ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 32

1981

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1982

Anlf1lual Report

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Nat~onal Institute of Genetics
No. 32, 1981

Published by

The National Institute of Genetics, Japan
1982

CONTENTS
General statement
Staff
Council
Association for propagation of the knowledge of genetics
Projects of research for 1981
Researches carried out in 1981
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Molecular Genetics
Methylation of the cap structure in mRNA by vaccinia associatedenzyme system. URUSHIBARA, T., KAMIMURA, T., YAMAGUCHI, K., HATA, T. and MIURA, K. -I
On the base-stacking in the 5'-terminal cap structure of mRNA:a fluorescence study. NISHIMURA, Yo, TsuBOI, Mo, YAMAGUCHI, K., HATA, To, HATTORI, M. and MIURA, K. -1.. . . . . .
The nucleotide sequence of the tobacco chloroplast gene for thelarge subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase. SHINOZAKI, K. and SUGIURA, M.. . . . . . . . . . .... . .
The complete nucleotide sequence of a 23S rRNA gene fromtobacco chloroplasts. TAKAIWA, F. and SUGIURA, M.. . .. . .
Nucleotide sequence of the l6S-23S spacer region in an rRNA genecluster from tobacco chloroplast DNA. TAKAIWA, F. and
SUGIURA, ]\II.
° ••••••••••••••• ° •The complete nucleotide sequence of a 16S ribosomal RNA genefrom a blue-green alga, Anacystis nidulans. TOMIOKA, N.
and SUGIURA, M
Cloning and characterization of a plasmid DNA from Anacystis
nidulans 6301. SHINOZAKI, K., TOMIOKA, N., YAMADA, C.
and SUGIURA, M,
Transformation with the DNA fragments encompassing thepolyoma virus promoter. SOEDA, Eo, MAKI, Yo, NAKANO,
Y. and TASHIRO, H...............
A total nucleotide sequence of murine papovavirus K DNA.
JIKUYA, H., TAKEMOTO, K. K., and SOEDA, E
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Genomic structure of human polyomavirus JC: nucleotide se0

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

quence of the region containing replication origin and small
T antigen gene. MIYAMURA, T., JIKUYA, H., SOEDA, E. and
YOSHIIKE, K," . . . . . . . . . . ..
Complete nucleotide sequence of Harvey murine sarcoma
virus genome. FuRUICHI, M., NAKANO, Y., CHAN, H. W.,
MARTIN, M. A. and SOEDA, E...
Construction of a gene library of silkworm and cloning its rRNA
genes. KUSUDA, J.

II.

Microbial Gelletics
Physical map of nrdA-nrdB-ftsB-glpT region of the chromosomal
DNA of Escherichia coli. YAMADA, M., TAKEDA, Y.,
OKAMOTO, K. and HIROTA, T...........................
Inhibitory effect of adenosine 3', 5'-phosphate on cell division ofEscherichia coli K-l2 mutant derivatives. UTSUMI, R, TANABE,
H., NAKAMOTO, Y., KAWAMUKAI, M., SAKAI, H., HIMENO,
M., KOMANO, T. and HIROTA, Y... . . . . . . . . . . . . . . . . . . . . . .
Cell surface charge and cell division in Escherichia coli after X
irradiation. SATO, c., KOJIMA, K., NISHIZAWA, K. and
HIROTA, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure of replication origin of the Escherichia coli K-12 chromosome: the presence of spacer sequences in the ori region carrying information for autonomous replication. ASADA, K.,
SUGIMOTO, K., OKA, A., TAKANAMI, M. and HIROTA, Y.....
Conjugative transfer of pBR322 carrying the chromosomal DNA
of Escherichia coli mediated by an Hfr. YAMADA, M. and
HIROTA, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Escherichia coli origin of replication: structural organization of theregion essential for autonomous replication and the recognition frame model. HIROTA, Y., OKA, A., SUGIMOTO, K.,
ASADA, K., SASAKI, H. and TAKANAMI, M, ..
On the process of cellular division in Escherichia coli: nucleotide
sequence of the gene for cellular division. NAKAMURA, M.,
MARUYAMA, 1., SOMA, M., KATO, J. -1., SUZUKI, H. and
HIROTA, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overproduction of Escherichia coli replication proteins by theuse of the runaway-replication plasmids. YASUDA, S.

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CONTENTS

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and TAKAGI, T.
Possible repressor of flagellar regulon in Escherichia coli K-12
KOMEDA, Y.
Coordination of flagellar formation and cell division in Escherichia
coli. 1. Flagellar formation and transcription of 'hag'gene in cell division mutants of Escherichia coli. NISHIMURA,
A. and HIROTA, Y.
Coordination of flagellar formation and cell division in Escherichia
coli. 2. Transcription of fla-operons in the mutants of cell
division in E. coli. NISHIMURA, A., KOMEDA, Y. and HIROTA,

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Biochemical Genetics and Immll1wgelzetics

A wild-derived new H-2 haplotype enhancing K-IA recombination.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.. . . . . . . . . . .. ..
Effect of major histocompatibility (H-2) gene complex on the susceptibility to urethan induced lung tumor in mice. MORIWAKI, K. and MIYASHITA, N...........................
The relationship between immune response genes and the sesceptibility to 4NQO-induced lung tumor. MIYASHITA, N. and
MORIWAKI, K.
Suppression of urethan-induced lung tumor development in the
hybrid between A strain and Japanese wild-derived MOL.
TEN2 strain. MORIWAKI, K. and MIYASHITA, N.........
Genetic divergence between European wild mice with Robertsonian translocations from view point of mitochondrialDNA. MORIWAKI, K., YONEKAWA, H., GOTOH, 0., WINKING, H. and GROPP, A...............................
Effect of major histocompatibility gene complex (MHC) on thegrowth of embryo-derived teratoma. TAYA, C. and MORI,VAKI, K.
Allelic constitution of Hbb locus in Chinese wild mice, Mus
musculus. MIYASHITA, N., SAKAIZUMI, M., WANG, C. H.,
MIGITA, S. and MORIWAKI, K...
Genic differentiation in the wild populations of the fish Oryzias
latipes. SAKAIZUMI, M., EGAMI, N. and MORIWAKI, K.....
Two-dimensional electrophoretic profiles of seed proteins from

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

one grain in wild and cultivated rice.
ENDO, T.

IV.

V.

SUN, C. R. andDevelopmental Genetics and Somatic Cell Genetics
In vitro differentiation of adult structures from embryonic cells
of Drosophila melanogaster. KURODA, Y.
Effect of chick endogenous lectin on cartilage differentiation ofchick limb-bud cells in culture. KURODA, Y., MATSUTANI,
E. and YAMAGATA, T.
Mutagenic activity of quercetin on embryonic human diploidcells in culture. KURODA, Y...........................
Co-mutagenic activity of non-mutagens in cultured Chinese
hamster cells. KURODA, Y. and ASAKURA, M.. . . . . . . . . . . .
Effect of TPA on cell-to-cell adhesion of Chinese hamster cellin culture. KURODA, Y. and MIKAMI, H.. . . . . . . . . . . . . . . .
An autosomal, non-maternal effect sex-differential lethal mutation in Drosophila melanogaster. OISHI, K. and WATANABE,
T.K.
Nematocyte differentiation and positional information in hydra.
FUJISAWA, T., NISHIMIYA, C. and SUGIYAMA, T...
Changes of the head activation and head inhibition potentials
during hydra regeneration. SUGIYAMA, T., ACHERMANN,
J. and TAKANO, J.....................................
Chimeric mouse embryos produced by aggregating testicular
teratocarcinoma cells and 8-cell stage embryos. HANAOKA,
K. and NOGUCHI, T...................................
Freeze preservation of mouse embryos. NOGUCHI, T. and SATO,
M.
Growth of nervous tissue of a Notch lethal, Df(l)N8 in Drosophila melanogaster. MINATO, K. .. . . . . . . . . . . . . . . .. . . . . . .

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Cytogenetics
Hypo-pentaploid chromosome constitution of a spontaneoustumor developed in the Oceanian type black rat. YOSIDA,

T.H.
Hypotrichotic mutant (ba) appeared in the inversion stock (LEM)of the Norway rat. YOSIDA, T. H. . . . . . . . . . . .. . . . . . . . . ..

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Quantitative changes of nucleolar organizer regions (NORs) in
3-methylcholanthrene induced tumors of the Indian spiny
mouse. YOSIDA, T. H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shift of the karyotypes in Indian spiny mouse tumor MPMC-53
after storage in deep freezing. YOSIDA, T. H.. . . . . . . . . . . .
Geographical variation of chromosomes numbers in the houseshrew, Suncus murinus (Soricidae) and its distribution in theworld. YOSIDA, T. H...
Geographical variation of Y-chromosomes in the house shrew,
Suncus murinus, and its distribution. YOSIDA, T. H.. . . . . .
Karyotype of a hybrid male between the domestic cat and theleopard cat. YOSIDA, T. H.
Effect of metabolic perturvation on induction of the sister chromatid exchange (SCE). SUZUKI, H., YOSIDA, T. H., KUSANO, H.
A re-examination of chiasma terminalization and chiasma frequency in male mice. IMAI, H. T. and MORIWAKI, K.......
X-Y chromosome dissociation in wild derived Mus musculus
subspecies, laboratory mice, and their F, hybrids. MATSUDA,
Y., IMAI, H. T., MORIWAKI, K., KONDO, K. and BONHOMME,
F.
Chromosome observations of some Uruguayan ants (Hymenoptera: Formicidae). GONI, B., de ZOLESSI, L. C. andIMAI, H. T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chromosome observations of tropical ants in western Malaysia
and Singapore. GONI, B., IMAI, H. T., KUBOTA, M.,
KONDO, M., YONG, H. -So and THOW, Y. P...............
Supernumerary Y chromosomes and its polymorphism in Drosophila punjabiensis. HANAKATA, A. and YAMAMOTO, M.. . . . . .
New series of segmental aneuploidy using transpositions between
X and Y chromosomes. HANAKATA, A. and YAMAMOTO,
M.
Cytogenetical studies on autosomal meiotic pairing in Drosophilamalanogaster male. YAMAMOTO, M. . . . . . . . . . . . . . . . . . . . . .
Constructions of T(2; 4), T(3; 4) autosomal translocations in
Drosophila melanogaster. YAMAMOTO, M. . . . . . . . . . . . . . .
Cytogenetical analyses of the gene altering sensitivity to trehalose

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in Drosophila melanogaster.

YAMAMOTO, M. and ISONO, K.

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The effects of the variaition in heterochromatin content on recombination frequencies in Drosophila melanogaster. YAMAMOTO, M
.

VI.

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Mutation and Mutagenesis in Animals
The mutagenic activity of N-ethyl-N-nitrosourea in various
germ-line cells of the silkworm. MURAKAMI, A.. . . . . . . . . .
Difference in mutability by the different administration routes ofchemical mutagens in the silkworm. MURAKAMI, A... . . . .
Relationship between chemical-dose and specific-locus mutations
by a mycotoxin aflatoxin B1 in pupal oocytes of the silkworm.
MURAKAMI, A.
Specific-locus mutations induced by p-emitter [methyl-3H] thymidine in primordial germ-cells of dormant silkworm embryos. MURAKAMI, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII.

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Radiation Genetics and Chemical Mutagenesis in Microorganism
and Plants
Enzymatic comparison of DNA damages induced by beta-rays
from tritiated water and by gamma-rays. INOUE, T. and
KADA, T.
Mechanisms of environmental antimutagenesis. KADA, T.. . . . . .
The effect of septum-initiation mutations on sporulation andcompetent cell formation in Bacillus subtilis. SADAIE, Y. and
KADA, T.
Purification and characterization of a human placental factor
which enhances the priming activity of r-irradiated DNA.
INOUE, T. and KADA, T................... ..
Inhibition of DNA-repair and radiosensitization. KADA, T.,
MOCHIZUKI, H. and YOKOIYAMA, A..
Mutagenic effect of ekatin (thiometon) in soybean test system.
FUJII, T. and INOUE, T.. . . . .. .. . . . . . . . . . . . . . . . . . . .. .. . .

VIIf.

Population Genetics (Theoretical)

Was globin evolution very rapid in its early stages?:

A dubious

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CONTENTS

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case against the rate-constancy hypothesis. KIMURA, M.. . . .
Possibility of extensive neutral evolution under stabilizing selection with special reference to nonrandom usage of synonymous codons. KIMURA, M.
Estimation of evolutionary distances between homologous nucleotide sequences. KIMURA, M.
Some calculations on the amout of selfish DNA. OHTA, T.
and KIMURA, M.
Two-locus problems in transmission genetics of mitochondria andchloroplasts. OHTA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic variation in small multigene families. OHTA...........
Genetic variability and rate of gene substitution in a finite population under mutation and fluctuating selection. TAKAHATA, N.
A model of evolutionary base substitutions and its application
with special reference to rapid change of pseudogenes.
TAKAHATA, N. and KIMURA, M.........................
A mathematical model of extranuclear genes and the genetic
variability maintained in a finite population. TAicAHATA,
N. and MARUYAMA, T.................................
A mathematical study on the distribution of the number of repeated genes per chromosome. TAKAHATA, N...........
Algebra of inclusive fitness. AOKI. K.......................
Estimating evolutionary distance from restriction maps of mitochondrial DNA with arbitrary G+C content. AOKI, K.,
TATENO, Y. and TAKAHATA, N.........................
One- and two-locus models of the origin of worker behavior in
Hymenoptera. AOKI, K. and MOODY, M...............

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Population Genetics (Experimental) and Evolutionary Genetics
Association of chromosome- and enzyme-polymorphism innatural populations of Drosophila melanogaster. INouE, Y.,
TSUNO, K. and WATANABE, T. K.
, 105
Typical hybrid dysgenesis is absent from the natural populationof Drosophila melanogaster in Japan. YAMAMOTO, A. and
WATANABE, T. K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Biochemical phylogeny of the Drosophila montium species sub-viii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

group.

X.

OHNISHI, S., KIM, K. W., WATANABE, T. K.. . . . . .

Human Genetics and Human Cytogenetics
Retinoblastoma: mutational mosaicism or host resistance?
MATSUNAGA. E.
Genetics of Wilms' tumor. MATSUNAGA, E. . . . . . . . . . . . . . . . . . .
The "loss" of kinetochores from chromosomes of aged individuals. NAKAGOME, Y. and ABE, T.. . . . . . . . . . . . . . . . . . ..

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Behavioral Genetics and Ecological Genetics
The behavioral changes in mice continuously reared under noise
condition. FUJISHIMA, T.
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Weed-mediated natural selection in a segregating populationof rice. MORISHIMA, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Demography and populational variability of Rumex obtusifolius
conditioned by biotic environment. TERAI, K. and MORISHIMA, H.
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Isozyme variation in Altingia forest at Ciwidey, West Java.
ENDO, T.
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Cross resistance to organophosphorous insecticides in Drosophila. INOUE, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 116

XII.

Applied Genetics
Numerical evaluation of the Indica-Japonica differentiation in rice
varieties. MORISHIMA, H. and OKA, H. 1.. . . . . . . . . . . . . . ..
Associative nitrogen fixation of rice plants with isolated bacteria.
FUJII, T. and OHTA, K...
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Diallel analysis of nitrogen fixation in the rhizosphere of rice.
IYAMA, S., SANO, Y. and FUJII, T.. . . . . . . . . . . . . . . . . . . . . . ..
Genetic variation of herbicide tolerance among F 5 lines of barnyard
grass. IYAMA, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Books and papers published in 1981 by staff members. . . . . . . . . . . . ..
Abstracts of diary for 1981
Foreign visitors in 1981
Author index

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GENERAL STATEMENT
The most memorable event in 1981 was the 3rd International Conference
on Environmental Mutagens held in Mishima, following Tokyo meeting.
At the 2nd ICEM Conference held in Edinburgh in 1977, we assumed a heavy
task to act as the host to the next Conference. Since then, we discussed
about the Conference site, either to be in Kyoto, Mishima or Tokyo. Finally
we decided to hold the meeting in two places, first in Tokyo for four days
and then in Mishima for the following two days. We earnestly desired tohold all meetings throughout in Mishima, but because of the insufficiencyof hotel accommodation we were obliged to split the meeting site. Main
events were scheduled in Tokyo, and special lectures and symposia related
to genetic effects were arranged in Mishima. Here two plenary lectures
were given by Honorary Presidents: Dr. A. Hollaender on "The quantitationof environmental mutagens" and Dr. Ch. Auerbach on "A view from theivory tower". Both farsighted pioneers in this field gave through their
enthusiastic talk deep impression to the audience, especially to young
scientists. The reception was held in a friendly atmosphere at Plaza Hotelunder the joint auspices of the Mayor of Mishima and the President of theThird International Conference on Environmental Mutagens, participatedby about 400 eminent scientists from all over the world. Among them were
Prof. P. Oftedal, the President of the International Environmental Mutagen
Society, and Prof. F. H. Sobels, the President of ICPEMC. It was, indeed,
one of the big events in the history of Mishima City. Thanks to the efforts
of many staff members of our Institute and other people concerned, theConference was closed in success.
With regard to the reorganization of the Institute of the present system
into an institute for joint use by the universities, the prospect is still dim
because of a tight financial policy of the government. However, we wish tocontinue our appeal to the government to realize the reorganization as earlyas possible so as to keep pace with the rapid progress in the science ofgenetics in the world.
To our regret, we lost Dr. T. Kawahara, a staff member of the Department
of Applied Genetics, on March 8 by a car accident. It is a great pity thatthe work on the domestication of wild quails has to be discontinued.

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We had many visitors from abroad. I, myself, visited People's Republicof China, accepting the invitation from the Ministry of Agriculture, forthree weeks from the middle of November for delivering lectures at theSouthwestern Agricultural College near Chongquing. I also gave talks at
Genetics Societies at Chongquing and Shanghai. Taking advantage ofthese opportunities I could get in touch with many young scientists of China.
I am happy that I was able to contribute to the scientific and cultural
exchange between China and Japan.
By the cooperation of the National Science Museum, the Institute held
public lectures at Tokyo on November 14. The speakers were Dr. Tomoko
Ohta, head of the 1st laboratory of the Department of Population Genetics,
who spoke on "Evolution of multigene families," and Dr. Ei Matsunaga,
head of the Department of Human Genetics, who talked on "Environmentand heredity in carcinogenesis." I also had a chance to talk to generalpublic at Tokyo Kyoiku Kaikan on a subject "What are genes?"
The budget for construction of sewage disposal was approved by thegovernment, and the works started at the end of the year.

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members

1. Department of Morphological Genetics
KURODA, Yukiaki, D. Sc., Head of the Department
The 1st Laboratory
MURAKAMI, Akio, D. Ag., D. Sc., Head of the Laboratory
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi, M. Sc.

2.

Department of Cytogenetics
YOSIDA, Tosihide R., D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Tosihide, H., D. Sc., Head of the Laboratory
YAMAMOTO, Masatoshi, Ph. D.
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, HIROTAMI, D. Sc.

3.

Department of Physiological Genetics
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
WATANABE, Takao, K., D. Sc., Head of the Laboratory
The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory

4.

Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory

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YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

5.

Department of Applied Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
The 3rd Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag., Head of the Laboratory

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Ag.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
SADAIE, Yoshito, D. Sc.
TEZUKA, Hideo

7.

Departmeut of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory

STAFF

8.

Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.
The 2nd Laboratory
YASUDA, Seiichi, D. Sc., Head of the Laboratory
YAMADA, Masao, D. Sc.

9. Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
OHTA, Tomoko, Ph. D., D. Sc., Head of the Laboratory
The 2nd Laboratory
KIMURA, Motoo, Ph, D., D. Sc., Head of the Laboratory
TAKAHATA, Naoyuki, D. Sc.
AOIG, Kenichi, Ph. D.
10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
The 1st Laboratory
MIURA, Kin-ichiro, D. Sc., Head of the Laboratory
SOEDA, Eiichi, D. Ag.
SHIMOTOHNO, Kunitada, D. Pha.
The 2nd Laboratory
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
SHINOZAKI, Kazuo, D. Sc.
U.

Genetic Stocks Center
YOSIDA, Tosihide H., D. Sc., Head of the Center
Animal Section
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.
INOUE, Yutaka, D. Sc.
KUSUDA, Jun, D. Ag.
Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory

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SANO, Yoshio, D. Ag.
Microbial Section
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
KOMEDA, Y oshibumi, D. Sc.
NISHIMURA, Akiko
12.

Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira

13.

Department of AdministrQtiOil
KITAHARA, Kunio, Head of the Department
lORI, Toshiteru, Chief of the General Affairs Section
OOIDE, Yukio, Chief of the Finance Section
Honorary Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
SAKAI, Kan-ichi, D. Ag.
MORIWAKI, Daigoro, D. Sc., Emeritus Professor of Tokyo Metropolitan
University
OSHIMA, Chozo, D. Sc.
OKA, Hiko-Ichi, D. Ag.

COUNCIL
Fum, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Director of Research Institute of Handicap to Growth
IINO, Tetsuo, Professor of University of Tokyo
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, Keisuke, Member of Atomic Energy Commission
MORIWAKI, Daigoro, Emeritus Professor of Tokyo Metropolitan University
MOROHOSHI, Seijiro, President of Tokyo University of Agriculture andTechnology
NAGAKURA, Saburo, Director of Institute of Molecular Sciences

COUNCIL7OOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, Emeritus Professor of University of TokyoSHINOZAKI, Nobuo, Director of Institute of Population Problems
TAKAHASHI, Man-emon, President of Musashi Women's Junior College
UMEZAWA, Hamao, Emeritus Professor of University of Tokyo
YAMAMURA, Yuichi, President of Osaka University
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Emeritus Professor of Tokyo Metropolitan
University
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager, Director of the National Institute of Genetics
OSHIMA, Chozo, Manager

PROJECTS OF RESEARCH FOR 1981
Department of Morphological Genetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA and MINATO)
Cytogenetics in the silkworm (MURAKAMI)
Studies on recombination in the silkworm (MURAKAMI)
Genetics of the silkworm (MURAKAMI, FUKASE and OHNUMA)
Radiation and chemical mutagenesis in the silkworm (TAZIMA and MURAKAMI)
Department of Cytogenetics

Studies on chromosome evolution and species differentiation in rodents andother small mammals (YosmA)
Chromosome study on experimental tumors (YOSIDA)
Experimental breeding and genetics of mice, rats and other wild rodents(YOSIDA and MORIWAKI)
Genetic study on the subspecies differentiation of mouse (MORIWAKI)
Immunogenetical study on the MHC functions in mice (MORIWAKl)
Cytogenetical study of ants (IMAI)
Cytogenetical studies on Drosophila (YAMAMOTO)
Cytogenetical study of fishes (YosmA)
Department of Physiological Genetics

Behavior genetics of Drosophila (WATANABE and OSHIMA)
Evolutionary and ecological genetics of Drosophila (WATANABE and KAWANISHI)
Population genetics of Drosophila (WATANABE)
Theory of population genetics and speciation (MARUYAMA)PROJECTS OF RESEARCH FOR 1981

Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA)
Analysis of gene action on cell differentiation in higher organisms (NAWA
and YAMADA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum proteins (OGAWA)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetics of isozymes in plants (ENDO)
Genetic analysis of developmental mechanisms in hydra (SUGIYAMA andFUJISAWA)
Department of Applied Genetics

Quantitative genetic studies in poultry (KAWAHARA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Behavioral genetic studies in mice (FUJISHIMA)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Evolutionary studies in wild and cultivated rice species (MORISHIMA andSANO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic effects of environmental pollution on plant population (IYAMA andMORISHIMA)
Department of Induced Mutation

Molecular mechanisms of radiation- and chemical-induced mutations(KADA, SADAIE and TEZUKA)
Environmental mutagens, desmutagens and antimutagens (KADA)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage andinduced mutagenesis (INOUE and KADA)
Genetics of Bacillus subtilis (SADAIE and KADA)
Molecular mechanisms of unicellular differentiation III Bacillus subtilis

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32(SADAIE)
Department of Human Genetics

Genetic studies on retinoblastoma and Wilms' tumor (MATSUNAGA)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME)
Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Department of Microbial Genetics

Genetic regulatory mechanisms of DNA replication in E. coli (YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA,NISHIMURA and YAMADA)
Molecular genetics on DNA replication (YASUDA and HIROTA)
Genetic studies on bacterial cell envelope (HIROTA and NISHIMURA)
Synthetic bacterial plasmid (YASUDA and NISHIMURA)
DNA replication origin of E. coli (YASUDA, YAMADA and HIROTA)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA, OHTA, TAKAHATA andAOKI)Studies on molecular evolution from the standpoint of population genetics(KIMURA, TAKAHATA and OHTA)
Theoretical studies 011 the evolution of multigene family (OHTA)
Theoretical studies on the evolution of altruism (AOKI)
Department of Molecular Genetics

Studies on the structure of genome RNA of viruses (SHIMOTOHNO andMIURA)
Studies on the primary structure of DNA (SOEDA and MIURA)
Studies on structure and function of messenger RNA (MIURA and SHIMOTOHNO)
Cloning of eukaryotic genes and their structural analysis (SUGIURA andSHINOZAKI)
Studies on molecular evolution of chloroplasts (SUGIURA and SHINOZAKI)PROJECTS OF RESEARCH FOR 1981

11

Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and SANO)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII, SAND
and IYAMA)
Studies on genetic differentiation in rice (SAND)
Cytogenetic studies of Norway rats and establishment of the chromosomal
mutant stocks (YOSIDA)
Developmental genetic studies on mouse teratomas (NOGUCHI)
Studies on preservation of rodent embryos by deep freezing (NOGUCHI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Genetical study of flagellar formation in Escherichia coli K-12 (KOMEDA)
Basic studies on the gene purification and the construction of DNA banks(SUGIURA)
Genetical study on photosynthesis in blue-green algae (SUGIURA)
Coordination of flagellar formation and cell division in E. coli (NISHIMURA,
KOMEDA and HIROTA)Electron microscopic study of cell division in E. coli (NISHIMURA)RESEARCHES CARRIED OUT IN 1981i.

MOLECULAR GENETICS

Methylation of the Cap Structure in mRNA by
Vaccinia Associated Enzyme System
Toshiyuki URUSHIBARA, Takashi KAMIMURA, Kazuo YAMAGUCHI,
Tsujiaki RATA and Kin-ichiro MIURA

Process of formation of the cap structure attached to the 5'-termini of
mRNAs synthesized in vitro by virion associated enzymes of vaccinia virus
was reported in 1979 of this report series (No. 30). It was suggested thatthe cap formation by vaccinia virion proceeds in the definite order as a beltconveyor system by using well orgarnized capping enzymes. However,when the enzymes solubilized from virion were used, the order of cap methylation was different. Here, substrate specificity of the 5'-5' confronting
dinuc1eotides was examined in vitro methylation by vaccinia virus particle
itself.
The rates of methylation of various confronting dinuc1eotides by vaccinia
virion depended on the number of the interposed phosphate groups. Maximum activity was observed in the material with three phosphate groups.
This number coincides with the interposed phosphate groups in the confronting nuc1eotides structure at the 5'-terminus of vaccinia virus mRNAs
synthesized in vitro as well as usual cap structure in a eukaryotic mRNA.
The rates of methylation of G 5'ppA and G 5'ppppA were 0.52 and 0.35,
No methylation occurred forrespectively, compared with G5'pppA.
G 5 'pA and G5'pppppG. GpA (3'-5') also was not methylated. Thus
the methylation enzyme recognizes 2 to 4 phosphates in the 5'-5' confronting
dinuc1eotides.
After methylation reaction with [methyl 3H] S-adenosyl-methionine, theadded confronting nuc1eotides were split into nuc1eotides or nuc1eosides andanalyzed. In any case only one radioactive material corresponding to 7methylaguanosine or 7-methylguanosine-5'-monophosphate was detected.
It is clear that methylation occurs only at the 7 position of guanosine in the

RESEARCHES CARRIED OUT IN 198113

confronting dinucleotides.
Comparing G5'pppA with G5'pppG, or G5'ppppA with G 5'ppppG, a
G-G structure were more effective than G-A structure as a substrate for virion
methylation. G5'pppG and G 5'ppppG have a symmetrical structure.
However, virion enzyme methylated only one guanine to yield 7-methylguanine. Methylation enzyme in a virion recognizes the 7-methylated
guanine in the confronting nucleotides, and it does not advance further methylation.
On tbe Base-Stacking in the 5 -Terminal Cap Structure of mRNA:a Fluorescence StudyfYoshifumi NISHIMURA, Masamichi TSUBOI, Kazuo YAMAGUCHI,
Tsujiaki HATA, Masao HATTORI, Kin-ichiro MIURA

With the use of a synthetic model compound, m 7G 5 'pppA and a few otherm G 5'pppN's, conformation of the 5 -terminal cap structure of mRNA wasstdied by fluorescence. The fluorescence at 370 nm of the 7-methylguanosine
residue (m 7 G) is found to be quenched when the base residue is involved in astacking interaction with the adenosine residue in the cap structure m 7 G 5'_
pppA of a eukaryotic mRNA. On the basis of the observed degree ofquenching, the amounts of the stacked and unstacked forms in the cap structure have been determined at various temperatures and pH's. It has beenfound that at pH 6.2 effective enthalpy and entropy in the unstackedstacked change are LlH=-4.4±O.l kcalJmole and LlS=-l4.3±0.2 e. u.,
respectively. The pKa value for the m 7 G residue is found to be 7.7 at lOoC
and 7.3 at 30°C. The stacked structure seems to be less favourable in the
deprotonated form that occurs in the higher pH solution. The similar results
were also obtained from the studies by circular dichroism and ultraviolet
absorption (M. Hattori, K. Miura: This report 1978 No. 29). A similar
analysis of some other cap structures indicated that the stacked form in
m 7 G5'pppN structure is favourable if N is a purine nucleoside or a 2'-0methyl-pyrimidine nucleoside but not for an unmethylated pyrimidine
nucleoside.
7

f

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

The Nucleotide Sequence of the Tobacco Chloroplast Gene forthe Large Subunit of Ribulose-l,5-bisphosphate
CarboxylasejOxygenase
Kazuo SHINOZAKI and Masahiro SUGlURAThe gene for the large subunit of ribulose-I,5-bisphosphate carboxylasej
oxygenase from tobacco has been cloned in pBR322 and sequenced (Fig. 1).
The coding region contains 1431 base-pairs (477 codons). The deduced
amino acid sequence of tobacco LS protein shows 90 %homology with thoseof maize and spinach LS. The positions in the gene corresponding to the
5' and the 3' ends of tobacco LS mRNA have been located on the DNA
sequence by a 81 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences and its terminator sequence is capable of forming a stem and loop structure. A sequence
GGAGG, which is complementary to a sequence near the 3' end of tobacco
chloroplast 168 rRNA and a putative ribosome binding site, occurs 6-10 bp
upstream from the initiation codon (Gene, 20, 91-102, 1982).
The Complete Nucleotide Sequence of a 23S rRNA Gene from
Tobacco Chloroplasts
Fumio TAKAIWA and Masahiro SUGIURA

The nucleotide sequence of a tobacco chloroplast 238 rRNA gene, including the spacer between it and the 4.58 rRNA gene, has been determined(Fig. 1). The 238 rRNA coding region is 2804 base-pairs long. A comparison with the 238 rRNA sequence of Escherichia coli reveals strong homology and further shows a similarity between the chloroplast 4.58 rRNA andthe 3'-terminal region of E. coli 238 rRNA. However, the 101 base-pairspacer sequence between the 238 and 4.58 rRNA genes has little homology
with E. coli 238 rRNA. This work has been reported in Eur. J. Biochem.
124, 13-19 (1982).

AATTCCG TATATTTTCA CATCTAGGAT TTACATATAC AACATATACC
ACTGTCAAGG GGGAAGTTCT TATTATTTAG GTTAGTCAGG TATTTCCATT TCAAAAAAAA AAAAAGTAAA AAAGAAAAAT TGGG~GC TATATATATG'"AAAGAGTAITA CAAT4ATGAT GTATTTGGCA AATCAAATAC CATGGTCTAA TAATcAAACA TTCTGATTAG TTGATAATAT TAGTATTAGT TGGAAATTTTGTGAAAGATT CCTATGAAAA GTTTCATTAA CACGGAATTC GTGTCGAGTA GACCTTGTTG TTGTGAGAAT TCTTAATTCA TGAGTTGTAG IGGAG~GATTT
A1GTCACCAC AAACAGAGAC TAAAGCAAGr GTTGGATTCA AAGCTGGTGT TAAAGAGTAC AAATTGACTT ATTATACTCC TGAGTACCAA ACCAAGGATACTGATATATT GGCAGCATTC CGAGTAACTC CTCAACCTGG AGTTCCAcCT GAAGAAGCAG GGGCCGCGGT AGCTGCCGAA TCTTCTACTG GTACATGGACIt"!

