ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT1988

No. 39Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1989

Annual Reportof theNational Institute of Genetics
No. 39, 1988

Published by

The National Institute of Genetics, Japan
1989

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council and Advisory Committee'" .. . ..
Association for propagation of the knowledge of genetics
Projects of research for 1988
Research activities in 1988

1
7
10
12
13
18

1. Molecular Genetics
Promoter selectivity of E. coli RNA polymerase: Cloning andcharacterization of E. coli promoters. KUBOTA, M., YAMAZAKI,Y., FUJITA, N. and ISHIHAMA, A.
Promoter selectivity of E. coli RNA polymerase: Subunit (j) plays
an essential role in stringent control. IGARASHI, K., FUJITA,
N., OKAMOTO, H. and ISHIHAMA, A.
RNA polymerase sigma-related protein in Escherichia coli: Purification and characterization of proteins which crossreact with
anti-sigma peptide antibodies. UESHIMA, R., FUJITA, N. and
ISHIHAMA, A.
RNA polymerase and transcription signals of Micrococcus luteus.
NAKAYAMA, M., FUJITA, N., OSAWA, S. and ISHIHAMA, A. '"Molecular mechanism of influenza virus RNA transcription. I.
Analysis of promoters for viral RNA polymerase. ISHIHAMA,
A., PARVIN, J. D., PALESE, P., KRYSTAL, M., HONDA, A.,
KATO, A., UEDA, S., YOKOIYAMA, A., YAMAZAKI, Y., YAMANAKA, K. and NAGATA, K.
Molecular mechanism of influenza virus RNA transcription. II.
Matrix protein epitopes involved in transcription inhibition.
HANKINS, R. W., NAGATA, K., BUCHER, D. J., and ISHIHAMA,A
Molecular mechanism of influenza virus RNA transcription. III.
Binding mode of influenza virus nucleoprotein to RNA.
YAMANAKA, K., OGASAWARA, N., YOSHIKAWA, H., NAGATA,
K. and ISHIHAMA, A.

18

19

20
20

21

22

23

ii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Transcription and replication of influenza virus RNA: An in vitro
system using isolation nuclei from virus-infected cells. NAGATA, K., SAKAGAMI, H. and ISHIHAMA, A.
Translational regulation of influenza virus mRNAs. YAMANAKA,
K., OGASAWARA, N., YOSHIKAWA, H., NAGATA, K. and ISHIHAMA. A
Mechanism of anti-influenza virus by mouse Mx gene. YOKOIYAMA, A., NAGATA, K., IWAKURA, Y., and ISHIHAMA, A. ....
Regulation of transcription by nuclear factor I, a protein essential
for adenovirus DNA replication. MATSUMOTO, K., NAGATA,
K., HANAOKA, F. and ISHIHAMA, A.
Soluble 5'-nucleotidases from rabbit heart. YAMAZAKI, Y.,
TRUONG, Y. L., LOWENSTEIN, J. M., and ISHIHAMA, A.
Structure and complete nucleotide sequence of the h~man thymidylate synthase gene. KANEDA, S., NALBANTOGLU, J.,
TAKEISHI, K., AYUSAWA, D., SHIMIZU, K., GOTOH, 0., HORIE,
N. and SENO, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Functional analysis of the human thymidylate synthase gene withthe use of a minigene. KANEDA, S., AYUSAWA, D. and
SENO, T.
Characterization of DNA double strand scission in mouse cells
induced by thymidylate deprivation. AYUSAWA, D., TEZUKA,
H. and SENO, T.
Coupling between replication and translation by E. coli singlestranded DNA binding protein. SHIMAMOTO, N.
Kinetic study on transcription by immobilized operons: Release of
E. coli RNA polymerase (J subunit requires f3-r phosphodiester
bond of nucleoside triphosphates. SHIMAMOTO, N. and
FUJIOKA, M.
Global variation in G+C content along vertebrate genome DNA,and chromosome band structures. IKEMURA, T., AOTA, S.,
ISHIBASHI, F. and MATSUMOTO, K.
Purification of RNA molecules with high resolution by twodimensional gel techniques. IKEMURA, T.
Computer analyses for searching for protein coding regions inhuman genome DNA sequences. WADA, K., AOTA, S. and
IKEMURA, T.

24

25
25

26
27

28

29

30
31

31

32
33

34

CONTENTS

Purification of a DNA supercoiling factor from the posterior silk
gland of Bombyx mori. OHTA, T. and HIROSE, S.
DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter. TABUCHI, H.
and HIROSE, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Mechanism of mRNA capping reaction. MIZUMOTO, K.
Messenger RNA synthesis of Sendai virus (HVJ). MIZUMOTO,
K
Regulation of chromosomal replication in bacteria. YOSHIKAWA,
H

II.

iii

34

35
36
37

38

Microbial Genetics
Gene organization in the region containing a new gene involvedin chromosome partition in Escherichia coli. KATO, J.,NISHIMURA, Y., YAMADA, M., SUZUKI, H. and HIROTA, Y. . ..
Isolation of novel mutants defective in DNA replication in Escherichia coli. YASUDA, S.
AIlelism of Escherichia coli parA to gyrB and some views on pardependent regulation of ceIl division. NISHIMURA, Y., KATO,
J. and SUZUKI, H. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Nucleotide sequence of the sufI gene responsible for the multicopy
suppression of a thermosensitive cell-division mutation in
E. coli. YAMAMOTO, Y., KATO, J., SUZUKI, H. and NISHIMURA, Y
C-terminal processing of penicillin-binding protein 3 of Escherichia
coli. I. Analysis of the degradation product that represents the
C-terminal half. SAKAGAMI, Y., NAGASAWA, H., SUZUKI, A.,
HARA, H. and SUZUKI, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C-terminal processing of penicillin-binding protein 3 of Escherichia
coli. II. Determination of the cleavage site. NAGASAWA, H.,
SUZUKI, A., HARA, H. and SUZUKI, H. .. . . . . . . . . . . . . . . . . ..
Requirement of the C-terminal region for the function of Escherichia coli penicillin-binding protein 3. HARA, H. and SUZUKI,
H.
Cloning of a gene involved in the C-terminal processing of penicillinbinding protein 3 of Escherichia coli. HARA, H. and SUZUKI,
H

41
43

45

47

48

49

51

52

iv

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Mapping of prc that causes a defect in the C-terminal processingin Escherichia coli. HARA, H., HIGASHITANI, A., NISHIMURA,
Y. and SUZUKI, H.
Accumulation of the precursor form of penicillin-binding protein
3 in Escherichia coli mutants defective in general protein export. HARA, H. and SUZUKI, H.
A new gene controlling the frequency of cell division per round of
DNA replication in Escherichia coli. NISHIMURA, A.
A cell division regulatory mechanism controls the flagellar regulon
in Escherichia coli. NISHIMURA, A. and HIROTA, Y.
Restriction map of a Bacillus subtilis gene involved in cell division,
sporulation and exoenzyme secretion. SADAIE, Y.

III.

55

56
57
58
59

Immunogenetics

Novel molecular organization of meiotic recombinational hotspots
in the murine major histocompatibility complex. SHIROISHI,
T., SAGAI, T., HANZAWA, N., STEINMETZ, M. and MORIWAKI,K
New evidence for trans-species evolution of the H-2 class I polymorphism. SAGAI, T., SHIROISHI, T. and MORIWAKI, K. ....
Steroid 21-hydroxylase deficiency in mice. GOTOH, H., SAGAI, T.,
RATA, J., SHIROISHI, T. and MORIWAKI, K.

60
61
62

IV. Developmental and Somatic Cell Genetics
Artificial expression of tissue-specific characteristics in embryoniclethal cells of Drosophila melanogaster. KURODA, Y.
Selective infection of SR spiroplasms in cultured embryonic cells of
Drosophila melanogaster. KURODA, Y., SAKAGUCHI, B.,
OISHI, K. and SHIMADA, Y.
Antimutagenic effects of safrole on chemically induced mutations
in cultured Chinese hamster cells. KURODA, Y., JAIN, A. K.
and TEZUKA, H.
Injury effect on hydra head regeneration. SHIMIZU, H. and SUGIYAMA, T
Random displacement of I-cells into buds and maintenance of the
I-cell population in Hydra. FUJISAWA, T.

63

64

65
66
67

CONTENTS

Genetic studies on the silkworm adult lifespan (a). Heredity ofthe short adult lifespan (sdi). MURAKAMI, A.
.
Genetic studies on the silkworm adult lifespan (b). A biological.
characteristic of the Daizo (sdi) strain. MURAKAMI, A
Genetic studies on the silkworm adult lifespan (c). Sex differences
and their biological significance. MURAKAMI, A.
.
The genetic nature of known mutants in the domesticated mulberrysilkworm, Bombyx mori. MURAKAMI, A. .
.
Decrease in hatchability of eggs laid by flies reared on excess yeast.
in Drosophila melanogaster. MINATO, K. .
Purification and structure analysis of an autocrine growth factor
in conditioned medium obtained from primary cultures ofscleral fibroblasts of the chick embryo. FUJIOKA, M., KAWAHARA, A., AMANO, M., WATANABE, K. and SHIMAMOTO, N . . .
The three-dimensional ultrastructure of intracellular membranous
system in cultured chick skeletal muscle cells. SHIMADA, Y.

v

67
69
70
71

73

74

75

v. Cytogenetics
Molecular analysis of complement factor H genes in wild mice(Mus musculus) and their relatives. HARADA, Y., BONHOMME,
F., NATSUUME-SAKAI, S., MORIWAKI, K. and TOMITA, T..... 76
Reproductive ability of hybrids between Mus musculus castaneusand M. m. subspecies in China. NIWA, M., WAKASUGI, N.,
MITA, A. and MORIWAKI, K. 77
Effect of the Y chromosome and H-2 complex of Japanese wild
mouse on sperm morphology. SUH, D.-S., STYRNA, J. and
MORIWAKI, K. 78
DNA sequencing analysis of rDNA in Robertsonian mice. KURIHARA, Y., SUZUKI, H., WINKING, H., BONHOMME, F. and MORIWAKI, K. 79
Geographical survey of wild mouse populations in Eastern Asia
based on restriction analysis and sequence analysis of mitochondrial DNA. YONEKAWA, H., MIYASHITA, N. and MORIWAKI, K
. 79

vi

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

VI.

Mutagenesis and Radiation Genetics

Tumor suppression gene in Asian wild-derived strains of mice.
MIYASHITA, N. and MORIWAKI, K.

VII.

Population Genetics

Further simulation studies on evolution by gene duplication.
OHTA, T.
Time for acquiring a new gene by duplication. OHTA, T.
Evolution by gene duplication and compensatory advantageous
mutations. OHTA, T.
More on the episodic clock. TAKAHATA, N.
The coalescent in two partially isolated diffusion populations.
TAKAHATA, N.
Dubious maternal inheritance of mitochondrial DNA in D.
simulans and evolution of D. mauritiana. SATTA, Y., ToyoHARA, N., OHTAKA, C., TATSUNO, Y., WATANABE, T. K.,
MATSUURA, E. T., CHIGUSA, S. I. and TAKAHATA, N. .. . . . . . .
Diffusion model of population genetics incorporating intergroup
selection and epistatic interaction in fitness. KIMURA, M. ...
A sexual selection model for the evolution of imitative learning ofsong in polygynous birds. AOKI, K.
Pleiotropy and preadaptation in the evolution of human language
capacity. AOKI, K. and FELDMAN, M. W.

VIII.

81

82
83
83
84
84

85
86
88

89

Evolutionary Genetics

Algorithmic analysis of the computer program for likelihood of ahuman pedigree. GOJOBORI, T. and YASUDA, N.
Molecular evolution of human and simian immunodeficiency
viruses. GOJOBORI, T., MORIYAMA, E. N. and YOKOYAMA,S
Molecular evolution of the human immunodeficiency and related
viruses. YOKOYAMA, S., CHUNG, L., MORIYAMA, E. N. and
GOJOBORI, T.
Evidence for species-to-species transfer in evolution of pathogenic
determinants of Escherichia coli and Vibrio cholerae 01.
YAMAMOTO, T., MORIYAMA, E. N. and GoJOBORI, T.

90

90

91

91

CONTENTS

Mitochondrial DNA polymorphism among five Asian populations. HARIHARA, S., SAITOU, N., GOJOBORI, T. and OMOTO,
K
Gametic disequilibrium for isozyme genes found in wild andcultivated rices. MORISHIMA, H.
Tropical and temperate types in Asian rice cultivars. TANG, L.-H.,
SATO, Y.-I. and MORISHIMA, H.
Plant opal analysis: Its significance to the origin and evolution ofrice. SATO, Y.-I., FUJIWARA, H. and UDATSU, T.
On the maximum likelihood method for estimating molecular
trees: Uniqueness of the likelihood point. FUKAMI, K. andTATENO, Y.
Is molecular evolution parsimonious?: Theoretical approach to the
problem. TATENO, Y.
Different rates of evolution of autosome-, X chromosome- and Y
chromosome-linked genes: Hypothesis of male-driven molecular evolution. MIYATA, T., KUMA, K., IWABE, N., HAYASHIDA, H. and YASUNAGA, T.
Immunoglobulin-like sequences in the extracellular domains ofproto-oncogene fms and platelet-derived growth factor receptor. HAYASHIDA, H., KUMA, K. and MIYATA, T. . . . . . . ..
Male-driven molecular evolution: A model and nucleotide sequence
analyses. MIYATA, T.

IX.

vii

92
92
94
95

95
96

96

97
98

Human Genetics

Cytogenetic studies on chromosome 18 polyploidy syndrome bynon-radioactive in situ hybridization techniques and probe
L1.84. IMAMURA, T., HASEGAWA, T., NAKASHIMA, H. and
FUJlYAMA, A
100
Population studies of gene conversion within the duplicated human
a-globin gene family. NAKASHIMA, H., FUJlYAMA, A. and
IMAMURA, T. .
101
Analysis of j3-globin gene mutation in Japanese patients with 13thalassemia reveals a substitution of thymine for cytosine in
the second intron. NARITOMI, Y., CHIFU, Y., NAKASHIMA,
H., FUJlYAMA, A. and IMAMURA, T
102

viii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Regulation of ras activity on membranes by post-translational processing modification. FUJIYAMA, A., TSUNASAWA, S. and
SAKIYAMA, F.
103
Molecular phylogeny and evolution of primate mitochondrialDNA. HAYASAKA, K., GOJOBORI, T. and HORAI, S.
104
Maternal inheritance of deleted mitochondrial DNA in a familywith mitochondrial myopathy. OZAWA, T., YONEDA, M.,
TANAKA, M., NONAKA, I. and HORAI, S
105
A mutation-epidemiologic study on sporadic retinoblastoma.
MATSUNAGA, E. and MINODA, K.
105
Ethnic differences in the incidence of childhood malignancies:
Comparison of Japanese and U. S. data. MATSUNAGA, E.
107
and NISHIHIRA, H. .

X.

Applied Genetics
Protein bands in morphological mutants of Japanese morning glory
detected by electrophoresis in immobilized pH gradients.
ENDO, T
110
Distribution of Wx alleles in rice. SANO, Y.
111
Molecular cloning of ribosomal RNA genes from two cultivated112
rice species, Oryza sativa and O. glaberrima. SANO, Y
Ribosomal DNA spacer-length polymorphisms in a wild rice
population from Dongxiang, China. SANO, Y., YI, H., SHAO,
Q. and IYAMA, S.
113
Selection strategies based on two different measurements of selection efficiency, expected genetic advance and chance of
success. YONEZAWA, K
114

XI. Database
Activities of the DNA data bank of Japan. MIYAZAWA, S. and
HAYASHIDA, H
117
Estimation of the average energy increments of protein native structures due to amino acid exchanges and its use in evaluatinga substitution probability matrix for homology search.
MIYAZAWA, S. and JERNIGAN, R. L.
120
Publications from Genetic Resources Section, Genetic Stocks
121
Research Center. IYAMA, S.

CONTENTS

ix

Publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 123
Abstracts of diary for 1988
130
Foreign visitors in 1988
133
Author index
136

GENERAL STATEMENT
This report presents an outline of research and related activities carriedout in our institute during the year 1988. Only new developments orchanges will be summarized here.
Support of research activities
Total expenditures, not including those for facilities, for 1988 FY (April
1988-March 1989) amounted to 1 billion 166 million yen, a half of which
was for personnel expenses. In addition, grants-in-aid amounting to 210
million yen were rendered to selected staff members by the Ministry of
Education, Science and Culture (Monbusho). A Gene Library Laboratory
was added to the DNA Research Center and the Radioisotope Center was
newly established. Accordingly, new positions for two associate professors,a research staff member (joshu), a technician, and an administrative official
were allotted. Thus the total number of positions available for regularstaffis 101, of which 59 are for research professionals. Concerning facilities,
a five-storied building (total floor area of 2389 centares) for the Radioisotope
Center and a central machinery house were completed on December 5.
The new building included two rooms for P3 level and three for P2 levelrecombinant DNA experiments. Now that molecular genetics has become
the main focus of genetic research, the completion of a modern building forhigh technology using radioisotopes is extraordinarily important for thefurther development of the institute.
Opening of the Graduate University for Advanced Studies
As was announced in the last issue, October marked the opening of the
Graduate University for Advanced Studies, which is based on six national
inter-university research institutes (Institutes of Statistical Mathematics,
High Energy Physics, Molecular Sciences, Genetics, Basic Biology, andPhysiological Sciences). Our institute will function as the Department of
Genetics, which, together with the Institute for Basic Biology and the Institute for Physiological Sciences, represent the Graduate School of "LifeSciences." We are preparing for the admission of about six students whowill major in genetics next April.

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

HonorsProf. M. Kimura, Head of the Department of Population Genetics,
retired at the end of March. He is continuing his research as a Visiting
Professor. Upon approval at a council meeting in June, Prof. M. Kimura
and Prof. Kin-ichiro Miura of the University of Tokyo, who held professorship from 1969 to 1984 at the institute, were conferred the title of Professor Emeritus, according to the regulations of the institute.
I am happy to note that three of our colleagues were honored in the past
year. Prof. Kimura was awarded the 4th International Prize for Biology,
for his outstanding contributions to the modern development of both thetheoretical aspects of population genetics with his study on diffusion models,
and to evolutionary genetics with his neutral theory of molecular evolution.
This prize was founded in order to encourage studies in biology, in commemoration of the Emperor's sixty year reign and his years of study inbiology. Prof. K. Miura was awarded the Japan Academy Prize for his"Molecular genetic studies on double-stranded DNA viruses, especially thediscovery of the cap structure of RNA." While he was in the institute, hediscovered that one end of RNA, to which genetic information of eukaryotic
cells and their viruses is transcribed, has a special structure called a "capstructure." Associate Prof. K. Tutikawa won an Encouragement Award
from the Environmental Mutagen Society of Japan for "The mouse spot
test: Development of the method with a new cross." This represents asystem to detect in vivo somatic mutations induced by mutagens, which iscurrently being used by several industries for mutagen testing of new drugs
and chemicals.
Personnel changes

The past year saw a number of personnel changes in the professional
staff of the institute. While Drs. Ryuji Fukuda (Laboratory of Molecular
Genetics), Tadashi Inoue (Laboratory of Mutagenesis), and Kenichi Aoki
(Laboratory of Population Genetics) left for universities as professors or
associate professors, the following ten appointments were made: Takeshi
Seno, professor; Dai Ayusawa, associate professor; and Sumiko Kaneda(Laboratory of Mutagenesis), Hidenori Tachida (Laboratory of Population
Genetics), Etsuko Moriyama (Laboratory of Evolutionary Genetics), Yoshio
Tateno and Hiroyuki Hirano (Genetic Stock Research Center), researchstaff members; Nobuo Shimamoto, associate professor; and Kenichi Matsu-

GENERAL STATEMENT

3

moto (DNA Research Center), and Ikuo Nakamura (Experimental Farm),
research staff members. Dr. Yoshio Sano and Dr. Takashi Gojobori were
promoted to associate professors. Associate Prof. Yoshito Sadaie wastransferred from the Laboratory of Mutagenesis to the newly establishedRadioisotope Center as its head.
International exchanges and symposia
The institute's staff were also very active in international personnel
exchanges in the past year. Ten staff members attended the XVIth International Congress of Genetics held in Toronto, Canada, in August.
Among them, M. Kimura, Professor Emeritus, was inivited to give a plenary
lecture on his neutral theory of molecular evolution, which evoked a great
deal of attention in local newspapers. Including that congress, membersof our staff went abroad on 41 occasions for the purpose of presenting
research results at various scientific meetings, exchanging information, andcarrying out collaborative studies or field investigations. On the other
hand, 58 scientists who visited our institute from abroad delivered lecturesat the Biological Symposium or exchanged information and views on recent
studies. Those who stayed longer than one month and carried out collaborative studies with our staff were: Miss Pascale Barbier, Universite desSciences et Techniques du Languedoc, Montpellier, France; Dr. Dong
Sang Suh, Genetic Engineering Center, Korean Advanced Institute ofTechnology, and Dr. Sam-Eun Kim, Sericultural Experiment Station, Rural
Development Administration, Republic of Korea; Dr. Ling Hua Tang,
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Miss
Xiao Mei Wu and Miss He Zhao, Lanzhou Institute of Biological Products,
Ministry of Public Health, People's Republic of China; Prof. W. B. Provine,
Cornell University, and Prof. D. M. Shankel, University of Kansas, U.S.A.,
and Prof. M. Rahat, Hebrew University, Israel.
Two international meetings were organized by our staff members. One
was the Fourth International Symposium on "Population Biology of Genes
and Molecules" held in conjunction with the Award of the InternationalPrize for Biology Nov. 28-Dec. 2 in Tokyo. With support from Monbusho,
ten scientists from abroad and ten Japanese were invited. Besides Prof.
Kimura's award lecture, recent results of studies by other top scientists
were presented to an audience of about one hundred. The other meeting
was the 2nd International Conference on Mechanisms of Antimutagenesis

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

and Anticarcinogenesis held in Ohito Dec. 4-8, organized by Prof. Yukiaki
Kuroda of the Laboratory of Phenogenetics and Prof. D. M. Shankel who
was in Japan for cooperative studies with Prof. Kuroda. Interest in environmental mutagens and carcinogens has recently been shifting to identifying desmutagens, antimutagens and anticarcinogens. This is a new
field initiated by the late Prof. Tsuneo Kada of the Laboratory of Mutagenesis (died Nov. 14, 1986). The conference was successful with 273
attendants, of which 99 were from abroad.
Service to scientific communities
The DNA Data Bank of Japan (DDBJ) continues to offer the following
services through the efforts of the staff of the Laboratory of DNA Data
Analysis: collection and input of data, sending data, on-line service bytelephone, and issuing newsletters (Nos. 7 & 8). Training courses anddemonstrations for beginners were carried out at general meetings of certain
academic societies such as the Japan Cancer Association, and the Biochemistry and Molecular Biology Associations. The input of data applies topapers mainly published in Japanese academic journals. The 3rd edition
issued in July, 1988, includes 230 entries and 345,850 bases, which corresponds to only one thirtieth of that of EMBL or GenBank; we have to
make every effort to increase our data bases. In the past year an International Advisory Committee was set up in order to give advice regarding theprocedure of the program. The committee recommended that DDBJ,
EMBL and GenBank should collaborate in maintaining close contacts witheach other. In order to assume a heavier responsibility in international
collaboration, a drastic strengthening of the man-power and increase in thebudget are indispensable. Prof. Hisao Uchida of Teikyo University, who
is a Japanese representative to the Advisory Committee, has been providing
advice to the operation of our DDBJ as a Visiting Professor since April.
Another project is to maintain and distribute the "E. coli Gene Library,"
which is a relational clone bank of the whole E. coli chromosome establishedby Drs. Yuji Kohara of Nagoya University and Katsumi Isono of Kobe
University. At present it is taken care of by the staff of the Laboratory of
Molecular Genetics. Since genome analysis of various organisms including
human beings has become a most challenging project in biology, the physicalmapping of the E. coli chromosome is very important as a first step. From
its start in September, 1987 till May, 1988, 17,433 clones in total were dis-

GENERAL STATEMENT

5tributed upon request for 37 domestic and 90 foreign projects.
The third project is to supply specific strains of mouse, Drosophila, E.
coli, B. subtilis, etc. The total number supplied in 1987 amounted to 369(42 sent abroad) and the total number of strains supplied was 4,388 (131sent abroad). The fourth is to systematize, with the staff of the Laboratoryof Genetic Resources, data-base information about location, characteristics,
availability of distribution, etc., of a variety of experimental organisms
preserved in universities and research institutes in Japan. "Silkworm
strains in Japan, 1988" and "Rice Genetics Newsletter, No.4" were also
issued and distributed.
Service to the public
The institute held its yearly open house for the public on April 23. Someof the research activities of each laboratory were exhibited using panels,
microscopes, microcomputers, and molecular models. Educational lectures, "Genetic individuality, natural environment, and the survival of
human beings" by Prof. Takashi Imamura and "Evolution at the molecularlevel" by Associate Prof. Takashi Gojobori were given. The double cherry
blossoms on the campus were at their best and some 3,000 visitors in theneighborhood enjoyed them. On November 12, public lectures were given
at the National Science Museum in Tokyo; the titles were "Regulation ofstem cell differentiation in Hydra" by Associate Prof. Toshitaka Fujisawa
and "The origin and molecular evolution of AIDS viruses" by Dr. Gojobori. In spite of it being a Saturday afternoon about 120 eager people
from universities and institutes listened to the lectures, which were followedby rather specialized questions and lively discussions.
Need for more facilities and man-pOll'er
Five years have passed since the institute was switched over to one forjoint use by universities. We have accepted upon application, 38 collaborative programs, II workshops, 5 graduate students, 7 research fellows
and 10 scholarships from industries, and 3 entrusted research projects fromprivate corporations. Yet, there remain a number of problems to be solvedin order to achieve the reality of reform. Among other things, constructionof a new building for research to accommodate laboratories for joint use
by visiting professors, and lodgings for visiting researchers with welfare
facilities are of the highest priority. We are all eager to do our best to6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

accomplish the missions of our institute. We hope to have continued
encouragement and support from all persons concerned.
At the completion of my term of directorship, I will retire on October 1st,
1989. I herewith express my heartfelt gratitude to all those who encouraged
and helped me during my six years of service.

STAFF
Director

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

1. Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Laboratory of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
NAGATA, Kyosuke, D. Pharm.
Laboratory of Mutagenesis
SENO, Takeshi, D. Sc., Professor
AYUSAWA, Dai, D. Ag., Associate professor
TEZUKA, Hideo
KANEDA, Sumiko, D. Pharm.
Laboratory of Nucleic Acid Chemistry (Guest member)
YOSHIKAWA, Hiroshi, D. Sc., Professor
UCHIDA, Hisao, D. Sc., Professor
MIZUMOTO, Kiyohisa, D. Sc., Associate professor
2. Department of Cell Genetics
MORIWAKI, Kazuo, D. Sc., Head of the Department
Laboratory of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami T., D. Sc., Associate professor
SHIROISHI, Toshihiko, D. Sc.
Laboratory of Microbial Genetics
YASUDA, Seiichi, D. Sc., Associate professor
NISHIMURA, Yukinobu, D. Sc.
HARA, Hiroshi, D. Sc.
Laboratory of Cytoplasmic Genetics (Guest member)
YONEKAWA, Hiromichi, D. Sc., Associate professor

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

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
IDE, Hiroyuki, D. Sc., Associate professor

4. Department of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Head of the Department
Laboratory of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKAHATA, Naoyuki, D. Sc., Associate professor
TACHIDA, Hidenori, D. Sc.
Laboratory of Evolutionary Genetics
TUTIKAWA, Kiyosi, Associate Professor
GOJOBORI, Takashi, D. Sc., Associate professor
MORIYAMA, Etsuko
Laboratory of Theoretical Genetics (Guest member)
KIMURA, Motoo, 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
IMAMURA, Takashi, D. Med., Professor

STAFF

FUJIYAMA, Asao, D. Sc., Associate professor
HORAI, Satoshi, D. Med.
NAKASHIMA, Hitoshi
Laboratory of Agricultural Genetics
MORISHIMA-OKINO, Hiroko, D. Ag., Professor
ENDO, Toru, D. Ag., Associate professor
SATO-HIRAOKA, Yoichiro, D. Ag.
Laboratory of Applied Genetics (Guest member)
WATANABE, Takeshi, D. Med., Professor
YONEZAWA, Katuei, D. Ag., Professor
6.

Research Facilities
Genetic Stock Research Center
SUGIYAMA, Tsutomu, Ph. D., Head of the Center
MIYASHITA, Nobumoto, D. Med.
WATANABE, Takao K., D. Sc., Associate professor
VEDA, Hitoshi, D. Ag.
SANa, Yoshio, D. Ag., Associate professor
HIRANO, Hiroyuki, D. Ag.
NISHIMURA, Akiko, D. Ag.
IYAMA, Shin-ya, D. Ag., Associate professor
TATENO, Yoshio, Ph. D., D. Sc.
l)lVA Research Center
SENO, Takeshi, D. Sc., Head of the Center
SHIMAMOTO, Nobuo, D. Sc., Associate professor
IKEMURA, Toshimichi, D. Sc., Associate professor
MATSUMOTO, Kenichi, D. Ag.
HIROSE, Susumu, D. Sc., Associate professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
HAYASHIDA, Hidenori
Radioisotope Center
SADAIE Yoshito, D. Sc., Head of the Center
Experimental Farm
IYAMA, Shin-ya, D. Ag., Head of the Farm
NAKAMURA, Ikuo, D. Ag.

9

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 3)

7.

Technical Section
ONIMARU, Kimiharu, Chief of the Section

8.

Department of Administration
HARA, Toshio, Head of the Department
UJIIE, Jun, Chief of the General Affairs Section
TANIGUCHI, Hiroshi, Chief of the Finance Section

COUNCIL
Chairman
YAMAMURA, Yuichi; Emeritus professor, Osaka University
Vice-chairman
EGAMI, Nobuo; Emeritus professor, University of Tokyo
Members (Alphabetical order)
HAYAISHI, Osamu; President, Osaka University of Medicine
HORIO, Takekazu; Director, Institute for Protein Research, Osaka
University
ICHIKAWA, Atsunobu; Dean, Graduate School at Nagatsuta, Tokyo
Institute of Technology
IINO, Tetsuo; Professor, Faculty of Science, University of TokyoIMAHORI, Kozo; President, Naruto University of Education
KIMURA, Motoo; Emeritus professor, National Institute of Genetics
KUWAHARA, Shogo; Director, Toho University
NAGAKURA, Saburo; President, Graduate University for Advanced
StudiesNAKAI, Junnosuke; President, Hamamatsu University School of
Medicine
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
OKADA, Tokindo S.; Director-General, Institute for Basic Biology
ONOE, Hisao; Dean, Faculty of Economics, Osaka Industrial University
OZEKI, Haruo; Professor, Faculty of Science, Kyoto University
SAKAI, Fuminori; Director, Japan Society for the Promotion of Science
TAKANAMI, Mitsuru; Director, Institute for Chemical Research, Kyoto
University
WATANABE, Itaru; Emeritus professor, Keio University

COUNCIL

11

YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University

ADVISORY COMMI'ITEE
Chairman
KURODA, Yukiaki; Professor, Laboratory of Phenogenetics
Vice-chairman
SASAKI, Motomichi; Professor, Faculty of Science, Hokkaido University
Members (Alphabetical order)
FUKUDA, !chiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
ISHIWA, Sadao; Professor, Faculty of Science, Ochanomizu Women's
University
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University of
Tokyo
NAKAJIMA, Tetsuo; Professor, Faculty of Agriculture, Tamagawa University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
OSAWA, Shozo; Professor, Faculty of Science, Nagoya University
TAKEUCHI, Takuji; Professor, Faculty of Science, Tohoku University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YOSHIKAWA, Hiroshi; Professor, Faculty of Medicine, Osaka University

IMAMURA, Takashi; Professor, Laboratory of Human Genetics
ISHIHAMA, Akira; Professor, Laboratory of Molecular Genetics
MORISHIMA-OKINO, Hiroko; Professor, Laboratory of Agricultural
Genetics
MORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
OHTA, Tomoko; Professor, Laboratory of Population Genetics
SENO, Takeshi; Professor, Laboratory of Mutagenesis
SUGIYAMA, Tsutomu; Professor, Laboratory of Developmental Genetics

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICSKONDO, Norio; President, Emeritus professor of Tokyo University ofAgriculture
KURODA, Yukiaki; Managing Director, Professor of National Institute of
Genetics
MORIWAKI, Kazuo; Managing Director, Professor of National Institute of
Genetics
ISHIHAMA, Akira; Manager, Professor of National Institute of Genetics
MATSUNAGA, Ei; Manager, Director of National Institute of Genetics
MIURA, Kin-ichiro; Manager, Professor of University of Tokyo
SENO, Takeshi; Manager, Professor of National Institute of Genetics
TAZIMA, Yataro, Manager, Director of Institute of Silkworm Genetics andBreeding
YAMAGUCHI, Hikoyuki; Manager, Professor of University of Tokyo

PROJECTS OF RESEARCH FOR 1988
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular Genetics

Studies on regulatory mechanisms of gene transcription (ISHIHAMA, FUJITA
and NAGATA)
Studies on molecular mechanisms of transcription and replication of animalviruses (ISHIHAMA and NAGATA)
Laboratory of Mutagenesis

Molecular and genetic studies of cell-cycle associated genes (SENO, AYUSAWA
and KANEDA)
Genetic consequences of nucleotide pool imbalance in mammalian cells(SENO, AYUSAWA and TEzuKA)
Radiation sensitivity in mammals (TEzuKA and AYUSAwA)
Laboratory of Nucleic Acid Chemisry

Studies on regulatory mechanisms of chromosome replication (YOSHIKAWA)
Molecular mechanisms of RNA 5'-cap formation (MIZUMOTO)
Studies on molecular mechanisms of transcription and replication of Sendai
virus (MIZUMOTO)
2.

DEPARTMENT OF CELL GENETICS
Laboratory of Cytogenetics

Studies on species differentiation of mouse from cyto- and molecular genetic
view points (MORIWAKI)
Immunogenetical studies on the mouse MHC (SHIROISHI and MORIWAKI)
Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Cyto- and molecular mechanism of meiotic recombination in mice (SHIRO-14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

ISHI, IMAI and MORIWAKI)
Laboratory of Microbial Genetics

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

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
and SANO)
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 the reproduction in Bombyx (MURAKAMI)
Ecogenetic studies on the nerve system characters in Bombyx (MURAKAMI)
Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)
Laboratory of Physiological Genetics

Electron microscopic studies on differentiation of animal cells (SHIMADA
and KURODA)
Effect of morphogens on pattern formation in hydra (IDE and SUGIYAMA)PROJECTS OF RESEARCH FOR 1988

4.

