worker ants have a vestigial ovary
whose function is completely reduced or inhibited by the queen in natural
condition. It can be, however, recovered in spring, and the workers
oviposite when they are isolated from the queen's inhibiting influence.
This report deals with the ovariole number of the workers of Aphaenogaster osimensis as compared with that of the queens. Each ovary of 9
normal queens of this species contained 12 to 18 (mean 13.6) ovarioles in
the pupal stage. On the other hand, only two ovaries having each one
ovariole were observed in the 20 adult workers (head width ranging from1.00 mm to 1.33 mm, while that of queens varied from 1.54 mm to 1.62 mm),as already observed by Brian (1954) in Myrmica.
Egg development in the ovaries of 20 vernal workers kept 17 days after
isolation from the queen was also observed. The two ovarioles of eachworker contained one to ten oocytes, on the average 4.5. These oocytes
were linked together along the ovariole in a row from a large basal oneof almost mature size (0.58 mm long) to the smallest distal one (0.17 mmlong). Oviposition by vernal workers was observed in other ants. The
development of the eggs was comfirmed.
From the above investigations it is clear that workers' ovaries are fully

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

functional or at least have the potential to produce eggs by parthenogenesis: they differ only in ovariole number from the queens' ovaries.

B.

38.

GI:NI:TICS, CYTOLOGY AND
BIOCHI:MISTRY OF PLANTS

Hereditary characteristics of three cytoplasms revealed
by long-time substitution experiments
Hitoshi KIHARA and Koichiro TSUNEWAKI

Nuclei of eight wheat species, five hexaploid and three tetraploid, wereintroduced by successive backcrosses into three different cytoplasms of
Aegilops caudata, Ae. ovata and Triticum timopheevi. The main characteristics of those nucleus-substitution lines are summarized in Table 1.
Table 1.

Hereditary characteristics of three cytoplasms revealed
by substitution experiments._---~

Cytoplasm
Nucleus donor

Ae. caudataI

Ae. ovataI

T. timopheevi

Hexaploid wheat

T. v. e.
P. 168
Salmonmale-sterile,
germless grainspartially male-fertile
male-sterile, haploidand twin seedlings
partially male-fertile
male-sterile, reduced
female fertility

male-sterile

male-sterile

completelymale-fertile
male-sterile

male-sterile
male-sterile

male-sterile
male-sterile

male-sterile
completelymale-fertilepolonicum vest.

pistillody, abortive
ovulespistillody

male-sterile,
delayed heading
male-sterile,
delayed heading

dicoccoides spont.

pistillody

male-sterile,
delayed heading

partially
male-fertile
completelymale-fertile
slightly
male-fertile

Compo 44spelta duh.
Tetraploid wheat

durum reich.

The response of a nucleus to the three cytoplasms was not the same.
The cytoplasm of Ae. caudata was, when functioning with the nucleus
of a hexaploid wheat, characterized by frequent formation of germless

RESEARCHES CARRIED OUT IN 1965

57

grains, haploid or twin seedlings, and by pistillody when the nucleus
belonged to emmer wheat. In some cases, female fertility was reduced.
P. 168 and Compo 44 acted as partial fertility-restorers. Ovata cytoplasm
induced male sterility with the nuclei of most wheat varieties andextremely prolonged vegetative growth with the nucleus of emmer. No
pistillody was caused. P 168 restored completely male fertility. Timopheevi
cytoplasm was different from the above two, because with the nuclei ofT. spelta and all emmer varieties male fertility was restored to a varying
level. This cytoplasm did not exhibit any other remarkable effect.
Hereditary characteristics of different cytoplasms are revealed only whena series of nucleus substitutions have been carried out and various different
nuclei of more or less closely related species have replaced their own.
This kind of approach provides a useful mean for the study of cytoplasmic
differentiation which was taking place in the course of plant evolution.

39. Preliminary study on free amino acids in the anthersof nucleus-substitution lines
Hitoshi KIHARA

Nucleus-substitution lines with the cytoplasm of Aegilops caudata, Ae.
ovata or Triticum timopheevi have been developed in many varieties of
6x and 4x wheats. In this experiment, those of six 6x wheats were
employed, i.e., Triticum vulgare var. erythrospermum (T. v. e. in abbrev.),
P 168 (a derivative of T. v. e.), T. compactum var. No. 44 (Comp. 44), T.
vulgare var. Salmon (Salmon), T. spelta var. duhamelianum (Spelta), anda synthesized hexaploid (ABD 13). The mature anthers were collected
before anthesis and the content of free amino acids was analyzed according
to the method described by FUKASAWA. An automatic amino acid analyzer
was used. No replication was made. The analysis was carried out by
Mr. S. SAKURAI of the Department of Biochemical Genetics, whose technicalhelp is greatly appreciated.
As the investigation is incomplete, I want to give only a short resumeof the results hitherto obtained.
The normal strain of P 168 is characterized by an accumulation of alarge quantity of free tyrosine. Among various nucleus-substitution lines,
no consistent tendency was observed to accumulation or deficiency ofamino acids due to a particular cytoplasm. However, in a majority ofcases (4 out of 6), proline was deficient in the substitution lines withovata cytoplasm. On the other hand, serine was accumulated in many
substitution lines. No clear relation was found between the content of
free amino acids and the restoration of fertility.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

40.

Comparative gene analysis of common wheat and its
ancestral species. II. Waxiness
Koichiro TSUNEWAKI

For the study of origin and differentiation of cultivated plants, the new
method of "Comparative gene analysis" has been applied to common wheat
and its relatives.
According to this method, gene analysis is carried out in a given
cultivated species and in parallel in its relatives in the same genetic
background. Due to the results obtained, the origin of genes which arefound in a cultivated species can be traced, and its coming into existence
can be based on the origin of those genes.
Adopting this working plan, a comparative gene analysis of common
wheat and its direct ancestors, emmer wheat and Aegilops squarrosa,
and, incidentally einkorn wheat, has been carried out for foliage waxiness.
The analysis consisted of three steps, i.e., (1) monosomic and conventional
gene analyses of common wheat in order to clarify the genic system
controlling this character, (2) monosomic analysis of various synthesized
6x wheats, from which the genotypes of their components, i.e., emmer
wheat and Ae. squarrosa were determined in comparison with those ofcommon wheat, and (3) survey of the distribution of those genes in alarge number of varieties of common wheat and its relatives by customarycrossing experiments or by simply observing their phenotypes.
Waxiness of common wheat is controlled by genes located in four loci,i.e., WI on chromosome 2A (formerly XIII), W2 in D genome (chromosome
remains unidentified), [I-Win A or B genome and [2- W on chromosome
2D (formerly XX). The W loci are for waxy genes and the [- W loci for
their epistatic inhibitors.
Almost all varieties of common wheat possess WI and, probably, W2
but lack both [1- Wand [2- W. In emmer wheat, almost all cultivatedvarieties have WI and i l- W except Triticum pyramidale that possesses[1- W together with WI.
On the other hand, wild emmer contains WI in
addition to [1- W. All varieties of einkorn wheat, including both wild andcultivated species, have WI but not [1- W. Waxy Ae. squarrosa strains
have the genotype, W2 i2- W, while waxless strains have W2 [2- W.
The presumable emmer and squarrosa parents of common wheat musthave been of the genotype WI il- Wand W 2i 2- W, respectively. T. dicoccoides and non-waxy squarrosa can be excluded from the list of probableparents of common wheat. The progenitor of common wheat must have
had the genotype WI W2 il- W i2- W. Since the occurrence of Ae. squarrosa
with the genotype W2 i2- W is restricted to north-western Iran, the birth-

RESEARCHES CARRIED OUT IN 196559

place of common wheat is assumed to have been in this region.
41.

Comparative gene analysis of common wheat and its
ancestral species. III. Growth habit and awnedness
Koichiro TSUNEWAKI

A comparative gene analysis has been carried out for two characters,i.e., growth habit and awnedness. The results are summarized as follows:
Growth habit of common wheat is mainly controlled by genes belonging
to three loci, Sgl, Sgz and Sga, located on chromosome 5D (formerly XVIII),
5A (IX) and 2A (XIII), respectively. Some multiple alleles are found inthese loci; Sgl, Sgz and Sga are alleles for typical spring habit, Sgl·, Sgz"
and sga for semi-spring habit, and Sgl and Sg2 for typical winter habit.
The Sgl allele is much more effective than sgz for the induction of wintergrowth habit. Most of those homologous alleles are found in the ancestorsof common wheat, i.e., Sgz, Sgz·, sgz, Sga and sga occur in emmer wheat
and Sgt· and Sgl in Ae. squarrosa.
All waxy strains of Ae. squarrosa, which is the D-genome donor to common
wheat, have Sgl. This fact suggests that common wheat received the mostpowerful gene for winter habit, Sgl, from the squarrosa, parent, acquiring
a better adaptability to high latitudes than that of the emmer parent.
As the geographical distribution of the Sgl gene in squarrosa populations
is mainly concentrated in northern Iran, the probable birthplace of common
wheat should be sought in this region.
Sgz alleles of emmer wheat are epistatic over Sgl alleles of Ae. squarrosa,
when brought together in synthesized 6x wheats. On the other hand,such a relation of Sg2 to Sg, alleles is not observed in common wheat.
Since the two pertinent loci are considered to be duplicated loci; their
genetic diploidization seems to have caused the loss of epistasis in thenatural polyploid.
Awnlessness or awnlettedness of common wheat is confirmed to bemainly controlled by three epistatic inhibitors, Hd on chromosome 4B(formerly VIII), B1 on 5A (IX) and B z on 6B (X). Awn promotion is causedby three genes, al on chromosome 2B (II), az on 2D (XX) and as on 2A(XIII). All varieties except Red Bobs have the three promoters. Morethan 30% of common wheat varieties have one or more epistatic inhibitors.
Almost all emmer varieties have the promoters, al and as. but noepistatic inhibitors. Apparently, all three inhibitors have been originatedat the hexaploid level. az of common wheat must have been derived fromthe squarrosa parent.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

42. Comparative gene analysis of common wheat and its
ancestral species. IV. Glume hairiness
Koichiro TSUNEWAKI

Monosomic and conventional gene analyses indicated that glume hairinessof common wheat is controlled by a single dominant gene, Hg, locatedon genome A chromosome, lA (formerly XIV). A similar analysis of asynthesized 6x wheat, ABD-VI, revealed that its glume hairiness is also
controlled by a single dominant gene, derived from Triticum durum
var. Golden Ball, which is located on chromosome lA, indicating that the
Hg gene of emmer is the same as that of common wheat. A dominant
gE'ne controls also glume hairiness of einkorn, the A genome wheat.
Thus, it is reasonable to assume that the Hg gene of einkorn wheat is
the same as that found in the homologous chromosomes of the ubiquitous
A genome of the tetra- and hexaploid wheats.
In common wheat both Hg and hg (allele for non-hairiness) are present,
but there is undoubtedly a significant difference between their frequencies
in various parts of the world. Primitive populations, both geographicallyand chronologically, contain more varieties with the dominant Hg thando advanced populations. Apparently owing to selection against hairy
glume the Hg allele is gradually being eliminated.
In emmer wheat hairy and hairless glumes are common both in wild
and cultivated species. Therefore, the responsible alleles found in common
wheat must have been obtained from emmer when the first 6x wheat was
produced or, later, through formation of pentaploid hybrids between thealready existing hexaploid and emmer wheats.
T. boeoticum, a wild einkorn wheat, has Hg, while cultivated einkorn
contains only hg. It is, therefore, assumed that the gene Hg of emmer
wheat was derived from wild einkorn wheat when the first tetraploid
wheat was produced. Since introgression of genes from einkorn to emmer
wheat through the formation of triploid hybrids is very difficult, the originof hg in emmer wheat can hardly be traced back to cultivated einkorn
wheat.
Taking all these considerations into account, we may assume that
T. dicoccoides, the wild emmer, was produced from a cross between
T. boeoticum, a wild einkorn, with Hg and some species of Sitopsis (mostlikely Ae. speltoides) , followed by amphidiploidization of the hybrid. In
this way, Hg of wild einkorn wheat was introduced into the wild emmer
wheat, in which mutation from Hg to hg took place. Both alleles weretransferred to cultivated emmer wheat during its differentiation from thewild form, and later incorporated into common wheat.

RESEARCHES CARRIED OUT IN 1965

43.

61

Awn inhibitor in Redman wheat
Koichiro TSUNEWAKI

Based on the F 1 data of a series of crosses between Redman mono'somics and Prelude, Campbell and McGinnis (1958) concluded that thecommon wheat variety, Redman, carries two complementary genes forawn suppression on chromosomes 5A (formerly IX) and 1D (XVII). One-of them located on chromosome 5A seems to be allelic to B 1 gene.
In order to confirm their result, Redman and two other awnless varieties,
Elgin and Jones Fife, which are known to carry only the B 1 gene, werecrossed to two awned varieties, Prelude and 5-615. Actual segregation
ratios of all six crosses fitted the 1: 3 ratio, disproving the 7: 9 ratio of
awned vs. awnless.
Mono-5A of Redman was crossed to 5-615 as male parent. In the F 1
generation, a cytological examination was carried out in order to distinguish the disomic from the monosomic hybrids. Their selfed progenies
were tested for the segregation of awn types. Similarly to the result ofCampbell and McGinnis, all disomic F 1 plants were awnless, while all F 1
mono-5A's were awned. In the F2 generation of the disomics, however,
1: 3 ratio of awned vs. awnless was obtained instead of 7: 9 ratio expectedfrom segregation of two complementary genes. No awnless plants were
found in the offspring of F 1 mon-5A.
From these results it can be said that the gene system for awnlessness
of Redman is not different from that of Elgin and Jones Fife, indicating
only one dominant inhibitor, B 1 • Person (1956) has already pointed outthat univalent shifts and other cytological changes may occur in monosomics due to occasional meiotic irregularities. Therefore, it is necessary
to carry out a monosomic analysis, at least, until the F2 generation, andto compare the F 1 record with the segregation data in F2. By suchprocedures a wrong conclusion due to an occasional cytological changethat occurred in a certain monosomic line may be avoided.

44. Genome analysis of the genus Eremopyrum
Sadao SAKAMOTO

Morphological and cytogenetical studies of Eremopyrum, a genus of
the tribe Triticeae, were carried out in 13 different interspecific hybrids
among three strains of two diploid species, Er. buonapartis (Spreng.)
Nevski and Er. triticeum (Gaertn.) Nevski and nine strains of two tetraploid.species, Er. buonapartis and Er. orientale (Linn.) Jaub. et Spach.
The main characteristics of the hybrids were as follows: (1) vigorous

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

growth of hybrids, and normal tillering and flowering; (2) intermediate
spikelet characters of the hybrids between parents; (3) very high pollen
sterility and complete seed sterility in all hybrid combinations.
Hybrids' average chromosome pairing per cell at MI of PMCs is shown
Table l.

Average chromosome pairing per cell in the PMCs ofinterspecific Eremopyrum hybrids

Cross combinations (Sj) X 0 )No. ofcells
observed

Average chromosome
pairing per cellIII

II

Er. buonapartis (4x)xEr. buonapartis (2x):7034 x 7035

Er. buonapartis (4x) X Er. triticeum (2x):7031*xEr. trit.-1
n7032 xn7033 xn7034 x7034 x Er. trit.-2
7036 X Er. trit.-1
n7038 xn7042 xn7043 xEr. buonapartis (4x) x Er. orientale (4x):7034 x 7037
Er. orientale (4x) x Er. buonapartis (4x):7037 x 7042
Er. orientale (4x) x Er. triticeum (2x) :7037xEr. trit.-1
Er. distans (2x) x Er. triticeum (2x)**:7041 x Er. trit.-2

189
156
306
183
408
139
115
425
273
346

0.01
0.02
0.02
0.01

276

6.95

7.11

1.37
1.35
0.97
1.13
1.73
0.97
1.35
0.76
1.01

18.27
18.30
19.06
18.70
17.48
19.05
18.28
19.48
18.70

6.22

15.55

445

0.00

6.62

14.76

725

0.00

6.82

7.34

263

0.01

0.70

12.59

* As to spikelet morphology this strain closely resembles Er. orientale.
** SAKAMOTO and MURAMATSU (1963).

in Table 1. From these data, the following conclusions are drawn: (1)the genome of diploid £1'. buonapartis is found in tetraploid £1'. buonapartis;(2) the genome of diploid £1'. triticeum occurs in £1'. orientale; (3) a commOngenome is shared by tetraploid £1'. buonapa1'tis and £1'. orientale.
The morphological characteristics and chromosome pairing of F 1 hybrids
between the diploid species £1'. distans (C. Koch) Nevski and £1'. triticeum
had been previously examined (SAKAMOTO and MURAMATSU 1963). Also akaryotypical comparison had been previously carried out for diploid andRESEARCHES CARRIED OUT IN 1965

63

tetraploid Er. buonapartis, Er. distans and Er. orientale (SAKAMOTO andMURAMATSU 1965). From those and the present studies, it is assumed
that the tetraploid Er. buonapartis has originated from an amphidiploid
between diploid Er. buonapartis and Er. distans, while the tetraploid Er.
orientale was derived from an amphidiploid between Er. distans and Er.

triticeum.
45.

An intergeneric hybrid between Eremopyrum orientale (Linn.)
jaub. et Spach. and Aegilops squarrosa Linn.
Sadao SAKAMOTO

An intergeneric triploid hybrid between Er. orientale (2n=28; Iran) andAe. squarrosa (2n=14; material received from Dr. H. KAPPERT) was,obtained in 1964.
The shape of the F, spikes was intermediate between the parents but
the spikelets showed characters of Eremopyrum.
Pollen- and seedsterility were complete. Average chromosome pairing per cell of the F,
was O.I I1 + 20.8!. All bivalents observed were associated loosely by aterminal chiasma. This indicates that the genomes of Er. orientale are,quite different from the D-genome of Ae. squarrosa.

46. Intergeneric hybrids between two Eremopyrum
and Agropyron species
Sadao SAKAMOTO

The following three intergeneric pentaploid hybrids were produced in
1964: (1) Er. buonapartis (Spreng.) Nevski var. buonapartis (2n=28; Iran)
X Ag. tsukushiense (Honda) Ohwi (2n=42; Japan), (2) Er. buonapartis var.
sublanuginosum (Drob.) Melderis (2n =28; Afghanistan) X Ag. tsukushienseand (3) Er. orientale (Linn.) Jaub. et Spach. (2n=28; Iran) X Ag. tsukushiense.
All F, plants were perennial, and the shape of their spikes and spikelets
was of Agropyron type. Complete pollen sterility was observed in all
hybrid combinations. Average chromosome pairing per cell of the F, wasin (1) O.Om+O.7 I1 +33.6!, in (2) O.8 u -f-33.5! and in (3) O.9 1l +34.0r . Bivalents:and trivalents were all loosely associated the partners connected only
terminallY. It was concluded that there is no genomic homology between
the two Eremopyrum species and Ag. tsukushiense.
Fifteen backcrossed plants were obtained when Ag. tsukushiense was,crossed as male parent to the third hybrid, Er. orientale X Ag. tsukushiense.
To its tiIIering clones colchicine solution was applied and two well-developed
seeds were obtained in 1965.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

47. Photoperiodic response of various Oryza species, VIII
Tadao C. KATAYAMA

A classification of Oryza strains into photoperiodically "sensitive" or"insensitive" to short day treatment was given in a previous report(KATAYAMA 1964). In the present article, further studies are reported
on 114 strains belonging to 10 species, which were recently collected fromseveral tropical countries, Dutch New Guinea, Brazil, Australia, Borneo,
Africa and Madagascar.
The seeds were sown on April 20 and the seedlings were transplanted
on May 20, 1965. They were grown under natural day light until June
22 (summer solstice) and thereafter, one half of the plants were exposed
to short day condition (12 hours light+ 12 hours dark cycles) and the otherhalf were kept under natural day length (control). The heading dates
observed in those two plots were compared. Plants whose heading was
earlier by 20 days or more under short day treatment than the control
were classified as photoperiodical:)' "sensitive", and those whose heading
was earlier by less than 10 days were classified as "insensitive".
Both sensitive and insensitive strains were found among 56 strains of
the cultivated species (Oryza sativa) collected from Borneo and Madagascar,
namely 26 were sensitive (46.4 per cent of the total).
Also 58 strains belonging to 10 wild species were tested, i.e., O. sativa
f. spontanea, O. perennis, O. barthii, O. breviligulata, O. ojjicinalis, O.
punctata, O. eichingeri, O. tisseranti and O. perrieri, collected from Dutch
New Guinea, Brazil, Borneo, Australia, Africa and Madagascar. Amongthem 57 strains (98.3 per cent) were photoperiodically sensitive. Only one
strain of O. barthii from South Africa was insensitive. Summarizing thepresent data and those reported in the previous paper (KATAYAMA 1964),
143 strains (70.4 per cent) of cultivated species were classified as sensitive
and the remaining 60 as insensitive, while 348 strains (90.9 per cent) ofwild species were sensitive and only 35 were insensitive.
Thus the great majority of wild Oryza species were photoperiodicallysensitive. This finding indicates that sensitive strains may have had in
the process of selection an advantage over the insensitive ones undernatural condition. On the other hand, many insensitive strains were
found in cultivated species, O. sativa and O. glaberrima. This is probably
due to that insensitive strains often carry such useful agronomic characteristics as a short growing period, hence their wide occurrence among thecultivated species resulting from breeders' selection.

RESEARCHES CARRIED OUT IN 1965

48.