AACTGTATGG TCCGATGGAC TTACCAGCCT TGATCGTTAC AAAGGGCGAT GCTACCGCAT CAGGCGTGTT GTTGGAGAAA AAGATCAATA TATTGCTTATW

GTAGCTTACC CTTTAGACCT TTTTGAAGAA GGTTCTGTTA CCAACATGTT TACTTCCATT GTAGGTAACG TATTTGGGTT CAAAGCCCTG CGCGCTCTACGTCTGGAAGA TCTGCGAATC CCTCCTGCTT ATGTTAAAAC TTTCCAAGGT CCGCCTCATG GGATCCAAGT TGAAAGAGAT AAATTGAACA AGTATGGTCG
TCCCCTGTTG GGATGTACTA CTAAACCTAA ATTGGGGTTA TCTGCTAAAA ACTACGGTAG AGTTGTTTAT GAATGTCTTC GCGGTGGACT TGATTTTACTAAAGATGATG AGAACGTGAA CTTATTATTT ATGCGTIGGA GAGGTCGTTT CTTATTTTGT GCCGAAGCAC TTTATAAAGC ACAGGCTGAA ACAGGTGAAATCAAAGGGCA TTACTTGAAT GCTACTGCAG GTACATGCGA AGAAATGATC AAAAGAGCTG TATTTGCTAG AGAATTGGGC GTTCCGATCG TAATGCATGA
CTACTTAACG GGGGGATTCA CCGCAAATAC TAGCTTGGCT CATTATTGCC GAGATAATGG TCTACTTCTT CACATCCACC GTGCAATGCA TGCGGTTATTGATAGACAGA AGAATCATGG TATCCACTTC CGGGTATTAG CAAAAGCGTT ACGTAIGTCT GGTGGAGATC ATATTCACTC TGGTACCGTA GTAGGTAAACTTGAAGGTGA AAGAGACATA ACTTTGGGCT TTGTTGATTT ACTGCGrGAr GATTTTGTTG AACAAGATCG AAGTCGCGGT ATTTATTTCA CTCAAGATTG

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GGTCTCTTTA CCAGGTGTTC TACCCGAGGC TTCAGGAGGT ATTCACGTTr GGCATATGCC TGCTCTGACC GAGATCTTTG GGGATGATTC CGTAC"ACAGTTCGGTGGAG GAACTTTAGG ACATCCTTGG GGTAATGCGC CAGGTGCCGT AGCTAATCGA GTAGCTCTAG AAGCATGTGT AAAAGCTCGT AATGAAGGAC

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00

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GTGATCTTGC TCAGGAAGGT AATGAAATIA ITCGCGAGGC TTGCAAATGG AGCCCGGAAC TAGCTGCTGC TTGTGAAGTA TGGAAAGAGA TCGTATTTAA
TTTTGCAGCA GTGGACGTTT TGGATAAGTA A AACAGTAG ACATTAGCAG ATAAATTAGC AGGAAATAAA GAAGGATAAG GAGAAAGAAC TCAAGTAATTV

ATCCTTCGTT CTCTTAAJTG AATTGCAATT AAACTCGGCC CAATcTTTTA CTA~GGAT TGAGCCGAAT ACAACAAAGA TTCTATTGCA TATATTTTGACTAAGTATAT ACTTACCTAG ATATACAAGA TTTGAAATAC AAAATCTAGA AAACTAAATC AAAATCTAAG AcTCAAATCT TTCTATTGTT GTCTTGGATC

Fig. 1. Nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphatecarboxylase/oxygenase.

---VI

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

TTC"-~CGAGGA.A.l\GGCTTACGGTGG.\TACCTAGGCACCCAGAGACGAG~AAGGGCGTA~TAATCGACGAAATGCTTCGGGGAGTTGAAiATAAGCATA~
1

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CCT.i\GGCGAAGACGCCCGA.WGCTGCA.CCCTAGATGGCGAAAGTCCAGTAGCCGAAAGCATCACTAGCTTATGCTCTGACCCG~.GTAGCATGGGGCACGT

200

300
400

GGMTCCCGTGTGA.;TCGAC;'.AGGACCACCTTGCA2\GGCTAA.;TACTCCTGGGTGACCGATAGCGA.\GTAGTACCGTGAGGG,\.\GGGTGAA.a.AGAACCCC

sao

ATCGGGGAGTG.;,;'l.ATAG;'.Ab.TG~.CCGTAAGCTCCCMGC.'\GTGGGAGGCCAGGGCTCTGACCGCGTGCCTGTTGA:.;GnATGAGCCGGCGACTCATAG

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•

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700

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ATGACCAGGATG.;AGCTTGGGTGAAACTAAGTGGAGGTCCGMCCGACTGATGTTGAAGAATCAGCGGATGAGTTGTGGTTAGGGGTGA.A.?.TGCC.n.CTCG

800

AACCCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAGTTGACTGGACATt.TAGGGGTAAAGCACTGTTT~GGTGCGGGC~GCGAGAGCGG
900
•

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TACC."..'!>,ATCGAGGCAAACTCTGAAT.:;CTAGATATGACCTCAAAATAACAGGGGTCAAGGTCGGCTAGTGAGACGATGGGGGA!AAGCTTCATCGTCGAGA
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GGGA.A..:;CAGCCCGGATCACC.:;GCTAAGGCCCCTAAATG;:I.TCGCTCAGTGATAA.~GG?.GGT.n.GGGGTGCAGAGACAGCCAGGAGGTTTGCCTAGMGCAGC,

CACCCTTGA...;;AGAGTGCGTAATAGCTCACTGATCGAGCGCTCTTGCGCCGAAGATGAACGGGGCTA.~GCG.\TCTGCCGAAGC':GTGGGATGTA.~TAC•

ATCGGTAGGGGriGCGTTCCGCCTTAGAGAGAAGCCTCCGCGCGAGCGGTGGTGGACGAAGCGG.::"",\GCGAGAATGTCGGCTTGAGTA..lI.CGCAAACATTGGT
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CTGCTTTGTCAGGGTAAGA..a.CGGGTAGAGAA,.lVI.TGCCTCGAGCCA...lI.TGTTCGAATACCAGGCGCT.'l.CGGCCGGA...n.GTAACCCATGCCATACTCCCAGGA...lI,.
I

AAGCTCGAACGAC'I'TTGAGCMGrlGGGTACCTGTACCCGAA.!l,CCGACACAGGTGGGTAGGTAGAGAATACCTAGGGGCGCGAGACAACTCTCTCTAAGGA
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•

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1600

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1900
2000

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GTCTCGGAGi".GAGGCTCGGTGAA..l\.T.l'I.GACA'l'GTCTGTGAAGATGCGGACTlI.CCTGCACCTAGACAGAAAGACCCTATGAAGCTTCACTGTTCCCTGGGAT

·

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CCCCGGTGAACGGCGGCCG'1'AACT.~TA.~CGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGCACGAAAGGCGTMCGATCTGGGCACT
,

1300

•

CTCCGCA.A1'I.GTCGTlI.AGACCATGT.\TGGGGCTG.\CGCCTGCCc..a.GTGCCGGAAGGTCAAGGA.'\GTTGGTGACCTGATCACAGGGGAGCCGGCGriCCGMC
•

1200

•

ACTCGGCA.,\.:;]>.TAGCCCCGTAACTTCGGGAGA.\GGGGTGCCTCCTCACA......a.GGGGGTCGCAGTGACCAGGCCCGGGCGACTGTTTACCAAAAACACAGGT
•

1100

•

GAGAATCC.::l.i'.TGCCCCGAA2'1..:'l.CCTriJ'IGGGTTCCTCCGC.:;AGGTTCGTCCACGGAGGGT<;;AGTCAGGGCCTMGA.TCAGGCCGA.a•.l'l.GGCTAGTCGATGG.\C
•

1000

2100
2200
2300

•

GG'I:TTCCTCGGGCCGGACGGAGATTGGCCCTCG.\GTGCAAAGGCAGAAGGGAGCTTGACTGCAAG.::..CCC.;CCCGTCGAGCAGGGACG.:'I.AA.GTCGGCCTT.!J...

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GTG.\TCCGACGGTGCCGAGTGGA...;GGGCCGrCGCTC.::I...i\CGGATA.!l,.AAGTTAC1'CTAGCGATAl'I.C.:l.GGCTGATCTTCCCCAAGAGCTCACATCGACG.GGAA"

GGTTTGGCACCTCG';TGTCGGCTCTTCGCC.;CCTGGGGCTGT.i\GTATGTTCCMGGGTTGGGCTGTTCGCCC.;TT.a......lI.GCGGT.!I,.CGTGAGCTGGGTTC.;G

2400

2500
2600

AACGTCGTG~GAC.n..GTTCGGTCO.TATCC~GTGTGGGCGTTAGAGCATT~AGAGGACCTTTCCCT.!I,.GTACGAGAGGACC~GGA...:l...GGACG~ACCTCTGGTT
2700

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TeeT

Fig. 1. Nucleotide sequence of tobacco chloroplast 23S rRNA gene.

2800

RESEARCHES CARRIED OUT IN 1981

17

Nucleotide Sequence of the 16S-23S Spacer Region in an rRNA
Gene Cluster from Tobacco Chloroplast DNA
Fumio TAKAIWA and Masahiro SUGIURA

The nucleotide sequence of a spacer region between 16S and 23S rRNA
genes from tobacco chloroplasts has been determined (Fig. 1). The spacer
region is 2080 bp long and contains tRNA IIe and tRNA Ala genes which haveintervening sequences of 707 bp and 710 bp, respectively. Strong homology
between the two intervening sequences is observed. These spacer tRNAs
are synthesized as part of an 8.2 kb precursor molecule containing 168 and
23S rRNA sequences. This work has been reported in Nucleic Acids
Research 10, 2665-2676 (1982).
The Complete Nucleotide Sequence of a 16S Ribosomal RNA Gene
from a Blue-Green Alga, Anacystis nidulans
Noboru TOMIOKA and Masahiro SUGIURA

The complete nucleotide sequence of a 16S ribosomal RNA gene from
Anacystis nidulans has been determined (Fig. 1). This nucleotide sequencehas 83 % homology with that of tobacco chloroplast 168 rRNA gene and74% homology with that of E. coli 168 rRNA gene. The 3I-terminal regionof this gene contains the sequence CCTCC which is complementary to
sequence found at the 5 ' -termini of prokaryotic mRNAs.
Cloning and Characterization of a Plasmid DNA from
Anacystis nidulans 6301
Kazua SHINOZAKI, Nabaru TOMIOKA, Chieka YAMADA and
Masahira SUGIURA

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCI density
gradient containing ethidium bromide. The size of the plasmid DNA,
named pBA1, was estimated to be 5.04±O.26 Md by electron microscopic
analysis and 5.2 Md by agarose gel electrophoresis. The pBAl was digested
with BamHI and cloned in pBR322 vector propagated in E. coli HBlOlcells. The recombinant DNA, named pBAS18, was digested with various
restriction endonucleases and its cleavage map was constructed. The
cleavage map of the pBAI plasmid DNA was made on the basis of the

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TG
A
TG
C
T A T
GC
1
GC

A

CCCCCGGGCG AT
G GGG GGCTCGC
T
A
A

AC

AG
GC
GC
GC
CG
TG
AT
TA

TGA

AT
CTCG
GAGC

G
GTA

G
C
T
C

C
G
T

C

G

G

ACG

g

C

GC
CG

72bp

'\

tRNA

73bp

-_...

lie

T
T

A

:;>::J

G
A

C
C
G A T
G
ATAC
T AT T
TA
T
CG
CG
T
C
TA
A TC
i CAT
AT
TG
TA
TA
G1
AT
iA
TA
AT
TG
GC
C C
iA
CG
CG
AT
TA
CG
GC
AC
TAGCA
CGGTTCGATCTAT
cAiee-- 235CG

707 bp ;'ATTG...

G ATA

C

A

CG
GC

'"

AC'"

CC GC

T

_
.., ......

CG

G
G

C

GC
GC
GT
GC
AT
TAG
AT
T
A
T T G
AT
TeGCC
G
A
CTCG
AGCGG
C
G
GAGC
Cr
TT
GrA
TCGATGG
GGACC T A
TCTGG
A
C
TT C

GCGAG~c:::;1>r,..",,"""'tI,
G T'"

CT G CTAT

T GC A

T\
710 b p iI
G ... _ _ .;'

trI

'"d

0

,..,

:;0

0

"Tl

Z

,..,

;1>
F-I

0

Z
;1>
r
F-I
Z
tn

,..,
,..,
,..,c:
F-I

trI

0

"Tl

0

trl

Z

,..,trI
F-I

(J
(J).

z

9
tRNA

Ala

Fig. 1. Nucleotide sequence of the 168-238 spacer region in an rRNA gene cluster from tobacco chloroplast DNA.

w

N

19

RESEARCHES CARRIED OUT IN 1981,0• ••••••••••••••••••• T. TC •• G••• A•••••• GA T. •• G••• , •T. •• TAC •••••••• GTA, •••••••• GC •• AACGT. ••• A. ACCA •• G. G
AGTGGCGGACGGGTGAGTAACGCGTGAGAATCTGCCTACAGGACGGGGACMCAG TTGGAAACGAC TGC TAATACCCGATGTGCCGAGAGG TGA C................... , •••• ,A, ••• C
CTTG ••• G••• A, ••••• AC
GT. •••••••••• CG.AG •• T••• GA. CA" ,G390

L,

GGGG. CCT. C. GGCe. CTTGCCATCG •AT. ••• CCAGATGG ••••••••••• A•••••••••• c, .. TC ••• T. •••••••••• CC ••••••••••••••~~~~~ TC~:::~:~~:~~G~:::::

e::~G::~:::::::~::~~~~~::~:~::~~:::~~~:~ ::~::~~:::~::~:~:~,::::•••••• C•••••••••••• A