15

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

Theoretical studies of population genetics (OHTA, TAKAHATA and AOKI)Studies on molecular evolution from the standpoint of population genetics(TAKAHATA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (TAKAHATA)
Theoretical studies on the evolution of altruism (AOKI)
Population genetical studies on gene-culture coevolution (AOKI)Laboratory of Evolutionary Genetics

Studies on molecular evolution (GOJOBORI)
Radiation genetics in mice (TUTIKAWA)
Laboratory of Theoretical Genetics

Theoretical studies of population genetics and molecular evolution (KIMURA)
Computer studies on the molecular evolution (MIYATA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Molecular genetics of growth and differentiation of human blood form;ng
cells (IMAMURA, FUJIYAMA and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA, FUJIYAMA
and NAKASHIMA)
Studies on DNA polymorphisms in human populations (HORAI and MATSUNAGA)
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16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

SATO)
Genecological studies on pollination in rice plants (SATO and MORISHIMA)
Genetic studies on rice reserve proteins (ENDO)
Laboratory of Applied Genetics

Molecular genetics of human immune mechanisms (WATANABE)
Theoretical studies on plant breeding (YONEZAWA)

6.

RESEARCH FACILITIESGenetic Stock Research Center

Genetic studies of speciation in rice (SANO and HIRANO)
Studies on plant gene expression (HIRANO and SANO)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Documentation of genetic stocks in Japan (IYAMA)
Studies on the management system of genetic stocks information (IYAMA)
Theoretical studies on molecular phylogeny (TATENO)
Evolutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA)
Coordination of DNA synthesis and cell division in E. coli (NISHIMURA)
Mapping of a whole set of cell division genes in Escherichia coli Kl2 (NISHIMURA)
Genetic mechanisms for regulating tumor development in the laboratory
and wild mice (MIYASHITA and MORIWAKI)
DNA Research Center

Interaction between proteins and nucleic acids (SHIMAMOTO)
Studies on codon usage (IKEMURA)
Studies on chromosome band structures at the DNA sequence level (IKEMURA and MATSUMOTO)
Sequence and structure analysis of DNA and proteins (MIYAZAWA and
HAYASHIDA)
Molecular genetics of insect development (VEDA and HIROSE)PROJECTS OF RESEARCH FOR 1988

17

Control of gene expression in eukaryotes (HIROSE andlUEDA)
Radioisotope Center

Radiation genetics of Caenorhabditis elegans (SADAIE)
Molecular mechanisms of unicellular differentiation in Bacillus subtilis(SADAIE)

RESEARCH ACTIVITIES IN 1988

I.

MOLECULAR GENETICS

Promoter Selectivity of E. coli RNA Polymerase: Cloning
and Characterization of E. coli Promoters
Masayo KUBOTA, Yukiko YAMAZAKI, Nobuyuki FUJITA and Akira ISHIHAMA

The two parameters of promoter strength, affinity to RNA polymerase
and rate of open complex formation, have been determined for a numberof E. coli promoters from previously identified genes (reviewed in Ishihama,A. (1988) Trends Genet. 4: 282-286). To make a collection of promoterswith yet unidentified unique properties, we have attempted, in parallel withthe above approach, to randomly isolate E. coli DNA sequences promoting
transcription of the ampC gene. Because a linear correlation exists between
ampC-encoded ~-lactamase production and the level of ampicillin resistance
expressed, the ampC gene has advantages over certain other indicator genes,
in particular for the direct selection of strong promoters or up-mutant
promoters.
For this purpose, we used pJAC4 (Jaurin, B. and Cohen, S. N. (1985)
Gene 39: 191-201) as a transcriptional fusion plasmid vector, into which
100-500 bp Sau3A fragments of total E. coli DNA were inserted. About
500 independent transformants were isolated, which were resistant to a low
level (10 JIg/ml) of ampicillin. Production of ~-lactamase encoded by
ampC could be determined accurately by measuring the maximum concentration of ampicillin that allowed colony formation. The values ranged
from 10 JIg/ml to over 5 mg/ml ampicillin.
The origins of these DNA fragments are being analyzed by hybridization
with a gene library of the whole E. coli chromosome. Using the samepromoter cloning system, the determination of in vivo strength is in progress
for the promoters so far characterized using the in vitro mixed transcription
system.

RESEARCH ACTIVITIES IN 1988

19

Promoter Selectivity of E. coli RNA Polymerase: Subunit (I)Plays an Essential Role in Stringent Control
Kazuhiko IGARASHI, Nobuyuki FUJITA, Hiroshi OKAMOTO·
and Akira ISHIHAMARNA polymerase of Escherichia coli is composed of at least four different
subunits, a, 1', 13' and one of various a subunits. In addition, subunit w,
10 KDa in size, is tightly associated with both the core and holoenzyme ata molar ratio of 0.5 to 2 per enzyme. The function of this subunit, however,remains unclear. Recently, the gene (rpoZ) coding for the w subunit has
been cloned and sequenced. On the basis of the DNA sequence, the rpoZ
gene was found to be located next to spoT, the gene involved in ppGpp
metabolism. This finding raised the possibility that the w subunit might
playa role in stringent control.
In order to examine whether the w subunit influences ppGpp-sensitivityof RNA polymerase, we isolated both the w subunit, and w-free core enzyme
from urea-treated RNA polymerase and reconstituted holoenzymes with orwithout the w subunit. RNA polymerase lacking the w subunit was foundinsensitive to ppGpp in an in vitro mixed transcription system (Igarashi, K.
et al., submitted for publication). Addition of the purified w subunit
restored ppGpp-sensitivity of w-free RNA polymerase. These results indicate that the w subunit is a regulatory subunit of RNA polymerase andthat it is involved in the ppGpp-mediated alteration of promoter selectivity.
Previously, we reported that some E. coli mutants with mutations in the
rpoB gene encoding I' subunit exhibited a relaxed phenotype and that the
mutant RNA polymerases were insensitive to inhibition by ppGpp (Glass,
R. E. et al., (1986) Mol. Gen. Genet. 203: 492-495). To link these two
independent observations together, we propose that the target of ppGpp
is subunit w, which associates with RNA polymerase at the stringent control domain on the 13 subunit. However, the possibility that ppGpp interacts with the I' subunit with this interaction being regulated by the w
subunit has not yet been excluded.

•

Department of Biochemistry, Tohoku University School of Medicine, Sendai.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

RNA Polymerase Sigma-related Protein in Escherichia Coli:
Purification and Characterization of Proteins which
Crossreact with Anti-sigma Peptide Antibodies
Rei UESHIMA, Nobuyuki FUJITA and Akira ISHIHAMA

Anti-sera against a synthetic tetradecameric peptide with the sequence
DLIQEGNIGLMKAV, which is highly conserved among 17 factors ofbacterial RNA polymerases, specifically crossreact with several E. coliproteins in addition to the known a factors (17 70 and (7 32 ) (Fujita, N. and
Ishihama, A. (1987) Mol. Gen. Genet. 210: 5-9). In order to examine thenature of these sigma-related proteins, we purified four major proteins withapparent molecular weights of 34 KDa, 27 KDa (A component), 27 KDa(B component) and 23 KDa. N-Terminal amino acid sequence analysis
revealed that three of these protein were as yet unidentified novel proteins
and that one was ribosomal protein S2. By glycerol gradient centrifugation
under both high- and low-salt conditions, however, these proteins did notstably associate with the RNA polymerase core enzyme.
The immunological crossreaction of 17 70 and all these sigma related protein~'with the anti-sigma peptide antibodies was interfered with the addition of
the 14-meric synthetic peptide. Among three hexameric peptides withsequences identical to portions of the 14-meric sigma consensus sequence,
GLMKAV could compete with all four sigma-related proteins, while noneof these peptides could effectively compete with 1770 • These observations
suggest that the anti-sigma peptide serum used in this study contained atleast three different species of antibodies, each recognizing a specific epitope
within the consensus sigma sequence. In addition, the antiserum might
have contained an antibody which recognizes a highly ordered structure
formed by the consensus sigma sequence within 17 70 • In order to detect
novel sigma factors, therefore, antibodies against such a conformational
epitope should be used.
RNA Polymerase and Transcription Signals of Micrococcus luteus
Manabu NAKAYAMA, Nobuyuki FUJITA, Syozo OSAWA*
and Akira ISHIHAMAThe G+C content (74 %) of Micrococcus luteus DNA is one of the highest* Department of Biology, Faculty of Science, Nagoya University, Nagoya.

RESEARCH ACTIVITIES IN 1988

21

in bacteria. Through analysis of transcription initiation sites for the spc
and str operons, we found that it nevertheless carries E. coli-type promoters(Nakayama, M. et al. (1989) Mol. Gen. Genet., in press). In order tocompare the promoter selectivity of RNA polymerase between M. luteus
and E. coli, we purified RNA polymerase from both M. luteus and E. coli.
Some E. coli promoters could be correctly transcribed by the M. luteus
enzyme as well as by the E. coli enzyme, but others could not. On thebasis of core enzyme-stimulating activity, we identified and purified one
sigma factor from M. luteus. This M. luteus sigma factor, with an apparent
molecular weight of 60 KDa on SDS-PAGE, could enhance transcription
by E. coli core RNA polymerase. The sigma factor of M. luteus is therefore
functionally exchangeable with that of E. coli.
In addition to this major sigma factor, we found a minor species, which
was different from the major one in both promoter selectivity and molecularweight. Because both sigma factors were identified in cells under nonstressed conditions, they might be vegetative sigma factors.
Molecular Mechanism of Influenza Virus RNA Transcription. I.
Analysis of Promoters for Viral RNA Polymerase
Akira ISHIHAMA, Jeffrey D. PARVIN*, Peter PALESE*, Mark KRYSTAL*,
Ayae HONDA**, Atsushi KATO***, Susumu VEDA***,
Akiko YOKOIYAMA, Yukiko YAMAZAKI,
Kunitoshi YAMANAKA and Kyosuke NAGATA

The RNA-dependent RNA polymerases of animal RNA viruses havebeen studied with regard to enzyme structure and reaction mechanisms.
Little is known, however, about the elements on template RNA which
promote transcription and replication by the RNA polymerases. Suchpromoter analysis is important for understanding how viral RNA polymerases recognize specific viral RNAs from among the many host-encoded
RNAs found in infected cells.
Previously we demonstrated that the genomic RNA of influenza virus wasin a panhandle structure with base-pairing of both termini (Honda, A et al.
(1987) J. Biochem 102: 1241-1249). Furthermore, it was found that

* Department of Microbiology, Mount Sinai Medical School, New York.
** Bristol-Meyer's Research Institute, Tokyo.
*** Nippon Institute of Biological Science, Ohme, Tokyo.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

influenza viral RNA polymerase is bound at the basepaired stem of thispanhandle, leading to the suggestion that the panhandle structure is required for recognition by the viral RNA polymerase.
In order to test this possibility, we prepared a short model template of
53 nucleotides in length, by transcribing plasmid DNA with T7 RNApolymerase. The termini of this model RNA are identical to the 15 and22 nucleotide sequences conserved at the 3' and 5' termini of RNA segment
8 from all influenza A viruses. In parallel, we prepared a mixture of RNApolymerase (PBI-PB2-PA complex) and NP by CsCl centrifugation of RNP
cores (Honda, A. et al. (1987) J. Biochem. 102: 1241-1249). The RNAfree RNA polymerase fraction specifically recognized the model RNA
template and, in the presence of ApG primer, transcribed it into templatesized complementary RNA (Parvin, J. D. et al., submitted for publication).
Additional template constructions containing only the 3' half of this model
template (and therefore the 3' end of genomic RNA) were shown to be as
efficiently transcribed as the original template. These results indicate thatthe promoter for primer-directed transcription lay solely within the 15
nucleotide 3' terminus of viral RNA. The role of individual nucleotides
within the putative promoter region was also analyzed by site-specificmutagenesis.
Molecular Mechanism of Influenza Virus RNA Transcription. II.
Matrix Protein Epitopes Involved in Transcription Inhibition
Raleigh W. HANKINS*, Kyosuke NAGATA, Doris J. BUCHER**
and Akira ISHIHAMA

Recent studies have revealed that influenza virus matrix (M) protein
inhibits vRNA transcription by binding to NP protein in the basic viraltranscribing unit (RNP cores) consisting of vRNA, PB2, PBl, PA and NP.
The inhibition mechanism is, however, not understood yet.
Experiments using monoclonal antibodies against the M protein enabled
us to identify the epitope(s) on the M protein involved in this transcription
inhibition. When a battery of 15 monoclonal anti-M antibodies was* Present address: Laboratory of Microbiology, Health Sciences Research Institute, Yokohama.
** Department of Microbiology and Immunology, New York Medical College, New
York, USA.

RESEARCH ACTIVITIES IN 1988

23

analyzed on western blot, five distinct banding patterns were observed.
A representative antibody of each group was analyzed as to the effect of
the M protein on transcription inhibition. From these data, the criticalsites on M for transcription inhibition were deduced. It appears now thatthe regions on M in the vicinity of amino acid residues #70 and #140 are
physically close together in the final 3-dimensional conformation of the Mprotein, and that the residues in between form a loop and are thus removed
from the functional site.
Molecular Mechanism of Influenza Virus RNA Transcription. III.
Binding Mode of Influenza Virus Nucleoprotein to RNA
Kunitoshi YAMANAKA, Naotake OGASAWARA*, Hiroshi YOSHIKAWA*,Kyosuke NAGATA and Akira ISHIHAMA

Ribonucleoprotein (RNP) cores (RNA polymerase-RNA-NP complexes)
were isolated from detergenet-treated influenza virus AjPRj8j34 by glycerol
gradient centrifugation (Honda, A. et al. (1987) J. Biochem. 101: 12411249). RNP cores were dissociated into P-RNA (RNA polymerase-RNA)complexes and nucleoprotein (NP) by CsCI centrifugation (Honda, A.
et al. (1988) J. Biochem. 104: 1021-1026). P-RNA complexes were capableof synthesizing only small-sized RNA. By adding NP to P-RNA complexes, RNP complexes were reconstituted, which supported the synthesisof template-sized RNA as did native RNP cores. Furthermore, RNP cores,
but not P-RNA complexes, were able to produce infections viruses, whentransfected into MDCK cells. These results suggest that NP is required for
efficient elongation of the RNA chain.
To elucidate the binding mode of NP to RNA, we optimized the conditions for binding of NP to RNA by using filter binding assay and gel shift
assay. NP binds to any RNA longer than 15 nucleotides; NP-RNA complexes formed at 30°C are more resistant to high concentrations of NaCI
than those formed at O°C; divalent cations are not required for the binding;treatment of NP with N-ethylmaleimide gives no effect on its RNA binding,whereas treatment with alkaline phosphatase enhances its RNA binding.
The mode of RNA binding was analyzed by the RNase "reverse-printing"method using RNase V1 • Reconstituted NP-RNA complexes as well as
native RNP cores carry RNase Vcsensitive sites for every 15 nucleotides.
'I<

Department of Genetics, School of Medicine, Osaka University, Osaka.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

The biological activity of these reconstituted complexes is being tested. In
parallel, we are developing a bacterial expression system for large scale
production of NP.
Transcription and Replication of Influenza Virus RNA:An in vitro System Using Isolation Nuclei
from Virus-Infected Cells
Kyosuke NAGATA, Hiroshi SAKAGAMI* 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). Fractionation and reconstitution of the system, indicated that a regulatory
factor(s) that can be dissociated from vRNA-RNA polymerase complexes
upon exposure to high ionic strength is involved in the switch from mRNA
to cRNA synthesis (Nagata, K., et al. (1989) J. Biochem. 106: 205-208). Sucha factor has been suggested as being either encoded by the virus genome or
induced by virus infection. To gain further insight into influenza virus
replication, attempts were made to discover specific inhibitors of viral
replication.
It has been shown that pine cone extract (PCE) contains not only a potent
antitumor substance(s) (Sakagami, H. et al. (1987) Anticancer Res. 7: 11531160), but also a substance(s) inhibitory to virus replication (Harada, H.
et al. (1988) Anticancer Res. 8: 581-588). We examined the effect of PCE
fractions on influenza virus replication. Acidic substances of PCE markedly
suppressed the growth of the influenza virus in MDCK cells. Significant
inhibition of both viral protein synthesis in infected cells and RNA-dependent RNA polymerase activity in virions was observed with these acidic
fractions. These results suggest that the PCE contains anti-influenza virus
substance(s). Using an in vitro system for transcription and replication,
we are attempting to accurately define the molecular mechanism for theaction of PCE.

* First Department of Biochemistry, School of Medicine, Showa University, Tokyo.

RESEARCH ACTIVITIES IN 1988

25

Translational Regulation of Influenza Virus mRNAs
Kunitoshi YAMANAKA, Naotake OGASAWARA*, Hiroshi YOSHIKAWA*,Kyosuke NAGATA and Akira ISHIHAMA

In influenza virus-infected cells, ten kinds of viral proteins are synthesized
and their synthesis is regulated with respect to total amount and timing.
Although recent studies revealed that transcription is the key step in thecontrol of gene expression in the influenza virus, we found that the translation efficiency of influenza viral mRNAs from at least non structural (NS)and neuraminidase (NA) genes is subject to temporal control following
viral infection (Yamanaka, K. et al. (1988) Virus Genes 2: 19-30).
In order to test whether this control is operative for translation of otherinfluenza viral mRNAs, cDNAs for each of the genome RNA segments ofinfluenza virus A/PR/8/34 were cloned and various portions of these cDNA
clones, containing the ATG initiation codon for translation, were inserted
into the 5'-leader sequence of the chloramphenicol acetyltransferase (CAT)gene in a pSV2cat vector. Cells were first transfected with one of therecombinant plasmids and then super-infected with influenza virus. Whenplasmids carrying the 5' proximal regions of the genes, which are expressed
early after virus infection, were transfected, maximal CAT activity was
obtained at an early stage of infection. In contrast, plasmids containing
cDNA segments for the late genes, directed the highest CAT activity at thelate stage of infection. These results suggest that translational regulation
takes place during differential control of gene expression among eight RNA
segments of the influenza viral genome. To identify a signal(s) and a
cognate trans-acting factor(s) responsible for such translational regulation,in vitro translation experiments using extracts from infected cells are currently ongoing.
Mechanism of Anti-Influenza Virus by Mouse Mx Gene
Akiko YOKOIYAMA, Kyosuke NAGATA, Yoichiro IWAKuRA**
and Akira ISHIHAMA

The inbred mouse strain A2G has been shown to be resistant to influenza
virus infection. This resistance is determined by a single dominant allele***

Department of Genetics, School of Medicine, Osaka University, Osaka.
Institute of Medical Science, University of Tokyo, Tokyo.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

designated Mx+. Several attempts have been made to determine themechanism(s) for induction of the anti-influenza viral state.
First, an attempt is being made to establish mouse cells producing theMx protein. For efficient expression of the Mx gene, we inserted human
metalothionein IIA promoter upstream of Mx cDNA. The recombinant
plasmid was transfected into mouse Cl27 cells (Mx-) with a pSV2neo by
the calcium-phosphate method and Neo-resistant colonies were selected by
G-418. Production of the Mx protein in transfected cells is being checkedby Northern and Western blottings. Second, we started the production oftransgenic mice carrying the Mx+ gene. In parallel with these in vivo experiments, we are purifying the Mx protein from E. coli cells carrying an
Mx plasmid, for analysis of its effect on in vitro transcription and replication of the influenza virus.
Regulation of Transcription by Nuclear Factor I, a Protein
Essential for Adenovirus DNA Replication
Ken MATSUMOTO*, Kyosuke NAGATA, Fumio HANAOKA*
and Akira ISHIHAMA

Nuclear Factor I (NFl) has been isolated from uninfected HeLa cell
nuclei as an essential factor for the initiation of adenovirus DNA replication,and shown to be a sequence-specific DNA-binding protein (Matsumoto, K.
et al. (1989) J. Biochem. 105: 927-932). NFl binding sites (FIB sites) possibly
include the CCAAT-box which often plays a role as a regulatory elementof transcription. Cloning of NFl cDNA is important for understanding
the physiological function of NFL
For this purpose, we have devised a novel strategy using a prokaryotic
transcriptions system. An oligonucleotide containing the FIB site wasinserted into the promoter region of the E. coli metZ gene so as to overlap
the transcription initiation site. Since the promoter -10 and - 35 regions
upstream from the initiation site remained intact in this construct, designatedZi12, we expected that the inserted FIB site might function as an operator.
In the in vitro transcription system using purified E. coli RNA polymerase,
purified NFl inhibited transcription from the Zil2 promoter by interferingwith the formation of the open complex. When a reporter gene is undercontrol of such an E. coli promoter-NFl operator sequence, it appears* Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo.

RESEARCH ACTIVITIES IN 1988

27

possible to clone NFl cDNA by monitoring the repression of the reporter
gene. This strategy is also applicable for the cloning of other sequencespecific DNA-binding proteins. Although NFl has been reported to be atranscriptional activator, it may also function as a transcriptional repressor
in eukaryotes.
Soluble 5'-Nucleotidases from Rabbit Heart
Yukiko YAMAZAKI, Y. L. TRUONG*, J. M. LOWENSTEIN*
and Akira ISHIHAMA

Two molecular species of soluble 5'-nucleotidases, termed N-I and N-II,
from rabbit heart, were separated using phosphocellulose chromatography
and characterized with regard to reaction properties and protein structure.
N-I has a preference for AMP over IMP as a substrate, while N-II prefers
IMP over AMP. Both enzymes require Mg2+ as a divalent cation, butdiffer in optimum Mg2+ concentrations. High concentrations of NaCI
inhibit N-I but activate N-II.
N-I has been purified to apparent homogeneity by phosphocellulose,
DEAE-Sepharose, AMP-agarose chromatography and PROTEIN-PAK 300
HPLC. Enzyme activity cosedimented with a 94,000-dalton polypeptide
in glycerol gradient. By polyacrylamide gel electrophoresis in the presenceof sodium dodecyl sulfate, N-I migrated as fast as a protein with the molecular weight of 42,000, indicating that N-I is a dimer of 42,000 dalton
subunit. Among the 5'-nucleotides tested, N-I showed the following orderof substrate preference: AMP>CMP> UMP>IMP=GMP. However,N-I displayed a sigmoidal saturation kinetics (h= 1.76, so.5=6.8 mM) in
the absence of ADP and a hyperbolic profile (h=1.0, so.5=3.4 mM) in thepresence of ADP. Neither ATP nor 2,3-diphosphoglycerate activate purified N-I.
These properties suggest that N-I is a novel species of soluble 5'-nucleotidase and is responsible for the release of adenosine from AMP under conditions of hypoxia or increased work load.

*

Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254,
USA.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Structure and Complete Nucleotide Sequence of the Human
Thymidylate Synthase Gene
Sumiko KANEDA, Josephine NALBANTOGLU*, Keiichi TAKEISHI**,Dai AYUSAWA, Kimiko SHIMIZU***, Osamu GOTOH***,
Nobuyuki HORIE** and Takeshi SENO

Using DNA mediated transformation of mouse cell mutant strains deficient in thymidylate synthase (TS), biologically active human genomic
DNA fragments for the enzyme were cloned and their nucleotide sequenceswere determined. Cloning, structure and functional analysis of human
TS cDNA have been previously reported (Ayusawa et al., 1984, J. Bioi.
Chem. 259, 14361; Takeishi et al., 1985, Nucl. Acids Res. 13,2035; Kaneda
et al., 1987, Nucl. Acids Res. 15, 1259). The human TS gene consists ofseven exons separated by six introns. The organization of exons and thesize of each imrons were consistent with those for mouse gene except for
intron 3 (6.3 kb) which is larger than its mouse gene counterpart (Deng, Li,
Jenh and Johnson, 1986, J. Bioi. Chem. 261, 16000). Within introns, a
total of 7 Alu-type sequences are distributed. However, no Alu-type
5equences are found in intron 3. Intron 3 of the human TS gene has aninteresting feature in that it contains a long L1 repetitive sequence. The
amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90 %) to th at encoded by the
known human teratocarcinoma L1 RNA species (Skowronski, Fanningand Singer, 1988, Mol. Cell. Bioi. 8, 1385) and contained several blocks ofthe sequences homologous to RNA-dependent DNA polymerases of various
origins. Intron 3 also contains a Hind III repeat sequence and 44 times
tandem repeats of the 6 bp element of GGT(or A)GAT. The 5' flanking
region of the human TS gene lacks the canonical 5' transcriptional regulatory
sequence elements "TATA", "CAAT" and "GC box" in the vicinity of thepresumed cap sites. In the 5'-untranslated region of cDNA, a consensus
GC box sequence (GGGCGG) for the binding site of the transcription
factor Spl, was found in reverse sequence. The gene is rich in GpC sequences that fit the general properties of the growth-associated mammaliangenes. The CpG sequence distribution is skewed and concentrated in the* INRS-Sante, Universite du Quebec, Pointe-Claire, Canada.
** School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka,
Shizuoka-ken.
*** Saitama Cancer Center Res. Inst., Saitama-ken.

RESEARCH ACTIVITIES IN 1988

29

5'-flanking region, exon 1 and intron 1. Comparison of the human andmouse sequences upstream from the ATG initiator codon revealed many
significant blocks of sequence homology, although the triple tandem repeatof a 28-bp sequence and its inverted sequence found in the human sequence
were not present in the mouse counterpart.
Functional Analysis of the Human Thymidylate Synthase
Gene with the Use of a Minigene
Sumiko KANEDA, Dai AYUSAWA and Takeshi SEND

We reported the total sequence of the human thymidy1ate synthase (TS)gene of about 23 kb that consists of 7 exons and 6 introns flanked by
5' and 3' sequences. In order to locate the regulatory sequences within
the gene, for its cell cycle-dependent expression, we analyzed the effect of
introns on the expression of the TS minigene when stably introduced into
TS-negative mutant cells from mouse FM3A and rat 3Y1 cell lines. The
minigene that contained only intron I transformed the TS-negative cells at
a frequency of about 2 x 10- 4 which was comparable to that obtained with
TS cDNA ligated to the expressible plasmid vector. The intronless minigene and the minigene that contained only intron 2, both transformed thecells at frequencies 2 orders of magnitude lower than the above value. We
also showed that at least 300 bp 5' upstream of the ATG initiation codon
are required for expression of the minigene with intron 1. We further
examined the intron I sequence of 1.6 kb in terms of enhancer activity, by
introducing a part of or whole sequence of intron 1 into recombinant plasmid
pAlO Cat 2 • Results showed that the 5' proximal half of intron 1 is enoughfor full enhancer activity. It is noted that within this 800 bp region, 3
consensus sequences (GGGCGG) for the binding site of transcription factor
Spl were located while no other GGGCGG sequence was found in the TS
gene. Further study is necessary, however, to show whether or not thefunction of intron I in the minigene is associated with the cell cycle-dependent expression of the gene.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Characterization of DNA Double Strand Scission in Mouse
Cells Induced by Thymidylate Deprivation
Dai AvusAwA, Hideo TEZUKA and Takeshi SEND

We previously isolated mouse cell mutants deficient in thymidylate synthase from the FM3A line. When deprived of thymidine, the mutant cells,
which are auxotrophic for thymidine, die rapidly. The phenomenon, so
called thymineless death, was originally studied in Escherichia coli. A
positive correlation between thymidylate deprivation (often called thymidylate stress for eukaryotic cell systems) and the induction of DNA
double stand breaks has been demonstrated in the mutant cells by analyzing
DNA size, using alkaline sucrose density gradient centrifugation and neutral
filter elution. It has been also demonstrated that cell death and DNA
breaks occur in S-phase cells. Thus, they are tightly associated with DNA
replication (Ayusawa et al., 1983, J. Bioi. Chern. 258, 12448). We have alsosuggested that an inducible endonuclease is involved in DNA double strand
scission (Ayusawa, Arai, Wataya and Seno, 1988, Mutat. Res. 200, 221).
By using pulsed-field gel electrophoresis we measured the size of chromosomal DNA fragments produced by thymidylate stress. The results showedthat the double strand scission has some specificity in nature: (a) DNA
fragments of a rather uniform size of 50-150 kb, were produced in thymidylate-deprived cells, thus migrating as a discrete band across the gelelectrophoresis that was apart from the remaining intact chromosomal
DNA, and (b) DNA fragments accumulated as a function of time of thymidylate stress, but the size of the fragments did not change. These results
indicate that the sites where DNA double strand breaks occur are notrandom, but predetermined. Since the size of the DNA fragments coincides
with the size of the mammalian replicon unit and the breaks are tightly
associated with ongoing DNA replication, we hypothesize that thymidylate
stress induces DNA double strand breaks at the origin or terminator regionof each of the clustered replicons, but not at the ongoing replication fork
regions. Characterization of the DNA fragments in terms of nucleotide
sequence in both termini, and nature of the inducible endonuclease involvedin the scission, are under investigation.

RESEARCH ACTIVITIES IN 1988

31

Coupling between Replication and Translation by E. coli
Single-Stranded DNA Binding Protein
Nobuo SHiMAMOTO

E. coli single-stranded DNA binding protein specifically binds to somegroup of mRNA in a cooperative manner. The importance of this binding
has been suggested as follows. First, the binding takes place at physiologicalconcentrations. Second, a significant amount of the binding protein was
found complexed with RNA in cell lysate. Third, the complexed form ofmRNA was an inactive template in a cell-free translation system in vitro.
Fourth, the copy number of the protein in a wild type cell was within therange where two types of complexes are formed, one with DNA and theother with RNA. From these lines of evidence the following picture
emerges. When cells grow in a favorable environment, replication forks isincreased, catching more binding protein. Thus more mRNA is used intranslation. The state of replication can be transmitted to a translation
system in this way.
To check this model, I identified three RNA segment responsible forthe specific and cooperative binding on ssb mRNA with homologous 12
base sequences, named SSB box, located outside of the coding regions.
Genes alkA and sec Y mRNA have such sequences. The in vitro translationof these genes was inhibited by the binding protein but that of recA and
nusA, with no such sequences, was not. Thus the binding protein may be
a pleiotropic translation factor for presumable SSB translational regulons.
An excess amount of the protein may be toxic by inhibiting expression of
the whole regulon. An overproduction of the binding protein by mRNA
without the SSB box should supply the excess amount of the protein. Results showed such an expression was lethal.

Kinetic Study on Transcription by Immobilized Operons:
Release of E. coli RNA Polymerase q Subunit
Requires fi-r Phosphodiester Bond of
Nucleoside Triphosphates
Nobuo SHiMAMOTO and Miki FUJIOKA

Transcription of genes is mediated by a protein complex which includes
the DNA-dependent RNA polymerase. One of the largest targets for

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

research is clarifying the composition of subunits of the transcription complex. To investigate the relationship between the structure of the complex
and the RNA product, one has to stop elongation and also to freeze thecomposition of subunits. We have devised a novel method for analyzing
the structure of the transcription complex and product RNA. This method
enables us to stop elongation without changing the ionic atmosphere.
A DNA fragment containing an operator and a promoter was fixed toagarose or acrylamide beads at its ends. The immobilized DNA was atemplate as good as the conventional free DNA for E. coli RNA polymerase.
The immobilization of APR operon caused little change in the distributionof synthesized RNA species.
We applied this procedure to sigma subunit release in the early step ofinitiation of transcription by E. coli RNA polymerase. Our previous
results can be interpreted in two ways. The release itself may need magnesium ion, or alternatively, nucleoside triphosphates might be needed forthe release. This problem can be solved if RNA synthesis is rapidly
quenched in the presence of magnesium ion by diluting with an excess
amount of the reaction buffer. The latter interpretation was the case because a release was quenched by such a dilution but not by dilution with
ATP. The use of ATP analogues revealed an obligatory breakdown of (3-rphosphodiester bond for a release. Thus nucleoside triphosphates play twv
roles in transcription, as substrates for elongation and for a release.
Global Variation in G+C Content along Vertebrate
Genome DNA, and Chromosome Band Structures
Toshimichi IKEMURA, Shin-ichi AOTA*, Fumie ISHIBASHI
and Ken-ichi MATSUMOTO

In higher eukaryotes such as vertebrates, the codon-choice patterns ofdifferent genes in a single organism often differ significantly. Some genes
are extremely G+C-rich at the codon 3rd position (e.g., 95 % G+C), whereassome others are rather A+T-rich (e.g., 30% G+C). The G+C content
of the 3rd letter was found to be correlated to the G+C content of a large
genome portion surrounding the gene; exons of genes with a high G+C %at the 3rd position (e.g., >80% G+C) are usually surrounded by G+C* Present address, Department of Health & Human Services, National Institute of
Health, U.S.A.

