NATIONAL INSTITUT~ OF G~N~TICS
JAPAN

ANNUAL RI:PORT
No. 131962

Publis1red by

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

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Annual Reportof theNational Institute of Genetics
No. 13, 1962

Published by

The National Institute of Genetics, Japan
1963

CONTENTS
General statement
Abstracts of diary for 1962........................................
Staff
Council............................................................
Projects of research for 1962
Foreign visitors in 1962............................................
Researches carried out in 1962

Genetics, cytology and biochemistry of animals
1. Pre-adult viability of heterozygous flies for lethal chromosomes (1) (OSHIMA)........................................
2. Pre-adult viability of heterozygous flies for lethal chromosomes (2) (OSHIMA and MINAMORI) . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Persistence of some recessive lethal genes in a natural population of Drosophila melanogaster (OSHIMA)
4. Pre-adult viability of heterozygous flies for semi-lethal chromosomes (OSHIMA).........................................
5. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 1. Method and representation ofcondensed data (MUKAI, CHIGUSA and YOSHIKAWA)
6. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 2. Variance increasing rate andmean decreasing rate (MUKAI).............................
7. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 3. Estimation of polygenic viability mutation rate (MUKAI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Studies on spontaneous polygenic mutations in Drosophilamelanogaster 4. Dominance in polygenic viability mutations(MUKAI, CHIGUSA and YOSHIKAWA)
9. Studies On spontaneous polygenic viability mutations in
Drosophila melanogaster 5. Polygenic mutations and genetic
structure of natural populations (MUKAI)
10. Persistence of two recessive genes in experimental populations of Drosophila melanogaster (CHIGUSA and MUKAI).....
11. Syntheses of Drosophila eye pigments (NAWA and FORREST)..
12. Melamine action in phenocopic pigment elimination fromhypodermal cells of the silkworm (TSUJITA and SAKURAI) . .

1
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6
9
11

A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.

26.

27.
28.

29.

Specific protein combining with yellow pigments (dihydropterin) in the silkworm (TSUJITA and SAKURAI)
Conversion from guanine to peteridine in the silkworm (SAKAGUCHI)
Analysis of marker genes in inbred strains of albino mice
(KURITA and SAKAKIBARA)
Genetical studies of post-axial polydactyly in the house mouse(NAKAMURA, SAKAMOTO and MORIWAKI)
Cytological study of a transplantable ascites plasma-cell
neoplasm in mice (KURITA and YOSIDA) . . . . . . . . . . . . . . . . . . . .
A characteristic chromosome found in the karyotype analysisof the mouse (NAKAMURA and TONOMURA) . . . . . . . . . . . . . . . . . .
Cytological studies of lymphogenic and hematogenic metastases in MH-134 mouse hepatoma (INUI)...................
Chromatographic analysis of coat coloring substance in mice
(NAKAMURA)
Biochemical changes in the skin of rhino mouse preceding
the hereditary hair loss (MORIWAKI)
Viscosity changes in the deoxyribonucleoprotein of L-strain
cells following treatment with a base analogue (MORIWAKI)
Endurance of one day old chicks under starvation (KAWAHARA)
Genetic studies on body weight at different stages of growthin the domestic fowl (KAWAHARA)
Use of rabbit anti-serum for muscle proteins of fowls forserological determination of homologous tissue proteins of
Triturus (OGAWA and KAWAHARA)
Effects of p-hydroxy-benzyl-methyl-ether, a component of
Citrullus colocynthis, on growth and differentiation of Triturus embryo (OGAWA and WATANABE)
Comparison of free amino acids among eye-color mutants of
Drosophila melanogaster (SHINODA) . . . . . . . . . . . . . . . . . . . . . . . . .
Spectrophotometric determination of trinitrophenyl derivatives of amino acids and peptides resolved by paper electrophoresis and chromatography (SHINODA) . . . . . . . . . . . . . . . . . . .
Paper electrophoretic heterogeneity of serum albumin (SRINODA)

B. Genetics, cytology and biochemistry of plants
30. Genome manifestation of wheat in Aegilops cytoplasm (KIHARA)
31.

Increased occurrence of haploids and twin seedlings due toan alien cytoplasm (KIHARA and TSUNEWAKI)...............

28
29
30
31
32
32
33
34
35
36
37
38

39

40
42

43
44

45
45

CONTENTS

iii

32. Comparative gene analysis of common wheat and its ancestral
species I. Progressive necrosis (TSUNEWAKI and KIHARA) .•33. Transmission of the monosomic condition through female

47

gametes in a common wheat, Chinese Spring (TSUNEWAKI).
Chromosome number of Gramineae species collected in Pakistan, Afghanistan and Iran (SAKAMOTO and MURAMATSU) ...
35. Reciprocal translocations in Emmer wheat (NISHIKAWA)......
36. Production of polyploid wheat by nitrous oxide, II. N 20treatment during fertilization with reference to culture
media (TSUNEWAKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37. Photoperiodic responses of Oryza species V (KATAyAMA).....
38. Developmental instability in tobacco-plants (SAKAI and SHlMAMOTO)
39. A method of estimation of genetic parameters in forest trees
without raising progeny (SAKAI and HATAKEYAMA)
40. Cytoplasmic effect on the inheritance of seed weight in
Pharbitis Nil (SAKAI and SUZUKI)
41. Effect of genotypes and cultural conditions on quantitative
characters of tobacco varieties (SAKAI and SHIMAMOTO).....
42. Studies on developmental instability of quantitative charactersin X-rayed progenies of rice (SAKAI and SUZUKI)...........
43. Intra-clonal variation of pathogenicity in Piricularia oryzae(GOTO and SAKAI)... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
44. Studies on blast-disease susceptibility of wild and cultivatedgroups of rice (GOTO and SAKAI)
45. Natural selection experiment in populations of wild rice,
Oryza perennis (OKA and CHANG)
46. Some considerations on the evolutionary dynamics of cultivated rice (HINATA and OKA)
47. F 2 segregation ratios in a hybrid between tetraploid Oryza
sativa and glaberrima (MORISHIMA and OKA)................
48. Comparison of evolution modes of cultivated forms derived
from two wild rice species, Oryza breviligulata and O.
perennis (MORISHIMA, HINATA and OKA) . . . . . . . . . . . . . . . . . . . . .
49. Cytogenetic studies in the genus Nicotiana XV. Reduction
divisions in three interspecific hybrids and one amphidiploid(TAKENAKA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50. Tumorous hybrids in Nicotiana (TAKENAKA and YONEDA) ....
51. Effects of extracts from Aralia elata Seem. and Artemisia
vulgaris L. var. indica Maxim. on cell division in living
plant tissue and growth of rat tumor (TAKENAKA and OGAWA)

47

34.

48
50

51
51
52

53
54
55
56
57
58
59
60
60

61

61
63

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

52.
53.
54.
55.
56.

Experiments with carnations (LILIENFELD and SUZUKI).
Comparison of flower color inheritance in pansy and morning
glory (ENDO)"" . . . .. .. . .
Mutations of dominant green to recessive chlorina in heterozygous plants (FuJII)......................................
Effect of nitrous oxide on germinating seeds of einkorn wheat(FUJII)
Cytological observations on chemically induced male sterility
in sugar beets (OHTA and MATSUMURA).....................

C. Mathematical genetics
57. On the probability of joint fixation of mutant genes (KIMURA)

65
68
69
70
71

71

D. Genetics and biochemistry of microorganisms
58.
59.
60.
61.
62.
63.
64.

Phase specific regulator of flagellin genes (HI and Hz) inSalmonella (IINO) .... " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Further studies on non-flagellated mutants of Salmonella(IINO and ENOMOTO)........................................
Effect of phenol on flagellation of Salmonella cells (lINO andMITANI)Grouping of paralyzed mutants in Salmonella (ENOMOTO) . . . .
Chromatography and electrophoresis of flagellin of Salmonellaabortus-equi (ENOMOTO and IINo)
Further studies on the nuclei of yeasts (YONEDA). . . . . . . . . . .
Temperature dependence of nitrous acid effects on phage T4(ISHIWA, YAN and KONDO)..................................

E. Radiation genetics in animals
65. Effect of radiation-induced mutations on fitness of Drosophilamelanogaster (HIRAIZUMI)...................................
66. Heterozygous effects of radiation-induced mutations on viability in homozygous and heterozygous genetic backgrounds
in Drosophila melanogaster (MUKAI, CHIGUSA and YOSHIKAWA)
67. Radiation-induced mutation rates of polygenes controllingthe sternopleural bristle number in Drosophila melanogaster(MUKAI, YOSHIKAWA and CHIGUSA)
68. Radiation sensitivity of infectious particles responsible for"sex-ratio" condition in Drosophila willistoni (SAKAGUCHI
and KONDO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
69. Revised interpretation of the non-recovery type dose-rate
dependence of radiation induced mutation rates of silkworm
gonia (TAZIMA and KONDO)

72
73
74
75
76
77
78

78

79

81

82

85

CONTENTS
70.

71.

72.

73.
74.
75.

Differential radiation-sensitivity of germ cells as a possible
interpretation of sex difference in dose-rate dependence of
induced mutation rates in the silkworm (TAZIMA and KONDO)
The increase in induced mutation frequency after fractionated irradiation of gonial cells of the silkworm (TAZIMA
and MURAKAMI)............................................
Modification of X-ray-induced mutation rate in the silkworm
by pre- or post-irradiation treatment with halogenated baseanalogues (MURAKAMI and T AZIMA) . . . . . . . . . . . . . . . . . . . . . . . . .
Cytological estimation of LD 50 X-ray dose for secondary
spermatogonia of the silkworm (SADO)Inactivation of Salmonella by 32p disintegration combinedwith UV (ISHIWA, YAN and KONDO)
Protective effect of sodium glucuronate on the differentiationof muscle tissue inhibited by X-irradiation (OGAWA).......

v

86

87

89
91
92
93

F. Radiation genetics in plants
76. Radiation-induced variegations in einkorn wheat and theirinheritance (MATSUMURA)
96
77. Effects of chronic irradiation on einkorn wheat (MATSUMURA
and FUJII).................................................
96
78. Relation of chlorophyll mutations to dose rates in r-irradiatedeinkorn wheat (MATSUMURA and MABUCHI)
97
79. Relation of radiation effects to dose rates of r-radiation inrice (MATSUMURA and MABUCHI) ..........................••
99
80. A red-stem mutant in einkorn wheat induced by X-rays(FUJII)100
81. Multi-subunit target model for RBE versus LET relationshipfor radiation damage to higher forms (KONDO)............ 101
G. Human genetics
82. Embryonal sex ratio in the Japanese determined by the sexchromatin test (MATSUNAGA, TONOMURA, INUI and HONDA) ..
83. Studies On selection in ABO blood groups (MATSUNAGA, HIRAIZUMI, FURUSHO and IZUMIYAMA).............................
84. Spontaneous and induced chromatid aberrations in humancells cultivated in vitro (TONOMURA).......................
85. Sex-chromosome conditions in four cases of true hermaphroditism (TONOMURA and HONDA)
86. Character of modified human hemoglobin (SHINODA) . . . . . . . . .
Books and papers published in 1962 by staff members.............

102
103
106

107
108
110

GENERAL STATEMENTAs shown in the photograph given above, two thirds of our mainbuilding were finished in 1962. The rest will be completed in the fallof 1963. Still we will need to add a few buildings, namely, an auditorium, an exhibit room, seed storage rooms and a dormitory for visiting
scientists.
To erect the new concrete building has taken four years. It was hightime to build it since the old one was partly destroyed by storms andwas liable to catch fire.
The recent advances of genetics in many fields make it necessary for
our Institute to accommodate to the diverse developments. We are planning to establish four new departments; namely, population genetics,
molecular genetics, biophysics and a research center for fine structure
investigations. However, we have to admit that the Institute should
keep its moderate size and not become too large. Therefore it is necessary to reorganize old departments hitherto established to fit our needs.
To keep a continuous flow of young people, it is desirable to havepost-graduate students who will be trained in our Institute. For thispurpose, the Institute should be converted into a research center ofgenetic sciences affiliated with all national universities.
It was customary to list on this page the past achievements. However as our building is near completion the future prospects have to bementioned.

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

ABSTRACT OF DIARY FOR 1962
Jan. 13. 100th meeting of Misima Geneticists' Club.
Feb. 14. 101st meeting of Misima Geneticists' Club.
Feb. 24. 102nd meeting of Misima Geneticists' Club.
Mar. 23. 103rd meeting of Misima Geneticists' Club.
Apr. 10. 43rd Biological Symposia.
Apr. 27. 104th meeting of Misima Geneticists' Club.
May 11. 44th Biological Symposia.
May 29. 105th meeting of Misima Geneticists' Club.
June 16. 106th meeting of Misima Geneticists' Club.
June 22. 45th Biological Symposia.
June 29. 107th meeting of Misima Geneticists' Club.
July 19-21. The 5th Summer Seminar on Genetics for High School
Teachers of Biology.
July 26. 46th Biological Symposia.
Aug. 6. 47th Biological Symposia.
Sept. 25. 108th meeting of Misima Geneticists' Club.
Oct. 17-19. The 34th Annual Meeting of the Genetics Society of Japan.
Nov. 9. 48th Biological Symposia.
Nov. 20. l09th meeting of Misima Geneticists' Club.
Dec. 21. 1l0th meeting of Misima Geneticists' Club.

STAFF
Director
Hitoshi KIHARA, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Members
Department of Morphological Genetics
Yataro TAzIMA, D. Ag., Head of Department
1st
Lab.

Yataro TAzIMA, D. Ag., Head of Lab.
Kimiharu ONIMARU, Akio MURAKAMI*

2nd
Lab.

Bungo SAKAGUCHI, D. Ag., Head of Lab.
Toshihiko SADO, D. Ag. (in U.S.A.)* Research member under grant from the Toyo Rayon Foundation

STAFF

3

Department of Cytogenetics
Yo TAKENAKA, D. Sc., Head of Department
1st
Lab.

Tosihide H. YOSIDA, D. Sc., Head of Lab. (in U.S.A.)
Kazuo MORIWAKI, D. Sc., Akira NAKAMURA*

2nd
Lab.

Yo TAKENAKA, D. Sc., Head of Lab.
Yoshiaki YONEDA, D. Sc.

* Visiting researcher
Department of Physiological Genetics
Chozo OSHIMA, D. Sc., Head of Department
1st
Lab.

Chozo OSHIMA, D. Sc., Head of Lab.
Toshifumi TAIRA, D. Sc. (in U.S.A.) Sumio MINAMORI, D. Sc.**

2nd
Lab.

Hitoshi KIHARA, D. Sc., Head of Lab.
Koichiro TSUNEWAKI, Ph. D., Sadao SAKAMOTO, Tadao C.
KATAYAMA*, Kozo NISHIKAWA*** Research member under grant from the Rockefeller Foundation** Visiting researcher

Department of Biochemical Genetics
Mitsuo TSUJITA, D. Ag., Head of Department
1st
Lab.

Saburo NAWA, D. Sc., Head of Lab. (in U.S.A.)

2nd
Lab.

Yoshito OGAWA, M. D., Head of Lab.
Toru ENDO, D. Ag.

3rd
Lab.

Mitsuo TSUJITA, D. Ag., Head of Lab.
Susumu SAKURAI

Department of Applied Genetics
Kan·Ichi SAKAI, D. Ag., Head of Department
1st
Lab.

Yukio YAMADA, D. Ag., Head of Lab. (in U.S.A.)
Takatada KAWAHARA, D. Ag.

2nd
Lab.

Kan·Ichi SAKAI, D. Ag., Head of Lab.
Shinya lYAMA, D. Ag. (in U.S.A.), Akira MIYAZAWA,
Akio SUSUKI, Iwasaburo GOTO*, Suekichi HATAKEYAMA*,
Yoshiya SHIMAMOTO*

3rd
Lab.

Hiko·Ichi OKA, D. Ag., Head of Lab.
Hiroko MORISHIMA* Visiting researcher

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

Department of Induced Mutation
Seiji MATSUMURA, D. Ag., Head of Department
1st
Lab.

Seiji MATSUMURA, D. Ag., Head of Lab.
Kiyosi TUTIKAWA, Terumi MUKAI, Ph. D., Sadao CHIGUSA*,
Isao YOSHIKAWA*

2nd
Lab.

Seiji MATSUMURA, D. Ag., Head of Lab.
Taro FUJII, D. Ag., Tomoo MABUCHI**

3rd
Lab.

Sohei KONDO, D. Sc., Head of Lab.
Hiromi ISHIWA, Yonhoi YAN**** Research member under grants from the N.LH. (U.S.A.)** Research member under grant from the Rockefeller Foundation*** Research member under grant from the Toyo Rayon Foundation
Department of Human Genetics
Ei MATSUNAGA, M. D., D. Sc., Head of Department
1st
Lab.

Ei MATSUNAGA, M. D., D. Sc., Head of Lab.
Akira TONOMURA, D. Sc., Tomotaka SHINODA

2nd
Lab.

Motoo KIMURA, Ph. D., D. Sc., Head of Lab. (in U.S.A.)
Yuichiro HIRAIZUMI, D. Sc.

Department of Microbial Genetics
Hitoshi KIHARA, D. Sc., Head of Department
1st
Lab.

I Tetsuo
IINO, Ph. D., D. Sc., Head of Lab.
Masatoshi ENOMOTO
Part-time Staff and Research Associates

Yoshinari KUWADA, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Kan OGUMA, D. Ag., Ex-Director, Emeritus Professor of Hokkaido University
Yoshimaro TANAKA, D. Ag., D., Sc., M.l.A., Emeritus Professor of
Kyushu University
Taku KOMAI, D. Sc., M.J.A., Emeritus Professor of Kyoto University
Flora A. LILIENFELD, Ph. D.
Tsuguo TATEOKA, D. Sc.

STAFF

5

Shiro SHIRATO, M. D., Izu Teishin Hospital

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

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

3

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

COUNCIL
YO K. OKADA, Director of National Science Museum, Chairman
Daigoro MORIWAKI, Professor of Tokyo Metropolitan University, Vice
ChairmanKan OGUMA, Ex-Director, Emeritus Professor of Hokkaido University
Masataka OMASA, Professor of Tokyo University
Seizo KATSUNUMA, Ex-President of Nagoya University
Minoru T ACHI, Director of Institute of Population Problems
Yakichi NOGUCHI, Professor of Tokyo University
Akira KAWADA, Director of National Institute of Agricultural Science
Toshio SAITO, Governor of Sizuoka Prefecture
Taiji SAKATA, President of Japan Monopoly Corporation
Takeo SAKATA, President of T. Sakata Company
Hiroshi TAMIYA, Director of Institute of Applied Microbiology, Univ. ofTokyo
Tanemoto FURUHATA, Director of Scientific Research Institute of Police
Bungo WADA, Professor of Sizuoka University
Kempo TSUKAMOTO, Director of National Institute of Radiological Sciences

6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

PROJECTS OF RESEARCH FOR 1962
Department of Morphological Genetics
Genetics of the silkworm (TAZIMA)
Studies on food preference of the silkworm (TAZIMA)
Chemical mutagenesis in the silkworm (TAZIMA, SADO and MURAKAMI)
Cytological study of silkworm germ cells (SADO)
Studies on dose-rate dependence of radiation-induced mutation rates(TAZIMA and ONIMARU)
RBE of radiations for induced mutation frequency in the silkworm(MURAKAMI)
Hereditary infections in Drosophila (SAKAGUCHI)

Department of Cytogenetics
Cytology and genetics of tumors (YOSIDA, MORIWAKI and KURITA)
Experimental breeding and genetics of mice and rats (YOSIDA, MORIWAKI,
KURITA and NAKAMURA)
Biochemical study on genetical abnormalities of mice (MORIWAKI and
NAKAMURA)
Determination and differentiation of sex in higher plants (TAKENAKA and
YONEDA)
Induction of abnormal mitosis and inhibition of growth by substances
extracted from certain plants (TAKENAKA and OGAWA)
Interspecific hybridization in Nicotiana (TAKENAKA and LILIENFELD)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus yedoensis (TAKENAKA)
Cytogenetics of Oryza species (TAKENAKA and YONEDA)
Comparative observations of chromosomes in haploid plants of Oryza(C. H. Hu and YONEDA)
Cytological studies on the yeast cell (YONEDA)

Department of Physiological Genetics
Genetical studies on insecticide-resistance in Drosophila (OSHIMA)
Physiological studies on eye-pigment formation in Drosophila (TAIRA andOSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila (OSHIMA and MINAMORI)PROJECTS OF RESEARCH FOR 1962

7

The persistence of some natural lethal genes in experimental populations (OSHIMA and FUWA)
Comparative gene analysis with reference to the origin of wheat (KIHARA,TSUNEWAKI and NISHIKAWA)
Nucleus substitution in wheat and related species (KIHARA)
Production of polyploids by N,O treatment (KIHARA and TSUNEWAKI)
Genetic studies of wheat aneuploids (TSUNEWAKI)
Genetic basis of ecological differentiation in Agropyron (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Morphological study of Oryza (KIHARA and KATAYAMA)
Investigation of photoperiodic responses of Oryza species (KATAYAMA)

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

Department of Applied Genetics
Studies on breeding and genetics in poultry (YAMADA and KAWAHARA)
Theoretical studies on plant breeding techniques (SAKAI)Studies on competition and migration in plants and animals (SAKAI, lYAMA
and NARISE)
Genetic studies of alkaloid content in tobacco plants (SAKAI and SHIMAMOTO)
Biometrical study of cytoplasmic inheritance (SAKAI and SUZUKI)
Genetic studies on developmental stability in plants (SAKAI, SUZUKI andSHIMAMOTO)
A statistic-genetical study in forest-trees (SAKAI and HATAKEYAMA)
Statistic-genetical studies in cultivated and wild rice (SAKAI, NARISE andSUZUKI)
Genetic studies on the blast-disease resistance in wild and cultivatedrice (SAKAI and GOTOH)
Comparison of evolutionary mode between series Sativa and series Glaber-

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

rima (MORISHIMA and OKA)
Survey of geographical variation in O. perennis (MORISHlMA, CHANG and
OKA)
Crossing-experiments and sterility of hybrids between wild and cultivatedrice strains (HINATA and OKA)
Studies of intermediate wild-cultivated forms in rice (OKA and CHANG)
Responses to growing conditions of wild and cultivated rice forms (OKAand CHANG)
Evolutionary-genetic studies in Oryza (OKA and MORISHIMA)
Department of Induced Mutation
Radiation genetics of mice (TUTIKAWA)
Population genetics of Drosophila (MUKAI and CHIGUSA)
Studies on the effects of irradiation on populations (MUKAI and YOSHIKAWA)
Estimation of polygenic mutation rates in Drosophila (MUKAI, CHIGUSAand YOSHIKAWA)
Relation between the quality of radiations and mutations (MATSUMURA
and KONDO)
Radiation genetics of cereals (MATSUMURA, FUJII and MABUCHI)
Radiation genetics and its practical application (FuJII and MABUCHI)
Biophysical studies of radiation genetics (KONDO, ISHlWA and Y AN)
Radiation dosimetry (KONDO and ISHlwA)
Male-sterility in sugar beets (MATSUMURA)
Genome analysis of Oryza species (MATSUMURA and MABUCHI)

Department of Human Genetics
Selection in ABO blood groups (MATSUNAGA and HIRAIZUMI)
Genetic studies on sporadic retinoblastoma in Japan (MATSUNAGA)
Studies on dimorphism in human normal ceru men (MATSUNAGA)
Cytogenetics in man (TONOMURA)
Sexual dimorphism in resting nuclei (TONOMURA (TOYOFUKU) and TONOMURA)
Biochemical studies on plasma proteins and haemoglobins (SHINODA)
Studies on individual difference in metabolism (SHINODA)
Theoretical studies in population genetics (KIMURA)
Effects of radiation-induced mutations on fitness (HIRAIZUMI)
Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)
Effect of radiation induced mutations on correlated characters of D.
melanogaster (HIRAIZUMI)FOREIGN VISITORS IN 1962

9

Department of Microbial Genetics
Immunogenetics of Salmonella (IINO and ENOMOTO)
Genetics of multi-drug resistance in bacteria (lINo)
Genetics of motility in bacteria (ENOMOTO)
Genetics of bacteriophages (IINO and SASAKI)FOREIGN VISITORS IN 1962
Feb.

16. S. E1. MOUKHTAR (Gawhary Embassy of the United Arab
Republic)
17. A. H. MOSEMEN and Jean MOSEMEN (The Pockefeller Foundation, New York, U.S.A.)
March 5. J. DRILON and Mercedes L. DRILON (The International Rice
Research Institute, Manila, The Philippines)
10. Edward KUCHLEWSKI (Elizabeth, N. J., U.S.A.)
April
10. Ady Raul da SILVA (Institute Agronomico do SuI Pelota,
Brazil)May
11. Joshua LEDERBERG and E. M. LEDERBERG (Dept. of Genetics,
Medical School, Stanford University, California, U.S.A.)
June
8. L. H. BAKER (Pioneer Hi-Bred Corn Co., Des Moines, Iowa,
U.S.A.)
21. E. R. DEMPSTER (Professor of Genetics, Genetics Department,
Univ. of California, Berkeley, California, U.S.A.)
July
13. H. M. BEACHELL (Rice Breeder, Rice Pasture Experiment
Station, Beaumont, Texas, U.S.A.)
13. Mrs. E. E. BEAcHELL (Iowa Technology, Beaumont, Texas,
U.S.A.)
13. B. R. GALGALI (Plant Breeder, Mysove State, India)
20. Te-Tzu CHANG (International Rke Research Institute, Los
Banos, Laguna, The Philippines)
26. Edward A. ADELBERG (Microbiology Department, Yale University, New Haven, Conn., U.S.A.)
27. Engracio BASIO (Principal Librarian, College of Agriculture
and Central Experiment Station, University of the Philippines, College, Laguna, The Philippines)
Aug.
6. Allen S. Fox (Department of Biochemistry, Michigan State
University, East Lansing, Michigan, U.S.A.)
Sept.
7. Selim NAZIF (Director General, Field Crops Research Div.,
Ministry of Agriculture, Cairo, U.A.R.)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

7. A. ABOUL-SEOUD (Director of Nutrition Section, Animal Pro7.
7.
15.
15.
Oct.

3.
3.
3.
3.
3.
9.

25.

Nov.

9.

duction Dept., Ministry of Agriculture, Cairo, U.A.R.)
I. M. IBRAHIM (Fruit Research Section, Horticultural Dept.,
Ministry of Agriculture, Cairo, U.A.R.)
G. E. DICKERSON (Kimber Farms Inst., Fremont, California,
U.S.A.)S. H. au (International Rice Research Institute, Los Banos.,
Laguna, The Philippines)
Jack A. SOULES (Office of Naval Research, Physics Branch,
Code 421, Washington 25, D.C., U.S.A.)S. S. SAINI (Rice Breeder, Regional Rice Research Station,
Kapurthola Punjab, India)
Fouad NAGUIB (Rice Breeder, Rice Research Station, Department of Agriculture, Giza-Orman, U.A.R.)
Mohammad MOOFIZAD (Rice Research Station, Departmentof Agriculture, Rasht, Iran)
U-Thai WONGVISES (Pimai Rice Experiment Station, Rice
Department, Ministry of Agriculture, Bangkok, Thailand)
Abdel-Wahab MUSTAFA (Rice Breeder, Rice Research Station,
Department of Agriculture, Sakha Experiment Station, Kafre
EL Shiekh, Egypt, U.A.R.)
Mihhail VASILJEVICH ORLOV, peti Efimovich B,OZKKO, Genrikh
Vyacheslanovich MILJINIKOV and Valentin Ivanovich KONOVALOY (Visiting Group of Specialists in Poultry Breeding,
U.S.S.R.)
Otto LAPORTE (Professor of Physics, University of Michigan,
Ann Arbor, Michigan, U.S.A. Currently, Science Attache,
American Embassy, Tokyo)
Arnold H. SPARROW (Brookhaven National Laboratory, Upton,
N.Y., U.S.A.)RESEARCHES CARRIED OUT IN 1962
A. GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF ANIMALS
1.