310

,

,

,

,

490

T•••••••••••• , ••••••••••••••••••• A•• G.A •••••••••••••• CT •• i.C ••• C. i ..

~~~:~~::~~::~:~::~~~~:::~~~:~ C: ~:::~~~: ::::~::~~~~::~~:~~:~:~~~~::::::~::~:~::::~~.~~:re:~~:'::~~~::
,

,

t

450
(

,

,

r

,

,

5()Q
,

• ••••• TAT. .A ••••• CC •• C•• GT. ••••• GTA•••• CAG. G•••• G•••• GGAGT .... TTAP.TACCT i TGCTCAT. •••• T•••• C. CA ••• G•••:::~:~:~::~~:~~~~~~~~~~:~~~::~~~:~~::::::~:~:~:~1~~~~
,

,

,

550

,

,

d:~~::~:~~:::~::~~:~":~
600

"

I

.AC
C
AG.GT. ••••••••• AT.
C••••••••••••••••• AC •••••••••• TG. T. •••• A
CCTCGG CTAATTCCGTGCCAGCAGC CGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAMGCG CCTG CAGGCGGTTAATCAAG TCT.A .• to., ,;C •. T..••• ; .•• , .•t. ' ; ••• t ••A.A~, ,r,'6~Q'" ,." ': ,G .••.••• : ••••.•• T.: .T .. ,T. ,C:TT,T,. "7~O

.A G T. CCC
UTGGGA. Ci TCT .. T
GCAAG •• T
C. C
A.. GG. GTA.
GTTGTCAAAG CGTGGGGCTCAACCTCATAC GCAATGGAAACTGAT TGACTAGAGTATGGTAGGOGTAGCGGGAATTCCAGGTGTAG CGGTGAMTec•CC
T. CCAerGG. " •• CGG
AC(AAG •• G
C
C.. A
T•C
G
..

' to

89 G

..... G
GII i ••• G. T.
G GC.CC
A,G.AG
G. TG
GT
A.. CAT
liTAGATATCTGGAltt AACACCAGCGG CGAAAGCGCGCTACTGGGCCATMCTGACGCTCATGGA,CGAAAGCTAGGG GAGCGAAAGGGATTAGATACCCC~'" .A,
A
A.T, .G,
G%o
A•• G.GAA•• T,';00
G••••••• A~ ••••••••• : ••• TCG ••• ~ •• A. GT.. T: CQjC. T.. G~ •• T. GQj. T: •• G T..:
~G,A ••••••• : •••••••• G~
TGTAG TCCTAG CCGTMACGATGMCACTAG GTGT GCGTGAATCGACCCG CG CAG TG CCeTAGC CMCG CGTTAAGTGTTCCG CCTGGGGilG TACGCA
A
G. T
C. •• T. C
T
T.
T
A, C
, ,.••• TT

9fo

lO?O

•••••• G.TA ••••••••• T
C
T•• TC ••••••CGCAAGTTGGAMCTCAMGGI1AHGACGGGGGCCCGCACMGCGGTGGr,GTATGTGGTTTAATTCGATGCMCGCGAAGAACCTTACCAGGGTTTG,ACA"'" .AAT
G
C
A
T•• C
..

.l.l~a10;0TC(( ~~';A~~T~Ti~GA~A;GAGA~ GTG((TT(GG GA~(~G~GA5,\(AG 5T 5~TGCAT55(TGTCGTCAS(TCST5T;GTGA~A~ GTT 5G6 TT.~AG

I

r" .c

.... G, L.JlTC .. CTTG •• A, ••• GGG,
,

,

I

I

c

11~O

C

,

,

,

A•• G

.

,

9

12 0

....................TTA. CC •• T. •••••. G. GG. C.G .cc ••• A•••• A.AG •• G•••••• A•••• T
T
G, •• T. ••••TCCCGCAACGAGCGCAACCCACGTTTTTAGTTGCCATCAT AGTTGGGCACTCTAG,\GAAACTGCCGGTGACA.AACCGeAGGMGGTGTGGACGACGT............. ,T••• GG
T.. A•• c. ,GA.C.G
TT •• GA••• T, ..
,

,

,

,

12;0

,

I

,

,

1:390

............ G••••••• GA •• A••••••••••••• G•••••••• c{J. GCAT. •• AA ••• 'A' ••• CCT. ••••• AGC, ••• 5G. C••• AT. •• GT. C
CAP,G TCATCATGCC cenACATCCTGG GCTACACACGTACTACAATG CTCCG ,CAGCG,AGACGCGAAGCCG CGAG GTGAAGCAMTCTCCCAMCCGG...................TGC T G
GGC, G
1'1. G. TTC
GTG ••• T.. C. C.AA ••••• C.

~=_~_-,c~14 09,-;--,.-_-'-,,.....-_'_3.;,.;O_ _-,,", ,.....,---i,.T.GT. •• C.G
G.. T
A.. C
T
A
A •• G.CA.
GG CTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGGCGGAATCGCTAGTAATCGCAGG CAGC TACTGCGGTGAATACGTTCCCGGGCCTT
Te. ••• ,., .GCC
e. ••• G
T.
,
.
1450

•

1.500

••••••••• : ••••••••• : ••••• G••
GTTGCAAAA
A.G: .G. T. •••• ~. cLJj
: .C.CTTA •• ~CT i TGllJ,A~ .c:
GTACACACCGCCCGTCACACCATGGMGnGGCCATGCC AAGTCGTTACCCTAACCGTTCGCGGAGGGGGG CGCCGAAG GTAG GGCTGATGACTGG....................T
G.. C
i5io .. TCA
CG , AG.

U:..

••••••••••••••••••• •A••••• GG
CC .. C•• T.
.
GGTGAAGTCG TMCAAGGTAGCCGTACCGGMGGTGTGGCTGGATCAC CTCen
A
T
C
..

Fig. 1. Nucleotide sequence of A. nidulans 168 rRNA gene. The non-coding
strand is shown (middle row). For comparison, sequences of E. coli rRNA gene
(top row) and of tobacco chloroplast rRNA gene (bottom row) are presented.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

cleavage map of the pBAS18 and pBR322 DNA.
reported in Gene 19, 221-224 (1982).

This work has been

Transformation with DNA Fragments Encompassing the
Polyoma Virus Promoter
Eiichi SOEDA" Y oshio MAKI, Yoshiro NAKANO and Hiroyuki TASHIRO

The polyoma Pst 1-2 fragments encompassing the promoter but not encoding functional tumor antigens were cloned in pBR 322 and applied to
3YI rat and NIH mouse fibroblast cells by using calcium phosphate technique.
The recipient cells were induced foci and exhibited some properties of transformed cells. Four clones of the transformed rat cells were transplanted to
new born Fisher rats to form tumors. The polyoma viral sequences weredetected in the DNAs prepared from the cells by Southern blotting hybridization as discrete Eco RI fragments. Some of these were isolated by using A
phage vector technique. The transformed phenotypes were transmissible
into normal recipients via DNA transfection.
These evidences have suggested that the polyoma virus promoter can induce
cellular transformation.
A Total Nucleotide Sequence of Murine Papovavirus K DNA
Hiroyuki JIKUYA, Kenneth K. TAKEMOT01) and Eiichi SOEDA

A total nucleotide sequence of murine papovavirus K (KV) genome has
been determined which consisted of 4994 nucleotides. Nucleotide comparison revealed that KV genome could code for small T antigen, large T
antigen, VP1, VP2, and VP3, of which sequences were homologous tothose of SV40 and polyoma virus, but not for the relevant of polyoma virus
middle T antigen. These evidences suggested that these viruses diverged
from a common origin, and enforced further that the polyoma virus middle T
antigen was implicated in tumor formation of animals.

1)
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases,
Bethesda, Maryland 20205 U.S.A..

RESEARCHES CARRIED OUT IN 1981

21

Genomic Structure of Human Polyomavirus JC: Nucleotide
Sequence of the Region Containing Replication
Origin and Small T Antigen Gene
Tatsuo MIYAMURA1 ), Hiroyuki JIKUYA, Eiichi SOEDA and
Kunito YOSHIIKE 1)

The nucleotide sequence of the region of human polyomavirus JC (JCV)DNA between 0.5 and 0.7 map units from a unique Eco RI cleavage site wasdetermined and compared with those of the corresponding regions ofanother human polyomavirus BK (BKV) and simian virus 40 (SV40) DNAs.
Within this region consisting of 945 base pairs, we located the origin of DNA
replication near 0.7 map units, the entire coding region for small T antigen,and splice junctions for large T antigen mRNA. The deduced aminoacid sequences for small T antigen and the part of large T antigen markedly
resembled those of BKV and SV40. The results strongly suggest that JCV
has the same organization of early genome as those of BKV and SV40 on thephysical map with the Eco RI site as a reference point.
Complete Nucleotide Sequence of Harvey Murine
Sarcoma Virus Genome
Masato FURUICHl, Yoshiro NAKANO, Hardy W. CHAN 2),
Malcolm A. MARTlN 2 ) and Eiichi SOEDA

The complete nucleotide sequence of Harvey murine sarcoma virus (HaMuSV) was determined and genetic elements were located on the nucleotide
sequence mainly by comparison with that of Moloney murine leukemia
virus (Mo-MuLV), progenitor of Ha-MuSV.
The viral genome is composed of 4,376 nucleotides inserts derived fromrat sequences flanked on the 5' and 3' ends by 141 and 907 nucleotides thesequences derived from Mo-MuLV, respectively. A topology of the genomic
organization has suggested that Ha-MuSV was generated as results of multiple recombinations between Mo-MuLV and rat cellular sequences, andbetween rat cellular sequences. The recombination sites between themwere tentatively assigned on the nucleotide sequence. The sequences close
1)
Department of Enteroviruses, National Institute of Health, Kamiosaki,Shinagawaku, Tokyo 141.
2)
DNA Recombinant Research Unit, National Institute of Anergy and Infectious
Diseases, National Institute of Health, Bethesda, Maryland 20205 U.S.A.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

to the recombination sites were characterized by the presence of somedirect repeat sequences.
The viral genome has a coding capacity for a transformation specificprotein, p21 src in only one open reading frame of the non-homologous region
to 30S RNA downstream from the left long terminal repeat (LTR) derived
from Mo-MuLV terminal sequences, intervening the non-coding region of
938 nucleotides in length which is highly homologous to 30S RNA. A
topology of the genomic organization of Ha-MuSV suggests that activation
elements for RNA synthesis resided in the LTR induce the enhanced expression of the p21 sre gene via approximately I kb non-coding region.
Construction of a Gene Library of Silkworm andCloning its rRNA Genes
lun KUSUDA

It has not been possible to extract intact whole genomic DNA from eukaryote cells using conventional biochemical methods because of its large
size. Recent advance of gene cloning techniques made possible to isolate
DNA fragments containing specific genes from genomic DNA.
This paper presents construction of a gene library of silkworm (Bombyxmori). Genomic DNA from posterior silkglands of 5th instar larvae(standard strain C-lOS) was digested partialy with restriction endonuclease
Sau3A and subjected to electrophoresis in 1% agarose gel. The DNA
fragments of I~2x 10 4 bp long eluted from agarose gel were joined to .Ie
phage vector. Charon30 DNA was cleaved into four fragments with BamHI
and removed two internal fragments with genes nonessential for phage viability. The Charon30 arms consist of BamHI fragments containing the .Ie cossite were ligated with genomic DNA fragments digested with Sau3A which
produced the same cohesive end as BamHI. Ligated DNAs were packaged
in vitro. Yield was about 1.4 X 10 5 infectious phages per /kg silkworm DNA.
Assuming an average size of 1.3 X 104 bp for replaceable fragments in
Charon30 and a genome size of I X 109 bp for silkworm, the number of independent recombinant phage needed to find any given single copy sequencein the library with a probability of 0.99 is calculated to be 3.5 x 10 5 phages on
the basis of an equation by Clarke and Carbon (Clarke, L. and Carbon, J.
1976 Cell 91-99). Approximately 4 X 10 5 recombinant phages were produced
for the gene library of silkworm. In order to examine the faithfullness for

RESEARCHES CARRIED OUT IN 198123

representation of silkworm genome in this library, the size of inserts and theprobability of finding clones carrying rRNA gene were determined. The
average size of insert DNAs from 35 independent plaques was lAx 10 4
bp as judged by restriction analysis. From this size, the number ofclones were proven to be sufficient for the gene library of silkworm. It is
known that the silkworm genome code for 240 copies of rRNA genes and therepeat unit of this gene is I X 104 bp long. We expected to detect one to two
rDNA clones in 400", 500 plaques from above values. The silkworm library
was screened for rDNA sequence using 32p labeled rRNA as a probe.
Positive signals were observed at the frequency of one per 360 plaques andthis agreed well with the estimated value. The size of inserts and a recovery
of clones containing rRNA gene suggest that this gene library is complete
and available as a gene pool of silkworm.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

II.

MICROBIAL GENETICS

Physical Map of nrdA-nrdB-ftsB-glpT Region of the Chromosomal
DNA of Escherichia coli
Masao YAMADA, Yutaka TAKEDA, Kazuo OKAMOTO and Yukinorr HiROTA

Seven pLC plasmids (pLC 3-46,8-12,8-24,8-29,14-12,19-24, and 42-17)
which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia
coli were analyzed. A restriction map of each plasmid was constructed andrestriction fragments were subcloned into pBR322. A physical map of
approx. 15 X 10 6 Md segment of the chromosomal DNA was deduced fromthe overlapping region of the pLC plasmids. The pLC plasmids and newly
constructed plasmids were examined for the ability to rescue the mutations.
The complementation tests defined the location of the genes in the 15 X
10 6 Md segment in the following order: nrdA-nrdB-ftsB-glpT. Functional
nrdA, B andftsB genes were located in the 3.1 X 10 6 Md EcoRI-PstIfragment.
(For detail, see Gene, 18,309-318, 1982).
Inhibitory Effect of Adenosine 3', 5'-phosphate on Cell Division ofEscherichia coli K-12 Mutant Derivatives
Ryutaro UTSUMI*, Hiroyuki TANABE*, Yumiko NAKAMOTO*,
Makoto KAWAMUKAI*, Hiroshi SAKAI*, Michio HIMENO*,
Tohru KOMANO*, and Yukinori HIROTA

Cell division of Escherichia coli K-12 strain PA3092 was inhibited by theaddition of adenosine 3',5'-phosphate (cAMP), and the cellular morphology
was changed from rods into filaments. Nucleoids in the filaments wereregularly distributed and septum formation was perfectly inhibited. This
inhibition of cell division by cAMP was reversed by the addition of guanosine
3',5'-monophosphate. To examine whether the inhibitory effect of cAMPon cell division in E. coli PA3092 was specific, its effect in several parental
strains was investigated. Induction of cell filamentation by cAMP was observed in E. coli PA309 and P678, but not in E. coli W595, WI, YlO, or the* Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto 606.

RESEARCHES CARRIED OUT IN 198125

wild type strain. This result suggests that filamentation by cAMP in E. coli
PA3092, PA309, and P678 was due to the mutagenesis by which E. coli P678
was derived from E. coli W595. (For detail, see Bacterial. 147(3), 11051109, 1981).
Cell SlJurface Clrnarge ami CeDi Division ill Escncftichia coli
after X Inadiatimll
Chikako SATO*, Kiyohide KOJIMA*, Kimiko NISHIZAWA*, andYukinori HIROTA

Simultaneous detection of electrophoretic mobility (EPM) and morphology
of individual irradiated Escherichia coli cells under the phase microscope
revealed a concurrent decrease in EPM and arrest of cell division. EPM
decreased with time and reached a minimum 15 min after irradiation withdoses ranging from 100 R to 80 kR. Cells
due to the division
block retained the minimum EPM. After a recovery
separated smallsized daughter cells and some long filamentous cells, which had a few
cleavages at the termini, returned to the normal EPM. This finding indicatesthat recovery in EPM, which represents recovery in the surface architecture,
precedes or coincides with the resumption of cell division. Nuclear stainingof the recovering cells leads to the suggestion that the cleavage of the celltakes place whenever the EPM has recovered, irrespective of the segregationof DNA, which gives rise to anuc1ear cells having normal EPM. It issuggested that the mechanism of EPM decrease is Ca2+-dependent conformational change of the mambrane accompanying vertical translocation of(For detail, see Radiation Research, 87, 646-656, 1981)* Laboratory of Experimantal Radiology, Aichi Cancer Center Research Institite,
Chikusa-ku, Nagoya 464, Japan.
** Laboratory of Oncological Pathology, Nara Medical University, Kashihara 634,
Japan.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Structure of Replication Origin of the Escherichia coli K-12
Chromosome: the Presence of Spacer Sequencesin the ori Region Carrying Information forAutonomous Replication
Kiyozo ASADA*, Kazunori SUGIMOTO*, Atsuhiro OKA*, Mituru TAKANAMI*,
and Yukinori HIROTA

The replication origin region of the Escherichia coli K-12 chromosome
has been cloned, and a region of 245 base-pairs has been shown to containall the information for autonomous replication (defined ori). In order toobtain further information on the sequence organization in the defined ori
stretch, various types of mutation were introduced by in vitro techniques
as a restriction site (AvaIl site) which is located near the middle of ori.
When the correlation between these mutations and replicating junction was
examined, different effects were obtained with the types of mutation: thereplicating function was completely destroyed by either insertion or deletionof short sequences, but not by base- substitutions. Based on these observations and on the fact that multi-gene products are involved in the initiation of
replication, we assume that two categories of sequences are present within
the ori stretch, one specifying interaction with initiation factors (recognition
sequences) and the other spacing the recognition sequences in appropriate
distances (spacer sequences), and that the AvaIl site is located in the spacer
region. (For detail, see Nucleic Acid Research 10(12), 3745-3754, 1982)
Conjugative Tnmsfer of pBR322 Canying the Chromosomal DNA of
Escherichia coli Mediated by alill Hfr
Masao YAMADA and Yukinori HIROTA

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and asegment of chromosomal DNA of Escherichia coli could be transferred froman Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of theHfr, the frequency of the transfer was high. The recA function of both
donor and recipient cells was required in the tansfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome between
the homologous sequence may cause the transfer of the non-mobilized* Institute for Chemical Research, Kyoto University, Uji, Kyoto.

RESEARCHES CARRIED OUT IN 1981

27

plasmid. This phenomemon is useful for the determination of the chromosomal site of an unidentified fragment cloned in a non-mobilized plasmid.
(For detail, see Gene, 1982).
Escherichia coli Origin of Replication: Structural Organization of
the Region Essential for Autonomous Replication and the
Recognition Frame Model
Yukinori HIROTA, Atsuhiro OKA*, Kazunori SUGIMOTO*, Kiyozo ASADA*,
Hitoshi SASAKI*, and Mituru TAKANAMI

The replication origin region of the E. coli chromosome has been cloned,
and the precise location of the region carrying autonomous replicating
function (defined ori) has been determined. The left boundary of ori is
between positions 23(A) and 35(T) and the right boundary is between positions 267(C) and 268(A). The maximum size of the ori segment is therefore
245 base pairs (bp) long.
A series of mutations having short sequences inserted or deleted was introduced in the vicinity of BglH, BamHI, AvaIl, mutations C to T (or G toA) were also introduced by in vitro, mutagenesis which used bisulfite, andthe correlation between the phenotypes and nucleotide sequences of mutant
was analyzed. We could identify two categories of regions: one is theregion in which even a single base substitution destroys ori function andthe other is the region in which insertion or deletion of short sequences,
but not base-substitutions, destroys ori function.
On the basis of these observations, we propose the following model forthe structural organization of ori (recognition frame model): the recognition
sequences that determine the sites where the components involved in thereplication initiation recognize and the spacer sequences (or distance sequences) that maintain the recognition saquences at precise destances areconnected with each other in a chimeric fashion.
According to this model, the striking effect caused by the shft of recognition sequences within ori can be explained. The ori stretch should provide
precise topological information for the formation of an initiation complex
by multiple components which are involved in the initiation of replication.
(For detail, see "The initiation of DNA replication, Academic Press., Ed.
Ray., pp 1-12.)*

Institute for Chemical Research, Kyoto University, Kyoto 611, JAPAN.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

On the Process of Cell11l1al." Division in Escherichia coli:
Nucleotide Sequence of the Gene for Cel.hdar Division
Masataka NAKAMURA, Ichiro N. MARUYAMA, Masaaki SOMA, Jun-ichi KATO*,
Hideho SUZUKI*, and Yukinori HIROTA

We determined the nucleotide sequence of a DNA fragment containing theftsI gene coding for penicillin-binding protein 3 (PBP-3) which is indispensable for the cell division of Escherichia coli. The DNA segment necessary forexpression of the fts! gene was contained in the PvuH restriction fragment

2.8 kilobase long derived from the chromosomal segment on pLC26-6.
The nucleotide sequence analysis showed that only one open reading frame,with this restriction fragment, allowed a region enough to code for the sizeof PBP-3 (60 kdal). The coding region for the PBP-3 was determined according to the N-terminal amino acid sequence of the in vitro synthesized
PBP"3, wich was mostto be the primary
product and had
the N-terminal sequence of (N) Met-Lys-Ala-Ala-Ala-Lys ..... The regionof the structural gene forftsI consisted of 1,764 base-pairs coding for a polypeptide of 588 amio acid residues and of 63,876 daltons in molecular weight.
The in vitro synthesized PBP-3 showed slower mobility in SDS-gel electrophoresis than the authentic PBP-3 and it wasthat the translational
product of the ftsI gene may be processed to yield the mature PBP-3. The
deduced amino-acid sequence indicates that the polypeptide of PBP-3 has atypical characteristic of a membranei. e. 79 % of amino-acidresidues is nonpolar or uncharged polar, and there are several hydflJpJllobic
regions. A possible promoter sequence and ribosome binding sequence ofthe ftsI gene are suggested. The pattern of codon usage for the ftsI gene
was similar to that reported for the ribosomal proteins in E. coli.

OverpJrodllJ!ctiOlJIl of Escherichia eoli Replication Proteins by the
Use of the RIDl!l1lH'l'llly-RepHcatiimJ! Piiasmids
Seiichi YASUDA and Tsutol11u TAKAGIA derivative of the runaway-replication plasmid was constructed. This
plasmid, pSY343, has the gene for kanamycin-resistance and single sites for
EeoRI, BamHI, HindUI, KpnI and XhoI which can be used as cloning sites* Laboratory of Genetics, Department of Biology, Faculty of Sciences, Universityof Tokyo, Hongo, Tokyo, 113 Japan.

RESEARCHES CARRIED OUT IN 198129

without inactivating the kanamycin gene or the replication genes. The
replication genes of Escherichia coli were cloned on the plasmid. Activityof dnaA, dnaZ and ssb gene products were 200, 90 and 60 fold greater,
respectively, in the cells containing these plasmids than in normal cells.
Possible Repressor of Flagellar Reguloll. ill
Escherichia coli K-12
Y oshibumi KOMEDA

We have examined Fla- effects onto transcription of each flagellar operon
using fla-lacZ fusion technique. Summarizing the results, operon "A" wassituated after operon "BOO whose gene products were responsible for transcription of operon "A" using arrow (as B-A, see this report of 1979 edition).
When we obtained results showing C-B, B-A, and C-A, we describedas C-B-A. This qualitative analysis did not specify molecular mechanism
between alternative manners: (i) "COO interacts with both "A" and "BOO and(ii) "COO interacts with "B" but not with "A", and "B" interacts with "A",consequently "COO indirectly interacts with "A".
This article focuses on the interaction between group (3) operons and (4),(5), (6) operons (designation, see 1979 report). The transcription of hag
gene, for example, requires all of flagellar gene products except flaS, flaT,
flaU and flbC gene products. There are three possible ways to carry out
this control: (i) All of the gene products are positive factors. (ii) There isone (or a few) repressor of hag transcription and the others are anti-repressors. (iii) Complex mechanism including the methods (i) and (ii).
Double mutants were constructed in order to examine these possibilities.
If the positive interaction is the only way as in (i), the transcription of hag,flaS, Mocha andflaZ operons is independent to flaU andflbC gene products
and only depends upon the existence of (3) operon gene products.
This mechanism predicts that flaU flaE or flbC flaE double mutants, forexample, would show the phenotype of single flaE mutant for transcriptionof (4), (5), (6)- operon- transcription. The phenotype ofthe flaE flbC double
mutant was the same as that of the flaE mutant itself; the phenotype of theflaEflaU double mutant was the same as that oftheflaUmutant itself. This
result showed that the function of flaE gene is not independent to flaU geneproduct. In order to extend this observation a whole set of double mutants
carryingflaU4511: Tn5 mutation was constructed. They were used as hosts

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

for examination of Lac phenotype of each ).jla-Iac phage. Almost all double mutants had the same phenotype as that of flaU4511 mutant. flaU4511
flaD, flaU4511 jlaI, and jlaU4511 jlbB double mutants showed the pheno.
types of the latter mutations. Next, the jlaD4512:: Tn5 mutation was
introduced to each jla mutant and its effect was examined. The result
showed that jlaD activity was required for transcription of (4), (5), (6)operons.
The simplest explanation of these data was that jlaU coded for the repressor of (4), (5), (6) operons and that it had interaction with (3) operon
products for its repressor action. The jlaU product amy repress (4), (5),(6) operon in the case of mutations in (3) operons (except jlaD, flaE andmotD genes). If a jlaU mutation occurs and leads to absence of repressor
molecules, (4), (5), (6) operons are transcribed even if there is a mutation in(3) operon (exceptjlaD product).
As already known, all of the jla genes are positive factors for the appearanceof a flagellum. The jlaU gene thus should be not only a repressor but also
a positive factor. Since we have identified all ofthejla genes by collection offlagellum-less phenotype mutants so far, this dual function may mutate tohave novel repressor function. Accordingly, all of the jlaU mutants were
examined for the capacity to support the lacZ expression after 'Ajla-IacZ infection. Out of five jlaU mutants, strain YK4435 was different to the othermutants. It had incomplete flagella as the others, but it did not permit
transcription of (4), (5), (6) operons. Insertion mutants, strains YK4511
and YK3440, were supposed to be null-phenotype and did not permit
transcription of above mentioned operons. Being different to these mutants,
strain YK4435 had a defect of flagellar formation still retaining repressor
function. The jlaU4435 jlaB double mutants did not permit transcriptionof (4), (5), (6) operons. Other double mutants carrying (3) operon defects
behaved as same way. ThejlaU4435jlaD4512:: Tn5 double mutants did notpromote transcription of 'Ajla-Iac phages as expected. The existence of thismutant (YK4435) favoured the idea that jlaU gene product has dual role:
Positive factor for appearance of flagellum and repressor of hag,jlaS, Mocha(motA and che) andjlaZ operon transcription.

RESEARCHES CARRIED OUT IN 1981

31

Coordination of Flagellar Formation and Ceil Divisionin Escherichia coli
1. Flagellar Formation and Transcription of 'bag' Genein Ceil Division Mutants of Escherichia coli
Akiko NISHIMURA and Yukinori HIROTA

The molecular process of cell division in E. coli was investigated by thefollowing experimental system. We document here a phenomenon thatflagellar formation of thermosensitive mutants of E. coli defective in cell
division is arrested when the mutant strain was grown under the nonpermissible temperature. We used ten thermosensitive mutants defectivein cell division at the different genes. Those are ftsB, C, D, E, F, G, I, or Z
(defective in cell division at 40° and form multinucleated filaments) and
parA, B (defective in partition of daughter nucleus at 40° and form filaments). These mutants were first grown in broth at 30° and then the incubation temperature was shifted to 40°. Cell division of the mutant was
immediately arrested upon temperature shift.
Flagellar Formation

Flagellar formation of the mutant strains at non-permissible temperature
was measured by counting the number of flagellar filaments per cell length.
Samples were taken from a culture after various times of incubation at 40°.
The number of flagella per cell and cell length in each sample were measured
by the electron microscopic observation (50-150 cells were examined in eachsample). The results showed that the seven fts mutant strains defective ineither ftsB, C, D, E, F, G, or Z immediately arrested flagellar formation at
40°. The two mutant strains defective in partition of daughter nuclei, parmutant, either parA or parB, arrested flagellar formation. However amutant class,ftsI, continued flagellar formation at the same rate to wild typestrain, PA3092. The ftsI mutant is a thermosensitive mutant defective inthe penicillin binding protein 3 (PBP-3) which is involved in the final step ofcell division (Suzuki, H. et aT. 1978: Proc. Natl. Acad. Sci. USA 75: 664)
Penicillin G inhibits cell division of E. coli by binding to PBP-3. The
effect of penicillin G on flagellar formation was therefore measured. A wild
type strain, PA3092, was cultivated at 40° in nutrient broth containing 2 units
per ml of penicillin G. The flagellar formation of the cell, thus inhibited
cell division, was continued as in the case of the mutant strain, ftsI, under

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

the non-permissible growth condition. This means that penicillin G inactivates PBP-3, the product offtsI gene, by binding, and inhibition offlagelJar formation did not take place.
From these results it is concluded that the sevenfts-genes or the two pargenes acts on the regulatory processes of cell division and flagellar formation.
Penicillin G or the TS-mutation occured in theftsI gene inhibits cell division
but the flagellar formation is not inhibited.
To elucidate the regulatory process acting in the coordination between
flagellar formation and cell division, the recovery of flagellar formation in
the mutant strain, ftsD, E or Z, at 30° was examined as follows. The cell
division and flagellar formation was arrested by growing the mutant cells at
40° for 60 min; the culture was then brought back to 30°. At various times,
aliqotes of sample were taken and flagellar formation was measured. The
results showed two kind of responses in those mutants. When a mutant
culure of ftsE or ftsZ was shifted to 30°, cell division resumed after a shortlag (~15 min) and flagellar formation was immediately recovered. Howevercell division and flagellar formation of the mutant strain,ftsD, did not resume
over 90 min incubation after the incubation temperature was shifted to 30°.
In conclution, the recovery of flagellar formation in those mutant strains at
30° accompany the recovery of cell division. These results support a mechanism of coordination is acting on the regulatory processes of cell divisionand flagellar formation.
Transcription offlagellin messanger RNA (fln-mRNA)
Flagellar filament is composed of monomer proteins, flagellin, which is
the gene products of a gene, hag. Transcription of fln-mRNA in the ftsmutant strains was therefore measured at 40° by DNA-RNA hybridization(Gillespie, D. and Spiegelman, S. 1965: J. Mol. BioI. 12: 829). The mutant
strain was grown at 30° and then the incubation temperature was shifted to40°. The culture was incubated at 40° and aliqotes of sample were teken
at various time and a 2 min pulse of 3H-uridine (10 ,uCijml final) was given tolabel RNA. RNA was extracted by phenol extraction from the sample. A hag
specific transducing A DNA (A hag DNA) was prepared and it was denatured
and fixed on cellulose-nitrate disc. Each disc carryed 1O,ug of denatured.Ie hag DNA. The disc was incubated (63° 24 hours) with RNA- 3H in 0.25 ml
2 X sse half saturated with phenol. Nonspecifically bound RNA was digested with RNase. The disc was then dried and the radioactivity was

RESEARCHES CARRIED OUT IN 1981

33

counted. The result showed that fln-mRNA synthesis of the nine mutants(ftsB through G, Z, parA and B) was inhibited at 40°. However fln-mRNAof ftsI mutant was continued to be synthesized under the non-permissible
temperature.
Two possibilities are involved in the result; when the incubation temperature was shifted from 30° to 40°, the cell division of these mutant strain
and transcription of fln-mRNA is arrested. Alternatively, rate of degradation of fln-mRNA is accelerated. Rate of degradation of fln-mRNA in the
mutant strain, ftsZ, and wild type strain, PA3092, was therefore compared,
as follows. The cells were grown at 30°, then a 2 min pulse of 3H-uridine
was given to a culture and rifampicine (l00 ,ugjml final) was added to inhibit
synthesis of fln-mRNA; the culture was then divided into two parts. The
one was brought to 40° and the other was kept at 30°. After various times
of incubation, aliqotes of sample were taken. Radioactivity of fln-mRNA
fraction of the samples was counted by the method mentioned above. The
results showed that the rate of degradation offln-mRNA in the mutant strain,
ftsZ, and wild type strain, PA3092, at 40° was approximately 6.5-8 min.
The rate of degradation offln-mRNA in both strains at 30° was 9 min. These
results agreed with the reported half life of fln-mRNA at 37° as 7.5 min(Suzuki, H. et at. 1974: Ann. Rep. Natl. lnst. Genet. (Japan) 24: 14). Thus
significant difference was not found in the degradation rate of fln-mRNA
between the mutant strain, ftsZ, and wild type strain, PA3092. From theseresults it is concluded that the arrest of flagellar formation in the fts-mutant
was due to the sessation of the transcription of fln-mRNA.
Accounting for the all of the experimental results, the following regulatory
mechanism was proposed. The nine fts-gene products (ftsB through G, Z,
parA and B) act on the transcription of genes of cell division and flagellar
formation. As a consequence of thermal inactivation of the fts-geneproducts at the non-permissible temperature, both cell division and flagellar
formation are arrested. In the case of the ftsI mutant it is known that thefunction offtsI gene product is to acton the enzymatic process of cell division,and that the thermal inactivation of the ftsI gene product does not affectthe processes of transcription.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Coordination of Flagellar Formation and Cell Divisionin Escherichia colin.

Transcription of jla-Operons in the Mutantsof Cell Division in Escherichia coli

Akiko NISHIMURA, Yoshibumi KOMEDA and Yukinori HIROTA

In the accompanying paper (in this issue), it is reported that the nine genes(ftsB, C, D, E, F, G, Z and parA, B) regulate transcription of fln-mRNA.
In this paper, we report the expression of a series of flagellar operons, flaZ,
hag and motA, are controlled under the same regulatory process.
In E. coli, over 40 genes are known to be required for synthesis of flagellaand functions of flagella including motility and chemotaxis of this organism.
Recently, Komeda (1982: J. Bact. 150: 16) analyzed these genes coding forthe components of flagella at the level of the transcription. He used "lacgene fusion technique" and 6 classes of flagellar operons were fused with
lacZ gene. In these strains, transcription-translation system of the fused
genes of lacZ operate via fla gene promoter so that the measurement of.a-galactosidase activity directly reflected the transcription of the fused
operon, in question.
A series of double mutant strains carrying fts-mutation also containing
these lac-fusion genes were constructed by bacterial mating experiment.
Therefore expression of fla-Iac gene of the fts-mutant strains under nonpermissible temperature can be quantitated by measuring the .a-galactosidase
activity of these strains. The .a-galactosidase activity of the fts derivatives
thus constructed was measured as follows.
The cells were grown at 30° and then incubation temperature was shifted to40°. Cell density was recorded by measuring the light absorbance at 600 m,u.
Aliqotes of cultures were taken after the temperature shift from 30° to 40°,then immediately one drop of toluene was added to permealize the cell. The
toluene was evaporated at 37° and .a-galactosidase activity of the sample wasmeasured (Miller, J. H. 1972). A 0.1 ml of samples were added to a 0.9 mlof assay medium (Z-buffer), then 0.2 ml of o-nitrophenyl-.a-D-galactoside(4 mgjml) was added. After 15 min of incubation at 28°, reaction was arrested by adding 0.5 ml of a lM-Na 2 CO a solution and optical density at
420 m,u was measured.
The result showed that .a-galactosidase activity of the fts-derivatives carrying both thefts-mutation (ftsZ,[tsD or parA) and the fused lacZ with either

RESEARCHES CARRIED OUT IN 1981

35

flaZ, hag or motA operon was decreased under the non-permissible temperature. However the derivative of JtsI mutant strains carrying the fusedlacZ with either flaZ, hag or motA operon had p-galactosidase activity under
the non-permissible temperature. In conclusion, JtsZ, JtsD and parA genes
regulates the expression of flaZ, hag and motA operons. However TSmutation occured in the JtsI gene does not effect on the expression of those
three genes. Expression of flbB, flaD, flaG, flaA or flbC operons in the
Jts-derivatives were examined. The result showed that the TS-mutation
occured in theJtsZ, D, I and parA genes does not effect on the expression of
those five fla-operons. These five fla-operons (flbB, flaD, flaG, flaA andflbC) are shown to be required for the completion of the basal structure offlagella, and it is also required in the early process of the flagellar formation.
The expression of the three aperons (flaZ, hag and motA) are shown to function at the late process of flagellar formation, and the expression of these
operons requires the expression of the other five fla-operons (Komeda, Y.
1982: J. Bact. 150: 16).
The Jts-gene products are required for transcription of genes essential forcell division and flagellar formation and affect on the expression of the flaoperons (flaZ, hag and motA) which are the components operating on thelate process of the flagellar formation.
Thus, we present here two alternative hypothesis: One hypothesis is that
the Jts-gene products act as catabolite activater proteins and control directly
the transcription of genes essential for cell division and flagellar formation(such as flaZ, hag and motA). Alternatively, it can be supposed that thethermal inactivation of Jts-gene products, which are involved in the cell division, changes the physical character of membrane of the mutants. As theresult, formation of basal structure of flagella can not be formed on the
mutant membrane and then transcription of the fla-genes, such as flaZ, hag
and motA, are arrested at the non-permissible temperature.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETiCS

A Wild-Derived New H-2 Haplotype Enhancing K-IA Recombination
Toshihiko SHIROISHI, Tomoko SAGAI and Kazuo MORIWAKI

It is generally accepted that the overall recombination frequency between
H-2K and H-2D loci is about 0.4 %. However, the value fluctuates depending upon the combinations of H-2 haplotypes in the heterozygote. We reexamined the intra-H-2 recombination frequency, using three BIO.W congenic
strains which carry the H-2 complex of Japanese wild mice. Of the three,
one strain, BIO.MOL-SGR (H_2 Will7) , showed an extremely high incidenceof recombination (about 3 %) in all combinations with three different inbred
RIO congenic strains (Table 1). Twenty-nine recombinants originated in theheterozygotes of these H-2 combinations. The serological typing of theserecombinants to map the crossing-over points revealed that most of therecombination events occurred between H-2K and IA loci (Table 2). These
results firmly suggest that the wm7 haplotype enhances the H-2K end specificrecombination irrespective of H-2 partners in heterozygous parents.