RESEARCH ACTIVITIES IN 198833

rich introns and G+C-rich flanking sequences (e.g., 60% G+C), and thosewith a low G+C% at the position (e.g., <50% G+C) are surrounded by
A+ T-rich introns and flanking sequences (e.g., 40% G+C). We arranged human DNA sequences in the GenBank according to the order ofthe Human Genetic Map (HGM9) and analyzed global variation in G+C
content along the DNA sequences thus ordered. The analysis showed thatthe human genome is a mosaic of regions with clear differences in G+C
content, and that the diversity at the codon 3rd position is related to themosaic structures. A correlation between the G+C % levels for humanDNA sequences and the chromosome band structures was observed. For
details, see Ikemura, T. and Aota, S. (1988) J. Mol. BioI., 203, 1-13.
Purification of RNA Molecules with High Resolution
by Two-dimensional Gel Techniques
Toshimichi IKEMURA

Most tRNA molecules of organisms such as E. coli, Salmonella typhimurium, Serratia marcescens, Bacillus subtilis, Mycoplasma capricolum,
Saccharomyces cerevisiae, Drosophila melanogaster, mouse and chicken,
could be purified by a two-dimensional gel system which we have developed.
The method is based on the combination of acrylamide- and urea-concentration shifts: The first dimension is on a 14 % acrylamide gel and the second
dimension on a 22 % acrylamide gel containing 40 % urea. Large RNAs
such as mRNAs also could be fractionated by other systems of two-dimensional gel electrophoresis, although the resolving power was far from satisfactory when compared with that for small RNAs. The first dimension is
on a composite gel of polyacrylamide (2.8-3.0 %) and agarose (1 %), and thesecond dimension on a acrylamide gel (5.5-10%) containing 40% urea.
P3~-labeled S. cerevisiae poly(A)+ RNAs, whose sizes are from 1600 to 3500
nucJeotides, can be separated with high resolution by this system. For
details, see Ikemura, T. Methods in Enzymology (Academic Press), Vol.
180, "RNA Processing" pp. 14-25, Purification of RNA molecules by gel
techniques'

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Computer Analyses for Searching for Protein Coding Regionsin Human Genome DNA Sequences
Ken-nosuke WADA*, Shin-ichi AOTA** and Toshimichi IKEMURA

Non-random usage of codons has been used by several researchers tosearch for protein coding regions in genome DNA sequences for organisms
such as E. coli, Drosophila and nematodes. As an attempt to establish ageneralized method for assigning protein coding regions in human genome
DNA sequences, solely dependent on computer analyses, we calculated andcharacterized codon usage patterns of about 600 human genes available
from the GenBank DNA Database. It was found that the G+C % at thecodon first position when averaged for the 600 human genes is about 55 %,that at the second position about 40 % and that at the third position
about 65 %. Utilizing this positional difference in the codon G+C % as
the criterion for searching for coding regions, a large portion of humanprotein coding regions in the GenBank sequences, when used for the test
sample, could be successfully assigned, especially for genes whose codon
third position is G+C-rich. About one fifth of the human genes, however,are known to be rather A+ T-rich at the third position, and the above
method turned out to be inapplicable for these A+T-rich genes. To establish a generalized method applicable for a much wider range of humangene sequences, we are now making extensive catalogs of the sequence
elements (di-, tri-, or higher oligonucleotide) for exons, introns and flanking
regions separately, for the purpose of introducing multiple criteria for theassignment of coding regions in human genome DNA sequences.
Purification of a DNA Supercoiling Factor from the
Posterior Silk Gland of Bombyx mor;
Tsutomu OHTA and Susumu HIROSE

The eukaryotic genome is organized into a series of looped domains thatare attached to a nonhistone protein scaffolding called the nuclear matrix.
DNA strands within the loops are not free for rotation, so that they can* International Institute for Advanced Study of Social Information Science, Fujitsu**

Limited.
Present Address, Department of Health & Human Services, National Institutes of
Health, USA.

RESEARCH ACTIVITIES IN 1988

35

form a superhelical state. These chromatin loops are thought to playa
role in the control of gene expression and replication by generating different
levels of DNA supercoiling within the genome. In order to test this possibility, we searched for a DNA gyrase-like activity in cell extracts fromvarious eukaryotic sources. Among these, a posterior silk gland cell
extract of Bombyx mori produced positive results. After phosphocellulose
fractionation of the posterior silk gland extract, a flow-through fraction
contained a factor which introduced negative supercoils into closed circular
DNA in conjunction with DNA topoisomerase II. The factor, termed
DNA supercoiling factor, was purified to homogeneity by a combination of chromatography on DEAE-Sephacel, Phenyl-Sepharose CL-4B andDNA cellulose. It was a heat-stable protein of a molecular mass of
50kD.
DNA Supercoiling Facilitates Formation of the Transcription
Initiation Complex on the Fibroin Gene Promoter
Hisahiro T ABUeR! and Susumu HIROSE

Transcription of the Bombyx mori fibroin gene in a posterior silk glandextract can be separated into three functional steps on the basis of sensitivity to Sarkosyl: I. formation of an initiation complex, which is blocked
by 0.025 % Sarkosyl; 2. conversion of the initiation complex to an elongation complex, a step sensitive to 0.05 % Sarkosyl; 3. the subsequent elongation of the RNA chain which occurs in the presence of 0.05 % SarkosyI.
While the last two steps are rapid and unaffected by template topology, thefirst step is slow and affected by DNA conformation. In the posterior silk
gland extract of Bombyx mori, closed circular DNA forms a superhelicalstate and supports more rapid assembly of the initiation complex than linear
DNA does. Both DNA supercoiling and rapid assembly of the initiation
complex require ATP and are abolished by the addition of a topoisomerase
II inhibiter VPI6. These results suggest that DNA supercoiling enhances
fibroin gene transcription by facilitating formation of the initiation complex.
For details, see J. Bioi. Chem., 263, 15282-15287.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Mechanism of mRNA Capping Reaction
Kiyohisa MIZUMOTO

Most of the cellular as well as viral mRNAs in eukaryotes contain a5'-terminal cap structure, m7GpppN. This cap structure is required f,::'r
the efficient initiation of translation and for RNA splicing. It has also
been suggested that the synthesis of the methylated cap structure may play
an important role in transcription itself. The cap structure is synthesized
at the initial stage of mRNA synthesis and conserved at the 5'-termini ofRNAs while they are processed in the nucleus and transported to the cytoplasm. Consequently, elucidation of the mechanism of the cap structure
is important for understanding the molecular mechanism of eukaryotic gene
expression.
We characterized the reactions catalyzed by the mRNA capping enzyme(mRNA guanylyltransferase), the key enzyme in the cap formation, fromvarious eukaryotic cells. The capping enzyme from animal cells consistsof a single polypeptide chain with an approximate Mr of 70,000 containing
two catalytic domains for each of the initial two consecutive reactions involved in the cap formation, i.e., those catalyzed by mRNA guanylyltransferase and RNA 5'-triphosphatase.
RNA5'.
pppN···.
-> ppN'" +Pl
tnphosphatase
ppN··· +GTP

mRNA guanylyl-> G(5')ppp(5')N'" +PPitransferase

In contrast, the highly purified yeast capping enzyme is composed of two
separate chains of 52 kDa (a) and 80 kDa ((3), responsible for the activities
of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively(Mizumoto, K. and Kaziro, Y. (1987), Prog. Nucl. Acid. Res. Mol. Bioi.,
34, 1).
To see whether the a and (3 chains of the yeast capping enzyme are derived
from a single polypeptide or are independent polypeptides encoded by two
separate genes, we attempted to isolate the gene(s) for the yeast capping
enzyme. A yeast genomic expression library in A gtll was screened with
antibodies against the yeast capping enzyme. One of the positive clones,
A C3, contained a 3,500 bp yeast DNA insert. From experiments based on

RESEARCH ACTIVITIES IN 1988

37

an epitope selection of antibodies by antigens produced with 2C3 in E. coli,
this clone was found to contain the gene for the a subunit. The identityof the gene was further confirmed by expressing the gene in E. coli to produce
catalytically active guanylyltransferase. The gene is present in one copy
per haploid genome, and encodes a protein of 459 amino acid residues.
From the primary structure of this gene as well as its mRNA size, we concluded that the a and (3 subunits of yeast capping enzyme are encoded by
two separate genes. Gene disruption experiments showed that the a geneis essential for growth (Mizumoto, K. et al. (1989), J. Cell. Biochem., 13D,
209).
We characterized an RNA polymerase II transcription initiation complex formed with HeLa cell extracts and a DNA fragment containing the
adenovirus major late promoter in the presence of ATP. An initiation
complex with an approximate size of 50 S could be isolated by glycerol
gradient centrifugation free from the bulk of RNA polymerase II and thecapping enzyme. Specific transcription was detected with this complex
when supplemented with the remaining nucleoside triphosphates. Analysesof the 5'-terminal structure of the transcript revealed the presence of a cap
structure m 7 GpppA in more than 50 % of the RNA chains. These results
indicate that at least two cap-related enzymes, mRNA guanylyltransferase
and mRNA(guanine-7-)methyltransferase, are specifically associated withthe complex. The precise step in transcription during which capping takes
place and the possible interaction between the capping enzyme system andtranscription factors remain to be studied.
Messenger RNA Synthesis of Sendai Virus (HV J)
Kiyohisa MIZUMOTO

Sendai virus (HVJ), a member of the paramyxovirus group, contains asingle nonsegmented negative strand RNA genome of approximately 15 kb
long, encoding at least seven proteins, NP, P, C, M, F, HN and L. The
genetic information of this negative strand RNA is expressed throughmonocistronic mRNAs that are transcribed by a virion associated RNAdependent RNA polymerase composed of P and L proteins. The precise
mechanism and the mode of transcription, however, have been unclear,since no efficient in vitro transcription system has previously been established.
We developed an efficient and faithful in vitro transcription system using

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

purified virus particles, and found that the HVJ mRNA synthesis is almost
entirely dependent on the presence of a host factor(s).
The host factor essential for the in vitro transcription was partially purified from mammalian cells and was shown to be separated into at least twocomplementary fractions, one of which, surprisingly, could be replaced byhighly purified tubulin.
The analysis of the cap-containing oligonucleotide from RNase T 2 digests
of in vitro-synthesized poly(A)+ RNA demonstrated that almost all RNA
molecules terminate in m7 GpppAmpGpGp···. This indicated that thetranscription of HVJ mRNA seems to start at the first U residue in the RI
consensus sequence (3'-UCCCNNUUNC-5') which is found in the upstream
regions of all six HVJ genes.
mRNA was synthesized in vitro in the presence of [a- or p_32P]ATP or GTP
as the labeled substrates, and the cap structure m7GpppAm was isolated
after nuclease PI digestion of RNA. The a and p phosphates of GTP
and the a phosphate of ATP, but not the p phosphate of ATP, were incorporated into the cap structure. These 32P-Iabeling data were consistent
with a unique mechanism of cap formation by condensation of GOP residue
from GTP with pA- at the 5'-termini of HVJ mRNAs.
Regulation of Chromosomal Replication in Bacteria
Hiroshi YOSHIKAWA

Regulation of initiation of chromosomal replication by OnaA-boxes in
the origin region of the Bacillus subtilis chromosome.
A gene homologous to the Escherichia coli dnaA gene and two flanking
regulatory regions which contain nine and four OnaA-boxes respectively,
are located in the replication origin region of the Bacillus subtilis chromosome. Attempts to isolate an autonomously replicating fragment from theseregulatory regions in order to identify oriC have been unsuccessful because
the DnaA-box-containing regions strongly inhibited plasmid transformation, particularly when inserted into a high-copy number plasmid, pUBllO.
Using two plasmids differing in copy number, the two regions were subdivided into three regions, A, Band C, each containing five, four and four
DnaA-boxes respectively, and differing in level of inhibition of transformation. Region C is downstream of the dnaA gene and inhibits transformation in high-copy, but not in low-copy number plasmids. When a part of1.

RESEARCH ACTIVITIES IN 198839

the DnaA-boxes were deleted from the incompatible plasmids, they became
transformable and produced slow-growing transformants in which theinitiation frequency of chromosomal replication was selectively reduced.
Fast-growing revertants were found containing the same number of plasmids
as the parent, but with single base changes in the DnaA-boxes. Thesemutations were in the most highly conserved bases of the DnaA-box sequence. This indicates that a sequence-specific interaction of the DnaAbox, probably with the B. subtilis DnaA protein, is responsible for theobserved incompatibility and thus appears to be involved in the control of
initiation frequency of chromosomal replication.
II. Structure of the dnaA region of Pseudomonas putida: conservation
among three bacteria, Bacillus subtilis, Escherichia coli and P. putida.
We cloned from Pseudomonas putida, a gene homologous to Escherichia
coli dnaA, and determined the sequence of the gene and its neighboring
region. The dnaA gene and at least three other genes, dnaA, recF and gyrB,
were found to be highly homologous to the genes in the dnaA regions of theE. coli and Bacillus subtilis chromosomes. A non-translatable region ofsome 600 bp immediately upstream of the dnaA gene, is also conserved in
the three bacteria and contains 3, 12, and 14 DnaA-boxes (TTATCCACA
and closely related sequences) in E. coli, P. putida, and B. subtilis, respectively. The present results confirm our hypothesis that the dnaA region is
the replication origin region of the ancestral bacterium and that the essential feature of the dnaA and DnaA-box combination is conserved in mosteubacteria and plays a central role in initiation of chromosomal replication.
III. Comparative anatomy of oriC of eubacteria.
We reported that at least six genes and their organization is conserved inthree bacteria, E. coli, P. putida and B. subtilis. In addition, we showedthat a gram positive bacteria, M. luteus, also had a dnaA and neighboring
genes highly homologous with those of the above three bacteria. We
therefore concluded that the dnaA region is conserved in most of the eubacteria. Comparison of dnaA genes among the four bacteria demonstrated clearly that the gene is composed of highly conserved and nonconserved domains. The remarkable feature of the dnaA region is the conservation of non-coding regions containing a cluster of DnaA-boxes.
Gram negative bacteria contain one region upstream of the dnaA gene,while gram positive bacteria contain two regions flanking the dnaA gene.
The combination of DnaA protein and DnaA-box plays a central role in

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

the initiation of chromosomal replication in E. coli. We showed that thesame combination is also essential in the initiation of chromosomal replication in B. subtilis. Thus, a temperature sensitive mutant which is
defective specifically in initiation of chromosomal replication, was isolated
by in vitro mutagenesis of the cloned dnaA gene. A single amino acidsubstitution was detected in the coding region of the dnaA gene of themutant cell. Secondly, introduction of an excess of DnaA-boxes into B.
subtilis cells resulted in the inhibition of initiation of chromosomal replIcation, probably through the titration of limited amounts of DnaA protein in
the cell. The DnaA-box region upstream of the dnaA gene in the P. putida
chromosome was shown to be active as oriC. These results show that the
DnaA protein-DnaA box interaction is universal in eubacteria and plays acentral role in the initiation of chromosomal replication.

RESEARCH ACTIVITIES IN 1988

II.

41

MICROBIAL GENETICS

Gene Organization in the Region Containing a New Gene
Involved in Chromosome Partition in Escherichia coli
Jun-ichi KATO*, Yukinobu NISHIMURA, Masao YAMADA**
Hideho SUZUKI*** and Yukinori HIROTA!

One goal of cell division is segregational transmission of a genome todaughter cells. To prevent production of chromosomeless cells, the processof cell division in Escherichia coli, as well as in other organisms, isplaced under elabrate control, to couple it with replication and partition ofchromosomes. For example, perturbation of chromosome replicationarrests cell division through the induction of a division inhibitor, the sulA
gene product, which targets the ftsZ (suIB) gene product, whose function isessential for the progression of the division process. Chromosome partition should be another process that regulates cell division. This process,although barely understood, seems to include two aspects: molecular separation (resolution of topological linkages), and topographical segregatIOnof the replicated chromosomes. The latter aspect has been postulated as
involving the attachment of replicons to the cell surface structure, thegrowth of which contributes to topographical segregation of the attachedreplicons.
Two kinds of Escherichia coli mutants defective in chromosome partition
have been described as parA and parB. They are thermosensitive andreveal the distinctive morphology of centrally congregated nucleoids at
restrictive temperatures. The par mutations have recently been investigatedconcerning their relationship to the termination of DNA replication, andparB is most probably allelic to dnaG (for primase) and located at 67 minon the genetic map (Norris, V. et al. 1986. J. Bacteriol. 168: 494-504; Nishimura, Y. et al. 1988. Ann. Rep. Natl. Inst. Genet. 38: 43-44). Anzlyses ofsuch mutations are indeed expected to provide clues to the mechanism of
*
**
***
!

Department of Bacteriology, National Institute of Health, Tokyo.
Present address: National Children's Medical Research Center, Tokyo.
Faculty of Sciences, University of Tokyo, Tokyo.
Deceased on December 23, 1986.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

chromosome partition and its control over the cell division process.
We identified a new mutation, parC, which causes abnormal chromosome
segregation, in two thermosensitive mutants of Escherichia coli. The
thermosensitive growth of the mutants was corrected by pLC4-14 in the
Clarke-Carbon collection, which is known to carry the metC region at
65 min (Dwivedi, C. M. et al. 1982. Biochemistry 21: 3064-3069). The
nearness of parC to metC was confirmed, and the cotransduction frequencyof parC was 59 % with metC and 20 % with glc. This plasmid carries aputative gene which can suppress the cell division defect due to ftsI (pbpB)
and has hence been termed sufI (Nishimura, Y. et al. 1977. Plasmid 1: 6777). The parC-sufI region was analyzed by subcloning the chromosomeof pLC4-14. The parC and the sufI gene products were electrophoretically
identified as proteins of 75 and 55 kilodaltons (kDa), respectively. The
allelism of parC+ on pLC4-14 to parC1215 was confirmed by cloning
parC1215. The sufI gene appeared to be dispensable to cell viability, andoverproduction of its product caused suppression of ftsI. An essential
gene coding for a 25-kDa protein was found between the parC and thesufI genes. These three genes were transcribed in the same direction andmay be organized into an operon, with parC to the proximal side and withinternal promoters, at least for the distal genes (Fig. 1). The localizationof the gene products was examined in maxicells. The sufI protein was
synthesized as a precursor which could be chased into a mature form. The
major part of the mature form was found in the soluble fraction. The
P

S B( 1) P

P B(2)P PSP

pLC4-14S

Hp B(l)Pv

P Pv<;===::J <~:J <:=====:::::J
sufi

25K

Hp

lkb

pare

Fig. 1. Possible location of the genes for parC, sufI, and the 25 K protein. The
arrows below the map indicate the roughly estimated location and directionof transcription of the parC gene, the sufI gene, and the gene for the 25 K
protein. B, BamHI; Hp, HpaI; P, PstI; Pv, PvuII; S, Sma!.

RESEARCH ACTIVITIES IN 1988

43

25-kDa protein was found almost exclusively in the membrane fraction.
The parC protein was associated with the membrane fraction in the presence of MgH, but found in the soluble fraction when MgH was sequesteredwith EDTA. The same MgH dependent localization has been found forthe sopB protein involved in the partition of a mini-F plasmid (K. Nagai,
et al. Annu. Meet. Mol. Bioi. Soc. Jpn. 1984, abstr. no. 7: B-17). Thissimilarity in MgH dependence may suggest a property common to theproteins engaged in the partitioning of replicons.
The parC gene product was identified as a 75 K protein by SDS-PAGE.
Proteins of similar size have been seen in OriC-membrane complexes and F
partition-specific complexes. The process of chromosome partition presumably involves interaction of the chromosome with the cell surface
structure, and the complex may be a structure for this interaction. The
75 K protein found in the F partition-specific complex was assumed tooriginate from the background synthesis in minicells. The parC productmight be identical to this protein and be shared by the chromosome andmini-F for their partitioning. A genetic analysis of these proteins would
be an effective approach to the problem, and analyses of par mutants mayhelp in understanding the roles of the proteins contained in the DNAmembrane complex. For details, see J. Bacterial. 1988. 170: 3967-3977.
Isolation of Novel Mutants Defective in DNA
Replication in Escherichia coli
Seiichi YASUDA

Although almost all protein factors involved in the replication of the
Escherichia coli chromosome have been identified, the mechanism of regulation of replication remains unsolved. I have been trying to find new
dna genes which are involved in regulation and hence, will give some clue
to the understanding of this mechanism.
E. coli Kl2 strain SY965 was treated with N-methyl-N'-nitro-N-nitrosoguanidine, and individual clones from the mutagenized culture were
tested for growth at a high temperature (42°). Clones which do not growat the high temperature were spotted on a plate in a grid pattern of 72 clones
per plate and incubated overnight at 30°. Colonies were transferred witha metallic apparatus with 72 needles arranged in a similar grid pattern onto
two nitrocellulose disks (82 mm), which were placed on L broth agar plates

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

and incubated at 30° for 2 to 3 hrs. When visible colonies grew on thefilters, one of the filters was transfered to labeling medium and the otherwas incubated further at 42° on a plate which had been kept at that temperature. The labeling medium was made in a fresh petri dish by adding
64 pCi of [3R] thyimidine to 3.2 ml of L broth and then placing twopieces of sterile No.2 filter paper (82 mm diameter) on it. After labeling,at 30° for 30 min. the nitrocellulose fiter was transferred to the top of a
stack of four pieces of No.2 filter paper, which had been soaked in 8 ml
of 1M RCi. Colonies grown at 42° were labeled at 42° similarly, usinglabeling medium prewarmed at 42°. Nitrocellulose filters with labeledcolonies were washed by suction with 70 %ethanol, dried in vacuum, soaked
in DPO solution (0.2 gjml DMSO), dried in air and then subjected toautoradiography at - 80° overnight. Colonies which showed lower incorporation at 42° than at 30° were saved as candidate dna mutants. Of about
800 temperature-sensitive mutants tested, 75 clones were classified as tentative dna mutants. 26 of 75 dna candidates were found to be mutants ofknown dna genes. The remaining 49 strains were analysed further to findnew dna genes.
Two mutants, 2614 and 2618, were found to have mutations in new dna
genes. Strain 2614 has a mutation in a gene closely linked to aroA andstrain 2618, to pyre. Both strains show residual DNA synthesis after
transferring cultures to a higher temperature, suggesting that these dna
genes function in the initiation step of chromosomal replication. Each ofthese mutations requires the presence of another mutation for expression of
typical dna characters. The nature of these secondary mutations, including
map positions, are not known. Crude enzyme fractions prepared fromthese dna strains had normal activity to support in vitro replication oforiC DNA. Thus, it is possible that these dna genes are involved in theregulation of initiation of chromosomal replication, rather than in catalytic
steps, but more extensive studies are necessary.

RESEARCH ACTIVITIES IN 1988

45

Allelism of Escherichia coli parA to gyrB and Some Views
on par-dependent Regulation of Cell Division
Yukinobu NISHIMURA, Jun-ichi KATO", and Hideho SUZUKI*"The Escherichia coli parA mutation was found originally in MFTllO andour studies on this mutant have suggested that MFTllO carries two thermosensitive (ts) mutations and the major ts mutation is not parA but mostprobably psd which lies at or very close to the locus where parA was originally mapped (Kato et al., 1988, Annu. Rep. Natl. [nst. Genet 38: 41-42).
The major ts mutation in MFTllO was displaced by the wild-type allel
cotransduced with a psd-linked Tn10 from ECL510 (zjd:: TnlO). Thermoresistant (tr) co-transductants were isolated on L agar plates from tetracycline resistant (Tc R ) transductants selected at 30°C. The tr derivatives
grew on L agar plates, but not on salt-free L agar, at 42°C, suggesting thatthe second ts mutation carried by MFTllO was repairable with 0.5% NaCIin the L medium. One of the tr derivatives was made Hfr by a cross with
P4 X 8 and used for mating experiments, the results of which suggested thatthe second ts mutation lay between ilvA and xyl. Then, tna:: TnlO wastransduced from a tna:: 10 recF strain (JCI2334), to one of the recombinants
that had the second ts mutation. Among the Tc R transductants, five were
tr and sensitive to UV (recF-) and one was ts and UV-insensitive (recF+).
Thus, the second mutation in MFTllO should be located close to recF and
tna at 83 min on the genetic map. This ts mutation was introduced into
W3110 by co-transduction with tna:: TnlO and the resultant W3110ts was
examined for the morphological phenotype. W3110ts manifested a typicalPar phenotype with an accompanying release of nucleoidless cells at 42°C
as observed with fluorescent nucleoids of DAPI (4',6-diamidino-2-phenylindole) stained cells. Obviously, the second ts mutation was responsible for
the Par phenotype as described for the original MFTllO (parAllO). Although the ts mutation was salt-repairable as to growth, the Par phenotype
was essentially the same in either the presence or absence of salt.
The chromosome region at 83 min contains a cluster of dnaA, dnaN,
recF and gyrB. parAllO was complemented by a plasmid pJBll (gyrB+) as
well as pJM2-9, carrying the whole cluster region, but not by pSY342(dnaA+) and pSY504 (dnaN+ recF+) (Fig. I). Therefore, parA 110 is most

* Department of Bacteriology, National Institute of Health, Tokyo.
** Faculty of Sciences, University of Tokyo, Tokyo.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

"0

;;:"

~
I

pJM2-9pJBll
pSYS04
pSY342

v>
..,c

t1

II

I

P
!

!

~'1Ii

I

,

dnaA

..

"'0"

0 ..

.c>
xc

•

0(

~
1

i

I

.'E

dnaN reeF

gyrll

~
I

:;;

Complementation ofsalt-repairable ts

I

+

;;:"

+

•!

Fig. 1.

Chromosome region that complements the salt-repairable ts mutation.
The 13 kb HindIIl fragment containing dnaA-gyrB region or part of thisfragment was carried by the plasmids as shown with rectangles. Complementation was examined with W3110parAlIO. The plasmids weregenerous gifts from Drs. J. Yamagishi and S. Yasuda, to whom we are
obliged.

probably an allele of gyrB and the ParA phenotype is likely to result froma defect in decatenation of daughter chromosomes.
Four par mutants have been described as parA-D in E. coli. The Par
phenotype, observed typically as elongated cells with centrally congregatednucleoids, appears to result from abnormal replication or failure in decatenation of DNA: parA and parB are allelic to gyrB and dnaG, respectively; pare probably represents a gene for a topoisomerase-like proteinthat interacts with DNA-membrane (Kato et al., 1988, J. Bacterial. 170:
3967; unpublished results); and ParD is due to gyrA (Hussain et al., 1987,
Mol. Microbiol. 1: 259). These facts and views suggest that the primaryeffects of par mutations may be attributed to a defect in resolution of replicated chromosomes. Topoisomerases would catalyze the decatenationof replicons. Besides the catalytic activities, the structural functions in
replicon partitioning have been suggested for a gyrase which binds the
pSClO1 par region (Wahle and Kornberg, 1988, EMBO J. 7: 1889). Someof the topoisomerases may mediate the DNA-membrane association responsible for chromosome segreagtion in cell division. Chromosomesmight then be bound to the prospective septation sites and the binding mightin turn inhibit unscheduled septations and consequently the production of
nucleoidless cells. In this respect, the temporal regulation of cell division
might involve the structural functions of a gyrase or another topoisomerase.
For details, see Mol. Gen. Genet. 1989,217: 178-181.

RESEARCH ACTIVITIES IN 1988

47

Nucleotide Sequence of the sufI Gene Responsible for theMulticopy Suppression of a Thermosensitive
Cell-division Mutation in E. coli
Yoshihiro YAMAMOTO*, Jun-ichi KATO**, Hideho SUZUKI***and Yukinobu NISHIMURA

The suppression of a mutation in one gene by amplified copies of a different wild-type gene has been termed "multicopy suppression" (Berg,
C. M. et al. 1988. Gene 65: 195-202). Growth thermosensitivity in an E.
coli Its1730 mutant defective in cell division, can be corrected by a cloned
sufI gene on a multicopy plasmid (Kato, J. et al. 1988. J. Bacteriol. 170:
3967-3977). To elucidate the molecular mechanism of the multicopy suppression of an ftsI mutation by sufI, we determined the nucleotide sequenceof a DNA fragment containing the sufI gene. The sequence analysis revealed that only one open reading frame allowed a region sufficient forcoding for the size of Sufi (55 kDa). The region of the putative structuralgene for sufI consisted of 1,476 basepairs, coding for a polypeptide of 492
amino acid residues and of 54,178 in molecular weight, in good agreementwith the size of the gene product as estimated by maxicell analysis. The
hydropathy profile of the deduced amino acid sequence suggested the existence of a possible signal peptide in the N-terminal region, consistent witha previous study showing that Sufi is probably a periplasmic protein, processed from the precursor into a mature form.
The amino acid sequences of Sufi and FtsI were compared. Some similarity was observed between the C-terminal region of Sufi and the Nterminal region of Ftsl (Fig. lA). FtsI is the penicillin-binding protein 3
participating in septal wall synthesis and its N-terminal half is supposed tocomprise the peptidoglycan transglycosylase domain. Thus, Sufi might
be a different type of transglycosylase and its overproduction could circumvent the ftsI lesion by compensating for a low catalytic reaction rate.
The mutation site in the fts1730 mutant protein is under investigation.
In addition, the N-terminal part of Sufi showed some similarity to the
middle part of creatine kinases of chick, rabbit, mouse and rat, which is
known to be highly conserved (Fig. IB).
* Hyogo College of Medicine.
** National Institute of Health, Tokyo.
*** Faculty of Sciences, University of Tokyo, Tokyo.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

A)

B)

Sufi

(409-467)

NGAMRSRKTEAGKIPFGLTDKRSCLFISVSLPGRTSRSTSTVRRGNGGPWL-DWATVGQS

Fts I(56-116)

EGDMRSLRVQQVSTSRGMITDRSGRPLAVSVPVKAIWADPKEVHDAGGISVGDRWKALANA

Suf I(22- B9)

CK-CM (175-235).*.****,H•. H.* ••

•.•..

*

.

PLKASAAGQQQPLPVPPLLESRRGQPLFMTVQRAH-WSFTPGTRASVWGINGRYLGPTIRVWKGDDVKL**** ... **.*.. *... H... .*. **..* ..* ...

*. ** .... *PLKAMTEQEQQQLI DDHFLFDKPVSPLLLASGMARDW--PDAR-G IWHNDNK---TFLVWVNEEDHL

Fig. 1. Similarity between Sufi and FtsI or creatine kinase. Regions in the aminoacid sequences of the proteins are indicated by the amino acid residue numbers in parentheses. Identical or structurally related amino acids are marked
by asterisks or dots, respectively.
CK-CM: chicken muscle creatine kinase.

C-Terminal Processing of Penicillin-binding Protein 3 ofEscherichia coli: I. Analysis of the Degradation
Product that Represents the C-Terminal HalfS
Youji SAKAGAMI*~, Hiromichi NAGASAwA*, Akinori SUZUKI*,
Hiroshi HARA and Hideho SUZUKI**

Penicillin-binding protein 3 (PBP 3) of Escherichia coli is an essential
enzyme involved in cell division. It is synthesized as a precursor form
distinguishable by electrophoretic mobility and then processed into a matureform. Investigations by gene manipulation for producing hybrid/truncated
PBP 3 molecules have shown that the C-terminal part, rather than the Nterminal part, appears to be responsible for the processing (Hara, H. et al.,
1988, Natl. Inst. Genet. Ann. Rep. 38: 49-51).
Preparations of purified PBP 3 contain a 40 kDa protein (40K fragment)
regarded as a degradation product of PBP 3. This fragment retains penicillin-binding ability and hence is considered to represent the C-terminal
half which constitutes the penicillin-binding domain of PBP 3. The 40K
fragment showed reduced electrophoretic mobility in PBP 3 preparations
from a mutant which is defective in the processing of PBP 3, suggesting thatthe processing occurred within this fragment and therefore in the C-terminal
region. Thus the 40K fragment was isolated from preparative gels and* Department of Agricultural Chemistry, University of Tokyo.
** Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo.
Present Address: Department of Agricultural Chemistry, Nagoya University.
This and the following report in series are dedicated to the memory of the lateProfessor Y. Hirota who laid the foundation of these c011aborative works.

RESEARCH ACTIVITIES IN 1988

49

analyzed for its amino acid sequence. The N-terminal part of the fragment
corresponded to the deduced sequence beginning with Lys21l. In sequencing the peptides of PBP 3 which were generated by Iysyl endopeptidase andfractioned by HPLC, we found that two peptides were identical with eachother in sequence except that the N-terminal Lys was present in one andabsent in the other. The identical part of the peptides corresponded to the
deduced sequence beginning with Asp212. Obviously, one peptide carrying
N-terminal Lys invulnerable to Iysyl endopeptidase was derived from the40K fragment and the other derived from the intact PBP 3 as a result of
Iysyl endopeptidase cleavage between Lys211 and Asp212. The identificationof these two peptides supports the idea that the N-terminus of the 40K
fragment is Lys211. These results confirm that the 40K fragment represents
the C-terminal half of PBP 3 and that the processing of PBP 3 occurs in
the C-terminal region.
The 40K fragment was associated with insoluble cell fractions and solubilized with a detergent along with intact PBP 3 molecules. This argues
that the C-terminal half of PBP 3 is associated with the membrane, although
it has been suggested that the C-terminal part of PBP 3 is extruded into
periplasm rather than anchored to membranes (Spratt, B. G. et al., 1988,
in Antibiotic inhibition of bacterial cell surface assembly and function (Actor,
P. et al., ed), American Society for Microbiology, p. 292-300).
C-Terminal Processing of Penicillin-binding Protein 3 ofEscherichia coli: II. Determinationof the Cleavage SiteS
Hiromichi NAGASAWA*, Akinori SUZlJKI*, Hiroshi HARA
and Hideho SUZUKI**

The maturation of penicillin-binding protein 3 (PBP 3), an enzyme essential for septum formation in Escherichia coli, involves a cleavage of the
C-terminal part of the precursor. Analyses of truncated PBP 3 molecules
which lost the C-terminal part in varieties of length have shown that theregion between residues 560 and 576 is essential for the processing (Hara,* Department of Agricultural Chemistry, University of Tokyo.
** Laboratory of Genetics, Department of Biology, Faculty of Science, Universityof Tokyo.
See footnote to p. 48.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

H. et al., 1988, Natl. Inst. Genet. Ann. Rep. 38: 49-51).
The cleavage site for processing was determined by peptide mapping andsequence analyses of the precursor and the mature PBP 3, which were
purified from a processing-defective mutant and a wild type strain, respectively. HPLC peptide mapping of lysyl endopeptidase digests of the precursor and mature forms revealed the peptides that differ in retention time
between the precursor and mature forms. These peptides were identified
by amino acid sequencing as the C-terminal region beginning with Tyr 540 .
Each of the C-terminal peptides was cleaved into two fragments by trypsin
digestion. By coupling with an aminopolystyrene resin, the resultant
carboxyl-side fragment derived from the mature form was sequenced successfully to the carboxyl end, which was found to be VaJ577 with free COOH.
Since it is unlikely that the Val-Ile bond was cleaved by lysyl endopeptidase
or trypsin, it was concluded that the VaI 577 _Ile 578 bond should be the cleavage
site for processing. This conclusion was supported by amino acid compositions determined for the relevant tryptic peptides which suggested thatthe peptide from the cleavage site to the end of the deduced sequence (Ile 578 Ser588) was present in the precursor but absent in the mature form.
The cleavage site at Va]577 lies one residue beyond the region of residues
561 to 576 which has been suggested to be essential for the processing byanalyses of the truncated PBP 3 molecules. The discrepancy might be
explained by the assumption that the processing enzyme is not strict withthe peptide residues to be cleaved but depends primarily on recognition ofa certain sequence within residues 561 to 576.
Curiously, the Lys573_Asn 574 bond resisted both trypsin and lysyl endopeptidase and remained unc1eaved in the peptides analyzed above. The
cause of resistance to tryptic cleavage is probably the structure of A Sp572_
Lys573, which is known to be hardly cleaved by trypsin. However, thereason for the inhibition of lysyl endopeptidase cleavage is unclear.
C-terminal processing has been described in several proteins of eukaryotic
and prokaryotic cells (e.g. Diner, B. A. et al., 1988, J. Bioi. Chern. 263:
8972-8980; Yanagida, N. et al., 1986, J. Bacteriol. 166: 937-944). No sequence similarity in peptides around processing sites has been found among
these proteins and PBP 3. The C-terminal processing of PBP 3 seems to
be unique and its significance in function is not obvious at present.

