ISSN 0077-4995

NATIONAL INSTITUT~ OF G~N~TICS
JAPAN

ANNUAL REPORT
No. 371986

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken. Japan1987

Annual Repor~of ~heNa~ional Ins~i~u~eof Gene~ics

No. 37, 1986

Published by

The National Institute of Genetics, Japan
1987

CONTENTS
General statement .
Staff
Council and Advisory Committee
Association for propagation of the knowledge of genetics .. ,Projects of research for 1986 .,Research activities in 1986 ..
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Molecular Genetics
The anatomy of Escherichia coli RNA polymerases: Mapping of thestructural domains involved in subunit-subunit interaction.
ISHIHAMA, A., GLASS, R. Eo, FUJITA, N. and NOMURA, T. .... 16
RNA polymerase sigma-related proteins in Escherichia coli. I.
Detection by antibodies against a synthetic peptide. FUJITA,
N., ISHIHAMA, A., NAGASAWA, Y. and UEDA, So .,. 0....... 17
RNA polymerase sigma-related proteins in Escherichia coli. II.
Heat-shock induction of sigma-32 synthesis. FUJITA, N. and
ISHIHAMA, A
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Stringent control signals of Escherichia coli: Determination of
signal strength in in vitro transcription. GLASS, R. E., ISHIHAMA, A., NOMURA, T. and FUJITA, N
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Expression of the leuX gene in Escherichia coli: Regulation at thetranscription and tRNA processing steps. NOMURA, To and
ISHIHAMA, A.
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Structure and function of genes for RNA polymerase binding proteins. I. Regulation of transcription of the SSP gene. SERIZAWA, H. and FUKUDA, R. .
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Structure and function of genes for RNA polymerase binding proteins. II. Examination of the physiological functions of SSP.
FUKUDA, R. and SERIZAWA, H.. . . . . . . . . . . . . . . . . . . . . . . . . .. 22
Molecular mechanism of transcription initiation by influenza virus
RNA polymerase. HONDA, A., MIZUMOTO, K., NAGATA, K.
and ISHIHAMA, A. .
00.... 23
Assignment of the physiological role of influenza virus NP protein.
UEDA, K., NAGATA, K. and ISHIHAMA, A. .
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

An in vitro system for transcription and replication of influenza
virus RNA. NAGATA, K., TAKEUCHI, K. and ISHIHAMA, A.

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Molecular cloning of cDNAs of influenza virus RNAs and theirexpression in mouse cells. YAMANAKA, K., NAGATA, K.,
HOSAKA, Y. and ISHIHAMA, A.
In vitro splicing of influenza viral mRNA. NAGATA, K. and ISHIHAMA, A.
Analysis of temperature-sensitive mutants of influenza virus. I.
Establishment of a system for quantitative analysis of viralRNAs. HATADA, E., MUKAIGAWA, J. and FUKUDA, R.
Analysis of temperature-sensitive mutants of influenza virus. II.
Analysis of influenza virus RNA synthesis in cells infected withts mutant viruses defective in RNA segments 1 and 8. HATADA, E., MUKAIGAWA, J., SHIMIZU, K. and FUKUDA, R.
Assignment of the function of nuclear factor I. NAGATA, K.,
MATSUMOTO, K. and HANAoKA, F. .
.
Organization of mitochondrial and nuclear DNA in cytoplasmic
male sterile rice. NAwA, S., SANO, Y. and FUJII, T.
DNA supercoiling enhances in vitro transcription of fibroin gene.
HIROSE, S., TABUCHI, H. and SUZUKI, Y. .
.
Participation of the upstream region of the fibroin gene in theformation of transcription complex in vitro. TSUDA, M.,
HIROSE, S. and SUZUKI, Y.
Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems. SUZUKI, Y., TSUDA, M., TAKIYA, S., HIROSE, S., SUZUKI, E., KAMEDA, M. and NINAKI, O.

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Diversity in G+C content at the third position of codons in vertebrate genes and chromosomal banding pattern. IKEMURA,
T. and AOTA, S.-I.
.
Nucleotide sequence and molecular evolution of mouse lAP elements. AOTA, S.-I., GOJOBORI, T. and IKEMURA, T. .
.

II. Microbial Genetics
On the process of cellular division in Escherichia coli: Penicillinbinding protein 3 of E. coli is a lipoprotein. HAYASHI, S.,

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CONTENTS

iii

HARA, H., SUZUKI, H. and HIROTA, Y. .
.
Conversion of the a component of penicillin-binding protein 1b
to the fi component in Escherichia coli. SUZUKI, H., KATO,
J.-I., SAKAGAMI, Y., MORI, M., SUZUKI, A. and HIROTA, Y.

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Molecular cloning of Bacillus subtilis gene involved in cell division,.
sporulation, and exoenzyme secretion. SADAIE, Y

III.

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Immunogenetics
Sexual preference of meiotic recombination within the H-2 complex. SHIROISHI, T., SAGAI, T. and MORIWAKI, K; ...
o. 38
Genetic polymorphism of a serum protein found in Japanese wild
mice. HARADA, Y., MORIWAKI, K. and TOMITA, T.
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Animal model for steroid 21-hydroxylase deficiency. GOTOH, H.,
SHIROISHI, T., SAGAI, T. and MORIWAKI, K. . 0. 0........... 40
The low polymorphic nature of the H-2 class I genes in Japanesewild mouse. SAGAI, To, SHIROISHI, T. and MORIWAKI, K.
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H-2 controlled genetic susceptibility to pulmonary adenomas induced by urethane and 4-nitroquinoline I-oxide in A/Wy congenic
strains. MIYASHITA, N. and MORIWAKI, K .. 0...• o' .... 00.. 43
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Developmental and Somatic Cell Genetics
Comparative studies on the antimutagenic actions of vitamin C and
its derivatives in cultured Chinese hamster cells. KURODA,
Y
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Combined effects of MMS and EMS after time lag treatment
on mutations in cultured Chinese hamster cells. KURODA,
Y., KOJIMA, H. and KONISHI, H ... 0.0 •.•.... 0....••. 0..... 46
Age-dependent accumulation of spontaneous mutations in humanembryonic organs. KURODA, Y. .. 0
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The fine structure of embryonic cells of Drosophila melanogastercultured in the presence of ecdysterone.
SHIMADA, Y. and
KURODA, Y. .
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Embryonic development of a maternally affected embryonic-lethal
mutant in Drosophila melanogaster. MINATO, K. and YAMADA, M. A
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Whole embryonic culture of spontaneous parthenogenetic diapauso' • • • • • • • • • • • • • •

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

ing-eggs in Bombyx mori. MURAKAMI, A., OHTSUKI, Y. and
KITAZAWA, T.
Studies on voltinism in Bombyx with special reference to extraembryonic membranes. MURAKAMI, A.
Genetic studies on voltinisms of a tropical race in Bombyx. MuRAKAMI, A.
Studies on the voltinism gene, pnd, in a tropical race of Bombyx.
MURAKAMI, A.
Rescue of the SRO-mediated male-specific lethal effect by cytoplasmic transplantation in Drosophila melanogaster. YAMADA, M-A and NAWA, S.
The mechanism of elimination of interstitial cell lineage in a mutant
hydra strain containing temperature-sensitive interstitial celllineage. TERADA, H. and SUGIYAMA, A.
Minimal critical size for hydra regeneration. SHIMIZU, H. and
SUGIYAMA, T.

V.

Cytogenetics
Theoretical bases for karyotype evolution. I. The mlmmum-interaction hypothesis. IMAI, H. T., MARUYAMA, T., GOJOBORI,
T., INOUE, Y. and CROZIER, R. H.
Polymorphism of a Y chromosome repeated sequence among
Asian Mus musculus and related European subspecies. BouRSOT, P. and MORIWAKI, K.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Are NORs (nucleolar organizer regions) necessary for the formation of Robertsonian translocations? KURIHARA, Y., WINKING, H., BONHOMME, F. and MORIWAKI, K. ..........•.....
Chromosomal locations of NORs (nucleolar organizer regions) instandard strains of the laboratory mouse. KURIHARA, Y.,
SUH, D. S. and MORIWAKI, K.
Effect of partial deletion of Y chromosome on morphology andprotein composition of mature mouse spermatozoa. STYRNA,J., SUR, D. S. and MORIWAKI, K.
Establishment of mouse strains congenic for mitochondrial DNA
for transplantation experiments. YONEKAWA, H. and TAYA,
C
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CONTENTS

VI.

VII.

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Mutagenesis and Radiation Genetics
Des-mutagenic action of refined corn bran. KADA, T., INOUE, T.,
HARA, M. and TAKEUCHI, M.
Cobaltous chloride, an antimutagen, enhances genetic recombination. INOUE, T. and KADA, T.
Bio-antimutagenic effect of L-ethionine on the spontaneous mutagenesis in Salmonella and Bacillus subtilis. INOUE, T. and
KADA, T.
Adenosine deaminase activity in wasted mouse, a radiation sensitive mutant. INOUE, T., TEZUKA, R. and KADA, T. . . . . . . .
Ultraviolet induction chromosome aberrations and separation inhibition in chromosomes of the rad-2 strain of Caenorhabditis
elegans. SADAIE, Y. and SADAIE, T.
A cytogenetic study on the radiosensitivity of bone marrow cellsof wasted mice. TEZUKA, R., TAMAI, K., INOUE, T. and
KADA, T.
The mode of action of vanillin, an food additive, on chemicallyinduced cytotoxicity and mutations in cultured Chinese
hamster cells. TAMAI, K., TEZUKA, R., KURODA, Y. and KADA,
T

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Population Genetics
Diffusion model of population genetics incorporating group selection, with special reference to an altruistic trait. KIMURA, M.
DNA and the neutral theory. KIMURA, M. .
Actual number of alleles contained in a multigene family.

T

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OHTA,

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Population genetics theory of multigene families with emphasis on
genetic variation contained in the family. OHTA, T. .
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Population genetics of an expanding family of mobile genetic
elements. OHTA, T. .
.
An attempt to estimate the effective size of the ancestral species
common to two extant species from which homologous genes
are sequenced. TAKAHATA, N.
Private alleles in a partially isolated population. II. Distributionof persistence time and probability of emigration. TAKAHATA,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

N. and SLATKIN, M.
A stochastic model of gene-culture coevolution suggested by the"culture-historical hypothesis" for the evolution of adultlactose absorption in humans. AOKI, K.. . . . . . . . . . . . . . . . . .
Stable polymorphic equilibria in a toy model of group selection.
AOKI, K.
Gene flow in natural populations of Drosophila melanogaster.
YAMAZAKI, T., CHOO, J-K., WATANABE, T. K. and TAKAHATA,N
The relationship between viability and number of P elements inthe second chromosome of Drosophila melanogaster. MUKAI, T., HARADA, K. and KUSAKABE, S.-I.

VIII.

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Evolutionary Genetics

Conservation of alleles and of genic heterozygosity in small managed populations. MARUYAMA, T.
Relative contributions of germline gene variation and somatic
mutation to immunoglobulin diversity. GOJOBORI, T. and
NEI, M.
A sequence homology between the pX genes of HTL v-lin andthe murine IL-3 gene. GOJOBORI, T., AOTA, S.-I., INOUE, T.
and SHIMOTOHNO, K.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions. NEI, M. and GOJOBORI,

T.

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Evolution of nested genes with special reference to cuticle proteinsin Drosophila melanogaster. MORIYAMA, E. N. and GOJOBORI,

T

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Higher rates of nucleotide substitution in Drosophila than in mammals. MORIYAMA, E. N. and GOJOBORI, T.
Expansion of Drosophila simulans in Japan. WATADA, M., INOUE,
Y. and WATANABE, T. K.
Computer study of molecular evolution. MIYATA, T.

IX.

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Human Genetics
Incidence of genetic disease in Japan. MATSUNAGA, E.
Paternity determination in two cases with hereditary disease.

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MA-CONTENTS

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TSUNAGA, E.
A T -+C transition in codon 110 of the human ,a-globin gene is a
novel cause of ,a-thalassemia phenotypes. IMAMURA, T., NAKASHIMA, H. and NARITOMI, Y. .
.
Distinct clustering of mitochondrial DNA types among Japanese,
Caucasians and Negroes. HORAI, S., GOJOBORI, T. and
MATSUNAGA, E
An apparent discrepancy between chain length and electrophoretic
mobility of restriction fragments: a case of human mitochondrial DNA. HORAI, S., INOUE, T. and MATSUNAGA, E.
Mitochondrial DNA polymorphisms in Japanese monkeys, Macaca
fuscata, derived from four local populations. HAYASAKA, K.,
HORAI, S., SHOTAKE, T., NOZAWA, K. and MATSUNAGA, E.

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Applied Genetics
Genic analysis of rice isozymes and their expression in calli. ISHIKAWA, R., KINOSHITA, T. and MORISHIMA, Ho o. o. o.
Latent differentiation toward the Indica and Japonica types in the
Asian common wild rice.
MORISHIMA, H. and GADRINAB, L.
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Gametic selection under different conditions in rice. SATO, Y. 1.,
ISHIKAWA, R. and MORISHIMA, H. .
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Ecological genetic studies in wild rice populations. BARBIER, P.
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and MORISHIMA, H.
Search for genes controlling photoperiodic sensitivity in rice using
linkage with isozyme genes. ISHIKAWA, Ro, SATO, Y. 1. and
MORISHIMA, H. .
95
Phylogenetic differentiation of the Japonica types of Asian cultivated rice. SATO, Y. 1. and MORISHIMA, H.
96
Wx protein of induced mutants in rice. SANO, Yo, KATSUMATA,
Mo and AMANO, E. .
96
Some comments on seed sampling from natural plant populations.
YONEZAWA, K. and ICHIHASHI, H. .
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Variations in protein fractions from rice endosperm. ENDO, T
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viii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

XI.

Database
Publications from Genetic Resources Section, Genetic Stocks Research Center. !YAMA, S.
101
Data base design of DNA base sequences. MIYAZAWA, S. .
102

Publication
Abstract of diary for 1986
Foreign visitors in 1986
Author index

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GENERAL STATEMENT
This report gives an outline of research activities carried out in our institute during the year 1986. The total expenditure for the FY 1986 (from
April 1986 to March 1987) amounted to 1 billion 76 million yen, 48 % ofwhich was for personnel expenses. In addition, grants-in-aid amounting
to 142 million yen were rendered to selected staff members by the Ministryof Education, Science and Culture. Changes in personnel allotments included new positions for three members approved in April; a research staff
member (joshu) for the DNA Research Center, and a technical and an administrative official for the Department of Administration. However, in
accordance with the 6th curtailment plan of the government, positions fortwo members were cut, so the total number of positions available for regular staff is 93 including 54 for research staff. Concerning facilities, construction work for the DNA Research Center started in July and the 80 per
cent of the building was completed by the end of the year; a greenhousefor plant quarantine and three automatic short-day paddy fields equippedwith dark chambers were also completed.
I am happy to note that six of our colleagues were honored in the past
year. Prof. Akira Ishihama was awarded the Inoue Prize by the Inoue
Foundation for the Promotion of Science for his "Studies on the transcriptional control of genes." Prof. Motoo Kimura was conferred an Honorary
Doctor of Science degree, from his alma mater, the University of Wisconsin,
for his world-famous contributions to population genetics. Prof. Tomoko
Ohta won the Avon Women's Grand Prix for her contributions to the promotion of women's social activities through her outstanding achievementin population genetics which serves as encouragment for younger women tofind career in science; Dr. Naoyuki Takahata was awarded an Encouragement Award of the Genetics Society of Japan for his "Theoretical studies
on population genetics on the molecular level." Prof. Yukinori Hirota
won the Fujiwara Award from the Fujiwara Science Foundation for his"Studies on the genes involved in bacterial cell division, and the foundationof a mutant bank of Escherichia coli." Finally, Dr. Ei Matsunaga, Director,
was awarded a Purple Ribbon Medal in the Emperor's autumn list of national awardees for his contributions to genetic and epidemiologic studies

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

on congenital anomalies and for his leadership in promoting human genetics
in this country.
We mourn the sudden loss of our two colleagues in the past year. Prof.
Tsuneo Kada died from esophageal cancer on November 14 at the age of 59,
and Prof. Y. Hirota from pneumonia following a stroke on December 23
at the age of 56.
While working in the institute for 19 years, Kada set up the researchsystem for the Laboratory of Mutagenesis and carried out a great deal ofpioneering research in his field. Among other things he developed andpropagated a simple new method (Rec assay), using repair-deficient mutants of Bacillus subtilis, to detect various environmental mutagens such as
AF-2 and phloxine which could not be detected by the conventional Ames
test. Both AF-2 and phloxine had been used widely as food additives at
one time in this country. Kada proposed the classification of antimutagenic
substances into "desmutagens" and "bio-antimutagens"; desmutagens refer to those substances which inactivate mutagens outside the cells, whilebio-antimutagens relate to factors that inhibit the process of mutation induction within cells. In so doing, he identified desmutagenic factors invarious fresh fruits and vegetables and bio-antimutagenic activities in CoCl 2
and green tea extracts.
Joining us at his post in 1973 from the Pasteur Institute, Hirota started
the K-project, aiming at elucidating all the genes of E. coli K-12, by picking
up every possible nitrosoguanidine-induced temperature-sensitive mutant.
He isolated thousands of independent mutants and characterized a numberof genes essential to cell division such as fts I. As soon as the recombinant
DNA technique was made available, Hirota cloned and determined thesequence of replication origin in E. coli. In recent years he initiated a joint
study to research all the cellular functions more comprehensively from theview point of cell division. He also had been a key person in the projectof nitrogen fixation in the rice rhizosphere.
With respect to personnel changes in the staff, Dr. Jun Kusuda of the
Invertebrate Section of the Genetic Stock Research Center, who had been
carrying out comparative studies on the molecular structure of the fibroin
gene in wild and domesticated silkworms, left in July for the National Institute of Health as a chief researcher to set up a human gene bank there.
Dr. Takashi Imamura of the Kyushu University was appointed as professor
for the Laboratory of Human Genetics in April, followed by the appoint-GENERAL STATEMENT

3

ment of Dr. Hitoshi Nakashima in Mayas a research staff member in thesame laboratory. Dr. Susumu Hirose, Associate professor, the National
Institute of Basic Biology, was transferred in June to the newly established
DNA Synthesis Section of the DNA Research Center. Dr. Toshihiko
Shiroishi, a research staff member, was transferred in July to the Laboratoryof Cytogenetics from the Mammalian Section of the Genetic Stock Research Center, his position being filled by Dr. Nobumoto Miyashita, agraduate student of Kanazawa University. Dr. Naoyuki Takahata, Laboratory of Population Genetics, and Dr. Toshitaka Fujisawa, Laboratoryof Developmental Genetics, were both promoted to associate professors inAugust. It is expected that the laboratories with newly appointed staff
will resume research activities as promptly as possible.
A working budget for a DNA data bank, which should be available forjoint use by researchers all over the country, was appropriated for the firsttime. Anticipating that the building for the DNA Research Center will
be completed by the end of January 1987, the budget for upgrading theelectronic computer was also appropriated. As in the previous year, Prof.
Takeo Maruyama, Dr. Sanzo Miyazawa and Dr. Takashi Gojobori continued to take charge of the construction and distribution of the DNA database, the development of analytical programs, and the issue of the DDBJ(DNA Data Bank of Japan) Newsletter (No.5). Laborious efforts weremade on the input of data and for selecting the type of computer. It is
expected that the project will get under way next spring with the appointmentof a new research staff member.
The Genetic Stock Research Center supplies, as far as possible, upon request of competent researchers in and outside of Japan, specific strains ofmouse, Drosophila, silkworm, E. coli, B. subtilis, etc. which are being kept
in the Center. The total number of cases supplied in 1985 amounted to 241(29 sent abroad) and the total number of strains supplied was 4723 (258 sent
abroad). Dr. Shin-ya Iyama continued to systematize data-base information about a variety of experimental organisms preserved in universities
and research institutes in Japan; in cooperation with other staff, he compiled the "Catalogue of Escherichia coli Genetic Stocks in National Institute of Genetics, 1986", updated the "Drosophila Genetics Stock List inJapan", and issued "Rice Genetics Newsletter No.2". These materials
were distributed among interested scientists in and outside of Japan.
On April 19th, the institute held its yearly open house for the public.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Some of the research activities of each laboratory were exhibited using
panels, microscopes, microcomputers, and molecular models. Scientificfilms were shown and a lecture, "How did ants become ants?", was given by
Dr. Hirotami T. Imai. The double cherry blossoms on the campus were attheir best and some 1300 visitors in the neighborhood enjoyed them. On
November 1, public lectures were given at the National Science Museum inTokyo; the titles were "Analysis of molecular mechanisms of cell proliferation in bacteria" by Dr. Seiichi Yasuda, and "Theoretical bases for chromosome evolution" by Dr. H. T. Imai. In spite of it being a Saturday afternoon about 150 eager people from universities and institutes listened to
the lectures, which were followed by rather specialized questions and lively
discussions. In addition, the 30th meeting of the directors of biomedical
research institutes, which are under the jurisdiction of the Ministry of Education, Science and Culture, or attached to national universities, took placein Mishima on October 27-28. Twenty-seven institutes participated anddiscussed various problems in advancing research activities under the present severe state of administrative and financial constraints.
International personnel exchanges were very active in the past year.
Members of our staff went abroad on 33 occasions for the purpose of presenting research results at various scientific meetings, exchanging information, and carrying out collaborative studies or field investigations, threemembers staying longer than 3 months. On the other hand, 41 scientists
visited our institute from abroad, with whom information and views on
recent studies were actively exchanged. Some distinguished guests, including Dr. Susumu Ohno of the City of Hope, delivered stimulating lectures at the Biological Symposium. Those who stayed longer than 3 months
and carried out collaborative studies with our staff members were: Dr.
Pierre Boursot, Universite Montpellier II, Miss Pascale Barbier, Universite des Sciences et Techniques du Languedoc, Montpellier, France; Dr.
Yi-de Huang, Shanghai Institute of Plant Physiology, the Chinese Academyof Sciences, and Dr. Yuan sheng Qiu, Guangdong Microbiology ResearchInstitute, the People's Republic of China; Dr. Lee Won Ho, Pusan National
University, Korea; Dr. Josefa Styrna, Jagiellonian University, Poland; Dr.
Tjan Kiauw Nio, Bandung Institute of Technology, Indonesia; and Dr.
Nguyen Xuan Hong, Hanoi University, Vietnam.
Three years have passed since the administrative category of our institutewas switched over to one for joint use by universities. Under the new sys-

GENERAL STATEMENT

5tern, we have accepted upon application, 33 collaborative programs, 13
workshops, 7 graduate students, 8 research fellows and 6 scholarships fromindustries, and 1 entrusted research from a private corporation. Yet,
there remain a number of problems to be solved in order to achieve thereality of reform. Among others, construction of the following facilities
is of the highest priority; an RI Center, which is indispensable for the expansion of research activities on the molecular level; a second main building
to accommodate laboratories for joint use by visiting professors, and lodgings for visiting researchers with welfare facilities. We are all eager to do
our best to accomplish the missions of the new institute. We hope to havecontinued encouragement and support from all persons concerned.

STAFF
Director

MATSUNAGA, Ei, D. Med., D. Sc.
Members

1. Department of Molecular Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
Laboratory of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUKUDA, Ryuji, D. Med., Associate professor
FUJITA, Nobuyuki, D. Sc.
NAGATA, Kyosuke, D. Pharm.
Laboratory of Mutagenesis
SADAIE, Yoshito, D. Sc., Associate professor
INOUE, Tadashi, D. Ag.
TEZUKA, Hideo
Laboratory of Nucleic Acid Chemistry (Guest member)
MIURA, Kin-ichiro, D. Sc., Professor
YAMANE, Kunio, D. Sc., Associate professor

2. Department of Cell Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
Laboratory of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami T., D. Sc., Associate professor
YAMAMOTO, Masatoshi, Ph. D.
SHIROISHI, Toshihiko, D. Sc.
Laboratory of Microbial Genetics
YASUDA, Seiichi, D. Sc., Associate professor
NISIDMURA, Yukinobu, D. Sc.
HARA, Hiroshi, D. Sc.
Laboratory of Cytoplasmic Genetics (Guest member)

STAFFSUZUKI, Hideho, D. Sc., Associate professor
YONEKAWA, Hiromichi, D. Sc., Associate professor

3. Department of Ontogenetics
KURODA, Yukiaki, D. Sc., Head of the Department
Laboratory of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
NAWA, Saburo, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
Laboratory of Phenogenetics
KURODA, Yukiaki, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Laboratory of Physiological Genetics (Guest member)
SHIMADA, Yutaka, D. Med., Professor
KIJIMA, Hiromasa, D. Sc., Associate professor
4. Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
Laboratory of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Professor
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKAHATA, Naoyuki, D. Sc., Associate professor
AOKI, Kenichi, Ph. D.
Laboratory of Evolutionary Genetics
MARUYAMA, Takeo, Ph. D., D. Sc., Professor
WATANABE, Takao K., D. Sc., Associate professor
TUTIKAWA, Kiyosi, Associate Professor
GOJOBORI, Takashi, D. Sc.
Laboratory of Theoretical Genetics (Guest member)
MUKAI, Terumi, Ph. D., D. Sc., Professor
MIYATA, Takashi, D. Sc., Associate professor
5. Department of Integrated Genetics
IMAMURA, Takashi, D. Med., Head of the Department
Laboratory of Human Genetics

7

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

IMAMURA, Takashi, D. Med., Professor
HORAI, Satoshi, D. Med.
NAKASHIMA, Hitoshi
Laboratory of Agricultural Genetics
MORISHIMA-OKINO, Hiroko, D. Ag., Professor
ENDo, Toru, D. Ag., Associate professor
FUJISHIMA, Tohru, D. Ag.
SATO-HIRAOKA, Yoichiro, D. Ag.
Laboratory of Applied Genetics (Guest member)

6.

Research Facilities
Genetic Stock Research Center
MORIWAKI, Kazuo, D. Sc., Head of the Center
FUJII, Taro, D. Ag., Associate professor
IYAMA, Shin-ya, D. Ag., Associate professor
MIYASHITA, Nobumoto, D. Med.
INOUE, Yutaka, D. Sc.
SANO, Yoshio, D. Ag.
NISHIMURA, Akiko
DNA Research Center
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Center
IKEMURA, Toshimichi, D. Sc., Associate professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
HIROSE, Susumu, D. Sc., Associate professor
Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira

7.

Technical Section
SUGIYAMA, Tsutomu, Ph. D., Chief of the Section

8.

Department of Administration
UEMATSU, Yoshihiro, Head of the Department
TAWARA, Koichi, Chief of the General Affairs Section
MATSUMURA, Tateo, Chief of the Finance Section

COUNCIL

9COUNCIL
Chairman
YAMAMURA, Yuichi; Emeritus professor, Osaka University
Vice-chairman
MOROHOSHI, Seijiro; Emeritus professor, Tokyo University of Agriculture and Technology
Members (Alphabetical order)
EGAMI, Nobuo; Director, National Institute for Environmental Studies
IINO, Tetsuo; Professor, Faculty of Science, University of TokyoINOUYE, Eiji; Emeritus professor, University of TokyoNAGAKURA, Saburo; President, Okazaki National Research Institutes
NAKAI, Junnosuke; President, Hamamatsu University School of
Medicine
NATORI, Reiji; Emeritus professor, Jikei University School of Medicine
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
OKADA, Tokindo S.; Director-General, Institute for Basic Biology
ONOE, Hisao; Director, Institute of Economic Research, Kyoto University
OZEKI, Haruo; Professor, Faculty of Science, Kyoto University
SAKAI, Fuminori; Director, Japan Society for the Promotion of Science
SASA, Manabu; President, Toyama Medical and Pharmaceutical University
SATO, Ryo; Director, Institute for Protein Research, Osaka University
TAZIMA, Yataro; Director, Institute of Silkworm Genetics and Breeding
WATANABE, Haru; Professor, Kitazato University
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto
University

ADVISORY COMMITTEE
Chairman
KIMURA, Motoo; Professor, Laboratory of Population Genetics
Vice-chairman
OHBA, Shigeru; Professor, Faculty of Science, Tokyo Metropolitan
University

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Members (Alphabetical order)
FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University ofTokyo
MUKAI, Terumi, Professor, Faculty of Science, Kyushu University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
OSAWA, Shozo; Professor, Faculty of Science, Nagoya University
SAKAGUCHI, Bungo; Professor, Faculty of Agriculture, Kyushu
University
SASAKI, Motomichi; Professor, Chromosome Research Unit, Facultyof Science, Hokkaido University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YAMADA, Yukio; Professor, Faculty of Agriculture, Kyoto University

IMAMURA, Takashi; Professor, Laboratory of Human Genetics
ISHIHAMA, Akira; Professor, Laboratory of Molecular Genetics
KURODA, Yukiaki; Professor, Laboratory of Phenogenetics
MARUYAMA, Takeo; Professor, Laboratory of Evolutionary Genetics
MORISHIMA-OKINO, Hiroko; Professor, Laboratory of Agricultural
Genetics
MORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
SUGIYAMA, Tsutomu; Professor, Laboratory of Developmental Genetics
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro; President, Emeritus professor of Tokyo Metropolitan
University
KURODA, Yukiaki; Managing Director, Professor of National Institute of
Genetics
SINOTO, Yosito; Manager
WADA, Bungo; Manager, Emeritus professor of University of TokyoTAZIMA, Yataro, Manager
OSHIMA, Chozo; Manager
MATSUNAGA, Ei; Manager, Director of National Institute of Genetics

PROJECTS OF RESEARCH FOR 1986
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular genetics

Studies on regulatory mechanisms of gene expression in E. coli (ISHIHAMA,FUKUDA and FUJITA)
Studies on molecular mechanisms of transcription and replication of animalviruses (ISHIHAMA, FUKUDA and NAGATA)
Laboratory of Mutagenesis

Molecular mechanisms of radiation- and chemical-induced mutations (KADA,SADAIE, INOUE and TEZUKA)
Environmental mutagens, desmutagens and antimutagens (KADA)
Biochemical factors involved in cellular repair of genetic damage and induced mutagenesis (INOUE and KADA)
Genetics of Bacillus subtilis (SADAIE and KADA)
Molecular mechanisms of unicellular differentiation in Bacillus subtilis(SADAIE)
Laboratory of Nucleic Acid Chemisry

Studies on the relationship between nucleic acid conformations and biological activities (MIURA)
Expression of B. subtilis genes (YAMANE)
Expression of the foreign genes in B. subtilis cell (YAMANE)
2.

