ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 41

1990

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1991

Annual Repor~of ~heNa~ional Ins~i~ute of Genetics
No. 41, 1990

Published by

The National Institute of Genetics, Japan
1991

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council and Advisory Committee. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Association for propagation of the knowledge of genetics
Projects of research for 1990
Reseach activities in 1990

I.

1
2
5
7
8
13

Molecular Genetics

The promoter selectivity of Escherichia coli RNA polymerase, I.
Involvement of the C-terminal region of a subunit in recognition of cAMP-CRP-dependent promoters. IGARASHI, K.,
HAYWARD, R. S., FUJITA, N. and ISHIHAMA, A.
13
The promoter selectivity of Escherichia coli RNA polymerase, II.
Comparison of three assay systems of the promoter strength.
KOBAYASHI, M., NAGATA, K. and ISHIHAMA, A.
14
The promoter selectivity of Escherichia coli RNA polymerase, III.
Structural and functional modulations of RNA polymerase
during phase transition of cell growth. OZAKI, M., FUJITA,
N., WADA, A. and ISHIHAMA, A.
15
The promoter selectivity of Micrococcus luteus RNA polymerase:
Identification and characterization of the major sigma factor.
NAKAYAMA, M., FUJITA, N., aSAWA, S. and ISHIHAMA, A..... 16
Cloning and sequence determination of the Schizosaccharomyces
pombe rpbl gene encoding the largest subunit of RNA polymerase II. AZUMA, Y., YAMAGISHI, M., UESHIMA, R. and
ISHIHAMA, A"
17
The promoter selectivity of influenza virus RNA polymerase: invitro and in vivo analysis of the promoter structure. YAMANAKA, K., NAGATA, K. and ISHIHAMA, A.
18
Mechanism of growth inhibition of influenza virus by the mouse
Mx gene. NAKAYAMA, M., NAGATA, K., IWAKURA, Y., KATO,
A. and ISHIHAMA, A.
19
Expression of human thymidylate synthase minigenes : Enhancer-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

like activity in intron 1. KANEDA, S., HORIE, N., TAKEISHI,
K., AYUSAWA, D. and SENO, T.
Cell-cycle directed regulation of human thymidylate synthase gene.
TAKAYANAGI, A., KANEDA, S., AYUSAWA, D. and SENO, T. ..
Mechanism of DNA degradation during thymidylate stress in mammalian cells. YAMAO, F., IMAI, N. and SENO, T.
Vbiquitin-activating enzyme as a determinant of cell-cycle progerssion. AYUSAWA, D., KANEDA, S. and SENO, T.
DNA supercoiling and eukaryotic transcription-Cause andeffect. HIROSE, S. and OHTA T.
Negative supercoiling of DNA with eukaryotic DNA topoisomerase II and dextran sulfate. OKADA, K., OHTA, T. and
HIROSE, S.
Core promoter of the mouse myelin basic protein gene governs
brain-specific transcription in vitro. TAMURA, T., SUMITA, K.,
HIROSE, S. and MIKOSHIBA, K.
Identification and purification of a Bombyx mori homologue of
FTZ-F1. VEDA, H. and HIROSE, S.
Search for target genes of Drosophila homeotic genes. HAYASHI,
S. and HIROSE, S.
Molecular cloning of fleabug, a gene controlling segmentation oflarva and adult. HAYASHI, S., SHIRRAS, A. and HIROSE, S. ..
Identification and characterization of a gene strawberry which is
required for organization of sensory organs. HAYASHI, S.,
WATANABE, J. and HIROSE, S.
Demonstration of extrinsic DNA from immune complexes in theplasma of a patient with systemic lupus erythematosus. TERADA, K., OKUHARA, E., KAWARADA, Y. and HIROSE, S. .. . . . ..
In vitro recombination system using nuclear extract from mouse
testis cells. KOIDE, T., SAGAI, T., SHIROISHI, T. and MORIWAKI, K.
Gene expression at the wx locus of rice (Oryza sativa). HIRANO,
H. Y. and SANO, Y.
Cloning and characterization of restriction fragments homologous
to B-1 sequences in cytoplasmic male sterile rice. YAMADA,

M

20
21

22
22

23

24

24
25
25
26

26

27

27
28

29

CONTENTS

II.

iii

Microbial Genetics

Mutational analysis of the minus strand origin of bacteriophagef1. HIGASHITANI, N., ROTH, A., HIGASHITANI, A. and HORIUCHI, K.
SOS induction in E. coli by infection with mutant filamentous
phages that are defective in complementary strand synthesis.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.
Replication of filamentous phage fl: Relationship between DNA
topology and the nicking activity of the initiator protein(gpII). HIGASHITANI, A. and HORIUCHI, K.
Cloning and nucleotide sequence of the ispA gene responsible forfarnesyl diphosphate synthase activity in Escherichia coli.
FUJISAKI, S., HARA, H., NISHIMURA, Y., HORIUCHI, K. andNISHINO, T.
Kinetic study on thranscription by immobilized operons: Requirement for 13, r-pyrophosphate bond of ATP in initiationby prokaryotic RNA polymerases. TERADA, H. and SHIMAMOTO, N.
Single-molecule dynamics of transcription by immobilized operons:
Sliding of RNA polymerase on DNA. SHIMAMOTO, N. .....
Structural study on E. coli single-stranded DNA binding protein.
SHIMAMOTO, N., TsuKIHARA, T., TAKAHASHI, K. and KAMATA,
K

31

33

34

36

37
37

38

111. Mammalian Genetics
Genetic and geographical differentiation of Mus musculus subspecies in China. MORIWAKI, K., MIYASHITA, N., SUZUKI,
H., TSUCHIYA, K., YONEKAWA, H., WANG, C. H., HE, X. Q.
and JIN, M. L.
A high incidence of visible mutation in intra-MHC recombinants.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.
Rescue of lethal adrenal-dysgenic mice through transgenesis.
GOTOH, H., SHIROISHI, T. and MORIWAKI, K.
Genetic control of susceptibility to urethane-induced lung tumor
in mice. MIYASHITA, N. and MORIWAKI, K.
Amplified EF2 related sequence (MER) in mouse genome. KOIDE,
T., SHIROISHI, T., ISHIURA, M. and MORIWAKI, K.

39
40
41
43
43

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

A new haplotype of the mouse j3-globin genes, Hbb w], in northwestern China. KAwASHIMA, T., MIYASHITA, N., SHIROISHI,
T., YAN, W. C., HUAI, W. C. and MORIWAKI, K.
44
Establishment of embryonic stem cell lines from BlO. H-2 congenic
strains. SAGAI, T., NAKATSUJI, N., SHIROISHI, T. and MORIWAKI, K.
4$
Comparison of the DNA sequence in the D-loop region of mousemitochondrial DNA. Four maternal lineages in the southern
population of Japanese "molossinus" mice. YONEKAWA, H.,
WATANABE, S., MIYASHITA, N. and MORIWAKI, K.
46Sperm head abnormalities in interspecific hybrids between Mus
musculus and M. spretus, and M. spicilegus. NIWA, M.,
MITA, A. and MORIWAKI, K.
47

IV.

Developmental and Somatic Cell Genetics

Head regeneration and budding in Hydra. SUGIYAMA, T.
49
A cDNA clone specific for stenotele differentiation in Hydra.
FUJISAWA, T.
50
Ovipositing behavior in Bombyx adults. a. Non-mated females.
MURAKAMI, A.
50
Oviposition behavior in Bombyx audlts. b. Mated females. MURAKAMI, A.
51
Genetic studies on growth and aging in the silkworm (BombyxMori). a. Growth rate in growth period. MURAKAMI, A. '"52
Genetic studies on growth and aging in the silkworm (BombyxMori). b. Growth rate in the pupal stage. MURAKAMI, A. " 54
Genetic studies on growth and aging in the silkworm (BombyxMori). c. Embryonic diapause as a prolongation of lifespan.
MURAKAMI, A.
55
A new approach to major genes which control lifespan in insects.
YONEMURA, I., HASEKURA, H., OKUNO, A. SHIMIZU, Y. and
MURAKAMI, A.
57
Premeability of eggs and abnormal development in Drosophila.
MINATO, K.
59'
Conditional radiation sensitivity in the wasted mouse mutant.
TEZUKA, H., AYUSAWA, D. and SENO, T.
60
Purification and structure analysis of an autocrine growth factor

CONTENTSv

in conditioned medium obtained from primary cultures ofscleral fibroblasts of the chick embryo. OHOYA, Y., WATANABE, K. and SHIMAMOTO, N.
61
In vitro amplification of whole cDNA from a single embryo of thenematode C. elegans and its application to differential screen61
ing. KOHARA, Y.

V.

Population Genetics

Theoretical study of near neutrality. I. Heterozygosity and rateof mutant substitution. OHTA, T. and TACHIDA, H.
64
How gene families evolve. OHTA, T.
64
Evolution of the multigene family: A case of dynamically evolving
genes in the major histocompatibility complex. OHTA,
T.
65
The major histocompatibility complex and the quest for origins.
KLEIN, J. and TAKAHATA, N.
66
Allelic genealogy and MHC polymorphisms. TAKAHATA, N.
66
Population biology of genes and molecules. TAKAHATA, N. and
CROW, J. F. (Eds.)
67
Allelic genealogy under overdominant and frequency-dependent
selection and polymorphism of major histocompatibility
complex loci. TAKAHATA, N. and NEI, M.
68
A simple genealogical structure of strongly balanced allelic lines
and trans-species evolution of polymorphism. TAKAHATA,N
69
Genealogy of neutral genes in two partially isolated populations.
TAKAHATA, N. and SLATKIN, M.
70
Incomplete maternal transmission of mitochondrial DNA in
Drosophila. KONDO, R., SATTA, Y., MATSUURA, E. T., ISHIWA, H., TAKAHATA, N. and CHIGUSA, S. I.
71
Evolution of Drosophila mitochondrial DNA and the history of themelanogaster subgroup. SATTA, Y. and TAKAHATA, N. ..... 71
A study on a nearly neutral mutation model in finite populations.
TACHIDA, H.
72
Fixation probability in spatially changing environments. TACHIDA, H. and IIZUKA, M.
73
Relationship between DNA polymorphism and fixation time.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

TAJIMA, F.
73
Relationship between migration and DNA polymorphism in a local
population. TAJIMA, F.
74
A simple graphic method for reconstructing phylogenetic trees
from molecular data. TAJIMA, F.
74
Some consideration on diversifying selection. TAJlMA, F. and
MUKAI, T75
An estimate for the total rate of mutations in humans. KIMURA,
M
75
Recessive hereditary deafness, assortative mating, and persistenceof a sign language. AOKI, K. and FELDMAN, M. W.
76
Some theoretical aspects of the origin of cultural transmission.
AOKI, K77

VI. Evolutionary Genetics
Evolutionary origin of human and SImIan immunodeficiency
viruses. GOJOBORI, T., MORIYAMA, E. N., IKEo, K. and INA,Y
Statistical methods for estimating sequence divergenece. GOJOBORI, T., MORIYAMA, E. N. and KIMURA, M.
Molecular clock of viral evolution, and the neutal theory. GoJOBORI, T., MORIYAMA, E. N. and KIMURA, M.
Patterns of amino acid substitutions in the envelope protein ofhuman immunodeficiency viruses and its application to vaccine
development. GOJOBORI, T. and MORIYAMA, E. N.
Rates and patterns of nucleotide substitutions for nuclear genes
of Drosophila species. MORIYAMA, E. N. and GOJOBORI,
T
Molecular evolution of human T-cell leukemia virus. INA, Y. and
GOJOBORI, T.
Host-independent evolution of the hepadnavirus family. INA,
Y., MIZOKAMI, M., ORITO, E., KAMESHIMA, N., YAMAMOTO, M.,
MORIYAMA, E. N. and GOJOBORI, T.
Molecular evolution of Kunitz-type protease inhibitor. IKEo,
K., TAKAHASHI, K. and GOJOBORI, T.
Patterns of nucleotide substitution in class I major histocompatibility complex genes. IMANISHI, T. and GOJOBORI, T. .. . . . . .

7'i!.
78
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80

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81

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8383

CONTENTS

Establishment of a phylogenetic survey system for AIDS-related
lentiviruses and demonstration of a new HIV-2 subgroup.
MIURA, T., FUKASAWA, M., MORIYAMA, E. N., GOJOBORI, T.,
ISHIKAWA, K. I., TSUJIMOTO, H. and HAYAMI, M.
.....
Maternal hybrid rescue (mhr) gene which rescues embryonic lethal
hybrids from Drosophila simulans females crossed with D.
melanogaster males. SAWAMURA, K. and WATANABE, T. K..
Zygotic hybrid rescue (Zhr) gene which rescues embryonic lethal
hybrids from Drosophila melanogaster males crossed withsibling species. SAWAMURA, K. and WATANABE, T. K.

VII.

vii

84

85

85

Human Genetics

Characterization of genetic polymorphisms in tandem repeatedsequence blocks. IMAMURA, T.
•.•.... ..
Analysis of genetic polymorphisms in the human Alu sequencefamily and the flanking regions for genetic mapping of chromosomes 18 and X. NAKASHIMA, H., SAKAI, M., HASEGAWA, T.
and IMAMURA, T.
Beta-thalassemia resulting from compound heterozygosity for
globin gene mutation in a Japanese family: Evidence formultiple origin of the thalassemia gene in Asian populations.
NAKASHIMA, H., CHIFU, Y., YOKOTA, E. and IMAMURA, T. '"Molecular phylogeny and evolution of human mitochondrial DNA.
HORAI, S.
Mitochondrial DNA evolution in primates: Transition rate has
been extremely low in the lemur. HASEGAWA, M., KISHINO,
H., HAYASAKA, K. and HORAI, S.
A common mitochondrial DNA mutation in the tRNALys of patients with myoclonus epilepsy associated with ragged-red
fibers. YONEDA, M., TANNO, Y., HORIA, S., OZAWA, T.,
MIYATAKE, T. and TSUJI, S.
A mutation in the tRNALeu<UR R) gene associated with the MELAS
subgroup of mitochondiral encephalomyopathies. GOTO, Y.,NONAKA, 1. and HORIA, S.
Chronic progressive external ophthalmoplegia: A correlative studyof mitochondrial DNA deletions and their phenotypic expression in muscle biopsies. GOTO, Y., KOGA, Y., HORIA, S. and

87

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90

91

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

NONAKA, 1.
93
Renal tubular involvement mimicking bartter syndrome in a patientwith kearns-sayre syndrome. GOTO, Y., ITAMI, N., KAlIl, N.,
TOCHIMARU, H., ENDO, M. and HORAI, S.
94
Mitochondiral myopathies: Morphological approach to molecularabnormalities. SATO, T., NAKAMURA, So, HIRAWAKE, H.,
UCHIDA, E., ISHIGAKI, Y., SEKI, K., KOBAYASHI, R., HORIA, S.
and OZAWA, T
0........................ 95
In situ hybridization of muscle mitochondiral mRNA in mitochondrial myopathies. NAKAMURA, So, SATO, T., HIRAWAKE,
Ho, KOBAYASHI, Ro and HORAI, S.
96
A tenascin-1ike gene found in the human major histocompatibility
complex class III region. MATSUMOTO, K., ARAI, Mo, ISHIHARA, N., ANDO, A., INOKO, H. and IKEMURA, T. ..
96
Evident diversity of codon usage patterns of human genes withrespect to chromosome banding patterns and chromosome
numbers: Relationship between nucleotide sequence data andcytogenetic data. IKEMURA, T. and WADA, K ...
97
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Applied Genetics

Spatial pattern of genetic variation within wild rice populations.
MORISHlMA, H. and BARBIER, P .....
98
A nuclear gene inducing fertility restoration in cytoplasmic male99
sterile rice. SANO, Y. .
Developmental genetics of phenotypic plasticity in rice. EIGUCHI,
M. and SANO, Y. . ....
101
Population biology of resistance to bacterial leaf blight disease102
in rice. HAMAMATSU, C. and MORISHlMA, H.
Size-Shape variation of the silica body in wild and cultivated rices.
FUJIWARA, H., SATO, Y. I. and MORISHlMA, H
103
New DNA fingerprinting procedure: Amplified fragment length
polymorphism of hazy associations (ALPHA). NAKAMURA, I.
o'
105
DNA markers specific to subspecies of Oryza sativa detected by the
ALPHA procedure. NAKAMURA, I., KANDA, Ko and SATO,
Y. I.
106
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CONTENTS

IX.

ix

Database

Consturuction of nucleotide sequence data base. UGAWA, Yo,
HAYASHIDA, H., MIYAZAWA, So, TATENO, Yo and GOJOBORI,
To ..
o'
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108
Codon usage tabulated from the GenBank genetic sequence data.
WADA, K., WADA, Yo, DOl, H., ISHIBASHI, F., GOJOBORI, To
and IKEMURA, To
o. 108
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Author index
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GENERAL STATEMENT
Our institute was established 40 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofgenetics research by members of our institute has made it the center ofgenetic study in our country, and a major institution with world-wide
recognition. In addition, seven years ago the institute was reorganized
into a National Inter-university Research Institute. With the foundationin 1988, of the Graduate University for Advanced Studies consisting ofseven inter-university research institutes, our institute has admitted a number of graduate students every year in the Department of Genetics of the
Graduate School of Life Science.
Recent rapid progress in the field of genetics with newly developed approaches has greatly shifted the course of study in biology. The establishment of the DNA Research Center some years ago was a timely and proper
step to meet changing demands. Our institute houses the DNA Data
Bank of Japan (DDBJ), one of the three banks in the world that gather,store and distribute information on DNA sequences. DDBJ will continue
to act as a regional center for such activities. It is obvious, however, that
this and other actions taken by the institute, are far from sufficient. Withsuggestions and guidance from people in and outside the institute, I would
like to lead the institute into a more fruitful and progressive future.
Regarding personnel changes in the past year, Prof. T. Ohta, Head of
the Department of Population Genetics, was appointed as vice-director in
June, while Prof. Y. Kuroda (Laboratory of Phenogenetics) and Associate
Professors K. Tutikawa (Laboratory of Evolutionary Genetics) and S.
Iyama (Genetic Stock Research Center) retired at the end of March, Drs.
S. Hayashi and Y. Ugawa joined us as research members in the DNA
Research Center, where Drs. T. Ikemura and T. Gojobori were promoted
to professors and Dr. Y. Tateno to associate professor.
It is a pleasure to note that Prof. A. Ishihama was honored by the Mochida Memorial Award for Scientific Research for his contribution to thedevelopment of molecular studies on the regulation of gene expression.

STAFF
Director

TOMIZAWA, Jun-ichi, Ph. D.
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.
YAMAGISHI, Masahiro, D. Sc.
Laboratory of Mutagenesis
SENO, Takeshi, D. Sc., Professor
YAMAO, Fumiaki, D. Sc., Associate professor
TEzuKA,Hideo
KANEDA, Sumiko, D. Pharm.
Laboratory of Nucleic Acid Chemistry (Guest member)
MIZUMOTO, Kiyohisa, D. Sc., Associate professor
AYUSAWA, Dai, D. Ag., 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.
GOTo, Hideo, D. Ag.
Laboratory of Microbial Genetics
HORIUCHI, Kensuke, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
NISHIMURA, Yukinobu, D. Sc.

STAFF

HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Laboratory of Cytoplasmic Genetics (Guest member)
OHTSUBO, Eiichi, D. Sc., Professor
YONEKAWA, Hiromichi, D. Sc., Associate professor

3. Department of Ontogenetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
Laboratory of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
Laboratory of Phenogenetics
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Laboratory of Physiological Genetics (Guest member)
SAWADA, Yasuji, D. Sc., 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.
TAJIMA, Fumio, Ph. D.
Laboratory of Evolutionary Geneitcs
MORIYAMA, Etsuko, Ph. D.
Laboratory of Theoretical Genetics (Guest member)
KIMURA, Motoo, Ph. D., D. Sc., Professor
AOKI, Kenichi, Ph. D., Associate professor
5. Department of Integrated Genetics
IMAMURA, Takashi, D. Med., Head of the Department
Laboratory of Human Genetics
IMAMURA, Takashi, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate professor

3

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETIC; NO. 41

HORAI, Satoshi, D. Med.
NAKASHIMA, Hitoshi, D. Med.
Laboratory of Agricultural Genetics
MORISHIMA, Hiroko, D. Ag., Professor
SANO, Yoshio, D. Ag., Associate professor
SATO, Yoichiro, D. Ag.
HIRANO, Hiroyuki, D. Ag.
Laboratory of Applied Genetics (Guest member)
WATANABE, Takeshi, D. Med., Professor
YONEZAWA, Katuei, D. Ag., Professor

6.1. Research Facilities
Genetic Stock Research Center
MORISHIMA, Hiroko, D. Ag., Head of the Center
MIYASHITA, Nobumoto, D. Med.
WATANABE, Takao K., D. Sc., Associate professor
VEDA, Hitoshi, D. Ag.
NISHIMURA, Akiko, D. Ag.
TATENO, Yoshio, Ph. D., D. Sc.
l)}{A Research Center
SENO, Takeshi, D. Sc., Head of the Center
SHIMAMOTO, Nobuo, D. Sc., Associate professor
IKEMURA, Toshimichi, D. Sc., Professor
MATSUMOTO, Kenichi, D. Ag.
HIROSE, Susumu, D. Sc., Associate professor
HAYASHI, Shigeo, D. Sc.
GOJOBORI, Takashi, D. Sc., Professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
HAYASHIDA, Hidenori, D. Sc.
VGAWA, Yoshihiro, D. Sc.
KOHARA, Yuji, D. Sc., Associate professor
Radioisotope Center
SADAlE Yoshito, D. Sc., Head of the Center
Experimental Farm
SANO, Yoshio, D. Ag., Head of the Farm
NAKAMURA, Ikuo, D. Ag.

COUNCIL

7.

5

Technical Section
ONIMARU, Kimiharu, Chief of the Section

8. Department of Administration
HARA, Toshio, Head of the Department
KANDA, Tokio, Chief of the General Affairs Section
TANIGUCHI, Hiroshi, Chief of the Finance Section

COUNCIL
Chairman
NAGAKURA, Saburo; President, Graduate University for Advanced
StudiesVice-chairman
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
Members (Alphabetical order)
HAMAl, Osamu; Dean, Faculty of Letters, University of TokyoICHIKAWA, Atsunobu; Deputy Director, The National Institutefor Environmental Sciences
IINO, Tetsuo; Professor, Faculty of Human Science, Waseda Universtiy
IMAHORI, Kozo; President, Naruto University of Education
KIMURA, Motoo; Professor emeritus, National Institute of Genetics
KUWAHARA, Shogo; Director, Toho University
NOHMA, Shoshichi; Dean, Faculty of Pharmaceutical Science, Teikyo
University
OKADA, Tokindo S.; President, Okazaki National Research Institutes
ONOE, Hisao; President, Shiga University
Om, Tatsuo; Professor, Faculty of Home Economics, Kyoto Women's
University
OSAKI, Ritoshi; Director, Japan Society for the Promotion of Science
SANO, Hirotoshi; President, Tokyo Metropolitan University
SUGANO, Raruo; Director, Cancer Institute
SUGIMURA, Takashi; President, National Cancer Center
TAKANAMI, Mitsuru; Professor, Institute for Chemical Research,
Kyoto University

6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

TAKEUCHI, Ikuo; Director-General, Institute for Basic Biology
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University
YURA, Takashi; Director, Institute for Virus Research, Kyoto University

ADVISORY COMMITTEE
ChairmanMORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
Vice-chairman
OSAWA, Shozo; Professor, Faculty of Science, Nagoya University
Members (Alphabetical order)
FUKUDA, Ichiro; 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
TAKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto 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

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

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE OF GENETICS 7

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
President
KONDO, Norio; Professor emeritus, Tokyo University of Agriculture
Managing Director
MORIWAKI, Kazuo; Professor, National Institute of Genetics
SENO, Takeshi; Professor, National Institute of Genetics
Manager
TOMIZAWA, Jun-ichi; Director, National Institute of Genetics
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
YAMAGUCHI, Hikoyuki; Professor emeritus, University of Tokyo
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University ofTokyo
KURODA, Yukiaki; Professor emeritus, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
Trustee
TAZIMA, Yataro; Director, Institute of Silkworm Genetics and Breeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Professor, Faculty of Medicine, Teikyo University
SHIGETO, Gakuji, Manager, Japan Society for the Promotion of
Science
MATSUNAGA, Ei; Professor emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, Shokabo Publishing Co.
Auditor
KIMURA, Motoo; Professor emeritus, National Institute of Genetics
IMAMURA, Takashi, Professor, National Institute of Genetics
MORISHIMA, Hiroko, Professor, National Institute of Genetics
Adviser
MORIWAKI, Daigoro; Honorary member, National Institute of Genetics

PROJECTS OF RESEARCH FOR 1990
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular Genetics

Studies on regulatory mechanisms of gene transcription in prokaryotes
and eukaryotes (ISHIHAMA, FUJITA, NAGATA and YAMAGISHI)
Studies on molecular mechanisms of transcription and replication of animaland plant viruses (ISHlHAMA, NAGATA and NAKAMURA)
Laboratory of Mutagenesis

Molecular and genetic studies of cell-cycle associated genes (SENO, YAMAO
and KANEDA)
Genetic consequences of thymidylate stress imbalance in mammalian cells(SENO and YAMAO)
Radiation sensitivity in mammals (TEZUKA)
Laboratory of Nucleic Acid Chemisry

Molecular mechanisms of RNA 5'-cap formation (MIZUMOTO)
Studies on molecular mechanisms of transcription and replication of Sendai
virus (MIZUMOTO)
Phenotypic diversity of mammalian cell cycle mutants defective in ubiquitin-activating enzyme (AYUSAwA)
2.

DEPARTMENT OF CELL GENETICS
Laboratory of Cytogenetics

Studies on species differentiation of house mouse from genetic view points(MORIWAKI)
Immunogenetical studies on the mouse MHC (SHIROISHI, GOTOH and
MORIWAKI)PROJECTS OF RESEARCH FOR 1990

9

Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Genetic mechanism of meiotic recombination in mice (SHIROISHI, IMAI
and MORIWAKI)
Laboratory of Microbial Genetics

DNA replication in E. coli (YASUDA, HIGASHITANI and HORIUCHI)
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)
StudIes on bacterial plasmids and transposons (OHTSUBO)
3.

DEPARTMENT OF ONTOGENETICS
Laboratory of Developmental Genetics

Genetic and molecular analysis of developmental mechanisms in hydra(SUGIYAMA, FUJISAWA and SHIMIZU)

Laboratory of Phenogenetics

Genetic studies on the life history characters in Bombyx (MURAKAMI)
Genetic studies on the nerve system characters in Bombyx (MURAKAMI)
Studies on development and growth of insect (MINATO)
Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)
Genetic studies on maternal effect lethality in higher organisms (YAMADA)
Laboratory of Physiological Genetics

Theoretical models for pattern formation in hydra (SAWADA, SHIMIZU and
SUGIYAMA)10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

4.

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

Theoretical studies of population genetics (OHTA, TAKAHATA, TACHIDA
and TAJIMA)
Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (TAKAHATA and TAJIMA)
Population genetical studies on quantitative characters (TACHlDA)
Statistics for DNA polymorphisms (TAJIMA)

Laboratory of Evolutionary Genetics

Studies on molecular evolution on viruses (GOJOBORI and MORIYAMA)
Studies on molecular evolution of Drosophila genes (MORIYAMA)

Laboratory of Theoretical Genetics

Theoretical studies of population genetics and molecular evolution (KIMURA)
Population genetical studies on gene-culture coevolution (AOKI)5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Genetic and physical mapping of human genome (IMAMURA, FUJIYAMA
and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA and NAKASHIMA)
Molecular biology of oncogenes (FuJlYAMA)
Studies on DNA polymorphisms on human populations (HORAI)

Laboratory of Agricultural Genetics

Evolutionary genetics in wild and cultivated rice species (MORISHlMA, SANO
and SATO)PROJECTS OF RESEARCH FOR 1990

11

Genetic studies of speciation in rice (SANO and HIRANO)
Studies on plant gene expression (HIRANO and SANO)
Bio-archaeological analysis of plant remains (SATO and NAKAMURA)

Laboratory of Applied Genetics

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

6.

RESEARCH FACILITIESGenetic Stock Research Center

Theoretical studies in molecular phylogeny (TATENO)
Evoutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA)
Genetic and Molecular studies of cell division mechanism in E. coli.
(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)
Studies on genes in HLA locus (MATSUMOTO and IKEMURA)
Computer analysis of DNA sequences (GOJOBORI and VGAWA)
Construction of DNA sequence database (GOJOBORI, VGAWA, HAYASHIDA,TATENO and MIYAZAWA)
Sequence and structure analysis of DNA and proteins (MIYAZAWA and
HAYASHIDA)
Molecular genetics of insect development (VEDA and HIROSE)
Control of gene exprestion in eukaryotes (HIROSE, HAYASHI and VEDA)
Molecular genetics of development of Caenorhabditis elegans (KOHARA)

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41
Radioisotope Center

Radiation genetics of Caenorhabditis elegans (SADAIE)
Molecular mechanisms of sporulation in Bacillus subtilis (SADAIE)
Experimental Farm

Molecular genetics of plant development (NAKAMURA)
Genetical analysis of physiological traits in plants (NAKAMURA)RESEARCH ACTIVITIES IN 1990

I.

MOLECULAR GENETICS

The Promoter Selectivity of Escherichia coli RNA Polymerase, I.
Involvement of the C-Terminal Region of a Subunit
in Recognition of cAMP-CRP-Dependent Promoters
Kazuhiko IGARASHI, Richard S. HAYWARD*, Nobuyuki FUJITA
and Akira ISHIHAMAThe RNA polymerase of E. coli is composed of four different subunits.
The core enzyme with the subunit structure a2f3f3' is fully active in RNA
polymerization, whereas the binding of one of the various species of q
subunits is required for specific initiation at promoters. Subunit a is composed of 329 amino acid residues, and its major role has been proposed topromote assembly of the two large subunits, 13 and 13', into enzyme comlexes (reviewed in Ishihama, A. (1881) Adv. Biophys. 14, 1-35). In fact.
in a temperature-sensitive E. coli strain carrying the rpoAl12 mutation in
the gene encoding the a subunit, the subunit assembly is blocked at an
early step (Kawakami, K. and Ishihama, A. (1880) Biochemistry 19, 34913495). Through sequence analysis of PCR-amplified DNA, the rpoA112
mutation was found to cause a substitution of Cys for Arg at amino acidresidue 45, indicating that the N-terminal region of a plays an importantrole in subunit assembly (Igarashi, K. et al. (1890) Nucleic Acids Res. 18.
5945-5948).
To test this possibility, we constructed a set of C-terminal deletion mutations of the rpoA gene. C-Terminal truncated a subunits were synthesizedin vitro and examined for their assembly competence using an in vitro reconstitution system. N-Terminal fragments of 296, 256, or 235 aminoacid residues in length were found to retain the ability to form pseudo-corecomplexes, while those truncated at residues 176 or 150 did not form suchcomplexes (Igarashi, K., Fujita, N. and Ishihama, A. (1991) J. Mol. Bioi.
* On leave of absence from Institute of Cell and Molecular Biology, University of
Edinburgh, Edinburgh, UK.

14

ANNUAL REPOPT OF NATIONAL INSTITUTE OF GENETICS NO. 41

218, 1-6).
Studies of E. coli strains producing the same set of truncted a subunits
showed that the a-296, a-256 and a-235 species can even form pseudoholoenzymes in vivo (Hayward, R., Igarashi, K. and Ishihama, A. (1991)
J. Mol. Bioi., in press). However, both rpoAlOl (Arg-to-Cys substitution
mutation at position 191) and rpoA1l2 could not be complemented by theexpression of anyone of these truncated a subunits, implying that the Cterminal region plays a yet unidentified essential role in transcription.
We then overproduced the C-terminal deletion derivatives lacking 73
or 94 amino acid residues, assembled in vitro into enzyme molecules, andtested for biological functions associated with the reconstituted enzymes.
To our surprise, the reconstituted core enzymes were as active in poly (A)'poly (dT)-dependent poly(A) synthesis as the native core enzyme. Furthermore, with the addition of a 70 , these core enzymes were reconstituted
into holoenzymes, which exhibited transcription activity at the lacUV5,
trp, and rplJ promoters as well as the native holoenzyme. The mutant
holoenzymes were, however, unable to catalyze cAMP-CRP activatedtranscription from the lac and uxuAB promoters (Igarashi, K. and Ishihama,A. (1991) Cell 65, 1015-1022). These results demonstrate that the Cterminal region of the a subunit plays an essential role in recognition of
the promoters which require cAMP-CRP. The simplest interpretation
would be that the C-terminal region of the a subunit proveides a contact
site for CRP, which binds at a site upstream of the promoter -35 signal
on the promoters examined.
The Promoter Selectivity of Escherichia coli RNA Polymerase, II.
Comparison of Three Assay Systems of the Promoter Strength
Makoto KOBAYASHI, Kyosuke NAGATA and Akira ISHIHAMADNA sequence analyses of a wide variety of prokaryotic promoters haveindicated that the promoters for the major form of E. coli RNA polymerase
holoenzyme (Ea 70 ) are composed of two conserved hexanucleotide sequences,
TTGACA and TATAAT, which are located at 35 and 10 bp (base pairs),
respectively, upstream of the transcription initiation site. Previously, wedeveloped the in vitro mixed transcription assay system to determine twoparameters governing promoter strength, i. e., binding affinity to RNA
polymerase (parameter I) and rate of open complex formation (parameter

RESEARCH ACTIVITY IN 1990

15II) (for a review see Ishihama, A. (1988) Trends Genet. 4, 282-288).