Pre-adult viability of heterozygous flies for lethal chromosomes (1)1)(By Chozo OSHIMA)

It was shown in the previous annual report (No.3) that the mean
relative viability of natural lethal heterozygotes was significantly lower
than that of normal heterozygotes and that of double lethal heterozygotes
within the same population was rather better than that of single lethal
heterozygotes. To confirm this complementary effect, similar experiments were carried out with ten normal and ten lethal chromosomes
isolated in 1961 from the Hiroshima and the Suyama-Juriki populations
respectively. Both populations are about 700 kilometers apart, and are
Table 1. Relative viabilities of normal, single and double lethaltrans-heterozygotes between the Hiroshima and the SuyamaJuriki populations, in comparison with those of normal
heterozygotes within the populations.
Pooled basis
Chromosome
combination

Line basis

No. ofcounted flies

Relative
viability

No. of lines

Relative
viability+h/+h+s-j/+s-j

15,858
13,9501. 119±O.0186-

45
45

1. 139±O. 0239

+h/+s-j

34,7131.086±0.0166

100

1.105±0.0202+ h/l,-j+s-j!lh

70,388l.083±0.0l1S- *200

1.100±0.O139

lh/ls- j

37,678

1. 117±0.0165] *100

1. 121±0.0179

The viability of Cy/Pm fly=l.OOOO The same for the other experiment.
+ h, lh: normal and lethal chromosomes isolated from the Hiroshima populations
respectively.
+8-j, ls-j: normal and lethal chromosomes isolated from the Suyama andJuriki populations, respectively.
* significance at the 5% level.
1) This work was supported by the United States Public Health Service Grant,
RG 7836.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

assumed to be genetically unrelated. The results are given in Table 1.
The mean viability of single lethal heterozygotes was lower thanthat of normal heterozygotes irrespective of whether any coadaptation
system existed or not. On the other hand, double lethal trans-heterozygotes were significantly superior in their mean viability to single lethal
heterozygotes and similar to normal heterozygotes.
During the last period of the breeding season, the population size isgradually reduced, but this might be not followed by a decrease inrelative frequency of lethals. If so, there may be chances that surviving lethal heterozygotes breed together and double lethal trans-heterozygotes are produced. Heterozygous flies for many lethal genes except
the heterotic ones would be eliminated, but those double lethal transheterozygotes of nearly normal viability would remain alive throughthe dormant season.

2.

Pre-adult viability of heterozygous flies for lethal chromosomes (2)1)(By Chozo OSHIMA and Sumio MINAMORI)

Several chromosomes on which two lethal genes are located, had been
isolated from the Hiroshima populations in early summer of 1961. Theselethal genes were separated by recombination and found again in theautumn lot of the same population. Sixteen normal, six single lethal
and three double lethal chromosomes were used and the viability ofheterozygotes for those chromosomes was estimated. The results are
given in Table 1.
Table 1. Relative viabilities of double lethal cis-heterozygotes in comparison
with those of normal and single lethal heterozygotes in the
Hiroshima populations.
Pooled basis
Chromosome
combination

+/+
+/ l
ll/++

No. ofcounted flies

Relative
viability

28,81625,97023.071

1.096±0.0185 -,1.072+0.0201-

1.148~0.0217-'* **Line basis
No. of lines

Relative
viability

60
60
60

1.093±0.0293-,
1. 171±0.0304- *1. 123±0.0284** significance at the 1% level, * significance at the 5% level.
1) This work was supported by the United States Public Health Service Grant
RG 7836.

RESEARCHES CARRIED OUT IN 1962

13

The mean viability of single lethal heterozygotes was a little less thanthat of normal heterozygotes, but double lethal cis-heterozygotes weresignificantly superior in their mean viability to single lethal heterozygotes
and even normal heterozygotes.
In the early period of the breeding season, double lethal cis-heterozygotes which might have been originated from double lethal transheterozygotes by recombination were actually found by Minamori in anatural population in Hiroshima and by Karlik and Sperlich (1962) in anatural population on Lipari Island.
The complementary effects on viability of double lethal heterozygotes
might be understood as an interaction between two loci carrying lethal
genes in the genetic background which was subjected to natural selection.

3.

Persistence of some recessive lethal genes in a natural populationof Drosophila melanogaster. 1 )(By Chozo OSHIMA)

Lethal second chromosomes have been isolated every year from 1959 fromthe Suyama-Juriki natural populations in Shizuoka prefecture. Thosechromosomes have been maintained by Cy balanced system in the following generations. Allelism test was performed between old and newlethal genes isolated during two successive years. The results showedthat the two lethal genes have been maintained at least for two years
and other two lethal genes have been retained at least for one year in
the same population. The former two lethal genes supposed to havehigh fitness in heterozygous state in the natural population were foundto be maintained with much higher frequencies for about forty generations in experimental populations (see previous annual report No.1),
as compared with the theoretical frequencies. The obtained results
show that the heterozygous flies for those lethal genes were heterotic
in both natural and experimental populations.

4.

Pre-adult viability of heterozygous flies for semi-lethal chromosomel l(By Chozo OSHIMA)

Fifteen semi-lethal and sixteen normal chromosomes isolated from the1) This work was supported by the United States Public Health Service Grant,
RG 7836.
2) This work was supported by the United States Public Health Service Grant,
RG 7836.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13
Table 1. Relative viabilities of normal, single and double semi-lethaltrans-heterozygotes within the Suyama-]uriki populations.
Pooled basis

Chromosome
combination

Line basis--

No. ofcounted flies

Relative
viability

+1+
sll+
sllsl

53,851
102,336
41,0691.160+0.0144
1. 166±0.0105-/
1. 128±0.0160-*- *

+1+
sll+
sllsl

55,30260,991
48,6250.999±0.0121
0.983+0.0114-

Relative
viability

No. of lines

-I

_.

-I

120
240
102

1.177 ±0.0184
1. 186±0.0137
1. 147±0.0214

110
121
106

1.006±0.0136
0.982+0.0134O.
0124J*- **

-I

~--

0.934~0.0122J** **942~0.

sl: semi-lethal chromosome* significance at the 5% level,
Table 2.

Relative viability of single semi-lethal heterozygotes.
First expo

Second expo

15
16

11
11

+1+
sll+

1. 160±0.0144
1. 166±0.0105

0.999±0.0121
0.983±0.0114

+1+
sl/+

1±0.0124
1.005±0.0091

1 ±0.0121
0.984±0.0114

No. of semi-lethal chr.
No. of normal chr.
Relative
viability

Pooled** significance at the 1% level.

+1+
sl/+

1
±0.0088
0.996±0.0071

Suyama-Juriki populations in 1961 were used in the experiment and theviability of single and double semi-lethal heterozygotes was estimatedand compared with that of normal heterozygotes. The results are given
in Table l.
The results obtained from the experiments of Table 1 were pooled
together and the mean viability of semi-lethal heterozygotes was standardized by that of normal heterozygotes. The results are shown in
Table 2.
The mean viability of single semi-lethal heterozygotes was not muchinferior to that of normal heterozygotes and detrimental effects on theviability of two non-allelic semi-lethal trans-heterozygotes were increased.
The frequency of semi-lethal chromosomes causing 20-30 per cent

RESEARCHES CARRIED OUT IN 1962

15

viability was lowest in the distribution curve manifested by individual
homozygous second chromosomes isolated from a natural population.
The causes for a similar finding have been discussed by Greenberg andCrow (1960) and Radom (1961). The semi-lethal chromosomes seem tohave accumulated numerous mildly detrimental genes and the intensified
deleterious effects on viability of double trans-heterozygotes may be due
to duplication of some common mild mutant genes.

5. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 1. Method and representationof condensed data
(By Terumi MUKAI. Sadao CHIGUSA and Isao YOSHIKAWA)

Studies on spontaneous polygenic mutations which slightly reduce theviability in homozygous condition are scanty. From the study of thiskind of mutations, a considerable amount of information could be drawn
with respect to the genetic structure of natural populations. Accordingly,
Table 1. Basic statistic and genetic parameters obtained
from data in Generations 10. 15, 20 and 25
25(21°C) (25°C)

10(25°C)

15(21°C)

20(25°C)

No. of lethal lines

2

5

13

15

15

No. of homozygously
detrimental lines

3

2

3

2

2

No. of quasi-normal lines tested

98

97

89

84

84

No. of missing lines

1

0

2

3

3

Estimated genetic variance(homozygote basis) (a~)

0.698

2.092

1.848

3.514

3.810

Average of the viability indicesof quasi-normal lines ('Ii)

31.60

27.89

30.93

28.36*

Estimated control viability
index (@)

32.94

29.69

32.36

32.45**

Total no. of flies counted inquasi-normal lines

140,328

240,473383.543 453,999 472.720

Generation(temperature)* Mean value of 21°C and 25°C experiments** Pooled estimate of 21°C and 25°C experiments

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

a series of experiments were started in May, 1961. The experiments
are still in progress, but results at hand are here reported.
A single male Pm/+ from a cross of Cy/Pmx +/+(an isogenic stockof Burdick: W160) was sampled and multiplied by the cross Cy/Pm(!? !? )xPm/+(l 0) and 104 lines of Cy/PmxPm/+i(i=l, 2,·····,104) were
established. In each line, the second chromosome has been maintained
through a single male by the cross Cy/Pm (5!? !? )xPm/+i(l 0) for the
purpose of accumulating spontaneous mutations affecting viability.
In Generations 10, 15, 20 and 25, homozygous viability of each second
chromosome was estimated by Wallace's Cy-method according towhich the expected ratio of wild-type flies to the total is 33.3 per centand 0 per cent in the absence of mutation and for recessive lethal mutations. Accordingly, the percentages of wild-type flies were employedas viability indices.
The basic statistic and genetic parameters were estimated by the aid ofanalysis of variance and other techniques. The results are presented
in Table 1. In this table, the control viability index implies the meanof the distribution of viability indices of quasi-normal lines (having alarger than 20 viability index in homozygous condition) in Generationo in the same environment as that of a certain generation in which thetest was conducted. Analysis was carried out with respect to quasinormal lines.

6. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 2. Variance increasing rate
and mean decreasing rate
(By Terumi MUKAI)

If the effects of polygenic viability mutations are additive or multiplicative, although in the latter case the effect of each mutation should
be small as compared with the viability of standard normal homozygotes,
the genetic variance among the lines increases approximately linearlyin the course of generations and the mean of quasi-normal homozygote
viabilities decreases also linearly. Although we do not have sufficient
data for testing the linearity of variances and means, it might be tested
graphically.
The control viability indices of Table 1 were estimated by the following procedure: for example, in the case of Generation 10, the line showing
the greatest viability and four lines following in the order of degree
were sampled in each of Generations 15, 20 and 25. In Generation 10,

RESEARCHES CARRIED OUT IN 1962

17

phenotypically Cy-flies and wild-type flies in those selected lines werepooled, respectively, and the viability index was estimated as the control
viability in this generation in terms of per cent wild-type flies on thepooled basis. Using this method, we assumed that the five lines selected
above in each of Generations 15, 20 and 25 did not contain any mutations and the effects of errors in those generations were not correlatedwith those in Generation 10. The control viabilities for Generations 15
and 20 were estimated by the same procedure as that in Generation 10.
That for Generation 25 was estimated on the basis of comparison between
the estimates of viabilities at 21°C and 25°C.
The relationships between reduction of means and generation numbers
and between variances and generation numbers are represented inTable 2. From Table 2, it might be said that the variances of viability
increase and the means decrease approximately linearly. Accordingly,
the following conclusions might be drawn: first, the average number ofmutations per individual increases linearly with generations, and secondly,
effects of interaction of new mutant genes with each other, and withalready existing genes are not of appreciably large magnitude in homozygous genetic background.
Table 2.

Genetic variances and decrements of the meansof viability indices
Regression coeffi-

Generation

0

10

15

20

25*
dent on generation

Genetic
variance00.69812.09181.8481

3.6622**0.1120±0.0185

Decrementof mean

0

1.34

1.80

1.43

4.09

** ±0.0207
0.1207* Average of the estimates at 21°C and 25°C*>t Significantly different from 0 at the one per cent level.

7. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 3. Estimation of polygenic
viability mutation rate
(By Terumi MUKAI)

According to Bateman's (1959) calculation, the following formula (1)can be obtained concerning the reduction of mean viability and the

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

increase of genetic variance caused by mutations under the assumptionthat spontaneous mutations are distributed on homogeneous chromosomes
according to Poisson.
iiP=@-v}(j;i2 +aa2)P=(IG2(1)

where @ is the mean of control viability indices, v is the mean viability
index of wild-type flies in quasi-normal lines, a is the effect of a single
mutation whose mean and variance are ii and a/, respectively, P indicates average number of mutations in the second chromosome, and (If}is the estimated genetic variance of viability indices among quasi-normal
homozygotic lines. For the sake of simplicity, @-v is equated to A and2(Ia to B. Consequently, A and B are the estimated values. From
formula (1), the maximum aa2 can be estimated. aa 2 should be 0 or positive. Thus, the range of aa 2 becomes as in formula (2).

(2)
The ranges of p and ii can be correspondingly calculated from formulae(1) and (2) under reasonable conditions; the results are presented informulae (3) and (4).
(3)

(4)
If we substitute the variance increasing rate and mean decreasing rate
for A and B in formulae (3) and (4), we can estimate the average numberof mutations per second chromosome per generation and the average
effect of mutations. The results are tabulated in Table 3.
From Table 3 it can be concluded that the mutation rate of polygenes
is of extremely large magnitude, when it is compared with that ofTable 3. The rate of polygenic mutations controlling
viability, their average effect and variance
Item

Estimate

Mutation rate
(per 2nd chromo per gen.)

Average effectRange ofvarianceof effect

0.130:O;;p:o;;O.260

0.464:o;;a:o;;O.928

O:O;;a~:o;;O.215

RESEARCHES CARRIED OUT IN 1962

19

recessive lethal genes (ca. 0.0063) per second chromosome per generation.

8. Studies on spontaneous polygenic mutations in Drosophilamelanogaster 4. Dominance in polygenic viability mutations(By Terumi MUKAI, Sadao CHIGUSA and Isao YOSHIKAWA)

When crosses in Generation 25 were conducted for estimating homozygous viabilities of the chromosomes in question, the viabilities of flies
carrying random combinations of those chromosomes were estimatedin the following combinations, 1x2, 2x3, 3x4, "', (n-1)xn, nx1, where
numbers indicate the line designations. The lines in which lethal orsemi-lethal mutations had taken place were not employed.
With the aid of analysis of variance, it was proven that there was adifference in heterozygous viability among random heterozygotes. In
addition, the genetic variance in heterozygous combination of thosechromosomes (a~,) was estimated. Phenotypic correlation (rpp')' genotypic correlation (rGG') and covariance between heterozygote viabilities
and the sums of the corresponding homozygote viabilities were estimatedboth in 25°C and 21°C experiments. These genetic statistic parameters
are presented in Table 4 together with the mean viabilities.
Suppose that the average reduction of viability per second chromosome
in homozygous condition is d and their average degree of dominance isii, then d and ii can be estimated on the basis of the mean viabilities
of control, homozygotes and heterozygotes; the results are d=4.09,
ii=0.37 on the average of the estimates at 21°C and 25°C in Generation
25. The estimated ii value, 0.37, is extremely large as compared withthose for recessive lethals estimated for natural-origin chromosomes(ii=0.02",0.04).
On the other hand, if hand d are independent, then the average
degree of dominance (h) can be estimated by the following formula:ii= Cov(Hetero and Sums of Homo)2(1,,2(5)

It we substitute the estimated parameters in this formula, then ii's at
21°C and 25°C become 0.72 and 0.80, respectively. Those values aremuch larger than the previous estimate (h=0.37), and might confirm
the large value of Ii. In addition, a large amount of the difference
between the two estimates might indicate that there is a positive correlations between the detrimental degree of viability mutations in

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

homozygous condition and the degree of dominance within the range ofquasi-normal viability.
Table 4.

Various genetic parameters in Generation 25

Temperature
Control viability
Mean viability of homozygotes
Mean viability of heterozygotes
Phenotypic correlation (rpp')*
Genotypic correlation (rGG')
Covariance*
Genetic variance of heterozygotes
Genetic variance of homozygotes

25"C
32.45
28.37
29.58
0.510
0.692
5.028
7.508
3.514

28.36
29.27
0.594
0.807
6.092
7.480
3.811

* Relationship between heterozygote viabilities and the sums of the corresponding
homozygote viabilities.

9. Studies on spontaneous polygenic viability mutations in
Drosophila melanogaster 5. Polygenic mutations andgenetic structure of natural populations(By Terumi MUKAI)

If the classical hypothesis is valid, the genetic load in the greater
fraction of loci is mutational, and its magnitude is 2p per locus (p=rateof mutation from wild-type gene to mutant gene) in autosomes inequilibrium of a random mating population (Muller 1950), disregarding
completely recessive genes with respect to viability. For the sex
chromosome, the magnitude is approximately 1.5 p (3 p in males and of
a negligible amount in females). It should be stressed in these casesthat the contribution of selection coefficients to the mutational load is
too small to be considered. On the other hand, if the balance hypothesis is true, the major part of the genetic load is segregational load,
and its magnitude is independent of the mutation rate and is determinedby selection coefficients.
Following Kimura (1960), a test can be conducted to see whether theclassical hypothesis is valid or not. Assuming this hypothesis, survival
rate of eggs (5) can be expressed by formula (6).
(6)

where 2,S Pi and 1.51'pj stand for the mutational loads in autosomes andRESEARCHES CARRIED OUT IN 196221

sex chromosomes, respectively.
In the present experiment, the mutation rate of polygenes in thesecond chromosome p is 0.130~p~0.260. We used the average of those
two extreme values and calculated the total mutational load to be 0.9760
on the basis of proportionality between chromosome length and mutation
frequency. The survival rate becomes e-O.9760=0.3768. This rate isinconsistent with the actual situation in natural or artificial randommating populations, where, for instance, the hatchability of eggs in Sy
and OR populations was found to be as high as 95 per cent (King 1961)
and consequently, this finding is unfavorable to the classical hypothesis,
if there is no epistasis favoring the coexistence of deleterious genes.
In the present experiment, we have detected that the average degreeof dominance of mutations which have not experienced natural selection(h) is large. This finding is inconsistent with that of Wallace andDobzhansky (1962). In their case, samples from artificial populations
were used. The difference might have been caused by natural selectionin the artificial populations. If this is the case, in natural populations,mutant genes having large h values should be eliminated rapidly bynatural selection and/or natural selection operates so as to produce a
genic system in which h is small or rather negative (overdominance).

10. Persistence of two recessive genes in experimental populationsof Drosophila melanogaster(By Sadao CHIGUSA and Terumi MUKAI)

Two cases of single gene heterosis have been tested associated withthe recessive genes sepia and expanded wing (formerly x, discovered in
Mukai and Burdick's (1959) Population 1, of Dichaete phenotype, thegene located at about 3-49±).
Four artificial populations (Pop. A-I and Pop. A-2 for se, and Pop.
B-1 and Pop. B-2 for ew) were established, and the gene frequencies
of Pop. (A or B)-1 and Pop. (A or B)-2 in the starting generation were
0.5 and 0.1, respectively. The homozygosities of the genetic backgrounds
in A-populations are extremely low, while those of B-populations are
relatively high. Each population consisting of 8 subpopulations has
been maintained by so-called Pearl's method, and the frequencies ofrecessive homozygotes were estimated in most of the generations. Four
populations seem to have reached equilibria since Generation 20-25,
which indicates the superiority of the heterozygote over either homozy·gote with respect to fitness; the equilibrium frequencies are given in

t-:l

t-)

>
Z

Table 1. Characteristics of artificial populations

Initialgene
Generfrequency ation
Pop. A-I
(lie)

0.5

39
40
41

5052
4591
3868

[+]
4659
4188
3549

3778
2445
3302

37

+ /se or
+/ew
85

28.0%

81

30.8

qtl

Numberof subpopulations

x 2t tJ8207.576>1-**

:=0
t"1

393
403
319

3244
2082
2861

534
363
441

14.13
14.85
13.36

81
68
73

85
71
93

36.0
36.5
37.8

7

334.332>1-**

>
>-3
::;
Z
>
t""'

2533
3746
3276

57
53

2.20
1.34
2.04

131
135
114

53
49
42

16.2
14.7
15.1

8

11.506

>-3

39

2590
3799
3335

32
33
35

3982
3003
3371

3820
2931
3234

162

4.07
2.34
4.06

71
96
98

60
80
83

26.3
24.4
26.2

-2

0.1

Pop. B-1
(ew)

0.5

38

0.1

Recessive
homozygote
frequency[se] or lew]+/+
7.78 % 108
8.78
8.25
84

31
33
34

-2

Population
size

Z

c::

>
t""'

Progeny test

Population count

68

72

137

'""0

~
>-3
o>'Ij

z

Z

(fJ

::J

c::

>-3

t"1

8

35.2957**

o
>'Ij
Cl

t"1

Z
t ):
tt):

**>1**

Recessive gene frequency. q is estimated by a simple method.
Test for heterogeneity among subpopulations.
Significant at a very low level.
Significant at the 1% level.

t"1

>-3

(=i
(fJ

z

9
......
w

RESEARCHES CARRIED OUT IN 1962

23

Table 1 in addition to the characteristics of the four populations. Fromthe progeny test, the goodness of fit of the observed data to thosewhich are expected can be calculated on the assumption that the threeclasses of flies are distributed in accordance with the Hardy-Weinberg
law. The results of the population counts show that heterozygote frequencies are larger than the expected ones in three counts out of fivein A-populations and in all counts in B-populations.
The relative values of the fitness components of the three genotypes
were estimated in other experiments and the results show that theheterozygote has been superior in each population to either homozygote
in female fecundity, male mating ability and zygotic viability.
It can also be seen from Table 1 that the recessive gene frequencies
have reached equilibria, but that the equilibrium frequencies of two
populations both in A-populations and B-populations are remarkably
different from each other. However, a more quantitative determinationof the trend is desired. This is now under way.
The x 2 values of heterogeneity test among subpopulations are verylarge in A-populations but those of B-populations do not indicate asignificant difference. The large x 2 values imply that subpopulations
have reached equilibria at various levels. As mentioned above, Apopulations have higher genetic variability in their genetic backgrounds
than B-populations. Therefore, the above mentioned phenomena might
have been caused by the effects of random genetic drifts of the genetic
backgrounds.
Weare still maintaining these populations, and details of the work
will be published elsewhere.

11. Syntheses of Drosophila eye pigments1)(By S. NAwA and H. S. FORREST)

Drosophila melanogaster has two kinds of yellow pigments and threekinds of red pigments. The structures of yellow compounds, isosepiapterin and sepiapterin, have been determined to be 2-amino-4-oxo-6acyltetrahydropteridines, the acyl group in isosepiapterin being propionyl
and in sepiapterin, lactyl (hydroxypropionyl). The red compounds,
drosopterins (drosopterin, isodrosopterin and neodrosopterin), are believed
to have structures related closely with sepiapterin, although their structures have not been confirmed.
Based on the findings that pyruvic acid gives a relatively stable1) This work was done in The University of Texas, U.S.A.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

carbanion (CHsCO-) in its decarboxylation by action of thiamine andthat a reduced form of 2-amino-4 hydroxypteridine adds anionic reagents
exclusively at the 6-position, formation of a pteridine having an acyl groupin its 6-position would be expected on reaction with reduced pteridine
and anion from a-keto acid. Indeed, incubation of reduced 2-amino-4hydroxypteridine, pyruvic acid and thiamine at
pH 9 yielded a yellow compound. It is sure thatthe compound has a substituent in the 6-position,
because permanganate oxidation of the compoundgave 2-amino-4-hydroxy-6-carboxypteridine. Its
structure is believed to be I (R=CH s). Isosepiapterin (I, R=CH 2CH s) was formed in the same
reaction at a reasonable yield, when a-ketobutyric acid was used insteadof pyruvic acid.
Considering the structure of sepiapterin (I, R=CH(OH)CH s), its synthesis should be possible using the same procedure with a-keto-j3hydroxybutyric acid as a source of the side chain. No sepiapterin was,however, obtained under conditions developed for maximal isosepiapterin
synthesis and even conditions modified variously. Nevertheless, themixture of a-keto-j3-hydroxybutyric acid and reduced pteridine became
gradually red on standing at room temperature (even in the absence ofthiamine). The reaction mixture contained three kinds of red compounds
which were found to be identical, spectrophotometrically and chromatographically, with drosopterin, isodrosopterin and neodrosopterin respectively. On the contrary, if hydroxypyruvic acid was used instead of
a-keto-j3-hydroxybutyric acid, no red pigment was formed but a yellow
compound was produced. The product was purified as a crystalline
form. The properties of the compound were very similar to those of
sepiapterin, and then its structure was identified to be I (R=CH 2(OH».
It is expected that in the reaction between reduced pteridine and acarbanion from a ketoacid an intermediate is a 6-acyl-5, 6, 7, 8-tetrahydropteridine. The intermediate will be then oxidized to a 7,8-dihydrocompound. This seems to be true if its acyl group is such one as
CHsCO-, CH sCH 2 CO- or CH 2 (OH)CO-. However, the intermediate having
CHsCH(OH)CO- as its acyl group was not oxidized to a corresponding
7,8-dihydro compound (sepiapterin) under conditions used. An unknown
different type of oxidation gave drosopterins. The mechanism is obscuresince structures of drosopterins have not been clarified.
The facts that the ratio of quantities of three kinds of drosopterins
produced in the reaction is approximately the same to that found in
Drosophila eye and that the reaction is carried out under mild conditions

RESEARCHES CARRIED OUT IN 196225

(at neutral pH and room temperature) suggest strongly a possibility of
their biosyntheses in this manner, although no accumulation of ketohydroxybutyric acid was detected in Drosophila. On this assumption, it
can be proposed that the mutant sepia which produces sepiapterin andnot the drosopteins, oxidises the intermediate exclusively to the 7,8dihydro compound whereas in the wild type, a divergent pathway withsome of the intermediate being converted to drosopterin and some tosepiapterin, is present.

12. Melamine action in phenocopic pigment elimination fromhypodermal cells of the silkworm(By Mitsuo TSUJITA and Susumu SAKURAI)

When mulberry leaves painted with 1% melamine solution were
continuously given to lemon larvae (yellow colored larvae) from the
beginning of 3rd, 4th or 5th instars, the hypodermis became translucent
2 days after the treatment and at the same time the yellow larval color
disappeared, because the yellow pigments were lost from the hypodermal
Table. 1. Loss of uric acid from the hypodermal cells by eating
mulberry leaves painted with melamine solution.
A.

Before the treatment with melamine~o. of Experiment
Strain?"--'_ _ _ _

B.

II

I

III

Mean

C124

mg/l g dry
matter86.00

mg/l g dry
matter84.00

mg/l g dry
matter85.00

mg/l g dry
matter85.00

1 em/l em

87.50

79.00

84.30

83.60

III

Mean

After the change to translucent hypodermis~tStrains

II

I

C124

mg/l g dry
matter5.00

mg/l g dry
matter3.00

mg/l g dry
matter8.00

mg/l g dry
matter5.30

1 em/l em

trace

trace

trace

trace

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

cells.
Each larva with translucent hypodermis was cut open from the dorsalside and almost all of the inner organs were removed and the remaining
hypodermal tissue was dried. Then the amount of uric acid and pteridine
compounds, such as yellow pigment or isoxanthopterin, was measured.
Experimental results are shown in the following tables.
As shown in Table 1, a large part of uric acid is lost from thehypodermal cells of larvae eating mulberry leaves painted with melamine
solution.
Table 2.

Strains

Loss of pteridine compounds from the hypodermal cells by
the effect of melamine
Pteridine
compounds

pg/individual

After the change
to transparent
hypodermis
pg/individual

Before treatment

C124

Isoxanthopterin

13.2

1.2

KHl9

Isoxanthopterin

15.0

6.0

yellow pigment

12.0

trace

Isoxanthopterin

3.0

trace

1 eml1 em

As shown in Table 2, under the influence of melamine the amountof isoxanthopterin decreases markedly and the remainder differs withthe three strains. On the contrary, yellow pigments are completely
lost from the hypodermal cells of the treated larvae.
Further, the hypodermal cells of larvae which lost uric acid andpteridine compounds were microscopically examined. It was found that
almost all chromogranules in their cytoplasm broke down and disappeared. To purify the chromogranules in the cytoplasmic layer of thehypodermis a procedure used in another experiment was applied. However we could not obtain the granular fraction. It may be said fromthese experimental results that pteridine compounds become released
from the chromogranules as the result of their collapse.
Effects of melamine treatment on the substances showing fluorescencein the cytoplasm of hypodermal cells and in the body fluid of larvae
were examined by paper electrophoresis and the following results wereobtained.
As shown in Table 3, there is a substance showing sky-blue fluorescence in addition to yellow pigment and isoxanthopterin in thecytoplasm of hypodermal cells of lemon larvae and traces of a similar

27

RESEARCHES CARRIED ONT IN 1962
Table 3.