RESEARCH ACTIVITIES IN 1988

51

Requirement of the C-Terminal Region for the Function ofEscherichia coli Penicillin-Binding Protein 3
Hiroshi HARA and Hideho SUZUKI*The C-terminal processing of penicillin-binding protein 3 (PBP 3) ofEscherichia coli has been investigated by constructing the plasmids that
direct the production of PBP 3 truncated at various sites in the C-terminal
part. The truncated product with a normal amino acid sequence to Thr560(PBP 3'-560) and the products with shorter normal sequences (PBP 3'-544
and -497) were unstable, but the product with a normal sequence to Phe576(PBP 3'-576) appeared to be as stable as normal PBP 3 (Hara, H. et al.,
1988, Nat!. Inst. Genet. Ann. Rep. 38: 49-51).
To investigate the functional aspect of the truncated products, those
p1asmids were examined for their ability to complement a temperaturesensitive (ts) PBP 3 mutation (its!), which causes the filamentation of cells
at a restrictive temperature. The ts growth of anftsI mutant on agar plates
was corrected by PBP 3'-576 and -560, but not by PBP 3'-544 and -497.
The C-terminal region to residue 560 is considered to be essential for thefunction of PBP 3, while the sequence of residues 561 to 576 is apparently
important to stability but dispensable in terms of function.
The morphological phenotype of the ftsI mutant at the restrictive temperature was normal when PBP 3'-576 was produced, showing that thistruncated product was fully functional. This product was stable andprocessed, although at a greatly reduced rate, into the mature form which
could be assumed to be identical with the normal mature PBP 3 except fora small span to the C-terminus (Nagawawa et al., in this Ann. Rep. p. 49-50).
With plasmids that directed the other truncated products, irrespective ofcomplementation-positive or negative, filamentous cells were found even at
the permissive temperature. Although PBP 3'-560 complemented thegrowth defect of the ftsI mutant, its function appears incomplete. WhenPBP 3'-497 or PBP 3'-544 was produced, many of the filamented cells became ghosts after 3 hr incubation at 42°C. The loss of the C-terminal
peptides not only impairs the activity of the truncated PBP 3 but may
produced an adverse effect on cell division and murein sacculus integrity.
Such an effect would be understood if PBP 3 functions in a (division) com* Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

plex as suggested previously (Takeda et al., 1981, Plasmid 6: 86-98). PBP 3'544 caused notable filamentation at 30°. This truncated product was so
unstable that the product could not be determined for processing capacity.
This product might be incorporated quite efficiently into the putative divisioncomplex, rendering the complex abortive through its rapid degradation.
PBP 3'-497 was less toxic compared with PBP 3'-544. The former might
be far less efficient in competition for the complex formation with the
product directed by the ftsPS gene on a chromosome.
These results show the importance of the C-terminal region to the function of PBP 3. This region contains four hydrophobic segments bracketedby basic residues, the last of which ends at Leu 558 • All of the four hydrophobic segments were required for the function of PBP 3, and the peptide
sequence that follows the last hydrophobic segment (residues 561 to 576) ispresumably required for stability and thus perfect functioning of PBP 3.
The complementation was examined under overproducing conditions whichmight have compensated for function insufficiency. The importance of theextreme C-terminal region of the mature PBP 3 molecule is in contrast with
the C-terminal region of PBP Ib, in which no processing is known and 64
residues from the C-terminus can be deleted without a detectable change inits function (Kato et al., 1984, Molec. Gen. Genet. 196: 449-457).
Cloning of a Gene Involved in the C-Terminal Processing of
Penicillin-Binding Protein 3 of Escherichia coli
Hiroshi HARA and Hideho SUZUKI*

The mature form of penicillin-binding protein 3, an enzyme essential forcell division in Escherichia coli, is produced by proteolytic cleavage of the
C-terminal part from its precursor form. We have found a mutant, JE7304,
defective in this unique processing reaction (Hara et al., 1988, Natl. [nst.
Genet, Ann. Rep. 38: 48-51). The mutation will be referred to as pre (processing in the C-terminal part). The pre mutant showed temperaturesensitive (ts) growth in salt-free L broth. Temperature-resistant transductants of JE7304 obtained through PI-transduction from a wild-typestrain revealed the normal processing of PBP 3 in all of the 12 clones tested,suggesting that the mutation causing the ts growth was identical or closely* Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo.

RESEARCH ACTIVITIES IN 1988

53
Growthat • low,1 kb,, ,PB

Salt Conen
30° 42°VectorE,

P, BH PEIIE,

P BH PI',

pACYC184

pHR53

+

+

+
+

p8R32261+ +pACYCl84

75

+

77

+

80

+

~-c

87

+

E
H
eatp~~

88

I

II

B

'4'
an
H

'ti'
P
::zS::!!

I'

H

E

Processing

+

:t

+

:t

+

pre

====:>

Fig. 1.

Analysis of the DNA region containing the pre gene.
Only the cloned chromosomal regions are shown. Wedges denote thedirection of the promoter of a cat gene, and an arrow denotes the inferred
location and orientation of the pre gene. Growth of J£7304 transformed
with each of the plasmids in salt-free L broth at 30°C and 42°C and the processing of PBP 3 in the transformants are shown by + or -. ± indicatespartial correction of the processing defect. B, BamHI; E, EcoRI; H,
HindIlI; and P, Pst!.

linked to pre. Therefore we tried cloning of the pre gene by selecting
recombinant plasmids that corrected the ts growth of JE7304.
Chromosomal DNA of a temperature-resistant transductant was digested
with EeoRI or partially with PstI and the fragments were inserted into thecorresponding sites of pBR322 or pACYC184. The recombinant plasmids
were introduced into JE7304 by transformation and tetracycline-resistanttransformants were selected at 42°C on salt-free L agar plates. All thetransform ants tested were found to be normal in the processing of PBP 3.
Two recombinant plasmids were reserved: pHR53 containing a 2.9 kb
EeoRI fragment and pHR61 containing an 11.1 kb PstI fragment. Restriction maps of these plasmids (Fig. 1) suggested that the cloned fragments were
derived from the same chromosomal region and that pre was contained in
the 2.6 kb EeoRI-PstI fragment. Insertion of a kanamycin resistance gene

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39(kan) into either the PstI or the BamHI site in this 2.6 kb fragment (pHR80

and pHR75) abolished the ability to complement the ts growth and to correct
the processing defect of JE7304, indicating that pre covers the PstI and
BamHI sites and is responsible for the ts growth. When kan was inserted
into the HindIIl site (pHR77) or the 0.9 kb HindIIl-EeoRI fragment wasdeleted (pHR87), the ts growth was complemented but the processing
defect was only partially corrected. Rec10ning of the 2.0 kb EeoRI-HindIU
fragment into the same vector but in the opposite orientation (pHR88)
restored the ability to completely correct the processing defect. However,this plasmid could not complement the ts growth and appeared rather inhibitory to growth: JE7304 harboring pHR88 did not grow in salt-free L
broth even at 30°C. The essential region for complementing the ts growth
and the processing defect is contained in the EeoRI-HindUI fragment. In
pHR88, the EeoRI site of this fragment was placed to the proximal side
within the cat gene of pACYCI84. Transcription from the cat promoter
may have affected the expression of the pre gene, and we infer that the pregene is probably transcribed in the direction from left to right in Fig. 1 anda little beyond the HindUI site. The disagreement between the complementing abilities for the ts growth and the processing defect in pHR88
suggests that overexpression of the pre gene may be deleterious to cell
growth, or alternatively that the two complementation phenomena may
result from different genetic factors.
The ts growth of the pre mutant and the inhibitory effect of the promoted
pre expression suggest that the pre gene is involved in an essential cellular
process. It is not certain at present whether such a process is directly
related or not to the vital function of PBP 3. The failure in PBP 3 processing did not always correspond with the growth defect. The pre mutant
failed to divide normally and became elongated at 42°C in salt-free L broth,but it grew normally even without detectable processing of PBP 3 underother conditions permissive to growth.

RESEARCH A CTIVITIES IN 1988

55

Mapping of prc That Causes a Defect in the C-Terminal
Processing in Escherichia coli
Hiroshi HARA, Atsushi HIGASHITANI*, Yukinobu NISHIMURA
and Hideho SUZUKI**

The prc mutation of Escherichia coli causes a defect in the unique processing reaction which cleaves the C-terminal part of the penicillinbinding protein 3 precursor. The mutation has been found in JE7304 andappeared to be linked with the temperature-sensitive (ts) growth of thisstrain (Hara et al., 1988, Natl. Inst. Genet. Ann. Rep. 38: 48-51). Althoughmapping of the ts mutation had been unsuccessful possibly because of thefrequent occurrence of a suppressor mutation for the ts growth but not forthe processing defect of the prc mutant, the success in cloning the prc gene
(Hara and Suzuki, in this Ann. Rep., p. 52-54) facilitated the mapping of
this gene.
The prc+-carrying pACYC184 derivative, pHR53 (tetracycline(Tc)-resistant) was introduced into an Hfr polAts strain which could not support
the replication of this plasmid at the restrictive temperature. The transformants were grown at 42°C in the presence of Tc and stable Tc-resistantclones were obtained in which the plasmid was integrated into the chromosome by homologous recombination around prc. An Hfr cross with amultiple auxotrophic, polAts mutant at 42°C suggested that the Tc-resistance(and thus the integrated prc+ plasmid) was located around his at min 44 on
the chromosome map. A Southern hybridization experiment with NotI
fragments of the whole chromosome, separated by pulse-field gel electrophoresis, showed that the cloned DNA fragment in pHR53 was hybridized
with a 106 kb fragment derived from a region around min 40 (Tabata, S.
et al., 1989, J. Bacteriol. 171: 1214-1218). The restriction patterns of thecloned fragments of pHR53 and pHR61, the prc+ -carrying pBR322 derivativethat contained a larger chromosomal fragment, were found to fit the patternaround map coordinate 1933 (kb) or min 40.3 on the physical map of thewhole E. coli chromosome by Kohara, Y. et al. (1987, Cell 50: 495-508).
This map position was confirmed by PI-transduction experiments that locatedthe ts mutation in JE7304 betweenfadD (min 39.9) and eda (min 40.7).
* Department of Biology, Faculty of Science, Nagoya University.

** Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Accumulation of the Precursor Form of Penicillin-Binding
Protein 3 in Escherichia coli Mutants Defectivein General Protein Export
Hiroshi HARA and Hideho SUZUKI*

The extracytoplasmic proteins in Escherichia coli are translocated acrossthe cytoplasmic membrane by a process that involves the sec Y and secA
gene function and the cleavage of an N-terminal signal sequence. Penicillinbinding protein 3 (PBP 3) participates in septal murein synthesis in E. coli
and appears to extend the bulk of its molecule into the periplasm where themurein lies. We suspected that this protein, although not an excreted
protein, may use the translocation and processing mechanism in common
with other extracytoplasmic proteins. However, the processing of PBP 3
was found to involve a unique reaction in which the C-terminal part, rather
than the N-terminal part, is cleaved (Hara et al., 1988, Natl. Inst. Genet.
Ann. Rep. 38: 49-51). Excretion of some extracellular proteins depends on
the interaction of the C-terminus with specialized machinery (e.g. Gray, L.
et al., 1986, Molec. Gen. Genet. 205: 127-133). Thus we examined whether
the maturation of PBP 3 is dependent on the translocation process involving
SecY and SecA proteins.
It has been shown that high-level synthesis of a MaIE-LacZ hybrid protein
inhibits the export of extracytoplasmic proteins, presumably by jamming
the translocation machinery. A strain which carries malE-IacZ genefusion and overproduces PBP 3 by plasmid pMS3l6 containing the PBP 3
gene was subjected to a pulse-chase experiment in the presence or absenceof maltose, an inducer for maIE-IacZ, and analyzed for the processing
kinetics of PBP 3 and an outer membrane protein OmpA. When the
hybrid protein was induced by maltose, the processing rate of PBP 3 as well
as of OmpA was greatly reduced. Similar retardation in the processing of
PBP 3 and OmpA was observed when temperature-sensitive secretionmutants (seePS, secAts and secAam supPS) harboring pMS3l6 were exposedto the restrictive temperature. These results indicate that PBP 3 or the
enzyme for C-terminal processing or both share the translocation machinerywith other extracytoplasmic proteins. The N-terminus of PBP 3 begins
with a basic and hydrophilic segment followed by a distinctly hydrophobic* Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo.

RESEARCH ACTIVITIES IN 1988

57

segment, which is characteristic of signal sequences. This N-terminal
region possibly functions as a signal for interaction with the translocation
machinery although not cleaved in the following processing step. PBP 3
molecules whose N-terminal 40 residues were artificially removed failed in
the translocation and accumulated in the cytoplasm (Bartholome-De Belder,
J. et al., 1988, Molec. Microbiol. 2: 519-525). The C-terminal processing
occurs rather slowly compared with the N-terminal processing for generalexported proteins, possibly after a substantial part of the molecule has beentranslocated through the membrane. The activity for the C-terminal processing was recovered in a membrane fraction (Hara et al., 1988, Natl. Inst.
Genet. Ann. Rep. 38: 52-53). The processing enzyme might reside on theouter surface of the cytoplasmic membrane.
When the translocation was blocked in the secretion mutants underrestrictive conditions, the cells became abnormally elongated, probably
because the function of PBP 3 for cell division was impaired. If the translocation of PBP 3 is independent of SecY and SecA, the defect of the secretion mutants in PBP 3 processing should be solely due to a failure in translocation of the processing enzyme for PBP 3. In the prc mutant, however,PBP 3 appeared to remain unprocessed under conditions that allowed normal
growth, suggesting that the processing defect did not necessarily lead to theloss of PBP 3 function. It is rather likely that PBP 3 uses the Sec translocation machinery for its proper localization in the cellular surface structure
and the translocation of PBP 3 in this sense may be of primary importanceto the function of septum formation.
A New Gene Controlling the Frequency of Cell Division per
Round of DNA Replication in Escherichia coli
Akiko NISHIMURA

A novel mutant of Escherichia coli, named cfcAl, was isolated from atemperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality, induced by the inhibition of
DNA replication, was reduced and delayed in the cfcAl dnaB42 strain, as
compared with the parental dnaB42 strain. Two types of inhibition ofdivision, induced by the addition of nalidixic acid or hydroxyurea, were
suppressed by the cfcAl mutation. Under permissive conditions for DNA
replication, the colony forming ability of cfcAl cells was significantly re-

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

duced as compared with that of cfc+ cells; conversely the division rate ofcfcA1 cells was higher than that of cfc+ cells. The cfcA1 mutation partiallyrestored division arrest induced in the thermosensitive ftsZ84 mutant at
restrictive temperatures and suppressed the UV sensitivity of the Ion mutation. The mutation was mapped at 79.2 min on the E. coli chromosome.
Taking these properties into account, it was hypothesized that the cfcA
gene is involved in determining the frequency of cell division per round of
DNA replication, by interacting with the FtsZ protein, which is essential
for cell division. For details, see Mol. Gen. Genet. 215: 286-293, 1989.
A Cell Division Regulatory Mechanism Controls theFlagellar Regulon in Escherichia coli
Akiko NISHIMURA and Yukinori HIROTA

The formation of flagella in various thermo-sensitive (Ts) cell divisionmutants of Escherichia coli was examined at nonpermissive temperatures.
The number of flagella per unit cell length, decreased sharply after shifting
the culture temperature from 30° to 40°C in the following Ts mutants:
ftsC108, ftsD1033 , ftsE1181, ftsFl141, ftsG29, ftsZ84 , parA110, dnaB42 ,nrdB, and dnaG. It was found that transcription of genes responsible forthe formation and/or function of flagella (hag, fla, mot, che), decreased
significantly at 40°C. However, in the fts1730 mutant at a nonpermissive
temperature, or in penicillin G treated wild-type cells, cell division was
blocked while formation of flagella continued. Moreover, when the cfcA1
mutation of a gene involved in coordinating DNA replication and cell
division, was introduced into the danB42 mutant strain, inhibition of cell
division and formation of flagella at 40°C was relaxed. These results indicate that the flagellar regulon is under the control of a cell division regulatory mechanism. If this kind of mechanism controls the biosynthesisof cellular constituents, it would be a fundamental reaction in the biologicalcontrol mechanism that coordinates cell growth with cell division. For
details, see Mol. Gen. Genet. 216: 340-346, 1989.

RESEARCH ACTIVITIES IN 1988

59

Restriction Map of a Bacillus subtilis Gene Involved in Cell
Division, Sporulation and Exoenzyme Secretion
Yoshito SADAIE

We established a partial restriction map of the wild type div-341+ geneof Bacillus subtilis which was cloned in a temperate phage, ¢105. The
order of restriction sites was Cfrl3I, ApaLI, MluI, StuI, ClaI, Cfrl3!. The
mutation div-341, located between sacU and uvrA loci, results in filamentous
growth at a restrictive temperature (42°C), and poor sporulation of typicalspoO type mutants at an intermediate temperature (37°C), where vegetative
growth is almost intact. The div-341 mutant showed reductions in extracellular protease and a-amylase secretion, competence development andautolysis at 37°C. It also showed a defective spore outgrowth at 37°C.
Therefore the wild type div-341+ gene seems to be involved in a common
step which is required for the excretion of extracellular proteins on the cellsurface, or of those which are involved in cell division, sporulation, autolysis,
and competence development. (Mol. Gen. Genet. 190, 176-178, J. Bacteriol.
163,648-653., Jpn. J. Genet. 64,111-119.)

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

III.

IMMUNOGENETICS

Novel Molecular Organization of Meiotic Recombinational
Hotspots in the Murine Major
Histocompatibility Complex
Toshihiko SHIROISHI" Tomoko SAGAI1 , Naoto HANZAWA 1,
Michael STEINMETZ 2 and Kazuo MORIWAKI 1

The physical distance between two genetic loci in the eukaryotic chromosomes is not always consistent with the genetic map distance deduced
from meiotic recombination frequencies. This difference suggests thatmeiotic recombination may not be random, but may rather occur more
frequently in certain specific regions. It has been established that meiotic
recombinations within the major histocompatibility complex (MHC) of
the mouse occur in clusters at restricted sites, so called, recombinational
hotspots. We identified a recombinational hotspot in the wm7 haplotypeof the MHC, which was derived from Japanese wild mouse, Mus musculs
molossinus. Recombinations involving the wm7 haplotype occur at a very
high frequency between H-2K and A(3 genes only in female meiosis but not
in male meiosis. Recombinational break points were confined to 1 kbp of
DNA between the A(33 and A(32 genes. Its location overlaps with a sexindependent hotspot previously identified in the CAS3 haplotype originatingfrom another Asian subspecies, M. m. castaneus. We cloned the DNA
fragment containing this recombinational hotspot from the wm7 haplotype
and the DNA fragments of corresponding regions from standard laboratory
strains, BlO.A and C57BL/lOJ, which do not have a recombinational hotspot
in this region. Nucleotide sequences surrounding this region from the
wm7 and from two hotspot-negative, standard laboratory strains indicatedthat no significant differences are present. However, a comparison of thenucleotide sequences of this A(3jA(32 hotspot and a previously characterized
hotspot in the E(3 gene clearly revealed that they have very similar molecular organizations. As shown in Figure 1, in each hotspot, the crossover
points clustered around two molecular elements: the consensus sequence
12

National Inst. of Genetics, Mishima, Japan.
Central Res. Unit, F. Hoffmann-La Roche & Co. Ltd., Basel, Switzerland.

RESEARCH ACTIVITIES IN 1988(a)

Bl0.A(4R)~r

Bl0.A(3R) ~Bl0.A(5R)I

CAGG I( GTCC
7-9

MT-consensus-L-H

AIJ 2 - - , - - EIJ"( IJ 1 exonl

61--....

(O~IlLsl0.Q

J'

;/

E.s
( IJ 2 exonl

Bl0.A(R207)(b)~:tg:~m~r.l

Bl0.A(R221)
CAS3(R18)
Bl0.A(R201)
CAS3(Rll
B l 0 . A ( R 2 0 6 ) I CAS3(RO)-------4

MT-consensus(

r8X% )

4-8

AIJ 3 -I~C-:::;::::J-;----~:;-_""':_--~z:...- AIJ 2
(orll

L L
Bl0(R231)
Bl0(R233)
Bl0(R236)
Bl0(R225)
CAS3(R25)

Bl0(R224)
11.10(11.237)
Bl0(R239)
CAS3(R23)

1 kbp

Fig. 1.

of the mouse middle repetitive MT-family, and the (CAGA)4_6 orThese two elements, separated from eachother by I kbp of DNA, probably serve as the molecular basis for the sitespecificity of recombination at the hotspots in the MHC.
(CAGG)7_9 repeat sequence.

New Evidence for Trans-Species Evolution of the H-2
Class I Polymorphism
Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

A serological survey using alloantisera specific for the H-2 class I antigens in Japanese wild mice, Mus musculus molossinus, revealed a highfrequency of the H-2Kf antigen. This antigen has also been found inEuropean wild mice, M. m. domesticus and M. m. musculus. In this survey,
the H-2Kf antigen was characterized through the use of ten newly isolated
monoclonal antibodies raised against cells of a Japanese wild mouse, andby Southern blot analysis using an H-2K locus-sepcific probe which hybridizes with the 3' end of the gene. The serologically identified H-2Kf
antigens revealed several minor variations in reactivities to the monoclonal antibodies. However, all the antigens examined could be clearly
separated into two types with respect to the RFLP pattern. The first type

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

found together with a single, characteristic RFLP pattern, was always associated with the presence of reactivity to one particular monoclonal antibody, MS54. The second type, found representing different RFLP patterns, is associated with the absence of reactivity to MS54. This concordance between the presence of an antigenic determinant and a particular
RFLP was observed not only within M.m. subspecies but also in a different
species: M. spretus, carrying the same antigenic determinant, which presented an identical RFLP to that of the other MS54-positive M.m. subspecies. The results suggest that the antigenic determinant specific for
MS54 is an ancient polymorphic structure which has survived the long
period of diversification of the Mus species (approximately 2-3 million
years) without alteration, and is associated with a stable DNA structure atthe 3' end of the H-2K gene.
Steroid 21-Hydroxylase Deficiency in Mice
Hideo GOTOH, Tomoko SAGAI, Jun-Ichi HATA,
Toshihiko SHIROISHI and Kazuo MORIWAKIThe enzyme steriod 2l-hydroxylase (2l-0Hase) plays a key role in adrenal
steroidogenesis. Defects in this enzyme are responsible for one of themost common inborn defects in metabolism in humans. Duplicated genes
for the enzyme are located in the class III region of the major histocompatibility complex (MHC), HLA. In the mouse, the genes encoding 21OHase have been mapped to the homologous region of the H-2 complex.
We previously described an H-2 recombinant haplotype aw18, in which thegene for the complement component C4 and one of the two genes for 31OHase in the H-2 Class III region have been deleted. We now report thatnewborn aw18 homozygous mice are deficient in 2l-0Hase activity, andthat homozygosity for the aw18 haplotype directly cause lethality at theearly postnatal stage. Morphological changes in the adrenal glands of thenewborn aw18 homozygotes are also observed. The aw18 recombinant
haplotype is expected to serve as a useful and, thus far, unique experimental
system to study adrenal steroidogenesis in vivo, and as an animal model forthe inherited human disease, congenital adrenal hyperplasia. (For details
see Endocrinology 123: 1923-1927, 1988).

RESEARCH ACTIVITIES IN 1988

IV.

63

DEVELOPMENTAL AND SOMATIC CELL GENETICS
Artificial Expression of Tissue-Specific Characteristics in
Embryonic Lethal Cells of Drosophila melanogasterYukiaki KURODA

In some sex-linked recessive lethal mutants of Drosophila melanogaster,embryos homozygous for the lethal genes die at specfiic stages in embryogenesis, due to defects in morphogenesis and in functions of specific tissues
or organs. One such mutant, the dar mutant showed specific defects insyncytium formation of muscle cells, formation of cellular spheres anddroplet formation on nerve fibers, when cells obtained from embryos at
an early stage in embryogenesis are cultured in vitro. The dar gene islocated in a region including six bands from IFl through 2A2 on the X
chromosome in salivary gland cells. In the present experiment, an attempt
was made to repair these defects in dar embryonic cells by introducing wildtype genes or their products which are responsible for these defects.
When dar embryonic cells were cultured in medium containing wild-type
egg extract, defects in muscle cells, cellular spheres and nerve cells were
found to be almost completely repaired. Embryos obtained from themating of dar/dar females and +/Y males were allowed to develop tovarious stages such as blastoderm formation (3 hours after fertilization),gastrulation (5 hours after fertilization), head and trunk segmentation (9hours after fertilization), and muscular movement (14 hours after fertilization). The extracts obtained from these embryos were tested for theirability to repair the defects of dar embyronic cells. An extract from embryos
at the stage of blastoderm formation showed no repair activity for thedefects of dar cells. Extracts from embryos at stages later than gastrulation were found to be effective in repairing the defects of dar cells.
This suggests that effective substances may be produced by the paternal
wild-type gene between 3 and 5 hours after fertilization. It seems likelythat the translation of the effective substances through mRNA transcribedby the paternal wild-type gene (DNA) takes place after blastoderm formation. The effective substances lost their activity after heat treatment as
80°C for 10 min, suggesting that they are enzymes or heat-labile proteins.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Further studies are under way to test the ability of DNA and mRNA,
isolated from embryos at various stages, to repair the defects of dor cells.
Selective Infection of SR Spiroplasms in Cultured
Embryonic Cells of Drosophila melanogasterYukiaki KURODA, Bungo SAKAGUCHI*, Kugao OISHI**
and Yutaka SHIMADA

Sex-ratio (SR) spiroplasms are known to selectively kill only male embryos of Drosophila, but do not affect female embryos. In the present experiment, how the SR spiroplasms recognize cells of male and female embryos and what types of cells or tissues of male embryos are infected by the
SR spiroplasms, were examined.
SR-containing body fluid was collected from 300-600 SR-infected adultfemales 7-10 days after eclosion by a capillary tube, diluted with the same
volume of physiological salt solution, sterilized through a Millipore filter
and added to cultured embryonic cells of Drosophila melanogaster. After
cultivation for one or two days, changes occurring in cultured cells were
examined under an inverted phase-microscope and an electron microscope.
Various types of cells differentiated from undifferentiated embryoniccells in culture. In muscle cells, nerve cells and cellular spheres, no detectable changes produced by SR spiroplasms were observed under a phasemicroscope. In epithelial cells, some brown necrotic changes were observed, which might have been produced through infection with SR spiroplasms.
In a control experiment, body fluid obtained from adult flies, which were
not infected with SR spiroplasms, or were infected with nonkiller mutant
SR spriroplasms, were not effective in producing necrotic changes in epithelial cells.
Under an electron microscope, it was observed that embryonic cells infected with SR spiroplasms contained many spiroplasm-like structures inintercellular spaces and the cytoplasm of necrotic cells. Whether cells withSR spiroplasms might be a specific type of cells, and whether SR spiroplasms
might proliferate under culture conditions, are under investigation.
* Department of Agriculture, Kyushu University.
** Department of Science, Kobe University.

RESEARCH ACTIVITIES IN 1988

65

Antimutagenic Effects of Safrole on Chemically Induced
Mutations in Cultured Chinese Hamster Cells
Yukiaki KURODA, Ajay K. JAIN* and Hideo TEZUKA

In our environment, more than two mutagens present together may act
to induce mutations, at a different frequency from those estimated theoretically from the effects of the single mutagens. Also, differential exposure
times of these mutagens may produce some different effects on the induced
mutation frequency. In our continuing experimental series, in which the
combined effects of two chemicals are being studied, the effects of safroleand cobalt chloride on the cytotoxic effects and mutations induced by
ICRl70 were examined in the present experiment.
ICRl70 alone had a dose-dependent cytotoxic effect on survival of
Chinese hamster V79 cells. The colony-forming activity of cells decreased
to 50 % of non-treated control cells at a concentration of 2.0 ,ugjml of
ICRI70. Safrole alone had a weak cytotoxic effect on cell survival,
producing 50 % survival at a concentration of 50 ,ugjml. In the presenceof 50 ,ugjml safrole, the survival of cells treated with 2,ugjml ICRl70 was
78 %, indicating that safrole reduced the cytotoxicity of ICRI70.
ICRl70 at a concentration of I ,ugjml induced 6-thioguanine (6TG)resistant mutations at a frequency of 46.8 x 10- 5 • On the other hand,safrole induced no detectable mutations at a concentration of 50 ,ugjml.
When cells were treated with safrole and ICRl70 together, the mutation
frequency of cells was similar to that induced by ICRl70 alone. However,when cells were treated with ICRI70, incubated for 3 hours, and thentreated with safrole, the induced mutation frequency was reduced to 30.0 X
10- 5 •
Cobalt chloride enhanced the cytotoxicity of ICRl70 in Chinese hamster
cells. On the other hand, cobalt chloride reduced the frequency of mutations induced by ICRl70 to 50 %, when cells were treated with these two
chemicals together. The reduction of the ICRl70-induced mutation frequency by cobalt chloride was also found when cells were treated withcobalt chloride 6 hours before ICRl70 treatment.
These results indicate that safrole and cobalt chloride had no detectable
activity in themselves for inducing mutations, but both were effective in
reducing the frequency of mutations induced by ICRI70.

* Central Drag Research Institute, Luchnow, India.

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Injury Effect on Hydra Head Regeneration
H. SHIMIZU and T. SUGIYAMA

Three different procedures were used to remove the original head fromhydra, and the regeneration processes of decapitated polyps were examined
and compared. Relatively young polyps bearing the first bud protrusionof a wild-type strain of Hydra magnipapillata (105) were used. The head
was removed by a cut made at the mid-point between the hypostome andbud protrusion.
In standard amputation, the head was cut off instantaneously using a sharp
surgical knife, whereas in ligation, the head was gradually removed byligation using a monofilament nylon fishing line. This procedure produced
a much smaller wound opening in the decapitated polyp than standard
amputation did. In mirror-image-grafting, the head was cut off as in standard amputation, followed by the grafting of two decapitated polyps in sucha manner as to make the wound opening of the two polyps face and covereach other.
Polyps subjected to standard amputation restored the original numberof tentacles in 2-3 days and 60-80 % more in 7 days. Polyps subjected toligation also restored the original number in 2-3 days, but only 30-40 %more in 7 days (cf. Newman, 1974). In contrast, about 40% of the polyps
subjected to mirror-image-grafting failed to regenerate any tentacles in 7
days.
Lateral tissue grafting was employed to examine the changes in head activation and inhibition potential in polyps subjected to standard amputationor
mirror-image-grafting. No significant differences were found in the decrease in inhibition levels in polyps subjected to the two procedures. In
contrast, increases in head activation levels were much smaller in polyps
subjected to mirror-image-grafting than in normally amputated polyps.
These observations show that, in addition to a decrease in inhibitionlevels, the state of the injured tissue produced by decapitation strongly
affects the increase in activation and subsequent head regeneration. Thissuggests that "injury effect" originally proposed by MacWilliams (1983) toexplain transplantation phenomena also plays an important role in headregeneration.

RESEARCH ACTIVITIES IN 1988

67

Random Displacement of I-Cells into Buds and Maintenanceof the I-Cell Population in Hydra
Toshitaka FUJISAWA

It has been believed that the interstitial cell (I-cell) population in hydra
is maintained by regulating its self-renewal probability (Bode and David,1978). Recently, however, Heimfeld (1986) reported that the rate of I-cellgrowth was relatively unchanged even when the I-cell density was loweredby hydroxyurea treatment and that the recovery of I-cells in the adulttissue was attained by increased retention at the expense of buds.
In the present study, the growth rate of I-cells and patterns of I-cell displacement into buds were examined in polyps oflow I-cell densities. Suchpolyps were produced by introducing a small piece of tissue from a normal
polyp into an epithelial polyp which lacks an I-cell lineage. The growthrate of I-cells was monitored by scoring the number of I-cells in macerated preparations. Patterns of I-cell displacement were analyzed byscoring cell numbers in adult and bud tissues and also by staining I-cells
in whole mounts with Toluidine blue. The results showed that the growthrate of I-cells was 2-fold higher at low densities than at normal ones. Furthermore, it was suggested that the displacement of I-cells from an adultinto buds was a random process. These results do not agree with those of
Heimfeld's but rather with the prevalent concept of self-renewal regulationof I-cells.
Although there were differences in experimental procedures, the exact
reason for differences in results between the present study and the
Heimfeld's is not known at present.

Genetic Studies on the Silkworm Adult Lifespan(a) Heredity of the Short Adult lifespan (sdi)Akio MURAKAMI

Because the duration of adult lifespan significantly differs among various
silkworm strams, It has been suggested that the adult lifetime may be genetically controlled (Murakami et al., 1986). The mean adult lifespan of theinsect is around 7-10 for females and 4-5 days for males. However, theadult lifespan of the Daizo strain is remarkably short (ca. 2 days) in both

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

sexes. In this experiment, using the Daizo (D) strain and the Japanese106 (J) strain which has an average lifespan various crosses (FH BFi andF 2 of both strains) were perfomed to clarify the genetic mechanism of theadult lifespan in the Daizo strain.
Experimental conditions throughout this series were controlled at atemperature of 25°C and at various relative humidities ranging from 50 to
80 % depending on the growth stage of the insect. The mean adult lifespans
for each group and sex in every strain were examined. At least 100 newly
emerged adult moths for each sex in every experimental group were used forobservations. The death of adult moths was judged when they did not
biologically respond to being poked softly by the point of a pencil.
The mean adult lifespan of F i hybrids between the Daizo and the JI06
strains was longer than for JI06 adults indicating that the adult lifespanincreases through hybridization. On the basis of these results, it was inferred that the short adult lifespan in the Daizo strain may be controlled bya single recessive factor named sdi (short duration of the imaginal lifetime)
for the convenience of the following experiments.
The lifespan of female adults of BFi or (D x J) x D showed the first peak
(48.4%) between the 1st and 7th days with a maximum on the 4th day andthe second peak (51.6 %) between the 8th and the 20th days with a maximum
on the 13th day. Although this distribution of the lifespan was slightly
prolonged, compared to those of the Daizo and the JI 06 strains (it isnecessary to take into account the difference in genetic background fromthe pure strains), the first and second peaks of the lifespan may have originated from sdi/sdi and sdi/+ individuals, respectively. A similar explanation could also be applied to the lifespan of (J x D) x D. For male moths,
the distribution pattern of their lifespan corresponded to the theoreticalvalue, although it was difficult to distinguish the distribution peaks fromeach other because of the possible existence of heterosis and recombinations,in addition to their generally short lifespans.
F 2 also seems to divide into sdi/sdi, sdi/ + and +/+ at the ratio of 1: 2: 1.
In the experiment with (D X J)F 2 , it was observed that the first peak (26.4 %)and the second and third peaks (73.6 %) were distributed between the 3rd
and 4th days after emergence and between the 5th and 18th days, respectively. Since the second peak (ca. 25 %) and the third peak (ca. 50 %) may
be produced by +/+ and sdi/ + moths, respectively, the distribution pattern in the lifespan corresponded almost to the theoretical value.