Thus the BIO.MOL-SGR strain will provide a powerful tool for investigating the structure and functions of H-2 genes, because we can obtain therecombinant derived from K-end specific crossing-over by screening at
most 30-40 mice, probably irrespecitive of H-2 haplotypes. Moreover, theresultant recombinants would be very useful in searching for unknown ClassI and Class II loci if they are in the position of the H-2K end, especially
Table l.
H-2 haplotype
of heterozygous
parentsa/wml
a/wmll
a/wm7
k/wm7
b/wm7
Total

Frequency of intra-H-2 recombination

Method ofscreening

No. of micescreened

No. ofrecombinants

Recombination
frequency (%)

HA
HA
HA
CT
CT

294
299
539
174
204
1510

1
0
16
5
8
30

0.3

HA: Hemagglutination test.
CT: Cytotoxicity method.

3.0
2.9
3.9
1.9

RESEARCHES CARRIED OUT IN 1981
Table 2.
ParentalH-2
haplotype
a/wm7

37

Genetic composition of the recombinant H-2 haplotypes
Origin of
H-2 subregions

EstablishedstrainRecombinant H-2
haplotype

K

A

E

S

D

BlO.A (R201)
BI0.A (R202)
BlO.A (R203)
BlO.A (R204)
BlO.A (R205)
BIO.A (R206)
BIO. A (R207)
BIO.A (R208)
BlO.A (R209)
BlO.A (R211)
BlO.A (R212)
BlO.A (R213)
BlO.A (R214)

aw 1
aw 2
aw 3
aw 4
aw 5
aw 6
aw 7
aw 8
aw 9
awl1
awl2
aw13
aw14
awlS
awl6
aw17

k
k
k
w
w
w
w
k
w
k
w
w
w
w
k
w

w
k
k
k
w
k
k
k
k
k
w
w
k
k

w
k
k
k
w
k
k
k
k
k
w
w
k
k

w
d
w
d
d
d
d
d
d
w
d
d
d
d

?

?

w

w

?
d

w
w
w
d
d
d
d
w
d
w
d
d
d
d
w
d

kwl
kw2
kw3
kw4
kw5

k
w
w
w
k

?

?

k
k
k
w

k
k
k
w

?
?
?
?
?

w
k
k
k
w

bwl
bw2
bw3
bw4
bw5
bw6
bw7
bw8

b
b
b
b
b
b
w
b

w
w
w
w
w
w
b
w

w

?
?
?
?
?
?
?
?

w
w
w
w
w
w
b
w

BlO.A (R217)
kjwm7

bjwm7

between the H-2K and IA loci.

?
?
?
?
?
?
?

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Effect of Major Histocompatibility (H~2) Gene Complex on the
Susceptibility to Uretban Induced Lung Tumor in Mice
Kazuo MORIWAKI and Nobumoto MIYASHITAIn addition to a ptr (pulmonary tumor resistance) gene, H-2 gene complex
seems to function in the processes of tumorigenesis in lung. This study aims
to reveal the effect of H-2 gene complex on the incidence of urethan induced
lung tumor in mice. A single subcutaneous injection of 15 %urethan solution
was carried out in a dose of 1.5 mg/g body weight using 21 day-old mice.
Number of tumor foci were scored after 5 months on the lungs fixed withformaldehyde-ethanol (1 :9). During the five months, the mice treated withurethan were bred in the lamina flow rack occasionally monitored by microbiological assay.
At first we examined A H-2 congenic strains. As shown in Table 1, thelung tumor incidence is clearly suppressed in A.SW (H-2 s ), A.CA (H-2 f ) and
A.BY (H-2 b ) comparing to A strain (H-2 a ). This H-2 effect was further demonstrated in BIO H-2 congenic mice (Table 2). BIO (H-2 b ) exhibited lower
susceptibility but BlO.A (H-2 a ) showed rather higher susceptibility. BlO.A(3R) which H-2 genes on K, A, E, S, D regions are b b k d d instead of k k k
d d in BlO.A has lower susceptibility. BlO.A (5R), b b k d d, showed lower
incidence as well. BlO.A (4R), k k b b b, is as similar as BlO.A (5R). These
data suggest that H-2 IA k and IEk genes are related to the high incidence ofurethan induced lung tumor. The similar effect of IAI' and IEk genes was
also observed as to the higher incidence of 4NQO-induced lung tumor(Miyashita & Moriwaki, this Annual Report). In the spontaneous lungtumor in mice, Feraldo et at. (Immunogent. 9: 383, 1979) demonstrated thepossible relevance of H-2 IRk gene to the higher incidence.
So far, urethan effect on the lung tumor development has been rather
considered to be an augmentation of spontaneous incidence (Shimkin andStoner, Adv. Cancer Res. 21: 1, 1975). But nobody knows the causation of
spontaneous lung tumor at this moment. Instead, the good accordance inthe effect of H-2 k genes on the higher incidence of lung tumors between thechemical-induced and spontaneous ones leads us to assume that certain
chemical factors in the environment may cause the spontaneous tumor
as well.

RESEARCHES CARRIED OUT IN 1981
Table 1.

39

Effect of H-2 complex on the incidence ofurethan-induced lung tumors

H-2 congenic
A strains
A

A.TL
A.TH
A.CA
A.SW
A.BY
( ):
Table 2.

Strains
BIO.BR
B10.A
BIO.A(2R)BIO
BIO ,A(3R)
B10.A(5R)
B10.A(4R)
BIO.D2BIO.M
BIO.S
BIO. RIll():

30.6 ± 1.3
25.8 ± 2.8
17.0 ± 0.6
17.0 ± 0.6
12.3 ± 0.7
13.8 ± 0.6

(24)
( 8)
(26)
(58)
(51)
(58)

Number of mice observed.

Effect of H-2 complex, especially I-region, on the susceptibility
to the urethan-induced lung tumors
Susceptibility
Tumors/lungto lung tumors
1.3 ± 0.2 (16 )High
1.2 ± 0.2 (26 )
High
(37)
1.2 ± 0.2High
0.7 ± 0.2 (37)Low
Low
0.7 ± 0.2 (31)0.6 ± 0.1 (59 )Low
Low
0.5 ± 0.1 (42)0.3 ± 0.1 (15)Low
0.5 ± 0.1 (46)Low0.7± 0.1 (24 )Low
0.4 ± 0.1 (2'1)
Low

H-2 complex
H-2
K A E S D haplotypek k k k kk

k k k d dk k k d bb b b b b
b b k d db b k d dk k b b b

a
h2
b
i3
i5
h4

d d d d df

dfs s s s sr r r r r

s
r

f

f

f

f

Number of mice observed.

The Relationship between Immune Response Genes andthe SusceptibHity to 4NQO-Indllced Lmllg Tumor
Nobumoto MIYASHITA and Kazuo MORIWAKI

We have previously reported (Moriwaki and Miyashita, A. R. N. I. G.
3.1: 37, 1981) that the H-2 gene complex is related to the development ofurethan-induced lung tumor in mice. But in vivo metabolic pathway ofurethan is still unknown. In the present study we examined A. H-2 congenic
strains of mice for their response to 4NQO in studying the effect of H-2 genecomplex to lung tumor induction. An avarage number of lung tumor fociwas 9.0 for H-2 a haplotype, and 2.6 to 5.0 for the other H-2 haplotypes (Table1). These results suggest that the I region genes control neither the metabolism nor the activation of the specific chemical carcinogen, but function as

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32
Table 1.

Strain
A
ATL
A.TH
ASW
A.CA
ABY

Effect of H-2 complex on the susceptibility to urethan- and
4NQO-induced lung tumors in AH-2 congenic miceH-2
haplotype

Ae:Eacomplex

a
t1
t2

k

E
Ek

f
b

Number of lung tumor foci per mouse
4NQO

Urethan9.0±2.l (13)
Not Tested
2.6±1.1 ( 8)5.0±O.5 (25)4.8±O.9 (37)5.0±O.9 (10)30.6±1.3 (24)25.8±2.8 ( 8)17.0±1.1 (26)12.3±O.7 (51)17.0±O.6 (58)13.8±O.6 (58)Urethan dose: 1.5 mg/g body weight.
4NQO dose: 12.5 ."g/g body weight.

the regulatory system for the more common steps
tumor.

the growth of lung§lllppressiolill of UJretlhiaIDl-lilliduced
l'nu1iloR' D~,vello)!!lmeil1lt
lID! tlIT:e
lbetweelTil. A Strai1TI1 !llli1liJl J2]~aJl]leS§
Derived MOL,l'EN2 Sinn],liKazuo MORIWAKI and Nobumoto MIYASHITA

Bloom and Falconer (J. N. C. 1. 33: 607, 1964) have suggested the presenceof a single recessive major gene (ptr) conferring low susceptibility to urethaninduced lung tumor in C57BL strain. In their experiment, F i hybrid between
A and C57BL exhibited nearly similar level of the higher incidence as thatof the susceptible parent, A strain. This tendency was also shown in ourstudy. To eliminate the effect of H-2 complex, we examined F i hybrids
between A and BlO.A which has C57BL background and H-2 a haplotype as
A. Their lung tumor incidence induced by urethan was almost the same as
A strain, which definitely confirmed the higher susceptibility to lung tumorin (Ax BlO)Fi (Table 1).
On the other hand, we have already observed that the susceptibility tourethan-induced lung tumor of MOL.TEN2 strain derived from Japanesewild mouse (Mus musculus molossinus) is significantly lower than that of
C57BLjIO (RIO). In the present study, we further found a novel mode ofinheritance in the hybrids between A and MOL.TEN2 strains. Differingfrom (AxBIO)F i , (Ax MOL.TEN2)F i exhibited the apparently lower

RESEARCHES CARRIED OUT IN 1981
Table 1.

41

Suppression of urethan-induced lung tumor development in theintersubspecies hybrids
Ptr
gene

StrainA
BI0(A X BI0)F1(A X BI0.A)F1
MOL.TEN2(Ax MOL.TEN2)F1
A X (A X MOL.TEN2)F1

Ptr
Ptr

Ptr/ptr
Ptr/ptr?
Ptr/?
Ptr, ?

H-2
No. of mice No. of tumor focihaplotype observed
per mouse
a
b
alb
alb
wml
a/wml
a, a/wml

MOL.TEN2 &(AxMOL. TEN2) F1

'0
ill

:>
;.;
ill

,.Q

0
ill
CJ
·M

{~

30.6±1.3
0.7±0.2
19.9±0.926.2±1.6
0.0
0.8±0.2
6.3±0.822.6±4.0

A

~

rn

24
37
28
10
13
12

1

S

'H

0

5

;.;
ill

~

::J

:z;

0-9

10-19

20-29

30-39

40-49

Fig. 1. Distribution of the lung tumor incidence in A x (A X MOL.TEN2)F1
backcrosses.

susceptibility to the lung tumor (Table 1). Pulmonary tumor resistance(ptr) seems to be dominant in this case. Backcross experiment between
A x (A X MOL.TEN2)F 1 demonstrated unexpected segregation. Though
the number of N 2 progenies with the lower susceptibility are seemingly equal
to those with the higher susceptibility as seen in Table 1, the frequency distribution shown in Fig. 1 obviously deviates to the lower susceptibility, instead of the bimodal peaks expected for a single locus.
These data possibly imply that in the hybrids between genetically remotesubspecies such as laboratory inbred mice (M. m. domesticus) and Japanesewild mice (M. m. molossinus), certain regulatroy locus or loci other than

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32'ptr' could function to suppress tumorigenesis. These loci seem to work
only under the heterozygous condition in the intersubspecies hybrids. The
lower susceptibility to lung tumor in (A X MOL.TEN2)F, hybrids is probably
explained by this notion. This phenomenon may not be generalized to thehybrids between laboratory inbred and wild mice, because F, hybrids between
A and BRV.MPL which is derived from European subspeices, M. m. brevi.
rostris, and relatively closer to the laboratory mice genetically. Ifwe assume
the mumber of such loci are more than two, the higher frequency of resistantindividuals in the backcross (N 2) progenies could be well understood.
The reason why these genetic loci could work only in the inter-subspecies
hybrids remains to be explored.
Genetic Divergence between Emrojpean Wild Mice wnth Robert§oJlll.an
Trmnsl.ocatioil.s {[![lID View Point of Mitodnonddal! DNA
Kazuo MORIWAKI, Hiromichi YONEKAWA'), Osamu GOTOH, Heinz VilINKING')
and Alfred GROpp2)

To estimate the genetic divergence between the European wild mice with
Robertsonian (Rb) translocations, their mitochondrial DNAs (mtDNAs)
isolated from liver were analysed by the agarose slab-gel electrophoresis afterdigestion with eight kinds of restriction endonucleases, BamHI, EcoRI,
HindU, HindIII, Pst!, HpaI, Hpall and BglI. These mtDNA preparations
were further subjected to make restriction map, from which approximate
divergence times between each Rb variations were estimated as the orderof 10 5 years (Table 1). Comparing these data with the divergence times
between various Mus musculus subspecies in Europe and Asia, the Rbvariations are apparently included within Mus 1 group. Five lines of Rb
variations were examined in the present study: Those from Poschiave Valley (Pos), Yougoslavea (Zadar), Milano (Mil-II), and two lines from Apennine (CD and CB). A linear relationship was observed between the divergence times and the number of shared translocations in those Rb variations, suggesting that the Rb translocation commonly observed in the Rbvariant populations should have occurred in a fairly old age such as 105
years ago.
1)

2)

Saitama Cancer Center Research Institute, Saitama-ken.
Medizinische Hochschule Lubeck, F. R. G.

Table 1.

Sequence divergence and time of divergence estimated from mtDNA among
five Robertsonian variations and six subspecies of Mus musculus

Robertsonian variations
Zadar

Mil-II

CD

i>:l
trl
trl

M. m. subspecies
CB

dom

brv

mus (D)mus (B)

IZl

mol

cas

bac

:ti>:l(J

Sequence divergence (%)
Pos
Zadar
Mil-II
CD
CB

1.5

0.8
2.2

1.5
2.1
1.4

1.1
1.8
0.4
1.0

0.8
1.4
1.5
1.4
1.1

0.0
1.5
0.8
1.5
1.1

1.8
2.4
1.0
0.3
0.7

4.0
4.9
4.8
4.5
4.2

3.4
4.0
4.2
4.0
3.6

6.1
6.1
4.9
4.6
4.3

7.5
7.4
6.1
5.9
5.5

1.40
1.72
1.68
1.58
1.47

1.19
1.40
1.47
1.40
1.26

2.13
2.13
1.72
1.61
1.51

2.63
2.59
2.14
2.06
1.93

Time of divergence (x 106 years)
Pos
Zadar
Mil-II
CD
CB

0.53

0.28
0.77

0.53
0.74
0.49

0.39
0.63
0.14
0.35

0.28
0.49
0.52
0.49
0.39

0.0
0.53
0.28
0.53
0.39

0.63
0.84
0.35
0.11
0.25

::c
01

IZl

(J

>i>:l

i>:l
trl

U
0

e>-I
I-<

-Z

\0

w

+w

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Effect of Major Histocompatibility Gene Complex (MHC)on the Growth of Embryo-Derived Teratoma
Choji TAYA and Kazuo MORIWAKI

Mouse teratoma can be experimentally induced by the transplantationof early embryos under the kidney capsule. In order to find out the genetic
factors governing the in vivo growth of embryo-derived teratoma, 6.5 and
7 day-old embryos of several strains were transplanted under the kidney
cpasule of syngeneic recipients. After 40 days the grown tumors were
weighed from the view point of the immune reaction of host aminals.
Strain A mouse was the greatest in the tumor weight among three kinds of
A. H-2 congenic mice. A. BY and A. SW strains developed rather smaller
weight of tumors. Teratoma produced in BlO.A strain was the smallest in
the weight. Tumors obtained from [A X BIO.A]F, were larger than those
produced in A strain (Table 1). This may imply that the susceptibility toteratoma in A strain mice is a dominant trait against that of BIO strain when
H-2 is the same.
These results suggest the genetic background of A strain plays an importantrole in the growth of embryo-derived teratoma and MHC seems to affectpartially on the tumor growth in this system.
Table l.

Effect of H-2 complex on the growth of embryo-derived teratoma
Weight of tumors (mg)±S. E.

StrainsA
A.BY
ASW[AxBIO.A]F,
BIO.A

H-2 haplotypes

a
b
a
a

6.5 day-old
embryo7 day-oldembryo633±134 (34)369± 74 ( 9)N.D.
881±193 (24)193± 29 ( 8)2033±567 ( 8)1562±417 (15)852±251 (13)N.D.
657±316 ( 5)RESEARCHES CARRIED OUT IN 1981

45

Allelic Constitution of Hbb Locus in Chinese
Wild Mice, Mus musculus
Nobumoto MIYASHITA, Mitsuru SAKAIZUMI, Cheng Rai WANG 1),
Shunsuke MIGITA 2) and Kazuo MORIWAKIIn order to investigate whether the high frequency of Hbb P allele is uniqueto Japanese wild mice, Mus musculus molossinus, a total of 49 mice werecollected at 9 localities of China. The restriction enzyme cleavage patternsof mitocondrial DNA of these mice were also examined (Yonekawa et al.
unpublished data). Electrophoretic survey of Hbb alleles in the Chinese
wild mice revealed a high frequency of Hbb P (93.9 %), whereas Hbb d was rare
(6.1 %) and Hbb S could not be found (Table 1.). Especially, in the sevenlocalities which mtDNAs were MOL type, Hbb P was solely observed. But
Hbb d allele was common (50.0 %) in the two localities which mtDNAs were
not MOL type. On the other hand, Hbb p allele is prevalent in the central
regions of Japan (Minezawa et al., Jap. J. Genetics 54: 165, 1979), whilewild mice in Philippines and Taiwan, A1us musculus castaneus, carries thelower frequency of Hbb P in addition to predominant Hbb d • These results
Table 1.

*

1)

2)

Phenotypes of hemoglobin beta-chain(Hbb) of wild mice from China
No. of phenotype

Locality ofcollection

No. ofmice

UrumuchiTurfanJiayuguang
Lanzhou
ChengtuChangchun
Beijing II
Beijing I
Shanghai
Total

10
1
3
13
5
7

10
1
3
13

4

4

2

1

1
3

44

4

p

PD

5
7

4

49

D

1
1

Y onekawa et at. unpublished.
MOL
Mus musculus molossinus (Japanese wild mouse) type.
BRV : Mus musculus brevirostris type.
CAS : Mus musculus castaneus type.

Institute of Biological Products, Lanzhou.
Cancer Research Institute, Kanazawa University.

Type of'"
mtDNA
MOL
MOL
MOL
MOL
MOL
MOL
MOL
BRV
CAS

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

suggest the close genetic relationship between M. n1. molossinus and Chinese
wild mice.
Genic Di:fferentiadml in the Wild PoplUllatiiom, of the Fish
Oryzias latipes
Mitsuru SAKAIZUMI, Nobuo EGAMIl), and Kazuo MORIWAKI

Allozyme studies at 21 loci revealed an extensive genic differentiation in
Japanese wild populations of the freshwater fish Oryzias lalipes. By meansof unique alleles at the Adh, Pgm, Idh, and Sod loci, the Japanese wild populations could be divided into two major groups, the 'Northern Population'and the 'Southern Population'. Crosses have shown that these four loci are
not closely linked. The Southern Population was further divided into several
subpopulations by the geographically specific alleles at the Aat, Acp, Amy,
Me, and Pgd loci. Concerning these nine loci, little dinal variation wasobserved. The boundary of allelic distribution was distinct and well correlated with mountain limit.
Two Chinese populatios, Beijin andstudied. Although
they were distinct from Japaneseallele at Ck-A
locus, they had the same alleles as the Southern
had at the Adh,
ldh, and Pgm loci, and shared the common allele with the northern ~~"U"HU
populations at Sod, and withof San-in district at Amy locus.
Beijin and Shanghai"vere similar (Table 1) in spite of the geographic distance between these tv/O sites
1000
No
mountainbarrier exists between theseand it is knovvn that the YeHow River
has changed its lower reach several times in these two thousand years.
Clinal variation at enzyme loci has been
for many fish ~I.!I~,",J."~.
These authors have argued for a correlation with water teJllj:1er,atlue In
case of Oryzias latipes little dinal distribution is observed. The distributionof allelic variation rather well coincides withfeatures. Thisphenomenon may be due to random drift and restricted mlgnltI()l1, rather
than to selection or the difference in environmental conditions.
The genetic distance value was calculated
the method of Nei (1972).
The estimates were presented in Table 1. The average D values for all
pairs of localities within a group were nearly O. This shows that the geneconstitution is fairly homogeneous within a group. On the other hand, the1)

Zoological Institute, Faculty of Science, University of Tokyo.

Table 1.

Estimates of gnetic distance among wild populations of medaka based on 21 lociSouthern Population:;0-----------------

Northern

Eastern
Subpop.

San-inSubpap.

Inland Sea
Subpop.

RyukyuSubpap.

Shanghai
population----.-

Northern
Population
Eastern
Subpop.
Inland Sea
Subpop.
San-inSubpop.
RyukyuSubpap.
Shanghai
population
Beijinpopulation

Beijinpopulation

t'11
t'11

(,fl

>-

:;0

(j

r:r:

(.01)

trl

(,fl

.35

(.03)

.33

.07

(.03)

.29

.14

.J

(.03)

.43

.19

.16

.19

(.01)

.47

.24

.21

.18

.39

-

.63

.24

.27

.24

.43

.03

(j

>-

:;0
:;0

"'t'1"1'

U
0

c:
"'"""
Z"'

\D
00

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

Mean values of genetic distance for all pairs of localities within populations are in parenthesis.

-l"-

-.j

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

D values among groups were markedly larger than them. Especially thevalues were large among the Northern Population, the Ryukyu Subpopulation, and the Chinese populations. These results suggest that this species
has much variation, and considerably differentiated regionally.
Two-dimensional Electrophoretic Profiles of Seed Proteins fromOne Grain in Wild and Cultivated Rice
Chong-rang SUN and Tom ENDO

It should be noted that protein profiles of dormant seeds are highly stable
qualitatively and quantitatively, though they are slightly affected by environmental conditions such as seasonal fluctuations during the protein synthesis.
Thus the profiles must be a useful tool for phylogenetic differentiation as
well as breeding problems in view point of nutritional improvement.
One grain, about 20 mg in cultivated and about 15 mg in wild rice, wasmacerated with 0.2 ml of the extractant containing 8 M urea, 1% Nonidet
P-40 and 2 % DTT, and resultant slurry was centrufuged. During the procedure, more than half amounts of whole proteins are generally possible to besolubilized. The supernatant was subjected to the two-dimensional polyacrylamide gel electrophoresis according to Brown et al. (1979, PNAS 76:
2381).
The extracts from one grain gave about 75 to 108 spots counted, most ofwhich were minor protein spots on the gels after staining with coomassie blue.
The major spots were classified arbitrarily into three groups, I, II and III,
which are located in the corresponding areas of the gels, respectively. GroupI consisted of five to eight major spots, but is not identified yet. Group II
comprised six to ten major spots, and was identified as glutelin. Group III
was composed of only one major spot, identified as globulin. Six Asian
perennis and 108 (Indica type of Oryza sativa) showed to have group I, II
and III. T65 (Japonica type of O. sativa) showed to have group II and III,
but lacked group 1. W025 (0. glaberrima) showed to have group I and II,
but lacked group III.
The present result suggests that Indica type is phylogenetically much nearer
to Asian perennis than Japonica type. Also, it may suggest that Japonica
type has a genetic margin for incorporation of genes specifying the group I
proteins.

RESEARCHES CARRIED OUT IN 1981

IV.

49

DEVELOPMENTAL GENETICS AND SOMATIC
CELL GENETICS

In Vitro Differentiation of Adult Structures from
Embryonic Cells of Drosophila melanogasterYukiaki KURODA

In Drosophila cells which will differentiate into adult tissues and organs arepredetermined at the stage of blastoderm formation in embryonic development. These cells proliferate in an undifferentiated state during embryonicand larval development and form imaginal discs. The final differentiationof the adult organs occurs under influence of ecdysterone secreted from thering gland during the pupal development. Recently I have succeeded informing the adult structures from embryonic cells of D. melanogaster inculture with ecdysterone-containing medium.
The wild-type Oregon-R strain was used. Newly laid eggs were dechorionated by treatment with sodium hypochloride solution. Embryos at thestage of post-gastrulation were surface-sterilized in 70 % ethyl alcohol, dissociated into small pieces of tissue fragments and cells, and cultured in T-5
flasks in medium K-17 supplemented with 15 %fetal calf serum and 0.1 mg/ml
fetuin at pH 6.7 at 28°C.
When tissues and cells from embryos were cultured in medium without
ecdysterone, various embryonic and larval tissues or cells differentiated.
Muscle cells, epithelial cells and nerve cells showed their characteristic morphology and function under these culture conditions employed. However,no adult tissues or organs were formed without ecdysterone.
When ecdysterone was added to culture medium at the concentrations of
1-10 ,ug/ml, some characteristic adult tissues and organs developed. Amongthem the structures which were very similar to the imaginal wing and antennal
discs were remarkable. These imaginal disc-like structures showed a typicalfeature, i. e. multiple concentric folded layer of thin cells. The eye disclike structures with a regular arrangement of ommatidium-forming cells
also developed. The bristles and trachea structures also differentiated very
well. In nerve cells many multiple branched nerve fibers were produced.
The most striking example of the development of adult organs was the

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

leg-like structures. After one day of cultivation, small leg disc-like buds
appeared, then they extended gradually, and finally every segments of theadult leg developed. They consisted of segments that corresponded to theadult segments of the legs, such as the coxa, the femur, the tibia and thetarsus. At the tip of the tarsus claw-like structures also developed.

Effect «lif Chick Enrrllogenoll!§ Lectin Olll CartHage Diifferemltiatimll«lif Chick Limb-Bud Cells in CuRhnre
Yukiaki KURODA, Etsuya MATSUTANI and Tatsuya YAMAGATA 1 )It has been reported that endogenous lectins occurred in a wide varietyof animal tissues. Among them, galactoside-specific lectins are widelydistributed in a variety of chick embryonic tissues, such as skeletal muscle,
brain, heart, lung, liver, retina, spinal cord, kidney and sterna. These arealso found in chick embryos at the earliest stage, blastoderm. However,their role in the development and differentiation of embryonic tissues still
remains unclear.
Mesenchyme cells obtanied from the limb-buds of chick embryos are
known to differentiate into cartilage cells in vitro. We reported that plant
lectins enhanced cartilage differentiation when added to the culture of mesenchyme cells dissociated from quail embryos (Matsutani, E. and Y. Kuroda,
1982
BioI.: 521-526). Since various chick embryo tissues so
far tested contained
lectin, we examined the activityof the chicklectin on the chondrogenesis of mesenchyme cells.
p-D-galactoside-specific lectin wasfrom 14chick embryos using
a lactamyl
column. The lectin
eluted from the second
affinity column was analyzed
SDS
gel electrophoresis,
and it showed one
band with a molecular weight of 16,000. Mesenchyme cells were dissociated from the limb-buds of White Leghorn chickembryos at the stage 24. Cells were incubated in medium 199 with 10 %serum,a modified micro-mass culture method. An inoculum of 1 X
10 5 cells was incubated for 12 hours in 0.5 ml of culture medium containing
5 X 10 4 units/ml of lectin.
The lectin-treated cells showed a slight increase in cell proliferation, formed
a greater number of cartilage nodules, and incorporated about twice as muchas 35S-sulfate per cell than cell in control cultures.
1)

Mitsubishi-Kasei Inst. of Life Sci., Machida, Tokyo.

RESEARCHES CARRIED OUT IN 1981

51

The results of this study indicates that the chick endogenous lectin, which
may have the activity to recognize the specific site(s) on cell membrane,
promotes the chondrogenesis of mesenchyme cells in vitro. We are now
carrying out more detailed study to clarify the mechanism by which thislectin promotes the chndrogenesis both in vitro and in vivo. For details,
see Develop. BioI. 92: 544-548, 1982.
Mutagenic Acltiv:ity of QllJleli'cetiHll on Emb"'y<tmk Hi-mum])iJPl~o:id CeHRs in CuUmre
Yukiaki KURODA

In the series of experiments, the mutagenicity of various chemicals on
cultured human diploid cens was compared with those in other test systems,
to find out the characteristics of human cells for these chemicals. In thepresent experiment, quercetin, which is a carcinogenic flavonoid in thebracken and had a strong mutagenic activity in the Salmonella test system,on mutation induction in cultured human diwas examined for its
ploid cells.
The effect of quercetin on cell survival was examinedthe colony forming activity of cells treated with quercetin at various concentrations for
4 hours. The surviving fraction was 0.73 in cells treated with 30 ,ug/ml
qUl~rceti]L1, and 0.64 in cells treated with 100 {.kg/ml, showing that quercetin
had not socytotoxicity. With higher concentrations of quercetin,
effect ofalcohol used as a solvent exceeded that of quercetin on cell
survival.
Theofresistant mutations induced
treatment
5
with 1 ,ug/ml
for 4 hours was 0.36 per 10 survivors. With 3 {.kg/ml
quen~etin, the mutation
was 0.66 per 10 5 survivors. Vllith higher
concentrations than 10 ,ug/ml, no more increase in induced mutation frequency was detected.
These results indicate that quercetin had a week mutagenicity, which was
about one tenth of that of tryptophan pyrolysis products, on cultured human
diploid cells. This is interesting with reference to the fact that quercetin
had no mutagenic activity in insect systems such as silkworm.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Co-Mutagenic Activity of Non-Mutagens in Cultured
Chinese Hamster Cells
Yukiaki KURODA and Masumi ASAKURA

Carcinogens present in human environment have been effectively detectedby their mutagenic activity in bacterial test systems. Results obtained insuch systems, however, can not directly apply to the assessment of these chemicals on human being. In the present experiment, some chemicals, whichhave not been detected for their mutagenicity in the Salmonella test systems,
were examined for their activity to induce 8-azaguanine (8AG)- or uabain(OUA)-resistant mutations in Chinese hamster V79 cells. Chemicals examined were aniline and norharman.
Aniline had a cytotoxic effect on V79 cells. The value of D oo for 4 hourtreatment was 1.7 mg/ml, and 1.2 mg/ml with metabolic activation by S-9
Mix.
For mutation experiments, cells were treated with chemicals for
4 hours, cultured for 6 or 2 days in normal medium for mutation expressiontime, and then replated on fresh dishes. The numbers of 8AG- or OUAresistant mutant colonies were scored after cultivation for another 14 or
10 days in respective selection medium.
The induced 8AG-resistant mutation frequency of cells treated withaniline at concentrations of 0.2-2.0 mg/ml was 7-10 per 105 survivors. No
increase in the mutation frequency was observed with addition of S-9 Mix.
OUA-resistant mutations were not significantly induced both by aniline alone
or with S-9 Mix. Norharman had a weak mutagenic activity on V79 cells.
When cells were treated with both aniline and norharman simultaneously
at low concentrations, which did not induce mutations by exposure of either
one chemical, 8AG-resistant mutations were effectively induced. The
induced mutation frequency increased by addition of S-9 Mix.
To analyze the mechanism of co-mutagenic effect of these two chemicals,
cells were separately treated with norharman before aniline treatment. The
induced mutation frequency was lower in cells treated with norharman 4hours or immediately before aniline treatment than that in cells treated withnorharman and aniline simultaneously. This suggests that the co-mutagenic
effect of aniline and norharman may be due to the inhibitory effect of norharman on the inactivation of aniline rather than due to the intercalationof norharman into the DNA molecule.

RESEARCHES CARRIED OUT IN 1981

53

Effect of TPA on CeU-to-CeU Adhesion of Chinese Hamster
Cells in Culture
Yukiaki KURODA and Hirokazu MIKAMI

A phorbol ester purified from seeds of Croton tiglius L., 12-0-tetradecanoyl
phorbol-13-acetate (TPA), had a strong promoter activity on the induction ofmouse skin tumor. TPA also had a variety of activities in the induction ofornithine dehydogenase activity in the cell membrane, the inhibition of themetabolic cooperation in cultured cells, and the modification of the cellulardifferentiation. These results suggest that the site of action of TPA may be
the cell membrane. In the present experiment, the effect of TPA on the cellto-cell adhesion of Chinese hamster V79 cells.
Cell suspensions containing 106 cells in 3 ml medium were rotated on agyratory shaker at 38°C. Under these culture conditions, cells in suspensions
come together, adhere to each other and form aggregates after 24 or 48 hours.
The shape and size of aggregates are constant and highly reproducible, andcan be used for examining quantitatively the effect of various chemicals on
the cell-to-cell adhesion.
In control cultures, V79 cells formed many spherical aggregates with adiameter of approximately 230 ,um, after rotation culture for 24 hours.
When TPA was added to the culture medium at concentrations of I to
100 ngjml, the average diameter of aggregates formed increased, indicatingthat TPA enhanced the cell-to-cell adhesion. This enhancing effect of TPA
on aggregation continued for another 24 hours in rotation culture, evenwhen TPA was removed from the medium. Phorbol had no effect on aggregate formation of Chinese hamster cells.
The adhesiveness of cell to the plastic surface of petri dishes was noaffected by TPA. The colony-forming activity and the population doublingtime of cells were also not affected by TPA. These results indicated that
TPA had a specific effect on the cell-to-cell adhesion.
. An Autosomal, Non-maternal Effect Sex-differential Lethal
Mutation in Drosophila melanogaster
Kugao OISHI and Takao K. WATANABE

Recent studies on the sex-specific lethals of D. melanogaster have indicated that all these lethals so far characterized are involved in dosage com-

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

pensation and/or sex determination in one way or another. They can beclassified into several groups by their modes of action: (1) A sex-linked,
non-maternal effect sex-specific locus (Sex-lethal, Sxl, which has both malespecific and female-specific alleles and plays a central role in sex determination and dosage compensation), (2) An autosomal, maternal-effect femalespecific locus (daughterless, da, which regulates the expression of Sxl+
locus), (3) Autosomal, non-maternal effect, male-specific loci (e. g., maleless, mle; four loci on the second chromosome and a locus on the third
have been characterized. These are involved in the regulation of dosage
compensation in males, and also regulate the expression of Sxl+ in females),and (4) A sex-linked, maternal-effect male-specific locus (sonless, snl,
which interacts with sex-transformation mutants).
We have recovered an autosomal, non-maternal effect, sex-differential
lethal which apparently represents a new class. Whether this mutant isinvolved in dosage compensation and/or sex determination, and if so how,
is thus of considerable interest. Preliminary results of examinations arepresented here. A third chromosome (# 100) extracted from a natural population at Katsunuma, Yamanashi Prefecture, was found to be female lethal
and male semi-lethal when made homozygous. The gene responsible forthis lethal effect, jle(3)lOO (femaleless), was mapped at 3-45. Homozygous
females were completely lethal, but males appeared at about 50 %the expected
number. It is not clear whether the male semi-lithality is due to jle(3)lOO or
to another gene tightly linked. Since the map position was close to sextransformation mutants transformer (tra, 3-45) and doublesex (dsx, 3-48.1),
a possibility was raised that homozygous males were lethal and the males
that appeared represented female-to-male sex-transformed flies. This was
negated as jle(3)lOO/tra and jle(3)lOO/dsx were normal and fertile fiies. At
least some homozygous jle(3)lOO females survived to pharate adults but
never eclosed. They had external morphology completely differentiated andnormal. No idication of abnormal dosage compensation was obtained in
the preparatins of salivary gland chromosomes of third-instar female larvae.
Further characterizations are underway.

RESEARCHES CARRIED OUT IN 1981

55

Nematocyte Dij[erentiation and Positional
Information illl. Hydra
Toshitaka FUJISAWA, Chiemi NISHIMIYA and Tsutomu SUGIYAMA

Nematocytes are hydra's stinging cells used for capturing preys and forprotection. There are 4 types of nematocytes (stenotele, desmoneme,
holotrichous isorhiza and atrichous isorhiza) classified by the morphology
of a stinging structure present in the cytoplasm of the cell.
The spatial distribution of the differentiating nematocyte along the bodycolumn is very characteristic. For example, desmonemes are differentiatedmainly in the distal part of the body column and stenoteles are differentiatedpredominantly in the proximal part of the body (Bode and Flick, 1977).
This position dependency of nematocyte differentiation suggests the possibility that positional information which controls pattern formation of hydra isalso involved in regulating nematocyte differentiation. In order to test
this possibility, 2 different sets of experiments were done: 1) The axial distribution of differentiating nematocytes was examined in the hydra strains
which have different levels of positional information. 2) An assay system
was devised to identify and characterize a factor(s) which controls nematocyte differentiation in the hydra tissue.
In the first experiments the patterns of nematocyte differentiation along
the body column were examined by the thiolacetic acid-lead nitrate method(David and Challoner, 1974) which specifically stains the nematocytes intheir final stage of differentiation from the interstitial stem cells. In thestandard wild type (105), stenoteles occupy 0-7 % of the total nematoblasts
in the regions near the head, 5-20 % in the middle and 50-60 % near the foot,whereas desmonemes occupy 65-75 %,55-65 % and 30-40 % in these regions,
respectively. These patterns of nematocyte differentiation are essentiallyunchanged in the mutant strains (reg-16 and mh-I) which have significantly
altered levels of both head activation and inhibition potentials. This suggests that, although nematocyte differentiation is position dependent, positional information involved in the head formation has no influence on theregulation of nematocyte differentiation.
In the second experiments, we found that the hydra tissue contains afactor(s) which preferentially inhibits stenotele differentiation when applied
to hydra externally. The factor is released into the culture solution by thehead regenerating animals. The kinetics of the release of the factor matches

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

well with the known kinetics of the release of "head inhibitor" from thehead regenerating hydra. This finding is consistent with the view thatstenotele differentiation is normally regulated by head inhibitor. However,the purified head inhibitor (gift of Dr. S. Berking) showed no inhibitory activity on the stenotele differentiation. In addition, the factor which is present
in the homogenates of normal hydra tissue was not found in the homogenatesof epithelial hydra (hydra consiting of only epithelial cells and lacking interstitial stem cells, their derivatives (nerves and nematocytes) and gland cells)
which has normal positional information. The identity of the factor ispresently unknown.
Changes of the Head Activation and Head Inhibition
Potentials during Hydra Regeneration
Tsutomu SUGIYAMA, Josef ACHERMANN* and Jun TAKANO**

Hydra has a strong regenerative capacity. After amputation of the head
and foot, a new head regenerates from the end of the body column which
originally had the head and a new foot regenerates from the other end of the
body column. This polarity of regeneration has been thought to be governedby some sort of polarity "gradient" which exists along the body axis (Child,1941).
Wolpert et al. (1974) and Gierer and Meinhardt (1974) proposed similar
models on the polarity gradient. According to them hydra tissue possesses
2 antagonistic morphogenetic potentials which control the formation of headstructure. They are the potential to activate the head formation (headactivation potential) and the potential to inhibit the head formation (headinhibition potential). Their levels are high in the head region and become
low in the foot region forming the "gradients" along the body column.
In order to test the validity of these models, we have examined the changesof the morphogenetic potentials during head regeneration of mutant strains
and compared them to those of the wild type strain (105). The relative
strengths of morphogenetic potentials were measured by the method of thelateral tissue grafting of Webster (1966). Mutant strains used were reg-16
which is defective in head regeneration and L4 which has a low budding rate.