Substances showing fluorescence in the hypodermal cells andin the body fluid examined by paper electrophoresis.
Pteridine compounds

Treatment

StrainBefore thetreatment

Tissues

Yellowpigment

Isoxanthopterin

Substance showing sky-blue
fluorescence

hypodermal
cells

++++

+

+

body fluid

±

-

±

hypodermal
cells

++

±

+++

body fluid

-

±

++

hypodermal
cells

+

+

+

body fluid

±

-

++

lemon

One day
after thetreatment

lemon

dilutelemon+:

Indicates relative amount.
Indicates the absence of the compound.
±: Indicates trace.

substance can be detected in their body fluid. One day after treatment with melamine the amount of yellow pigments decreases and thatof the substance showing sky-blue fluorescence increases in the hypodermal cells. According to Table 3 the amount of the latter compoundshows the tendency to increase in the body fluid of the treated larvae.
The same increase in the amount of substance showing sky-blue fluorescence could be observed in the body fluid of dilute lemon larvae ascompared with lemon larvae. It is inferred from the experimental
results that yellow pigments released from the chromogranules areconverted into other pteridine compounds, such as the substance showing
sky-blue colored fluorescence, which are excreted from the hypodermal
cells into the body cavity.
We mentioned in a previous report (Tsujita 1963) that a largepart of yellow pigments produced in the hypodermal cells of dilutelemon larvae is lost from the cells by the action of d-Iem gene. The
finding that owing to melamin treatment yellow pigments are lostfrom the hypodermal cells of lemon larvae can be considered as an
example of phenocopy. However, it should be noticed that there is

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

remarkable difference between the action of d-lem gene and that ofmelamine; d-lem gene controls a small specific change, namely "theproduction of an altered specific protein" within the chromogranules,
while melamine induces a drastic change, namely "disintegration ofchromogranules.' ,13. Specific protein combining with yellow pigments(dihydropterin) in the silkworm(By Mitsuo TSUJITA and Susumu SAKURAI)

A large amount of spherical granules that contain yellow pigments
and isoxanthopterin occur in fresh hypodermal cells of larvae of thestrains lemon and dilute lemon. The presence of these chromogranules
in the hypodermal cells gives a yellow color to the larvae.
The yellow granules were purified by a new method devised by us.
The chromogranules obtained from the lemon and the dilute lemon
larvae show almost the same shape, an irregularly spherical one. The
granules obtained from the lemon larvae are much darker yellow
in color than those from the dilute lemon larvae. Similarly shaped
granules containing isoxanthopterin but smaller in size than those of
the lemon and the dilute lemon larvae are present in the hypodermal
cells of normal larvae.
The protein composition of the three types of chromogranules obtained from normal, lemon and dilute lemon larvae has been examined bypaper electrophoresis, and characteristic differences have been found intheir electrophoretic mobility. It was at first thought that these electrophoretic differences might reflect small structural dissimilarities among
the three types of chromoprotein, comparable to those that have beenfound in mutant human hemoglobin (Ingram 1958). However, the digestion of the chromoprotein with trypsin showed differences in the peptide
composition; the chromoprotein of the lemon sample contained two
supernumerary peptides in addition to those commonly observed in thethree types of chromoprotein, while the chromoprotein of the dilutelemon sample had only one additional peptide that had a different Rfvalue when compared with the supernumerary one produced by the 2-Pof the lemon sample. The two supernumerary peptides found in thelemon sample are believed to be components of the specific protein
which has the ability to combine with yellow pigments. The single
supernumerary peptide found in the dilute lemon sample is apparently
a component of the specific but altered protein.

RESEARCHES CARRIED OUT IN 1962

29

On the basis of the present study, the biochemical interactions between
lem and +d-l,m or between lem and d-lem genes may be explained asfollows. As reported in a previous paper (Tsujita 1961), the yellow
pigments are produced abundantly in hypodermal cells in which thestep leading from dihydropterin to tetrahydropterin is blocked by thelem gene. A specific protein is produced under the control of +d-l'm
gene action in the presence of yellow pigments. As the chromogranules
in hypodermal cells of larvae with the genotype lem/lem;+d-l,m/+d-Iem
contain this protein, they are able to absorb yellow pigments and keepthem within the cells. On the other hand, the altered protein produced
under the control of d-lem gene action is present in the chromogranulesof the dilute lemon larvae. Consequently in the dilute lemon larvae,
the ability of the granules to absorb and retain the yellow pigments ismarkedly decreased, and such larvae have a dilute yellow color.
On the basis of other experimental results, it is believed that a largepart of the yellow pigments which are not absorbed in the chromogranules
are converted into other compounds, such as substances which show
sky-blue fluorescence, and are excreted from the hypodermal cells into
the body fluid.

14. Conversion from guanine to peteridine in the silkworm(Bt Bungo SAKAGUCHI)

It is generally considered that the pteridine ring is biosynthesizedfrom a purine precursor. But a direct evidence of the biosynthesis of
the compound has not been found yet for the silkworm.
Guanine-2- 14C was injected 1,uc per head into females of early pupaeof a normal strain (C 108). Young larvae were collected in the following
generation from the adult insects. Pteridine and purine compounds
were extracted from the young larvae with boiling water and the protein
was removed from the extract by acidification. Paper chromatography
and column chromatography (Dowex I-Formate) were used for the isolation of pteridine and purine compounds which were detected by use of
an ultraviolet light source and a spectrophotometer. For paper chromatography radioactive compounds were determined by autoradiography andscanning gas flow counter.
Purification of isoxanthopterin extracted from young larvae was achievedby performing four times paper chromatography with different solvents.
Radioactivity of purified isoxanthopterin was tested by the gas flow
counter and it was found to be radioactive. It is suggested from the30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

result that HC atom in carbon 2 of guanine molecule is contributed toisoxanthopterin. The turnover rate from the same precursor of guanine_HC to isoxanthopterin-HC and uric acid-HC was shown to be one
to seventeen.
In order to find out whether guanine-HC injected into pupae wasbeing incorporated into nucleic acid components in the young larvae
derived from the following next generation of the pupae, acid soluble,
RNA and DNA fractions were separated from the young larvae by themethod of Schmit et al (1945). The result is given in the following
Table.
Table 1. Distribution of HC in the nucleic acid and otherfractions prepared from young larvae

~
cpm*

%

Lipid

Acid soluble

RNA

DNA

Protein

1617
12.8

2468
23.6

7419
58.9

433
3.5

150
1.2

* Counting value of extraction from one gram of wet weight.

As the Table shows high activity of HC originated from labeled
guanine was found in both RNA and acid soluble fractions. The labeled
components of nucleic acid in the fractions were separated and analyzed
by the above method. It was found from the results that guanosine,
guanylic acid and a small amount of pteridine derivatives were holding
HC atoms liberated from guanine-2- HC.
Weygand et al (1961) have considered guanosine-Sf-phosphate to be
the precursor of leucopterin in the butterfly Pieris brassicae. Present
results also indicate that guanine and guanylic acid directly contributeto the biosynthesis of the pteridine ring in the silkworm.

15.

Analysis of marker genes in inbred strains of albino mice
(By Yoshinori KURITA and Katsumi SAKAKIBARA)

The marker genes of the main inbred strains of albino mice whichhave been maintained in Misima were analyzed. All albino mice weremated with C57BR and NH mice. It was found that the A/HeMs substrain consisted of genotypically three different families. The resultsof these analyses are summarized as follows:

RESEARCHES CARRIED OUT IN 1962
Albino strain

Genotype

A/HeMsAABBeeAAbbeeaabbeeaaBBeeSSPPDD
AAbbeeAAbbeeAAbbeeSSPPDDAABBceAABBeeaaBBeeAABBeeAABBeeAKR/JMs
BALB/eJMs
CFW/Ms
DM/Ms
DI03/Ms
MA/JMs
RF/Ms
SLIMs
SMA/Ms31

16. Genetical studies of post-axial polydactyly in the house mouse(By Akira NAKAMURA, Hitoshi SAKAMOTO and Kazuo MORIWAKI)

Post-axial polydactyly characterized by the presence of supernumerary
fingers on the forefeet was found in an albino mouse obtained from afancier in 1958. Since then it has been maintained by sister-brothermatings. Strong (1934) has already reported similar features in his brief
notes. Later, Phillips (1949) described a somewhat comparable condition
which seemed to be heritable, but a detailed analysis has not yet beenmade.
In our experiments in which outcrosses were carried out of thepolydactylous strain to various inbred strains (CBA, DM, A and C57BR),
the abnormality in the forefeet appeared in various numbers in Fl' But
in a cross between Swiss albino and the polydactylous strain, the condition could be observed in 95% of F 1 and a 3:1 segregation was demonstrated in F 2 • Moreover, the backcross of F 1 to Swiss albino showed a
1:1 ratio.
In view of the above results it is probable that this character depends
on a major dominant gene and possibly some minor genes which may
modify its manifestation. The major dominant gene was named postaxial polydactyly gene (symbol Po).

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

17. Cytological study of a transplantable ascites plasma-cell
neoplasm in mice
(By Yoshinori KURITA and Tosihide H. YOSIDA)

A transplantable ascites plasma-cell neoplasm in C3H mice has beenobtained from an experimental intraperitoneal injection of X5563 solid
plasma-cell neoplasm, and has been maintained by means of serial intraperitoneal transplantations. The averaged life span of the mice inoculatedwith the ascites was 15.3±1.7 days. The modal chromosome numberof the ascites neoplastic cells was 83, and four marker chromosomes
have been ascertained, namely two large mediocentric, one metacentricand a diminutive one. The characteristic feature of this neoplasm isthat it has a stable tetraploid cell population, consisting of more than
99% tetraploid cells. Its origin in connection with its stable heritable
globulin production is under investigation.

18.

A characteristic chromosome found in the karyotype
analysis of the mouse(By Akira NAKAMURA and Akira TONOMURA)

For a long time it has been believed that, in mice, all chromosomes
are telocentric and morphologically indistinguishable from one another.
Recently, the improved techniques for mammalian chromosome cytology
has revealed that certain mouse chromosomes are characterized by thepresence of a small knob near the centromeric region. The evaluationof this finding led us to re-examination of the classical chromosomal
studies of the mouse.
The cells of normal mouse (DM/Ms strain) embryos were cultivatedin media containing 20% of calf serum. Chromosome preparations fromthe cultured cells were made by acetic-dahlia squash technique, after
pretreatment with 0.0001% colchicine solution and distilled water.
Most of the chromosomes were telocentric; a second arm was entirely
imperceptible (Fig. 1). A small knob was observed in some chromosomes(Fig. 2), but it could not be always distinguished when the chromosomes
were somewhat condensed by colchicine treatment. A detailed observation, however, disclosed one characteristic chromosome shown in Figure 3.
It had a well-defined round or oval head separated from the main bodyof the chromosome by a thin chromatin strand. The head was almost
1/4 as long as the body, and its diameter was considerably smaller than

RESEARCHES CARRIED OUT IN 1962

33

Fig. 1-3. Higher magnification of three structurally
distinct chromosomes in the mouse. 7,000 x .
Fig. 1; Ordinary telocentric chromosome. Fig. 2;
Chromosome with small knobs. Fig. 3; A pair ofchromosomes with well-defined oval heads. The
heads are much larger than the knobs of Fig. 2.

that of the body. A detailed analysis of five cells revealed that thischromosome pair may be placed between the 14th to 16th pairs.
On morphological grounds, it is impossible to confuse this characteristic
chromosome with the others, although whether its prominent head refers
to the second arm of a subtelocentric chromosome or is only a part ofthe chromosome due to a secondary constriction is still unknown.

19.

Cytological studies of lymphogenic and hematogenic metastases
in MH-134 mouse hepatoma(By Naomichi INUI)

MH-134 mouse ascites tumor contains two different types of tumor
cells having 40 (2n) and 80 (4n) chromosomes, their ratio being 1 to 0.97.
In the present study, the proportion of diplo- to tetraploid tumor cells
in metastases of the lymphnodes and parenchymal organs was examined
and compared with that found in the original ascites tumor. Metastases
to lymphnodes were induced by intracutaneous injection of ascites tumor
cells into the tail of the mouse (Exp. A). Metastases to parenchymalorgans were established by subcutaneous injection into the back.
Experiment A: Seven to ten days after transfer of the tumor cells,
the tail was amputated at the base. About 20 days after the amputation, the mice were killed and caudal, lumbar and sciatic lymphnodes
were removed. These lymphnodes showed a notable enlargement andcontained a great number of tumor cells in 48 out of 50 animals. In
seven mice remote metastases occurred in brachial, axillary and superfi-

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

cial lymphnodes. The ratios of diploid to tetraploid cells were almost
similar to those found in the original ascites tumor.
Experiment B: About one month after the removal of the transplanted
tumors, the animals were killed. In 24 out of 30 mice, metastases wereobserved in the parenchymal organs, such as lung, liver and kidney.
The ratios of diplo- to tetraploid cells were found to be 1 : 1,06 in lung,1 : 1.01 in liver and 1 : 0.86 in kidney. Detailed karyotype analysis
revealed that 38 acrocentric chromosomes were in diploid cells and 76
acrocentric and 4 metacentric were in tetraploid cells. No chromosomalchange could be found in the two types of tumor cells, as comparedwith those of the original ascites tumor cells.
The results here obtained suggest that, in MH-134 mouse hepatoma,
the diplo- and tetraploid tumor cells invade both lymphnodes and parenchymal organs through the lymphatic and blood systems, and there is
no preference for either diplo- or tetraploid cells in the specific organs.

20. ChromCltographic analysis of coat coloring substance in mice
(By Akira NAKAMURA)

Rebell et al. (1957) have performed the extraction and chromatographic
separation of L-kynurenine from the hairs of albino rats, and in the1

Fig. 1. Six typical spots on the two-dimensional
paperchromatogram.

RESEARCHES CARRIED OUT IN 1962

35

same year Hashimoto has observed kynurenine in mice skin. On theother hand Nachimas (1959) reported that kynurenine is not utilized for
pigment formation in the skin of yellow mice (AY).
In this study, the hair coat of 24 strains of mice was analysed by
one-dimensional and two-dimensional paper chromatography. A summaryof the results obtained is presented, and the positions of six typicalspots (none for kynurenine) on the paperchromatogram are mapped in
Figure 1.
1) Kynurenine was not observed in any strain.
2) Spot 1 was present in all strains, and exhibited a yellowish whiteblue fluorescence.
3) Spot 2 was present in all strains and turned rose-red in the presence of Ehrlich's diazo reagent. The color appeared immediately after
spraying with the reagent and then gradually fainted.
4) Spot 3 was present in all strains, especially in AY strain, A and
CFW. Ehrlich's aldehyde reaction of the spot showed yellowish color.
5) Spot 4 was positive in AY strain, CBA, C57BR, D103, C57BL, A,
C3H, CFW and DM, but negative in C h strain and Swiss albino. Colorof the spot in Ehrlich's aldehyde reaction was purple.
6) Spot 5 was present in C57BL and CC\ and exhibited purple fluorescence.
7) Spot 6 was positive only in AY and turned red in the presenceof Ehrlich's diazo reagent.

21. Biochemical changes in the skin of rhino mousepreceding the hereditary hair loss
(By Kazuo MORIWAKI)

A mouse homozygous for rhino gene (hr rh ) begins to lose the hair coat
about 15 days after birth and becomes completely naked within One
month. At that time acid phosphatase (acid Pase) and inorganic pyrophosphatase (PPase) activities in the skin show a markedly higher level
than in the heterozygotes, as recently reported by the author (1962).
Whether these changes in enzyme activity precede the beginning ofdepilation or not is an interesting problem for studying the manifestation mechanism of rhino gene in terms of biochemical genetics.
From this view point, the activities of acid Pase and PPase in theskin have been compared between homo- and heterozygotes at several
stages of development. Some other biochemical characters, Le., lipid
phosphorus content, cathepsin activity and cystine content, which seem

36

ANNUAL REPORT OF NANIONAL INSTITUTE OF GENETICS NO. 13

to have certain intimate relations to the regulation of hair formation,
were also compared in similar fashion. Among them, acid Pase activity
has shown an apparent difference at the earliest stage, namely, an appreciable increase in enzyme activity could be observed in homozygous
skin 8 days after birth. Differences in the other characters became recognizable after 10 days. Lipid phosphorus content began to increase tosome extent on the 10th day in the skin of homozygotes, and PPase andcathepsin activities were also increased around the 25th day. Cystinecontent in the skins of both homo- and heterozygotes remained constantthroughout the development.
Considering the precedence of skin acid Pase in the sequential biochemical changes accompanying the manifestation of rhino gene, aqualitative analysis of the enzyme was carried out using agar electrophoresis technique. In both homo- and heterozygotes, similar patterns
consisting of two isozymes were obtained. Furthermore, a comparison
between homo- and heterozygotes was performed on zymograms of esterases which have probably to some extent a substrate range in common
with acid Pase. Also in this case, there was no difference between
them, though seven isozymes, at least, could be separated.
These results suggest that acid Pase is an intermediate step in thebiochemical sequence leading to the manifestation of rhino gene, though
the change in activity certainly precedes the appearance of the phenotype.

22.

Viscosity changes in the deoxyribonucleoprotein of L-strain
cells following treatment with a base analogue(By Kazuo MORIWAKI)

A crude unpurified deoxyribonucleoprotein (DNP) extracted with 1 M
sodium chloride from rat thymus exhibits some viscosity losses after
low X-ray exposures, which are understood as breakdowns in the fundamental units of chromosome structure (Fisher et aI, 1959). On the other
hand, Hsu and Somers (1961) reported that a base analogue, 5-bromodeoxyuridine (BUDR), can induce chromosome aberrations in mammaliancell cultures similarly to ionizing radiations.
The present study was an attempt to find out a relationship between
chromosome breaks and viscosity changes in DNP of cultured mammaliancells treated with a base analogue. L-strain cells treated with BUDR
(25 ,ug/m!) for 3 days showed chromatid breaks and simultaneously viscosity loss in DNP extracted with 1 M sodium chloride from the cells.
Viscosity of DNP was measured using Cannon-Fenske viscometer modi-

37

RESEARCHES CARRIED OUT IN 1962

fied by Fisher. Cell homogenates for DNP extraction were adjusted at
the concentration of 500 mg% in wet weight. Specific viscosity in DNP
decreased from 0.052 to 0.017 following treatment with BUDR.
This result suggests the possibility that chromosome aberrations induced by BUDR may be accounted for the breakdowns in the fundamental DNP units of chromosome structure.

23.

Endurance of one day old chicks under starvation(By Takatada KAWAHARA)

The time of surviving in starvation of one day old chicks was comparatively investigated in two purebreds, White Leghorns (WL) andBarred Plymouth Rocks (BPR) and their reciprocal hybrids. The starvation treatment was given in an incubator under the condition of relative humidity of about 55 percent and temperature of 37.6°C. Data
were collected from 108 purebred and 138 crossbred chicks. The hatchedbody weight and average endurance of tested chicks are shown in
Table 1.
The results of this investigation are summarized as follows:1). The coefficient of correlation between the surviving time andTable 1.
Breed or crossEndurance under starvation of two breeds and their hybrids.
Sex

28
26
54

38.23±O.88
37.00±O.60
37.64±O.55

156.14±2.93164.77 ±3.62159.63±1.93

4.13±O.O9
4,44±O.O8
4.28±O.07

28
26
54

35.86±O.53
35.37±O.51
35.62±O.37

172.14±2.88
183.04±5.86
180.69±3.26

4.82±O.O9
5.21±O.18
5.1O±O.1O

Both

44
45
89

38.09±O.50
37.71±O,4437.90±O.19

166.73±1.60
168.76±3.32167.75±2.06

4.39±O.O7
4,49±O.O9
4,44±O.O6

0

23

~

26
49

35,48±O.93
35.13±O.66
35.30±O.45

169.26±4.12170.85±5.33
170.1O±3.34

4.79±O.12
4.88±O.14
4.83±O.O9

0

WL

~

Both
0

BPR

~

Both
0
WL~ xBPR

BPR~ xWLooEndurance
Number Body weightof(g)Surviving time \Survivin g time (hours)chicks(hours)Body weight

~

Both

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

hatched body weight was -0.063 in the purebreds, while in the hybrids
it was +0.284.
2). The difference between strains was remarkable, BPR showing avery high endurance in comparison with WL. BPR lived 21.06 hours
longer than WL.
3). In all strains, females survived longer than males.
These results suggest that the endurance of chicks under starvation
may be controlled at least partly by genes.

24.

Genetic studies on body weight at different
stages of growth in the domestic fowl
(By Takatada KAWAHARA)

Heritability of body weights during development and genetic andphenotypic correlations between weights at various growth stages were
investigated in a closed flock of White Leghorns. Measurements of bodyweight were taken 4, 8, 12 and 18 weeks after hatching, at first egg
laying, and after 48 weeks in adults. The estimates of heritabilities
from analyses of variance are shown in Table 1.
Table 1.

Heritabilities of body weights in various stages of growth.

Body weight at:
4 weeks
8 weeks
12 weeks
18 weeks
Age at 1st egg
Adult stage
Table 2.

0.51
0.99
0.77
0.76
0.53
0.40

0.73
0.29
0.34
0.55
0.73
0.48

0.62
0.64
0.55
0.66
0.63
0.44

Correlations between body weights at different growth stages.

4 weeks-Adult
8 weeks-Adult
12 weeks-Adult18 weeks-Adult
Age at first egg-Adult*: Significant at the 1% level.

Genetic correlation

Phenotypic correlation

0.573
0.727
1.100
0.803
0.984

0.445*
0.515*
0.590*
0.589*
0.705*RESEARCHES CARRIED OUT IN 1962

39

The estimate of heritability of (h~+d) averaged for body weights at various stages of growth is 0.59.
Table 2 presents the estimates of coefficients of genetic and phenotypic correlations between adult body weight and body weight at various younger stages of growth.

25.

Use of rabbit anti-serum for muscle proteins offowls for serological determination ofhomologous tissue proteins of Triturusl)(By Yoshito OGAWA and Takatada KAWAHARA)

For immuno-chemical researches 21 ,3) ,41 the embryo of Triturus pyrrhogaster is a suitable material, because the developmental process is relatively slow and very typical. But it is very difficult to obtain enoughpure actin or myosin for the preparation of the anti-serum, since Triturusis a very small animal and has scanty muscle tissue. The possibility
to determine muscle proteins of Triturus by using the anti-sera formuscle proteins of White Leghorn was examined. Pure actin 5 ) andmyosin 61 were isolated from muscle tissue of Triturus or White Leghornby the method of Szent-Gyorgyi and the anti-sera of both proteins wereprepared by injecting them into the rabbit intravenously. The precipitin
test with anti-sera for Triturus muscle proteins was made as control.
The titre of both anti-sera was adjusted to 1:512.
Triturus embryos from 120 to 192 hrs. after fertilization were mashedin ice cold potassium chloride solution (0.6 M). The homogenate wasallowed to stand for one hour at ODC under constant stirring and thenwas centrifuged. Before carrying out the precipitin reaction with theanti-serum, the clear supernatant extract was placed in a collodiummembrane tube and treated with large amounts of distilled water for
48 hrs. at oDe, to eliminate the potassium chloride. Constant volume ofanti-sera and progressively decreasing amounts of antigen, ranging from
1:10 to 1:160 in weight, were used for preventing a possible failure due1) This work was supported by a Grant-in-Aid for Fundamental Scientific
Research (No. 710267) of the Ministry of Education in Japan
2) Ogawa, Y.: Nature, 181: 621, 1958.
3) Ogawa, Y.: Nature 186: 1312, 1958.
4) Ogawa, Y.: Exptl. Cell Res., 26: 269, 1962.
5) Szent-Gyorgyi, A. G.: J. Biol. Chern., 192: 361, 1951.
6) Szent-Gyorgyi, A. G.: Chemistry of Muscular Contraction, (1951) Acad. Press,
New York.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

Table 1.

Determination of developing skeletal muscle proteins in Trituru8 embryo
Lowest antigen concentration giving positive results

Hours
after
fertilization

Results given by the anti-sera
for Trituru8 muscle proteins(Control)

108
120
132
144
156
168
176
180
192

Actin

I

MyosinResults given by theanti-sera for fowls muscle
proteins
Actin

-

-

-*

-*

1 : 40
1 : 80<1: 160

1 : 80
1: 160< 1: 160-

I

Myosin--*-*

1: 160<1: 160< 1: 160

1: 160< 1: 160< 1: 160* Reaction at high antigen concentration (1:2) was added to confirm thenegative result.

to excess of antigen. The experimental results are given in Table 1.
The results obtained by the anti-serum for White Leghorn's muscle
proteins showed no marked difference from those found by the antiserum for Triturus muscle proteins. This finding may be useful inresearch work on muscle protein development in Amphibian embryos.

26.

Effects of p-hydroxy-benzyl-methyl-ether, a component of Citrullus
colocynthis, on growth and differentiation of Triturus embryo(By Yoshito OGAWA and Hiroyasu WATANABE)

P-hydroxy-benzyl-methyl-ether is a new compound. It was first isolated
in 1961 1 ) from the glycosid fraction of the fruits of Citrullus colocynthis
SCHRAD.
The influence of this substance on the early embryos of Trituruspyrrhogaster was examined, because this glucosid fraction shows an inhibiting action on the growth of animal tumors 2 ).
The embryos were raised in solutions of this compound (0.5%, 0.05%,
0.005% and 0.0005%) at 20°C from the 6th day after fertilization (Stage 20),and their body weight and length were measured. Furthermore, thegrowth progress with respect to organ formation was histologically1) Watanabe, H., A. Ide and Y. Ogawa,: Agr. BioI. Chern., 25: 269, 1961.
2) Ogawa, Y.: Ann. Rep. Natl. Inst. Genet., 9: 94, 1959.

41

RESEARCHES CARRIED OUT IN 1962

investigated. Non-treated groups were prepared for control.
It was found that p-hydroxy-benzyl-methyl-ether showed a significant
promoting effect on the growth of embryos while a normal balance inorgan formation was maintained. Its most effective concentration was
0.5 mg per 1000 cc. This promoting effect was observed approximately
on the 6th day after treatment (Stage 34), but it did not last longer
than 5 days. The difference between treated and non-treated plots
seemed to correspond to about two developmental stages, representing
a period equivalent to 54 hours at 20°C. The solutions of higher concentrations (0.5% and 0.05%) suppressed the growth of embryos andlower concentration (0.005%) showed no influence.
In order to examine the influence of the new chemical on the differentiation of early embryos, those of Triturus were raised in 0.005% solution
at 20°C (most effective concentration to promote the growth) and theformation of muscle proteins, actin and myosin, in the developing
embryos was investigated. The identification of muscle proteins was
carried out by serological technique as already reported 31 •In the non-treated control group actin and myosin first became detectable in early embryos 132 and 176 hrs. after fertilization, respectively.
But in the treated embryos, actin and myosin were first detected after
Table 1.

Muscle protein development in the Triturus embryo
treated with p-hydroxy-benzyl-methyl-ether
Lowest antigen concentration giving positive results

Hr. after
fertilization

Control
Actin

60
84
108
132
156
180
204
220
252

I

Treated
MyosinActin-

1 : 20
1 : 80<1: 160

Myosin

-

-

-*

I

-

-*

-

-

-*

-

1: 160<1: 160<1: 160-*

1 : 80
1: 160<1: 160

1: 10
1: 20
1: 40

* Reaction at high antigen concentration (1:2) was added to confirm thenegative results.
3) Ogawa, Y.:

Nature, 182: 1312, 1958.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

204 and 132 hrs., respectively. Thus, this chemical suppressed markedly
the synthesis of actin and promoted that of myosin, concerning formation rate and amount as shown in Table 1.
The order of formation of both proteins in embryos treated with phydroxy-benzyl-methyl-ether is therefore, reversed to that found in nontreated material. This result shows a similar tendency to my findings
concerning the effect of glucuronolacton 4J , x-irradiation 5J and temperature 6J on the development of muscle proteins during early embryonal
stages. Thus, another new evidence was found of the independence ofthe formation of actin and myosin.
4) Ogawa, Y.:5) Ogawa, Y.:
6) Ogawa, Y.:

Ann. Rep. Nat1. Inst. Genet., 11: 40, 1961.
Nature, 186: 77, 1960.
Nature, 194: 978, 1962.

27. Comparison of free amino acids among eye-color
mutants of Drosophila melanogaster(By Tomotaka SHINODA)

To obtain some information on the differences concerning utilizationof amino acids among eye-color mutants of Drosophila melonogaster,
free amino acids were compared.
Three mutant strains of Drosophila melanogaster, en, bw and en bw,
were cultured in the standard medium at 25°C. By homogenizing wholebodies of five day old flies with cold 0.7 M TCA at 4°C, free amino acids
were extracted. The resulting samples were analyzed by paper chromatography followed by paper electrophoresis at pH 6.6. Trinitrobenzene
sulfonic acid was employed in the quantitative determination of aminoacids. As a control, flies of a wild strain were treated in the sameway.
The result shows that (a) in en mutants alanine and glutamine are increased while serine and glycine decreased as compared with the wild
strain, (b) in bw mutants no significant increase of amino acid was foundwhile glutamic acid, glycine, histidine and proline were decreased, (c)in en bw mutants the values of cystine, glutamine and threonine wereincreased while proline, glycine and histidine were decreased.
Thus, there might be some differences in the utilization of amino acids
between wild and eye-color mutant strains of Drosophila melanogaster.