65

Diallel crosses among Sikkimese rice types, II
Tadao C. KATAYAMAIn Sikkimese rice, the occurrence of indica and japonica types was
ascertained in the recent three years from the examination of several
characters and behavior in crosses. One of the most important charactersfor the classification into indica and japonica types is the hybrid sterility
between them. Therefore, diallel crosses were carried out using sixteen
strains, namely, fourteen of Sikkimese rice, one indica type and one
japonica type strain (KATAYAMA 1965). Most of the crosses were successful and 2,072 hybrid seeds were obtained during the last two years.
Some of them were sown in April, 1965. Several characters of the hybrids,
such as germination rate, vigor, number of tillers, plant height, heading
date, length of panicles, number of rachises, pollen- and seed-fertilities,
number of seeds, grain weight, were compared with those of the parents.
The results hitherto obtained indicate the occurrence of both indica andjaponica types in Sikkimese rice. Of 14 strains studied, 10 strains, C7716,
C7717, C7718, C7725, C7727, C7729, C7732, C7734, C7754 and C7757, wereof indica type, while, C7707, C7719, C7722 and C7735 were of japonica
type. The average pollen fertilities of 20 japonicaxjaponica, 110 indica
x indica, 55 indicaxjaponica and 55 japonicaxindica hybrids were 85.5,
89.8, 72.0 and 68.3 per cent, respectively. The pollen-fertility of thelatter inter-sterile two groups was considerably lower than of the former
intra-fertile two groups.
From the present results, it is assumed that the differentiation ofcultivated Oryza sativa into indica and japonica types might have takenplace in the Indian subcontinent, particularly in Sikkim or adjacentregions, and that both types have been cultivated together in the Sikkimese paddy fields.

49.

Embryo transplantation in the genus Oryza
Tadao C. KATAYAMA

In order to investigate the compatibility relation between embryo andendosperm of Oryza species, embryo transplantations between rice species J
were made. The method was described in the previous paper (KATAYAMA 1965).
Nine strains of five species, namely O. sativa (two indica and twojaponica types and a strain of o. sativa f. spontanea) , o. perennis, O.
barthii, o. glaberrima and O. breviligulata, were used. Seedlings from
intact seeds developed besides the coleoptile, the 1st, 2nd and 3rd leaves

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

and one to four roots. Embryos alone without endosperm produced only
the coleoptile, no root appeared except in "Kyoto Asahi", a japonica
type of O. sativa.
Seedlings from homogeneous transplantation (embryo transplanted totheir own endosperms) produced the coleoptile, 1st, 2nd and 3rd leaves
and three roots. In heterogeneous transplantation, embryos of "Kyoto
Asahi" (KA) were grafted upon the endosperms of other strains of O.
sativa, O. sativa f. spontanea, O. perennis, all Asian species. In this casethe coleoptile, 1st and 2nd leaves and a root have developed. In otherheterogeneous transplantations, between" Kyoto Asahi" (KA) and African
species, KA/barthii, KA/glaberrima and KA/breviligulata, only the coleoptile
grew but no root was found. In all reciprocal transplantations, sativa/KA,
spontanea/KA, perennis/KA, barthii/KA, glaberrima/KA and breviligulata/
KA, the coleoptile, 1st leaf and a root developed.
In heterogeneous transplantation, "108" (indica type of O. sativa) toother strains of O. sativa, only the coleoptile and 1st leaf appeared.
However, in other heterogeneous transplantations, 108/spontanea, 108/
perennis, 108/barthii, 108/glaberrima, 108/breviligulata, only the coleoptile
grew. In the reciprocal combinations, sativa/108, spontanea/108, perennis/
108, barthii/108, glaberrima/108, breviligulata/108, coleoptile, 1st and 2nd
leaves developed.
From these results, the following conclusions were drawn. The growthof embryos grafted upon endosperms differs according to the combination.
Embryos of japonica type grafted upon endosperms of Asian species weremore vigorous than the same embryos grafted upon the endosperms ofAfrican species. Such differences were not found with embryos of indica
type. Nutrient absorption from endosperms of indica type by embryosof other species, was more remarkable than that from endosperms ofjaponica.

50.

Anatomical changes in the seminal root of Oryza sativa
after the removal of adventitious roots
Tadao C. KATAYAMA

It has been observed in several Gramineae crops that the seminal root
dies after the development of many adventitious roots. However, it was
found in rice that the seminal root alives very long and exhibits structuraland developmental changes. To examine them, the following experiment
was made. A common variety of Japanese rice, "Kyoto Asahi", wasused as experimental material.
The plants were cultured in WAGNER'S pots. In order to keep the

RESEARCHES CARRIED OUT IN 196567

materials alive by the seminal root alone, all adventitious roots developed
from the nodes were cut off in succession. During the period from 30
to 100 days after sowing, the seminal roots were fixed 24 hours withformalin-acetic acid mixture, dehydrated with ethyl alcohol and N-buthylalcohol and embedded in paraffin. Ten to 20 p thick transverse andlongitudinal sections were made and Delafield's haematoxylin was used
for staining.
The first change in the seminal root was an enlargement of root diameter due to a remarkable secondary growth and an increase in thexylem and phloem strands. Under normal condition, six strands of xylem
and phloem were observed as shown in Fig. 1, but more than seven of

Fig. 1.

Fig. 2.

both were found in the present experiment. Thirteen strands of Fig. 2
are the extreme case observed.
Further, more lateral roots developed as compared with the control.
The secondary growth of the seminal root is an adaptive change ofthis root performing in my experiment the function of the normal rootsystem.

51.

Variations in the breeding system of a wild nee,
Oryza perennis
Hiko-Ichi aKA and Hiroko MORISHIMA

A number of genetic stocks of Oryza perennis Moench, collected fromvarious tropical countries of the world, were investigated regarding avariety of characters related to the breeding system of the species. O.
breviligulata and cultivated rice species, sativa and glaberrima, were also
observed for comparison. Two variation axes were assumed, one concerning the pollination system, and other concerning the relative extent

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

of seed and vegetative propagation. The former was expressed by a
combination of measurements for the time interval from flowering to pollen emission, stigma size and pollen-grain number per anther; latter fromthe regenerative ability of stem cuts, seed number per plant, "awn-development index" and "seed-dormancy index". Variations in all these characters were correlated, and it was concluded from the correlations thatstrains propagated mainly by seeds tend to be relatively autogamous.
For vegetative propagation were found three methods, cespitose growth,rhizome formation and tiller separation. Most African and a few Asianas well as Oceanian strains were rhizomatous, while some Amazonianstrains showed tiller separation. Not only the Asian forms were differentiated into perennial (perennis) and annual (spontanea) types as previously pointed out by the writers, but O. perennis as a whole seemed
to be differentiated in the same manner. (Evolution, in press)

52.

Estimation of indices showing genetic plant types and theircorrelations with yielding capacity in a segregating
populatzon of rice
Hiroko MORISHIMA, Hiko-Ichi OKA and Te-Tzu CHANG

A genetic plant type was defined as a genetically conditioned association of morphological features. A number of Fa lines obtained from across between two rice varieties, Peta (tall) x IGT (short), were measured
regarding various agronomic characters, in the wet and dry crop-seasons,at the International Rice Research Institute, Philippines. As the parents
differ in a major gene controlling height which gives a 3: 1 F 2 ratio, Fa
lines homozygous for the tall and the short alleles were used for observation. The major height gene seemed to pleiotropically modify various
characters bringing about a contrast between "tall" and" short" types.
We assume that polygenes may also bring about axes of genetic variationin plant type. Regarding polygenic variations, genetic correlations ofcharacters within the tall and short line-groups were analyzed by using
the technique of principal component analysis. After rotation to "simple
structure" of component vectors, two axes of variation in plant typewere obtained, one (a axis) showing the variation between "paniclenumber" and" panicle-length" types, and the other ((3 axis) showing thevariation between "internode-length" and "internode-number" types.
The panicle-number type has a Jarger number of shorter panicles andsmaller leaves than the panicle-length type. The internode-length typehas a smaller number of longer internodes and more erect leaves than
the internode-number type. These axes of genetic variation in plant type

RESEARCHES CARRIED OUT IN 196569

were considered as showing latent phases of character association due topolygenic effects. Potential yielding capacity of the Fa lines in an intensive cultural condition was estimated from the same data in terms of
total panicle length per plant and light transmission ratio (- K. LAI). Shortlines with the major gene for short stature were more productive thantall plants. Within the tall and short line-groups, plants of "paniclenumber type", as well as of "internode-length type", seemed to havehigher yielding potential than those of the opposite types. These planttype scores had relatively high heritability values, and were considered
to be useful criteria for selection.

53. Seasonal change in genetic plant type of rice
Hiroko MORISHIMA, Hiko-Ichi OKA and Te-Tzu CHANG

Genotype-season interactional variations in various agronomic charactersand in plant types were investigated using the data for Fa lines of across between two rice varieties, Peta X IGT, which were grown in duplicate in the wet and dry crop-seasons at the International Rice ResearchInstitute, Philippines. As mentioned in our previous paper, the a and (3axes of genetic variation in plant type were obtained from genetic correlations (due to polygeness) of characters, by rotation to "simple structure"of vectors of principal components. The a axis shows the variation between" panicle-number" and" panicle-length" types, the former having
a larger number of shorter panicles and shorter leaves than the latter.
The f3 axis shows the variation between" internode-length" and "internode-number" types, the former having a smaller number of longer
internodes and more erect leaves than the latter. The results of varianceanalysis showed that in leaf and stem characters as well as in the a andf3 plant-type scores, the variance due to genotype-season interaction was
significantly large, indicating that their response to growing seasons is
genetically controlled. Variations in the magnitude of seasonal differencesin various characters were estimated by using a response index, R=Xwet-Xdry/Xwe,+Xdry (in%). Regarding seasonal change in plant type, thosealong the a and f3 axes of genetic variation and those of the axes weredistinguished. It was found that the complex of co-variations of characters due to genotype-season interaction could be for a greater part
accounted for by seasonal changes in the a and f3 plant-type scores. It
was concluded that so far as the data of the present study are concerned,
selection of "panicle-number type" in the wet season and of "internodelength type" in the dry season may bring about not only a stability ofgrowth in different seasons but also a high yielding potential.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

54.

Analysis of genes controlling heading time in Taichung
65 and other rice varieties
Kuo-Hai TSAI and Hiko-Ichi OKA

A representative Ponlai variety of Taiwan, Taichung 65, and an extremely
early variety from Northern China, Tatong-tsailai, were compared regarding their differences in genes controlling the heading date; bothvarieties belong to the Japonica type and are insensitive to photoperiod.
After a biometrical survey by Mather's method, genic analysis was madeby selecting certain homozygous progeny lines from the original crossand crossing them with the parental strains or with one another, so thatthe effects of invidual genes could be observed in a relatively uniform
genetic background. In addition to the earliness gene, E, which was
carried by our isogenic lines of Taichung 65, at least two modifiers promoting flower initiation, M 1 and m2, were found to be involved. Under
the high temperature of the second crop-season, M 1 and m2 exert theireffect only when E is present, but in lower temperature of the firstcrop, they promote flower initiation without E. They were considered
to be genes lowering the temperature response in respect to flower
initiation. It was found further that polygenes would exert a buffering
action on the effect of major genes, accelerating the heading of latelines and delaying that of early lines. The earliness gene E was foundto be distributed in various early varieties of the Japonica type, even ina photoperiod sensitive variety, Kissin. (Bot. Bull. Acad. Sinica, 7 [2]:54-70, 1966)

55.

Weakness of Fl hybrids in the Sativa rice group
Yaw-En CHU, Hiko-Ichi OKA and Yo TAKENAKA

Various crosses within the Sativa group containing series sativa andseries glaberrima were observed. In general, the frequency of occurrenceof Fl weakness was low within series sativa and high within series
glaberrima as well as between the two series. The F 1 plants of threedifferent crosses were used for culturing separately shoots and roots inagar and liquid medium, respectively. The Fl between two sativa varieties (414 x419) was characterized by leaf necrosis which appeared 19
days after germination. The F 1 between an Asian perennis and a barthii
strain (W106xAf111) showed a reduced plant weight; shoot elongation
seemed to be retarded, though the roots grew normally. In anothercross between a glaberrima and a barthii strain (W025 X W031), bothshoots and roots were poor, showing unbalanced growth.

RESEARCHES CARRIED OUT IN 1965

56.

71

Deterioration of Fl embryos in hybrids between Oryza sativa
and O. perennis subsp. barthii
Yaw-En CHU and Hiko-Ichi aKA

Anatomical studies of deteriorating Fl embryos in the cross sativax
barthii were reported last year. Reciprocal cross experiments showedthat when sativa was used as the female parent, the cross ability wasalmost normal (53.8% on the average), but germination was as low as
16.9%. In contrast, when barthii was used as the female parent, thecross ability dropped to 9.7%, while germination was normal (100%).
From anatomical observations of the embryos at early stages of development (1-3 day after pollination) fertilization was found to take placenormally in both directions of the cross. It then seems that the reciprocal crosses differ in the stage at which deterioration occurs. In sativa
xbarthii deterioration occurs 5-7 days after fertilization, while in barthii
X sativa it occurs 3 days after fertilization. The deterioration of embryos
was always accompanied by that of the endosperm. These observations
suggest that the inviability of Fl seeds was primarily due to the degeneration of the endosperm.
57.

Fl pollen and embryosac sterilities in the Sative rice group
Yaw-En CHU and Hiko-Ichi aKA

The Fl plants between series sativa and series glaberrima generally
show a high pollen sterility. The F 1 plants showing various degrees ofpollen sterility were investigated regarding the frequencies of chiasmata,lagging chromosomes and chromosome bridges. Some crosses with highpollen sterilities showed a reduced number of chiasmata (e .g. 16.9 percell in W042 X W106 and 23.3 in the parents). But no significant correlation was found between chiasma frequency and pollen sterlity. Some
Fl plants showed at low frequency lagging chromosomes and bridges(3.2% in Fl plants and 1.5% in parents).
Further, the embryosacs of Fl hybrids were observed. The pollen
sterile Fl hybrids between sativa and glaberrima had morphologicallynormal embryosacs at a high percentage (26.9-82.4%). Traces of degenerating cells found in the embryosacs suggested that the degeneration
was taking place after meiosis.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

58. Developmental instability in leaves, panicle
and culm internodes of rice
Kan-Ichi SAKAI and M. S. BALAL

The present report includes some results of investigations on developmental instability in the length of several organs, namely first, second andthird upper leaves, panicle and first, second, third and fourth culm internodes. The materials used were 24 lines taken at random from each of
the two populations, control and X-irradiated, originated from the same
variety Norin No. 8 and propagated according to the "one plant-one
offspring" scheme for five generations. The length of the upper threeleaves, panicle and four culm internodes was measured on an individual
culm basis and the developmental instability was determined for eachorgan in terms of intraplant variability.
Table 1.

Mean and genetic variance of developmental instability in length ofleaves, panicle and culm internodes of rice.
LeavesCulm internodes

Population

Paniclefirstsecond third

Control (C 6)
X-irradiated (X 6)

6.70
6.79

8.53
8.52

Control (Co)X-irradiated (X o)

0.1245 .4690 .4236 .0717.2386 .1356 .7213 .0516

firstsecondthird

fourth

3.54
3.57

1.74
1.72

1.91
1.89

2.24
2.19

-

Mean

7.64
7.85

1.72
1.74

Genetic variance.0038t .0137.1116 .0403.0038t .0320.0392 .0675

t Between lines variance is not statistically significant.
Mean values of developmental instability are given III Table 1. It isseen from the table that leaves in general have higher developmental
instability than culm internodes and panicle. There is, however, a definite difference with regard to developmental instability among homologous organs, e.g. developmental instability in the first of the three leaves
is the lowest, while that in the first of the four culm internodes is thehighest. No remarkable difference was observed between the values of
the control (C) and the X-irradiated (X) populations. Genetic varianceis also shown in Table 1, which demonstrates that, with the exception ofpanicle and second leaf, it was higher in the X-irradiated than in thecontrol population. Genetic correlations among developmental instabilitiesof panicle and four culm internodes reveal that neighboring organs are

RESEARCHES CARRIED OUT IN 196573

highly correlated while distant ones are not, and the closer the location
the higher the values.
Conclusions drawn from the present study are:
1. Degree of developmental instability is not the same among different
organs and even among homologous parts in rice.
2. X-ray treatment seems to have induced genetic variation in themagnitude of developmental instability of different organs.
3. Genetic correlation among developmental instability of neighboring
internodes is higher than that among distant ones.

59.

Genetical studies on clustered spikelets in rice
Katuyoshi MORIMURA and Kan-Ichi SASAKIThe dominant gene Cl for clustered spikelets belongs to the firstlinkage group. This gene is characterized by producing clustered spikelets
in the top part of rachises. There might, however, be polygenes which
could modify the effect of Cl gene in plus or minus direction. In thepresent study the relation between the gene Cl and the presumed polygenes was investigated. The material for this study were two normal
strains, seven F 4 lines possessing the Cl gene derived from the samecross, six Fl hybrids and two F 2 populations between the two normal
and some derived lines. In the latter, intraline variation in rachis length,
grain number per plant, grain density in a panicle and the degree ofclustering was observed, indicating that they are still segregating. In
addition to this intraline variations, seven lines were found to be significantly different with respect to the degree of clustering. On the other
hand, normal lines lacking the Cl gene were not always free from theoccurrence of clustering. Two normal lines in our material differed inthis respect, and they also behaved differently in crosses with the sameCl-line. This may suggest the presence of polygenes besides the major gene.
In the F2 population, normal and clustered segregate in 3 : 1 ratio. A
comparison between the normal plants with those segregating ones,
revealed that the first rachis as well as second rachises in the clusteredplants were apparently shorter than in normal plants, although there
was little difference with regard to grain number per plant. Thus, grain
density on clustered plants tends to be higher than in normal plants, but
the correlation between the degree of clustering and grain density was
not as high as was expected from the above observation. There is a highpositive correlation between the two following measurements of clustering; a, number of grains in clusters divided by total number of grains,and b, number of nodes from which the grains develop in clusters divided

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

by total node number.
It is found from this study that the Cl-gene or the polygenes concerned
produce shortening of rachises and clustering of grain near the top ofrachises, though the number of grains per panicle remains constant.
Thus, it is concluded that the effect of Cl-gene as well as of polygenes,concerned might be a growth inhibition of the top parts of rachises
which should otherwise continue growing.

60. Biometric-genetic study on rachis deficiency in rice
Kikuo WASANO and Kan-Ichi SAKAI

A mutant character in rice, rachis deficiency, is controlled by an allele,sp, a recessive major gene effecting partial failure of rachis formation on
the panicles. The sp gene was introduced into ten strains differing from'each other with regard to their genetic backgrounds. From examinationof those strains and their hybrids with normals it was found that:
1) The rachis deficiency tended to occur mostly in the lower half ofa panicle.
2) In the normal strain, there was a tendency in the primary branches
to be longest in the middle part of a panicle, whereas in the sp strain,
the primary branches were longest near the top and the length decreased
downward.
3) The expressivity of sp gene was variable among the strains: the expressivity in the lowest strain was 37.5, whereas it was in the highest
strain 76.5%.
4) The expressivity was found to be genetically negatively correlatedwith the length of the nearest internode of the culm, length of culm andpanicle, and also number of panicles, but it was positively correlatedwith the number of non-bearing tillers; the gene seems to prohibit normal
development and growth of panicles.
5) In the Fl hybrids between normal and sp strains, the dominance,effect of the normal gene was not complete; i. e., the Fl appeared moreor less affected by the sp gene. In the F 2 population, it was found thatin addition to the 3 : 1 segregation of normal and the deficient character,quantitative variation in expressivity took place. This may mean thatbesides the major gene, there must be polygenes responsible for the
expression of the mutant character.

RESEARCHES CARRIED OUT IN 1965

61.

75

Studies on genetic relationships among leaves, culm
internodes and the panicle in rice
M. S. BALAL and Kan·Ichi SAKAI

The rice variety, Norin No.8, revealed, though naturally self-fertilizing,
genetic variability for quantitative characters. A number of lines selected
at random from the variety proved to differ significantly with regard toleaf size, internode length and panicle size. The present paper reports theresults of an investigation on genetic correlations among those charactersin two groups of lines selected at random. The results and conclusions
drawn from this study are as follows:(1) It was found that lines from the same variety, Norin No.8, weredifferent with respect to the characters investigated.
(2) Genetic correlation between first and second leaf was as high as
approximating 0.9, while that between first and third leaves as low as
approximating zero. The genetic correlation between second and third
leaves was about 0.4.
(3) With regard to internode length, it was found that genetic correlation between two adjacent parts, that is, between panicle and firstinternode, or between first and second internode and so on, were generally
very high, the average being 0.765, while those between parts adjacentseparated by one, two or three organs became increasingly lower, thecorresponding values being 0.370, -0.010 and -0.415, respectively.
(4) Genetic correlations between panicle or first internode and firstleaf, between third or fourth internode and third leaf were very high,whereas genetic correlations among distant organs tended to be low ornegative.
(5) It is thus concluded that adjacent organs of the rice plant areunder stronger control of common genes than distant ones irrespectiveof whether they are leaves, internodes or panicles.

62. Cytogenetic studies in the genus Nicotiana
Yo TAKENAKA

The reduction division in PMC's was studied in F 1 hybrids of Nicotiana
species. Taxonomically, some of them were crosses within sections Alatae,
Aluminatae, Paniculatae and Suaveolentes while others were crosses between
different sections. The haploid chromosome number of parental strains
and the number of bivalent chromosomes found in the Fl hybrids are
given in Table 1. The Fl hybrids within section Alatae and Acuminatae

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

showed normal pairing, but other crosses had a few bivalents or none,
most chromosomes remaining as univalents.
Table 1. Nicotiana F 1 hybrids and the number of bivalent chrosomes in meiosis
of the hybrids (haploid number of parental species in brackets)
Cross combination

Bivalent No.