DEPARTMENT OF CELL GENETICS
Laboratory of Cytogenetics

Studies on species differentiation of mouse from cyto- and molecular genetic
view points (MORIWAKI)
Cyto- and immunogenetical mechanisms for regulating tumor developmentin the laboratory and wild mice (MIYASHITA* and MORIWAKI)• Genetic Stock Research Center

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Immunogenetical studies on the mouse MHC (SHIROISHI and MORIWAKI)
Development of new mouse strains from wild populations (MORIWAKI andSHIROISHI)
Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Cytogenetical analysis of meiotic mechanisms in mice (IMAI and MORIWAKI)
Cytogenetical studies on Drosophila (YAMAMOTO)
Laboratory of Microbial Genetics

DNA replication in E. coli (YASUDA and HIROTA)
Cellular division in E. coli (HIROTA, NISHIMURA and HARA)
Penicillin-binding proteins in E. coli (HIROTA, HARA and NISHIMURA)
Laboratory of Cytoplasmic Genetics

Peptidoglycan biosynthesis in E. coli (SUZUKI and HIROTA)
Studies on cytoplasmic genes during subspecies differentiation of house
mouse Mus musculus (YONEKAWA)
3.

DEPARTMENT OF ONTOGENETICS
Laboratory of Developmental Genetics

Genetic analysis of developmental mechanisms in hydra (SUGIYAMA, FUJISAWA and SHIMIZU)
Studies on transformation and cell differentiation in higher organisms (NAWA
and YAMADA)
Mitochondrial DNA organization in male-sterile cytoplasms of rice (NAWA,
SANO and FUJII)
Laboratory of Phenogenetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on animal cells in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA)
Genetic studies on abnormal fertilization in the silkworm (MURAKAMI)
Mutagenesis in germ cells of the silkworm (MURAKAMI)
Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)PROJECTS OF RESEARCH FOR 1986

13

Laboratory of Physiological Genetics

Electron microscopic studies on differentiation of animal cells (SHIMADA
and KURODA)
Nerve network formation in Hydra (KIJIMA)
Genetic analysis of nervous system development in hydra (KIJIMA andSUGIYAMA)

4.

DEPARTMENT OF POPULATION GENETICS
Laboratory 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 on the evolution of multigene family (OHTA)
Theoretical studies on variation and evolution of extranuclear DNA (TAKAHATA)
Theoretical studies on the evolution of altruism (KIMURA and AOKI)
Population genetical studies on gene-culture coevolution (AOKI)Laboratory of Evolutionary Genetics

Theory of population genetics and evolution (MARUYAMA)
Studies on molecular evolution (GOJOBORI)
Evolutionary genetics of Drosophila (WATANABE)
Radiation genetics in mice (TuTlKAwA)
Laboratory of Theoretical Genetics

Theoretical and experimental studies of transposons in population of Drosophila (MUKAI)
Computer studies on the molecular evolution (MIYATA)

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Molecular genetics of growth and differentiation of human blood forming
cells (IMAMURA and NAKASHIMA)
Studies on DNA polymorphisms in human populations (HORAI and MATSUNAGA)
Genetic studies on retinoblastoma (MATSUNAGA and HORAI)
Mitochondrial DNA restriction analysis of non-human primates (HORAI,
MATSUNAGA, HAYASAKA, SHOTAKE and NOZAWA)
Laboratory of Agricultural Genetics

Evolutionary studies in wild and cultivated rice species (MORISHIMA and
SATO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic studies on rice reserve proteins (ENDo)
Behavioral genetic studies in animals (FUJISHIMA)
Laboratory of Applied Genetics

Methodology for the conservation of genetic resource populations (YONEZAWA)
6.

RESEARCH FACILITIESGenetic Stock Research 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 Gramineae (FUJII,SANO and IYAMA)
Studies on genetic differentiation in rice (SANO)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Studies on gene transfer in Gramineae (SANO and FUJII)PROJECTS OF RESEARCH FOR 1986

15

Documentation of genetic stocks in Japan (lYAMA)
Studies on the management system of genetic stocks information (IYAMA)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Molecular studies on the origin of silkworm (KUSUDA)
Coordination of DNA synthesis and cell division in E. coli (NISHIMURA)
Synthetic ColEI plasmids carrying genes for cell division in E. coli (NISHIMURA)
Development of mouse embryo freezing system (SHIROISHI, MIYASHITA andMORIWAKI)
Molecular mechanism of high frequency recombination in mouse MHC
region (SHIROISHI and MORIWAKI)
Genetic mechanisms for regulating tumor development in the laboratory
and wild mice (MIYASHITA and MORIWAKI)
DNA Research Center

Regulatory mechanisms of gene transcription (lSHIHAMA)DNA data analysis (MARUYAMA)
Studies on codon usage (IKEMURA)
Studies on RNA molecules of mouse brain (IKEMURA)
Database design for DNA base sequences (MIYAZAWA)
Control of gene expression in eukaryotes (HIROSE)RESEARCH ACTIVITIES IN 1986
I.

MOLECULAR GENETICS

The Anatomy of Escherichia coli RNA Polymerase:
Mapping of Structural Domains Involvedin Subunit-Subunit Interaction
Akira ISHIHAMA, Robert E. GLASS*, Nobuyuki FUJITA
and Teruaki NOMURARNA polymerase of E. coli is a multimeric enzyme consisting of a core
enzyme with a subunit composition of (X 2(3(3, and one of the sigma factors.
The holoenzyme is assembled in a step wise fashion under a defined pathway:(X+(X~(X2~(X2(3~(X2(3(3'~(X2(3(3'a.
Subunit-subunit interaction is absolutely
required for the expression of intrinsic activities associated with RNA polymerase. An understanding of protein-protein interaction is therefore ofparamount importance to the structure-function characteristics of this complex enzyme.
To detect RNA polymerase subunits and assembly intermediates in wholecell lysates, we empoyed a combination of immunoprecipitation of radiolabeled cell lysates against anti-subunit antibodies and polyacrylamide gelelectrophoresis of immunoprecipitates. Using this method, we detected not
only intact subunits but also various degradation fragments of RNA polymerase in mutants defective in the assembly of RNA polymerase (Ishihama,
A. et al. (1987) Proteins, in press). The subunit fragments detected differedamong the assembly-defective mutants. By analyzing the degradation patterns, we were able to estimate the intermediate species of RNA polymerase
assembly accumulated in those mutant cells. In addition, we found thatsome of the intact subunits were degraded during the stationary-phase ofcell growth, presumably due to conformational change of preexisting RNA
polymerase as well as to inhibition of the assembly of newly synthesizedsubunits.
* Department of Biochemistry, Queen's Medical Centre, Nottingham, England.

RESEARCH ACTIVITIES IN 1986

17

In order to detect unassembled subunits and assembly intermediates in
unlabeled cell lysates, we further developed a rapid and sensitive method
using the same collection of anti-subunit antibodies. In brief, whole celllysates of unlabeled cells are fractionated by glycerol gradient centrifugation
under non-denaturing conditions; glycerol fractions are separated on polyacrylamide gel electrophoresis under denaturing conditions; proteins aretransferred onto nitrocellulose filters; and, finally, filters are immunoblottedagainst a series of different anti-subunit antibodies (Ishihama, A. et al. (1987)
Proteins, in press; Fujita, N. et al. (1987) Mol. Gen. Genet., in press; Glass,
R. E. et al., submitted for publication). The same filters can be reused toimmunostain consecutively against a series of different antibodies. Our
procedure for studying protein-protein interaction can be generally applied
to a variety of studies.
Using this method, we found several pseudo-complexes containing degradation fragments of RNA polymerase. Analysis of these complexes permits the identification of the sites (or domains) involved in subunit-subunit
interaction. We then performed a systematic study to identify structural
domains on the 13 subunit involved in subunit-subunit interaction and analyzed the assembly of eleven N-terminal amber fragments of the 13 subunit
ranging in size from 97 to 23 % of the length of the inteact 13 polypeptide(1,342 amino acids) (Glass, R. E. et aI., submitted for publication). In thisway, we were able to define regions on the 13 subunit in binding a, p' and a.
This method was also successfuly employed for the identification of theheat-shock sigma factor, a 32 , and other sigma-related proteins in whole celllysates (Fujita, N. et al. (1987), Mol. Gen. Genet., in press).
RNA Polymerase Sigma-Related Proteins in Escherichia coli, I.
Detection by Antibodies against a Synthetic Peptide
Nobuyuki FUJITA, Akira ISHIHAMA, Yoji NAGASAwA*
and Susumu VEDA*

E. coli RNA polymerase is a complex enzyme consisting of the common
core enzyme subunits (a, 13 and 13') and one of the sigma factors. Eachsigma factor confers on the core enzyme an ability to recognize a specificgroup of promoters. So far at least three sigma factors have been identified,* Nippon Institute for Biological Science, Ohme, Tokyo.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

which are designated as (J70, (J60 and (J32 on the basis of respective molecularweight. The major sigma factor «(J70) is encoded by the rpoD gene and theholoenzyme containing (J70 recognizes the majority of E. coli promoters.
The minor species, (J32, is encoded by the rpoH (htpR) gene and the holoenzyme (E(J32) recognizes a small group of promoters, known as the promotersfor heat-shock genes. Previously, we purified two forms of the holoenzyme,
E(J70 and E(J32, and compared their promoter selectivities in the in vitro mixed
transcription system using our collection of E. coli promoters (Fujita, N.
et al. (1987), J. BioI. Chern., 262, 1855-1859). These studies showed that astrict difference exists between the two holoenzymes with respect to promoter
selectivity.
In order to detect other sigma-related proteins in E. coli, we prepared anantiserum against a synthetic tetradecameric peptide with a sequence,
DLIQEGNIGLMKAV, which is present in both (J70 and (J32. In a Westernblot analysis of whole cell lysates, the antiserum reacted specifically with
(J70, (J32 and, additionaly, with at least three proteins with apparent molecularweights of 75, 27 and 23 kilodaltons. Major portions of (J70 and (J32 wererecovered as associated forms with RNA polymerase, by glycerol gradientcentrifugation of whole cell lysates while the other three cross-reacting proteins were not associated with RNA polymerase at least under the conditions empolyed. Purification and characterization of these proteins are in
progress.
RNA Polymerase Sigma-Related Proteins in Escherichia coli, II.
Heat-Shock Induction of Sigma-32 Synthesis
Nobuyuki FUJITA and Akira ISHIHAMA

The induction of heat-shock genes in Escherichia coli is under positive
control of the rpoH (htpR) gene, which encodes (J32, one of the sigma subunits of RNA polymerase. Purified holoenzyme containing (J32 (E(J32) iscapable of transcribing heat-shock genes in vitro while E(J70 is virtually inactive (Fujita, N. et al. (1987) J. BioI. Chern., 262, 1955-1859). Likewise,
the genes transcribed by E(J70 can not be transcribed by E(J32.
A Western-blotting analysis using an antiserum raised against a tetradecameric peptide with the consensus sigma sequence revealed that the intracellular level of (J32 increases rapidly under heat shock (Fujita, N. et al. (1987),

RESEARCH ACTIVITIES IN 1986

19

Mol. Gen. Genet., in press). To reveal the induction mechanism of a 32 synthesis, we analyzed the transcription of the rpoH gene before and after heatshock treatment.
Sl nuclease mapping and reverse transcriptase mapping indicated that thetranscription of the rpoH gene is initiated, both in vivo and in vitro, fromtwo major (PI and P2) and one minor (P2*) promoters (Fujita, N. et al.
(1987), Mol. Gen. Genet., in press). The level of rpoH transcript from thedownstream P2 promoter increases rapidly when E. coli cells are transferredfrom 30 to 42°C, while the transcript from the upstream PI promoter remains
at a constant level. Under these conditions, the metabolic stabilities ofrpoH mRNAs are virtually unaffected. These observations indicate that
heat-shock induction of a 32 synthesis is due to an increased transcription of
the rpoH gene.
In vitro synthesis of rpoH mRNA was found to be absolutely dependent
on the major species of RNA polymerase holoenzyme containing a 70 but
not on the minor one (Ea 32 ). The mechanism of Ea 7°-dependent transcription enhancement of the rpoH gene by heat shock is being currently
analyzed.
Stringent Control Signals of Escherichia coli:
Determination of Signal Strength inin vitro Transcription
Robert E. GLASS·, Akira ISHIHAMA, Teruaki NOMURA
and Nobuyuki FUJITA

Several global control circuits responsible for regulating groups of genes
are present in Escherichia coli. Stringent control is clearly part of an adaptive response to a short supply of substrates for translation. The presence of uncharged tRNAs in the ribosomal decoding site leads to the synthesis of two unusual nucleotides, ppGpp and pppGpp, which are theeffectors of stringent control. Transcription in vitro of stringently controlledgenes by purified RNA polymerase is subject to repression by ppGpp (Kajitani, K. and Ishihama, A. (1985) J. BioI. Chern., 159, 1951-1957). Somemutations in the gene (rpoB) encoding the f3 subunit of RNA polymerase,
render E. coli insensitive to amino acid starvation, i.e., relaxed phenotype•

Department of Biochemistry, Queen's Medical Centre, Noittingham, England.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37(Nene, V. and Glass, R. E. (1983), FEBS Lett., 153, 307-310) and the mutants produce ppGpp-insensitive RNA polymerases (Glass, R. E. et al.
(1986), Mol. Gen. Genet., 203, 265-268). These observations indicate thatthe target for ppGpp is RNA polymerase. Among the stringently controlled genes, a GC-rich sequence known as 'discriminator' is conserved,
spanning the region between the -10 signal and the transcription start site.
It therefore appears that ppGpp-bound RNA polymerase recognizes thissingnal and as a result is unable to initiate transcription.
This year, we measured the quantitative effect of ppGpp on various E.
coli promoters with different discriminators, using both wild-type and relaxed RNA polymerases. This has allowed us to classify promoters basedon their susceptibility to ppGpp. With the wild-type RNA polymerase, thehierarchy of susceptibility to ppGpp was: rplJ, leuX>dnaQplo dnaQp2>trp,
alsS>lacUV5; but, with the relaxed RNA polymerases, it was: leuX, dnaQpl>rplJ, dnaQpl>trp, alaS>lacUV5 (Glass, R. E. et al. (1987) Mol. Gen.
Genet., in press). It was therefore concluded that the strength of the stringent control signal depends on the nature of the RNA polymerase as wellas that of the promoter. It is interesting to note that the relaxed RNA
polymerases are more sensitive to inhibition by ppGpp when measured
with a promoter such as the dnaQp2 promoter which is not known to besusceptible to ppGpp.
Expression of tbe leuX Gene in Escherichia coli:
Regulation at tbe Transcription and tRNA
Processing Steps
Teruaki NOMURA and Akira ISHIHAMA

The leuX gene of E. coli confers the Su+ phenotype and encodes a supressor,
tRNA, which inserts leucine at the amber codon. By mapping with Sl
nuclease and reverse transcriptase of the initiation sites for both in vitro
and in vivo transcription, we found that this gene was organized into a singlegene operon carrying its own promoter and p-independent terminator (Nomura, T. et al. (1987), J. Mol. Bioi., in press). In spite of the accepted concept that in E. coli cells, primary transcripts of tRNA genes are rapidly
processed into mature forms, we found that varying levels of the primaryleuX transcript were accumulated in wild-type cells with respect to tRNAprocessing enzymes, but that the levels of mature tRNA stayed rather con-

RESEARCH ACTIVITIES IN 1986

21

stant under the culture conditions analyzed.
The quantitative measurement of both primary transcript and maturetRNA revealed that transcription of this gene was under both stringent andgrowth rate-dependent controls. These observations indicate that the expression of the leuX gene is regulated not only at the transcription step but
also at the tRNA processing step.
Structure and Function of Genes for RNA Polymerase
Binding Proteins. I. Regulation of Transcription
for the SSP Gene
Hiroaki SERIZAWA and Ryuji FUKUDA

A set of transcription factors appear to participate in the control of transcription by interacting with RNA polymerase. Up to ten polypeptides
which associate with RNA polymerase and may modulate the function of
RNA polymerase during transcription have been identified in this laboratory.
We isolated some of them, and have been studying their effects on transcription in the in vitro system. We have also been trying to isolate their respective genes to perform genetic analyses and study their physiological functions.
Stringent starvation protein (SSP) is one such polypeptide. In the previous issue, we reported on the structural analysis and genetic mapping of the
SSP gene, as well as on the cloning of the gene (Fukuda et al., Mol. Gen.
Genet, 201, 151, 1985). This year, we studied the regulation of the expressionof the gene, and tried to identify its physiological functions.
As described previously, we have determined the 5' end of the in vivotranscript of the SSP gene, which was identical for both transcripts preparedfrom cells grown under normal, or stringently starved conditions. However, the consensus promoter sequences could not be found at the -10 and-35 regions upstream from the transcription initiation site (Serizawa andFukuda, Nucleic Acids Research, 15, 1153, 1987). In order to identify promoter signals of the gene, deletion mapping of the 5' flanking region was
performed to test promoter activity. For this study, various fragments ofthat region were fused to lacZ genes cloned in a plasmid, and the activity of.a-galactosidase was measured in the cells carrying the plasmids. As withordinary promoters of E. coli, a fragment 40 bp upstream from the transcription initiation site was essentially required. In addition, the presence ofan additional fragment 40 bp upstream from that essential region stimulated

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

the transcription seveal fold. This cis-acting region has AT clusters of 10
bp length, and the 3' half of the region is enriched with pyrimidine residues.
The DNA fragment containing this region exhibits abnormal mobility onpolyacrylamide gel electrophoresis. This cis-acting region did not stimulate
the in vitro transcription with purified RNA polymerase, suggesting a transacting factor(s) interacting with the region to enhance transcription.
Using purified RNA polymerase, we analysed the in vitro transcripts ofthe cloned SSP gene. We detected two kinds of transcripts, both of whichwere produced with RNA polymerase containing a 70 , but not with the enzyme containing a 32 • The 5' ends of both in vitro products were extremely
near to the site determined for the in vivo transcripts. The promoter activity
in vitro of the gene is only 1/20 of that of the lacUV5 promoter, and seemed
to be very weak compared to the activity in vivo, also suggesting the presenceof some transcription stimulation factor(s). We next investigated the effectof ppGpp on these in vitro promoters. One of them is inhibited by ppGpp,
whereas the other is not. At present we are trying to determine which of the
5' ends of the in vitro products is identical with that of the in vivo transcript.
Structure and Function of Genes for RNA Polymerase
Binding Proteins. II. Examination of the
Physiological Functions of SSP
Ryuji FUKUDA and Hiroaki SERIZAWA

To elucidate whether SSP is essential for cell growth or not, we tried toconstruct an E. coli strain in which SSP is not synthesized. As a preliminary
step, we constructed an E. coli strain in which SSP is synthesized under thecontrol of the lac promoter. In this strain, SSP is synthesized in the presence of IPTG, but not in the absence of the inducer, as shown by labelingthe cellular proteins and detecting SSP, using the anti-SSP antibody. As
such cells were able to grow normally in the absence of the inducer, it appears that SSP is dispensable for cell growth at least under normal culture
conditions. We are now isolating another deletion mutant of the SSP gene,and studying the effect of SSP deletions on cell growth, when the cells are
exposed to various stresses, such as amino acid strarvation or heat shock.

RESEARCH ACTIVITIES IN 1986

23

Molecular Mechanism of Transcription Initiation
by Influenza Virus RNA Polymerase
Ayae HONDA, Kiyohisa MrZUMOTO·, Kyosuke NAGATA
and Akira ISHIHAMA

The influenza virus genome consists of eight single-strand RNA segmentsof negative polarity. The RNA-dependent RNA polymerase associatedwith the RNP cores in virions is responsible for primary transcription of theviral genome into plus-strand mRNAs. The transcription initiation positions and the RNA polymerase-binding sites on viral RNAs were analyzed.
To determine the transcription initiation positions, we measured thepriming activities of trinucleotide formation by dinucleotides of all possible
base sequences (Honda, A. et al. (1986), J. BioI. Chem., 261, 5987-5991).
Dinucleotide ApG, complementary to positions 1-2 from the 3' termini ofviral RNA segments, was found to be most active primer. Dinucleotide
GpC, complementary over position 2-3, was also an active primer anddirected the formation of either GpCpG or GpCpA. However, both dinucleotide CpG and CpU, complementary over position 3-4, were virtuallyinactive. These results indicate that transcription is initiated within thefirst four nucleotides at the 3' termini of viral RNAs.
To determine the binding sites of RNA polymerase on the genome RNA
segments, the ribonucleoprotein (RNP) cores were isolated from detergenttreated virions by glycerol gradient centrifugation. Subsequently, RNApolymerase-RNA (P-RNA) complexes were isolated from RNP by centrifugation on a double gradient of cesium trifluoroacetate (CsTFA) andglycerol. The RNP cores and the P-RNA complexes were subjected to 3'terminal labeling with (32P]pCp and T4 RNA ligase, and then to foot-printing
analysis using double-strand-specific RNase VI and single-strand-specific
RNase T2. The results indicate that the 3' termini of viral RNAs form
intramolecular base-pairs with the respective 5' termini and that the RNA
polymerase is associated with a section approximately 18 bases from the 3'
termini of viral RNA segments (Honda, A. et al. (1987), J. Biochem., inpress).
In further experiments, RNA polymerase was dissociated from viral RNAsby centrifugation on CsTFA or CsCI gradients. The results of preliminary
reconstitution experiments supported the model that RNA polymerase re•Institute of Medical Science, University of Tokyo, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

cognizes and associates at the termini of viral RNAs.
Assignment of the Physiological Role of Influenza
Virus NP Protein
Kenji UEDA, Kyosuke NAGATA and Akira ISHIHAMA

Genetic studies have indicated that the three P proteins (PB 1, PB2 andPA) and the NP protein of influenza virus are absolutely required for viral
RNA synthesis. In purifying the viral RNA polymerase, we obtained the
RNP complex (a complex of all these proteins and viral RNAs) and the PRNA complex devoid of the NP protein (Kato et al. (1986), Virus Res., 3,115-127; Honda et al. (1987) J. Biochem., in press). The RNP complex
produces full-sized RNAs but the P-RNA complex only supports the synthesis of oligonucleotides. Reconstituted complexes from isolated P proteinsand viral RNAs were found to catalyze only the limited elongation reaction(Honda, A., unpublished observations). These observations suggest that
the NP protein is required for efficient elongation of RNA chains but notfor initiation of RNA synthesis.
To confirm this hypothesis and to identify the physiological role of the
NP protein, we established a transfection assay system of ribonucleoprotein complexes. For this purpose, mixtures of various RNA-protein complexes and DEAE-dextran were transfected into cultured cells and possible
production of infectious viruses was examined. Transfection of purified
viral RNAs did not produce viruses. On the other hand, the RNP complex
could produce infectious virus (Veda, K. et al., submitted for publication).
Infectivity of the P-RNA complex and the reconsituted RNA-protein complexes is presently being tested.
An in vitro System for Transcription and Replicationof Influenza Virus RNA
Kyosuke NAGATA, Kaoru TAKEUCHI* and Akira ISHIHAMA

We developed an in vitro system for transcription and replication of influenza virus RNA using isolated nuclei from influenza virus-infected HeLa
cells (Takeuchi, K. et al. (1987), J. Biochem., 101, 837-845). In this system,* Department of Measles Virus, National Institute of Health, Tokyo.

RESEARCH ACTIVITIES IN 1986

25

two species of positive-sense RNA transcripts, i.e., mRNA and cRNA(complete copy of vRNA) and one negative-sense RNA transcript, vRNA,
are synthesized. We carried out a detailed analysis of the molecular mechanism of the synthesis of positive-sense RNA transcripts.
While optimizing the isolated nuclear system, it was found that in thepresence of low concentrations of salt, both mRNA and cRNA were synthesized, while in the presence of high concentrations of salt, mRNA wasa predominant product. Thus, we prepared a salt-extract of nuclei (nuclear
salt-extract=NE) and salt-treated nuclei (L1N). The NE fraction mainly
produced cRNA while mRNA was a major product synthesized by L1N.
The NE fraction was further fractionated by centrifugation. The precipitate(NEP) exhibited mRNA synthesis activity but with the addition of thesupernatant fraction (NES) cRNA was the main product. The NES fraction
alone exhibited a low level of cRNA synthesis activity. These observations
strongly suggest that a regulatory factor present in the NES fraction is involved in the switch from mRNA to cRNA synthesis. Attempts are being
made to isolate and characterize the putative regulatory factor(s).
Molecular Cloning of cDNAs of Influenza Virus RNAs
and their Expression in Mouse Cells
Kunitoshi YAMANAKA, Kyosuke NAGATA, Yasuhiro HOSAKA*
and Akira ISHIHAMA

Recent studies have revealed that cellular immunity plays a crucial rolein protection against influenza virus infection. In order to identify protein(s)and epitopes which are recognized by cytotoxic T lymphocytes, we are trying
to establish a collection of cell lines, a single species of viral proteins by meansof transfection of a specific cDNA to influenza virus (RNAs).
Ifluenza virus RNA were extracted from purified virions by a SDSphenol method and full-length cDNAs were synthesized using synthetic
primers and reverse transcriptase. Double-strand cDNAs were joined toeither an EcoRI or Sma! linker, and then inserted into the respective cloningsite of the pSP65 vector. cDNAs encoding the M2 or NS2 proteins werealso constructed from full-sized M and NS eDNA, respectively.
For efficient expression of eDNA in mouse cells, a bovine papilloma
virus DNA was used as an expression vector. It was equipped with an* Research Institute of Microbial Diseases, Osaka University, Suita, Osaka.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

eukaryotic transcription promoter (either mouse metalothionein I promoteror SV40 early region promoter) including splicing signals as well as a signal
for poly(A) addition. Recombinants containing influenza viral cDNAs
were tranfected into mouse cells and their expression is presently being examined using immunological methods.
In l'itro Splicing of Influenza Viral mRNA
Kyosuke NAGATA and Akira ISHIHAMA

Segment 7 RNA of the influenza virus genome codes for at least two
polypeptides, MI and M2 which are translated from separate mRNA species.
Segment 8 also codes for two non-structural polypeptides, NSI and NS2,translated from separate mRNA species. MI and NSI mRNAs are unspliced primary transcripts of viral RNA segment 7 and 8, respectively,
whereas M2 and NS2 mRNAs are generated from MI and NSI mRNAs after
splicing. In influenza virus-infected cells, the ratio of spliced to unspliced
mRNAs changes depending on the time after infection. To elucidate themolecular mechanism of splicing and its control, we attempted to develop
an in vitro splicing system using nuclear extracts prepared from either influenza virus-infected or unifected human cells.
To prepare the substrate RNA for splicing, a segment 8-specific eDNA
was synthesized by AMV reverse transcriptase, cloned into a pSP vector andtranscribed in vitro by SP6 RNA polymerase. Nuclear extracts preparedfrom infected cells exhibited a low level of splicing activity and yileded specific splicing intermediates only when ATP was supplemented. Nuclear
extracts from uninfected cells, however, did not exhibit this activity. Theseobservations suggest that a factor(s) exists in influenza virus-infected cells
which affects the level of splicing. Studies to identify such a factor(s) arein progress.

