ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT1989

No. 40Published by

THE NATIONAL INSTITUTE OF GENETICS
Mi.ima, Sizuoka-ken, Japan1990

Annual Reportof theNational Institute of Genetics
No. 40, 1989

Published by

The National Institute of Genetics, Japan
1990

CONTENTS
General statement.........................................
Staff
Council and Advisory Committee. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Association for propagation of the knowledge of genetics
Projects of research for 1989
Research activities in 1989

I.

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Molecular Genetics
Promoter selectivity of Escherichia coli RNA polymerase, I. Effectof base substitutions in promoter - 35 region on the promoter
strength. KOBAYASHI, M., NAGATA, K. and ISHIHAMA, A. . . . 14
The promoter selectivity of Escherichia coli RNA polymerase,
II. Random screening of promoters and classification
based on the promoter strength. KUBOTA, M., YAMAZAKI,Y. and ISHIHAMA, A.
15
Promoter selectivity of Escherichia coli RNA polymerase, III.
Structural and functional modulations of RNA polymerase
during growth phase transition. OZAKI, M., FUJITA, N.,
WADA, A. and ISHIHAMA, A.
. . . . . 16
Structural modulation of ribosomes during growth phase transitionof Escherichia coli. WADA, A., YAMAZAKI, Y., FUJITA, N.
and ISHIHAMA, A.
17
The molecular anatomy of Escherichia coli RNA polymerase: Functional mapping of the a subunit. IGARASHI, K., HAYWARD,
R. S., FUJITA, N., YAMAGISHI, M. and ISHIHAMA, A.
18
Promoter selectivity of Micrococcus luteus RNA polymerase:
Identification and characterization of the major sigma factor.
NAKAYAMA, M., FUJITA, N., OSAWA, S. and ISHIHAMA, A..... 19
Molecular mechanisms of transcription and replication of phage
Qfi: Cloning, sequencing and mapping of host factor. KAJITANI, M. and ISHIHAMA, A.
20
Molecular mechanisms of transcription and replication of influenza virus RNA, I. Molecular structure of virus-associatedRNA polymerase. ISHIHAMA, A., HONDA, A., MUKAIGAWA,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

J., KATO, A., NAGATA, K., NAYAK, D., UEDA, S. and KRySTAL, M
Molecular mechanisms of transcription and replication of influenza
virus RNA, II. Function of influenza viral proteins, PB2 andNP, in viral RNA transcription. YAMANAKA, K., OGASAWARA, N., UEDA, M., YOSHIKAWA, R., ISHIHAMA, A. and
NAGATA, K. .
Molecular mechanisms of transcription and replication of influenza
virus RNA, III. Analysis of transcription promoters andreplication origins of viral genomes. YAMANAKA, K., OGASAWARA, N., YOSHIKAWA, R., ISHIHAMA, A. and NAGATA, K. ..
Molecular mechanisms of transcription and replication of influenza virus, IV. Mechanism of anti-influenza virus bymouse Mx gene. NAKAYAMA, M., NAGATA, K., YOKOIYAMA,
A., IWAKURA, Y., KATO, A. and ISHIHAMA,A
0...
Molecular mechanisms of transcription and replication of adenovirus: Involvement of nuclear factor I in transcription regulation. MATSUMOTO, K., RANAOKA, F., ISHIHAMA, A. and
NAGATA, K
Molecular mechanisms of transcription and replication of rice
stripe virus. ISHIHAMA, Ao, BARBIER, P., TAKAHASHI, M.
and TORIYAMA, S. .
Molecular studies on evolution, I. Annual and perennial differentiation in wild rice Oryza rufipogon. BARBIER, P. and
ISHIHAMA, A.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Molecular studies on evolution, II. Variation in the sequence ofa storage protein gene family in wild rice. BARBIER, P. and
ISHIHAMA, A.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Molecular studies on evolution, III. Introgression of mitochondrial DNA in land snail Luchuphaedusa (Ophaedusa) ophidoon.
UESHIMA, R. and ISHIHAMA, A .....
Structural and functional analysis of the human thymidylate
synthase gene. KANEDA, S., NALBANTOGLU, J., AYUSAWA,
Do, TAKEISHI, K., SHIMIZU, K., GOTOH, O. and SENO, T. ....
Functional analysis of the human thymidylate synthase gene bymolecular dissection. TAKAYANAGI, A., KANEDA, S., AYUSAWA, D. and SENO, T..... 0............................
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CONTENTS