RESEARCHES CARRIED OUT IN 1962

43

Table 1. Free amino acid content in several mutant strainsof Drosophila melanogaster (/1 mol/lOO mg wet weight)
Mutant Strains
Amino acids

wd
en

Asp
Glu
Cys/2
Glun
Gly
Ser
Thr
Ala
j3-AlaPro
r- ABA
Tyr
Try
Phe
Met
Val
Leu/ILeu
His
Lys
Arg
Xl*
X2*

.44
1.56
.66
.74
2.04
1.35
.66
2.02
1.38
2.15
.38
.02
trace
.31
.17

.24
.11
1.35
.60
1.18
.57
.64

I

.34
1.26
.84
1.33
1.41
.60
.60
2.85
1.07
1.80
.42
.08
.04
.23
.21
.31
.16
1.05
.69
1.03
.51
.42

bw

.51
.95
.64
.68
.96
1.38
.46
2.03
.97
1.57
.45
trace
trace

.28
.08
.19
.21
.80
.50
1.08
.38
.30

I

enbw

.36
1.94
1.22
2.10
1.51
1.27
1.20
2.18
1.24
.68
.31
.04
.02
.21
.13
.27
.08
.93
.64
.98
.60
.39

* Not identified yet

28. Spectrophotometric determination of trinitrophenyl derivativesof amino acids and peptides resolved by paper
electrophoresis and chromatography(By Tomotaka SHINODA)As already reported by the present author, amino acids and peptide
contents in solution (0.01 to 1 fl mol) can be determined after quantitative trinitrophenylation with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS).
The present paper deals with an application of the TNBS technique

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

to quantitative determination of minute amounts of amino acids andpeptides resolved on paper by electrophoresis and chromatography.
Paper electrophoresis was performed at a constant potential gradientof 20 volt per centimeter, using a medium of pyridine-acetate system(pH 6.65), and n-butanol-acetic acid-water system was used as a solvent
for paper chromatography. The paper so developed was treated withbufferized TNBS solution, and the optical density of the solution wasdetermined at 340 mil in N HeI. The molar extinction coefficients of
TNP-amino acids were 1.2 to 1.5x 10', while in the case of peptides thecorresponding value was 1.05 X 104 for an amino group.
The amino acid composition of insulin and fractionated globins was
assayed by TNBS method. The result was in good agreement with thetheoretical expectation for the insulin with an error of ±4 per cent.
The present method was also applicable for the quantitative determination of total free amino groups in a polypeptide chain, and for limited
modification of proteins under physiological conditions. Further, thismethod was effective for modifying the bases in RNA and DNA.

29.

Paper electrophoretic heterogeneity of serum albumin(By Tomotaka SHINODA)

Homogeneity of serum albumin has been reported by many investigators. However, its heterogeneous behaviour in acidic medium has
also been reported. In order to study the heterogeneity of serum albuminin the alkaline range, the author employed paper electrophoresis combined with mediums which contained various concentrations of urea.
The pH range tested was from 7.4 to 11.6. The materials subjected
were crystallized human serum albumin (HSA) and crystallized bovine
serum albumin (BSA). Analytical paper electrophoresis of the materials
was carried out at constant potential gradient of six volts per centimeterfor three hours at 100e. The total ionic strength of the media was 0.1
in most cases and the concentration of protein solution to be analyzed
was 10-15 mg per milliliter in each case.
Under the conditions employed, both serum albumins were resolved
into two components, F and S, where F represents the fast moving
component toward the anode side, while S is the slow one.
The amounts of the two components varied according to urea concentration or to pH variation. Their "finger-printing" indicated that there
are no apparent differences in the tryptic digests between F and S.
The above results indicate that there are two components in albumins

RESEARCHES CARRIED OUT IN 196245

with different stability or reactivity to some chemical agents such asurea, guanidine etc., although these differences could not be detectedeven by "finger printing".

B.

GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF PLANTS

30. Genome manifestation of wheat in Aegilops cytoplasm(By Hitoshi KIHARA)

In order to investigate the effect of alien cytoplasm on genome manifestation, genome complements of 4x and 6x wheat have been introducedby successive backcrosses into the cytoplasm of Aegilops caudata or Ae.
ovata. The backcrosses have been carried out 14 times for the mostadvanced materials and only twice in the least advanced ones.
Influence of the alien plasma has been noticed in morphological andphysiological wheat characters. It was most remarkable in the floralorgans. When their genomes were introduced into caudata cytoplasm,
two varieties of 6x and four of 4x wheat showed complete or incomplete
pistillody and all became male sterile. Fourteen other varieties or strains
became also male sterile but no pistillody occurred. Those were sevenvarieties of 6x wheat, three of 4x wheat, three strains of synthesized
6x wheat and one Triticale. A 6x wheat, Triticum compactum var. No.
44, differed from the others in being normally male fertile. In general,genome manifestation of 4x wheat in this respect was more influenced
by caudata cytoplasm than that of 6x wheat. This was critically shownin the difference between 4x wheat (pistilloid) and the hexaploids (malesterile but not pistilloid) synthesized from the former as one of theparents.
Genomes of four 4x wheats were introduced into ovata cytoplasm, andall became male sterile. Pistillody did not occur in any strains.
Effect of the alien cytoplasm was also noticed in the increased occurrence of haploid and twin seedlings. The extent of the increase varied
greatly with the genotype.
31. Increased occurrence of haploids and twin
seedlings due to an alien cytoplasm(By Hitoshi KIHARA and Koichiro TSUNEWAKI)

The authors have been placing by successive backcrosses nuclei of46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

various wheats, including Triticale, in the cytoplasm of Aegilops caudata.
During these experiments it has been noticed that the alien cytoplasm
increased the frequency of haploids and twin seedlings. For convenience's sake wheat strains with their own cytoplasm are termed "autoplasmic" and those with Aegilops cytoplasm "alloplasmic".
Records on the occurrence of haploids are at present available for twovarieties, Triticum aestivum ssp. vulgare var. erythrospermum (T. vulgare
erythr.) and Taylor's Triticale, which are summarized in Table 1.
Table 1. Occurrence of haploids in auto- and alloplasmiclines of T. vulgare var. erythr. and Taylor's Triticale

Materials

T. vulgare erythr.: auto plasmic: alloplasmic
Taylor's Triticale: autoplasmic
II: alloplasmic
II

No. ofNo. ofNo. of Haploid freq.(%)
plantshaploids
plantsgrown (N) hecked (n)
(M)
MIN
Min

I

1,633
1,649
19
17

312
402
19
17

0
11
0
9

0.0
0.7
0.0
52.9

0.0
2.7
0.0
52.9

The occurrence of twin seedlings in auto- and alloplasmic lines of wheat
and Taylor's Triticale was studied in detail this year. The results are
summarized in Table 2.
Table 2.

Occurrence of twin seedlings in auto- and alloplasmiclines of wheat and Taylor's Triticale
No. of seedlings

Materials

Freq. of twin (%)
Total

Wheat: autoplasmic
II: alloplasmic
Triticale: autoplasmic
II: alloplasmic

13,644
580
54
98

I

Twin
10
5
0
7*

0.07
0.86
0.00
7.14

* One of the seedlings was a triplet.
The present results clearly indicate that the occurrence of both
haploid and twin seedlings in wheat and Triticale was increased by thealien cytoplasm introduced from Ae. caudata. The extent of the increase
varied greatly with the genotype.

RESEARCHES CARRIED OUT IN 1962

32.

47

Comparative gene analysis of common wheat andits ancestral species, I. Progressive necrosis(By Koichiro TSUNEWAKI and Hitoshi KIHARA)

Progressive necrosis in common wheat is controlled by three genes,
Nel located in chromosome 5B, Nez in 2A and Nea in 3D. Distribution'of those genes in common wheat and its ancestors, Emmer wheat andAegilops squarrosa, was investigated using Triticum aestivum Prelude'(haploid genotype: Ne1nezNe a), T. aestivum Kharkov (netNezNea) and T.
macha subletshchumicum (Ne1Neznea) as test varieties.
In Emmer wheat a majority of varieties were found to have the genotype Netnez, while minor fractions were either nelneZ or Ne1Ne z.
All strains of Ae. squarrosa so far tested had Nea.
In common wheat, most varieties were either nelne2Nea or netNezNea,
while a small fraction were Ne1nezNea. One variety only was found to
be NelneZneg and another only one to be NetNe2neg, both belonging to T.
macha.
From these results, the genotypes of Emmer wheat, that supplied theAB genomes to common wheat, are assumed to be NelneZ or ne1nez. T.
dicoccoides spontaneo-nigrum, some forms of T. dicoccum, T. turgidum,
T. persicum and T. orientale, and many varieties of T. durum have thesegenotypes.
The donor of the D genome to common wheat must have possessed
Neg. All strains of Ae. squarrosa so far tested had this allele.
The presumable hexaploid progenitor must have had either Ne1nezNeg
or netne2Neg. In common wheat, some forms of T. spelta, T. sphaerococcum, T. compactum and T. aestivum have these genotypes. T. macha,that is an exception in possessing the neg allele, is considered to be an
isolated species among the hexaploids and seems not to have contributed
to the origin of common wheat.
33.

Transmission of the monosomic condition through female
gametes in a common wheat, Chinese Spring(By Koichiro TSUNEWAKI)

Since 1955 the monosomics of the Chinese Spring series have been<:Tossed as female parents to a number of hexaploid wheats. In eachcross a limited number of F 1 plants were cytologically examined andmonosomic plants were selected. Up to the present about 150 plants
have been examined in each monosomic line.
In order to know the transmission rate of the monosomic condition

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13'
Table 1. Frequency of monosomics in the F 1 generationof crosses between 21 monosomic Iines* of Chinese
Spring (9) and various 6x wheat varieties (0)Freq. of monosomics (%)
Homoeologous
group
A
1
2
3
4
5
6
7

Within-groupheterogeneity'

Genome

78.1
63.4
75.2
66.7
79.7
75.5
66.7

I

B
64.8
68.4
77.2
72.7
69.4
55.5
68.8

I

D

I Average

(X 2)

75.8
66.4
79.7
68.8
70.7
77.9
69.3

72.9
66.1
77.4
69.4
73.3
69.6
68.3

7.04>1"*
0.94
0.90
1.33
4.75
23.37***
0.28* Monosomic lines are indicated by the genome and the homoeologous group.
to which the monosomic chromosomes belong. ** Significant at the 5% level.
*** Significant at the 1% level.

through the female, accumulated data on the occurrence of monosomics.
have been analyzed. The results are summarized in Table 1.
The three monosomic lines belonging to homoeologous groups 2, 4, 7,.
5 and 3 showed similar frequencies, namely low in the first three groups,
intermediate in the fourth, and high in the last group.
Two homoeologues of groups 1 and 6 showed high frequencies of monosomics, while the third member of both, namely 1B and 6B showed very
low frequencies. Nucleolus-organizers of common wheat are located on
chromosomes 1B and 6B. This fact seems to suggest that gametes or
zygotes lacking either of the nucleolar chromosomes are less viable thanthe other normal ones.
From the present results the following conclusions can be drawn: (1)Transmission rate of the monosomic condition through female gametes
is determined mainly by homoeology of the monosomes. (2) Nucleolusorganizing chromosomes show very low transmission of monosomic condition as compared with their homoeologues.

34. Chromosome number of Gramineae species collected
in Pakistan, Afghanistan and Iran(By Sadao SAKAMOTO and Mikio MURAMATSU*)

During the Kyoto University Scientific Expedition to the Karakoram* Curtis Hall, Univ. of Missouri, Columbia, Mo., U.S.A.

RESEARCHES CARRIED OUT IN 1962

SpeciesCollected
locality

49

I

No..ofChromosome
stra!ns number (2n)exammedI

Agropyron intermedium (Host) P.

Iran

1

56

A. trichophorum (Link) Richt

Afghanistan
and Iran
Iran

2

42

1

56

Iran

1

14

Brachypodium sp.

Iran

1

18

Bromus brizaeformis Fisch. et Meyer
B. Commutatus Schrader
B. Danthoniae (Des£.) Trin.

Iran
Iran
Pakistan,
Afghanistan
and Iran
Afghanistan
Iran
Iran
Iran
Iran
Afghanistan
Pakistan,
Afghanistan
and Iran
Pakistan and
Afghanistan
Afghanistan
Pakistan and
Iran
Iran
Iran

1
3
7

14
14
14

2
1
5
1
1
1
6

28
14
14

2

28

1

14
28

2
3

14,42

Iran

2

14

Afghanistan
and Iran
Afghanistan
Afghanistan
Afghanistan
Afghanistan
Iran

3

14

1
2
1
1
1

26
28
28

Beauv.?

A. sp. probably A. repens (Linn.)
P. Beauv. or A. intermedium
Alopecurus mysuroides Huds.

B. macrostachys Des£.
B. madritensis Linn.
B. racemosus Huds.
Cynosurus echinatus Linn.
Dactylis glomerata Linn.
Elymus dahuricus Turcz.
Eremopyrum buonapartis (Spreng.)
Nevski var. buonapartis
E. buonapartis (Spreng.) Nevski var.
sublanugin08um (Drob.) Melderis
E. distans (C. Koch) Nevski
E. orientale (Linn.) Jaub. et SpachFestuca elatior Linn.
Henrardia persica (Boiss.) C. E.
Hubbard var. persica
H. persica (Boiss.) C. E. Hubbard
var. glaberrima (Hausskn.) C. E.

2

16

28
42
14,28

14

Hubbard

Heteranthelium piliferum (Banks etSoland.) Hochst.

Koeleria phleoides (Vil!.) Pers.
Phalaris minor Retz.
Polypogon mon08peliensis (Linn.) Desf.
Taeniatherum asperum Nevski
T. crinitum (Schreb.) Nevski

14
14

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

and Hindukush in 1955, considerable numbers of wild and cultivatedGramineae species were collected. In this article, the chromosome numbers of those species, excluding Aegilops, Echinochloa, Hordeum, Lolium,
Secale and Triticum, are reported.

35.

Reciprocal translocations in Emmer wheat(By Kozo NISHIKAWA)

The meiotic chromosome pairing was analyzed in ten F hybrids among
five Emmer wheat varieties, T. dicoccoides spontaneo-nigrum, T. dicoccum
liguliforme, T. dicoccum arras (Khapli), T. dicoccum (Vernal) and T.
durum reichenbachii.
As shown in Table 1 in hybrids of spontaneo-nigrum and liguliforme
with Khapli, Vernal and reichenbachii, a quadrivalent which was eithera ring of four (55-72%) or a chain of four (45-28%) was found in morethan 85% of PMC's. Two quadrivalents which were also either of ring
shape (71.8%) or chain shape (28.2%) were observed in 94% of PMC's of
the hybrid between spontaneo-nigrum and liguliforme. The percentagesof quadrivalents presented here are much higher than those reported byprevious authors for comparable hybrids. In the other F/s which wereobtained among Khapli, Vernal and reichenbachii no quadrivalent was
found.
j

Table 1.

Chromosome pairing at first metaphase in ten Emmer wheat hybrids

I bachii
ntgrum
I spo!"taneo-

reichen-

KhapliVernalIliguliforme
2lV +lO ll
l rv + 12n
bv+ 12Il

spontaneo-nigrum
reichenbachii-

l rv +12n--

l rv + 12Il
14Il

KhapliVernal---bv+ 12 1I
14Il
1411

-

-

-

-

bv+12nliguliforme

-

-

-

-

-

From these results, it is concluded that the quadrivalents are attributable to reciprocal translocations. Thus, four cultivated varieties of
Emmer wheat used here have at least one reciprocal translocation incommon which is not found in the variety of wild Emmer, T. dicoccoides spontaneo-nigrum, and in addition to this one liguliforme carries
another reciprocal translocation.

RESEARCHES CARRIED OUT IN 1962

36.

51

Production of polyploid wheat by nitrous oxide, II. NzO-treatment
during fertilization with reference to culture media(By Koichiro TSUNEW AKI)

For the improvement of the practical use of nitrous oxide, the following three points have been investigated in wheat: (1) Effect of NzO
applied in the course of fertilization, (2) production of amphidiploids
directly from cross-pollinated florets, and (3) supplementation of culture
medium for treated spikes by glucose and penicillin.
NzO-treatment of pollinated florets did not disturb fertilization, which
seemed to proceed rather normally in NzO. Polysperm fertilization resulting in triploidy was not induced. NzO appeared to inhibit specifically
cell divisions after fertilization, producing polyploid plants. By suitable
treatment, all resulting seedlings became polyploid.
Amphidiploids can be directly produced by treating cross-pollinatedflorets with NzO. This was demonstrated in the cross, Triticum aestivum
x Secale cereale.
Supplement of glucose as a carbon source to culture media of the NzOtreated spikes had only a slight effect on seed setting but improved
markedly the endosperm development and, consequently, germination.
This, in turn, allowed polyploid embryos a better chance to survive.
Supplement of penicillin as an antibiotic to the culture media improved
neither seed setting, endosperm development nor germination, but anincrease in polyploid occurrence was noticed. Its cause must be further
investigated.

37.

Photoperiodic responses of Oryza species V ll
(By Tadao C. KATAYAMA)

Several factors are responsible for different photoperiodic sensitivities
among various Oryza strains. Among them, the aging effect and thecritical day length were considered in the previous reports (Ann. Rep.
Nat. Inst. Genet. 11: 48-49, and Ibid. 12: 57-58). In this report, the difference of photoperiodic sensitivity was analyzed from the viewpoint ofdifferential heading acceleration of various strains under short day condition. 68 strains belonging to eleven Oryza species were used in thisinvestigation.
1) This work was supported by Grant RF 62027 from The Rockefeller Foundation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

Three different methods for expressing the acceleration rate of heading
date were devised: (1) by the coefficient of linear regression of growing
period on sowing time (denoted by LRC-l), (2) by the coefficient of linear
regression of growing period on day length at the time of flower bud
formation (LRC-2), and (3) by the angle of the slope of the regression
line used in (2) (A-LRC-2).
The three methods were applied to the data obtained from 16 strains
and they were all found to agree well with the previous classificationof those strains into sensitive and insensitive. The border line between
the two groups was found to be 0.5 for both LRC-l and LRC-2 and 30°
for A-LRC-2.
LRC-l indicates acceleration rate of heading date (in number of days)
due to delaying the sowing time by one day, while LRC-2 or A-LRC-2
represents acceleration rate of heading due to shortening the day length
by one minute; the latter is biologically more significant than the former.
It was found that LRC-2 is less suitable than A-LRC-2 for estimating
differences in photoperiodic sensitivity. For these reasons, it was concluded that A-LRC-2 is the best index among the three. With the helpof this method, the correct degree of photoperiodic sensitivity was determined for each of the 16 rice strains.
Using index A-LRC-2, the acceleration rate of heading was investigatedin another experiment with 54 strains. It was found that 39 strains
were sensitive and the rest were insensitive. This classification is inperfect agreement with the previous one based on a different method.
Regardless of the species, strains that have short critical day length
showed in general greater sensitivity than those having long critical day
length. This finding explains why in low latitudes sensitive strains withshort critical day length predominate; their sensitive response to a smallchange in day length gives them a selective advantage.

38. Developmental instability in tobacco-plants(By Kan-Ichi SAKAI and Yoshiya SHIMAMOTO)

Investigation of developmental instability was conducted on foliar andfloral characters in Nicotiana tabacum L.
Using eleven varieties, three foliar and three floral characters wererecorded as measures of instability in organ development. They are: (1)fluctuation in vein distance, i.e., within-leaf fluctuation in distance between two adjacent veins, (2) indentation index, i.e., degree of non-

RESEARCHES CARRIED OUT IN 1962

53

parallelism of two adjoining veins measured by the difference between
the widest part and the narrowest one divided by the average length of
two veins, (3) asymmetry, Le., the absolute value of difference between
left and right half of leaf blade, (4) intra-plant variability of style length,
Le., flower to flower variation in length of styles, (5) intra-plant variabilityof filament length, Le., flower to flower variation in average length offive filaments and (6) intra-flower variability of filament length. Measurements in the first character were expressed in terms of coefficient ofvariability while those in the last three in terms of standard deviations.
Three plants of each variety were taken at random. Measurement offoliar attributes was made on all leaves from the bottom to top of eachplant, while for floral attributes, twenty flowers were used which werecollected at random from each plant.
By the analysis of variance of data obtained, it was shown that variation among varieties was highly significant for all characters, except
intra-plant variability of filament length. Developmental instabilities inthe three foliar characters were found to be positively correlated witheach other and the same was the case for the three floral characters.
It was found, however, that instability in foliar characters and that infloral characters did not appear to be parallel, the repeatability being
so low as zero.
In order to find out correlation, if any, between foliar and floral instabilities, instability-indices for foliar and for floral characters werecomputed by the principle of the discriminant function. The correlation
coefficient between the two instability-indices was negative and low with
r= -0.3327, suggesting that the degree of instability in foliar charactersand that in floral characters may be independent from each other.
Of interest is that the examination of varieties in respect of developmental instability in foliar characters showed that high stability was
found in two tobacco varieties used for cigar production while our commercial varieties widely grown and highly productive were rather unstable in foliar characters.

39. A method of estimation of genetic parameters
in forest trees without raising progeny(By Kan-Ichi SAKAI and Suekichi HATAKEYAMA)

A method of estimating heritability and genetic correlations of quantitative characters in forest trees has been investigated. The underlying
principle is that in hybrid forests, variation between cluster means

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

consists of one-x th of t he genetic variance and one- x b th of the environmental variance, x being number of trees included in each cluster andb being a function of the variation pattern of the environmental conditions in the forest. b is expected to lie between zero and unity.

GE

V(;)=-+bx xV,X) denotes the variance of means of clusters, each including x trees,and G and E stand for genotypic and environmental variances, respectively. The formula can be rewritten as
xV(X)=G+X1-bE=G+x B E

where B= I-b. By repeated grouping of adjoining trees into clustersof various sizes, we get a number of simultaneous equations involving
three unknown quantities, G, E and B. The trial and error method isadopted to find B o value which gives the best fit between observed meansquares and expected ones which are computed from the values of G
and E obtained by the least squares method with a given value of B.
If Go and Eo with B o are obtained, then we can estimate the value ofheritability. Genetic and environmental components of covariance are
obtained in the same manner by replacing variances with corresponding
covariances, and genetic correlations are computed therefrom.
Our investigations of three forests, one being a clone of Populus euramericana, the remaining two consisting of seed-propagated Abies sachalinensis, gave the following results: G values calculated from the Populus
clone were so small as regarded as zero, suggesting the validity of themethod. Heritability values calculated for five characters in the two
Abies forests which were genetically not related at all, were reasonably
comparable for the same characters though b values were rather different.
Genetic correlations could also be computed in the two forests. Thus,
this method is considered to be usable for genetic investigation of forest
trees. The advantage of this method over others is that it requires
neither clonal forests nor families grown from randomly selected mothertrees. Accordingly, hardships involved in genetic studies with forest
trees due to extremely long time periods required for growth can besurmounted by this method of analysis. The details will appear in
Silvae Genetica before long.

40. Cytoplasmic effect on the inheritance of seed weight in Pharbitis Nil(By Kan-Ichi SAKAI and Akio SUZUKI)

We have noticed that the weight per seed of morning glory, Pharbitis

RESEARCHES CARRIED OUT IN 1962

55

Nil., is matroclinously inherited. This report deals with the result ofa breeding experiment of reciprocal crosses between two varieties U and
H, differing from each other with respect to seed size. The weights
per seed in miligrams in different generations after the hybridization
were as follows:

P
F1
F2
F3

x(Hx U)y(UxH)

xo=26.9xl=35.5x2=42.5x3=45.8

yo=52.6
Yl=42.6
Y2=46.6
Y3=47.4

Second degree statistics in different generations were calculated andestimates of the coefficient of maternal effect, m, were computed therefrom. The formulas used for this estimation were those given by
Chandraratna and Sakai (1960: Heredity 14: 365-373). It was found thatm for H was 0.51 while that for U was 0.38. The regression of F 3 line
means upon F 2 measurements was also computed. The regression coefficient in Hx U was 0.658 while that in Ux H was 0.438, again suggestingstronger effect of H than of U cytoplasm.
Weight per seed in F 2 plants was found not to be correlated with thenumber of seeds per capsule, but negatively correlated with the total
number of seeds per plant. The correlation coefficients were as follows:
Weight per seed and

UxH

HxU

Average

Number of seed per capsule
Total number of seed

-0.17
-0.31*'"

0.20-0.37**

0.014
-0.34

It seems rather reasonable to conclude that seed weight may beaffected by cytoplasm, which may bring about increase or decrease in
the total number of seeds produced per plant.

41.

Effect of genotypes and cultural conditions on quantitative
characters of tobacco varieties(By Kan-Ichi SAKAI and Yoshiya SHIMAMOTO)

Seven varieties of tobacco plants, Nicotiana tabacum L., were tested
for their response to four growing conditions in two successive years,
1961 and 1962. The purpose of this experiment was primarily finding

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

out the relation between different characters in different genotypes
exposed to different growing conditions.
The treatment variables were: (1) spacing between hills, (2) doses offertilizer application, (3) topping effects and (4) sowing dates. Charactersmeasured were the total yield in grams of fresh leaves, content ofalkaloids in dry leaves, number of leaves per plant, length and width oflargest fresh leaf, thickness of dry leaves and diameter of stem.
Analysis of variance of data revealed that varietal differences as wellas treatment effects were statistically significant for every character.
Interaction between treatment and variety was in some cases significant while not in others.
Computation of genotypic correlation showed that alkaloid contents
and leaf thickness were positively highly correlated with r=0.7022-0.9048
while alkaloid contents and yield were negatively correlated with r=-0.8043--0.8876.
Environmental correlations among different characters in different
genotypes under different growing conditions were investigated by partial
correlations. It was concluded that alkaloid contents were in most cases
positively and highly correlated with leaf thickness, suggesting thatgenetic as well as environmental factors producing thicker leaves may
bring about a higher content of alkaloids, in other words, a common
factor might be responsible genetically as well as environmentally forthe two characters.

42. Studies on developmental instability of quantitative charactersin X-rayed progenies of rice
(By Kan-Ichi SAKAI and Akio SUZUKI)

One hundred and sixty X, lines derived from a single rice variety
were grown in comparison with the same number of control lines selected
at random from the same variety in two replicated plots with twelve
investigated plants in each plot. The present report describes the occurrence of increased developmental instability in various quantitative
characters due to X-ray treatment. The characters investigated werelength and width of seed, plant height, weight per plant, culm number
per plant, number and length of panicles per plant and heading date.
It was found that intra-plant variability of seed length and seed width
measured in terms of within plant standard deviation increased significantly in the X-rayed lines. The same was found for intra-plot variabilityof other quantitative characters in X-rayed lines. The between-line

RESEARCHES CARRIED OUT IN 1962

57

genetic variability in these was significantly higher than in control
lines, suggesting polygenic mutations occurring in all characters.
There was a general impression that with respect to polygenic mutations, X-ray treatment caused them to occur in minus direction,
while developmental instability was mostly directed toward increase by
X-ray treatment. In addition, mutations in developmental instability
were not necessarily associated with quantitative ones of the given
character.
From the result of this experiment, we conclude that there should begenes or genic systems responsible for developmental stability which
can mutate by the effect of X-rays resulting in deficient regulation oforgan development or "buffering against developmental noise," according
to Waddington's terminology (1957).

43. Intra-clonal variation of pathogenicity in Piricularia oryzae(By Iwasaburo GOTO and Kan-Ichi SAKAI)

Pathogenicity of rice blast fungus, Piricularia oryzae, has been known
to be not a stable character, but variable according to the culture conditions of the fungus. It is the purpose of this report to describe resultsof our observations concerning seemingly intra-clonal pathogenicity
segregation in the fungus. The method of investigation consisted ofisolating a certain number of subclones or sub-subclones from clonalraces and examining intra-racially their pathogenicity each on five or
more plants of a rice variety. The number of initial clonal races was
six, each started from single spore isolates. They were different fromeach other with respect to pathogenic reaction of the host plants. Fromeach clone were thirteen subclones isolated in the first experiment byplate-culturing and they were tested for their pathogenicity under
microscope by sheath inoculation. The seedlings to be inoculated weregrown under as uniform conditions as possible in zinc cases kept in thegreenhouse. Analysis of variance of the data obtained showed that infour of the six clonal races, variation among subclones within the sameclone was statistically significant. One of the four clonal races, named
K-373, which showed significant variation among subclones was selected
and again tested for intra-clonal variability with 15 subclones by three
replications. The result of experiment was quite in accord with thefirst test in that the between-subclone variation was statistically highly
significant.
Three subclones of K-373 were again divided into 7 or 10 sub-subclones

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

each and inter-sub-subclone variation was tested. It was then foundthat the inter-sub-subclone variation was highly significant, the variation
being so great that variation among parental subclones tested against it
failed to reach the level of significance. The examination of correlation
between pathogenicity of subclones and that of their sub-subclones isleft to future investigation.
From the results of this experiment, we may state that a segregation
seems to occur between vegetative isolates from a clone. For the present,
we are unable to explain the phenomenon, but it is doubtless that it
deserves further study not only from the standpoint of plant pathology
but also genetics.