Within section Alatae,
pl1tmbaginijolia (10) xjorgetiana (9)9Iln
X langsdorjfii (9)9Il
Within section Acuminatae,
pauciflora (12)X acuminata (12)12 IIWithin section Paniculatae,
X glauca (12)paniculata (12)0-1 II
Within section Suaveolens,suaveolens (16)
X excelsior (19)OIl
0-1 II
goodspeedii (20)
X suaveolens (16)benthamiana (19)
X gossei (18)
1-4 II
Between sections,suaveolens (16, Suaveolens) X glutinosa (12, Tomentosae)
OIlrustica (24, Paniculatae)
X suaveolens (16)OIl
0-4 IIn
X megalosiphon Suaveolens)
0-1 IIH
X gossei (18, Suaveolens)
0-1 II
excelsior (19, Suaveolens) X longiflora (10, Alatae)
0-1 II
undulata (12, Paniculatae) X occidentalis (21)
1-3 II
debneyi (24, Suaveolens)
X tomentosijormis (12, Tomentosae)
2-5 II
knightiana (12, Attenuatae) xalata (9, Alatae)
0-1 II
paniculata (12, Paniculatae) x glutinosa (12, Tomentosae)
0-1 II
glauca (12, Paniculatae)
x sylvestris (12, Tomentosae)

63. Karyotypes in the genus Nicotiana
Yaw-En CHU and Yo TAKENAKAT AKENAKA, one of authors, had devoted himself to studies incytogenetics of Nicotiana. His main lines of work were observations ofchromosome pairing and tumor formation in species hybrids (TAKENAKA
1950 in Cytologia; 1953, 1956, 1962 in Bot. Mag., Tokyo; 1953 in Chromosoma; 1962a, 1962b in Jap. J. Breed. 1955, 1962c, 1962d, 1963 in Jap. Jour.
Genet.). In addition to these, the karyotypes of somatic chromosome havebeen observed. Strains belonging to four diploid species were used as
the materials. The root tips of adult plants were fixed in Carnoy fluid,
and acetocarmine smear technique was employed. The results so far
obtained are as follows:

RESEARCHES CARRIED OUT IN 1965

Speciesundulata
corymbosatrigonophylla
paniculata

77

Median Sub-median Sub-terminal Total

8
4
12
6
5
1
12
9
2
1
12
2
3
7
12
Hereby it was confirmed that the above diploid species of Nicotiana havedifferen t karyotypes.

64. Genetical studies on vein formation in
Nicotiana tabacum
Kan-Ichi SAKAI and Kikuo W ASANO

Two lots of seeds from the same variety of tobacco, one for control
and the other X-rayed, were propagated up to the third filial generationby the "one plant-one offspring" method. In the Ca (control) and Xa(X-rayed) populations, 44 lines selected at random from each population weregrown and the vein number per leaf was counted in two leaves fromeach of 20 plants in each line. The within-line variance or the environmental variance estimated in Ca and Xa lines was 1.71 and 1.86, while thegenetic variances were 1.62 and 2.33, respectively. The variation rangeof line means was greater in the X population than in the control,
especially toward a lower vein number.
Selection experiment was conducted in the X-line population for higher
as well as lower vein number. In the next generation, X4, the selection
was found to be ineffective for higher, but effective for lower veinnumber. Genetic and environmental correlation between vein number
and other leaf characters were investigated. It was found that veinnumber per leaf was not correlated with leaf number per plant neither
genetically nor environmentally. Vein number, however, was found to
be positively correlated genetically with leaf length but negatively withleaf width, while it was environmentally positively correlated with either
leaf length or leaf width. Thus, the vein number was found to be notcorrelated genetically with leaf area but positively correlated environmentally.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

65.

Inheritance of developmental instability inleaves of tobacco
Yoshiya SHIMAMOTO and Kan·Ichi SAKAI

Inheritance of vein distance variability and of bilateral asymmetry ofleaves was investigated in hybrids between a developmentally stabletobacco variety, Connecticut Broad Leaf, and an unstable variety,Ambalema. The F l hybrid was approximately intermediate between thetwo parents without any noticeable sign of dominance. The segregation
was apparent in the F 2 population, its variance being significantly larger
than in PI, P 2 and Fl. The heritability was found to be 0.7279 for veindistance variability and 0.4900 for bilateral asymmetry. It was found ina variety·trial conducted before that those two values were closely associatedamong 11 varieties, whereas no such association was observed in thepresent F2 population, the coefficient of the genetic correlation being
0.2325. The vein distance variability, however, was found to be positively
highly correlated genetically with the vein number per leaf with rG=0.8230.
Investigation of F 4 lines revealed that lines with higher variability in veindistance had longer but narrower leaves than others. The correlation
between Fa and F 4 lines of vein distance variability was 0.6712.

66. Competition in forests and an evidence that plantsof the same genotype do not compete
Kan-Ichi SAKAI, Hiromasa MUKAIDE and Koji TOMITAIntertree competition in a standing forest can be investigated bycomparing the growth of adjoining trees since environmental or soil
similarity may tend to make their growth similar while competition
produces differences. Accordingly, detection of intertree competition inforests may be possible by examining the correlation between adjacent
two trees or between two differences among three adjoining trees; thatis, correlation between Yi and Yi+l or that between (Yi-Yi+J) and (Yi+l-Yi+2), where y stands for stem diameter. (In our study hitherto conducted, it has been found that tree height does not tell much about
competition). Theoretically speaking, rYiYi+1 would be positive if there isno competition, but smaller or negative if intertree competition takes
place. The corresponding values of rCYi-Yi+l) (Yi+I- Yi+2) would be larger than
-0.5 in the absence of competition, but would become smaller in case ofcompetition.
Twelve forests of Cryptomeria japonica 1., nine of which raised from

RESEARCHES CARRIED OUT IN 196579

seed and the remaining three isogenic clones afforested by cuttings, were
investigated with regard to the two correlations above mentioned. Of
importance for this study was that the forest consists of trees of thesame age. The results are presented in Table 1.
Table 1.

Competition in forests of Cryptomeria japonica L.
StrainClone

KyushuHyogo (1)Hyogo (2)Average

Seedpropagatedforest1) ra= rY i Y i+1

NumberI of trees

Hokkaido (1)Hokkaido (2)Hokkaido (3)Akita
MiyagiShizuoka (1)Shizuoka (2)Hyogo (1)Hyogo (2)
2)

144
169
146

I
128
106
147
140
202
170
167
198
208

rb=r(Y-Yi+l) (Yi+1- Y i+2)I

Competition3)
effect
Nil
Nil
Nil

0.57
0.31
0.43

-0.32
-0.51
-0.37

0.436

-0.4660.08**-0.28**
0.05**-0.21**
0.06**-0.09**-0.12**
0.33-0.02**-0.75**-0.90**
-0.58
-0.65
-0.25
-0.51
-0.53

-Ht

-

Nil

-

+

+
-Ht

+
+
+

*

3) Conclusion drawn from ra'

It is found from Table 1 that:r a proved to be a better criterion than rb for the detection of competition
effect.
(2) The r a values are all highly positive in the three clone forests wheretrees of the same genotype are growing. The X2 test of the three correlations obtained from the clone forests allowed to regard them as estimatesfrom the same p(P=0.10- 0.20).
(3) Forests raised from seed showed apparently lower values of the correlation
except for one forest from Hyogo.
It is hereby demonstrated that trees of the same genotype do not compete
against each other. Information from other studies on the estimation of genetic,·environmental and competitional parameters in Cryptomeria forests has shownthat clone forests are completely free from competition. It is thus concluded
that competition is the result of interference between different genotypes, atleast in plants.
(1)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

67. Callus tissue culture in Japanese morning glory
Yo TAKENAKA, Yoshiaki YONEDA and Yaw-En CHU

A strain of Japanese morning glory which frequently produced tumors
was reported in 1964 (Ann. Rep. 15: No. 73). This strain (MT), threestrains derived from it (B, Ie and dl, carrying the genes for Blown,feather and delicate, respectively) and a wild strain (Tks) were compared
regarding the growth in vitro of callus tissues which have been preservedin culture for two years. Pieces of tissue weighing 50 mg each wereplaced in the following three media and were observed at 28°-30°C for
28 days.
a) White's basal mediumb) White's + Casein hydrolyzate (1000 ppm)c) White's + Casein + Kinetin (0.1 ppm) + 2.4 D (0.1 ppm)
In White's basal medium, the callus tissue from MT grew slowly, but
those from the other strains did not show any perceptible growth. When
Table 1. Growth of callus tissues from five morning glory strains
Fresh weight of tissue block in mg.
Culture medium

7 daysI

14 days

basic
MT
dl

Ie
B
Tks
basic+Casein
MT
dl

Ie
B
Tks
modified
MT
dl

Ie
B
Tks

90
66
61
56
77

212
142
65
70
86

95
76
73

59
79
72

69
85
82
84

I

I

21 daysI

28 days

376
304
81
160
113

410
119
102
144
104

211
74
206
95
142

434
130
280
164
272

894
384
478
334
516

157
244
133
301
230

514
570
394
478
562

1090
1232
680
605
860

RESEARCHES CARRIED OUT IN 196581

-casein hydrolyzate was added to the medium, MT showed a rapid growth.and the others were gradually growing. When kinetin and 2.4 D were
further added, the tissues from all the five strains grew rapidly. The
fresh weights of the growing tissue blocks taken at one-week intervals
are given in Table 1. It seems that the callus tissue of the tumor-producing strain was growing in a minimum medium in which the otherfour could not grow.

68.

Genetic analysis of a tumorous strain in Japanesemorning glory!)
Yoshiaki YONEDA

Tumors in Japanese morning glory occur in association with certain-combinations of several genes (Takenaka and Yoneda, 1963, 1965). Amongthese, a tumorous strain was further analysed in respect to the Blown{B), feathered (fe) and delicate (dl) genes.
Seeds of a plant showing characteristics of gene B were sowed and its,offsprings were analysed. Among them, there were found four phenotypes, namely 1) B fe dl, 2) B fe, 3) B dl and 4) B. Each phenotype,could be clearly distinguished by the shape of the cotyledons, leaves andflowers. The exception was type B fe dl which produced small flower
buds which failed to bloom. Numbers of segregants of B fe dl, B fe,
B dl and B were 5, 17, 14, 59, respectively, which almost matched the
expected ratio of 1:3:3:9, if the original individual analysed had the
genotype BB+fe+dl. Phenotypes B fe dl and B fe were completely
sterile because of the presence of gene fe. Only a few seeds were obtained from B dl type and its offsprings segregated either 1) B dl or 2)
B dl and B fe dl. This indicates that the genotypes of B dl are BB+ +dldl or BB+fe dldl. 58 of the 59 type B plants obtained (one plant died
before seed setting) were analysed further for their segregation. 25 plants
produced all four phenotypes, while 13 plants yielded Band B fe, 14 plants
yielded Band B dl, and 6 plants produed only phenotype B. This result
is consistent with the expected ratio of 4:2:2: 1, if assumed as above.
These data can be interpreted if we assume the original plant used had
the genotype BB+fe+dl. It is impossible to determine the genotypesof B fe dl and B fe because of their sterility, but the segregation data
coupled with the morphological features suggests that B fe dl had the
genotype BB fefe dldl, and B fe had either BB fefe+ + or BB fefe+dl.
1) This work was supported by a fellowship from the Damon Runyon Fund(DRF-378).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

All the B fe dl types showed tumorous growth, and some of the B fe
also showed slightly tumorous growth. From these results, tumor formation in this strain seems to be predominantly high in BB fefe dldl andto be slightly in BB fefe+ + or BB fefe+dl.
In conclusion, tumrous growth in this strain seems to be dependent on
the combination of the Band fe genes, and to be enhanced by the dl
gene.

69.

Auxin destruction and growth in Japanesemorning glory stems!)
T. STONIER and Y. YONEDA

A wild-type strain of the Japanese morning glory (Pharbitis nil Choisy)
was investigated to ascertain the normal relationship between auxin andstem internode elongation. In young plants an age gradient was discovered in which the decreasing rate of elongation of older internodes
correlated with an increasing ability of such tissue to destroy indoleacetic
acid. Fragments of tissue from old internodes when incubated withindoleacetic acid, destroyed the hormone immediately and rapidly; incontrast, young rapidly elongating internode tissue destroyed IAA only
after a lag of several hours. In older plants the gradient was more
erratic towards the middle of the plant but old and young tissue behave
as in young plants i. e., old internodes destroyed IAA rapidly whereas
young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing
an internal environment in which auxin is rapidly destroyed.
Further investigations to ascertain this relationship between stem
internode maturation and increasing auxin destruction, established thefollowing: There exists in young, elongating internode tissue, substances
which prevent the destruction of indoleacetic acid by enzymes normallyfound in stem tissue. Most of the activity can be attributed to a substance whose molecular weight is in the 5,000 to 10,000 gm/mole range.
A second inhibitor of auxin destruction has a molecular weight in the
1,500 to 5,000 gm/mole range. These substances are water soluble, andappear to be readily destroyed by heating, at least in vitro. It is further
suggested that in the wild type, associated with stem maturation, is theloss of auxin-protecting substances, and as a consequence of this loss theloss of further endogenous auxin-induced elongation.
1) This work was supported by U.S. Public Health Service grant j\:CA-06957 to
the senior author, and Damon Runyon Fellowship DRF-378 to the second author.

RESEARCHES CARRIED OUT IN 1965

70.

83

Induction of maize seedling catechol oxidase by maleic hydrazide
Toru ENDO and Drew SCHWARTZI)

Investigations on the control of catechol oxidase in plumules of maize
seedlings have indicated the existence of two distinct classes. One classshows high enzyme activity while in the second class activity is almost
nil. Enzyme activity was measured photometrically by the oxidation of
catechol. The determinations were made on homogenates of 10 plumules
from 4 day-old seedlings. As is evident in Table 1 striking differences
Table 1.

Catechol oxidase activity per plumule at 4 day-old incontrol and MH pretreatment

Active line
F536
K201
K4

Control
213 ± 171316±92512±201

MH pretreatment539±45664± 131823±29Inactive line
N541
S535

10±8
13±8

10±10
15±10

F I hybrid (Active X Inactive)FxS and SxF

163±35194±43in enzyme levels exist even between those lines which are active in producing the enzyme. These differences reflect variability in the proportion of seedlings in each line which are actively producing enzyme aswell as differences in enzyme levels in the individual seedlings. In strain
K 4 about 73 % of the seedlings are active while in strain F 536 only 22 %are active. This was determined by testing individual seedlings zymographically with starch gel electrophoresis. The enzyme appears as a
sharp band which migrates to the cathode in pH 8.5 borate buffer system.
Pretreatment of seeds with 0.01 M maleic hydrazide (MH) for 24 hours
resulted in a significant increase in catechol oxidase activity in the activelines (Table 1). MH had no effect on enzyme level in the inactive lines.
Almost all of the seedlings in the active lines showed catechol oxidase
activity with MH pretreatment. Thus in the active lines seedlings which
do not normally produce the enzyme have the capacity to do so if treatedwith MH. The seedlings in the inactive lines can not be similarly induced.
No increase in enzyme activity was noted when seeds were presoaked inII

Department of Botany, Indiana University, U.S.A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

solutions containing catechol oxidase substrates such as catechol or
dihydroxyphenylalnine.
Preliminary analyses on F 1 hybrids between active and inactive lines
are interesting and may shed some light on the control mechanism.
Whereas enzyme activity is observed in the untreated controls characteristic of the active parent, the hybrids resemble the inactive parent inthat no induction occurs with MH pretreatment. Increased enzyme
activity in the active lines with MH pretreatment colud reflect either
gene induction with increased enzyme synthesis or activation of latent
enzymes by MH. The former is a more likely explanation since no
increase in activity occurs in pretreated hybrids.

71. Tulip breaking virus andsynthesis of water soluble proteins in tuliP petals
Yoshito OGAWA

In order to examine the relation between virus infection and injection ofRNA, we applied pure RNA isolated from virus infected petals of William
Pitt to normal tulip leaves using the carborandam method. The influenceof RNA on the host plant was examined at the level of protein synthesisin the petals. Besides the experimental group, three control groups werearranged, 1) non-treated normal group, 2) virus inoculated group and 3)
group treated by RNA, extracted from normal tulip petals.
Fresh tulip petals were mashed with the same volume of distilled waterin a glass homogenizer at ace. Immediately after centrifuging thehomogenate at 1,500 g for 10 minutes, electrophoretic analysis of theobtained supernatant was carried out at room temperature on cellulose
acetate strips (Separax) of 2.5 x9.0 cm. size, using veronal buffer (pH 8.6,
Table 1. Influence of RNA isolated from virus inoculated tulip petalson the synthesis of soluble proteins in the petals of William Pitt(%)

I
ControlgroupsNormal group
Virus infected group
Treated with RNA isolated fromnormalpetals groups

with RNA isoExperimental Treatedlated from virus inocugrouplated petals

A

B

C

D

E

F

G

H

4.2 4.2 12.6 19.4 30.4 20.8 5.6 2.8
1.6 3.2 12.8 0.0 47.2 22.4 9.6 3.2
2.1

2.1

27.3

3.6 3.6 25.2

16.8 32.8 14.7 2.1

2.1

0.0 29.9 21.6 12.5 3.6

RESEARCHES CARRIED OUT IN 196583

[1=0.06) at 0.6 mA/cm for two hours.

Nigrosin staining was used for theestimation of the analyzed proteins.
The analytical results are given in Table 1. The soluble proteinsextracted from normal tulip petals were separated into eight fractions(A, B, C, D, E, F, G, and H). In the virus inoculated group fraction D
disappeared and only seven fractions were recognized, though no remarkable change was found in the group treated with RNA extracted fromnormal tulip petals. In the experimental group, treated with RNA isolated
from virus infected petals, fraction D also disappeared just like in thevirus inoculated group.
The assumption that tulip breaking virus could modify the gen.ic action
on the synthesis of water soluble proteins of tulip petals seems to be
acceptable.

C.

72.

MATHEMATiCAL GENETICS

A stochastic model concerning the maintenance ofgenetic variability in quantitative characters
Motoo KIMURA

A new model is proposed to explain the maintenance of genetic variability in quantitative characters. The basic assumptions of the model
are: (1) At every locus involved with the quantitative character, mutation
can produce an infinite sequence of alleles. (2) The effect of a new allele
on the quantitative character is only slightly different from the parent
allele from which it was derived by a single mutational step. (3) The
genes are additive with respect to their effect on the quantitative character.
(4) The optimum phenotype is fixed, and fitness decreases in proportion
to the squared deviation from the optimum.
Based on those assumptions, the properties of the model were worked
out, enabling one to make predictions about the relation between mutation
rates, genotypic variance and mutational load.
The present model is in sharp contrast with the conventional models
in which mutation is assumed to occur only between a pair of alleles, say
A. or a. For details, see Kimura (1965).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

73. Quasi linkage equilibrium
Motoo KIMURA

Recently, a number of papers on population genetics have been published treating the effect of linkage and epistasis on selection.
The purpose of this note is to report a remarkable property inherent
in the linked gene system, which may be summarized as foIlows:
In a large random mating population, if gene frequencies are changing
by natural selection, under loose linkage and relatively weak epistaticinteractions, a state is quickly realized in which chromosome frequencieschange in such a way that

R= XIX. =constant ,X2Xa

where XI, X2, Xa and x. are respectively the frequencies of 4 types ofchromosomes, AIBI, A2BI, A IB 2 and A2B2, in which Al and A2 are a pairof alleles in the first locus and B I and B 2 are a pair in the second locus.
In other words, R is the ratio between the frequencies of coupling andrepulsion phases.
Such a state was termed quasi linkage equilibrium and it was shownthat several important conclusions follow from its formulation, namely:(1) The rate of change in the mean fitness of a population is equal to theadditive genetic variance in fitness. So, Fisher's fundamental theoremof natural selection holds. (2) The direction of change in gene frequencies by natural selection is such that the mean fitness is increased. (3)The stable equilibrium of gene frequencies corresponds to the local
maximum of the mean fitness and Wright's classical picture of " adaptive
topography" continues to be useful if the concept of quasi linkage equilibrium is taken into account.
In order to corroborate the above points, an extensive numerical study
was carried out with the help of computers. For details, see KIMURA(1965).
7 4.

Simulation studies on the number of self-sterility
alleles maintained in a small population
Motoo KIMURAThe number of self-sterility alleles (S alleles) maintained in a plant
population of finite size was first studied mathematically by Wright (1939)
and later by Fisher (1958). Their treatments were based on approximations
and the scientific validity of their results were recently questioned by

87

RESEARCHES CARRIED OUT IN 1965

Ewens (1964), who claims that the alleles are lost at much slower rate
than indicated by the previous treatments.
The purpose of the present work is to check these points by carryingout simulation experiments with the help of a high speed computer (IBM7090). In each generation, the process of mutation, random sampling of
gametes, testing of compatibility between pollen and stigma, fertilizationand the sampling of zygotes for the next generation was simulated bygenerating pseudo-random numbers, until a given number of offspringwere produced, and the experiments were carried out for many generations.
SELF-STERILITY ALLELES
DETERMINISTiC MUTATICN
N.5OD,jJ'IO·'

00. . - MEAN (ACTUAUNUMBER<> .... EFFECTIVE NUMBER

C/l

'"
'"

. .J 40

. .J
..J
<[

l5 '0

a:

'"~'"

20

Z

I~

_

~

~

~

1100

~

I~

~

I~

2~

2~

2~

2~

l~

GENERATION

Fig. 1.