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Enhancement by I-leucovorin of thymidylate stress-induced DNA
double strand breaks in mouse cells. AKAZAWA, S.,
AYUSAWA, D., WATAYA, Y., TEZUKA, H. and SENO, T.
31
Genetic analysis of mammalian cell cycle mutants in a uniquecomplementation group. AYUSAWA, D., KANEDA, S. and
SENO, T.
32
Coupling between replication and translation by E. coli singlestranded DNA binding protein. SHIMAMOTO, N.
32
Kinetic study on transcription by immobilized operons: Requirement for {3, r-phosphodiester bond of ATP in initiation.
SHIMAMOTO, N. and FUJIOKA, M. . . . . . . . . . . . . . . . . . . . . . . . . . 33
Kinetic study on transcription by immobilized operons: Slidingof RNA polymerase on DNA. SHIMAMOTO, N.
34
Giant G+C %mosaic structures of human genome found at a levelof DNA sequences. IKEMURA, T., WADA, K., AOTA, S. and
MATSUMOTO, K.
34
Codon usage tabulated from GenBank genetic sequence data.
WADA, K., AOTA, S., TSUCHIYA, R., ISHIBASHI, F., GOJOBORI,
T. and IKEMURA, T.
35
G+C % mosaic structures of the higher vertebrate genome anddistribution of dinucleotide frequencies. WADA, K., WATANABE, I., TSUCHIYA, R. and IKEMURA, T.
36
Effect of DNA supercoiling on in vitro transcription from theadenovirus early region 4. HANDA, H., WATANABE, H.,
SUZUKI, Y. and HIROSE, S.
37
Supercoiling of DNA by DNA topoisomerase II and a supercoiling factor purified from the posterior silk gland of Bombyx
mori. OHTA, T. and HIROSE, S.
38
A sequence-specific DNA-binding protein that activates lushi
tarazu segmentation gene expression. VEDA, H., SONODA, S.,
BROWN, J. L., SCOTT, M. P. and WV, C.
38
Cloning and structural analysis of the waxy (wx) locus gene of rice
(Oryza sativa). HIRANO, H. Y. and SANO, Y.
39
Studies on the mechanism of mRNA capping and the process oftranscription initiation by RNA polymerase II. MIZUMOTO,
K
40
Transcription of Sendai virus (HVJ) genome. MIZUMOTO, K. '"42

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iv

II.

Microbial Genetics

Replication of filamentous phage f1: Enhancer-independent mutation of an initiator protein (gpII). HIGASHITANI, A., HIGASHITANI, N. and HORIUCHI, K.
Multicopy suppression in E. coli: Cloning of sufB, a dosagedependent suppressor of a thermosensitive cell-divisionmutation, ftsB. HIGASHITANI, A., YAMAMOTO, Y. and NISHIMURA, Y.
A novel DNA topoisomerase essential for the E. coli chromosome
partition. KATO, J., NISHIMURA, Y., HIRAGA, S. and SUZUKI,
H
Identification and cellular localization of the product of prc, a gene
involved in the C-terminal processing of penicillin-binding protein 3 of Escherichia coli. HARA, H. and SUZUKI, H.
Nucleotide sequence of the prc gene of Escherichia coli. YAMAMOTO, Y., NISHIMURA, Y. and HARA, H.
A mutant producing temperature-sensitive farnesyl diphosphatesynthase of Escherichia coli. FUJISAKI, S., NISHINO, T., KATSUKI, H., HARA, H. and NISHIMURA, Y.
Mapping of a whole set of cell division genes in Escherichia coli K12. NISHIMURA, A., IROBE, R., SUZUKI, K. and UEYAMA,K
The secA gene of Bacillus subtilis. SADAIE, Y., TAKAMATSU, H.,
NAKAMURA, K. and YAMANE, K.