To
reveal the role of individual bases within these two regions, we constructeda set of single base substituted variant promoters derived from the lacUV5
promoter, and determined their promoter strengths using the mixed transcription system (Kobayashi, M. et al. (1990) Nucleic Acids Res. 18, 73677372).
Since the abortive initiation assay is widely used for measurement ofpromoter strength, we also compared these two in vitro assay systems ofpromoter strength using a set of variant promoters with all possible substitutions within the promoter -35 region. No significant difference wasobserved in the promoter strengths determined by the two independent
methods.
Next, we attempted to measure promoter strength in vivo. For thispurpose, we inserted DNA fragments carrying the variant promoters into
plasmid pMS4342 at a site between the ara promoter and the lacZ gene.
Promoter strength in vivo was determined by measuring p-galactosidase
activity in the absence of arabinose. The results indicate that: i) the promoter containing the consensus sequence TTGACA at the -35 region
was the strongest in both in vitro and in vivo assays; and ii) promoter
strengths measured in vivo were generally in good agreement with thosemeasured in vitro, especially with the values of parameter I. Thus, atleast for the promoters examined, the strength in vivo appears to correlatewith binding affinity to RNA polymerase.
The Promoter Selectivity of Escherichia coli RNA Polymerase, III.
Structural and Functional Modulations of RNA Polymerase
during Phase Transition of Cell Growth
Miwako OZAKI, Nobuyuki FUJITA, Akira WADA* and Akira ISHIHAMAIn exponentially growing cells of E. coli, the synthesis of core enzyme
subuntis, lX, p and p', of RNA polymerase is coordinated and under growthrate-dependent control (for reviews see Ishihama and Fukuda (1980) Mol.
Cell. Biochem. 31, 489-496; Ishihama (1981) Adv. Biophys. 14, 1-35). This
coordination is, however, disrupted in stationary-phase cells and, as aresult, the newly systhesized subunits are degraded without being assembled,* Department of Physics, Faculty of Science, Kyoto University, Kyoto.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

even though RNA polymerase is one of the metabolically stable proteinsin exponetially growing cells. Last year, we reported that, during growthtransition of E. coli culture from exponential growing to stationary phase,the pre-existing RNA polymerase is modified into at least three different
forms, which can be separated by phosphocellulose column chromatography(for details see Ozaki, M. et al. (1991) Mol. Gen. Genet., in press).
In order to determine the altered functions associated with the stationaryphase RNA polymerases, we examined the promoter selectivity for thesemodified RNA polymerases using the in vitro mixed transcription systemand our collection of natural and systhetic E. coli promoters. Results
indicated that each of the three RNA polymerases carries different promoter
selectivity from that of the regular holoenzyme (Ea 7D ) from exponentiallygrowing cells.
As an attempt to identify the structural basis of the observed functional
modulation, we carried out holoenzyme reconstitution experiments fromthe core enzyme and a 7D subunit, which were isolated from each enzyme
form. The hybrid enzymes from the exponential phase a7D and the stationary-phase core enzyme showed essentially the same promoter recognition properties as those of the stationary-phase holoenzymes. This indicates that the core enzyme is modified during growth transition fromexponential to stationary phase. Modification of RNA polymerase might
be a major mechanism for the global control of gene transcription at least
during growth phase transition.
The Promoter Selectivity of Micrococcus luteus RNA Polymerase:
Identification and Characterization of the Major Sigma Factor
Manabu NAKAYAMA, Nobuyuki FUJITA, Syozo OSAWA· and Akira ISHIHAMA

The promoters of Micrococcus luteus, a bacterium whose chromosomal
DNA has a high G+C content (74 %), diverge from the consensus prookaryotic promoter in having GC-rich DNA sequences at less important
positions (Nakayama, M. et al. (1989) Mol. Gen. Genet. 218, 384-389).
In order to compare the promoter selectivity of RNA polymerase between
M. luteus and E. coli, we purified the enzme from both organisms andexamined their promoter recognition properties in the in vitro mixed tran-·•

Department of Biology, Faculty of Science, Nagoya University, Nagoya.

RESEARCH ACTIVITY IN 1990

17

scription system using promoter collections from both organisms.
The sets of E. coli and M. luteus promoters recognized by two purified
RNA polymerases were found to overlap partly. Some E. coli promoterswere correctly transcribed by the M. luteus RNA polymerase as well as theE. coli enzyme. To identify the structural unit involved in promoter recognition, we purified the M. luteu (] subunit with a molecular weight ofabout 60 kDa (Nakayama, M. et al. (1991) J. Bioi. Chem. 266, 2911-2916).
With the addition of either M. luteus or E. coli core enzyme, it was reconstituted into holoenzymes. Likewise, the M. luteus core enzyme wasreconstituted into a hybrid holoenzyme through the addition of the E.
coli (]70 subunit. Both hybrid holoenzymes were, however, able to initiate transcription only from a set of promoters which were recognized byboth of the native holoenzymes. These observations indicate that not
only the (] subunit but also the core enzyme participates in promoter
selection.
In addition to the (]60 subunit, we found a minor species of M. luteus (]subunit which differs from the major species in both molecular size andpromoter selectivity.
Cloning and Sequence Determination of the Schizosaccharomyces pombe
rpbl Gene Encoding the Largest Subunit of RNA Polymerase II
Y oshinao AzuMA, Masahiro Y AMAGISHI, Rei U ESHIMA and Akira ISHIHAMA

Extensive studies are being carried out on cis-acting DNA signals andtrans-acting protein factors involved in transcription regulation in eukaryotes. By contrast, little is known on the basic machinery of RNA
synthesis, except that three types of nuclear RNA polymerases, I, II, andIII, exist, each being composed of more that 10 different subunits. In
order to get detailed knowledge of the structure and function of RNApolymerases, we started cloning and DNA sequencing of the Schizosaccharomyces pombe genes encoding the subunits of RNA polymerase II.
This year, we cloned both the genomic DNA and cDNA coding for thelargest subunit using the corresponding gene, RPB1, of Saccharomyces
cerevisiae as a cross-hybridization probe, and determined the completesequence of genomic DNA and some of the peR-amplified cDNA (Azuma,
Y. et al. (1991) Nucleic Acids Res. 19, 461-468).
The predicted protein coding sequence, interrupted by six introns in the

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

genomic clone, encodes a polypeptide of 1,752 amino acid residues in lengthwith the molecular weight of 194 kDa. The polypeptide contains theeight structural domains conserved among the largest subunits of RNApolymerases from other eukaryotes. Both the metal binding motif in the
N-terminal proximal first domain and the DNA binding motif in the third
domain exist in the S. pombe protein. The C-terminal domain (CTD) is
composed of 29 repetitions of the heptapeptide motif, Tyr-Ser-Pro-ThrSer-Pro-Ser.
Northern analysis of RNA from exponentially growing cells showedthat the size of the rpbI transcript is about 5.6 kb in length. The sites oftranscription initiation and polyadenylation were determined by primer
extension experiments and cDNA sequencing. In a gene disruption experiment and Southern analyses of total S. pombe DNA, the rpbI gene was
identified as an essential gene consisting of a single copy on the S. pombe
chromosome.
The Promoter Selectivity of Influenza Virus RNA Polymerase:in vitro and in vivo Analysis of the Promoter Structure
Kunitoshi YAMANAKA *, Kyosuke NAGATA and Akira ISHIHAMA

The genome of influeza virus consists of eight single-stranded RNA segments of negative polarity. Both 5'- and 3'-terminal sequences of 12-13
nucleotides in length, which are complementary to each other, are conserved among the eight segments and various virus strains. In virus particles, RNA polymerase (a complex of three P proteins, PBl, PB2 and PA)
binds to each RNA segment at the stem of a panhandle-like structure(Honda, A. et al. (1987) J. Biochem. 102, 1241-1249), while the nucleprotein(NP) binds to viral RNA every 15-20 nucleotides (Yamanaka, M. et al.
(1990) J. BioI. Chem. 256, 11151-11155), altogether forming ribonucleoprotein (RNP) cores. In agreement with this structural model, transcription
in vitro catalyzed by RNP cores is initiated from the 3' terminus of each
RNA segment (Honda, A. et al. (1986) J. BioI. Chem. 261, 5987-5991).
These observations suggested that the promoter for transcription includes
the 3'-terminal conserved sequence of viral RNA.
Previous in vitro studies using isolated RNA polymerase and synthetic* Present address:

Kumamoto University School of Medicine, Kumamoto.

RESEARCH ACTIVITY IN 1990

19

model RNA templates showed that the 3' terminal conserved sequence isindeed needed for promoter activity (Parvin, J. et al. (1989) J. Viral. 63,
5142-5152). Mutations in this region more or less result in the reductionof promoter activity in vitro.
To examine the structure needed for in vivo promoter function, we established a transfection system of reconstituted RNP cores. For thispurpose, we first made cDNA for segment 8 RNA, replaced the coding
region for the nonstructural protein (NS) with the chloramphenicol acetyltransferase (CAT) gene, and inserted the resulting recombinant gene ata site downstream from the promoter for T7 RNA polymerase. The antisense CAT RNA was synthesized in vitro by transcribing this DNA with
T7 RNA polymerase, and reconstituted into model RNP cores by mixing
with viral proteins, RNA polymerase and NP, which were isolated frompurified RNP cores by CsCI centrifugation. Transfection of HeLa cellswith this reconstituted CAT RNP in the presence of the helper ribonucleoprotein cores led to significant expression of the recombinant CAT geneas detected by measuring plus-strand CAT mRNA using a combination ofreverse transcription and RCR, and also by immunostaining of the CAT
protein using monospecific antibodies (Yamanaka, K. et al. (1991) Proc.
Natl. Acad. Sci. USA, in press). This transfection system was used foranalysis of the promoter structure for influenza viral RNA polymerase.
Mechanism of Growth Inhibition of Influenza Virus by the Mouse Mx
Gene
Manabu NAKAYAMA, Kyosuke NAGATA, Yoichiro IWAKURA*,
Atsushi KATO** and Akira ISHIHAMA

The inbred mose strain A2G is resistant to infection by influenza virus.
This resistance has been shown to be determined by a single dominant allele
designated Mx, and both cDNA and genomic DNA encoding the Mx
protein have been cloned and sequenced. The resistance mechanism of
the Mx protein, however, remains known.
In order to reveal the action mechanism of the Mx protein, we examinedthe effect of Mx protein, induced in primary cultures of A2G mouse embryos* Institute of Medical Science, University of Tokyo, Tokyo.
•• Nippon Institute of Biological Science, Ohme, Tokyo.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

by interferon treatment, on each step of the growth cycle of influenza virus.
The absorption of viruses, the uncoating of envelope and membrane, andthe transport of viral RNA-protein cores from cytoplasm to nuclei all were
found to take place in the presence of the Mx protein as well as in its
absence. In contrast, transcription of viral genes, translation of viralmRNAs, and the production of infectious viruses were reduced by morethan 30 fold. These results indicate that the primary inhibition site for theinterferon-induced Mx protein is at the step of viral mRNA synthesis.
To examine in detail the mechanism of the Mx protein action, we purified
the Mx protein by successive chromatography on DEAE-cellulose, phosphocellulose, protein-pak 300 and SP-TOYOPEARL columns from both cell
extract from Escherichia coli expressing the Mx eDNA, and liver extractfrom A2G mouse previously treated with poly(I)'poly(C) for interferon
induction. The purified Mx protein showed GTP-binding activity with
GTPase activity. A mutation in the nucleotide binding motif reduced theactivities of both GTP binding and GTPase.
Expression of Human Thymidylate Synthase Minigenes:
Enhancer-like Activity in Intron 1
Sumiko KANEDA, Nobuyuki HORIE, Keiichi TAKEISHI, Dai AYUsAwA
and Takeshi SENO

Thymidylate synthase (TS) is a housekeeping enzyme responsible forthe formation of thymidylic acid in the de novo pathway. The expressionof the TS gene is cell-cycle regulated. The human TS gene is about 16
kilo bases in length and composed of seven exons and six introns. In
order to locate the essential sequence responsible for normal expression of
the gene, we constructed a series of minigenes, consisting of about 4 kilobases of the 5 -flanking sequence, 1.7 kilo bases of the 3'-flanking sequence,
and all the exons with or without the intron at normal positions. The
activities of minigenes were determined by transfecting each of them into
TS-negative thymidine auxotrophic mouse cell mutant FSthy21 and measuring the frequency of TS-positive stable transformants.
The minigene pmHTSI, which has intron 1 showed a high transforming
activity compared to that of pcHTSI, a human TS eDNA containing the
SV40 promoter region. However, the minigene pmHTSO which lacks all
the introns or pmHTS2 which has only intron 2 showed activity one-tenthfRESEARCH ACTIVITY IN 199021

and one-hundredth the level of that of pmHTSI, respectively. These
results suggested that intron I contains an enhancer-like sequence. The
enhancer activity was located in the 5' half of intron I and was active regardless of their position and direction. Notably, this enhancer has promoter specificity. The CAT activity was stimulated by the construct withthe 5' half of intron I and 5' flanking sequence of the TS gene, whereas theactivity was not stimulated with the SV40 promoter. This observation
indicates that the 5' half of intron I and 5' promoter region stimulate thetranscription of TS gene in a cooperative manner.
Cell-Cycle Directed Regulation of Human
Thymidylate Synthase Gene
Atsushi TAKAYANAGJ, Sumiko KANEDA, Dai AYUSAWA and Takeshi SENO

In order to locate the regulatory sequences responsible for cell-cycle
dependent expression of the human thymidylate synthase (TS) gene, weconstructed a series of minigenes lacking some or all introns, and related
chimeric genes partially replaced by corresponding SV40 promoter sequencesor 3' regulatory sequences in the Okayama-Berg expression vector. The
constructs were introduced into a TS-negative mutant of rat 3YI cells toestablish stable transformant lines.
Each transformant line was synchronized in the GOjG I phase by serum
starvation, and then stimulated to traverse into the S phase by the additionof serum. The level of TS mRNAs was compared between cells in GOjG I
and those in S phases by Northern hybridization analysis with a fragmentof human TS cDNA as a probe. The results showed that both genomic
5'-flanking sequences and intron 1 were responsible for the S phase-specificincrease of the mRNA. Each of the sequence alone was able to stimulate
the expression of the TS gene at S phase but to a lesser extent. The rate oftranscription of each transformant line was almost the same for the twophases as measured by a nuclear run-off experiment. Therefore, the cellcycle dependent expression of the TS gene is regulated posttrascriptionally.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

Mechanism of DNA Degradation during Thymidylate Stressin Mammalian Cells
Fumiaki Y AMAO, Nobuyuki IMAI and Takeshi SENO

Thymidylate stress is known to cause rapid cell death, so called thymineless death. In mammalian cells chromosomal DNA degradation occurs
during this stress, resulting in DNA fragments ranging from 50 to 200 kb
in length. Previous studies suggest that the DNA cleavage is coupled with
DNA replication. In order to investigate the molecular basis of the DNA
degradation, temperature-sensitive growth mutants of mouse mammary
carcinoma cell line FM3A were isolated by selecting thymineless death
resistants at high temperature (39.5°C). Among the ts mutants we screened
those whose chromosomal DNA is not degradable under thymidylate stresseven at a permissive temperature (33.5°C). Consequently, four mutants
were established and classified into two complementing groups by cell fusion
analysis. The mutated gene in one complementation group was identified
as that for ubiquitin-activating enzyme £1 since mutants were not complemented with the authentic £1 mutant. Previous analysis of £1 mutantsof mouse cell lines suggested that ubiquitination is somehow involved in
DNA synthesis and cell cycle regulation of the SjG2 phase. Thus, it could
be supposed that dynamic regulation of the chromatin structure during DNA
replication is coupled with ubiquitination of some nuclear protein (s) which
may be affiliated with chromatin structure of replication machinery. The
chromatin associated with the ubiquitinated protein (s) would be sensitive tonucleolytic activities during its replication under thymidylate stress, resulting in the instability of the chromosomal DNA.
Ubiquitin-Activating Enzyme as a Determinant of
Cell-Cycle Progression
Dai AYUSAWA, Sumiko KANEDA and Takeshi SENO

Ubiquitin is a conserved, small protein found in all eukaryotes. The
covalent ligation of ubiquitin to various cellular target proteins is implicatedin various cellular functions. Ubiquitin-activating enzyme £1 catalyzes
the first step of sequential transfer of ubiquitin to target proteins. However, it is unclear how each target protein is involved in a particular biological function, especially in terms of cell-cycle progression, due in partRESEARCH ACTIVITY IN 199023

to lack of genetic analysis.
Human ubiquitin-activating enzyme £1 was cloned by genetic complementation of a temperature-sensitive (ts) mouse FM3A cell mutant FS20
which has a thermolabile £1 enzyme, is defective in DNA synthesis, andarrested in the S phase upon shift-up in temperature. The cloned £1full-length cDNA (3,428bp) in a mammalian expression vector was able totransform FS20 to non-ts cells with a frequency of 10- 3 to 10- 4 per surviving cell. The determined nucleotide sequence of the cDNA encodes aputative protein of 108kDa, the amino acid sequence of which was confirmed in part by direct sequencing of the peptide fragments obtained fromthe purified human £1 protein. With cell-cell hybridization, FS20 did
not complement with the ts85 mutant which has been characterized as a
G2-specific cell-cycle mutant and to have the thermolabile £1 enzyme.
Thus, enzyme £1 appears to playa multiple role in cell-cycle regulation.
DNA Supercoiling and Eukaryotic Transcription-Cause and EffectSusumu HIROSE and TsuToMU OHTA

Both underwinding and overwinding of the DNA double helix induce
twisting and coiling of the helix unless the DNA strands can rotate freely.
The coils thus formed are termed negative and positive supercoils, respectively. An enzyme that changes the topological form of DNA is called topoisomerase. There are two types of topoisomerase in eukaryotes: DNA
topoisomerase I and II. Both of these enzymes can relax negative and positive supercoils but neither can introduce supercoils into relaxed DNA.
Though chromatin DNA in eukaryotic cells is topologically underwound,bulk DNA is not under superhelical tension because the supercoils are constrained in histone-DNA interactions. This does not necessarily exclude
the possibility that a small fraction of the chromatin has unconstrained
supercoils. Recent progress in the field of DNA topology highlights alinkage between unconstrained supercoils and eukaryotic transcription:
supercoiling affects transcription and transcription can cause supercoiling.
For details, see Cell Struct. Funct. 15, 133-135, 1990.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

Negative Supercoiling of DNA with Eukaryotic DNA
Topoisomerase II and Dextran Sulfate
Kohichi OKADA, Tsutomu OHTA and Susumu HIROSE

In the presence of a molar excess of eukaryotic DNA topoisomerase II
and an appropriate concentration of dextran sulfate, relaxed closed circular
DNA is converted to a negatively supercoiled form. The reaction isdependent on ATP. Neither adenosine 5'-[fi,r-imido]triphosphate nor
adenosine 5'-[r-thio] triphosphate can substitute for ATP. The negative
supercoils formed are relaxed with the subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of anicked circular DNA in the presence of DNA topoisomerase II and dextran
sulfate but in the absence of ATP, causes a small decrease in the linking
number. These results suggest that when appropriate concentrations ofdextran sulfate are present, the binding of a molar excess of eukaryotic
DNA topoisomerase II constrains a small number of negative supercoils
in DNA, which in turn generates unconstratined negative supercoils at theexpense of ATP. For detailes, see J. Biochem. 109, 365-369, 1991.
Core Promoter of the Mouse Myelin Basic Protein Gene Governs
Brain-Specific Transcription in Vitro
Taka-aki TAMURA*, Kohsuke SUMITA*, Susumu HIROSE and Katsuhiko MIKOSHIBA*

The core promoter of the mouse myelin basic protein (MBP) gene from-36 to + 12 was preferentially transcribed in brain nuclear extracts. Boththe TATA at -34 and downstream elements to +12, were required for
efficient, accurate and brain-specific transcription. From brain and liver
nuclear extracts, we have partially purified the general transcription factor
TFIID. The partially purified fractions contained TATA element binding
factors of the MBP promoter as well as adenovirus major late promoter(MLP). The tissue-derived TFIID was functionally exchangeable for the
HeLa TFIID, and directed transcription from the MLP. Surprisingly,
brain THIID activated transcription from the MBP core promoter whileliver TFIID did so to a much lesser extent. Exchange of the TATA-containing short DNA stretch to the MBP core promoter for a corresponding* National Institute for Basic Biology, Myodaiji-cho, Okazaki.

RESEARCH ACTIVITY IN 1990

25

region of the mouse albumin promoter or MLP abolished the brain specificity. We found that several tissue-specific promoters other than MBP,
such as mouse neurofilament and human a-I-antitrypsin promoters werealso transcribed much more efficiently with brain and liver TFIID, respectively. We suggest that different tissues contain functionally nonequivalent TFIID or TFIID-like activities. For details, see EMBO J.
9,3101-3108, 1990.
Identification and Purification of a Bombyx mori Homologue of FIZ-F1
Hitoshi VEDA and Susumu HIROSE

Extracts from embryos and from the posterior silk glands of the silkworm,
Bombyx mori contain a sequence specific DNA binding factor termed
BmFTZ-Fl. The factor binds to the recognition site of FTZ-Fl, a positive
regulator of the fushi tarazu gene in Drosophila melanogaster (Ueda, H.,
et al., 1990, Genes Dev. 4, 624-635). BmFTZFl and FTZ-Fl share thesame methylation interference patterns, the same chromatographic behaviors
and similar protease digestion profiles. Anti-FTZ-Fl cross-reacts with
BmFTZ-Fl. These results indicate that BmFTZ-Fl is a B. mori homologue
of FTZ-Fl. The mobility of the factor-DNA complex formed in the silk
gland extract changes depending on developmental stages. Purificationof BmFTZ-Fl to an almost homogeneous state reveals that the factor is
73 kd protein. For details, see Nucleic Acids Res. 18, 7229-7234, 1990.
Search for Target Genes of Drosophila Homeotic Genes
Shigeo HAYASHI and Susumu HIROSE

Homeotic genes of Drosophila play a central role in the specification ofbody segments. Genes that are controlled by homeotic genes, the target
genes, are not well understood. We are using a genetic and molecular approach to identify such genes. Formation of the anterior spiracle, which ispart of the tracheal system, is controlled by homeotic genes in the bithorax
complex. Study of genes required for the formation of the anterior spiracle
should lead to identification of the target genes. Using the "enhancer
trap" screen, we have identified three lines which express p-galactosidase
specifically in the precursor of the anterior spiracle. Using those lines,

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

genetic and molecular characterizations of genes expressed and requiredin the anterior spiracle are in progress.
Molecular Cloning offleabug, a Gene Controlling
Segmentation of Larva and Adult
Shigeo HAYASHI, Allan SHIRRAS and Susumu HIROSE

One of the enhancer trap lines #2605 has studied in detail. p-galactosidase
is expressed in stripes in every segment during mid embryogenesis andlater in the anterior spiracle. A lethal gene was identified which is closely
associated with the P-element insert and was mapped cytologically to polytenband 35C '" D. Loss of function mutations affect segmentation of thelarva and the adult abdomen. We are currently cloning the gene named
fleabug (fig) in collaboration with Dr. Allan Shirras at Lancaster University. So far, ",60 kb of genomic DNA flanking the P-element insert
has been cloned. Mapping of the fig mutation and fig transcript are in
progress.
Identification and Characterization of a Gene strawberry which is
Required for Organization of Sensory Organs
Shigeo HAYASHI, lira WATANABE and Susumu HIROSE

We have identified a new lethal gene strawberry located in the left arm of
the third chromosome. A loss of function mutation of the gene affects theassembly of cells composing the larval sense organ in the antenno-maxillarycomplex and the adult compound eye. In the larva, duplication and lossof the sense organ is observed. In the adult, fusion of several ommatidias
in the compound eye ocurred. Presumably the gene controls proper cell
division and differentiation of cells in external sense organs. About 17
kb genomic DNA flanking the gene has been cloned by virtue of a P-element
plasmid rescue. We are currently cloning the cDNA of the gene.

RESEARCH ACTIVITY IN 1990

27

Demonstration of Extrinsic DNA from Immune Complexes in the
Plasma of a Patient with Systemic Lupus Erythematosus
Kunihiko TERADA*, Eiji OKUHARA*, Yoshihiko KAWARADA*
and Susumu HIROSE

Antigen DNA isolated from immune complexes, present in the plasma of
three patients with active systemic lupus erythematosus, was cloned andsequenced. One clone, designated pKS7, was found to have a region homologous with that of the E. coli metK gene, and another, designated pKS8, hada region homologous with a sequence including the replication origin ofbacteriophage f1. A gel retardation assay revealed that pKS7 and pKS8
interacted with the patient's IgG fraction to form immune complexes. The
affinity-purified antigen DNA proved to originate from bacteria or a bacteriophage. For details, see Biochem. Biophys. Res. Commun. 174,323-330,
1991.
In vitro Recombination System Using Nuclear Extract from Mouse
Testis cells
Tsuyoshi KOIDE, Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Homologus recombination within the mouse MHC region during meiosis is not random, but occurs preferentially at restricted sites, so-calledrecombinational hotspots. So far, four different hotspots have been identified in this region. The molecular mechanism responsible for the highfrequency of recombination at the hotspots is not clear. Further studyof site-specific recombination at the molecular level requires an efficient
assay system. If an in vitro assay system is established, it can be used tosearch for protein factors which may interact with the hotspots and to
elucidate target DNA sequence. Thus, we are attempting to develop an invitro recombination system. To evaluate the recombination frequency, weconstructed plasmids, termed pBPlacRB, which contain an indictor gene,
lacZ, located between two identical DNA fragments harboring the recombinational hotspot identified between the Ab3 and Ab2 genes in the MHC.
A similar plasmid, named pBPlacCB, containing two different DNA fragments, one harboring the hotspot and the other irrelevant to the hotspot*

Department of Biochemistry, Akita University School of Medicine, 1-1-1 Hondo,
Akita.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

sequence, was also prepared as a control construct. We used nuclear
extracts from mouse testis cells to evaluate the homologous recombination
frequency between two DNA fragments in vitro. After incubation with thenuclear ectracts, the plasmids were used for transfomation of E. coli. deficient in lacZ activity. Plasmids with recombination between two DNA
fragments loop out the indictor lacZ gene. The recombinant and nonrecombinant plasmids can be easily scored on the basis of color of colonies.
As a result, approximately 10 % of colonies transformed by pBPlacRB
plasmids were scored as lacZ negative. More than 90 % of them wererevealed to be recombinants between two homologous DNA fragments.
In contrast, the control plasmid construct, pBPlacCB, exhibited a very
low frequency of lacZ negative colonies. Only 0.3 % of colonies werescored as lacZ-. These results indicated that this assay system is efficientfor evaluating homologous recombination frequency in vitro. At present,
we are preparing other plasmid constructs in order to test the feasibilityof using this system to reproduce the site-specificity of recombination isobserved in in vivo within the mouse MHC region.
Gene Expression at the wx Locus of Rice (Oryza Sativa)
Hiro-Yuki HIRANO and Yoshio SANa

The wx locus controls amylose synthesis in rice endosperm. The amylose
content in total starch greatly affects the quality of rice. Mutants at thewx locus are easily screened by seed phenotype and the genotype of the locus
is determined by the I 2/KI staining of the pollens. Thus the gene at the wx
locus is one of the best genes for molecular genetic research among the genesof higher plants.
We cloned the wx+ gene from rice and determined the entire sequence ofthe coding region. The deduced amino acid sequence indicated a putativetransit peptide of 77 amino acids and a mature protein region of 532 aminoacids. The putative transit peptide, which may be essential for transporting
the product of the wx+ gene (wx+ protein) into the amyloplast, has somecharacteristic features similar to those of the transit peptide associated withproteins to be transported into the chloroplast.
An antiserum against the rice wx+ protein (anti-WXP serum) was preparedand the expression of the wx locus was examined using DNA cloned fromthe wx locus and the anti-WXP serum. The wx+ gene was expressed spe-

RESEARCH ACTIVITY IN 1990

29

cifically in only the endosperm and pollen, although the amount of the wx+
protein in pollen was about 2 % of that in the endosperm. The wx+ gene
was active at the early stage and middle stages but almost inactive at the latestage of seed development. During active transcription of the gene, thewx+ protein and total starch appear to accumulate linearly. The level of
the wx+ protein in plants grown at semi-low temperatures (l8°C) during
seed maturation was higher than that of plants grown at normal temperatures (28°C). The endosperm grown at 18°C contained an elevated level ofamylose content. This suggests that the low quality of rice grown at low
temperature in the northern area of Japan is due to a higher amylose content,
which is caused by the enhancement of wx+ gene expression.
Cloning and Characterization of Restriction Fragments Homologous
to B-1 Sequences in Cytoplasmic Male Sterile Rice
Masa-Aki YAMADA

Cytoplasmic male sterility (ems) has been reported in various higher
plants. Two supercoiled circular DNAs, B-1 and B-2, were found in the
mitochondria of a cytoplasmic male sterile strain in rice, and cloned into
plasmid pUC 12. Using these as probes, it was previously reported thathybridization with restriction fragments revealed the presence of various
sequences homologous to B-1 and B-2 in mitochondrial DNA and in nuclear
DNA, both in the ems strain and in the normal strain (Nawa et al., 1987).
DNA fragments of length 5.1 kb homologous to B-1, produced by digestion with EeoRI and Hind III of the mitochondrial DNA from the ems
strain, were respectively cloned into plasmid pUC 13. Digestion by Hinc
II of the 5.1 kb EeoRI fragment (EM5-1I3) produced two fragments, oflength 1.6 kb homologous to B-1 and of length 3.5 kb fragment (EM5113-1) not homologous to B-1. Digestion by EeoRI of the 5.1 kb Hind
III fragment (HM5-32) produced two fragments, of length 0.4 kb homologous to B-1 and of length 4.7 kb not homologous to it.
To test for contaminations of nuclear DNA into mitochondrial DNA
during the preparations of the mitochondrial fraction, digests with EeoRIof DNAs from the nuclear fraction and the mitochondrial fraction of the ems
strain were analyzed by Southern hybridization with EM5-1I3-1 fragments,
which were not homologous to B-1. Sequences homologous to EM5113-1 fragments were present in the digests with the EeoRI of both nuclear

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

DNA and mitochondrial DNA, but in the nuclear DNA were present at
lower levels. In the digests with EeaRl of DNAs from the nuclear fraction
and the mitochondrial fraction of callus cells, additionally cultuted inshaking culture for 2 months, sequences homologous to EM5-113-1 were
present in both DNAs, but at lower levels in the nuclear DNA, while inthe case of sequences homologous to B-1, a weakly labeled band was detected in nuclear DNA but undetectable in mitochondrial DNA.
These results suggested that the EM5-ll3 fragments detected in the mitochondrial DNA were not contaminations of the nuclear DNA, and thesequences homologous to B-1 in the mitochondrial genome DNA varied in
the state of callus cells cultured.

RESEARCH ACTIVITY IN 1990

II.

31

MICROBIAL GENETICS

Mutational Analysis of the Minus Strand Origin ofBacteriophage f1
Nahoko HIGASHITANI, Amy ROTH*, Atsushi HIGASHITANI and Kensuke HORIUCHI

Upon infection, the single-stranded DNA of bacteriophage fl is converted
to double-stranded replicative form (RF) through synthesis of the minus
strand, which is carried out by the combined action of RNA polymerase
and DNA polymerase III of the host. The (-) strand origin has been
defined as the region of single-stranded DNA that is protected by RNA
polymerase against nuclease digestion in the presence of E. coli single strand
binding protein (SSB). The protected region contains two hairpin structures [B] and [C] (Gray et al. (1978) Proc. Natl. A cad. Sci. USA. 75, 5053). An RNA primer is synthesized from a site downstream (in terms ofthe template DNA strand) of the hairpin [C] by RNA polymerase (Geider
et al. (1978) Proc. Nat!. Acad. Sci. USA. 75, 645-649). However, this
5950

5~O

Wild Type

5700
I

3'

5'

R36S
R377
R410
R411
R412
R413
R227
R414
R41S
R416
R417
R41S
R419
R420
R421

Fig. 1.

* Rockefeller University.

Mutations of the minus strand origin.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41
Table l.

Activities of mutant origins
in vivowild-type
R368
R377
R410
R411
R412
R413
R227
R414
R415
R416
R41/
R418
R419
R420
R421

in vitro

Conversion
to RF( - ) ori plasmid
replication

primer RNA
synthesis

+
+

+
+

+
+

+
+

+
+

+
+

+

+

+

+

+

+

+
+

,+

+
+

+

+

+

region does not contain any sequences similar to the consensus sequence fortranscriptional promoters.
In order to determine which sequence(s) is essential for the origin function,
we constructed a number of deletion and insertion mutants in hairpins[B) and [C] (Fig. 1). The mutants were examined for activity of the origin.
We used three assays: (1) rate of conversion of 32P-labeled phage DNA todouble-stranded RF upon infection, (2) novel in vivo system of plasmid
replication that is dependent on the function of the phage ( - ) strand origin,and (3) an in vitro reaction of primer synthesis, which consists of viral singlestranded DNA, E. coli RNA polymerase and E. coli SSB.
The results of all three assays were consistent with each other (Table 1),
and suggested that [1] deletion of regions in the 3' end of hairpin [C], whichcontain the initiation point of the primer RNA, does not seriously affectthe ( - ) strand origin function, [2] the entire region of hairpin [B) is essential,
and [3] the distance between hairpins [B) and [C] is important.
We thank Drs. A. Ishihama, K. Igarashi, N. Shimamoto, and K. Takahashi (National Institute of Genetics) for providing us with purified RNA
polymerase and SSB.