Fig. 1 shows the result of an experiment with a population of 500
individuals in which exactly one new mutation is produced in each generation until generation 2,100 after which mutation is stopped but simulation
was carried out for another 800 generations. The initial population was
assumed to contain 1,000 different S alleles. Equilibrium between mutation and random extinction appears to be reached by generation 100.
In the figure, both the mean and the effective numbers of alleles are
given at intervals of 40 generations starting from generation 140. The
mean number (na) is the one which is obtained by the reciprocal of themean frequency (i) of the alleles contained in the population. It is thesame as the actual number of alleles in the population. The effective
number (ne) is computed by the reciprocal of the sum of squares of theallelic frequencies and it is usually smaller than the actual number. The
average value of na computed from 50 outputs (generations 140~2,100) is
na=36.2,
and this should be compared with the theoretical value obtained by Wright,
i. e. na=33.8. Similarly, the average value of n e computed from 50 outputs is

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

ne=25.3,
and this should be compared with the theoretical value predicted by
Fisher's theory, i.e. ne=21.4.
Thus, the agreement between the computer outputs and the theoreticalpredictions is sufficiently good for practical purposes. The figure also
shows the results of stopped mutation and it may be seen that the mean
number of alleles drops rather sharply during at least the first couple ofhundreds of generations, while the effective number drops less markedly.
This is due to the fact that, after the cessation of mutations the rare
alleles are lost rather rapidly but this has little effect on reducing theeffective number.
The same type of simulation experiments were also performed by Dr.
T. Maruyama at the University of Wisconsin, assuming stochastic mu·
tations, namely, assuming that the actual number of mutations follows
Poisson distribution. The results obtained were essentially the same as
those obtained by assuming deterministic mutations.
Hereby the validity of the results obtained by Wright and Fisher on the
self-sterility alleles is confirmed and the arguments put forward by Ewens(1964), based on the extrapolation from his study of the 3 allelic system
are disproved.

D.

GI:N~TICS

75.

AND B'OCH~MISTRY OF MICROORGANISMS

Leaky mutants of flagellation genes in Salmonella
Tetsuo lIND and Michiko MITANI

Strains SJ82, SJ122 and SJ376, originated from Salmonella typhimurium
TM2, were found to be leaky non·flagellate mutants. By transduction
analysis their mutant sites were located in flaA, flaB, and flaC cistrons
respectively. On semisolid medium, these mutant clones formed satellite
colonies; that is, a large colony was surrounded by many minute colonies,
each formed by non·motile descendants of a motile multi·flagellate cell.
Flagella of these mutants were observed under electron microscope. In
all of these strains, flagellated cells were present at the frequencies of 5
to 10% of the populations in broth culture. Their average number or
flagella per flagellated cell was one, while there were 7 flagella in the wild
strain, TM2. It is worth noting that flagella of the leaky mutant cells
were much less numerous but were as long as those of the wild
strain.

RESEARCHES CARRIED OUT IN 196589

From these observations the events occurring in the culture of theleaky mutants are described as follows. In the cell population of themutants, cells which have recovered the ability to synthesize flagella lessefficiently than the wild strain appeared at the frequency of one per 102
to 103 cells. They carried multi-flagella as long as the normal strain.
Each minute colony of the satellites on semisolid media is inferred to beoriginated from such a cell. The ability to produce flagella lasted only
one or at most a few cell generations, and the number of flagella perbacterium was decreasing until finally the offspring turned out to be
single-flagellated or non-flagellated due to dilution during successive cell
divisions.

76.

Heteromorphism of flagella in a Salmonella mutant
Tetsuo IINO and Michiko MITANIThe heteromorphous mutant of Salmonella abortus-equi differs from the'wild strain in the structural gene of phase-2 flagellin. It predominantly
produces normal flagella, but at the frequency of 2%, cells with bothnormal and curly flagella appear in the broth culture. Often a flagellum
has mixed normal and curly waves. No cells with curly flagella only
have been ever detected. Each subclone started from a single cell of
the mutant clones showed the same character. Therefore the heteromorphism is not explained by frequent mutation from normal to curlyin the mutant culture. The strain was stable in flagellar phase-2.
Consequently, the heteromorphism cannot be attributed to flagellar
phase-variation. The remaining possibilities are that (1) both normal andcurly flagellins are produced by coding degeneracy in the mutant, or (2)flagellin produced by the mutant is of the same kind regarding amino acidcomposition but has the potentiality to take either of the two alternative
conformations depending on the condition of its polymerization.

77.

Dimorphic nature of bundled flagella in a small-amplitude
mutant of Salmonella abortus-equiMichiko MITANI and Tetsuo IINO

By both darkfield microscopy and electronmicroscopy, morphologicalconversion of bundled flagella was found to appear in a small-amplitude
mutant of Salmonella abortus-equi when the cells were suspended in 0.25%
methylcellulose solution. The change in the shape of bundled flagellafrom small-amplitude to curly was observed about 40 minutes after the

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

cells had been suspended in the solution, and thereafter the fraction of
curly cells increased in the course of time up to 80% of the cell population.
Dispersed small-amplitude flagella did not undergo morphological change
in the solution. Tight association of the component flagella in eachbundle caused by viscous solution of methylcellulose may produce a stressamong them resulting in the alteration of their shape. The process ofthe transition in the flagellar shape was observed on a cell under darkfieldmicroscope: the twirl of the bundle accompanied by a sudden change
from small-amplitude to curly started to occur from the tip of the bundled
flagella and proceeded to the base. As the small-amplitude mutant differsfrom the normal in the structural gene of flagellin, the dimorphism isprimarily attributed to the elastic bipotential conformation of the flagellin
produced by the mutant.

78.

An assay for newly-synthesized flagellin of Salmonella
Hideho SUZUKI and Tetsuo IINO

An attempt was made to apply the reconstitution of flagella for·separating flagellin from a cell-free extract which might include a
newly-synthesized fraction of flagellin.
Flagellin monomers produced by treatment with dilute HCI at pH 2
were reconstituted into flagellar filaments with efficiency of more than
80% in the following mixture: flagellin 4-10mg/ml flagella fragments asseeds 1/5-1/4 amount of flagellin, phosphate buffer (pH 6.8) 20 mM, KCI
150 mM, MgCb 5 mM.
Cell-free extract from HC-Iabelled culture of Salmonella abortus-equistrain SL23 (enx) was subjected to heat treatment in order to dissociate
all flagellar fragments into monomers and to denature the bulk of cellular
protein, then the extract was added to a reconstituting mixture which
consisted of flagellin and seeds originated from Salmonella strain 8J25
(1,2). The reconstituted flagella were analyzed by starch gel zone
electrophoresis. Flagellin enx and 1, 2 were separable electrophoreticallyat pH 8.4 (buffered with 10mM tris); i. e. flagella reconstituted incorporated
labelled material and gave a peak of radioactivity at the position
corresponding to enx. When an aliquot of the sample was mixed witha r-globulin fraction containing antibody for enx, prior to electrophoresis,
this peak practically disappeared and almost all of the radioactivity
remained unmoved at the original point, indicating that the radioactive
material was reactive specifically with anti-enx globulin.
A similar method was applied to detect intracellular flagellin. The

RESEARCHES CARRIED OUT IN 196591

labelled cell-free suspension prepared from cells of SJ28, the non-flagellatemutant strain which was known to produce enx flagellin CRM (IINO andHARUNA: Annual Report No. 11, 1960), was acidified to pH 2 with HCI
and the resulting denatured protein was removed. The acid-treatedextract was brought into the reconstituting system with 1,·2 flagellin andseed, after the volume of the extract had decreased to less than 0.5 ml
by freeze-drying. An electrophoretic analysis again displayed the presenceof the radioactive material at the position in which flagellin enx should
reside. This radioactive material was also reactive with r-globulin fractionof anti-enx serum.
Flagellin produced by the CRM-type mutant, SJ28, is incorporated into
flagella in the reconstituting system, so that it is inferred that lack of amechanism to organize its flagellin into a flagellum, rather than anabnormality of flagellin, is responsible for the loss of flagella in thismutant.

79. Composition of chromosome fragments participating in P22 mediatedtransduction of fla·H1·mot loci in Salmonella typhimurium
Masatoshi ENOMOTO

Based on the hypothesis proposed by PEARCE and STOCKER (1965) thatexogenotic Hi allele in transductional heterogenote involving cotransducible
fla·Hl or mot· Hi loci could be expressed when it was carried with mot+or fla+ locus, the composition of the transducing fragments participatingin transduction of fla, Hi, and mot loci was estimated. Three Hi-linked
nonmotile mutants, mot C-244, fla-48, and mot C-244·fla-48 derived from
Salmonella tYPhimurium TM2 (i: 1.2), were used as recipients. The orderof the genes in this chromosomal region is fla-48·Hl·motC-244. Phage
P22 was propagated on line SJ697 of TM2 strain whose phase-1 flagellar
antigen was replaced by gp by transduction of Hi from Salmonella dublin
SJ11 (gp:-). Overnight broth cultures of the recipients in phase-1(mot C-244) or in latent phase-1 (fla-48 and double mutant) were mixed
with phage at moi 10- 2 (single mutant) or at 5 (double mutant). The
mixtures were plated, if necessary after dilution, on semisolid medium
containing anti-1.2 or anti-1.2 and -gp sera. After incubation for 9-10 hr
at 37°, the number of trails was counted under binocular microscope.
Frequency of cotransduction of fla and / or mot with Hi was determinedby slide agglutination test of swarms obtained from another transduction
test. The results are shown in Table 1.
It is necessary for trail production in the transduction to the double
mutant (fia-· Hi· mot-) that the donor allele Hlgp is carried together with

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
Table 1.

Transduction from SJ697 (gp: 1.2) to H1-linked nonmotile mutants.
Trail (abortive transductant)

Transductant (per lOS PFU)
Recipient Medium No. of transexpressing..
ductants percontammg
10 sPFUi (recipient)
gp (donor)
No.
%
No.
%
anti-1.2
anti-1.2, gp
anti-1.2
motC-244.
antI-1.2, gp
fia-M
anti-1.2
mot C-244 anti-1.2, gp
fia-48

5.27
4.97
67.3 3.00x 10- 1 4.1
4.97
2.10
1.40 x 10- 1 1.9 1.96
26.5
1.40 x 10- 1
1.56x 10- 2
1.56 x 10- 2 0.2
2.60x 10- 4

Swarm (complete
transductant)
Percent No. ofof H1- transc ts ductants
o r::m - per lOs
ductIon PFU
2.1 4.77x 10- 1
36.0 1.36 x 10- 1
74.7 7.20 x 10- 4

both fla+ and mot+ genes. When the transducing fragments participatingin transduction of fla and / or mot were taken as 100, those carrying fla,
fla·Hi, fla· Hi· mot, Hi· mot, and mot loci were 67.3,4.1,0.2,26.5, and 1.9%
respectively. The transducing fragments ending between fla and Hi were
93.8% and those between Hi and mot were 6.0%. This indicates thatmost of the transducing fragments are cut off at a predetermined
position.
The frequency of wild type recombinants in the transduction to the
double mutant was less than 2 X 10-2 as compared with that of single
mutants. The frequency of Hi-cotransduction with fla-48 was very low.
These low frequencies may be explained by the above estimation thatchromosome fragments carrying either fla·Hi·mot or fla·Hi are few.

80.

Dependency of argos mutant of Salmonella typhimurium
not only on uracil but also on arginine
during arginine inhibition
Jun-ichi ISHIDSU

Among various physiological characteristics of arg-s-i, an arginine sensitive mutant of S. typhimurium, it has been reported that transfer of cells
from a medium containing both arginine and uracil to the same medium
which contains only arginine at any stage of growth brings about immediate and absolute growth inhibition (ISHIDSU, Annual Report No. 15,
1964). This suggests that, during the normal growth in the first medium,
formation of uracil synthesizing enzymes, or of one of them at least, isstrongly repressed and cannot be recovered even after removal of uracil
from the medium.

RESEARCHES CARRIED OUT IN 1965

93

Effect of similar transfer to other media was examined. Cells weregrown in a medium supplemented with both arginine and uracil as far
as middle log phase. They were washed twice by saline and transferred
to fresh media supplemented with (1) both arginine and uracil (control),(2) only uracil and (3) neither (minimal medium). Care was taken not tochange markedly the titer of cells upon transfer. When medium (1) wasused, no interruption of logarithmical growth was observed. But transfer
to medium (2), as well as to (3), affected the cells and a 1 to 2 hour lag period
was brought about before the cells could recover logarithmical growth.
Additionally, it was found that this lag disappears if medium (2) is supplemented with citrulline, one of the biosynthetic precursors of arginine.
Another precursor, ornithine, could not save the cells from the lag following the transfer to the medium (2).
These findings suggest the following considerations: (1) The mutant
requires arginine as well as uracil during arginine inhibition. (2) Thisdependency on arginine is due to the inhibition of the conversion ofornithine to citrulline by arginine. (3) The inhibition at this specific stepis the result of the inhibition of carbamyl phosphate formation which acts
as a common substrate of ornithine- and aspartate-transcarbamylase inthis organism.
Enzymatical studies of carbamyl phosphate synthetase of this mutant
are now under way.

81. Subunit analysis of g-complex antigens of Salmonella
Shigeru YAMAGUCHIIt is known that the flagellar antigens of Salmonella are composed ofseveral antigenic subunits. Among them, the so-called g-complex antigens,all of which belonging to phase-I, have been studied most accurately inrespect of their subunit composition. The already known subunits of gcomplex antigens are f, g, m, s, t, p, q, u, and ZSl. However, it has often
been recognized in the process of serum absorption when preparing single
factor antisera that subunit g may be further subdivided.
Table 1. Subunit compositions of g-complex antigens.
Antigen type

Strains
Salmonella abortu8-equi Tr6

4, 12; gt : (enx)

Trll
Tr16
Tr17

4, 12; gp : (enx)hhh4, 12; fg : (enx)4, 12; gm: (enx)Subunit compositionof phase-1
gl g2 g3 t
gl g4 gs P
g3 g4 fgl g2 g4 gs m

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

A subunit analysis of g-complex antigens was carried out by means ofreciprocal absorption tests of rabbit antisera and slide agglutination tests
among four strains of phase-1 stable Salmonella abortus-equi, each having
H1 transduced from one of the four serotypes of g-group Salmonella, that
is, Salmonella sp. (gt:-), S. dublin (gp:-), S. derby (fg:-), and S. enteritidis(gm:-). As shown in Table 1, it was found that subunit g is not single
but can be further subdivided into at least five.
82.

Mutation of g-complex antigen in Salmonella
Shigeru YAMAGUCHI and Tetsuo II NO

Flagellin, flagellar protein of Salmonella, has complex antigenicities. Itsantigenic specifficity is determined by a single gene, H1 in phase-1 andH2 in phase-2. It is well known that mutants slightly altered in theserological character can be selected for by incubating the cells on asemi-solid medium containing homologous or cross-reacting antiserum.
Serological and genetical investigations of such serologically altered mutantsof Salmonella having the g-complex antigen were carried out.
Tr16 (4,12; gsg.j: (enx)) is a phase-1 stable strain of S. abortus-equi. H1gene of the strain was transduced from S. derby (lg: -). This strain was
cultivated on semi-solid medium containing rabbit anti-f serum at theminimum concentration in order to immobilize the Tr16 cells. After 48 hr
incubation five mutants (Tr16-fM1 to -fM5) were obtained as swarms spreading faster in the medium than the parental type cells. Tr16 was also
cultivated on semi-solid medium containing anti-gst serum or anti-g.g5P
serum and each of five mutants (Tr16-gsM1 to -gsM5, and Tr16-g.M1 tog.M5) were isolated from the respective medium.
Agglutinabilities of these fifteen mutants with anti-f, anti-gsm, and antig.g5P sera were compared with those of the wild type cells by tube agglutination test. In nine mutants (Tr16-fM1 to -fM5, -gsM1, -g.M2, -g.M4,
and -g.M5), only agglutinabilities of the antigenic subunits corresponding
to the agglutinins used for the selection have decreased to one fourth to
one tenth of those of the wild type cells in agglutination titer, and thoseof the other antigenic subunits have remained unchanged. In the othersix mutants, agglutinabilities not only of the subunits corresponding to
the agglutinins used for selection but also of all the other subunits decreased to one tenth of those of the wild type cells. Since the rabbit
antisera against mutants Tr16-fM2, -gsM1, -gsM2, -g.M1, and -g.M2 werecompletely absorbed by the wild type cells, it is thought that the antigenic
specificities of these mutants are not qualitatively different from those of
the parental strain.

RESEARCHES CARRIED OUT IN 1965

95

Transduction of Hi genes from Trl6-fM2, -g3M1, -g3M2, -g4M1 and -g4M2
to S. abortus-equi strain SJ241 (4,12; a: (enx» was carried out by using
phage P22 as mediator and a semi-solid medium containing anti-a serum
as selecting medium. All the transductants obtained had the antigenicity
exactly the same as the donor mutants, indicating that the mutation sitesof these serological mutants are within Hi. Tryptic peptide maps offtagellins of Trl6-g3M1 was compared with that of the wild typeftagellin. In the peptide map of Trl6-g3M1 ftagellin, a neutral spot of wild
type ftagellin was substituted by a somewhat basic spot. The other 33-34
spots were identical between these maps.

83.

Lack of host cell reactivation of bacteriophage
Tl inactivated by 32p decay
Mituo IKENAGA and Sohei KONDO

In ultraviolet (UV) inactivated phage T1, a fraction of the UV lesions
can be reactivated by the host cell (GAREN and ZINDER 1955). The reactivation, later called" host cell reactivation" (HCR), is believed to result
from elimination of UV damage of the phage's DNA, due to some repair
enzyme of the host cell. According to a recent report by SAUERBIER(1964), phage T1 damaged by ionizing radiation also undergoes repair byhost cell reactivation. Thus, HCR might be a versatile repair mechanism
capable of eliminating various kinds of damage sustained by irradiatedphage DNA.
In order to gain more insight into the mechanism of HCR, the survivalof 32p decay inactivated T1 was determined under HCR conditions and inabsence of HCR. Heavily 32p labeled T1 was prepared as reported by
STENT and FUERST (1960), and stored in air or in nitrogen saturated
glycerol casaminoacid medium at 4°C. The survivors were counted day
by day by parallel plating on the host bacterium E. coli H/r, capable of performing HCR, and E. coli Bs- l , a mutant lacking the reactivation enzyme.
Fig. 1 shows the survival curve of 32p decay inactivated T1 in air or
nitrogen saturated medium when plated on E. coli H/r and B.-l, respectively. Apparently, survivals of 32p labeled T1 are the same in both hostbacteria, irrespective of having or not the HCR enzyme. HCR of T1 inactivated by 32p decay was completely inhibited even in nitrogen saturated
medium in which HCR efficiency of r-rayed T1 is found to be about two
times larger than in air. The lack of HCR in the present study indicatesthat 32p decay induced lesions in phage DNA are not host cell reactivable.
The lesions responsible for T1 inactivation may be double strand scissions,of T1 DNA molecule most probably due to 32p transmutation.

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 1&

in ni {,rogensaturated
llJedium

10 -4 uL--o:-'-,'":'"c-----::0.....
' .,:------::(:-',,'":'"G--~o--O;;.l,7,"-----;;O~, 4;--~0 ,';;6---'FracLion of

3~p Atoms Decayed

Fig. 1. Survival curve of phage Tl inactivated by 32p decay (300 mCi/mg-P}
plated either on E. coli H/r (open circle) or BS _ 1 (solid circle).

E. RADIATION GENETICS IN ANIMALS

84. Mechanisms underlying the enhancing effect of induced-mutation
frequency after fractionated r-irradiation of silkworm gonia
Yataro TAZIMA and Kimiharu ONIMARU

As reported previously, it was found that fractionated exposure to rrays enhances the induced mutation frequency in silkworm gonia. As apossible interpretation of this phenomenon some kind of modification*in the cellular metabolism was assumed presumably being caused by a disturbance, which hindered the repair of premutational lesions until after acertain while the condition disappeared. Hence, experiments were carriedout to seek supporting evidences for this assumption. In an experiment,
in which irradiated insects had been kept at high or low temperaturesduring the period between two exposures, it was found that the duration*) In a previous report the term "metastasis" was used for representing a"modified state" of the irradiated cell. Since in medical science "metastasis"
is used for representing "transfer of a disease-producing agency", the term
"modified state" will be used hereafter instead of "metastasis".

RESEARCHES CARRIED OUT IN 196597<:>f the persistenc of the modified state was temperature dependent. Posttreatment of irradiated cells with agents that are known to affect cellular
metabolism proved to be effective in modifying mutation frequencies.
Those results were consistent with the expectation that the post-irradiation modification may be a change in the metabolic activity of the cell.
However, a finding that the enhancing effect did not give a decay curve,
but instead exhibited a peak at 18-24 hours after the initial exposure, did
not support the view of a biochemical disturbance, but seemed to suggest cell synchronization. Nevertheless, this latter view contradicted ourprevious finding that mutation frequencies never fell below a single acuteexposure level for 48 hours. In other words, no effect in opposite direction was observed during this period. Questions arose, therefore, if thetested interval was not short enough or had we not tested a period longenough to reveal an opposite effect due to cell synchronization. In orderto answer those questions, the following experiments have been carriedout.
a)Fractionation experiment with different initial doses.
Total 1000 r were given in two fractions, 250 rand 750 r, to two experimental series in eversed order at various time intervals. In one series
250 r were given first followed by the second exposure of 750 r at 6 hourintervals. Whereas, in the other group the initial dose was 750 r and thenG52, L.Ct'3 stage, Spermatogonia., ,-t:""",..:'~ // ~~~,,,~\',,<::---: : 50

~g

~
.2

/ /

1----- ,.
012--------0------------3.