III.

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Immunogenetics

Fine mapping of recombinational hotspots in the mouse MHC
by direct sequencing of PCR amplified DNA. SHIROISHI,
T., SAGAI, T., GOTOH, H. and MORIWAKI, K.
Characterization of Asian specific H-2Ku class I antigen. SAGAI,
T., SHIROISHI, T. and MORIWAKI, K.

56
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IV. Developmental and Somatic Cell Genetics
Studies on freeze preservation of embryos of Drosophila melanogaster. KURODA, Y. and TAKADA, Y.

58

CONTENTS

Detection on SR spiroplasms in embryonic tissues of Drosophila
me/anogaster by an electron microscope. KURODA, Y.,OISHI, K. and SHIMADA, Y.
Developmental genetics of E-pseudoallelic domain in Bombyx.
1. Genetic characteristics. MURAKAMI, A.
Developmental genetics of E-pseudoallelic domain in bombyx. 2.
Developmental characteristics. MURAKAMI, A.
Genetic studies on the diapause phenomenon in Bombyx. 1.
Seasonal adaptation of the tropical multivoltine race in thetemperate zone. MURAKAMI, A. .... . . . . . . . . . . . . . . . . . . . ..
Genetic studies on the diapause phenomenon in Bombyx. 2. A
possible role of the central nerve center system on adaptation.
MURAKAMI, A.
Genetic studies on the diapause phenomenon in Bombyx. 3.
Artificial modification of the diapause state in temperate races
to nondiapause type. MURAKAMI, A.
The mutagenic activity of a carcinostatic drug, Neocarzinostatin*,in germ cells of Bombyx mori L. MURAKAMI, A.
Egg permeability and development of embryo in Drosophila.
MINATO, K.
Axial and lateral head formation in hydra. SHIMIZU, H., SUGIYAMA, T. and SAWADA, Y.
Two way controls of interstitial stem cell proliferation in Hydra.
FUJISAWA, T.
Purification and structure analysis of an autocrine growth factor
in conditioned medium obtained from primary cultures ofscleral fibroblasts of the chick embryo. FUJIOKA, M., OHOYA,
Y., WATANABE, K. and SHIMAMOTO, N.
Assembly of connectin (titin) in relation to myosin and a-actinin
in cultured cardiac myocytes. SHIMADA, Y.
Translational control of troponin C gene expression in chickenskeletal muscles. SHIMADA, Y.

V.

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Cytogenetics
Effect of H-2 complex on morphological abnormality of mouse
spermatozoa. MORIWAKI, K. and MITA, A.
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A new variant of the mouse j3-globin gene, Hbb q , in northwestern
China. KAWASHIMA, T., MIYASHITA, N., SHIROISHI, T.,
YAN, W. C., HUAI, W. C. and MORIWAKI, K.
In vitro development of preimplantation embryos derived frominter subspecific hybrids between Mus musculus molossinus
and M. m. domesticus. NIWA, M., WAKASUGI, N. and
MORIWAKI, K.
Studies of adrenal function in mouse development by transgene.
GOTOH, H., SHIROISHI, T. and MORIWAKI, K.
Antigenic polymorphism of a serum protein found in a strain
derived from Indonesian wild mice (Mus musculus castaneus).
HARADA, Y., SUMANTRI, C., TOMITA, T. and MORIWAKI, K. ..
DNA sequence comparison in the D-loop region of mitocondrial
DNA within and among subspecies of the species Mus musculus. YONEKAWA, H., MIYASHITA, N. and MORIWAKI,
K

VI.

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Mutagenesis and Radiation Genetics

Genetic analysis of pulmonary adenoma susceptibility (Pas) inmice. MIYASHITA, N. and MORIWAKI, K.