RESEARCH ACTIVITY IN 1990

33

SOS Induction in E. coli by Infection with Mutant Filamentous
Phages that are Defective in Complementary Strand Synthesis
Nahoko HIGASHITANI, Atsushi HIGASHITANI and Kensuke HORIUCHI

SOS responses in E. coli are controlled by a complex circuitry involving
the RecA and LexA proteins. When DNA in a cell is damaged, an inducing signal is generated, which activates the RecA protein. The activated
RecA protein causes proteolytic cleavage of the LexA protein, and thisleads to derepression of the SOS genes, which are repressed by the LexA
protein. In vitro studies have revealed that the RecA protein is activatedwhen it forms a ternary complex with single-stranded DNA and ATP(Craig, N. L. et al. (1980) Nature 283, 26-30). In vivo, however, it hasremained unclear what constitutes the inducing signal.
The fl mutant phage R377 lacks the entire region of the minus strand
origin, and thus is defective in complementary strand synthesis (see theaccompanying report). We found that E. coli cells infected with R377
formed filaments, while those infected with wild-type fl did not. It is known
that E. coli cells exposed to SOS-inducing treatment continue to divide,
but are inhibited from septating, and thus form filaments (Gottesman, S.
et al. (1981) J. Bacterial. 148, 265-273). Whether R377 infection causes
the SOS induction was studied using an E. coli test strain that carried alacZ gene under the control of a promoter of an SOS-responsive gene
(dinD). The activity of j3-galactosidase in this strain increased by infection
with R377, but not with the wild-type fl phage. To further confirm thispoint we measured the level of RecA protein in extracts of phage-infected
cells using the anti-RecA antibody. The results of Western blotting experiments showed that R377, but not the wild-type fl, induced synthesisof the 40 kDa RecA protein. The activated RecA mediates proteolytic
cleavage of the lambda repressor, which in turn induces growth of lambda
increased by infection of lambda lysogens by R377.
Another (-) ori deletion mutant, M13.JEIOI, has been described byKim et al. ((1981) Proc. Natl. Acad. Sci. USA. 78, 6768-6788). They
have also constructed M13GorilOl, in which the (-) strand origin of G4,
another single-stranded DNA phage, was inserted into M13.JElOl. We
found that infection with M13.JElOl results in the SOS induction in E.
coli, while infection with M13GoriiOI did not.
From these results we conclude that infection of E. coli cells with mutant

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

filamentous phages which are defective in the origin of minus strand synthesis induces the SOS functions. This observation strongly suggests thatstingle-stranded DNA by itself induces the SOS response in vivo.
Replication of Filamentous Phage f1: Relationship between DNA
Topology and the Nicking Activity of the Initiator Protein (gpII)
Atushi HIGASHITANI and Kensuke HORIUCHI

Gene II protein (gpII), which is coded for by the filamentous phage fl
genome, is a multifunctional protein that participates in DNA replication
at a number of levels. It introduces a single-strand break at a specificsite on the plus strand of negatively supercoiled replicative-form (RF-I)(Meyer et al. (1979) Nature 278, 365-367). The 3'hydroxyl end of thenick serves as the primer for initiation of plus strand rolling circle replication (Gilbert & Dressler (1968) C.S.H. Syrnp. Quant. BioI. 32, 473-484).
gpII functions also at a step beyond nicking; unwinding of origin DNA at
the initiation step (Geider et al. (1982) J. BioI. Chern. 257, 6488-6493), andcleavage and circularization of the displaced single strand at the termination
step (Harth et al. (1981) Eur. J. Biochern. 119, 663-668). When incubatedwith gpII in vitro, approximately 60 % of RF I molecules are nicked toyield RF II, while the other 40 % are converted to a relaxed, closed form
(RF IV) as the result of nicking and joining (Meyer & Geider (1979) J.
BioI. Chern. 254, 12642-12646). Thus, gpII has a sequence-specific topoisomerase activity.
In vivo studies have shown that E. coli IHF (integration host factor)
stimulates filamentous phage DNA replication approximately 30-foldthrough its action on the replication enhancer sequence (Dotto et al. (1984)
J. Mol. BioI. 172, 507-521; Johnston & Ray (1984) J. Mol. BioI. 177, 685700; Greenstein et al. (1988) Proc. Natl. A cad. Sci. USA. 85, 6262-6266).
Several enhancer independent mutants of gpII have been isolated, whichallow wild-type levels of replication in the absence of either the enhancer
sequence or IHF (Dotto & Zinder (1984) Nature 311, 279-280; Kim & Ray(1985) J. Viol. 53, 871-878). Two of these mutants were mapped at codon
40 and 73, respectively. Both M40I (Met40~Ile) and G73A (Gly73~
Ala) mutations increase the co-operativity with which the protein binds tothe origin to form a functional complex for the nicking reaction (complex
II: Greenstein & Horiuchi (1990) J. Mol. Bio!' 221, 91-101; Higashitani

RESEARCH ACTIVITY IN 199035

et al. (1990) Ann. Rep. N. I. G. 40,43-44).
In this study, in order to determine the level of negative-supercoil of RF I
required for the nicking reaction, a series of RF-topoisomers with different
negative-superhelical densities was prepared and used as substrates. The
results shown in Fig. 1 demonstrate that the in vitro nicking reaction by thewild-type gpII requires a high level of RF I superhelicity, while an enhancerindependent mutant version, G73A, does not require such a high level ofsupercoiling. When substrate DNA has a negative-superhelical density of
0.025, which corresponds to the value estimated for the in vivo superhelicaldensity in E. coli (Bliska & Cozzarelli (1987) J. Mol. Bioi. 194, 205-218),
the wild-type gpII converted about 35 % of the substrate DNA to RF II
and 65 % to RF IV. The G73A mutant gpII converted this substrate to a
mixture of 80 % RF II and 20 % RFIV under the same experimental conditions (Fig. 1). When the substrate consisted of completely relaxed circles,
G73A gpII still converted about 35 %of the molecules to RF II. The wildtype gpII converted only 8 % of this substrate to RF II (Fig. 1). Furthermore, the G73A gpII showed an approximately 5-fold greater nicking
activity compared to the wild-type gpII on a linear DNA fragment containing the origin.
The nicking activity of gpII on purified f1 DNA was not affected by theaddition of IHF, regardless of the superhelical density of the substrate.
How do the IHF protein and the enhancer sequence enhance the replication
offl DNA in vivo? One possibility is that IHF may reduce the requirement(%)

negative superhelical density
0.2
1.5
3.0
5.4

(x101

100

DRFIV
I RFII+ : wild type gpll
M: G73A

Fig. 1. Reduced superhelicity requirement in the nicking activity of the mutant
gpII.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

for negative superhelicity, as has been suggested for the strand transfer
reaction of the phage Mu in vitro (Surette & Chaconas (1989) J. BioI. Chern.
264, 3028-3034). The only known step in fl DNA replication that requires
superhelicity is the initial nicking reaction by gpII. Stimulation of fl
DNA replication in vivo by the replication enhancer sequence and IHF
is well established, but the conventional in vitro replication system forphage DNA is not affected by either the enhancer sequence or IHF. A
possilbe explanation for this would be that there is a factor(s) in the cellwhich is necessary for manifestation of the IHF effect and that the factor(s)is missing in the in vitro system. Such a factor(s) can be an IHF-dependent
stimulator of replication or an inhibitor that can be nullified by IHF. Identification of the factor(s) requires further study.
Cloning and Nucleotide Sequence of the ispA Gene Responsible for
Farnesyl Diphosphate Synthase Activity in Escherichia coli
Shingo FUJISAKI"', Hiroshi HARA, Yukinobu NISHIMURA,
Kensuke HORIUCHI and Tokuzo NISHINO**

Escherichia coli ispA strains have temperature-sensitive farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity, and the defective gene is locatedat about min 10 on the chromosome. The wild-type ispA gene was subdoned from a A phage clone containing a chromosomal fragment aroundmin 10, which we picled up from the aligned genomic library of Kohara
et al. [Kohara, Y, Akiyama, K. & Isono, K. (1987) Cel/50, 495-508]. The
doned gene was identified as the ispA gene on the basis of recovery andamplification of FPP synthase activity in an ispA strain. A 1,542-nucleotide
sequence of the cloned fragment was determined. This sequence specifies
two open reading frames, ORF-1 and ORF-2, encoding proteins with the
expected molecular weights of 8,951 and 32,158, respectively. A part ofthe deduced amino acid sequence of ORF-2 showed a similarity to thesequences of eucaryotic FPP synthases and the crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of thelac promoter complemented the defect of FPP sunthase activity in the'"*'"

Department of Chemistry, Faculty of General Education, Gifu University, Gifu,
Gifu.
Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku
University, Sendai.

RESEARCH ACTIVITY IN 1990

37

ispA mutant. This implies that the product encoded by ORF-2 is theispA product. Maxicell analyses indicated that a protein of molecularweight 36,000, approximately consistent with the molecular weight of thededuced ORF-2-encoded protein, is the gene product.

Kinetic Study on Transcription by Immobilized Operons: Requirementfor {J, r-pyrophosphate Bond of ATP in Initiation
by Prokaryotic RNA Polymerases
Hiroyuki TERADA* and Nobuo SmMAMoTo

The requirements ofr the 13, r-pyrophosphate bond of ATP in transcription
initiation by prokaryotic RNA polymerases has long been questioned.
We devised a novel method for showing these requirements and this method
enabled us to substitute substrates during elongation without changing theionic atmosphere.
A DNA fragment containing an operator and a promoter was fixed toacrylamide beads at its ends. The immobilized DNA was a template as
good as free DNA for E. coli, bacteriophage T7, T3, and SP6 RNA polymerases. Transcription was started by adding a set of substrates, and theninterrupted rapidly diluting substrates to 0.1 pM. Elongation was resumed
by adding the second set of substrates. Using either ATP or an ATP
analogue as one of the four substrates, we clarified that the (3, r-pyrophosphate bond of ATP increased the yield of long transcripts in the early stageof elongation but did not affect it much in the late stage. Moreover, abortive initiation was activated with such an ATP analogue. The requirement
for intact ATP in productive initiation was found in all the transcription
systems examined. Thus ATP is likely to be used to keep the elongation
complex in productive form in the early preiod of initiation.

Single-Molecule Dynamics of Transcription by Immobilized Operons:
Sliding of RNA Polymerase on DNA
Nobuo SHIMAMOTO

The first step of transcription is the search for and binding of RNApolymerase to a promoter, and this promoter search is the major determinant* Faculty of Meeicire Kyushu University.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

in selective expression of genes. The enzyme could directly bind to a promoter on a long DNA, or alternatively, first bind to DNA in a nonspecificmanner and then form the promoter complex by one-dimensional diffusion
along the DNA chain. Unfortunately, kinetic evidence for both of theconflicting models has been reported and further study should be based on
more confident evidence. The immobilized operon provides a criticallyimportant method for determining which case is true. Linear DNA immobilized on a slide glass was prepared. Fluorescently labeled RNA
polymerase was added to the linearly fixed DNA. The movement of asingle molecule of RNA polymerase was followed through microscopy andrecorded on a video-tape. The one-dimensional movement was detected
and its contribution to the promoter binding is now being evaluated.
Structural Study on E. coli Single-Stranded DNA Binding Protein
Nobuo SHiMAMOTO, Taketomi TSUKIHARA*, Khoichi TAKAHASHI*,
and Katsuhiko KAMATA*

E. coli Single-Stranded DNA Binding Protein (SSB) is a 19kda protein

and essential to replication and recombination. Under physiologicalconditions, it exists as a tetramer and binds to both single-stranded DNA
and a group of mRNAs which have a homologous RNA element called
SSB box. Both of the bindings are cooperative and the protein molecules
form clusters on such nucleic acid molecules. These bindings suggest a
coupling between replication and translation by exchanging SSB between
replication forks and mRNAs.
As a first step fo understanding the mechanism of these characteristic
bindings, the protein was over-produced and purified to homogeneity.
Crystals of the protein were formed with ammonium sulfate or ethanol
and a larger crystal suitable for X-ray crystallography is being developed.
The protein seems to have two structural domains although it is a small
peptide.

*Faculty of Technology Tottori University.

RESEARCH ACTIVITY IN 1990

III.

39

MAMMALIAN GENETICS

Genetic and Geographical Differentiation of Mus musculus Subspecies
in China
Kazuo MORIWAKI, Nobumoto MIYASHITA, Hitoshi SUZUKI*,
Kimiyuki TSUCHIYA**, Hiromichi YONEKAWA***, Cheng-huai WANG****,
Xin-quao HE***** and Mei-Iei JIN*****

Since 1984, genetic and geographical surveys of house mouse, Mus musculus, in China have been carried out. Molecular genetic data on the
Chinese wild mice accumulated so far indicated that Mus musculus musculus subspecies inhabit areas north of the Chang Jiang River and M. m.
castaneus to the south of it. To confirm this genetic and geographicaldistribution more precisely, a fifth survey on Chinese wild mice was carriedout in December, 1990. RFLPs of their ribosomal and mitochondrial
DNAs were analysed using wild mice collected from various areas in China,
as summarized in Table 1.
As already indicated by our previous survey, the Chang Jiang River canbe seen as a good geographical barrier between musculus and castaneus
subspecies in the eastern half of China. In the western China, castaneus
type mitochondrial DNAs seem to ne predominant, suggesting a possibility
that in ancient times, the castaneus subspecies had inhabited this area andlater the area was invaded by the musculus subspecies, especially theirmales, from the eastern region. The intersubspecies hybrid whose nuclear
genome is the musculus type and mitochondrial genome is from the castaneus subspecies, seem to be quite similar to the molossinus subspecies inJapan as already reported by us (Yonekawa et al., 1988).

*
**
***
****
*****

Jikeikai Medical University, Tokyo.
Miyazaki Medical College, Miyazaki.
Tokyo Metropolitan Institute of Medical Science, Tokyo.
Lanzhou Institute of Biological Products, Lanzhou.
Shanghai Laboratory Animal Center, Shanghai.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41
Table 1. Geographical distribution of RFLPs in the ribosomal and mitochondrial
DNAs as subspecies markers
Place of collection
Western China
Aksu (Xinjian Uygur prov.))
Tacheng (")
Kashgar ("II
Urumqi ()
Manasu ()"
Lasa (Tibet prov.)
Northern China
Xinin (Qinhai prov.)
Jiayugan (Gansu prov.))
Lanzhou ("Yinchuan (Ningsia prov.)
Chengdu (Sichuan prov.)
BeijinYangchou (Anhwei prov.)
Changchun (Jilin prov.)
Mohe (Heilung Kiang prov.)
Southern Chin
Shanghai
Zheknjian (Jiangsu prov.)
Kunrning (Yunnan)
Chongqing (Sichuan prov.)
Guilin (Gangxi prov.)
Hoikou (Hainan Is.)
Sanya ("

Subspecies specificrDNA with EcoRI

type of RFLPs in:
mtDNA with BamHI

MUS
MUS
MUS
MUS/CAS
MUS
MUS

CAS
CAS
n. t.
CAS
MUS/CASn. t.

MUS/CAS
MUS
MUS
MUS
MUS
MUS
MUS
MUS
MUS

MUS
MUS
n. t.
MUS
MUS
MUS
n. t.
MUS
MUS

MUS
MUS/CAS
CAS
CAS
CAS
CAS
CAS

MUS
n. t.
CAS
CAS
CAS
n. t.
n. t.

A High Incidence of Visible Mutation in Intra-MHC Recombinants
Toshihiko SHIROISHI, Tomoko SAGAI and Kazuo MORIWAKIThe BlO.MOL-H-2 lines are congenic strains which carry the MHC
region derived from Japanese wild mouse. We have produced a number
ofintra-MHC recombinants from crosses between one ofsuch BlO.MOL-H-2
lines, BIO.MOL-SGR, and standard inbred strains, BlO.A and BlO. We
found that these recombinants yielded multiple visible mutations. So far,six independent mutations have been detected in screening of approximately
2500 mice. Mating experiment indicated that three of them are recessive

RESEARCH ACTIVITY IN 1990

41

single mutations and the remaining three are dominant single mutations. In
mice, it is reported that the average rate of recessive and dominant visible
muctation is 3.8 X 10- 6 and 4.4 X 10- 7 per locus per gamete, respectively.
Thus, the rate of visible mutation observed in the intra-MHC recombinantis extremely high compared to the standard value. Further genetic analysis revealed no clear likage of the mutated genes with the MHC on chromosome 17. Because parental strains, BIO.MOL-SGR and standard
inbred strains have not yielded mutations, the high incidence of the mutation is a unique genetic trait peculiar to intra-MHC recombinants. In
order to reveal the molecular mechanism of this high incidence of themutation, we are attempting to map the mutated genes by an analysis oflikage of mutation phenotypes with a number of multilocus DNA markers,
such as endogenous murine leukemia proviruses.
Rescue of Lethal Adrenal-dysgenic Mice through Transgenesis
Hideo GOTOH, Toshihiko SHIROISHI and Kazuo MORIWAKIThe chromosome of an H-2 haplotype aw18 has a recessive lethal deletionencompassing genes for the steroid 21-hydroxylase and the complement
component C4 in the class III region. Homozygotes for the aw18 haplotype
die within two weeks after birth.
Following chemotherapy experiments in which crude afrenal homogenate
was injected into such mice postnatally, an adult-aged aw18 homozygous
female was obtained. The results of these preliminary experiments indicated that the cause of the lethality is adrenal dysfunction. The deletion of
the steroid 21-hydroxylase gene is thought to be the most probable causeof this lethality.
To save the life of aw18/aw18 homozygote, the trait of a transgenic mouse
was adopted. We prepared a 6.6 kb recombinant DNA fragment whichis constituted from a MMTV-LTR sequence and the murine steroid 21hydroxylase genomic gene. Using this DNA fragment, we produced six
transgenic mice, and four of them were fertile.
The exogeneous transgene was introduced into the mutant mouse genome
through mating. Firstly, a transgenic +/+ mouse was crossed with aaw18/+ heterozygote to obtain a transgenic aw18/+ moues. That mouse
was then crossed with a aw18/+ heterozygote. A transgenic aw18/aw18
mouse should be produced with a second cross. Several crosses have been

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

() d

II~jj

OODD(I[j0: H-2+/+(I: H-2..,,18/+.:..,,18/. .,,18H-2. '--. .,~+..•...-,-"j,...-.rI-r()1

od()

()

...;'T""I-(1"-1-(lIr-•

o:
o:
o• :

femalemaletransgenic

Fig. 1. Generation of transgenic awlS/awlS mice.

performed for each transgenic lineage, and the genotype of offspring wasdetermined at 14 days after birth by Southern analysis. Among the progenyof two independent transgenic lines, several live aw18 homozygotes carrying
the transgene have been obtained. A pedigree tree illustrating the production of transgenic aw18/aw18 mice is shown in Fig. 1. All the transgenic
aw18/aw18 mice surviving the first two weeks of postnatal life have shownnormal development to date.
These results showed that the deficiency of steroid 21-hydroxylase is thecause of the lethality of aw18 homozygotes, and demonstrated the potentialof gene therapy through transgenesis. This is the first demonstration of
the genetic rescue of a lethal mutation in mammals. Further analyses are
now being undertaken.

RESEARCH ACTIVITY IN 1990

43

Genetic Control of Susceptibility to Urethane-Induced
Lung Tumor in Mice
Nobumoto MIYASHITA and Kazuo MORIWAKI

Many kinds of inbred, H-2 congenic, recombinant inbred (RI) strains,a newly-established strain derived from Japanese wild mice, M.MOLTEN2, and the crosses among these strains of mice were investigated fortheir susceptibility to the induction of lung tumor by urethane. In H-2
congenic strains with a C57BL/IO (BIO) background, genetic polymorphisms
between K-E(3 and E(3-D contributed to differences in susceptibility to thedevelopment of the lung tumor. In the H-2-matched crosses between
lung tumor sensitive A/WySnJ and the resistant BIO.A/SgSnJ mice, theresistant BlO.A phenotype behaved as a completely recessive. This indicated the presence of a single recessive major gene conferring resistanceto the induction of lung tumor in the BlO.A strain. This gene was identicalto those already reported as 'pulmonary tumor resistance (ptr)' or 'pulmonary adenoma susceptibility-l (Pas-J)'. In the crosses between theintermediately tumor-sensitive BALB/cByJ strain with the resistant C57BLI
6ByJ strain and in CXB RI strains, more than 2 genes contributed to differences in susceptibility between these strains. Experiments on the
crosses of the M.MOL-TEN2 strain, which was derived from wild mice(Mus musculus molossinus), with A/J mice indicated that the allelic differences
for at least 2 genes contributed to resistance to the induction of lungtumor. These genes were epistatic to A/J Pas alleles.
Amplified EF2 Related Sequence (MER) in Mouse Genome
Tsuyoshi KOIDE, Toshihiko SHIROISHI, Masahiro ISHIURA* and Kazuo MORIWAKIElongation factor 2 (EF2) is an essential protein in the steps involved in
the elongation of polypeptide chains during protein synthesis in eukaryotes.
We have found that cells from standard laboratory mouse strains contain
about 70 copies of amplified EF2-related sequences, called MERs, per
haploid genome, in addition to a single copy of the EF2 gene. To characterize the structure of the MER and its flanking sequence, three independent clones were sequenced. The sequence of the MER is conserved well
* Division of Cell Fusion, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Aichi 444.

44

ANNUAL REPOPT OF NATIONAL INSTITUTE OF GENETICS NO. 41(more than 99 %) in all three, except for the polymorphisms of number of
repeats in (GAAA(A))n sequence located in the 3' region of the MER.
Furthermore, outer regions which surround the MERs are also highly
conserved among these MERs. Therefore, it seems likely that one copyof the MER was integrated into a specific site in the mouse genome and then
the number of copies of the MER, together with its large flanking sequence,increased about 70-fold during the evolution of the mouse. To determine
when the MERs was amplified in the process of speciation in genus Mus,the degree of amplification of the MER was analyzed by Southern blot
hybridization. In this study, we used genomic DNAs prepared fromdifferent subspecies of M. musculus, including wild mice collected in mainland China and Japan, and from different species of genus Mus. As aresult, the copy number was found to be similar in different subspecies ofmusculus except for wild mice in Lanzhou, China, which showed a remarkably low copy number for the MER. In contrast, different species in genus
Mus showed variations in degree of amplification of the MER. M. spicilegus
yielded a more intensed band than M. musculus and M. spretoides, and thelatter two showed almost the same number of copies. On the other hand,the existence of multicopies of MER was not detected in M. spretus, M.
caroN, M. platythrix and M. leggada. These data indicated that the MER
were amplified after differentiation of genus Mus and before subspecies
differentiation of M. musculus.
A New Haplotype of the Mouse .a-Globin Genes, Hbb w1 ,in Northwestern China
Tsuyoshi KAWASHIMA, Nobumoto MIYASHITA, Toshihiko SHIROISHI,
Wang Chog YAN*, Wang Cheng HUAI* and Kazuo MORIWAKI

We have studied the genetic polymorphism of the hemoglobin beta chain(Hbb) of wild mice, Mus musculus, mostly in Asia. The wild populationin the northwestern pars of China, Korea and the central part of Japan
exhibited an almost monomorphic distribution of the Hbb p haplotype.
The southern part of Asia shows lower frequency of Hbb p and a predominant distribution of Hbb d (Miyashita et al., 1985). In the process of thesurvey of Hbb polymorphism in China, we found another new haplotype*

Lanzhou Institute of Biological Products China.

RESEARCH ACTIVITY IN 199045

of Hbb, assigned Hbb wl • It is distributed in a wide area in northwestern
China. Southern blot analysis of Hbb wl and other Hbb haplotypes werecarried out with Hbb' DNA probes using nine restriction enzymes. Based on
differences in restriction sites, sequence divergence was estimated. It
was revealed that the distance betwee Hbb wl and Hbb' haplotype was 2.64 %diversity, but the distance betwee Hbb wl and Hbb p haplotype was only
0.011 % diversity. These results indicate that the Hbb wl haplotype may
be generated from the Hbb p haplotype. Hbb-bl wl and Hbb-b2 wl werecloned from genomic DNA and are in the process of being sequenced.
Establishment of Embryonic Stem Cell Lines from
BIO. H-2 Congenic Strains
Tornoko SAGAI, Norio NAKATSUJI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Embryonic stem (ES) cells are cell lines derived from the inner cell massof a mouse blastocyst. When ES cells are microinjected into a host blastocyst, they can differenciate into any tissue, including germ cells andgenerate chimeric animals. Modified ES cells by homologous recombination in vitro can be used to introduce any mutaions into the germ lines ofchimeric mice.
The ES cell should provide a useful system for our various studies on
BlO.H-2 congenic strains. In particular, the study of the molecular mechanism of meiotic recombination in the H-2 region definitely requires
this system. Thus far, ES cell lines heve been produced from limited mousestrains, for example l29jSv. At present, there are no ES lines derived
from BlO.H-2 congenic strains. Therefore we started to establish new ES
cell lines from several different BlO.H-2 congenic strains. Now we arecloning the first ES cell line from one of the congenic strains, BlO.A (R209),
which showed high recombination frequency in the H-2 region during meiosis. The cells of this line grow very rapidly on the feeder cells and themorphology is quite similar to a typical ES cell line, E14. A karyotype
analysis showed that a majority of the cell population has normal diploidand the clone was derived from a male blastocyst suitable for chimera
formation.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

Comparison of the DNA Sequence in the D-Ioop Region of Mouse
Mitochondrial DNA. Four maternal Lineages in the Southern
Population of Japanese "molossinus" Mice
Hiromichi YONEKAWA, Sumiyo WATANABE, Nobumoto MIYASHITA
and Kazuo MORIWAKI

Based on mtDNA haplotype analysis with restriction enzymes, we previously proposed the hypothesis that Japanese wild mouse Mus musculus
molossinus, is a hybrid from crosses between ancestral colonies, possibly
very small, of M. m. musculus and M. m. castaneus (Yonekawa et al. (1988)
Mol. BioI. Evol. 5, 63). To prove this hypothesis, it is necessary to analyze
more precisely the population structure of Japanese "molossinus" mouse.
To do so, however, there was a problem: Japanese wild mice showed norestriction polymorphism in the southern population with a musculus typemitochondrial DNA (mtDNA) and a very small degree of polymorphismin the northern population with castaneus type mtDNA. The problem wassolved by introduction of a new technique, DNA sequence analysis combinedwith PCR technique.
The D-Ioop region of mouse mtDNA was amplified by PCR and thencloned in MI3mpl8 or MI3mpl9 RF plasmids. DNA sequencing was
carried out at on least two recombinant MI3 phage clones for every mouse
mtDNA in order to eliminate misreading of the mtDNA sequence caused bymisincorporation of nucleotides by Taq polymerase during PCR. Six
sequencing primes were sued for the sequencing.
We first sequenced 19 mtDNAs obtained from four Mus musculus subspecies and from one closely related species Mus caroli. Based on thesequence data, we constructed a phylogenic tree using the UPG method
and the NJ method. Comparing the tree with that constructed using
restriction enzyme site variation, we found that both trees are very similar
to each other in topology. This suggests that substituted nucleotides in
the mtDNA D-Ioop region, like restriction site variation, can be used asa diagnostic marker for subspecies identification.
Then we sequenced I I mtDNAs obtained from mice belonging to thesouthern population. Although restriction analysis failed to detect any
polymorphism in these mtDNA, sequence analysis showed that at least
four different mtDNAs exist in the population. We designated them as
major type (7 mtDNAs out of II), Miyazaki type (2 out of I I), Ogasawara

RESEARCH ACTIVITY IN 19;047

type (1 out of 11) and Tsushima type (1 out of 11). In the previous issue(pp. 80-81, 1989), we described the existence of two highly variable segmentsin the D-loop region. The nucleotide substitutions detected among thefour types were also found in the segments.
Sperm Head Abnormalities in Interspecific Hybrids between
Mus musculus and M. spretus, and M. spicilegus
Michiko NlwA*, Akihiko MITA and Kazuo MORIWAKI

Mus spretus originated in the Western Mediterranean region includingSouthern France, Iberia and Northern Africa. This mouse can interbreed
under laboratory conditions with inbred strains of M. musculus. Offspring can be otained in both directions of this interspecific cross. The Fl
females are fertile and they can be backcrossed to males of either species.
On the other hand, the Fl males are sterile. M. spicilegus originated ineastern Europe where there is sympatry with M. musculus. Hybrids between them may be obtained, but have not been studied in detail about theirreproductive ability.
We observed the morphology of sperm from Fl hybrids between inbred
strains and SEG (M. spretus), and ZBN (M. spicilegus) (Table 1). Almost
all sperm from Fl males between inbred mouse and M. spretus was abnormal. The number of sperm was low and testis weight was significantly
reduced (almost half of parents). We observed 15 male offspring bred
Table 1.

The rate of abnormal shaped sperm head in interspecific hybrids
No. of 60'
observed

abnorma rate ofsperm (%)

C57BL/6 (B6)C3H/He (C3H)
M. MOL-MSM
SEG (M. spretus)
ZBN (M. spicilegus)

5

11.1
21.4

5

2.1
4.3
0.4

(B6xSEG) F1(C3H x SEG) F1(MSM x SEG) F1(B6xZBN) Fl

3
6
3
4

95.4
98.9
91.4
100.0

strain or
hybrid3
7
4* Saitama Cancer Center Research Institute, Saitama.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

from a (B6 x SEG) FI ~ x B6 6 cross. Four males ahd normal values
for sperm morphology and testis size, 4 had abnormal sperm and normal
testis size, and 7 had abnormal sperm and small testis. It may be said
that the sperm abnormality do not related tightly to the testis size, andthat 4 males out of 15 are fertile. Guenet et al. (1990) reported that no
more than two genes are related to hybrid sterility between laboratory
mice and M. spretus and at least one is X-linked. Our results indicate thesperm abnormality of hybrids between the inbred strain and M. spretus
may cause hybrid sterility in this case. FI males between C57BLj6 (aninbred strain) and ZBN (M. spicilegus) also produced abnormal sperm,
but the number of sperm and testis weight were similar to their parents.
We cannot take offspring from the crosses between these FI progeny and
B6.
Further study on more backcross progeny from hybrids between laboratory mice and M. spretus, and M. spicilegus is necessary to learn themechanisms for hybrid sterility.

RESEARCH ACTIVITY IN

IV.

990

49

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Head Regeneration and Budding in Hydra
TstOffiU SUGIYAMA

Hydra tissue has four types of morphogenetic capacities involved in forming "head-type structures". They are the capacities (1) to form a secondary
head, (2) to inhibit secondary head formation, (3) to form a bud, and (4)to inhibit bud formation. Evidence exists which indicates that a secondary
head-forming/inhibiting mechanism is involved in determining head regeneration, whereas a bud-forming/inhibiting mechanism is involved indetermining bud formation. At present, however, the relationship between
the two mechanisms is not clear.
In order to gain an understanding of the relationship of the two mechanisms, lateral transplantation procedure (Browne, 1909) and ring transplantation procedure (Ando et al., 1989) were employed to examine andcompare the relative levels of the four morphogenetic capacities in the tissueof Hydra magnipapillata (strain 105). The following observations were
made. (1) Secondary head-forming capacity forms a geadient from thehead to the foot along the body column of intact hydra, whereas bud-forming capacity forms a gradient in the opposite direction. (2) Both the secondary head-inhibiting and the bud-inhibiting capacities form gradients fromthe head to the foot in intact hydra. However, the secondary head-inhibiting capacity is unstable and falls significantly immediately after headremoval in the regenerating tissue, whereas the bud-inhibiting capacity
does not decrease under the same condition. (3) Repeated treatment of
intact hydra with diacylglycerol produces a significant increase in the secondary head-forming capacity, but no changes in the bud-forming capacity.
These observations provide strong evidence for the view that, although
head-type structures are formed by both head regeneration and budding,these two processes are regulated by two different mechanisms.