44. Studies on blast-disease susceptibility of wild andcultivated groups of rice
(By Iwasaburo GOTO and Ken·lchi SAKAI)

Two cultivated species of rice, Oryza sativa, including indica andjaponica groups, and O. glaberrima, are thought to have developed fromO. perennts-O. spontanea and O. breviligulata, respectively. This paper
deals with a comparative study of those species with respect to susceptibility to the blast-disease fungus, Piricularia oryzae. The strain of
fungus used is 53-33, kindly supplied by the National Institute ofAgricultural Sciences, Tokyo. This strain has been isolated from a
population collected in Japan, and accordingly, it seems to be more
adapted to Japanese varieties of rice than to other forms. In descriptions
to follow, Japanese varieties will be dealt as a control, and comparison
will be made among the other groups of species. The evaluation ofsusceptibility of rice plants was made by the method of leaf-sheathinoculation.
Comparison among 90 varieties of O. sativa and O. glaberrima showedthat O. sativa indica was less susceptible than O. sativa japonica andO. glaberrima. Among different local groups of O. sativa indica, varieties
from South America tended to be more susceptible than those collectedfrom Asiatic countries.
Comparison between cultivated and wild forms revealed no substantial
difference in general. A distinct difference, however, was observed between
two species groups, glaberrima-breviligulata and sativa-perennis, the former
being apparently more susceptible than the latter.
Genetic variability between varieties or strains within species orsubspecies was not much different from that observed between species,RESEARCHES CARRIED OUT IN 1962

59

except for O. sativa indzca which showed the least variability. Thismeans that so far as susceptibility to the present fungus race is con·cerned, varieties belonging to indica-group were rather uniform though
they were collected from different continents.
Three populations of wild rice of perennis-spontanea group were
investigated for disease susceptibility. They differed from each other,but variability in each population was quite apparent. The effect ofindividual selection was investigated. It was then found that variation
contained in each population was heritable, the heritability being so highas 0.7.
By testing individuals of a wild population of O. perennis for threeraces of Piricularia oryzae, it was found that a wild population involved
different genotypes with respect to reaction against different races. Theinteraction between genotypes and pathogenic races was statisticallysignificant.
Degree of inherent susceptibility, that of environment-respondent
susceptibility and the degree of interaction between the two were investigated (For details see the preceding issue of this Report, No. 12,1962). Degree of inherent susceptibility (Dc) was very low in japonicagroup. Among other groups, indica-group was the highest, breviligulata
and glaberrzma being intermediate and perennis-spontanea group was thelowest.
The same tendency was again found for the degree ofenvironment-respondent susceptibility (Db)' Degree of interaction between
the two was positive in japonica group, but negative in all other groups.
Conclusions drawn from this study are: (1) O. glaberrima and its wild
ancestor species are more susceptible on the average than O. sativa andits ancestral wild species. (2) Wild populations of O. perennis-spontanea
involved a good deal of genetic variability. (3) They also involved
different genotypes with respect to reaction to different races of Piricularia oryzae. (4) Variation in the inherent susceptibility was small inJapanese varieties, while indica varieties involved the highest variability.
(5) Indica varieties were again variable with respect to environmentrespondent susceptibility.

45.

Natural selection experiment in populations of wild rice,
Oryza perennis(By Hiko-Ichi aKA and Wen-Tsai CHANG)

This experiment was carried out at Taichung, Taiwan, with three
populations of Oryza perennis, i. e., a spontanea type (India), a perennis

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

type (India), and a hybrid-swarm type (Taiwan) population. They weregrown in bulk for three generations in an experimental field, and werecompared with the populations raised from the original seeds, regarding
the degree of grain shedding, grain weight, seed dormancy, and othercharacters. The results showed that the population genotype of perennis
type, as well as that of the hybrid swarm, had changed toward cultivated rice in three generations, but that of spontanea type remained
unchanged. This indicates that populations of wild rice readily respondto natural selection under cultivation, if they contain a large enoughamount of genetic variation.

46. Some considerations on the evolutionary dynamics of cultivated rice
(By Kokichi HINATA and Hiko-Ichi OKA)This study was attempted with the view to looking into the evolutionary
dynamics of cultivated rice. A review of the previous theoretical studies
on partly self-pollinated plant populations was given. Further, thewriters' computations were reported dealing with the effects of seed
dormancy and vegetative propagation on population structure, and thechange in frequency of a gene controlling selfing probability. It waspointed out that the breeding system of the perennis type of wild rice,which is partly cross-pollinated, capable of propagating vegetatively andhas long seed dormancy, would be quite suitable for accumulating genetic
variation in its populations. It was also suggested that selfing genes
can increase by themselves unless counteracted by another force favoring
the heterozygotes. Therefore, if the habitat assumes conditions ofcultivated fields and homozygous plants have relatively an advantage,
the populations will tend toward selfing. (Pub!. in Jap. Jour. Genet. 37:
329-342, 1962).

47. F 2 segregation ratios in a hybrid between tetraploid Oryza
sativa and glaberrima(By Hiroko MORISHIMA and Hiko-Ichi OKA)

Observations of the F 1 hybrid between induced tetraploid strains of
the two species were reported by Hinata and Oka (Ann. Rep. 12: 1962).
This year, about 500 F 2 plants were observed with regard to endosperm
character, phenol reaction, pollen fertility and other traits. Few recessive homozygotes were found, their frequency being lower than 1/36

RESEARCHES CARRIED OUT IN 1962

61

expected from random chromosome pairing. The frequency of simplex
heterozygotes for the glutinous gene, estimated from the frequency ofglutinous pollen grains in each plant, was also lower than expected.
There might be a slight tendency to preferential pairing of chromosomes
derived from the same parent. Pollen fertility of most of the plants
ranged between 35% and 70%.

48. Comparison of evolution modes of cultivated forms derived fromtwo wild rice species, Oryza breviligulata and O. perennis(By Hiroko MORISHIMA, Kokichi HINATA and Hiko-Ichi OKA)

Two cultivated rice species, Oryza sativa L. and O. glaberrima Steud.,seem to have independently arisen from their respective wild progenitors, O. perennis Moench (Asian perennis type) and O. breviligulata Chev.
and Roehr., for the following reasons: First, the two species series,
called Sativa (sativa and perennis) and Glaberrima (glaberrima and breviligulata), are separated by a pronounced F, sterility barrier, and secondly, in certain particular regions continuous arrays of intergrades arefound connecting perennis with sativa and breviligulata with glaberrima.
The patterns of intra-specific variations and population structure of O.
breviligulata were compared with those of the Asian forms of O. perennis.
The results suggest that between the two species series certain similarities and differences may be pointed out regarding the mode of evolutionof cultivated forms, which seem to be partly attributable to different
breeding systems of the wild progenitors. (Publ. in Evolution 17: 170181)

49. Cytogenetic studies in the genus nicotiana XV.
Reduction divisions in three interspecific hybrids and one amphidiploid(By Y6 TAKENAKA)

The reduction divisions in PMC's were studied in three hybrids, N.
paniculataxN. benavidessi, N. debneyixN. glutinosa and N. tabacumxN.
forgetiana, and one amphidiploid of N. rustica X N. tabacum produced bycolchicine treatment.
1) F, of N. paniculata (n= 12) X N. benavidesii (n= 12).
At MI of PMC's of this hybrid, twelve bivalents were usually counted
whose partners were of same size and shape and very seldom elevenbivalents and two univalents were found. At 2nd division, a few ir-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

regularities were very rarely observed such as a chromosome bridge
and one to two chromosomes or a fragment strayed in the cytoplasm.
In the same hybrid, Goodspeed (1954) found that twelve bivalents
were the norm and univalents, trivalents and quadrivalents occurredvery rarely. Although there are some differences between Goodspeed's
observations and mine, considering the findings mentioned above, thetwo species are closely related but it is thought that the genomes ofN. paniculata and of N. benavidesii are somewhat differentiated by Inversions, translocations and other irregularities.
2) F 1 of N. debneyi (n=24)xN. glutinosa (n=12).
So far as I know, this hybrid has been previously reported. The
nuclear plate in MI of PMC's of this hybrid consisted of bivalents andmany univalents and it was somewhat difficult to count them clearly.
But the number of univalents did not reach generally to one half ofthe total chromosome number of 36 and many bivalents were observed.
Therefore it is assumed that N. debneyi is somewhat related to N.
glutinosa on one hand but on the other hand intra-genomic bivalents
may be formed in the former, since it is probably of amphidiploidorigin.
3) F 1 of N. tabacum (n=24)xN. forgetiana (n=9).
So far as I know, no report on this hybrid had been published. At
MI of PMC's of this plant, the bivalent range was from 1 to 8, withthe mode at 3. The frequency of PMC's with 4 and 5 bivalents followedthat of PMC's with 3 bivalents. PMC's with 2, 6, and 7 bivalents wereoccasionally found but those with 1 and 8 bivalents were very rare.
Some of them may be due to autosyndetic affinities between the twosubgenomes of N. tabacum which is of amphidiploid origin and others
may be caused by allosyndetic affinities between the genomes of theparents. Accordingly N. tabacum is assumed to be slightly related to

N. forgetina.
4) Amphidiploid hybrid of N. rustica (n=24)xN. tabacum (n=24).
This amphidiploid was produced by colchicine treatment of F 1 of N.
rustica x N. tabacum with the purpose of breeding a new strain containing the genomes of N. tabacum and N. rustica. But this plant was
highly sterile and the offspring showed segregation for various characters.
At MI of PMC's of this amphidiploid, the number of univalents ranged
from to 12, with the mode at 4. The frequency of PMC's with 2, 3,
5, 6, 7, 8 and 9 univalents followed that of PMC's with 4 univalents,
and PMC's with 0, 1, 10, 11 and 12 univalents were rare.
F 1 of N. tabacum x N. rustica showed the bivalent range from to 10°°

RESEARCHES CARRIED OUT IN 196263

with the mode at 4 (Takenaka, 1955). Accordingly, it is assumed thatin the amphidiploid allosyndetic affininity between the genomes of theparents leads to conjugation.

50.

Tumorous hybrids in nicotiana(By YO TAKENAKA and Yoshiaki YONEDA)

For researches on phyletic relationships of Nicotiana species and introduction of useful genes into tobacoo breeding, we have produced
many interspecific hybrids from 1950 to 1962. Among them the following ten tumorous hybrids were found, four of which are new (Asterisk),
as far as we know.
( 1) F, N. glauca X N. langsdorffii( 2) F, N. glauca x N. longiflora( 3) F, N. glauca X N. plumbaginifolia( 4) F, N. paniculata X N. langsdorffii( 5 ) F 1 N. rustica X N. cavanillesii(6)

F 1 N. suaveolensxN. langsdorffii(7) *F, N. suaveolensxN. plumbaginifolia( 8) *F, N. gossei X N. alata( 9) *F 1 N. gossei X N. longiflora(10) *F, 4x N. tabacum X N. alata
Kostoff (1930) first observed tumorous hybrids in Nicotiana and reported sixteen of them (1941-1943). Since his observations, some researchers found new non-parasitic tumors as well as those observed byhim in seven interspecific Nicotiana hybrids (Whitaker 1930, Brieger
and Roster 1942, Kehr 1950, Hilter and Izard 1951).
Nat (1958) also found four tumorous hybrids in Nicotiana and summarized all such hybrids in a table from the results mentioned above.
He advocated the establishment of one plus- and one minus-group
among Nicotiana species, indicating that specific hybrids between thetwo groups produce non-parasitic tumors, whereas the hybrids withineach group do not.
Although his classification is not yet completely confirmed, it is very
interesting. According to NiH, N. alata and N. longiflora belong to theplus-group and therefore N. gossei must be placed in the minus-group,
since we newly obtained the following two tumorous hybrids, F 1 N.
gosseixN. alata and F, N. gosseixN. longiflora. In fact, N. gossei isclosely related to N. suaveolens which is a minus-species. Although
Nat observed no tumor in a hybrid between N. suaveolens (minus-species)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

and N. plumbaginifolia (plus-species), the same hybrid produced by ourselves showed marked tumors on stems and roots.
Kostoff (1930) found non-parasitic tumors in F 1 of N. tabacum X N.
alata, and we also found some tumors in F 1 of 4x N. tabacum X N. alata.
We succeeded in obtaining in vitro cultures of tumorous tissue fromstem of F 1 N. glauca X N. plumbaginifolia and preliminary studies havebeen started with this material.
The tissues grew slowly and were rather compact in texture when
they were cultured on White or White+NAA agar media, while they
grew rapidly and had soft and crumbling texture on White + yeast ex·
tract, White+yeast extract+2,4-D and White+coconut milk+2,4-D agar
media.
Differentiations of shoot-like structures were observed mainly on
White or White+NAA agar media in the dark at 25°C after second to
third transfers from isolation. On rare occasions, some roots were produced directly from the cultured mass on semi-solid medium containing
NAA. This phenomenon is now under study. Concerning this problem
Skoog (1944) reported that roots only arose secondarily on differentiatedstems of the cultured tumor tissue of N. glauca X N. langsdorjfii. In
light, the cultured tissues became green to pale green and showed nodifferentiation on all media tested.

51. Effects of extracts from Aralia elata Seem.
and Artemisia vulgaris L. var. indica Maxim. on cell dzvisionin living plant tissue and growth of rat tumor.
(By Y6 TAKENAKA and Yoshito OGAWA)

The extracts of Aralia elata Seem. and Artemisia vulgaris L. var.
indica Maxim. were investigated regarding their effects on cell divisions
in root tips of Allium scorodoprasum var. viviparum and on the growthof Yoshida sarcoma.
Leaves of Aralia elata were treated with methanol and stems andleaves of Artemisia vulgaris were extracted with ethy-ether at room
temperature. The yields of methanol and ethy-ether fractions of bothplantsl were 7.6% and 4.8% of dried original tissue weight, respectively.
Warler solutions of various concentrations were prepared from theabove two extracts and living roots of A. scorodoprasum var. viviparum
were placed in the solutions for 4 to 24 hours. Living root-tips weretreated according to the hydrocloric-acetic acid-orcein method and observed under the microscope. Water solutions of Aralia elata extract

RESEARCHES CARRIED OUT IN 1962

65

caused faint radiomimetic phenomena after 4 hour treatment with 4%
solution; they were remarkable after 4 hour treatment with 8% solution.
Using the extract of Artemisia vulgaris var. indica, remarkable radiomimetic phenomena were found in the dividing cells after only 4 hourtreatment with 0.5% water solution.
24 hrs. after transplantation of the ascites tumor, both fractions were
injected subcutaneously at the axilla (0.5 g per 100 g body weight ofrat) and their influence on the sarcoma was observed in respect toitoxicity, systemic symptoms (lack of appetite, ascites, icterus andmetastasis) and prolongation of the host's life.
The extract of Aralia elata produced some inflammation at the siteof injection and some collapse of the regional tissue was found in thefirst 7 days after injection. But the group treated with this extract
showed a remarkable recovery in systemic symptoms 5 days after injection and 50% of the treated animals remained alive for 14 days,
though the non-treated control group died out within 10 days after
transplantation.
The extract of Artemisia vulgaris showed no remarkable toxicity except for a slight inflammation at the injected site observed only in thefirst three days after injection, but no effect was found on the prolongation of the life of rats bearing the sarcoma.

52. Experiments with carnations(By F.A. LILIENFELD and K. SUZUKI)

1. A case of albo-variegation
In 1958 a commercial seed sample of Dianthus chinensis L. was sownas No. 51. Fifteen plants raised had fairly uniform appearance and one
was selected for further propagation. Among its offspring (No. 72) a
considerable number of plants, namely 14 of 63, were in varying degree
albo-variegated. Variegation started with the first leaf whorl (cotyledons were green) and continued upward through several whorls
with increasing participation of green, until apparently uniform green
pigmentation was reached. Only exceptionally a plant remained invariegated condition throughout the whole development. Later experience showed that such plants sometimes formed green, colorlessand variegated branches.
Offspring of a green, a medium variegated and 2 highly variegatedplants of No. 72 are given in the table. As the table shows, even an
apparently green plant like 72.16 was throwing off variegated plants,

~

~
Z
c:
~

Table
Material

Offspring of No. 72, selting and intra-cross

::tl
t'j

No. 72(commercial sample)
49 green, 14 var.

72.16 green, self

72.2 var., self

72.8 high var.,
self

I

I

I

No. 77

No. 7518 green, 5 var.

1 green, 20 var.

No. 76o green, 20 var.

76B high var.,self

72.16x72.8 F 1

It

No. 84o green, 15 highvar.

No. 7810 green, 7 var.

~
~

~
Z

~

~
~

Other crossesZ(fl

No. 80(another commercial
sample) green
D.8uperbu8

green

76B(var.)xSO.l
green

I

80.1x76BI

76Bx80.5I

80.5x76BI

72.1(var.)xD.8up.

I

~

::J

c:
~

No. 85 (F1)

No. 86 (F1)

No. 81 (F 1)

No. 87 (F 1)

No. 79 (F 1)t'j

all green

all gre,en

2 pits green

all green

all green

o"%j

I

I

G"l

t'j

No. 88 (F2)

No. 83 (F2)Z

31 green, 9 var.

12 green, 5 var.,
3 albino(fl~......

(")

z9......

w

RESEARCHES CARRIED OUT IN 196267

while the 2 throughout variegated gave only variegated offspring (72.8
and 76B). The green 72.16 was crossed by high variegated 72.8; thecross yielded green and variegated, more of the latter than were obtained from 72.16 selfed. When branches of a highly variegated plant
like 76B showed different degrees in variegation, the differences werereflected in the progeny (colorless branches gave no seeds).
The above observations indicated the presence of a highly mutablegene in the whole original sample. This was confirmed by further
crosses with D. chinensis from another source and D. superbus L. As
may be seen from the table, the F /s obtained from both directions ofcross 76B (high variegated) X chinensis (80.1 and 80.5) were entirely green.
In F 2 31 green and 9 variegated were obtained. Likewise, 72.1 (variegated) x D. su perbus gave a green F I and in F 2 variegated plants weresegregated. The investigation will be continued.
2. Prickly leaf
In one of the further generations from crossing experiments with" ise-nadeshiko" (D. chinensis L. var. laciniatus Koern.), a plant was
found with many scattered projections on the upper leaf side. They
remembered of prickles or spines except for the blunt tips. Theseemergences were due to local hyperplasia of parenchyma and palisadetissues. A fully developed prickle consisted of a green base and acolorless blunt tip from which sometimes secondary small processes
were arising. Inside, a well developed system of spiral tracheary elements was found. The whole plant had stunted habit and dark green
leaves. Its seeds did not germinate. The plant was crossed by atypical chinensis, of very different growth habit, having long stems andlight green leaves. Only two seeds germinated and the two F I plants
showed predominance of the chinensis parent. They had no prickles
but only once in one of them a very weak indication of the abnormality
was recorded. This F I plant is still alive and all its leaves are normal.
In F 2 (No. 92) 24 plants were obtained; their growth habit was ingeneral like that of Fl' No plant approximating the prickly parent
was found in the small F 2 • Since the first observations revealed insome plants only very weak indications of the abnormality, records
were taken 10 times in succession. The plants which continuously, or
almost so, showed sure, though weak, indications of prickles were classified
as corresponding to prickly homozygotes. Six such plants were foundand five more were alternately recorded as with or without indicationof prickles. On this assumption the abnormality was simply segregating (18 normal + doubtful: 6 with indications). The fact that in F 2 theabnormality was only weakly expressed may be attributed to the entire-

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

ly different genetic background in respect to growth habit introducedby the cross.

53. Comparison of flower color inheritance in pansy
and morning glory(By Tom ENDO)

It has been well-known that pansy and morning glory exhibit anabundant variation in flower colors, the former varying in bright cyanic
and acyanic colors and the latter in dull cyanic ones. The historicalsurvey of Wittrock (1896) undoubtedly shows that most of flower color
variations in pansy resulted from hybridization among some Viola species,
mainly V. lutea and V. tricolor, in the early days of nineteenth century.
On the other hand, the flower color variations in the Japanese morning
glory, Pharbitis nil, have originated from a prototype with blue flowers
through natural mutations and their combinations from seventeenth tothe present century, while hybridization with other Pharbitis species
was not tried at all until recently.
It was observed in twenty-one combinations among seven pansy varieties (Endo 1959 and 1961) that there were complicated F 1 and F 2 segregations in general, especially continuous quantitative variations in theamount of pigment in most of the combinations between cyanic andacyanic varieties. This observation suggests that each pansy variety
examined has a unique genetic background, derived from original hybridization, contributing to flower color. It is noteworthy that there
are among the varieties, in quality and/or quantity, mutually additive
relations among three kinds of pigments, anthocyanins, flavonols andxanthophylls (Endo 1954).
In contrast to this, it has been established by Imai (1931), Hagiwara(1931) and others that inheritance of flower color in morning glory isalways subjected to simple Mendelian ratio among numerous combinations. The results of gene-analysis obtained by them are summarized
in Fig. 1. It seems that there is no detectable differentiation of thegenetic background so far as flower color is concerned. However, there
were found considerable effects of some mutant genes on glycosylationof anthocyanins. For example, the genotype, i dy pr mg (brownish red),produces at least seven peonidin-glycosides, most of which are glycosylated to a lower level than those produced by i + pr mg (purplish red),
in which eight peonidin-glycosides are produced.

RESEARCHES CARRIED OUT IN 1962

69

Fig. 1. Flower color genes in the Japanese morning glory.
I~X: Linkage groups

Accordingly, it is noted that appreciable variations in the genetic
background contributing to flower color do not always result in intraspecific irregular relations in the pigment system and, otherwise, thateven one mutant gene causes a comparatively large modification on theanthocyanin system as a whole.

54. Mutations of dominant green to recessive chlorina
in heterozygous plants ll(By Tar5 FUJII)

Dormant seeds from the cross between normal-500l and chlorina-5040
were r-irradiated at lOkr. Germination rate in the irradiated lot was alittle inferior to that of the non-irradiated control but no marked difference was observed in the survival rate and average number of tillers
per individual. No mutation was detected at an early seedling stage,
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

but chlorina stripes from somatic mutation appeared in some tillers of
201 among 1,809 plants. The mutation appeared on leaf and stem aslongitudinal stripes. Only one plant had striped and pure chlorina tillers,
together with green tillers within the same individual. In some cases,
width of leaf and chlorina stripes was measured in the middle of thesecond, third or following leaves. Some tillers had only one stripe ineach leaf, and two or more stripes were observed in other cases. The
area of stripes shows large variation.
The chlorina stripes mostly appeared in one tiller of each individual;
two or more tillers with chlorina stripes appeared in some cases in successive offshoots from striped primary tillers. One tiller only of one
individual had chlorina stripes in 118 plants; two or more striped tillers
within the same individual were found in 83 plants and the maximum
number of striped tillers was 12.
From the results, the number of initial cells which contributed totillering was estimated to be about 100 in the embryonic stage.

55. Effect of nitrous oxide on germinating seeds of einkorn wheat(By Tar/) FUJII)

60 seeds for each lot were soaked in water at room temperature.
Germinating seeds were picked up 24 and 48 hours after the start ofthe experiment and were treated with nitrous oxide for 8, 16 and 24
hours, each treatment at the pressure of 3 and 6 atmospheres. Nitrous
oxide treatment apparently does not affect either germination or survival
rates and seedling length or the number of offshoots. Induction of polyploidy was examined by the observation of chromosomes at MI withsmear technique. Each spike per most of the surviving plants waschecked. All spike, except two, had seven bivalents like those of thecontrol lots. Thus tetraploid or any kinds of chromosome irreguralities
were not obtained. One plant in each of the lots subjected to 6 atm.
for 24 hr. and 3 atm. for 8 hr. was haploid but this was not due tothe effect of nitrous oxide because sporadical appearance of haploid is
known in T. monococcum. On the basis of cytological observation theupper confidence limit on the proportion of induced polyploids at the
95% level was calculated as 0.8%. Nitrous oxide did not produce polyploids when it was applied to germinating seeds for up to 24 hours atpressures of 3 and 6 atm.

RESEARCHES CARRIED OUT IN 196271

56. Cytological observations on chemically induced
male sterility in sugar beets(By Tomoko OHTAl) and Seiji MATSUMURA)

According to F. WIT'S method, 0.4% solution of Na-2.3-dichloroisobutyrate was sprayed on the leaves of sugar beet variety GW 359, when
the beets began to head and had young inflorescences with small flower
buds. Black spots were observed on the leaves and young tissues
around the growing point began to etiolate 5 days after the treatment.
Degeneration of pollen grains began after a week and completely
sterile pollen was found after 2.-4 weeks. Later pollen fertility wasslightly recovered, while on the female side no degeneration occurred.
Therefore, this method should be useful in sugar beet breeding.
The induced male sterility was investigated cytologically and histologically. In the treated inflorescence young anthers had a normal appearance before meiosis, but later the tapetal cells developed abnormallyand showed hypertrophy, as observed in the case of genetical malesterility. They stained very deeply and thickened and remained unchanged. The pollen grains, not being able to absorb nutrients fromthe tapetum, finally degenerated. In this case the abnormal development of tapetal cells was most striking. Thus, the relation between
pollen grains and the tapetal tissue is thought to be the main cause ofmale sterility.
1) Kihara Institute for Biological Research, Yokohama.

C.

MATHEMATICAL GENETICS

57. On the probability of joint fixation of mutant genes(By Motoo KIMURA)

Let us consider n independent loci each with a pair of alleles, conveniently called the normal and the mutant genes and denote by pli)the initial frequency of the mutant gene in the i tk locus (i= 1,2, ... , n).
Let u(p(l),pI2), ••• , pin); t) be the probability that all the n mutant genes
become fixed in the population by the ttk generation given that theirinitial frequencies at t=O are p(l), p(2), ... , pin) respectively. As in the
single locus case, we will be mainly concerned with the ultimate probability of joint fixation defined by

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

U(pll), p(2), ... , p(nl)=lim U(p(l), p(2), ... , p(n); t) .
t-=

Under the assumption of random mating and constant (but not necessarily equal) fitness of individual genotypes, it is possible to show that

(1)

U(plll, p(2), ... , plnl)

where a=a(x(l), X 121 , ... , x(n» is the average fitness of the population
measured in Malthusian parameters and is a function of gene frequencies(x(l), ... , X lnl ), in which Xiii (i= 1, ... , n) stands for the frequency ofthe mutant gene in the itk locus in the population. If selective values
are used to measure the fitness of individual genotypes (discrete modelof generation time), log iiJ may be substituted for a in the above formula,where w is the relative selective value in the sense used by Wright; w
coincides with relative viability if no fertility differences are involved.
The above formula (1) can be obtained as the steady state solution of
the Kolmogorov backward equation for n variables by assuming randommating and constant fitness of individual genotypes. The formula enables us to study the effect of epistasis in fitness on the chance of joint
fixation of mutant genes.

D. GENETICS AND BIOCHEMISTRYOF MICROORGANISMS
58. Phase specific regulator of jlagellin genes (H1 and H 2)
in Salmonella 1 )(By Tetsuo IINO)

H 1 and H 2 are the structure genes of Salmonella flagellar protein(flagellin), in antigenic phase-l and phase-2, respectively. The presenceof an H 1 -specific regulator, designated ah lJ was reported previously (lino,
Annual Report, No.8, 1958). The functional relationships between ah 1
and H 1 were studied in detail. The heterogenotes occurring by transduction of an ah1-H1 segment from a diphasic strain to an ah 1 - monophasic-2 strain produce trails on the semisolid plate containing the anti1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Diseases (E·2872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1962sera against phase-2 antigens of both donor and recipient. But they do
not produce trails when the antiserum against phase-1 antigen of thedonor together with the anti-phase-2 sera is added to the medium.
These results indicate that ah l + is dominant to ah i - , but the functionof ah l + is effective to HI only when it is in cis-position with it. It was
further shown that ah l - mutants do not produce phase-1 flagellar protein at all even in phase-I. HI and ah l may behave as two component
parts of a genetic transcription unit analogous to the operator-structure
gene system in the lac-region of Escherichia coli (Jacob and Monod,
1961).
Transductional analysis of monophasic-1 mutants of S. typhimuriumand S. paratyphi B disclosed a phase-2 specific regulator ah 2 • The function of ah 2 to H 2 is in parallel with that of ah l to HI' Diphasic strains
carry an allele, ah 2 +, which switches on the production of phase-Z
flagellin. A mutant allele ah 2 - switches off the production. Consequently.
the mutant carrying ah 2- is stable in phase-I. Ah 2 is adjacent to H'l;.
on the chromosome.