VII.

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Population Genetics

Role of gene duplication in evolution. OHTA, T.
The mutational load of a multigene family with uniform members.
OHTA, T.
Time for spreading of compensatory mutations under gene duplica..
tion. OHTA, T. ..
Gene genealogy in three related populations: Consistency probability between gene and population trees. TAKAHATA, N.
Stochastic models for molecular clocks. TAKAHATA, N.
A population genetic model of selection that maintains specific trinucleotides at a specific location. TACHIDA, H. . . . . . . . . . . . .
A study on fixation probabilities in a subdivided population
with varying environments. TACHIDA, H. .. . . . . . . . . . . . . . ..
Theoretical studies on DNA polymorphism and statistical method
for testing the neutral mutation hypothesis. TAJIMA, F. ....

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CONTENTS

The neutral theory of molecular evolution and the world viewof the neutralists. KIMURA, M.
A shifting balance type model for the origin of cultural transmission.
AOKI, K

VIII.

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Evolutionary Genetics

Pattern of nucleotide substitutions and its implications for the
immunological diversity of human immunodeficiency virus.
GOJOBORI, T., MORIYAMA, E. N. and SHIMIZU, N.
Evolutionary relationships between human and simian immunodeficiency viruses. GOJOBORI, T., MORIYAMA, E. N., TSUJIMOTO,
H. and HAYAMI, M.
Rates of nucleotide substitution for Drosophila species. MORIYAMA, E. N. and GOJOBORI, T.
Evolutionary origin of kringle structure in apolipoprotein (a).
IKEO, K., TAKAHASHI, K. and GOJOBORI, T.
Molecular evolution of hepadnaviruses. INA, Y., ORITO, E.,
MIZOKAMI, M., MORIYAMA, E. N. and GOJOBORI, T.
A method for molecular phylogeny construction by direct use
of nucleotide sequence data. TATENO, Y.
Robustness of maximum likelihood tree estimation against different patterns of base substitutions. FUKAMI, K. andTATENO, Y.
Publication from genetic resources section, Genetic Stock ResearchCenter. IYAMA, S. and TATENO, Y.

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Human Genetics

Characterization of the telomeres and sub-telomeric DNA sequences of human chromosomes. IMAMURA, T., NAKASHIMA,
H., FUJIYAMA, A. and HASEGAWA, T.
99
Genetic polymorphisms of gene conversion within the duplicatedhuman a-globin loci. NAKASHIMA, H., FUJIYAMA, A.,
KAGIYAMA, S. and IMAMURA, T.
100
A common Chinese .a-thalassemia mutation found in a Japanesefamily. NARITOMI, Y., NAKASHIMA, H., KAGIMOTO, M.,
NAITO, Y., YOKOTA, E. and IMAMURA, T. .. . . . . . . . . . . . . . . .. 101

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Intraspecific nucleotide sequence differences in the major noncoding
region of human mitochondiral DNA. HORAI, S. and
HAYASAKA, K.
102
DNA amplification from ancient human skeletal remains andtheir sequence analysis. HORAI, S., HAYASAKA, K., MURAYAMA, K., WATE, N., KOIKE, H. and NAKAI, N.
102
Heteroplasmies and polymorphisms in the major noncoding
region of mitochondiral DNA in Japanese monkeys associatedwith the tandem repeated sequences. HAYASAKA, K., ISHIDA,
T. and HORAI, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 104
Mitochondrial DNA mutation in a family with Leber's hereditary
optic neuropathy. YONEDA, M., TSUJI, S., YAMAUCHI, T.,
INUZUKA, T., MIYATAKE, T., HORAI, S. and OZAWA, T
104
Specific amplification of deleted mitochondrial DNA from amyopathic patient and analysis of deleted region with Sl
nuclease. TANAKA-YAMAMOTO, T., TANAKA, M., OHNO, K.,
SATO, W., HORAI, S. and OZAWA, T
105
Increased level of mitochondrial gene expression in polyps offamilial polyposis coli patients. YAMAMOTO, A., HORAI, S.
and YUASA, Y.
106