50

ANNUAL REPOPT OF NATIONAL INSTITUTE OF GENETICS NO. 41

A cDNA Clone Specific for Stenotele Differentiation in Hydra
Toshitaka FUJISAWA

Nematocytes are hydra's stinger cells which are primarily used for capturing prey and for protection. There are 4 types of nematocytes (stenoteles, holotrichous isorhizas, atrichous isorhizas and desmonemes), all ofwhich are differentiated from interstitial stem cells. The pathways ofnematocyte differentiation from stem cells are well characterized at thecellular level. The main objective of this study was to analyze nematocyte
differentiation at the molecular level.
An attempt was made to isolate genes which are specifically expressed
during nematocyte differentiation. First, a cDNA library was produced
from fractionated cells in the interstitial cell lineage. Using this libraryclones specific to interstitial cells were differentially screened. Individualclones were tested for specific expression through in situ hybridization.
One of the clones called 5/5 was proven to be stenotele specific. In situ
hybridization combined with BUdR labeling showed that the gene is expressed starting from about 66 h after the stenotele commitment and lastingfor 12 h. This is the time when stenotele capsules become visible. The
nucleotide sequence of the cDNA reveasl that the gene codes a proteinwith 56 amino acids and that its function, however, is unknown since it
is a new sequence which does not have homology with known genes.
Ovipositing Behavior in Bombyx Adults
a. Non-mated Females
Akio MURAKAMI

Concerning the life-history of the silkworm (Bombyx mori L.), not a
few number of characteristics including voltinism (number of generations
per year), hatching and eclosion are subjected to considerable influencesfrom the circadian rhythms of gene expression involved in them. For
the hatching and eclosion of these moths, the circadian rhythms have been
noted at the population level but not at the individual level. As reported
herein, however, the ovipositing behavior of unmated females follows a
distinct circadian rhythm. We conducted observations of the ovipositing
process by making use of the females' habit of secreting a cohesive substance
together with their eggs: on a record paper moving 10 or 20 mm per unit

RESEARCH ACTIVITY IN 1990

51

time was placed an acryl box of 3 cm each side which would not interferewith the movement of the paper; one female each was fixed in the box andthe number of eggs laid per individual and per unit of time was determined.
In the case of mated females, since oviposition is completed four or five
hours after sunset within a day of emergence, it is difficult to refer to it asperiodic behavior in the strict sense. In contrast to this, freshly emerged(non-mated) females show almost no sign of ovipositing behavior on thatday but start to lay eggs about two hours after sunset on the second dayof eclosion; before or after sunrise of the following day, the oviposition
is discontinued once and, as on the preceding day, reopened about twohours after sunset; eggs are laid down slowly and constantly until the dawnof the following day. A similar rhythm, though varied according to thenumber of eggs in the body of each female, continues to be observed for
four or five days after eclosion. To sum this up, from these ovipositing
behaviors of unmated females, the presence of a circadian rhythm wasdemonstrated at the individual level of this insect.
Oviposition Behavior in Bomhyx Adults
b. Mated Females
Akio MURAKAMI

Eclosion of adults occurs within a period of time from thirty minutes totwo hours after sunrise regardless of the strain. Males tend to emerge
significantly earlier than females. Twenty to thirty minutes after eclosion,
moths of each sex initiate the courtship behavior followed by copulation;
naturally, the male and female remain in this state for two or three hours
and then part from each other spontaneously to dissolve the copulation. In
the case of artificial dissolution of copulation, although copulating moths
are separated usually any time between one and three o'clock in the afternoon, the mated females begin to deposit about two hours after sunset
regardless of the time of the separation. During the subsequent four tofive hours, the females keep on laying eggs actively and all the eggs in the
body are laid down within this period of time. These observations, confirm
that the ovipositing behavior of mated females also follows a circadianrhythm as in non-mated females.
As mentioned in the preceding report, in the case of non-mated females,
the deposition of unfertilized eggs lasts [intermittently] for four to five

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

days. It is supposed that the difference in the oviposition rate observed
between unmated- and mated females can be attributed to the presence/
absence of effects from oviposition-stimulating factors (OSF) which are
secreted accompanying copulation. The above finding, however, suggests
the possibility that the secretion of OSF is regulated by a circadian rhythm.
When 20 to 35 mated females are kept together in a small styrol container(dianeter: 10 cm; hight: 5 cm), considerably more active ovipositing behaviors can be seen as early as 20 minutes after dissolution of copulation
no matter what the time it is. The females continue to lay their eggs actively
for about 1.5 hours and almost all the eggs in their body are laid down
within two hours. These observations seem to indicate the involvementof an instinctive aspect in ovipositing behaviors: under such conditions
as mentioned above, the neurosystem for oviposition of each female isstimulated by the competitive instinct to assure the survival of their respective
descendants-probably owing to visual recognition of rivals or physicalcontact with them-so that a fairly high degree nervous activity come into
action in a different manner from instructions by intrinsic circadian rhythmor the induction of oviposition accompanying copulation. That is, although
the secretion of OSF occurs during a time period from sunset to sunrise
essentially according to the biorhythm, the time of secretion can be easily
modified through the nervous system by giving it powerful stimuli such as
the competitive instinct. One can infer that hormone-like oviposition
stimulating factors (OSF) are already accumulated in a sufficient amountin the body at the time of eclosion of females. It appears that the oviposition time is determined by a time measurement/regulation mechanism
located in the central nervous system as is the case for vOltinism and hatching/eclosion which are based on circadian rhythm and photoperiodism.
Genetic Studies on Growth and Aging in the Silkworm (Bombyx Mori)a. Growth Rate in Growth Period
Akio MURAKAMI

A consistent report on the duration time of the whole growth period,
from embryo through larva to pupa, has not yet been published on the silkworm. For this insect, a relatively detailed observation on the larval
period has been conducted, breeding records at the National Sericultural
Experiment Station, M. A. F. F. are complete for a number of preserved

RESEARCH ACTIVITY IN 1990

53

silkworm stocks. We analyed duration time in the larval stage making
use of registered data in the record. Consequently, the distribution of larval
periods is shown to occur in a clear binominal fashion with a mean value of
23.5 days under standard rearing conditions. There are a few extreme
cases: 16 days at the minimum (in the case of the bimoulter strain) and 30
days at the maximum (in the case of the tetramoulter strain). However,the larval period is markedly not influenced by moultinism or the numberof moulting times. The present findings and other observations clearlysuggest that duration and/or growth rate in larval stages are under thecontrol of polymeric genes. It is a widely known fact that rearing temperatures influence on the growth rate or growth period in the larval stage,
but there are no serious effects with the standard culture temperatures ofaround 19-25°C on growth. A similar situation is also observed in theembryonic stage. In other words, the growth rate is essentially under thecontrol of a certain genetic system, but not markedly influenced by environmental conditions. These findings suggest that a strong function related
to homeostasis operates throughout the whole larval (and/or embryonic)
stage. It is likely that in Bombyx the growth period and growth rate in thelarval stage are subjected to relatively small variation.
With the courtesy of the Taiwan Sericultural Improvement Station(Taipei), we received the breeding records of the year 1986 in which there
were registering 172 tetramoulter strains of Chinese, European, Japanese,and tropical races reared for three seasons (spring, summer and autumn)
under natural climatic condition in Taipei and Taigo. Utilizing the observation data registered on the record, we analyzed a duration time of thewhole growth period from the embryo stage to pupa. The result of theanalyses indicated that the whole growth period in the temperate race isestimated to be about 50 days without any extreme fluctuation, while the
14 tropical tetramoulter strains are divided into two groups in response to
the period of growth: one is about 43 days and the other is 35 days. Accordingly, growth rate during the whole growth period in the tropical racecomes to be about 1.2 and 1.4 times faster than for the temperate ones.
In addition, duration time in any stage comprising the growth period ina tropical race is intrinsically shorter than for the temperate race. Although the whole larval period fluctuates to some extent among the strains
analyzed, the ratio of duration time in each developmental stage to thewhole lifespan in the insect is almost the same. Consequently, it can be

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said that the tropical race has a certain genetic factor promoting growthrate through the whole growth period. A preliminary attempt to detectsuch a presumptive genetic factor, pgr, for example, has been carried out
a plan making use of several tropical races preserved in our country and inconsequence a few candidates were obtained. The genetic characterizationof the candidates is in progress. It is worthwhile to note that the following
mutants, pre (or pgr), are perform their whole lifespan without any pathological symptoms. It is also clear that the function of these mutants responsible for the growth period is axiomatically different from the mutant,sdi (short duration of imaginal lifespan), which only expresses its functionin the post growth period in adults. Accordingly, it became evident thatthe mechanism of aging in Bombyx is made up of at least two steps, thegrowth period concerned in growth rate and the post growth period related
to senescence rate.
Genetic Studies on Growth and Aging in the Silkworm (Bombyx Mori)b. Growth Rate in the Pupal Stage
Akio MURAKAMI

Genetic analyses in the growth of silkworms, a lepidopteran insect, havebeen carried out to better understand mechanisms concerned in aging
and/or senescence. There are drastic changes in morphological as well
as physiological activity with the developmental stage from embryos toadults as it is a holometabolic insect. Growth periods can be distinguishedfrom aging periods on the developmental process: the growth period iscomposed of the following three developmental stages, embryo, larva andpupa, and the aging period is composed on only the adult stage. There isrelatively abundant biological information on the growth period, the embryoand larval stages, while there is scanty information on the pupal and adultstages. Such being the situation, the duration of the adult stage was investigated and results show the adult stage in females is about 1.5 times
longer than that in males, regardless of strain. In addition, it was clearlyshown that the duration of adult lifetime, 5 days to 8 days depending on
strain and sex, is under the control of a particular genetix system and in
addition, an autosomal recessive gene mutant sdi (short duration of imaginal
lifespan), was detected.
Male adults emerged usually about one day earlier from pupae than

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55

females regardless of strain, indicating that the pupal period in males is,
as a rule, shorter to some extent than that in females of this insect. It
should be noted that the growth rate of the male is several hours fasterthan the female. But female moths in an F 1 hybrid from a cross between
one of the common bivoltine strain, JI06, and a tropical multivoltine strain,
Cambodge, emerged a few days earlier than males. A similar event wasobserved in the F , hybrid moths obtained from a cross between a Japanesebivoltine strain, JI06, and a Chinese univoltine strain, Santosanmin. Of
course, the adult lifespan for F , females, was about 1.5 times longer thanfor males. These findings clearly indicated that the genetic system involvedin the pupal lifespan is different from that in the adult lifetime. From theresults of genetic analyses on the phenomenon, it became clear that thegenetic factor responsible for the early emergence of the female moths is arecessive gene pre (precocious), which is located on the X-chromosome.
It appears that the gene pre operates mainly on the pupal stage, since thelarval period is almost the same regardless of sex or genetic constitution.
Known X-linked gene (Lm) alleles, which are responsible for maturationin the silkworm, are sensitive to change in environmental conditions, while
the pre gene is indifferent to them. As stated already, however, the growthrate in male pupae of the silkworm is slightly but significantly faster thanfor females or the eclosion of female moths is delayed only for a day ascompared with the males regardless of either strain or hybrid line. Thisdifference seems to originate in the function of the Y-chromosome which
determines femaleness.
In the silkworm, both embryonic and larval stages are sensitive to changes
in environmental conditions, while both pupal and adult stages are insensitive to them. Thus, it is possible to say that pupal growth rate isprincipally under the control of a particular genetic system rather than theneuro-endocrine system which is under the influence of some environmental
conditions. In brief, the gene pre is not identical to Lm gene-alleles.
Genetic Studies on Growth and Aging in the Silkworm (Bombyx Mori)c. Embryonic Diapause as a Prolongation of Lifespan
Akio MURAKAMI

Genetic studies on growth in the silkworm, Bombyx mod, have mainly
focused so far on two main life history characteristics, voltinism (the number

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of generation times per year) and moultinism (the number of ecdysial
times in the larval stage). The former has the function of adaptation forthe maintenance of survival of individual life. Looking at voltinism fromanother angle, this characteristic is also acts upon the determination of
the diapause period or when the insect enters into the diapause state. In
Bombyx, the diapause-a period of inactivity and suspension of growthtakes place in the embryo stage. The event is accompanied by greatly
decreased metabolic activity. The diapause state in the insect lasts a
considerably long time ranging from a few months up to about one year
partly depending on genetic background. The diapausing eggs in all
strains hatch the following spring. It is important to note that the diapause
characteristic fluctuates according to several environmental conditions,
including photoperiodism experienced in the parental generation (orrearing season). In fact, the diapause event is under the control of a genetic
system connected with the central nervous system (brain) through which
the management of various environmental information is performed. In
other words, the diapause period is determined by some genetic and environmental factors, which determine the time for entering the dormant
state.
In the univoltine race, for example, the whole growth period is about
50 days under standard rearing conditions, exclusive of the embryonicdiapause term. Even if the adult lifetime which ranges from about two
to ten days in the race is added to the duration time of the growth period.
The whole lifespan lasts no more than 60 days, while the diapause period
in this race is at least 10 months. The situation is complicated in thetropical multivoltine race: the diapause period is subjected to a wide fluctuation from zero to about 10 months depending on what time the embryoenters into the dormant state. Also, the determination of diapause periods
is partly depends on the rearing place or latitude of the parental generation.
In the multivoltine race, an early stage diapause event (estivation and/or
hibernation) is observable even late in June and is a late stage event in
December under the climatic conditions in Japan. In addition, there arecases of the insect being completely free of the diapause state. In general,
the tropical multivoltine race has a short growth period, ca.40 days, exclusive of the duration of the diapause state, but the duration of the adultstage does not deviated from the standard lifetime in the insect. It should
be aslo emphasized that awakened embryos after the dormant state proceed

RESEARCH ACTIVITY IN 1990

57

in their development without any abnormal symptoms and have normal
lifespan and life functions. Certain genetic factors leading to a low
metabolic state will produce a long-lived silkworm strain.
As referred to an early part of the communication, moultinism is also
related to the growth events in Bombyx. This life history characteristic
is under the control of some major genes as well as under the influence ofenvironmental conditions. In Bombyx, there are known to be various
types of moultinisms, bimoulters, pentamoulters and tetramoulters, among
which the tetramoulters are the most common type followed by the trimoulters. It is a well-known fact that in this insect, a change of the numberof moulting times per larval stage is no more than once regardless of strain:
for example, from tetramoulters to trimoulters, and vice versa. Prior tomoult, about two days are required to make the new epidermis and cuticle
for each ecdysis, so larval growth is not apparently observed during the
preparation for ecdysis. It is also a well-known fact that marked metabolic
activity occurs in preparation for ecdysis. From a series of analyses on
the relationship between moultinism and the duration time in a given larval
instar (or between the two moults), it was revealed that a reduction of moulting times compensates for an increase in duration time in the instar between
the two moult events. The increase and decrease of moult times per larvalstage does not have a great influence on the duration time in the larvalstage. Accordingly, the ecdysial event is no more than a secondary factor
in prolonging the entire lifespan of the silkworm.
A New Approach to Major Genes which Control Lifespanin Insects
Isamu YONEMURA*, Hayato HA%KURA*, Akira OKUNO**,
Yoshiharu SHIMIZU*** and Akio MURAKAMI

Longevity is one of the most fascinating and challenging research subjects
in life science, but had been left out of genetic studies at the molecular level
because of the general opinion that the biological event is under the control
of polygenic factors. However, a pioneering work by Yonemura et al.
(1986) has clearly shown that a certain major gene controls lifespan in* Dept. of Legal Medincine, Tokyo Ika Shika Univ. School of Medicine, Tokyo.
** Dept. of Lipid Biochemistry, Shinshu Univ. School of Medicine, Matsumoto.
*** Dept. of Public Health, School of Medicine, Tokai Univ., Isehara.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

purebred strains of Drosophila melanogaster. A similar finding was also
reported in D. melanogaster by Luckinbill et al. (1987). Recent experiments
by Yonemura et al. (1989, 1990) revealed the mode of major gene inheritances in relationship to the lifespan in adult Drosophila; they identified
genes concerned in longevity, which are designated as a Jm (Jumyo gene).
The Jm alleles also control developmental speed in larvae, indicating thatthe alleles express their first function in the late larval stage to the early
pupal one. In other words, the longevity potential of the alleles is differentiated during this period (Yonemura et al., 1991).
In Bombyx, Murakami (1887) detected certain major genes concernedin the lifespan: a recessive gene, sdi, which controls the short duration in
the imaginal lifetime and a sex-linked recessive gene pre (precocious),
which accelerates growth rate in pupal stages (Murakami, in this Report).
He also found a strain preserving a genetic factor, which influences a highgeowth rate throughout the whole growth period, ranging from the embryonic to pupal stages. There is generally observed a typical binominal distribution pattern on duration time in larval as well as pupal stages in 179
Bombyx strains analyzed so far, which may show that the lifespan characteris still influenced by a number of polymeric genes. However, it seems to
be impossible to completely deny a possibility that the effect of some major
genes on the character in question decides the mode of lifespan distribution
originated in the polymeric gene system.
So far we have analyzed (Yonemura et al., in preparation), a remarkablechange in the protein profile of both the short- and long-lived strains of
D. melanogaster during the first few days after emergence. Only one protein
was shown to be different between the strains. The protein quickly disappeared after emergence in the short-lived strain, while it remained for twodays or more in the long-lived strain. The protein in question was extracted from young pupae of the long-life strain by phosphate bufferedsaline and purified by column chromatography procedures. The chemical
characteristics of the protein were as follows: M. W. 76,600 (by SDSPAGE), isoelectric point pH 6.5, molecular extinction coefficient A(1~~o)=18.3, and a glycoprotein containing 3 % hexoses (Okano et al., in preparation). This protein was termed Ju-myo protein (JP).
When D. melanogaster adults were fed with JP mixed in food every day
after day 5 of emergence, a significant life-prolongation effect was observed
on both short- and long-lived strains, even with the extreme dilution of

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59

0.0005 ug/ml (Yonemura et al., unpublished data), while all the other proteins, devoid of lP, had no effect at all. Details of this series of experiments
will be published elsewhere.
Thus, we obtained a clue to the molecular approach for longevity making
use of lP and/or a pure line with known Jm-alleles in D. melanogaster.
Furthermore, an antibody against lP has now been produced, which will
facilitate cloning of the Jm genes. Use of the antibody will make possible
a search for the presence of the Jm gene or lP in animals other than D.
melanogaster, such as Bombyx, mice, etc. This line approach will makea strong contribution to elucidating genetic mechanisms of the determination of lifespan and aging.
Permeability of Eggs and Abnormal Development in Drosophila
Kiyoshi MINATO

In general, Drosophila eggs are impermeable to aqueous solutions evenafter dechorionation. However, "premature eggs" forced to be laid in
rapid succession (Yoon & Fox, '65) or eggs laid spontaneously but still
being at an early stage of development (Limboug & Zalokar, '73) are known
to be, when dechorionated, permeable to aqueous solutions. Previously,
we also found that eggs laid freshly by females and collected successively
for short time intervals in Drosophila melanogaster failed to develop normally and did not hatch when dechorionated and incubated in aqueous media
such as water and 0.9 %NaCI, but they developed normally and hatchedsuccessfully when incubated in non-aqueous media such as paraffin-oil.
The findings suggested that the above abnormal development of eggs occured as a result of permeability to aqueous solutions of the eggs in "premature" or early stage of embryonic development.
The time dependencies of the phenomenon and the characteristics of thedevelopmental anomalies induced were investigated in detail. Consequently, eggs which were incubated after the cynsytial blastoderm stage (2 h at25°C) developed normally and hatched successfully, but eggs before thecynsytial blastoderm stage developed abnormally and never hatched.
Thus, there was a critical difference in the reaction of eggs incubated inaqueous media before and after the blastoderm stage, and therefore a layerof cells formed on the surface of the egg in this stage seemed to be responsible for the impermeability of eggs or the establishment of a waxy coating

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on the vitelline membrane.
As to the differences in incubation media, when eggs were incubated inwater, the eggs showed little sign of development and became graduallyopaque, and after several hours swelled, and didn't hatch. Incubated in
0.9 %NaCl, eggs developed to a later stage and in most cases formed astructure like the cellular blastoderm only in the posterior-half of the embryo. They developed to the embryo stage with the amorphous andopaque structure anteriorly and a herniated bag-like structure posteriorly.
These embryos became somewhat shrunken and hence detached from thevitelline membrane at both anterior and posterior ends, and did not hatch.
Conditional Radiation Sensitivity in the Wasted Mouse Mutant
Hideo TEZUKA, Dai AYUSAWA and Takeshi SENO

A quantitative study on the alteration in development of wasted marrow
CFU-S and CFU-E revealed that (a) at weaning 21-22 days of age, the number of CFU-S per femur (bone marrow) and per spleen had already changed,
while that of CFU-E per femur did not change, (b) at 26-27 days of age,
the numbers of all these stem cells and progenitor cells decreased markedly.
A qualitative study on the developmental alteration of CFU-E revealed
that (a) at the weaning age, the responsiveness of wasted marrow CFU-E toerythropoietin (epo) did not change, as shown by the colony forming ability of the cell in culture under different epo concentration, (b) at 26-27
days of age, however, CFU-E from wasted marrow required 8-10 foldconcentrations of epo to form the same number of colonies as the control
did. The number of wasted marrow CFU-E colonies formed significantly
decreased, compared with the control, under the same epo concentrationsat any level. This indicates that the responsiveness of wasted marrow
CFU-E to epo at 26-27 days of age is lower than that at 21-22 days of age.
The time course in changes of CFU-E responsiveness to epo coincidedwith the appearance of radiation sensitivity in the wasted mouse, indicatingthat the change in CFU-E responsiveness to epo may be the cause of theradiation sensitivity of the cell.
To investigate this hypothesis, the radiation sensitivity of bone marrow
CFU-E from wasted and control mice was determined under different
epo concentrations. Results showed that rediation sensitivity of both
CFU-Es from wasted and control mice was dependent on epo concen-

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tration.
In addition, CFU-E from wasted marrow and from control, formed asimilar number of colonies under different epo concentrations, when unirradiated (IO-fold concentration of epo for wasted CFU-E). Under theseculture conditions, these two cells showed similar radiation sensitivity.
This suggests that the radiation sensitivity of marrow CFU-E may be
determined by how epo interacts on the cell and consequently how thecell is stimulated to proliferate and differentiate.
Purification and Structure Analysis of an Autocrine Growth Factor
in Conditioned Medium Obtained from Primary Cultures of
Scleral Fibroblasts of the Chick Embryo
Yoshie OHOYA*, Kazuo WATANABE* and Nobuo SHIMAMOTO

The time course of cell proliferation and cell differentiation may be determined by enviroment, including growth factors. Most simply, local
concentrations of growth factors could control a spatial distribution ofcells. One candidate for such a situation is the scleral fibroblast of thechick embryo which forms a thin layer around the eye balls. As a firststep in checking 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-phasecultures by DEAE-Sepharose column chromatography and non-denaturing
polyacrylamide gel electrophoresis. A new purification procedure wasdeveloped by using centrifugal evaporation as the first concentrating method.
The specific activity was increased 1,000. fold in this step. The yield andthe specific activity of the purified growth factor was improved. A cDNA
library was constructed from mRNA prepared from the primary-cultured
cell to clone the cDNA of the growth factor.

•

Faculty of Integrated Arts and Sciences Hiroshima University.

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In Vitro Amplification of whole cDNA from a Single Embryoof the Nematode C. elegans and Its Application
to Differential Screening
Yuji KOHARAThe nematode C. elegans is one of the best systems for studying the
network of gene expression regulation during development at the level ofindividual cells. Aiming to isolate genes which are expressed at specificstages or in specific cells, a new method to make a series of cDNA probes
from single embryos at various stages was developed. The method consists of (1) disruption of a staged-single embryo under a dissecting microscope and extration of total RNA, (2) first strand cDNA synthesis using
a specific primer of oligo (dT)12 linked with a 20 mer tag sequence at theSf-side and [a_ 32 P] dCTP at high specific activity instead of cold dCTP,(3) shortening of the cDNA to about I Kb on average by disintegration ofincorporated 32p (4) dG tailing (20-30 mer) by Terminal transferase, (5)amplification of the cDNA by PCR using the tag sequence used in thefirst strand synthesis and oligo (dC)12 linked with another tag sequence
as primers.
Through this method, cDNA were amplified from individual embryos
every 2 hr after the first cleavage. To assess uniform amplification, aliquots of amplified cDNA were dot-blotted onto strips of nylon membraneand probed with various genes whose expression patterns are known:
myo-2 (pharyngeal muscle specific myosin heavy chain), vit-2 (vitellogenin,
one of the most abundant mRNA but not present in embryo and unc-30(essential gene for differentiation of two motor neurons, which was kindly
provided by Dr. R. Hoskins, MRC-LMB). The myo-2 probe gave signals
at the dots from 9.5, 11.5 and 13.5 hr embryos, vit-2 gave no signal andunc-30 gave signals at the dots from 7.5, 9.5 and 11.5 hr embryos. These
results coincide with the expression patterns of these genes perfectly.
The set of amplified cDNA were 32P-labeled and probed a embryoniccDNA library made by J. Ahlinger (Univ. of Wisconsin). Although there
were not any big differences in the patterns of positive signals between far
different stages (probably due to the artificial rearrangement of the eDNA
insert during library construction), by comparing the signals in autoradiograms very carefully, several clones seemed to be differentially expressed
To examine these, the dot-blot strips were hybridized with 32P-labeled

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eDNA inserts from individual clones. For example, clone 4-3 gave a verystrong signal at the dot from the 1.5 hr embryo, a weak signal for the 3.5 hrembryo and virtually no signal at 0 hr and other stages. Clone 4-1 gavea strong signal for the 7.5 hr embryo. A control clone 1-1 which did notshow differential expression gave strong signals at almost the same level
through out all the stages. These hybridization patterns were essentially
reproducible in another experiment using amplified cDNA from anotherseries of embryos. The cDNA in clone 4-1 and 4-3 were mapped near
zyg-ll (on chromosome II) and mec-l (on chromosome V), respectively, by
probing so-called "polytene filters", membrane filters blotted with a set ofordered YAC (Yeast Artificial Chromosome) clones which covers virtually
the whole genome. Characterization of these cDNA is in progress.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

V.

POPULAliON GENETICS

Theoretical Study of Near Neutrality. I. Heterozygosity andRate of Mutant Substitution
Tomoko OHTA and Hidenori TACHIDA

In order to clarify the nature of "near neutrality" in molecular evolution
and polymorphism, extensive simulation studies were performed. Selection
coefficients of new mutations are assumed to be small so that both random
genetic drift and selection contribute to determining the behavior of mutants.
The model aslo incorporates normally distributed spatial fluctuation of
selection coefficients. If the system starts from "average neutrality", itwill move to a better adapted state, and most new mutations will become"slightly deleterious". Monte Carlo simulations have indicated that such
adaptation is attained, but that the rate of such "progress" is very low for
weak selection. In general, the larger the population size, the more effective
the selection becomes. Also, as selection becomes weaker, the behaviorof the mutants approaches that of completely neutral genes. Thus, theweaker the selection, the smaller is the effect of population size on mutant
dynamics. An increase in heterozygosity with population size is very
pronounced for subdivided populations. The significance of these results
is discussed in relation to various observed facts on molecular evolution
and polymorphism, such as generation-time dependency and overdispersionof the molecular clock, or contrasting patterns of DNA and protein polymorphism among some closely related species. For details, see Genetics
126, 219-229.
How Gene Families Evolve
Tomoko OHTA

Theories and facts of gene family evolution are reviewed. Concerted
evolution is commonly observed for gene families which originated a longtime ago, however there are many different types of multigene families,
from uniform to diverse. The rate of homogenization caused by unequalcrossing-over, gene conversion, etc. has been evolutionarily adjusted for

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each gene family. When new functions are needed by organisms,
gene families may evolve into superfamilies, in which no further concerted
evolution takes place, and each member of the family may acquire an
indispensable function. The homeobox-containing gene family is a most
exciting example of such a superfamily. For details see Theor. Pop. Bioi.
37, 213-219.
Evolution of the Multigene Family: A Case of Dynamically
Evolving Genes in the Major Histocompatibility Complex
Tomoko OHTA

It is now known that many multigene families exist in eukaryote genomes.
Multigene families that are known to be evolving under continued occurrences of unequal crossing-over and gene conversion provide a different
picture of evolution from that of conventional population genetics. As
an example of an evolving multigene family, evolution and variation at
major histocompatibility complex loci were reviewed. The exceptionallyhigh polymorphisms at the class I and class II loci of the major histocompatibility complex (MHC) have been of great interest for many years. In
addition, recent studies indicate that amino acid substitution at antigen
recognition sites (ARS) is more rapid than synonymous substitution, contrary to the general pattern of nucleotide substitution in evolution. However, such acceleration seems to be limited to a certain period after gene
duplication. In order to explain such an unusual pattern, a population
genetic model of diversifying selection was constructed. To make themodel fit the data, diversifying selection needs to work on enhancing diversity not only between alleles at the same locus, but also between genes
at different loci belonging to the gene family. Gene conversion among
genes is also incorporated by choosing parameter values that are thought
to be realistic. Simulation studies reveal that very weak selection at individual amino acid sites can explain the unusual pattern of evolution andpolymorphism at MHC loci under this model. The applicability of thepresent model is discussed by surveying other examples, such as evolutionof protease inhibitors and immunogloblin variable regions which show
a similar pattern of acceleration of amino acid substitutions. For details,
see "Evolution of Life," eds. S. Osawa and T. Honjo, Springer-Verlag,
Tokyo, pp. 145-159.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

The Major Histocompatibility Complex and the Quest for Origins
Jan KLEIN and Naoyuki TAKAHATA

Two lines of pursuit in biological research hold the promise of particularly great satisfaction if drawn to a successful conclusion. One has thepotential of helping the unfortunate among us who are stricken by somephysical or mental impairment. The other touches upon the timelessquestion au venons-nous? Whence do we come? Unlike Paul Gauguin,those of us engaged in finding answers to this question do not paint broad
canvases that appeal to our senses and feelings or evoke a vague awarenessof an answer. Our work can rather be compared to a mosaic to which
each of us contributes but a single piece and on which the answer emerges
in sharply delineated contours as the effort continues over generations.
Some of us are lucky enough to be able to place a piece in a prominent
position, others contribute dispensable or less conspicuous chips, whileothers still are among the unfortunate ones who see the pieces they laboriously placed in the mosaic removed again and replaced by better fitting
fragments. But we are all working together on this venture in a collective effort to create a composition which will affect others. As for ourselves, we would not trade the opportunity to place a small piece into
the mosaic au venons-nous? for anything in the world. In this article,
we discuss one genetic system where evidence for trans-species transfer ofpolymorphism is unassailable, with the hope that once we learn where wecome from, we may begin to fathom the enigma of our existence. Thissystem is the major histocompatibility complex. See Immunological Reviews
13, 5-25, 1990 for details.
Allelic Genealogy and MHC Polymorphisms
Naoyuki TAKAHATA

Allelic genealogy, the genealogical relationships among different alleles,
exhibits very different patterns depending on the underlying classic (neutral,deleterious, advantageous and overdominant) selection regimes and is used
to elucidate the evolutionary mechanisms responsible for the extraordinary
polymorphisms observed at the MHC (Major Histocompatibility Complex)
loci. The polymorphisms can be most easily accounted for by overdominant selection. Two models of frequency dependent selection often
invoked in relation to MHC polymorphisms actually cannot explain either

RESEARCH ACTIVITY IN 199067

the long separation time of different alleles or a large number of segregating
alleles under realistic circumstances, and hence they are incompatible with
MHC polymorphisms. If we apply a strict minority advantage model,
however, it is possible to generate exactly the same pattern of allelic genealogy as that of overdominant alleles, but in practice it is not easy to justify
such a model on biological grounds. See Takahata, N. and Crow, J. F. (ed.)
Population Biology of Genes and Molecules (Proceedings of the 4th Symposium of the International Prize), pp. 267-286, Baifukan, Tokyo 1990 for details..
Population Biology of Genes and Molecules
Proceedings of the 4th Symposium of the International Prize for Biology
Edited by Naoyuki TAKAHATA and James F. CROW

The International Prize for Biology was established in April 1985 III
celebration of the sixtieth year of the reign of the Emperor of Japan. It
commemorates his long devotion to biological research. The first two
Prizes were awarded in systematics and taxonomy, fields in which His
Majesty was personally interested. In the following year the field wasdevelopmental biology. The current Prize is in population biology andwas awarded to Professor Motoo Kimura on November 28, 1988. The
serious condition of His Majesty prevented the Emperor from receiving
Professor Kimura following the Prize ceremony. The award was grantedin the presence of the then Crown Prince, now Emperor of Japan.
For the past thirty years Motoo Kimura has made many important contributions to mathematical, population, and evolutionary biology.
One of his most important contributions has been the development ofdiffusion models and applying these to significant problems in genetics
and evolution. Starting as a graduate student he published a series of
papers treating the transient distribution of gene frequencies in finite populations. These papers have provided a basis for studying the evolutionarychange of natural populations and are now regarded as classics in population
genetics. He has since studied additional problems including the probabilityof fixation of mutant genes, the dynamics of linked loci, the infinite allele
and infinite site models, and the genetic consequences of geographical structure. With the advent of molecular biology, he pioneered a new field inwhich principles of both population and molecular genetics are synthesized
and the resulting theory serves as a basis for investigating the mechanism

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of evolution at the molecular level.
In 1968 Kimura proposed the neutral theory of molecular evolution,suggesting that evolutionary changes of proteins occur mostly by random
fixation of neutral or nearly neutral mutations rather than positive Darwinian selection. Furthermore, the theory implies that a large fraction of theprotein polymorphism in natural populations represents a transient phaseof molecular evolution, because those genotypic mutants that are destinedto spread into a species take a long time until fixation and on their way
take the phenotypic form of portein polymorphism. The neutral theory
has had an enormous impact not only in population genetics, but in evolutionary biology in general. It has stimulated research throughout theworld. During the past twenty years the evolutionary study of DNA andproteins has generated a large amount of new data to support Kimura's
hypothesis, and the theory is now widely regarded as substantially correct.
It has become the guiding principle, the starting point for analyzing andinterpreting molecular data. His 1983 book, The Neutral Theory of Molecular Evolution, is already regarded as a milestone in evolutionary biology.
Kimura's work in unifying the theory of population genetics and the dataof molecular evolution will undoubtedly remain as one of the most importantachievements of twentieth century biology.
This Symposium, held in Tokyo the day after the Prize was awarded,emphasized population genetics, quantitative genetics, and molecular
evolution, subjects of great interest to Kimura and to which he has madesuch important contributions.
Although the International Prize is awarded by Japan, the recipient ischosen on a world-wide basis by an international committee. It is especiallypleasing that this year's recipient is from Japan. See Takahata, N. andCrow, J. F. (ed.) Population Biology of Genes and Molecules (Proceedings ofthe 4th Symposium of the International Prize for Biology), Baifukan, Tokyo
1990 for details.
Allelic Genealogy under Overdominant and FrequencyDependent Selection and Polymorphism of Major
Histocompatibility Complex Loci
Naoyuki TAKAHATA and Masatoshi NEI

To explain the long-term persistence of polymorphic alleles (trans-specific

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polymorphism) at the major histocompatibility complex (MHC) lociin rodents and primates, a computer simulation study was conducted on
the coalescence time of different alleles sampled under various forms of
selection. At the same time, average heterozygosity, the number of alleles
in a sample, and the rate of codon substitution were examined to explain
the mechanism of maintenance of polymorphism at the MHC loci. The
results obtained as follows. (1) The coalescence time for neutral alleles
is too short to explain the trans-specific polymorphism at the MHC loci.
(2) Under overdominant selection, the coalescence time can be tens of
millions of years, depending on the parameter values used. The average
heterozygosity and the number of alleles observed are also high enough to
explain MHC polymorphism. (3) The pathogen adaptation model proposedby Snell is incapable of explaining MHC polymorphism, since the coalescence time for this model is too short and the expected heterozygosity andthe expected number of alleles are too small. (4) From the mathematicalpoint of view, the minority advantage model of frequency-dependent selection is capable of explaining a high degree of polymorphism and transspecific polymorphism. (5) The molecular mimicry hypothesis also givesa sufficiently long coalescence time when the mutation rate is low in thehost but very high in the parasite. However, the expected heterozygosity
and the expected number of alleles tend to be too small. (6) Considerationof the molecular mechanism of the function of MHC molecules and otherbiological observations suggest that the most important factor for the maintenance of MHC polymorphism is overdominant selection. However,further studies are necessary to compare the overdominance and frequencydependent selection hypotheses. See Genetics 124, 967-978, 1990 for details.
A Simple Genealogical Structure of Strongly Balanced AllelicLines and Trans-Species Evolution of Polymorphism
Naoyuki TAKAHATA

Different alleles undergoing strong symmetric balancing selection show
a simple genealogical structure (allelic genealogy), similar to a gene genealogy
describted by the coalescence process for a sample of neutral genes randomly
drawn from a panmictic population at equilibrium. The only difference
between the two genealogies lies in different time scales. An approximate
scaling factor of the allelic genealogy relative to that of the neutral gene

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genealogy is {,vSj(2M)}[ln{Sj(16rrM2)}]-s/2, where M=Nu and S=2Ns(N; effective population size, u; mutation rate to selected alleles per locus
per generation, s; selection coefficient). The larger the value of ,vS j M(~100), the larger the scaling factor. These findings, supported by simulation results, allow us to apply all theoretical results of the coalescence process
directly to the allelic genealogy. Combined with trans-species evolutionof the MHC (major histocompatibility complex) polymorphism, for whichbalancing selection is believed to be responsible, the allelic genealogy predicts
that the number of breeding individuals in the human population could not
be as small as 50 ~ 100 at any time of its evolutionary history. The analysis
appears to contradict the founder principle as being important in recent
mammalian evolution. See Proc. Natl. Acad. Sci. USA 87, 2419-2423, 1990
for details.
Genealogy of Neutral Genes in Two Partially Isolated Populations
Naoyuki TAKAHATA and Montgomery SLATKIN

Gene genealogy in two partially isolated populations which diverged
at a given time t in the past and have since been exchanging individuals at
a constant rate m is studied based upon an analytic method for large t anda simulation method for any t. Particular attention paid to the conditions
under which neutral genes sampled from populations are mono-, para-,and polyphyletic in terms of coalescence (divergence) times of genes. It
is shown that the probability of monophyly is high if M=2Nm<0.5 andT=tj(2N) > 1 where N is the size of ancestral and descendant haploid populations, in which case most gene genealogies are likely to be concordant
with population relatedness. This probability decreases as the sample
size of genes increases. On the other hand the case where the probabilityof monophyly is low implies either a situation where M> 1 and any T or
where M < 1 and T < 1, but the clear distinction between these conditions
appears very difficult to make. These results were also examined for a genegenealogy reconstructed from nucleotide differences. It is then shown that
results based upon coalescence times remain valid if the number of nucleotide
differences between a pair of genes is not much smaller than 10. To observe
such large nucleotide differences in small populations and therefore infera reliable gene genealogy, we must examine a fairly long stretch of DNA
sequences. See Theor. Pop. BioI. 38, 331-350, 1990 for details.