Both mutants were shown to have lower head activation potentials and higher* Zoological Institute, University of Zurich, Switzerland.
** Department of Biology, Nagoya University.

RESEARCHES CARRIED OUT IN 1981

57

head inhibition potentials comparing to 105 under the steady state growth
conditions (Achermann and Sugiyama, 1982; Takano and Sugiyama, 1982).
After head amputation, 105 polyp shows an immediate and drastic decrease of the head inhibition potential and a gradual increase of the headactivation potential at the distal regenerating tip. In reg-16, the decrease of
the head inhibition potential occurs very slowly and in steps, and the increase of the activation potential does not take place. L4 shows a gradualand stepwise decrease of the head inhibition potential (similar to reg-16),and small and gradual increase of the head activation potential (intermediate
between 105 and reg-16).
These observations suggest that the head activation and inhibition potentials play crucial roles in hydra head regeneration, and that whether or noteven a small increase of the head activation potential takes place at the regenerating tip determines success (in L4) or failure (in reg-16) of the subsequent head regeneration.
Chimeric MOllse Embryos Produced by Aggregating Testicular
Teratocarcinoma Cells and 8-ceH Stage Embryos
Kazunori HANAOKA and Tahehiko NOGUCHI

We reported in the last year that chimeric aggregates formed between
cleaving stage embryos and embryonal carcinoma cells (ECC) of a testicular
teratocarcinoma (STT-2) developed at a high rate into blastocysts.
This year we studied the capacity of these chimeric blastocysts to develop
into normal conceptuses in utero. One hundred blastocysts formed between
cleaving stage embryos and ECC of STT-2, and 130 blastocysts formed
between the embryos and ECC of 311 (a clonal line derived from STT-2)
were transfered into uterus of pseudopregnant females of random bred ICR
mice. As STT-2 has type "a" Gpi-l, strains of mice having type "b" Gpi-lwere chosen as the embryo donors so that chimerism may be determined by
the allozyme marker. They were sacrificed at 6 and 7 days after transfer(corresponding 8 and 9 day of gestation). Chimerism of the embryos was
examined by electrophoretic analysis of Gpi-l allozymes using the whole
homogenate of the embryos. The results are summarized in Table l.
Seven (30 %) of the 24 embryos with normal morphology, and 35(82 %)of the 43 abnormal ones were chimeras. Contribution rate of ECC-derived
cells in the normal chimeric embryos were estimated by densitometrical

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32
Table 1. Developmental capacity in utero of blastocysts madebetween ECC and 8-ceII stage embryos
No. implanted
Teratocarcinoma
STT-2
311

No. transfered

Total

Dead

Abnormal(Chimeric)Normal(Chimeric)

100
130

66
58

31
26

21 (17)22 (18)14 (4)
10 (3)

analysis of electrophoregram of PGI-l. The contribution rates of ECCderived cells in the seven chimeric embryos were estimated to be ].0, 0.89,
0.42, 0.33, 0.24, 0.20, 0.]6.
Freeze Preservation of Mouse Embryos
Takehiko NOGUCHI and Masahiro SATO

Mouse embryos similar to tissue cells show no deterioration in viability
during prolonged storage in liquid nitrogen (Whittingham, and Whitten,1974). Freeze preservation of mouse embryos has been applied as insurance
against loss of valuable and irrevocable mouse strains and mutants. It has
been also applied as a method to prevent genetic drifts which may occur
during maintenance of mice in live state. In addition, it offers great economic benifit in the husbandry of mice.
As these benifits of freeze preservation of embryos meet well to the purposesof this laboratory, we started freezing embryos from this year. Eighthundreds of 8-cell stage embryos obtained from inbred strains including
C57BL/6, C57BL/1O, 129/Sv and A/He were frozen essentially by the methodof Whittingham, Leiba and Mazur (1972). To estimate the viability, 366
embryos were thawed 1 to 3 months after freezing, and cultured in Biggers
medium Within 2 days in culture, 232 embryos (63 %) developed into expanded blastocysts. To check the developmental capacity in utero of theseblastocysts, 80 of them were transfered into uterus of pseudopregnant ICR
mice. Twenty two (30 %) new born mice were obtained. They grew into
fertile adults.

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59

Growth of Nervous Tissue of a Notch Lethal, DjU)N8 in
Drosophila melanogaster
Kiyoshi MINATO

The lethal alleles of Notch locus in Drosophila melanogaster are known tobe embryonically lethal, producing an extensive hypertrophy of nervous tissue
at the expense of hypoderm. Using the recently developed techniques ofmore facilitated fixation (Zalokar, 1976) and embedding resins enabling easyorientation of embryo, one of Notch lethals, Df(l)N8 was alayzed quantitatively for the growth of nervous and other tissues with the histologicalpreparations.
As the results, some abnormalities were observed in the first time inembryos at 6-hours of development, in which neuroblasts as about four times
in number as those in the normal embryos were seen, probably segregatedfrom the ectoderm and leaving fewer hypodermal cells (about a half of normal
embryos). These hypertrophied neuroblasts were accompanied with thetypical assymetrical mitosis giving rise to relatively smaller ganglionic cells
so frequently as in normal embryos. At more advanced stage of 8-hours,
still many neuroblasts were observed in lethal embryos, while the numbersof those in normal embryos decreased conciderably through divisions producing ganglionic cells. While, at this stage, few mitoses of neuroblast
themselves were seen in normal embryos, more frequent mitoses were observed in abundant neuroblasts in lethal embryos.
At the succeeding stages of 10- and 12 hours, the fully condensed central
nervous system was formed in normal embryos, while only some groups ofloosely condensed ganglionic cells were observed in lethal embryos. Although these poorly differentiated ganglionic cells occupied much more areasof the body cavity in lethal embryos, they showed, on the basis of the cellnumber, only less extent of hypertrophy (at most, two times or less of normalones) than expected from the presence of far many neuroblasts in the initial
phase. These results suggest that the accumulation of neuroblasts in theinitial phase may be produced by a delayed and prolonged process of mitoses
which might also explain more mitotic figures observed at the later stage
in lethal embryos.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32v.

CYTOGENETICS

Hypo~pentaploid

Chromosome Constitution of a Spontaneous
Tumor Developed in the Oceanian Type Black Rat
Tosihide H. YOSIDA

A tumor (fibrosarcoma) developed spontaneously in the dorsal side of one
male in the Oceanian type black rats (Rattus rattus rattus) obtained from ourbreeding colony. This black rat was originally obtained from the UnitedStates, and at the third generation in the sister-brother mating the tumor
developed. The chromosomes were observed in the primary culture by
conventional and G-banding stainings. The normal karyotype of the tumor
bearing rat was observed from cells of the tail culture by our routine technique.
The normal karyotype of this rat showed 38 chromosomes in all 50 cells
analysed. Among them two large metacentric autosome pairs, M 1 and M 2
were identified as the Oceanian type black rat. The large M 1 chromosome
is consisted of the centromeric fusion of pair nos. 4 and 7, and the other Me
chromosome by pair nos. 11 and 12.
Chromosome numbers counted in 50 tumor cells were distributed from38 to 150. Among them cells with 91 to 95 chromosomes occurred mostfrequently (60 %), those with 71 to 75 chromosomes were 12 %in occurrence,and the other cells were very few (6 to 2 %). This tumor, thus, is characterized by having the hypo-pentaploid stemline. Karyotype of the tumor
cells is not always constant in cells under study, eventhough they have thesame chromosome number. Interesting is that the number of the chromosomes differs according to the chromosome pair. For instance, in one
hypo-pentaploid cell with 93 chromosomes, 8 chromosome pairs vvere pentasomic, 4 pairs were hexasomic, 6 pairs were tetrasomic and X and Y were
disomic. In another cell with a hypo-tetraploid constitution (72 chromosomes), 13 pairs were tetrasomic, 3 were disomic, one was trisomic and theremaining one was pentasomic. Another interesting feature in this tumor
was that the centromeric fission was observed in the large metacentric M 2chromosomes. For example, hypo-pentaploid cells are expected to have6 M 2 chromosomes, but they had 3 metacentric M 2 , 3 acrocentric no. 11 andRESEARCHES CARRIED OUT IN 1981

61

3 acrocentric no. 12 by fission of 3 M 2 chromosomes. In contrast, M 1
metacentric chromosomes were always stable. By the polyploidization,
the number of pair no. I showed 4.6± 1.00. The number of M 2 chromosomes, however, showed 2.3±0.62 in average. This suggests strongly
that about the half number of the metacentric Me elements dealt with thecentromeric fission.
Hypotrichotic Mutant (ba) Appeared in the Inversion Stock(LEM) of the Norway Rat
Tosihide H. YOSIDA

A rat stock "LEM" has been derived from the rats with inversion chromosome no. I (Yosida 1980, Jap. J. Genet. 55: 397). These rats are characterized by the presence of the large metacentric pair no. 1, though each ofwhich is subteIocentric in the normal karyotype of the other strains. A hypotrichotic mutant occurred spontaneously in the course of inbreeding in thisstock. The present paper deals with a process of establishment of the mutant shock, and the genetical and chromosomeal analyses of the new mutant.
The LEM-stock here concerned originated from the hybrids between the
Lewis and Long Evans strain rats. In a female of the Lewis-strain rat (LEWIe), a translocation occurred between pair nos. I and 12. This rat wasmated to a normal Lewis male. One female out of the offspring was a mosaic
consisting of two cell types, such as translocation/normal pair no. I andtranslocation/inversion pair no. 1. This female was crossed to a normal
Long Evans-strain rat. In the offspring thus obtained the rats carrying
inversion/normal pair no. 1 appeared by segregation. Among hybrids
between these rats the inversion homozygotes were obtained. Two female
hypotrichotic rats appeared in the third generation from a mating between
a inversion homologous female and a male. Further, 3 females and 3 malesof a similar appearance were yielded in the other two litters. The hypotrichotic rats were easily identified soon after birth because of slightly smaller
size than the normal ones. They did not show normal hair growth in theyoung stage, though some fuzzy hairs were often observed in young andadult stages showing somewhat an irregular periodic cycle from fuzzy hair toalmost complete hairless. The hairs, however, sometimes remained thinlyon the face appearing as striped pattern in the adult rat. The hypotrichotic
rats were similar in general appearance to the nude rat, but the thymus was

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

present in both new born and adult rats. The thymus, however, was slightly
smaller in size than the normal one, being remarkable by rather yellowish
colouration. The male and female hypotrichotic rats were fertile, and thefemale parents were able to lactate to the new born, though its ability seemed
to be slightly inferior. Based on the genetical nalysis, it is shown that thepresent hypotrichotic character is due to a simple and recessive mutant gene
bared, (ba). In the cross between the hypotrichotic female and male, fiverats were obtained, all of which showed the hypotrichosis. The chromosome constitution of the bared rats studied so far was characterized by atypical inversion pair no. 1, the pattern being the same as that of other
LEM-stock rats. Except the no. 1 chromosome inversion, all other chromosomes of the bared rats were normal. This means that the bared (ba)rats should be produced by gene mutation in the LEM-stock, but is not due
to the new chromosomal mutation.
Quantitative Changes of Nucleolar Organizer Regions(NORs) in 3-methyicbolanthrene Induced Tumorsof the Indian Spiny Mouse
Tosihide H. YOSIDA

Indian spiny mouse, Mus platythrix, is characterixed by 26 acrocentricchromosomes, and the size of the chromosome is generally larger than thatof the house mouse. The nucleolar organizers of this animal are locatednear centromeres of three chromosome pairs, nos. 5, 8 and 12 (Yosida 1980,
Cytologia 45: 753). In the methylcholanthrene induced tumors (fibrosarcomas), Yosida (Cancer Genet. Cytogenet. 3: 211, 1981) reported the frequent occurrence of chromosome anomalies in the autosome pairs with thenucleolar organizer regions (NORs). In order to inquire into the problem
as to whether the quantitative variation of the NORs occurs in these tumor
cells, the NORs were observed in two tumors (MPMC-53 and MPMC-55)
newly induced in the animals by subcutaneous injection of 3-emthylcholanthrene. The tumor cells in early culture passages were used for the chromosome study. Cells of the tail tip cultured from the animal bearing
MPMC-55 were used as the normal control in the present study.
The NORs in the normal cells were found to occur in chromosome nos. 5,
8 and 12 with considerably different frequencies, being 88.3 % in pair no. 5,
14.2 % in no. 8 and 47.2 % in no. 12. The tumor MPMC-55 was charac-

RESEARCHES CARRIED OUT IN 1981

63

terized by a near-diploid stemline karyotype. In 22 karyotypes analysed,
16 cells (73 %) had 26 chromosomes, leaving two cells with 25 chromosomes,
three cells with 27 chromosomes and one cell with 28 chromosomes. Amongthem 12 cells showed translocation. They are consisted of nos. 5 and 12(8 cells), nos. 5 and 8 (one cell), and some other chromosomes (3 cells). The
frequencies of the NORs observed in 22 cells were 84.0% in no. 5 and 13.6%
in no. 8, while 109.1 % in no. 12. The latter high frequency value is due tothe occurrence of trisomy in this chromosome. In 26 cells of the MPMC-53
tumor examined, 15 cells (57.6 %) were in a diploid range (24 to 27 chromosomes). Among them 10 cells showed translocations. The trnslocation
occurring between no. 5 and no. 8 was found in 10 cells, and that between
no. 8 and no. 12 in 2 cells. The latter translocation was found to be duplicated with the fomer one. The number of the NOR in nos. 5, 8 and 12
increased generally to 86.6, 76.6 and 80.0 %, respectively. They are also
higher than the control frequencies.
Shift of the Karyotypes in Indian Spiny Mouse Tumor
MPMC-53 after Storage in Deep Freezing
Tosihide H. YOSlDA

Indian spiny mouse tumor, MPMC-53, which induced by 3-methylcholanthreene was characterized by having diploid (57.7%), triploid (3.8 %) andtetraploid ranged cells (38.5 %) in in vitro cultivation. Among 15 diploid
ranged cells which analysed their katyotype by G-bandings, 10 cells had
translocation between chromosome pairs 5 and 8, and 2 cells between nos. 8
and 12. One batch of cultures of this tumor was stored in the deep freezer(-80°C). Ten days after storage the cells were again cultured in vitro andthe katyotypes of these cells were analysed.
A shift of the ploidy in tumor cell population was found to be diploid(83.8 %), triploid (1.5 %) and tetraploid ranged cells (14.7 %). It is clearlyshown that the frequencies of diploid and tetraploid ranged cells reversed
from those in non-freezing bathes. The karyotype of tumor cells after
freezing also changed markedly from that in non-frozen batches. A newtranslocation between homologous chromosomes of no. 6 was found in all
25 cells analysed, and the translocation between nos. 5 and 8, which were
frequent in the non-frozen cells, was never observed in the cell population
developed after freezing.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

The frequencies of the necleolar organizer in pair nos. 5, 8 and 12 in the
non-frozen cells were 86.6, 76.6 and 80.8 per cent, respectively, but thoseafter freezing changed to 100.0, 8.0 and 90.0 %, respectively. The shift ofkaryotypes seems to be attributable to a preferential selection in cell population due to deep freezing.
Geographical Variation of Chromosomes Numbers in theHouse Shrew, Suncus murinus (Soricidae) amI. its
Distribution in the World
Tosihide H. YOSIDA

Karyotypes of the house shrew (Suncus murinus) collected from 8 different
localities in East, Southeast and Southwest Asia were examined with an interest of the geographical variation and distribution of this animal. The
chromosome number of the house shrews collected from 7 localities; Okinoerabu in East Asia, Tainan, Bogor and Makassar in Southeast Asia andKanpur, Kalyani and Bhubaneswar in Southwest Asia; was constantly 40 in
the somatic cells. This seems to be the basic number in this species.
Karyotypes of all specimens with 40 chromosomes were identical except theshape of Y chromosomes. The autosomes were classified into 3 groups;
acrocentrics (nos. 1 to 12), subtelocentrics (nos. 13 to 15) and meta- orsubmentacentric (nos. 16 to 19). The X chromosome was the largest submetacentric, while the Y chromosome was a rather smaller submeta- or
metacentric which showed apparent variations in its size and shape among
specimens collected from different localities.
Specimens collected from Trincomalee in Sri Lanka had 32 chromosomes,
among which 4 autosome pairs (nos. 1 to 4) were acrocentrics, three autosome
pairs (nos. 5 to 7) were subtelocentrics, and the other 8 autosome pairs(nos. 8 to 15) were metacentric or submetacentric. Among the latter group
4 pairs (nos. 8 to 11) were conspicuously large meta- or submetacentrics.
The other 4 pairs (nos. 12 to 15) appeared to correspond to the pair nos. 16
to 19 seen in the basic karyotype with 40 chromosomes. Similarly, nos. 5
to 7 in the Sri Lanka karyotype were thought to be homologous to nos. 13
to 15 in the basic karyotype. The X chromosome was a large submetacentric, and the Y a smaller submetacentric. Based on the comparison
between the karyotypes of the house shrew in Sri Lanka and the specimens
collected from the other localities, it is strongly suggested that the 4 pairs of

RESEARCHES CARRIED OUT IN 1981

65

large biarmed elements in the 32-chromosome specimens were diviatedthrough Robertsonian fusions among 8 acrocentric pairs in the animal
with 40 chromosomes. Variation of chromosome numbdrs from 40 to 36
has been reported by Yong (1974) in specimens collected in the central partof West Malaysia. House shrews with 32 and 30 chromosomes have beenreported by Satya Parakash and Aswathanarayana (1976) in the specimens
collected from southern India.
According to some taxonimists the wild form of the house shrew has been
distributed originally in the forest of central India, and then they moved tothe other parts of Southwest, Southeast and East Asia with the migrationof man. They are suggested to be the original type which should be characterized by having the basic karyotype with 2n=40. On the other hand, thesequential Robertsonian rearrangements should have taken place in thehouse shrews moved to southern India from the central India. The animals
with 32 chromosomes have developed by the Robertsonian fusion between
8 chromosome pairs, and those with 30 chromosomes by the Robertsonian
rearrangement of two more chromosome pairs than the above karyotype.
The former animals seemed to have migrated from southern India to Sri
Lanka. These animals, would migrate to some islans in the Indian Ocean
and to the west coast of the Malayan peninsula. The Malayan population
with 40 to 36 chromosomes seems to have arisen through hybridization
between the animals with 40 chromosomes originally migrated there andthose with reduced chromosome numbers which migrated through theIndian Ocean.
Geographical Variation of Y-Chromosomes in the House Shrew,
Suncus murinus, and its Distribution
Tosihide H. YOSIDA

Basic karyotype of the house shrew, Suncus murinus, was 2n=40. The
chromosome numbers of this animal distributed in southern India, however,showed 30 to 32 by Robertsonian translocation between acrocentrics included in the basic karyotype. The X-chromosome in this animal wasinvariably large metacentric, while the Y-chromosome showed a wide
geographical variation in its size and shape.
The Y-chromosome in specimens from Java and Celebes (2n=40) was aconsiderably small submetacentric, which was shorter than the long arm of66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

the X. The Y in the Sri Lanka taxon with 2n=32 was similar in size andshape to that in Indonesia, but that in the Okinoerabu specimens was themetacentric, though its size was similar to the above specimens. The
Kalyani specimens had a slightly larger Y, nearly comparable to the long armof the X. In contrast, that in the Kanpur population was an unusually large
submeta- or subtelocentric element, being much longer than the long arm of
the X. The Y chromosomes, thus, are classified roughly into 5 types; (A),the large submentacentric (or subtelocentric); (B), the median sized submetacentric; (C), the small submetacentric; (D), the small metacentric and (E),the small acrocentric. The type A found in Kanpur and Delhi seemed toshow a distribution in northern India. The type B was observed in northern(Kalyani) and central India (Hyderabed) to southern Malaysia (Malacca).
The type C and D were also found rather widely in Southeast and East Asia.
The type E was found in northern India (Kalyani) and South Vietnam. If
a suggestion on the evolution of the Y is allowed, the largest type A which
distributed widely in northern India is the original form, and the types BandC occurred by sequential deletions of the long arm. Type D is resulted
from an inversion in the type C, and type E from a deletion of one arm intype C or D.
Karyotype of Hybrid Male between the Domestic Cat
and the Leopard Cat
Tosihide H. YOSIDA

As a hybrid male born from mating between the female domestic Siamesecat (Felis catus) and a male leopard cat (F. bengalensis) was obtained, thehybrid was cytogenerically examined. The chromosomes were studied
based on leukocytes cultured by the routine procedure.
The chromosome number and the karyotype of the domestic cat were
fully consistent with those by the previous studies. Eighteen autosome
pairs were classified into A to F groups; the group A is consisted of 3 large
submetacentric pairs (A-l to A-3); the group B of 4 large subtelocentric
pairs (B-1 to B-4); the group C of 2 large metacentric pairs (C-l and C-2);
the group D of 4 considerably small subtelocentric pairs (D-l to D-4); thegroup E of 3 small metacentric pairs (E-l to E-3) and the group F of 2 small
acrocentric pairs (F-l and F-2). The E-l pair was remarkable by having thesecondary constriction. The X was metacentric corresponding in size toRESEARCHES CARRIED OUT IN 198167

the C group, while the Y was of small subtelocentric structure.
Although karyotype of the leopard cat could not be examined, it has beenstudied by Makino and Tateishi (1952) and Hsu and Benirschke (1968).
According to them, the leopard cat had 38 chromosomes similar to the domestic cat, but the karyotype is slightly different between them. The
karyotype of the leopard cat was similarly classified into A to F, but the
number in the groups Band F was different between these two species. In
the leopard cat the B group was consisted of 5 pairs (B-1 to B-5), and the
F group was represent by a single pair (F-I). The other autosome pairs
were apparently similar between the two species. The X and Y chromosomes were identical between them, except the size of Y which is slightly
smaller in the leopard cat.
The chromosome number of the hybrid between the domestic cat and theleopard cat was the same as that of the parents (2n=38), but its karyotype
was slightly different from those of both parents. The autosomes wereclassified into A- to F-groups corresponding to parents. Three A-pairs(A-l to A-3), 2 C-pairs (C-I and C-2), 4 D-pairs (D-l to D-4) and 3 E-pairs(E-l to E-3) were closely identical with parent karyotypes, but the chromosome numbers of the groups Band F were different in the hybrid. In thegroup B, 4 pairs (B-1 to B-4) were the same as those of both parents, while
B-5 was single without homologous pair in the hybrid. Apparently thesingle B-5 was of the leopard cat origin. In the group F, 3 small acrocentics were observed. The F-l was characterized by normal pair, but theF-2 was single without homologue in the hybrid. Comparison revealed
that the unpaired F-2 element was a derivative from the domestic cat.
Effect of Metabolic Perturvation on Induction of the
Sister Chromatid Exchange (SCE)
Hitoshi SUZUKI, Tosihide H. YOSIDA and Toshihisa KUSANO*

Although 5-bromodeoxy uridine (BrdU) is incorporated into DNA strands,
the effect of BrdU on SCE induction seems to be due to the metabolic perturbation of DNA nucleotide synthesis (Davidson et al. 1980). It is interesting
to know whether metabolic perturbation induced by other drugs can also
increase the SCE frequency. SeE induction by treatment with 5-fluorodeoxyuridine, known as a inhibitor of thymidine kinase, could be com* Dept. of Life Science, Hiroshima Women's University,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

pletely suppressed by addition of thymidine or BrdU, but not suppressed bydeoxycytidine, deoxyuridine or uridine. Thus the effect of 5-flurodeoxyuridine on SCE induction may be due to depletion of dTTP pool.
The SCE frequency increased by treatment with excess amount of deoxyribonucleosides such as dT, dA and dG. These chemicals are known to
inhibit the ribonucleotide reductase and cause the metabolic perturbation
in DNA nucleotide synthesis. Induction of SCE by these chemicals waspartially suppressed by the addition of the other deoxyribonucleosides suchas dC, which could rescue the metabolic perturbation.
Hydroxyurea also known as a inhibitor of the ribonucleotide reductase
can induce SCE (Ishii and Bender, 1981), but the induction could not be suppressed by the addition of the four deoxyribonuleosides. Thus, it is questionable to say that the SCE induction by HU is depended on metabolic
perturbation.
We also examined whether the SCE was induced in the uridine requiringmutant UR-216 cells (Kusano et aI., 1976) when they were cultured in uridine
insuficient medium. When the UR-2l6 cells were cultured in low concentration of uridine for two cell cycles, the SeE frequency was about two times
higher than that of control. In these studies a conclusion may be drown thatmetabolic perturbation in DNA synthesis is one of the factors of SCE
induction.
A Re-Examination of Chiasma TerminaHzation and Chiasma
Frequency in Male Mice
Hirotami T. IMAI and Kazuo MORIWAKIThe distribution and frequency of chiasmata have been analyzed in male
BALB/c mice. Bivalents were classified in terms of the number of interstitial chiasmata (CH) and terminal associations (TA) present as follows;
lCH, 2CH, ITA, lCH'lTA, lCH·2TA, 2CH·1TA, 2CH·2TA and O.
We provide evidence that the TA frequently dissociates during 1st meiotic
prophase. Consequently six of the observed bivalents may be derived fromthree basic bivalent types (namely 2CH· 2TA, 1CH ·2TA and 2TA) by dissociation of the TA according to the following schemas: (1) 2CH ·2TA----?
2CH 'lTA----?2CH, (2) lCH ·2TA----?lCH 'lTA----?lCH, and (3) 2TA----?lTA----?O. We also provide evidence that interstitial chiasmata do not move,which implies that a TA can not be formed by chiasma terminalization. The

RESEARCHES CARRIED OUT IN 1981

69

chiasma frequency estimated by assuming that terminal associations do not
result from terminalized chiasmata is 17.2±2.4 compared to a value of
25.4±2.2 calculated on the assumption of chiasma terminalization. For
details, see Chromosoma (Berlin) 85: 439-452, 1982.
X-Y Chromosome Dissociation in Wild Derived Mus musculus

Subspecies, Laboratory Mice, and Their Ii'1 Hybrids
Yoichi MATSUDA1), Hirotami T. IMAI, Kazuo MORIWAKI,
Kyoji KOND0 1), and F. BONHOMME 2)

The frequencies of X and Y chromosome dissociation in primary spermatocytes of wild-caught specimens of six Mus musculus subspecies, fifteen
inbred strains, and their F 1 hybrids were examined. Three different types(I, II, and nI) ofX-Y dissociation were identified. Type I was found in intersubspecies hybrids (BALB/c X M. m. molossinus, M. m. bactrianus, M. m.
castaneus, or M. m. urbanus), in which the X-V dissociation was less than
20 % in each subspecies but 60-80 % in the F 1 hybrids. Type II was seen in
the DBA/2J and DDK strains, in which the dissociation frequency was about
30 % in the homoxygous condition and increased up to 50 % in inter-strain
hybrids (C57BL/6J X DBA/2J or DDK). Type III found in the WB/Re
strain also showed high dissociation frequency (51.1 %) in the homozygous
condition but the value was remarkably lower (11.3 %) in (C57BL/6J X
WB/Re)F1s. Some Asian wild mice seem to have type n or III as well astype 1. For details, see Cytogenetics and Cell Genetics 34: 241-252, 1982.
Chromosome Observations of Some Umgl1ayam Allts(Hymenoptera: JFormicidae)
Beatriz GONI, Lucrecia C. de ZOLESSI 3), and Hirotami T. IMAI

Although more than 300 species of ants including European, Indian,Japanese, Australian and New World fauna have been cytologically investigated (Crozier 1975, Anim. Cytogenet. 3, Insect 7; Imai et at. 1977,
Chromosoma 59: 341-393; Imai and Kubota 1981, Nucleus 24: 93-96),
Faculty of Agriculture, Nagoya University, Nagoya.
Laboratoire d'Evolution des Vertebres, Universite des Sciences et Techniques duLanguedoc, Mnotpellier.
3)
Departmento de Artropodos, Facultad de Humanidades y Ciencias, Universidad dela Repllblica O. del Uruguay, Montevideo, Uruguay.
1)

2)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

chromosomes of neotropical ants have rarely been observed (Crozier 1970,
Can. J. Genet. Cytol. 12: 109-128). As the Neotropical region is one ofthe
centers of ant's distribution and differentiation, and as a lot of endemic species
are found in there, karyological survey of neotropical ants is very important
for analysing the karyotype evolution of ants. Recently, we had an oppportunity to observe Uruguayan ants. This paper is a preliminary reporton chromosomes of thirteen Uruguayan ants involving three subfamilies(Myrmicinae, Dolichoderinae and Formicinae).
Myrmicinae: Four species of Pheidole, one species of Solenopsis, andthree species of Acromyrmex were examined. It was revealed that all the
species of Pheidole (cornutula, fallax, spinninodis and strobeli) had the same
chromosome number (2n=20 or n= 10, and 2AN =40) and showed a nearly
similar karyotype. According to Crozier (1975) and Imai et al. (1977),
most members of the genus Pheidole reported (more than thirty) have 2n=20.
Our present observations also suggest that Pheidole is a karyotypically conservative genus. A large fire ant species Solenopsis saevissima had 2n=32(or n = 16) and 2AN= 62, which is almost identical with those of S. aurea(Crozier, 1970) and S. invicta (Glancey et al. 1976, Ann. Ent. Soc. Am. 69:
469-470) having many metacentrics and submetacentrics. Two New World
attine ants Acromyrmex (Acromyrmex) ambiguus and A. (A.) hispidus had thesame karyotype 2n=38 and 2AN=62. Another attine species belonging toa different subgenus Acromyrmex (Moellerius) heyeri also showed a very
similar karyotype to the previous two species mentioned above (i. e., 2n=38
and 2AN =62), suggesting that they are karyologically closely related. Thisis the first report on the chromosomes of the tribe Attini.
Dolichoderinae: One species (Conomyrma pyramica) was examined.
This species is one of the common ants in South America, but this is thefirst cytological report of the genus. The diploid chromosome number was
2n=18. As all chromosomes excepting one pair were hi-armed, the diploidarm number was 2AN=34.
Formicinae: Four species of the cosmopolitan genus Camponotus wereobserved. The results were as follows: C. mus (2n=26, 2AN=42), C.
bonariensis (2n=40, 2AN=48), C. punctulatus (2n=40, 2AN=44), andC. rujipes (2n=40, 2AN=44). The karyotypes of the latter three species
were almost the same excepting a minor change in arm number. On theother hand, the karyotype of C. mus was quite different from the othermembers in chromosome number. In spite of such a remarkable difference

RESEARCHES CARRIED OUT IN 1981

71

in chromosome number, their arm numbers were fairly conservative (2AN=
42-48), suggesting that Robertsonian rearrangements would have played asignificant role in their karyotype differentiation.
Chromosome Observations of Tropical Ants in Western
Malaysia and Singapore
Beatriz GONl, Hirotami T. IMAI, Masao KUBOTA1 ), Masaki KOND0 2),
Hoi-Sen YONG 3), and Yow Pong TH0 4)

In order to investigate the karyotype evolution of ants by using the karyograph method devised by Imai and Crozier (Am. Nat. :H6: 537-569, 1980),
the second author is collecting ants from all over the world. As a part ofthe project Imai, Kubota and Kondo had recently the chance to collecttropical ants of Malaysia and Singapore. This is a preliminary report ofchromosome numbers of 73 ant species placed in 36 genera, 20 tribes and
5 subfamilies. As the precise identification of species name is practicallydifficult in ants, we tentatively classified them as sp. 1, 2, 3, ... based on external morphologies and karyotypes. For the convenience of researchers
who are interested in the taxonomy of these ants, we deposited one set ofthe dry specimens to the Australian National Insect Collection (CSIRO,
Canberra, Australia) and also to the Basel Museum (Switzerland). The
results are summarized in Table 1.
Table 1.
Taxa

Chromosome numbers of Malaysian ants
Chromonumber(n) 2nSUBFAMILY PONERINAE
Tribe P1atythyreini
Probolomyrmex sp.
28
Tribe Ectatommini
Gnamptogenys sp. 1
42
Gnamptogenys sp. 2
36
Tribe Ponerini

Chromonumber(n) 2n
Leptogenys sp. 1
38
Leptogenys sp. 2
48(18) 36
Diacamma sp.
(11,1l+1B)j
Pachycondyla sp.
22+2B(B-chrom. polymorphism)
28
lvlesoponera sp. 1
Taxa

Nakasone 13, Odawara, Kanagawa-ken, 250.
Shiraume Gakuen College, Kodaira, Tokyo.
3)
Department of Genetics and Cellular Biology, Universiti Malaya. Kuala Lumpur,
Malaysia.
4)
Forest Research Institute, Kepong, Malaysia
1)

2)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32
Table 1.

Chromonumber(n) 2nMesoponera sp. 2
22
Mesoponera sp. 3
36
Hypoponera sp.
38
Brachyponera sp.
22(Reciprocal translocation)
Tribe Odontomachini
Anochetus sp. 1
24
Anochetus sp. 2(19)
Odontomachus sp. 1(22) 44
Odontomachus sp. 2
30,30+lB(B-chrom. polymorphism)
Odontomachus sp. 3(22) 44
Tribe Cerapachyini
Cerapachys sp.
(25) 50SUBFAMILY PSEUDOMYRMICINAE
Tribe Pseudomyrmini
Tetraponera sp.
44
SUBFAMILY MYRMICINAE
Tribe Pheidolini
Aphaenogaster sp.
30
Pheidole sp. 1
20
Pheidole sp. 2(10) 20
Pheidole sp. 3
20
Pheidole sp. 4
20
Pheidole sp. 5(16, 17)(Robertsonian polymorphism)
Pheidole sp. 6
20
Pheidole sp. 7
16
Pheidole sp. 8
38
Pheidole sp. 9
18
Pheidole sp. 10
20
Tribe Cardiocondylini
Cardiocondyla sp.
40
Tribe Crematogastrini
Crematogaster sp. 1
26
Crematogaster sp. 2
36
Crematogaster sp. 3
36
Crematogaster sp. 4
24
Tribe Solenopsidini
Monomorium sp. 1
22
Taxa

Continued
Taxa

Chromonumber(n) 2n22
38
36

Monomorium sp. 2
Diplorhoptrum sp.
Oligomyrmex sp. 1
Tribe Myrmecinini
Acanthomyrmex sp. 1(11)
Acanthomyrmex sp. 2
22
Pristomyrmex sp.
22
Tribe Myrmicariini
Myrmicaria sp. 1
44Myrmicaria sp. 2
44
Myrmicaria sp. 3
44
Tribe Tetramoriini
Triglyphotrix sp. 1
20
Triglyphotrix sp. 2
18
Tetramorium sp. 1
22
Tetramorium sp. 3
26
Tetramorium sp. 4
14
Tribe Attini (?)
Proatta sp.
32
Tribe Dacetini
Smithistruma sp.
16
SUBFAMILY DOLICHODERINAE
Tribe Dolichoderini
Dolichoderus sp.
18
Tribe Tapinomini
Iridomyrmex sp.
18
Tapinmna sp.
(5) 10Technomyrmex sp.
30
SUBFAMILY FORMICINAE
Tribe Plagiolepidini(17) 34
Anoplolepis sp.
Acropyga sp.
(15)
Tribe Camponotini(19)
Camponotus sp. 1(20) 40
Camponotus sp. 2
38
Camponotus sp. 3(18) 36
Camponotus sp. 4(20) 40
Camponotus sp. 5
Camponotus sp. 6
44(21)
Polyrhachis sp. 1

RESEARCHES CARRIED OUT IN 1981
Table 1.
Chromonumber(n) 2nPolyrhachis sp. 2(21)
Polyrhachis sp. 3
20(Pericentric inversion polymorphism)
Tribe Lasiini
Paratrechina sp. 1
16
Paratrechina sp. 2
26
Taxa

73

Continued
Chromonumber(n) 2nParatrechina sp. 3
28
Paratrechina sp. 4
16
Paratrechina sp. 5
28
Prenolepis sp.
34, 36(Robertsonian polymorphism)
Taxa

Supernumerary Y Chromosomes and its Polymorphism inDrosophila punjabiensis
Akiko HANAKATA and Masatoshi YAMAMOTO

Extra sex chromosomes are normally detrimental to the individuals carrying them. In Drosophila melanogaster an extra X chromosome causes
inviability, on the other hand extra Y chromosomes are less deleterious.
The presence of an extra Y chromosome in both sexes hardly causes any
clear phenotypic effect. A female can endure two extra Y chromosomes tobe fertile, while a male having two extra Ys (XYYY) become sterile. Theirfitness is lower than normal males and females and further they show meiotic
drive through abnormal meiotic segregation of sex chromosomes. Thus it
could be suspected that the number of Y chromosome aneuploids in natural
condition should be rare. In fact there has been no report to date on thevariation in Y chromosome numbers in natural populations of any organisms
even those having sex determination mechanisms similar to Drosophila.
We now report here the first finding of polymorphism of Y chromosome
numbers in Drosophila punjabiensis isofemale lines collected from natural
populations.
D. punjabiensis is classified into a Drosophila montiumsubgroup which
belongs to Drosophila melanogaster group. This species is widely spread andcommonly found in South East Asia. Ten isofemale lines collected fromseven local populations in India, Malaysia and Burma were cytologicallyexamined. The genome of D. punjabiensis cosists of two pairs of metacentricchromosomes (2nd & 3rd), one pair of dot-shaped chromosomes (4th) andsex chromosomes of acrocentric X and acrocentric Y chromosome. Twolines among ten, namely Texas 3033.4 from Malaysia and MYS 172 from

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

India, had supernumerary chromosomes. These supernumerary chromosomes were not distinguishable from the Y chromosome found in XY karyotypic male, judging from either conventional staining of Giemsa or Quinacrine-Hoechst staining. A variety of Y chromosome numbers wasobserved in the two lines. They were XX, XXY, XXYY in the female and
XY, XYY, XYYY in the male. The frequencies of individuals carrying extra
Y chromosome(s) were ca. 70% in MYSI72 and ca. 40% in Texas3033.4.
This extremaly high frequency of supernumerary Y chromosomes should
indicate that the additional Y chromosomes are very stable in the labolatory