RESEARCH ACTIVITIES IN 1988

69

On the basis of these experimental results, it was clear that the short adult
lifespan of the Daizo strain was controlled by the single recessive sdi gene.
In addition, it was also found the adult lifespan has a Mendelian monogenic
hereditary mode and the responsible gene is located on a specific certain
autosome.
Genetic Studies on the Silkworm Adult Lifespan(b) A Biological Characteristic of the Daizo (sdi) Strain
Akio MURAKAMI

Although the adult lifespan of the Daizo (sdi) strain is quite short (ca.
a days) regardless of sex, reproductive behaviour, and the life history
throughout fertilization and embryonic, larval and pupal stages proceedssmoothly. Therefore, the sdi gene factor may control the short life (microbiotic imago), but not adult lethality. However, this is not certain without
further investigation.
The Daizo strain moths showed no prolongation effect on lifespan with
low temperature storage, which is usually found in silkworm moths ofother strains or lines. However, it is of interest to note that the mutant
moths showed a prolongation effect under high humidity conditions (90 %or more of relative humidity), which is also a generally common feature
in Bombyx adults. Unlike moths of ther strains or hybrids lines, death of
Daizo (sdi) strain moths occurs immediately after oviposition, thus hardly
causing a decrease in the adult fresh weight. On the other hand, a possibility that the nervous system may be concerned in the duration of adult
lifespan has been indicated by the fact that the adult lifespan was shortened
to about 2 days when the brain was removed at the early pupal stage (Murakami and Shimada, 1988). Taking the non-feeding habit of silkworm
adults into consideration, the short adult life charactestic in the Daizo(sdi) strain seems to be due to a certain disorder of the nervous system causedby an abnormal nitrogen metabolism (or excretion) rather than an abnormalenergy-intake.

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Genetic Studies on the Silkworm Adult Lifespan(c) Sex Differences and their Biological SignificanceAkio MURAKAMI

It was reported that female adults in the silkworm Bombyx mori have along lifespan compared with males in observations on sexual differences ina dozen pure lines designed to clarify the genetic mechanism of adult
lifespans (or the length of moth stages) (Murakami, 1986). The present
experiment was carried out to confirm the finding by using single and triple
hybrids. Hybrids obtained from either dual or ternary crossbreeding with22 combinations of the Cambodge, the Daizo, the J106 and the od strains
were used. Rearing conditions and methods for the observation of survival
days were set up in accordance with the previous communication describedabove. For the measurement of lifespans, at least 100 nonmated moths
were used for female or male groups in each hybrid group. The number ofmoths that naturally ceased their biological activity was counted two times
a day (10 a.m. and 3 p.m.) and the mean lifespan was calculated in eachhybrid group.
The mean lifespans of male and female moths in the experiments varied
from 2.7 to 6.7 days and from 4.1 to 11.6 days, respectively. When themean lifespans of female and male moths in each experimental group werecompared, it was found that the average length of the adult stage was 1.7
times longer in females than in males, although the ratio varied from 1.1 to
3.1 depending on each hybrid group. These observations corresponded wellwith results obtained using pure lines (Murakami, 1986). These results
confirm that in the Bombyx silkworm, the lifespan of female moths is significantly longer than that of males.
In the silkworm, males have a higher a oxygen consumption per bodyweight basis, index of metabolic activity, than females throughout thewhole life cycle (Itaya, 1944). The wing-beat frequency of male moths ishigher than that of female moths for the first several days after eclosion
until the last day of reproduction time. After the completion of voiposition in females, their wing-beat activity is markedly reduced. In the silkworm, the nominal body-weight of females is about 1.3 to 1.5 times heavier
than that of males. However, if the contribution of the total weight ofeggs or oocytes in the body cavity is subtracted from the body-weight, thenet body-weight offemale moths may not be much heavier than that of males

RESEARCH ACTIVITIES IN 1988

71

or may be almost the same as males. From this it can be suggested that the
metabolic rate or energy consumption may be one of the principal mechanisms
for sex differences in adult lifespan rather than other possibilities.
In Bombyx silkworms, a member of the lepidopteran order, the male ishomogametic ZZ (XX) and the female is heterogametic ZW (XY), which
different from other insect orders including diptera with the XY type. In
several lepidopteran species (e.g., Woodruffe, 1951) other than Bombyx
and some saturniid species (McArther and BailIe, 1932), female adults also
have longer lifespans than males. Rockstein and Miquel (1972) reported
an adult insect lifespan list including five orders or 47 species. They indicated that females in most species surveyed live much longer than males.
Thus, it becomes clear that the sexual difference in the insect adult lifespanmay be not dependent on the sex-chromosome constitution of either ZW
or XY types, but may be a secondary sexual characteristic. This deny theparticipation of sex-chromosomes, either Z(X) or W(Y), in sex differencesin the adult lifespan. In addition, the possibility of involvement of certain
autosomal factor(s) in the length of insect adult stages can not be excluded.
In a ZW-type bird, the lifespan of both sexes is similar, or that of females islonger than that of males, but the reverse case also occurs, (Landaur andLandaur, 1931). Therefore, further studies are required to draw a final
conclusion about whether ZW females have longer lifespans than ZZ
males.
The Genetic Nature of Known Mutants in the DomesticatedMulberry Silkworm, Bombyx Mori
Akio MURAKAMI

A cursory glance over the mutant list or the linkage map of the Bombyx
silkworm indicates that this insect abounds with what are called dominant
mutations symbolized by a capital letter compared to other species, forexample, D. melanogaster where recessive mutants are frequent. In B. moriapproximately 85 % or more of artificially-induced mutations and about
45 % of spontaneous mutations are indicated as dominant characters. In
addition to these features, more than a half of the induced mutations andabout one-quarter of the spontaneous mutations are lethals when homozygous, suggesting that they are caused by chromosomal aberrations (either
deficiency or deletion). The genetic nature of some artificially-induced

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

mutants symbolized by capital letters in the original paper is clearly specified
as a deficiency of chromosomes. For example, a radiation-induced mutation,
Rg, is found to be a large deficiency in chromosome 3. In the silkworm,
artificially-induced recessive mutants other than lethal mutations are few,
but some of them are also accompanied with lethality. In the linkage map,
we have rarely witnessed translocations which are distinctly classified as
chromosomal aberrations. It has been well documented that many spontaneous as well as some artificially-induced translucent (oily) skin mutations
are located over a dozen linkage groups in the silkworm. Most of thesemanifest a weak lethality and/or sometimes inviability in the homozygous
state, indicating that the genetic nature of those mutations is analogous to
Minute mutations which have small deletions in the ribosomal gene loci of
D. melanogaster. It can be said that the large part of the induced mutations is not genine gene mutations, but chromosomal aberrations cover awide range of deficiencies.
While, for the spontaneous mutations, there are problems in an inadequate
manner of the gene nomenclature for several main characters because of theunsettled wild-type in this insect and for isozyme loci which are currently
marked by a capital letter regardless of the characters either dominant orrecessive. The standard phenotype has been selected from a wide rangeof popular phenotypes available among commercial strains. It should berecalled, however, that B. mori mandarina Moore, a non-domesticated silkworm, is widely accepted as the possible ancestor of B. mori L. If we
accept that B. mori mandarina is a wild-type of B. mori, then many current
phenotypes for several genetic characters symbolized by the capital letterlike cocoon colour, cocoon shape, larval marking pattern, moultinism andvoltinism would have to be rewritten with a small letter as recessive characters. All isozyme loci documented as spontaneous mutations are currently
symbolized by capital letters regardless of what are called recessive anddominant characters. This nomenclature, however, is inconventional. To
avoid confusion between the conventional nomenclature system and theisozyme one, it is logical to adopt a uniform nomenclature. In conformity
with the conventional nomenclature the isozyme should be redesignated as
the case of a locus controlling alkaline phosphatase. For example, Aphn
(or Aph- and Aph o : a null type) should be rewritten as aph and the otherco-dominant alleles, Aph s (a slow type) and Aph F (a fast type) as +Aph-S
and +Aph-F, respectively.

RESEARCH ACTIVITIES IN 1988

73

It thus appears that the great part of the mutations symbolized by acapital letter is not a gene mutation and some of them are not dominant
types. In this connection, it is desirable to be separated then the chromosomal mutations from what we call the gene mutations in the mutant listof the Bombyx silkworm.

Decrease in Hatchability of Eggs Laid by Flies Reared on
Excess Yeast in Drosophila melanogaster
Kiyoshi MINATO

In the previous study (1979), it was found that female flies laid three tofour times more eggs (30-40 eggs/day/female), when reared on agar medium(agar, cornmeal, dry dead-yeast, sugar, and propionic acid) with dailychanges, and with the surface partly coated with paste-like live yeast, thanflies reared on agar medium without any coating with yeast. However, thehatchability of eggs decreased gradually as time went on (90-95 % at a days
and 50-60 % at 10 days after eclosion), whereas eggs laid by flies reared onagar medium without any coating with yeast showed little decrease inhatchability. Further analyses of this phenomenon showed that when heattreated (90°C, 10 min.) yeast was used in place of live yeast, flies laid nearly
as many eggs as flies reared on agar medium coated with live yeast. The
hatchability of eggs, however, decreased little during the culture of flies
unlike the intense decrease in the hatchability of eggs in flies fed on live
yeast.
This shows that the decreased hatchability of eggs laid by flies fed on
excessive live yeast was not due to the increased rate of egg laying which
may give rise to unfertilization and, hence, unhatched eggs owing to thefailure of sperm to enter eggs and/or the shortage of sperm supply. On theother hand, in flies showing decreased hatchability, an insufficient supplyof sperm from male flies is, also, unlikely to occur, because neither increasein the male to female ratio in population, nor exchange of aged males byyoung ones improved the hatchability of eggs. Since the above unhatchedeggs did not show any internal morphological changes under a light microscope, they appeared to be either unfertilized due to reasons other thaninsufficient sperm-supply, or to be fertilized but undeveloped.
A similar phenomenon of an increase in oviposition and a decrease inhatchability of eggs was sometimes seen in stock cultures too, when the agar

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

medium was supplemented excessively with either paste-like or dry live
yeast. The use of antibiotics in agar medium, however, improved hatchability in these cultures, suggesting that bacteria might have proliferated
inside the thick sheet of paste-like live yeast and this was confirmed byexamination after Gram staining. Live yeast remained non-growing andthe flies fed on such agar medium might produce eggs with low hatchability.
This posibility appears to be confirmed by the following findings: the hatchability of eggs decreased little when yeast was used after heat-treatment
or sterilization, and agar medium coated with yeast was reserved at a lowtemperature (4°C), or when fresh live yeast was added to the agar
medium only ujst before use. Then, it is physiologically of interest why
the eggs laid by flies fed on highly infected medium often don't hatchin spite of high egg production of parent flies.
Purification and Structure Analysis of an Autocrine Growth
Factor in Conditioned Medium Obtained from Primary
Cultures of Scleral Fibroblasts of the Chick Embryo
Miki FUJIOKA, Akira KAWAHARA, Minoru AMANO,
Kazuo WATANABE and Nobuo SHlMAMOTO

The time course of a cell proliferation and cell differentiation may be
determined by the initial state of the cell and by its envioronment including
growth factors. Most simply, local concentrations of growth factors could
control a spatial distribution of cells. One of candidates for such situationis the scleral fibroblast of the chick embryo which forms a thin layer surrounding eye balls. As the first step to check this hypothesis, we purified
a related growth factor.
We found that the cells secrete autocrine growth factors. One factor
was purified from conditioned medium collected from growing-phase cultures by DEAE-Sepharose column chromatography and non-denaturing
polyacrylamide gel electrophoresis. The specific activity was increased
1,lOO-fold through purification. The purified growth factor was heatstable, and resistant to acidic or basic treatment. Removal of the glycosidechain did not alter activity but reduction by dithiothreitol or digestion bytrypsin hampered activity. These characters are similar to those of theplatelet-derived growth factor (PDGF) but its molecular weight, 32 kd, ismuch larger than PDGF. Thus this factor might be a new family of growth

RESEARCH ACTIVITIES IN 1988

75

factors. The primary sequence was determined for 20 amino acid at N
terminal.
The Three-Dimensional Ultrastructure of Intracellular
Membranous System in Cultured Chick
Skeletal Muscle Cells
Yutaka SHIMADA

The development of the transverse tubular system (T system) and thesarcoplasmic reticulum (SR) in embryonic chick skeletal muscle cells in
vitro was studied by (1) scanning electron microscopy of freeze-polished,osmium-macerated cells and (2) transmission electron microscopy of rotary
replicas of critical-point dried and physically reptured cells. Intracellular
membranous organelles (T system and SR) were arranged periodicallyalong the length of myofibers. The T system was tubular in appearancewith many warts, and it followed a tortuous course. In the cortical regionof the cell, a complex anastomonic network of T system tubules was observed. Occasionally, coated vesicles were associated with T systemtubules. Many SR appeared to be continuous with each other and often
exhibited swellings. At such dilated areas of the SR, T system tubules
associated to form early triatic structures. At early stages of development,these two functionally related organelles were not arranged regularly and,thus, their distinction was sometimes difficult. Mitochondria were longrod-shaped with occasional branchings. At the cracked surface of
mitochondria, mitochondrial cristae could be observed.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39v.

CYTOGENETICS

Molecular Analysis of Complement Factor H Genes in Wild
Mice (Mus musculus) and Their Relatives
Yoshi-nobu HARADA, Fran90is BONHOMME"', Shunnosuke NATSUUME-SAKAI"'''',
Kazuo MORIWAKI and Takeshi TOMITA*"''''

Complement factor H is a plasma glycoprotein which functions as a
cofactor in the conversion of C3b to iC3b by serine protease I. Threeallotypes of factor H, H.I, H.2 and H.3, were reported in mice (Musmusculus). A serological survey of factor H allotypes in wild mice fromall over the world revealed that geographical distributions of factor H allotypes were specific to the subspecies. The survey also revealed that thethird allotype, H.3, was able to be found only in BFM/2Ms and BFM/IMpl
strains which were derived from M. m. domesticus captured in Montpellier,France. On the other hand, a serological survey of mouse relatives showedthat all six strains derived from Mus spretus had factor H which strongly
cross-reacted with H.3-specific antiserum. Consequently a molecularanalysis was carried out on factor H genes from BFM/2Ms, BFM/IMpl andspretus strains. RFLPs of factor H genes were detected by a Southern
analysis using a 0.7 Kb cDNA probe of mouse factor H gene establishedby S. N.-S. A 5Kb DNA fragment of Msp I digestion and a 5.4 Kb fragmentof Hae III digestion were specific to BFM/2Ms and BFM/IMpl. All six
strains derived from M. spretus also had these DNA fragments. No mice
without the H.3 allotype had these DNA fragments. In October 1988, an
additional serological survey was carried out on some wild mice captured in
the Montpellier area where the original mice of the BFM/2Ms and BFM/
IMpl strains were collected. Two of the eight mice from Montbazin andall eight mice from the campus of Montpellier University had the H.3
allotype. This observation shows that the H.3 allotype of the BFM/2Ms
and BFM/IMpl strains is not a mutation which occurred in the laboratorywhen these strains were established.
Results of the molecular analysis and the serological survey may suggest
'"

*'"
"'*'"

Montpellier University.
Kanazawa University.
Nagoya University.

RESEARCH ACTIVITIES IN 1988

77

that the H.3 gene in BFM/2Ms and BFM/IMpl is derived from M. spretus
by gene introgression after the differentiation of the genus Mus.
Reproductive Ability of Hybrids between Mus musculus
castaneus and M. m. Subspecies in China
Michiko NIWA, Noboru WAKASUGI"', Akihiko MlTA
and Kazuo MORIWAKI

The reproductive performance of hybrids between Mus musculus castaneusand M. sub-Cht, derived from M. m. subspecies in Chetzu (China) whichclosely resembles M. m. musculus, was observed. The inbreeding generation of this Cht-strain was Fll-FI4 and the mean litter size was 4.7±O.2(n=50, mean±sem). We made reciprocal matings between M.sub-Cht
and the following 3 strains derived from M. m. castaneus: M.cas-Hmi,
M.cas-Mal and M.cas-Bgr whose ancestors were captured in Hemei(Taiwan), Malaysia and Bogor (Indonesia), respectively. The numbers ofinbreeding generations of M.cas-Hmi, M.cas-Mal and M.cas-Bgr were
F2-F7, FI-F4 and F9-FI3. The mean litter sizes were 5.8±O.2 (M.casHmi, n=46), 6.6±0.4 (M.cas-Mal, n=33) and 5.9±O.2 (M.cas-Bgr, n=50).
Table 1. Litter size in the matings between 1'.1. m. casfaneus and 1\-1. m. subspecies in China.
Matings
Female x MaleNo. oflitters

Litter size
mean ± sem

Hmi x Cht
Cht x Hmi
Mal x Cht
Cht x Mal
Bgrx Cht
Cht x Bgr

9
4
15
4
10
1

5.3±0.5
7.5±0.9
5.5±0.3
4.5±1.2
5.1±0.6
5.0(Hmi x Cht)Fl x (Hmi x Cht)FI(Cht x Hmi)Fl x (Cht x Hmi)Fl(Mal x Cht)FI x (Mal x Cht)Fl(Cht x Mal)FI x (Cht x Mal)Fl(Bgr x Cht)FI x (Bgr x Cht)Fl(Cht x Bgr)FI x (Cht x Bgr)Fl

33
38
35
20
17
3

8.0 ±O. 3
6.2±0.3
5.6±0.3
6.5 ±0.4
4.6±0.5
3.3 ±O. 7

Hmi: M. cas-Hmi, Mal: M. cas-Mal, Bgr: M. car-Bgr and Cht: M. sub-Cht.
'" Faculty of Agriculture, Nagoya University.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Each reciprocal mating between the two subspecies produced progeny.
Fl hybrids were fully fertile and showed no reproductive break down
(Table I), and sex ratios were normal in the progeny. Japanese wild mice,
M. m. molossinus, are thought to be originated from hybrids between M. m.
castaneus and a Chinese race of M. m. musculus (Yonekawa, H. et al., 1988,
Mol BioI. Evol. 5: 63). The results of this study support the possibility thatthe intersubspecific hybrids occurred under natural conditions.
Effect of the Y Chromosome and H-2 Complex of JapaneseWild Mouse on Sperm Morphology
Dong-Sang SUH*, Jozefa STYRNA** and Kazuo MORIWAKIThe spermatozoan head abnormalities in adult male mice are known to
be determined by Y-linked factors and a small number of autosomal genes(Krzanowska, 1969, 1972, 1976). Two possible locations of these autosomal
genes were chosen for investigation: the central portion of chromosome 4,
and the proximity of the H-2 complex on chromosome 17. Both of theseregions show homology to the sex-related sequences of the Y chromosome(Kiel-Metzger and Erickson, 1984).
The purpose of the present study was to investigate the relationshipbetween sperm abnormalities and H-2 haplotypes. Segregation of sperm
abnormality levels and H-2 haplotypes was investigated in F2 hybrid males
obtained from reciprocal crosses involving two BIO. congenic strains ofJapanese wild mice carrying H-2 and the Y chromosome: BIO.MOL-OHM(H-2 wm 4, 23.1 % of sperm abnormalities) and BlO.MOL-OKB (H_2wm8 ,ll.l % of sperm abnormalities). In both types of crosses mean levels ofabnormal spermatozoa were significantly higher for males typed as H_2 wm4 j
H_2wm4 than for heterozygous H_2wm4jH_2wm8 or homozygous H_2 wm8 j
H_2wm8 . These results suggest that the gene for high sperm abnormality
is linked to the H-2 complex of the BlO.MOL-OHM strain. For details,
5ee Genet. Res. 53: 17-19 (1989).

* Genetic Engineering Center, Korea Advanced Institute of Science and Technology,
Seoul, Korea.
** Department of Genetics and Evolution, Institute of Zoology, JagieJlonian University, Krakow, Poland.

RESEARCH ACTIVITIES IN 1988

79

DNA Sequencing Analysis of rDNA in Robertsonian Mice
Yasuyuki KURIHARA, Hitoshi SUZUKI*, Heinz WINKING**,
Franc:;ois BONHOMME*** and Kazuo MORIWAKI

Previously we reported DNA restriction fragment length polymorphisms(RFLPs) of ribosomal RNA genes (rDNA) in wild mice with Robertsoniantranslocations (Rb). As Rb mice carrying RFLP patterns specific to eachwild mouse subspecies, M. m. domesticus, M. m. brevirostris, M. m.
bactrianus and M. m. musculus, were observed, we assumed that Rb miceoriginated from intersubspecies hybrid between each of the above four
subspecies. In order to confirm this hypothesis, the following rDNA from
Rb and normal wild mice were cloned to analyse their nucleotide sequences;
6.5 Kb EcoRI fragment from M. m. domesticus (SK), 5.5 Kb EcoRI fragment from M. m. brevirostris, 5. I Kb EcoRI fragment from M. m. bactrianus,
5.5 and 5.1 Kb EcoRI fragments from Rb mice (WMP and Ta, respectively).
DNA sequence analysis demonstrated that European wild mouse subspecies,
M. m. domesticus and M. m. brevirostris, seem to be the closest to the two
Rb mice. The origin of Rb mice suspected from this finding did not coincidewith the RFLP data mentioned above. Further study is needed to specify
the origin of Rb mice.
Geographical Survey of Wild Mouse Populations in Eastern
Asia Based on Restriction Analysis andSequence Analysis of Mitochondrial DNA
H. YONEKAWA, N. MIYASHlTA and K. MORIWAKI

Analyzing mtDNA polymorphisms detected by restriction enzyme digestion, we plotted the geographical distribution pattern of the Mus musculus
subspecies in China. The Chinese populations consist mainly of two subspecies, M. m. castaneus and M. m. molossinus. M. m. castaneus is boundin Southern China, whereas M. m. musculus is found in Northern China.
The subspecies boundary lies along the Yang-tzu river (Yonekawa et al.
1989).
To confirm these results we collected 36 additional wild mice from 5 new* Jikei Medical School.
** Medizinische Hochschule Lubeck, Federal Republic of Germany.
*** Universite Montpellier, France.

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

localities in China such as Guilin, Manasi, Mohe, Menhai, Xinin and one
locality, Urmuchi, which has been examined before. We found that the
haplotype from Urmuchi mice is identical to that reported previously andalso identical to that from the Xinin mice. Furthermore, the Xinin micealso have two other different haplotypes.
Only one exception was found on the geographical distribution pattern;
the mice trapped at Menhai, which was expected to be castaneus territory,
have the mtDNA haplotype specific to M. m. domensticus.
The mice trapped at Mohe showed the following interesting features;
their morphologies showed several characteristics specific to M. m. castaneus
such as tail/body ratio and coat color, suggesting that the mice belong to
M. m. castaneus. Allozyme analysis also supported this idea with somediagnostic markers such as Hbb d which is specific to castaneus mice inEastern Asia (Miyashita et al. 1984). The restriction analysis of mtDNA
from the mice, however, showed that they had the musculus haplotype.
This suggests that cytoplasmic gene flow occurs in the mice of Northern
China and it should be noticed that this is reverse of the situation observedin Japan (Yonekawa et al., 1989).
Although restriction analysis is useful, as mentioned above, to deduce
what subspecies of Mus musculus the wild mice collected belong to, it is notsuitable for examining the method nucleotide and amino acid substitutions
during subspeciation of M. musculus. To examine this subject, we sequenced entirely, three haplotypes of mtDNA; two mtDNAs from M. m.
domesticus and one from M. m. molossinus (H. Yonekawa, E. Harmel,c.-W. Wang, K. Fischer Lindahl, unpublished). Comparing the entire
sequence among the three mtDNA and another one which has been alreadypublished (Bibb et al., 1981), we found that there were not many base substitutions in the D-loop regions within domesticus mtDNA. This, however,
was not true when we compared molossinus and domesticus mtDNA. We
found two distinct hot spots of base substitutions in the D-loop regions.
We are now proceeding with sequence analysis of the D-loop regions among
the various kinds of subspecies which we have so for collected.

RESEARCH ACTIVITIES IN 1983

VI.

81

MUTAGENESIS AND RADIATION GENETICS

Tumor Suppression Gene in Asian Wild-derived Strains of Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIWe determined susceptibility to urethane-induced pulmonary adenoma
for 8 strains derived from wild mice. The animals were sacrificed in the 5thmonth after a single subcutaneous injection of 1.5 mg urethane/g bodyweight. After the lungs were fixed in ethanol/formaldehyde (9: I), thenumber of adenoma foci was assessed. Since all of the wild-derived strains
had a mean of less than I adenoma per mouse, it is apparent these strains
are resistant to the development of pulmonary adenoma.
In order to analyze the genetic factor for tumor suppression, we crossed the
adenoma-resistant BGR strain, which was derived from wild mice (MUSmusculus castaneus) collected in Indonesia, with adenoma-sensitive A/Wy
mice. The FI progeny between these strains exhibited adenoma induction
similar to that of the BGR parent. This finding suggests that the resistant
BGR phenotype was completely dominant to the sensitive strain A/Wy
phenotype. Because A/Wy-backcross progenies showed a statisticallysignificant difference from the I: I distribution, the possibility of multiple
genes' being dominant for the suppression to adenoma formation wasapparent. A two-gene model can be proposed from the results with F2progenies in which the predicted ratio of 9: 6: I =resistant: intermediate:sensitive was found. These findings indicate that the suppression of
adenoma formation is regulated by at least two genes in the BGR strain.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

VII.

POPULATION GENETICS

Further Simulation Studies on Evolution by Gene Duplication
Tomoko OHTA

In order to understand the origin of multigene families, Monte Carlosimulations were performed to see how a genetic system evolves underunequal crossing-over, mutation, random genetic drift and natural selection, starting from a single gene copy. Both haploid and diploid models
were examined. Beneficial, neutral, and detrimental mutations were incorporated, and "positive" selection favored those chromosomes (haploid)
or individuals (diploid) with more beneficial mutations than others. The
same model for haploids was previously investigated with special reference
to the evolution of gene organization, and the ratio of numbers of beneficial
genes to pseudogenes was found to be a rough indicator of the relative
strengths of positive and negative (against deleterious alleles) natural selection.
In the present study the evolution of gene organization and sequence
divergence among genes in the multigene family was examined. It was
found that positive selection accelerates the accumulation of arrays containing different beneficial mutations, but that total divergence, including
both neutral and beneficial mutations, is not very sensitive to positive
selection, under this model. The proportion of beneficial mutations tototal mutations accumulated is a better indicator of positive selection thanis total divergence. Results indicated that various observed examples inwhich amino-acid substitutions are accelerated, when compared withsynonymous substitutions in duplicated genes, reflect the effect of selection
in a manner similar to that of the present scheme. The diploid model was
shown to be more efficient for accumulating beneficial mutations in duplicated genes than the haploid one, and the relevance of this finding toadvantages for sexual reproduction was discussed. For details, see Evolution 42: 375-386.

RESEARCH ACTIVITIES IN 1988

83

Time for Acquiring a New Gene by Duplication
Tomoko OHTA

In view of the widespread occurrence of gene families in eukaryotic
genomes suggesting the importance of gene duplication in evolution, a
population genetics model incorporating unequal crossing-over was formulated. By using this model, the time needed for acquiring a new gene
was investigated by an approximate analytical method and by computer
simulations. The model assumes that natural selection favors those chromosomes with more beneficial genes than other chromosomes in the population, as well as random genetic drift, mutation, and unequal crossingover. Starting from a single gene copy, it was found that the time foracquiring another gene with a new function is dependent on the rates ofoccurrence of unequal crossing-over and mutation. Within a realistic rangeof parameter values, the required time was at least several times 4N generations, where N is the effective population size. Interchromosomal unequalcrossing-over at meiosis was found to be more effective than intrachromosomal (between sister chromatids) unequal crossing-over for obtaining a
new gene, provided that other parameters were the same. However, thegenetic load for acquiring a gene is larger under the model of interchromosomal crossing-over. The relevance of this finding to advantages forsexual reproduction was discussed. For details, see Proc. Nat!. Acad. Sci.
USA 85: 3509-3512.
Evolution by Gene Duplication and Compensatory
Advantageous Mutations
Tomoko OHTA

Relaxation of selective constraint is thought to play an important rolefor evolution by gene duplication, in connection with compensatory advantageous mutant substitutions. Models were investigated by incorporating
gene duplication through unequal crossing-over, selection, mutation andrandom genetic drift into Monte Carlo simulations. Compensatory advantageous mutations were introduced, and simulations were carried out
with and without relaxation, where genes were redundant on chromosomes.
Relaxation was introduced by assuming that deleterious mutants have noeffect on fitness, so long as one or more genes free of such mutations remain

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

in the array. Compensatory mutations are characterized by an intermediatedeleterious step in their substitutions, and therefore relaxation by generedundancy is important. Through extensive Monte Carlo simulations, it
was found that compensatory mutant substitutions require relaxation in
addition to gene duplication, when mutant effects are large. However whenmutant effects are small, such that the product of selection coefficient andpopulation size is around unity, evolution by compensatory mutation isenhanced by gene duplication even without relaxation. For details, see
Genetics 120: 841-847.
More on the Episodic ClockNaoyuki TAKAHATA

Previously, I (Genetics 1987, 116: 169-179) quantitatively studied three
conceivable causes for elevated variance in the rate of molecular evolution:(1) multiple substitutions, (2) deleterious mutations and changing population sizes, and (3) changes in the degree of selective constraints against agene through successive substitutions. In addition, I made a cautionary
note for a fourth case, varying mutation rates among lineages, because thiscase violates the usual assumption that all lineages under study share common rules for molecular evolution. Based on statistical models for (1)-(4),I found (1) and (2) to be unsound, but (3) and (4) as likely causes. Gillespie(1988), however, raised question on the motivation for (1), the evaluationof (2), and the strong model dependence of (3). I replied to these questions
as a letter to the Editor in Genetics. For details, see Genetics 118: 387-388.
The Coalescent in Two Partially Isolated Diffusion Populations
Naoyuki TAKAHATA

The no coalescent of Kingman (1982) describes the family relationships
among a sample of no individuals drawn from a panmictic species. It is astochastic process resulting from no-l independent random events(coalescences) from each of which n (2~n~no) ancestral lineages of a sample
are descended from n-l distinct ancestors for the first time. A similar
genealogical process was studied for a species consisting of two populations
with migration between them. The main point of interest is with the probability density of the time length between two successive coalescences andRESEARCH ACTIVITIES IN 198885

the spatial distribution of n-l ancestral lineages over two populations
when the n to n-l coalescence takes place. These are formulated basedon a non-linear birth and death process with killing, and are used to derive
several explicit formulae in selectively neutral population genetics models.
To confirm and supplement the analytical results, a simulation method was
proposed based on the underlying bivariate Markov chain. This method
provides a general method for solving the present problem even when an
analytical approach appears very difficult. It becomes clear that the effectsof the present population structure are most conspicuous with a 2 to 1
coalescence, and to a lesser extent with a n to n-l (3~n) coalescence.
This implies that in a more general model of population structure, the number of populations and the way in which a sample is drawn are importantfactors in determining the no coalescent. For details, see Genet. Res.
Camb. 52: 213-222.
Dubious Maternal Inheritance of Mitochondrial DNA in
D. simulans and Evolution of D. mauritiana
Yoko SATTA, Nobue TOYOHARA, Chiaki OHTAKA, Yumi TATSUNO,
Takao K. WATANABE, Etsuko T. MATSUURA, Sadao I. CHIGUSA
and Naoyuki TAKAHATA

Within-line heterogeneity was found in the mitochondrial DNA (mtDNA)
in two isofemale lines of D. simulans. The co-existing types, Sand M,
were typical of the mtDNA in D. simulans and in D. mauritiana, respectively,
with their nucleotide divergence per site being ca. 2.1 %. Segregation
analysis confirmed that some individuals in these lines were heteroplasmic
and suggested incomplete maternal inheritance of mtDNA in Drosophila.
Examination of other lines of D. simulans revealed that the M type of D.
mauritiana occurs with frequency 71 % in Reunion, 38 % in Madagascar
and 0 % in Kenya. This finding and interspecific sequence comparisons ofboth M types indicate that D. mauritiana diverged from D. simulans probably less than 240,000 years ago. For details, see Genet. Res. Camb. 52:
1-6.

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Diffusion Model of Population Genetics Incorporating
Intergroup Selection and EpistaticInteraction in Fitness
Motoo KIMURA

In order to treat Wright's "shifting-balance theory" of evolution inquantitative terms, I propose a diffusion model (diffusion equation method)in which intergroup selection (or competition between demes) and epistatic(i.e., non-additive) interaction in fitness are incorporated.
Let us assume a hypothetical population (species) consisting of an infinite number of competing subgroups (demes) each with N e breeding
diploid individuals (or 2Ne haploid individuals) mating at random. Consider two independent gene loci and assume that a pair of alleles A and A'
are segregating in the first locus, and alleles Band B' are segregating in thesecond locus. Let x and y be respectively the relative frequencies of A'
and B' within a deme. We shall denote by ¢i(x, y; t) the distribution function of x and y at time t such that ¢i(x, y; t)LlxLly represents the fraction ofdemes whose frequencies of A' and B' lie in the ranges (x, x + Llx) and(y, y + Lly) respectively, where O<x, y< 1.
Mutation is assumed to occur at the first locus at the rate v/ per generation from A to A', and in the reverse direction at the rate v. Similarly,
at the second locus, the mutation rate from B to B' is v 2', and in the reverse
direction v 2 • With respect to natural selection within demes, we assume"genic selection" and denote the relative fitnesses (in terms of Wright's
selective values) of the four haploid genotypes AB, A'B, AB' and A'B'
respectively by W u , W 21 ' W 12 and W 22 • Migration is assumed to occur following Wright's island model so that in each generation, individual demes
contribute emigrants to the entire gene pool of the species at the rate m,
and receive immigrants from the pool at the same rate.
As to intergroup selection, we denote by c(x, y) the fitness, relative tointer-deme competition, of the deme with gene frequency pair (x, y). Thismeans that during a short time interval LIt, the change of ¢i(x, y; t) by intergroup selection isLI¢i = {c(x, y) - c}¢iLlt ,(Llt-+O) ,where c is the mean of c(x, y) over the whole population (species).
Then, we have the following diffusion equation for ¢i(x, y; t).