RESEARCH ACTIVITIES IN 1986

27

Analysis of Temperature-sensitive Mutants of Inftuenza Virus. I.
Establishment of a System for Quantitative Analysis of Viral RNAs
Erika HATADA, Jun MUKAIGAWA* and Ryuji FUKUDA

To study the function of viral proteins participating in the transcription
and replication of influenza virus, we have been analysing a collection oftemperature-sensitive (ts) mutants of influenza AjUdornj72, which havedefects in viral RNA synthesis (Shimizu et al., Virology, 117, 38, 1982). In
the previous issue, we reported the changes in protein synthesis exhibited bythese mutants in MDCK cells. This year, we analysed viral RNA synthesis
in MDCK cells infected with these mutant viruses.
The genome of influenza virus A comprises eight RNA segments of negative
polarity. In the infected cell, three different types of viral RNA are synthesized, virion RNA (vRNA) and two types of transcripts ofvRNA, mRNAand cRNA (the template for vRNA replication). mRNA contains a 3'
poly (A) tail and a 5' capped end of 10 to 13 nucleotides derived from hostcell RNA primers, and is an incomplete copy of vRNA in that it lacks acopy of the last 17 to 22 nucleotides at the 5' end of vRNA. The other typeof viral transcript, cRNA is a complete copy ofvRNA, and is initiated without a primer. In addition, a portion of mRNA from segment 7 and 8 isprocessed to give two to three, and two species of spliced mRNA, respectively. Therefore 28 to 29 species of RNA in all are found in the infected
cell and synthesis of each species is regulated temporally during the infectious
process. To study the mechanism of viral RNA synthesis and its regulation,
it is essential to develop a system for measuring the quantities of these RNA
species individually. For this purpose, we established a quantitative hybridization system in which RNA probes intensely labeled with 32p wereused in a molar excess sufficient to overpower complementary RNAs present
in the viral RNA samples. To get the RNA probes, 170-500 bp framgentsindividual viral ds-cDNAs were recloned in the SP-6 vectors, and the DNA
fragments were transcribed with SP-6 RNA polymerase. The probes formRNA and cRNA were obtained by transcribing the SP-6 recombinantDNAs, which gave the intact 5' end sequence ofvRNA. As mRNA deletes
16 nucleotides at the 5' end of vRNA (see above), the RNA hybrid formed
with mRNA is shorter than the cRNA hybrid. We can thus discriminate* Tohoku University, School of Medicine, Sendai.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

cRNA from mRNA on polyacrylamide gel electrophoresis for base sequencing. Transcripts of the opposite strand of cDNAs give the probes forvRNA.
To digest out the single-stranded region of the hybridized RNAs, wefound nuclease T 1 is superior to nuclease Sl, which is required in large
amounts to digest RNA, and often cuts the double-stranded region of RNA.
As the nucleotide lengths of hybrids formed with each probe differ from one
another, the probes for eight segments can be combined into two groups.
We can thus measure the quantities of 24 RNA species in four test tubes.
In this way, we elucidated the time course of the synthesis of individual
RNA species in cells infected with wild-type virus with new results.
Analysis of Temperature-sensitive Mutants of Influenza Virus. II.
Analysis of Influenza Virus RNA Synthesis in Cells
Infected with ts Mutant Viruses Defective in RNA Segments 1 and 8
Eriko HATADA, lun MUKAIGAWA*, Kazufumi SHIMIZU**
and Ryuji FUKUDA

RNA segment I of influenza virus encodes the PB2 protein which is oneof the components of viral RNA polymerase and exhibits cap binding
activity.
One of the ts mutants of this segment, JCRC2?, exhibited severely reduced
synthesis of Ml and HA, which are classified as late proteins at nonpermissive temperatures. In contrast, no decrease was seen in the synthesis ofearly proteins, NP and three P proteins.
RNA segment 8 encodes two proteins, NSI and NS2 which are foundonly in infected cells but not in virions. NS2 mRNA is produced by splicing
NSI mRNA, but the functions of these proteins remains unknown. As
previously reported, two NSI mutants, ICRl629 and SPC45 (as determinedby nucleotide sequencing) exhibited greatly reduced synthesis of the MI
protein as well as the HA protein at the nonpermissive temperature of 40°C.
A reduced synthesis of NSI protein was also observed in both mutants, andNS2 synthesis was greatly decreased in SPC45, whereas reductions in thesynthesis of NP on the three P proteins were not obvious. Therefore, it* Tohoku University, School of Medicine, Sendai.
** Nihon University, School of Medicine, Tokyo.

RESEARCH ACTIVITIES IN 1986

29

appears that the two NSI mutants have a defect in late viral protein synthesis
as ICRC27 does.
No significant differences were seen in the viral protein synthesis for theNS2 mutant, ICR516.
Using the quantitative hybridization method as desribed above, we measured the quantities of mRNA, cRNAs and vRNAs for the eight genome segments which were synthesized in MDCK cells infected with these mutant
viruses. At nonpermissive temperatures, the syntheses of all vRNAs wereseverely reduced in three ts mutants, ICRC27, ICR1629 and SPC45, whichhave defects in late protein synthesis. The quantities of cRNA as well as
mRNA, except for those of NP and three P proteins, also decreased in thesemutants. The results indicate that the primary block in these mutants is in
the synthesis of cRNA or vRNA synthesis. Accordingly, the secondarytranscription which depends on vRNA synthesis, does not occur, resulting
in the defective synthesis of the late proteins. This suggests that NSI aswell as PB2 plays an essential role in the replicase complex. Strangely, thePB2 mutant exhibited an elevated synthesis of cRNA in segments 1 and 2
at permissive temperatures.
In the NS2 mutant, ICR516, the quantities of mRNA as well as cRNA
were not reduced, but the synthesis of all vRNAs decreased at nonpermissive temperatures, suggesting the participation of the NS2 protein at somestep of replication.
Assignment of the Function of Nuclear Factor I
Kyosuke NAGATA, Ken MATSUMOTO* and Fumio HANAOKA*

Nuclear factor I (NFl) has been purified as an essential factor involvedin the initiation of adenovirus DNA relpication and shown to be a site-specific DNA binding protein (Hurwitz, J. et al. (1985) In: Genetics, Cell Differentiation and Cancer, pp. 15-14). Using an in vitro reconstituted chromatin, we found that DNAse-hypersensitive sites, which are often found in
the transcriptionally active chromatin and the origins of some replicons,
were generated in the neighbourhood of NFl-binding sites. To gain insight
into the in vivo function(s) of NFl, we prepared a monoclonal antibody
against NF-I from HeLa cells and performed an antibody-blotting analysis(Western blotting) of various celllysates fractionated by gel electrophoresis.

* Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

In parallel, we carried out a DNA-blotting analysis (South-Western blotting)of the same cell lysates using specifically labeled DNA probes.
The protein-blotting analysis revealed that NFl or NFl-like protein waspresent not only in HeLa cells (human) but also FM3A cells (mouse) and
MDCK cells (canine), suggesting that NF-I is a ubiquitous and, therefore,
essential protein. Although the molecular weight of purfied NF-I has been
estimated to be 47 kdal, a cross-reactive protein with a molecular weight ofabout 100 kdal was also found in partially purified preparations of NFL
The interrelationship between the two proteins awaits further study.
Organization of Mitochondrial and Nuclear DNA in
Cytoplasmic Male Sterile Rice
Saburo NAWA, Yoshio SANO and Taro FUJIITwo supercoiled circular DNAs, B-1 and B-2, found in rice mitochondria
of male sterile cytoplasm, (ems-bora) rl,rl" were cloned in the plasmid PUC.
Using these as probes, the organization of mitochondrial and nuclear DNA
in regard to sequences homologous to B-1 and B-2 was studied. It was
found that the nuclear fraction from male sterile strains contained open
circle forms of B-1 and B-2. These oe existed irrespective of the presenceor absence of nuclear fertility-restoring genes. Although the supercoiled
forms were not detected, it is not clear at present whether the circular molecules were not present naturally or changed to oe during preparation procedures of the nuclear fraction. No such DNAs were present in nuclear
fractions from the strain having normal fertile cytoplasm, (n-boro)rl,rl" orfrom cytoplasmic revertants.
EeoR 1 digestion of the high molecular weight DNA of mitochondria
from ems strains gave four fragments homologous to B-1, while DNA fromthe normal strain and revertants lacked the most strongly labeled one of thefour fragments. In the cae of B-2 homologus sequences, EeoR 1 digestionof mitochondrial DNA from ems strains produced five fragments, while
DNA from the normal strain and revertants had only one of the five. Thus,
the reversion to fertility in the male sterile cytoplasm resulted in the loss ofsome of the mitochondrial chromosomal sequences homologous to B-1 and
B-2, as well as the loss of the free plasmids.
Sequences homologous to B-1 and B-2 were also present in the nuclear
chromosomal DNA. EeoR 1 digestion of nuclear DNA from ems strains

RESEARCH ACTIVITIES IN 1986

31

produced at least six fragments which hybridized to B-1, while five B-1
homologous fragments were produced from the normal strain and revertants.
Two of these fragments were conserved in both ems and the normal strain
or revertants. Common six fragments homologous to B-2 were obtainedin EeoR 1 digestion of nuclear chromosomal DNA from both ems and thenormal strain or revertants, with additional three fragments being obtained
from ems strains. These results show that a complex change in the organization of nuclear chromosomal sequences occurred in the cytoplasmic reversion. The presence of common DNA sequences in rice nuclear and mitochondrial genomes is also suggested.
DNA Supercoiling Enhances In Vitro Transcriptionof Fibroin Gene
Susumu HIROSE, Hisahiro TABUCHI and Yoshiaki SUZUKI*

In prokaryotes, DNA supercoiling is often an important requirement for
proper gene expression. But little is known in eukaryotes. Using cellfree systems of transcription, we tested whether topological tension is also
required for eukaryotic gene expression or not. A modified method for
Sl nuclease assay was developed to quantitate amounts of radioactive
transcripts in the presence of unlabeled homologus RNA. Using thismethod, we assayed in vitro transcription of the fibroin gene on circular andlinear templates. In a posterior silk gland cell extract, covalently closed
circular (ccc) DNA forms the superhelical state and supports three to 10
times more transcription than nicked circular or linear DNA does. (fordetails, see J. Bioi. Chem. 260: 10557-10562) A HeLa cell extract shows
neither the supercoiling nor the preference of ccc DNA over the linear one.
Upon fractionation of the posterior silk gland cell extract through phosphocellulose, both the flow-through and the 0.6 M KCI eluate fraction are
required for supercoiling and efficient transcription on ccc template in theacceptor HeLa cell extract. While the 0.6 M KCI fraction has DNA topoisomerase II activity, the flow-through fraction contains a supercoilingfactor which introduces the negative supercoiling into ccc DNA with theaid of topoisomerase II.

* Department of Developmental Biology, National Institute for Basic Biology, Okazaki.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Participation of the Upstream Region of the Fibroin
Gene in the Formation of Transcription
Complex In Vitro
Masaaki TSUDA"', Susumu HIROSE and Yoshiaki SUZUKI'"

In posterior silk gland extracts, a region covering about 200 base pairs of
DNA sequences upstream from the TATA box of the fibroin gene enhancestranscription efficiency. In an attempt to examine the mechanism of thisenhancement, the following studies were made. The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructedwith preincubation of the templates with the extracts, the inhibitory effectof histones was greatly reduced. Transcription of a fibroin gene construct,
pFb5'LI-238, with the upstream region beyond the TATA box was relatively
less inhibited than that of pFb5' LI-44 which lacks the upstream region. Thistendency toward differential inhibition was observed in silk gland extracts
but not in a HeLa cell extract and persisted even after complex formationin the silk gland extracts, suggesting a specific interaction of the upstream
region with some factors in the extracts. The complexes formed on pFb5'LI44 are probably more susceptible to the inhibitory effect of histones. These
results suggest a participation of the upstream region of the fibroin gene in
the formation of stable transcription complexes in the promoter through aninteraction with specific factors in the silk gland. Since the transcriptionenhancing effect via the upstream region is augmented at a high histone/DNA
ratio, it may mimic the in vivo situation in which the fibroin gene can betranscribed in the posterior silk gland even in the presence of excess suppressive materials. For details, see Mol. Cell. Biol., 6, 3928-3933.

'"

Department of Developmental Biology, National Institute for Basic Biology, Okazaki.

RESEARCH ACTIVITIES IN 1986

33

Tissue-Specific Transcription Enhancement of the
Fibroin Gene Characterized by Cell-Free Systems
Yoshiaki SUZUKI*, Masaaki TSUDA*, Shigeharu TAKIYA*,
Susumu HIROSE, Etsuko SUZUKI*, Mayumi KAMEDA*
and Osamu NINAKlt

An upstream region of the Bombyx mori fibroin gene enhances transcription efficiency. Six cell-free extracts of transcription were used to characterize the nature of DNA signals and trans-acting factors responsible forthe transcription enhencement of the fibroin gene. The upstream elementof the fibroin gene involved in the enhancement can be divided into two
regions. The proximal region, -72 to -32, is recognized as a common
enhancing signal by all B. mori extracts from the posterior silk gland, middle
silk gland, ovarian tissue, and an embryonic cell line. It is weakly recognized by an Antheraea silkworm cell line extract but not by a HeLa cell extract.
The distal region, -238 to - 73, appears to be a tissue-specific enhancing
signal that is recognized more effectively by the posterior silk gland extract
than by the middle silk gland extract. These observations suggest that theuse of these cell-free systems can offer a means for the biochemical characterization of the trans-acting factors involved in the tissue-specific regulation of the fibroin gene. For details, see Proc. Natl. Acad. Sci. USA, 83,9522-9526.
Diversity in G+ C Content at the Third Position of
Codons in Vertebrate Genes and Chromosomal
Banding Pattern
Toshimichi IKEMURA and Shin-ichi AOTA

In higher eukaryotes such as vertebrates, the codon-choice patterns of different genes in a single organism often differ significantly. Some genes are
extremely G+ C-rich at the codon 3rd position, whereas others are rather
A+ T-rich. We found that this G+C content of the 3rd letter was correlated to the G+C content of a large genome portion surrounding the gene;exons of genes with a high G+C %at the codon 3rd position are surroundedby G+C-rich introns and G+C-rich flanking sequences, and those with a* Department of Developmental Biology, National Institute for Basic Biology, Okazaki.
t Sericultural Experiment Station, Tsukuba.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

low G+C% at the position by A+T-rich introns and flanking sequences.
Analysis of G+C content distribution along DNA sequences using a DNA
Sequence Data Bank supported the view that the vertebrate genome is amosaic of regions with clear differences in their G+C content. The global
variation in the G+C content throughout human genome thus found was
attributed to the chromosomal G (Giemsa) banding or R (Reverse) banding,since the analysis on the GenBank sequence showed that the genes being
mapped on R bands in the Human Genetic Map (the 8th edition) usually
have the G+C content higher than 50% and those mapped on G bands havethe content lower than 50 %; on the basis of cytogenetic studies at the microscopic level, G bands have been believed to correspond to A+T-rich, latereplicating DNA segments, and R bands to G+C-rich early-replicating
ones. For details, see Nucl. Acids Res., 14 (1986): 6345-6355 and 8702.
Nucleotide Sequence and Molecular Evolution of
Mouse lAP Elements
Shin-ichi AOTA, Takashi GOJOBORI, and Toshimichi IKEMURA

We determined the nucleotide sequences of cDNA and genomic clones
for murine intracisternal type A particle (lAP) elements, which are retrovirus-like repetitive sequences in rodent genomes. The sequence of thecDNA resembled that of retrovirus RNA genomes in its lack of the U5
sequence within the 3' long terminal repeat. By sequence comparison of
our clones with reported rodent lAP elements, we located the probablegag, pol, and env gene regions. The sequences for the pol, env, and the 3'
two-thirds of the gag region were conserved among the lAP elements. In
the regions, synonymous substitutions occurred more frequently than nonsynonymous ones, which suggested that the regions in question were functionally constrained until fairly recently. The rate of nucleotide substitutionsin the regions was estimated to be 6-10x 10- 9 per site per year, and significantly higher than that of the cellular genes. These rates may exemplifya characteristic of the nucleotide substitutions for an endogenous retrovirus.
Gene (1987),56,1-17.

RESEARCH ACTIVITIES IN 1986

II.

35

MICROBIAL GENETICSOn the Process of Cellular Division in Escherichia coli:
Penicillin-Binding Protein 3 of E. coli is a Lipoprotein
Shigeru HAYASHI, Hiroshi HARA, Hideho SUZUKI
and Yukinori HIROTA

Penicillin-binding protein 3 (PBP-3) of Escherichia coli is a membraneprotein that plays an indispensable role in cell division. It functions in theformation of a septum of the peptidoglycan sacculus and is one of the lethal
tragets of p-Iactam antibiotics. During the examination of the primarystructure of PBP-3 deduced from the nucleotide sequence of the fts! gene
(Nakamura M. et al. 1983, Molec. Gen. Genet. 191: 1), we noticed the existence of the amino acid sequence, Leu-Leu-Cys-Gly-Cys, in the NH 2terminal region, which shows striking homology with the Leu-Leu-AlaGly-Cys consensus sequence for modification and processing of the precursor
forms of Braun's lipoprotein and other bacterial lipoproteins. These lipoprotein precusors are modified with diglyceride in the SH group of cycteineat the last position in the consensus sequence, cleaved between glycine andcysteine residues by a specific processing enzyme, and acylated with fatty
acid in the NH 2 group of the cysteine rsidue.
As expected from the existence of the consensus sequence, PBP-3 wascovalently modified in vivo with [2- 3 H]glycerol and (3H]palmitic acid. Glycerylcysteine residue resulting from the modification of cysteine residue withthioester-linked diglyceride was detected in the acid hydrolysate of purified
PBP-3, although such modification occurred only in a small fraction (about
8 %) of total PBP-3 molecules. Fatty acids incroporated into PBP-3 were
found in both mild alkali-labile and mild alkali-resistant linkages. Thus
PBP-3 contains fatty acids ester-linked to glycerol and amide-linked to the
NH 2-terminal cysteine residue. These results indicate that a portion of
PBP-3 is a lipoportein which has the same NH 2-terminal modification as
found in Braun's lipoprotein and other bacterial lipoproteins.
In Braun's lipoprotein, the fatty acid residues modifying the NH 2-terminal
cysteine serve to anchor this protein to the outer membrane. It is probablethat PBP-3 is also anchored to the outer membrane with its fatty acid-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

modified NH 2-terminus. PBP-3 has several highly hydrophobic sequencesin the COOH-terminal region and they are probably embedded in the cytoplasmic membrane. It seems likely that lipid-modified molecules of PBP-3
span the periplasmic space from the cytoplasmic membrane to the outermembrane. In this topography, the active site serine that occurs toward
the middle of the molecule (Houba-H6rin et al. 1985, Molec. Gen. Genet.
201: 499) would be located close to the peptidoglycan layer. PBP-3 is an
enzyme acting on the peptidoglycan sacculus to change its structure for
septum formation. The function of this enzyme might be regulated bymodification with lipids. It is possible that only PBP-3 molecules arranged
at the presumptive septal site are modified to become functional, and thus
the modified molecules could be a small fraction.
Conversion of the a Component of Penicillin-Binding
Protein Ib to the fi Component in Escherichia coli
Hideho SUZUKI, Jun-ichi KATO, Youji SAKAGAMI,
Masaaki MORI, Akinori SUZUKI
and Yukinori HIROTA

Penicillin-binding protein (PBP) I b of Escherichia coli is one of the essential enzymes involved in the process for extention of the murein sacculus.
This protein is composed of three components as detected among proteinsbound to (14C]benzylpenicillin in membrane fractions and the component
proteins are termed a, I' and r in order of decreasing apparent molecularweight. It has been suggested that the a and r components are the primary
products of the ponB (PBP I b) gene and that their difference arises from theexistence of different translation initiation codons within the same coding
frame.
To confirm the two initiation codons and to elucidate the origin of the I'
component, the a component and the I' component were purified as preparations uncontaminated by each other; the r component was isolated; andthe amino terminal sequences of the three components were determined.
The N-terminal amino acid sequences for a and r matched the reported
nucleotide sequences following the 1st and the 2nd ATG in the ponB coding
region, respectively, and also revealed that the methionine for translation
initiation was removed. The N-terminal sequence of I' indicated that I' isderived with the removal of 24 amino acids from the N terminus of a.

RESEARCH ACTIVITIES IN 1986

37

The amount of fi was found to increase with a simultaneous decrease in aduring incubation of membrane fractions. Therefore, the origin of fi wasinvestigated to see if this component might be a product incidental to the
preparation of membrane fractions. When the PBP 1b components wereisolated from cells subjected to instantaneous protein denaturation by direct
addition of trichloroacetic acid to the culture, essentially no fi component
was found. On the other hand, a substantial amount of fi was detectedaccompanied by a decrease in a, in PBP 1b prepared from membrane fractions. The results were essentially the same Jor cells harvested at different
growth phases, for those grown overnight, and for minicells; and led to theconclusion that fi was formed from a primarily after cell distruption. (Fordetails see J. Bacteriol. 169: 891-893, 1987).
Molecular Cloning of Bacillus subtilis Gene Involved inCell Division, Sporulation, and Exoenzyme Secretion
Yoshito SADAIE

The wild type div-341 gene of Bacillus subtiis was cloned with a temperate
phage rho-ll and recloned in a smaller temperate phage ¢105. The resulting transducing phage ¢105-div-341+ carries the Cfrl3I chromosomal
fragment containing the entire div-341 gene which is involved in cell division,
sporulation, exoenzyme secretion, competent cell formation, and autolysis(Mol. Gen. Genet., 190, 176-178; J. Bacteriol., 163, 648). The div-341 strain
carrying ¢105-div-341+ recovered wild type growth at restrictive temperatures
suggesting that the wild type allele is dominant over the mutant allele. The
div-341 mutational site is very close to restriction sites produced by AluI,
BcnI, HapII, HhaI, HinfI, MspI, EcoRI, EcoRV, HindIII, EcoT14I, MvaI,and TthHB8I as the transforming activity of the cloned fragment was completely lost with these restriction endonucleases.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

III.

IMMUNOGENETICS

Sexual Preference of Meiotic Recombination
within the H-2 Complex
Toshihiko SHIROISHI, Tomoko SAGAI and Kazuo MORIWAKIWe previously reported that the BIO. MOL-SGR congenic strain, H_2 wm7
haplotype of which was derived from Japanese wild mouse, exhibits marked
recombination enhancement within the H-2 complex (Shiroishi et al. Nature
300: 370-372, 1982). In contrast to the standard recombination frequencyof 0.3 % (Klein, 1975), a tenfold higher value was calculated between the
H-2K and H-2D marker loci, based on the screening of backcross progeny
from females heterozygous for the wm7 haplotype. In that experiment,
however, the recombination frequency was measured only in female mice.
Here we present recently obtained data on recombination frequencies measured in males that are heterozygous for the H_2wm7 haplotype. The results are
summarized in Table 1. In contrast to a higher recombination frequency
in female mice, only one recombinant between C57BLjlOJ and BIO. MOLSGR was detected in the screening of 650 progeny generated from heterozygous males. No recombinants were found in crosses of BIO. A X BIO.
MOL-SGR and AjWyx BIO. MOL-SGR, although heterozygous female
with the same combination of H-2 haplotypes, gave rise to a number of
intra-H-2 recombinants.
Table 1. Frequency of intra-H-2 recombination between H_2 wm7 and common
H-2 haplotypes of inbred mice in females and males.
Females
Heterozygous
Frequency
partner with
No.
BI0. MOL-SGR recombinants/±S.E. (%)
Total no. mice
BI0. A (H-2&)
BIO. BR (H-2 k )BI0 (H-2 b )A/wy (H-2&)
Total

16/539
5/174
8/204
3/117
32/1,034

3.0±0.7
2.9±1.3
3.9±1.4
2.6±1.5
3.1±O.5

Males
No.
recombinants/
Total no. mice

Frequency±S.E. (%)

0/301

0.0

1/176
0/173
1/650

0.5±0.5
0.0
O.2±O.2

RESEARCH ACTIVITIES IN 1986

39

The present data thus indicates that male recombination in heterozygotes
for the wm7 haplotype occurs at almost the same rate (0.2 %) as was obsserved in heterozygotes between the common H-2 haplotypes of inbred mice.
A genealogical study of recombination indicated that the post-meiotic stage
is not involved in the generation of sexual preference of recombination
enhancement, suggesting that the preference is a result of mexiotic-drive anda female specific mechanism is involved in the enhancement of meiotic recombination mediated by the H_2 wm7 haplotype. For details, see Immunogenetics 25: 258-262, 1987.
Genetic Polymorphism of a Serum Protein Found inJapanese Wild Mice
Yoshi-nobu HARADA, Kazuo MORIWAKI, and Takeshi TOMITA'"

Genetic polymorphism of a serum protein in mice was studied using gel
precipitation with alloantisera produced by reciprocal alloimmunization
between BALB/c and MOM (a stranin derived from Japanese wild mice,
Mus musculus molossinus). A protein migrating in the a region of serum
proteins was identified as alphaprotein-2B (APH-2B). A gene locus controllong the serological variation of the APH-2 molecule was designated as
Aph-2 (alphaprotein-2), in which two alleles were proposed, Aph-2b allele forthe positive reaction of APH-2B, and Aph-2 a for the negative reaction. The
strain distribution of APH-2B is shown in Table I. All common laboratory
strains examined in this study had Aph-2 a , while three strains, MOM, MolNis, and Mol-Tsm also had the Aph-2b allele. In addition the BFM/2Ms
strain established from European wild mice also possessed Aph-2b • APH2B was antigenically homologous to alphaprotein-IB (APH-IB), a serum
protein reported previously (Immunogenetics 24: 47-50, 1986).
Linkage analysis between the Aph-2 and Aph-l loci controlling APH-IB
was carried out using backcross progeny obtained from a cross of (BALB/c x
MOM)F 1 mated to BALB/c. No recombination between the two loci wasobserved in 307 backcross progeny. These results suggest that the Aph-2
locus is closely linked to the Aph-l locus.

'"

Faculty of Augriculture, Nagoya University.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37
Table 1. Strain distribution of the serum component APH-2B in different
strains of mice.
Phenotypesof APH-2B

AllelePositive
Negative

Aph-2 bAph-2 aStrains
MOM, Mol-Nis, Mol-Tsm, BFM/2Ms
A/HeN, AKR/J, AU/SsJ, BALB/cJ, BALA/cAn,
CBA/J, CBA/StMs, CE/J, CS, C3H/HeJ, C3H. OL,
C57BL/6J, C57BL/IOJ, C57BL/10Sn, C57L, C58/J,
DBA/lJ, DBA/2J, DD, DM, HRS/J, HTG, HTH,
HTI, ITES, lITES, IXBL, KK, KR, KR/C, KSB,
MOA, MOL-ANJ, MOL-MSM, MOL-NEM,
MA/MyJ, NC, NZB, NZW/San, P/J, PL/J, RFM,
RIIIs/J, SJL/J, SK/Cam, SM/J, SWR/J, WB/J, WN,
YBR, 129/J, 129/Sv

Animal Model for Steroid 21-Hydroxylase Deficiency
Hideo GOTOH, Toshihiko SHIROISHI, Tomoko SAGAI
and Kazuo MORIWAKI

So far, a number of H-2 recombinants have been produced from a crossofBlO. A and wild-derived BlO. MOL-SGR strains. One such recombinant,designated aw18, has a deletion of the complement component C4 and oneof the steroid 2l-hydroxylase genes, possibly due to unequal recombination.
The mouse homozygous for the aw18 haplotype is lethal in the neonatal
period (Shiroishi et al. Proc. Nati. Acad. Sci. USA, 84: 2819-2823, 1987).
To determine 2l-hydroxylase activity of animals homozygous for the
aw18 haplotype, we tested the level of serum l7a-hydroxyprogesterone (17OHP) which is a cortisol precursor for 2l-hydroxylase. A radioimmunoassay system using the l7a-hydroxy-progesterone radioimmunoassay-kit (CSISorin) was utilized.
Animals to be tested were generated from an intercross of aw18/b heterozygotes. We collected serum samples by heart puncture from 0.5-daynewborn mice, and determined the H-2 haplotype of these individuals bothby the cytotoxicity test for thymocytes and by the Southern blotting analysis
using a class II gene probe. To measure the level of l7-0HP, 5 ,ul of serum
was assayed for each individual.
The serum l7-0HP concentration of aw18/b and bib haplotypes ranged
from 2.0 ng/dl to 5.7 ng/dI, and was considered normal. No differences

RESEARCH ACTIVITIES IN 198641

were observed in 17-0HP levels between awl8/b and bib haplotypes. On
the other hand, awl8/awl8 indicated markedly high levels of serum 17-0HP,
ranging from 5.1 ng/dl to 46.7 ng/d/. These results clearly demonstrated
that the awl8/awl8 mouse is deficient for 21-hydroxylase, and the 21-0Hase
genes which still remained in the awl8 haplotype was biologically inactive.
In human, deficiency of steroid 21-hydroxylase is one of most common
inborn error of metabolism. The awl8 recombinant haplotype is expected
to provide useful and sole animal model for this disease.
Clinical characterization for C4 deficiency of awl8/awl8 homozygous
mouse is still unknown.
The Low Polymorphic Nature of the H-2 Class I
Genes in Japanese Wild Mouse
Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKIIn spite of extremely high polymorphism in H-2 class I genes, relatively
low polymorphism or oligomorphic features of class I genes has been observed in some geographically isolated populations of European and North
American wild mice (Klein, J., 1981). We made a serological survey ofJapanese wild mouse populations with respect to genetic polymorphism of
the H-2K antigen. This study revealed that two private alloantisera reacted
predominantly with Japanese wild mice. One of them, anti-H-2Kf, reacted with 34% of the mice tested. While another, anti-H-2Ku, reactedwith 19% of the mice. To more precisely characterize H-2Kf antigenictyof Japanese wild mice, we established monoclonal antibodies against the
H-2Kf antigen using the BlO. MOL-TEN1 congenic strain as an immunization donor. The H-2 complex of this strain was derived from the Japanesewild mouse having a previously determined H-2K locus of f haplotype. Of
11 monoclonal antibodies obtained, six appeared to be H-2 f specific using
the panel test on standard H-2 haplotypes of indbred mice. These 6 antibodies, however, showed different reactivities to wild mice. The other 5
antibodies exhibited crosseractivities with some inbred strains (Table 1).
With these antibodies, most Japanese wild mice testing positive to an antiH-2Kf private alloantiserum, H-2K.26, showed identical reaction patterns
to those of the BlO. MOL-TEN1 and BlO. M strain. An RFLP analysis
using two DNA probes which hybridize to the 3' and 5' ends of the H-2K
gene respectively, did not indicate any differences in autoradiogram patterns

.l>N

>
Z
Z

c::

>
t""
:;tl
tTl

Table 1. Similar reactivities with anti BI0. MOL-TEN1 H-2 class I monoclonal antibodies
in Japanese wild mice
Monocloal antibodies and Specificities
Mice

BIO. M and A. CA
BIO. MOL-TEN1
M. m. mol. Aziro
NiigataIiyamaShizuoka
Kyoto
Omiya
Mito-lMito-2

MS
51
f

+
+
+
+
+
+
+
+

MS
52
f

+
+
+
+
+
+
+
+

MS
53
f

+
+
+
+
+
+
+
+

MS
55
f

+
+
+
+
+
+
+
+

-

±

±

±

+

+

+

+

MS
56
f

+
+
+
+
+
+
+

MS
68
f

+
+
+
+
+
+
+

±

±

-

+
±

MS
54
f,p

+
+
+
+
+
+
+
+
+
+

MS
59
f,p

+
+
+
+
+
+
+
+
+
+

MS
60
f,Kb

+
+
+
+
+
+
+

-

n.t.