conditions at least. Although similar observation concerned with Y chromosome-number polymorphism has been made in a wild population oftsetse fly (Maudlin, 1. 1979), the frequency of Y chromosome aneuploids wasvery low both in males and in females.
In order to understand the mechanism by which such high frequency of
Y chromosome naeuploids can be maintained in Drosophila punjabiensis,
the following questions are now being examined.
1) Meiotic segregation of XYY, XYYY in the male and XXY, XXYY
in the female.
2) The effect of extra Y chromosomes on viability and fertility.
New Series of Segmental Aneuploidy Using Transpositions
between X and Y Cbromosomes
Akiko HANAKATA and Masatoshi YAMAMOTOIn Drosophila melanogaster, chromosomal duplications and deficiencies
are of great use for not only cytological gene mapping, studies of genefunctions and its dose effect, but also molecular biological studies. Manyefforts have been made for their production and application for geneticalreseach since the begining of Drosophila genetics. With regard to X chromosome, a lot of deficiencies and free duplications have already been produced. But there are at least ten regions where no deficiency has beenobtained because of haplo-lethal or halpo-sterile in the female, and most of
the free duplications are limited in either proximal or distal regions.
Translocations between X and Y [T(X; Y)] are the most powerful tools
to generate deficiencies and duplications in the X chromosome. When flies
possessing T(X; Y) translocations with different X chromosome breakpoints
are crossed, they produce aneuploids of deficiency and duplication besides

RESEARCHES CARRIED OUT IN 198175

the euploids. Although the progenies carry extra Y chromosome fragmentsin their genome, it can be assumed that the Y chromosome aneuploidy does
not give a serious damage to the individual because of the inertness of theheterochromatic Y chromosome. By this method, it is theoretically possible
to produce adequate deficiencies and duplications in any region of the X.
Although the system of segmental aneuploidy is very efficient and useful in
Drosophila genetics, it certainly have the following two disadvantages forhigher techniques of chromosome manipulation. (1) Because two translocation fragments are newly combined to synthesize a duplication and a deficiency
as a segmental aneuploidy, a portion or all of the Y chromosome is duplicated in the genome. Though an Y chromosome hardly affects the external
morphological phenotype, it reduces viability and fertility of the fly. (2)A duplication can be made at any region of the X with segmental aneuploidy,
but the chromosomes used must be the ones of T(X ; Y). It is impossible
at present to make a duplication between a particular X chromosome and asmall segment which covers a speicfic regions in question.
The most useful and simple chromosomal rearrangement for this purpose
could be a free Y chromosome carrying a small portion of the X. We have
tried to generate such chromosomes as follows: males carrying T(X; Y),y+BSjYwere irradiated with 3600R of X-rays and were crossed to C(l)DX,
ywf females. y+BS progeny males were individually crossed to C(l)DX,
ywf females and their offsprings were examined. If a transposition of a X
fragment into Y chromosome occurred, phenotypic changes can be easily
observed. From a single cross, males carrying T(X ----7 Y) transposition andfemales carrying the extra X fragment as a free duplication on the Y appearedas brothers and sisters. After the screening, they were examined cytologically. Up to date, the original T(X; Y)B146, T(X; Y)B146 L B133 R (breakpoint l2E9) and T(X; Y)D12 (7Cl) gave seven T(X ----7 Y)s from 10,854 y+BS
screened males. They are:Transposition
Cytological breakpoints
T(X----7Y)M04
12E9:14A
T(X----7Y)M57
12E9:15A
T(X----7Y)H4
l2E9:l5A
T(X----7Y)H5
12E9 :13Al
T(X----7Y)H16
lOD:l2E9
T(X----7Y)H17
l2E9:13E2
T(X----7Y)H31
7Cl:8C

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

It is conceivable that free Y chromosomes carrying a X segment of any
region of the X can be obtained by the same method.
Cytogenetical Studies on Autosomal Meiotic Pairing in
Drosophila melanogaster Male
Masatoshi YAMAMOTO

Conventional pairing at meiosis is regarded as complete in the sense thatpartner homologues lie in close parallel alignment throughout their entire
length. This assumes that pairing is by strict homology and that there areno pairing sites as such.
It has long been appreciated that conventional pairing does not apply tomany sex chromosome systems. The clearest example is in Drosophila
where X and Y chromosomes were argued for the occurrence of speicfic
pairing sites within the heterochromatin. Here pairing is not by heterochromatic homology but rather by specialised entities called pairing sites or
collochores.
Since the autosomes in D. melanogaster males do not engage in crossing
over one can ask whether the autosomal pairing mechanisms are conventional. It is known that the conventional stages during which pairingnormally occurs appear to be absent in meiosis of D. melanogster as too is
synaptonemal complex. Male meiosis is thus thoroughly unconventional.
In the study of heterochromatin function on chromosome pairing, I foundthat the heterochromatin of autosomes are not involved in homologue
recognition and subsequent pairing. The finding that euchromatic homology is of importance to normal meiotic pairing in the male leads to the next
logical question; Is all euchromatin equally efficient in bringing about pairing and disjunction, or are some euchromatic segments are more important
than others?
I have asked the above question on chromosomes 2, 3 and most extensively
4. I describe here the results obtained from the study of chromosome 4.
The translocation T(X; 4)B' and T(2 ; 4)b both have breakpoints on theeuchromatin of chromosome 4. The breakpoint of T(X; 4)B' is at 1/4
proximal from the euchromatic distal end and that of T(2 ;4)b is at the middleof euchromatin. In both cases the proximal portion containing heterochromatin is a partner for meiotic pairing with a nomra14. T(X;4)e15 is broken
in the heterochromatin, but the size of translocated segment is minimal me-

RESEARCHES CARRIED OUT IN 198177

asured by Q-H banding techniques. In this translocation too, the proximal
fragment is involved in pairing. When, however, almost entire 4th chromosome heterochromatin is translocated onto chromosome 2 as is r(2; 4) Y64
for example, a normal chromosome 4 always pairs with the translocated 4th
fragment, and never pairs with the proximal portion of the 4th. Similarly,
reX; 4)w m5 and rex; 4)wm25S-i8 whose breakpoints are in the heterochromatin and the distal 1/3 is translocated onto the distal end of X chromosome
were applied to cytological examination. As a result a normal chromosome
4 failes to pair with either distal or proximal fragments in almost all meiocytes. Interestingly enough however, at low frequency the translocateddistal portion of 4 which is now on the X is involved in association witha normal 4.
This finding clearly indicates that there is a discrete priring site in the 4thchromosome heterochromatin as found in sex chromosome systems, and thesite is located at the close visinity to the breakpoint of T(X; 4)w m5 . This is
the first experimental demonstration to show the existence of pairing site in
an autosome. It should be interesting to study the nature of this pairing
site on the molecular basis and also ask if there exist similar entities as pairing
sites in chiasmate organisms as well which show the conventional meiotic
pairing processes as such.

OmstmctioFi of T(2; 4), T(3; 4) Autosomal TraIDl§!ocaHoIDls
in D/I'osophila me!cmogaste/l'
Masatoshi YAMAMOTO

Meiotic chromosome pairing has been extensively studied in sex chromosomes because of ease in manipulating chromosomal abberations. Beginning the study of autosomal meiotic pairing, I faced the serious difficultythat not enough chromosomal rearrangements involving autosomes wereavailable.
In order to carry out cytological studies of meiotic pairing of chromosomes
2 and 3, I firstly constructed T(2; 4) and T(3; 4) translocations. The screening procedure employed was as follows; Wild-type males of Canton-S strain
were exposed to 3600R of X-rays. Then the X-rayed males were crossed to
ei/ei virgin females in a mass and kept in 18°C incubator in order to enhance
penetrance of the ei phenotype. Most F i ei+ /ei males showed wild-type
phenotype, but at a low frequency males having ci phenotype appeared. The

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

T(2; 4)Y2
T(2; 4)Y7
T(2; 4)Y22
T(2; 4)Y34
T(2; 4)Y40
T(2; 4)Y47
T(2; 4)Y64
T(2; 4)Y76
T(2;4)Y86
T(2; 4)Y92
T(2; 4)Y100
T(2; 4)Y106
T(2; 4)Y109
T(214)Y141
T(2; 4)Y142
T(2; 4)Y164
T(2; 4)Y185
T(2; 4)Y209
T(2; 4)Y220
T(2; 4)Y231
T(2; 4)Y241
T(2; 4)Y308
T(2; 4)Y318
T(2; 4)Y325
T(2; 4)Y344
T(2; 4)Y375
T(2; 4)Y376
T(2; 4)Y476
T(2; 4)Y492[In(2LR)]T(2; 4)Y496
T(2; 4)Y517
T(2; 4)BIlI 17
T(2; 4)K10
T(2;4)K12
T(2; 4)K13
T(3; 4)Y9
T(3; 4)Y13
T(3; 4)Y25[In(3R)]

Translocations and their breakpoints are listed below;
80-81; 10lF
T(3; 4)Y37
35D3-4; 101F
75F; 102C
T(3; 4)Y57
50C14-D1; 102C-D
92A; 101F
T(3; 4)Y61
40-41;101F
80-81; 101F
T(3;4)Y63
36C-D; 101F
81F; 101F
T(3; 4)Y71
56B-C; 101F
100F; 102C14
T(3;4)Y78
31D-E; 101F
64E; 101F
T(3; 4)Y81
35C; 101F
96Al-4; 101F
T(3; 4)Y104
40-41; 101F
80-81;
101F
T(3;4)Y121
21E1-2; 101F
96A20-25; 101F
T(3; 4)Y140
57B4-6; 101F
88C; 101F
T(3; 4)Y144
21D; 101F
78B; 101F
T(3; 4)Y151
55A; 101F
nc; 101F
T(3; 4)Y154
40-41; 59B; 101F
99E; 101F
T(3; 4)Y161
40-41; 101F
76Bl; 102D
T(3; 4)Y168
52E; 102C
9lD; 101F
T(3; 4)Y175
33A; 101F
77B; 101F
T(3; 4)Y177
52D; lOlF
68D;
101F
T(3;
4)Y183
36C-D; lOlF
67E; 101F
T(3; 4)Y210
56F6-8; 101F
63D; 101F
T(3; 4)Y226
52F; l02D
80-81; 101F
T(3; 4)Y249
54D-E; 10lF
80-81; 101F
T(3; 4)Y252
49FIO-15; 101F
94B; 101F
T(3; 4)Y255
40-41; 101F
71F; 101F
T(3; 4)Y262
34E-F; 101F
96D-E; 101F
T(3; 4)Y285
59F; 101F
80-81; 101F
T(3; 4)Y291
60F5; 101F
88D; 101F
T(3; 4)Y320
33C; 101F
98Fl1-12; 101F
T(3; 4)Y391
29D; 101F
67E3-4; 101F
T(3; 4)Y403
30A7-9; 56F8-9;
83A; lOIF
T(3; 4)Y425
10lF
80-81; 101F
T(3; 4)Y434
22Al-2; 101F
70D;
101F
T(3;
4)Y446
54F; lOlF
88E4-6; lOlF
T(3; 4)Y449
40-41; 101F
75C; 101F
T(3; 4)Y477
57F3-6; 101F
98B; 101F
T(3; 4)Y494
49B; 101F
70C; 101F
T(3; 4)Y495
25A3-4; lOlF
94B-C; 96E; 101F
T(3; 4)Y512
65B4; 101F
[In(3R)]
80-81; 101F
77A; 101F
T(3; 4)B118
90C; 98C; 101F
93E; 101F
T(3; 4)BlI 13

RESEARCHES CARRIED OUT IN 198179

ci phenotype is known to come out when a breakage occured in the visinityof ci locus on chromosome 4. F i males showing ci phenotype, thus indicatethat the individual was very likely to have a breakage on the 4th at least.
The ci males were then individually allowed to mate with yf:=; bw; e; ci
eyR females. F 2 males heterozygous for the markers were testcrossed to the
yf: = ; bw; e; ci eyR females and checked their segregation of marker characteristics. A translocation heterozygote can be easily identified since it shows
new linkage groups. The translocations newly synthesized were kept as
stocks over 8M], Cyand TM3, 8b.
Cytogenetical Analyses of the Gene Altering Sensitivity toTrehalose in Drosophila melanogaster
Masatashi YAMAMOTO and Kunia IsoNo

Various behaviours of organisms, such as locomotion, flight, mating,phototactics, circadian activity, eclosion and learning are believed to beunder the genic control. In D. melanogaster feeding behaviour is primarily
determined by the sensitivity at the chemosensory hairs which have taste
receptor cells for water, salts, and sugars. Behavioural and electrophisiological studies have revealed that there are at least four groups of sugar receptor
sites for glucose, fructose, sucrose and trehalose.
Recently Isono et al. found a feeding behaviour mutant which showed
insensitivity to trehalose. It is already known about the mutant that (1)the gene Tre+ controls sensitivity specific to trehalose not to other sugars
and (2) Tre+ gene functions at the site of sensory organs, chemosensory
hairs, not in the nurvous system, because the mutant does not make any
alterations on feeding behaviour and sensitivity to other sugars.
We have analysed the mutant by sucrose-trehalose feeding preference test.
Wild type flies show similar degrees of sensitivity to 2 mM sucrose and 20 mM
trehalose. Naturally the flies ingest approximately equal amount of eachsugar when they are allowed to choose either 2 mM sucrose or 20 mM trehalose. Because sucrose solution contains red dye and trehalose does blue,
wild type flies show purple (red +blue) abdomens after ingestion and themutants show red abdomen.
Genetic dissections of Tre mutant using several X chromosome deficiencies
and T(X; Y) translocations for segmental aneuploidy has given the following
results;

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32(1) The sensitivity to trehalose is determined by the single gene Tre+ on theX, differing from other behavioural genes that are often polygenic.
(2) The Tre+ locus was mapped cytologically between 5A8-5B3 according
to the salivary chromosome bands.
(3) The sensitivity to trehalose measured by the ratio between the numberof flies ingested trehalose and that of those ingested sucrose showed a strong
correlation with the gene dose of Tre+. The order of sensitivity goes Tre/
0= Tre/Tre< Tre+ /Tre=cTre+ /0< Tre+ /Y=cTre+ /Tre+ < Tre+ /Tre+ /Tre+.
Here 0 means a deficiency for the gene. The number of wild type alleles
induces higher sensitivity proportional to the gene dose in the female. In
the male, on the other hand, a single gene dose of Tre+ showed the same sensitivity to the female homozygous for Tre+, but no hypersensitivity wasobserved in the male carrying an extra Tre+ as a duplication.
That the intensity of a certain sensitivity leading to an animal behaviour
can be modified by gene dose has not been reported earlier. The phenomenon as above clearly indicate that the gene Tre+ produces a protein underquantitative control through dosage compensation mechanisms and theamount of the gene product is a determinant for sugar sensitivity and feeding
behaviour.
The Effects of the Variatimll ilm Heterochromatin Content mll
Recombination Frequencies in Drosophila melanogaster
Masatoshi YAMAMOTO

Heterochromatin has long been an enigma when it comes to the natureof its function. Heterochromatin is a commonly found nuclear entity inalmost all eukaryotic organisms. An interesting common finding amongvast variety of organisms is "variation". The most of eukaryotes so far
studied showed extensive variation not only in the amount of heterochromatin but also in the nucleotide sequences of heterochromatic DNA (satelliteDNA) between species as well as within a species. Cytologically too, thevariation of constitutive heterochromatin was observed more prominently.
The positions where heterochromatic blocks existed in a genome were sovariable in both between and within species. Constitutive heterochromatin
can be found at centromeric, telomeric, interstitial regions or as free heterochromatic chromosomes, or every possible combinations of the above.
Recently it has been demonstrated that the alteration of the centromeric

RESEARCHES CARRIED OUT IN 1981

81

heterochromatin content in D. melanogaster females causes decrease or increase in the amount of recombination intra- as well as inter-chromosomally.
The degree of alteration in recombination frequency was strongly correlatedwith the changes of centromeric heterochromatin content. The more wasthe centromeric heterochromatin of the X deleted, the less occured therecombination on the X. On the contrary, the frequency of cross-overs on
the 3rd chromosomes was higher if the X chromosome had larger deficiencies
in the heterochromatin. Similar heterochromatic effect on chiasmata has
been argued to be a general function of heterochromatin based on mainly
cytological studies of meiosis in grasshoppers and several plants.
Many observations obtained from a variety of eukaryotes can strongly
suggest a general rule or function of heterochromatin, but no more than that.
They have totally lacking experimental tests for the speculations. The mosteukaryotic organisms but Drosophila melanogaster are trojJblesome tohandle for genetic studies and hardly meets this demand. D. melanogasteris only an organism we can use to study extensively the relationship between
the variation of heterochromatin content at different chromosomal positions
and the changes in cross-over frequencies. This is so we have constructed
every class of heterochromatic chromosomes.
Interstitially heterochromatic chromosomes are a novel chromosomal
rearrangement in D. melanogaster. This rearrangement was constructedfrom males of T(X; Y)B146 of which chromosomal breakpoints are 12F9
on the X and on the long arm of the Y. The males were irradiated with
3600R of X-rays and then allowed to mate with C(l)DX, y'v';j females. F 1
males were checked for phenotypic characteristic of yellow. The yellow
males were likely to have a fragment of heterochromatic Y chromosome at
12F9 of the X. The chromosomes were then cytologically examined andconfirmed that they were interstitially heterochromatic (Y origin) chromosomes.
Recombination on such interstitially heterochromatic chromosomes has
not been studied in any organisms, although the ones having heterochromatin
inserted were found in widely defferent species. In our experiment recombination was measured in the females either heterozygous or homozygous
for the experimental chromosomes whose genetic backgrounds were all thesame and the X chromosome euchromatin was replaced with the same origin.
Under such stringent conditions, it has been demonstrated that the inserted
heterochromatin has a drastic effect on cross-over frequencies. The females

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

heterozygous for an X chromosome containing comparatively large hetero~chromatin showed a big reduction in the amount of recombination, from58 % of control value to the mere 14 % as a total map distance. In a smallinterval the effect was more prominent as from 24 % to only 1 %. The levelof reduction in recombination frequency was clearly correlated with thesize of heterochromatin inserted. The females homozygous for the heterochromatically duplicated X, however, hardly showed any alteration eventhough the heterochromatic size was altered.

RESEARCHES CARRIED OUT IN 1981

VI.

83

MUTATION AND MUTAGENESIS IN ANIMALSThe Mutagenic Activity of N~Ethyl-N-Nitrosoureain Various
Germ-Line Cells of the Silkworm
Akio MURAKAMI

The mutagenic activity of N-ethyl-N-nitrosourea (END) has been welldemonstrated in many species including mice. In mice, this carcinogenic
chemical has been reported to be supramutagenic in spermatogonia (Russelland his co-workers, Proc. Natl. Acad. Sci. (USA), 76: 5818-5819 (1979),
but the result of mutagenicity tests in female germ-cells is not yet reported.
This gap may be attributed to some technical limitations : though it can beeasily obtained a large number of offspring from small numbers oftreated male mice, but not from the females. In the assessment of chemically-induced genetic hazards in man, it is an accepted practice to consider
spermatogonia in males and oocytes in females as the germ-cell stages mostat risk. In this respect, the silkworm specific-locus test method using eggcolour gene markers has an advantage to easily collect a large number of F loffspring from mating of the treated wild-type females as well as males withthe tester stock. In addition, there is possible to test the mutagenicity ofchemicals to whole germ-cells in gametogenesis for both sexes. The purposeof the present experiment is to find out whether or not ENU is mutagenic for
induction of the recessive visible mutations by the specific-locus method in
oocytes and post-spermatogonial germ-cells of the silk moth.
The silk moths used in this experiment were (C108 X Aojuku) F l hybrids
homozygous for the wild-type at two egg-colour gene loci marked in the test
stock. Freshly prepared END solution (0.1, 0.5, 1.0 and 10.0 /kg/mg bodyweight) in pH 6.0 were administered by the injection method into the bodycavity of the wild-type males at the mid-pupal stage to detect the mutagenic
activity of this chemical for both oocytes and spermatozoa. For the 4th- and5th-instar wild-type F l male larvae END solutions in two different doses in
84.9 and 170.0 /kg/mg body-weight were administered by the injection method
to detect the mutagenic activity in growing spennatocytes and the early phaseof meiotic spermatocytes, respectively. The mulberry leaves spread with
END solutions were given by the feeding method to the 2nd and 3rd instar

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

male larvae in doses of lOO and 200 ,ltg/larva. The corresponding germ-cellstages are the late spermatogonia and very early phase of spermatocytes,
respectively. In this experiment, actual fed doses were calculated to be
69.0 and 138.7 ,ltg/larva or 2.1 and 4.3 ,ltg/mg body-weight. Controls wereapplied with 0.85 % solution alone. After emergence, the treated silkworms
were mated with the tester stocks to detect for the egg-colour specific-locus
mutations at the two loci.
END treatments by the injection method produced significant increases
in mutation frequencies compared to controls for spermatocytes, meiotic
spermatocytes, spermatids in larvae and spermatozoa and oocytes in pupae.
A striking difference between spermatocytes and spermatozoa in the frequency of induced mutations was observed: spermatozoa were more sensitive
to END than spermatocytes, by a factor of 50-60. It is of inereest to notethat the mutagenic activity of END in spermatozoa was simalar to that in
oocytes. In the feeding experiments for young larvae, no significant increase of mutations was detected in the corresponding germ-cell stages,
spermatogonia and immature spermatocytes. This finding may indicatethat END is unstable in digestive juice and/or in body-fluid of the larvae.
It can not exclude a possibility, however, that the incorporated dose might
be not sufficient to induce the specific-locus mutations. Additional research
will be necessary to clarify the mechanism(s) involved. From this silk mothspecific-locus test of END, it is clear that this carcinogenic mutagen END
was very mutagenic in oocytes, spermatocytes, spermatids, and spermatozoa,
but not in spermatogonia contrary to the observation in mouse spermatogonia shown the most-potent mutagenicity among several mutagens
detected in mice (Russell et al., 1979). The mechanism(s) responsible forthis differential sensitivity in interspecies is not clear, but it is likely thatdifferential metabolic activity may be concerned in this difference.
DHfereJDlce in Mutabmty by the Different Administnatioll
Rmlltes of Chemical MUltageJnlS in the §i!rrworm
Akio MURAKAMI

The intensity of chemically-induced mutagenicity is markeldy influenced
by a number offactors. The metabolic activity in the body and in the target
cell seems to be one of important factors regardless of directly- or indirectlyacting chemicals. This factor would be mainly concerned in the administra-

RESEARCHES CARRIED OUT IN 1981

85

tion routes (and/or manners) of the chemical. From the standpoint of risk
evaluations ofthe possible hazards to man of many environmental chemicals
with mutagenic potential, it should take into consideration on the exposure
pattern to the chemicals.
Silkworm larvae on the day 1 of the 5th instar were used in this experimentbecause the stage larvae are suitable for the administration of chemicals byeither the feeding method or the injection method. Accordingly, it is possible
to compare the mutagenic activity of the chemical mutagens to the germ-cellsof the last instar larvae treated by both methods. In the male, the corresponding germ-cell stage is the early phase of meiotic spermatocytes. In thisexperiment, Mitomycin C (Me) was chosen for the model chemical agent.
This antibiotic agent is an indirectly-acting mutagen which required metabolic activation to mutagenic derivatives and that is a strong mutagen
followed by Aflatoxin B1 in pupal oocytes and spermatozoa of the silkworm.
The egg-colour specific-locus test was used as the method for detecting andquantifying the induction of recessive visible mutations. Freshly prepared
MC in 0.85 % NaCl solution was administered by the injection method into
the body-cavity of the 5th instar wild-type (C108 X Aojuku) F 1 hybrid malelarvae in doses of 5, 10, and 20,ug/larva corresponding to 7.9, 15.7, and31.5 X 10- 3 ,ug/mg body-weight. The mulberry leaves with the MC solution
were applied by the feeding method to the larvae in nominal applied doses of
20,40, and 60,ug/larva or in effective fed doses of 2.13,4.15, and 6.28x
1O- 2 ,ug/mg body-weight, respectively. Control larvae were treated with
0.85 % NaCl solution alone.
The results demonstrated that the incidence of specific-locus mutationsin the spermatocytes of the last instar larvae was induced with the administration of MC by both injection and feeding methods. The shape of
dose-response curves obtained by either parenternally or perorally administered routes was a linear fashion, but the slope was remarkably different
from each other. The mutagenic activity of MC when given parenternally
was about 130 times as active as when given perorally. This weak mutagenicity of MC by the feeding method may be the result of a detoxic activity
for the antibiotic agent in digestive organs and/or in hemolymph. Fromthese findings, it can be confirmed that the mutagenic activity of the chemical
agent may be remarkably under the influence of the manner of administrations.
By the way, growing spermatocytes in the 4th instar larvae were more

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

sensitive to MC than meiotic spermatocytes in the last instar larvae. However, MC-induced mutational events in pupal spermatozoa were slightly but
significantly less frequency than those in the spermatocytes. The present
finding seems to be different from the well-accepted general view that postmeiotic germ-cells are more mutagenic to alkylating agents including MC
than pre-meiotic ones. A similar observation has been reported in micethat post-spermatogonial stages are less sensitive to MC than spermatotonial stages [Russell and his co-workers, Mut. Res. 86: 329-354 (1980)J.
In the silkworm, contrary to those observations, Ethylmethanesulphonate
was quite active for the induction of the specific-locus mutations in spermatozoa, but did not induce a high incidence of the mutational events inspermatocytes. Such being the case, further studies with a variety of alkylating agents are necessary to draw the final conclusioll for this topics.
Relationship between Chemical-Dose and Specific-Locus
Mutations by a Mycotoxin Aflatoxin HI in Pupal
Oocytes of the Silkworm
Akio MURAKAMI

To evaluate the genetic risk with chemical as well as physical agents theinvestigation of the dose-response curve is of dominant importance. The
major object of the analysis of dose-response kinetics is to answer the point
at issue whether or not the threshold dose for the induction of mutations isexist. For this purpose, clear dose-response curves should be obtained in therange of low dose levels. It is well known by our experiences, however, thatthe frequency of mutations induced with low dose levels tends to be coveredby the spontaneous mutation frequency. Consequently, the sample size inan experiment point at low-dose levels has to be extremely large to establish
significant differences between induced and spontaneous mutation frequencies. From this point of view, we considered that the silkworm specificlocus mutation test system using the egg-colour loci seems to be content withsuch the requirement: it can easily handle a large number of F 1 offspring andrapidly score the mutational incidence.
The present communication deals with the experimental results on theeffect of low-dose levels, 0.01, 0.5, and 0.1 ,ug/capita on specific-locus mutations inducing from Aflatoxin BI (AFTB I ) applied to the oocytes of midstage pupal females which have been previously been demonstrated to be the

87

RESEARCHES CARRIED OUT IN 1981

most potent mutagen in the silkworm and shown to be a linear dose-response
kinetics at relatively high dose ranges between 0.1 and 5.0 ,ug/capita. In theincorporation experiment with [3H] AFTBr, it could be estimated that an
oocyte (or ovium) took in about 0.037 %of the total injected dose of AFTB 1
per female pupa: 0.01, 0.5 and 0.1 ,ug/pupa were corresponded to ca. 3.7,
37.0 and 370 pg/oocyte, respectively. (CI08 X Aojuku) F 1 wild-type females
at the mid-stage pupa were injected with a single dose of the three different
doses of AFTB 1 dissolved in 0.85 % NaCl solution. After emergence they
were then mated with untreated marker strain male moths homozygous for
egg-colour genes, pe and re and the offspring were submitted to score therecessive visible mutations at the two loci, pe+ and re+. For each dosepoint, 500,000 or more of F 1 offspring were used to estimate the frequencyof mutations.
The dose-response curves between 0.01 and 5,ug/pupa of AFTB 1 wereshown in Figure 1 based on the present finding in the low-dose range together
with the previous one in the relatively high-dose range from 0.1 to 5.0 ,ug/

~

1000

/@/
/t/

i

III

J

...o

~
~ 100

X

::l

i

....~

O

c::

Cl)

@/

pe

l~OCUS@l _@l"el0cUS

co""'-----@-@

c::

o

+:;

/~

....<0

~

10

-@

0.01

0.1

5

Dose in 1l9!pupa

Fig. 1. The dose-response curves for mutagenicity of Aflatoxin Hl in silkworm
oocytes. Vertical lines indicate the 95 % confidence intervals for each mutation
frequency.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

pupa. As can be seen from the figure, the fashion of a dose-response curveat the pe+ locus is observed no difference between the induced mutation
frequency obtained with a moderate dose at 0.1 ,ug/pupa and the spontaneous
mutation frequency within the 95 % confidence intervals. Whereas at there+ locus, the dose-response curve goes linearly down to by a dose at 0.01 ,ug/
pupa or 3.7 pg/oocyte and the frequency of mutations induced with this dose
is significantly above the control level. This finding indicates that a threshold dose will be at a dose less than 0.01 ,ug/pupa. This difference in sensitivity between the loci will be responsible for the frequency of spontaneous
mutations for both loci: the spontaneous mutation frequency at the pe+locus was about one order as high as the re+ locus.
Specific-Locus Mutatimns Induced by .a-Emitter [MethyPH]
Thymidine in Primordial Germ-Cells of Dormant
Silkworm Embryos
Akio MURAKAMI

Environmental contamination from internal p-emitters arises in various
nuclear operations, such as fuel processing, nuclear power stations, nuclear
fusion devices, et cetera. Consequently, there is increasing concerns overthe possible mutagnicity as well as other biological effects of these internal
radioemitters. In general, human exposure to p-radiation derived fromvarious internal radiation sources in which were naturally or accidentally
accumulated in the body or in the tissues will be low dose and low dose-rate.
In this connection, silk moth (Bombyx mori) may have a biological advantage
for detecting some genetical effects in germ-cells by incorporated radioactive
substances at the above-mentioned exposure condition. The embryos ofsilkworm eggs usually take diapause in the early phase of embryogenesis forthe maximum over one year. The duration of diapause state can be regulated
easily by the control of incubation temperatures for the dormant eggs. The
gonad in this embryonic stage consist of primordial germ-cells and thenumber of the cell is known to be at least 20 per embryo. In the silkworm,
the mid-stage female pupae are suitable for the administration of radioisotopes because of the oocytes in this pupal stage can actively take invarious nutritional materials as well as radioisotopes from hemolymph forvitellogenesis. In this investigation, [Methyl-3H] thymidine was used asa model radionucleide for the internal p-emitters.

RESEARCHES CARRIED OUT IN 198189

Wild-type C108 strain mid-stage female pupae treated with one of DNA
precursors of 3H-thymidine of 25 ,uCijcapita were mated to an another wildtype Aojuku strain males to allow deposition of the radioactive eggs. The
duration for diapause period of the eggs was 263 days from Oct. 10th 1980 toJuly 1st 1981. After hatching, offspring were raised and then mated to thecorresponding an opposite sex of marker stock moths homozygous at the two
egg-colour genes (pe and re) scored for the recessive visible mutations.
Five eggs sampled randomly from each batch were determined radioactivities(counts per minute) by the liquid scintillation counter. An average radioactivity per egg for a given batch was calculated to be ranging from 2500 to
3800 cpm. Each egg used in this experiment was about 0.45 mg in weight.
As has been anticipated from a nature of the test system adopted in thisexperiment, a number of mutant clusters with a large size e.g., 10 to 20
mutants per batch (or 400 to 500 F i offspring) were often detected, indicatingthat an accurate determination of p-ray-induced mutation frequencies isdifficult as the result of the marked cluster effect. However, data obtained
clearly indicated that p-ray from [3H] thymidine deposited in the dormant
egg were highly mutagenic to embryonic primordial germ-cells. For (3ray-induced mutational events in these germ-cell stages, males were 2-3 times
as high sensitive compared with females and pe locus was more sensitive thanre locus. These finding are quite the same to external radiations, r- or Xrays. The incidnece of mutations was markedly increased up to 100-300 X
10- 5 per locus when the embryos which would have more actively incorporated the isotopes were used. This suggests that mutational incidence induced with p-rays from [3H] thymidine seemed to be proportional to the isotope doses deposited in the egg. The incidence of mutations in the highly
radioactive group with ca. 3800 cpmjegg produced approximately the same
frequency in dormant eggs irradiated with around 2500 R (or more) of rrays at a dose-rate of 300 Rjmin. The dominant lethal test by the reductionof egg-hatchability revealed, however, that in the 25 ,uCijpupa of PH] thymidine treated groups the embryonic lethality was not significantly affected,
whereas in 2500 R of r-rayed dormant eggs the lethality was remarkable.
This observation indicates that p-ray-induced cell-killing effect was clearly
different from r-rays. The differential sensitivity in genetic end-points may
be due to the dose-rate effects of p- and r-rays on the germ-cells in thedormant stage eggs. Not yet determined what quantities of [3H] thymidine
were actually deposited in the nucleus of the germ-cells. In a prelimianry

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

experiment, however, tritium water (THO), which is not a precursor of DNA,
induced almost the same mutational incidence in an equivalent dose level ofPH] thymidine. This finding and the others suggest that [3H] thymidine
might be predominantly deposited in the cytoplasm rather than in the nucleusof the germ-cells.
The experiment in this series is not yet completed, but it is likely to saythat the method described here is a relevant test system to evaluate thegenetic risk from the internal p-emitters at the low dose-rate and at the lowdose level.

RESEARCHES CARRIED OUT IN 1981

VII.

91

RADIAnON GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Enzymatic Comparison of DNA Damages Im:luced by Beta-Raysfrom Tritiated Water and by Gamma-Rays
Tadashi INOUE and Tsuneo KADA

Quantitation of the biological effects of tritium is of practical interest
because this nuclide will be released into environment when nuclear fission
reactor is peacefully developed in near future. Reactor-generated tritium
enters the biosphere as tritiated water and the main concern for purposes ofradiation safety is the effectiveness of the beta particle produced when it
decays.
In order to assess the effectiveness, we compared the nature of the DNA
lesions produced by internal beta-irradiation by tritiated water with those
produced by external irradiation by 137Cs-gamma-rays using several enzymes
specific for DNA lesions. First of all, we examined effectiveness to introduce
single strand scissions in covalently closed circular DNA of colicin EI.
From the results of anlyses by neutral sucrose density gradient centrifugation, the RBE of tritium beta particles relative to the gamma-rays was estimed to be approximately 3. Terminals of DNA strand scissions inducedby the irradiation were analyzed with two kinds of exonuclease, namely
venom phosphodiesterase and spleen phosphodiesterase. The former
enzyme initiates hydrolysis of oligonucleotides at a 3'-OH terminal andyields 5'-P mononucleotides, while the latter enzyme requires 3'-P terminal
yielding 3'-P mononucleotides. When the DNA with avarage 25 single
strand scissions per molecule was subjected to hydrolysis by venom phosphodiesterase, no difference was detected between two types of radiation
which create the strand scissions in substrate DNA, suggesting that bothtypes of radiation produce 3'-OH terminals in comparable efficiency. On
the other hand, neither type of radiation produced lesion susceptible tospleen phosphodiesterase, indicating that very few number of 3'-P terminals
were produced by both types of radiation.
In addition to strand sessions, ionizing radiation introduces apurinic sites(AP-sites) in DNA. Number of AP-sites in irradiated DNA was measured

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

by using AP-endonuclease of B. subtilis. The experiments yielded theresults that while gamma-irradiation introduced 0.8 AP-site per one strand
scission, only 0.3 AP-site per one strand scission was made by beta-irradiation.
These data suggest that lesions produced by beta-irradiation are bothquantitatively and qualitatively different from those produced by gammairradiation.
Mechanisms of Environmental Antimutagenesis
Tsuneo KADAIn these several years, our knowledges on environmental mutagens
increased greatly. We know that mutagens showing potent activities
in vitro are often modulated in the body of mammals, indicatingpresence of in vivo factors antagonizing with the mutagens. We defined
desmutagens as agents inactivating directly mutagens in vitro (T. Kada, 1982,
Proc. 3rd Intern. Conf. Env. Mutagen). On the other hand, we call antimutagens those agents working as antagonists in cellular mutagenesis in vivo.
Systematic trials have been made to detect antimutagens in our environment.
For this purpose, indicator bacteria were first mutagenicaed by one of chemical mutagens or radiations and exposed to test agents for several generations.