RESEARCH ACTIVITIES IN 1988

87~~ = L(¢) + {c (x, y) - c}¢In this equation ¢ stands for ¢(x, y; t) and
1 02
1 02
L = 2 ox2 Vax + 2 oy2 Vay -00
ox Max - oy May

is the two dimensional Kolmogorov forward operator, whereV- x(l - x)
ax IN. 'V_ y(l - y)
IN. '

ay -

,
x(l - x) ow_
Max = V l (l - x) - VlX +~ + m(x - x)

w

uX

and
May = v/(l - y) - v 2y + y(l - y) ~w + m(y - y)w
uy

in which w=w l1 (l-x)(l- y) + w21 x(1- y)+ Wl2(l-X)Y+ W22 xy.
If the competitive ability of a deme is proportional to the mean fitness ofthe deme, thenc(x, y)=kw(x, y) ,where k is a positive constant. This means that genotypes which areadvantageous for individual selection are also favorable for group selection, and vice versa.
Of particular interest, in relation to Wright's shifting-balance theory, isthe case in which single mutants are deleterious while the double mutant isadvantageous so that wl1 =l, w21 =I-sa', w12 =1-Sb' and w22 =1+sab'
where sa'>O, Sb'>O, Sab>O. Assuming that these selection coefficients aremuch smaller than unity, and neglecting higher order terms, we havex(l- x)(-sa' + ey) and y(l- y)(-Sb' + ex) respectively for the changes ofthe frequencies of A' and B' per generation due to intra-deme selection,
where e=sa' +Sb' +sab' The numerical analysis of the diffusion equation
for such a case is under way.

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

A Sexual Selection Model for the Evolution of Imitative
Learning of Song in Polygynous Birds
Kenichi AOKI

While normal song development in oscines depends on imitative learning,
it presumably was innately determined (directly inherited, non-modifiable)in the ancestral species. Evolution of imitative song from innate song wasinvestigated theoretically, based on a genetic model of sexual selection byfemale choice in a polygynous population. Since song is usually limited
to sexually mature males, and females exhibit preferences with respect tovarious aspects of song, sexual selection may well have been involved.
Specifically, it was assumed that imitative learning may give rise to a variant
song that some females prefer over innate song; and also that female preference is not affected by early song experience. However, the assumptionof polygyny restricts the applicability of the results obtained here, sincemost living bird species are monogamous. It was found that coevolutionof imitative song and female preference from low initial frequencies requires that the preference be strong. The available data, although not of
direct relevance, suggest that such strong preference may sometimes occur.
When females that show preference are more common than those that mateindiscriminately, a two-fold difference in preference may still be necessary
for imitative song to be favored. If female preference is assumed to be
fixed, the effect of various modes of song transmission can be compared.
It was shown that transmission from father to son is critical and must be
efficient, unless compensated for by strong female preference. Moreover,an initial increase in imitative song is less likely to occur if paternal songis forgotten to be replaced by a new song learned from another adult male.
These predictions may be difficult to reconcile with the assumption ofpolygyny, which may imply limited opportunity for interaction between
father and son. They would certainly seem to rule out the possibility ofthe evolution of imitative song in a lekking species. Finally, the conditions
for the evolution of imitative song were compared with those for the evolution of human speech. The latter were obtained previously on a model
assuming that speech serves to communicate adaptive cultural traits fromparent to child. The conditions are highly analogous in spite of the disparity in the selective forces assumed. For details, see Amer. Natur., inpress.

RESEARCH ACTIVITIES IN 198889

Pleiotropy and Preadaptation in the Evolution of
Human Language Capacity
Kenichi AOKI and Marcus W. FELDMAN

The capacity for spoken language in the human is a genetic trait, but the
information communicated by this means is to a large extent culturally determined. Using a gene-culture coevolutionary approach, we modeled the
hypothesis that speech evolved as a channel for the communication ofadaptive cultural traits from parent to offspring. The motivation for thiswork was a condition obtained previously, that an initial increase in communication would require at least a two-fold advantage for the transmitted
trait. In the present study, we showed that under reasonable assumptions
the invasion condition becomes less stringent. In Model 1, we assumed
that two adaptive cultural traits could be transmitted. A gene that permits
communication of one adaptive trait modifies the expression of anothergene that permits communication of the second adaptive trait. In Model2, we assumed that a related function such as greater memory capacity wasa prerequisite for speech, and that this function conferred an advantage
independent of its association with speech. In both models we assumed
haploid sexual genetics and a simple scheme of vertical transmission. The
stability properties of all corner and edge equilibria of the models wereanalyzed. The two models taken together suggest a possible scenario for
the initial stages of the evolution of speech. For details, see Theor. Populo
BioI. 35: 181-194.

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

VIII.

EVOLUTIONARY GENETICS

Algorithmic Analysis of the Computer Program for
Likelihood of a Human Pedigree
Takashi GOJOBORI and Norikazu YASUDA

The computer program "LIPED", for computing the likelihood of ahuman pedigree, was originally made by J. Otto. The program is based on
the so-called "Elston-Stewart algorithm," in which the conditional probabilities of a phenotype, given a particular genotype, are computed.
LIPED can be a powerful tool for linkage analysis, since in the analysis, thelikelihood of a human pedigree must be computed for a given value of therecombination rate. In particular, LIPED is very useful for the study ofhuman genome mapping, because it can be used for the linkage analysis
of genes responsible for genetic diseases. However, it is quite difficult tomanage this program because too many input parameters are involved.
To avoid this difficulty, we modified LIPED by incorporating a menu
system into the computer program. Since the menu system can work as afriendly man-machine interface, our modified version of LIPED is moremanageable than the original one.

Molecular Evolution of Human and Simian Immunodeficiency Viruses
Takashi GOJOBORI, Etsuko N. MORIYAMA and Shozo YOKOYAMA

To construct phylogenetic trees of human immunodeficiency viruses(HIVs) and simian immunodeficiency viruses (SIVs), we estimated numbersof nucleotide substitutions between viral isolates. From these numbers,
phylogenetic trees were constructed by unweighted pair-group (UPO) andneighbor-joining (NJ) methods. The trees obtained clearly show two majorgroups of viruses; the HIV-l and HIV-2 groups. The HIV-l group contains virus isolates from North America and Central Africa. The HIV-2
group consists of HIV-2 isolates from West Africa and all SIV isolatespresently available. The divergence times at all branching points of thetree were estimated, using rates of nucleotide substitution. In particular,
the divergence time between the HIV-l and HIV-2 groups were estimated

RESEARCH ACTIVITIES IN 1988

91

to be 150-200 years. Using the obtained trees, we also estimated thepatterns of amino acid (and nucleotide) substitutions. This may be usefulfor vaccine development using prediction of the direction of amino acidsubstitutions. For details, see IV International Conference on AIDS,Book I, p142.
Molecular Evolution of the Human Immunodeficiency
and Related Viruses
Shozo YOKOYAMA, Lyinn CHUNG, Etsuko N. MORIYAMA
and Takashi GOJODORIA phylogenetic tree for different human immunodeficiency viruses types
1 (HIV-l) and types 2 (HIV-2), lentiviruses, and oncoviruses has been
constructed by comparing the nucleotide sequences of the two regions of
their pol genes which encode reverse transcriptase and endonuclease/integrase. The results obtained indicate that (1) different HIV-l isolates form
one cluster and their common ancestor diverged from the ancestor of HIV-2,(2) the common ancestor of HIV-l and HlV-2 isolates diverged from thatof the lentiviruses, and (3) the lentivirus group and the oncoviruses diverged
from a common ancestor. In particular, the divergence time between the
HIV-l and HIV-2 groups suggests that AIDS has existed for several hundredyears but went undetected. For details, see Mol. Bioi. Evol. 5(3): 237-251
and "Gann '88" pp 55-62.
Evidence for Species-to-Species Transfer in Evolution of
Pathogenic Determinants of Escherichia coli
and Vibrio cholerae 01
Tatuo YAMAMOTO, Etsuko N. MORIYAMA and Takashi GmODORITo clarify the origin and evolution of the pathogenic determinants inbacteria, particularly enterotoxigenic Escherichia coli and Vibrio cholerae 01,
we constructed a phylogenetic tree for heat-labile toxin (LT). LT has
structural and functional features in common with cholera toxin (CT)produced from Vibrio cholerae 01 which is the causative agent of cholera.
Moreover, LT is subdivided into two types (LTh and LTp) having different
epitopes; LTh is found only in human isolates of enterotoxigenic E. coli,
whereas LTp is found only in porcine isolates. The phylogenetic tree

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

showed that the divergence between the CT gene and the two LT genes
occurred much more recently than the species divergence between V.
cholerae 01 and E. coli. The species divergence was inferred from thephylogenetic tree for 5S rRNA genes. Moreover, the codon usage of theLT gene is similar to other genes of V. cholerae rather than those of E. coli.
It suggests that the LT genes might have resulted from the species-to-species
transfer of the CT gene from V. cholerae 01 to E. coli.
For details, see Advances in Research on Cholera and Related Diarrheas
06, Pp 195-212.
Mitochondrial DNA Polymorphism among Five Asian
Populations
Shinji HARIHARA, Naruya SAITOU, Takashi GOJOBORI
and Keiichi OMOTO

Mitochondrial DNA (mtDNA) polymorph isms were examined using 13
different kinds of restriction enzymes on the total DNA obtained fromblood samples of five Asian populations: Japanese, Ainu of northern
Japan, Koreans, Negritos (Aeta) of the Philippines, and the Yedda of Sri
Lanka. As a result, 28 restriction-enzyme morphs were detected and combinations of the morphs enabled us to classify the mtDNAs of 243 individuals
into 20 mtDNA types. We constructed a phylogenetic tree for these
mtDNA types. It showed that the Japanese, Ainu, and Korean populations were closely related to each other. Although the Aeta were found toshow a relatively close relationship to these three populations, the Yedda
were quite different from the other four populations.
For details, see Am. J. Hum. Genet. 43: 134-143.
Gametic Disequilibrium for Isozyme Genes Found in Wild
and Cultivated Rices
Hiroko MORISHIMA

Isozyme data in rice cultivars (Oryza sativa) and their wild relatives (0.
rufipogon), accumulated with the collaboration of a number of my colleagues,
were analyzed in relation to multilocus association patterns. Nonrandom
association of alleles at two loci ordered within gametes, usually called"linkage disequilibrium", is termed "gamefc disequilibrium" (GD), as thisRESEARCH ACTIVITIES IN 1988

93

phenomenon is affected by many factors other than linkage. In the present
study, deviation from random association was measured by D' =D/Dmax(D=X,-P,q,), assuming two loci A and B; X" the gamete frequency of
A,B, ; P, and q" the frequencies of alleles A, and B" and D mm the maximum
D possible for a given set of gene frequencies. The following results wereobtained:
1) Among cultivated strains, a strong GD was frequently found in 12
loci assayed. The D' values did not show relationships to the degree ofchromosomal linkages between the relevant loci. This nonrandom association results in differentiation into two major enzymatic groups corresponding
to the Indica and Japonica types.
2) Sixty wild strains assayed in the same manner showed generally
smaller values of D' than cultivars, as expected from their mating systemof partial outbreeding. Yet, certain loci-combination (Cat-l vs. Pgi-2,
Cat-l vs. Est-2, Cat-l vs. Acp-l, etc.) showed nonrandom association in thesame direction as in cultivars, suggesting that Indica-Japonica differentiation at the isozymic level occurred in wild progenitors though not as conspicuous as in cultivars. Further, a locus Pox-l which is polymorphic inwild rices but monomorphic in cultivated rices, showed GD with someloci. This seemed to reflect ecotypic differentiation into perennial andannual types occurring within this species.
3) GD observed among individual plants within natural populations
was inconsistent in different populations. It was not certain whether intrapopulational GD is in a stable state or not.
4) In three experimental populations obtained from crosses between
wild rice strains, it was observed that D' values between two loosely linked
loci, Pox-l and Acp-l, tended to increase as generations proceeded.
A part of the GD observed in rice cultivars possibly originated fromrandom drift or the founder effect and has been maintained by the systemof predominant inbreeding. However, the increasing trend of GD at
certain loci over time and GD which are found consistently in different
regions characterizing ecotypes, shows that the possibility of GD being
molded by the coadaptation of interacting loci can not be ruled out.

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Tropical and Temperate Types in Asian Rice Cultivars
Lin-Hua TANG*, Yo-Ichiro SATO and Hiroko MORISHIMA

It is generally accepted from variation studies on characteristics andisozymes in rice that Asian rice cultivars tend to be differentiated into two
major varietal groups, Indica and Japonica. To examine whether or not
differentiation towards a direction other than the Indica-Japonica line
occurs in Asia, isozyme variations were extensively surveyed, using 226
cultivars considered to be a random sample of Asian rices. Data of 12
polymorphic loci were analyzed by a multivariate analysis called "Quantification III", with each allele being treated as a variate. The first axis
distinguished two varietal groups. This division as expected, coincidedwith the Indica and Japonica types classified by three diagnostic characteristics, with a few exceptions. The variation revealed by the second axis
showed a trend of differentiation between cultivars according to tropicaland temperate origins and it was found in Indicas as well as in Japonicas.
The isozyme loci which contributed much to this axis of variation wereAmp-3, Est-2 and Pgd-l. Reexamination of our old data of 11 characteri
stics (Morishima & Oka 1981, Jpn. J. Breed. 31: 402-413) for Asian rices
revealed a similar trend in differentiation between tropical and temperatevarietal groups in Indica as well as in Japonica types along a third axis of
the principal component analysis.
Previously, Oka (1958, Ind. J. Genet. & PI. Breed. 8: 79-89) argued that
Japonica types can be further divided into tropical and temperate types bya characteristic association pattern. More recently, Nakagahra (1978,
TARS 11: 77-82) reported that Chinese Indicas are genetically different
from Indicas in tropical countries. From the results obtained in the present
study, it may be concluded that differentiation into tropical and temperate
types was found in Asian rices, and that it occurred within both Indica andJaponica types, bringing about some genetic differences at the isozymic andphenotypic levels common to both types.

* Food Crop Inst., Jiangsu Acad. of Agric. Sci., China.

RESEARCH ACTIVITIES IN 1988

95

Plant Opal Analysis: its Significance to the Origin and
Evolution of RiceYo-Ichiro SATO, Hiroshi FUJIWARA* and Tetsuo UDATSU*

The origin of cultivated rice (Oryza sativa L.) has been disputed for many
years, beginning with de Candolle. Its differentiation into two ecogeographic races, indica and japonica, has also been controversial, because
bio-archaeological evidence is scanty. In this study, the silica body (accumulated Si0 2 in particular cells) removed from living rice plants, andthat excavated from soil (plant opal) were discussed as evidence for supportof such arguments. The plant opal has been used for assessment of pastvegetation, and as evidence for rice cultivation at archaeological sites,
based upon species-specific morphology. It has not been however, used
for studies of intraspecific variation. We showed indica and japonica weredistinguishable by the shape of the silica body removed from 96 cultivars,
with a few inconsistencies. Studies on plant opal from excavated samples
may enable us to indicate whether they were indica or japonica cultivars.
Accumulated data on the shape of plant opals would be helpful in assessing
the origin and evolution of indica and japonica types.
On the Maximum Likelihood Method for Estimating
Molecular Trees: Uniqueness of the
Likelihood Point
Kaoru FUKAMI** and Yoshio TATENO

Studies were carried out on the uniqueness of the stationary point on thelikelihood surface for estimating molecular phylogenetic trees, yielding
proof that there exists at most one stationary point, i.e., the maximum
point, in the parameter range for the one-parameter model of nucleotide
substitution. The proof is simple, yet applicable to any type of tree
topology with an arbitrary number of taxa. The poof ensures that any
valid approximation algorithm is able to reach the unique maximum point
under the above mentioned conditions. An algorithm developed incorporating Newton's approximation method was then compared with theconventional one by means of computer simulation. The results showed* Faculty of Agriculture, Miyazaki University.
** Ochanomizu University.

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

that the newly developed algorithm requires less computer time than theconventional one, whereas both algorithms lead to identical molecularphylogenetic trees in accordance with the proof. (J. Mol. Evol., 28: 460,1989)
Is Molecular Evolution Parsimonious? Theoretical
Approach to the Problem
Yoshio TATENO

The parsimony concept in phylogeny was reviewed first, which confirmedthat no scientific basis had been established to support or deny the concept.
Nevertheless, it is also true that the concept is quite useful in a practicalsense for constructing phylogenetic trees. Next, a method for constructing
molecular phylogenetic trees which was developed on the basis of the parsimony concept was introduced. It directly makes use of nucleotide
sequences. The parsimony concept is incorporated in the method in sucha way that it always chooses the tree which, on the whole, requires the
fewest possible nucleotide changes among the candidates examined. The
performance of the method was then examined by conducting computer
simulation that imitates an evolving gene along a model phylogenetic tree.
The results quantitatively confirmed the usefulness of the parsimony concept under the simulation model. There are, however, cases in which theparsimony criterion gives plural tree topologies including the correct one.
There seems to be no way to single out the correct topology among them,unless employing other criteria. (In: Population Biology of Genes andMolecules-Proceedings of the Fourth Symposium for the InternationalPrize for Biology-Edited by J. F. Crow and N. Takahata, in press)
Different Rates of Evolution of Autosome-, X Chromosomeand Y Chromosome-linked Genes: Hypothesis
of Male-driven Molecular Evolution
Takashi MIYATA, Kei-ichi KUMA, N. IWABE, Hidenori HAYASHIDA
and T. YASUNAGA

The relationships between different numbers of germ-cell divisions between males and females, and different mutation frequencies between
autosomes and sex chromosomes, were examined under the assumptionthat errors in DNA replication are the major factor in mutations con-

RESEARCH ACTIVITIES IN 1988

97

tributing to molecular evolution. The expected mutation frequency of the
X chromosome relative to that of autosomes (R(X/A)) was shown to be 2/3
in the XX female/XY male system, if the male to female ratio of the numberof germ-cell divisions (a) is very large. Similarly, for the relative mutation
frequency R(Y/A) of the Y chromosome, we have R(Y/A)=2 for a~I.
To test this, we compared nucleotide sequences of autosome- and X-linked
genes for human and mouse (or rat), and analysed the evolutionary rate ofsilent substitutions, a weakly constrained component that would reflectmutation frequency directly. The rate of silent substitution for X-linked
genes relative to that for autosome-linked genes (R'(X/A)), was shown tobe 0.60, being close to the expected value 2/3. Similarly, for the relative
evolutionary rate R'(Y/A) of the Y-linked gene, we observed R'(Y/A)=2.2,
which again is close to the expected value 2. The marked correlation between the observed rates R' and the expected rates R for a-I suggests thatmales serve as mutation generators. The relative mutation frequency of
the ZW female/ZZ male system was shown to the reverse of that of the
XX/YY system: For a~I, R(Z/A)=4/3~R(W/A). Surprisingly smallvalue of R(W/A) is expected. An analysis of avian genes would provides
clear-cut proof for the present model.
Immunoglobulin-like Sequences in the Extracellular Domainsof Proto-oncogene fms and Platelet-derived
Growth Factor Receptor
Hidenori HAYASHIDA, Kei-ichi KUMA and Takashi MIYATA

The McDough strain of feline sarcoma virus (SM-FeSY) carries the transforming gene, v-fms, which belongs to the family of src-related oncogenes
with tyrosine-specific protein kinase activity. The sequence analyses of the
products of the v-fms and its cellular homologue (c-fms), revealed structuralproperties of a cell-surface receptor consisting of an extracellular domain,a transmembrane region and a cytoplasmic tyrosine kinase domain. Furthermore, c-fms was shown to be identical or related to, the receptor for
macrophage colony-stimulating factor (CSF-I). The platelet-derived growth
factor (PDGF) receptor previously has been shown to be closely related insequence to the c-fms protein over the entire sequence and has been identified as a member of the src family. We found that the extracellular domains of the c-fms protein and the PDGF receptor show marked sequence

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

homologies with known immunoglobulin-related sequences. Thus c-fms
and the PDGF receptor belong to two distinct gene families; the cytoplasmic coding sequence belongs to the src family and the extracellularcoding sequence to the immunoglobulin gene superfamily. This uniquestructure of the c-fms and PDGF receptor possibly evolved through the
shuffling of primodial genes from distmct gene families. Proc. lpn. A cad.
64B: 113-118, 1988.
Male-Driven Molecular Evolution: A Model andNucleotide Sequence Analyses
Takashi MIYATA

There are several lines of evidence suggesting that errors in DNA replication are the major source of mutations contributing to molecular evolution.
If this is really the case, there is another important factor which should beconsidered explicitly in relation to the rate of molecular evolution. It is
generally thought that the number of germ cell divisions in sperm differsfrom that in eggs. Inclusion of this factor with possible sources of mutations may be important for a comprehensive understanding of mechanisms
underlying molecular evolution.
Under the assumption that errors in DNA replication are the major
factor in mutations contributing to molecular evolution, we showed thatdifferent numbers of germ-cell divisions between males and females resultin different mutation frequencies between autosomes and sex chromosomes.
The expected mutation frequencies R X / A , of the X chromosome and R y / A ,of the Y chromosome, relative to the mutation frequency of autosomes,
were respectively shown to be 2/3 and 2 in the XX female/XY male system,
if the male to female ratio of the number of germ-cell division (a) is verylarge. To test this, we compared nucleotide sequences of autosome- andX-linked genes, human and mouse, (or rat) and analyzed the evolutionary
rates of silent substitutions. The average rate of silent substitutions of
X-linked genes relative to that of autosome-linked genes (R' X/A) was shown
to be 0.58, being close to the expected value 2/3. For the relative evolutionary rate, R'/ A, of the Y-linked gene, we observed R'Y/A=2.2, which again
is close to the expected value 2. From the strong correlation between theobserved R' and the expected R for o'~ I, we proposed that males possibly
serve as a mutation generator. We also found that, in X-linked genes, theRESEARCH ACTIVITIES IN 198899

rate of pseudogene evolution is 1.54 times higher than the functional counterpart and rodents evolve with a rate 2.3 times higher than those of nonrodent mammals. These properties quantitatively agree well with thosefound in autosome-linked genes, suggesting a uniformly reduced mutation
frequency over the whole X chromosomal region. The relative mutation
frequencies of the ZW femalejZZ male system was shown to be the reverseof those of the XXjXY system: For a~l, RZ/A(=4j3»I~Rw/A. Surprisingly small value of R wIA is expected. An analysis of avian genes
would provide clear-cut proof for the present model.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

IX.

HUMAN GENETICS

Cytogenetic Studies on Chromosome 18 Polyploidy Syndromeby Non-radioactive in Situ Hybridization
Techniques and Probe Ll.84
Takashi IMAMURA, Tomoko HASEGAWA, Hitoshi NAKASHIMA
and Asao FUJIYAMA

The development of improved non-radioactive in Situ hybridization techniques with chromosome-specific probes for cytogenetic analysis, has madethis method an increasingly important molecular tool for directly detecting
specific nucleic ac_d sequences within cells or genomes. These probes, now
available for over half of the human chromosomes, hybridize to repeatedsequences on compact regions of the target chromosome near the centromere. This permits the rapid identification of chromosomes in metaphase spreads, determination of copies of selected chromosomes in interphase nuclei, and the determination of the relative positions of chromosomes in ;nterphase nuclei. We used highly sensitive methodology for the
localization of indIvidual DNA sequences using fluorescence detection of
biotinated probes hybridized in situ. Because of the unusually low background and high hybridization efficiency achieved, this methodology allows
direct, high resolution localization of single sequences not only on chromosomes, but, more importantly, within the interphase nucleus. We used
this approach to investigate the origin and structure of a tiny extra chromosome in five patients who showed syndrome of chromosome 18 polyploidy.
All five (two males and three females) examined showed psychomotor
retardations, dolichocephalies, delicate, but only slightly anomalous, facial
appearances, and small, low set ears. Limited joint movements were observed in four patients. Chromosomal studies by the G-banding method
revealed one tiny extra-metacentric chromosome in all cases. Both armsof each extra-metacentric chromosome were very similar to the short armof chromosome 18. Biotm-labeled probe L1.84 (DevI1ee, 1986), specificfor the pericentric region of chromosome 18, was hybridized in situ tometaphase chromosomes and interphase nuclei of lymphoblast cells from

RESEARCH ACTIVITIES IN 1988

101

these patients, which resulted in three bright fluorescein spots in eachmetaphase figure and nucleus. All exhibited labelling on the centromere
of both chromosomes 18 and that of the minute extra-chromosome. Thus,
the minute extra-metacentric chromosome could be i(l8p). One female
patient without joint limitations had a mosaicism of 46, XXj47, XX,+i(l8p). By the combined use of the procedures of chromosome microdissection and polymerase chain reaction, we are cloning for use as probes,
a number of unique DNA sequences from the tiny extra-metacentric chromosome. Our goal is to identify the genes that cause tri- or tetrasomy 18
syndromes.
Population Studies of Gene Conversion within the DuplicatedHuman a-Globin Gene Family
Hitoshi NAKASHIMA, Asao FUJIYAMA and Takashi IMAMURA

The identification of individuals possessing one or three adult a-globin
genes on a single chromosome instead of the normal two a-gene haplotype
provides strong genetic evidence for the occurrence of unequal crossingover in the human a-gene complex. The alignment of the restriction mapsof the a-loci residing on the one-, two-, and three-gene chromosomes reinforces this hypothesis. Additional evidence that sequence homology inthe a-gene cluster promotes unequal recombination, is the production ofdeletions, indistinguishable from those found in human populations, throughthe propagation of the cloned a-gene region in Escherichia coli.
Although the duplicated human a-globin genes encode identical polypeptides, this and other previous studies have established that the a 1 anda2 genes are not identical at the DNA level. Whereas the 5'-untranslated
regions, the three coding blocks, all of the first and the 5' four-fifths of thesecond intervening sequences (lVSl and IVS2) are highly homologous, the3'-ends have markedly diverged. This finding must be reconciled by aJ;lymechanism purported to affect the concerted evolution of the a-globin
genes. We studied frequencies of gene conversions producing single or
triplicated a-gene genotypes in the Japanese population. To precisely
identify the 5'-homology boundary of the a-genes, we cloned and sequenced
a naturally occurring product of unequal crossing-over and recombination
between the normal a-globin genes, in an attempt to define the molecularmechanism which underlies this process in the Japanese population.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

High molecular weight nuclear DNAs were prepared from white blood
cells of 645 unrelated individuals residing in West Kyushu, followed bydigestion with restriction endonucleases, BamHI and EcoR!. The firstscreening was carried out by electrophoresis of digests on an agarose gelplate and samples showing an abnormal digesting pattern were examinedfurther by restriction with BglII, HpaI and SacI. We found that 10 of
the 645 samples showed a triplicated aaa loci on a single chromosome, but
none showed the presence of a single a haplotype. Thus, the gene frequency of the triplicated a-gene cluster in a Japanese population might beassessed as 0.008 (10/1290), while that of the single gene locus is less than
0.0008. This study provides the opportunity to compare the frequencies
of a-globin gene deletion (a+ -thalassemia), as well as those of triplication,in the Japanese with those in Melanesians exposed to different malaria
endemicities. Although the mechanism involved remains obscure, epidemiological data provide some indications of the magnitude of the apparent selective advantage conferred by a-globin gene deletion with respect
to malaria. It is notable that while the trplicated aaa genotype was present at a gene frequency of 1 %, irrespective of the region, there is a strong
association between the endemicities of malaria and the frequencies of agene deletion in different parts of melanesia. Thus far, the situation in
Japan suggests that the genes responsible for the hemoglobin variants
encountered here do not confer a marked selective advantage, but are approximately neutral and owe their low frequency to a simple mutation
process and random genetic drift which has been occurring in a relatively
small number of founders in this population.
Analysis of ,8-Globin Gene Mutation in Japanese Patientswith ,8-Thalassemia Reveals a Substitution ofThymine for Cytosine in the Second Intron
Yuji NARITOMI, Yutaka CHIFU, Hitoshi NAKASHIMA
Asao FUJIYAMA and Takashi IMAMURA

We identified the substitution of a thymine for a cytosine at nucleotide
position 654 in the second intron of the .a-globin gene that causes .a-thalassemia in two Japanese families. The molecular basis for the thalassemia
syndrome in our Japanese patients can be explained by aberrant m-RNA
splicing in IVS-2. One patient was apparently homozygous for the thalas-

RESEARCH ACTIVITIES IN 1988

103

semia gene, but analysis of both of the aIIelic p-globin genes revealed thatthe patient was a compound heterozygote for different .a-globin gene mutations. Another aIIele had a normal sequence in the region of 1,994 basesof the p-globin locus so far examined. Therefore, we thought that theother mutation occurred outside the region sequenced at the site controIIing
the gene expression, which remained to be studied further. This mutation
was reported to occur rather frequently in patients of Chinese origin, but
has rarely been found in other ethnic groups.
p-Thalassemia is a heterogeneous inherited disorder of p-globin gene
expression and one of the most common single gene defects. Althoughvarious mutations have been identified in defective p-globin genes, a limited
number of specific mutations are prevalent in a given ethnic group in which
the thalassemia occurs rather frequently. In the Japanese population,
heterozygous p-thalassemia occurs with a frequency of approximately one
in 1000 individuals, while only a few patients with homozygous p-thalassemia have been observed so far. This is the first molecular identificationin Japanese patients of the p-globin gene mutation. This mutation in
IYS-2 has only been found in Chinese patients. The p-thalassemia genein this Japanese family and those in Chinese may be of the same origin as
they are both framework I genes. The spread of the mutation to thepresent day geographical pattern has been explained on the basis of population migration. However, since the family record indicated no apparent
evidence of this family being related to Chinese, the possibility of an independent occurrence of specific mutations cannot be excluded.
Regulation of ras Activity on Membranes by Post-Translational
Processing Modification
Asao FUJIYAMA, Susume TSUNASAWA and Fumio SAKIYAMA

The ras oncogene was first identified as a transforming gene of Harvey/
Kirstein sarcoma viruses. The identification and isolation of ras genes
from the DNA of human cancer tissue and the analyses of those genesequences, along with that of normal tissue, revealed that a single point
mutation changed the normal ras gene into a transforming ras gene. Thisfinding of the difference between normal and transforming ras genes is thefirst experimental evidence that ceIIular transformation is induced by asingle mutation on one gene. Utilyzing recombinant DNA technique,104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

further biochemical analyses have been carried out using p21 proteins produced in E. coli cells. Although the E. coli product exhibits biochemicalfunctions such as GTP binding and GTPase, these proteins are non-functioning as transforming proteins. We as well as others have reported that
ost-translational modification, including modification by fatty acid acylation, is essential for the transforming activity of ras proteins. Our goal is
to understand the mechanism of cellular transformation by ras through theanalyses of post-translational regulation of ras protein.
Molecular Phylogeny and Evolution of Primate
Mitochondrial DNA
Kenji HAYASAKA, Takashi GOJOBORI and Satoshi HORAI

We determined nucleotide sequences of homologous 0.9 kb fragmentsof mitochondrial DNAs (mtDNAs) derived from four species of old-worldmonkeys (Japanese, rhesus, crab-eating, and Barbary macaques), one
species of new-world monkey (common squirrel monkey), and two speciesof prosimians (Philippine tarsier and ring-tailed lemur). Using these
nucleotide sequences and homologous sequences for five species ofhominoids (human, chimpanzee, gorilla, orangutan, and gibbon), a phylogenetic tree for the four groups of primates was constructed. The phylogeny obtained is generally consistent with evolutionary trees constructedin previous studies, except for the position of the tarsier. Although most
molecular studies have suggested that the tarsier is more closely related toanthropoids than to prosimians, our analysis of mtDNA showed that thetarsier is more closely related to the lemur than to anthropoids.
To estimate the rate of nucleotide substitution, we examined the relationship between between-species numbers of nucleotide substitutions anddivergence times. The estimated rate of nucleotide substitution for
mtDNAs in hominines (human, chimpanzee, and gorilla) is (0.53-1.05) X
10-s/site/year/lineage and that in old-world monkeys is (1.12-2.24) X
lO-s/site/year/lineage. A relative rate test using the squirrel monkey as areference species also revealed that more nucleotide substitutions haveoccurred in the lineage of old-world monkeys than in hominoids. These
results may suggest that the rate of nucleotide substitution for mtDNA has
slowed down in hominines compared with that for old-world monkeys.
This evolutionary feature of mitochondrial genes is similar to one found in

RESEARCH ACTIVITIES IN 1988

105

nuclear genes.
For details, see Mol. Bioi. Evol. 5: 626-644, 1988.
Maternal Inheritance of Deleted Mitochondrial DNA in a
Family with Mitochondrial Myopathy
Takayuki OZAWA*, Makoto YONEDA*, Masashi TANAKA*,
Ikuya NONAKA** and Satoshi HORAI

Skeletal muscles from a mother and her daughter, both with chronic
progressive ophthalmoplegia, were analyzed. Histological and biochemical
analyses of their muscle samples showed typical features of this type of
mitochondrial myopathy. Southern blot anlysis revealed that, in bothpatients, there were two species of mitochondrial DNA (mtDNA): a
normal one and a partially deleted one. The sizes of the deletion weredifferent; the mutant mtDNAs from the mother and the daughter had
about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs
shared a common deleted region of 1.2-kilobases. However, both the startand the end of deletion were different between them, implying a novel modeof inheritance. This is the first report that mutant mtDNA is responsible
for the maternal inheritance of a human disease. For details, see Biochem.
Biophy. Res. Commun. 154(3): 1240-1247.
A Mutation-Epidemiologic Study on Sporadic Retinoblastoma
Ei MATSUNAGA and Kensei MINoDA***

Whereas our knowledge about the molecular basis of genetics and genesis of retinoblastoma has increased tremendously during the past years,
few epidemiologic studies have been conducted to investigate possible
environmental factors associated with germinal or somatic mutations
leading to heritable or nonheritable retinoblastoma. Obviously, the rarityof this disease makes it difficult to collect a sufficiently large number ofsporadic bilateral or unilateral cases to provide an epidemiologically soundconclusion. Of particular interest are the possibilities of a viral etiology
*
**
***

Department of Biomedical Chemistry, Faculty of Medicine, University of
Nagoya, Nagoya.
National Institute of Neuroscience, Tokyo.
Teikyo University Ichihara Hospital.