MS
61
f,s

+
+
+
+
+
+
+
+
+
+

MS
69
f,p,s

+
+
+
+
+
+
+
+

±

+

"tl

0

:;tl
>-I

Reactivities
to anti
H-2K.26

0

"r1

Z

>
::!
0

+
+
+
+
+
+
+
+
+
±

Z

>
t""
.....
Z

VI

::!

>-I

c::
>-I
tTl

0

"r1

0

tTl

Z

tTl

>-I
.....

(j
VI

z

9

t#>
......

43

RESEARCH ACTIVITIES IN 1986

among these wild mice. This wider distribution of stable H-2Kf antigenicity
suggests that the Japanese wild mouse population has a relatively low polymorphic nature in regards to the H-2K gene.
H-2 Controlled Genetic Susceptibility to Pulmonary
Adenomas Induced by Urethane and 4-nitroquinoline
I-oxide in AjWy Congenic Strains
Nobumoto MIYASHITA and Kazuo MORIWAKIThe genetic effect of H-2 complex on the development of chemically induced pulmonary adenomas was clearly demonstrated in H-2 congenic
strains with an AjWy background having a high susceptibility to pulmonary
adenomas. A single subcutaneous injection of either urethane or 4-nitroquinoline I-oxide (4NQO) was given to nine strains of mice. Among them,
the number of adenoma foci per mouse was significantly higher in AjWy
(haplotype H-2&), AjJ (H-2&), ATL (H_2 t1 ) and A.AL (H-2&1), than in ABY(H-2 b ), A.SW (H-2S), ACA (H-2 f ) and A.TH (H_2 t2 ) strains. In addition,
Table 1. Effect of H-2 complex on urethane- or 4NQO-induced pulmonary
adenoma in H-2 congenic strains with an A/Wy background
Urethane treatment')
StrainA/WySnJ
A/J
A.SW
A.TH
A. AL
A. TL
A.CA
A.BY
BI0.A

Tumor No.,H-2
haplotype mean±S.E.M.

a
a
s
t2
al
tl
f
b
a

26.8±1.3
30.6±1.4
12.5±0.8*b)
18.0±1.0*
26.3±1.3
29.0±1.5
16.8±0.6*
13.8±0.6*
1.2±0.2*

4NQO treatment a)

Incidence:
No. of micewith tumors/
total no. ofsurviving mice

Tumor No.,
mean±S.E.M.

Incidence:
No. of micewith tumors/
total no. ofsurviving mice

35/35
24/24
50/50
39/39
36/36
20/20
58/58
58/58
20/26

9.5±2.1
9.0±2.2
5.0±0.5*5.4±1.1*
11.0±2.99.0±1.7
4.8±0.9*
5.3±0.9*
NTo)

11/13
10/13
24/25
20/26
11/11
14/14
30/37
12/13
NT

Mice received an injection of 1.5 mg urethane/g of body weight or 12.5 f'g 4NQO/gof body weight.
b) Student's t-test was applied. *P<O.OOI, in comparison with A/Wy strain.
0) Not tested.
a)44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

the average number of adenoma foci in A/Wy (H-2&) was more than 20times that in the BlO. A (H-2&) strain (Table 1). Thus, multiplicity of adenoma foci appeared to be regulated by at least two genes, one located in
the I or S region in the H-2 complex and the other in the non-H-2 genetic
background. The genes in the H-2 complex were distinct from that for
pulmonary adenoma susceptibility (Pas)-!. For details, see lpn. I. Cancer
Res. 78: 494--498 (1987).

RESEARCH ACTIVITIES IN 1986

IV.

45

DEVELOPMENTAL AND SOMATIC CELL GENETICS
Comparative Studies on the Antimutagenic Actions ofVitamin C and its derivatives in Cultured
Chinese Hamster Cells
Yukiaki KURODA

In a previous study, it was reported that vitamin C had strong inhibitory
effects on the cytotoxic action and 6-thioguanine (6TG) resistant mutations
induced by ethyl methanesulfonate (EMS) in cultured Chinese hamster V79
cells. In the present study, the effects of vitamin C and its derivatives oncytotoxicity and mutagenicity of EMS were compared in Chinese hamster
V79 cells.
EMS had a cytotoxic effect on the cells, showing an LD~o of 541 pg/m/.
In the presence of vitamin C or its derivatives at concentrations of 50 or
100 pg/m/, the cytotoxicity of EMS expressed at LD 50 was reduced to morethan 1,000 pg/ml for vitamin C, and 639 pg/ml for iso-vitamin C. Dehydro-vitamin C enhanced the cytotoxicity of EMS, giving an LD 50 of 234 pg/
mi. EMS showed a strong activity for inducing 6TG-resistant mutations
in V79 cells. At a concentration of 1,000 pg/m/, EMS induced 6TG-resistant mutations at a frequency of 88 x 1O-~. Vitamin C decreased EMSinduced mutations by 2/3-3/4. Dehydro-vitamin C and iso-vitamin C also
decreased the EMS-induced mutations by 1/2 to 2/3.
When cells were treated with vitamin C after treatment with EMS, theinduced mutation frequency was not affected, while pretreatment withvitamin C was effective in reducing EMS-induced mutations. Vitamin C
mixed with EMS and incubated for 2 hours in the absence of cells, also hada strong effect in reducing mutations. This suggests that vitamin C wasdesmutagenic rather than antimutagenic against induction of mutations by
EMS.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Combined Effects of MMS and EMS after Time Lag
Treatment on Mutations in Cultured Chinese
Hamster Cells
Yukiaki KURODA, Hajime KOJIMA * and Hiroaki KONISHl*

It was reported in the previous study that the combined effects of simultaneous treatments with methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on 6-thioguanine (6TG)-resistant mutations in Chinese
hamster V79 cells were additional or synergistic, depending on the sequenceof treatments with both chemicals.
In the present work, to elucidate more details concerning the mechanismsof the combined effects of both chemicals, cells were treated with the two
chemicals successively at different times for 3 hours, or incubated in normal
medium for 3 hours between two treatments of both chemicals.
The sequence of treatments with two chemicals as well as incubation innormal medium markedly affected cell survival. Especially, in the case of
MMS treatment, cell survival after posttreatment with EMS showed agreater decrease than after pretreatment with EMS. In most cases, cell
survival after successive treatments was lower than that after simultaneous
treatments, and increased by incubation in normal medium for 3 hours between two treatments with EMS and MMS.
On the other hand, the induced mutation frequency after successive treatments was higher than that after simultaneous treatments, while the sequenceof treatments with two chemicals showed no detectable effect. Incubationin normal medium between two treatments with EMS and MMS decreased
induced mutation frequency, suggesting that a repair mechanism for mutational damages caused by the initial treatment may be operating during the
incubation period.

Age-dependent Accumulation of Spontaneous Mutationsin Human Embryonic Organs
Yukiaki KURODA

Many theories have been proposed on the mechanism of aging. In mostof these theories, it is suggested that some changers or modifications in the* Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., Ogaki, Gifu.

RESEARCH ACTIVITIES IN 1986

47

genetic information of cells may be related to the mechanism of aging. In
the present work, changes in the frequency of spontaneous mutations incells of organs in human embryos at various gestation stages were examined.
With the cooperation of several sanitary hospitals in the Mishima area,various organs such as lungs, hearts, livers, kidneys and skins were dissectedfrom artificially aborted human embryos at various stages ranging from 6
weeks to 24 weeks. These organs were dissociated into single cells by treatment with trypsin. Inocula of 105 cells in petri dishes were incubated in
8-azaguanine (8AG)-containing medium. The number of colonies of 8AGresistant mutant cells were counted.
Among cells obtained from various organs in 6 week embryos, only lungcells formed colonies in normal medium, whereas cells obtained from otherorgans did not form colonies. Cells obtained from other organs in embryos
at more advanced stages did form colonies. Lung cells showed the highest
colony-forming activity among cells obtained from various organs. The
colony-forming activity of lung cells obtained from various ages was compared. Such activity was low in cells obtained from embryos at early gestation stages, increased gradually with the development of embryos, reaching
its height in 13 week embryos and went down again to a low activity in cells
obtained from 24 week embryos.
On the other hand, the frequency of 8AG-resistant mutant cells was also
low in lung cells obtained from early embryos, reaching a peak in 13 weekembryos, and again went down in more advanced stages.
The ratios of the number of colonies of 8AG-resistant mutant cells to thoseof normal cells were compared among cells obtained from embryos atvarious stages. Ratios increased lineally with the progress of the embryonicdevelopment, indicating that mutations may be accumulated in normal organs
with the progress of embryonic development. Results also suggest that theratios obtained in a certain district may be an indicator of the all over strengthof mutagenic factors in that district.
The Fine Structure of Embryonic Cells of Drosophilamelanogaster Cultured in the Presence ofEcdysterone
Yutaka SHIMADA and Yukiaki KURODA

In a series of tissue culture studies of embryonic cells for Drosophila mela-

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

nogaster, we examined the fine structure of these cells cultured with ecdysterone.
Undifferentiated cells dissociated from post-gastrula embryos of the wildtype strain (Oregon-R of Drosophila melanogaster) were cultured in medium
K-17 supplemented with 15 %fetal calf serum, 0.1 ,Ltg/ml fetuin, and 10 ,Ltg/ml
ecdysterone, fixed in glutaraldehyde and osmium, and thin sections were
examined with an electron microscope.
In addition to cell types previously reported (muscle cells, nerve cells, andelectron-dark and light cells of unidentified nature), we observed the formation of cuticule and trachea in the cultured cells.
The cuticle was a homogeneous structure with a dense line lining multilayered lamellar structures. Each lamella was composed of simple epithelial-like cells. Between lamellae, obliquely running fibrillar structures
were found. Around the lamellae, dense cells with many vacuoles wereseen.
The trachea was a hellow-like structure found within parenchymal cells.
It appeared to be formed initially by a similar mechanism as that in thedevelopment of nerve myelin. The center of onion-like running membranes
disappeared to canalize the cell and, thus, the lumen of trachea was formed.
These results suggest that the fine structure of insect cells cultured withecdysterone my be useful in analyzing the more detailed characteristic actionsof specific genes during development.

Embryonic Development of a Maternally Affected
Embryonic-Lethal Mutant in Drosophilamelanogaster
Kiyoshi MINATO and Masa-Aki YAMADA

Embryonic development of a maternally affected embryonic-lethal mutant,fs(l) MAY-263 (Yamada, M. A., 1978), in Drosophila melanogaster, wasobserved by time-lapse photographs taken through an optical microscope.
Eggs laid by female flies homozygous for the above mutant gene developed
normally to the end of the cellular blastoderm stage, but thereafter the formation of the ventral furrow was very weak compared to a normal embryoand, hence, a little dorsal extension of germ band was oberved. Instead,many inward invaginations of the external layer were found dorsally andventrally in these embryos. Consequently, at the last stage of embryonic

RESEARCH ACTIVITIES IN 198649

developemt, they had some compartments formed by epidermal layers andhad less developed internal organs of the gut, trachea, and Malpighiantubule. Because the embryos moved actively within the vitelline membraneat that time, they appeared to have some muscle development. Although
the embryos never hatched, they had nearly consumed the yolks. Theseobservations suggest that the embryos die not from metabolic defects but
from some teratogenic anomalies.
Whole Embryonic Culture of Spontaneous Parthenogenetic
Diapausing-Eggs in Bombyx mori
A. MURAKAMI, Y. OHTSUKI* and T. KITAZAWA*

It is comparatively easy to artificially initiate the development of eggs
without fertilization in the Bombyx silkworm. So it is assumed that spontaneous activation commonly occurs in nature. In fact, we observed thatvirgin female moths frequently produced spontaneous parthenogenetic eggs
in almost all races of the silkworm. We reported that the frequency ofnaturally-occurring parthenogenesis is markedly different among several
strains, ranging from 3.6 % to 32.1 %, with rates flucutating from batch tobatch. Only a small proportion of the well developed eggs survive beyond
the diapause period. It is well known that when a normally-fertilized diapausing egg is taken from its chorion and explanted in Grace's medium,the embryo instantly begins to develop to the stage immediately preceding
hatching. In such a case, spontaneous parthenotes could be rescued byreleasing the diapause state and allowing development to the last stage.
In the present experiment, eggs laid from F 1 female moths in a cross J
106~ x Cambodge 6' were used, because females in this cross showed thehighest rate (32.1 %) of natural parthenogenesis in the silkworm. Whenvirgin females were placed on the egg-card, they began their oviposition a
few days after emergence and it lasted to about the 10th day. The eggs,
obtained in such a manner were kept at 25°C to make diapause proceed forabout 4-12 weeks after oviposition until the time when they were used for
culturing. For the culture the whole embryo and yolk, enclosed by theserosal membrane, was taken from the egg-chorion in a 0.85 % NaCI solution
and then cultured in hanging-drops of Grace's medium at 25°C tor 2 weeks
according to Takami et al. (1966) without renewing the medium. After* Sericultural Experiment Station, Tsukuba.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

cultivation for 2 weeks some eggs were dissected in 0.85 % NaCl solution
to examine the development of embryos.
Results showed that the growth of spontaneous parthenogenetic embryosin vitro varied among different ages of embryos after oviposition. Most four
to five week-old parthenogenetic diapausing-eggs grew to the stage of bristle
formation (or the late stage of morphogeneisis) as partial embryos, while
the majority of twelve week-old eggs developed completely at least up to thestage of bristle formation as normally-fertilized diapausing eggs did. A
few others grew to the stage of larvae ready to hatch. It is of interest tonote that the post-diapause embryonic development was observed even insome poorly pigmented parthenogenetic diapausing-eggs to an extent similar to well pigmented eggs.
The present finding and others clearly indicate that in whole embryo culture, spontaneous parthenogenetic diapausing eggs were able to be partly
rescured from unavoidable genetic death. It is also suggestive that naturalparthenogenesis is common in the Bombyx silkworm as it is in the order
Hymenoptera.
Studies on Voltinism in Bombyx with Special Reference
to Extra-Embryonic Membranes
Akio MURAKAMI

Diapausing eggs of the silkworm are easily reactivated by various means,electric shock, brushing, immersion in warm HCl solution, and so on, Umeya
1937, 1939) pointed out that prickling of diapausing eggs served as a stimulus for termination of diapause and some eggs immediately resumed theirpost-diapause embryonic growth. Also, deprivation of chorions from normally-fertilized diapausing eggs leads to the termination of diapause asreported by many researchers. In this insect the diapause phenomenon is
generally dependent upon maternal traits. The eggs of silkworms are surrounded by three main extra-embryonic membranes, chorion, vitelline membrane and serosal memberane. The former two membranes are maternal
products and the latter one is a zygotic product. On the other hand, allthree membranes in parthenogenetic eggs are maternal in origin differingfrom fertilized-eggs. In spite of such differences, when spontaneous parthenogenetic whole embryos are explanted in Grace's medium after they deprivation of chorions they instantly resume their embryonic growth as fertiliz-

RESEARCH ACTIVITIES IN 1986

51

ed diapausing-eggs do. This finding clearly suggests that the deprivation ofchorions may stimulate the termination of diapause, while the serosal membrane may not be concerned in the event. Various artificial treatments to
disrupt diapause may change the property of chorions by increasing thepermeability of oxygen into the embryo and yolk complex covered by serosal
membranes. It seems that mechanical wounding may modify physiologicalconditions of the embryo-yolk complex.
In the previous report it was found that most spontaneous parthenogenetic
diapausing-eggs in nature could not survive beyond the diapause period incontrast to fertilized diapausing-eggs. When parthenogenetic eggs were
provided with shelter prior to the termination of diapause, most of them wereable to develop to a fairly advanced stage as seen in the fertilized eggs. In
general, diapausing eggs of the silkworm are remarkably resistant to unfavorable environments because they are highly inactive physiologically. Although parthenogenetic eggs, which may have a certain genetic defect inserosal membranes, could not overcome an early step in the termination of
diapause in vivo, they could do so under in vitro culture conditions. Among
various liquid culture media, Grace's medium appeared the best sited forwhole embryonic development, probably owing to its pertinent osmotic pressure on the embryo-yolk complex rather than as a nutritional source. In
brief, a high mortality of spontaneous parthenogenetic diapausing-eggs innature may be caused by genetic defects in functions of a serosal membrane
and/or its secondary product, a serosal cuticle.
Genetic Studies on Voltinism of a TropicalRace in Bombyx
Akio MURAKAMI

A tropical polyvoltine race, Cambodge, produces generally nondiapausing
eggs in the Tropics throughout the year. In Japan, however, this polyvoltine
race is giving rise to moths which lay nondiapausing eggs during the mild
climate season, from spring to early autumn, under natural conditions, whilesome individuals are often giving rise to moths which lay diapausing eggs.
This latter phenomenon particularly appears in the early part of autumn, suggesting that this egg diapause is a physiological and developmental adaptation for increasing the probability of survival during periods of low temperatures or a shortage of feed in the Temperate Zone. In the climate of Japan,

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

it was once difficult to preserve such a tropical silkworm race, because of theshortage of mulberry leaves as feed. At present it is possible to keep various
tropical races throughout the year due to the development of an artificial
diet. Accordingly, the tropical race has been preserved with diapasuring
eggs having a low hibernating ability. An attempt to selecet and/or establish a highly stable polyvoltine line from the race Cambodge has been
made, taking advantage of the artificial diet. The experimental temperature
throughout the present study was constantly kept at 25°C and light conditions in the laboratory were controlled by natural sun light through two
small windows in addition to fluorescent lighting.
A highly stable polyvoltine line was obtained in the course of only three
generations. At the same time, a low line was also selected. However, thethe high line rarely produced nondiapause batches regardless of season.
The unstable line both diapause and nondiapause batches, 1106, Aojuku,
and ClO8 are typical divoltine races giving rise to moths which lay diapausing
eggs under the natural conditions in Japan. To further clarify the genetic
constitution of the tropical polyvoltine race, the F 1 eggs of a cross between
Cambodge females and 1106 males, were analyzed for the extent of hibernation. Results showed that most F 1 eggs were nondiapause. Similartendencies were observed in crosses between Cambodge females and malesof various divoltine lines, CI08, Aojuku, and Daizo.
When females of various lines were crossed with a fixed Cambodge male,the F 1 eggs were diapause without exception. These observations clearly
confirmed that a genetic behavior of voltinism in Cambodge is maternal inheritance as observed by Nagatomo (1926) and by Umeya (1925). The nondiapausing eggs laid by Cambodge females were colorless or non-pigmented
and consequently they were designated as a non-pigmented and nondiapause(npnd), while the color of diapausing eggs was yellow ocher, peculiar various
tropical races. The F 1 moths from a cross between Cambodge females and1106 males laid only diapausing eggs, while those of 1106 females and Cambodge males laid mostly nondiapausing eggs. The situation was also confirmed for several different crosses using males of various strains, such as
C108, Aojuku, and Daizo. These findings indicate that the nondiapause
trait depends on the male parent, suggesting that the genetic factor responsible
for the polyvoltine and non-pigmented egg traits in Cambodge is located on
the sex-chromosome of the Cambodge race as suggested by Nagatomo (1926)
and Umeya (1925).

RESEARCH ACTIVITIES IN 1986

53

Studies on the Voltinism Gene, pnd, in a
Tropical Race of Bomhyx
Akio MURAKAMI

In a previous study, two different lines, a highly stable polyvoltine line andan unstable polyvoltine line were developed from a tropical polyvoltine race,
Cambodge. The unstable line includes a line with a low polyvoltine tendency. In Bombyx, it has been shown that the Cambodge race is a stable polyvoltine mutant, which lays pigmented but non-diapause (pnd) eggs. It originated from an Indonesian polyvoltine race (Katsumata, 1968). Eggs, homozygous for pnd, are continuously nondiapause throughout the year regardless of environmental conditions, while heterozygous eggs (pnd/ +) arediapause. The gene having such voltinism is located on the 11th (pnd) and12th (pnd-2) linkage groups. Accordingly, it is of interest to analyze therelationships between the highly stable polyvoltine line in Cambodge andthe pnd gene.
When pnd females were crossed with highly stable polyvoltine line malesof Cambodge, most female moths laid diapausing eggs, but some laid nondiapausing eggs. As a matter of course, F 1 eggs of a reciprocal cross, Cambodge femalesxpnd males, showed non-hibernation depending on physiological conditions already experienced by females. Thus, it is clear that someCambodge lines preserved in our laboratory may have a pnd gene in heterozygous as well as homozygous states. Accordingly, it can be presumed
that genetic constitutions concerning nondiapause traits in Cambodge areexpressed as follows: Y/npnd: +/+, Y/npnd: pnd/+ and Y/npnd: pnd. The
first two lines may correspond to unstable lines, while the last one to thehighly stable polyvoltine line. The stable line would have been selected out
under either natural or laboratory conditions in Japan. Such being the case,the stock of Cambodge seems to have been maintained as Y/npnd: pnd/+
and Y/npnd: +/+. A similar situation would be observed for all stocks of
Cambodge in Japan, because this tropical race was originally introduced by
Dr. Y. Tanaka from Taiwan to the University of Kyushu in 1935 and subsequently transferred to the National Sericultural Experiment Station, fromwhich the stock has generally been distributed to several research institutions.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Rescue of the SRO-mediated Male-specific Lethal
Effect by Cytoplasmic Transplantation in
Drosophila melanogaster
Masa-Aki YAMADA and Saburo NAWA

Sex-ratio organisms (Spiroplasma SRO) are transovarially transmitted into
progeny of Drosophila and specifically kill male zygotes. It has been shownthat in crosses of females carrying SROs and ring X-males, fewer gynandromorphs were obtained than with controls and those obtained had only small
patches of XO-tissues (Tsuchiyama et al. 1978, Genetics 89, 711). SROs
microinjection experiments suggested that SROs affected male zygotes (withone X-chromosome) at early developmental stages (Omura-Tsuchiyama etal. 1980, lpn, I. Genet. 55, 484;
, 1981, ibid. 56, 628).
In order to study the male killing action of SROs, We tested whether or
not SRO-mediated lethal male zygotes were rescued by microinjection of
normal egg cytoplasms. About 2 nl (1-2 % of egg volume) of egg cytoplasm
was injected into SRO-infected eggs at the cleavage stage. Eggs carrying fourrecessive genes, y cv v f, on the X chromosome were used to discriminate
males rescued by co-transplanted female nuclei (with two X chromosomes).
In the injections with normal egg cytoplasm at the cleavage stage, 11 of 77
adults obtained (14 %) were males carrying SROs in hemolymphes. They
had no mosaics exhibiting normal characteristics, indicating that the males
had no cells with nuclei from the normal donor. In the case where the cytoplasm was injected into blastodermal embryos, no males were obtained.
On the other hand, in injection experiments either with cytoplasms of normal
non-cleavage eggs or with SRO-infected cleavage eggs, all adults obtained(131 and 126, respectively) were females. Injections of the cytoplasm of
SRO-infected cleavage eggs into normal eggs at the same stage was used asa control and females and males were obtained at a 1: 1 ratio.
These results suggest that the cytoplasm of normal eggs at the cleavage
stage contain a substance essential to the normal development of male zygotes,
but which is inactivated by SROs.

RESEARCH ACTIVITIES IN 1986

55

The Mechanism of Elimination Of Interstitial Cell
Lineage in a Mutant Hydra Strain Containing
Temperature-Sensitive Interstitial
Cell Lineage
Hiroyuki TERADA and Tsutomu SUGIYAMA

The interstitial cell lineage in the mutant strain sf-l hydra is temperaturesensitive. When these hydra are grown at a restrictive temperature (23°C
or higher) they lose nearly all interstitial cells and some of their differentiation products (nematoblasts, namatocytes, nerve cells and gland cells) within
48 hours. The mechanism responsible for this cell elimination process wasinvestigated.
Hydra were macerated, the resultant dissociated cells stained with Feulgen,counterstained with Fast green, and then examined for evidence of phagocytosis of interstitial cells by epithelial cells. In some epithelial cells maintained at the elevated temperature, the cytoplasm had large phagocytic vacuoles
that contained partially degraded cells, whose nuclei had highly-condensed
and intensely Feulgen-positive chromatin granules. This indicates that, as
in colchicine-treated (Campbell, 1976) or starved (Bosch and David, 1984)
wild-type hydra, epithelial cells in the mutant strain sf-l eliminate interstitialcells by phagocytosis.
The incidence of phagocytosis was higher in sf-l tissue maintained at theelevated temperature than that at the normal temperature. However, theobserved incidence was relatively low (maximally 0.14 phagocytosed cell
per epithelial cell) and appeared to be insufficient to account for the very
rapid loss of the interstitial cell lineage at the elevated temperature in thisstrain. We concluded that elimination of the interstitial cell lineage instrain sf-l takes place in part by phagocytosis and in part by other yetunidentified mechanisms (ef., Marcum et al., 1980).
Minimal Critical Size for Hydra Regeneration
Hiroshi SHIMIZU and Tsutomu SUGIYAMA

In hydra, a piece of tissue with approximately 1/30 of the total number ofcells of a normal polys can regenerate an entire organism while a smaller
piece cannot. Although hydra has a great capacity for regeneration, factors
which determine minimal tissue size necessary for regeneration are presently

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

unknown. In the present study we investigated in detail the process ofregeneration from a small picece of hydra tissue, in order to identify thefactor(s) determining minimal regeneration size and to gain instight into theunderlying principle of hydra pattern formation.
A small square tissue was excised from the body column of a wild strainof H. magnipapillata (l05) and allowed to regenerate at 18°C. The tissue
excised was initially a two layered cell sheet, consisting of an ectodermal andan endodermal layer. Some tissues disintegrated shortly after excision.
Within a day, others developed into spheres having a continuous ectodermal
layer outside, a continuous endodermallayer inside, and an empty cavity at
the center. The entire surface of these spheres was covered by mucous
material. Regeneration occurred from these spheres in the next several
days.
Twenty-four hours after excision, the diameter of the sphere was measured
by a micrometer under a dissecting microscope. It was found that the diameter of the smallest spheres produced was 0.2 mm. These spheres contained an average of about 250 epithelial cells. Various attempts to produce
spheres less than 0.2 mm in diameter all failed. This suggests that 0.2 mm
represents the minimal tissue mass necessary to form a stable sphericalstructure. Any tissue too small to form this structure apparently died anddisintegrat within 24 hr after excision, presumably due to ion leakage fromthe tissue.
Spheres ranging in size form 0.20 to 0.45 mm were all found to be capableof regenerating normal hydra. The rate of regeneration, however, varied
greatly, depending on the tissue size. It was about 5 % for the smallest size
(0.2 mm), gradually increasing as size increased, and reaching a value of 100%
at 0.45 mm. In addition, time required for regeneration also varied depending on tissue size, ranging from about 8 days for 0.2 mm to about 3.5 daysfor 4.5 mm.
These features of regeneration were compared to similar features in computer-simulated pattern formation processes based on the reaction-diffusion
mechanism.
In the reaction-diffusion system, consisting of a line of cells with homogeneous morphogen distribution, the gradients of the morphogens are notformed if the system size is below a finite critical limit. On passing throughthis critical limit, gradients of the morphogens appear with the following
two features. First, as the system size increases, the gradient level increases

RESEARCH ACTIVITIES IN 1986

57

in proportion to (L-L c), where L is the system size and L c is the critical limit
size. Secondly, the time steps required to reach the final steady state decreases in proportion to (L-L c). These two features are qualitatively similar
to the two experimentally obtained features of the regeneration describedabove, suggesting that a chemical reaction-diffusion mechanism plays an important role in hydra regeneration, and that 0.2 mm is close to the minimal
critiacal size for pattern formation.
It is probable that two independent factors are involved in determining theminimal size of hydra regeneration. One is the survival factor. Excised
tissue too small to form a stable spherical structure can not survive. The
other is the space factor for prepattern formation. Prepattern for regeneration (presumably by the reaction-diffusion mechanism) can not be established if the system size is too small. The spherical tissue mass 0.2 mm indiameter and containing about 250 total epithelial cells corresponds, within
the accuracy of the analyses, to the minimal limit for both factors in thestandard wild type strain.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37v.