59. Further studies on non-jlagellated mutants of Salmonella ll(By Tetsuo IINO and Masatoshi ENOMOTO)

The jla-genes of Salmonella are antigenic phase non-specific regulators:of flagella production. They are complementary with the structure
genes, HI and H 2 , in trans-position. A jla- mutation in anyone of these'
genes causes the loss of the ability to produce flagella in both phase-1
and phase-2. The wild type allele, jla+, is dominant to all of the known
jla- alleles. The genetic complementation test reported in the Annual
Report No. 8 (lino, 1958) was extended to fourty three jla- mutants
obtained from S. typhimurium TM2 and S. abortus-equi SL23. They
are classified into twelve complementation units. At least three of them
are closely linked to HI'
Production of 'flagellin-CRM', which immunologically cross-reacts
with wild type flagellin, was examined on twenty five stable mutants
among them. They included both deletion types and point mutants.
The procedure of immunological tests was the same as that describedin the Annual Report No. 12 (lino and Haruna, 1961). Among the
mutants examined, one strain, SJ28, of S. abortus-equi produced flagellinCRM which is immunologically indistinguishable from wild strain
1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

flagellin (Annual Report No. 12). The remaining twenty four strains
did not produce flagellin-CRM at all.
From these results, it is inferred that the fla-gene mutated in SJ28 is
responsible for the formation of flagella from their component protein
molecules. The remaining fla-genes may regulate the synthesis of flagellin, presumably either producing an internal inducer of flagellin or controlling the production of ribosomes specific for flagellin synthesis.

60.

Effect of phenol on flagellation of Salmonella cells ll(By Tetsuo IINO and Michiko MITANI2J)

The cells of Salmonella tYPhimurium TM2 grown in the broth containing 0.13% phenol lose the ability to synthesize flagella. They can divide,and the number of flagella per bacterium decreases as the cells multiply, although the preformed flagella are not destroyed at that concentration. At the concentration of 0.2% not only the de novo synthesis offlagella is stopped but also the preformed flagella are paralyzed, and thecells become non-motile. The paralyzed cells recover motility immediately after they are transferred to phenol-free broth: the paralysis is reversible. Bacterio-static effect appears at the concentration of phenol
higher than 0.3% in the broth. The non-flagellated H-agglutination
negative cells produce protein which is immunologically indistinguishable from the flagellar protein of the bacteria.
Regeneration of flagella in cells deflagellated beforehand by phenol
treatment occurs in the standard regeneration medium, which contains
a 0.4% amino acid mixture and 0.2% glucose in phosphate buffer, during
3 hours at 3TC, whereas a uracil-less mutant of TM2 regenerates flagellaonly when uracil is supplemented to the standard regeneration medium
under the same experimental condition. Cells deflagellated by sonicvibration regenerate flagella in the standard medium without uracil inuracil-less mutants as well as in prototrophic strains.
These results may be explained as follows: the multiplication of thenormal flagella forming apparatus in a bacterial cell is inhibited byphenol, consequently the flagella proteins synthesized by the cell cannot be organized into a flagellum under the presence of phenol but are
accumulated in the cell. For the recovery of the flagella forming apparatus, de novo synthesis of RNA seems to be a prerequisite.
1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.
2) Department of Biology, Faculty of Science, Tokyo Metropolitan University.

RESEARCHES CARRIED OUT IN 196275

61. Grouping of paralyzed mutants in Salmonella')(By Masatoshi ENOMOTO)

Sixty paralyzed mutants of independent origin were isolated from
Salmonella typhimurium, TM2, by the method reported previously (Enomoto and lino, Annual Report No. 12, 1961). Transductions of the
motility genes (mot) were carried out with phage P22 in all pairwise
combinations of the mutants. Production of swarm or trail on semisolid
nutrient medium was recognized as the proof of allelic nonidentity or
genetic complementation, respectively. The paralyzed mutants examined
could be classified into three groups, that is, thirty mutants belonging
to group-A, eighteen to group-B and twelve to group-C. The mutantsof group-A are non-complementary with each other, but are complementary with any mutant of group-B. Similary, a mutant of group-B
is non-complementary with any other mutant of group-B but complementary with every mutant of group-A. Thus group-A and -B represent
each a genetic functional unit, a 'cistron'. The behavior of the mutantsin group-C is somewhat peculiar. Many mutants in this group show
partial complementation with each other; a few are complementary withthe mutants of both group-A and -B, while others are non-complementary with them. It may be worth noting that the absence of complementation in the latter combination is not explained as a result ofdeletion extending over C-region and A- or B-cistron. Syntrophic recovery of motility does not occur in the mixed culture of the represnttative mutants of these groups either in broth or on semi-solid medium.
Measurements of ATP content and ATPase activity of the representative mutants in these groups and of wild type were carried out. For
the estimation of ATP, the energy rich phosphate esters extracted from
cells with TCA were calculated as hydrolysable-P in IN-HCI for 10 min.
at lOODC. In order to search for ATP in the flagella, chromatographyon column of Dowex-l 8X was used. ATPase activity was determinedby measuring free inorganic-P liberated from added ATP after it was
treated with crude enzyme solution. The chemical analyses have shownthat there is no significant difference in ATP content and ATPase
activity per cell between the mutants of the different groups. Eachvalue is, however, slightly lower than the comparable value in the
motile wild type strain, TM2. The flagella of the three paralyzed
1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Diseases (E·2872), Public Health Service, U.S.A. to T.
lino.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

groups as well as the motile parent strains did not show any ATPase
activity.

62.

Chromatography and electrophoresis of jlagellinof Salmonella abortus·equil)(By Masatoshi ENOMOTO and Tetsuo IINO)

Flagellins of S. abortus-equi, SL23 and its curly flagellar mutant, SJ30
were isolated and purified. Cells incubated on nutrient agar medium
for 20-25 hrs. at 30°C were collected in 0.9% saline. The saline suspensions were centrifuged at 3000 g for 15 min. and washed first withsaline, then with distilled water. The cells were resuspended in distilled water to concentration of ca. 5x 10 lo /ml and shaken (ca. 750 stroke/min., 25 mm amplitude) for 30 min. with fine glass beads and a drop ofsilicon defoamer. The suspension was centrifuged at 1500 g for 60 min.
and the supernatant liquid was centrifuged at 90000 g for 60 min. The
precipitate was disintegrated in 0.005N-HCI for 1 hr., then the suspension
was centrifuged at 90000 g for 60 min. at 2°C. The supernatant was
dialyzed at 2°C against 3 changes of distilled water for 5 hrs. This wasused as flagellin specimen. Column chromatography was performed on
DEAE-cellulose (Brown). Prior to packing the cellulose was adjusted topH 8.0 by a KH 2PO. solution and equilibrated with starting buffer.
The flagellin sample equilibrated with the same buffer was applied tothe column (1 X 10 cm). A gradient was established with 100 ml of 0.005Mphosphate buffer (pH 8.0) in the mixing chamber and first 80 ml of 0.5Mphosphate buffer (pH 6.0) was added followed by 80 ml 1M-NaCI in 0.05MKH 2PO. (pH 4.2) in the reservoir. The rate of flow of buffer was 20 ml/hr
and 3.2 ml fractions were collected. Protein was measured spectrophotometrically at 280 m,u or/and 750 m,u after folin-phenol reaction. In
the flagellin sample of SL23, only one peak (peak B) was observed in
the effluent corresponding to 25 ml-50 ml. The test of H(enx)-antigenicityof this peak by means of Ouchterlony plate was positive. In somefractionations a small peak (peak A) came off before the major peak B
and in this time a shoulder (peak C) always appeared behind peak B.
In order to determine whether peak A and C are caused by contamination with other proteins or denaturation of flagellin, the dialyzed sample
was divided into two portions and one was applied to the column immediately, while the other was stored in O°C and applied after three1) This work was supported by a research grant from the National Institute ofAllergy and Infectious Disease (E-2872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1962

77

days. In the chromatogram after three days, the small peak A increased, peak B decreased compared to the first fraction and the shoulder(peak C) grew to one large peak. The antigenicity of peak A was
negative to both anti-H(enx) and anti-O(4, 12) serum and the other two
peaks (B and C) showed positive reactions to anti-H(enx) serum. Moreover, the rechromatogram of peak B showed the fractionation patternof three peaks. Thus it seems that peak A is a degradation product offlagellin and peak C represents a flagellin polymer. The chromatogramof flagellin sample of SJ30 showed the same profile as SL23; the fractionation pattern of one large peak or three peaks depend on the time
when the dialyzed sample is applied on column. Dialyzed flagellinsample of SL23 and SJ30 was lyophilyzed in vacuo and used for paper
electrophoresis. Electrophoresis was carried out on Toyo No. 51 paperwith verona 1 buffer (pH 8.6, I 0.05) and 8M-urea containing veronalbuffer to avoid polymerization of flagellin. In both buffers, flagellin of
SL23 and SJ30 showed one band at the same distance from the original
point.'

63.

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

Five species of yeasts (strains of Saccharomyces, Schizosaccharomyces
pombe, Lipomyces starkey£, Torula utilis and Torula rubra) contained
single conspicuous nucleoli within the nuclei. These nucleoli stained
densely with haematoxylin. Feulgen-carmine and Giemsa stainings differentiated the nucleoli from the chromatin. Such nucleoli might becomparable with those of other fungi observed by Robinow (1957) andBakerspigel (1957, '59, '60).
In the course of nuclear divisions, a spindle-like structure was observed in Torula utilis. This structure appeared as a transparent zone,
while the nucleoli became red by staining witn aceto-carmine after
fixation with alcohol-formalin-acetic acid. Since metal salt solutions
were reported to fix well spindle structures in cells of higher plants(Wada and Fukunaga. 1957), the present author tested various kinds ofmetal salts as fixatives. Although the spindle-like bodies were not yet
confirmed with these fixatives, the nuclear boundary and the nucleoli
were clearly differentiated after treatment with 1j10M CuCl z , HgClz and
CdCl z aqueous solutions. In this case, aceto-carmine or acid fuchsin
stained the nucleolus, and the chromatin was assumed to be located in
the hyaline region around the nucleolus.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

64.

Temperature dependence of nitrous acid effects on phage T4(By Hiromi ISHIWA, Yonhoi YAN!) and Sohei KONDO)

Nitrous acid is one of the chemical mutagens most thoroughly studied
concerning its reaction with nucleic acid and mutagenic action on phages.
To obtain a better knowledge of its mutagenic action, temperature dependences were studied with respect to its lethal and mutational effects
on T4 phages. Free phages were treated with nitrous acid in an acetic
acid-sodium acetate buffer, 0.2 M in acetate ions, at pH 4.0 or 5.0.
Nitrous acid was produced by adding KN0 2 at the final concentrationof 0.2M.
The surviving fraction 5/50, 50 being the initial phage titer, decreased
exponentially with time t of treatment and mutation frequency f increased linearly with t in the range of inactivation down to 10- 3 :( 1)

where fl.l and fl.m are, respectively, killing and mutation rates per unit
time. By changing the temperature from 2°e to 27°e, both fl.l and fl.m
were confirmed to follow the Arrhenius equation
fl.moce-Elk'I',(2 )where k is the Boltzmann constant, T absolute temperature and E denotes activation energy. Differences in E between inactivation andmutation either at pH 4.0 or 5.0 were too small to be detected significantly beyond experimental errors. Average Evalue was O.71±O.06eV.
1) Department of Physics, Faculty of Science, Kyoto University, Kyoto.

E.
65.

RADIATION GENETICS IN ANIMALS

Effect of radiation-induced mutations on fitness of
Drosophila melanogaster ll(By Yuichiro HIRAIZUMI)

Several males of D. melanogaster (+/cn L 2 bw 0 's) were x-rayed inorder to find out whether flies heterozygous for the induced mutations
on second chromosome show any demonstrable increase in fitness. The
exposure consisted of 500r given at a rate of 100r per minute for 5
minutes. The irradiated males were kept for two days together with

79

RESEARCHES CARRIED OUT IN 1962

the females of standard, cn bw, stocks, and then they were mated toheterozygous females of +fSMl (SMl: balancer) for additional two days,
to isolate each irradiated and control second chromosome. Therefore
most of the chromosomes thus isolated were exposed to x-rays at the-stage of sperm. All the second chromosomes used in this study were,originated from a single chromosome, and the genetic background otherthan that of the second chromosome had been replaced by that of thestandard stock.
Ninty-seven x-rayed and the same number of control chromosomeswer~ tested for their effects on female fertility, developmental rate(measured by the period from egg till eclosion) and viability (only 74
chromosomes were tested for viability). The results are summarized in
the table.
Table,

Effect of x-rayed second chromosomes (=EB) on several
components of fitness

Female fertility
Developmental rate
Viability

+1+

+/EB

EB/EB

1.000
1.000
1.000

0.981
0.998
1.003

0.957*
1.003
0.972* Significant at 5% level. The relative values shown in this table are basedon the average of 97 (74 for viability) chromosome lines.

Since most of the values in the experimental classes do not deviatesignificantly from those in control, the results are inconclusive. Taken
at face values, however, it is interesting to note that the heterozygote(+ fEB) shows, on the average, faster developmental rate and higher
viability than the control homozygote (+ f +), although female fertilityof + fEB is reduced. This experiment is still continuing for conclusive
results.
I) This work was supported by a PHS research grant No. RH-00034-01 fromthe Division of Radiological Health, Bureau of State Services, Public Health Service,
U.S.A.

66. Heterozygous effects of radiation-induced mutations on
viability in homozygous and heterozygous genetic
backgrounds in Drosophila melanogaster(By Terumi MUKAI, Sadao CHIGUSA and Isao YOSHIKAWA)

It has been recognized that a unique co-adaptation system of genes

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

has been established within a population of Drosophila as a result of
natural selection. In order to test the difference of heterozygous effectof radiation-induced mutations on viability between individuals having acomplete coadapted genic system and hybrids originated from two unrelated populations, the following experiments were conducted.
Two isogenic lines (AA: Burdick's W160, and BB: Burdick's WI09,
were extracted from unrelated natural populations. A and B indicate
the genomes. After X-ray irradiation at the dose of 150 r, (irradiation
is indicated by superscript [' D, heterozygotes AA' and AB' were produced by the aid of Cy/Pm Ubx/Sb and Xa/+ + strains. The relative
viability of irradiated heterozygotes with respect to the second and thethird chromosomes were estimated in comparison with that of Xa/+A+A.
The results are presented in terms of mean viability in Table 1.
Table 1. Heterozygous effects of radiation-induced
mutations on viability (Acute 150 r)

Genotype

No. ofchromosomes
testedAverageviabilityAA

330

0.9843AA'

330

1.0021AB

430

1.2831AB'

430

1.2531(irradiated) - (control)+0.0178-0.0310*Viability of Xal+A +A=1.0000* Significant at the 5% level

From Table 1, it can be seen that radiation-induced mutations which
occurred in homozygous individuals were heterozygously beneficIal to·their carriers, i.e., the average viability of AA' was larger than thatof AA by 0.0178 viability index although it is not significantly different
from 0, while those that occurred in the hybrids between individuals
originated from the two unrelated populations were detrimental to theircarriers, i.e., the average viability of AB' was smaller than that of AB'
by 0.0310.
It is a further problem to clarify whether this difference in effects ofradiation-induced mutations is caused by the difference in the genic
system of the genetic background or by the difference in heterozygosity
per se.

RESEARCHES CARRIED OUT IN 1962

81

67. Radiation-induced mutation rates of polygenes controllingthe sternopleural bristle number in Drosophila melanogaster(By Terumi MUKAI, Isao YOSHIKAWA and Sadao CHIGUSA)In order to obtain a fundamental information on the genetic influenceof radiation on human populations and on the estimation of spontaneous
mutation rates of polygenes, radiation-induced mutation rates of polygenes controlling the sternopleural bristle number have been estimated.
The experiments have been conducted since 1961, and the results obtained in 1962 are here reported.
The males of an isogenic line extracted from a wild population ofErie, Pa. (U.S.A.) were irradiated with X-rays at 250 rand 500 r. Immediately after irradiation, the irradiated males were mated in milkbottles to the females of the same line. The number of sternopleuralbristles in females and males which had hatched on or before the 13th
day after the mating were scored and the means and the variances were
calculated to test the heterozygous effects of radiation-induced mutations.
The results obtained in 1962 are presented in Table 1.
Table 1.

Means and variances of sternopleural bristle numbers (1962)

250 r

Control

500 r

Q

0

Q

0

Mean

17.661416.9815

17.6713

16.9775

17.5450** 16.9512

Variance

1.39691.2993

1.44881.29371.4964*

1.2999No. ofirradiatedgenomes

2519

2111

2422

2000

2517

1928

Q

0

* Significant at the 5% level** Significant at the 1% level
The variances of the irradiated groups have approximately linearly
increased with the dose in females, but have rather decreased in males
as compared with the controls. This difference might be attributed tochanges of hetero-chromatic parts in Y-chromosomes.
Thus, the mutation rates were estimated by using the data for females
only. A modified method for the estimation of polygenic mutation rate
developed by Mukai (1961) with reference to Bateman's work (1959) was
employed. For the reason that the third moment about the mean is
influenced a great deal by the effect of error, it was not used in thepresent analysis. Accordingly, the heterozygous effects of each mutation

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

were assumed to be equal to one half the standard deviations (Oka etal. 1958). Thus, the mutation rates were estimated on the assumptionof 500 loci which control the number of sternopleural bristles. This.
assumption may not be an underestimate considering Falconer's estimation as 100 of the number of loci controlling the abdominal bristles.
The results are shown in Table 2.
Table 2. Estimation of radiation-induced mutation rates of polygenes
controlling the number of sterno-pleural bristles (1962)
250 r
experiments

500 r
experiments

Mean number of positive mutations per genome

0.083

0.044

Mean number of negative mutations per genome

0.066

0.241

Mean number of mutations per genome

0.149

0.285

1.19x 10- 6

1.14x 10- 6

Mutation rate per locus per r*
Variance increasing rate per r on heterozygote
basis

*

2.02xlO-'(rAssuming 500 loci

The estimated polygenic mutation rates were of the order of 10- 6/
locus/r both in 250 rand 500 r experiments as well as the experiments.
of 1961.
Thus, it may be concluded that radiation-induced polygenic mutation
rates are substantially higher than those for major genes. This result
may support the view that spontaneous polygenic mutation rates arehigher than those for major genes (Mukai and Chigusa 1962).

68. Radiation sensitivity of infectious particles responsible for"sex-ratio" condition in Drosophila willistoni 1 )(By Bungo SAKAGUCHI and Sohei KONDO)The abnormal condition referred to as "sex-ratio" (SR) in D. willistoni
is characterized by extreme departure from the normal 1: 1 ratio of thesexes, i.e. by either completely or nearly completely, female progenies.
(Malogolowkin 1957). Recently Poulson and Sakaguchi (1960, 1961) haveshown that the SR agent is a spirochete-like infectious microorganism.
1) This work was supported by a grant from the National Institute of Health,
Education and Welfare (GM 10238-1), Public Health Service, U.S.A.

83

RESEARCHES CARRIED OUT IN 1962

In order to clarify its properties, and experiment on the determinationof the agent's sensitive volume was carried out by r-ray irradiation.
The dose-effect curve for inactivation of the infectious SR agent was
obtained as follows. The hemolymph taken from females of D. willistoni in which the infectious agent is present at high level, was injected into female of D. virilis, which served in the place of a test tube
culture of SR agent, and then the SR infected females were exposed to
Cs-137 r rays with doses from 4 to 32 kr. The hemolymph containing
a large amount of the irradiated infectious agent was transferred fromthe irradiated flies into females of an inbred strain, Oregon-R, D. melanogaster, then the latter were mated with males of the same strain andobservation was made on the manifestation of SR condition in the following next generation. The degree of inactivation of the infectious
microorganisms was estimated from the prolongation of the latent period,
which is defined as the incubation period between injection and SR
manifestation.

t

30

•

...'"~
...

20

...
0

.

~

'"

•

+'
<l

.."

+'

'"

10

o

/I

8

16

32

Dose ( Kr )Fig. 1. Relationship between r-ray dose and latent period of SR agent.

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

The experimental data are summarized in the curve of latent periodt versus dose D (Fig. 1). The curve follows a clear straight line relationship expressed by the following equation:t(days)= 1.5+0.75D(kr)(1 )Time t required for manifestation of SR condition should depend on
the concentration C of SR agent at the time of injection. The relation
between t and C was experimentally determined by dilution tests ofhemolymph of SR female adults of D. willistoni. The following empirical
relation was obtained:
C2 t /2.1=const.

or C=Coe-~(t-1.2)(2)

where Co is the SR agent concentration of the control not subjected todilution. This is interpreted to mean that the infectious microorganisms
duplicate in 2.1 days and when their concentration reaches a constantthreshold value SR condition appears, irrespective of the initial concentration of infectious microorganisms at the injection time.
On the other hand, if inactivation follows single hit response, thenconcentration C of microorganisms surviving dose D at the time of
injection should be related to concentration Co of the control used inequation (2) as follows:

C=Coe-OlJ)
where a is inactivation probability per unit dose.
and (3), we obtain
t=1.2+3aD(3)

From equations (2)(4)

Close agreement in t value at D=O between equations (2) and (3) supports the validitity of the assumptions used which lead to the equivalence between (1) and (4). Thus, from the identity of the two equations, we obtain l/a=4(kr) which is the so-called inactivation dose D 37
corresponding to 37% survival of the infectious agent.
According to the target theory, the target size in molecular weightis given by 0.7x 109 jD37 (kr). Hence from the experimental value of D37 ,the molecular weight of the infectious microorganisms is estimated tobe 2x 108 (M.W.). This result supports the previous experimental conclusion (Poulson and Sakaguchi 1960) that SR agent is rather spirochetal
than viral in nature.

RESEARCHES CARRIED OUT IN 1962

69.

85

Revised interpretation of the non-recovery type dose-rate
dependence of radiation-induced mutation
rates of silkworm gonia(By Yataro TAZIMA and Sohei KONDO)As reported in our previous paper (Tazima, Kondo, Sado, 1961), twotypes of dose-rate dependence of radiation-induced mutation frequency
were observed in early gonia I cells of the silkworm. In one type (type I),
mutagenic effectiveness of chronic irradiation was lower than that ofacute irradiation and in the other type (type II), the situation was completely reversed. The occurrence of either type depends upon the stageof irradiated germ cells.
The type I dose-rate dependence has been interpreted in the light of
the repair hypothesis proposed by Russell et al. (1958) on the groundsof their findings in the mouse. This hypothesis, however, can not explain the type II dose-rate dependence, because acute irradiation gives
rise to lower mutation frequency than chronic irradiation. Therefore,
we have made the following provisional assumptions. 1) Primary mutation rates differ according to the developmental stage of gonial cells,2) highly mutable cells, in particular the secondary spermatogonia, areselectively killed after acute exposure because of their high sensitivity
to radiation but can survive chronic irradiation.
Cytological studies of irradiated gonial cells, which was carried out
by Sado in parallel with our work, failed to obtain clear-cut evidencesin support of our hypothesis. Both acutely and chronically irradiatedsecondary spermatogonia were observed to undergo necrosis, althoughdegeneration was less in the latter. Sado also found that after degeneration of primary and secondary spermatogonia, usually less in theformer, young gonial cells regenerate from irradiated cells that havebeen presumably dormant at the time of irradiation.
Taking into account these cytological findings together with our recent
results, we have modified our previous interpretation as follows. Radiation sensitivity of primary and secondary spermatogonia is much higher
than we had previously assumed. Survival fraction of secondary spermatogonia depends solely upon radiation intensity; almost all cells are
killed after acute irradiation within the dose range from 150 r to 1000 r
but a few cells can survive chronic irradiation although the majority
are killed. Dormant spermatogonia can survive both acute and chronic
irradiations in the dose range used. Sensitivity of primary spermatogonia may be intermediate. The deficit, caused by Rilling of most ofdeveloping gonial cells in the irradiated gonad, is soon compensated by

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

young germ cells regenerated from the survived dormant cells. Thus
change will be brought about in the proportion of cell types in the
gonad after irradiation, their amount depending upon the degree of the
deficit. Acute irradiation may cause more severe deficit than chronic one
and hence will give rise to a higher proportion of young germ cells
regenerated from dormant cells. This will result in a lower mutation
frequency at acute than at chronic treatment as observed for type II
dose-rate dependence in our experiment.
Thus modified hypothesis dose not seem to contradict with our previous one but explains more satisfactorily all the genetical and cytologicalexperimental results so far obtained. Further studies, however, arenecessary for obtaining direct support of the proposed hypothesis.
(Presented before the 2nd International Congress of Radiation Research.
Harrogate, 1962.)

70.

Differential radiation-sensitivity of germ cells as a possible
interpretation of sex difference in dose-rate dependence
of induced mutation rates in the silkworm(By Yataro TAZIMA and Sohei KONDO)

During the course of our studies (Tazima, Kondo and Sado, 1961) on
dose-rate dependence of radiation-induced mutation rates in the silkworm, we noticed that it was more pronounced in the oogonia than in
the spermatogonia at an early stage at which type I dose-rate dependence was observed. The ratio of the observed mutation frequency foracute irradiation (320 r/min.) to that for chronic irradiation (0.1 r/min.)was 2 : 1 in the oogonia and 1.5: 1 in the spermatogonia.
Our recent studies on mutation frequency versus dose curves, carriedout by the specific locus method using pe and re loci, have confirmedthat there was, indeed, a marked difference in mutation response between oogonia and spermatogonia, though cells of both cytologicallydid not show yet at that stage any appreciable difference. First, themutation frequency was about twice as high in spermatogonia as inoogonia. Second, the shape of their dose-frequency curves was different.
In oogonia, a linear relationship was clearly observed within the range
from 500 to 1500 r both for acute (320 r/min.) and chronic (0.07 to 0.21 r/min.) irradiations, and acute exposures were significantly more effective
than chronic ones over the whole dose range studied. In contrast, inspermatogonia the frequencies for acute and chronic exposures increased
more rapidly than expected for linearity with increasing dose, and the

RESEARCHES CARRIED OUT IN 196287

difference between them became smaller in the high dose range. At
1500 r, the frequency for chronic was almost equal to that for acuteirradiation.
These findings seemed to indicate a close relationship between mutation induction and sensitivity of germ cells. Therefore, to evaluate thecontribution of killing effect on germ cells to the mutation mechanism
involved, we made an estimation of the difference in radiation sensitivity
between spermatogonia and oogonia from our earlier data, by using thetheoretical formula proposed by the junior author, Kondo (Ann. Rep.
Nat. Inst. Genet. 11: 96, 12: 93-94).
From the experimental probability distribution of the number ofmutants per mating pair, estimation was made of surviving germ cells
at the time of mutative events, and survival versus dose curves wereconstructed for acutely irradiated spermatogonia and oogonia at various
doses.
The LD,o was about 1000 rand 2000 r for spermatogonia and oogonia,
respectively. The difference in radiation sensitivity is clear between
both sexes. Thus it was concluded that the mutation frequency isclosely related to the killing effect.
These findings appear to suggest that recovery of mutational damage,if any, becomes rather difficult in the highly sensitive cells, not only
after acute but also after chronic exposure. Hence, the differences in
the observed mutation frequencies in the spermatogonia between acuteand chronic irradiations become smaller with increasing dose, although
the difference may exist to some extent. On the contrary, in oogonia,
which are rather resistant to radiation, the recovery process seems tooperate with less interference by the killing effect.
From these considerations, we assume that the repair process is, even
if it works under normal cellular conditions, masked or eliminated tosome extent by cellular radiation damage and that the degree of doserate dependence becomes much smaller in sensitive cells such as earlyspermatogonia.
(Presented before Symp. on Repair from the Genetic Radiation Damage,
Leiden, 1962 and will appear on the Proceedings.)

71.