2•~·I-~'
.k:
_-c_

I,
o
6

12

18

,

24

'8

.~;

'

36

.

48

Interv'Jls In HourFig. 1. Comparison of induced mutaiton frequencies
between two fractionation experiments of total 1000 r
with eversed first and second doses.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

250 l' were given at the same intervals. The results are given in Fig. l.
It may be seen from the figure that the peak appeared earlier for the
initial 250 l' series than for the initial 750 l' series, although it was lower.
Those results can better be interpreted by assuming cell-synchronization
than by postulating a biochemical disturbance.
b) Fractionation experiment with protracted intervals.
If cell synchronization had actually occurred after the initial exposure,the opposite effect of fractionated irradiation could be revealed whenlonger intervals were examined. Furthermore, such experiment mightreveal even a second peak of the next synchronized cell cycle. Since one
cell cycle has been estimated to last 34 hours for non-irradiated primary
spermatogonia (Sado, this Report), our previous observation for a 48 hour
period seemed to be insufficient for such purpose.
An experiment was, therefore, undertaken by extending the intervals
between two exposures up to 72 hours. The initial and the second doses
were the same, 500 r. The results are given in Fig. 2.

30

36

42

48

54

eo

88

72

Intervals in HourFig. 2. Results of a fractionation experiment of
500 r+500 r with extended intervals up to 72 hours.

As seen from the figure, the enhancing effect of fractionated exposure
disappeared after 48 hours and thereafter the mutation frequency wasmaintained almost at the single exposure level. The apparent decline of
the curve after 48 hours, seems, instead of representing an effect in opposite direction, to show a general trend of frequency decrease in accordance with the progress of the development of the larvae. Why, then,
was the opposite effect not observed? Since cell synchronization became
plausible from experiment (a), how could we interpret the result of experiment (b)? The contradiction seems to be resolved by assuming thatsynchronization is destroyed immediately in the subsequent cell cycle.

RESEARCHES CARRIED OUT IN 1965

99

In this regard the phenomenon is not cell synchronization in true sence,but a temporary accumulation of cells that are blocked at a certain phaseof their cycle. Presumably, the blockade is gradually released after acertain while.
If the above view is correct, the nature of the" modified state" of theirradiated cell can be interpreted as a shift of cell compartment in regard
to the DNA synthetic cycle among germ cell population. Thus theenhancing effect of fractionated irradiation may be explained by assuming an accumulation of cells at a certain phase in which a repair of
premutational lesions can hardly occur.

85.

Time of reversal of radiation does-rate effect on mutationinduction in silkworm gonia
Yataro TAZIMA and Yosoji FUKASE

With the purpose of studying in more detail at what stage of development the dose-rate effect on mutation induction becomes reversed, an
experiment has been carried out with silkworm gonia within the period
from 3 days before to 5 days after hatching. Total 1000 r r-rays were
given either acutely or chronically. Dose rates used were 100r/min for
acute and 0.3 r/min for chronic treatment. In the acute series irradiation
was given in succession every 24 hours to seven groups, whereas the
exposure of the chronic series lasted 3 days and was started every 24
hours in order to examine the variation in mutation response during the
8 day period indicated above. The results are shown in Fig. 1. In thisfigure the response curve for the chronic treatment was obtained byconnecting the observed mutation frequencies plotted at the start of eachexposure, since the cytological observation revealed that the progressionof the cell cycle is arrested shortly after the onset of chronic exposure
at a dose-rate of 0.16 r/min (SUGAI, in preparation and SADO, this Report).
It can be seen from the figure, that for the male germ cells the mutation
frequency at chronic exposure rushed ahead of the acute as early as one
day afrer hatching. In female germ cells mutation response to different
dose rates was somewhat different from that of male germ cells. Yet, asimilar overshoot was observed two days after hatching. These findings
suggest that the critical time of development at which the dose-rate effectbecomes reversed is closely correlated with the metabolic state of thegerm cells. Presumably, the cells become active after hatching.
It was found in our previous experiments (TAZIMA, 1965) that theenhancing effect of fractionated irradiation is stronger for advanced stage
gonia (7 day old larva) than in their younger stages (hatching day larva).

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
Male germ cells
250 .:0-'

~

/

/

/

:..

::

germ

cells

"',,

/'

\

\\

I

~~ :!CI'

Female\-" "'"

po

~

~I

-2

~r

~:s

t

~

0

,-, ...,,

g
~

,
-l

'00

\
\

i

,
ro

I

so~

I

r
-,

-.

'-,

•

~l
0
I
Development 'n days

L~=::~~;--"

1,--3

-~

..

b

2

Development in days

Fig. 1. Changes in mutation frequency after acute and chronic exposures ofsilkworm gonia with their development.
Solid line: acute,Broken line: chronic

Since a chronic exposure can be regarded as an integrated form of
fractionated exposures, it may give rise, in analogy to fractionatedirradiation (preceding article in this Report), to higher mutation frequencies in advanced stage gonia than the acute treatment.

86. Further data on post-irradiation treatment of silkworm
spermatogonia with various metabolic inhibitors
Yotaro T AZIMA and Kimiharu ONIMARU

Experiments have been continued with various metabolic inhibitors anda radiation-protecting chemical in order to confirm the effect of posttreatment on modifying mutation frequency (cl. Annual Report No. 15).
Agents tested in this experiment were nitrogen gas, 8-azaguanine, mitomycin C, chloramphenicol and a known radiation protecting chemical,
AET. Except for nitrogen gas, chemicals were administered to the larvae
by feeding them with fresh mulberry leaves, which had been coated withan aqueous solution of the chemicals. Wild type larvae of C108 strain
were irradiated with 1,000 r r-rays at 7 day age. Two hours after irradiation, the insects were allowed to feed on the prepared leaves for 9-21

RESEARCHES CARRIED OUT IN 1965

101

hours. For gas treatment, the insects were placed immediately after
irradiation in a vial filled with nitrogen gas. The condition for eachtreatment was as follows:Cont. =l,OOOr r-rays only.
Gas l=Raised 24 hours in a current of nitrogen gas mixed with aminimum quantity of air in order to avoid asphyxia.
Gas 2=Same gas as above, but in tightly shut vials. Opened twice tointroduce air when asphyxia observed.
AG 1=8-azaguanine (" Azan", Tanabe Seiyaku Co., Ltd.), 105 mg per
1,000 larvae.
AG 2=8-azaguanine, 210 mg per 1,000 larvae. Given twice in two half
doses.
MMC =Mitomycin C (Kyowa Hakko Co., Ltd.), 6mg per 1,000 larvae.
CAP =Chloramphenicol (" Chloromycetin ", Sankyo Seiyaku Co., Ltd.),
100 mg per 1,000 larvae. Given twice in two half doses.
AET =2-Aminoethyl isothiouronium bromide (Koch-Light Laboratories,Ltd.), 150 mg per 1,000 larvae. Given twice in half doses.
The results, given in Table 1, show that treatment with those agents,
Table 1.

Treatment

Results of post-irradiation treatment of spermatogoniaof 7 day old larvae (653).

ObservednumberNumber of mutants
---

~

..

Mutation frequency (x 10- 5)_._--_._-_._-_.-

pe pe,+mos re re,+mos

pe pe,+mos re re,+mos

Cant.
Gas 1
Gas 2

177,599
173,255
173,258

69
93
99

0
1
2

18
41
36

2
2
3

38.85 0.00 10.14 1.13
53.68 0.58 23.66 1.15
57.14 1.15 20.78 1.73

AG 1
AG 2

173,998
172,507

107
96

0
0

38
40

3
3

61.49 0.00 21.84 1.72
55.65 0.00 23.19 1.74

MMC
CAP
AET

163,445
172,515
164,803

152
101
80

2
3
1

56
25
22

5
0
0

93.00 1.22 34.26 3.06
58.55 1.74 14.49 0.00
48.54 0.61 13.35 0.00

except for AET, increased mutation frequency to a considerable extent.
Considering that those agents either disturb respiration or inhibit nucleic
acid synthesis or protein synthesis, it can be surmised that they had
suppressed the repair of radiation induced premutational lesions resulting
in increased mutation frequencies. An alternative interpretation is also
possible, by assuming that those agents affected the progression of the102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

cell cycle by prolonging the duration of radiation-caused blockade (cf. anarticle by SADO in this Report).
.
In conclusion, the present results are consistent with both hypothesesof metabolic disturbance and progression-blockade, but they do not furnish
a conclusive clue which would allow us to determine which of the two is
more reasonable.

87.

Further studies on proliferation kinetics of silkworm
spermatogonia during chronic r-irradiations
Toshihiko SADO

In a previous report (SADO 1965), the kinetics of proliferation and killingof spermatogonia of young silkworm larvae (2.5-7.5 day old) during chronic
exposure was studied. This stage was selected because the number ofcells at the beginning of exposure was not very large and they were morehomogeneous than at more advanced stages. It was thought that thiswas advantageous for the kinetic studies of primary spermatogonial
populations during chronic irradiation. In the present experiment,
however, the irradiation of the larvae was performed between 5 and 10
days after hatching. This is the interval where Type II dose-rate effecton mutation induction is observed. The following kinetic indices were
determined at various intervals after the onset of chronic exposure (Cs-137
r-rays, 0.130r/min for 5 days): (a) total viable cell (or gonocyst) counts,(b) incidence of pycnosis, (c) mitotic index, and (d) S index, or the percentage of cells that take up H3-thymidine in 1 hour after isotope
administration. It was hoped that this will make possible the assessmentof dynamic changes in a spermatogonial population with regard to the
distribution of irradiated cell stages during chronic exposure.
The results show that there was a considerable difference in various
indices from those obtained in the previous experiment. Roughly speaking,the cells were more sensitive to radiation killing and mitotic inhibition inthis experiment (5-10 day old larvae) than in the previous study (2.5-7.5
day old larvae). 1) The majority of secondary spermatogonia present at
the beginning of the exposure were killed by the end of exposure. Thus,
only a few secondary spermatogonia (12 cysts per testis) survived theexposure (total dose: 900 r). 2) The maximum depletion of primary
spermatogonia was observed 3 days after the onset of exposure. 3) Incidence of pycnosis of primary spermatogonia reached the maximum
values of 33.3 and 34.0% at 48 and 72 hours, respectively, after the onsetof exposure, or after accumulated doses of 360 rand 540 r, respectively.
At the termination of the exposure, only 4.4% was found to be pycnotic.

RESEARCHES CARRIED OUT IN 1965

103

In contrast, 10.4% of secondary spermatogonia (cyst) was still pycnotic at
this time. 4) Mitotic activity of primary spermatogonia was considerably
suppressed during the treatment. Maximum suppression was observedon the third day in one experiment (17% of the control), whereas it was
found on the fourth day (12% of the control) in another experiment. It
is obvious that the maximum depletion period corresponds to the time
when mitotic activity is extremely low and incidence of pycnosis is veryhigh. These results suggest that there was a significant, but not complete,
block in cells at premitotic stage (Gz) entering into mitosis (M) during
chronic irradiations. 5) S index of primary spermatogonia also decreased
to 10% in contrast to 30% of the control as early as 12 hours after the
onset of the exposure (accumulated dose: 90 r) and was constant for 36
hours. This indicates that a fraction of cells in S phase decreased to 1/3of the control soon after onset of chronic exposure. This may have been
due to a partial block of cells in Gl entering into S phase.
These results show that there was a considerable accumulation of cellsin (late?) Gl as well as in Gz period during chronic irradiation. It is
obvious that the distribution of cells at various phases of the cell cycle
during irradiation is significantly different from that of unirradiated cell
population to which a single acute dose is generally given to compare
the dose rate effect on mutation induction. As we know that radiosensitivity of cells is not constant throughout the cell cycle, the change
in distribution of primary spermatogonia at various phases of the cell
cycle should no doubt affect the mutation frequency. We cannot determine at this moment, however, which of the two i.e., accumulation ofcells in Gl or in G2, is more important in enhancing the mutation rate bychronic irradiation.

88.

Change in distribution of primary spermatogonia of
the silkworm at various phases of the cell
cycle after irradiation
Toshihiko SADO and Kugao OISHI

It has been held by many workers that the effect of dose fractionation
on cell survival or on mutation frequency should be similar to the effectof reducing the dose-rate of chronic irradiation. TAZIMA's (1965) recent
finding that acute doses given to silkworm larvae in two or more fractions
give a much higher mutation frequency than that given in a single acuteexposure raised the question if synchronization of cells by the first dosemight be responsible for the result. Because of the great significancein the interpretation of the mechanisms involved in Type II dose-rate104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

effect on mutation induction in silkworm gonia, experiments were undertaken to examine how the distribution of spermatogonia throughout
various phases of the cell cycle would be affected by radiation.
First, the distribution of primary spermatogonia of unirradiated larvae
at various phases of the cell cycle was studied. This was done byestimating the relative time occupied by each phase of the cell cycle, on
the assumption that the cells are distributed randomly throughout the cell
cycle. As will be reported elsewhere, the generation time of these cells
is approximately 34 hours, the distribution of cells in various phases
being roughly as follows: G1 , 40%; S, 40%; G2, 18%; and M, 2%.
In the following experiment, 6 day old male larvae were exposed to asingle acute dose of 1,000 l' (100r/min) and various kinetic indices such as(a) total surviving cells, (b) incidence of pycnosis, (c) mitotic activity, and(d) S index, as a function of time after irradiation were studied. 1) It
was found that primary spermatogonia were not homogeneous in respect
to radiosensitivity, possibly due to difference in celll generations or inphases of the cell cycle. (These cells are indistinguishable morphologically from each other but there is no doubt that they represent a mixed
population comprising stem cells, or predefinitive spermatogonia, andindefinitive spermatogonia, i.e., intermediate generations between the stem
cells and the first generation of secondary, or definitive spermatogonia(ct. HANNAH-ALAvA 1965)). 2) Mitotic activity dropped abruptly following
irradiation and no mitoses were observed between 3 and 12 hours.
Recovery of mitoses proceeded very slowly. Thus after 24 and 36 hours
the frequency of mitoses was still 5 and 25% of the control, respectively.
These observations indicate that there was a complete block of cells in
G2 entering into M phase (G2 block) immediately following irradiationand that recovery from this block went on a very slow rate. 3) S index
also decreased linearly during the first 12 hours, from 40% at time 0 to
9% after 12 hours. This slope can be extrapolated to 0% at 15 hours on
the time axis. After 24 hours S index was 3.6% and after 36 hours it
was 14%. Extrapolation of these two values goes back to 0% at 20 hours
on the time axis.
These results suggest a nearly complete block of cells in GI entering
into S phase immediately following irradiation on the one hand and a normal transit of cells from S into G2 phase on the other. Since mitosis wasnearly completely blocked during the first 24 hours, there was a decreaseof the cell fraction in S phase and concomitant increase of G2 population
during this period. G1 block seems to have been lost after about 20 hours
when S index began to increase. Distribution of cells in various phasesof the cell cycle at different intervals after irradiation can, therefore, be

RESEARCHES CARRIED OUT IN 1965

At time 0

12 hr affer

1000r

105

24 hr after
1000r

Fig. 1.

represented schematically as in Fig. 1. It is clear from this figure that
there is a high correlation between the size of Gz population and theenhancement of mutation frequency by fractionated doses given at intervals after the first dose (cf. TAZIMA 1965). However, we can not rule out thepossibility that accumulation of cells in late Gl might also be responsible,
because Gl block was nearly complete immediately following irradiationand continued for as long as 20 hours.

89. Radiosensitivity of silkworm spermatogonia during the cell cycle
I. Fate of primary spermatogonia irradiated in S phaseToshihiko SADO

The foregoing experiment showed that as many as 58% of primary
spermatogonia in contrast to 18% of the control was occupied by Gz population 24 hours after irradiation with 1,000 r, whereas the proportion of
Gl population was not significantly different from that in the unirradiatedgroup, i. e., approximately 40%. This is because radiation caused an immediate block of cells in Gl entering into S (Gl block) and of cells in G2
going into M phase (G2 block), without affecting the transit of cells from
S into G2. For the evaluation of these results in relation to the enhancement of mutation frequency by dose fractionation, it is essential toascertain the radiosensitivity of cells during various phases of the cell
cycle. This includes sensitivity to mutation induction, induction ofchromosome aberrations and radiation killing.
In the following experiment, the fate of primary spermatogonia irradiated in S phase was studied with H3-thymidine (H3-Tdr) as cell marker.
6 day old male larvae were injected with H3-Tdr (0.2 pc/larva). One hourlater the injected larvae were exposed to a single acute dose of 1,000 r.
At intervals, frequencies of label among survivors, pycnotic cells andmitotic cells were studied. The results are presented in Table 1.
1) Frequency of label among survivors is relatively constant during thefirst 60 hours. Thereafter, the frequency begins to fall and after 120 hours.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
Table 1.
Time after
irradiation
30 min
24hrs
36 n

48

n

60 n
72 n

120 n

Dose:

Survivals

Pycnosis

MitosisLabeled/total (%)

Labeled/total (%)

Labeled/total (%)

271/ 849 (30.3)
475/1069 (44.4)
166/ 404 (41.1)
251/ 399 (38.0)
74/ 157 (47.1)
80/ 392 (20.4)
51/ 744 ( 6.9)

0/0
0/0
( 0.0)
0/1
26/78 (33.3)
42/102 (41.2)
12/40 (30.0)
5/13 (38.5)

0/0
0/0
1/4 ( 25.0)
1/1 (100.0)
0/0
0/2 ( 0.0)
0/15 ( 0.0)

1,000 r(5th day) it was only 7%. At a first glance this result might be understood to mean that the majority of cells irradiated in S period were killed
within 5 days and, therefore, did not contribute to the repopulation of
spermatogonia in the irradiated testes. However, if we take into consideration the number of grains/cell as a function of time after irradiation,
we will see that this is not the case. The number of grains/cell was as
low as 8.8± 1.39 five days after irradiation in contrast to 30.9±4.04 after
48 hours. This can be best explained by the fact that normal mitotic
activity was restored between 48 and 60 hours after irradiation which
resulted in the dilution of the label thereafter. 2) Using a half-time of
the mean grain count/cell between 48 and 120 hours, the generation timeof primary spermatogonia in irradiated testes was estimated to be approximately 35 hours which is not significantly different from that known
for unirradiated cells. In other words, irradiation caused inhibition ofmitosis for nearly 24 hours after irradiation but once the mitoses were
restored the generation time was not affected to a noticeable degree. 3)
Pycnosis of primary spermatogonia was almost absent during the first 36
hours after irradiation with 1,000 r. During the next 24 hours its incidence increased rapidly. Thus, after 48 and 60 hours it was nearly 20
and 40%, respectively. During the next 12 hours it fell down suddenly
to 8%. Throughout the intervals studied in this experiment the frequencyof labeled pycnosis was not much different from that of the surviving
cells. This suggests that the cells irradiated in S phase have the same
sensitivity to radiation killing as cells irradiated in other phases of thecell cycle. An experiment is now in progress to examine the sensitivityof cells irradiated in Gl or in Gz periods.

RESEARCHES CARRIED OUT IN 1965

90.

107

Variation in radiosensitivity during the early developmental
stage of the silkworm egg
Akio MURAKAMI

In order to elucidate the differences in radiosensitivity at various phasesof DNA synthetic cycle of the cell, experiments have been carried out
with newly deposited eggs of the silkworm using the hatchability as acriterion. The materials used were eggs from wild type C108 female
moths, mated with males having the double recessive egg colour genes,
pe and reo Moths were allowed to lay eggs which were collected at definite
intervals at 25°C.
Expt. 1. The eggs were collected at 30 minute intervals. They were
irradiated with 1,000 r of 137Cs gamma-rays at a dose-rate of 100 r per
minute at their various age. The results are given in Fig. 1. No cyclicchanges were observed in hatchability among different age groups. Presumably this result might have been due to the heterogeneity of thematerial with regard to the division cycle of the eggs.
Expt. 2. Eggs were collected every 15 minutes and irradiation wasapplied at 15 minute intervals from 60 to 300 minutes after oviposition.
1,000 r of 173 kVp X-rays were administered at a dose-rate of 1,000 r per
minute. As shown in Fig. 2, a rhythmical change in embryonic mortality
was observed with a 45 to 50 minute cycle. It is known in this species
that the 2nd maturation division of oocyte begins approximately 60 minutes
after oviposition and is completed about 20 minutes later, and that thefusion of female and male pronuclei occurs about 120 minutes after oviposition. The first peak of the curve may, therefore, correspond to the endof the 2nd maturation division, and the 2nd, 3rd and 4th peaks of thecurve correspond to cleavage I, II, and III, respectively. It is also clear
from the figure that the degree of radiosensitivity is essentially the samefor every cycle regardless of the increase in the number of cleaved nuclei.
The same sensitivity pattern has been reported for Drosophila (Wiirgler
et al., 1963).
Expt. 3. The eggs were collected at 10 minute intervals at 30-31°C andwere exposed to 1,000 r of X-rays at a dose-rate of 1,000 r per minuteduring the period from 0 to 500 minutes after oviposition. As shown in
Fig. 3, the cyclic change in radiosensitivity was again demonstrated clearly
during the first 500 minutes of egg development. Each cycle was repeatedafter 30 minutes and its length was kept almost constant at least up to
the 10th division.
Cytological observation by squash method showed that the time required(%,)

Fig. 1

70

·

·

40

t'

30

~

~

.~

..'"•

55
>

2c:::

60

.

t-

60

~

......