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Incomplete Maternal Transmission of Mitochondrial DNA in Drosophila
Rumi KONDO, Yoko SATTA, Etsuko T. MATSUURA, Hiromi ISHIWA,
Naoyuki TAKAHATA and Sadao I. CHIGUSA

The possibility of incomplete maternal transmission of mitochondrial
DNA in Drosophila, previously suggested by the presence of heteroplasmy,
was examined by intra- and interspecific backcrosses of D. simulans andits closest relative, D. mauritiana. mtDNAs of offspring in these crosses
were characterized by Southern hybridization with two a 32 P-labeled probesthat are specific to paternal mtDNAs. This method was sufficient for
detecting as little as as 0.03 % paternal mtDNA in a sample. Among 331
lines that had been backcrossed for ten generations, four lines from theinterspecific cross D. simulans (female) X D. mauritiana (male) showed
clear evidence for paternal leakage of mtDNA. In three of these, thematernal type was completely replaced while the fourth was heteroplasmic.
Since the total number of fertilization carried out in this experiment was
331 X 10=3310, the proportion of paternal mtDNA per fertilization was
estimated as about 0.1 %. The mechanisms and evolutionary significancefor paternal leakage are discussed in light of this finding. See Genetics 126,657-663, 1990 for details.
Evolution of Drosophila Mitochondrial DNA and the History of
the Melanogaster Subgroup
Yoko SATTA and Naoyuki TAKAHATA

The nucleotide sequences of a common region of 15 mitochondrial DNAs
(mtDNAs) sampled from the Drosophila melanogaster subgroup were determined. The region is 2527 bp long, encoding most of the NADH dehydrogenase subunit 2 (ND2) and cytochrome oxidase subunit 1 (COI)
genes punctuated by three tRNAs. The comparative study revealed (i)the extremely low saturation level of transitional differences, (ii) recombination or variable substitution rates even within species, (iii) long persistence times of distinct types of mtDNA in D. simulans and D. mauritiana,
and (iv) an apparent lack of within-type variations in island species. Also
found was the high correlation among transitional rate, saturation level,
and GC content (or codon usage). It appears that D. simulans and D.
mauritiana have maintained highly structured populations for more than

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one million years. Such structures are consistent with the originationof D. sechellia from D. simulans. Yet, geographic isolation is so weak
as to show no evidence for further speciation. Moreover, one type of
mtDNA shared by D. simulans and D. mauritiana suggests either theirrecent divergence or ongoing introgression. See Proc. Natl. Acad. Sci. USA
87, 9558-9562, 1990 for details.
A Study on a Nearly Neutral Mutation Model in Finite Populations
Hidenori T ACHIDA

As a nearly neutral mutation model, the house-of-cards model wasstudied in finite populations using computer simulations. The distributionof the mutant effect was assumed to be normal. Behavior was mainly
determined by the product of population size, N, and standard deviation,a, of the distribution of the mutant effect. If 4Na is larger than one, a
few advantageous mutants are quickly fixed in early generations. Then,
most mutation become deleterious and a very slow increase of the average
selection coefficient follows. It takes a long time for the population toreach an equilibrium state. Substitutions of alleles occur very infrequently in the later stage. If 4Na is the order of one or less, the behavior is qualitatively similar to that of the strict neutral case. A gradual increasein the average selection coefficient occurs and in generations several times
the inverse of the mutation rate, the population almost reaches an equilibrium state. Both advantageous and neutral (including slightly deleterious)
mutations are fixed. Except in the early stage, an increase in standard
deviation of the distribution of the mutant effect decreases the average
heterozygosity. The substitution rate is reduced as 4Na is increased.
Three tests of neutrality, one using the relationship between the averageand the variance of heterozygosity, another using the relationship between
average heterozygosity and the average number of substitutions and Watterson's homozygosity test were applied to the results of the present model.
It was found that deviation from the neutral expectation becomes apparent
only when 4Na is more than two. Also a simple approximation for themodel was developed which works well when the mutation rate is verysmall. For details, see Genetics 128, 183-192.

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Fixation Probability in Spatially Changing Environments
Hidenori TACHIDA and Masaru IrzuKA *The fixation probability of a mutant in a subdivided population withspatially varying environments was investigated using a finite island model.
This probability is different from that in a panmictic population if selection
is intermediate to strong and migration is weak. An approximation wasdeveloped to compute the fixation probability when migration among
subpopulations is very weak. By numerically solving the two-dimensional
partial differential equation for the fixation probability in the two subpopulation case, the approximation was shown to give fairly accurate values.
With this approximation, we show in the case of two subpopulations thatthe fixation probability in subdivided populations is generally greater thanthat in panmictic populations. The increase is most pronounced whenthe mutant is selected for in one subpopulation and is selected against in
the other subpopulation. Also it was shown that when there are two
types of environments, further subdivision of subpopulations does notcause much change in the fixation probability unless the product of theselection coefficient and the local population size is less than one.
Relationship between DNA Polymorphism and Fixation Time
Fumio TAJIMA

When there is no recombination among nucleotide sites in DNA sequences, DNA polymorphism and fixation of mutants at nucleotide sites
are mutually related. Using the method of gene genealogy, the relationshipbetween DNA polymorphism and the fixation of mutant nucleotide wasquantitatively investigated under the assumption that mutants are selectively neutral, that there is no recombination among nucleotide sites, and thatthe population is a random mating population with N diploid individuals.
The results obtained indicate that the expected number of nucleotide differences between two DNA sequences randomly sampled from the population is 42 % less when a mutant at a particular nucleotide site reaches
fixation than at a random time, and that heterozygosity is also expected tobe less when fixation takes place than at a random time. However, the* General Education Course, Chikushi Jogakuen Junior College, Fukuoka.

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amount of reduction depends on the value of 4Nl.! in this case, where l.! isthe mutation rate per DNA sequence per generation. The formula forobtaining the expected number of nucleotide differences between the two
DNA sequences for a given fixation time was also derived, and indicates
that, even when it takes a large number of generations for a mutant to reachfixation, this number is 33 % less than at a random time. The computer
simulation conducted suggests that the expected number of nucleotide
differences between the two DNA sequences at the time when a advantageous mutant becomes fixed is essentially the same as that of a neutral mutant
if the fixation time is the same. The effect of recombination on the amountof DNA polymorphism was also investigated using computer simulation.
For details, see Genetics 125, 447-454.
Relationship between Migration and DNA Polymorphism
in a Local Population
Fumio TAJIMA

The expected amount of DNA polymorphism, measured in terms of thenumber of nucleotide differences between the two DNA sequences randomly
sampled from subpopulations, was studied using the stepping-stone modeland the finite island model, under the assumption that the migration rate
is not the same among different subpopulations. The results obtained
indicate that the expected amount of DNA polymorphism in the subpopulation with a lower migration rate is smaller than that with a higher migration
rate. This suggests that marginal populations tend to have lower levels
of DNA polymorphism than central populations, if the migration ratein the marginal populations is lower than that of the central populations.
For details, see Genetics 126, 231-234.

A Simple Graphic Method for Reconstructing Phylogenetic
Trees from Molecular Data
Fumio TAJIMA

A simple graphic method was proposed for reconstructing phylogenetic
trees from molecular data. This method is similar to the unweighted
pair-group method with arithmetic mean (UPGMA), but the process of

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computation of average distances and reconstruction of new matrices,
required in the latter method, was eliminated from this new method, sothat one can reconstruct a phylogenetic tree without a computer unless thenumber of operational taxonomic units (OTUs) is very large. Furthermore,this method allows a phylogenetic tree to have multifurcating branches
whenever there is ambiguity with bifurcation. For details, see Mol. Bioi.
Evol. 7, 578-588.
Some Consideration on Diversifying Selection
Fumio T AJIMA and Terumi MUKAIDiversifying selection due to genotype-environment interaction can
increase genetic variation in natural populations. It is known, however,that the conditions for stable genetic polymorphism or marginal overdominance are quite restricted in this selection model. In this study asimple model of diversifying selection was examined, and the following
results were obtained: (1) Even when the conditions for marginal overdominance are not satisfied, if diversifying selection is operating, the frequency of mutants can be higher than that in the case of simple mutation-selection
balance. (2) This selection model causes a large amount of genetic load
(environment load), even when the conditions for marginal overdominanceare not satisfied, namely even when the equilibrium frequency of mutant
is very low. From these results it can be concluded that the number ofloci on which this type of diversifying selection is operating is very small,
if any. For details, see lpn. l. Genet. 65, 193-200.
An Estimate for the Total Rate of Mutations in Humans
Motoo KIMURA

Applying the neutral theory of molecular evolution, I intend to showthat the total mutation rate per genome per generation in man must bevery much higher than earlier estimates. To show this, I use the estimatedvalue of the evolutionary rate of the mouse globin pseudogene, which isapproximately k,=5x 10- 9 per nucleotide site per year (Li et al. (1981)
Nature 292, 237-239). If the neutral theory is valid, this represents the total
mutation rate per site per year in the mouse. This is because, according
to the neutral theory, the rate of mutant substitution at the molecular level

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is equal to the neutral mutation rate, and also, all the mutations of thepseudogene ("dead gene") are selectively neutral. I will also utilize theobservation (Li and Tanimura (1987) Nature 226, 93-96) that the rate ofsynonymous substitutions in rodents is about 7 times higher than that inhigher primates.
The human genome consists of about three billion (3 X 109) nucleotide
sites, so if we assume that the average human generation span is 20 years,
we can obtain the total mutation rate per genome per generation as follows.

or approximately VT =43, meaning that the total number of new mutations
per generation due to base substitutions amounts to 43 per gamete, andtwice as many per zygote. This is a very high value compared with thetraditional estimates of the genomic mutation rate (i. e., much smaller
than unity). According to the genetic load theory, the mutational load
becomes intolerably high unless the great majority (say, 99 % or more) of
these mutations are selectively neutral (i. e., nondeleterious).
Recessive Hereditary Deafness, Assortative Mating,and Persistence of a Sign Language
Kenichi AOKI and Marcus W. FELDMAN

We modelled the cultural transmission of a sign language when there is
one-locus genetic variation for deafness and hearing. Our premises arethat the deaf are more motivated to learn sign language than the hearing,and that a vertically transmitted sign language, unlike recessive hereditarydeafness, cannot "jump a generation". Conditions were obtained for
persistence (i. e., protection from loss) of signers. These conditions are
more easily satisfied the greater is the fraction of the hearing who also
learn sign language and as the frequency of the recessive gene for deafuess
increases. Persistence is also facilitated by assortative mating for deafness,but not by assortment for signing. With vertical transmission only, it isnecessary that one signer parent be able to transmit sign language withgreater than one-half the efficiency of two. Under the assumption thanthe hearing do not learn sign language, the following additional results
were obtained. Persistence is more likely with dominant as opposed torecessive inheritance. When recessive hereditary and acquired deafness

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co-occur, increasing the frequency of the latter has an opposite effect,
depending on the degree of assortment. Opportunities for the deaf tolearn sign language outside the family do not seem to affect the conditions
for persistence. For details, see Theor. Populo BioI. 39, 358-372.
Some Theoretical Aspects of the Origin of Cultural Transmission
Kenichi AOKI

Cultural transmission can be roughly defined as the transfer of information
between individuals by social learning. Despite its manifest importancein the determination of various aspects of human behavior, the questionof its origin(s) has not received due attention. Previous theoretical studies,notably by Cavalli-Sforza and Feldman, have indicated that there may beserious obstacles to the evolution of a genetically determined capacity forcultural transmission. This study reviews some new theoretical resultsthat extend their work and notes the conditions that may favor the occurrence of cultural transmission. Basically, our claim is that an increase infrequency of communicators was linked to the spread, by vertical transmission, of a specific adaptive trait. Hence, if cultural processes are constrained by evolutionary history, we expect that a propensity should exist forchildren to acquire useful innovations from their parents. An attempt
was made to interpret the observations of cultural transmission in birds,
nonhuman primates, and a hunter-gratherer group in terms of this and otherpredictions. The theoretical results presented here may also be useful insuggesting further empirical studies that can improve our understandingof the origin(s) of cultural transmission. For details, see Evolution ofLife (S. Osawa and T. Honjo, eds.), pp. 439-449, Springer-Verlag, Tokyo,
1991.

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VI.

EVOLUTIONARY GENETICS

Evolutionary Origin of Human and Simian
Immunodeficiency Viruses
Takashi GOJOBORI, Etsuko N. MORIYAMA, Kazuho IKEO and Yasuo INAControversy continues to exist on which viruses the human immunodeficiency viruses (HIVs) originated from. To settle this controversy, we
analyzed nucleotide sequences of simian immunodeficiency viruses (SIVs)
and HIVs from the view point of molecular evolution. In particular, we
compared the nucleotide sequences of the entire genomes, gene region bygene region, between a given pair of viruses. The viral strains used in thisstudy include HIVs and four types of SIVs; SIVs isolated from mandrills(SIVmnd), African green monkeys (SIV agm ), sooty mangabeys (SIV sm ),and rhesus macaques (SIV mac)' The phylogenetic trees obtained showedthat the HIV-1 and HIV-2 groups, SIVagm , and SIVmnd all diverged fromone ancestor at almost the same time, although SIV sm and SIV mac definitely
belong to the HIV-2 group. Moreover, the evolutionary position of SIVagm
and SIV mnd on the phylogenetic tree depended on the gene examined.
From these results, we concluded that the present HIVs may have emerged
as different variants of SIVs in Old World monkeys, possibly from recombination between different strains of viruses related to SIVs. For details,
see Froc. Natl. Acad. Sci. USA. 87,4108-4111, 1990 and Sixth International
Conference on AIDS 2, 124.
Statistical Methods for Estimating Sequence Divergence
Takashi GOJOBORI, Etsuko N. MORIYAMA and Motoo KIMURA

Methods for estimating the number of nucleotide base substitutions are
crucial for studies of molecular evolution. Knowledge of the number ofbase substitutions is particularly important for computing the evolutionaryrate and constructing phylogenetic trees at the DNA level.
From the perspective of population genetics, nucleotide and amino acidsubstitutions can both be treated as stochastic processes. Methods for
estimating the number of DNA base substitutions, however, are different

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79

from those for estimating the number of amino acid substitutions. Because
only four kinds of nucleotide bases exist (usually denoted A, T, C, andG), multiple and superimposed nucleotide substitutions at the same site
may occur undetected especially when sequence divergence is great. For
example, when two comparable DNA sequences have different bases, say
A and G, at the corresponding (i.e., homologous) site, a change of Ao+To+G
may have occurred for one sequence and no change for the other at thatsite. The observed number of nucleotide differences between the two
DNA sequences is thus frequently different from the total number of nucleotide substitutions that have actually occurred during their divergence.
Statistical methods for estimating the number of nucleotide substitutions
are therefore required for comparative studies of DNA sequences. We
described various methods for estimating the number of nucleotide substitutions. In particular, we discussed the advantages and disadvantagesof these methods. For details, see Methods in Enzymology (Ed. R. F.
Doolitte) 183, 531-550.
Molecular Clock of Viral Evolution, and the Neutral Theory
Takashi GOJOBORI, Etsuko N. MORIYAMA and Motoo KIMURA

Evolution of viral genes is characterized by an enormously high speed
compared with that for nuclear genes of eukaryotic organisms. The evolutionary rates and patterns of base substitutions were examined for retroviraloncogenes, human immunodeficiency viruses (HIV), hepatitis B viruses(HBV), and influenza A viruses. Our results show that the evolutionary
process of these viral genes can readily be explained by the neutral theoryof molecular evolution. In particular, the neutral theory is supported
by our observation that synonymous substitutions always greatly predominate over nonsynonymous substitutions, even though the substitution ratevaries consideranly among the viruses. Furthermore, the exact correspondence between the high rates of evolutionary bese substitutions andthe rates of production of mutants in RNA viruses fits very nicely withpredictions of the theory. The linear relationship between substitution
numbers and time was examined to evaluate the clocklike property of viralevolution. The clock appears to be quite accurate in the influenza A
VIruses 111 man. For details, see Proc. Natl. Acad Sci. USA. 87, 1001510018.

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Patterns of Amino Acid Substitutions in the Envelope Proteinof Human Immunodeficiency Viruses and Its Application
to Vaccine Development
Takashi GOJOBORI and Etsuko N. MORIYAMA

A major obstacle to the development of a HIV vaccine is the variabilityof the env gene region in HIVs. Although the env region is variable, certain
sub-regions may have less variation than others. To identify these subregions, we first computed the frequency for each amino acid, site by site,
in the amino acid sequences of the envelope protein for various isolates of
HIV-Is. We then identified four conserved sub-regions within the gpl20
region of the envelope protein, and these were also found to be relativelyhydrophilic. Moreover, we estimated the direction of amino acid substitutions, using the phylogenetic tree for the envelope protein of HIVs.
We then made a 20 x 20 matrix for amino acid substitutions which can beused to predict the future direction of amino acid substitutions in HIVs.
In fact, it is possible to synthesize peptides with various combinations of
the predicted amino acids, taking into account the possible directions ofamino acid substitutions in the conserved sub-region. For detail, see
Population Biology of Genetics and Molecules (eds. N. Takahata and J. F.
Crow), Baifu-Kan, Tokyo, pp. 323-340, 1990.
Rates and Patterns of Nucleotide Substitutions for
Nuclear Genes of Drosophila Species
Etsuko N. MORIYAMA and Takashi GOJOBORI

It is known that the nuclear genes of the Drosophila species evolve at
rates two to four times higher than those of mammals. These rates arealso known to vary with the genes (1O.8-20.2x 1O-9!site!year) (see Jpn.
J. Genet. 52, 139-147, 1987 and Genetics 122, s41, 1989). To examine therelationships between the evolutionary rate and the mutation mechanism
in Drosophila, we estimated the patterns of nucleotide substitutions fornuclear genes in Drosophila. We then compared the patterns of Drosophilawith those for rodents, artiodactyls and primates. In Drosophila, mostnuclear genes had almost the same patterns of nucleotide substitutions at
the third codon position, although at the second codon position the patternvaried with genes examined. Furthermore, the relative frequency of trans-

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version substitutions was nearly equal to that of transition substitutions
in Drosophila. For other organisms, on the other hand, transitions occurred
more frequently than transversions. Therefore, the mutation mechanismin the Drosophila genome may be different from that in the mammalian
genome. Nevertheless, we cannot exclude the effect of the shorter generation time of Drosophila on the evolutionary rate. For details, see Jpn.
J. Genet. 55, 529-531, 1990.
Molecular Evolution of Human T-Cell Leukemia Virus
Yasuo INA and Takashi GOJOnORI

Phylogenetic trees for the human T-cell leukemia virus type I (HTLV-I)
and its related viruses were constructed using of nucleotide sequences ofthe long terminal repeat (LTR) and the tax gene. The trees showed that theviruses diverged from a common ancestor and that they are classified into
two groups whose hosts are either primates or bovines. However, thetopology of the trees for the viruses differed from that for the hosts. Thissuggests that HTLV-I and HTLV-I-related viruses evolved independentlyof host-species divergence. It also implies that interspecies transmission
between human and monkeys occurred in the past. The nucleotide diversityof the tax genes of HTLV-I was estimated to be 0.025. This value is morethan 10 times larger than that of human globin genes, but it is about 20
times smaller than that of human influenza A viruses. Thus, the genetic
variability of the HTLV-I genes seems to be higher than that of nuclear
genes but much lower than the genes of typical RNA viruses. Furthermore, we examined functional constratints on the overlapping region of
the rex and tax genes of HTLV-I. The results obtained show that forthe overlapping region, the tax gene has much stronger constraints against
amino acid changes than the rex gene. For details, see J. Mol. Evol. 31,
493-499.

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Host-independent Evolution of the Hepadnavirus Family
Yasuo INA, Masashi MIZOKAMI*, Etsuro ORITO*, Nobutoshi KAMESHIMA*,
Masahiko YAMAMOTO*, Etsuko N. MORIYAMA
and Takashi GOJOBORI

Nucleotide sequences of 25 hepatitis B viruses (HBVs), 6 woodchuck
hepatitis viruses (WHVs), 1 ground squirrel hepatitis virus (GSHV), 2
duck hepatitis B viruses (DHBVs) and 1 heron hepatitis B virus (HHBV)
have been determined to date. To examine whether the evolution of thehepadnavirus family is host-dependent, we constructed phylogenetic trees
by use of the nucleotide sequences. Although the phylogenetic trees forthe viruses had the same topology of that for their host-species, the trees
for the viruses showed that the divergence times of the viruses are much
more recent than those of their host-species. These results strongly support
our previous study (Proc. Natl. Acad. Sci. USA. 86, 7059-7062), in which
we used 18 sequences of the hepadnavirus family. Thus, the host-independent evolution of the hepadnavirus family was confirmed.
Furthermore, we estimated the pattern of nucleotide substitutions inorder to know characteristics of the mutation mechanism. The rate ofnucleotide substitution between C and T was the highest for HBV. Thisfeature is very different from those of retroviral genes (the rate of nucleotide
substitution between G and A is the highest) but similar to those of eukaryotic genes, although it is believed that HBV replicates itself by reverse
transcriptase. Based on the results, we proposed a model of mutation
mechanism of HBV. The model suggests that pyrimidine analogues could
be more effective anti-HBV drugs than purine analogues. Veda et al.,have recently reported that oxetanocin A (OXT-A), analogue of aranine,
inhibited HBV replication more effectively than 3'-azido-3'-deoxythymidine(AZT), analogue of thymidine. For details, see the Abstracts of the FourthInternational Congress of Systematic and Evolutionary Biology (ICSEBIV) 154.

* Second Department of Internal Medicine, Nagoya City University Medical School,
Nagoya.

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Molecular Evolution of Kunitz-type Protease Inhibitor
Kazuho IKEO, Kei TAKAHASHI and Takashi GOJOBORIThe Kunitz-type protease inhibitor is known as a serine protease inhibitor.
It is also known that this inhibitor is mainly found in blood or saliva as akind of soluble protein. Recently, an insertion of the Kunitz-type sequence
was found in the protein sequence of the amyloid beta precursor. It is
known that this protein accumulates in the neuritic plaques and cerebrovascular deposits in the patients with Altzheimer's disease. Collagen type VI
in chicken also has an insertion of Kunitz-type sequence. To elucidate
the evolutionary origin of these insertion sequences, we constructed aphylogenetic tree using all the available sequences of the Kunitz-type inhibitors. This tree showed that the ancestral gene of the Kunitz-typeinhibitor appeared about 500 million years ago. Thereafter, this gene
duplicated itself many times and some of these duplications were inserted
into other protein-coding genes. During this process, the insertion sequencein the amyloid precursor diverged from its ancestral gene about 350 million
years ago, and was inserted into the amyloid beta gene after duplication.
Although the function of these insertion sequences is unknown, we haveshown that these insertion sequences have an evolutionarily close relationshipwith each other.
Patterns of Nucleotide Substitution in Class I Major
Histocompatibility Complex Genes
Tadashi IMANISHI and Takashi GmOBORIThe major histocompatibility complex (MHC) genes are highly polymorphic in humans so that the value for heterozygosity exceeds 0.9. There
has been controversy over the proposed causes of such extremely highpolymorphism in MHC genes. Recently, it was proposed that MHC polymorphism is probably generated by overdominant selection. If this is
the case, selective forces may have significantly affected the patterns of
nucleotide substitution in MHC genes. To examine this possibility, we
examined the patterns of nucleotide substitution in MHC genes.
To estimate the substitution patterns, we used the following method.
First, we reconstructed the phylogenetic trees of human class I MHC genes.
Second, we estimated the nucleotide sequences of the ancestral genes by

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using the maximum parsimony method, and determined the direction of
nucleotide changes from a particular nucleotide (A, T, C, or G) to another.
Finally, we calculated 12 parameters of relative substitution frequencies,
which comprised the substitution patterns.
The substitution pattern at the antigen recognition sites (ARS) in functional MHC genes was remarkably different from that at the remaining
coding region (Non-ARS). In particular, the proportion of transitions(P.) among all substitutions was extremely low at the third codon positionsof ARS. In the HLA-A genes, p. at the third codon positions was only
6% in ARS, whereas it was 69% in Non-ARS. In HLA-B, the corresponding values were 30% in ARS and 80% in Non-ARS, respectively. On
the other hand, p. in a class I pseudogene (HLA-AR) was 57 % showing
a strong similarity to p. in other pseudogenes.
Since pseudogenes are selectively neutral, the pattern in pseudogenes isalmost identical with the pattern for spontaneous substitution mutations.
The pattern in functional genes usually deviates from the pattern in pseudogenes because they are subject to natural selection or functional constraints. In protein coding genes, transitions at the third codon positions
rarely cause amino acid replacements, while about half of all transversions
do so. Accordingly, p. at the third codon positions decreases if the rateof amino acid replacements is enhanced by natural selection, but increases
if amino acids are conserved by functional constraint. Our observations
imply that the ARS region is subject to natural selection favoring aminoacid replacements while the Non-ARS region is subject to functional constraints.
Establishment of a Phylogenetic Survey System for AIDS-related
Lentiviruses and Demonstration of a New HIV-2 Subgroup
Tomoyuki MIURA, Masashi FUKASAWA, Etsuko N. MORIYAMA, Takashi GOJOBORI,
Koh-ichi ISHIKAWA, Hajime TSUJIMOTO and Masanori HAYAMI

We designed a universal primer (UNIPOL) for DNA amplification of
AIDS-related viruses. The phylogenetic tree constructed from the presumed sequences amplified with UNIPOL was representative of the tree calculated
from whole pol gene sequences so far reported. UNIPOL was able toamplify the sequences of all four major groups of primate lentiviruses andalso that of a distinct virus from a Ghanaian patient with an AIDS-related

RESEARCH ACTIVITY IN 1990

85

complex, designated GH-2. This strain rarely hybridizes with known
HIV/simian immunodeficiency virus (SIV) DNA probes. Sequence analysisof the only amplified fragment revealed quickly that GH-2 was quite similar
to the recently reported HIV-2ALT (D205) and that these two viruses forma new subgroup distinct from known HIV-2 and SIVmac/SIVsm in the large
HIV-2 group. This system will be useful for further phylogenetic study ofvarious primate lentiviruses. For details, see AIDS 4, 1257-1261, 1990.
Maternal hybrid rescue (mhr) Gene which Rescues Embryonic
Lethal Hybrids from Drosophila simulans Females Crossedwith D. melanogaster Males
Kyoichi SAWAMURA and Takao K. WATANABE

Hybrid females from the crosses of D. simulans ~ X D. melanogaster 6
are embryonic lethal, while hybrid males from the reciprocal crosses arelarval lethal. We found new D. simulans strains which resued the former
embryonic lethality. The rescue gene(s) was named maternal hybrid
rescue (mhr), since it had a maternal effect. It was recessive and located on
the second chromosome. Embryonic lethality was caused by the incompatibility between the cytoplasm of D. simulans (C.) and the X chromosomeof D. melanogaster (Xm ) , whereas the larval lethality was caused by the
non-existence of the X chromosome of D. simulans (X.). Therefore, hybrid/'..

males (C.XmY.) from the crosses of D. simulans XX/Y ~ xD. melanogaster6 should be doubly lethal, for both the embryonic and larval stages. The
embryonic lethality was rescued by mhr, and the larval lethality was rescued
independently by the Hybrid male rescue (Hmr) gene of D. melanogaster.
Zygotic hybrid rescue (Zhr) Gene which Rescues Embryonic
Lethal Hybrids from Drosophila melanogaster Males
Crossed with Sibling Species
Kyoichi SAWAMURA and Takao K. WATANABEInterspecific hybrid females from the corsses of D. simulans ~ X D.
melanogaster 6 are generally embryonic lethal. They were zygoticallyrescued by two paternal strains. We analyzed them genetically, and named
their rescue genes Zygotic hybrid rescue (Zhr) and Zygotic hybrid rescue-

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temperature sensitive (Zhr tS ). They were located on the X chromosome of
D. melanogaster, proximal to 1-62.6 and to 1-56.7, respectively. Zhr also
rescued the embryonic lethalities of hybrid females from the crosses of D.
mauritiana ~ x D. melanogaster <5 and of D. sechellia ~ x D. melanogaster<5, but Zhr ts did not. However, if some chromosomal parts of D. mauritiana
or D. sechellia were replaced by the D. simulans chromosome homozygously,
they were also rescued by Zhr ts • Finally, we demonstrated the independenceof three hybrid lethalities in crosses between D. melanogaster and D. simulans: (1) embryonic lethality caused by incompatibility between thecytoplasm of D. simulans and the X chromosome of D. melanogaster; (2)larval lethality caused by the non-existence of the X chromosome of D.
simulans; and (3) temperature sensitive pupal lethality caused by incompatibility between the X chromosomes of these two species.

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VII.