CYTOGENETICS

Theoretical Bases for Karyotype Evolution
1. The Minimum-Interaction Hypothesis
Hirotami T. IMAI, Takeo MARUYAMA, Takashi GOJOBORI,
Yutaka INOUE, and Ross H. CROZIER

Recent literature has shown that the relative probabilities of occurrence forvarious categories of spontaneous chromsomal mutations do not match thosepredicted by either the random-contact-and-exchange model or the randombreakage-and -reunion model. These models do not take into account recent findings that, during the meiotic prophase and especially at pachytene,
eukaryote chromsomes are attached by each end to the nuclear membrane,
leading to a configuration we term the "suspension-arch structure".
We recalculated the relative probabilities of the occurrence of spontaneouschromosomal mutations given the suspension-arch structure and assumingthat these rearrangments arise from errors in the resolution of interlockings
between bivalents and a special type of crossover that we call the "heterosite" crossover. From these calculations we found that the relative probability of the occurrence of reciprocal translocations (the most fitnessdamaging rearrangement) declines with increases in chromosome number
and in nuclear volume. We also found that paracentric inversions occur
increasingly more oftern than pericentric ones as the centromere position
becomes more terminal and the distance between attachments to the nuclear
membrane becomes greater (this distance increases as nuclear volume increases). These results are in accord with cytogenetic data from Drosophila,
humans, and ants.
A puzzling phenomenon is that the relative rates of radiation-induced
chromosomal mutations differ greatly from those calculated under the suspension-arch structure model and from rates of spontaneous chromsomal
mutations. We propose the testable hypothesis that (1) most spontaneouschromosomal mutations occur in synaptonemal complexes and involve crossovers and errors in the resolution of interlockings, and (2) radiation andchemical mutagens allow rearrangements when chromsomes intersect at
any stage.

RESEARCH ACTIVITIES IN 1986

59

These considerations lead to the "minimum-interaction hypothesis", whichstates that karyotype evolution has been in large part shaped by selection to
reduce the occurrence of such fitness-reducting spontaneous chromosomal
mutations as reciprocal translocations. Some of the response to this selection is the result of an improvement in DNA-repair mechanisms, an increasein contraction of chromosomes caused by higher-order helix formation, andthe development of sex-chromosome heteropycnosis. We concentrate on
examining two further interacting responses in the light of this hypothesis.
One of these is increase in nuclear volume, but if the ratio of genome size tonuclear volume is high, then an increase in chromosome number, caused bysuch factors as centric fission, is adaptive because it recduces the occurrenceof reciprocal translocation. Although chromsome number can be reducedby centric fusion, such instance seem to be "back eddies" in the main streamof karyotype evolution. For details see Am. Nat. 128: 900-920 (1986).
Polymorphism of a Y Chromosome Repeated Sequenceamong Asian Mus musculus and Related
European Subspecies
Pierre BOURSOT* and Kazuo MORIWAKIPY353/B, a sequence specific to the mouse Y chromosome and representing
part of a small multisequence family (Bishop et al. 1985, Nature 315: 70-72),
was used to proble Southern blots of DNA in males from a variety of Asianmice. Sixteen wild derived strains (maintained at the National Institute of
Genetics) of various geographical origins (China, Korea, Taiwan, The Philippines and Indonesia) were tested. They represent a sample of the two parapatric Asian subspecies, M. m. musculus (northern type) and M. m. castaneus(southern type). Among the 13 restriction enzymes used, three (Eco RI,
Hae III and Hind II) did not show any interstrain differences. The otherten (Hind III, Bgl II, Kpn I, Bam HI, Taq I, Hinf I, Alu I, Hpa I, Pvu II
and Pst I) produced two patterns, one found in a strain from Bogor (Indonesia), the other found in all other strains. The unique pattern for the
first 3 enzymes and the pattern common to the 10 others were found identicalto those shown by a European M. m. musculus strain (MBT, from Bulgaria).
Five strains of Japanese mice of various origins ranging from Hokkaido to
Kyushu also displayed the same patterns.

* Laboratoire de Genetique, Institut des Sciences de J'Evolution, U.S.T.L., France.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Using enzymes that best discriminate Bogor from the other Asian strains(Pst I, Hap I, Alu I and Pvu II), we compared the Bogor strain to two otherM. musculus subspecies, M. m. bactrianus (one strain from Iran) and M. m.
domesticus (4 wild derived strains from England, Canada, France and Bulgaria). The patterns obtained were very similar, but small differences with
Hpa I, Pvu II and Pst I showed Bogor and bactrianus to be identical but
different from the 4 domesticus, which were indistinguishable among themselves.
The main conclusions are as follows:(1) Among the four subspecies of the complex species M. musculus,
pY353jB identifies two subsets which are well differentiated from each other:M. m. domesticus and M. m. bactrianus on the one side, M. m. musculus andM. m. castaneus on the other. Slight differences were detected between thetwo first subspecies, but the two last are indistinguishable with this probe.
(2) The Bogor strain, believed to belong to M m. castaneus, carries a M.
m. bactrianus Y chromosome. This indicates that genetic introgression has
occurred between these two subspecies in Indonesia.
Acknowledgments: We thank C. E. Bishop for providing the probe
pY353jB.
Are NORs (Nucleolar Organizer Regions) Necessary
for the Formation of Robertsonian
Translocations?
Yasuyuki KURIHARA, Heinz WINKING*, Fran~ois BONHOMME**
and Kazuo MORIWAKIThe standard karyotype of Mus musculus, including the laboratory mouse,consists of 20 pairs of acrocentric chromosomes, but some European wild
mice have a reduced chromosome number (2n=22-39) due to fusions of acrocentric chromosomes at their centromeres (Robertsonian translocations).
The mechanism which caused these centric fusions in European wild mice iscontroversial. In some cases, NORs, cytologically defined ribosomal RNA
genes, have been shown to play an essential role in Rb translocations in
man and mouse. From the accumulated data already reported, we examined all chromosome combinations so far involved in Rb fusions to de***Medizinische Universitat zu Liibeck, Federal Republic of Germany.
Universite Montpellier, France.

RESEARCH ACTIVITIES IN 1986

61

termine whether NORs are prerequisite for the formations of metacentricchromosomes in European w,ld mice. Statistical examination suggested
that NOR-bearing chromosomes are not necessarily required for the Rb
exchanges, but probably also inhibit them when two NOR-bearing chromosomes are concerned. This is inconsistent with the above hypothesis.
Previously we reported that some Rb-mice are probably an intersubspecies
hybrid between M. m. domesticus and M. m. bactrianus based on the restriction fragment length polymorphisms (RFLPs) of ribosomal RNA genes.
Further analysis of RFLPs in genomic DNAs of European wild mice carrying either the normal karyotypes or Rb-exchanges confirmed the uniquegenetic character of Rb-mice as such.
Chromosomal Locations of NORs (Nucleolar Organizer
Regions) in Standard Strains of theLaboratory Mouse
Yasuyuki KURIHARA, Dong Sang SUR'" and Kazuo MORIWAKIGenes coding for ribosomal RNA are cytogenetically demonstrated bysilver staining. On the average, a mouse has about 200 copies of ribosomal
RNA genes, which are repeated tandemly and located on several different
chromosomes. Their chromosomal locations have so far been reported only
in four laboratory mouse strains among hundreds. We attempted to survey
widely the distribution of NOR in various mouse strains.
Spleen cell cultures stimulated by concanavalin-A and lipopolysaccharide
were employed for the chromosome preparations. After chromosome identifications by G-banding, a silver staining procedure modified from Howell andBlack was applied to the samples. At least 35 well spread karyotypes wereanalyzed in each strain. As summarized in Table 1, chromsomal10cationsof NORs in the 10 inbred strains were restricted to five pairs of chromosomes,#12, #15, #16, #18 and #19, but the frequencies of the appearance varied. For
example, the 129/J strain had NORs on chromosomes #12, #16, #18 and #19.
Most cells (>80%) had NORs on #12, #18 and #19. Only 20% of the cells
had NOR on #16, in addition to #12, #18 and #19. Similar results were reported previously on chromosome #16 in the BALB/c strain. This phenomenon was explained by the reduced number of ribosome RNA genes on#16. Alternatively, we can also assume that the regulated expression of'"

Korea Advanced Institute of Science and Technology, Korea.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37
Table 1. NORs distributions and frequencies of appearance in inbred strainsof miceNo. ofkaryotypes
analyzed

Strain

SM/J
CBA/J
CE/J
C3H/J
129/J
RIII/J
SJL/J
I/J
WB/ReJ·W
NZB/BINJ

40
40
45
38
35
41
41
39
38
38

Chromosome number and appearanceof NORs (%)
10 11

12 13

14 15

67
90
93
31
85
82
89

16 17

96
95
86
97 68
20
83
73
67
38

18
94
98
42
67
83
70
96
77
37
93

19
41

87
96
91
89
89
66

ribosome RNA genes might cause this phenomenon.
Effect of Partial Deletion of Y Chromosome onMorphology and Protein Composition of
Mature Mouse Spermatozoa
J6zefa STYRNA*, Dong Sang SUH** and Kazuo MORIWAKI

Spermatozoa of males from the inbred mouse strains differ widely in thepercentage of abnormal heads and in efficiency of fertilization. The differences in sperm abnormality were found to be polygenically determinedby a small number of autosomal genes and the role of Y chromosome was
detected (Krzanowska H., 1976 Genet. Res. 28: 189). To get more insight
into the function of Y chromosomal genes, B1OBR/SgSn and BlOBR-ydel/Ms
congenic males originated from B10BR/SgSn by partial deletion ofY chromo
some were used for comparative morphological and biochemical analysis ofmature sperm.
The two congenic strains differ considerably with respect to the proportionof abnormal spermatozoa which amounts to 21.9 % in B10BR/SgSn and asmuch as 59.6% in BlOBR-Ydel/Ms strain. In the F 1 generation a heterosis
effect was observed if BlOBR/SgSn males were crossed to females from* Jagironian Univerity, Krakow, Poland.
** Korean Advanced Institute of Science and Technology, Seoul, Korea.

RESEARCH ACTIVITIES IN 1986

63

SJLjJ, AKRjJ. C3HjHeJ, CBAjJ and BALBjcAnN strain (0.6-3.6 % ofabnormalities) but not if BIOBR-YdeljMs males were used for matings (l9.962.7% of abnormalities respectively). It appeared that not only the total
percentage of abnormal sperm, but also the frequency distribution of abnormality classes were characteristic for a given strain. In BIOBR-ypeljMs
males the most frequent is class with flat acrosomal part (49.4 % of all abnormalities), but in BIOBRjSgSn males only 1.2 % abnormal heads belong tothis category.
Three distinct sperm fractions were recovered following density gradientcentrifugation in 1.6 M sucrose: SDS-soluble proteins mostly derived fromplasma membrane and acrosome, SDS-insoluble tail components and SDSinsoluble head components. A significant reduction of a major-39.000
molecular weight component in SDS-soluble fraction of BlOBR-YdeljMs
sperm was detected by polyacrylamide gel electrophoresis. Triple staining
procedure used for evaluation of site of acrosomal enzymes activity showeda failure of acrosome staining for about 30% of BIOBR-Ydel/Ms sperm.
The silver staining for proteinase activity was performed for sperm separated
by sedimentation in Percoll density gradient. Upper layers containing
mostly Y-bearing sperm exhibit 87.3 % negatively stained cells.
These results suggest that deleted fragment of Y chromosome contains
gene(s) exerts a regulatory function on the synthesis of acrosomal proteinase.
Low production of the proteinase can cause inability of Y-bearing sperm for
fertilization. This conclusion is also supported by evidence that progeny
derived from matings with BlOBR-YdeljMs males shows significantly distorted sex ratio with lower proportion of males (x2 =31.3 p>0.02).
High sperm abnormality in mutant males and absence of heterosis
effect indicate that a factor affecting head shaping of spermatozoa is also
located on the Y chromosome and can be separated from the other polygenes
influencing the same character.
Establishment of Mouse Strains Congenic for Mitochondrial
DNA for Transplantation Experiments
Hiromichi YONEKAWA and Choji TAYA*

Transplantation of cells and tissues is a useful technique in biomedical
reseach, especially in developmental biology, immunology and oncology. It* Instituet of Biomedical Research

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

is often important to be able to easily distinguish donor from host cells of
any organ with high sensitivity and reliability in quantitative analysis. For
this purpose, we have developed unique congenic strains that carry mitochondrial DNA (mtDNA) of a restriction enzyme phenotype rarely foundamong laboratory mice. The mtDNA-congentic strains have at least two
advantages: The restriction enzyme phenotype of mtDNA can easily bedetected with high sensitivity from any tissue or cell samples, and its amount
can be precisely determined by Southern blot hybridization (P. Bursot, H.
Yonekawa and F. Bonhomme, Mol. BioI. Evol., in press).
We have bred and established four mtDNA-congenic mouse strains by
successive backcrosses, using ddY as maternal progenitor: C57BLj6J-mtJ,
C57BLjlOSn-mtJ, DBAj2Yok-mtJ and DDYjYok-mtJ. The closed colonyof the ddY stock contains two types of mtDNA: the common type of standard
laboratory mice and the mtJ (for Japan) type that differs by its restriction
enzyme phenotype. Comparison of ddY mice with wild subspecies of thehouse mouse, Mus musculus, suggests that the mtJ type of mtDNA comesfrom an Asian race of M. m. musculus (previously termed M. m. molossinus)(H. Yonekawa et al., Curro Top. Microbiol. Immunol. 127, 62, 1986). The
mtJ type has so far been found only in the ddY and RR laboratory strains
established in Japan, and not in any wild mice (H. Yonekawa et al., Differentiation 22, 222, 1984).
It is known that mtDNA can affect cell surface antigenicity throught Mta,
the maternally transmitted antigen of mice, which can cause reiection oftransplanted cells by a host of different mtDNA type (K. Fischer Lindahl,
Trends Genet. 1, 135, 1985). Fortunately, the mtJ type mtDNA shares thecommon a phenotype with more than 95 % of all laboratory strains andwild mice, and thus we avoid the complications that would follow an immune
response against cells congenic for other types of mtDNA (K. Fischer
Lindahl, H. Yonekawa and K. Moriwaki, to be published).
At generations N9 to N12, we examined enzyme markers from 12 chromosomes and found, as expected, that they had all become fixed for theallele of the paternal backcross parent. We therefore concluded that thenuclear genes of all four mtDNA-congenic strains were now identical tothose of their respective inbred partner strain.
To investigate whether mtDNA-congenic mice are useful for transplantation experiments, we made two chimeric mice by aggretation of embryosfrom strains C57BLj6J-mtJ and DBAj2Yok. At 8 weeks of age, these spot-RESEARCH ACTIVITIES IN 1986

65

ted mice were killed and 15 organs (brain, kidney, testis, heart, muscle,
thymus, spleen, peripheral blood, liver, pancreas, salivary gland, lung,stomach, small intestine, and large intestine) were removed for mtDNA
typing with restriction enzymes. From brain, liver, kidney, testis, heart andmuscle, mtDNA was isolated by the cleared lysate method and the restriction fragments patterns detected by ethidium bromide staining and bySouthern blot analysis. The restriction phenotype of the mtDNA of theother organs was examined by Southern blot analysis only, using total DNA
from these organs and purified mtDNA, nick-translated with 32P-a-CTP,
as a probe. All organs showed both mtDNA restriction phenotypes, but
the composition varied from 5 to 50 % of the mtJ type between organs.
These results show that mtDNA can be typed in any tissue and the mtDNAcongenic strains can be usefully applied in transplantation experiments.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

VI.

MUTAGENESIS AND RADIATION GENETICS

Des-mutagenic Action of Refined Corn Bran
Tsuneo KADA,t Tadashi INOUE, Masako HARA
and Masayasu TAKEUCHI*

Many kinds of desmutagens, which are defined as agents that inactivate
mutagens by reacting directly with them, have been identified in various
natural sources and the possible role in environmental mutagenesis has beensuggested by many investigators.
We report here that refined corn bran (RCB), a dietary fiber derived fromthe mechanical refining of corn hulls. irreversibly adsorbed and inactivatedvarious environmental mutagens. When RCB was added at a concentration of 10 mg/ml to an aqueous solution of dinitropyrene, one of the mostabundant environmental carcinogen, 91.6 % of the mutagenicity towards
Salmonella tester strain T A-98 disappeared. Under similar conditions decreases in mutagenicity of dinitropyrene using wheat bran and cellulose
powder were 58.4 % and 43.0 %, respectively. The adsorption of dinitropyrene to the fibers appeared irreversible since little mutagenicity was recovered by washing the treated fibers with aqueous buffer solutions of various
pH's. Even with an organic solvent (methanol: ammonium hydroxide; 50:1), only two thirds of the mutagenicity of dinitropyrene was recovered. RCB
was also able to inactivate mutagenic heterocyclic amines such as IQ, TrpP-1, Trp-P-2, Glu-P-1, and Glu-P-2.
Considering that RCB is not digested in the human body, it can adsorb
and inactivate mutagens/carcinogens and cause them to be rapidly excreted.
RCB may therefore have a protective effect against dangerous carcinogens
in the human body.

t Deceased on November 14, 1986.
* Japan Maize Products Co., Ltd.

RESEARCH ACTIVITIES IN 1986

67

Cobaltous Chloride, an Antimutagen, Enhances Genetic
Recombination
Tadashi INOUE and Tsuneo KADA

In earlier studies, we demonstrated that cobaltous chloride, a potent antimutagenic metal compound, stimulated the RecA protein in vitro enhancing the formation ofD-loop which is thought to be an intermediate in genetic
recombination. The metal compound also elevated the ATPase activity of
the RecA protein, which is expressed during the unwinding of doublestranded DNA. These data suggest that this metal compound exhibits its
antimutagenicity elevating recombinational activity in the cells. In order
to confirm this concept, we examined whether the metal compound stimulates genetic recombination in vivo using a newly developed assay system.
Plasmid pMW334 (AmRTc S ) and pTH4 (CmRTcS ) were derived from
pBR322 (AmRTc R) and pACYCl84 (CmRTcR), respectively. Plasmids
pMW334 and pTH4 were made Tc s through in vitro mutagenesis at different
sites in the identical Tc genes of parental plasmids. In cells harboring
both pMW334 and pTH4, genetic recombination between the Tc gene of
pMW334 and pTH4 yields a TcRAmRCmR phenotype which can easily bedetected.
In this system, cobaltous chloride increased recombination frequency byseveral-fold suggesting that the antimutagenicity of the metal compound
can be ascribed to the stimulation of error-free recombination repair activity
catalyzed by the RecA protein.
Bio-antimutagenic Effect of L-ethionine on the Spontaneous
Mutagenesis in Salmonella and Bacillus subtilis
Tadashi INOUE and Tsuneo KADA

Ethionine, the ethyl analogue of the essential amino acid methionine, is
one of the well-known hepatocarcinogen. Recent results reported fromthis and other laboratories suggest that ethionine interferes with DNA
metabloism resulting in a alteration of mutation frequency in treated cells.
In order to gain more insight into the genetic action of ethionine, we examined the genetic effect of ethionine using bacterial systems which exhibit
high spontaneous mutation frequencies due to known molecular mechanisms.
Salmonella TA98 and TAIOO are known to exhibit relatively high spon-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

taneous mutation rates due to the presence of plasmid pKM101 which encodes genes for error-prone repair enzymes. When applied to these strains,
L-ethionine diminished spontaneous mutation frequencies in a dose-dependent manner, and at 5 p,g/mi of L-ethionine every few mutant colonies deeveloped whereas D-ethionine did not exhibit such an antimutagenic effectat all. L-ethionine also abolished spontaneous mutations in the parental
strains. TA1538 and TA1535, which do not harbor the plasmids, indicatingthat plasmid-mediated mutagenesis is not specifically involved in the expression of the bio-antimutagenic activity of L-ethionine.
Another system we examined was the spontaneous mutagenesis in Bacillus
subtilis NIG 1125 in which an error-prone DNA replicating enzyme causes
the induction of a high spontaneous mutation rate. Also in this system,
L-ethionine was very effective in reducing spontaneous mutation induction,
indicating a possible involvement of the error-prone DNA replicating enzyme in the expression of bio-antimutagenic activity of this amino acid.
One of the most probable targets for the action of L-ethionine is themethionine in cells. Ethionine may be incrorporated into protein in placeof methionine or these two amino acids may compete with each other during
methylation of biomolecules including proteins and nucleic acids. Thisidea is supported by the fact that the bio-antimutagenic effect of L-ethionine
was no longer observed when L-methionine, but not D-methionine, waspresent in the medium. From these results, we suggest that modificationby L-ethionine of an enzyme functioning in repair/replication is a causal
factor for the bioantimutagenic action of this amino acid.
Adenosine Deaminase Activity in Wasted Mouse, aRadiation Sensitive Mutant
Tadashi INOUE, Hideo TEZUKA and Tsuneo KADA

The mouse mutant "wasted" (wst/wst) is a putative animal model for
ataxia-telangiectasia (A-T), a human genetic disease characterized by chromosomal instability, radiation-hypersensitivity, immune-deficiency andincreased predisposition to cancer. Although the wasted mouse shares
many characteristics with A-T, it also resembles another human genetic
disease SCID (severe combined immunodeficiency syndrome) whose causal
factor is known to be anomaly of adensoine deaminase (ADA). Recently
Abotte et ai. reported the decreased level of ADA in erythrocytes of the

RESEARCH ACTIVITIES IN 1986

69

wasted mouse indicating that wst is the structural gene for ADA. Accord
ing to our previous observations, however, target organs of wasted mutation
are restricted to lymphoid organs such as spleen or thymus, and the wasted
phenotype appears age-dependently. We therefore examined ADA activity
in the extracts from spleens of wasted and control animals of various ages
in order to confirm the observation of Abotte et al. However, differencesin the ADA activity between wasted and control animals could not be detected throughout the developmental stages examined. Simple interpretation that reduced ADA activity in erythrocytes in wasted mouse is a resultof mutation in the structural gene for ADA should therefore be re-examined.
We are now comparing the ADA gene from wasted mouse with that of normalmouse at sequence level.
Ultraviolet Induction of Chromosome Aberrations andSeparation Inhibition in Chromosomes of therad-2 Strain of Caenorhabditis elegans
Yoshito SADAlE and Tamiko SADAIE

The nematode Caenorhabditis elegans is transparent and has less than 1000
somatic cells with established cell lineage. Transparency permits studies
on the mechanisms of DNA repair and mutation induction using ultraviolet
irradiation. In preceding reports (No. 35, No. 36) we described the gammaray induced chromosome aberrations in early embryonic cells and a repairof damages responsible for the aberrations, depending on rad-2 gene activity.
In this study we examined the induction of chromsome aberrations by ultraviolet irradiation. Results showed that ultraviolet irradiation induced aberrations in the rad-2 strain but not in the wild type strain. Furthermore,
ultraviolet strongly inhibited separation of metaphase chromosomes onlyin the rad-2 strain. This might suggest that ultraviolet suppresses DNA
synthesis in the rad-2 strain followed by a defective separation of the centromeric regions of the chromosomes. Therefore the rad-2 gene might bedirectly involed in DNA repair, and the aberrations induced by ultraviolet
might result from a defective DNA repair process in the rad-2 strain.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

A Cytogenetic Study on the Radiosensitivity of Bone
Marrow Cells of Wasted Mice
Hideo TEZUKA, Koichi TAMAI, Tadashi INOUE
and Tsuneo KADA

The induction kinetics of chromsomal aberrations and of micronuclei inbone marrow cells of wasted mice were compared with those in controls.
Dose-response studies 24 hours after gamma-ray irradiation revealed thepresence of numerous aberrations in metaphases of bone marrow cells in
wasted mice but only a few micronuclei in polychromatic erythrocytes, incontrast to what was observed in controls. Time-course studies conducted
at a constant gamma-ray dosage of 1 Gy revealed delayed recovery fromradiation-induced chromosomal aberrations and a low and delayed appearance of micronuclei in wasted mouse bone marrow.
These results suggest either the presence of a sensitive stage to radiationinduced damage in the wasted mouse cell cycle or a severe delay during thecondensation and the expulsion of nuclei from erythroblasts.
The Mode of Action of Vanillin, an Food Additive,
on Chemically Induced Cytotoxicity and Mutations
in Cultured Chinese Hamster Cells
Koichi TAMAI, Hideo TEZUKA, Yukiaki KURODA
and Tsuneo KADA

Some chemicals in our environment have significant effects on the biologicalproperties of living cells. Vanillin is frequently used as a flavoring in food,and this chemical has been found to have a potential of suppressing chemically induced mutations by enhancing a function of rec A-dependent errorfree DNA repair in E. coli (Ohta et aI., 1986, Food Chern. Toxic. 24: 51-54).
We examined possible modifying effects of the chemical, vanillin, on cytotoxicity and genetic alterations in cultured Chinese hamster V79 cells
treated with two different types of mutagens, EMS (ethylmethanesulfonate)and H 20 2 (hydrogen peroxide). In the case of EMS treatment, a significant
decrease in cell numbers survived and a significant increase in induced 6thioguanine-resistant mutants have been found in cells by co-treatment withvanillin and EMS when compared with cells treated with EMS alone. These
effects were observed as a function of vanillin dosage. In contrast, we foundRESEARCH ACTIVITIES IN 1986

71

a different kind of action of H 20 2 from that of vanillin on V79 cells. Vanillin shows a pronounced effect on recovery from H 20 2-caused cytotoxicity.
A preliminary result showed that vanillin treatment decreased the frequencyof chromosomal aberrations induced by H 2 0 2 •These results suggest that biological effects of vanillin are dependent on
the types of mutagens used and that some different mechanisms may operate
in cellular repair of chemically induced DNA lesions in cultured mammaliancells.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

VII.

POPULATION GENETICS

Diffusion Model of Population Genetics IncorporatingGroup Selection, with Special Reference to an
Altruistic Trait
Motoo KIMURA

In order to investigate under what conditions an altruistic trait evolves
through group selection, the following diffusion model was formulated.
Consider a species consisting of an infinite number of competing groups(demes), each having a constant number of reproducing members and inwhich mating is at random. Then consider a gene locus and assume apair of alleles A and AI, where AI is the "altruistic allele." Let x be therelative frequency of A' within a deme, and let ¢=tjJ(x; t) be the density
function of x at time t such that tjJ(x; t)i/x represents the fraction of demes
whose frequency of AI lies in the range (x, x+i/x). Then, we havewhere M Jz and VJz stand for the mean and variance of change in x per
generation (due to mutation, migration, individual selection and random
sampling of gametes) within demes, and c is a positive constant (called thecoefficient of interdeme competition) and x is the mean of x over the species,
i.e. x=~: x¢dx.

By studying the above diffusion equation at a steadystate (atjJ/at=O), a condition is obtained for group selection to prevail overindividual selection in the evolution of an altruistic trait. For details, see
Stochastic Processes and Their Applications (Ed. K. Ito and T. Hida) pp.
101-118. Lecture Notes in Mathematics, Springer-Verlag, Berlin.
DNA and the Neutral Theory
Motoo KiMURA

The neutral theory claims that the great majority of evolutionary changes
at the molecular (DNA) level are caused not by Darwinian selection but by

RESEARCH ACTIVITIES IN 1986

73

random fixation of selectively neutral or nearly neutral mutants. This theory
also asserts that the majority of protein and DNA polymorphisms are selectively neutral and that they are maintained in the species by mutational
input balanced by random extinction. In conjunction with diffusion models(the stochastic theory), of gene frequencies in finite populations, it treatsthese phenomena in quantitative terms based on actual observations.
Although the theory has been strongly criticized by the 'selectionists',
supporting evidence has accumulated over the years. Particularly, therecent outburst of DNA sequence data lends strong support to the theory,both with respect to evolutionary base substitutions and DNA polymorphism, including rapid evolutionary base substitutions in pseudogenes. In
addition, the observed pattern of synonymous codon choice can now bereadily explained in the framework of this theory. I reviewed these recent
findings in the light of the neutral theory. For details, see Phil. Trans. R.
Soc. London B 312: 343-354.
Actual Number of Alleles Contained in a Multigene Family
Tomoko OHTA

By using a simple model of gene conversion, the actual number of alleles
contained in a multigene family was theoretically studied. It was shownthat the Ewens' sampling theory is applicable in predicting the actual numberof alleles in a gene family of a genome. However, the actual number ofgene families forming the total population becomes larger or smaller thanthe predicted value by the sampling theory, depending upon the relative
magnitude of the rates of two homogenization processes; intra-genome andwithin the population. For details, see Genet. Res., Camb. 48: 119-123.
Population Genetics Theory of Multigene Families with
Emphasis on Genetic Variation Contained in the Family
Tomoko OHTA

The significance of repetitive DNA families in evolution has three aspects;
to increase genetic information, to regulate expression of structural genes,
and to increase themselves as selfish DNA. Various existing multigene
families (or gene clusters, supergenes and gene complexes) are real examples

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

of the first aspect. Their evolution is characterized by concerted change
due to a continued occurrence of unequal crossing-over, gene conversion andtransposition. A population genetics theory concerning evolution wasdeveloped based on identity coefficients. Transitional equations of allelism,
an allelic identity coefficient, and two nonallelic coefficients are presented
for a model of gene conversion and duplicative transposition under the assumption of constant copy number per genome. Results of numericalstudies show that by adjusting parameter values such as copy number, conversion rate and transposition rate, any desired gene family may be attainedwith respect to genetic variability contained in the family. Natural selection on these parameters is termed "indirect selection." Actual gene families
range from those with highly uniform members to those made of quite diverse gene copies, and they are likely to be attained by indirect selection.
For details, see Evolutionary Processes and Theory, (Eds. S. Karlin and E.
Nevo) 239-253, Academic Press, New York.
Population Genetics of an Expanding Family of
Mobile Genetic Elements
Tomoko OHTA

A model of an expanding family of dispersed repetitive DNA was studied.
Based on previous results using the model of duplicative transposition, an
approximate solution for presenting allelisms and identity coefficients asfunctions of time was obtained, and theoretical predictions were verified byMonte Carlo experiments. Results showed that even if the copy number
per genome increases very rapidly, allelism and identity coefficients may
take a long time to reach equilibrium. Changes in allelism and allelicidentity are similar to those of homozygosity at an ordinary single locus,
whereas those of nonallelic identity can be much slower, particularly when
the copy number per genome is large. Thus, many existing families of highly
repetitive sequences may represent nonequilibrium states for nonallelicidentity. The present model may be extended to include other evolutionary
forces such as gene conversion or the recurrent insertion from normal genecopies. For details, see Genetics 113: 145-159.