70 '

60

~

Fig. 2

(~)

.

60

~

~

•"
o

:::0
t:rj

~

~

c

o

..::'il

20
10•

Control level

60

1(10

160

200

200

300

360

400

~
Z

tT

60

780 1,600

100Ti•• after depoai tioa. of the .Ii (aiD)

200

160

>

260

j

Ti •• after d.podti02 of the -ill

~
>
t'"'

Z
~

>,

(~ )

~

Fig. 3

c:::

>-:l

."

.. 100

.~

t:rj

~

."

"a
"
=
0

~

50

0

~

>,

"

""
.3

I
0

50

,
100

,

150

200

250

,
300

350

,
400

450

,
500 (min)

n
Ul
z

9

......

Time after dep08ition of the egg

Figs. 1-3. Observed change in X.ray induced embryonic mortality during meiosis and early cleavage of theilkworm egg.

O"lRESEARCHES CARRIED OUT IN 1965109

for one division cycle was 40-50 minutes at 25°C, and 30 minute at30-31°C. The results were in good agreement with those obtained fromhatchability data, indicating that the radiosensitivity pattern may well
be correlated with a cyclic change in the cell division cycle. The cytological studies now in progress indicate that the peak of radiosensitivity
appears around late prophase and metaphase.

91. Enhancing effect of fractionated irradiation with 14 MeV
neutrons on the induction of visible recessive mutations
in silkworm gonia
Akio MURAKAMI, Sohei KONDO and Yataro TAZIMA

Since 1962, the effect of fractionated doses on the induction of mutations
has. been investigated in silkworm gonia by one of the authors, Y. TAZIMA,
using low LET radiations. The results showed that the induced mutation
rates were approximately 2 to 2.5 times higher in the dose fractionationgroup than in the single exposure group, for both spermatogonia andoogonia, when irradiations were given around the stage of hatching. In
the present experiment it was confirmed that the similar enhancing
effect of fractionated irradiation was also observable even in 14 MeV
neutron irradiation. Our materials were the same as described in theprevious paper (TAZIMA, 1963). Larvae of the wild type C108 strain were
exposed at one to three days after hatching to 14 MeV neutrons of T (d, n)
He reaction at the Research Institute of Nuclear Medicine and Biology,
Hiroshima University. Neutrons of total 1,000 rad were administered in
two fractions (500+500) 10 to 12 hours apart at a dose-rate of 0.8 rad per
minute. Owing to technical limitations the 1,000 rad single exposure group
was not examined.
The results are shown in Figs. 1 and 2 for spermatogonia and oogonia,
separately. As seen from these figures, an enhancing effect of dosefractionation is more marked for spermatogonia than for oogonia. The
phenomenon is quite similar to those reported previously by Y. Tazima
for X- and gamma-rays. The mutation frequencies were about two times
as high as those at a single exposure. The highest enhancing effect wasobserved in spermatogonia when the second dose was given 36 hours
after the initial exposure, in contrast to the 18 hours for X- and gammarays (Tazima, 1963). It is noted, therefore, that a delay of 18 hours
occurred in the manifestation of the peak effect in case of 14 MeV neutrons compared with r-irradiation. A possible cause of this delay may bethat the 14 MeV neutrons have higher efficiency to affect cell divisions
than X- or gamma-rays.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

300

150
~

~

1 100
c

o

c

.!

.:'l

i

~100
::l:

H.tch

15

30

50

o

45

Hatch

Time in hour

15

30

45

Time in hourFig. 1. Spermatogonia

150

c

o

~ 50

o

15

Hatch

Time in hour

30

15

15
Hatch

Fig. 2.

30

45Time in hour

OogoniaFig. 1-2. Variation in induced mutation frequency with time interval between
two neutron exposures.

92. Chromosome aberrations induced in mouse bone marrowcells by gamma irradiation
Yoshinori KURITA, Reiko TSURUTA, Tosihide H. YOSIDA
and Katsumi SAKAKIBARA

Many mouse leukemias induced by ionizing radiation are characterized
by aberrant karyotypes (Ford, 1958). It is also known that irradiation
causes a persistent damage to the chromosomes of living cells. To
investigate the mechanism of the persistence of the damage, primarychromosome aberrations induced by ionizing radiations were examined inbone marrow cells of C57BL/6 male mice within 24 hours after a single
450 r gamma irradiation. Eight hours after irradiation, four different typesof aberrations were observed; namely (1) chromatid breaks, (2) chromatidtranslocations, (3) isochromatid aberrations, and (4) chromosome type aberrations. Their frequencies were 0.42/cell, 0.08/cell, 00.5/cell, and 0.35/cell,RESEARCHES CARRIED OUT IN 1965

111

respectively. Minute chromosomes only were observed 8 hours after
treatment. They seem to have resulted from breakage in the secondary
constriction of certain chromosomes. The most striking feature of chromosome aberrations 24 hours after irradiation was the disappearance ofchromatid translocations. The types of aberrations that appeared 24 hours:after irradiation were; chromatid fragments (0.15/cell), ring chromosomes(0.04/cell), and minutes (0.37/cell). Nonrandom distribution of those chromosome aberrations was evident.

93. Persistent chromosome aberrations in mouse bonemarrow cells after gamma irradiation 1)
Tosihide H. YOSIDA, Yoshinori KURITA, Reiko TSURUTA
and Katsumi SAKAKIBARA

Although the significance of chromosomal abnormalities in relation toleukemia pathogenesis remains uncertain, it was of utmost importanceto investigate the persisting type of chromosome abnormalities inducedby irradiation. In order to supply the badly needed information, thetypes and incidence of long-time persisting chromosomal aberrations werestudied in bone marrow cells of C57BL/6 and RF strain mice after threefractionated 450 r gamma treatments.
It was observed that the persisting aberrations were of chromosome
type only. Four kinds were observed, namely (1) minute chromosomes,(2) D-type chromosomes, closely linked at the middle part, (3) subtelocentric chromosomes, arisen from secondary chromosome rearrangement, and(4) giant chromosomes. None of them were found in the control spe,cimens. 12 out of 18 mice examined showed persisting chromosome abnormalities. Their incidence was slightly lower than that observed in LAf
mice by Nowell and Cole (1963), but considerably higher than that obtainedin C57BL/6J mice irradiated with four fractionated 600 r of X-ray byNadler (1963). From these results, it seems highly probable that the cellswith persisting abnormal karyotypes participate in the development ofmouse leukemias after irradiation.

1) This work was supported by a research
grant from the National Cancer Institute (CA 07798-02), Public Health Service, U.S.A.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 1&

F. RADIATION GENETICS IN PLANTS

94. Photoreactivation of ultraviolet-induced
mutation in maize pollen
Seiji MATSUMURA and Tomoo MABUCHI

From our preliminary experiment (ct. Ann. Rep. No. 15:137-138) it was·assumed that photoreactivation of ultraviolet (UV)-induced mutation inhigher plants really exists. In order to confirm this assumption, a similar
experiment was repeated with the same material. Pollen grains of normal'
maize (Su) were irradiated in a single layer in a Petri dish with UV of
254 mf! at a dose rate of 90.3 ergs/mm 2 /sec for 30, 60 and 90 seconds.
Immediately after UV exposure, the irradiated pollen grains were divided
into two batches, one was respread on a Petri dish and exposed to visible
light (7,400 lux) from 440W high pressure mercury lamp for 30 minutes atthe distance of 110 cm (VL treatment) and the other batch was held for
30 minutes is darkroom (dark treatment). After VL and dark treatment,.
the pollen grains were dusted on the silks of a recessive sugary line (su}3
VL- visible lightO-darkO(W+C).::/9

VL(w+c~l':/·//.
/./:{~/'''''

;e
~2
v

....

c:

'""

f

...

... VL-W

#:p'

5~~

o~

550

!

1100

r-ray dose (R)Fig. 1. Effect of visible light on r-ray induced mutations in maize pollen•.

RESEARCHES CARRIED OUT IN 1965113

in the field under sun light and in the darkroom under red light, respectively. For comparison they were irradiated with 550 and 1,100 R of
r-rays from 137CS and dusted on the silks in the same way.
The percentage of seeds set was likewise high in both doses of r-rays,
while it decreased with the increase of UV-dose and was about 50% of thecontrol lot at the highest dose. Induced whole and chimeral endospermmutants were scored separately, and the obtained mutation rates from r-ray
irradiation are given in Fig. 1. There were no marked differences in thefrequency of whole and chimeral mutants between VL and dark treatment.
As to the frequency of the two types, whole mutation was more frequent
than partly mutated chimeras. The frequency of chimeras fits roughly
a linear relation with increasing doses, while this relation is rather exponential than linear for whole mutation. It is assumed that the VL
treatment was completely ineffective in r-ray irradiation.
On the other hand, the frequency of whole and chimeral mutations in
UV irradiation showed almost linear relation, as shown in Fig. 2. It was3

VL-visible Iighf
O-dark
W-whaleC-chimeros

~ 2

.

>-

":<>:

0-

~

<:
0

.0'

)2

~

VL-W

...•....130

•

-

I

I

60

90

UV - dose in sec. (90ergs/mrm-sec.)

Fig. 2. Effect of visible light on UV·induced mutations inmaize pollen.

considerably decreased by exposure to visible light, while there was nodifference in the frequency of chimeras between VL and dark treatment.
It may be assumed that chimeral mutation may be caused by UV-induced

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

alteration in one or a half chromatid at Su locus and whole mutation byalteration in two chromatids. The fact that whole mutation is reversibleby visible light more frequently than the chimeras suggests that photoreactivation may proceed in some way from whole to chimeral mutationand from there to normal endosperm.

95. Effects of dose fractionation of r-rays on
mutation in maize pollen
Tomoo MABUCHI

In order to elucidate the radiological response of the sperm chromosomes in pollen grains, an experiment was carried out on the effects ofdose fractionation of r-rays on mutation rate. Mature pollen grains of adominant line (Su) of maize were irradiated by 137Cs r-rays and dusted on
the [silks of a recessive line (su). A single dose of 550 or 1,100 R was
applied in acute (917 R/min) irradiation. Fractionated doses (550 R) were
given twice with intervals of 0.5, 1.0, 2.5, 5.0, 10, 30, 60 and 120 minutes.
Single irrad.
ilOOR
0 Total (whole + chimeras)
Bose line
2x single 550R • Whole endosperm mutated
Fractional irrad. 550R+ 550R 0 Chimeras, po",

3 a-q.-.- 0 - - - - - - - . ( ; 0 1 - - - - - - - - - - - 0
II

.

~

,!t-••'" ...
•,:i-\~· b·---_________

~

\

>-

\,!

;;;

::J
0-

~
C

,

2

.

.----------~

0\\----.- ----·-----·-----------------·o----~----?
-----_..!_-.:.-_:::=:'=:::.~::=:"= ..~.._-----_....... :.=-=~:::-=---.::-~::;-_-:~::;:;-=--~

0

.~

:=;
~

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

10

30

60

120

Duration of storage or interval (min)Fig. 1. The relation between storage or radiation fractionation and the frequency of mutation in maize.

RESEARCHES CARRIED OUT IN 1965

115

The base line was obtained from single 550 R dose by multiplying themutation frequency by two. Mutations (Su---+su) were detected in the
kernels developed from the fertilization by the treated pollen. The results
are shown in Fig. 1.
There was no significant difference in the frequency of chimeral (partial)
endosperm mutations between the base line and a single 1,100 R dose or
fractionated doses with time intervals 0.5~ 120 min. But whole mutations
in a single 1,100 R dose were produced twice as high as the base line, as
did the fractionated doses with 0.5 to 5 min intervals. In the experimentwith fractionated doses, whole mutations dropped rapidly at 5 to 10 min
intervals and slightly decreased with increasing intervals (1O~120 min).
The results of the dose fractionation experiment indicate that most of
the breaks induced in the sperm chromosome of pollen grains remained
open at least for 10 minutes.

96. Storage and oxygen effects on r-ray induced
mutation in einkorn wheat
Tomoo MABUCHI and Seiji MATSUMURA

Dry seeds of Triticum monococcum flavescens containing 13% water
were sealed in ampules filled with oxygen, nitrogen and air, exposed to
10 and 15 kR r·rays. For acute and chronic irradiation dose rates of
10,000 R/hr with 137CS and 19.6 R/hr with 60CO were used, respectively.
_

Aeut. Irroct. + !504 hours storage_

Acuff irrad. + 762hours StorQge

c:J Acute irTOd.+ no storooe(Ae.lrrad. IO.OOOR/hr)_( Ch. irrad.

e:tntW: irrad.+ no sforage

19.6R'hr)

10

~

.go"

8

<7

~ 6
co

o

~::;: 42

.n::l....

o l-ILL
I.L..
1------l;2LJLi.J....·..J::a~LJ::"':-:-I2;LAir O. N.

t>Jr

O.

~.

':-:-"-."'.. --~"'--'~~-

Air

O.

N..

~
IOkR
IS'kR
Fig. 1. Oxygen and storage effects on chlorophyll mutation in einkorn wheat(1965).

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

Two chronic irradiations (10 and 15 kR) were terminated simultaneously
before sowing. Acute irradiations were given at the time coinciding withthe beginning and termination of the chronic ones. The data for chlorophyll mutations in X2 seedlings are shown in Fig. 1. In general, chronic
irradiations were found to be more effective than the acute ones, especially at higher irradiation with 15 kR, as already found for the inhibitionof seedling growth (ct. Ann. Rep. No. 15:139-141). There were no clear
differences between air and oxygen treatment either in acute or chronic
irradiation. On the other hand, nitrogen treatments clearly showed aprotective effect on mutation frequencies. Also post-irradiation storage
was effective on inducing mutation, especially in acute irradiations withnitrogen treatments.
These results might be explained by the relationship between production
and decay of free radicals and radiation damage, as already mentioned.
It is assumed that the storage effect is due to the decay of long-lived free
radicals which combine with biologically important molecules, like watermolecules, and the nitrogen treatment decreases the radical yield, andproduction and decay of the radicals are more gradual and slower inchronic than in acute irradiation.

97. Genetic effects of X-irradiated pollen in wheats
Tomoo MABUCHI and Seiji MATSUMURA

Anthers before dehiscence of T. monococcum, T. durum and T. Spelta
were irradiated by X-rays at 1 kR (200 R/min). The pollen grains were
dusted on emasculated florets of the same species. Reciprocal translocation (RT), plant height at maturity, seed fertility in Xl and chlorophyllmutations in X2 were investigated. The results are given in Table 1.
Plants with RT in T. monococcum and T. durum were observed in highfrequency in irradiated lots, but not in the non-irradiated control. In
diploid species, there was no significant difference in plant height between
irradiated and non-irradiated lots. Plant height of polyploid species,
however, was considerably inhibited by irradiation. No correlation between plant height and RT was found in T. monococcum, but it was
found in T. durum. Seed fertility was reduced markedly by irradiationin all species tested and there was a positive correlation between seedsterility and RT. Chlorophyll mutations in X2 were also found to be induced by pollen irradiation like from seed irradiation.
From these results, it is assumed that in polyploid wheats plants with
RT and other chromosome aberrations which reduce plant height have

RESEARCHES CARRIED OUT IN 1965

117

considerable viability, but not in diploid wheats.
Table 1. Effects of X-irradiated pollen on chromosome aberration at
MI, plant height and fertility in Xl and mutation
rate in Xz of wheats
Species% of plants
with RT at
MI

Plant height Seed fertility
(em)
(%)

Chlorophyllmutation rate
per spike (%)

T. monococcum(2n=14){~0.0 ( 0/24)
40.0 (26/65)

124.0± 9.8
127.5±10.5

74.1± 8.542.2±24.6

0.0 (0/32)
3.9 (2/51)

T. durum(2n=28){~0.0 ( 0/11)
61.3 (27/44)

158.0± 6.1
141.5±20.9

84.8±12.443.9±35.6

0.0 (0/26)
1.4 (1/70)

T. Spelta(2n=42){~134.6± 9.5
107.8±18.5

54.7±20.1
14.8±21.1

0.0 (0/33)
0.0 (0/46)

C:

Control,X:-

X-irradiation

98. Relation between polYPloidy and radio-sensitivity
under chronic condition
Seiji MATSUMURA and Taro FUJIIThe facility for chronic r-ray irradiation of growing plants, the so-called
gamma-greenhouse, was built in our institute in 1964. Effects of chronic
irradiation on growing wheat were examined in the gamma-greenhouse.
Two diploid, 3 tetraploid and 2 hexaploid species were used in thisexperiment. The employed seven species constituted one block. Four
blocks were set up for irradiation at various distances from the source or
at various dose rates. Radiation intensity was 0.31~3.06R/hr. Therefore,
total dosages varied considerably from 870 R at the minimum to 8,700 R
at the maximum.
For examination of seed fertility, spikes of the tallest, main tiller of
each individual were collected from all surviving plants and the average
seed set was calculated. Decrease of fertility was clear in each species
with increasing dosage, and unexpectedly, there were no marked differences among the three ploids. But when the decreasing rate was compared
between the cultivated and wild types at each ploidy level, in general therate for the wild type was slightly lower than for the cultivated types.
From our experiments, we would say roughly that the difference inradio-sensitivity between ploids became smaller under chronic than underacute condition. The smaller radiation effect in chronic than in acutecondition may be due to recovery phenomena during a long irradiation

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

period at low intensity. A protective effect in polyploids is also an
important factor in radiation tolerance. Therefore, in polyploid plants
radiation effects under chronic condition may be influenced by thesefactors, and differences in mitotic rate and genome complexity in allopolyploids should be additional factors in the appearance of radiation
damage.

99.

Development of mutated cells in diPloid wheat
Taro FUJII

Normals and recessive chlorina mutants of diploid wheat were used as
female and male parents, respectively. 500 R of r-rays were given to
young embryos 24, 28, 72 and 96 hrs after pollinations. The F l seeds
were harvested and the mutated stripes were scored in the Fl or Xl
generation. Germination rate in all irradiated lots were lower than in thecontrol and that of the 24 hr lot was as low as 35%. TSUNEWAKI (1955)
examined the number of cells several times after pollination and foundthat it was 24, 48, 72 and 96 hrs after pollination about 1.0, 8.2, 37.8 and105.7, respectively. When the cell at one-cell stage were damaged byirradiation, all cells in the ripe embryo would have the same kind ofaberration which could affect germinability.
Longitudinal chlorina and necrotic stripes caused by somatic mutations
were detected in young seedling having one or two leaves. The widest
chlorina stripes occupied about half the area of a leaf and the narrowest
stripes were restricted to about 1/4 leaf area. One seedling in the 48 hr
lot was chlorina without any trace of green. The number of mutated
spikes in the present experiment was 10 times larger than after seed
exposures. Of course, if mutations occurred in some cells among a small
number of cells in the young embryo, mutated cells should be incorporated
into a large part of tissues through many steps of development.

100. Effects of ultraviolet-rays on Arabidopsis thaliana
Taro FUJIIF2 seeds obtained from the F l of crosses between the hairless mutant
and the wild hairy strain were used because it is somewhat difficult toobtain many F l seeds. Seeds were sown on wet filter paper in Petridishes. About 5mm long seedlings were exposed to UV-rays. Wave
length of the UV-rays was 253.7 mp; the absorbed energy was 90.3
ergs/mm 2 /sec. About 1,500 seedlings were used in each lot and 0.5 andRESEARCHES CARRIED OUT IN 1965

119

1 min exposures were applied. Irradiated seedlings were kept in a darkroom for 24 hours.
During the growth of seedlings, the segregation ratio of hairless homozygotes was examined. In the 0.5 min lot, hairless homozygotes segregatedaccording to the ratio 3: 1 as expected. Therefore, two thirds of hairy
plants were expected to be heterozygotes. Hairless sectors appeared in
two plants in the 0.5 min lot among 581 presumably heterozygotic plants.
From the results, mutation rate was calculated as 0.34 per cent. Segregation ratio of hairless homozygotes in the 1 min lot did not fit the 3: 1
ratio. The small number of hairless plants in the 1 min lot might be dueto their low survival rate.
Only one mutated plant was observed in the 1 min lot among 552 hairy
plants. The low mutation rate in this lot seems to be the result of a toosmall number of surviving plants. Photoreactivation effect and doseversus-mutation curve are now under examination.

101.

Mutagenic efficiency of heavy ionizing particles in
Arabidopsis thaliana
Taro FUJII, Mituo IKENAGA and John T. LYMANIl

F2 seeds obtained from F 1 hybrid between a hairless mutant and thewild hairy strain were subjected to 1~5 krad He 4 and C12-ions, respectively, and 3 and 5 krad of Ar 4 °-ions, initial particle energy being 41.6,
124.8 and 416 MeV, respectively. For comparison of RBE values andestablishment of a standard, 27 and 47 krad r-ray exposures were given
to the same material using 6 kCi of 137Cs source. About 67% of hairy
plants may be assumed to be heterozygotes for the hairy-hairless allele
pair. When a leaf or a part of a leaf was hairless it was scored as one
mutational event occurring in the dominant hairy allele.
The frequency of plants with hairless sectors increased with increasing
dosage in r-ray, C-ion and Ar-ion lots. Somatic mutation rates in C-ion
lots were very high; those of Ar-ion lots were very much lower. On theother hand, only one mutated plant was observed in the He-ion 2 kradlot. Dose-versus-mutation curves for the C- and Ar-ion lots were fairly
linear, while a non-linear relation taking the shape of an exponential curve
was observed between mutation frequency and r-ray dosage. Thus theshapes of dose-response curves are different, and therefore the RBE valueof heavy ionizing particles cannot be expressed by a single parameter.
1) Donner Laboratory and Lawrence Radiation Laboratory, University of California, California, U.S.A.