The increase in induced mutation frequency after fractionatedirradiation of gonial cells of the silkworm(By Yataro TAZIMA and Akio MURAKAMI)

Since the dose-rate dependence of radiation induced mutation-frequency

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

had been observed in the silkworm, it was considered of some interest
that induced mutation frequency could be modified by fractionating theadministered radiation dose. For this purpose experiments have been
carried out during the early larval stage from the 7th to 9th day after
hatching. For the estimation of mutation frequency the specific locus
method for pe and re loci was used.
The design of the experiments was as follows. In one group (Fr 1)
total X-ray dose 1000 r was given in two halves separated by 48 hourinterval, the first 500 r on the 7th day and the second 500 r on the 9thday. In another group (Fr 2) the same dose was given in three fractions; the first 333 r on the 7th day, the second 333 r on the 8th day,and the remaining 333 r on the 9th day, with 24 hour intervals. In thethird group (Fr 3) total dose was reduced by one half to 500 r, and 10 r
was given 50 times every hour during the same larval stages. For
comparison's sake three single dose groups were arranged with 1000 r
given in single shots at different developmental stages, i.e. on the 7th,
8th and 9th day (Sl, S2 and S3) respectively. A non-irradiated group(Cont.) was simultaneously examined. The results are shown in Table
1.
Table 1.

Fractionated X-irradiation of spermatogonia and oogonia
Irradiated stage

Groupdays after hatching
~

7
Fr 1
Fr 2
Fr 3
S 1
S 2
S 3
Cont.

8

Mutation frequency (x 10- 5)
Total
dose

9

500 r
500r
333 r
333 r
333 r
10 rx50 1 hour interval
1000 r
1000r
1000r

1000 r999 r
500r
1000 r1000 r
1000r
Or

Spermatogonia

Oogonia

pe

re

pe

re

87.2
221.8
57.5
73.6
31.5
36.7
8.9

31.4
90.4
35.2
31.5
9.2
5.6
0.0

53.6
256.2
43.6
20.7
37.2
26.9
9.2

33.3
156.8
26.8
14.2
32.3
20.9
8.8

It may be seen from the table that fractionated groups give higher
mutation frequencies than the three single shot groups; group Fr 2, inparticular, gives extraordinarily high frequency. The frequency ingroup Fr 3 is also high when it is multiplied by two.
These findings seem to suggest that fractionated irradiation with 24
hour interval (group Fr 2) is more effective than that with 48 hourinterval (group Fr 1) in increasing mutation frequency.

RESEARCHES CARRIED OUT IN 196289

But shortening of the interval has a limit: fractionated irradiation
with 1 hour interval showed lower mutation frequency than when 24
hour interval was applied, even when it is adjusted at 1000 r. Further
experimental data, which are currently being accumulated, indicate that
irradiation with 24 hour intervals is of all the most effective in increasing mutation frequency.

72. Modification of Xray-induced mutation rate in the silkworm
by pre- or post-irradiation treatment withhalogenated base analogues(By Akio MURAKAMI and Yataro TAZIMA)

It has been known that certain halogenated base analogues are mutagenic in bacteria when supplemented to the culture media. It has also
been reported that mammalian and bacterial cells grown in the presenceof halogenated thymidine analogues become highly sensitive to killing
effects of X-rays and ultraviolet light. Nevertheless, for higher organisms mutagenicity of those substances was still uncertain. In orderto test it a series of experiments has been carried out with the silkworm
by feeding larvae on 5-Bromouracil (5BU), 5-Bromodeoxyuridine (5BdUR)or 2-Aminopurine at early instars. Thus far more than 1.5 million individuals have been tested for induced mutations at specific loci for peo
and reo However, no significant increase, if any, has been observed.
Therefore, the mutagenicity of those analogues has been tested in combination with X-irradiation. The results were definitely positive.
1. Experiment in the first instar.
In this series 5-BdUR was administered to wild type larvae by feedingthem on leaves supplemented with the chemical (30 mg per 1500 larvae)
for about one and a half day after X-irradiation at 48 hour age of thefirst instar. The results are given in Table 1.
It may be seen from the table that 5-BdUR treatment, when combinedwith X-irradiation, enhances markedly the mutation frequency above
the expected level from X-irradiation only, although 5-BdUR alone has
no effect on increasing mutation frequency. The enhancement is moremarked in the male than in the female for both pe and re loci. Spontaneous mutation frequency in the control group is seen to be in thisexperiment at the ordinary level.
2. Experiment in the second instar.
In the second series of experiments it was tested whether 5-BU is
also effective in modifying X-ray induced mutation frequency. The

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13
Table 1.

Combined experiment with 5·BdUR and X-irradiation(622, 1st instar)

pe

re

Total
no.
observed Mut.
detd.

Mut. freq.

Male
Cont.
X·rays*
BdUR**
X+BdUR

121,800
101,850
122,850
127,400

20
45
14
163

16.4xlO- 5
8.4- 25.4
44.2
21. 7- 59.2
11.4
4.4- 19.4
107.4-149.2
127.9

2
31
0
89

1.6xl0- 5 0.1- 3.9
30.4
18.8-43.2
054.6-85.9
69.9

FemaleCont.
X-rays*
BdUR**
X+BdUR

110,700
62,10074,25036,900

9
16
4
10

8.1
25.8
5.4
27.1

3.5- 15.8
11.4- 43.0
0.6- 8.8
7.0- 50.0

3
7
2
6

2.7
11.2
2.7
16.3

TreatmentCon£. into

Mut.
detd. Mut. freq. Con£.int.

0.5- 5.3
3.8-14.4
0.1- 3.9
1.6-30.0* 1000 r (1 rlsec.)** 30 mg/1500 head.

X-rays.t.

~atoh1ng

Table 2.

Treatment

BdUR

I~

ibM

5-BU feeding before or after X-irradiation(623, 2nd instar male)

Total
pe
no.
Mut.
observed
detd. Mut. freq.

BU*(pre)X-rays**
BU+X-rays
Bu(post)X-rays+BU

120,400
116,900
104,650
107,450
125,300

15
133
173
26
213

re
Conf. into

Mut.
Con£. into
detd. Mut. freq.

12.4x 10- 5
5.1- 20.8
2
113.8
93.2-135.2 75
165.3
139.1-192.5 71
24.2
13.8- 35.6
2
170.0
146.6-194.8 112* 120 mg/1500 head.
** 1000 r (1 rlsec.)

Hatohing

-rays
pre.
C::;:->Z::::!::;::>
i
post.

I

1.6x 10- 5 0.1- 3.9
48.4- 80.8
64.2
50.2- 85.9
67.8
0.1- 3.9
1.8
71.7-108.0
89.4

RESEARCHES CARRIED OUT IN 196291

difference between pre- and post-irradiation treatment was also studied.
Second instar larvae were raised for about one day on leaves supplemented with 120 mg 5-BU (per 1500 individuals) either before or after
X-irradiation. The X-ray dose was 1000 r. The results are shown in
Table 2.
Although a control plot was not used in this experiment, mutation
frequency in the 5-BU group is approximately at the spontaneous level
and could be treated as control. Both pre- and post-treatment groups
represent higher mutation frequency than that group of X-irradiation
alone, although the frequency in the pre-treatment group is slightly
lower than that in the post-treatment group. These results do notallow to assume the different mechanism for both pre- and post-treatmentgroups in respect to modification of mutation frequencies, because thechemical administered before irradiation might have remained for somewhile within the insect body and become effective after X-irradiation.
It may, however, be inferred with almost certainty that the enhancement is not due to the sensitizing effect of the analogues but to some
sort of effect that acts upon post-irradiation recovery process.
It is tempting to speculate that the analogue is incorporated into
X-rayed DNA and gives rise to a copying error in newly formed DNA.
However, supporting evidences are still meagre for this conclusion.

73. Cytological estimation of LD50 X-ray dose for secondary
spermatogonia of th~ silkworm(By Toshihiko SADO)

Earlier experiments have shown that the secondary spermatogonia
are the most radiosensitive cell type in the silkworm testis and the
maximum depression of these cells usually occurs 3 days after irradiation (Sado 1959, 1961). During the second instar, the testis contains
primary and secondary spermatogonia predominantly. Therefore, when
the silkworm males are irradiated during this period with doses higher
than 500 r, which is generally lethal to secondary spermatogonia, mutations are induced only in primary spermatogonia. If these cells are
exposed to doses much lower than 500 r, since some of the secondary
spermatogonia, may not be killed, we should expect mutations induced
in both primary and secondary spermatogonia. For this reason, we felt
it necessary to estimate the approximate LD so dose for these cells.
Male silkworms from the C108 strain, which has been routinely used
for mutagenesis work in this laboratory, were exposed to 25 r, 50 r, 100 r,

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

150r, 200r, 250r and 500r of acute X-rays on the first day of the second
instar, or 5 days after hatching. One and three days after the exposure,the abdominal portion of the treated insects were fixed in Bouin's fluid,
7 f.l thick paraffin sections were made, stained with Delafield's hematoxylin and the number of necrotic as well as surviving cells in thetestis were estimated semiquantitatively.
Following irradiation with 25 rand 50 r we could find little, if any,detectable difference between irradiated and control testes. With anincrease in the dose administered, the number of necrotic cells at 24hours increased and the number of surviving cells on day 3 decreased.
100 rand 150 r were not enough to kill half of the secondary spermatogonia. When the insects were irradiated with 200 r or more more than
50% of the cells were killed.
These results suggest that LD 50 dose for secondary spermatogonia of
the silkworm lies between 150-200 r, which is 6-8 times higher thanthat reported for intermediate and type B spermatogonia of the mouse(Oakberg, 1955).

74. Inactivation of Salmonella by 82 P disintegration
combined with UV ll(By Hiromi ISHIWA, Yonhoi YAN2) and Sohei KONDO)

Salmonella grown in H medium plus 82p lost their colony forming
ability during storage at -20°C and the curve of the surviving fraction
versus fraction of decayed 82p atoms incorporated into their DNA followed one hit response (e.g. Curve NO UV in Fig. 1). Calculation fromexperimental data gave the killing efficiency a=0.04 per disintegrationof 82p atoms per nuclear DNA which is very close to the a value forE. coil (Fuerst and Stent 1956). This value was compared with thekilling efficiency for cells surviving pre- or post-treatment with UV(Fig. 1, Curves PRE and POST). It should be noted that synergetic
action of UV and 82p disintegration is clear only for posttreatment with
UV as seen from comparison of slopes of inactivation curves in Fig. 1.
This work was undertaken to obtain information concerning the mechanism of inactivation by UV and 82p disintegration.
1) This work was supported by Grant No. 91003 for Intramural Cooperative
Scientific Research from the Ministry of Education.
2) Department of Physics, Faculty of Science, Kyoto University, Kyoto.

RESEARCHES CARRIED OUT IN 1962

93

10

I

10'

:;Itl.

!l

Q.

?; c

10-2

l!'.2

~ :
~ ~

~

'0

~

~ 10

3

.8 g'

uv Exposure~ :~0" sec-+- -0-

8--A-- -l:r-

E i.ll

~ ~

~ l1

POST

16

-..-D-

24"

__

-4

10

.,

PRE

+.. -<>-10

15 (days)-5~~~~~=
01
0.2
0.3
0.4
0_5

'0

L-

Fraction of

32p atoms decoyed

Fig. 1. Comparison of killing efficiencies of 32p disintegration for cells subjected to various UV treatment at ca. 13
erg/mm 2/sec; No UV-32P treatment only, PRE-UV before
storage for 32p disintegration, POST-UV after storage.

75.

Protective effect of sodium glucuronate on the differentiationof muscle tissue inhibited by X-irradiation!)(By Yoshito OGAWA)

Last year, the protective effect of sodium glucuronate on abnormal
formation of actin in X-irradiated Triturus embryos was reported2 ).
This paper deals with the influence of the same chemical on the synthesis of myosin, another important contractile protein of muscle tissue
and contributes some essential data on the practical value of the protective action. The experimental method was the same as before 213 )4).
Immediately after fertilization, the embryos were raised at 20°C in
1) This work was supported by Grant-in-Aid from Tokyo Biochemical Research
Foundation.

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

0.01% solution of sodium glucuronate (most effective concentration topromote the growth of Triturus embryos5), and X-irradiation with 50 r,
200 rand 500 r was carried out 108 hrs. after fertilization. After irradiation, treatment with the same solution was continued and theanalysis of myosin was immediately carried out. Myosin, isolated fromthe skeletal muscle tissue of adult Triturus, was injected into rabbits
and the obtained anti-serum was heated with saline extracts of liver,spleen and skin tissue of Triturus to eliminate non-specific antibodies.
The titre of the anti-serum was adjusted to 1: 512 before carrying out
the precipitin reaction with the saline extract of X-irradiated embryos.
Non-treated but irradiated, treated and non-irradiated, and normal (nontreated and non-irradiated) groups were prepared as controls.
In normal embryos, myosin first became detectable 176 hrs. after
fertilization 3 ). In the present experiment, myosin formation in thenormal group was first recognized 180 hrs. after fertilization since theexamination was carried out in periods of 24 hrs. following fertilization.
In the case of the non-treated but irradiated group, myosin was detected 156 hrs. after fertilization with 50 rand 200 r, and 132 hrs. with
500 r 4 ) as shown in Fig. 1. Thus the synthesis of myosin was markedly
promoted not only as to the rate of formation but also as to the amount,and the acceleration was proportional to X-ray dosage. In irradiatedand treated embryos the tendency to promote the rate of myosin formation was markedly accelerated, though no remarkable difference was

TreA t ed

Z'rea,t ed

cLowest antigen
centration giving
os1tive result9'500r

Irradiate4
Hr. aner 'fert1l1zatiof)
Bon-irradiated contJ"(llFig. 1. Effect of sodium glucuronate on the synthesis of myosinin X-irradiated Trituru8 embryos.

95

RESEARCHES CARRIED OUT IN 1962

found in embryos irradiated with 50 rand 200 r in the amount of synthesized myosin from that of the treated but non-irradiated group. However a heavy dose of 500 r, suppressed the amount of myosin formation.
(Fig. 1).
The differentiation of muscle tissue is not accomplished before bothactin and myosin formations coincide. The experimental results of bothprotein syntheses are summarized in Fig. 2. As shown in this figure,
TreatedTreatedon-treate

Lowest ntlgenconcentra ion giving
positlv results'5001"

252
228

204
180

Irra41ated

Hr. after fertilization

Non.::-irradiated cOntrol

Fig. 2. Effect of sodium glucuronate on the chemo-differentiationof muscle tissue in X·irradiated Triturus embryo.

the chemo-differentiation of muscle tissue is markedly suppressed by
X-irradiations. This suppression is due to the inhibition of actin synthesis. Sodium glucuronate markedly protects from the obstruction ofactin synthesis due to X-irradiation, the main cause of the suppression·of muscle tissue differentiation 2 ), and accelerates myosin synthesis during
early embryonal development (Fig. 1).
Thus, it was proved that sodium glucuronate has a significant protective effect in abnormal chemo-differentiation of muscle tissue of Xirradiated embryos.
2)
3)
4)
5)

OGAWA. Y.:

Ann. Rep. Nat!. Inst. Genet.. 12: 36. 1962.
Y.: Nature. 182: 1312, 1958.
OGAWA. Y.: Nature, 186: 77. 1960.
OGAWA, Y.: Ann. Rep. Nat!. Inst. Genet., 10: 150, 1960.
OGAWA.

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

F. RADIATION GENETICS IN PLANTS
76. Radiation-induced variegations in einkorn wheat
and their inheritance l )(By Seiji MATSUMURA)

Seeds of Triticum monococcum flavescens were exposed to X- and rrays by 60Co, thermal and fast neutrons (14 MeV) and j3-rays by S2p_
and ISII-solution. In the treated XI and later generations white and/or
yellow stripes were often observed and their mode of inheritance wasstudied. The striping was divided into the following types. 1) Mostof white- or yellow-striping found in XI was maternally or cytoplasmically inherited and was due to plastid mutation. The first appearance of
this type occurred seldom in the X 2-generation. 2) In the X 2 head progeny
segregating albina in a simple Mendelian ratio, white-striped leaves
were often observed in a few plants. They might be mostly due to asomatic mutation in a heterozygous plant concerning albina (Aa). 3)
Some white stripes which could be distinguished from others by thefine nature of striping occurring in all leaves of a plant were controlled
by a mutated recessive gene. 4) Special variegation was found in three
Xcplants. Their progeny contained some white seedlings. The basiviridis (or virido-albina) seedlings, other than albinas, invariably grew
up to be variegated; they were controlled by a mutated recessive gene.
Evidence indicated that the recessive basi-viridis gene for variegation,
which was stable, stimulated plastids to mutate (irreversibly) fromgreen to white. Plastid "exomutation", or mutation from green towhite was affected by environmental factors, such as temperature. This
type of variegation was already reported for "Okina-barley by IMAl
(1929).

77. Effects of chronic irradiation on einkorn wheat(By Seiji MATSUMURA and Tar6 FUJII)The facility for chronic r-irradiation of growing plants (r-field) was
built first in 1961 by the Ministry of Agriculture and Forestry in the
National Institute of Radiation Breeding, Hitachi-Omiya, Ibaragi-ken,
Japan. The radiation source there is radio-cobalt ca. 2,000 curies. Our
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

RESEARCHES CARRIED OUT IN 196297

experiment was carried out with seedlings of einkorn wheat (Triticum
monococcum jlavescens), planted at the stage of 3.-4 foliage leaves on Feb.
23rd in several distances from the source and irradiated with varying
dose rates and dosages. The plants were irradiated during their growthand harvested June 21st. Regular irradiation followed the routine of ther-field, namely it was applied 20 hours per day starting from April 1st,
but before that time, irradiation was given occasionally for measurements of dosage or dose rates and checks of the apparatus. Therefore,
total irradiation time from planting to harvest amounted 1,843 hours.
And total dosages were calculated as 369 r at minimum and 4,608 r at
maximum, intensity being 4.-50 r/20 hr. Survival rate was clearly decreased at the highest dosage, while there was no marked difference
between the other five irradiated lots and the non-irradiated one. On
the other hand, seed fertility was non-linearly (gradua,lly at higher dosages) decreased with increasing dosage and reached· to about one halfof that of the control lot at the highest dosage.
For comparison, seedlings of the same material were irradiated by 0.5
and 1 kr of acute r-rays (1 kr/hr) 15 days after sowing. Survival rate
and fertility were markedly decreased at 1 kr irradiation. The resultsfrom these lots were not easily comparable with those of chronic irradiation because acute irradiation was done only in early seedling stage,
but some information may be obtained on the difference of effects between acute and chronic irradiation. Acute 1 kr irradiation showed
severe effects and a similar effect was seen at the highest dosage ofchronic irradiation (4,608 r). Thus, the plants must be more tolerant tohigher dosage when radiation intensity is low.

78. Relation of chlorophyll mutations to dose rates
in r-irradiated einkorn wheat!)(By Seiji MATSUMURA and Tomoo MABUCHI)

Dry seeds of Triticum monococcum jlavescens were exposed to r-rays
at 4.-15kr. For acute and chronic irradiation the dose rates, 10,OOOr/hrwith mCs and 35.6 r/hr with 60Co, were used, respectively, as shown inthe diagram of Fig. 1. The germination rate, seedling height 20 days
after sowing, seed fertility in Xl and chlorophyll mutations in X 2 were
investigated.
Chronic irradiation was slightly more effective in inhibiting seedling
1) This work was supported by Grant from the Toyo Rayon Foundation for
the Promotion of Science and Technology.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13
Chronic irrad.
( 3O.6r/hr)"Coj

4 kr Ie12t-~----;:::===:::~

I_

1-----------.. . .

-----1:-Acute IrrOll'/ 5':., (10.000 r/hr)··Cs
Control

10

•

15

_

•

•

0
336

S"'rCllle (hralFIQ. I.

112•o

DiaQram of acute and chronic irradlatlonl

Stara,. (,.,.)

2

224

6

C;"'Oftk: { ~_._.-o I I ~t{ : - .......• : : :

Acue.

112

.-.

0

.
. . . »?

/~................•

/: .

•l·

. . <:.l& J~:"'",..0",:11-~°O~---J4;--:5;--~e:---:':IO::---7.:12:---~1:=5Do.e (kr)Fig. 2. Relation between chlorophyll mutations anddoses or dose rate in einkorn wheat

growth than the acute one applied at the beginning of the chronic one.
and clearly less effective than that applied at the end of the latter.
The later was the irradiation at the same dose, the more reduced wasseedling growth, especially at acute irradiation. Thus, almost no intensification of radiation damage due to storage effects was found. It is
supposed that recovery phenomena were involved.

RESEARCHES CARRIED OUT IN 1962

99

The frequency of chlorophyll lllUtations in the X 2 -gcneration is shownin Fig. 2. For acute irradiations which were applied just before sowing,
it increased roughly in a linear relation to dose. Acute irradiation
showed clearly a higher mutation rate than the chronic ones, whichwere terminated just before sowing. This indicates a dose rate dependency in the frequency of chlorophyll mutations. On f e other hand,
there was no marked difference between chronic and at te irradiation
at the beginning of the chronic 12 kr irradiation after the k ~ ~st storage.
Unexpectedly, the later was the irradiation at acute 10 kr, the higher
was the mutation rate. It is supposed that recovery phenomena wereinvolved.

79.

Relation of radiation effects to dose rates ofr-radiation in ric/ l(By Seiji MATSUMURA and Tomoo MABUCHI)

A series of experiments with rice were initiated to expand and verify
the earlier studies (d. Ann. Rep. No. 11: 100 and No. 12: 106). Dry
seeds of upland rice were irradiated by r-rays from a 60Co or I3,CS
source at the doses of 10, 20, 30 and 40 kr. For acute and chronic
irradiations the dose rates, 10,000 r/hr with 137Cs and 20 r/hr with 60Co,
were used, respectively. All acute and chronic 10 kr treatments were
given in three ways; namely at the beginning, in the middle and at theend of the chronic 40 kr irradiation. Also the chronic 20 and 30 kr irradiations were done in two ways; namely at the beginning and at theend of the chronic 40 kr treatment. The germination rate, seedlingheight 13 days after sowing, seed fertility in Xl and chlorophyll mutations in X 2 were investigated.
The results are shown in Fig. 1 for seedling height. An intensification of radiation damage due to storage effects was clearly observed.
For instance, at 20 and 30 kr acute irradiations only the batches given
the treatment at the end of the chronic ones showed moderate germination while others did not germinate. The earlier was the irradiation
with 10 kr, the more reduced were seedling growth and seed fertility atacute and chronic irradiations. Furthermore, chronic irradiations wereclearly more effective in radiation damage than the acute ones administered at the end of the chronic ones, and less effective than those given
at the beginning of the chronic ones. Thus it is supposed that almost
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

I

I

J

. . . Uft)
6
11100
CIllo....{ 0
1110
«>--0
0

..... I~t:..- 100:1000

0~1 _ _---:~--~!~--_:fl~
o
10
20
30
DoeeI (II,)

Fig. 1. Relation between seedling height and doses ordose rates in upland rice (1961)

no recovery phenomena were involved.
As to chlorophyll mutations, a slight increase of mutation rate due tostorage was also found. Further, the acute irradiations showed mostly
a higher mutation rate at 10 kr than the chronic ones, indicating probably
a dose rate dependency in the frequency of chlorophyll mutations.

80. A red-stem mutant in einkorn wheat induced by X-rays(By Taril FUJII)In 1957, a mutant with red stem was found in the Xs-generation of
Triticum monococcum flavescens after 10 kr irradiation by X-rays. It appeared among 8 Xs-plants, and 50 normals and 22 red-stem plants weresegregated in the Xs-generation from the green Xs heterozygotes. They
showed almost normal green color at the seedling stage. The red pigment appeared around the middle of April in the main veins of the
3rd or 4th leaf and successively in the veins of the following leaves andstems. The red color of the stems was more distinct within the leaf
sheaths than in the non·sheathed upper parts of the stem which develop

RESEARCHES CARRIED OUT IN 1962

101

much more chlorophyll. According to the microscopical examination ofcross sections of stems, the pigment is found in the cell walls of thestem epidermis. Some mutant seedlings were placed in the greenhouse;
the red pigment appeared from the 3rd or 4th leaf like in those grownin the field during the winter. The appearance of red pigment was not
influenced by temperature.
The plant height was measured at heading time in 1959; it was 115.8
em in mutants and 126.9 cm in normals. Germination and fertility ofnormal and mutant plants were examined; they were slightly lower inmutant strain than in the norm2.ls. Crossing experiments with normals
and several other mutants were carried out. All F 1 plants showednormal green color and relatively high fertility, showing no marked difference from the normal strain. Segregation ratios were examined in theF 2 -generation. Linkage relationship between chlorophyll mutant genes
and the gene for red stem could not be determined because of highmortality in winter of chlorophyll mutant seedlings, which made it
impossible to recognize the red-stem mutant. But genes for irregularear and red stem may be linked.

81.

Multi-subunit target model for RBE versus LET relationshipfor radiation damage to higher forms 1)(By Sohei KONDO)

Assume that RBE depends only on LET. Then, the RBE of radiation
A.i to Ao is expressed by (Quastler: 1960) RBE (Ai: Ao)=(LoP;/PoLi ) where
Land p are, respectively, the mean LET (energy E divided by chargedparticle range R) and the mean reaction probability to give rise to agiven type of biological response when a target molecule is traversed
by a particle. Since p is expressed as R-1~P(L)dR where P(L) is thereaction probability of a particle at stopping power L, assuming RccE18
we have(1)
P(L)= P(L)-0.44L(dP/dL) .
A multi-subunit target model is assumed for P(L) as follows:

P(L)=(1_e- Lt / W )m ,(2)

where w( =75 eV) is mean energy per primary ionization, m the numberof subunits per target each of which must be inactivated by one or
1) This work was supported by Grant from the Toyo Rayon Foundation for the
Promotion of Science and Technology.

102

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

more primary ionizations and t the average diameter of each subunit.
In other words, chromosomes are assumed to be made up of m or morethan m subunits of t in diameter. All the curves of p versus L calculated
from the published aerobic RBE values with respect to radiation damages
to mice and Tradescantia show striking similarity. By adjusting m andt, equation (2) for the intermediate LET can be fitted very well to theempirical p versus L curves obtained by applying (1) to these p versus
L curves. In conclusion, all the RBE versus LET curves in the intermediate LET can be explained by the assumption that chromosomes inhigher forms are made up of ca. 10 or more subunits and that primarylesions of radiobiological damages to higher forms are due to chromosomal damage whose production requires one or more primary ionizationin each subunit of ca. 60A in diameter.

G.

HUMAN GENETICS

82. Embryonal sex ratio in the Japanese
determined by the sex-chromatin test
(By Ei MATSUNAGA, Akira TONOMURA, Naornichi INUI and Takeo HONDA)

Fresh epithelial tissues obtained from 218 medical abortions in apparently normal early pregnancy (about 4 to 18 weeks) were investigated.
Preparations for the sex-chromatin test were made according to theordinary histological method, after fixation in alcohol-acetic (3:1), andstained by Feulgen technique. One hundred cells from each specimen
were examined by the use of a standard microscope at a magnificationof 2000 X. Specimen encountered with 30 to over 90 per cent of sexchromatin positive nuclei were classified as female, while those with lessthan 10 per cent were regarded as male.
In the first examination of the 218 specimens, 14 were discarded fromthe present study, since the staining of tissue cells was unfavorable forthe detection of sex. Of the remaining 204, 76 specimens were easily
identified as male and 73 as female. The remaining 55 specimens exhibited some discrepancy in the percentages of sex-chromatin positive
nuclei between the investigators, so that these were re-examined in an
additional hundred cells from each specimen. The result of the second
examination showed that 19 specimens were male, 14 female, and in theremaining 22 the percentages of sex-chromatin positive nuclei ranged
from 11 to 29. In the third examination 14 out of the 22 specimens had
11 to 20 per cent of sex-chromatin positive nuclei and 5 ranged from 21

103

RESEARCHES CARRIED OUT IN 1962

to 29 per cent. The remaining 3 specimens showed 30 per cent of sexchromatin positive nuclei and were determined as female. The resultsof the three successive examinations are summarized in the following
table.
Table.

Result of sex-chromatin test in 204 human embryos
Percentage of sex-chromatin
positive cells

Examination

1-10
First
Second
Third

76
19
-

I

11-20

I

21-29

-

-

--

-

14

5

95
Total

95

14

5

I

Total

Sex ratio

73
14
3

149
33
22

104.1:100

90

(185)

105.6:100

90

204

30-

In the present investigation 185 out of 218 specimens (84.9%) wereavailable for the determination of sex; namely 95 specimens were classified as male and 90 as female. Disregarding the remaining 19 cases
which failed to provide unambiguous results in sex determination, thisgives a ratio of 105.6 males: 100 females. Though the material here
examined is too small to be conclusive, the result of our preliminary
investigation may approximate the early embryonal sex ratio in theJapanese.