RESEARCH ACTIVITIES IN 198675

An Attempt to Estimate the Effective Size of an Ancestral
Species Common to Two Extant Species from which
Homologous Genes are Sequenced
Naoyuki TAKAHATA

When DNA sequence data on various kinds of homologous genes sampledfrom two related species are available, the effective size of their ancestral
species can be inferred from a simple consequence of gene genealogical considerations. This method, when applied to the common ancestral speciesof human and rat, human and mouse, human and bovine, or rodents andbovine estimates their effective sizes all to be of the order of 10 7 , supporting
the view that these species indeed shared, around 75 million years ago, acommon ancestral species from which they are descended. The effective
size thus estimated would imply that the ancestral species was abundantenough to have ample opportunity for adaptive radiation. The extent ofsilent polymorphism in that species might have been very large, possiblycomparable to the number of silent substitutions accumulated in a gene after
mammalian divergence. Some causes that may alter these results and require a more elaborated statistical analysis were discussed. For details,
see Genet. Res., Camb. 48: 187-190.
Private Alleles in a Partially Isolated Population
II. Distribution of Persistence Time andProbability of Emigration
Naoyuki TAKAHATA and Montgomery SLATKIN

To study statistically useful properties of "private" alleles in partially
isolated populations, we developed and analyzed a general Wright-Fisher
model of migration, mutation, and selection with an arbitrary degree ofdominance. Instantaneous killing of the process due to emigration of amutant leads to one of two diffusion processes with a killing term. One(weak gene flow) is the boundary case of the other (strong gene flow), which
can cover a wide range of gene flow. The diffusion process subject tostrong gene flow is similar to that studied by S. Karlin and S. Tavare (1983,
SIAM J. Appl. Math. 43: 31-41). The spectral decompostition ofthe transition probability density of mutant allele frequencies is presented in the caseof strong gene flow. The fate of mutants in a deme is governed largely by

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

random sampling drift and emigration. When the emigration rate is high,the quantitative features of "private" alleles are due mostly to the distribution of alleles after their initial spread to other populations and may havefairly long histories since their first appearance. For details, see Theor.
Pop. BioI. 30: 180-193.
A Stochastic Model of Gene-Culture Coevolution
Suggested by the "Culture-Historical Hypothesis"
for the Evolution of Adult Lactose
Absorption in Humans
Kenichi AOKIA stochastic model of gene-culture coevolution, suggested by the "culture
historical hypothesis" of Simoons and McCracken, was presented. According to this hypothesis, adult lactose absorption, believed to be an autosomal dominant trait, attained a high frequency in some human populations due to positive selection pressure induced by culturally determined
milk use in those populations. Two-dimensional Kolmogorov backward
equations with appropriate boundary conditions were derived for the ultimate fixation probability of milk users, of the gene for adult lactose absorption, and of both jointly, and for the average time until fixation of the gene.
These boundary value problems were solved numerically by the GaussSeidel method. I defined a theoretical measure of the correlation between
gene and culture in terms of the three ultimate fixation probabilities. Monte
Carlo simulations were conducted to check and extend the numerical results
and also to obtain the first arrival time at gene frequency 0.70, which isapproximately the highest observed frequency in any population. Tworesults that pertain to the culture historical hypothesis were obtained. First,
the incomplete correlation observed between adult lactose absorption andmilk use does not necessarily constitute evidence against the hypothesis.
Secondly, for the postulated genetic change to have occurred within the
6000-year period since the advent of dairying, either the effective population
size was of the order of 100, or, if it was of a larger order, the selection coefficient probably had to exceed 5 %. For details, see Proc. Natl. Acad. Sci.
USA 83: 2929-2933.

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77

Stable Polymorphic Equilibria in a Toy Modelof Group Selection
Kenichi AOKI

A basic model of group selection was formulated by Eshel (1972) and corrected for an "unnatural" assumption by Roughgarden (1979). The model
assumes two counteracting selective forces, group selection and individual
selection, in a deme-structured population; and was proposed in relation to
the evolution of altruistic behavior. In this study, I derived further properties of this model, concentrating on the special case of two haploid individuals per deme. Specifically, I demonstrated the existence and stabilityof polymorphic equilibria in the extreme case of migration rate 1. The
existence and stability of other equilibria were studied systematically bydefining four regions in the (m, k)-parameter space for fixed s. Here, k isa measure of group selection, s is the selection coefficient against the individually deleterious type, and m is the migration rate. One region appears
to correspond to stable polymorphic equilibria. Results were discussed
in relation to the critical equality k=2Nms where N is the deme size (Aoki,
1982), and also in relation to polymorphic equilibria in the diffusion approximation when mutation is ignored (Kimura, 1983, 1984; Ogura andShimakura, submitted). For details, see lpn. l. Genet. 61: 481-490.
Gene Flow in Natural Populations of Drosophilamelanogaster
Tsuneyuki YAMAZAKI, Jong-Kil CHOO, Takao K. WATANABE
and Naoyuki TAKAHATA

Neutral or nearly neutral genes may not always be appropriate for estimating the level of gene flow in a geographically structured population
unless the level is rather low, or the population size is small. In this experiment, we dealt with natural and cage populations of D. melanogaster andstudied lethal allelism rates and protein variation simulataneously. A surveyof 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal second chromosomes, was carried out in five
(four Japanese and one Korean) natural populations and one cage population. It was found that lethal allelism rates decreased rapidly as geographical distance between two populations increased, while variation at protein

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

loci showed a remarkable similarity over all populations examined. Thesefindings suggest that there are very high levels of gene flow in these natural
populations and that selection at protein loci which can maintain substatial
geographic variation, if present, is overshadowed by gene flow. There is no
indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromsomes are still in nonequiribrium.
For details, see Genetics 113, 73-89.
The Relationship between Viability and Number of P
Elements in the Second Chromosome of
Drosophila melanogaster
Terumi MUKAI, Ko HARADA and Shin-ichi KUSAKABE

The copy number of one kind of transposable element is limited to acertain range in one individual. There must be some factor by which the
maximum number per individual is determined. The P element, one of thetransposable elements in D. melanogaster, induces deleterious mutations
when this active element moves on the chromosome. However, it appears
that activity of transposition gradully disappears when the P type changes
to the Q and from Q to M'. We examined the relationship between the
copy number of all types of P elements and the viability of their carriers.
Two populations were chosen, the Raleigh, N. C. population and theNagasaki population. Results of the gonad sterility test for the P-M hybrid
dysgenesis are shown below as the percentage of each type: In this table,n stands for the number of tested lines. These results indicate that P elements in vaded the Nagasaki population earlier than the Raleigh populationand in the former no active P element can be seen.
Type

P

Q

MorM'

OthersNagasaki

0.0%

60.5%

37.0%

2.5%

50.0%

26.5%

10.3%

13.2%

(n=81)

Raleigh(n=68)

The average number of P elements in the second chromosomes were estimated in homozygotes by an in situ hybridization method: The Nagasaki
population: 13.20±4.26 (1984); The Raleigh population: 15.22±4.76 (1984),
17.50±4.36 (1970). The figures in parentheses indicate the years when the

RESEARCH ACTIVITIES IN 1986

79

samples were taken. It is likely that as the number of active P elements
descreases, the copy number of the element slightly decreases but not significantly so.
Finally, the relative viabilities of second chromosomes were estimated by
the Cy method, and a correlation was calculated between viability and the
copy number of P elements. This test has not been completed yet, but,thus far, the following results have been obtained:
The Nagasaki r= -0.12 (n=23)
The Raleighr= -0.24 (n= 15)
Both estimates are not significantly different from 0 but they are negative.
Studies are continuing.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

VIII.

EVOLUTIONARY GENETICS

Conservation of Alleles and of Genic Heterozygosity
in Small Managed Populations
Takeo MARUYAMA

Data has been presented on the loss of alleles from populations of small
fixed size. Emphasis has been placed on the effect which the allele frequency
distribution and the interlocus distribution of heterozygosity will have on
the pattern of allelic loss. Rare alleles are rapidly lost during the initialsampling period, and continue to be lost for several generations following
the establishment of small population size. The nonequilibrium nature of
the process of loss of genetic variability is stressed. The rapid loss of rare
alleles and the preservation of intermediate and high frequency alleles will
result in (1) heterozygosity declining much more slowly than allele number,and (2) the establishment of genetically similar populations when sampledfrom the same base population. For details, see Zoo Biology 5: 171-179.
Relative Contributions of Germline Gene Variationand Somatic Mutation to Immunoglobulin Diversity
Takashi GOJOBORI and Masatoshi NEI

The relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity were studied by comparing gene sequenceswith their rearranged counterparts for mouse V H' V., and V). genes. The
mutation rate at the amino acid level was estimated to be 7.0 % in the firstand second complementarity-determining regions (CDRs) and 2.0 % in theframework regions (FRs). Differences in mutation rates at the nucleotide
level between the CDRs and FRs were of the same order of magnitude as
those for the amino acid level. Analysis of amino acid diversity or nucleotide diversity indicated that the contribution of somatic mutation to immunoglobulin diversity is about 5 %. However, the contribution of somatic
mutation to the number of different amino acid sequences of immunoglobulins is much larger than that estimated by the analysis of amino acid diversity,and more than 90 % of the different immunoglobulins seem to be generated

RESEARCH ACTIVITIES IN 1986

81

by somatic mutation. Examination of the pattern of nucleotide substitution
has suggested that clonal selection after somatic mutation may not be as
strong as generally believed. For details, see Mol. Bioi. Evol. 3: 156-167(1986).
A Sequence Homology between the pX Genes of
HTLV-IjII and the Murine IL-3 Gene
Takashi GOJOBORI, Shin-ichi AOTA, Tadashi INOUE
and Kunitada SHIMOTOHNO

Searching the protein sequence database for amino acid sequences homologous to the x-lor sequence in the pX region of human T-cell leukemia
virus types I and II (HTLV-IjII), we found that there is a region of 38 aminoacids where the murine interleukin 3 (IL-3) sequence has a 40 % homology
with the x-lor sequence. A statistical analysis shows that this homology ishighly significant with a probability of 1.57x 10- 1 °. Since various T-celllines established by HTLV-induced transformation have been demonstrated
to produce several lymphokines including IL-3, it is possible that the activation of the IL-3 gene may have been an important step in the productionof this leukemia. Thus, biological functions of the pX gene involved in themechanism of HTLV-induced leukemogenesis may be related to those of
IL-3. For details, see FEBS letters 208: 231-235 (1986).
Simple Methods for Estimating the Number of Synonymous
and Nonsynonymous Nucleotide Substitutions
Masatoshi NEI and Takashi GOJOBORI

Two simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions were presented. Although they give
no weights to different types of codon substitutions, these methods give essentially the same results as those obtained by Miyata and Yasunaga (1980)
and by Li et al. (1985). Computer simulation indicated that estimatesof synonymous substitutions obtained by the two methods are quite accurate
unless the number of nucleotide substitutions per site is very large. It was
shown that all available methods tend to give an underestimate of the numberof nonsynonymous substitutions when the number is large. For details, see
Mol. BioI. Evol. 3: 418-426 (1986).

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Evolution of Nested Genes with Special Reference to
Cuticle Proteins in Drosophila melanogaster
Etsuko N. MORIYAMA and Takashi GOJOBORIOne of the pupal cuticle protein (PCP) genes has been found within an
intron of a Drosophila house keeping gene, Gart locus, which encodes three
enzymes in the purine pathway. This intronic gene has been described asa gene within a gene, and the gene is now called a "nested" gene. This
"nested" gene structure has been taken as an unambiguous exception to theclassical linear model of gene organization, particularly in the nuclear genomes of eukaryotes. Since the intronic PCP gene has a sequence homology
with larval cuticle protein (LCP) genes, it should have been derived fromone of the LCP genes or their ancestral gene. Therefore, we attempted toelucidate the evolutionary origin of the "nested" gene structure using thesegenes. Comparing nucleotide sequences of four LCP genes with those of
the PCP genes, we studied possible phylogenetic relationships among these
five genes. The results obtained suggest that the PCP gene may haveoriginated from its ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we thanestimated the divergence time between the PCP gene and LCP genes to beabout 70 million years (Myrs). The estimated divergence time between
these genes is much larger than that of D. melanogaster's sibling species (about
2.5 Myrs). Thus, it is probable that the "nested" gene structure can be seen
not only in D. melanogaster, but also in other Dorsophila species.
Higher Rates of Nucleotide Substitution
in Drosophila than in Mammals
Etsuko N. MORIYAMA and Takashi GOJOBORI

To examine whether the rate of nucleotide substitution is affected by thegeneration time of organisms, we attempted to estimate an accurate rate ofsynonymous (silent) substitution in Drosophila lineages. We used the nucleotide sequences of alcohol dehydrogenase (Adh) and heat shock protein
82 (hsp 82) genes for a total of eight species of Drosophila and estimatedthe number of synonymous substitutions. From paleontological, paleobiogeographical and electrophoretic data, divergence times between D. melangasterand its sibling species, between D. melanogaster and D. pseudoobscura andRESEARCH ACTIVITIES IN 1986

83

between D. melanogaster and D. virilis have been estimated to be about 2.3,
30, and 40 Myrs, respectively. Using these values, we estimated the rateof synonymous substitution in Drosophila lineages to be 10.5x IO-Djsitej
year. This rate is approximately two times higher than that of rodents andten times greater than higher primates. The higher rate in Drosophila may
be explained by the shorter generation times of the Drosophila species,
though the possibility that the mutation mechanism in Drosophila may
differ from that in mammals cannot be excluded. For details, see lpn.
I. Genet. 62: 139-147.
Expansion of Drosophila simulans in Japan
Masayoshi WATADA, Yutaka INOUE
and Takao K. WATANABE

Drosophila simulans is distributed throughout the world in association withhuman habitation. This species, however, had never been collected in Japan
before 1972 except on Ogasawara Island. In 1974 and 1975, many D. simufans individuals were collected in Kitakyushu and the Tokai district. In
1976 the species was found to be distributed in two separated areas; Kyushu
and the Kanto-Tokai districts. The intervening area comprising the Kinki
and Chugoku districts, was almost free of D. simulans.
Domestic and semi-domestic species of Drosophila were collected in Japan
from 1977 to 1985 in order to study the expansion of D. simufans and its
effect on other species. Newly established populations of D. simufans wererecorded in the western mainland (Kinki and Chugoku). In addition, someisolated populations of D. simulans were found in Sapporo, Akita and Miyakojima. The frequency of D. simufans temporarily changed in the newly
invading localities before its successful colonization. Comparisons of the
proportions of collected flies suggested that the expansion of D. simulans
affected D. melanogaster severely in domestic sites and D. futescens slightly
in semi-domestic sites. For details, see Zool. Sci. 3, 873-883.

Computer Study of Molecular Evolution
Takashi MIYATA

The proto-oncogne c:fmsjmacrophage colony-stimulating factor receptor
and platelet-derived growth factor (PDGF) receptor are closely related in

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

sequence. Both have a structual property of cell-surface receptors consisting of an extracellular domain of putative five repeat units, a transmembraneregion and a cytoplasmic tyrosine kinase domain homologous to the src
family (Coussens et ai., 1986; Yarden et ai., 1986). We showed that theextracellular domains of these growth factor receptors exhibit significant
homologies in sequence with myelin-associated glycoprotein (MAG) and theneuronal cell adhesion molecule (N-CAM), known to be members of theimmungoglobulin superfamily. The degrees of homologies are in the rangeof 34 % for the c-fmsjMAG pair to 19 % for the PDGF receptorjN-CAM
pair, and the probabilities that these homologies are realized by chance areless than 6.3 x 10- 5 • Furthermore the three repeat units of c-fms and thetwo repeat units of the PDGF receptor show statistically significant homologies with 33 known immunoglobulin-related sequences for regions aroundinvariant cysteine residues involved in disulfide bonding. Thus these growth
factor receptor genes are a new subgroup of the immunoglobulin gene superfamily encoding an immunoglobulin-related extracellular domain and an
src-related cytoplasmic protein kinase domain, and possibly evolving throughthe shuffling of primordial genes from distinct gene families. An additional
homology of a Drosophila neuron-specific glycoprotein with Thy-l was also
shown.

RESEARCH ACTIVITIES IN 1986

IX.

85

HUMAN GENETICS

Incidence of Genetic Disease in JapanEi MATSUNAGA

In 1934, T. Komai, who compiled 741 pedigrees of some 80 kinds of hereditary diseases and abnormalities published in Japanese medical literature,
was struck by the finding that, when compared with what was known for
Europeans, there was very little difference either in the kinds of disease orin the mode of transmission. As instances of rare exception, he mentionedthat Oguchi's disease and Leber's optic-nerve atrophy were much commonerin Japan than in Europe while there was no authentic case of Huntington
disease known in this country. Since then, numerous hereditary disorders
have been documented in Japan, and as far as phenotypic similarity of themorbid genes is concerned, the general principle first pointed out by Komai,that there is very little ethnic difference, was substantiated, even for mental
disorders such as schizophrenia and Down's syndrome. However, information about the frequency of individual disorders in the general populationis still limited. The following are a summary based on available data collected from literature.
The incidence at birth of various types of chromosomal abnormalities
is virtually the same in both Japanese and European populations. Most
Mendelian disorders so far investigated, including monogenic hypercholesterolemia and neurofibromatosis, are as common in Japan as inEurope and North America. As instances of rare exception, tuberous
sclerosis, congenital cataract (dominant and recessive), congenital muscular
dystrophy (Fukuyama type), Oguchi's disease, acatalasemia, corneal amyloidosis and xeroderma pigmentosum, seem more frequent in Japan thanin Europe and North America, while Huntington disease, phenylketonuria
and cystic fibrosis are much less frequent in Japan. The overall frequencyof chromosomal and some Mendelian disorders is estimated at about 1 in 100
newborn infants. There are two foci of familial amyloid neuropathy, one
in Kyushu and the other, the second largest concentration of this disorder,
in central Japan. With declining frequencies of consanguineous marriages
and the breaking down of geographical and social isolates in this country,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

it is expected that the incidence of all kinds of recessive disorders will decrease gradually to some extent. However, the prevalence rate of patientswith hereditary diseases and disabilities in the general population is generally
increasing owing to an improvement in medical care and therapy. Details
are in press in Human Genetics, Proceedings of the 7th International Congress, Berlin, 1986.
Paternity Determination in Two Cases with
Hereditary Disease
Ei MATSUNAGA

If, in paternity determination, a child is affected with a rare dominantdisorder and the same condition is also found in a putative father, it will
constitute strong evidence that he is the biological father of the child. However, if the genetic disease in the child is of a kind which arises usually asa result of fresh mutation, then the determination of paternity will be very
difficult.
We recently experienced a case of paternity determination in which theplaintiff, a 25-year-old woman, and the defendant, a 50-year-old man, wereboth affected with congenital aniridia, whereas the mother of the plaintiff, aclassmate of the defendant in primary school, was mentally retarded but
had normal irises. It was ascertained that aniridia ran in the family of the
defendant; this condition was inherited from his father, and two sisters andone niece were also affected, showing an autosomal dominant pattern ofinheritance. Examination of 13 genetic markers in the three persons gave
results consistent with the paternity of the defendant. Since the frequencyof this condition in Japan seems to be of the order of 10-5 , it was concluded
that the defendant is the father of the plaintiff.
In the second case, the plaintiff, a 19-year-old boy, was affected with typical
Down's syndrome. Because the boy showed distinct features peculiar to
Down's syndrome, it was difficult to detect phenotypic resemblances between
the plaintiff and the defendant, a 50-year-old man. However, examinationof genetic markers revealed that there was no inconsistency with the paternity of the defendant. In particular, both the plaintiff and the defendant possessed a rare Gc variant (plaintiff 2-ICI, mother 2-IS and defendant IAIICI). It was concluded that the defendant was in all probability the fatherof the plaintiff. Details were reported in the lpn. l. Legal Med. 40: 520,

RESEARCH ACTIVITIES IN 1986

87

1986.
A T --+C Transition in Codon 110 of the Human p-Globin
Gene is a Novel Cause of P-Thalassemia Phenotypes
Takashi IMAMURA, Hitoshi NAKASHIMA and Yuji NARITOMI'"

Thalassemia results from inherited defects in the rate of synthesis of one
or more of the globin chains, which leads to clinical manifestations of anemia,
hemolysis and ineffective erythropoiesis. The similar but genetically distinct hemolytic syndrome, unstable hemoglobin-hemoglobinopathies, result
from an inherited structural alteration in one of the globin chains, throughwhich physical stability of the molecule is affected. The presence of one ofthese particular abnormal hemoglobins in a patient is associated with chronic
hemolysis and anemica, due to the denatured hemoglobin precipitates formedin vivo.
We now report evidence that a single nucleotide substitution in the third
exon of the p-globin locus of an affected Japanese patient produced the pthalassemia phenotype through a post-translational mechanism. This novel
human globin gene mutation resulted in the substitution of proline for leucine
at position 110 in the G-helix of the p-globin chain. Both molecular stabilityof the p-globin subunit and IXIPI dimer formation, the first step in hemoglobin
tetramer formation, were impeded resulting in the p-thalassemia phenotype.
Although the common molecular mechanisms for thaIasemia are produced
by abnormal mRNA processing and by early or late termination, the molecular lesion which is defined in this study was not due to gross mRNA deficiency. The structural variant was so unstable that it could not be detectedin cells by any means. Thus, the globin chain deficiency, apparently similar
to that in thalassemia, resulted from synthesis of the variant hemoglobin subunits which were extremely unstable in amounts equal to their gene dose (orslightly less), having a greatly accelerated rate of denaturation and removal.
The mutation could be identified at the DNA level upon digestion with arestriction enzyme Msp I. This detection of the mutation on the gene level
is of significant advantage for differential diagnostic purposes.

'"

Faculty of Medicine, Kyushu University, Fukuoka.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Distinct Clustering of Mitochondrial DNA Types
among Japanese, Caucasians and Negroes
Satoshi HORAI, Takashi GOJOBORI and Ei MATSUNAGA

A phylogenetic tree constructed by genetic distances among the 62 mitochondrial DNA types indicated that at least two distinct lineages exist in theJapanese population. In order to evaluate the evolutionary significance of
this finding, a phylogenetic tree of mtDNAs among three major racial
groups was constructed by combining our data with those for Caucasians
and Negroes analyzed by Cann (1982) using 11 restriction enzymes. A totalof 117 restriction types were observed among the three major racial groups.
It is to be noted that any restriction type in one racial group was unique tothat group. The average number (d) of nucleotide substitutions for Japanese
was almost the same as that for Caucasians, whereas the value for Negroes
was about two times larger than those for the other two races. This implies
that Negroes are much more diverse with respect to mtDNA than Caucasians
or Japanese.
We also estimated the number of nucleotide substitutions for all pairs ofindividuals in the three races and constructed a phylogenetic tree for the 117
types by the unweighted pair group method (Sokal and Sneath 1963; Nei
1975). We tentatively classified all lineages into 8 clusters designated as Cl
to C8. While three of them (C3, C7 and C8) represent interminglings ofindividuals from different racial groups, the remaining five show distinct
clustering of the same racial group: Cl (consisting of 9 types) may be calleda Negroid cluster, C2 (13 types), C4 (6 types) and C6 (24 types) Japanese,and C5 Caucasian, although one out of 19 types of the C5 was derived from
Negroes.
In the tree, the CI and C2 clusters diverged first from the rest of the clusters.
As indicated in a previous paper (Rorai and Matsunaga, 1986), C2 (group I),
which represented 18 % of the Japanese, was quite different from the rest
(group II) of the Japanese in two ways; individuals of C2 showed a higher
level of polymorphism as revealed by several restriction enzymes, and they
all showed a RaeIII variant (morph 2), due to a length polymorphism characterized by a 7 bp deletion in a 141 bp restriction fragment. None of theother Japanese clusters exhibited this length polymorphism. In the data
analyzed by Cann (1982) the Rae III morph 2 was found in six Asians andone black individual, but none was detected among the Caucasians. In the

RESEARCH ACTIVITIES IN 198689

case of this length polymorphism, an independent occurrence of variation
seems unlikely, because exactly the same amount of electrophoretic mobility
shift was identified in the variant individuals. Some Mongoloids and apart of Negroids exhibiting the HeaIII morph2 may share a common ancestor.
In our analysis, the branching point of Cl and C2 clusters goes back toabout 170,000 years ago, if we assume that the rate of nucleotide substitution
in mtDNA is 2x 10-Sjsitejyear. On the other hand, Nei and Roychoudhury(1974), using gene frequency data for 28 protein loci, estimated that the divergence time between the Caucasoid and the Mongoloid (Japanese) races
is 50,000 years ago, whereas that between the Negroid and the CaucasoidMongoloid group is about 120,000 years ago. The standard errors involvedin the estimation of divergence times must be large in both cases. Moreover, our analysis referred to gene phylogeny, whereas Nei and Roychoudhury's was concerned with the time of population splitting. However, ourfinding may suggest that the time of gene divergence was much earlier than
the time of population splitting. In other words, prior to the racial divergence there were two ancestral types of mitochondrial genes, one for Cl andthe other for C2. An alternative hypothesis is that the time of divergence
between some parts of the Negroid and Japanese was still earlier than that
estimated from the protein data.
With respect to C3, C7 and C8, the intermingling of different racial groups
may be due to the ancient polymorphism of human mtDNAs. This intermingling may also be explained by gene migration. In particular, theintermingling between American whites and blacks could be due to recent
admixtures (Reed, 1969). However, such gene admixtures between Japaneseand the other two races are unlikely because the Japanese population seems
to have been isolated for a long time.
Althogh there are several possible explanations for the interpretation of
the overall clustering patterns in the tree, the most pertinent explanation isthat the ancestral human population was already polymorphic in the mitochondrial genome before the divergence of the three major races. For
details, see Jpn. J. Genet. 61: 271-275.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

An Apparent Discrepancy between Chain Length andElectrophoretic Mobility of Restriction Fragments:
a Case of Human Mitochondrial DNA
Satoshi HORAI, Tadashi INOUE and Ei MATSUNAGA

Restriction enzyme analysis has been used as a powerful tool for detecting
genetic variations in human mitochondrial (mt) DNA. We previously analyzed mtDNA from 116 Japanese with restriction enzymes of four or five basepair recognition and postulated that there was an insertion of about 60 bp
in the region form URF2 to tRNA Asu as compared to the published sequence(Anderson et al. 1981, Nature 290: 457). The insertion was common to allindividuals screened, and was clearly demonstrated by three different enzyme
digestions. The apparent presence of the insertion was also confirmed bydouble digestion experiments with enzymes of six base pair recognition(Horai and Matsunaga 1986, Hum. Genet. 72: 105).
In order to localize the insertion in the fragment, we sequenced the fragment in question. Contrary to expectations, there was no insertion in the
fragment and the actua11ength of the fragment was found to be exactly thesame as that derived from the sequence data reported by Anderson et al.
(1981). To clarify this discrepancy between the sequence data and the gelelectrophoretic analysis, we compared the mobility of the fragments inquestion in native gel with those in a denaturing 'gel. Results showed thatthe mobility of the restriction fragments in native gel does not necessarilyreflect real molecular weight. These findings suggest that certain fragments
had peculiarly higher structures resulting in slower mobility in the native gelelectrophoresis. However, a computer analysis of the sequence data of thefragment in question failed to show any specific secondary structures orbiases of base compositions.
In conclusion, we should be sufficiently careful when assessing molecularweight of restriction fragments by native gel electrophoresis, assuming alinear relationship between mobility and the logarithm of the molecularweight. These data were published in Hum. Genet. 75: 73-74.