106 ANNUAL REPORT OF NATIONAL INSTI TUTE OF GENETICS NO. 39

for the somatic mutations and a paternal age effect upon the germinal
mutations. A viral etiology has been suggested from time to time but has
never been tested by epidemiologic studies or by an examination of patientsfor antiviral antibodies. A paternal age effect may suggest an accumulated
and overall effect of various kinds of environmental mutagens.
Based on medical records of more than one thousand patients withretinoblastoma as ascertained by a nationwide registry, we were able tocollect data concerning dates of births for 753 sporadic unilateral cases;
about 90 % of them can be assumed to be due to somatic mutations which
occurred some time during the fetal or early postnatal periods. These
children were born in the period from 1965 to 1982, and the control wasconstructed on the basis of the vital statistics data for all Japan, adjusted
by the year of births of the patients. The frequency of these cases fluctuates to some extent by months of births, but there was no statisticallysignificant deviations from the control. Thus, the occurrence of nonheritable retinoblastoma is not likely to be associated with certain viruses
such as human adenovirus 12 whose activity varies markedly with season.
We also analyzed the paternal ages for 225 patients with sporadic bilateralretinoblastoma, which can be assumed to have arisen from germinal mutations. These patients were born in the periods from 1965 to 1968 andfrom 1975 to 1982, for which the vital statistics data provided paternal age
distributions for all legitimate live-births in Japan. The mean paternal ageof the patients was 30.2 years, which was close to the 30.1 for the control
population as adjusted by the year of births of the patients. Thus, there
was no evidence for a paternal age effect. This finding may be due, in part,to a diminishing variance in parental ages during the last 30 years in thiscountry. However, it does suggest that paternal exposure to ionizing radiation or chemical mutagens does not play a major role in the production ofgerminal mutations for retinoblastoma.
In conclusion, our epidemiologic survey based on a large body of data
failed to catch any clue for environmental risk factors in sporadic retinoblastoma. The negative results, together with fairly uniform pattern in theincidence of this tumor among different populations (Parkin et al. 1988, Int.
J. Cancer 42: 511), suggest that most, if not all, cases of sporadic retinoblastoma are caused by some intrinsic biological mechanisms common to allindividuals, and not by mutagens found in our daily environment that arevariable from place to place and from time to time.

RESEARCH ACTIVITIES IN 1988

107

Ethnic Differences in the Incidence of Childhood Malignancies:
Comparison of Japanese and U. S. Data
Ei MATSUNAGA and Hirokazu NISHIHIRA*

It is generally accepted that the first step in the carcinogenic process isa genic or chromosomal mutation in a somatic cell. In fact, in a numberof malignancies, specific cellular changes have recently been identified at
the chromosomal or DNA level. In order to search for genetic and/or
environmental factors that are responsible for the induction of these mutations, it is imperative to know the incidence of individual malignancies in
the general population. This, however, is not always easy. In Kanagawa
Prefecture, with a population of nearly 7 million, a new approach to childhood cancer registry was initiated in 1972 by making use of an official
record. This was made possible because since 1971, childhood malignancies
have been among a number of "designated diseases" for which the cost of
medical treatment is defrayed, upon application, by central and local governments. By reviewing the application forms submitted by patients tothe prefectural government, it was possible to achieve nearly complete
ascertainment of new cases of childhood malignancies within the prefecture.
Table 1. Incidence rates (per million per year) of malignant neoplasms inchildren under 15 years of age. U.S. data are from Young and Miller(1975).
U.S. (1969-'71)

Kanagawa(1975-'82)

White

Black

Leukemia
LymphomaBrain tumors
Retinoblastoma
Wilms' tumors
Neuroblastoma
Liver tumors
Bone tumors
Testis tumors
Soft tissue sarcomas
Others

39.7
7.2
24.4
4.2
3.5
8.3
2.1
4.7
2.1
3.3
8.0

42.1
13.2
23.9
3.4
7.6
9.6
1.9
5.6
1.0
8.4
7.8

24.3
13.9
23.9
3.0
7.8
7.0
0.4
4.8
0.4
3.9
8.4

Total

107.5

124.5

97.8

Diagnosis* Kanagawa Children's Medical Center

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Analysis was made of data (published by Nishihira in Japanese in a
local journal) concerning new cases of malignancies in children under 15
years of age for which the diagnosis was first made within the respective
years from 1975-'82. The number of all malignancies that occurred ineach year ranged from 168 to 196, with the ratio of male to female patients
being 1.30. As shown in Table 1, the most frequent malignancy was acuteleukemia, followed by brain tumors, neuroblastoma, lymphoma, bonetumors, retinoblastoma, Wilms' tumors, soft tissue sarcomas, liver tumors
and testis tumors. Compared with U.S. data for white and black children(Young, J. L. and R. W. Miller, 1975, J. Pediat. 86: 254), no ethnic difference can be seen in the incidence rates for brain tumors, retinoblastoma,
neuroblastoma, and bone tumors. Leukemia is as common among Japanese as in U.S. white children but in blacks it is less common. The rates
for lymphoma and Wilms' tumors in Japan are about one-half that in U.S.
white and black children. The rate for liver tumors in Japan is close tothat in U.S. white children, but in U.S. black children it is much lower.
The rate for testis tumors is the highest in Japan and it is lowest in U.S.
black children. Soft tissue sarcomas seem as common in Japan as in U.S.
black children but it is much more common in U.S. white children. The
overall incidence rate was 107.5 in Japanese children, which is intermediate
between U.S. white (124.5) and black children (97.8). These differences
Table 2. Incidence rates (per million per year) of lymphoma and soft tissue
sarcoma in children under 15 years of age. U.S. data are from Youngand Miller (1975).

Lymphoma
Lymphosarcoma
Reticulosarcoma
Hodgkin's disease
Malignant 1ymphomas*
Soft tissue sarcomas
Rhabdomyosarcoma
Fibrosarcoma
Hemangiosarcoma
Others c. unspecified* Not otherwise specified.

U.S. (1969-71)

Japan(1975-'82)

White

Black

7.2
2.6
1.2
0.8
2.7
3.3
2.0
0.2
0.2
0.9

13.2
4.7
0.8
5.8
1.1
8.4
4.5
0.9
0.1
2.9

13.9
5.2
1.3
6.1
0.4
3.9
1.3
0.4
2.2

RESEARCH ACTIVITIES IN 1988

109

Table 3. Incidence rates (per million per year) of retinoblastoma and Wilms'
tumor by laterality in children under 15 years of age.

Retinoblastoma
Bilateral
UnilateralWilms' tumor
Bilateral
UnilateralJapan(1975-'82)

U.S. white(1969-'71)

4.2
1.4
2.8

3.4
1.2
2.2
7.6
0.4
7.2

3.5
0.1
3.4

appear to be largely genetic.
With regard to lymphoma and soft tissue sarcomas, it is interesting toinvestigate which subcategories differ in incidence among the three ethnic
groups. Because our original data did not provide information about thehistological classification of tumors, we estimated the incidence of respective subcategories according to the relative frequencies in the All Japan
Children's Cancer Registration (Vol. II, edited by N. Kobayashi, 1982).
As shown in Table 2, Hodgkin's disease is rare in Japan; its incidence isabout 1/7 that in U.S. white or black children, and lymphosarcoma also
appears less common in Japan. Regarding soft tissue sarcomas, both
rhabdomyosarcoma and fibrosarcoma appear less common in Japanese thanin U.S. white children.
It is well known that for sporadic retinoblastoma or Wilms' tumors,bilateral cases almost always arise from de novo germinal mutation, whilemost unilateral cases are due to somatic mutations. For sporadic cases ofretinoblastoma, about 1/3 are bilaterally affected and there are no ethnic
differences in this respect. For Wilms' tumor, the proportion of bilateralcases is about 3 % in Japan (Hanawa, Y., 1988, Gann Monog. 36: 71) andabout 5 % in the U.S. (Breslow, N. E. and Beckwith, J. B., 1982, JNC! 68:429). We partitioned the overall incidence rates of both tumors into rates
for bilateral and unilateral cases respectively and compared them between
Japanese and U.S. white children (Table 3). With respect to retinoblastoma
locus, there are no ethnic differences in the germinal or somatic mutation
rate, but for Wilms' tumor locus both germinal and somatic mutations
appear to occur more frequently in U.S. white children than in Japanesechildren. This seems to be the first case in human genetics suggesting an
ethnic difference in mutation rates.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

X.

APPLIED GENETICS

Protein Bands in Morphological Mutants of JapaneseMorning Glory Detected by Electrophoresis
in Immobilized pH Gradients
Toru ENDO

A number of horticulturally valuable morphological mutants in Japanesemorning glory have been recognized since 16th century. Several loci ofthese mutants were used in the present study, acuminate (ae), cordate (co),Hederacea (eo H ), dwarf (dw) , maple (m), polymorph (py) , side-reduced(sr), weeping (we) and a standard type, Tokyo-Kokei (TK), with the firstsix mutants being detectable at the cotyledonary stage. They were successively backcrossed with TK for at least three generations, and semi-isolinesof them were obtained. Dry seeds of each line were crushed to 100 meshpowder, 200 mg of which was washed with 100 ml of a mixture of ethoxyethanol/2-mercaptoethanol, 4: 1 (v/v), to remove polysaccharides. This
pretreatment was essential to apply the seed protein to electrophoresis withimmobilized pH gradients (lPG). After pretreatment, the seed protein

RESEARCH ACTIVITIES IN 1988111

was extracted with a 100 ml buffer of 10 mM ammonium carbonate containing 0.1 M 2-mercaptoethanol (pH 8.6), freezed dried, dissolved in 1 mlof 8 M urea and then 20 ,ul of the suppernatants was analysed. The regionsof IPG used were a wide type, pH 4-10, as well as narrow types, pH 4-5,
pH 5-6 and pH 6-7. The former showed about fifty bands and each of thelatter three about thirty, with most of them extremely fine. Bands wereinclined to become wider or more obscure in regions with pH 7-8 or pH 8-9.
In the photograph shown (pH 6-7 region), the dominant mutant strain of
COR showed a unique band near pH 6.7, which was not located in the other
other strains.
Distribution of Wx Alleles in Rice
Yoshio SANO

The waxy (wx) locus specifies a starch-bound glucosyl transferase that is
responsible for the production of amylose in the endosperm. Amylose
content in the endosperm affects grain quality and it varies among nonglutinous rice cultivars. As reported already, multiple alleles at the wx
locus occur in rice. wx a and Wx b as well as wx were found in Asian cultivars (Oryza sativa). Wx a and Wx b control the quantitative level of bothamylose and the gene product (called Wx protein) in the endosperm, indicating that the allelic difference is a major factor regulating amylose
content among nonglutinous cultivars. In addition, some cultivars showed
an intermediate amount of both amylose and Wx protein between wx aand Wx b • Backcrossing experiments using these cultivars revealed that anadditional Wx allele contributes to an amount of amylose and Wx protein
intermediate to that observed in the original strains. Thus, the wx locus
gives an opportunitiy to look into the evolutionary significance of gene
regulation and dynamic changes of allelic frequency in response to farmers'
selection of cultivated rice.
Modern rice cultivars in Japan mostly carry Wx b while all wild relatives
with the AA genome carry wx a only. Indica type rice predominantly
carries Wx a , showing a higher amylose content as do its wild relatives.
The intermediate allele between Wx a and Wx b seems to be frequent inJavanica type rice as recognized by the quantitative level of the Wx protein.
Such an intermediate allele was not found in five wild species (66 strains
surveyed in total). All allelic states except for Wx a seem to be specific to112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Asian cultivars. The distribution of Wx alleles was surveyed in traditionalold cultivars from southern parts of Japan (16 from Kyushu and 20 from
Okinawa) in addition to 8 weedy strains from Korea and Japan. Results
indicated that Wx" was not detected in those from Kyushu but was frequently detected in those from Okinawa and the weedy strains. In contrast, traditional cultivars from Kyushu frequently carried an intermediate
allele as well as Wxb, suggesting that the fixation of Wx b took place recently
in modern Japanese cultivars after the elimination of Wx... Wx" itself
appeared to have no adverse effect, since weedy strains often carried wx...
Thus alleic changes might have been brought about through artificial selection by farmers.
Molecular Cloning of Ribosomal RNA Genes from Two
Cultivated Rice Species, Oryza sativa
and O. glaherrima
Yoshio SANO

Ribosomal RNA (rRNA) genes are organized as families of tandemly
repeated genes, which may comprise the nucleolar organizer (Nor) regions.
The intergenic spacer region between rRNA coding sequences shows extensive sequence divergence suggesting that the intergenic spacer region
might have rapidly evolved. In rice, differential expression of rRNA
genes have been suggested since the number of Nor regions varies among
different species or taxa. Therefore, the intergenic spacer region might beuseful for reexamining complex relationships among rice species. In order
to compare the molecular organizations of rRNA genes, the repeat unit
was cloned from two cultivated rice species; Oryza sativa and O. glaberrima.
Two strains, Taichung 65 (Japonica type of O. sativa) and W025 (0.
glaberrima) were used for cloning of rDNA repeats. Taichung 65 carriedhomogeneous rDNA repeats of 8.3 kb, and W025 homogeneous repeats of
7.95 kb. BamHI cleavage yields two fragments of DNA from each rDNA
repeat unit as detected by southernblotting hybridization. Although a
BamHI fragment of 3.8 kb was constant in size, the other fragment which
included the intergenic spacer region was highly variable among species.
Taichung 65 had a 4.5 kb BamHI fragment of DNA while W025 had a
4.15 kb fragment in addition to an invariable fragment of 3.8 kb.
DNA was isolated from lO-day etiolated seedlings by phenol-chloroform

RESEARCH ACTIVITIES IN 1988

113

extraction and was banded in a CsCl-ethidium bromide gradient. Purified
DNA was partially digested with Sau3A, then 15-20 kb fragments of DNA
were separated in a sucrose gradient and ligated to AEMBL4 DNA cleavedwith EcoRI and BamHI. After in vitro packaging, recombinant phages
were screened by plaque hybridization of 32p labelled RNAs (a mixture ofl7S RNAs and 25S RNAs from rice seedlings). Then, rDNA repeats were
subcloned from the AEMBL4 genomic clones into the BamHI site ofpUC13.
Finally, four plasmids, pRY12, pRYI8, pRY42 and pRY44 were obtained,
pRY12 and pRY18 carry a 4.5 kb insert from Taichung 65 and a 4.15 kb
insert from W025, respectively, which contain the intergenic spacer region.
pRY18 and pRY44 carry invariable fragments of3.8 kb from the two species.
The intergenic spacer region in pRY12 and pRY42 was extensively compared by means of a series of single and double digests using KpnI, XbaI,
SmaI and SaIl since the coding region seemed to be highly conservative.
Further, TaqI, SmaI, SaIl and HinfI sites were mapped by partial digests ofend-labeled DNA. Partial digests of end-labeled DNA revealed that HinfIand SaIl sites greatly differed in the middle of the intergenic spacer region
between the two species. Thus, comparisons of the intergenic spacer region
might be useful for reevaluation of species' relation ships at the molecularlevel.
Ribosomal DNA Spacer-Length Polymorphisms in a Wild
Rice Population from Dongxiang, China
Yoshio SANO, Haoxiong X. YIl), Qiqan Q. SHAO O) and Shinya IYAMA

The northernmost population of wild rice (Oryza rufipogon) occurs in
Dongxiang (28.1 ON), Jiangxi, China. The plants are perennial and give
fertile progenies when crossed with cultivars. Interestingly, they survive
temperatures below O°C in winter and regrow the following season. In
order to compare their genetic constitution with those from tropical andsubtropical areas, we examined rDNA spacer-length (sl) variations in the
Dongxiang population.
Southernblotting analysis revealed that the intergenic spacer is highlyvariable in size and is differentiated among morphological and geographicalgroups of wild and cultivated rices, indicating its rapid change in evolution.
1)

0)

Jiangxi Univ., Nanchang, Jiangxi, China.
Inst. Genet., Beijing, China.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Among mouy sl variants detected so far, two (4.5 kb and 5.0 kb BamHI
fragments) were found to predominate in Japonica but not in Indica rice.
The two variants were seldom found in wild rice strains from tropical andsubtropical areas, but they frequently occurred in the Dongxiang population. No difference was observed in the subrepeat structure within theintergenic spacer between Dongxiang wild rice and Japonica, suggestingthat they carry rDNA of the same origin.
Selection Strategies Based on Two Different Measurementsof Selection Efficiency, Expected Genetic
Advance and Chance of Success
Katsuei YONEZAWA

Selection methodology in plant breeding has been discussed in terms ofthe genetic advance expected to be attained by selection and a selection
method (selection index, field design, etc.) expected to give the largest genetic
advance has been regarded as the most efficient. The size of the genetic
advance, however, is not always the most appropriate criterion for measuring the efficiency of selection. However, a selection method which does
not produce the greatest genetic advance may provide the largest chance of
success of obtaining genotypes with sufficient desirability as a new cultivar.
The chance of success rather than the expected genetic gain may fit thebreeder's concern in many situations, especially in breeding of self-pollinating crops. The optimal selection strategy varies according to whether
efficiency of selection is measured by expected genetic advance or chanceof obtaining desirable genotypes.
As an example, a very simple modelled population as presented in Table
1 was assumed to be a target population in which selection is carried out.
Now suppose that N plants of this population are grown and assessed forTable 1. Genetic constitution of a target population for selection.
Genotype
Desirable

Undesirable

Expected frequency
Genotypic value/

1-/

Population mean

1'1+(1-/)'0=/IoRESEARCH ACTIVITIES IN 1988115

selection, with plants showing phenotypic value (assessment scores) rated
as desirable as or more desirable than a critical value, being selected. Whenthe amount of resources such as time and labour is limited, as usually is thecase in practical breeding projects, the number of plants triable, N, should
be small if an assessment with high precision is applied. Introducing thefollowing variables,
Pd=the probability that a desirable genotype is selected, i.e., shows aphenotypic value (assessment score) surpassing the critical value forselection,
pu=the probability that an undesirable genotype is selected due to mistaken assessment,
and assuming that genetic segregation does not occur in the next generation(as assumable for a genetically fixed population of a self-pollinating crop),
the expected genetic advance, which for simplicity is expressed here by theaverage genetic value of the selected plants, is formulated as,

The chance of success, i.e., the probability that at least one desirable genotype is obtained by the selection, is presented as,

Pr= 1- 2: (~) (i(l_f)S-i(l-Pd)i
,

c

]
~

'0
~

c
.ill
o

~

Pr
Low

•

Large •.. High ••. Precision of assessment(Assessment cost per plant).. Small.. ·Population size N

Fig. 1. Relationship between efficiency of selection and precision of assessment witha fixed amount of resources expendable for selection.

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

The probabilities Pd and P" were formulated, though not presented here, interms of the precision of assessment of plants. The probability Pd becomeslarger, and in contrast, P" becomes smaller, as the precision of the plant
assessment gets higher. Assessments with a higher precision, require greater
expense of resources.
For many probable situations, relationships, as shown in Fig. 1, between
the precision of assessment and the efficiency of selection as measured by
M and Pr were determined. M incerases, while in contrast, Pr decreases
with the increasing precision of the assessment. Thus, when measured byM, an assessment with high precision, i.e., high-input assessment, produces
a better result. When measured by Pr, an assessment with a rather simple
method, i.e., low-input assessment, is recommended.

RESEARCH ACTIVITIES IN 1988

XI.

117

DATABASE

Activities of the DNA Data Bank of Japan
Sanzo MIYAZAWA and Hidenori HAYASHIDA

The primary task of the DDBJ is, of course, DNA sequence collection.
However, in addition to that, we have a wide range of activities; 1) DNA
data collection and data entry in collaboration with other databanks, 2)data distribution, including the secondary distribution of the GenBank
and EMBL databases in Japan, 3) to provide on-line access to DNA andrelated databases, 4) to develop research tools for sequence analysis, 5) toregularly publish newsletters to inform people of the activities of the DDBJ,
and 6) to provide training courses for users of the DDBJ computer system.
We have been developing a data entry system to manage data collection, asearch/retrieval system for sequence databases, and research tools for
DNA and protein information analysis. To let people know of such activities of the DDBJ, we published newsletter No.7, and had a training
course in 1988. Newsletters contained articles that described the state ofinternational collaboration among databanks and how to submit data to
databanks as well as matters of interest specific for Japanese scientists
such as available databases at the DDBJ, how to access the DDBJ computer system and how to use the databases. The activities of the DDBJ
are also conveyed through the on-line service of the DDBJ computer system.
All these activities provided by the DDBJ are open to anybody irrespectiveof whether one works for a non-profit organization or not. In the following, I will briefly report this year's activities of the DDBJ.
1) Data Entry and Management
Sanzo MIYAZAWA and Hidenori HAYASHIDA
Our data collection began in December, 1986 and is carried out in collaboration with the GenBank and the EMBL Data Library. The collaboration includes projects of designing a new feature table and rebuilding a
DNA database. Data is currently entered in the GenBank format and fully
annotated. Since we released the first version of the DDBJ database in
July, 1987, our database has been released every half year; version 3, which
included 345,850 bases in 230 entries, was released in July, 1988, and version

118

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

4 including 535,985 bases in 302 entries was released in January, 1989.
The DDBJ collected about 240,000 bases in the year from July 1987 toJuly 1988. About 8,000,000 bases were collected during the same periodby the EMBL Data Library and the GenBank. In other words, the DDBJ
processed about 1/30 of the total collection of DNA sequences in a year;
this number may be reasonable, if the number of staff of the DDBJ is compared with those of GenBank and the EMBL Data Library. Each release
included a coding sequence database and a peptide sequence database thatwere extracted and translated from the original DNA sequence database.
Release 2 and later releases included the files of journal index, accession
number index, short directory, and data submission form.
2) FLAT Database and Sequence Analysis System for DNA and Proteins:
Release 1.0 p.
Sanzo MIYAZAWA
We have been developing a search and retrieval system for flat file databases in order to provide simple tools for using DNA and protein sequence
databases. This system called FLAT consists of primitives, most of which
perform a single operation and work as filters in the UNIX system; a filter
program reads a line from standard input, processes it and then writes some
output into standard output. Some basic commands available in FLAT
perform single operations such as I) extracting specified types of records
from database files, 2) searching for strings in each entry of database and,if found, outputing those entry names, 3) performing "and", "or", and"xor" in respect of entry names, and 4) extracting specified entries fromdatabase files. These filters may be combined with the UNIX pipe toperform complicated jobs; one may search and retrieve entries from databases by key words such as author name, journal name, title, organism
name, source name, and any combination of such items. This is a typicalapproach for designing programs in the UNIX system. Strings for these
programs are specified in the regular expression, so that one can search andretrieve entries in databases by fuzzy key words and entry names. The"seqgrep" program also allows users to use the regular expression to specify
sequence patterns to be searched for in databases. Some of these filters
were programmed in the Bourne shell and use UNIX tools such as sed,
egrep, sort, and awk, so that they are flexible enough to support many
formats of databases and to easily keep up with format changes which
often occur. At present, the Genbank, EMBL, PIR (Protein Identification
Resource) and PRF (Protein Research Foundation) data formats are sup-RESEARCH ACTIVITIES IN 1988

119

ported. However, this approach tends to trade computational speed forflexibility, and so applications whose processing speed is critical are written
in the C language. A program "getgb", which extracts specified entries
from databases, uses a pseud index file to quickly find the location of entries
in a flat database file. This FLAT search/retrieval system for sequence
databases is designed to be portable among UNIX systems which areavailable for a wide range of computers from super to micro computers.
Its f3 version of release 1.0 was released in 1988.
3) The Qanalys Sequence Analysis System for Molecular Evolution:
Version 1.0 f3
Hidenori HAYASHIDA
Programs for sequence analysis were developed to study molecular
evolution, including programs for the calculations of base or amino acidcomposition and similarity between sequences, a sequence alignment program, and a display program for postprocessing. The f3 version of release
1.0 was released in 1988.
4) A Guide to the DDBJ Computer System: the "getinfo" Command
Sanzo MIYAZA W A
An online help program called "getinfo" was made to provide databankstaff and users an easy way to get necessary information. One may use
the "getinfo" to learn how to submit DNA data and to which databank thedata should be submitted, and even to get a data submission form. The"getinfo" apparently mimics the help utility of the VAX/VMS system.
However, unlike the VMS help utility, each help information is stored asa flat file and organized into a tree-like structure, if necessary, by using
symbolic links or pseud symbolic links. The pseud symbolic link was devised
because the symbolic link is not available in the System V UNIX. The"getinfo" displays a specific topic and, if available, a list of help items at
the next level and prompts users to choose one of them. A pager program,pg or less, which is available in the UNIX system is used to print files ona terminal, so that one may read a file page by page, and may save it ina file, if necessary. A hard-copy version of the guide to the DDBJ computer system was published in 1988.
For details, see "DNA Data Bank of Japan: present status and futureplans" by Sanzo Miyazawa in "The interface between Computational Scienceand Nucleic Acid Sequencing, Santa Fe Institute Studies in the Sciences ofComplexity, Eds. G. Bell and T. Marr (Reading, MA: Addison-Wesley),vol. VIII, 1989".

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

Estimation of the Average Energy Increments of Protein Native Structuresdue to Amino Acid Exchanges and Its Use in Evaluating a
Substitution Probability Matrix for Homology Search
S. MIYAZAWA and R. L. JERNIGAN·

The energy change due to an amino acid exchange in protein structures wasestimated on average, and used to evaluate a substitution probability matirix,
which was then used to measure the similarity between protein sequences.
In a statistical sense, each type of amino acid residue is found at a particular location in the three dimensional structure of proteins; non-polar
residues are more often found in the non-polar environment of the proteincore and polar residues on the protein surface. Residues surrounding anamino acid in protein structures are specific to the type of amino acid.
We consider a typical or average protein which satisfies statistical features
observed in a large set of protein structures, and the average energy increment due to an amino acid exchange in such an average protein. In aprevious study (Miyazawa & Jernigan, 1985), we estimated the effective interresidue contact energy of each type of amino acid pair for proteins in solution
from 18,192 residue-residue contacts observed in 42 globular proteins, andalso compiled the types of residues and their average numbers in contact witheach type of amino acid. By using their contact energies and statistical
data of surrounding residues, we evaluated the average energy increment
due to an amino acid exchange in the average protein for each pair of
the 20 kinds of amino acid residues. These estimates show reasonablecharacteristics of physico-chemical similarities of amino acids. The averageenergy increment due to an amino acid exchange would be a good measureof structural instability caused by an amino acid exchange. We assume
that the fitness of an amino acid exchange in the evolutionaly process is
proportional to the Boltzmann factor of the average energy increment due
to the amino acid exchange. The substitution process of codons consists of
two steps, mutation and selection at the DNA level, and selection at theprotein level. The process of codon substitution is assumed to be in equilibrium and for simplicity its rate at the DNA level is assumed to be proportional to the equilibrium frequency of the codon. Thus a transition
matrix of amino acid substitution for a long time interval which corresponds
to 250 PAM (accepted point mutations per 100 residues) is generated from* National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

RESEARCH ACTIVITIES IN 1988

121

that of codon substitution for. a short time interval, and the log of eachelement of the 250 PAM matrix divided by the amino acid frequency is
calculated as a scoring matrix in the same way as Dayhoff et al. (1978).
The correlation coefficient between this scoring matrix and Dayhoff's scoring
matrix (MDM 7s), which corresponds to the 250 PAM substitution probability
matrix calculated from amino acid substitutions observed in closely related
proteins, is about 0.55 for all off-diagonals but 0.82 when infrequent aminoacids of met, trp, cys and tyr are excluded. The poor correlation for all
off-diagonals may result from statistical errors due to small numbers of substitutions. This scoring matrix has demonstrated the same degree of detection power of sequence homology as the Dayhoff's scoring matrix. These
results indicate that the average energy increments due to an amino acidexchange as estimated here reflect the structural instability caused by theamino acid exchange. This manuscript is in preparation.
Publications from Genetic Resources Section, Genetic
Stocks Reseach Center
Shin-ya IYAMA

The following publications were released from the Genetic Resources
Section this year.
1. "Silkworm Strains in Japan". (Compiled by S. Iyama, H. Doira andA. Murakami) 181 pages (in Japanese). This was completed in cooperation with the Silkworm Genetic Resources Subcommittee, the JapaneseSociety of Sericulture. The catalogue listed 943 entries from 18 locationsin Japan, with special emphasis on experimental strains. Each stock was
described with its strain name, location kept, genotype, historical record of
establishment and acquisition, genetic characteristics, and information on
maintainance and distribution. A list of gene symbols with the description of their characteristics was added for the convenience of research
workers in using materials.
2. "Rice Genetics Newsletter Vol. 5, 1988". 162 pages. (in English).
This volume contained the following:(I) Report of the Committee on Gene Symbolization, Nomenclatureand Linkage Groups, describing 8 newly registered genes, 13 newly adopted
gene symbols and 16 marker genes newly confirmed for their linkage relations.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39(2) Two reports from coordinators describing 1) current status ofanalysis and symbols for male sterile cytoplasms and fertility-restoring
genes and 2) gene symbols for leaf and culm traits.
(3) List of 73 recently published papers on rice genetics.
(4) 60 research notes of various fields of rice genetics, the first five of
which concerned chromosome studies related to the chromosome numbering
for linkage groups.

PUBLICATIONS

123

PUBLICATlONSABDULAEV, N. G., ARTAMONOV, 1. D., ZOLOTAREV, A. S., BESPALOV, 1. A., DERGACHEV,
A. E., IWABE, N., MIYATA, T. and TSUDA, M.: Octopus rhodopsin. Amino acidsequence and membrane topology. In "Molecular Physiology of Retinal Proteins(Proc. Yamada Conf. 21)" (T. Hara, ed.), pp. 1-7, Yamada Sci. Found., Osaka, 1988.
AOKI, K. and FELDMAN, M. W.: Pleiotropy and preadaptation in the evolution of humanlanguage capacity. Theor. Pop. BioI. (in press).
AOKI, K.: A sexual selection model for the evolution of imitative learning of song inpolygynous birds. Amer. Natur. (in press)
AOTA, S., GOJOBORI, T., ISHIBASHI, F., MARUYAMA, T. and IKEMURA, T.: Codon usage
tabulated from the GenBank genetic sequence data. Nucl. Acids Res. 16 Suppl.:r315-r402, 1988.
AYUSAWA, D., ARAl, H., WATAYA, Y. and SENO, T.: A specialized form of chromosomal
DNA degradation induced by thymidylate stress in mouse FM3A cells. Mutation
Res. 200: 221-230, 1988.
AYUSAWA, D., KANEDA, S., SHIMIZU, K., TAKEISHI, K. and SENO, T.: Structure of thehuman thymidylate synthase gene and its cell cycle dependent expression in normal
human diploid fibroblasts and transformants of rat 3Yl fibroblasts stimulated toproliferate. Cell Struct. Funct. 13: 348-349, 1988.
BALTHOLOME-De BELDER, J., Nguyen-DIsEsHE, M., Houba-HERIN, N., GHUYSEN, J. M.,
MARUYAMA, 1. N., HARA, H., HIROTA, Y. and INOUYE, M.: Overproduction, solubilization and refolding of a genetically engineered derivative of the penicillin-binding
protein 3 of Escherichia coli K12. Molec. Microbiol. 2: 519-525, 1988.
CARSTEN, A. L., BENZ, R. D., HUGHES, W. P., IcHIMASA, Y., IKUSHlMA, T. and TEZUKA,
H. : Summary update of the Brookhaven tritium toxicity program with emphasis
on recent cytogenetic and lifetime-shortening studies. In "Proceedings of the 3rdWorkshop on Tritium Radiobiology and Health Physics" (in press.)
CROSLAND, M. W. J., CROZIER, R. H., and IMAI, H. T.: Evidence for several sibling
biological species centered on Myrmecia pilosula (F. Smith) (Hymenoptera: Formicidae). J. Aust. Entomol. Soc. 27: 13-14, 1988.
FUJIOKA, M., SHIMAMOTO, N., KAWAHARA, A., AMANO, M. and WATANABE, K.: Purification of an autocrine growth factor in conditioned medium obtained from primarycultures of scleral fibroblasts of the chick embryo. Exp. Cell Res. (in press)
FUJISAWA, T.: Inhibition of stenotele commitment by an endogenous factor in Hydra.
J. Cell Sci. 91: 361-366, 1988.
FUJITA, Q. M., YOSHIKAWA, H. and OGASAWARA, N.: Structure of the dnaA region of
Pseudomonas putida: Conservation among three bacteria, Bacillus subtilis, Escherichia
coli and P. putida. Mol. Gen. Genet. 215: 381-387, 1989.
FUKAMI, K. and TATENO, Y.: On the maximum likelihood method for estimating molecular trees: Uniqueness of the likelihood point. J. Mol. Evol. 28: 460-464, 1989.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39
FUKUDA, R., NISHIMURA, A. and SERIZAWA, H.: Genetic mapping of the Escherichia
coli gene for the stringent starvation protein and its dispensability for normal cell
growth. Mol. Gen. Genet. 211: 515-519, 1988.
GLASS, R. E., RALPHS, N. T., FUJITA, N. and ISHIHAMA, A.: Assembly of amber fragments of the f3 subunit of Escherichia coli RNA polymerase. Eur. J. Biochem. 176:
403-407, 1988.
GOJOBORI, T., MORIYAMA, E. N. and YOKOYAMA, S.: Molecular evolutionary analysis
of AIDS viruses. IV Int. Conf. on AIDS. Book 1: 142, Stockholm, 1988.
GOODMAN, L. E., PEROU, C. M., FUJIYAMA. A. and TAMANOI, F.: Structure and expression of yeast DPRI, a gene essential for the processing and intracellular localizationof ras proteins. Yeast (in press)
GOTOH, H., SAGAI, T., HATA, J. I., SHIROISHI, T. and MORIWAKI. K.: Steroid 21-hydroxylase deficiency in mice. Endocrinology 123: 1923-1927, 1988.
GOTOH, H., SHIODA, T., SAKAI, Y., MIZUMOTO, K. and SHIBUTA, H.: Rescue of Sendai
virus from viral ribonuc\eoprotein-transfected cells by infection with recombinantvaccinia viruses carrying Sendai virus L and PIC genes. Virology (in press)
HARADA, Y., MORIWAKI, K. and TOMITA, T.: Genetic polymorphism of serum protein
APH-2 found in the Japanese wild mouse (Mus musculus molossinus). Immunogenetics 27: 153-156, 1988.
HARIHARA, S., SAITOU, N., HIRAI, M., GOJOBORI, T., PARK, K. S., MISAWA, S., ELLEPOLA,
S. B., ISHIDA, T., and OMOTO, K.: Mitochondrial DNA polymorphism among five
Asian populations. Amer. J. Hum. Genet. 43: 134-143, 1988.
HAYASAKA, K., HORAI, S., GOJOBORI, T., SHOTAKE, T., NOZAWA, K. and MATSUNAGA, E.:
Phylogenetic relationships among Japanese, rhesus, Formosan, and crab-eatingmonkeys inferred from restriction enzyme analysis of mitochondrial DNAs. Mol.
BioI. Evol. 5: 270-281, 1988.
HAYASAKA, K., GOJOBORI, T. and HORAI, S.: Molecular phylogeny and evolution ofprimate mitochondrial DNA. Mol. BioI. Evol. 5: 626-644, 1988.
HAYASHI, S., HARA, H., SUZUKI, H. and HIROTA. Y.: Lipid modification of Escherichia
coli penicillin-binding protein 3. J. Bacteriol. 170: 5392-5395, 1988.
HAYASHIDA, H., KUMA, K., and MIYATA, T.: Immunoglobulin-like sequences in theextracellular domains of proto-oncogene fms and platelet-derived growth factor
receptor. Proc. Jpn. Acad. 64B: 113-118,1988.
HIRANO, H., KOMEDA, Y. and lINO, T.: Highly repetitive tandem arrays of a short 32 bp
unit in the Pharbitis nil genome: the Rsa I family. Plant Cell Physiol. 29: 11531157, 1988.
HIROSE. S. and SUZUKI, Y.: In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling. Proc. Nat. Acad. Sci. USA 85: 718722, 1988.
HIROSE, S., TABUCHI. H. and YOSHINAGA, K.: GTP induces knotting, catenation, andrelaxation of DNA by stoichiometric amounts of DNA topoisomerase II from
Bombyx mori and HeLa cells. J. BioI. Chern. 263: 3805-3810, 1988.
HONDA, A., UEDA, K., NAGATA, K. and ISHIHAMA, A.: RNA polymerase of influenza
virus: Role of NP in RNA chain elongation. J. Biochem. 104: 1021-1026,1988.