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

The frequencies of somatic mutations of r-rays and heavy ionizing
particles were compared at 0.5% mutation rates from their dose-versusfrequency curves. About 33 krad of r-rays was necessary for producing
the 0.5% mutation rate, and about 0.9 and 6.7 krad of C-ions and Ar-ions
could produce the same mutation frequency. From the results, RBE forsomatic mutation at 0.5% frequency was roughly estimated as 35 for
C-ions and 5 for Ar-ions. Neither could we decide about He-ions' RBE,
but if the mutation rate at 2 krad lot was sustained their RBE for somatic
mutation might be about 10.

G.

HUMAN GENETICS

102. Genetic epidemiology of Down's syndrome in Japan l )Ei MATSUNAGA

It is now known that the overwhelming majority of cases with Down's
syndrome are due to primary non-disjunction of the 21st autosomes,
either at gametogenesis or at an early cleavage of the fertilized egg.
Theoretically, this event may be caused by factors either on paternal ormaternal side, or on both sides, but it is generally believed, from the
Caucasian data, that the maternal side is of relative importance, since
there is a strong dependency of the occurrence of this abnormality upon
the maternal, not the paternal age if the two are appropriately separated.
Consequently, it is natural to inquire whether such agents as hormones
acting upon ovarial function may affect the aging process or the tendencyof the stocked ova to chromosomal non-disjunction. This question may
best be answered by testing the effects of birth order and of pregnancy
free intervals preceding the births of the patients.
Another question of genetic interest is concerned with whether recessive genes are involved in non-disjunction in man. In Drosophilamelanogaster and in Zea mays the existence of such genes is well known.
If recessive genes were involved in the etiology of Down's syndrome, ahigher incidence of consanguinity among maternal grandparents would
be expected.
Keeping the above considerations in mind, data obtained from 834 casesof Down's syndrome in institutions for mental defectives were comparedI} This work was supported by a grant from the Toyo Rayon Foundation for the
Promotion of Science and Technology.
I

RESEARCHES CARRIED OUT IN 1965

121

with various sets of matched controls including standardized vital statistics.
The rise of relative incidence of this abnormality with maternal age
showed a pattern quite similar to that found for the Caucasians; this.
pattern did not vary by the socio-economic conditions of the mothers.
When the maternal age effect was eliminated, the paternal age had inpractice no effect, while a significant decrease in the relative incidence
was noted according to live-birth rank, irrespective of the socio-economic
conditions. This is likely to be due, however, not to birth order effect assuch, but to some selection, practiced probably by the parents in bringing
their children to institutions, because the same tendency was found forother mental defectives than those with Down's syndrome. As to pregnancy free intervals preceding the births of the patients, there was no
indication that either a too short or a too long interval is of etiologicalsignificance.
Consanguinity was investigated, by referring to "koseki" records,
among grandparents of 104 patients born to younger mothers. There was
no increase in frequency of consanguineous marriages among maternal
grandparents as compared with that among the paternal ones, suggestingthat recessive genes of the mothers, either autosomal or sex-linked, play
no role in chromosomal aberrations leading to Down's syndrome.
Details of this work will be published elsewhere.

103. Parental age and sporadic retinoblastoma 1 )Ei MATSUNAGA

It has been pointed out by some workers, notably Penrose, that for
certain hereditary diseases such as chondrodystrophy and acrocephalosyndactyly the mean paternal age is characteristically increased above thepopulation's average for sporadic cases attributable to fresh mutation,
indicating that the site of the genic mutation is mainly in the paternal
gonads. For another group of diseases, e.g., retinoblastoma and neurofibromatosis, the mean paternal and maternal ages are both only slightly
raised, and it had been impossible to make any inference as to which of
the parental ages is of primary significance. Those observations werebased on Caucasian data.
Using data from 65 cases of sporadic retinoblastoma previously ascertainedin Hokkaido, the parental ages have been carefully checked against" koseki" records. Of the 65 cases 21 were bilateral and 45 unilateral.
Although the year of birth of the patients range from 1930 to 1958, all1)This work was supported by a grant from the Ministry of Education.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

but four were born after 1940, for which, with the exception of 1944-46,
the distributions of maternal ages in quinquenniums are available for all
Japan from vital statistics data. Their percentage distributions for eachyear of the period were then weighted by the number of cases born in
the respective year, and from them the mean maternal age for the control
group was estimated. As to controls for paternal age, the vital statistics
provide only data for 1952 and thereafter. Because the mean paternal age
in the vital statistics has been gradually decreasing, the control was takenfrom the data for 1952.
Table 1.

Mean parental ages of sporadic retinoblastoma cases
compared with those of control group
Paternal ages

Affected eyes I
Bilateral
Unilateral
Combined~~~e~f I Pl
21
45
66

34.4
33.0
33.4

I
32.0
32.0
32.0

+2.4
+1.0
+1.4

39.05
34.77
35.97

(one failed
test)

P<0.05
P>O.1
P<0.02

1.76
1.14
1.79

Maternal ages
Affected eyes I
Bilateral
Unilateral
Combined~~~e~f I ml
21
45
66

28.9
28.9
28.9

I
28.2
28.5
28.4

+0.7
+0.4
+0.5

31.19
29.87
29.82

0.61
0.54
0.79

(one failed
test)

P>0.2
P>0.2
P>O.1

Key:
pl, ml =mean parental ages for retinoblastoma.
po, mo = mean parental ages for controls.

V 1 =variance of parental age for retinobastoma.
Control for paternal age=vital statistics data for 1952.
Control for maternal age=weighted average from 1940-58 for all Japan.

As can be seen from Table 1, the mean paternal age shows a significant
increase for the bilateral but not for the unilateral cases, while the meanmaternal ages for both bilateral and unilateral cases remain almost thesame as the mean ages of the mothers in control group.
A sensitive indication of the primary importance of paternal or maternal.age is obtained by comparing the mean difference in ages, p-m, of theparents of the patients with that in the general population. If the disease
were primarily associated with maternal age, the mean difference should

123

RESEARCHES CARRIED OUT IN 1965

be considerably lower than the difference in the general population, while
the reverse would be expected if the disease were primarily dependent
upon paternal age. Table 2 shows the mean difference between parental
Table 2. Mean of the difference between parental ages, p-m forthe retinoblastoma cases compared with a control population
Affected eyes
Bilateral
Unilateral
Combined

Mean of p-m Mean of
No. of for
the retino- p-m forcases blastoma
cases controls
21
45
66

+5.1
+4.0
+4.3

+3.9
+3.9
+3.9

t

V1
8.45
10.77
10.13

1.75
0.12
1.00 IP(one tailed
test)
P<0.05
P>0.9
P>0.15Key:
V1 =variance of p-m for retinoblastoma.
Control for p-m was based on the vital statistics for 1952.

ages for our retinoblastoma cases compared with a control value calculated
from the vital statistics data for 1952. The mean difference is significantly
raised for the bilateral cases, while for the unilateral cases this figure
scarcely differs from the control's.
In our previour report (1961), we presented some evidence, based on thefollowed-up studies of patients with retinoblastoma, that most cases ofsporadic bilateral retinoblastoma appear to be due to new mutation, while
the great majority of unilateral sporadic cases are to be regarded as
phenocopies. The results given here are consistent with this view.
Furthermore, they suggest that the primary site of the mutation is mainlyin the paternal gonads, as is the case for chondrodystrophy.
References
E.: Genetic studies on sporadic retinoblastoma in Japan.
Ann. Rep. Nat. Inst. Genet. No. 12: 116-117, 1961.
PENROSE, L. S.: Mutation. In Recent Advances in Human Genetics:
1-19, Churchill, London, 1961.
MATSUNAGA

104. Genetically controlled variations of red
cell acid phosphatase
Tomotaka SHINODA

Attempts have been made to estimate frequency of variations of theacid phosphatase types in human red cells. When hemolyzates are tested
by means of starch gel electrophoresis, at least three zones of acid124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

phosphatase activity can be obtained. These patterns are detectable ascolored zones by incubation of the gel with a substrate solution.
Blood samples were collected in Numazu City and in Tokyo during thesummer of 1965. A total of 437 samples were tested. In all individuals,
ABO blood types and enzyme activity were determined. Table 1 sum·
marizes the data.
Table 1. Numbers of individuals of the different enzyme phenotypes
Phenotypes

No. of individual

Incidence

A
AB
B
AC
BC
C

77
202
146
3
8
1

0.176
0.462
0.334
0.007
0.018
0.003

Total

437

1.000

It has been suggested that these phenotypic differences are determinedby three allelic autosomal genes, pa, pb, and pc. If this hypothesis
is correct, then the frequencies P=O,4lO, q=0.574, and r=0.016 of thegenes pa, pb, and pc may be obtained from the results given in Table 1.
By the appropriate calculations it will be seen that there is a good agreement between the observed numbers of the various phenotypes and thenumbers expected.
Further analysis of the data is now in progress.

105.

Alkaline phosphatase in human serum
Tomotaka SHINODA

It has been demonstrated that alkaline phosphatase in human serum
fell into two groups, type 1 and type 2. One of these was found to bestrongly associated with certain red-cell antigens. Type 1 had a single
zone of activity, while type 2 had an additional slower migrating zone
the intensity of which varied considerably from sample to sample.
Attempts were made to obtain some population data, by using part ofthe samples collected for classification of acid phosphatases in red cells.
Total activity of serum alkaline phosphatase was determined at 410 mf-l
using p-nitrophenyl phosphate as a substrate. Some sera were found to125

RESEARCHES CARRIED OUT IN 1965

have a slow migrating band which was quite weak compared with thatof type 2, especially in the case of undiluted sera. These were not,however, used for classification, because the resolution of zone on the gel
was inferior to that obtained with diluted specimens. Other zones withenzyme activity are occasionally seen in normal serum. One of thesemigrates between the fast and the slow bands seen in the type 2. Another migrates very slowly and appears just ahead of the origin.
Among 438 sera tested in all, 92 were found to be type 2, giving afrequency of 21 per cent. The relation between the phosphatase groups
and the ABO blood groups is summarized in Table 1.
Table 1.

Relation between phosphatase groups
and ABO blood groups
Blood group

Phenotypes

Total

A

B

a

AB

1

176

59

83

28

346

2

5

37

43

7

92

It is apparent that there is a much higher incidence of type 2 sera withgroups B and a than with A. The AB individuals constitute a small
sample (n=30) with an intermediate frequency.
There is considerable variation in level of enzyme activity from serum
to serum within each of the two groups. The average level of enzyme
activity in type 2 sera is about 27 per cent greater than in type 1.
Two different hypotheses have been advanced concerning the nature of
the slower migrating component. The more likely of these suggests thatthe two enzyme zones in normal serum are qualitatively different.

106. Studies on haptoglobin
Tomotaka SHINODA

Haptoglobin, a serum protein with hemoglobin-combining ability, usually
falls into three common types 1-1, 2-1, and 2-2, the relative frequenciesof which vary among ethnic groups so far studied.
In order to obtain more population data, a search was carried out in1965.
Blood samples were collected from unrelated persons in Izu area.
Among 694 sera so far tested the frequencies of the types 1-1, 2-1, and126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

2-2 were respectively 38, 271, and 381. Accordingly, the frequency of
the gene Hpl may be estimated to be 0.251. In this study three sera were
found to be of type O. One sample appeared to contain a variant of type2-1. The second and third fastest haptoglobin zones of this variant wereless intense than normal, and seemed to overlap. Some attempts weremade to find out the difference between the variant and type 2-1.
However, it could not be shown that the variant contained an alteredpolypeptide chain in the molecule. There was no significant differencein hemoglobin-combining capacity between type 2-1 and the variant so
far examined. The overall frequency of the gene Hpl was 0.254, and nodifferences in distribution have so far been obtained among the samples
collected from different areas in Japan. Further analyses are in progress.

107. Cytogenetic survey of 127 cases with Down's syndrome
Akira TONOMURA, Hidetsune OISHI and Yasumoto KIKUCHI

The incidence of translocation and mosaicism among patients with
Down's syndrome is about 8% in Western Europe and North America, but
comparable figures from other parts of the world have so far been very
limited. The purpose of the present investigation was to estimate theincidence of translocation and mosaicism in a sample of Japanese patientswith Down's syndrome. The karyotypes of 127 cases with this abnormality (76 males and 51 females) have been examined by the use of
leucocyte culture of peripheral blood. Clinical examination and diagnosisof the patients have been made at the Pediatric Clinic of the Konodai
National Hospital in Ichikawa and a few other University Hospitals inTokyo. The patients' age at the time of chromosome examination varied
from a few months after birth to 12 years, but about 85% of these were
under five years. There was no case in the present series which contained
a second affected child in the sibships.
In 127 cases with Down's syndrome, 118 (92.9%) were found to be of
the standard 21-trisomy type. There were six cases with translocations,
three of them showed 13-15/21 translocation and the other three had
21-22/21 translocation or an isochromosome for the long arm of chromosome no. 21. Analysis of cultured blood cells from their parents showedapparently normal karyotypes. Mosaicism was observed in three patients.
One case was a chromosomal mosaic with 46 (normal)/47 (trisomic 21)/48(tetrasomic 21) chromosomes. This patient has been described before.
The other two cases consisted of two different cell types, one with 46(normal)/47 (trisomic 21) and the other with 47 (trisomic 21)/48 (tetrasomic21) chromosomes.

RESEARCHES CARRIED OUT IN 1965

127

The results obtained are summarized in Table 1. In the present series
the frequency of Down's syndrome with chromosome constitutions otherthan the standard 21-trisomy type is about 7.2%. This figure may becomparable to those obtained in Western Europe and North America, andthere is no indication of any difference between Japanese and Caucasiansin the relative rate of Down's syndrome with translocation and mosaicism.
Table 1.

Karyotypes
Numberof patients(%)

Results of chromosome survey of 127
cases of Down's syndrome

I 21 trisomy I 13-15:21
trans!.
118(92.9%)

3(2.4%)

21-22:21trans!.

mosaics

Total

3(2.4%)

3*(2.4%)

127(100.1%)* 46/47, 46/47/48, 47/48
108. Identification of human somatic chromosomesfrom measurements of their areas
Hidetsune OISHI and Akira TONOMURA

In general, the Denver classification proposed in 1960 by the Denverstudy group is now widely accepted as the standard system of nomenclature of human somatic chromosomes. However, there are some important·differences of views on specific points, particularly on the morphological
identification of the X chromosome. In that study group, some participants considered that the X was larger than no. 6 autosome, while theothers thought that it was smaller. In this system, classification ofindividual chromosomes was based on the unidimensional measurement,
which was concerned with length and arm ratio of the chromosomes.
This method is not reliable, since the larger chromosomes tend to contract
more in later stages of the metaphase than the smaller ones. In thepresent study, an attempt has been made to give to distinguish the·chromosomes more accurately by measuring their area rather than length
only.
Chromosome preparations from human peripheralleucocytes were madeby standard culture technique. The cells were spread by air-drying
method and stained with Giemsa. A suitable metaphase figure was obtained
from each of ten normal males; the distinction of the X from the auto'somes, especially nos. 6 and 7, is usually very difficult in female cells,
while it is less difficult in male cells where pairs of nos. 6 and 7 aredearly ascertained so that an unpaired chromosome can be identified as

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

the X. All chromosomes were magnified about 18,000 times from photomicrographs by a photographic enlarger. The projected image was traced
on a sheet of white typing paper, and the figures were then measured bya planimeter. The lengths of the chromosomes were also recorded in thesame cells. The measurement for each chromosome relative to the totalof a normal X-containing haploid set was expressed per thousand.
The results are summarized in Table 1. There is good agreement
between the two numberings of the chromosomes obtained by area andthose by length measurements. However, values obtained by area
Table 1.

Relative lengths and areas in normal human chromosomes

No.

Relative area

1
2
3

84.7±3.72
82.2±3.28
67.9±3.49

1.1
1.6
1.2

81.8±5.47
78.3±6.42
67.1±3.25

1.1
1.6
1.1

4
5

63.4±2.16
59.2±3.132.8
2.860.5±3.09
56.5±1.68

2.5
2.5

6
X
7
8
9
10
11
12

57.5±2.59
56.7±2.49
51.0±3.68
46.9±2.37
46.2±1.2144.2±2.1342.4±1.88
42.0±1.25

1.7
1.7
1.6
1.8
2.5
2.4
1.5
2.4

57.3±3.00
51.9±1.55
49.8±1.81
46.7±1.1145.4±1.50
43.8±0.98
42.6±0.93
41.6± 1. 71

1.5
1.6
1.5
1.7
2.2
2.1
1.5
2.2

13
14
15

36.4±2.36
34.1±2.1431.3±1.14

10.9
11.5
9.0

37.5±1.54
35.0±2.1133.4±1.70

5.5
5.5
4.6

16
17
18

29.3±1. 71
27.4±2.1424.3±1.89

1.4
2.6
3.5

31.4±1.60
29.1±1.24
26.1±1.48

1.3
2.1
2.6

19
20

21.9±1.58
19.5±1.44

1.3
1.2

24.4±1.28
22.7±0.86

1.2
1.2

21
22

16.7±2.03
14.7±1.08

5.9
4.6

19.2±2.45
18.0±1.27

3.4
2.6

y

19.6±3.02

8.3

22.6±3.59

4.9

RESEARCHES CARRIED OUT IN 1965

129

measurements tended to be somewhat larger for the larger chromosomes
than the values from length measurements, while the relation was reversed
for the smaller chromosomes. Further, the estimates of the arm ratios
in terms of relative areas were found to be larger for the submetacentrics,
such as nos. 6-12 and 16-18, than the corresponding estimates based on
length measurement.
In comparison with the data of length measurements by the Denverstudy group, the presents results are very similar to the data of LEVAN
and Hsu, and BUCKTON, JACOBS and HARNDEN, who suggested that the X
was smaller than no. 6. In the present study, the value of the X was
56.7±2.49 by area measurement and 51.9±1.55 by length measurement.
An independent test for the accuracy of our identification of the X
chromosome may be made by referring to the so-called drumstick appendage of the polymorphonuclear leucocytes, which is generally considered,on the basis of recent cytogenetic discoveries, to be an expression of an
X in somatic cells of the female. Therefore, we can estimate, from theobserved ratio of the size of the drumstick to the total nuclear size of thepolymorphs, the expected ratio of an X chromosome to the total of an
X-containing haploid set in terms of area measurement. From theobservation of 60 polymorphonuclear cells with drumsticks, the ratio ofthe drumstick to the total nucleus had been found to be about 0.03, andthis is equivalent to 0.058 for the ratio of an X to the haploid set. Thus,
the latter is rather close to the value of 0.057 obtained by the direct
measurement of the area.
Briefly the area measurement may sometimes be useful for the identification of individual chromosomes, especially, for the measurement of.quantitative changes involved in the X chromosome material.

109. Clinical conditions of patients with apparently
normal chromosomes, II
Hidetsune OISHI, Yasuffioto KIKUCHI and Akira TONOMuRA

Based on leucocyte cultures of peripheral blood, patients with the following clinical conditions were found to have 46 chromosomes of apparently
normal karyotype.
Name
Age
Legal sex
Clinical conditions
26 S. M.
2 years
M
Congenital cataracta
27 Y. T.
3 yearsF
Sjogren's syndrome
28 T. Y.
5 years
Male pseudohermaphroditism
1\1
29 N. I.
10 years
M
Retentio testis abdominalis

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46

T.S.
H.A.
N.A.
M.O.
A.S.
K. T.
Y.Y.
-.M.
M.Y.
M.Y.
T.O.
A.S.
A.H.
T.K.
K.K.
K.K.
T.H.
110.

1 year19 years
17 years
3 months
7 years
6 years
14 years
1 day
6 months
10 years
3 years
46 years
12 years
2 years
3 years
7 years
3 years

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

True hermaphroditism
Testicular feminization
Hirschsprung's disease
Turner's syndrome in the male
Female intersex
Eunuchoidism
Anencephaly
Pterygium coli
Gonadal disorder
Chondro-osteo dystrophy
Eunuchoidism
Pterygium coli
Mental retardation
Klippel-Feil's diseasede Lange's syndrome
Female pseudohermaphroditism

DNA replication pattern of the human Y chromosome
Yasumoto KIKUCHI

In human karyotype, certain structural characteristics make it easy to
distinguish the Y chromosame from the acrocentric chromosomes of G
group (nos. 21-22), namely absence of satellites on the Y chromosome,
its longer size, lower arm ratio, and condensed or pycnotic condition. In
a considerable number of metaphases, however, it may not be possible
to distinguish the Y chromosome from the acrocentrics of G group.
Hence, we made a special effort to ascertain the autoradiographic patternof the Y chromosome as compared to that of the small acrocentricchromosomes, hoping to find out if the DNA replication pattern can beused for identification.
Leucocytes obtained from venous blood secured from male subjects
with XXY, XYY, X·long Y, and normal XY were cultured acording to amodified method of MOORHEAD et al. (1960). DNA replication was analysedby means of autoradiography, tritiated thymidine, and pulse labelingtechnique (KIKUCHI and SANDBERG 1964, 1965).
To demonstrate possible differences in grain content of the Y and the
G chromosomes, 20 metaphase plates of each subject were examined.
The results are shown in Table 1. In normal male cells, the averagegrain number of Y, 4 G's, and the most heavily labeled G chromosome
were 10.0, 2.3, and 4.2, respectively. Grain distribution over the Y

131

RESEARCHES CARRIED OUT IN 1965
Table 1. Summary of the result of grain counts for the Y and thechromosomes of G group from 5 male subjects. The data are
based on counts obtained from 20 metaphases in each case.
Normal(XY)XXY

I X-long Y I

XYY

XYY

Average no. of grainson 1 Y chromosome

10.0

11. 7

12.4

8.2

8.4

Average no. of grainson 1 G chromosome

2.3

2.2

2.8

2.4

2.6

4.2

5.0

5.6

4.5

4.7

Average no. of grainson the most heavily
labeled G chromosome I
Ichromosome was about four times heavier than over the G chromosomes.
Further, the Y chromosome was about two times heavier labeled as themost heavily labeled G chromosome. Similar results were obtained in cellsof XXY, X-long Y, and XYY subjects.
In general, it was noted that the human Y chromosome has a markedly
late DNA replicating pattern which differs from that of G group. The
results obtained from detail analysis of grain content may indicate thatthe DNA replication of the Y chromosome is later than that of any
chromosome of G group, and that the grain count by autoradiography isa useful tool for distinguishing the Y chromosome from the chromosomesof G group.