RESEARCH ACTIVITIES IN 198691

Mitochondrial DNA Polymorpbisms in Japanese Monkeys,
Macaca fuscata, Derived from Four Local Population
Kenji HAYASAKA, Satoshi HORAI, Takayoshi SHOTAKE,
Ken NozAwA and Ei MATSUNAGA

We analyzed mitochondrial DNA polymorphisms of 10 Japanese monkeys,
Macaca fuscata, derived from four local populations using 18 restriction
enzymes. Of the 18 enzymes used, polymorphisms of cleavage patterns
were observed in 12 enzyme digestions. By combining the cleavage patterns
for each enzyme, the 10 samples were classified into four restriction types.
Each of the four types was found exclusively in the respective population.
The four types of mtDNAs had 43 to 49 restriction sites for the 18 enzymes.
The number of nucleotide substitutions per site among the 10 samples wasestimated to range from 0 to 0.0236 and the average was 0.0132. Thisaverage value is several times larger than that observed in human populations
but within the range of those observed in great apes and rodents.
The phylogenetic tree constructed using the number of nucleotide substitutions revealed that the four types were divided into two clusters. If
we assume the rate of nucleotide substitution to be 2 X 10- 8 per year per
site, these two clusters diverged approximately 1 X 106 years ago. From
the data of blood protein polymorphisms, the divergence time between
Japanese monkeys and rhesus monkeys, which are considered to be the closest
relatives of the former, was estimated at 5 X 1O~ years. This discrepancy between divergence times may result from either a higher rate of nucleotide substitutions or the divergence of mtDNAs before the divergence of species.
Our observation that each local population has its own unique restriction
type of mtDNA is in agreement with both the maternal inheritance of mtDNA
and the life history of this species whose females stay in their native populations for their entire lives. This observation is also in agreement with thestudy of blood protein polymorphisms which revealed a low genetic variability within local populations and restricted distribution of variant alleles.
For details, see Jpn. J. Genet. 61: 345-359.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

X.

APPLIED GENETICS

Genic Analysis of Rice Isozymes and Their
Expression in Calli
Ryuji ISHIKAWA*, Toshiro KINOSHITA* and Hiroko MORISHIMA

About 40 isozyme loci have so far been identified in rice. To assign thoseisozyme loci to the respective chromsomes, a series of trisomies derived form
a Japanese variety were crossed with some Indica varieties and wild strains.
Segregations in F 2 populations so far examined revealed that Cat-l belongs
to linkage group I (chromsome 6), Amp-2 to linkagegroup su (chromosome
12), Pgd-l to linkage group VIII (chromsome 9), Pox-2, Acp-l and Sdh-l tolinkage group d33 (chromosome 4), respectively. Together with the locialready known, 12 isozyme loci were assigned to six chromsomes.
To verify the expression of isozymes in calli, allelic expression of 14 lociwere examined in the embryo-derived calli. Calli cultured for two to threemonths and those cultured for more than one year were compared to evaluate
the effect of age. Six loci (Pgi-l, Pgi-2, Cat-I, Pgd-l, Sdh-l, Est-2) showed
the same phenotypes as in the original plants and their expression was quite
stable, so that they could be used as useful genetic markers selection in vitro.
Pox-l and Pox-2 never appeared in the calli. Phenotypic expression of theother six loci was altered or modified during the culture, and it seemed difficult
to distinguish allelic differences. Tissue specificity observed in the originalplants tended to disappear in calli.
Latent Differentiation towards the Indica and Japonica
Types in the Asian Common Wild Rice
Hiroko MORISHIMA and Lilian GADRINAB**

In our earlier studies we accepted evidence that the wild ancestors of the
Asian cultivated rice did not differentiate into the Indica and Japonica types,
which represent the two major phylogenetic groups in cultivars, and that
this differentiation occurred along with the process of domestication. This
* Hokkaido University.
** BIOTROP, Indonesia.

RESEARCH ACTIVITIES IN 1986

93

problem was reexamined using a wider range of materials and characteristics(both phenotypic traits and isozymes).
Analysis of variation and covariation of 23 characteristics and 12 isozyme
loci revealed that distinct differentiation into the Indica and Japanica types
as found in the cultivars is not fully developed among wild strains.
Yet, a trace of non-random character association characterizing the Indica
and Japonica types was detected. Moreover, this trend towards differentiation found in phenotypic characteristics and that found in isozymes weresignificantly related to one other. Most wild strains formed a continuous
array between typical Indica and Japonica types. Chinese wild rices werecloser to the Japonica type than the strains from other regions. It was also
found that among the Asian wild rices the perennial type (supposedly primitive type) was more Japonica-like than the annual type (advanced type).
Indica vs. Japonica differentiation must reflect a complex combination ofhistorical incidence and consequences of selection during evolutionary divergence.
Gametic Selection under Different Conditions in RiceY. I. SATO, R. ISHIKAWA and H. MORISHIMA

Mendelian segregation assumes that all pollens or sperms are given equalchance of fertilization. Is this assumption always correct? We sometimes
observe significant segregation distortions and fluctuations in F 2 populationsof rice, though no particular genes causing segregation distortion are involved.
This suggests that a relative proportion of fertilization by pollen grains withdifferent genotypes is influenced by the environment. To confirm this, allelicfrequencies at nine isozyme loci were compared among four samples of F 2
seeds born on F 1 plants obtained from the same cross but grown at different
places (Sapporo, Mishima, Kyoto and Taichung). The data indicated that
segregation ratios of three loci were significantly different among F 2S derived
from F1s grown at different places. In addition, F 1 plants of another crosswere grown in the summer (outdoor) and the winer (greenhouse) seasons in
Mishima. In this cross, a marker gene (gl) controlling hairiness of hull
segregated. Frequencies of gl gl hommozygous plants were 0.169 and
0.112 in the F 2 populations which were obtained from F 1 plants grown inthe summer and winter seasons, respectively. Since these experiments were
only preliminary, we were unable to detect a general tendency. However,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

some important questions were raised.
1. Selection in gametic phase must be more a general phenomenon thanpreviously expected. Population genetic studies taking the genetic expression of the gametes into consideration is needed to elucidate its
consequences in the sporophyte generation.
2. Breeding materials in early generations are often grown in greenhouses
during winter for generation acceleration. Is it truly good for the efficiency of breeding?
Ecological Genetic Studies in Wild Rice Populations
P. BARBIER* and H. MORISHIMAThe Asian common wild rice Oryza perennis shows an intraspecific differentiation into annual and perennial ecotypes. Both types differ by several
life-history traits, particularly in the use of sexual vs asexual reproduction
and the mating system. Annuals are predominantly seIfers that reproduce
by seeds, and perennials are predominantly outbreeders but reproduce largely
by asexual means.
Seven natural populations located in Thailand were studied regarding
various phenotypic charactes and isozymic loci, in order to examine therelationships between life-history traits (sensu lato) and the distribution ofgenetic variability between and within populations.
Outcrossing rates were estimated from progeny of single mother plants
sampled from three populations, using nine isozyme loci. Maximum likelihood estimates were about 10 % for annuals and 50-60 % in perennials. In
outbreeders, inbreeding coefficients were generally higher than expectedequilibrium F values. This could be explained by the existence of consanguineous matings between genotypes that are spatially distributed in a patchymanner.
The selfing annual populations exhibited low intra-population genic diversities, and the outbreeding perennial populations high diversities, in mostcases both in the seeds and juvenile plants sampled. However, significantly
lower diversities in juveniles as compared with those in seeds were foundin one annual and one perennial population. We tried to interpret theseresults by looking at the demographical records available for these populations. In the annual, the decrease in genic diversity was related to a decrease* NAGOYA Univ.

RESEARCH ACTIVITIES IN 198695

in the number of individuals (due to mortality of juvenile plants). Such annual populations are likely to be subject to drift when exposed, for instance,
to unexpected drought after recruitment of seedlings. In the perennial
population, the variability observed in seeds, which is generated by pollen
flow, possibly including pollen from outside of the population and sexual
recombination, is probably seldom used, since newly recruited plants aremainly asexual propagules of the founders.
On the other hand, in another perennial weedy population which propagates both by seeds and asexually, a high genic diversity was found bothin seeds and in juvenile plants. This indicates that the level of variability
in natural populations is strongly connected to the mating system (opportunities for gene flow), but also depends on other life-history traits such as
thb mode of recruitment.
Search for Genes Controlling Photoperiodic Sensitivity in
Rice Using Linkage with Isozyme Genes
R. ISHIKAWA, Y. I. SATO and H. MORISHIMA

Rice is a shortday plant in which flowering time is accelerated undershortday conditions. The photoperiodic sensitivity (PS) of the rice plant
has not been fully studied from the viewpoint of genetics. In order to understand the genetic variablility of this trait in various cultivars, three F 2
populations derived from three cultivars with differing degrees of PS and a
non PS tester strain were each divided into two groups and grown undershortday and longday conditions respectively. Some of the segregants grownunder longday condition did not head due to their strong PS. Therefore,among obtained progeny, the frequencies of alleles derived from PS cultivars
at loci linked with PS genes were expected to be lower under longday condition than those observed under shortday condition. In this experiment,eight isozyme loci were analyzed using the progeny seeds of headed F 2 plants.
Results obtained suggest the existence of four PS loci. Strong PS seemed
to be controlled by combinations of PS alleles at two or more loci. Allelicfrequencies of three isozyme loci belonging to linkage group I (chromosome6), were consistently different between shortday and longday conditions inthree populations. A PS locus (Se-I) already reported belongs to thislinkage group. The PS alleles detected here may be on the Se-l locus. It
was assumed that another PS locus was located on the same chromosome

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

being linked with Cat-I.
mosome 5.

An additional one was revealed to be on chro-

Phylogenetic Differentiation of the Japanica Types ofAsian Cultivated RiceY. I. SATO and H. MORISHIMA

Asian cultivated rice (Oryza sativa L.) is classified into two major varietalgroups, the Indica and Japonica types. Analysis of genetic structure within
the Japonica type was attempted by principal component analysis using 13
morphological and physiological characters. The first principal component
showed a latent phase represented by the variation axis of 'panicle length
vs. panicle number' type. Scores of this component (Zl score) showed abimodal distribution. The Japonica type is apparently differentiated into
two types. Judging from the obtained larg loadings of the certain charactersused by Oka (1958) for discrimination of his tropical and temperate Japonicas,
the two types extracted by peA seemed to correspond with his tropical andtemperate types. Tropical Japonicas were panicle length type, while temperate Japonicas were panicle number types. These two types were fairly
different in geographical distribution pattern; as their names indicate troipcal
and temperate Japonica type tend to distribute in the tropical and temperatezones, respectively. They also showed differences in the frequency of the
Hwc-2 gene for F 1 weakness. The Japonica types have been differentiatedthrough various selections and adapted to different environments.

Wx protein of Induced Mutants in Rice
Yoshio SANO, Mitsuko KATSUMATA and Etsuo AMANOAs previously reported, a cis-acting regulatory mutation produced Wx bfrom wx a • The frequency of Wx b which drastically reduces the Wx protein
level as well as amylose content in rice endosperm seems to have increased
during domestication. Thus, naturally occurring variants were useful forrecognizing the importance of regulatory functions at the waxy locus of rice.
Unfortunately, naturally occurring variants are difficult to use for interpreting regulatory functions without the use of near isogenic lines, since their
genetic backgrounds may lead to misinterpretation and their evolutionay
origins are often obscure. A number of induced variants including waxy

RESEARCH ACTIVITIES IN 198697

and the low amylose mutatuion may give us a great opportunity for a better
understanding of the Wx gene expression. When the induced mutants sharea clear parent-progeny relationship, any biochemical changes detected arelikely to be the cause of the mutant gene(s).
The waxy and low amylose mutants obtained were readily distinguishable
from their endosperm appearances when compared with the original line of
Norin 8. All mutants gave a 3 normal: 1 mutant ratio when crossed withT65 (nonwaxy), indicating that their mutant endosperms are each controlled
by a single recessive gene. When the 11 waxy mutants were crossed with awaxy strain (T65wx), no normal endosperm segregated in F 2 , showing thatthe mutant genes involved are allelic to wx. On the other hand, the 5 low
amylose mutants gave normal, intermediate and waxy segregants in a ratioof 9: 3: 4, 3: 1 or 9: 7 in crosses with T65wx. The endosperm appearanceof a low amylose mutant (74LA5) was close to its normal counterpart, sointermediate segregants were not distinguishable from normal segregantsin F 2 • Another mutant (76LA3), showed an endosperm appearance close
to waxy and gave a segregation ratio of 9: 7 in F 2' These results indicatethat the genes involved in low amylose mutants are inherited indepndentlyof wx and they have different effects on endosperm appearances although
the allelism test among the mutants remains to be studied.
Amylose content and Wx protein level were examined in these mutants tolook into regulatory changes at the wx locus. Out of 11 waxy mutants, only
3 almost completely lacked amylose and the Wx protein. It should be notedthat naturally occurring waxy variants all belong to this type. The other 8
waxy mutants produced the Wx protein more or less and 3 of them produced
traces of amylose, suggesting that some mutants produce an inactive form of
the Wx protein. A mutant gene of 74wx2 seemed to affect only the level ofthe Wx protein, since the degree of reduction was similar in both amylose
and the Wx protein when compared with to those of N8. Further evidence
regarding the inactivation of the Wx protein was gained from the 75wxl
mutant. The mutant had a Wx protein about 1,500 daltons larger than thatof the Norin 8 as determined by comparisons of migration rates. An inactive
form of the Wx protein larger than normal size was also detected in maize,
which resulted from an insertion of a controlling element into wx locus.
Low amylose mutants affected the quantitative level of Wx protein as wellas amylose. Endosperm appearance seemed to be closely associated withamylose content. The mutant genes obtained were regulatory in nature.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Since they show different genic action and phenotypic expression, different
types of regulatory genes might be involved.
Some Comments on Seed Sampling form Natural
Plant Populations
K. YONEZAWA and H. ICHIHASHI*

A seed sampling method for field collection was theoretically investigatedwith respect to the number of plants per site (population), m, and seeds perplant, n. Populations which are at genetic equilibrium with mixed selfing
and outcrossing were assumed as the target populations to be collected, andthe probability that all (two or three) of the alleles existing in the target
populations are taken up was calculated for different rates of selfing anddifferent combinations of rna and n. It was shown that in predominantly
selfing populations, the results of sampling primarily depend on the plant
number m, but a defect due to a shortage in plant number can well be compensated for by an increase in the seed number per plant, unless the selfing
rate is as large as or larger than 0.95. In predominantly outcrossing populations, on the other hand, no substancial difference is found regardless ofwhether the plant number or the seed number per plant is increased. The
optimum size of sampling is not much affected by the number of(indepenent)loci and alleles per locus to be collected, but is largely determined by thefrequency of the rareest allele at any locus. Sampling about ten plants,
with 10 to 20 seeds being collected from each plant, is estimated to be sufficient unless very infrequent (0.05 or fewer) alleles are to be collected fromhighly selfing populations. (Proceedings of the fifth International Symposium on Crop Exploration and Utilization of Genetic Resources, Taichung,1986).
Variations in Protein Fractions from Rice Endosperm
Toru ENDO

Many aspects of the differences between Indica and Japonica types ofrice cultivars have been studied, including seed protein contents. For sequentially extracted protein fractions from rice endosperm, the content* Fac. Agr., KYOTO University

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RESEARCH ACTIVITIES IN 1986

ratios of albumin, globulin, prolamin and glutelin of some cultivars havealso been described (Padhy and Salunkhe 1979, Cereal Chern. 56: 389).
Here I report on the content ratios of five Indica and five Japonica cultivars(see table).
Varieties
Indica group
Peiku
130
419
713
717
Japonica group
Taichung
Kosihikari
HarebareMurasaki
Kochi-aka

Albumin %Globulin %Prolamin %Glutelin %

8.6
10.9
19.4
21.3
23.9

11.6
4.3
2.3
3.3
3.8

1.9
11.4
17.9
2.6
4.1

77.9
73.4
60.0
72.8
68.1

23.0
17.6
37.0
12.2
14.3

9.8
8.5
7.8
4.4
2.4

3.3
6.7
1.5
8.9
12.6

63.9
67.2
53.7
74.5
70.7

Albumin, globulin and prolamin fractions were extracted from 10 g of
100-mesh endosperm powder with specific extractants, 0.01 M Tris-HC1, pH
8.5, for albumin; 0.5 M NaCl, pH 6.5, containing 0.2 M Tris and 0.2 M ascorbic acid for globulin; and 80% t-butanol for prolamin. In all cases theextraction was carried out overnight at 4°C in the presence of 0.1 M 2mercaptoethanol. Glutelin was extracted with 0.1 M NaOH for 15 min
and precipitated with ascorbic acid at about pH 6.
It can be noted from the table that there are no significant difference incontent ratios between Indica and Japonica groups and that there are considerable differences among the cultivars of each group. For instance,
albumin content is much higher in Harebare and 717 than the others, andglobulin content is appreciable higher in Peiku and Taichung. Prolamincontent is variable within both groups. Although the endosperm protein
contents depend on agrotechnical, climatic and storage conditions, theabove-mentioned differences must be partially due to differential genetic
systems among the cultivars empolyed.
It shoud be noted, however, that isoelectrofocusing polyacrylamide gelelectrophoresis (IEF-PAGE), covering the pH 2-11 range, gave blurred banding patterns for albumin, globulin and prolamin samples, but gave clear patterns for glutelin. On the other hand, immobilized pH gradient polyacryl-100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

amide gel-electrophoresis (lPG-PAGE), covering the pH 4-10 range, gave
sharp bandmorphs for globulin and glutelin samples, and gave clear but
weak bands, small in number, for the albumin and prolamin samples. These
results suggest that the overnight-extraction method caused a denaturationof most protein fractions resulting in blurred bands by IEF-PAGE andleading to a few sharp bandmorphs by lPG-PAGE. Although the overnightextraction method is not necessary for qualitative experiments, it is desirable
for a quantitative examination of the protein content ratio. Comparative
analysis of the bandmorphs among the cultivars is now under investigation.

RESEARCH ACTIVITIES IN 1986

XI.

101

DATABASE

Publications from Genetic Resources Section,
Genetic Stocks Reseach Center
Shin-ya IYAMA

The following publications were released from Genetic Resources Section
this year.
1. "Catalogue of Escherichia coli Genetic Stocks in National Instituteof Genetics". (Compiled by A. Nishimura and S. Iyama) 181 pages.
This was completed in cooperation with the Microbial Stocks Section.
The catalogue listed 2,177 main genetic stocks of E. coli out of about 10,000
those maintained in the Institute. Each stock was described with its accession number, strain name, sex, containing genetic markers together withprophage and plasmid, if any. A list of 1353 marker genes and chromosomal deficiencies was added. Under each item, the accession numbers ofstocks possessing the marker were indicated so as to be used as an index for
finding the stocks interested.
2. "Drosophila Stock List in Japan 1986". 56 pages.
Previous list published in 1985 was updated. This issue included 1250
Drosophila stocks kept in various places in Japan. Each stock was indicatedwith the place of maintenance. Wild and mutant stocks of D. melanogaster,D. simulans, D. ananassae, D. hydei, D. virilis and other 115 species were
included.
3. "Rice Genetics Newsletter Vol. 3". 128 pages. (in English).
This volume contained the followings:(1) Rules for gene symbolization in rice proposed by the Committee on
Gene Symbolization, Nomenclature and Linkage Groups of Rice Genetics
Cooperative.
(2) Lists of recommended gene symbols for chlorophyll deficiencies
and leaf spots, heading behavior, anthocyanin coloration, grain size andshape, and isozymes.
(3) Lists of gene symbols and gene markers supplemented to the previous
ones.
(4) List of about 200 recent publicaitons on rice genetics.
(5) 62 research notes on rice genetics.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

Data Base Design of DNA Base Sequences
Sanzo MIYAZAWA

DNA base sequencing have become so easy that DNA base sequences
analyzed are rapidly growing. This recent trend prompts biologists to use
computer to analyze DNA base sequences. Typical examples of common
analyses are those such as homology search, restriction enzyme site map,and open reading frame search. Search and retrieval of specific base sequences from DNA sequence data are prerequisite to such analyses. Thus,a DNA data base is demanded to efficiently manipulate sequence data.
The unix operating system has been chosen as a target system to develope
a data base, because of a portable operating system. By taking advantage
of superior tools for interprocess communication in unix such as sharedmemory and pipeline, basic tools for data manupulation are being implemented as filters at the shell level.
Basic tools are provided such as- to output specified entries,- to output specified types of records in specified entries,- to output the name of entries with strings specified in regular expression,- to output base sequences specified in regular expression with their entry
names by searching a whole data base, and also
- to convert formats.
These tools are used with unix tools such as "sort", "uniq" and so on to
search and retrieve entries by keywords such as author names, the titles of
papers, species names, locus names, and others. In addtion, a wide variety
of application programs for sequence analyses will be incorporated into thedata base system.
This type of simple search and retrieval system has an advantage to
be portable among unix systems whether computer is a large, mini or
personal computer. Although portability is lost, an alternative method tobuild data bases is to use one of data base management systems that arecommercially available. Relational data base is advantageous, because itis easy to build and also it may allow detailed search and retrieval. We
are planning to build a relational data base to manage data entry in the
DNA Data Bank of Japan as well as for its use as a research tool.

PUBLICAnONS

103

PUBLICATlONSAKETAGAWA, J., MIYATA, T., OHTSUBO, S., NAKAMURA, T., MORITA, T., HAYASHIDA, H.,
MIYATA, TAKASHI, and IWANAGA, S.: Primary structure of limulus anticoagulant
anti-lipopolysaccharide factor. J. BioI. Chem., 261: 7357-7365, 1986.
AKETAGAWA, J., MIYATA, T., OHTSUBO, S., NAKAMURA, T., MORITA, T., HAYASHIDA,
H., MIYATA, TAKASHI, IWANAGA, S., TAKAo, T. and SHiMONISHI, Y.: Primarystructure of anticoagulant, anti-lipopolysaccharide factor, from the limulus Tachypleus
tridentatus. J. BioI. Chem. (in press).
AOKI, K.: A stochastic model of gene-culture coevolution suggested by the "culture
historical hypothesis" for the evolution of adult lactose absorption in humans. Proc.
Natl. Acad. Sci. USA, 83: 2929-2933, 1986.
AOKI, K.: Stable polymorphic equilibria in a toy model of group selection. Jpn. J.
Genet. 61: 481-490,1986.
Aota, S. and Ikemura, T.: Diversity in G +C content at the third position of codons invertebrate genes and its cause. Nucl. Acids Res. 16: 6345-6355 & 8702, 1986.
BEHRENS, P. Q., NAKASHIMA, H., YOKOTA, E. and RIGGS, A. F.: The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. J. BioI. Chem. 261 (23):10520-10525, 1986.
CARSTEN, A. L., BENZ, R. D., COMMERFORD, S. L., HUGHES, W., ICHIMASA, Y., IKUSHIMA,
T., and TEZUKA, H.: Further studies on the genetic damage to bone marrow andother somatic effects following exposure to low level tritium. NIRS-M-52 (Proc.
2nd Workshop on Tritium Radiobiology and Health Physics), 258-275, 1985.
FUERST, P. A. and MARUYAMA, T.: Fate of genetic variation in captive 300 populations.
Zoo Biology 5: 171-179, 1986.
FUJII, T. and TANO S.: Mutagenic activities of EMS on somatic (M,) and recessive (M 2)mutations in the soybean test system. Environ. Exp. Botany 26: 191-195, 1986.
FUKUDA, R, and HATADA, E.: RNA synthesis of influenza A viruses: Temperaturesensitive mutants defective in RNA synthesis. (in Japanese) Uirus (Virus) 36: 203219, 1986.
FURUSATO, T., TAKANO, J., JIGAMI, Y., TANAKA, H., and YAMANE, K.: Two tandemly
located promoters, artificially constructed, are active in a Bacillus subtilis a-amylase
secretion vector. J. Biochem., 99: 1181-1190, 1986.
GLASS, R. E., JONES, S. T. and ISHIHAMA, A.: Genetic studies on the fJ subunit of Escherichia coli RNA polymerase, VII. RNA polymerase is a target for ppGpp. Mol. Gen.
Genet. 203: 265-268, 1986.
GLASS, R. E., JONES, S. T., NENE, V., NOMURA, T., FUJITA, N. and ISHIHAMA, A.: Genetic
studies on the fJ subunit of Escherichia coli RNA polymerase, VIII. Localisation ofa region involved in promoter selectivity. Mol. Gen. Genet. 203: 487-491, 1986.
GLASS, R. E., HONDA, A. and ISHIHAMA, A.: Genetic studies on the fJ subunit of Escherichia coli RNA polymerase, IX. The role of the carboxyterminus in enzyme assembly.
Mol. Gen. Genet. 203: 492-495, 1986.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37
GOJOBORI, T., and NEI, M.: Relative contributions of germline gene variation and somatic
mutation to immunoglobulin diversity. Mol. BioI. Evol. 3: 156-167, 1986.
GOJOBORI, T., AOTA, S., INOUE, T. and SHIMOTOHNO, K.: A sequence homology between
the pX genes of HTLV-Ijll and the murine IL-3 gene. FEBS Let. 208: 231-235,
1986.
HATTORI, M., BUSE, J. B., JACKSON, R. A., GLIMCHER, L., FORF, M. E., MINAMI, M.,
MAKINO, S., MORIWAKI, K., KAZUYA, H., IMURA, H., STRAUS, W. M., SEIDMAN, J.
G. and EISENBARTH, G. S.: The NOD mouse: Recessive diabetogenic gene in the
major histocompatibility complex. Science 231: 733-735, 1986.
HAYASAKA, K., HORA!, S., SHOTAKE, T., NOZAWA, K. and MATSUNAGA, E.: Mitochondrial DNA polymorphism in Japanese monkeys, Macaca fuscata. Jpn. J. Genet.
61: 345-359, 1986.
HAYASHIDA, H., KUMA, K. and MIYATA, T.: Sequence homology between mas oncogene
product and rhodopsin family. Nature 323: 116,1986.
HIRAO, I., ISHIKAWA, M., and MIURA, K.: Partial synthesis of leader sequence of phage
f1 coat protein mRNA. Chemistry Letters. (1986): 1929-1932.
HONDA, A., MIZUMOTO, K. and ISHIHAMA, A.: RNA polymerase of influenza virus:
Dinucleotide-primed initiation of transcription at specific positions on viral RNA.
J. BioI. Chern., 261: 5987-5991, 1986.
HORAI, S. and MATSUNAGA, E.: Mitochondrial DNA polymorphism in Japanese II.
Analysis with restriction enzymes of four or five base pair recognition. Human
Genetics 72: 105-117, 1986.
HORA!, S., GOJOBORI, T. and MATSUNAGA, E.: Distinct clustering of mitochondrial DNA
types among Japanese, Caucasians and Negroes. Jpn. J. Genet. 61: 271-275, 1986.
HOUBA-HERIN, N., HARA, H., INOUYE, M. and HIROTA, Y.: Binding of penicillin to thiolpenicillin-binding protein 3 of Escherichia coli. Identification of its active site.
Molec. Gen. Genet. 201: 499-504,1985.
IHARA, M., GADRINAB, L. U., SIREGAR, U. J., and IYAMA, S.: Genetic control of alcohol
dehydrogenase and estimation of some population parameters in Hopea odorata
Roxb. (Dipterocarpaceae). Jpn. J. Genet. 61: 127-136, 1986.
IKEGAMI, M., MORINAGA, T., and MIURA, K.: Infectivity of virus-specific double-stranded
DNA from tissue infected by bean golden mosaic virus, a geminivirus. Ann. Phytopathol. Soc. Jap. 52: 735-739, 1986.
IMAI, H. T.: Modes of species differentiation and karyotype alteration in ants and mammals. In "Modern Aspects of Species" (ed. by K. Iwatsuki, P. H. Ravan, and W. J.
Bock), 87-105, Univ. of Tokyo Press, Tokyo, 1986.
IMAI, H. T., MARUYAMA, T., GOJOBORI, T., INOUE, Y. and CROZIER, R. H.: Theoreticalbases for karyotype evolution. I. The minimum interaction hypothesis. Amer.
Nat. 128: 900-920, 1986.
IMAMURA, T.: The thalassemia syndromes. (in Japanese) Handbook of Internal Medicine,Vol. 86-A (ed. by Y. Yoshida, S. Oguma and H. Uchino), 99-113, Nakayama-Shoten,
Tokyo, 1986.
IMAMURA, T.: Thalassemia syndromes. (in Japanese) Clinical Investigations 30: 381-388,
1986.