PUBLICATIONS125

IKEMURA, T.: Purification of RNA molecules by gel techniques. In "Methods in Enzymology (RNA Processing)," Academic Press. (in press)
IKEMURA, T. and AOTA, S.: Global variation in G+C' content along vertebrate genome
DNA: Possible correlation with chromosome band structure. J. Mol. BioI. 203:
1-13, 1988.
IMAI, H. T.: Centric fission in man and other mammals. In "The Cytogenetics ofMammalian Autosomal Rearrangements" (A. Daniel, ed.), pp. 551-582, Alan R. Liss,
Inc., New York, 1988.
IMAI, H. T., TAKAHATA, N., MARUYAMA, T., DANIEL, A., HONDA, T., MATSUDA, Y., and
MORIWAKI, K.: Theoretical bases for karyotype evolution. II. The fusion burst in
man and mouse. Jpn. J. Genet. 63: 313-342, 1988.
IMAI, H. T., TAYLOR, R. W., CROSLAND, M. R. J. and CROZIER, R. H.: Modes of spontaneous chromosomal mutation and karyotype evolution in ants with reference tothe minimum interaction hypothesis. Jpn. J. Genet. 63: 159-185, 1988.
ISHIHAMA, A. and NAGATA, K.: Viral RNA polymerases. In "CRC Critical Reviewsin Biochemistry", pp. 27-76, CRC Press, Florida, 1988.
ISHIHAMA, A.: Promoter selectivity of prokaryotic RNA polymerases. Trends in
Genet. 4: 282-286, 1988.
ISHIHAMA, A. and KAWAKAMI, K.: Transcription and replication of RNA viruses. (inJapanese) In "Modern Medical Sciences", Vol. 8A, pp. 279-318, Nakayama-shoten,
Tokyo, 1988.
KATO, J., NISHIMURA, Y., YAMADA, M., SUZUKI, H. and HIROTA, Y.: Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli. J. Bacteriol. 170: 3967-3977, 1988.
KAWAGUCHI, T., YOKOTA, E., NIIw, Y. and IMAMURA, T.: Complement receptor 1(CRl) on erythrocytes of patients with systemic lupus erythematosus. Jpn. J.
Human Genet. 33: 451-460, 1988.
KIMURA, M.: Natural selection and neutral evolution, with special reference to evolution and variation at the molecular level. In "L'Evolution dans sa Realitf} et ses
Diverses Modalites" (Fondation Singer-Polignac, ed.), pp. 269-284, Masson, Paris,
1988.
KIMURA, M.: Thirty years of population genetics with Dr. Crow. Jpn. J. Genet. 63:
1-10, 1988.
KOBATAKE, E. and SUGIYAMA, T.: Genetic analysis of developmental mechanisms in
hydra. XIX. Stimulation of regeneration by injury in the regeneration-deficientmutant strain, reg-16. Development, 1988. (in press)
KUKITA, A., MUKAI, T., MIYATA, T. and HORI, K.: The structure of brain-specific rat
aldolase C mRNA and the evolution of aldolase isozyme genes. Eur. J. Biochem.
171: 471-478, 1988.
KUMA, K., HAYASHIDA, H. and MIYATA, T.: Recent gene conversion between genes
encoding human red and green visual pigments. Jpn. J. Genet. 63: 367-371, 1988.
KURIHARA, Y., SAKAIZUMI, M., YONG, H. S., KANEHISA, T. and MORIWAKI, K.: Ly-2.3
antigen derived from subspecies of the Asian mouse (Mus musculus castaneus). Immunogenetics 28: 289-291, 1988.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39
KURODA, Y. and SHIMADA, Y.: Differentiation of embryonic cells of Drosophila studied
with electron microscope. In "Invertebrate and Fish Tissue Culture" (Y. Kuroda,
et al., eds.), pp. 95-99, Jpn. Sci. Soc. Press, Tokyo and Springer-Verlag, Berlin, 1988.
KURODA, Y., KURSTAK, E. and MARAMOROSCH, K. (eds.); Invertebrate and Fish Tissue
Culture. 294 p. Jpn. Sci. Soc. Press, Tokyo and Springer-Verlag, Berlin, 1988.
KURODA, Y.: Mutagen-modifying effects of vitamin A and vitamin E in mammaliancells in culture. Mutation Res. 203: 377, 1988.
MAKINO, K., SHINAGAWA, H., AMEMURA, M., KIMURA, S., NAKATA, A. and ISHIHAMA, A.:
Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro. J. Mol. BioI. 203: 85-95, 1988.
MATSUMOTO, K., NAGATA, K., HANAOKA, F. and th, M.; Tissue-specific DNA bindingof nuclear proteins that bind to the adenovirus inverted terminal repeat. J. Biochem(in press)
MATSUNAGA, E. and MINODA, K.: Use of retinoblastoma and Wilms' tumor as sentinel
phenotype for population surveillance. In "Genetics of Human Tumors in Japan(Gann Monogr. Cancer Res. 35)" (H. Takebe, et al., eds.), pp. 127-133, 1988.
MIYAMOTO, M., FUJITA, T., KIMURA, Y., MARUYAMA, M., HARADA, H., SUDO, Y., MIYATA, T. and TANIGUCHI, T.: Regulated expression of a gene encoding a nuclear
factor, IRF-l, that specifically binds to IFN-p gene regulatory elements. Cell 54;
903-913, 1988.
MIYATA, T., HAYASHIDA, H., KUMA, K., MITSUYASU, K. and YASUNAGA, T.: Male-driven
molecular evolution: A model and nucleotide sequence analysis. Cold Spring
Harbor Symp. Quant. BioI. 52: 863-867, 1988.
MIZUMOTO, K. and KAZIRO, Y.: Messenger RNA capping enzymes from eukaryotic
cells. Prog. Nucl. Acid Res. Mol. BioI. 34: 1-28, 1987.
MIZlJMOTO, K., SHIBAGAKI, Y., !TOH, N., YAMADA, H., NAGATA, S. and KAZIRO, Y.:
Structure and function of yeast capping enzyme. J. Cell Biochem. 13D: 209, 1989.
MORIYA, S., FUKUOKA, T., OGASAWARA, N. and YOSHIKAWA, H.: Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the
Bacillus subtilis chromosomes. EMBO J. 7: 2911-2917, 1988.
MORIYAMA, E. N., and GOJOBORI, T.: Evolution of nested genes with special reference
to cuticle proteins in Drosophila melanogaster. J. Mol. Evol. (in press)
MURAKAMI, A., OHTSUKI, Y. and KITAZAWA, T.: Whole embryonic culture of spontaneous parthenogenetic embryos in the silkworm, Bombyx mori. In "Invertebrate
and Fish Tissue Culture" (Y. Kuroda, et al., eds.), pp. 64-67, Jpn. Sci. Soc. Press,
Tokyo and Springer-Verlag, Berlin, 1988.
NAGATA, K., TAKEUCHI, K. and ISHIHAMA, A.: In vitro synthesis of influenza viral RNA:
Biochemical complementation assay of factors required for influenza virus replication. J. Biochem. (in press)
NARITOMI, Y., NAITO, Y., NAKASHIMA, H., YOKOTA, E., and IMAMURA, T.: A substitution of cytosine for thymine in codon 110 of the human p-globin gene is a novel
cause of p-thalassemia phenotypes. Hum. Genet. 80: 11-15, 1988.
NISHIMURA, A.: A new gene controlling the frequency of cell division per round of
DNA replication in Escherichia coli. Mol. Gen. Genet. 215: 286-293,1989.

PUBLICATIONS127

NISHIMURA, A. and HIROTA, Y.: A regulatory mechanism of cell division controls theflagellar regulon in Escherichia coli. Mol. Gen. Genet. 216: 340-346, 1989.
NOMURA, T. and ISHIHAMA, A.: A novel function of RNase P from Escherichia coli:
Processing of a suppressor tRNA precursor. EMBO J. 7: 3539-3545, 1988.
OGASAWARA, N., FUJITA, Q. M., MORIYA, S., FUKUOKA, T., HIRANO, M. and YOSHIKAWA,
H. : Comparative anatomy of oriC of eubacteria. In" The Bacterial Chromosome"(K. Drlica and M. Riley, eds.), John Wiley and Sons, New York, 1989.
OHTA, T.: Multigene and supergene families. In "Oxford Surveys in Evolutionary
Biology, V." (P. H. Harvey, et al., eds.), pp. 41-65, Oxford Vniv. Press, 1988.
OHTA, T.: Further simulation studies on evolution by gene duplication. Evolution 42:375-386, 1988.
OHTA, T.: Time for acquiring a new gene by duplication. Proc. Natl. Acad. Sci. USA
85: 3509-3512, 1988.
OHTA, T.: Evolution by gene duplication and compensatory advantageous mutations.
Genetics 120: 841-847, 1988.
OZAWA, T., YONEDA, M., TANAKA, M., OHNO, K., SATO, W., SUZUKI, H., NISHIKIMI,
M., YAMAMOTO, M., NONAKA, 1. and HORAI, S.: Maternal inheritance of deleted
mitochondrial DNA in a family with mitochondrial myopathy. Biochem. Biophys.
Res. Commun. 154(3): 1240-1247, 1988.
SAKAI, K. 1., ENDO, T., IYAMA, S., MIYAZAKI, Y., HAYASHI, S., SHIMAMOTO, Y., GADRINBAB,
L. V., and JUNIARTI, U.: Studies on the breeding structure of tree species in thetropical rain forest. 1. Family clumps and intra population differentiation. BIOTROPIA
1(1): 1-25, 1987.
SAKAIZUMI, M., HASHIMOTO, Y., SUZUKI, A., YAMAKAWA, T., KIUCHI, Y. and MORIWAKI,
K.: The locus controlling liver GM1(NeuGc) expression in mapped 1 cM centromeric to H-2K. Immunogenetics 27: 57-60, 1988.
SATO, Y. 1., and MORISHIMA, H.: Distribution of the genes for F 2 chlorosis in rice cultivars of the Indica and Japonica types. Theor. Appl. Genet. 75: 723-727, 1988.
SATTA, Y., TOYOHARA, N., OHTAKA, C., TATSUNO, Y., WATANABE, T.-K., MATSUURA,
E. T., CHIGUSA, S. 1. and TAKAHATA, N.: Dubious maternal inheritance of mitochondrial DNA in D. simulans and evolution of D. mauritiana. Genet. Res., Camb.
52: 1-6, 1988.
SEKIGUCHI, T., MIYATA, T. and NISHIMOTO, T.: Molecular cloning of the cDNA ofhuman X-chromosomal gene (CCG1) which complements the temperature-sensitive
Gl mutations, tsBN462 and ts13, of the BHK cell line. EMBO J. 7: 1683-1687, 1988.
SUGIMOTO, K., MIYASAKA, T., FUJIYAMA, A., KOHARA, Y. and OKAZAKI, T.: Change inpriming sites for discontinuous DNA synthesis between the monomeric and concatemeric stages of phage T7 replication. Mol. Gen. Genet. 211: 400-406, 1988.
TABUCHI, H. and HIROSE, S.: DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter. J. BioI. Chern. 263: 1528215287, 1988.
TAKAHATA, N.: The coalescent in two partially isolated diffusion populations. Genet.
Res., Camb., 52: 213-222, 1988.
TAKAHATA, N.: More on the episodic clock (Letter to the editor). Genetics 118: 387-

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39
388, 1988.
TAMAI, K., TEZUKA, H., KURODA, Y. and KADA, T.: Studies on the combined effects ofenvironmental mutagens on mammalian cells in culture. IV. Mutation Res. 203:
191, 1988.
TAMANOI, F., HSUEH, L. E., GOODMAN, L. E., COBITZ, A. R., DETRICK, R. J., BROWN
W. R. and FUJIYAMA, A.: Posttranslational modification of ras proteins: Detectionof a modification prior to fatty acid acylation and cloning of a gene responsible forthe modification. J. Cellul. Biochem. 36: 57-69, 1988.
TAMURA, T., INOUE, T., NAGATA, K. and MIKOSHIBA, K.: Enhancer of human polyoma
JC virus contains nuclear factor I-binding sequences: Analysis using mouse brain
nuclear extracts. Biochem. Biophys. Res. Commun. 157: 419-425, 1988.
TERADA, H., SUGIYAMA, T. and SHIGENAKA, Y.: Genetic analysis of developmental mechanisms in hydra. XVIII. Mechanism for elimination of the interstitial cell lineage
in the mutant strain sf-I. Developm. BioI. 126: 263-269, 1988.
TSURUTA, H., ISHIHAMA, A., FUJITA, N., AMEMIYA, Y. and KIHARA, H.: Dissociation
and association kinetics of E. coli ribosomes. Stopped flow X-ray scattering study
at subzero temperature. Photon Factory Activity Report 6: 131, 1988.
TUTIKAWA, K.: T alleles from population of wild Japanese mice. Shizuoka Exp.
Anim. Res. Associat. Rep. 29: 19-28, 1988.
UESHIMA, R., FUJITA, N. and ISHIHAMA, A.: DNA supercoiling and temperature affectthe promoter activity of Escherichia coli rpoH gene encoding the heat-shock sigma
subunit of RNA polymerase. Mol. Gen. Genet. 215: 185-189, 1989.
UESHIMA, R. and KUROZUMI, T.: Anatomical features of Hirasea (s.s.) diplomphalus andtaxonomic position of the genus Hirasea Pilsbry, 1902 (Pulmanata: Sigmurethra).
Venus, Jpn. J. Malac. 47(4): 261-270, 1988.
YAMAMOTO, T., GOJOBORI, T., MORIYAMA, E. N., YOKOTA, T. and KUWAHARA, S.: Discontinuous evolution of bacterial pathogens: Evidence for species-species transfer
in evolution of pathogenic determinants of Escherichia coli and Vibrio cholerae 01.
In "Advances in Research on Cholera and Related Diarrheas. Vol. 6" (N. Ohtomo,
et al., eds.), pp. 195-212, KTK Sci. Publ., Tokyo.
YAMANAKA, K., ISHIHAMA, A. and NAGATA, K.: Translational regulation of influenza
virus mRNAs. Virus Genes 2: 19-30, 1988.
YAMAO, F., IWAGAMI, S., AZUMI, Y., MUTO, A., OSAWA, S., FUJITA, N. and ISHIHAMA, A.:
Evolutionary dynamics of tryptophan tRNAs in Mycoplasma capricolum. Mol.
Gen. Genet. 212: 364-369, 1988.
YAMAUCHI, M., AYUSAWA, D., SHIMIZU, K., SENO, T. and MATSUHASHI, M.: Two typesof mouse FM3A cell mutants deficient in 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and their transformants isolated by human chromosomemediated gene transfer. Somat. Cell Mol. Genet. 15: 39-48, 1989.
YOKOYAMA, S., CHUNG, L. and GOJOBORI, T.: Molecular evolution of the human immunodeficiency and related viruses. Mol. BioI. Evol. 5: 237-251, 1988.
YONEKAWA, H., MORIWAKI, K., GOTOH, 0., MIYASHITA, N., MATSUSHIMA, Y., SHI, L.,
CHO, W.-c., ZHEN, x.-L. and TAGASHIRA, Y.: Hybrid origin of Japanese mice"Mus musculus molossinus"; Evidence from restriction analysis of mitochondrialPUBLICAnONS
DNA.

129

Mol. BioI. Evol. 5: 63-78,1988.
Sample size for collecting germplasms from natural
plant populations in consideration of seed embryos borne on a single plant.
Euphytica, 1988. (in press)
YONEZAWA, K.: Appropriate size of sample for collecting germplasms from natural
plant populations. JICA, REF. 2, 1988. (in press)
YONEZAWA, K. and ICHIHASHI, H.:

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

ABSTRACTS OF DIARY FOR 1988
Biological Symposium
269th meeting Mar. 18

270th

Apr. 26

271stApr. 27
Apr. 27272nd

May 26273rd

May 27274th

May 30275th

July 15

276th

Aug. 11277th

Oct. 17278th

Oct. 19279th

Oct. 24280th

Oct. 31

Restriction endonucleases for pulsed field
mapping of chromosomes(Michael McClelland)
Translational control by phosphorylation of
mammalian initiation factors (John W. B.
Hershey)
Molecular genetics in mouse development(William F. Dove)
Molecular genetics of the t-region of themouse genome (Alexandra Shedlovsky)
How sick are we genetically-speaking? Genetic and partially-genetic diseases in man(K. Sankaranarayanan)
Mouse gene mapping using R.F.L.P.'s: recent data (Guenet Jean-Louis)
Mammalian X-chromosome: Some comparative aspects (James R. Miller)
Recent developments and insight in the antiHIV activity and metabolism of novel purine
and pyrimidine nucleoside analogues (Ir. Jan
Balzarini)
Mutation affecting the extra chromosomal
replication of the Tetrahymena ribosomal
RNA gene (E. arias)
Algal/hydra symbioses-a model for hostsymbiont interactions (Menashem Rahat)
Nucleic acid databases-Vision for the future(James C. Cassatt)
EMBNet: Network for molecular biology inEurope (Chris Sander)
Neurons and neuropeptides in hydra andother coelenterates

ABSTRACTS OF DIARY FOR 1988

131

(C. J. P. Grimmelikhuijzen)
Nov. 1 Identification of a novel E. coli sigma factor
involved in high temperature gene expression.
(James W. Erickson)
282nd
Nov.25 Antimutagenesis in microbial systems(Delbert M. Shankel)
Dec. 12 Col El DNA replication (Jun-ichi Tomizawa)
283rd
284th
Dec. 14 Regulation of ribosome synthesis in E. coli:translational regulation, retroregulation, feedback loops and cooperativity in assembly(Masayasu Nomura)
285th
Dec. 25 Statistical analysis of DNA sequences(Kotoko Nakada)
286thDec. 28 Molecular basis of mutagenesis (Laurence A.
Loeb)
Mishima Geneticists' Club
333rd meeting Jan. 6 Function of en v-related leukemogenic geneencoded by Friend spleen focus-forming virus(Yoji Ikawa)
334th
Jan. 8 Interactions between repressor and operator(Akira Sarai)
335th
Jan. 14 Molecular genetics of urea biosynthesis(Masataka Mori)
336th
Jan. 20 Gene regulation and evolution of highly repetitive and transcribable sequence in thesalmon genome (Kenichi Matsumoto)
337th
Feb. 22 Expression of the storage-protein gene ofsoybean in transgenic petunia (Ikuo
Nakamura)
338th
Apr. 13 Gene regulation in teratocarcinoma stem
cells: Analysis using recombinant retrovirus(Makoto Taketo)339thApr. 22 Somatic cell genetic study on the mammalian
thymidylate synthase gene (Dai Ayusawa)
340th
Apr. 30 Study on the structure and function of humanthymidylate synthase gene (Sumiko Kaneda)
341st
May 9 Purification of a transcription factor for
281st

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

342nd

May 9

343rd

May 25344th

May 31

345th

July 25

346th

Oct. 14347th

Oct. 19348th

Oct. 19349th

Oct. 25350th

Nov. 26

immunoglobulin genes and transcriptional
activation from HIV promoters (Kiyoshi
Kawakami)
New insight into mechanisms of DNA enzymes (Samuel Wilson)
Mechanisms of speciation in historical perspective (William B. Provine)
Intracellar concentration and cooperative
binding of single-stranded DNA binding
protein (Nobuo Shimamoto)
Chromosomal recombination of eukaryote(Kenzo Sakaguchi)
Control of ribosome synthesis in E. coli and
S. pombe (Masahiro Yamagishi)
Studies on transposon-like elements and repetitive sequences in plant genomes(Hiroyuki Hirano)
Low temperature stress and gene expression
in rice (Yasunori Ban)
Molecular evolutionary analysis of Drosophila genes (Etsuko Moriyama)
Regulation of transcription by cAMP (Koji
Aeba)

FOREIGN VISITORS IN 1988

133

FOREIGN VISITORS IN 1988
April 9, 1984-

Pascale Barbier, Universite des Sciences et Techniques
du Languedoc, Montpellier, France
April 1, 1987Dong Sang Suh, Genetic Engineering Center,
March 31, 1988 Korea Advanced Institute of Technology, Korea
August 4, 1987Ling Hua Tang, Institute of Food Crops, Jiangsu
Academy of Agricultural Sciences, China
September 1, 1987- Sam-Eun Kim, Sericultural Experiment Station, Rural
February 10, 1988 Development Administration, Korea
December 4, 1987- Xiao Mei Wu, Lanzhou Institute of Biological Product, Ministry of Public Health, China
December 4, 1987- He Zhao, Lanzhou Institute of Biological Product,
Ministry of Public Health, China
February 20, 1988 Song Wang, Animal Research Institute, Chinese
Academy of Sciences, China
March 18-19
Michael McClelland, University of Chicago, U.S.A.
April 14
Makoto Taketo, The Jackson Laboratory, U.S.A.
April 19
Debao Li, National Rice Research Institute, China
April 19
Zhingmin Xiong, National Rice Research Institute,
China
April 19
Guowen Hu, National Rice Research Institute, China
Apri119Yi Du, National Rice Research Institute, China
April 19
Xiaoan Zhang, Ministry of Agriculture, Animal Husbandry and Fishery, China
April 26
John W. B. Harshey, University of California, U.S.A.
April 27
William F. Dove, University of Wisconsin, U.S.A.
April 27
Alexandra Sheldlovsky, University of Wisconsin,
U.S.A.
May 2-30
William B. Provine, Cornell University, U.S.A.
May 9
Samuel Wilson, National Institutes of Health, U.S.A.
May 9
Kiyoshi Kawakami, The Rockefeller University,
U.S.A.
May 25
K. Sankaranarayanan, State University of Leiden,
The Netherlands

134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

May 27
May 30June 2
July 15
August 11
September 2December 20
September 2
September 9
September 16November 16
September 17-19
September 27October 14
October 19
October 24
October 31
November 1
November 4
November 14
November 14
November 14
November 14
November 14
November 14
November 14

Jean-Louis Gu6net, Institut Pasteur, France
James R. Miller, University of British Columbia,
CanadaKazutoshi Maeda, Wayne State University, U.S.A.
Ir. Jan Balzarini, Rega Institute, Belgium
Edward Orias, University of California, U.S.A.
Delbert M. Shankel, University of Kansas, U.S.A.
Marney Thone, CSIRO Division of Animal Production, Australia
Usha Goswami, National Institute of Oceanography,
India
Menashem Rahat, The Hebrew University, Israel
James F. Crow, University of Wisconsin, U.S.A.
Guan Cheng Sun, Sericultural Research Institute,
Chinese Academy of Agricultural Sciences, China
Masahiro Yamagishi, University of California, U.S.A.
James C. Cassatt, National Institutes of Health, U.S.A.
Chris Sander, Europe Molecular Biology Laboratory,
West Germany
C. J. P. Grimme1ikhuijzen, UniversiHit der Hamburg,West Germany
James W. Erickson, University of Wisconsin, U.S.A.
Amauri B. Simonetti, University of Sao Paulo, Brazil
Jin-ming Qian, Wenzhou Agricultural Research Institute, Zhejiang Province, China
Rolando Saborit Reyes, Rafael Sanzo Mancebo RiceExperimental Station, Cuba
Wagdy Mohamed Khalil Elswaidy, Agriculture Department, Dumiat Governorate, Egypt
Emmanuel Tetteh-Bio, Kpong Farms' Project, Ghana
Md. Jasin Bin Ab. Rahman, Muda Agricultural Development Authority, Malaysia
Agustin Antonio Martinez Lopez, National Institutefor Agricultural Research, Mexico
Rafiqullah Shah, Agricultural Research Institute,FOREIGN VISITORS IN 1988

November 14
December 2
December 2
December 2
December 2
December 2
December 2
December 2
December 2
December 7
December 12
December 14
December 24
December 28

135

Pakistan
Saul Antonio Orrego Puelles, Enterprise of Rice Commercialization, North-Coast, Peru
James F. Crow, University of Wisconsin, U.S.A.
W. G. Hill, Institute of Animal Genetics, Scotland
Charles Langley, National Institute of Environmental
Health Sciences, U.S.A.
Masatoshi Nei, University of Texas, U.S.A.
Montgomery Slatkin, University of California, U.S.A.
Geoffrey A. Watterson, Monash University, Australia
B. S. Weir, North Carolina State University, U.S.A.
Shozo Yokoyama, University of Illinois, U.S.A.
Abraham Eisenstark, University of Missouri, U.S.A.
Jun-ichi Tomizawa, National Institutes of Health,
U.S.A.
Masayasu Nomura, University of California, U.S.A.
Kotoko Nakada, National Cancer Institute, U.S.A.
Laurence A. Loeb, University of Washington, U.S.A.

136

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 39

AUTHOR INDEXAMANO, M!SHIHAMA, A. .... 18, 19, 20, 21,22,23,24,

AOKI, K.

25,26,27
96
IWAKURA, Y
25!YAMA, S
113, 121
JAIN, A. K • . . . . . . . . . . . . . . . . . . . . . . . . . 65
JERNIGAN, R. L.
120
KANEDA, S
28, 29
KATO, A
21
KATO, J
.41, 45, 47
KAWAHARA, A. .
74
KIMURA, M. .
86
KRYSTAL, M • . . . . . . . . . . . . . . . . . . . . . . . 21
KUBOTA, M
18
KUMA, K.
96, 97KURIHARA, Y
79
KURODA, Y. .
63, 64, 65
LOWENSTEIN, J. M
27
MATSUMOTO, K • . . . . . . . . . . . . . . . . . 26,32
MATSUNAGA, E
105, 107
MATSUURA, E. T
85
MINATO, K. .
73
MINODA, K • . . . . . . . . . . . . . . . . . . . . . . . . 105
MITA, A77MIYASHITA, N • . . . . . . . . . . . . . . . . . . . 79, 81
MIYATA, T" .96,97,98
MIYAZAWA, S • . . . . . . . . . . . . . . . . . . . . . . 120
MIZUMOTO, K • . . . . . . . . . . . . . . . . . . . 36, 37
MORISHIMA, H. .
92, 94MORIWAKI, K •...... 60,61,62,76,77,78.
79,81
MORIYAMA, E. N
90, 91
MURAKAMI, A
67, 69, 70, 71
NAGASAWA, H. .
.48, 49
NAGATA, K
2\, 22, 23, 24, 25, 26
NAKASHIMA, H
100,101,102
NAKAYAMA, M
20
NALBANTOGLU, J
28
NARITOMI, Y. .
102
NATSUUME-SAKAI, S. .
76

74
88, 89
AOTA, S. .
32, 34AYUSAWA, D
28, 29,30
BONHOMME, F
76,79
BUCHER, D. J • . . . . . . . . . . . . . . . . . . . . . . . 22
CHIFU, Y. .
102
CHIGUSA, S. I.
85
CHUNG, L
91
ENDo, T
110
FELDMAN, M. W
89
FUJIOKA, M • . . . . . . . . . . . . . . . . . . . . 31, 74
FUJISAWA, T
67
FUJITA, N
18, 19,20
FUJIWARA, H
95
FUJIYAMA, A •.......... 100, 101, 102, 103
FuKAMI, K
95
GOJOBORI, T
90, 91, 92,104
GOTOH, H • . . . . . . . . . . . . . . . . . . . . . . . . . 62
GOTOH, O
28
HANAOKA, F. .
26
HANKINS, R. W
22
HANZAWA, N
60
HARA, H. .
.48, 49, 51, 52, 55, 56
HARADA, Y
76
HARIHARA, S. .
92
HASEGAWA, T
100
HATA, J • • . . . . . . . . . . . . . . . . . . . . . . . . . . 62
HAYASAKA, K • . . . . . . . . . . . . . . . . . . . . . 104
HAYASHIDA, H • . . . . . . . . . . . . . 96, 97, 117
HIGASHITANI, A • . . . . . . . . . . . . . . . . . . . . . 55
HIROSE, S • . . . . . . . . . . . . . . . . . . . . . . . 34, 35
HIROTA, Y. .
.41, 58
HONDA, A.
2\
HORAI, S.
.
104, 105
HORIE, N
28
IGARASHI, K
19
IKEMURA, T
32, 33, 34
IMAMURA, T. .
100, 101, 102
ISHIBASHI, F. .
32

IWABE, N

AUTHOR INDEX137

NISHIHIRA, H

SUZUKI, H ..... .41, 45,47,48,49,51,52,

NISHIMURA, A

55, 56, 57, 79
35
TAKAHATA, N
84, 85
T AKEISHI, K.
28
TANAKA, M
105
TANG, L.-H
94
TATENO, Y
95,96
TATSUNO. Y
85
TEZUKA. H. .
30, 65TOMITA, T. .
76
TOYOHARA, N
85
TRUONG, Y. L.
27
TSUNASAWA, S
103
UDATSU, T • . . . . . . . . . . . . . . . . . . . . . . . . . 95
UEDA, S
21
UESHIMA, R. .
20
WADA, K
34
WAKASUGI, N • . . . . . . . . . . . . . . . . . . . . . . 77
WATANABE, K. .
74
WATANABE, T. K
85
WINKING, H
79
YAMADA, M.41
YAMAMOTO, T. .
91
YAMAMOTO, Y.47
YAMANAKA, K
21, 23, 25
YAMAZAKI, Y
18, 21, 27
YASUDA, N
90
YASUDA, S. .
43
YASUNAGA, T
96
YI, H
113
YOKOIYAMA, A
21, 25YOKOYAMA, S • . . . . . . . . . . . . . . . . . . . 90, 91
YONEDA, M
105
YONEKAWA, H
79
YONEZAWA, K.
114
YOSHIKAWA, H. "23, 25, 38

107
57, 58NISHIMURA, Y.41, 45, 47,55
NIWA, M
77
NONAKA,1.
105
OGASAWARA, N. .
23, 25
OHTA, T. .
34, 82, 83
OHTAKA, C. .
85
OISHI, K
64
OKAMOTO, H. .
19
OMOTO, K.
92
OSAWA, S
20
OZAWA, T
105
PALESE, P
21
PARVIN, J. D
21
SADAIE, Y
59
SAGAI, T
60, 61, 62
SAITOU, N • . . . . . . . . . . . . . . . . . . . . . . . . . 92
SAKAGAMI, H
24
SAKAGAMI, Y
48
SAKAGUCHI, B.
64
SAKIYAMA, F
103
SANO, Y • . . . . . . . . . . . . . . . . . . 111,112,113
SATO, Y.-I.
94, 95
SATTA, Y • . . . . . . . . . . . . . . . . . . . . . . . . . . 85
SENO, T28, 29, 30
SHAO, Q113
SHIMADA, Y. .
64, 75SHIMAMOTO, N • . . . . . . . . . . . . . . . . . . 31, 74
SHIMIZU, H
66
SHIMIZU, K
28
SHIROISHI, T
60, 61, 62
STEINMETZ, M. .
60
STYRNA, J
78
SUGIYAMA, T
66
SUH, D.-S • . . . . . . . . . . . . . . . . . . . . . . . . . . 78
SUZUKI, A. .
48, 49TABUCHI, H

m
?err

lE:lz:iltta~i'f~ffl:¥fa
39 ~3jlRl(;5f;~ 9 J'I 25 13 IlI.lPU
3jlRl(;5f;~ 9 J'I 31 13
?err~

tl

7k

?k

1E.it~~~ijIf~ffl~

au~~~ EEl 1'J IJB • !'l' ~H£

1E.it5l~~ijIf~ffl~

'!1.
#
m 5A
*J?:mtrmgiflj BU!i~ 3-8-8
III.lPUffl :~ 00 I~t)( mU=P .ffl1Jt±* J?: mtr mg iflj ffi.\!i~ 3-8-8
Je rr ffl IE 3L ~ ffi ~ iJf ~ PJT
IlI.lPU~T411

m.llill!Ji!<=~iP1:i-ffi 1111

'lit 1m ft~ (0559) (75) 077 1

to HakoneNt
1:25,000