87

HUMAN GENETICS

Characterization of Genetic Polymorphisms in
Tandem Repeated Sequence Blocks
Takashi IMAMURA

Tandemly repetitive DNA sequences are thought to have the potential toexpress the locus-specific organization of their folding structure in the nucleus. Because of the inherent possibilites of rapid evolutionary sequence
alterations, including a tolerance for mutation events, repetitive sequence. units are predestined to develop and balance locus-specific repetitive higher
order structures. This potential may create a specific chromatin folding
structure whenever there is a selection force at the position of this reptitive
DNA sequence in the genome. Human tandemly repetitive sequences
are of interest for two reasons. First they define the boundaries or the
signposts of the physical and genetic maps of human chromosomes andthus particularly important in the physical and genetic mapping of the humangenome that is now being attempted. Second, some of them are essential
for human chromosome function. In this study, I attempted to explore
the principal organization of human telomere-associated (or proterminal)
DNA at 48 distinct telomeres and that of an apparently new repeat sequencefamily which was found in PeR-amplified genomic DNA. The preliminary
results suggest that these DNAs and organized as a string of distinct repeatedsequence elements. The chromosomal distribution of these sequences
could be highly polymorphic. These data thus identify a new source ofhuman genetic variation, emphasizing the evolutionary conservation of
the organization of tandemly repeated sequence family.
Human telomeres are similar to those of simple organisms. It has beenshown that the telomere repeat of (TTGGGG)n from Tetrahymena thermophila will corss-hybridize with human telomeres, and that the cloned
Arabidopsis telomere repeat (TTAGGGG)n also recognizes humantelomeres. Human telomeres have repeating units of (TTAGGG), equivalent to those of the trypanosome telomere, which suggest that in humans,
as in yeast, DNA sequences that are significant for telomere function aretelomeric simple repeated sequences. I used a series of synthetic oligonu-

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cleotides and restriction enzymes to clone directly this sequence from humanchromosomal DNA, mainly by using a ploymerase chain reaction. The
present and previous reports suggest that human telomeres are not uniform
throughout their length, comprising several multiple, different, but related
tandem motifs. In somatic tissue cells, the major, most distal, component
consists of 5-10 kb of repeats that are very similar to the sequence(TTAGGG)n. A (TTAGGG)4 probe can now be used to identify cloned
telomeric DNA fragments and the flanking sequences derived from bothends of each human chromosome. Study is now being directed towards
cloning and sequencing of the subtelomeric or so-called proterminal single
copy fragments adjacent to these repeating units, which should prove to
be invaluable genetic markers for completing the linkage map of the humangenome and for characterizing chromosome rearrangements involving.
telomeres, which may have been stabilized during evolution.
In the course of this study an unrelated new family of repetitive DNA
was identified. A tight genomic likage was observed through the use of
non-radioactive in situ hybridization assay between this repetitive DNA andacrocentric chromosomes 13, 14, 15, 21 and 22; the centric region of chromosome 1 also had this repeated sequence block, identified by the strong
fluorescent signal on metaphase chromosome preparations. These results
suggest that the long range sequence organization is not related to anysatellite or alphoid DNA sequence. From the sequence analysis of a seriesof these repeat units, the polymorphic sequence divergence will be estimatedin due course.
Analysis of Genetic Polymorphisms in the Human Alu Sequence
Family and the Flanking Regions for Genetic
Mapping of Chromosomes 18 and X
Hitoshi NAKASHIMA, Masako SAKAI, Tomoko HASEGAWA
and Takashi IMAMURA

As a step towards constructing the complete genetic linkage map ofchromosome 18, we made a gene library from a mouse hybrid cell line withan extra human chromosome 18, using the pWE15 cosmid cloning vector.
We attempted to place these clones at distinct bands of chromosome 18
using in situ hybridization techniques and high sensitivity microscopic
fluorescent imaging apparatus. Genetic polymorphisms in regions that

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89

are identified with these clones were analysed with orne of the clone locatedeither on the short or on the long arm of chromosome 18. For this purpose
we isolated several fragments from each cosmid clone that contains the Alu
family sequence and the flanking DNA including the poly (A) tail sequence.
The results suggest that the Alu family sequence and the flanking region
are polymorphic and could be site-specifically identified. We are continuing
this line of work, aiming at the construction of a high resolution genetic
linkage map for chromosome 18 with a series of at least 100 well spaced
polymorphic markers.
One of the clones was found to be hybridized specifically with DNA atthe centromere of chromosome X. Using this clone as a probe, both thegenetic polymorphism in the centric DNA and the parental origin of chromosom X could be identified through in situ hybridization.
Beta-Thalassemia Resulting from Compound Heterozygosity for
Globin Gene Mutation in a Japanese Family: Evidence forMultiple Origin of the Thalassemia gene in
Asian Populations
Hitoshi NAKASHIMA, Yutaka CHlFU, Eisuke YOKOTA
and Takashi IMAMURA

Thalassemias are a heterogeneous group of inherited disorders of hemoglobin synthesis, all being characterized by the absence or reduced output
of one or more of the globin chains of hemoglobin. This leads to an imbalanced globin chain synthesis. One of the most remarkable aspects ofthalassemia is how it has been possible to relate the diverse clinical manifestations to primary genetic defects in the a- or p-globin genes. Thalassemias are the most common single gene disorder in the world population.
Genetic studies at the DNA level disclosed more than 40 different molecularvarieties of thalassemia. Thus, it is common for individuals to receive
genes for more than one type of thalassemia.
Although thalassemias are not frequent in Japanese, we encountered apatient with relatively severe disease resulting from heterozygosity for
two types of p-thalassemia. One mutation is a C to T substitution at IVS-2
position 654 and the second is a G to A substitution at IVS-2 position 1.
These two different mutations in the p-globin allele are associated with therestriction fragment length polymorphism (RFLPs) haplotype IX, defined

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in Mediterranean population, and X not yet defined, respectively. The
IYS-2 654 mutation is commonly found in the Chinese population,
associated with haplotype 1. The IYS-2 I mutation was found in a Mediterranean family, associated with haplotype IX, but as yet there is no
known case of this mutation in Asiatic populations. Thus, studies ofmutation at the DNA level and the linked polymorphisms in and aroundthe ~-globin gene suggest that these mutations have arisen independently
in different populations.
Before it was possible to understand the remarkable heterogeneity of~-thalassemia by analysing human DNA directly, a great deal was written
about the distribution of ~-thalassemia mutations, as was the case for the
sickle cell gene, under the assumption that population movements could
be discerned from the distribution of genetic markers of this type. Morerecently, however, it has been possible to define precise molecular lesions
and ~-globin gene haplotypes by examining the pattern of restriction fragment length polymorphisms (RFLPs) in and around the ~-globin genecarrying a thalassemia mutation. The particular arrangement of RFLPs
is referred to as the ~-globin gene haplotype. These results suggest thatthe mutation may have had multiple origins.
Molecular Phylogeny and Evolution of Human
Mitochondrial DNA
Satoshi HORAI

Using restriction fragment length polymorphism of mitochondrial DNA(mtDNA), my colleagues and I first showed that the Japanese popuation
could be separated into two distinct groups; A group with smaller frequency(group I) first diverged from the other group with larger frequency (group
II). By a more exhaustive survey, the existence of the two groups was
confirmed in two other Japanese populations, though the frequencies of
the groups are different among populations. A phylogenetic analysis among
three major racial groups indicated that the group I Japanese and the majority of Negroids first diverged from the rest of Japanese (group II) and Caucasoids. Later, we extended our analysis to various ethnic groups ofhumans to study their evolutionary relationships by the sequence analysisof the major noncoding region of mtDNA. Based on the sequence comparison of over a hundred of individuals, remarkable features of nucleotide

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91

'Substitutions and insertion/deletion events have been revealed. The nucleotide diversity among the sequences is estimated as 1.45 %, which is three
to six-fold higher than the corresponding value estimated from restriction
enzyme analysis of the whole mtDNA genome. More recently we applied
polymerase chain reaction (PCR) to molecular evolutionary studies on not
only contemporary but archaeological samples. We have succeeded toamplify mtDNA extracted from an ancient Japanese bone, whose age isestimated at about 6000 years B.P., and determined the nucleotide sequenceof part of the major noncoding region. Sequence comparison shows thatthe ancient individual has a close phylogenetic affiliation to Southeast
Asians.
It is obvious that the Mongoloids can be separated into two subpopulations in the present sequence analysis. This observation confirmed ourearlier study which clearly demonstrated the existence of two distinct groups
in the Japanese by restriction enzyme analysis. Most of those who belong
to the group I cluster in the Japanese have the deletion of 9-bp in region V(Cann and Wilson 1983). This deletion is observed not only in East Asians,
Indonesians, Taiwan Chinese but also in Polynesians with a very high frequency. The same deletion has never found in Caucasoids and Negroids.
Thus, the existence of two major groups is one of characteristics foundcommonly among Mongoloid populations. Nowadays Mongoloid descendants live in North and South America, Oceania, South East Asia, East Asia
and Siberia, adapting themselves to various environment on the earth. An
extended study of mtDNA polymorphism and its genealogy by using bothcontemporary and archaeological samples will be required to confirm theorigins and dispersal of the two subpopulations of Mongoloids.
For details, see New Aspects of the Genetics of Molecular Evolution,
Edited by M. Kimura and N. Takahata, pp. 135-152 (1991).
Mitochondrial DNA Evolution in Primates:
Transition Rate Has Been Extremely Low in the Lemur
Masami HASEGAwA*, Hirohisa K!SIDNO*, Kenji HAYASAKA
and Satoshi HORA!

Based on mitochondrial DNA (mtDNA) sequence data from a wide
range of primate species, branching order in the evolution of primates was*

The Institute of Statistical Mathematics.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

inferred by the maximum likelihood method of Felsenstein without assuming rate constancy among lineages. Bootstrap probabilities for being
the maximum likelihood tree topology among alternatives were estimatedwithout performimg a maximum likelihood estimation for each resampled
data set. Variation in the evolutionary rate among lineages was examined
for the maximum likelihood tree by a method developed by Kishino andHasegawa. From these analyses it appears that the transition rate of
mtDNA evolution in the lemur has been extremely low, only about 1/10
that in other primate lines, whereas the transversion rate does not differ
significantly from that of other primates. Furthermore, the transition ratein catarrhines, except the gibbon, is higher than those in the tarsier and inplatyrrhines, and the transition rate in the gibbon is lower than those inother catarrhines. Branching dates in primate evolution were estimatedby a molecular clock analysis of mtDNA, taking into account the rate ofvariation among different lines, and the results were compared with thoseestimated from nuclear DNA. Under the most likely model, where theevolutionary rate of mtDNA has been uniform within a great apes/humanclade, human/chimpanzee clustering is preferred to the alternative branching orders among human, chimpanzee, and gorilla. For details, see J.
Mol. Evol. 31, 113-121 (1990).
A Common Mitochondrial DNA Mutaion in the tRNA Lysof Patients with Myoclonus Epilepsy Associatedwith Ragged-Red Fibers
Makoto YONEDA*, Yoshinori TANNO*, Satoshi HORAI,
Takayuki OZAWA**, Tadashi MIYATAKE*
and Shoji TSUJI*

Nucleotide sequence analyses of muscle mitochondrial DNA (mtDNA)
from a patient with myoclonus epilepsy associated with ragged-red fibers(MERRF) revealed 33 single base substitutions, including 23 in coding
regions for mitochondrial polypeptides and 10 in non-coding regions, ascompared with the normal human mtDNA sequence. Three substitutions,in cor, ND4, and Cytb, would result in amino acid substitutions, which
are conserved among species. Of three patients with MERRF, all had an* Brain Research Institute, Niigata University.
** Faculty of Medicine, University of Nagoya.

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93

indentical A to G base substitution only at nucleotide position 8344 in thetRNALys region. The substitution was not found in 15 controls. Various
degrees of the combined enzymic defects in the oxidative phosphorylation
system of mitochondria were found in the MERRF patients. The defects
could be explained by altered founction or processing of the mutant tRNALYs.
This mutation in the tRNALyS is the most probable cause of MERRF. For
details, see Biochemistry International, 21(5), 789-796 (1990).
A Mutation in the tRNALeuIUUR) Gene Associated with the
MELAS Subgroup of Mitochondrial
Encephalomyopathies
Yu-ichi GOTO*, Ikuya NONAKA* and Satoshi HORAI

Mitochondrial encephalomyopathies are usually divided into three distinct clinical subgroups: (1) mitochondrial myopathy, encephalopathy,
lactic acidosis and stroke-like episodes (MELAS); (2) myoclonus epilepsy
associated with ragged-red fibers (MERRF); and (3) chronic progressive
external ophthalmoplegia (CPEO) including Kearns-Sayre syndrome. Large
deletions of human mitochondrial DNA and a transition mutation at the
mitochondrial transfer RNALys gene give rise to CPEO including KearnsSayre syndrome and MERRF, respectively. Here, we report an A-to-Gtransition mutation at nucleotide pair 3,243 in the dihydrouridine loop of
mitochondrial tRNALeuIUUR) that is specific to patients with MELAS.
Because this mutation creates an Apa I restriction site, we could perform asimple molecular diagnostic test for the disease. The mutation was present
in 26 out of 31 independent MELAS patients and lout of 29 CPEO patients,
but absent in the 5 MERRF and 50 controls tested. Southern blot analysis
confirmed that the mutant DNA always coexists with the wild-type DNA(heteloplasmy). For details, see Nature 348, 651-653 (1990).
Chronic Progressive External Ophthalmoplegia: aCorrelative Study of Mitochondrial DNA Deletions
and Their Phenotypic Expression in Muscle Biopsies
Yu-ichi GOTO*, Yasutomi KOGA*, Satoshi HORAI
and Ikuya NONAKA*

Deleted mitochondrial DNA (mtDNA) has been shown to coexist with* National Institute of Neuroscience.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

normal mtDNA (heteroplasmy) in muscles from chronic progressive external ophthalmoplegia, including Kerans-Sayre syndrome. In this study,
we correlated heteroplasmic mtDNA abnormality with clinical, biochemicaland histological findings with the following results: (a) large deletions
ranging from 1.8 to 8.8 kb in 22 muscle specimens from 28 patients who had
ophthalmoplegia clinically and focal cytochrome c oxidase (CCO) deficiency
by histochemistry, (2) no difference in clinical and biochemical findings
between patients with and without mtDNA deletions, (3) no relationshipbetween the size, site or populations of deleted mtDNA and respiratory
chain enzyme activities in muscles, (4) positive correlation between thenumber of CCO-deficient fibers and the populations of deleted mtDNA,
and (5) higher incidence of COO-negative fibers in patients with deleted
mtDNZ than in those with no deletion of mtDNA. These results suggestthat deleted mtDNA is, at least in part, responsible for focal CCO deficiency
as a phenotypic expression and that the investigation on pathogengetic
mechanism of focal CCO deficiency may provide a clue to understanding
the underlying pathophysiology in this disorder. For details, see Journalof Neurological Science, 100, 63-69 (1990).
Renal Tubular Involvement Mimicking Bartter
Syndrome in a Patient with Kearns-Sayre
Syndrome
Yu-ichi GOTO*. Noritomo ITAMI**, Naofumi KAJII**,
Hiroyuki TocHIMARu**, Machiko ENDo**and Satoshi HORAI

A 10-year-old boy had short stature, external opthalmoplegia, atypicalretinal pigmentary degeneration, and sensorineural hearing loss (KearnsSayre syndrome). In addition to ragged-red fibers observed on modified
Gomori trichrome staining, there were scattered fibers exhibiting no cytochrome c oxidase activity, indicating a focal deficiency. Cytochrome coxidase and other respiratory chain enzyme activities were normal biochemically. The patient also had renal tubular dysfunction, including isosthenuria, decreased urine-concentrating ability, and excessive excretion of potassium and magnesium. In addition, he had hyperreninemia and hyper* National Institute of Neuroscience.
** Hokkaido University School of Medicine.

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95

aldosteronism but no hypertension. The renal dysfunction was thought
to have resulted from a primary defect in the thick ascending limb of theloop of Henle, mimicking Bartter syndrome. In contrast to previously
described cases of cytochrome c oxidase deficiency with de Toni-Fanconi
Debre syndrome, the patient had less intensive muscle abnormalities. A
renal biopsy specimen showed ultrastructural changes in mitochondria thatwere similar to those seen in biopsy specimens of muscle. A large-scaledeletion (8.8 kilobases) in mitochondrial DNA was found in biopsy specimens of muscle and kidney. For details, see The Journal of Pediatrics,
116(6),904-910 (1990).
Mitochondrial Myopathies: Morphological Approachto Molecular Abnormalities
Takeshi SATO*, Shinji NAKAMURA*, Hiroko HIRAWAKE*, Etsuko UCHIDA*,
Yasunori ISHIGAKI*, Koichi SEKI*, Ryo KOBAYASHI*,
Satoshi HORAI and Takayuki OZAWA**

Cytochrome c oxidase (CCO) activity was shown by biochemical andhistochemical examination to be decreased in the skeletal muscles of twelve
patients with mitochondrial myopathies, especially in 8 chronic progressive
external ophthalmoplegia (CPEO) cases, which included 2 Kearns-Sayre
syndrome and 6 ocular myopathy patients. In 4 MELAS patients, NADH
cytochrome c reductase activity was decreased. Immunocytochemical examination, using anti-CCO, anti-complex I and III rabbit sera revealed thatCCO was stained more weakly in the muscle fibers of one of the CPEO
patients than in those of the control. Immuno-electron microscopic examination of CCO, complex I and III, using a method of gold labeling, wasalso performed. Extensive labeling by gold particles, representing the
localization of respiratory enzymes, could be seen in close vicinity to thecristae and inner mitochondrial membrane of normal shaped mitochondria.
The concentration of gold particles was markedly decreased in one of the
CPEO patients. To detect the localization of mitochondrial DNA or
mRNA, in situ hybridization was performed on human biopsied muscles
using a 35S labeled mitochondrial DNA probe. The wide distribution of
autoradiographic grains for mRNA over the sarcoplasm of all muscle fibers* Juntendo University School of Medicine.
** Faculty of Medicine, Nagoya University.

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was correlated with the distribution of immno-stained mitochondria. Southern blotting revealed large deletions of mitochondrial DNA in six of thepatients with CPEO. In one of these patients, in situ hybridization showeda marked decrease in density of autoradiographic grains in muscle fibers.
For details, see Bioenergetics, Edited by C. H. Kim and T. Ozawa, pp.
429-439 (1990).
In Situ Hybridization of Muscle MitochondrialmRNA in Mitochondrial Myopathies
Shinji NAKAMURA*, Takeshi SATO*, Hiroko HIRAWAKE*,
Ryo KOBAYASHI* and Satoshi HORA!

To determine whether a mitochondrial mRNA deficiency exsits in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial
myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)
were studied using in situ hybridizarion. Histochemistry and immunohistochemistry were performed along with hybridization. Hyridization reactions were widely distributed over the sarcoplasm of all muscle fibers in
the patient with MELAS. In the patient with CPEO, 80% of the fibers
showed a marked decrease in density of autoradiographic grains. This
marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochrome c oxidase (CCO).
The isotope-labeled mtDNA probe used in in situ hybridization in this study
complements a part of subunit I of CCO and a part of subunit II of complex
I in the mitochondrial gene. Our results suggest a defect in the mRNA in
this CPEO patient. For details, see Acta Neuropathol. 81, 1-6 (1990).
A Tenascin-like Gene Found in the Human Major Histocompatibility
Complex Class III Region
Ken-ichi MATSUMOTO, Miyuki ARAI, Nanayo ISHIHARA, Asako ANDo u ,Hidetoshi INoKo** and Toshimichi IKEMURA

Walking and sequencing a genome portion centromeric to CYP2lB in
the human MHC class III disclosed a clustered occurrence of fibronectin
type-III repeats in a ca. 50 kb DNA segment. The repeats are known to* Juntendo University School of Medicine.
** Department of Transplantation II, School of Medicine, Tokai University, Kanagawa.

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consist of ca. 90 amino acid residues and are present in a wide range ofprotein species. A homology search of Protein Databases showed ourrepeats to have the highest homology with repeats of tenascin, an extracellular matrix protein. Furthermore, we found that the OSG cDNA sequence, a portion of which was mapped on the opposite strand of CYP21B,
had six type-III repeats followed by a fibrinogen domain and these andour repeats showed the same polarity. Upstream of the type-III repeats(at least 14 repeats in total), we found 18.5 EGF-like repeats and 4 heptad
repeats. The organization of these repeats and of the fibrinogen domain at
the C-terminal part was closely related with that of tenascin. Thus weconcluded that there exists a tenascin-like gene in the HLA locus (Genomic>
in press).
Evident Diversity of Codon Usage Patterns of Human Genes with
Respect to Chromosome Banding Patterns and Chromosome
Numbers; Relationship between Nucleotide Sequence
Data and Cytogenetic Data
Toshimichi IKEMURA and Ken-nosuke WADA*

The sequences of the human genome compiled in DNA databases are
now about 10 megabase pairs (Mb), and thus the size of the sequences isseveral times the average size of chromosome bands at high resolution.
By surveying this sequence data, it may be possible to clarify the global
characteristics of the human genome, through correlation of gene sequence
data (kb-level) with cytogenetic data (Mb-level). By extensively searching
the GenBank database, we calculated codon usages in about 2000 humansequences. The highest G+C percentage at the third codon position was
97 %, and that of about 250 sequences was 80 % or more. The lowest
G+ C % was 27 %, and that in about 150 sequences was 40 % or less. A
major portion of the GC-rich genes was found to be on special subsets of
R-bands (T-bands and/or terminal R-bands). AT-rich genes, however,were mainly on G-bands or non-T-type internal R-bands. Average G+C %at the third position for individual chromosomes significantly differedamong chromosomes, and was related to T-band density, quinacrine dullness, and mitotic chiasmata density in the respective chromosomes (see
Ikemura, T., Wada, K., and Aota, S., Genomics 8,207-216 (1990); Ikemura,T., and Wada, K., Nucl. Acids Res. 19,4333-4339 (1991)).
01<

Chukyo University, School of Computer Sciences and Cognitive Sciences, Nagoya.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

VIII.

APPLIED GENETICS

Spatial Pattern of Genetic Variation within Wild Rice Populations
Hiroko MORISHIMA and Pascale BARBIER*

The spatial pattern of genetic variation within natural populations is
an important determinant of evolutionary and ecological genetic processes
in plants. It is also a key factor in considering strategies for sampling
natural populations. Fine scale spatial distribution of genetic variation
within natural populations of Asian common wild rice (Oryza rufipogon)
was investigated using isozyme markers.
Two wild rice populations examined were a perennial-type population
growing in a roadside ditch (NE88) and a weedy type population growing
at the fringe of a rice field (CP20, perennial-annual intermediate type), bothbeing from the Central Plain of Thailand. Seeds were sampled on an
individual basis with the locations of respective plants being recorded ona map. In the perennial population, sampling was done in a long line-transect along the ditch. In the weedy population, sampling was done along
five smaller line transects. Progenies were grown in a plant-to-row experiment at Mishima, and allozyme variations at 7 polymorphic loci were
examined using plumules and leaves at the tillering stage. The genotypeof each mother plant (individual in natural population) was estimated byallelic segregation in progeny. In the perennial population, 39 different
multilocus genotypes were identified among 60 plants. In the weedy
population, 42 different mutilocus genotypes were found among 54 plants.
To obtain quantitative measures representing the degree of similarity inmultilocus genotypes among individuals within populations, allozyme
data were anayzed in each population by a multivariate technique called"Quantification-III" or pattern analysis. Inspection of the geographicaldistribution of the score for each plant on the map revealed the contrasting
spatial patterns for the perennial and weedy populations. In the perennial
population, distribution of the scores was structured forming "genetic
neighbors", in other words similar genotypes were closely distributed. It
was also found that a number of "genets" existed although the plants prop-* Present Address:

CENS, 91191 Gif sur Yvett, France.

RESEARCH ACTIVITY IN 199099

agate mainly by vegetative means in this population. In the weedy population which propagates both by seeds and vegetative means, however, distribution of genotypes seemed to be rather random.
Spatial distribution patterns of neutral genes such as isozymes may be
a function of reproductive biology of the paInts and the degree of habitat
disturbance. Outcrossing rates of these two populations, which were
estimated by the same set of data, proved to be about 50-55 % for both
populations. Habitat is relatively stable in the perennial population, but
strongly disturbed in the weedy population. Under such circumstances,
identities of the neighbors are easily preserved and genetic variation tends
to be geographically structured in the former population. In the latter
population, gene flow efficiently functions through a high rate of seedpropagation blurring genetic neighbors. Further, strong habitat disturbancemight aid in the decay of structured genetic variation.
A Nuclear Gene Inducing Fertility Restoration in
Cytoplasmic Male Sterile Rice
Yoshio SAND

Cytoplasmic male sterility (CMS) is characterized by a failure of affected
plants to produce functional pollen. The trait is maternally inherited,
and most evidence associates it with changes in the mitochondorial genome.
Althogh maternally inherited traits are stably transmitted to progenies
in most cases, Laughnan and his coworkers found that spontaneous reversions to male fertility including cytoplasmic and nuclear changes occur
not infrequently in S type male-sterile cytoplasm of maize. The nuclear
genotype governs the frequency of reversions as well as the relative frequency
of cytoplasmic and nuclear reversions. Recent molecular studies revealed
that cytoplasmic reversions are also highly associated with reoganizationof mitochondrial genomes. However, the genetic mechanism inducing
spontaneous reversions remains to be studied. The present study gives
evidence on the presence of a single nuclear gene which induces nuclear
reversion in CMS rice.
Rice cytoplasms which exhibit CMS are classified into different types
on the basis of the effects of different nuclear genes which can restore fertility(Rf genes). Normal cytoplasm allows fertile pollen development in all
nuclear backgrounds. The (cms-bo) cytoplasm of Chinsurah boro II

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

exhibits male sterility when combined with the nucleus of Taichung 65(T65A) as reported by Shinjo (1975). Fertility restoration of (cms-bo)cytoplasm is gametophytically controlled by RII' Although male sterility
is stably expressed in the genetic background of T65A, Taichung 65 (T65B)
preserved in our laboratory showed about 8 % seed fertility when pollinatedto male-sterile plants (cms-bo)rllr];. Unexpectedly, a strange phenomenon
was detected in later generations of the hybrid, namely seed fertility gradually
increased with repeated seltings and tinally almost fully fertile plants wereobtained in the Fe generation. In this experiment, seed fertility was examined by strictly bagging to eliminate cross pollination. The occurrenceof fertile segregants might be simply explained by the assumption that
T65B carries recessive restorers and their accumlation gives rise to fertile
plants in later generation of the hybrid. The assumption was tested byestablishing plants with (cms-bo) cytoplasm and a T65B nucleus by successive
backcrossings.
The assumption was ruled out since resultant plants
still showed about 6 %seed fertility. Accordingly, the fertile plants observedin Fe might have resulted from mutational events such as found in the
S-type of maize cytoplasm.
The cytoplasm of the fertile revertant (FR37-4) was examined in relation
to cytoplasmic reversion. FR37-4 was repeatedly backcrossed with T65A
possessing rflrll . Results indicated that no cytoplasmic change was detected in FR-4 since the cytoplasm was completely male sterile in the T65A
genetic background. Therefore, mutational events seem to have occurredin the nuclear genes. A gradual increase of fertility strongly suggested thatmultiple genes are involved in the fertile revertant. FR37-4 x T65B F 2gave more fertile plants than (cms-bo) rllrll X T65B F 2 supporting the assumption that FR37-4 carries mutant nuclear genes. Conventional genic analysis for them, however, seems to be impossible because of instability.
The segrgation manners observed in later generations of (cms-bo) rl,rll X
T65B suggsted that genes controlling partial fertility in the hybrid areassociated with the induction of fertility reversion since F 2 plants showing
different degrees of fertility similarly exhibited a gradual increase in fertility
with seltings. In other words, partially fertile plants seemed to produce
fertility restoration with repeated seltings. Therefore, the genetic control
for partial fertility was examined. When (cms-bo) rf,rf, was pollinatedwith the pollens of T65A x T65B F" completely sterile and partially fertile
plants segregated into a 1: 1 ratio, suggesting that partial fertility is con-

RESEARCH ACTIVITY IN 1990

101

trolled by a single dominant gene. From the assumption, when T65A X
T65B F 2 individuals were crossed to (cms-bo) rfIrf!> three different typesof F 2 individuals which produce only sterile plants, only partially fertile
ones or both would arise. Results supported the above assumption showing
the expected ratio of I: I : 2. In addition, an allelism test revealed thatthe gene seemed to be independent of Rfl' The gene detected was designated Ifr (t). At present, whether Ifr (t) gives rise to mutational events
only in the presence of (cms-bo) cytoplasm is under investigation.
Developmental Genetics of Phenotypic Plasticity in Rice
Mitsgu EIGUCHI and Yoshio SANO

Plants utilize environmental signals for altering the timing of gene expression so as to respond to changing environments. A drastic change
in morphology due to changing environments is known as phenotypic
plasticity which is particularly important in plants because of their sessile
life style. An example of plastic response is deepwater or floating rice,
whose internodal elongation starts with increasing water depth as a meansfor escaping submergence, resulting in various changes in morphology.
However, no or little internodal elongation occurs in non-deepwater rice
as well as in air-grown deepwater rice. A number of recent experiments
indicated that deepwater tolerance is related to enhanced internodal elongation resulting from the action of the plant hormones ethlene and gibberellin. Although the inheritance of deepwater tolerance seems to be complex,
genetic and developmental regulation gives an excellent oppotunity forunderstanding the mechanisms of phenotypic plasticity in plants. In thepresent study we successfully detected a major gene which is responsiblefor deepwater tolerance in rice. We report here that responding to flood,
the gene for deepwater tolerance induces elongation of basal internodes
whose intercalary meristem produces no elongation without the gene.
The materials used were a perennial type of Oryza rujipogon (W120
from India) and a near isogenic line of Taichung 65 with wx (T65wx).
Generally, perennial types of O. rujipogon shows tolerance for deepwater
since they prefer deepwater and stable habitats while Taichung 65 is a nondeepwater rice cultivar intolerant of flood. In order to introduce an alienfactor(s) responsible for deepwater tolerance, the T65wxxWl20 F I was
successively backcrossed with T65wx as the recurrent parent. In each102 ANNUAL REPOPT OF NATIONAL INSTITUTE OF GENETICS NO. 41

generation, F 2 plants were grown in a deepwater tank and subjected toflood at 6 weeks of age. The water level was raised 10 cm every other day
to a maximum depth of 100 cm. Under those conditions, only 1/5 to 1/4
of F 2 plants survived and came to flowering and these were used for further
backcrossings. Segregation data in BC5 F" BC5 F 2 and BC4 F a consistently
supported the assumption that deepwater tolerance is controlled by a single
recessive gene which was designated dw a•
To examine gene expression of dW a during the course of development,
plants homozygous for dW a were subjected to flood at different stages.
When subjected to flood after 6 weeks of age the homozygotes survived
with elongation of internodes. However, they produced no internodal
elongation and died when subjected to flood before 4 weeks of age, suggesting that internodal elongation capability in response to flood is age
specific. The elongation of internodes begins around the time of floral
initiation in the recurrent parent T65wx. The homozygote for dW a exhibitedinternodal elongation before the time of floral initiation only when subjected
to flood. Thus, the dW a gene changes the patterns of internodal elongation
rather than causing an increase in the rate of internodal growth depending
on environmental conditions.
Population Biology of Resistance to Bacterial Leaf Blight
Disease in Rice
Chika HAMAMATSU and Hiroko MORISHIMA

Leaf blight is a serious bacterial disease in rice caused by Xanthomonas
Racial differentiation in virulence is known
among Xco isolates. In rice, on the other hand, about 20 resistance genes
which show differential responses to infection by different Xco races wereidentified. Our objective is to draw a coevolutionary scenario of riceand Xco by investigating how the genetic structure of host populations
affects that of pathogen populations, and vice versa. Our target system
is primitive or non-agricultural communities. Understanding plant-pathogen
interactions in non-agricultural ecosystems, which seem to be well balanced
without causing a serious damage to plants, may help us in considering
strategies for controlling disease epidemics in agro-ecosystems.
Using wild and landrace (native cultivars) populations of rice collectedin Thailand, Bangladesh and China, we surveyed between- and withincampestris pv. oryzae (Xco).