Those agents reducing remarkably induced mutabilities without affecting
considerably the cellular lethality were studied further in details.
Certain antimutagens are working probably by reducing errors involved
in SOS repair of DNA damage induced by UV or 4NQO in E. coli strains.
They include cobaltous chloride (Kada, Proc. 6th Int. Congr. Rad. Res,
1979), cinamldehyde (Ohta et at., Mutation Res., in press), enmein (Kakinuma et at., Ann. Meeting Abstr. Agr. Chern. Soc. 3B18, 1982) or protoanemonin (Komura et at., Mutation Res., in press). Such a mechanism may
explain part of antimutagenic actions of the human placenta factor (Kadaand Mochizuki, J. Rad. Res. 22: 297, 1981; Mochizuki and Kada, Mutatoin
Res. 95, 457, 1982). On the other hand, some of other antimutagens areworking in mutageneis induced by ionizing radiation, MNNG or Trp-P-l.
They inculde cobaltous chloride (Kada and Kanematsu, Proc. Japan Acad.
54B, 234, 1978, Mochizuki and Kada, Mutation Res. 95, 145, 1982) andcarboxy-ethylgermanium sesquioxide (Mochizuki and Kada, Int. J. Radiat.
BioI. in press). Studies are under way to elucidate mechanisms involved

RESEARCHES CARRIED OUT IN 198193

in them.
The Effect of Septum-Initiation Mutations on Sporulation
and Competent Cell Formation in Bacillus subtilis
Yoshito SADAIE and Tsuneo KADA

Sporulation of many gram-positive bacilli begins with the formation of anasymmetric forespore septum upon starvation for nutrients, followed bydifferential expression of genes on the chromosomes segregated into two cytoplasms, indicating the most simple system of differentiation. As theprocess of forespore septum formation may share common steps withvegetative cell division (Hitchin and Slepecky 1969), the elucidation offorespore septum formation in relation to vegetative division is required to
study how nutritional starvation leads cells to divide asymmetrically. Bacillus
subtilis is genetically well characterized and offers the best organism to study
microbial differentiation. We studied the effect of septum-initiation mutations on sporulation with four Bacillus subtilis mutants carrying septuminitiation mutations of different loci, div-3I , div-34I , div-12, and div-355
which showed filamentous growth at 45°C (Miyakawa and Komano 1981).
Cells were grown to T 1 stage before forespore septum formation at 30°C
and transferred to 37°C or 42°C. These four strains sporulated well at
permissive temperature (30°C) and showed poor sporulation at higher temperatures (37°C and/or 42°C). Thus the initial steps of septation mediatedby div-3I+, div-34I+, div-12+, and div-355+ allels are required for sporulation, although we do not know whether they are required for forespore
septum formation or for some later processes in sporulation.
On the other hand, it was suggested that common steps were involve bothin competent cell formation and in forespore septum formation (Sadaie andKada 1982, IV Genetics of Industrial Microorganisms, Kyoto). Therefore,
it is possible that vegetative septation is involved in competent cell formation
as well. A competent culture was induced by diluting early stationary phaseculture grown at 30°C with fresh medium and incubating for 90 min at 37°C
and 40°C. The strain carrying the div-3I mutation lost competence at any
temperatures examined while the div-34I and div-355 strains showed lesscompetence at higher temperatures (37°C and/or 40°C). Vegetative growthof these strains was not affected at these temperatures. The mutation div-12
made cells slightly poor for competence at any temperatures examined.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

Therefore, the mutations div-31, div-341, and div-355 may affect forespore
septum formation because these mutations affected competent cell formation
which seemed to share common steps with forespore septum formation.
The div-12 mutation may affect sporulation in the stage later than forespore
septum formation.
Div mutations employed in the present study affected competent cell formation and sporulation at intermidiate temperatures where vegetative growth
was not affected. This fact suggests a qualitative difference which discriminates vegetative septation from asymmetric sporulation specific septation
and a "division" in competent cell formation. (1. Bacteriol., Mol. gen.
Genet. in press)
Purificatiolll lUld Characterization of a Humlm Placental
Factor which E.IllnmllCeS the PriJ:niJ!llg Activity()If r-Irradiated DNA
Tadashi INOUE and Tsuneo KADA

A human placental factor which specifically enhances the priming activity
or r-irradiated DNA was purified near homogeniety by successive application of the extracts to r-irradiated DNA cellulose, Sephacryl S200, andPhenyl-Sepharose column chromatographies. The factor was protein innature and had a molecular weight of arround 48,500. In addition to ther-irradiated DNA, the priming activity of neocartinostatin-treated DNA
was also enhanced by the factor. Niether exonuclease nor endonucleaseactivity was detected toward intact DNA while r-irradiated DNA was
rendered acid-soluble to some extent. These results suggest that the factor
is a r-exonuclease acting on damages in DNA treated with r-rays or certain
chemicals to make the DNA suitable primer for repair synthesis by DNA
polymerase.

Tsuneo KADA, Hajime MOCHIZUKI, and Aleio YOKOlYAMA

Damage induced by gamma-ray-irradiation from a 137CS source in DNA
of Bacillus subtilis was repaired to certain extent by in vitro treatment withthe highly diluted extract obtained from human placenta tissues, as estimatedfrom the increase in the transforming activity. This repair activity was

RESEARCHES CARRIED OUT IN 198195

sensitive to heating (lOO°e) or to treatment with EDTA (5 mM), suggestingsome enzymes involved (Mochizuki and Kada, in preparation). Nakatsugawa and Sugahara (Rad. Res. 84, 265, 1980) showed that cordycepin inhibits
potentially lethal damage (PLR) of X-irradiated Chinese hamster hai cells
and can be used as a radiosensitizing agent. That cordycepin is really acting
as a repair inhibitior was shown using our in vitro reapir system where
gamma-ray (10 kr) exposed transforming DNA of Bacillus subtilis was incubated with extracts of human placenta and the effects of cordycepin wereobserved. The placental extracts remarkably recovered the transforming
capacity. Such a recovery was gradually lost by presence of the radiosensitizer of increasing concentrations. This system may offer a good one forscreening radiosensitizers working in human (Kada et at., in preparation).
Cordycepin was confirmed to be a real inhibitor working in the PLD
repair. Results of radiosensitizing experiments using the DNA-repair inhibitors in tumor-bearing animals are so far promising and further developments in this field are expected.
Mutagenic Effect of Ekatin (tlhiometon) in SoybeaJl1l. Test System
Taro FUJII and Tadashi INOUE

Agricultural chemicals which are used commonly in our farming wereapplied to soybean test system. No increase of spotting frequency wasobserved with five chemicals (Antio, Baycid, Diazinon, EPN and Karphos)
indicating that they may not have any mutagenic effects, at least at the concentrations examined (500-2000 times dilution). However, increase of mutational spots were observed by Ekatin treatment even at 2000 times dilution
which is the usual concentration in practical use for pest control. Ekatin
is a mixture of 25 % thiometon and 75 % solvent; the former is the essential
component of this pesticide. Because Ekatin showed some mutagenic
activity, effects of thiometon were examined with the same treatment procedure. An increase of mutational spots per leaf was evidently observed at1000 and 500 times dilutions. From these results, it is apparent that thiometon, essence of Ekatin, has a mutagenic activity.
The activation of chemicals into mutagens by higher plants is a recently
discovered hazard. Chemicals that are themselves not mutagenic but got
convereted into mutagens through plants metabolism have been noticed inseveral plant materials such as maize and sybean. Since Ekatin and its

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

essence, thiometon, exhibited an unequivocal mutagenicity in the soybean
test system, we examined the mutagenicity of this sampel on Salmonella
TA98 and TAlOO. Thiometon has no mutagenic activity toward Salmonellain the absence or presence of rat liver S9 fraction. We also examined whether
thiometon was activated by the soybean drug metabolizing system. However, the 59 fraction obtained from the soybean seedlings did not activate
the pesticide. Furthermore, we could not detect any mutagenicity of thematerial extracted from the soybean seeds which had been incubated withthiometon for 24 hr. These results suggest that the mutagenic activityof Ekatin and thiometon is specific for plant cells.
Use of pesticide is a common procedure for crop production in modern
farming. Therefore, if pesticides possess mutagenicity, even though theapparent hazard is minor, retention and accumulation of induced damage
may eventually have detrimental effect upon the natural ecosystem. Additionally, residues or derivatives of these substances in the edible plants may
affect human health. Systematic mutagenicity studies of agricultural chemicals using the plant systems are, therfeore, necessary.

RESEARCHES CARRIED OUT IN 1981

VIII.

97

POPULATION GENETICS (THEORETICAL)Was Globin Evolution Very Rapid in Its Early Stages?:
A Dubious Case Against the Rate-Constancy Hypothesis
Motoo KIMURA

Goodman et al. 's (1975) claim of accelerated evolution in the early stages ofglobin evolution (see Nature 253, 603-608) is based on an erroneous assignment of the time of divergence of vertebrate myoglobin and hemoglobin.
When this is corrected, there is no basis for their claim. The data are much
more consistent with the nearly constant rate expected on the neutral mutation-random drift hypothesis than with the uneven rates expected if mostamino acid changes were caused by substitution offavorable mutants throughDarwinian selection. In addition, the majority of the codons determined bytheir maximum parsimony method have turned out to be wrong whencompared to the actual nucleotide sequences of rabbit Q' and human phemoglobins determined by direct sequencing. For details, see J. Mol. Evol.
17, 110-113.
Possibility of Extensive Neutral Evolution under Stabilizing
Selection with Special Reference to Nonrandom Usageof Synonymous Codons
Motoa KIMURA

The rate of evolution in terms of the number of mutant substitutions in a
finite population is investigated assuming a quantitative character subject
to stabilizing selection, which is known to be the most prevalent type ofnatural selection. It is shown that, if a large number of segregating loci(or 'sites) are involved, the average selection coefficient per mutant understabilizing selection may be exceedingly small. These mutants are veryslightly deleterious but nearly neutral, so that mutant substitutions aremainly controlled by random drift, although the rate of evolution may belower as compared with the situation in which all the mutations are strictly
neutral. This is treated quantitatively by using the diffusion equation
method in population genetics. A model of random drift under stabilizing

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

selection is then applied to the problem of "nonrandom" or unequal usageof synonymous codons, and it is shown that such nonrandomness canreadily be understood within the framework of the neutral mutation-random
drift hypothesis (the neutral theory, for short) of molecular evolution. For
details, see Proc. Nat!. A cad. Sci. USA 78, 5773-5777.
Estimation of Evolutionary Distances between Homologous
Nucleotide Sequences
Motoo KIMURA

By using two models of evolutionary base substitutions-"three-substitution-type" and "two-frequency-class" models-some formulae arederived which permit a simple estimation of the evolutionary distances (andalso the evolutionary rates when the divergence times are known) throughcomparative studies of DNA (and RNA) sequences. These formulae areapplied to estimate the base substitution rates at the first, second, and third
positions of codons in genes for presomatotropins, preproinsulins, and (tand p-globins (using comparisons involving mammals). Also, formulaefor estimating the synonymous component (at the third codon position)and the standard errors are obtained. It is pointed out that the rates ofsynonymous base substitutions not only are very high but also are roughly
equal to each other between genes even when amino acid-altering substitution rates are quite different and that this is consistent with the neutral
theory of molecular evolution. For details, see Proc. Nat!. A cad. Sci. USA
78, 454-458.
Some CalkuJiatio1i15 OID! the AmolillJl1lt of Se!1fiJ§1!1 DNA
Tornoko OHTA and Motoo KIMURA

A population genetical theory is developed to treat the amount of selfish
DNA in a genome. We assume that the selfish DNA consists of replicating
units and that it evolves by multiplication within a genome, exchange
between genomes, and random genetic drift at reproduction. Special reference is made to the mean and variance of the number of replicating units
per genome in the population. Under the assumption of no systematic
evolutionary pressure, the number of units changes randomly with time, andits variance increases by replication process. Although under certain cir-RESEARCHES CARRIED OUT IN 198199

cumstances the variance increases also by exchange process, under ordinary
circumstances this process tends to decrease the variance. Random genetic
drift also reduces the variance. The relationship between the mean andvariance at equilibrium of the number of replicating units per genome in the
population was derived. The analysis is extended to derive equations andsimulate distributions to show how the numbers of tandem and dispersed
repeat units of selfish DNA in a population change with time. It was foundthat whereas the mean number of clustered and dispersed units remains
constant, the distribution of the population mean becomes increasingly dispersed with time. For details, see Proc. Natl. A cad. Sci. USA 78, 11291132 and Nature 292, 648-649.
Two-Locus Problems in Transmission Genetics ofMitochondria and ChlOlroplasts
Tomoko OHTA

A problem in transmission genetics was studied theoretically by regarding
a cell as a population of organelle chromosomes. A hybrid cell of the twostrains is assumed to consist initially of equal numbers of the two types of
nuc1eoids, each containing an equal number of chromosomes. The twoparent strains have different alleles at the two loci, and the two-locus dynamics of the hybrid cell line was analyzed. Within each nucleoid, stochastic matching and gene conversion are assumed to occur between the chromosomes, and at a certain rate, fusion and division are assumed to take place
between the uncleoids within the cell. Under this model, the changes of
average genotype frequencies at the two loci within a nucleoid and within acell were formulated, and the final frequencies of recombinant chromosomes
were obtained. Random segregation of nucIeoids at cell division was incorporated into the analyses; in addition, the increase of homozygosity inthe cell was formulated. The results may be useful for interpreting the observed data on organelle genetics of some species of algae and yeast. For
details, see Genetics
543-555,
Genetic Variation lin Sman Muhligene Families
TOl11oko OHTA

In order to understand the evolution of genetic systems in which two genes

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

are tandemly repeated (small multigene family) such as has been recently
found in the haemoglobin IX loci of primates, haemoglobin (3 loci of mOUse
and rabbit and other proteins, a population genetics approach was used.
Special reference was made to the probability of gene identity (identity coe.
fficient), when unequal crossing-over is continuously occurring as well as
random genetic drift, inter-chromosomal recombination and mutation.
Two models were studied, cycle and selection models. The former assumes
that unequal crossing-over occurs in cycles of duplication and deletion, andthat the equilibrium identity coefficients were obtained. The latter is basedon more realistic biological phenomena, and in this model it is assumed
that natural selection is responsible for eliminating chromosomes withextra or deficient gene dose. Unequal crossing-over, inter-chromosomal
recombination and natural selection lead to a duplication-deletion balance,
which can then be treated as though it were a cycle model. The basic
parameter is the rate of duplication-deletion which is shown to be approximately equal to 2(u+2(3)X, where u is the unequal crossing-over rate, 2(3is the inter-chromosomal recombination rate and X is the frequency of chromosomes with three genes or of that with one gene. Genetic variation of
the globin gene family, of which gene organization is known in most detail,is discussed in the light of the present analyses. For details, see Genet.
Res. 37, 133-149.
Genetic Variability and Rate of Gene Substitution
in a Finite Population under Mutation
and Fluctuating Selection
Naoyuki TAKAHATA

By using numerical method of solving stochastic difference equations,the level of gnetic variability maintained in a finite population and the rateof gene substitution under several models of fluctuating selection intensities
were studied. It is shown that mutation and random genetic drift bothplay an important role in determining genetic variability and the rate of genesubstitution. Compared with the case of neutral mutations, the fluctuationof selection intensity caused by temporal and spatial heterogeneity of environments generally increases the rate of gene substitution, but the level ofgenetic variability may be increased or decreased, depending upon the modeland the parameters used. Although such a type of selection per se can not

RESEARCHES CARRIED OUT IN 1981101

be ruled out, when mutation is taken into account, it is difficult to explain
both the observed amount of genetic variability and the rough constancy of
evolutionary rate within a framework of fluctuating selection models. For
details, see Genetics 98, 427-440.
A Model of Evolutionary Base Substitutions and
Its Application with Special Reference to
Rapid Change of Pseudogenes
Naoyuki TAKAHATA and Motoo KIMURA

A model of evolutionary base substitutions that can incorporate different
substitutional rates between the four bases and that takes into account unequal composition of bases in DNA sequences is proposed. Using thismodel, we derived formulae that enable us to estimate the evolutionary
distances in terms of the number of nucleotide substitutions through comparative studies of nucleotide sequences. In order to check the validity ofvarious formulae, Monte Carlo experiments were performed. These formulae were applied to analyze data on DNA sequences from diverse organisms. Particular attention was paid to problems concerning a globin
pseudogene in the mouse and the time of its origin through duplication.
We obtained a result suggesting that the evolutionary rates of substitution in
the first and second codon positions of the pseudogene were roughly 10 times
faster than those in the normal globin genes; whereas, the rate in the third
position remained almost unchanged. Application of our formulae tohistone genes H2B and H3 of the sea urchin showed that, in each of thesegenes, the rate in the third codon position is tremendously higher than thatin the second position. All of these observations can easily and consistently be interpreted by the neutral theory of molecular evolution. For details,
see Genetics 98, 641-657.
A Mathematical Model of Extranuclear Genes and the Genetic
Variability Maintained in a Finite Population
Naoyuki TAKAHATA and Takeo MARUYAMA

A mathematical theory of population genetics accounting for the genes
transmitted through mitochondria or chloroplasts has been studied. In themodel it is assumed that a population consists of N m males and Nt females,

102

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

the genetic contribution from a male is p and that from a female 1- p, andeach cell line contains n effective copies of a gene in its cytoplasm. Assuming
selective neutrality and an infinite alleles model, it is shown that the sum(H) of squares of allelic frequencies within an individual and the corresponding sum (Q) for the entire population are, at equilibrium, given by
~

H~{

1

(1-1jnY'
2NeVA}
1+p 1-(1-1jn).l 1+2Ne VA (1+2nv)

and
Q~fIj(1 +2NeVA)

where p=2p(1-p), N e={p2jNm +(1_p)2jNf }-1, A is the number of somatic
cell divisions in one generation, and v is the mutation rate per cell division.
If the genes are transmitted entirely through the female the formulae reduce
Nonequilibrium beto fI~1j(1+2nv) and Q~1j{1+(2NeA+2n)v}.
haviours of Hand Q are also studied in the case of a panmictic population.
These results are extended to geographically structured models, and applied
to existing experimental data. For details, see Genet. Res. 37, 291-302.
A Mathematical Study on the Distribution of the Number of
Repeated Genes per Chromosome
Naoyuki TAKAHATA

I develop a mathematical model which can account for a distribution of
the number of repeated genes per chromosome under the joint effects of sister
chromatid exchange (SCE), inter-chromosomal crossing-over (ICC), andselection. The model can be applied not only to the cases of small geneclusters but also to multigene families. Based on this model, an appropriate
mathematical formula is derived and used to obtain the equilibrium distribution. Assuming stabilizing selection and two simple schemes concerning
SCE and ICC, I numerically calculate the equilibrium distribution and compare the result with observations on frequencies of single and triple ahaemoglobin genes in primates. It is also shown that if SCE and ICC occur
according to the same probabilistic law, the distinction between them does
not make much sense in the equilibrium distribution. For details, see
Genet. Res. 38, 97-102.

RESEARCHES CARRIED OUT IN 1981

103

Algebra of Inclusive Fitness
Kenichi AOKI

Using the Cotterman k-coefficients in a one-locus two allele model, I showthat under inbreeding Hamilton's rule (br>c) for the increase of an altruistic
allele does not generally hold unless dominance is absent. If selection is
weak in a random mating population, Hamilton's rule holds for altruistic
interactions among any relatives of the same generation, not only for fullsibs or other particular relationships as has been shown by previous authors.
See Evolution 35, 659-663 for particulars.
Estimating Evolutionary Distance from Restriction Mapsof Mitochondrial DNA with Arbitrary G+C Content
Kenichi AOKI, Yoshio TATENO, and Naoyuki TAKAHATA

We develop a mathematical model for estimating evolutionary distancefrom restriction enzyme maps, which incorporates back and parallel mutation and non-uniformity of the rate of base substitution into the theory andallows for an arbitrary G+C content at equilibrium. When the G+C
content differs significantly from 1/2, the traditional model of base changes
can introduce a systematic bias even for phylogenetically close comparisons
which depends upon the base composition of the restriction site. In addition,
the accuracy of estimated evolutionary distance depends heavily upon the
choice of restriction enzyme in that the expected number of sites is also
affected. Monte Carlo experiments are conducted to check the validity ofthe present theoretical treatment and from which we draw several cautionary
notes on estimation. An application is made to the available data on
restriction enzyme maps of human mitochondrial DNA, and we suggestthat Brown's estimate of racial differentiation may be an overestimate.
See J. Mol. Evol. 18, 1-8 for particulars.
One- and Two-Locus Models of the Origin of Worker
Behavior in Hymenoptera
Kenichi AOKI and Michael MOODY

Hamilton suggested that, if females are singly inseminated, the asymmetries
in intra-familial relationships inherent in haplo-diploidy should predispose

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

the Hymenoptera to evolve female sex-limited worker behavior, if either aworker raises sibs as reproductives but raises a bias of sisters (pathway 1),
or she raises a combination of sibs and sons as reproductives but substitutes
sons for brothers preferentially (pathway 2). To investigate these hypotheses when mating is random, we present mathematical and numericalanalyses of the one-locus two-allele model of Charnov which we generalize.
Furthermore, we extend the Charnov model to a more realistic two-locus
situation where we postulate that the ability of a worker to raise a bias ofsisters or to substitute sons for brothers preferentially is controlled by alocus separate from the locus controlling worker behavior, rather than beinga pleiotropic effect of that locus. Considerations of the relative stringenciesof the conditions for substitution of the allele for worker behavior and theconstraints on linkage suggest that pathway 2 is more likely than pathway 1
for the origin of worker behavior. For pathway 1, we compare the Charnov
model with its diplo-diploid analogue. This comparison reveals that,
depending on the penetrance of the allele for worker behavior in the heterozygous state, both models permit the evolution of worker behavior even when
workers raise the ratio of sisters to brothers laid by the queens, and that anadvantage to haplo-diploidy becomes apparent only when workers raise abias of sisters. For pathway 1, we also consider a generalization of the
Craig model which is quite similar to the Charnov model. See J. Theor.
BioI. 89, 449-474 for particulars.

RESEARCHES CARRIED OUT IN 1981

IX,

105

POPULATiON GENETICS (EXPERIMENTAL) AND
EVOLUTIONARY GENETICS

Association of Chromosome- and Enzyme-Polymorphism inNatural Populations of Drosophila melanogastel!
Yutaka INOUE, Kendo TSUNO and Takao K. WATANABE

The second chromosome of this species carries a lot of variations innature. A chromosome inversion, In(2L)t, is frequently found as well as
the standard sequence chromosome, ST. Enzyme loci, Adh and a-Gpdh,
carry F and S alleles which are fairly frequent in nature. The frequencies of
In(2L)t, Adh s and a-Gpdh F were surveyed in three Japanese populations;Katsunuma (Yamanashi), Akayu (Yamagata), Ishigaki (Okinawa) as shownin Table 1. Frequencies of these variations were yearly stable in each population. However, they were different from population to population.
Adh and a-Gpdh loci are located in the left arm and two alleles (F, S) of these
two loci are in linkage equilibrium. But these allozymes show significantly
nonrandom association with In(2L)t. An almost complete linkage(97 ~98 %) of In(2L)t with Adh s and In(2L)t with a-Gpdh F have often been
confirmed by several natural populations.
The correlation coefficients between latitude and the frequencies of In(2L)t,
Adh s, ST-Adh s (=Standard chromosome with Adh S ), a-Gpdh F , ST-a-Gpdh F
were calculated by the data of Voelker et al. 1978, Knibb et al. 1981,
Oakeshott et al. 1982, and the present study. A significantly negative correlation was observed in the cases for In(2L)t and Adh s, but not for ST-Adh s .
On the other hand, it was significantly positive in the case for ST-a-Gpdh F ,but not for a-Gpdh F • These results suggest that the two allozyme themselves may respond to the natural selection of each latitudinal populationin different manner.

....
o
0\

I
t'"'
:;0

Table 1.

The frequencies of /n(2L)t, Adh s and a-Gpdh F in three Japanese natural populationsof Drosophila melanogaster. Sample size is given in parenthesis/n(2L)tAdh s

a-Gpdh F

Reference

1969
1970
1972
1976
1979
1980
1981

.236 (123).180 (233).125 (200).115 (200).134 (232).147 (238).160 (244).466 (1050).395 (263).421 (197).374 (240).341 (232).382 (238).361 (244).847 (396).833 (264).777 (197).749 (240).759 (232).761 (238).803 (244)Kojima, Gillespie and Tobari 1970
Langley, Tobari and Kojima 1974
Watanabe and Watanabe 1977
The present study
The present study
The present study
The present study

Akayu

1974
1977

.263 (300).225 (200).493 (215).407 (240).874 (215).916 (240)

Watanabe and Watanabe 1977
The present study

Ishigaki-Jima

1973
1974
1976

.531 ( 81).579 (252).545 (200).744 ( 78).784 (292).841 (240).859 ( 78).928 (292).893 (240)

Watanabe and Watanabe 1977
Watanabe and Watanabe 1977
The present study

Population
Katsunuma

~

o
~

o
"rj

~

o

~

~
~
c
tri

o
"rj

Cl
tIl

Z

tIl

::l

(')
(,f.l

z

9

'.>J

IV

RESEARCHES CARRIED OUT IN 1981

107

Typical Hybrid Dysgenesis is Absent from the Natural Populationof Drosophila melanogaster in Japan
Akihiko YAMAMOTO and Takao K. WATANABE

Hybrid dysgenesis was investigated in a natural population of Japanese
Drosophila melanogaster. Hybrid females with rudimentary ovaries emerge
from some interstrain crosses, when they are developed under high temperature, e. g., 29°C. These gonadal dysgenesis are observed in one of thetwo reciprocal crosses, in which paternal strains are called "P" strains andmaternal ones are referred to as "M" strains. Most strains from natural
populations in U. S. A. and Australia were judged P by Kidwell et al. (1977),
since males of them caused dysgenic daughters when they were mated withfemales from the M strain.
However, 46 isofemale strains from Katsunuma, Japan (strains kept in
our laboratory for 6 months at l8°C), did not cause dysgenesis both whenmales of them were mated with the M strain and females with the P strain.
Then, an of these strains were judged "Q" strains (Engels and Preston, 1981)
and the nature of the wild population at Katsunuma differred apparently
from the populations in U. S. A. and Australia.
Among 11 long-established laboratory strains (8 to 30 years) derived fromJapan, 6 were M strains and others were Q or possibly in the intermediate
state between Q and M. Therefore, Q strains discovered at Katsunuma willturn into M when they are kept in the laboratory.

Biochemical Phylogeny of tbe Drosophila mOlltium
Species Subgrm1p
Seido OHNISHI, Ki Won KIM and Takao K. WATANABEThe D. montium species subgroup to which 61 species belong is the biggest
subgroup in the D. melanogaster species group (Tsacas, 1979). Recently,
Baimai (1980) surveyed karyotypes of twenty species including new species
such as D. leontia and D. bocki,and found much variation in the fourth andY chromosomes, showing that most of the species can be distinguished by the
karyotypes. Although morphological characters (sexcombs and genitalia
of males) as well as karyotypes are useful tools for the qualitative identification of these species, quantitative relationships (e. g. genetic distances)
between these species have never been shown in this subgroup.

108

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

In this study, a phylogenic tree among 21 members (14 different species)of the subgroup was constructed, based on the data set obtanied by twodimensional electrophoresis (2DE). The results are as follows; The 21
members were classified into four groups. The first group is the D. kikkawai
complex to which six species belong (D. bocki, D. pennae, D. kikkawai,
D. leontia, D. lini and D. barbarae). The second one includes three species,
D. jambulina, D. punjabiensis-like and D. punjabiensis, whose systematics wasreported in Jap. J. Genet. (in press) in more details. The third one is theD. auraria complex including four species (D. auraria, D. biauraria, D.
triauraria and D. quadraria). The last one is D. rufa which is distantly related
to the other species. However, an analysis of starch gel electrophoresis
showed that D. rufa was more closely related to the D. kikkawai complex,
and that only D. jambulina was classified into a separate group. In the 2DE
analysis, it was found that D. quadraria was very similar to D. triauraria,
although these two species are different in geographical distribution. It
suggests that D. quadraria seems to be a variation of D. triauraria. In addition, an unclassified species which had been collected on Iriomote Island(Japan) in 1979 was found to be the same as D. bocki in the 2DE analysis.
This was confirmed by an interspecific hybridization between them. Thus,
it was evidenced that these electrophoretic techniques are useful as adjunctive
tools in the study of systematics.

RESEARCHES CARRIED OUT IN 1981

X.

109

HUMAN GENETICS

Retinoblastoma: Mutational Mosaicism Of
Host Resistance?
Ei MATSUNAGACarlson and Desnick's proposals (Am. J. Med. Genet. 4: 365-381, 1979)
of multiple allelism and mutational mosaicism to account for the observed
variability in penetrance and expressivity in retinoblastoma do not seem togive a clear-cut explanation for the facts that 1.) a bilaterally affected patient
could have an unaffected carrier child who in turn produced bilaterally affected offspring, and 2.) carriers of the retinoblastoma gene are prone to nonradiogenic osteosarcoma irrespective of their phenotypes. However, theseobservations are readily explained by assuming that inherited host resistanceplays an important role in the manifestation of a single major gene forretinoblastoma. Based on their model, Carlson and Desnick offer recurrence risk figures for genetic counseling, but their figures concerning thetransmissible types ofretinoblastoma do not seem more accurate, as assumed
by them, than those previously estimated from the family data.
We present evidence that host resistance may be regarded as a multifactorial threshold character with high heritability, and we propose thattissue-specific genes, which we presume to be involved in normal differentiation of retinoblasts and osteoblasts, playa crucial role in malignant transformation. Two lines of suggestions are made for future studies: one,
to search for polymorphic genes associated with retinoblastoma, and theother, to see whether the principle of almost synchronous appearance of multiple tumors in persons at high risk applies to other cancers as well. For
details, see Am. J. Med. Genet. 8: 375-387, 1981.
Genetics of Wilms' tumor
Ei MATSUNAGA

The analyses of published data for Wilms' tumor show that the genetics
and genesis of this tumor is essentially similar to that of retinoblastoma.
Main results obtained are as follows:110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

1) The mode of inheritance of familial cases, which constitute less than
1 % of all Wilms' tumors, is autosomal dominant with variable penetrance
and expressivity. The familial pattern reveals no evidence that this tumor
is associated with a vertically transmitting tumor virus.
2) There is a significant correlation between the first-degree relatives
affected with Wilms' tumor in regard to expressivity as measured by the
proportion of bilaterally affected or by the age of the patients at diagnosis.
In particular, expressivity in the children affected varies consistently withexpressivity in the carrier parents, indicating that inherited host resistanceplays an important role in the manifestation of the major gene. The averagerisk of developing Wilms' tumor for offspring of unaffected carriers, who
may be regarded as inherently resistant to tumor formation, is estimated atabout 0.30. Although the data are limited, the risk for offspring of survivorsof hereditary unilateral or bilateral cases may be as high as 0.40 or more.
The data from seven pairs of monozygotic twins, including the discordant
one associated with del (11 p) and aniridia, are consistent with the host resistance model.
3) In sporadic cases, the proportion of bilateral involvement is about
3 %, in contrast with 20 %in the familial cases. For bilateral cases, however,the distribution of ages of the patients at diagnosis is virtually the same
whether sporadic or familial, and in two-thirds of the cases the disease isalready bilateral when the first tumor is noted. These findings suggest thatbilateral cases are probably always hereditary. Ages of the patients at
diagnosis of sporadic unilateral cases tend to be much higher than in thepatients with familial unilateral cases, indicating that most sporadic unilateralcases are nonhereditary. The overall risk for survivors of sporadic unilateralWilms' tumor to transmit the disease to children may be as low as 2", 4 %.
For details, see Hum. Genet. 57: 231-246, 1981.
The "loss" of Kinetochores flWm Chromosomesof Aged Individuals
Yasuo NAKAGOME and Tatsuo ABE')

An increase in the proportion of aneuploid cells in aged women is wellknown (Jacobs, P. A. et al. Ann. Hum. Genet. 27: 353, 1964). A phenomenon called premature centromere division (peD) also increases with ad1)

Dept. of Internal Med., Kyoto Prefectural Univ. Kyoto.

RESEARCHES CARRIED OUT IN 1981

111

vancing age (Fitzgerald, P. H. et at. Ann. Hum. Genet. 38: 417, 1975). It isalso widely known that the frequency of meiotic non-disjunction increases
with advancing maternal age resulting in both autosomal trisomies and a
few numerical sex chromosome abnormalities (Trimble, B. K. and Baird,
P. A. Amer. J. Med. Genet. 2: 1, 1978). Causes of these phenomena havenot been known.
In 1974, Eiberg (Nature 248: 55, 1974) described a technique called Cdbanding which stains two small dark spots on both sides of the kinetochore.
Cd denotes a centric dot; however, there was a controversy as to whether
it, in fact, represented the kinetochore (Roos, U. P. Nature 254: 463, 1975).
Later, it was established that a positive Cd reaction represented the presenceof an active kinetochore and a negative reaction either its inactivation (supression) or loss (Nakagome, Y. et at. Clin. Genet. 9: 621, 1976; Daniel, A.
Hum. Genet. 48: 85, 1979; Nakagome Y. this report, 30: 97, 1980; Maraschio, P. et at. Hum. Genet. 54: 265, 1980).
In the present study, the Cd-band technique was applied on mitotic
spreads obtained from aged women. Blood samples were obtained from 14
females over 60 years of age (66 to 87y, average=78y4m). Two aged males,
84 and 90 years, were also examined for comparison. In Cd-stained preparations, each chromosome showed small dots on both sides of the kinetochore.
To avoid effects of possible technical variables, only cells in which all 6
members of A-group chromosomes showed positive Cd spots were includedin the study. The remaining 40 chromosomes, B through G groups, werescored except for those overlapping at the kinetochore. For comparison,some of the preparations were treated by either the C-band technique or the
distamycin-DAPI (dD) method (Schweizer, D. et at. Exp. Cell Res. 111:
327, 1978). Only cells in which all 6 members of the A group showed adistinct constriction at the kinetochore were included in the study. The