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

In order to investigate the selective mechanism operating on ABO
blood groups, an extensive survey was carried out in the City of Ohdate,in Akita Prefecture, in collaboration with the local government and the
Ohdate Health Center. About 3,400 couples with wife ranging in age
from 30 to 40 years at the time of the investigation, were located irrespective of whether they had children or not. With the aid of some twenty
trained midwives who were acquainted with the individual families, information with respect to reproductive histories was obtained by interviewing the wives. An effort was made to examine the blood groups
1) This work was supported by a grant (RF 61113) from the Rockefeller Foundation.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13
Table 1. Summary of pooled data, showing segregationof blood groups in children.
A. Compatible matings<;1 X (')

No. ofmatings

AxA
BxB
ABxAB
AxO
BxO
ABxO
ABxA
ABxB
OxO

256
159
18
287
204
75
67
48
275

ChildlesscouplesNo. Ipn?porhon
10
0.039
2
0.013
0
0
13
0.045
0.029
6
1
0.013
2
0.030
0
0
0.025
7

Total

1389

41

Mating0.030

No. of living children
0

103
70
289
211

Total
milledI A I B I AB IU~exa-I
531
-

8
401
-

96
84
33

-

-

325
16
308
99
44
63
-

18

26
19
3

-

29

-

24
-

14
11
9
8
23

660
414
45
719
533
206
178
128
715

83

142

3598

49

126
190
57
32

20
18
7
14
21
8
2

713
536
196
407
542
211
111

669

405

90

2716

- -- 1365 1153 855
692

-

-

41

B. Incompatible matings
OxA
OxB
OxAB
BxA
AxB
AxAB
BxAB

274
215
78
162
202
81
44

5
7
4
3
7
6
2

0.018
0.033
0.051
0.019
0.035
0.074
0.045

258
214
78
93
-

98
108
123
117
28

Total

1056

34

0.032

643

909

-

435

-

-

-

304
91
81
115

-

29

of as many couples and as many children as possible. Thus, appropriate
data were collected from about 2,500 couples including some six thousandchildren. Some results of preliminary nature are summarized in Tables
1 and 2.
As to segregation ratio of the blood groups of children, comparisons
between two reciprocals in AxO, BxO, ABxA and ABxB matings,
respectively, revealed no significant deficit of incompatible children.
There were no differences in the proportion of childless couples or in
the mean number of living children between compatible and incompatible
mating groups. The mean number of postnatal deaths of children wasalmost the same in these two mating groups.
As was anticipated from the general trend toward a sudden increase

105

RESEARCHES CARRIED OUT IN 1962
Table 2. Summary of pooled data, showing mortality of children.
Proportions refer to the mean number per mating
A. Compatible matings

MatingMeanno. oflivingchildren

AxA
BxB
ABxAB
AxO
BxO
ABxO
ABxA
ABxB
OxO
Total

Mortality of children

Mean no. of

1 - - - - - - - , - - - - - - - 1 pregnancies,

excluding
inducedabortions

2.578
2.604
2.500
2.505
2.613
2.747
2.657
2.667
2.600

Postnatal
ProporNo.
tion
57
0.223
61
0.384
3
0.167
108
0.376
0.353
72
37
0.493
16
0.239
17
0.354
0.382
105

Prenatal
ProporNo.
tion
50
0.195
43
0.270
0.389
7
70
0.244
51
0.250
30
0.400
17
0.254
17
0.354
0.251
69

2.590

476

0.343

354

0.255

3.188

59
62
18
43
53
23
8

0.215
0.288
0.231
0'264
0.262
0.284
0.181

3.091
3.153
3.051
3.117
3.252
3.210
2.932

266

0.252

3.139

I

I

2.996
3.258
3.056
3.125
3.217
3.640
3.149
3.375
3.233

B. Incompatible matings
OxA
OxB
OxAB
BxA
AxB
AxAB
BxAB

2.602
2.493
2.593
2.512
2.683
2.605
2.523

75
81
55
62
26
10

0.274
0.377
0.308
0.340
0.307
0.321
0.227

Total

2.572

333

0.315

24

of induced abortions during the past ten years in this country, the present
survey revealed that the local area here concerned was not an exception
in this respect. If the answers of the wives are taken at their facevalue, the average frequency of such abortions was about 1.02 per wife.
In most cases the wives had experienced induced abortions after they
had given birth to two or more children. However, the distribution of
induced abortions was homogeneous in different matings, so that thiswas omitted from our calculation and the numbers of prenatal deaths
presented in Table 2 included only spontaneous miscarriages and stillbirths. Because of the uncertainty of marital histories these figures are106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

less reliable than the postnatal mortality data, but it is evident that
there was no significant difference in the mean number of prenatal deaths
recorded between compatible and incompatible mating.
To sum up, the statistical analysis of the pooled data so far indicatedno evidence of elimination of children due to ABO incompatibility. The
result shows a remarkable contradiction to the previous ones obtained
either by segregation studies of family data published by various investigators or by fertility surveys carried out in Hokkaido. The reason
for this contradiction is at present not clear: the statistical analysis
provides no hints of interference of the frequent occurrence of induced
abortions with a possible effect of maternal-fetal incompatibility.
Analyses of the data are in progress by means of more rigorous methods.

84. Spontaneous and induced chromatid aberrations
in human cells cultivated in vitro(By Akira TONOMURA)

Breakages in chromosome regions induced by 5-bromodeoxyuridine(BUDR) treatment were analysed in cultured human cells, and comparedwith those occurring spontaneously. The cells obtained from a threemonth-old fetus were cultured in McCoy's synthetic medium supplemented with 20% of calf serum. The three-day culture cells were treatedwith BUDR (25,ug/ml) for 3 and 5 days. In scoring the chromatid aberrations, the chromosomes were classified according to the generally
accepted Denver system of nomenclature.
1) Spontaneous aberrations: In the control culture spontaneous chromatidaberrations occurred with a frequency of 0.06 breaks/cell. Most of theseaberrations were found on the large chromosomes of group I. Single
chromatid breaks were rarely detectable in the medium-sized submedian chromosomes of group III; some were found probably in one of
the three pairs of chromosomes nos. 8-10. No single chromatid or isochromatid deletion was observed in the chromosomes of groups II andIV.
2) Induced aberrations: Following the treatment with BUDR for 3
and 5 days, the aberration rates increased to 0.40 to 0.63 breaks/cell.
One of the most striking features of the chromosomes was the lengthening of the secondary constriction and centromeric region. In control cells
a secondary constriction located in the long arm near the centromere
can be detected in chromosome no. 1. In the BUDR-treated series, thesecondary constriction of this chromosome was exaggerated in a large

107

RESEARCHES CARRIED OUT IN 1962

number of cells. Similar responses were also noted in the centromericregions of the short arm of chromosome no. 1 and of the long arm of
no. 2. Single chromatid breaks were characteristic of the long arm ofchromosome nos. 1, 4, 8, 9 and 11. Furthermore, an obvious chromatid
breakage was observed at the middle part of the long arm in one of thethree pairs of group IV.
If the specific regions with high frequency of breaks are closely associated with incorporation of BUDR into the DNA, the chromosome
analysis, using such base analogues, may help in the investigation of
the structure of the human chromosomes in more detail.

85.

Sex-chromosome conditions in four cases of true hermaphroditism(By Akira TONOMURA and Takeo HONDA)

The importance of sex-chromosome conditions in chromatin-negativetrue hermaphrodites has aroused specific interest not only in relation to
the clinical features but also to the specific role of the sex-chromosomes
in sex differentiation. During our current survey of human chromosomes
in various types of congenital disorders and sex anomalies, the authors
examined the chromosomes in four cases of true hermaphroditism whose
nuclei of the oral mucosa cells showed no sex-chromatin bodies. The
clinical and histopathological examinations were carried out at the Department of Urology, Faculty of Medicine, University of Tokyo. The
results are summarized briefly in the following table 1.
The number of chromosomes and karyotypes from each case were
examined in cultured leucocytes from peripheral blood. All of the cells
here examined showed 46 chromosomes. They were found to contain 5
Table 1.

Some clinical and pathological data in four patients under study

Case
no.

Age Rearing
Sex

1

12

M

2

13

M

3

18

4

6

Gonad

Vaginalopening

Uterusperineoscrotal
hypospadias
penoscrotal
hypospadias

present
separately

"

M

"

M

"

in theposteriorurethrano
opening

UrethraRight

I Leftpresent

testisovary

welldeveloped
rudimentary"IIovotestis

testis

ovary

"

small

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

small acrocentric chromosomes in group VII and 15 medium-sized submedian chromosomes in group III. The Y chromosome was readily
identified as the largest one in group VII. It is, therefore, beyond doubtthat the present four true hermaphrodites have male chromosomal constitutions with an XY complex.
So far as we know, the presence of cells with XY complex in truehermaphrodites has been recorded only in XO/XYand XX/XY mosaics.
The latter case, however, showed a chromatin-positive pattern and wasinterpreted as another possibility of XX/Xx mosaicism (Waxman et al.,1962). One of the XO/XY mosaics revealed a partial deletion of the Y
chromosome (Conen et al., 1961). It is, therefore, a possibility of mosaicism in tissues from different parts of the body but the possibility of
undetectable small deletion on the Y chromosome is not eliminated in
the present cases. On morphological basis, however, it does not seem
possible to prove the latter possibility. Consequently, the present fourcases may be concluded to be the first examples of chromatin-negativetrue hermaphroditism in which cells with XY complex are predominant.

86. Character of modified human hemoglobin(By Tomotaka SHINODA)~

In order to obtain a fundamental information on the biological functionof the prosthetic groups in native protein in relation to its chemical
structure and reactivity to chemical agents, native human hemoglobin(Hb-A) was used as the model substance.
Crystallized Hb-A was treated with trinitrobenzenesulfonic acid (TNBS),
which was previously found by the author to be an effective modifying
agent of primary amino group in aqueous solution. Under several conditions employed, three steps of the reaction were observed. At thefirst step, f3-di-TNP-HbA was produced, at the second a, f3-tetra TNP-HbA
and at the third step a, f3octa-TNP-HbA appeared. Thus modified
hemoglobin has a specific UV-absorption spectrum of trinitrophenylated
protein (A max 355 mil, pH 6.8) in addition to that within the range of
500 mil to 600 mil which corresponds to absorption normally observed innative hemoglobin.
However, this modified substance was homogeneous in agar-gel andstarch-gel electrophoresis or CM-cellulose column chromatography. Its
oxygen affinity was decreased, and it was unstable in acidic medium,
being easily changed into methemoglobin. In the alkaline denaturation
test, it was also less stable compared with untreated hemoglobin. By

RESEARCHES CARRIED OUT IN 1962

109

dissociation-recombination experiment of the TNP-HbA with normal one,formation of new hemoglobin molecules, which were assumed to be
a2 A fJ2 TNP and a2 TNP fJ2 A , was observed electrophoretically. From structural analyses, the following results were obtained.
i) fJ-di-TNP-HbA corresponded to hemoglobin in which lysine No.
144 placed in the fJ-chain was modified with TNBS.
ii) a, fJ-tetra TNP-HbA was the substance in which the terminal
amino group of the a-chain and lysine No. 144 in the fJ-chain were modified.
iii) a, fJ-octa- TNP-HbA was the substance in which additional trinitrophenylation occurred:on lysine No. 60 or 61 in a-chain and No. 65 or 66
in fJ-chain.
To follow up this conclusion, an experiment on hemoglobins fromdifferent species is now in progress. Details of this work will be
published elsewhere.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13

BOOKS AND PAPERS PUBLISHED IN 1962
BY STAFF MEMBERS
CHIGUSA, S. and T. MUKAI 1962 Two cases of heterosis associated with the recessive genes sepia (se) and expanded wing (ew) in D. melanogaster (in Japanese).
Jap. J. Genet. 37: 381-382
ENDO, T. 1962 Inheritance of anthocyanin concentration in flowers of Torenia
fournieri. Jap. J. Genet. 37: 284-290
1962 Biochemical genetics of flower color variation (in Japanese). Nucleus
and Cytoplasm 4: 30-35
FUJII, T. 1962 Radiosensitivity in plants. V. Experiments with several cultivatedand wild rices. Jap. J. Breed. 12: 63-68- - - 1962 Chlorophyll mutations in einkorn wheat induced by radiation. Seiken
Zih6 13: 138-143- - - 1962 Chlorophyll mutations induced by radiation in einkorn wheat andtheir occurrence in clusters. Seiken Zih6 14: 1-11
1962 A red-stem mutant in einkorn wheat induced by X-rays. Seiken
ZiM 14: 51-53
HINATA, K. and H. 1. OKA 1962 A survey of hybrid sterility relationships in the
Asian forms of Oryza perennis and O. sativa. Jap. J. Genet. 37: 314-328- - - and
1962 Some considerations on the evolutionary dynamics of
cultivated rice. Jap. J. Genet. 37: 329-342
HIRAIZUMI, Y. 1962 Low viability induction by the segregation-distorter (SD)locus; preliminary note. D. 1. S. 36: 77
1962 Distorted segregation and genetic load. Jap. J. Genet. 37: 147-154
NOVITSKI, E.,
and E. MATSUNAGA 1962 Meiotic drive. Science 137: 861862
IINO, T. 1962 Curly flagellar mutants in Salmonella. J. Gen. Microbiol. 27: 167175
ISHIWA, H., E. Ryo and S. KONDO 1962 Synergetic action of 32p transmutation
and radiations on lethal effect in Salmonella (in Japanese). Jap. J. Genet. 37:
389-390
KATAYAMA, T. C. 1962 Some considerations on rice cultivation in New Guinea.
Jap. J. Trop. Agr. 5: 80-86
KIHARA, H. 1962 Comparative gene analysis in wheat and its relatives. Jap. J.
Genet. 37: 363-373
and K. TSUNEWAKI 1962 Polyploids and aneuploids of Triticum dicoccum
var. Khapli produced by N 20-treatment. W. 1. S. 14: 1-3
and1962 Use of an alien cytoplasm as a new method of producing haploids. Jap. J. Genet. 37: 310-313- - - , T. C. KATAYAMA and K. TSUNEWAKI 1962 Floating habit of 10 strainsof wild and cultivated rice. Jap. J. Genet. 37: 1-9
KIMURA, M. 1961 Some calculations on the mutational load. Proc. Symp. Genetic
Effect of Radiation. Jap. J. Genet. 36 (Suppl): 179-190

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

111

1962 On the probability of fixation of mutant genes in a population.
Genetics 47: 713-719
1962 A suggestion on the experimental approach to the origin of supernumerary chromosomes. Amer. Nat. 96: 319-320
KONDO, S. 1961 Response of silver-activated phosphate glass to a-, p- and r-rays
and neutrons. Health Phys. 7: 25-35
1962 Physical aspects of radiation biology (in Japanese). TanpakushitsuKakusan-Koso 7: 94-102
KURITA, Y. 1962 Viren als Ursache von Leukamien (in Japanese). Iden 16: 15-19
LILIENFELD, F. A. 1962 Plastid behavior in reciprocally different crosses between
two races of Medicago truncatula Gaertn. Seiken Zih6 13: 3-38
MATSUMURA, S. (ed.) 1962 Environment·controlled growth rooms in Japan. Comm.
Environ.-Cont. Growth Rooms, Tokyo: pp. 67
1962 Radiation genetics in wheat. VII. Comparison of radiation effectsof beta· and gamma-rays on diploid wheat. Radiation Botany 1: 155-165
1962 Radiation-induced variegations in Einkorn wheat and their inheritance (lap. with Eng. summ.). Seiken Zih6 13: 75-88
1962 RBE and dose rate for radiation effects in wheat. Seiken Zih6 13:
155-159- - - , T. FUJII and T. MABUCHI 1962 Relation between radiation effects anddose rates in rice (in Japanese). Proc. 3rd Conf. Radioisotopes, Tokyo: 10641067- - - , S. KONDO and T. MABUCHI 1962 Boron effects upon gamma-ray andthermal neutron irradiations in einkorn wheat; RBE of heavy particles fromlOB(n, a)7Li reaction. Seiken Zih6 14: 86-92- - - and T. MABUCHI 1962 Relation between radiation effects and dose rates
of gamma-rays in rice and einkorn wheat. Seiken Zih6 14: 66-73
OHTA, T. and1962 Cytological observations on chemically induced malesterility in sugar beet (lap. with Eng. summ.). Seiken Zih6 14: 53-56
MATSUNAGA, E. and Y. HIRAIZUMI 1962 Prezygotic selection in ABO blood groups.
Science 135: 432-434
1962 Selective mechanisms operating on ABO blood groups. HOMO 13:
73-81
1962 Some evidences of heterozygote advantage in the polymorphism of
MN blood groups. Proc. 8th Congr. Int. Soc. Blood Transf., Tokyo 1960: 126-130- - - and K. MURAl 1962 Genetic study of haptoglobin types in a Japanese
population. Proc. 8th Congr. Int. Soc. Blood Transf., Tokyo 1960: 397-400
1962 The dimorphism in human normal cerumen. Ann. Human Genet.
25: 273-286- - - 1962 Selective mechanisms operating on ABO and MN blood groups withspecial reference to prezygotic selection. Eugen. Quart. 9: 36-43- - - 1962 Population genetics on the selection due to ABO incompatibility
between mother and fetus (in Japanese). Jap. J. Human Genet. 7: 1-9
1962 An inert allele Hpo at the Hp locus. Jap. J. Human Genet. 7: 133136

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13- - - , K. MURAl and E. MATSUDA 1962 Inheritance of haptoglobin types in
51 Japanese families. Acta genet. 12: 262-280
MORISHIMA, H., K. HINATA and H. I. OKA 1962 Comparison between two cultivatedrice species, Oryza sativa L. and O. glaberrima Steud. Jap. J. Breed. 12:

153-165- - - , and1962 Floating ability and drought resistance inwild and cultivated species of rice. Indian J. Genet. and PI. Breed. 22: 1-11
MORIWAKI, K. 1962 Activity change of phosphatases in the skin of hairless mutant
mice during development. Physiol. Zool. 35: 193-200
MUKAI, T. 1962 A study of the genetic structure of natural populations in Drosophila melanogaster by means of spontaneous polygenic mutation rates 1.
Theoretical and statistical. Proc. Jap. Acad. 38: 741-746
1962 On the mutation rates (in Japanese). Iden 16: 19-23- - - and A. B. BURDICK 1962 Behavior of sex·linked recessive lethals in
populations. Seiken Zih6 14: 35-44
and S. CHIGUSA 1962 Radiation-induced mutation rates of polygenes
controlling the number of sternopleural bristles in D. melanogaster. D. I. S.
36: 93-94- - - and S. CHIGUSA 1962 Spontaneous mutation rate of polygenes controllingthe viability, and the genetic structure of natural populations in D. melano·
gaster (in Japanese). Jap. J. Genet. 37: 400- - - and
1962 A study of the genetic structure of natural populations
in Drosophila melanogaster by means of spontaneous polygenic mutation rates
2. Experimental. Proc. Jap. Acad. 38: 747-752- - - and I. YOSHIKAWA 1962 A radiation genetical consideration
concerning the genetic structure of natural populations in Drosophila melano·
gaster. Abstracts of the papers presented in the 4th meeting of The Japan
Radiation Research Society: 41-42- - - , I. YOSHIKAWA and S. CHIGUSA 1962 Radiation·induced mutation rate ofpolygenes controlling the sternopleural bristle number in Drosophila melanogaster (in Japanese). Jap. J. Genet. 37: 400
NARISE, T. 1962 Studies on competition in plants and animals, X. Genetic
variability of migratory activity in natural populations of Drosophila melanogaster. Jap. J. Genet. 37: 451-461
NAWA, S. and H. S. FORREST 1962 Synthesis of the yellow pteridine, isopepiapterin.
Nature, 196: 169-170- - - and
1962 Structures of sepiapterin and isosepiapterin. Nature
196: 372~373
NISHIKAWA, K. 1962 Hybrid lethality in crosses between Emmer wheats andAegilops squarrosa, II. Synthesized 6x wheats employed as test varieties. Jap.
J. Genet. 37: 227-236- - - 1962 Hybrid lethality in crosses between Emmer wheats and Aegilops
squarrosa, III. Gene analysis of Type 2 necrosis. Seiken Zih6 14: 45-50
HIRAYOSHI, I.,
and T. FUJII 1962 Studies on homologous inhibitors, III.
Growth inhibitors extracted from culture solution of pea, upland- and paddy-

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

113

rice plants. Res. Bull. Fac. Agr. Gifu Univ. 17: 99-103
OGAWA, Y. 1962 Temperature and development of muscle protein in Triturusembryos (in Japanese). Kagaku 32: 34- - - 1962 Growth, differentiation and regeneration XXVIII. Influence of temperature on the synthesis of myosin in early Triturus embryo (in Japanese).
Med. and Bio1. 62: 4-6- - - 1962 Growth, differentiation and regeneration XXIX. Effect of sodiumglucuronate on the synthesis of contractile protein, myosin, in the X-irradiatedTriturus embryo (in Japanese). Med. and Bio1. 62: 33-36- - - 1962 Abnormal taste response to phenyl-thio-carbamide in Japanese (inJapanese). Med. and Bio1. 62: 116-119- - - 1962 Protective action of sodium glucuronate in abnormal differentiationof skeletal muscle tissue in the X-irradiated Triturus embryo. Proc. of the
7th Symp. on Glucuronic acid 128-137- - - and H. WATANABE 1962 Growth, differentiation and regeneration XXX: Effects of p-hydroxy-benzyl-methl-ether, a component of Citrullus colocynthis, on
the growth and differentiation of Triturus embryo (in Japanese). Med. andBio1. 63: 1-5
1962 Synthesis of skeletal muscle proteins in early embryos and regenerating tissue of chick and Triturus. Expt1. Cell Research 26: 269-274
1962 Biochemical relation between actin and myosin syntheses during thedifferentiation of skeletal muscle tissue of Triturus. Exptl. Cell Research
26: 526-533- - - 1962 Hyper sensitive and sweet taste response to phenyl-thio-carbamide(PTC) (in Japanese). Med. and Bio1. 63: 57-60- - - 1962 Growth, differentiation and regeneration XXXI. Similarity of the
response to X-ray of myosin synthesis in early embryo and regenerating limb
tissue of Triturus (in Japanese). Med. and Bio1. 63: 112-115- - - 1962 Influence of temperature on the syntheses of contractile proteinsin Triturus embryos. Nature 194: 978-979
OKA, H. I. 1961 A review of rice culture in Tropical America (in Japanese). Jap.
J. Trop. Agr. 5: 32-35- - - and W. T. CHANG 1962 Rice varieties intermediate between wild andcultivated forms and the origin of the Japonica type. Bot. Bull. Acad. Sinica
3: 109-131- - - and Y. DoIDA 1962 Phylogenetic differentiation of cultivated rice, XX.
Analysis of the genetic basis of hybrid breakdown in rice. Jap. J. Genet.
37: 24-35
OSHIMA, C. 1962 The persistence of some recessive lethal genes in natural populations of Drosophila melanogaster. n. Proc. Jap. Acad. 38: 278-283
1962 The persistence of deleterious genes in natural populations of D.
melanogaster. D. I. S. 36: 105- - - and S. MINAMORI 1962 Study of deleterious genes in natural populationsof Drosophila melanogaster. Jap. J. Genet. 37: 408- - - 1963 The persistence of some recessive lethal genes in natural populationsof Drosophila melanogaster. III. Proc. Jap. Acad. 39: 125-130

114

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 13
1963 The persistence of deleterious genes in natural populations of D,

melanogaster. D. 1. S. 37: 115
SAKAGUCHI, B. and D. F. POULSON 1962 Distribution of "sex-ratio" agent intissues of D. willistoni. Genetics 46: 1665-1676- - - 1962 Cytoplasmic factors of abnormal "sex-ratio" condition in Drosophila(in Japanese). Nucleus and Cytoplasm 3: 27-34
SAKAI, K. 1. 1962 Recommendations for the coconut palm investigations. The
Indian Coconut Journal. 15: 111-112
SAKAI, K. 1. and S. IYAMA 1962 Genetics. In "Comprehensive Dictionary for
Statistical Science" (ed. by NAKAYAMA, 1.) (in Japanese). Toy6 Keizai-Shimp6.
Sha, Tokyo: pp. 536-540
SAKAMOTO, S. 1961 Arisaema triphyllum, Jack-in-the-Pulpit, in Minnesota, especiallyat the Cedar Creek Natural History Area. Proc. Minn. Acad. Sci. 29: 153-168
SHINOHARA, M. 1962 Variation in the number of nucleoli in rice species having
the AA genome (in Japanese). Jap. J. Genet. 37: 243-247
TAKENAKA, Y. 1962 Cytogenetic Studies in Nicotiana, XV. Reduction divisions
in five interspecific hybrids between sections Alatae and Suaveolentes (inJapanese). Jap. J. Genet. 37: 80-85- - - 1962 Cytogenetic studies in Nicotiana, XVI. Reduction divisions in six
interspecific hybrids between N. tabacum and other six species (in Japanese) ..
Bot. Mag. Tokyo 75: 237-241- - - 1962 Cytogenetic studies in Nicotiana, XVII. Reduction divisions in fiveinterspecific hybrids (in Japanese). Jap. J. Genet. 37: 343-347- - - 1962 Cytogenetic studies in Nicotiana, XVIII. Reduction divisions in.
three interspecific hybrids between autotetraploid N. tabacum and three other'
species (in Japanese). Jap. J. Breed. 12: 275-277
1962 Cytogenetic studies in Nicotiana, XIX. The reduction divisions infour hybrids between N. paniculata and other species of section Alatae (inJapanese). Jap. J. Breed. 12: 278-280- - - 1962 Studies on the genus Prunus, 1. The origin of Prunus yedoensis'(in Japanese). Bot. Mag. Tokyo 75: 278-287
TATEOKA, T. 1962 Starch grains of endosperm in grass systematics. Bot. Mag.
Tokyo 75: 377-383
T AZIMA, Y. 1962 The genetics of the silkworm. Logos press, London (in press)- - - and S. KONDO 1962 Differential radiation-sensitivity of germ cells as apossible interpretation of sex difference in dose-rate dependence of induced
mutation rates in the silkworm. Proceedings of a symposium on "Repair
from genetic radiation damage and differential radiosensitivity in germ cells".
Pergamon Press, Oxford (in press).
TONOMURA, A., Y. TOYOFUKU and E. MATSUNAGA 1962 The frequency of so·called
drumsticks in the polymorphonuclear neutrophilleucocytes of Japanese females.
Jap. J. Human Genet. 7: 60-66_ _ _ , T. HONDA and T. KURITA 1962 Chromosome abnormalities in a child'with Down's syndrome and in its father. Proc. Japan Acad. 38: 526-530
TOYOFUKU, Y. 1962 Drosophila survey of Hokkaido, XVII. A study of chromo-

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

115

somal polymorphism in natural populations of Drosophila nigromaculata. Jap.
J. Genet. 37: 291-301
TSUJlTA, M. 1962 Changes in the amount of isoxanthopterin with developmental
stages of the silkworm larvae (Abst). J. Seric. Sci. Jap. 13: 212- - - 1962 Emission mechanism of alkaloids from tobacco plants (I) Changes
in the amount of alkaloids with the growth of two "Bright yellow" strains,
ordinary and cherry red, and emission of alkaloids from their leaves (Abst).
Proc. Ann. Meet. Tokai Branch Seric. Soc. Jap. 10: 25-26
1962 Emission mechanism of alkaloids from tobacco plants (II) The
amount of alkaloids obtained from fresh leaves by different distillating method(Abst). Ibid: 26- - - 1962 Tumour formation by multiplication of abnormal small cells on
the mid-gut wall of the silkworm larvae (Abst). Ibid: 29-30- - - 1962 Manifestation mechanism of yellow larval color in the silkworm,
with special regard to d-lem gene. Jap. J. Genet. (in press)
TSUNEWAKI, K. 1962 Monosomic analysis of synthesized hexaploid wheats. Jap.
J. Genet. 37: 155-168- - - 1962 Functional differentiation among the homoeologous chromosomesof common wheat. Seiken ZiM 13: 112-118- - - 1962 Production of polyploid wheat by nitrous oxide, II. N20-treatment
during fertilization with reference to culture media. Seiken Zih/) 14: 12-20- - - and H. KIHARA 1962 Comparative gene analysis of common wheat andits ancestral species, I. Necrosis. Jap. J. Genet. 37: 474-484
YAMADA, Y. 1962 Genotype by environment interaction and genetic correlationof the same trait under different environments. Jap. J. Genet. 37: 498-509
YOSIDA, T. H., K. MORIWAKI, Y. MATANO and K. UTSUMI (1961) Cytological studies
on the effect of radiation, II. Modification of frequency of X-ray induced
chromatid breaks in Ehrlich tumor cells by pretreatment with dinitrophenol.
Jap. J. Genet. 36 (Suppl.): 94-104

ACKNOWLEDGMENT·The editor wishes to acknowledge Dr. F. A. LILIENFELD for reading the manuscripts (Sohei KONDO).

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