RESEARCH ACTIVITY IN 1990

103

populational variability in resistance to four Japanese Xco races. The
following results were obtained.
1) Wild rice plants are generally more resistant to Xco than cultivars,
although response patterns differ in respect to host genotypes and pathogen
races.
2) Response to Xco is highly variable among individuals within thesame population in wild rice as well as in landrace rice. The level of diversity in resistance is not correlated to diversity in isozymes nor to thatin quantitative traits.
3) Intrapopulational diversities in resistance revealed in the present
study seem to be partly due to segregation of major genes (race specificresponse, discontinuous variation) and partly due to polygenes (race nonspecific response, continuous variation).
4) In the samples collected in Bangladesh, populations of cultivars
adapted to deepwater conditions frequently contain individuals which are
more resistant than those adapted to shallow-water conditions and coexisting wild rice populations. This suggests that deepwater conditions favoured epidemics of Xco and that under such environment disease-mediatedselection works on host populations.
The mechanism for preserving genetic variability in resistance of hosts(and most probably in virulence of pathogens too) should be elucidated.
The fitness cost of resistance in plants as well as of virulence in pathogens
might be the core issue to be discussed in this argument. We are planningto examine variability in natural populations of pathogens. Further,several F 2 populations are under study in order to examine whether or notpresumed major genes segregating in our materials are identical to alreadyknown genes.
Size-Shape Variation of the Silica Body in Wild and Cultivated Rices
Hiroshi FUJIWARA*, Yo-Ichiro SATO and Hiroko MORISHIMA

Silica (Si0 2) absorbed by plants and accumulated on the surface of cell
walls is called "plant opal", forming replicas of plant cells. It remains
in the soil for a long time after the plants are deposited and decayed. Sincea plant-derived silica body has a species-specific morphology, analysis of*

Miyazaki University, Faculty of Agriculture, Miyazaki.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

plant opal excavated from the soil enables us to assess old vegetation formedon the soil in the past.
Hoping to shed light on the evolutionary process of rice species and thehistory of rice cultivation, we are studying plant opal obtained from thesoil in excavation sites in Japan and China. In order to identify the typeof rice which carried the excavated plant opal, we have to have information
on inter- and intra-specific variation of plant opal morphology in wild
(Oryza rufipogon) and cultivated (0. sativa) rice species.
In the present study, 53 strains of O. rufipogon collected in various Asian
countries were surveyed regarding size and shape of plant opal, and data
were analyzed together with those previously obtained for O. sativa. With
the purified silica bodies, three size measurements (length, width and thickness) and two ratios derived from the above measurements were obtainedfor each strain. As no single characteristic could discriminate clearly wildfrom cultivated types, the preceding five characteristics were collectively
analyzed using multivariate techniques. On the two-dimensional plane
defined by the first and second multivariate axes mathematically extracted,
wild strains shared a distribution range with two thirds of the cultivatedstrains. The remaining Indica and Japonica cultivars were distributed
outside this wild-cultivar complex range showing their respective distribution ranges.
The present study indicated that from plant opal data alone it is notpossible to discriminate wild from cultivated types, but samples which
are distributed outside of the wild-cultivar complex range when examinedin a multivariate plane could be judged as a cultivated type and furthermore
as either Indica or Japonica type. Variation among wild rice strains
did not show clear distinctions between two major ecotypes, perennial andannual types, but a trend in geographical variation was detected. Morphological variation observed in the present study might be largely due
to genetic factors because samples were taken from plants grown underuniform conditions. Environmental factors as well as intrapopulationalvariability should be examined. According to our preliminary results,
shape components, rather than size components, seem to be more reliablein the morphological analysis of plant opal obtained from the soil.

RESEARCH ACTIVITY IN 1990

105

New DNA Fingerprinting Procedure: Amplifed Fragment
Length Polymorphism of Hazy Associations (ALPHA)
Ikuo NAKAMURA

A new DNA fingerpriniting procedure based on PCR technique (Mullis
et al., 1986) has been developed in our laboratory. The procedure, named
ALPHA (Amplified fragment Length Polymorphism of Hazy Associations),
is designed to detect the size polymorphism of DNA fragments amplified
by a standard PCR reaction using a single primer with a lower annealing
temperature which causes a hazy association (allowing for incomplete
homology) between primer and template. Thus, the ALPHA procedure
could amplify and analyze unknown fragments in the genome without any
sequence information. In this regard, the ALPHA procedure is different
from RFLP (Botstein et al., 1980) and other DNA fingerprint methods
such as VNTR (Jeffrey et al., 1985).
In a model experiment, four different oligonucleotides (#1; 5'-GATGTCTTCTTCAAGACCTACTCAA-3', #2; 5'-TGTACCAGTTGTCCA-GAGGATGC-3', #3; 5'-CAGATCTGCTGGGACTCCTTT-3', #4; 5'-CATGATTATTTTGCTGGAGC-3') used as primer in PCR reactions were arbitrarily taken from the DNA sequence of the rice phytochrome gene reported by
Kay et al. (1989). PCR reactions were carried out in a 25 .ul reaction mix,
which contained 2.5.ul of lOx PCR buffer (100 mM Tris-HCl, 15 mM MgCI 2 ,800 mM KCl, 0.5 % BSA, 1 % Na-cholate, 1 % Triton X-lOO), 200.uM offour dNTPs, 4.uM of primer, 5 ng of rice total DNA as a template, and 1
unit of Thermus thermophilus DNA polymerase. The reactions were carriedout in a thermal programer for 42 cycles under these conditions: 1 min.
denaturation at 92°C; 1 min. annealing at 37°C; and 2 min. polymerization
at noc. Aliquots of four .ul were analyzed by 0.8 % agarose gel electrophoresis.
No amplified product was found when the #1 or #2 primers were used
for the PCR reaction. On the other hand, some fragments were amplified
when the #3 or #4 primers were used. If single 20-mer nucleotides wereused as the primer for amplification, the association possibility of the primer
with the completely complementary sequence of template DNA would
be 1 x 10- 12 • This value corresponds to a genome size as large as 2 X 1013
bp., which exceeds a rice genome of 5 x 108 bp. by long. Some primers havebeen known to associate with template DNA even with a sequence homology

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

of 80 %. Supposing it were true, the primer should theoretically associatewith the template at an average distance of 500 bp. In this case, nofragment could be amplified by the PCR reaction. But some amplified
fragments were actually found, depending on the primer sequence, in our
experiment. This means that nucleotide sequences are not completely
random in the rice genome.
The stringency of associations between the primer and template could
be controlled by the changing annealing temperature. When the annealing
temperature was raised, the number of amplified fragments decreased andthe size of fragments tended to be reduced. These changes of amplification pattern in the ALPHA procedure might reflect the secondary structureof template DNA. This is advantage of the ALPHA procedure because
different levels of polymorphism could be detected using the same primer
by changing the annealing temperatures. Two important factors might beresponsible for the amplification events of ALPHA procedure: 1) few basesat the 3' end of the primer sequence need to be perfectly matched with thetemplate in order to start DNA polymerization with a thermostable DNApolymerate. 2) the remaining part of the primer has the key for determining the thermostability of the primer-template complex. How hazy associations occur in the PCR reaction and what primer sequence is suitable for
the ALPHA procedure remain to be investigated.
The ALPHA procedure is not only a useful DNA fingerprint techniquefor detection of polymorphisms between closely related strains in verious
species, but also a convenient technique for cloning of DNA markers usefulfor RFLP analysis.
DNA Markers Specific to Subspecies of Oryza sativa
Detected by the ALPHA Procedure
Ikuo NAKAMURA, Katsuhiro KANDA* and Yo-ichiro SATO

Asian cultivated rice (Oryza sativa) is classified into two suspecies, indica
and japonica according to characteristics found in morphological andphysiological analyses (Oka. Ind. J. Ganet. Plant Breed 18, 79-89, 1958).
Recent studies carried out by many researchers indicated that this classification holds true at the molecular level. (isozymes: Graszmann. TAG, 74,* Gakken Plant Technology Institute.

RESEARCH ACTIVITY IN 1990

107

21-30, 1987; rDNA spacer: Sano and Sano. Genome 33, 209-218, 1990;
Chloroplast DNA: Ishii et al., Japan J. Genet. 63, 523-536, 1988, Dally andSecond. TAG 80, 209-222, 1990; RFLPs: Wang and Tanksley. Genome
32,1113-118,1989, Kawase. RFLP Link 1, 8-18,1991)
We were able to identify new DNA markers specific to indica ecospecies
using the ALPHA procedure (see Nakamura in this report). Typicaljaponica (Taichung 65) and indica (Ac 419) cultivars were analyzed by the
ALPHA procedure where a single primer (5'-CATGATTATTTTGCTGGAGC-3') and 5 ng of genomic DNA from each cultivar were includedin the reaction mixture, and reactions of 42 cycles were carried out withtemperatures of 94°C for denaturation, 37°C for annealing and noc forextension.
Three DNA markers (a'r-J-l, a'r_r2 and a'r_J-3) were identified as the sizesof ca. 750, 720 and 670 bp fragments, respectively. As these markers were
found only when template DNA was extracted from typical indica cultivars,
they might be useful for distinguishing the two subspecies. We made a surveyof variations in these markers among native rice cultivars using the ALPHA
procedure. Among 60 tested cultivars, 20 had all three markers, 30 had
none of them and 10 had one or two of them. They were classified asindica, japonica and "recombinant" types, respectively (Table 1). These
Table 1. Relationships between indica-japonica classifications with Sato's discriminant function and ALPHA markers
ALPHA markers
indicajaponica

recombinant

sum

20
0
20

0
30
30

6
4

26
34
60

Sato'sindicajaponica

sum

10

results showed a good correspondence to classifications done according
to Sato's discriminant function (Sato et al., In New Front. Breed. Res.
pp.185-l9l, 1986). Furthermore, this procedure is a useful tool for
analyzing the evolution of the genome structure in the indica-japonica
complex, because it can clearly identify recombinant types. These markers
were also useful for analysis of ancient rice seeds excavated in archaeologicalsites (Ann Rep. 40).

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

IX.

OATABASE

Construction of Nucleotide Sequence Data Base
Yoshihiro UGAWA, Hidenori HAYASHIDA, Sanzo MIYAZAWA,
Yoshio TATENo, and TAKASHI GOJOBORI

As one of various activities at the DNA Data Bank of Japan, we continued
construction of the database of nucleotide sequences and distribution ofthese data to the academic community in Japan. In particular, we started
a reorganization of DDBJ and a program for improvement of data bankservices. At the same time, we spent considerable time preparing the
DDBJ database Release 8. This release will contain more than 870 entries
that correspond to more than 1,570,000 nucleotide bases. We also developed several UNIX programs which were necessary for constructionof the detabase. Moreover, we started improving the management ofour computer network system by reorganizing the reiavant software.
We collaborated on construction of the internationally unified databasewith GenBank in the United States and the EMBL Data Library in EC.
International collaboration is essential for not only contributing to theacademic community in the world but also maintaining the present activitiesof DDBJ. We will definitely continue collaborative works with GenBank
and EMBL.
Codon Usage Tabulated from the GenBank Genetic Sequence Data
Ken-nosuke WADA"', Yoshiko WADA"'''', Hirofumi DOl"''''''', Fumie ISHffiASHI,
Takashi GOJOBORI and Toshimichi IKEMURA

Now the codon usages in 15137 genes can be analyzed using the nucleotide
sequence data obtained from the GenBank Genetic Sequence Data Bank
(Release 65.0, Sep., 1990). This codon database was registerd as CUTG
by LiMB Database (Listing of Molecular Biology databases, contructed by'""''''"'''''''

From Chukyo University, School of Computer and Cognitive Sciences, Nagoya.
Niigata University, School of Medicine, Niigata.
From International Institute for Advanced Study of Social Information Science,
Fujitsu Limited.

RESEARCH ACTIVITY IN 1990

109

Los Alamos National Laboratory). Because of the growing size of thedetabase, it had become impossible to print the data. Distribution of thedatabase with the electric version of the Sequence Supplement of Nucleic
Acids Research using a CD ROM, is planned possibly beginning in 1991.
This year was a transition year, and thus we have sent upon request, amagnetic tape or a hard copy listing the codon usages in 15137 genes. For
details, see Kennoske Wada, Yoshiko Wada, Hirofumi Doi, Fumie Ishibashi, Takashi Gojobori and Toshimichi Ikemura (1991) Nucl. Acids Res.
19 (Supplement), 1981-1986.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

PUBLICAnONSAMEMURA, J., ICHIKAWA, H. and OHTSUBO, E.: Tn3 Transposition immunity is conferredby the transposase-binding domain in the terminal invertedrepeat sequence of Tn3.
Gene 88: 21-24, 1990.
AOKI, K.: Some theoretical aspects of the origin of cultural transmission. In "Evolutionof Life" (S. Osawa and T. Honjo, eds.). Springer-Verlag, Tokyo, in press.
AOKI, K. and FELDMAN, M. W.: Recessive hereditary deafness, assortative mating, andpersistence of a sign language. Theor. Popul. BioI. (in press).
AzuMA, Y., YAMAGISHI, M., UESHIMA, R. and ISHIHAMA, A.: Cloning and sequence
determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest
subunit of RNA polymerase II. Nucleic Acids Res. 19: 461-468, 1991.
BARBIER, P. and ISHIHAMA, A.: Annual and perennial differentiation in wild rice: Analysis by direct sequencing of PCR-amplified DNA. Rice Genet. Newslett. 7: 138140, 1990.
BARBIER, P. and ISHIHAMA, A.: Relatedness of annual and perennial strains of Oryza
rufipogon as determined by nuclear DNA sequences. Proc. Internatl. Rice Genet.
Symp. 81: 693-702, 1991.
BARBIER, P. and ISHIHAMA, A.: Variation in the nucleotide sequence of a prolamin genefamily in wild rice. Plant Mol. BioI. 15: 191-195, 1990.
BARBIER, P., MORISHIMA, H. and ISHIHAMA, A.: Relatediess of annual and perennial
strains of Oryza rufipogon investigated with nuclear DNA sequences. Theor. Appl.
Genet. 81: 693-702, 1991.
EOUCHI, Y. and TOMIZAWA, J.: Complex formed by complementary RNA stem-loops
and its stabilization by a protein: Function of ColEI Rom protein. Cell 60: 199-209,
1990.
EOUCHI, Y. and TOMIZAWA, J.: Control of ColE1 plasmid replication: Complex formed
by RNA I and RNA II and its stabilization by Rom protein. In "Molecular Mechanisms in DNA Replication and Recombination" (c. C. Richardson and I. R. Lehman,eds.) pp. 203-214, Wiley-Liss, New York, 1990.
FUJISAKI, S., HARA, H., NISHIMURA, Y., HORIUCHI, K. and NISHINO, T.: Cloning andnucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthaseactivity in Escherichia coli. J. Biochem. 108: 995-1000, 1990.
FUJISAWA, T., DAVID, C. N. and BoscH, T. C. G.: Transplantation stimulates interstitialcell migration in Hydra. Dev. BioI. 138: 509-512, 1990.
FUJIYAMA, A. and TAMANOI, F.: RAS2 Protein of S. cerevisiae undergoes removal ofmethionine at N-terminus and removal of three amino acids at C-terminus. J.
BioI. Chern. 265: 3362-3368, 1990.
FUKAI,U-KoBAYASHI, K. and Y. TATENO: Robustness of maximum likelihood tree estimation against different patterns of base substitutions. J. Mol. Evol. 32: 79-91, 1991.
GOJOBORI, T. and MORIYAMA, E. N.: Molecular phylogeny of AIDS viruses and its

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application to vaccine development. In "Population Biology of Genes and Molecules"(N. Takahata and J. F. Crow, eds.), pp. 323-340, Baifukan, 1990.
GOJOBORI, T., MORIYAMA, E. N., INA, Y., IKEO, K., MIURA, T., TSUJIMOTO, H., HAYAMI,
M. and YOKOYAMA, S.: Evolutionary origin of human and simian immunodeficiency
viruses. Proc. Natl. Acad. Sci. USA. 87: 4108-4111, 1990.
GOJOBORI, T., MORIYAMA, E. N. and KIMURA, M.: Molecular Clock of Viral Evolution,and the Neutral Theory. Proc. Natl. Acad. Sci. USA. 87: 10015-10018, 1990.
GOJOBORI, T., MORIYAMA, E. N. and KIMURA, M.: Statistical methods for estimating
sequence divergence. In "Methods in Enzymology", Vol. 183 (R. F. Doolittle, ed.),pp. 531-550, Academic Press, San Diego, 1990.
GOTO, Y., ITAMI, N., KAJII, N., TOCHIMARU, H., ENDO, M. and HORAI, S.: Renal tubular
involvement mimicking Bartter's syndrome in a patient with Kearns-Sayre syndrome.
J. Pediatr. 116: 904-910, 1990.
GOTO, Y., KOGA, Y., HORAI, S. and NONAKA, I.: Chronic progressive external ophthalmoplegia: A correlative study of mitochondrial DNA deletions and theirphenotypic expression in muscle biopsies. J. Neurol. Sci. 100: 63-69, 1990.
GOTO, Y., NONAKA, I. and HORAI, S.: A mutation in the tRNALeu(UUR) gene associatedwith the MELAS subgroup of mitochondrial encephalopathies. Nature 348: 651653, 1990.
GREENSTEIN, D. and Horiuchi, K.: Replication enhancer-independent mutation increases
the co-operativity with which an initiator protein binds its origin. J. Mol. BioI.
211: 91-101,1990.
HANKINS, R. W., NAGATA, K., KATO, A. and ISHIHAMA, A.: Mechanism of influenza
virus transcription inhibition by matrix (M!) protein. Res. Virol. 141: 305-314,
1990.
HARADA, H., SAKAGAMI, H., NAGATA, K., Oh-HARA, T., KAWAZOE, Y., ISHIHAMA, A.,
HATA, N., MISAWA, Y., TERADA, H. and KONNO, K.: Possible involvement oflignin structure in anti-influenza virus activity. Antiviral Res. 15: 41-50, 1991.
HASEGAWA, M., KISHINO, H., HAYASAKA, K. and HORAI, S.: Mitochondrial DNA
evolution in primates: Transition rate has been extremely low in lemur. J. Mol.
Evol. 31: 113-121, 1990.
HAYASHI, J. I., TANAKA, M., SATO, W., OZAWA, T., YONEKAWA, H., KAGAWA, Y. and
OHTA, S.: Effects of ethidium bromide treatment of mouse cells on expression andassembly of nuclear-coded subunits of complexes involved in the oxidative phosphoIylation. Biochem. Biophys. Res. Commun. 167: 216-221, 1990.
HAYASHI S.: Transcriptional regulation during development Jikkenn-igaku 9: 252-258,
1991 (in japanese).
HAYASHI, S. and SCOTT, M.: What determines the specificity of action of Drosophila
homeodomain Protein. Cell 63: 883-894, 1990.
HIROSE, S. and OHTA, T.: DNA supercoiling and eukaryotic transcription-cause andeffect. Cell Struct. Funct. 15: 133-135,1990.
HONDA, A., MUKAIGAWA, J., YOKOlYAMA, A., KATO, A., UEDA, S., NAGATA, K., KRYSTAL,
M., NAYAK., D. P. and ISHIHAMA, A.: Purification and molecular structure of
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HORAI, S. and HAYASAKA, K.: Intraspecific nucleotide sequence differences in the major
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HORAI, S. and HAYASAKA, K.: Molecular Evolution and Biology of Human Mitochondrial DNA. In "Bioenergetics: Molecular Biology, Biochemistry and Pathology"(c. H. Kim and T. Ozawa, eds.), pp. 373-387, Plenum Press, New York, 1990.
HORI, T., TAKAHAsHI, E., AYUSAWA, D., TAKElsHI, K., KANEDA, S. and SENO, T.: Regional assignment of the human thymidylate synthase (TS) gene to chromosome band
18 p. 11.32 by nonisotopic in situ hybridization. Human Genet. 85: 576-580, 1990.
ICHIKAWA, H., IKEDA, K., AMEMURA, J. and OHTSUBO, E.: Two domains in the terminal
inverted repeat sequence of transposon Tn3. Gene 86: 11-17, 1990.
ICHIKAWA H. and OHTSUBO, E.: In vitro transposition of transposon Tn3. J. BioI.
Chern., 265: 18829-18832,1990.
IGARASHI, K., FUJITA, N. and ISHIHAMA, A.: Identification of a subunit assembly domain
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IGARASHI, K., FUJITA, N. and ISHIHAMA, A: Sequence analysis of two temperaturesensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase. Nucleic Acids Res. 18: 5945-5948, 1990.
IKEMURA, T.: Genome organization and codon usage & molecular biology databasesin the world. In "RNA JForld" (S. Osawa and Y. Shimura, eds.), pp. 205-232,
pp. 233-259, Kodansha Scientific, 1990 (in Japanese).
IKEMURA, T.: Life as evolutionary products. In "Frontier in Biophsics" (The biophysical society of Japan, ed.), pp. 268-271, Kodansha Scientific, 1990, (in Japanese).
IKEMURA, T., WADA, K-N. and AOTA, S.: Giant G+C% mosaic structures of the humangenome found by arrangement of GenBank human DNA sequences according to
genetic positions. Genomics 8: 207-216, 1990.
IMAI, H. T., TAYLOR, R. W., KUBOTA, M., OGATA, K. and WADA, M.: Notes on theremarkable karyology of the primitive ant Nothomyrmecia macrops, and of the related
genus Myrmecia (Hymenoptera: Formicidae). Psyche 97: 133-140, 1990.
IMAI, H. T., WADA, M. Y. and MORIWAKI, K.: The sex chromosome association (Sxa)gene is located on the X-chromosome in mice. Jpn. J. Genet. 65: 65-69, 1990.
INA, Y. and GOJOBORI, T.: Molecular evolution of human T-cell leukemia virus. J.
Mol. Evol. 31: 493-499, 1990.
INAMoTo, S., ABO, T. and OHTSUBO, E.: Binding sites of integration host factor in oriTof plasmid RIOO. J. Gen. Appl Microbiol. 36: 287-293, 1990.
INAMoTo, S. and OHTSUBO, E.: Specific binding of the TraY protein to oriT and thepromoter region for the traY gene of plasmid RlOO. J. BioI. Chern. 265: 64616466,1990.
INAMOTO, S., YOSHIOKA, Y. and OHTSUBO, E.: Site- and strand-specific nicking in vitro
into oriTby the TraY-TraI endonuclease of plasmid RI00. J. BioI. Chern. (in press).

PUBLICAnONS113

INOUE, Y., WATANABE, T. K. and WATADA, M.: Natural and laboratory hybridization
between Drosophila melanogaster and D. simulans. Jpn. J. Genet. 65: 47-51, 1990.
ISHIHAMA, A.: Duplication of appratuses for gene expression. In "Japanese ScientificMonthly", 44: 493-497, 1991 (in Japanese).
ISHIHAMA, A.: Genes for cell growth in Escherichia coli. In "Control of Cell Growthand Division" (A. Ishihama and H. Yoshikawa, eds.), pp. 199-204, Jpn. Sci. Soc.
Press/Springer-Verlag, 1991.
IsHIHAMA, A.: Global control of gene expression in bacteria. In "Control of Cell
Growth and Division" (A. Ishihama and H. Yoshikawa, eds.), pp. 121-140, Jpn.
Sci. Soc. Press/Springer-Verlag, 1991.
ISHIHAMA, A.: Molecular assembly and functional modulation of Escherichia coli RNA
polymerase. In "Adv. Biophys", Vol. 26, pp. 19-31, 1990.
ISHIHAMA, A.: The promoter selectivity of Escherichia coli RNA polymerase. In "GeneExpression", Mochida Memorial Found., Tokyo, 1990.
ISHIHAMA, A.: Structure and transcription-replication of the influenza virus genome.
In" Virus", 40: 33-37, 1990 (in Japanese).
ISHIHAMA, A.: Transcription and RNA processing. In "l\fedical Molecular Biology"(M. Muramatsu, ed.), Nankodo, Tokyo, 1991 (in Japanese).
ISHIHAMA, A.: FUJITA, N., IGARASHI, K. and UESHIMA, R.: Structural and functional
modulation of Escherichia coli RNA polymerase. In "Structure and Function of
Nucleic Acids and Proteins" (C-W. Wu and F. H. Wu, eds.), Raven Press, New
York, pp. 145-152, 1990.
ISHIHAMA, A., FUJITA, N., IGARASHI, K., UESHIMA, R., NAKAYAMA, M. and YAMAZAKI,
Y.: Molecular mechanisms of transcription regulation: Promoter selectivity of RNApolymerase. In "Recent Advances in Biochemistry" (S. Y. Byun et al., eds.) pp. 179185, 1991.
ISHIHAMA, A. and HONDA, A.: Markers for RNA viruses. In "Research Manuals in
Bioscience" (T. Seno et al., eds.), pp. 349-358, Kyoritsu Shuppan, Tokyo, 1990 (inJapanese).
ISHIHAMA, A. and KAJITANI, M.: Markers for gene expression: transcription apparatuses.
In "Research Manuals in Bioscience" (T. Seno et al., eds.), pp 564-576, Kyoritsu
Shuppan, Tokyo, 1990 (in Japanese).
ITO, M., MATSUHASHI, M., SENO, T. and AYUSAWA, D.: High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants. Somat. Cell. Mol.
Genet. 16: 443-450, 1990.
KAJITANI, M. and ISHIHAMA, A.: Identification and sequence determination of the hostfactor gene for bacteriophage Qf3. Nucleic Acids Res. 19: 1063-1066, 1991.
KANEDA, S., NALBANTOGLU, J., TAKEISHI, K., SHIMIZU, K., GOTOH, 0., SENO, T. andAYUSAWA, D.: Structural and functional analysis of the human thymidylate synthase gene. J. BioI. Chern. 265: 20277-20284,1990.
KATO, H., MORrwAKI, K. and NOMURA, T.: Genetic monitoring: with emphasis on theusefulness of the critical subsets. In "Laboratory Animal and Health for All"
(Erichsen, S., Coates, M. E. and Chatikavanij, P., eds.), Thai Watana Panich, pp.
261-278, 1990.

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KATO, J., NISHIMURA, Y., IMAMURA, R., NIKI, H., HIRAGA, S. and SUZUKI. H.: New
topoisomerase essential for chromosome segregation in E. coli. Cell 63: 393--404,
1990.
KATOH, H. UTSU, S., CHEN, T. and MORIWAKI, K.: H-2 polymorphisms in outbred
strains of mice. Laboratory Animal Science 40: 490--494,1990.
Kmo, Y., AONO, M., YAMAKI, T., MATSUMOTO, K., MURATA, S., SANEYOSHI, M. and
OKADA, N.: Shaping and reshaping of salmonid genomes by amplification of tRNAderived retroposons during evolution. Proc. Natl. Acad. Sci. USA 88: 2326-2330,
1991.
KIMURA, M.: Some models of neutral evolution, compensatory evolution, and the shifting balance process. Theor. Popul. BioI. 37: 15D-158, 1990.
KIMURA, M.: The present status of the neutral theory. In "Population Biology of Genes
and Molecules" (N. Takahata and J. F. Crow, eds.), pp. 1-16, Baifukan, Tokyo, 1990.
KIMURA, M.: Theorie Neutraliste de l' Evolution. (Preface de Jacques Ruffle), Flammarion, Paris, 1990.
KLEIN, J. and TAKAHATA, N. 1990 The Major Histocompatibility Complex and the Quest
for Origins. Immunological Review 113: 5-25.
KOBAYASHI, M., NAGATA, K. and ISHIHAMA, A.: Promoter selectivity of Escherichia coli
RNA polymerase: Effect of base substitutions in the promoter - 35 region on promoter strength. Nucleic Acids Res. 18: 7367-7372, 1990.
KOIKEDA, S., IBUKI, R., SAWADA, Y., NAGATA, K., SHIBATA, H., MASAMUNE, Y. and
NAKANISHI, Y.: Nuclear factor I stimulates transcription of the adenovirus 12 EIA
gene in a cell-free system. Biochem. Biophys. Acta, 1048: 85-92, 1990.
KOHARA, Y.: Correlation between the physical and the genetic map of E. coli K-12
chromosome. In "The Bacterial Chromosome" (M. Riley and K. Drlica, eds.) 29--42,
Am. Soc. Microbiol., Washington D. c., 1990.
KONDO, R., SATTA, Y., MATSUURA, E. T., ISHIWA, H., TAKAHATA, N. and CHIGUSA, S. I.:
Incomplete maternal inheritance of Drosophila mtDNA. Genetics 126: 657-663,
1990.
KOSEKI, H., IMAI, K., NAKAYAMA, F., SADO T., MORIWAKI, K. and TANIGUCHI, M.:
Homogenous junctional sequence of the V14 + T-cell antigen receptor J chain expanded in unprimed mice. Proc. NatI. Acad. Sci. 87: 5248-5252, 1990.
LOVELAND, B. E., WANG, C.-R., YONEKAWA, H., HARMEL, E. and FISCHER FISCHERFINDAHL, K.: Maternally transmitted histocompatibility antigen of mice: A hydrophobic peptide of a mitochondrially encoded protein. Cell 60: 971-980, 1990.
MAEDA, Y., NUNOMURA, K. and OHTSUBO, E.: Differential scanning calorimetric studyof the effect of intercalators and other kinds of DNA-binding drugs on the stepwise
melting of plasmid DNA. J. Mol. BioI. 215: 321-329, 1990.
MAEDA, Y., NUNOMURA, K. and OHTSUBO, E.: Interaction of a plasmid pJL3-TB5 DNA
and an anthracycline antibiotic 4'-O-tetrahydropyranyladriamycin (pyrarubicin).
Thermochimica Acta. 163: 129-132, 1990.
MASUKATA, H. and TOMIZAWA, J.: A mechanism of formation of a persistent hybrid
between elongating RNA and template DNA. Cell 62: 331-338, 1990.
MATSUMOTO, K., ARAl, M., ISHIHARA, N., ANDO, A., INOKO, H. and IKEMURA, T.: Cluster

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of fibronectin type-III repeats found in the human major histocompatibility complex
class III region shows the highest homology with the repeats in an extracellular matrix
protein, tenascin. Genomics (in press).
MATSUTANI, S. and OHTSUBO, E.: Nucleotide sequence of insertion element IS629.
Nucl. Acids Res. 18: 1899, 1990.
MISHINA, Y. AYUSAWA, D., SENO T. and KOYAMA, H.: Thymidylate stress induces
homologous recombination activity in mammalian cells. Mutat. Res. 246: 215220, 1991.
MIURA, S., OHTANI, K., MURATA, N., NIKI, M., OHBO, K., INA, Y., GOJOBORI, T., TANAKA,
Y., TOZAWA, H., NAKAMURA M. and SUGAMURA, K.: Molecular cloning andcharacterization of a novel glycoprotein, gp34 that is specificaly induced by thehuman T-cellienkemia virus type I transactivator p40tax. Mol. Cell. BioI. (in press).
MIURA, T, SAKURAGI, J., KAWAMURA, M., FUKASAWA, M., MORIYAMA, E. N. GOJOBORI,
T., ISHIKAWA, K., MINGLE, J. A. A., NETTEY, V. B. A., AKARI, H., ENAKI, M., TsuJIMOTO H. and HAYAMI, M.: Establishment of a phylogenetic survey system for
AIDS-related lentiviruses and demonstration of a new HIV-2 subgroup. AIDS 4:
1257-1261, 1990.
MIYAUCHI, T., KANEKURA, T., YAMAOKA, A., OZAWA, M., MIYASAWA, S. and MURAMATSU, T.: Basigen, a new, broadly distributed member of the immunoglobulin
superfamily, has strong homology with both the immunoglobulin V domain andbeta-chain of major histocompatibility complex class II antigen. J. Biochem. 107:316-323, 1990.
MIZUMOTO, K., SHIBAGAKI, Y., IToH, N., YAMADA, H., NAGATA, S. and KAZIRO, Y.:
Structure and function of yeast capping enzyme. In "Nucleic Acid Methylation"(Eds.: G. Clawson, D. Willis, A. Weissbach & P. Jones), Alan R. Liss, Inc., NY,
pp. 45-56, 1990.
MIZUTANI, M. OHTA, T., WATANABE, H., HANDA, H. and HIROSE, S.: Negative supercoiing of DNA facilitates an interaction between transcription factor lID and thefibroin gene promoter. Proc. Nat. Acad. Sci. USA. 88 (in press).
MORISHIMA, H. and BARBIER, P.: Mating system and genetic structure of natural popula-·
tions in wild rice Oryza rufipogon. Plant Species Biology 5: 31-40, 1990.
MORIWAKI, K., SAGAI, T., SHIROSHI, T., BONHOMME, F., WANG, C., HE, X., JIN, M. and
Wu, Z.: Mouse subspecies differentiation and H-2 polymorphism. Biological
Journal of Linnean Society 41: 125-139, 1990.
MORIYAMA, E. N. and Gojobori, T.: Molecular Evolution of Human and Simian Immunodeficiency Viruses. In "New Aspects of the Genetics of Molecular Evolution".
(M. Kimura and N. Takahata, eds.) Japan Scientific Societes Press/Springer-Verlag,
Berlin, pp. 291-301, 1991.
MORIYAMA, E. N., INA, Y., IKEO, K., SHIMIZU, N. and GOJOBORI, T.: Mutation pattern of
human immunodeficiency virus genes. J. Mol. Evol. 32: 360-363, 1991.
MORIYAMA, E. N., INA, Y., IKEO, K., MIURA, T., TSUJIMOTo, H., YOKOYAMA, S., HAYAMI,
M. and GOJOBORI, T.: The origin of human and simian immunodeficiency viruses.
Sixth International Conference on AIDS, vol. 2, 124, 1990.
MUTo, A., ANDACHI, Y., YUZAWA, H., YAMAO, F. and OSAWA, S.: The organization

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and evolution of transfer RNA genes in Mycoplasma capricolum. Nucl. Acids
Res. 18: 5037-5043, 1990.
NAGATA, K. and MATSUMOTO, K.: Markers for gene expression-transcription factors.
In "Research Manuals in Bioscience" (T. Seno et al., eds.), pp. 57&-583, Kyoritsu
Shuppan, Tokyo, 1990 (in Japanese).
NAGATA, K., SAKAGAMI, H., HARADA, H., NONOYAMA, M., ISHIHAMA, A. and KONNO,
K.: Inhibition of influenza virus infection by pine cone antitumor substances.
Antiviral Res. 13: 11-22, 1990.
NAKAMURA, I. and SATO, Y.-I.: Amplification of chloroplast DNA fragment from asingle ancient rice seed. Proc. of the Intnl. Rice Symp. (in press).
NAKAMURA, K., HASHIMOTO, Y., MORIWAKI, K., YAMAKAWA, T. and SUZUKI, A.: Genetic regulation of CM4 (NeuAc) expression in mouse erythrocytes. J. Biochem.
107: 3-7, 1990.
NAKAMURA, K., SASAKI, M., FUJIMOTO, J., ENOMOTO, Y., KANEKO, Y., OZAKI, M., MIYASHITA, T., TSUNEMATSU, Y, HATA, J., KOBAYASHI, N. and MIZUTANI, S.: Moleculardiversity of precursor B acute lymphoblastic leukemias identified by the immunoglobulin heavy chain gene organization. Leukemia, 2: 10&-110,1990.
NAKAMURA, S., SATO, T., HIRAWAKE, H., KOBAYASHI, R., FUKUDA, Y., KAWAMURA, J.,
UJIKE, H. and HORAI, S.: In situ hybridization of muscle mitochondrial mRNAin mitochondrial myopathies. Acta. Neuropathol. 81: 1-6, 1990.
NAKAMURA, T., IHARA, T., NAGATA, T., ISHIHAMA, A. and UEDA, S.: A complementdependent neutralizing monoclonal antibody against glycoprotein II of pseudorabies
virus. Veterinary Microbiol. 24: 193-198, 1990.
NAKASHIMA, H., FUJIYAMA, A. KAGIYAMA, S. and Imamura, T.: Genetic polymorphismsof gene conversion within the duplicated human a-globin loci Hum. Genet. 84:
568-570, 1990.
NAKAYAMA, M., FUJITA, N., OSAWA, S. and ISHIHAMA, A.: Identification and characterization of RNA polymerase sigma factor from Micrococcus luteus. J. BioI.
Chem. 266: 2911-2916, 1991.
NARITOMI, Y., NAKASHIMA, H., KAGIMOTO, M., NAITO, Y., YOKOTA, E. and IMAMURA,
T.: A common Chinese fJ-thalassemia mutation found in a Japanese family. Hum.
Genet. 84: 480-482, 1990.
NATSUUME-SAKAI, S., NONAKA, M., NONAKA, M., HARADA, Y., SHREFFLER, D. C. andMoriwaki, K: Demonstration of an unusual allelic variation of mouse factor H
by the complete cDNA sequence of the H.2 allotype. Jour. Immunol. 144: 358-362,
1990.
NISHIMURA, A., AKIYAMA, K., KOHARA, Y., TAKEDA, Y., NISHIMURA, Y., HIGASHITANI,A., YASUDA, S., HORIUeH, K. and HIROTA, Y.: Mapping of a whoe set of cell
division genes in Escherichia coli K-12. In "Control of cell growth and division"(Ishihama, A. and Yoshikawa, H., eds.), Japan Sci. Soc. Press, Tokyo(SpringerVerlag, Berlin, pp. 205-223, 1991.
NISHIMURA, A., MORITA, M., NISHIMURA, Y. and SUGINO, Y.: A rapid and highly efficientmethod for preparation of competent Escherichia coli cells. Nucl. Acids. Res. 18:
6169,1990.