PUBLICATIONS105

INOUE, T., TEZUKA, H., KADA, T., AIKAWA, K. & SHULTZ, L. D.: The mouse mutant"wasted": An animal model for ataxia-telangiectasia. In" Antimutagenesis andAnticarcinogenesis: Mechanisms" (ed. by D. M. Shankel & others), 323-335, Plenum
Press, New York, 1986.
INOUE, T., MURAKAMI, K. and FUJII, T.: Mutagenic potential of cordycepin (3'-deoxyadenosine) in Salmonella and soybean tester strains. Mutation Res. 174: 179-182,
1986.
INOUE, T., AIKAWA, K., TEZUKA, H., KAnA, T. and SHULTZ, L. D.: Effect of DNA damaging agents on isolated spleen cells and lung fibroblasts from the mouse mutant "wasted", a putative animal model for ataxia-telangiectasia. Cancer Res. 46: 3979-3982,
1986.
INOUE, T., SUZUKI, 0., KADA, T. and FUJII, T.: Bio-antimutagenic effect of L-ethionine
on the spontaneous mutagenesis in Salmonella and Bacillus subtilis. Jpn. J. Genet.
61: 461-467, 1986.
ISHIHAMA, A.: Transcription signals and factors in Escherichia coli. In "Adv. Biophy.",
21, 163-173, 1986.
ISHIHAMA, A., FUJITA, N. and NOMURA, T.: Promoter selectivity of Escherichia coli RNA
polymerase. In "RNA Polymerase and the Regulation of Transcription", Elsevier
Sci. Publ. Comp., New York, 1986.
ISHIHAMA, A., MIZUMOTO, K., KAWAKAMI, K., KATO, A. and HONDA, A.: Proofreading
function associated with the RNA-dependent RNA polymerase from influenza virus.
J. BioI. Chern. 261: 10417-10421, 1986.
ISHIHAMA, A.: A proposal for promotion of virus research. (in Japanese) Virus (Virus)
36: 181-183, 1986.
ISOBE, Y. and SHIMADA, Y.: Organization of filaments underneath the plasma membraneof developing chicken skeletal muscle cells in vitro revealed by the freeze-dry androtary replica method. Cell Tissue Res. 244: 47-56, 1986.
IsOBE, Y. and SHIMADA, Y.: Cytoskeleton in embryonic skeletal muscle cells. Bio Essays
4: 167-171, 1986.
KADA, T., INOUE, T., OHTA, T. and SHIRASU, Y.: Antimutagens and their modes ofaction. In "Antimutagenesis and Anticarcinogenesis: Mechanisms"(ed. by D. M.
Shankel & others), 181-196, Plenum Press, New York, 1986.
KADA, T., INOUE, T., MORITA, K. and NAMIKI, M.: Dietary desmutagens. In "Genetic
Toxicology of the Diet" (ed. by I. Knudsen), 245-253, Alan R. Liss, Inc., New York,1986.
KATOH, Y., HASEGAWA, T., SUZUKI, T. and FUJII, T.: Plant regeneration from the callus
derived from mature embryo of Hiproly barley, Hordeum distichum L. Agric. BioI.
Chern. 50: 761-762, 1986.
KI1IMA, S. and KrJIMA, H.: Statistical analysis of channel current from a membrane patch.
I. Some stochastic properties of ion channels or molecular systems in equilibrium.
J. Theor. BioI. (in press)
KI1IMA, S. and KrJIMA, H.: Statistical analysis of channel current from a membrane patch.
II. A stochastic theory of a multi-channel system in the steady-state. J. Theor.
BioI. (in press)

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37
KIKUNO, R. and MIYATA, T.: Slowly evolving Drosophila mitochondrial genes. Mol.
BioI. Evol. (in press)
KIMURA, M.: Diffusion model of population genetics incorporating group selection, withspecial reference to an altruistic trait. "Stochastic Processes and Their Applications",(ed. by K. Ito and T. Hida), 101-118, Lecture Notes in Mathematics, Springer-Verlag,
Berlin, 1986.
KIMURA, M.: Diffusion models of population genetics in the age of molecular biology.
"The Craft of Probabilistic Modelling", (ed. by J. Gani and C. C. Heyde), 150-165,
Springer-Verlag, Berlin, 1986.
KIMURA, M.: DNA and the neutral theory. Phil. Trans. Roy. Soc. London, Series B,
312: 343-354, 1986.
KITAGAWA, H., KITAMURA, N., HAYASHIDA, H., MIYATA, T. and NAKASHIMA, S.: Structural relationship of the three rat kininogen genes and their evolution. J. BioI. Chern.
(in press)
KOJIMA, H., KONISHI, H. and KURODA, Y.: Combined effects of chemicals on mammaliancells in culture. I. Effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the mutation induction. Mutation Res. 164: 272, 1986.
KURODA, Y.: Genetic and chemical factors affecting chemical mutagenesis in cultured
mammalian cells. In "Antimutagenesis and Anticarcinogenesis: Mechanisms" (ed. by
D. M. Shankel & others), 359-375, Plenum Press, New York, 1986.
KURODA, Y.: In vitro studies on the spermatogenesis of Drosophila melanogaster. In"Techniques in the Life Sciences, Techniques in Vitro Invertebrate Hormones andGenes" (ed. by E. Kurstak & H. Oberlander), C124, 1-6, Elsevier Biomedical, 1986.
KURODA, Y.: Differentiation of adult structures in cultures of embryonic tissues from
Drosophila melanogaster. In "Techniques in the Life Sciences, Techniques in Vitro
Invertebrate Hormones and Genes" (ed. by E. Kurstak & H. Oberlander), C215, 1-5,
Elsevier Biomedical, 1986.
KURODA, Y.: Antimutagenic activity of vitamin C in cultured mammalian cells. Mutation Res. 164: 273, 1986.
KUSUDA, J., TAZIMA, Y., ONIMARU, K., NINAKI, O. and SUZUKI, Y.: The sequence aroundthe 5' end of the fibroin gene from the wild silkworm, Bombyx mandarina, and comparison with that of the domesticated species, B. mori. Mol. Gen. Gent. 203: 359364, 1986.
MARUYAMA, T., GOJOBORI, T., AOTA, S. and IKEMURA, T.: Codon usage tabulated fromthe GenBank genetic sequence data. Nucl. Acids Res. 14: Suppl. r 151-197, 1986.
MATSUNAGA, E.: Paternity examination of two cases with hereditary disease. (in Japanese) Jpn. J. Legal Med. 40: 520, 1986.
MIYASHITA, N. and MORIWAKI, K. : Genetic mechanism governing development of tumors
in mammalian forms. (in Japanese) In "Progress in Cancer Research '86" (ed. by Y.
Yamamura & others), Vol. 23, 183-190, Nakayama-shoten, Tokyo, 1986.
MIYASHITA, N.: Effect of major histocompatibility complex on the development of carcinogen-induced pulmonary adenomas in mice. (Abstract in English) J. Juzen Med.
Soc. 95: 161-169, 1986.
MIYATA, T., TOH, H. and ONO, M.: Homology between IgE-binding factor and re-PUBLICATIONS

101

trovirus genes. Nature 322: 484, 1986.
MORISHIMA, H.: Wild progenitors of cultivated rice and their population dynamics. In"Rice Genetics", 3-14, IRRI, Manila, 1986.
MORIWAKI, K.: Molecular and cytogenetical differentiation of Mus musculus species.
In "Modern Aspects of Species" (ed. by K. Iwatsuki, P. Raven and W. J. Bock), 221225, Univ. of Tokyo Press, Tokyo, 1986.
MORIWAKI, K., MIYASHITA, N., SUZUKI, H., KURIHARA, Y. and YONEKAWA, H.: Genetic
features of major geographical isolates of Mus musculus. In" The Wild Mouse in
Immunology" (ed. by M. Potter, J. H. Nadeau and M. P. Cancro), Current Topics in
Microbiology and Immnuology 127,55-61, Springer-Verlag, Berlin. 1986.
NAKASHIMA, H., BEHRENS, P. Q., Moore, M. D., Yokota, E. and RIGGS, A. F.: Structureof hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of theoverlapping peptides, ordering the CNBr fragments, and the complete amino acidsequence. J. BioI. Chern. 261 (23): 10526-10533, 1986.
NAKASHIMA, H., TOKUNAGA, S., KONISHI, H., NISHIDA, H., TSURU, A., NAKAMURA, S. and
MATSUNAGA, A: A case of small bowel ileus strangulated by falciform ligament. (inJapanese) Med. J. Mutual Assoc. 35: 449-451,1986.
NEr, M., and GOJOBORI, T.: A simple method for estimating the number of synonymousand nonsynonymous nucleotide substitutions. Mol. BioI. Evol. 3: 418-426, 1986.
NOMIYAMA, H., JINNO, Y., MATSUDA, I., SHIMADA, K. and MIYATA, T.: Amplification ofhuman argininosuccinate synthetase pseudogenes. J. Mol. BioI. 192: 221-233, 1986.
NOMURA, T., FUJITA, N. and ISHIHAMA, A.: Promoter selectivity of Escherichia coli RNA
polymerase: Alteration by fMet-tRNAfMe'. Nucleic Acids Res. 14: 6857-6810,
1986.
OHTA, T.: Population genetics theory of multigene families with emphasis on genetic
variation contained in the family. "Evolutionary Processes and Theory", (ed. by S.
Karlin & E. Nevo), 239-253, Academic Press, New York, 1986.
OHTA, T.: Population genetics of an expanding family of mobile genetic elements. Genetics
113: 145-159, 1986.
OHTA, T.: Actual number of alleles contained in a multigene family. Genet. Res. 48:
119-123, 1986.
OKAMURA, H., YANAGI, Y., IMARI, Y., KUDO, J., ISHIBASHI, N. and IMAMURA, T.: Werner's
syndrome associated with cholangiocarcinoma. Jap. J. Med. 25 (2): 179-183, 1986.
ONO, M., YASUNAGA, T., MIYATA, T. and USHIKUBO, H.: Nucleotide sequence of thehuman endogenous retrovirus gene related to the mouse mammary tumor virus gene.
J. Virol. 60: 589-598, 1986.
SAGAI, T., SHIROISHI, T., MORIWAKI, K., BONHOMME, F., PETRAS, M. L., THOHARI, M.,
Yu, Z. C., Lu, D. Y. and CHO, W. S.: Characterization of newly isolated monoclonal
antibodies against MHC of a Japanese wild mouse. Immunogenetics 24: 361-361,
1986.
SAKOYAMA, Y., HONG, K-J., BYUN, S. M., HISAJIMA, H., UEDA, S., YAOITA, Y., HAYASHIDA,
H., MIYATA, T. and HONJO, T.: Nucleotide sequences of immunogloblin epsilon
genes of chimpanzee and orang utan: DNA molecular clock and hominoid evolution.
Proc. Natl. Acad. Sci. USA 84: 1080-1084, 1986.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37
SANO, Y.: Sterility barriers between Oryza sativa and O. glaberrima. In" Rice Genetics",
IRRI, Manila, 1986.
SANO, Y., KATSUMATA, M. and OKUNO, K.: Genetic studies of speciation in cultivatedrice. 5. Inter- and intraspecific differentiation in the waxy gene expression of rice.
Euphytica 35: 1-9, 1986.
SASAKI, Y. F., IMANISHI, H., WATANABE, M., SEKIGUCHI, A., MORIYA, M., SHIRASU, Y. andTutikawa, K.: Mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) in the mouse
spot test. Mutation Res. 174: 145-147, 1986.
SATO, Y. I., CHITRAKORN, S. and MORISHIMA, H.: The Indica-Japonica differentiationof rice cultivars in Thailand and its neighboring countries. In "New Frontiers in
Breeding Researches" (ed. by B. Napompeth & S. Subhadrabandhu), 185-194, Kasetsart Univ., Bangkok, 1986.
SHIMADA, Y. and ISOBE, Y.: Cytoskeletal organization in embryonic chick skeletal muscle
cells in vitro revealed by detergent-extraction, freeze-dry method. In "Molecular
Biology of Muscle Development" (ed. by C. Emerson, D. A., Fischman, B., NadalGinard and M. A. O. Sidiqui), pp. 725-739. Alan R. Liss, Inc., New York, 1986.
SHINKAWA-TACHI, K., MORIMOTO, K., KOIZUMI, A. and Kuroda, Y: Combined effects
of chemicals on mammalian cells in culture. II. Effects of ethyl methanesulfonate(EMS) and arabinosyl cytosine (Ara-C) on mutation induction. Mutation Res. 164:
281, 1986.
SHIRASU, Y., MORIYA, M., TEzuKA, H., TERAMoTo, S., OHTA, T. and INOUE, T.: Mutagenicity of pesticides. In "Mutation, Cancer and Malformation" (ed. by E. H. CHU
and W. M. GENEROSO), 617-624, Plenum Press, New York, 1984.
SHIROISHI, T., SAGAI, T., SAKAI, S. and MORIWAKI, K.: Lethal deletion of the C4 and 21hydroxylase genes in the mouse H-2 class III regions, caused by meiotic recombination.
Proc. Natl. Acad. Sci. USA 84: 2819-2823, 1987.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.: Sexual preference of meiotic recombination
within the H-2 complex. Immunogenet. (in press)
SUZUKI, H., MIYASHITA, N., MORIWAKI, K., KOMINAMI, R. MURAMATsu, M., KANEHISA,
T., BONHoMME, F., PETRAS, M. L., Yu, Z. C. and Lu, D. Y.: Evolutionary implicationof heterogeneity of the nontranscribed spacer region of ribosomal DNA repeating
units in various subspecies of Mus musculus. Mol. BioI. Evol. 3: 126-137, 1986.
SUZUKI, H.: Penicillin-binding proteins. (in Japanese) Biseibutsu 2: 81-89, 1986.
SUZUKI, Y., TSUDA, M., TAKIYA, S., HIROSE, S., SUZUKI, E. KAMEDA, M. and NINAKI, 0.:
Tissue-specific transcription enhancement of the fibroin gene chraracterized by cellfree systems. Proc. Natl. Acad. Sci., USA, 83: 9522-9526, 1986.
TACHIBANA, H. and ISHIHAMA, A.: Correlation between the rate of productive transcription initiation and the strand-melting property of Escherichia coli promoters. Nucleic
Acids Res. 13: 9031-9042, 1986.
TAKAHATA, N.: An attempt to estimate the effective size of the ancestral species common
to two extant species from which homologous genes are sequenced. Genet. Res. 48:
187-190, 1986.
TAKAHATA, N. and SLATKIN, M.: Private alleles in a partially isolated population. II.
Distribution of persistence time and probability of emigration. Theor. Pop. BioI.

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30: 180-193, 1986.
TANABE, N., KIJIMA, H., and MARuHAsHI, J.: Trinitrobenzen sulfonic acid (TNBS) increases transmitter release at the frog neuromuscular junction. Proc. Japan Acad.
63B (in press)
TAYA, C. and MORIWAKI, K.: Effect of H-2 complex on the growth of embryo-derived
teratomas in mice. Am. J. Reprod. Immunol. 10: 35-38, 1986.
TEZUKA, H., INOUE, T., NOOUTI, T., KADA, T. and SHULTZ, L. D.: Evaluation of themouse mutant "wasted" as an animal model for ataxia telangiectasia. I. Age-dependent and tissue-specific effects. Mutation Res. 161: 83-90, 1986.
TOMITA, I., NAKAMURA, Y., YAGI, Y. and TUTIKAWA, K.: Fetotoxic effects of mono-2ethylhexyl phthalate (MEHP) in mice. Environ. Health Perspect. 65: 249-254, 1986.
TSUDA, M., HIROSE, S. and SUZUKI, Y.: Participation of the upstream region of the fibroin
gene in the formation of transcription complex in vitro. Mol. Cell. BioI. 6: 39283933, 1986.
VEDA, S., WATANABE, Y., HAYASHIDA, H., MIYATA, T., MATSUDA, F. and HONJO, T.:
Hominoid evolution based on the structures of immunoglobulin epsilon and alphagenes. Cold Spring Harbor Symp. 51: 429-432, 1986.
WATADA, M., INOUE, Y. and WATANABE, T. K.: Expansion of Drosophila simulans inJapan. Zoo1. Sci. 3: 873-883, 1986.
YAMANE, K., NAKAzAWA, K., NAKAMURA, K., MINEKURA, M., TAKANO, J., SHIROZA, T.,
SoHME, A. and FUJITA, T.: Secretion activity of the Bacillus subtilis a-amylase signal
peptides with different lengths in Bacillus subtilis and Escherichia coli cells. In"Bacillus Molecular Genetics and Biotechnology Application" (ed. by A. T. Ganesan
and J. A. Hoch), p. 411, Academic Press, New York, 1986.
YAMAZAKI, T., CHao, J-K., WATANABE, T. K. and TAKAHATA, N.: Gene flow in natural
populations of Drosophila melanogaster with special reference to lethal allelism rates
and protein variation. Genetics 113: 73-89, 1986.
YONEKAWA, H., GOTOH, 0., TAGASHIRA, Y., MATSUSHIMA, Y., SHI, L.-I., CHO, W. S.,MIYASHITA, N. and MORIWAKI, K.: A hybrid origin of Japanese mice "Mus musculus
molossinus". In "The Wild Mouse in Immunology" (ed. by M. Potter, J. H. Nadear
and M. P. Cancro), Current Topics in Microbiology and Immunology 127, 62-67,
Springer-Verlag, Berlin, 1986.
YONEZAWA, K. and OKA, H. I.: Conservation methods of crop populations with mixed
selfing and outcrossing. Proc. of 5th SABRAO Int. Cong.: 575-584, 1986.
YONEZAWA, K., TATEMATSU, N., SPETSOV, P. and TSUNEWAKI, K.: Increasing genetic
variability in common wheat by utilizing alien cytoplasms--Effects of four Aegilops
cytoplasms on the cross, Norin 26 x Norin 61. Jpn. J. Breed. 36: 262-273, 1986.
Yoo, I. D., FUJII, T., SANO, Y., KOMAGATA, K., YONEYAMA, T., IYAMA, S. and HIROTA,
Y.: Dinitrogen fixation of rice-Klebsiella associations. Crop Sci. 26: 297-301, 1986.
YUKUHIRO, K. and MUKAI, T.: Increased determental load possibly caused by a transposon in a local population of Drosophila melanogaster. Jpn. J. Genet. 61: 25-43,
1986.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

ABSTRACTS OF DIARY FOR 1986
Biological Symposium
247th meeting Jan. 23

248th
249th
250th

25lst
252nd
253rd
254th
255th
256th

The mechanism of RNA polymerase: Evi·dence in support of rotationary translocation
model for transcription (D. Dennis)
Apr. 3Ecosystem assembly (G. Sugihara)
Apr. 30 Immortal genes (S. Ohno)
June 11 Structure and regulation of Bacillus subtilis
RNA polymerase sigma-43 operon (R. H.
Doi)
July 7
Mitochondrial DNA studies on Australianresella parrots (R. H. Crozier)
July 29 Genetic-epidemiologic studies in Hungary(A. Czeizel)
July 30 Sex and sexual selection: a role for parasites?
(W. D. Hamilton)
Sept. 1 Genetics of susceptibility to mammary cancer
in mice (J. Hilgers)
Nov. 10 The Neutralismjpanselectionism controversy
and some geckos (G. Pasteur)Nov. 27 In vitro transcription of eukaryotic genes (J.
L. Manley)

Mishima Geneticists' Club
3l0th meeting Jan. 23
3llth
3l2th
3 13th

314th
315th

Protein evolution and the partitioned struc·ture of genes (M. Go)
Feb. 14 Invertebrate taste reception (H. Kijima)
Mar. 4 The molecular structure, function and evolution of hemocyanins (H. Nakashima)Mar. 13 Structure and function of Heliozoan micro·
tubule (Y. Shigenaka)
Mar. 28 Analysis of human rotavirus genome (Y.
Furuichi)Mar. 31 Construction of transforming retrovirus

ABSTRACTS OF DIARY FOR 1986

316th meeting

Apr. 11317th

May 29Special program Oct. 17

318th

Oct. 23

111

carrying ras induced cellular gene, TGF-a(S. Watanabe)
Role of DNA conformation in the transcription (S. Hirose)
Genetics of mouse complement system (S.
Sakai)New methods of homology search for nonconservative proteins: Its applications to immunoglobulin superfamily and rhodopsin
family (H. Hayashida)
Hominoid evolution inferred from DNA sequence data (N. Saito)

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

FOREIGN VISITORS IN 1986

April 9, 1984October 10, 1986
February 26, 1985July 25, 1986
March 1, 1985September 11, 1985September 10, 1986
October 15, 1985January 14
January 24
January 30March 7
March 15-June 12
April 3
April 22April 30
June 11
July 7
July 29
July 30-31
September 1-2
September 2-29

Pascale Barbier, Universite des Sciences et Techniques du Languedoc Montpellier, France
Pierre Boursot, Universite Montpellier, France
Yi-de Huang, Shanghai Institute of Plant Physiology, China
Won Ho Lee, Pusan National University, Korea
Yuan shen Qiu, Guangdong Microbiology Research Institute, China
Don Dennis, University of Delaware, U.S.A.
Sydney Brenner, MRC Laboratory of Molecular
Biology, U.K.
Jozefa Styrna, Jagiellonian University, Poland
Robert Fujimura, Oak Ridge National Laboratory, U.S.A.
Tjan Kiauw Nio, Bandung Institute of Technology, Indonesia
George Sugihara, Scripps Institute of Oceanography, UCSD, U.S.A.
Nguyen Xuan Hong, Hanoi University, Vietnam
Susumu Ohno, Beckman Res. Inst. of the Cityof Hope, U.S.A.
Roy H. Doi, University of California, Davis,
U.S.A.
R. H. Crozier, University of New South Wales,
Australia
Andrew Czeizel, National Institute of Hygiene,
Hungary
W. D. Hamilton, Oxford University, U.K.
Jo Hilgers, Netherlands Cancer Institute, Netherlands
Robert E. Glass, University of Nottingham, U.K.

FOREIGN VISITORS IN 1986

113

R. T. Walker, University of Birmingham, U.K.
J. A. Richards, British Council, Tokyo
Songkran Chitrakon, Department of AgricuSeptember 10-24
lture, Thailand
September 29-30
Arne Hagberg, The Swedish University ofAgricultural Sciences, Sweden
September 10-October 9 Peng Luo, Sichuan University, China
Chi Yen, Sichuan Agricultural University, China
October 1-2
Qi quan Shao, Institute of Genetics, Academia
October 2-5
Sinica, China
Rusdy E. Nasution, National Biological Institute,
October 3-7
Indonesia
Tibor Sik, University of Agricultural Sciences,
October 8
Hungary
Bengt O. Bengtsson, University of Lund, Sweden
October 15-16
Ganesh Prasad, University of Gorakhpur, India
October 15-24
Li ming Shi, K~nming Animal Research InOctober 15
stitute, Academia Sinica, China
Ulfur Amason, University of Lund, Sweden
October 15
Udda Lundquist, Swedish University of AgriOctober 17-18
cultural Sciences, Sweden
F. Scholz, Zentralinstitut fUr Genetik und KuOctober 18-19
lturpflanzenforschung, German Democratic Republic
Georges Pasteur, Universite Montpellier-H,
November 10
FranceShozo Yokoyama, Washington University School
November 12
of Medicine, U.S.A.
James L. Manley, Columbia University, U.S.A.
November 27
Nai Ai Liang, Academia Sinica Guangzhou
December 9
Branch, China
David M. Lambert, University of Auckland,
December 12
New Zealand; Anthony J. Huges, Auckland
Hospital, New Zealand
December 19-20
Masatoshi Nei, The University of Texas, U.S.A.
September 4

114

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 37

AUTHOR INDEXAMANO, E ••.•.•...••...••..••....•. 96

KAoA, T. ..

AOKI, K •.......•..••••••••••.... 76,

KAMEDA, M ••••••••••••••••••.•••••• 33

77
AOTA, S.-I. .•.•••••..•••.... 33, 34, 81
BARBIER, P. . •..•••.•.•••.•••.•..... 94
BONHOMME, F. • •••••.••••....•...... 60
BOURSoT, P • . . . . . . . . . . . . . . . . . . • . . . . . 59
CHoo, J.-K. • •.••••..........•...... 77
CROZIER, R. H. • ...•.......•..•...•• 58
ENDO, T •.•.•.•••••••....•.••....... 98
FUJII, T
30
FUJITA, N
16, 17, 18, 19
FUKUDA, R. .
21, 22, 27, 28
GADRINAB, L. U. .
92
GLASS, R. E. .
16, 19GOJOBORI, T. • .. 34, 58, 80, 81, 82, 88
GOTOH, H • . . . . . . . . . . . . . . . . . . . . . • . . . 40
HANAOKA, F. . .....................• 29
HARA, H. .
'35
HARA, M . . • . . . . . . . . . . . . . . . . . . . . . . . . 66
HARADA, K. . •••••...••••....•...... 78
HARADA, Y. .
39
HATADA, E •..•.•...•..••........ 27, 28
HAYASAKA, K •.•...•...........•.... 91
HAYASHI, S •••..•...•• 1 ••••••••••••• 35
HIROSE, S • . . . . . . . . . . . . . . . . . • 31, 32, 33
HIROTA, Y
35, 36HONDA, A ••.••..••.••..•........... 23
HORAI, S. . ••....•..•.••.•.. 88, 90, 91
HOSAKA, Y
25
ICHIHASHI, H. • •....••..•.....••..... 98
IKEMURA, T. . ....•.••........... 33, 34
IMAI, H. T
58
IMAMURA, T •••.....•••.•..••....•... 87
INOUE, T •.••..•• 66, 67, 68, 70, 81, 90
INOUE, Y. .
58, 83
IsHIHAMA, A.

.. 16, 17, 18, 19, 20, 23, 24, 25, 26
93, 95IYAMA, S •..•.•••••.•............... 101
ISHIKAWA, R •••.•••••....... 92,

KATO, J.-I.

66, 67, 68, 70••••••••••••..••••••••••• 36

KATSUMATA, M •...•.••.•••••••.••..• 96
KIMuRA, M.

.

72

KINOSHITA, T •.........•........••.•. 92
KITAZAWA, T.

• ••••••.••........... .49

KOJIMA, H •.......•••.•.....•........ 46
KONISHI, H.

. .......•....••.•...... .46

60, 61.45, 46, 47, 70
KUSAKABE, S.-I. .......•.....•...••..• 78
MARUYAMA, T. ..
58, 80
MATSUMOTO, K. • ........•..•.....•.. 29
MATSUNAGA, E
85, 86, 88, 90, 91
MINATO, K. . ................•...... 48
MIYASHITA, N.43
MIYATA, T •...........•...•.•.•.•..• 83
MIYAZAWA, S • . . . . . . . . . . . . . . . . . . . . . 102
MIZUMOTO, K
23
MORI, M • . . . . . . . • . . . . . . . . . . . . . . . . . . 36
MORISHIMA, H. . •... 92, 93, 94, 95, 96
KURIHARA, Y.

KURODA, Y.

.

.

MORIWAKI, K .

.. 38, 39, 40, 41, 43, 59, 60, 61, 62
MORIYAMA, E. N • . . . . . . . . . . . . . . . . . . . 82
MUKAI, T • . . . . . . . . . . . . . . . . . . . . . . . . • 78
MUKAIGAWA,J........•.......... 27,

28
50, 51, 53
NAGASAWA, Y
17
NAGATA, K • . . . . . . . . 23, 24, 25, 26, 29
NAKASHIMA, H. . •..•••......•......• 87
NARITOMI, Y
87
NAwA. S. •30, 54
NEI, M •......................... 80, 81
NINAKI, O. • .................•...... 33
NOMURA, T. . ......•....•••. 16, 19, 20
NozAwA, K •••.......••........•...• 91
OHTA, T ....•.•..•........••...• 73, 74
MURAKAMI, A • . . . . . . . . . .49,

OHTSUKI, Y

49

AUTHOR INDEXSADAIE, T ••••.•.••.•..••••....•••... 69
SADAIE, Y •.•.••••.....••••••..•• 37,

69
SAGAI, T
38, 40, 41
SAKAGAMI, Y •••••.•.•.•••••..•••.•.• 36
SANO, Y •.•..•..•...•.•.•••...••• 30, 96
SATO, Y. I
93, 95, 96
SERIZAWA, H
21, 22SHIMADA, Y
47
SHIMIZU, H. .
55
SHIMIZU, K
28
SHIMOTOHNO, K. .
81
SHIROISHI, T •..•...••.•••.... 38, 40, 41
SHOTAKE, T •............•.••.•••.•.. 91
SLATKIN, M
75
STYRNA, J'" .. 62
SUGIYAMA, Too •.....•.•......... '" .. 55
SUH, D. S •......••........••.... 61, 62
SUZUKI, A. .
36
SUZUKI, E. .
33
SUZUKI, H
35, 36SUZUKI, Y •..........•.....• 31, 32, 33

115

TABUCHI, H

31
75, 77
TAKEUCHI, K
24
TAKEUCHI, M. .
66
TAKIYA, S •..•.•••••.•.•••••.•.•••••• 33
TAMAI, K
70
TAYA, Coo •••••.•.••••••.•.••••••.•.. 63
TERADA, H
55
TEZUKA, H
68, 70TOMITA, T •.•••.•.•••...•••...••.••• 39
TSUDA, M. .
32, 33
UllOA, K
24
UEOA, S . . . . . . . . . . . . . . . . . . • . • • . • . . . • 17
WATAOA, M
83
WATANABE, T. K
77, 83
WINKING, H
60
YAMADA, M. A. .
48, 54YAMANAKA, K
25
YAMAZAKI, T •.•..............•.....• 77
YONEKAWA, H
63
YONEZAWA, K"
98
TAKAHATA, N.

.

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