rest of the chromosomes were examined to determine whether both chromatids were separated or attached at the kinetochore.
In aged individuals, 56 were Cd negative out of a total of 6476 examinedchromosomes. In controls, 11 were Cd negative out of a total of 3057.
The difference was highly significant (X 2 =7.6, d. f.= 1, p<O.Ol). Fourty
seven of the former and nine of the latter assumed parallel-bar appearance.
The rest showed constriction at the kinetochore although they were Cd
negative. Thirty one of the Cd-negative chromosomes belonged to theC group which was higher than the expected 22.4. Six of the 14 aged females

112

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

were examined by either the C or the dD method. Thirteen out of a totalof 2080 chromosomes showed separation of their kinetochores. In 11
controls, only 7 showed separation out of 5080 chromosomes examined.
The difference was highly significant. In two aged males, 8 Cd-negative
chromosomes were detected out of 1426 examined. The frequency (0.0056)
was between the levels of aged females (0.0087) and control females (0.0036),
It was concluded that the kinetochore tended to deteriorate and lose Cdpositive material in aged individuals implicating its causative relationshipwith the increase of both aneuploid cells in mitoses and possibly nondisjunction in meioses.

RESEARCHES CARRIED OUT IN 1981

XI.

113

BEHAVIORAL GENETICS AND ECOLOGICAL GENETICS
The Behavioral Changes in Mice Coutim.l.ously Reared
under Noise Conwtion*
Tohru FUJISHIMA

Mice continuously reared under noise conditions have been examined
for the behavioral changes in comparison with those reared under normal,
or non-noise conditions as a control. The mice of the two groups wereoriginally derived from the same full sib families. They were kept at 25°C
with 12 hour light and 12 hour darkness. The mice in the noise treatedgroup were exposed six times to noise (pink, 100 phon) for one hour each at
one hour interval during the dark period (6 p. m. to 6 a. m.). The avoidanceand discriminatory learning performances of mice approximately 70 days
old were measured with an automated Y-type maze apparatus. Their activity and body weight were also recorded.
The result of the fourth generation obtained this year showed significant
differences in avtivities and body weights between the treated and the control
groups, coinciding with the result of the previous generation. This suggested
that the effect of noise might arouse the emotionality of mice. Then, toexamine whether mice recover from the effect of noise by their removalfrom noise to normal conditions, a portion of the mice of the noise treatedgroup was removed to the normal condition at birth. The result showedthat no behavioral change occurred by the removal, indicating no recovery
from noise effects. These results suggested that the behavioral changes
occurred in the noise treated group might have resulted from the effect ofnoise given to mice before birth.
Weed-Mediated Natural Selection in a Segregating
Population of Rice
Hiroko MORISHIMA

The response of rice to weeding or weed tolerance is known to differ according to varieties, hence it is genetically controlled. In an attempt to look*

This work was supported by a grant from the Ministry of Environment.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

into the effect of natural selection imposed by weeds, a segregating population
derived from a cross between Indica and Japonica rice varieties was divided
into three parts, and one was grown in a weed-free condition while the othertwo were reared with weeds (Echinochloa crus-galli, barnyard grass, andC}perus difformis, a sedge, were respectively sown in the first year). After
two successive generations of natural selection, twenty successful plants
were selected from each plot, and their progeny lines were tested in the above
three conditions, respectively. The tolerance to weed was evaluated by thedifference in log-transformed plant weight of rice between weed-free andsedge-sown plots, or between weed-free and barnyard grass-sown plots.
The results showed that lines from weedy plots had higher tolerance to weed
those from weed-free plots. Although the lines varied in a wide range, theanalysis of variance of line-group means proved that the differences among
the three treatments were highly significant. Three lines from the sedge
plot gave a higher performance with sedge than in weed-free condition.
Although this experiment was incomplete, the data proved that plants wererapidly selected for the ability to coexist with their neighboring plants. This
may promote niche differentiation between coexisting species in a community, resulting in genotypic differences in niche dimension within species populations.
Demography and Populational Variability of Rumex ohtusifolius
Conditioned by Biotic Environment
Kenji TERAI!) and Hiroko MORISHIMA

To gain an understanding of the effect of biotic environment on population
dynamics of plant species, five populations of a perennial weed, Rumex
obtusifolius, were observed with regard to emergence, mortality, density,
growth performance, and some other traits. Composition of coexisting
species in the community was also observed. The biotic environment wasrepresented by the diversity of life-form in the coexisting species. Life-formof each species was categorized based on its reproductive system and plantform. Each study-site was taken so as to cross over zones differing in theintensity of habitat disturbance and other conditions, for instance, covering
dike of arable land, grassland and roadside. Various demographic parameters as well as measurements of community diversity were compared1)

Faculty of Education, Akita University.

RESEARCHES CARRIED OUT IN 1981

115

among sub-sites within site. We found that mortality and recruitment
rates, standing density, distributional pattern of R. obtusifolius differedwithin a site according to the community diversity. Inter-plant variability ofplant size of R. obtusifolius tended to be larger in the community with medium
degree of diversity than in either of the community with high and low diversity. It was also found that R. obtusifolius and its two relatives, R. acetosaand R. acetosera, had differential zones of distribution along the variationin community diversity.
Further, germination behavior under different temperatures, characteristics
of seed morphology and various traits of seedlings grown in a uniform condition were studied, using seed samples of 30 populations collected on an
individual basis from various habitats, such as waste places or roadside intown, dikes of crop fields, and semi-natural grassland. Variation in each
trait was partitioned into between- and within-populations, as well as into
between- and within- (between progeny plants derived from a mother)individuals in a population. Low-temperature response of germination
behavior varied between and within populations and its variation pattern
seemed to reflect proportion of bare-land in the collection site (degree ofvegetational saturation). Populations collected in town showed larger interpopulational and smaller intra-populational variation in many charactersthan those collected in semi-natural sites outside town, suggesting that the
populations distributed in strongly disturbed habitats have higher selfing
rate than those in less-disturbed habitats.
The above observations indicate that population dynamics is, at least
partly, conditioned by the intensity of habitat disturbance and by consequently resulted changes in biotic environment. To what extent genetic
variation is related to interaction with biotic environment remains unsolved.
Isozyme Variation in Altingia Forest at Ciwidey, West Jay£!
Toru ENDO

Nature of tropical natural forests has been characterized with high species
diversity and low genetic diversity per unit area from the view point of plant
ecology. The genetic diversity per unit number of any plant species, however, must not always be similar or low, because of much more favorable
environmental conditions for various mutants in tropical zone than thosein temperate or arctic zones. Such a problem has not yet been examined

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

in tropical forests.
From February 3 to 5, 1982, a teem of five Japanese members and threestaffs of Tropical Forest Biology Section of BIOTROP, Bogor, conducted astudy in natural forests of Altingia excelsa at the foot of Mt Patuha, Ciwidey.
Thirty eight trees of Altingia at Plot 2 (50 X 50 m) were mapped and characterized, and leaves of all Altingia trees, including three poles and one sapling
were collected for estimation of morphological characters as well as for thezymographic analysis of peroxidase isozymes.
Using the zymograms from 38 trees totally 703 combinations were carriedout, and disagreement number counts (DNC) were obtained in each tree.
The DNC ranged from 2.53 to 5.79, and the mean was 3.44. Although thelocus number of the isozymes is not estimated, the value of Altingia was
unexpectedly low, being compared with the value 12.99 from a sample plotof Thujopsis dolabrata at Aomori (Sakai and Miyazaki 1972, Silv. Genet.
21: 5).
Cross Resistance to OrganophospborolJls
Insecticides in Drosophila
Yutaka INOUE

From the Tochigi population of D. melanogaster, a Diazinon resistantstrain (TG20) and a Sumithion resistant strain (TG44) were obtained. Adult
survival tests against Diazinon (40 ppm, 24 hrs), Sumithion (80 ppm, 48 hrs)
and DDT (0.25 %, 2 hrs) were carried out using the TG20 and TG44 strains
and additional DDT-resistant (Hikone-R), Ether-resistant (Eth-29) andOregon-R (OR) strains. The results are shown in Table 1. OR and Eth-29
were not resistant to any insecticide. TG44 and Hikone-R were resistantto both Sumithion and DDT. TG20 showed moderate resistance to Sumithion and DDT. However, there was no strain showing strong resistanceto all three insecticides. The tendency of cross resistance was observed
between different insecticide-resistant strains. Hikone-R has never been
exposed to Sumithion, but it was resistant to Sumithion as well as to DDT.
Flies were cultured in the food containing Diazinon (30 ppm) or Sumithion(45 ppm) for long generations. Diazinon-treated flies showed significant
Diazinon-resistance at the 12th generation, and they also showed Sumithionresistance at the 24th generation. Sumithion-treated flies were significantly
Sumithion-resistant at the 24th generation and their Diazinon-resistance117

RESEARCHES CARRIED OUT IN 1981
Table 1. The comparison of cross-resistances of the several strains to DDT,
Diazinon and Sumithion. Number of tested females in each experimentwere 80 for DDT, and 60 for Diazinon and Sumithion
Mortality
Treatment
DDT
Diazinon
Sumithion

TG20

TG44

Hikone-R

OR

Eth-29

0.26
0.50
0.46

0.16
0.75
0.08

0.16
1.00
0.12

0.69
1.00
1.00

0.84
1.00
1.00

was observed at the 36th generation. Thus, the corss resistances seemed to
be grown up in a few generations after the occurrence of the drug specificresistance. The amount of deleterious genes maintained in the resistantpopulations was compared. Diazinon(30 ppm)-treated and Sumithion(45 ppm)-treated populations did not show any difference from a control
population in the frequency of lethal, semilethal and sterile genes on the
X and the second chromosomes.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

XII.

APPLIED GENETICS

NmnerkaH Evaluation of the Indica-Japmlica Differentiationin Rice Varieties
Hiroko MORISHIMA and Hiko-ichi OKA1)

Multivariate analysis of the data for 11 characters of 89 cultivars of
Oryza sativa sampled at random from a large collection of Asian origin hasreconfirmed the previous assertion by the junior author that the IndicaJaponica differentiation of rice cultivars is recognizable on the basis of character-association patterns, although there is no key character discriminating
between the two types. The probability of misclassificationby a single
character ranged from about 3 percent by the potassium chlorate resistanceof seedling to about 40 percent by the length/width ratio of spikelets. Discriminant functions combining 3 or 4 character values gave lower probabilities of misclassification, although there still remained a few intermediate
forms having recombined characteristics. The F 1 sterility relationships
were almost useless for the purpose of classification. About a half of thetotal character variance among the cultivars was attributable to IndicaJaponica differentiation. The normalized genetic identity between the two
types was estimated to be 0.62 from data for 28 alleles at 12 loci. A great
number of genic differences seem to be involved in the Indica-Japonica
differentiation. For details, see Japan. J. Breed. 31: 402-413.
Associative Nitrogen Fixation of Rice Plants with
Isolated Bacteria
Taro FUJll and Koki OHTA

In our previous experiment, bacterial inoculation onto germ-free seedlings
growing in the test tube filled with sterilized soil has been carried out to investigate the interaction of rice and bacterial strains in associative nitrogen
fixation. Two strains of nitrogen fixing bacteria isolated from rhizospheresoil, COC-8 (Azospirillum lipoferum) and NG-13 (Klebsiella oxytoca) showed
appreciable acetylene reduction activity (ARA). In the present experiment,
1)

Chung Hsing University, Taichung, Taiwan

RESEARCHES CARRIED OUT IN 1981

119

synthesized medium was used for systematic study of nitrogen fixation by
association of rice plants with particular bacterial species. Medium used
is basically the same as White's but nitrogen and carbon sources have been
omitted since nitrogen source inhibit nitrogenase activity and bacterial growthis promoted by carbon source.
Germ-free seedlings growing in the medium for one week after germination were inoculated with COC-8 and NG-13. ARA was measured weeklyintervals during one to 4 weeks after inoculation. ARA was increased up
to 3 weeks after inoculation and decreased the 4th week. Number of inoculated bacteria was also measured. Sudden increase in number was observed
one week after inoculation, namely about 102 inoculum was reached to orderof 10 7, and almost same number was maintained up to 4th week. On theother hand, seedling growth was arrested about 4 weeks after germination due
to lack of nitrogen and carbon sources in the medium. The results indicatedthat the inoculated bacteria propagated by supply of carbon source(s) excreated from roots, exhibited nitrogenase activity. However, when plant
growth stopped ARA decrease because of the lost of the supply of carbon
source.
To sustain the seedling growth, a small amount of KNOB and Ca(NO s)2'
equivalnt to one-tenth of White's basal medium was added to the medium
and same experiment was again carried out. When a small amount of
nitrogen is added to the medium slightly better seedling growth occurs
and there was very little change in the ARA. Exploitation of modified
medium for much better plant growth is now being studied.
Diallel Amll.Yi'lis iilf Nitrogen Fixation in tile
Rbiz<ilsphere of Rice
Shinya IYAMA, Yoshio SANO and Taro FUJI!

The genetics of nitrogen fixing ability in the rhizosphere of rice was investigated by diallel analysis. Diallelic crosses were made among 11 strainsof rice chosen from the collection which was kept at the Genetic Stock Centerof the Institute. They were chosen to cover a wide range of origins fromSouth Asia to Japan. F , plants and their parent strains were grown individually in a small plastic pot containing 180 g of soil. Nitrogen fixing
activity in the rhizosphere of them were measured by the acetylene reduction
method with the aid of gas chromatography. The measurement was done

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

at the heading time of each plant.
In an 11 X 11 diallel set, presence of non-allelic interaction was revealed.
Excluding the interacting parents, a 7 X 7 diallel set which fitted to theadditive-dominance model indicated the average degree of dominace being
partial to complete dominance and the excess of dominant alleles over recessive alleles. Relationship between parental performance and (Wr+Vr)
value calculated from the diallel table suggested that there was no difinite
relation between nitrogen fixing activity in the rhizosphere and direction of
dominance but that of the highest parent was likely governed by recessive
alleles. Positive correlation between nitrogen fixing activity of parent andthat of F 1 was statistically significant (r=0.88). It was concluded from theabove results that the nitrogen fixing activity in the rhizosphere are inherited to the progeny through hybridization and can be improved by selection among the progeny of hybridization.
Genetic Vmriatioll of Herbicide Tolerance among
F 5 Lines of Barnyard Grass
Shinya IYAMA

Crosses between barnyard grass (Echinochloa crus-galli) strans of different
tolerance to propanil were made and kept by single seed decent procedurefrom F 2 to F 4 • Their F 5 lines were examined for the tolerance to propanil.
Seedlings of 2 weeks old were sprayed by a given amount of propanil solution
and the number of plants survived in each line was counted after 2 weeks.
The result indicated that the segregation regarding the tolerance was foundamong the F 5 lines. It was observed in most cross combinations thattolerance of some lines exceeded the range of the parents, suggesting that
more tolerant genotypes may appear through recombination of responsible
genes among progenies of natural hybridization in a field.

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121

BOOKS AND PAPERS PUBLISHED IN 1981

BY STAFF MEMBERS
AMANo, E. 1981: Genetic and biochemical characterization of waxy mutants in cereals.
Environmental Health Perspectives 37: 35-41.
AMANo. E. 1981: Flow system for automated analysis of maize pollen. Environmental
Health Perspectives 37: 165-168.
AOKI, K. and M. MOODY* 1981: One- and two-locus models of the origin of worker
behavior in Hymenoptera. J. Theor. BioI. 89: 449-474.
AOKI, K. 1981: Algebra of inclusive fitness. Evolution 35: 659-663.
AOKI, K., Y. TATENO* and N. TAKAHATA 1981: Estimating evolutionary distance fromrestriction maps of mitochondrial DNA with arbitrary G+C content. J. Mol. Evol.
18: 1-8.
ENDo, T. 1981: Differential regulation of peroxidase isozymes coded by Px-l locus inrice. Jap. J. Genet. 56: 175-183.
ENDo, T. 1981: Developmental modification and hybridization of allelic acid phosphataseisozymes in homo- and heterozygotes for the Acp-l locus in rice. Biochem. Genet.
19: 373-384.
FuJII, T. 1981: Mutagenic effect of L-ethionine in soybean and maize. Exp. Environ.
Botany 21: 127-131.
FUJISAWA, T. and C. N. David* 1981: Commitment during nematocyte differentiation in
Hydra. J. Cell Sci. 48: 207-222.
HIRoTA, Y., M. YAMADA, A. NIsmMuRA, A.OKA*, K. SUGIMOTO*, K. ASADA* and M.
TAKANAMI* 1981: The DNA replication origin of Escherichia coli: Structure andfunction of the ori-containing DNA fragment. Progress Nucleic Acid Res. Mol. BioI.
26: 33-48.
SATO, C*., K. KOJIMA*, K. NIsmzAwA* and Y. HIROTA 1981: Cell surface charge andcell division in Escherichia coli after X irradiation. Radiation Research 87: 646-656.
UTSUMI, R*., H. TANABE*, Y. NAKAMoTO*, M. KAWAMUKAI*, Y. SAKAI*, M. HlMENO*;
T. KOMANO* and Y. HIROTA 1981: Inhibitory effect of adenosine 3' 5'-phosphate on
cell division of Escherichia coli mutant derivatives. J. Bacteriol. 147: 1105-1109.
IMAI, H., Y. MATSUDA*, T. SHIROISHI and K. MORIWAKI 1981: High frequency of X-V
chromosome dissociation in primary spermatocytes of F 1 hybrids between Japanesewild mice (Mus musculus molossinus) and inbred laboratory mice. Cytogenet. Cell
Genet. 29: 166-175.
HIRAI, H*., Y. SAKAGUCHI* and H. IMAI 1981: C-band polymorphism in a Japanese lung
fluke Paragonimus ohirai (Trematoda; Platyhelmintes). Heredity 47: 249-252.
INOUE, T. 1981: Ataxia-telangiectasia. Jap. J. Genet. 56: 315-334.
INOUE, T., K. MORITA* and T. KADA 1981: Purification and properties of a plant desmutagenic factor for the mutagenic principle of tryptophan pyrolysate. Agric. BioI.
Chern. 45: 345-353.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32
INOUE, T., S. OTA*, Y. SADAIE and T. KADA 1981: Effect of cobaltous chloride on SPon.
taneous mutation induction in a Bacillus subtilis mutator strain. Mutation Res.
91: 41-45.
INOUE, T., A. YOKOIYAMA and T. KADA 1981: DNA repair enzyme deficiency and invitro complementation of the enzyme activity in cell-free extracts from ataxia telan.
giectasia fibroblasts. Biochimica et Biophysica Acta 655: 49-53.
KADA, T. and H. MOCBIZUKI 1981: Antimutagenic activities of human placental extract
on ultraviolet light- and gamma-ray-induced mutation in Escherichia coli WP 2B/r trp.
J. Radiat. Res. 22: 297-302.
LEIFER, Z*., T. KADA, M. MENDEL*, E. ZEIGER*, R. STAFFORD* and H. S. ROSENKRANZ*
1981: An evaluation of tests using DNA repair-deficient bacteria for predicting
genotoxicity and carcinogenicity. Mutation Res. 87: 211-297.
KARUBE, M*., T. MATSUNAGA*, T. NAKAHARA*, S. SUZUKI* and T. KADA 1981: Pre.
liminary screening of mutagens with a microbial sensor. Anal. Chem. 53: 1024-1026.
KAWANISHI, M. and T. K. WATANABE 1981: Genes affecting courtship song and mating
preference in Drosophila melanogaster, Drosophila simulans and their hybrids. Evolu.
tion 35: 1128-1133.
OHNISHI, S. and M. KAWANISHI 1981: The sexcomb of Drosophila simulans: Geogra_
phical variation and genetic analysis. Kontyu 49: 37-44.
KIMURA, M. 1981: Estimation of evolutionary distances between homologous nucleotide sequences. Proc. Natl. Acad. Sci. USA 78: 454-458.
KIMuRA, M. 1981: Was globin evolution very rapid in its early stages?: a dubious caseagainst the rate-constancy hypothesis. J. Mol. Evol. 17: 110-113.
KIMURA, M. 1981: Doubt about studies of globin evolution based on maximum parsimony
codons and the augmentation procedure. J. Mol. Evo!. 17: 121-122.
KIMuRA, M. 1981: Possibility of extensive neutral evolution under stabilizing selection
with special reference to nonrandom usage of synonymous codons. Proc. Natl. Acad.
Sci. USA 78: 5773-5777.
SUZUKI, T*. and Y. KOMEDA 1981: Incomplete fiagellar structures in Escherichia coli
mutants. J. Bacteriol. 145: 1036-1041.
KURODA, Y. (ed.) 1981: Genetic Experiments Using Cultured Cells. (in Japanese) Kyoritsu-Shuppan (Tokyo), 380p.
KURODA, Y. 1981: In vitro cells. In "Ageing of Cells" (ed. by Tauchi & Kuroda). (inJapanese) Kyoritsu-Shuppan (Tokyo), pp. 121-217.
MARUYAMA, T. and E. SOEDA 1980: Molecular evolution in papova viruses and their hostspecies, and in bacteriophages. Proc. Stadler Genetics Symp. 12: 83-96.
MARUYAMA, T. and N. TAKAHATA 1981: Numerical studies of the frequency trajectories
in the process of fixation of null genes at duplicated loci. Heredity 46: 49-57.
MARUYAMA, T. and H. IMAI 1981: Evolutionary rate of the mammalian karyotype. J.
Theor. BioI. 90: 111-121.
MARUYAMA, T. 1981: Stochastic problems in population genetics: Applications of Ito's
stochastic integrals. In "Stochastic Nonlinear Systems in Physics, Chemistry, andBiology" (eds. L. Arnold and R. Lefever), 154-161. Springer-Verlag, Berlin.
MARUYAMA, T. and M. NEI 1981: Genetic variability maintained by mutation and over-

BOOKS AND PAPERS PUBLISHED IN 1981 BY STAFF MEMBERS

123

dominant selection in finite populations. Genetics 98: 441-459.
MATSUNAGA, E. 1981: Genetics of Wilms' tumor. Hum Genet. 57: 231-246.
MATSUNAGA, E. 1981: Retinoblastoma: Mutational mosaicism or host resistance? Am.
J. Med. Genet. 8: 375-387.
URUSHIBARA, T*., C. NISHIMURA* and K. MIURA 1981: Process of cap formation of messenger RNA by vaccinia virus particles carrying an organized enzyme system. J.
gen. Virol. 52: 49-59.
URUSHIBARA, T*., K. OKAMOTO*, C. NISHIMURA, T. KAMIMURA*, Y. OZAKI*, K. YAMAGUCHI*, T. HATA* and K. MIURA 1981: Methylation of the 5'-5' confronting
dinucleotides by vaccinia associated enzyme system. Nucleic Acids Res. Symp.
Ser. No. 10: 237-240.
INoUE, A*., M. NAKAMURA, S. NAKANISHI*, S. HIDAKA*, K. MIURA and S. NUMA* 1981:
5'-Terminal nucleotide sequence of the messenger RNA coding for bovine corticotropin/f3-Iipotropin precursor. Europ. J. Biochem. 113: 531-539.
MORISHIMA, H. and H. I. OKA 1981: Phylogenetic differentiation of cultivated rice, XXII.
Numerical evaluation of the Indica-Japonica differentiation. Jap. J. Breed. 31(4):402-413.
ASSEMAT, L*., H. MORISHIMA and H. 1. OKA 1981: Neighbor effects between rice (Oryza
sativa L.) and barnyard grass (Echinochloa crus-galli Beauv.) strains, n. Some experiments on the mechanisms of interaction between plants. OEcol. Plant. 2(16),No.1: 63-78.
MORIWAKI, K., H. YONEKAWA*, M. MINEZAWA*, Y. MATSUDA, T. SHIROISHI and N.
MIYASHITA 1981: Genetic status of Japanese wild mouse, Mus musculus molossinus. Hereditas 94: 15.
MIZUOCHI, T*., Y. YAMASHITA*, T. HAMAOKA* and K. MORIWAKI 1981: The role of laantigens in the activation of T cells to concanavalin A. Cell. Immunol. 57: 28-41.
SUZUKI, S*., K. TSUJI* and K. MORIWAKI 1981: Friend murine leukemia virus resistancein Japanese wild mice: Possible allelism with Fv-4 in FRG mice. J. Nat. Cancer
lust. 66: 729-731.
ODAKA, T*., H. lKEDA*, H. YOSHIKURA*, K. MORIWAKI and S. SUZUKI* 1981: Fv-4:
Gene controlling resistance to NB-tropic murine leukemia virus. Distribution in wild
mice, introduction into genetic background of BALB. C mice and mapping of chromosomes. J. Nat. Cancer Inst. 67: 1123-1127.
MINEZAWA, M*., K. MORIWAKI and K. KONDo* 1981: Geographical survey of protein
variations in wild populations of Japanese house mouse, Mus musculus molossinus.
Jap. J. Genet. 56: 27-39.
lKAwA, Y*., G. KIYAMA*, K. SUDO*, K. SUZUKI* and K. MORIWAKI 1981: Strainspecific appearance of a possible fetal a-globin polypeptide chain in mice. Develop.,
Growth and Differ. 23: 1-8.
YONEKAWA, H*., K. MORIWAKI, O. GOTOH,* J. HAYASHI*, J. WATANABE*, N. MIYASHITA,
M. L. PETRAS* and Y. TAGASHIRA* 1981: Evolutionary relationships among fivesubspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA. Genetics 98: 801-816.
SHIROISHI, T., T. SAGAI and K. MORIWAKI 1981: A simplified micro-method for cyto-

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32
xicity testing using a flat-type titration plate for the detection of H--2 antigens. MicrobioI. Immunol. 25: 1327-1334.
MATSUBARA, T.*, K. lINUMA*, Y. NAKAGOME and T. YOKOCHI* 1981: Familial case of
Down's syndrome. A psu dic (21) (q 22) and a rob (14q21q) in cousins. Jpn. J. Hum.
Genet. 26: 55-59.
TAKEDA, Y.*, A. NISHIMURA, Y. NISHIMURA, M. YAMADA, S. YASUDA, H. SUZUKI* andY. HIROTA 1981: Synthetic ColEl plasmids carrying genes for penicillin-binding proteins in Escherichia coli. Plasmid. 6: 86-98.
STERNGLANZ, R. *, S. DINARDO*. K.A. VOELKEL*, Y. NISHIMURA, Y. HIROTA, K. BECHERER*,
L. ZUMSTEIN* and J. C. WANG* 1981: Mutations in the gene coding for Escherichia
coli DNA topoisomerase I affect transcription and transposition. Proc. Natl. Acad.
Sci. USA 78: 2747-2751.
NOGUCHI, T. 1981: Mouse teratomas. (in Japanese) Gan to kagakuryoho 8: 979-988.
OZAWA, M*., J. FUJIYAMA*, T. FURUKAWA*, K. SATO*, T. NOGucm and T. MURAMATsu*
1981: Glycoproteins from teratocarcinomas and organs of adult mice: Analysis byaffinity chromatography on lectin-conjugated agarose. Develop. Growth and Differ.
23: 515-522.
OHTA, T. 1980: Two-locus problems in transmission genetics of mitochondria and chloroplasts. Genetics 96: 543-555.
OHTA, T. 1981: Genetic variation in small multigene families. Genet. Res. 37: 133-149.
OHTA, T. 1981: Population genetics of selfish DNA. Nature 292: 648-649.
OHTA, T. and M. KIMURA 1981: Some calculations on the amount of selfish DNA. Proc.
Nat!. Acad. Sci. USA 78: 1129-1132.
SADAIE, Y., T. INOUE, H. MOCHIZUKI and T. KADA 1981: Efficiencies of DNA Inactivation and mutation induction by tritiated glycerol in bacterial systems. J. Radiat.
Res. 22: 387-394.
SANO, Y., T. FUJII, S. IYAMA, Y. HIROTA and K. KOMAGATA* 1981: Nitrogen fixation in
the rhizosphere of cultivated and wild rice strains. Crop Science 21: 758-761.
TODOH, N., K. SHINOZAKI and M. SUGIURA 1981 : Sequence of a putative promoter region
for the rRNA genes of tobacco chloroplast DNA. Nucleic Acids Research 9: 53995406.
TOMioKA, N., K. SmNOZAKI and M. SUGIURA 1981: Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans. Mol. Gen.
Genet. 184: 359-363.
SUGIURA M. 1981: An Escherichia coli acid phosphatase which hydrolyzes preferentiallynucleoside 3', 5'-diphosphates. FEBS Lett. 123: 285-286.
TAKAIWA, F. and M. SUGIURA 1981: Heterogeneity of 5S RNA species in tobacco chloroplasts. Mol. Gen. Genet. 182: 385-389.
KATO, A., H. SHIMADA, M. KUSUDA and M. SUGIURA 1981: The nucleotide sequencesof two tRNAAsn genes from tobacco chloroplasts. Nucleic Acids Research 9: 56015607.
TAKAHATA, N. 1981: A mathematical study on the distribution of the number of repeatedgenes per chromosome. Genet. Res. 38: 97-102.
TAKAHATA, N. 1981: Genetic variability and rate of gene substitution in a finite population

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125

under mutation and fluctuating selection. Genetics 98: 427-440.
TAKAHATA, N. and T. MARUYAMA 1981: A mathematical model of extranuclear genes andthe genetic variability maintained in a finite population. Genet. Res. 37: 291-302.
TAKAHATA, N. and M. KIMURA 1981:: A model of evolutionary base substitutions and its
application with special reference to rapid change of pseudogenes. Genetics 98:
641-657.
YAGI, Y*., K. NAKAMURA*, I. TOMITA*, K. TUTIKAWA and N. SHIMOI* 1980: Teratogenic
potential of di- and mono-(2-ethylhexyl) phthalate in mice. J. Environ. Pathol.
Toxicol. 4-2, 3: 533-544.
WATANABE, T. K. and M. KAWANISHI 1981: Asymmetrical mating success and the phylogeny of Drosophila. Zool. Mag. 90: 317-324.
NAKAMURA, M., M. YAMADA, Y. HIROTA, K. SUGIMOTO*, A.OKA* and M. TAKANAMI*
1981: Nucleotide sequence of the asnA-gene coding for asparagin synthetase of E. coli
K-12. Nucleic Acids Res. 9: 4669-4679.
YAMAMOTO, M. 1981: Mechanism of meiotic chromosome pairing in the D. melanagaster.
(in Japanese) Jap. J. Genet. 56: 79-96.
YOSIDA, T. H. 1981: Studies on the karyotype differentiation of the Norway rat, V.
Hypotrichotic mutant rats appeared in the inversion stock (LEM). Proc. Jap. Acad.
57(B): 29-34.
YOSIDA, T. H. 1981: Chromosome alteration and the development of tumors, XXIII.
Banding karyotype analysis of methylcholanthrene-induced tumors in the Indianspiny mouse, Mus platythrix, with special ragard to the anomalies of chromosomeswith nucleolar organizer regions. Cancer Genet. Cytogenet. 3: 211-220.
YOSIDA, T. H. 1981: Chromosome alteration and development of tumors, XXIV. Hypopentaploid chromosome constitution of a spontaneous tumors developed in the
Oceanian type black rat, with particular reference to centromeric fission. Proc. Jap.
Acad. 57(B): 260-265.
YOSIDA, T. H. 1981: Chromosome polymorphism of the large naked-soled gerbil, Tatera
indica (Rodentia, Muridae). Jap. J. Genet. 56: 241-248.
YOSIDA, T. H. and T. INOUE* 1981: Cytogenetic studies on the Indian spiny mouse, I.
Frequency of sister chromatid exchanges in lung primary culture. Proc. Jap. Acad.
57(B): 13-17.
YOSIDA, T. H. 1981: Population cytogenetics of the black rat, Rattus rattus. Proc. XIV
Intern. Congo Genet. (Symposium), Vol. II: 96-117.
TAKATSUKI, K*., H. MASAI*, A. UBUKATA*, J. KAMAMATSU* and T. H. YOSIDA 1981:
Quantitative variation in cytoarchitectures of the retine and the striate cortex amongsome species of Myomorpha (suborder) in relation to their habits. Zool. Anz. Jena
206(198): 263-272.

* Author not on NIG staff.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

ABSTRACTS OF DIARY FOR 1981
January
March

April
May
July
AugustSeptember
OctoberNovember

December

29
7
17
20
26
4
20
12
16
24
28
12
5
9
15
23
24
28
12
20
20
19

270th Meeting of Misima Geneticists' Club
271st Meeting of Misima Geneticists' Club175th Biological Symposium
176th Biological Symposium
272nd Meeting of Misima Geneticists' Club
177th Biological Symposium
178th Biological Symposium
273rd Meeting of Misima Geneticists' Club
274th Meeting of Misima Geneticists' Club
275th Meeting of Misima Geneticists' Club
179th Biological Symposium
180th Biological Symposium
18lst Biological Symposium
182nd Biological Symposium
276th Meeting of Misima Geneticists' Club
183rd Biological Symposium
184th Meeting of Misima Geneticists' Club
185th Meeting of Misima Geneticists' Club
186th Meeting of Misima Geneticists' Club
187th Meeting of Misima Geneticists' Club
188th Meeting of Misima Geneticists' Club
277th Meeting of Misima Geneticists' ClubFOREIGN VISITORS IN 1981

127

FOREIGN VISITORS IN 1981
February
March

16-Sept. 30, '82
16-20

17

20
21-23
30-Apr. 23
April

I-Jul. 29
2

10-11
April

17-20
20
24-May 15

May

2
13-16

27
29

SUN, Chong-ron, Futan University, China
Fujimura, Robert K., Oak Ridge National
Laboratory, U. S. A.
Yamazaki, R., International Agency for
Research on Cancer, World Health Organization, France
PETROV, Spetrov P., Institute for Wheat andSunflower, Bulgaria
CROW, James F., University of Wisconsin,
U. S.A.
DYER, Tristan A., Plant Breeding InstituteEngland
KIM, K. W., Chonnam Nat. University,
Korea
Xu, Y. and Others, Inst. Developm. BioI.,
China
JEFFERSON, Roland M., U. S. National
Arboretum, U. S. A.
WHITFELD, Paul R., Commonwealth Scientific and Industrial Research Organization,
Australia
HIRTH, Leon, Universite de Strasbourg,
FranceSCHWARZ, VIi, Max-Planck-Institlit, West
Germany
WALKER, D. A., University of Sheffield,
England
STEVENS, L. C., The Jackson Laboratory,
U. S.A.
Nasoetion, Andi H., Institute Pertanian
Bogor, Indonesia
TONEGAWA, S., Basel Institute for Immunology, Switzerland

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

June

19

August19
20-Sept. 4
28

September

10-26
16-0ct.31
17-Dec. 7
24-0ct. 28
24
26

PEACOCKE, A. Robert, Clare College, Cam~bridge, England
Kuo, Chun-Yen, South China Inst. of
Botany, Academia Sinica, China
NEI, M., University of Texas, U. S. A.
GARTLAND, W. J., Jr., National Institutesof Health, U. S. A.
SHARMA, A. K., University of Calcutta,
India
SHANKEL, Delbert M., University of Kansas,
U.S.A.
WANEK, Nancy Lynn, University of California, U. S. A.
CROW, James P., University of Wisconsin,
U. S.A.
KIHLMAN, B. A., University of Uppsala,SwedenROH, J. K., Korea Advanced Institute of
Science and Technology, Korea
OKAMOTO, K., University of New South
Wales, Australia
SMUTKUPT, S., Kasetsart University, Thailand
BHUNYA, S. P., Utkal University, India
McKENNA, P. G., The University of Ulster,
Northern Ireland
BOGAJEWSKI, J., Institute of Human Genetics, Polish Academy of Sciences, Poland
CHOUROULINKOV, 1., Institut de Recherches
Scientifiques sur la Cancer, France
AUERBACH, c., The University of Edinburgh,EnglandOMENN, G. S., University of Washington,
U. S. A.
KIHLMAN, B. A., University of Uppsala,SwedenSCHULL, W. J., University of Texas HealthFOREIGN VISITORS IN 1981

October

28-0ct. 1
15
15

19

20-21
21
23

23-24
31-Nov.

11

17-18

20-21
20

129

Science Center, U. S. A.
RUSSELL, W. L., RUSSELL, L. B., Selby, P. B.,
Oak Ridge National Laboratory, U. S. A.
LIM-SYLIANCO, C. Y., University of thePhilippines, Philippines
HASHEM, N., Ain-Shams University, Egypt
WOOD, R. D., University of California,
U.S.A.
FISCHMAN, H. K., New York State Psychiatric Institute, U. S. A.
TAN, C. c., Futan University, China
RANFT, Dietrich and NICKEL, Dietmar;
Max-Pa1nck Gesellschaft, West Germany
KAJI, A., University of Pennsylvania,
U.S.A.
FREY, Kenneth J., Iowa State University,
U.S.A.
STRONINGER, Jack, Harvard University,
U.S.A.
JOLLY, M. S. and others, Central Sericultural Research and Training Institute, India
OHNO, S., City of Hope Research Institute,
U.S.A.
Gross, Hans J., Wiirtzburg UniversiHit,
West Germany
ARBER, Werner, Basel Universitiit, Switzerland
RIEGER, R., Institute of Genetics and Research in Cultivated PLANTS, Academy of
Sciences, German Democratic Republic
ALBERTS, Bruce, University of California,
U.S.A.
FRAENKEL-Conrat, Heinz, University ofCalifornia, U. S. A.
BODMER, Walter F., Imperial Cancer Research Fund, England130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

20

28

FIERS, Walter, Rijks Universiteit-Gent,
Belgium
BEAKWITH, Jon, Harvard University, U. S. A.

AUTHOR INDEX131

AUTHOR INDEX
ABE, TACHERMANN, J
AOKI, K.
ASADA, K.

.

ASAKURA, M
BONHOMME, F. .
CHAN,

H. W

DE ZOLESSI, L. C

110
56
103
26, 2752
69
21
69

EGAMI, N.46

ENDO, T48, 11595, 118, 119
55
113
21
69,71
42

FUJII, T
FUJISAWA,T

FUJISHIMA, T
FURUICHI, M
GONI, B.
GOTOH,O

GROPP, A
HANAKATA, A.

.42

73,74
HANAOKA, K.
57
HATA, T12, 13
HATTORI, M
13
HIMENO, M. .
24
HIROTA, Y
24,25,26,27,28,31,34
IMAI, H. T. .
68,69,71
INOUE, T. .
91, 94, 95
INOUE, Y
105, 116ISONO, K
79
IYAMA, S
119, 120JIKUYA, H. .
20,21
KADA, T.
91,92,93,94
KAMIMURA, T. .
12
KATO, J. 1.
'"
28
KAWAMUKAI, M
24
KIM, K. W. .
107
KIMURA, M
97, 98,101
KOJIMA, K. .
25
KOMANO, T • . . . . . . . . . . . . . . . . . . . . . . . . 24
KOMEDA, Y
29, 34KONDO, K. . . . . . . . . . . . . . . . • . . . . . . 69, 71

71
71
KURODA, Y
49,50,51,52,53,71
KUSANO, T
67
KUSUDA, J. .
22
MAKI, Y20
MARTJN, M. A . . . . . . . . . . • . . . . . . . . . . 21
MARUYAMA, 1.
28
MARUYAMA, T. .
101
MATSUDA, Y. .
69
MATSUNAGA, E.
109
MATSUTANI, E
50
MIGITA, S. .
45
MIKAMI, H.
53
MINATO, K.
59
MIURA, K. 1.
12, 13MIYAMURA, T
21
MIYASHITA, N
38, 39, 40, 45
MOCHIZUKI, H
94
MOODY, M . . . . . . . . . . . . • . . . . . . . . . . . . 103
MORISHlMA, H.
113, 114, 118
MORIWAKI, K.
36, 38, 39,40,
42,44,45,46,68,69
MURAKAMI, A. .
83, 84, 86, 88
NAKAGOME, Y. .
110
NAKAMOTO, Y
24
NAKAMURA, M
28
NAKANO, Y
20,21
NISHIMIYA, C
55
NISHIMURA, A.
30, 33NISHIMURA, Y. .
13
NISHIZAWA, K • . . . . . . . . . . . . . . . . . . 25,68
NOGUCHI, T
58
OHNISHI, S. .
107
OHTA, K
118
OHTA, T
98,99
OISHI, K
53
OKA, A.
26, 27
OKA, H. 1.
118
KONDO, M

KUBOTA, M

132

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 32

OKAMOTO,K. .

SADAlE, Y
SAKAI, H
SAGAI, T
SAKAIZUMI, M.

.

SANO, YSASAKI, H.
SATO, CSATO,

M

SHINOZAKI, K.
SHIROISHI, T.
SOEDA, E.
SOMA,

.

.

M

SUGIMOTO, K.
SUGIURA, M.

.

SUGIYAMA, T
SUN, C, R
SUZUKI, H
TAKAGI, T
TAKAHATA, N
TAKAIWA, F
TAKANAMI, M
TAKANO, J
TAKEDA, Y.

.

TAKEMOTO, K. K.
TANABE,

H

24
93
24
36
.45,46119
27
25, 5958
14, 1736
20, 21
28
26, 2714, 1755,56
48
28, 6728
100, 101, 102, 103
14, 17
26,27
56
24
20
24

H. "

"

20
103
TAYA, C.
44TERAI, K. .
114
THOW, Y. P
71
TOMIOKA, N
17
TSUBOI, M
13
TSUNO, K.
105
URUSHIBARA,1'. . . . . . . . . . • . . . . . . . . . . . 12
UTSUMI, R
24
WANG, C. H
45
WATANABE, T. K
53, 105, 107
WINKING, H
42
YAMADA, C.
17
YAMADA, M. ..
24, 26YAMAGATA, T. .
50
YAMAGUCHI, K.
12, 13
YAMAMOTO, A
107
YAMAMOTO, M
73,74,76,77,79,80
YASUDA, S
28
YOKOIYAMA, A
94
YONEKAWA, H
42
YONG, H. S
71
YOSHIIKE, K
21
YOSIDA, T. H
60, 61, 62, 63,
64,65,66,67
TASHIRO,TATENO, Y

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