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NOBUSAWA, E., AOYAMA, T., KATO, H., SUZUKI, Y., TATENO, Y. and NAKAJIMA, K.:
Comparison of complete amino acid sequences and receptor binding properites
among 13 serotype hemagglutinins of influenza A viruses. Virology 182: 475-485,
1991.
NOMURA, T. and YONEZAWA, K.: Genetic correlations among life history charactersof adult females in the azki bean weevil Callosobruchus chinensis (L.) (Coleoptera:
Bruchidae). App!. Ent. Zoo!. 25: 423-430, 1990.
OHTA, T.: How gene families evolve. Theor. Pop. Bio!. 37: 213-219, 1990.
OHTA, T. and HIROSE, S.: Purification of DNA supercoliling factor from the posteriorsilk gland of Bombyx mori. Proc. Nat!. Acad. Sci. USA. 87: 5307-5311, 1990.
OHTA, T. and TACHIDA, H.: Theoretical study of near neutrality. I. Heterozygosity
and rate of mutant substitution. Genetics 126: 219-229, 1990.
OKADA, K., OHTA, T. and HIROSE, S.: Negative supercoiling of DNA by eularyotic
DNA topoisomerase II and dextran sulfate. J. Biochem. 109: 365-369, 1991.
OKADA, N., AONO, M., ENDOH, H., KIDO, Y., KOISHI, R., MATSUMOTO, K., MATsuo, M.,
MURATA, S., NAGAHASHI, S. and YAMAKI, T.: Evolution of repetitive sequences. In"Evolution of life" (S. Osawa and T. Honjo, eds.), Springer-Verlag, Tokyo/Berlin,
pp. 175-186, 1991.
OZAKI, M., ASADA, M., TAMURA, C., ZHONG, W. K., NAKAMURA, K., FUJIMOTO, J., MIYASHITA, T., KOBAYASHI, N. and MIZUTANI, S.: Molecular analysis of 5'J region of
immunoglobulin heavy chain gene in human acute leukemias. Leukemia 6: 415418, 1990.
SAKAGAMI, H., NAGATA, K., ISHIHAMA, A., OH-HARA, T. and KAWAZOE, Y.: Antiinfluenza virus activity of synthetically polymerized phenylpropenoids. Biochem.
Biophys. Res. Commun. 172: 1267-1272, 1990.
SANO, Y.: The genic nature of gamete eliminator in rice. Genetics 125: 183-191, 1990.
SANO, Y. and SANO, R.: Variation of the intergenic spacer region of riboso mal DNA
in cultivated and wild rice species. Genome 33: 209-218, 1990.
SATO, T., NAKAMURA, S., HIRAWAKE, H., UCHIDA, E., ISHIGAKI, Y., SEKI, K., KOBAYASHI,
R., HORAI, S. and OZAWA, T.: Mitochondrial myopathies: Morphological approach
to molecular abnormalities. In "Bioenergetics: Molecular Biology Biochemistry
and Pathology" (c. H. Kim and T. Ozawa, eds), Plenum Press, New York, pp. 429439, 1990.
SATO, Y. I., ISHIKAWA R. and MORISHIMA, H.: Nonrandom association of genes andcharacters found in an indicaxjaponica hybrid of rice. Heredity 65: 75-79, 1990.
SATTA, Y. and TAKAHATA, N.: Evolution of Drosophila mitochondrial DNA and thehistory of the melanogaster subgroup. Proc. Nat!. Acad. Sci. USA. 87: 9558-9562,
1990.
SEKINE, Y. and OHTSUBO, E.: Translational frameshifting in IS elements and in othergenetic systems. In "New Aspects of The Genetics of Molecular Evolution". Eds.
M. Kimura and N. Takahata. Japan Scientific Societies Press, Tokyo/SpringerVerlag, Berlin (in press).
SHIMIZU, N., TAKEUCHI, Y., NARUSE, T., INAGAKI, M., MORIYAMA, E. N., GOJOBORI, T.
and HOSHINO, H.: Six strains of human immunodeficiency virus type-l isolated in

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Japan and their molecular phylogeny. J. Mol. Evol. (in press).
SHIROISHI, T., HANZAWA, N., SAGAI, T., ISHIURA, M., GOJOBORI, T., STEINMETZ, M. and
MORIWAKI, K.: Recombinational specific to female meiosis in the mouse major
histocompatibility compex. Immunogenet. 31: 79-88, 1990.
SHIROSHI, T., SAGAI, T., HANZAWA, N., GOTOH, H. and MORlwAKI, K.: Genetic control
of sex-dependent meiotic recombination in the major histocompatibility complex.
EMBO J. 10 (in press).
SUGITA, S., YOSHIOKA, Y., ITAMURA, S., KANEGAE, Y., OGUCHI, K., GOJOBORI, T., Nerome,
K. and OYA, A.: Differences in evolutionary patterns of swine and human influenza
virus hemagglutinin (HI subtype) gene: A quantitative and qualitative analysis ofmolecular evolution. J. Mol. Evol. (in press).
SUZUKI, H., KURIHARA, Y., KANEHISA, T. and MORIWAKI, K.: Variation in the distribution of silver-staining nucleolar organizer regions on the chromosomes of thewild mouse, Mus musculus. Mol. BioI. Evol. 7: 271-282, 1990.
SUZUKI, H., TSUCHIYA, K., SAKAIZUMI, M., WAKANA, S., GOTOH, 0., SAITOU, N., MORIWAKI, K. and SAKURAI, S.: Differentiation of restriction sites in ribosomal DNA
in the genus Apodemus. Biochemical Genetics 28: 137-149, 1990.
TACHIDA, H.: A population genetic model of selection that maintains specific trinucleotides at a specific location. J. Mol. Evol. 31: 10-17, 1990.
TAJIMA, F.: Relationship between DNA polymorphism and fixation time. Genetics
125: 447-454, 1990.
TAJIMA, F.: Relationship between migration and DNA polymorphism in a local population. Genetics 126: 231-234, 1990.
TAJIMA, F.: A simple graphic method for reconstructing phylogenetic trees from molecular data. Mol. BioI. Evol. 7: 578-588, 1990.
TAJIMA, F. and MUKAI, T.: Some consideration on diversifying selection. Jpn. J.
Genet. 65: 193-200, 1990.
TAKAHASHI, M., TORIYAMA, S., KIKUCHI, Y., HAYAKAWA, T. and ISHIHAMA, A.: Complementary structure in terminal sequences of rice stripe virus RNAs. J. Gen.
Virol. 71: 2817-2821,1990.
TAKAHATA, N.: Allelic genealogy and MHC polymorphisms. In "Population Biology ofGenes and Molecules" (Proceedings of the 4th International Symposium in conjunction with the Award of the International Prize for Biology), (N. Takahata and J. F.
Crow, eds.), pp. 267-286, Baifukan, Tokyo, 1990.
TAKAHATA, N.,: A simple genealogcal structure of strongly balanced allelic lines andtrans-species evolution of polymorphism. Proc. Natl. Acad. Sci. USA. 87: 24192423, 1990.
TAKAHATA, N. and CROW, J F. (eds.): Population Biology of Genes and Molecules.
(Proceedings for the 4th International Prize for Biology). Baifukan, Tokyo, 1990.
TAKAHATA, N. and NEI, M.: Allelic genealogy under overdominant and frequencydependent selection and polymorphism of major histocompatibility complex loci.
Genetics 124: 967-978, 1990.
TAKAHATA, N. and SLATKIN, M.: Genealogy of neutral genes in two partialy isolated
populations. Theor. Pop. BioI. 38: 331-350, 1990.

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TAMURA T., SUMITA, K., HIROSE, S. and MIKOSHIBA, K.: Core promoter of the mouse
myelin basic protein gene governs brain-specific transcription in vitro. EMBO J.
9: 3101-3108, 1990.
TENZEN, T., MATSUTANI, S. and OHTSUBO, E.: Site-specific transposition of insertion
sequence IS630. J. Bacteriol. 172: 3830-3836, 1990.
TERADA, K., OKUHARA, E., KAWARADA, Y. and HIROSE, S.: Demonstration of extrinsic
DNA from immune complexes in plasma of a patient with systemic lupas erythematosus. Biochem. Biophys. Res. Commun. 174: 323-330, 1991.
TOMIZAwA, J.: Control of ColE1 plasmid replication: Intermediates in the bindingof RNA I and RNA II. J. Mol. BioI. 212: 683-694, 1990.
TOMIZAwA, J.: Control of ColEl plasmid replication: Interaction of Rom protein withan unstable complex formed by RNA I and RNA II. J. Mol. BioI. 212: 695-708,
1990.
UEDA, H. and HIROSE, S.: Identification and purification of a Bombyx mori homologue
of FTZ-F1. Nucleic Acids Res. 18: 7229-7234, 1990.
UEDA, H., SONODA, S., BROWN, J. L., SCOTT, M. P. and Wu, c.: A sequence-specificDNA-binding protein that activates fushi tarazu segmentation gene expression.
Genes Dev. 4: 624-635, 1990.
UMEDA, M. and OHTSUBO, E.: Mapping of insertion element ISS in the Escherichia coli
K-12 chromosome: Chromosomal rearrangements mediated by IS5. J. Mol. BioI.
213: 229-237, 1990.
UMEDA, M. and OHTSUBO, E.: Mapping of insertion element IS30 on the Escherichia
coli K12 chromosome. Mol. Gen. Genet. 222: 317-322, 1990.
UMEDA, M. and OHTSUBO, E.: Four types of lSI with difference in nucleotide sequences
reside in the Escherichia coli K-12 chromosome. Gene (in press)
WADA, A., YAMAZAKI, Y., FUJITA, N. and ISHIHAMA, A.: Structure and probable genetic
location of a "ribosome modulation factor" associated with 100S ribosomes in stationary-phase Escherichia coli cells. Proc. Natl. Acad. Sci. USA. 87: 2657-2661,
1990.
WADA, K-N., WATANABE, I., TSUCHIYA, R. and IKEMURA, T.: G+C% mosaic structuresof the higher vertebrate genome and distribution of dinucleotide frequencies. In"New Aspects of the Genetics of Molecular Evolution" (Kimura, M., et al., eds.),
Springer-Verlag, Berlin, pp. 195-210,1991.
YAMADA, H., HAYATA, S., OMATA-YAMADA, T., TARA, H., MIZUMOTO, K. and IEASAKI,
K.: Association of the Sendai virus C protein with nucleocapsids. Archiv. Virol.
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YAMANAKA, K., ISHIHAMA, A. and NAGATA, K.: Reconstitution of influenza virus RNAnucleoprotein complexes structuraly resembling native viral ribonucleoprotein cores.
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YAMANAKA, K., OGASAWARA, N., UEDA, N., YOSHIKAWA, H., ISHIHAMA, A. and NAGATA,
K.: Characterization of a temperature-sensitive mutant in the RNA polymerase
PB2 subunit gene of influenza AjWSNj33 virus. Arch. Virol. 114: 65-73, 1990.
YONEDA M., TANNO Y., HORM S., OZAWA, T., MIYATAKE, T. and TSUJI, S.: A common
mitochondrial DNA mutation in the t-RNALY' of patients with myoclonus epilepsy120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41
associated with ragged-red fibers. Biochem. Int. 21: 789-796, 1990.
YONEZAWA, K., SATO, Y. I., NOMURA, T. and MORISHIMA, H.: Computer-simulated
evaluation of the hybrid weakness gene system as a means of preventing genetic
contamination of rice cultivars. Plant Breeding 104: 241-247, 1990.
YOSHIOKA, Y., FUJITA, Y. and OHTSUBO, E.: Nucleotide sequence of the promoter-distal
region of the tra operon including tra! (DNA helicase I) and traD genes of plasmid
RI00. J. Mol. BioI. 214: 39-53, 1990.

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ABSTRACTS OF DIARY FOR 1990
Biological Symposium
304th meeting Jan. 21

305th

Jan. 27

306th

Mar. 2307th

Mar. 12308th

Mar. 20309th

Mar. 22310th

Mar. 28

3Ust

Mar. 30

312nd

Apr.

313rd

Apr. 23

314th

Apr. 27

6

The development and use of a human genetic
linkage map to locate genes responsiblefor inherited disorders (Tim P. Keith)
DNA methylation: How important inplant gene control? (Jarunya Ngernprasirtsiri)
How RNA splicing enables the expressionof a maize gene with a transposable element
insertion in an exon (0. E. Nelson Jr.)
Molecular genetics of Alzheimer's disease(Josephine Nalbantoglu)
Engineering virus resistance in transgenic
plants (Gregg Clark)
Mini collagens a major component ofhydra nematocyte capsules (Charles N.
David)
Regulation of heat shock genes in Drosophila (Carl Wn)
The neutral mutation theory of molecular
evolution and the disunity of evolutionary
biology (William B. Provine)
Transcriptional termination in Escherichia
coli: Effects of translation and of sequence
hypersymmetry on rho-independent termination (Richard S. Hayward)
Intercellular signalling molecules regulate
gene expression in myxococcus development (A. D. Kaiser)
The heat shock response in Hydra: Characterization of species unable to synthesize
a major heat shock protein in response tostress (Thomas C. G. Bosch)122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

3l5th
3l6th

3l7th
3l8th
3l9th

320th
322lst

322nd
323rd
324th
325th

326th
327th

328th

329th

May 11

Genetic map of the mouse X chromosome(Verne M. Chapman)
May 17 Renaturation of bacteriophage A repressor
requires heat shock proteins (Max E. Gottesman)
May 30 A New technology of mouse gene mapping(Jean-Louis Guenet)
June 8 The role of DNA topology in adenovirus
replication (Ming-Ta Hsu)
June 19 Regulation of gene expression by minor
codons in Escherichia coli: Minor codon
modulator hypothesis (Masayori Inoue)
July 19 Molecular evolution of visual pigment genes(Shozo Yokoyama)
July 26 Molecular evolution in the descent of
humans and other primates (Morris Goodman)
Aug. 8 Negative control mechanism of DNA replication in E. coli (Masamichi Kohiyama)Aug. 20 Hemoglobinopathies in India (K. C. Malhotra)
Aug. 29 Statistical and molecular analysis of fitness
variation (Thomas Mitchell-Olds)
Sept. 21 Transcription and replication of viral RNA
genomes: Vesicualr stomatitis virus andhuman parainfluenza virus 3 (Amiya K.
Banerjee)Sept. 25 Some features of DNA mismatch repair in
E. coli (Maurice S. Fox)
Sept. 27 Structure and function of Escherichia coli
RNA polymerase sigma factors (Richard
R. Burgess)Sept. 29 Directional mutation pressure DNA compositional equilibrium and molecular evolution (Noboru Sueoka)
Oct.
The mechanism of chemical steps in Mu
DNA strand transfer: Comparison with

ABSTRACTS OF DIARY FOR 1990

123

A integration reaction (Kiyoshi Mizuuchi)
Oct. 18 Towards the molecular basis of the gradientsin hyrda (Hans R. Bode)
Oct.
29
Repetitive
sequences organization and func33lst
tions in the genome of Caenorhabditis
elegans (Adriana La Volpe)
Nov. 8 Biological correlates of oncogene expression
332ndand function in chemically induced transformation (Mukkattu R. Das)
Nov. 26 Two problems in gonoalogy: Oldest and333rdcommon ancestor alleles (Geoff Watterson)
334th
Dec. 5 The genome of Caenorhabditis elegans (John
Sulston)Dec. 5 Genetic recombination: Chi and the Rec
335th
BCD pathway (Franklin W. Stahl)
336th
Dec. 17 The taxonomic status of guinea pig based onprotein data (Dan Graur)
Mishima Geneticists' Club
365th meeting Feb. 16 Long-distance restriction mapping of humanchromosome 21 with Notl linking clones(Misao Ohki)
336th
Mar. 9 Amino acid sequence homology between
prostaglandin D synthetase and gramicidin
S synthetase in relation to their physiological
function (Hiroyuki Toh)
367th
Mar. 26 A three-way Needleman-Wunsch algorithm(Mitsuo Mita)
368th
Apr. 19 Expression and functions of stress proteinstumorgenesis . differentiation' heat shock(Kazuhiro Nagata)
Sept. 6 ACTINOBASE: An image database of
369th
actinomycetes (Yoshihiro Ugawa)
370th
Sept. 7 Biochemistry of the SV40 DNA replication(Toshiki Tsurimoto)
37lst
Nov. 9 Morphogenesis and expression of tissuespecific genes (Sadao Yasugi)
372ndNov. 19 Roles of cell-cell interaction in determination
330th

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO 41

373rd
374th
375th

of developmental fate: Cell interaction insexual reproduction of slime mold (Hideko
Urusibara)
Nov. 15 Mouse embryogenesis and X chromosome(Nobuo Takagi)
Dec. 11 Mechanism of length determination inbacteriophage lambda tails (Isao Katsura)
Dec. 19 Regulation of the phosphate regulon in
Escherichia coli (Kozo Makino)

FOREIGN VISITORS IN 1990

125

FOREIGN VISITORS IN 1990

April 9, 1884August 20, 1990
Septemer 1, 1888January 7, 1889September 25, 1990
April 10, 1989March 31, 1990
January 10, 1990
February 3
February 21
February 27
March I-May 31
March 2
March 9-28
March 12
March 20
March 28
March 30
April 5-6
April 23
April 23-May 14
April 17
May 11
May 30

Pascale Barbier; Universite des Sciences et Techniques du Languedoc, Montpellier, France
Guan Cheng Sun; Sericultural Research Institute,
Chinese Academy of Agricultural Sciences, China
Yong Hong Wang; Lanzhou Institute of Biological
Product, Ministry of Public Health, China
Fengshan Wang; Tianjin Institute of Industrial
Hygiene and Occupational Diseases, China
Guangyu Zhou; Shanghai Institute of Biology andChemistry, Academia Sinica, China
Yutaka Eguchi; National Institutes of Health,
U.S.A.
Tim P. Keith; Collaborative Research Inc., U. S. A.
Jarunya Ngernprasirtsiri, Nagoya University
Richard S. Hayward; University of Edinburgh,
U.K.
O. E. Nelson, Jr.; University of Wisconsin,
U.S.A.
Charles N. David; University of Munich, W.
Germany
Josephine Nalbantoglu; Institut national de larecherche Scientifique Quebec, Canada
Gregg Clark; Washington University, U. S. A.
Carl Wn; National Cancer Institute, NIH, U. S. A.
William B. Provine; Cornell University, U. S. A.
Masatoshi Nei; University of Texas, U. S. A.
A. D. Kaiser; Stanford University, U. S. A.
Thomas C. G. Bosch; University of Munich, W.
Germany
Max E. Gottesman; Columbia University, U. S. A.
Verne M. Chapman; Roswell Park Cancer Institute,
U.S.A.
Jean-Louis Guenet; Institut Pasteur, France

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

Ming-Ta Hsu; Mount Sinai School of Medicine,
U.S.A.
Masayori Inouye; University of Medicine &June 19
Dentistry of New Jersey, U. S. A.
Shozo Yokoyama; University of Illinois at UrbanaJuly 19
Champaign, U. S. A.
Morris Goodman; Wayne State University,
July 26-27
U.S.A.
August I-December 31 Song Mong Lee; Sericultura1 Experiment Station,
Rural Development Adminitration, Korea
Masamichi Kohiyama; Institut Jacques Monod,
August 8
FranceK. C. Malhotra; Indian Statistical Institute, India
August 20
August 29-30
Thomas Mitchell-Olds; University of Montana,
U.S.A.
September 14
V. Hemleben; University of Tubingen, E. Germany
Amiya K. Banerjee; Cleveland Clinic Foundation
September 21
Research Institute, U. S. A.
September 25
Maurice S. Fox; Massachusetts Institute of Technology, U. S. A.
Richard R. Burgess; University of Wisconsin,
September 27
U.S.A.
September 29
Noboru Sueoka; University of Colorado at Boulder,
U.S.A.
October 1
Kiyoshi Mizuuchi; National Institute of Diabetes
and Digestive and Kidney Diseases, NIH, U. S. A.
October 8-10
Masatoshi Nei; Pennsylvania State University,
U.S.A.
October 16-28
Hans R. Bode; University of California at Irvine,
U.S.A.
October 29
Adriana La Volpe; Istituto Internazionale di
Genetica e Biofisica, Italy
October 29
John Pulitzer; University of Naples, Italy
November 1
V. Marc Nigon; Universite Claud Bernard Lyon I,
FranceNovember 8
Mukkattu R. Das; Center for Cellular and Molecular Biology, India
June 8

FOREIGN VISITORS IN 1990

November 8
November 11-12
November 12
November 21-29
December 3
December 5
December 5
December 17
December 17

127

Marcus W. Feldman; Stanford University, U. S. A.
S. N. Ethier; Utah University, U. S. A.
Woong Jik Lee; Professor Emeritus, Seoul National
University, Korea
Geoff Watterson; Monash University, Australia
Peter G. Condliffe; Scientist Emeritus, National
Institutes of Health, U. S. A.
John Sulston; MRC Laboratory of Molecular
Biology, V. K.
Franklin W. Stahl; University of Oregon, U. S. A.
Dan Grauer; Tel Aviv University, Israel
Shen Zhang; Da Sheng Wang; Zi jian Wang;
Nai-Kai Zhu; Committee of Environment, Academia Sinica, China

128

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41

AUTHOR INDEXANDO, A •....•.•.•...•.....•••.•.•. 96

HORIE, N ••.•.•.•••••.•.•••.••.••••• 20

AOKI, K. . .....•.•••.••....•..... 76, 77

HORIUCHI, K ••.•.•••.•.•.•••• 31,33,34

96

HUAI, W. C ••••••••••.•••••••.•••••• 44

AYUSAVVA,I)•..•.••....•.. 20, 21,22, 60

IGARASHI, K. • .•.•.•••.•.•.•••.•.•••. 13

AZUMA, YIIZUKA, M.

ARAI, M

17.

73

BARBIER, P ••.•••.•.•.•.•.•.•••••.••• 98

IKEMURA, T •..•.•.•.•••.••••• 96, 97,108

CHIFU, H •..•...........•.....•••... 89

IKEO, K. . .....•••...•.•.•••••••• 78, 83. ••.............•..... 71

IMAI, N ••.•••.•••••••••••••••••••••• 22

CRovv, J. F ••.•.....•...•.•......... 67

IMAMURA, T •••••••••••••••••. 87,88,89

CHIGUSA, S. I.
1)01, HIMANISHI, T ••••••••••••••••••••••••• 83

EIGUCHI, M

INA, Y108
101
ENDo, M
94
FELDMAN, M. W
76
FUJISAKI, S •...............•......... 36
FUJISAVVA, T • . . . . . . . . . . . . . . . . . . • . . . . 50
FUJITA, N ••••...........•.... 13, 15, 16
FUJIVVARA, H. • .•...•.•...........•. 103
FUKASAVVA, M
84
GOJOBORI, T ••.•....... 78,79,80,81,82,
83,84, 108
GOTO, Y93, 94
GOTOH, H •••..•...•.......•.•..••.. 41
HAMAMATSU, C.
102
HARA, H ••••••.•.......•.....•.•.•. 36
HASEGAVVA, M
91
HASEGAVVA, T .•••.•.........•...•••. 88
HASEKURA, H. . .......•••.•.......•. 57
HAYAMI, M
84
HAYASAKA, K.
91
HAYASID, S. . ....••.•.•.•...•.... 25, 26
HAYASHIDA, H •••.............•. 13, 108
HAYVVARD, R. S ...•.•...•••.•••••••. 13
HE, X. Q
39
HIGASHITANI, A ••....•••••.••. 31, 33, 34
HIGASIDTANI, N ••..•.••••.•...... 31, 33
HIRANO, H. Y. .
28
HIRAVVAKE, H •...•.•..••••.•.••.. 95, 96
HIROSE, S ••......•.• 23, 24, 25, 26, 27, 36
HORAI, S •.....•. 90, 91, 92, 93, 94, 95, 96

78,81,82
96
ISHBAsHI, F. • .•.••••••••••••••••••. 108
ISIDGAKI, Y
95
ISHIHAMA, A.
13, 14, 15, 16, 17, 18, 19
ISHIHARA, N ••.•••••••••.•.•......... 96
ISHIKAVVA, K. I.
84
INOKO, H

ISHlURA, M.

.

ISHIVVA, H.

. ••.•...•.•.•••••.•....•. 71.43

ITAMI, N •...•••••••••.•.•••••.•.•••• 94
IvvAKURA, Y

19
39
KAlIl, N
94
KAMATA, K
38
KAMESHIMA, N. . ...•...•.•.•.•.•..•. 82
KANDA, K . . . . . . . • . . . • . . . . . . . . . . . . . 106
KANEDA, S ..••.•.•..•..•..... 20,21,22
KATo, A .•.•...................••••. 19
KAVVARADA, Y. .
27
KAVVASmMA, T •.•......•.•.•.•.•.•.. 44
KIMuRA, M. .
75, 79KISHINO, H .••••.••.•...•...•.•.•..• 91
KLEIN, J
66
KOBAYASHI, M. .
14
KOBAYASHI, R ••.•••.•.•••.•.•••.• 95, 96
KOGA, Y93
KOHARA, Y
62
KOIDE, T •...••.•••••••••..•..•.. 27,43
KONDO, R •..•••••••••••••.......... 71
JIN, M. L

129

AUTHOR INDEXMATSUMOTO, K

96

MATSUURA, E. T •.••..•..•.•...•..... 71
MncOSIHBA, K

24
59
MITA, A ••••.•.•••.•••••.•••.•••... .47
MIURA, T ••••.•.....•.•.•.•.•••.•... 84
MIYASHITA, N. . .•••••••••• 39, 43, 44, 46
MIYATAKE, T •.•.•••••••.•••.•••••.•• 92
MIYAZAWA, S
108
MIZOKAMI, M. . •••.•.......••..••.•. 82
MORISHIMA, H. • ••••••••••.. 98, 102, 103
MORIWAKI, K. . .... 27, 39,40,41,43,44,
45,46,47
MORIYAMA, E. N ••• , •.•. 78,79,80,82,84
MUKAI, T ••.••.........•..••..••••.. 75
MURAKAMI, A.
50, 51, 52, 54, 55, 57
NAGATA, K.
14, 18, 19
NAKAMURA, I ..•.....•.•.•..... 105,106
NAKAMURA, S • . . • . . . . . . . . . . . . . . . . 95, 96
NAKASHIMA, H. . .••.............. 88, 89
NAKATSUJI, N. • ..•••............... .45
NAKAYAMA, M . • • • . . . . . . . . . . . . . . . 16, 19
NEI, M ...................•••.•.•.•. 68
NfflHIMURA,1'• . . . . . . . . . . . . . . . . . . . . . . 36
NISHINO, T • . . . . . . . . . . . . . . . . . . . . . . • . . 36
NIWA, M • . . . . . . . . . . . . • . . . . . . . . . . . . .47
NONAKA, I. .
93
OHOYA,1'. . . . . . . . . . . . . . . . . . . . . . . • . . 61
OHTA, T •.......••••••.....•.•... 23, 24
OHTA, T • . . . . . . . . . . • . • . • . . . . . . . . . . . . 64
OKADA, K
24
OKUHARA, E. . ...•••.•.•...•........ 27
OKUNO, A •..••.••.•................ 57
ORITO, E
82
OSAWA, S ...........•...•..••.•...•. 16
OZAKI M ••.......•.....•••.•.•...•. 15
OZAWA, T . . . . . . . . . . . . . • . • . . . . . . . 92, 95
ROTH, A
31
SAGAI, T •....•.•...•.•..••.... 27, 40, 45
SAKAI, M .•.•.•...•.•••••.•.•......•. 88
SANO,1'. . . . . • . . . . . . . . . . . . • • . 28, 98, 101
SATO, T. . .......•..•....•.....•. 95, 96
SAT01'.-I. . .•.....•••.•.•.•.•.. 103, 106
SATTA, 1'. . .•.....................•. 71
MINATO, K.

.

85
95
SENO, T
20,21,22,60
SHIMAMOTO, N ••.•.•.•.•.••••.... 37,61
SHIMIZU, 1'••.•.•.•.•.••••••••.....•. 57
SHIROISm, T ••.•••.• 27, 40, 41, 43, 44, 45
SHIRRAS, A
26
SLATKIN, M. • .•.•••.•.•••••••....... 70
SUGIYAMA, T. • •••••••••.....••.•.•. .49
SUMITA, K
24
SUZUKI, H. • .•••••.••••.•......•.••. 39
TACHIDA, H •..•..•.•.•••.•.•. 64, 72, 73
TAJIMA, F
73,74,75
TAKAHASHI, K
38
TAKAHATA, N • . • . . . . 66, 67, 68, 69, 70, 71
TAKAYANAGI, A •..••••••••..••....... 21
T AKEISHI, K. .
20
TAMURA, T
24
TANNO,1'••.•.•...••••••........•.•. 92
TATENO, 1'•.•••••••••.•••....•..... 108
TERADA, H. . ...••.•..•.............. 37
TERADA, K
27
TEZUKA, H ••.•.•.•••••••••.•.•••.... 60
TOCHIMARU, H .••..•••••••••••••.•... 94
TSUCHIYA, K. .
39
TSUJI, S ....•....•...•.•••.•...•.•... 92
TSUJIMOTO, H
84
TSUKIHARA, T
38
UCHIDA, E •••..••••..•.•..•..•...... 95
UEDA, H
25
UESHIMA, R. . ...•.•.•.•...•.•.•..... 17
UGAWA, 1'••••.••..•.••.•...•••.•.. 108
WADA, A •......•.....•...•.••••..•• 15
WADA, K.
97,108
WADA, 1'••.•.•••••••••.•...•.•.•.• 108
WANG, C. H
39
WATANABE, J
26
WATANABE, K. .
61
WATANABE, S
46
WATANABE, T. K. .
85
'rAMADA, M. ..
29
l'AMAGISHI, M. . .••.••••••.••••••••.. 17
1'AMAMOTO, M •..•.••.•.............. 82
l'AMANAKA, K. ..
18
SAWAMURA,

SEKI. KK

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 41
F
YAN, W.C
YOKOTA, E

YAMAO,

22
44
89

YONEDA, M.

. ...................••.•92
H.................•• 39,46
YONEMURA, I. ....................••. 57
YONEKAWA,ffiI.n:}I~~iPf~m~¥ll.>fRlt 3 ~ 10 }j 25 B.>fRlt 3 ~ 10 }j 31 B
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