H.
111.

TI:CHNICAL NOTI:

Separax, a new Japanese cellulose acetate strip
for electrophoresis
Yoshito OGAWA

Cellulose scetate strips for zone electrophoresis manufactured by Fuji
Photo Film Company in Japan under the guidance of the present writerand sold giving the name of "Separax" have several advantages over thepresently used kinds, namely Oxoid (England), Membranfolien (Germany),
Millipore, Sepraphore III (U.S.A.) and Cellogel (Italy).
The benefits of Separax strips are as follows:
1. Separax is not fragile and does not roll.
2. It does not take color, e.g. Ponceau 3R or Nigrosin, and remains

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

completely colorless.
3. Its ability of separation is excellent (Fig. 1).
4. Rapid separation and drying, with a great saving of time and labor.
On Separax strips, electro-osmotic phenomena are clearly observed. The
material must be applied near the anode (Fig. 1).
alb

pre-

y

alb

+
< : - - - - - - 3 .7 cm---------o>/:>

i"'1

I

Fig. 1. Curve obtained by scanning a Ponceau 3R stained Separax(1 X6cm.) cleared with paraffin oil. az and jS-fractions wereseparated into three and two fractions respectively.
Results:
Total protein
7.0 gm%Albumin59.5%
al globulin
3.2%
az globulin
10.1%
jS globulin
8.2%
r globulin
19.0%
A/G
1.47

Separax is opaque-white and is usually sold in 6x 22 em. strips, but othr
sizes can also be obtained*.
The physical and chemical characteristics are as follows:
Thickness: approximately 130-150 fl.
Wet to dry ratio, after blotting: 2.6: 1.
Refractive index of strips immersed in paraffin oil: Approximately
1.477 (Nii\* Jo-Ko Sangyo CO. LTD.:

Tamagawa Okuzawa 3-164, Setagaya, Tokyo. Japan.

Table 1. Technical errors in the analysis of human sera on Separax(%J

Size ofstrips(em)

Numberof
ex periments

Albumin

Scanning

1x6

561.02±1.149

2.33±0.149

9,40±0.584

7.37±0.343

19.98±0.355

Separax

Elution

2x6

5

61.09±0.894

4.91±0.609

9.18±0.308

7.81±0.264

17.01±0.397

Separax
Oxoid(Control)Elution

4x6

562.89 ± 0 ,429

3.22±0.009

9.26±0.285

6.95±0.012

17.68±0.230

Scanning

1x6

563,40±1.151

4,45±0.645

8.95±0.388

7.94±0.613

15.26±0.641

Kindof
strips

Method ofquantitative
determination

Separax

Globulin
(¥l

(¥2

I

(3

I

r

I

Examination was carried out using the same sample under optimal experimental condition of each strip.
Separax: Electric run, 0.8 rnA/em, 33minutes at 28°C., Staining 90sec. with 0.8% ponceau 3R solution.
Oxoid: Electric run, 0.6 rnA/em, 50 minutes at 28°C., Staining 60 sec. with 0.4% ponceau 3R solution.

:::0

~

:(.I:j
:t:
Ul
(')

?;
C!
~

Table 2.
Method ofquantitative
determination

Normal values of Japanese serum analysed on Separax(%)

Globulin
Blood donors

Albumin
(¥l

I

(¥2

I

(3

I

r

10 persons

68.3±2.2

3.3±0.98.0±1.3

7.6±0.912.8±2.4

Female 10 persons

67.8H,4

3.7±0.3

7.7±1.1

8.2±1.5

12.6±2.8Male

10 persons

70.3±0.9

3,4±0.3

6.8±0.7

7.0±0.812.5±0.6

Female 10 persons

69.3±0.7

3.6±0.3

6.8±0.2

7.7±0.5

12.6±0.6

Male

8>-3
52
......

8l

Scanning

Elution......

t:5

134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16

Electrical resistance of strips immersed in barbitone buffer pH 8.6, ionicstrength 0.06: 323,000 ohms/em. length/em. width.
Chemical composition and solubility are generally similar to those of theother commercial kinds.
The optimal experimental conditions for analysing human serum forclinical examination are as follows:
1. 1x6cm. strips for scanning and 3x6cm. strips for elution are convenient. When many samples have to be analysed on the same strip wider
strips may be convenient.
2. Buffer solution: Barbitone buffer pH 8.6, ionic strength 0,06 isrecommended. (Barbitone 1.62g., Sodium diethyl-barbitone 12.38g., Distilled water to 1 liter).
3. Application of sample: Samples at normal protein concentration
should be applied on aproximately 0.8 pI/em. strips.
4. Electrophoretic run: 0.8 mA/cm. strips should be applied whenusing current constant power stabilizer until an adequate separation of
3.7 em. length is produced (ca. 33 minutes at 28°C.).
5. Staining: The strips are treated with 0.8 per cent Ponceau 3R solution of 6 per cent aqueous trichloracetic acid for abuot 60 seconds.
6. Washing: Continue washing with 1 per cent aqueous acetic aciduntil the background is quite white. This takes only 5-10 minutes.
7. Densitometry: Cleared strips by paraffine oil are measured withdensitometer using 0.5 X 8 mm. slit adapter with 500 mp wavelength.
8. Elution method: Immediately after blotting off the washing solution,
the wet strip is cut in separate portions. Each part is treated with
N/100 NaOH solution separately for 5 minutes under constant stirring andthe extracted Ponceau 3R is estimated in the usual manner by colorimeterwith 500 mp wavelength. White part of the strip of 1 em. length, usuallyon the cathode side of r-globulin fraction, is utilized for control.
Standard error of this technique and normal values of Japanese sera on
Separax are given in Tables 1 and 2.

BOOKS AND PAPERS PUBLISHED IN 1965 BY STAFF MEMBERS

135

BOOKS AND PAPERS PUBLISHED IN 1965
BY STAFF MEMBERS
CASPARI, E. and S. NAWA, 1965. A method to demonstrate transformation in
Ephestia. Zeit. Naturforschung 20b: 281-284.
CROW, J. F. and M. KIMURA, 1965. Evolution in sexual and asexual populations.
Amer. Nat. 99: 439-450.
DAVE, M. J" N. TAKAGI, H. OISHI and Y. KIKUCHI, 1965. Chromosome studies on
the hare and the rabbit. Proc. Japan Acad. 41: 244-248.
ENOMOTO, M., 1965. Slow motile mutant in Salmonella typhimurium. J. Bacteriol.
90: 1696-1702.
FUJII, T., 1965. Effects of 14 MeV neutrons in heterozygotic einkorn wheat. Jap.
J. Genet. 40: 209-218.
FUJII, T., 1965. Effects of UV-rays on Arabidopsis seedling. Arabidopsis Research147-151.
FUJII, T., 1965. Development of mutated cells in wheat embryo. Seiken Zih6 17:
39-43.
HIRAIZUMI, Y. and K. NAKAZIMA, 1965. SD in a natural population of Drosophilamelanogaster in Japan. D.I.S. 40: 72.
HIYAMA, Y., Y. TAZIMA, Y. MIYAKE and T. TOYODA, 1965. Problems in the studiesof radiation-hazard (in Japanese). Kagaku 35: 298-308.
IINO, T., 1965. Nucleic acid and gene (in Japanese). J. Crin. Sci. 1: 1410-1418.
IINO, T., 1965. Structure of gene (in Japanese). Genetic Information I. Yoshioka
Shoten, Kyoto, pp. 319-377.
IKENAGA, M. and S. KONDO, 1965. Comparative studies of mutation frequencies
induced by 32p treatment and r-irradiation in the male silkworm. Mutation
Research 2: 534-543.
KATAYAMA, T. C., 1965. Photoperiodism in rice (in Japanese). Control in Biology
2: 75-87.
KATAYAMA, T. C., 1965. Differentiation of photoperiodic sensitivity in the genus
Oryza (in Japanese with English summary). Jap. J. Trop. Agric. 8: 126-131.
KAWAHARA, T. and Y. ITO, 1965. Genetic analysis of growth of early chickembryos (in Japanese with English summary). Proc. Commemo. 15th Yr. Found.,Lab. Anim. Breed., Tokyo Univ. of Agric. pp. 66-71.
KAWAHARA, T. and M. NAKAMURA, 1965. Effect of heterosis and sex-linkage on
sexual maturity in the domestic fowl (in Japanese with English summary).
Proc. Commemo. 15th Yr. Found., Lab. Anim. Breed., Tokyo Univ. of Agric.
pp. 75-81.
KIHARA, H., 1965. The origin of wheat in the light of comparative genetics. Jap.
J. Genet. 40: 45-54.
KIHARA, H., 1965. Stoneless pomegranates from Pakistan and Afghanistan. Resultsof the Kyoto University Scientific Expedition to the Karakoram and Hindukush,
1955, I: 287-289.
KIHARA, H., K. YAMASHITA and M. TANAKA, 1965. Morphological, physiological.

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
genetical and cytological studies in Aegilops and Triticum collected from
Pakistan, Afghanistan and Iran. Results of the Kyoto University Scientific
Expedition to the Karakoram and Hindukush, 1955, I: 1-118.
KIHARA, H. and K. TSUNEWAKI, 1965. How crop plants expand the area of cultivation? Seiken Ziho 17: 1-10.
KIKUCHI, Y. and A. A. SANDBERG, 1965. Chronology and Pattern of human chromosome replication. II. Autoradiographic behavior of various Y and X chromosomes. J. Nat. Cancer Inst. 34: 795-813.
KIMURA, M., 1965. Attainment of quasi linkage equilibrium when gene frequencies
are changing by natural selection. Genetics 52: 875-890.
KIMURA, M., 1965. Some recent advances in the theory of population genetics.
Jap. Jour. Human Genet. 10: 43-48.
KOBAYASHI, S. and B. SAKAGUCHI, 1965. Morphological observations of "sex-ratio"
agents of D. willistoni and D. nebulosa. D.1.S. 40: 53.
KURITA, Y., T. H. YOSIDA and K. MORIWAKI, 1965. Nonrandomness in the distribution of chromosome aberrations induced by a radiomimetic chemical, 4-nitroquinoline-I-oxide, in tumor cells. Jap. J. Genet. 40: 365-376.
MAKINODAN, T., 1. HOPPE, T. SADO, E. E. CAPALBO and M. R. LEONARD, 1965. The
suppressive effect of supraoptimum doses of antigen on the secondary antibodyforming response of spleen cells cultured in cell-impermeable diffusion chambers.
J. Immunol. 95: 466-479.
MATSUMURA, S., 1965. Relation between radiation effects and dose rates of X- andT-rays in cereals. Mechanisms of the Dose Rate Effect of Radiation at the
Genetic and Cellular Levels. Jap. J. Genet. 40 (Suppl.): 1-11.
MATSUMURA, S. and T. FUJII, 1965. Radiosensitivity in plants. VII. Relation
between polyploidy and radiosensitivity under chronic condition. S~iken Ziho
17: 17-23.
MATSUMURA, S. and T. MABUCHI, 1965. Differencies in effects of r-rays and fastneutrons from Po-Be source on paddy rice. Seiken Ziho 17: 37-45.
MATSUMURA, S. and T. OHTA, 1965. Beets collected by KUSE, 1955. Results of the
Kyoto University Scientific Expedition to the Karakoram and Hindukush, 1955,
I: 26-29.
MATSUNAGA, E., 1965. Measures affecting population trends and possible genetic
consequences. United Nations World Population Conference.
MATSUNAGA, E., T. SHINODA and Y. HANDA, 1965. A genetic study of the quantitative variation in erythrocyte glucose-6-phosphate dehydrogenase activity ofapparently healthy Japanese. Jap. Jour. Human Genet. 10: 1-12.
MATSUNAGA, E., 1965. Biology of sexual differences in man (in Japanese). Popular
Medicine 4: 44-48.
MITANI, M. and T. IrNO, 1965. Electron microscopy of bundled flagella of the curlymutant of Salmonella abortivoequina. J. Bacteriol. 90: 1096-1101.
MUKAI, T., 1965. Position effect of spontaneous mutant polygenes controlling
viability in D. melanogaster. D.I.S. 40: 53.
MUKAI, T., 1965. Probable factors suppressing the manifestation of overdominancein natural population of Drosophila melanogaster. Genetics 52: 460-461.

BOOKS AND PAPERS PUBLISHED IN 1965 BY STAFF MEMBERS
MUKAI, T., 1965.

137

Polygenic mutations affecting quantitative characters of Drosophilamelanogaster. Gamma Field Symposia 3: 13-29.
MUKAI, T., S. CHIGUSA and I. YOSHIKAWA, 1965. The genetic structure of natural
populations of Drosophila melanogaster. III. Dominance effect of spontaneous
mutant polygenes controlling viability in heterozygous genetic backgrounds.
Genetics 52: 493-501.
MURAKAMI, A., S. KONDO and Y. TAZIMA, 1965. Comparison of fission neutrons andr-rays in respect of their efficiency in inducing mutations in silkworm gonia.
Jap. J. Genet. 40: 113-124.
OGAWA, Y., 1965. Growth, differentiation and regeneration, XLI. Radiosensitivityof muscle formation in Triturus embryos (in Japanese). Med. and BioI. 70:
28-31.
OGAWA, Y., 1965. The cellulose acetate electrophoretic technique, I. Cellulose
acetate strips and Ponceau 3R staining (in Japanese). Med. and BioI. 70: 46-49.
OGAWA, Y., 1965. The cellulose acetate electrophoretic technique, II. Ponceau 3R
staining of serum proteins on Oxid strips (in Japanese). Med. and BioI. 70:
58-61.
OGAWA, Y., 1965. Cellulose acetate electrophoresis and immunodiffusion techniques(in Japanese). Metabolism and Diasease 2: 514-532.
OGAWA, Y., 1965. Separax, a new Japanese cellulose acetate strip for electrophoresis(in Japanese). Physico-Chemical BioI. 11: 31.
OGAWA, Y., 1965. On the bitter taste response to phenyl-thio-carbamide (PTC) (inJapanese). The heredity 19: 12-15.
OGAWA, Y., 1965. Development of skeletal muscle proteins during early embryonal
stages (in Japanese). Acta Anatomica Nipponica 40: 396-398.
OKA, H. 1., 1965. Some considerations on gem- mutations and the genetic background.
Gamma Field Symposia 3: 1-11.
OKA, H.1., 1965. "Rice culture in West Africa" (in Japanese). Jap. Jour. Trop.
Agr. 9: 10-14.
OKA, H.1., 1965. "Origin of cultivated types and evolution of cultivated varieties
in rice" (in Japanese). Recent Advances in Breeding Science 6: 3-7.
OISHI, H. and C. M. Pomerat, 1964. Chromosomal studies on human leucocytes
following treatment with radioactive iodine in vivo and in vitro. Symp. Intern.
Soc. Cell BioI. 3: 137-154.
OSHIMA, C., 1965. Persistence of some recessive lethal genes in natural populationsof Drosophila melanogaster. Proc. Mendel Memorial Symposium in Prague, 41-48.
OSHIMA, C. and T. K. WATANABE, 1965. Persistence of some recessive lethal genes
in natural populations of D. melanogaster. D.I.S. 41: 103-105.
OSHIMA, C. and T. WATANABE, 1965. The effect of insecticide selection on experimental populations of Drosophila pseudoobscura. D.I.S. 41: 140-141.
SAKAGUCHI, B., K. OISHI and S. KOBAYASHI, 1965. Interference between" Sex-ratio"
agents of Drosophila willistoni and Drosophila nebulosa. Science 147: 160-162.
SAKAI, K. 1. 1965. Contributions to the problem of species colonization from theviewpoint of competition and migration. The Genetics of Colonizing Species.
Academic Press, N. Y., pp. 215-241.

138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 16
SAKAI, K. 1. 1965. Developmental genetics of quantitative characters (in Japanese).
Recent Advances in Breeding Sciences. Jap. Soc. Breed. pp. 62-69.
SAKAI, K. 1. and Y. SHIMAMOTO, 1965. A developmental-genetic study on panicle
characters in rice, Oryza sativa L. Genet. Res., Camb. 6: 93-103.
SAKAI, K. 1. and Y. SHIMAMOTO, 1965. Developmental instability in leaves and flowersof Nicotiana tabacum. Genetics 51: 801-813.
SAKAMOTO, S. and M. MURAMATSU, 1965. Gramineae collected by the KUSE (1955)
from Pakistan, Afghanistan and Iran. W.1.S. Nos. 19, 20: 24-28.
SAKAMOTO, S. and M. MURAMATSU, 1965. Morphological and cytological studies on
various species of Gramineae collected in Pakistan, Afghanistan and Iran.
Results of the Kyoto University Scientific Expedition to the Karakoram and
Hindukush, 1955, I: 119-140.
SAKURAI, S. and M. TSUJITA, 1965. Genetical and biochemical studies of thechromogranules in larval hypodermis of the silkworm, Bombyx mori. (llI).
Lipid contents in the chromogranules. J. Seric. Sci. Japan 34: 230-234.
SHINODA, T., 1965. The role of amino groups in the formation of hemoglobinhaptoglobin complex. J. Biochem. 57: 100-102.
SHINODA, T., 1965. The apparent high reactivity of some amino groups of native
hemoglobin. Bioch. et Bioph. Acta 97: 382-384.
SUZUKI, H., 1965. Synthesis of Ribosome (in Japanese). Genetic Information 11.
Yoshioka Shoten, Kyoto, pp. 319-377.
TAZIMA, Y., 1965. Mechanisms controlling two types of dose-rate dependence ofradiation-induced mutation frequencies in silkworm gonia. Jap. J. Genet. 40(Supp!.): 68-82.
TAZIMA, Y., 1965. From the conference on mechanisms of dose rate effect of radiation at genetic and cellular levels (in Japanese). Atoms in Japan 9: 8-10.
TAKENAKA, Y. and Y. YONEDA, 1965. Hereditary tumor in Japanese morning glory(in Japanese). Jap. J. Genet. 40: 141-145.
TAKENAKA, Y. and Y. YONEDA, 1965. Tumor formation in interspecific hybrids ofNicotiana, especially its relation to wounding (in Japanese). Jap. J. Genet. 40:
227-232.
TAKENAKA, Y., 1965. Studies on the Genus Prunus. II. The origin of Prunus
yedoensis, Continued (in Japanese). Bota. Mag. Tokyo 78: 310-331.
TSAI, K. H. and H.1. OKA, 1965. Genetic studies of yielding capacity and adaptability in crop plants, 1. Characters of isogenic lines in rice. Bot. Bull. Acad.
Sinica 6: 19-31.
TSUJITA, M. and S. SAKURAI, 1965. Purification of the three specific soluble chromoproteins from chromogranules in hypodermal cells of the silkworm larva. Proc.
Japan Acad. 41: 225-229.
TSUJITA, 1M. and S. SAKURAI, 1965. Amino acid analysis of three chromoproteins
purified from chromogranules in hypodermal cells of the silkworm larva. Proc.
Japan Acad. 41: 230-235.
TSUNEWAKI, K., 1965. The awn inhibitor in Redman wheat. Seiken ZihiJ 17: 4345.
TUTIKAWA, K. and A. AKAHORI, 1965. Strain differences of susceptibility to induced

BOOKS AND PAPERS PUBLISHED IN 1965 BY STAFF MEMBERS

139

congenital anomalies in mice (in Japanese). Official Journal of the Congenital
Anomalies Research Association of Japan 5: 4-12.
WEISS, G. H. and M. KIMURA, 1965. A mathematical analysis of the stepping stone
model of genetic correlation. Jour. Applied Probability 2: 129-149.
YOSIDA, T. H., 1964. Chromosomal alteration and the development of tumors. XI.
Karyological observations on the Misima subline of hyperdiploid Ehrlich tumor
and its three clones. Cytologia 29: 359-369.
YOSIDA, T. H., A. NAKAMURA and T. FUKAYA, 1965. Chromosomal polymorphism in
Rattu8 rattu8 L. collected in Kusudomari and Misima. Chromosoma (Berl.) 16:
70-78.
YOSIDA, T. H. and K. AMANO, 1965. Autosomal polymorphism in laboratory bred
and wild Norway rats, Rattu8 norvegicu8, found in Misima. Chromosoma (Berl.)
16: 658-667.
YOSIDA, T. H., Y. KURITA and K. MORIWAKI, 1965. Chromosomal aberrations in
YOSIDA sarcoma cells treated with 4-nitroquinoline-1-oxide. Gann 56: 523-528.

ACKNOWLEDGMENT
The editor wishes to express his sincere gratitude to Dr. F. A. LILIENFELD forher kindness in reading the original manuscripts. (Saburo NAWA)

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