x. Applied Genetics
Association between Pox-] variation and seed productivity po107
tential in wild rice. MORISHlMA, H.
Variation in resistance to bacterial leaf blight between and withinwild rice populations. HAMAMATSU, C., SATO, Y.-I. and
MORISHIMA, H.
108
Genetics oflife-history trait associations in wild rice from Thailand.
BARBIER, P. and MORISHIMA, H. .... . . . . . . . . . . . . . . . . . . . . .. 109
A new hypothesis on the parentage and dissemination of Japaneserice. SATO, Y.-I.
110
Toward a rationale of genetic system in the flowering responseof the rice plant to photoperiod. SANO, Y.
III
A gene complex responsible for seed shedding and spreading
panicle found in wild rice species. EIGUCHI, M. and SAND,
Y
112

CONTENTS

ix

Extraction and amplification of a chloroplast DNA fragment froma single ancient rice seed. NAKAMURA, I. and SATO, Y.-1. ... 114
Anomalous 3: 4: 1 segregation of a- and ;S-subunits of seed conglycinin in mutant soybean. NAKAMURA, I., ODANAKA, H.
and KAIZUMA, N.
115
Construction of a targeting signal for plant mitochondria. NAKAMURA, I., KYOZUKA, J. and SHIMAMOTO, K
116
On the conditions causing the periphery effect in genotype dis116
tribution within plant populations. YONEZAWA, K
Computer simulated evaluation of the hybrid weakness gene
system as a means of preventing genetic contamination of ricecultivars. YONEZAWA, K., SATO, Y.-I., NOMURA, T. and
MORISHIMA, H.
117

XI.

Database

Activities of the DNA Data Bank of Japan. MIYAZAWA, S. and
HAYASHIDA, H.
119
Basigin, a new, broadly distributed member of the immunoglobulin
superfamily, has strong homology with both the immunoglobulin V domain and the ;S-chain of major histocompatibility
complex class II antigen. MIYAUCHI, T., KANEKURA, T.,
YAMAOKA, A., OZAWA, M., MIYAZAWA, S. and MURAMATSU,
T
121
Publications
Abstracts of diary for 1989
Foreign visitors in 1989
Author index

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GENERAL STATEMENT
Our institute was established for comprehensive research on genetics
and we celebrated our 40th anniversary in June, 1989. The outstanding
accomplishments in the advancement of genetics by members of our institute has made it the center of genetic study in our country, and a major
institution with world-wide recognition. In addition, six years ago theinstitute was reorganized into a National Inter-university Research Institute. With the foundation of the Graduate University for Advanced
Studies consisting of seven inter-university research institutes, our instituteadmitted the first graduate students this April, in the Department of Genetics of the Graduate School of Life Science.
Recent rapid progress in 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 tomeet the changing demands. It is obvious, however, that this and otheractions taken by the institute, are far from sufficient. We shall have many
research related problems remaining to be handled. With proper advice
and guidance of people in and outside the institute, I would like to lead theinstitute into more prosperous years ahead.
Regarding personnel changes in the past year, Dr. E. Matsunaga, thefifth director, retired on October 1st. From 1983 to 1989 Dr. Matsunaga
had led the members of our institute in six years of drastic reform in theinstitute. Succeeding him, I accepted the appointment as director. While
Prof. S. Nawa (Laboratory of Developmental Genetics) and AssociateProf. T. Endo (Laboratory of Agricultural Genetics) retired, and Associate Prof. D. Ayusawa was transferred to the University of Tokyo,
the following six researchers joined us: Dr. K. Horiuchi as professor (Laboratory of Microbial Genetics); Dr. Y. Kohara (DNA Research Center)
and Dr. F. Yamao (Laboratory of Mutagenesis) as associate professor,
Drs. H. Goto (Laboratory of Cytogenetics), F. Tajima (Laboratory of
Population Genetics), and M. Yamagishi (Laboratory of Molelcular Genetics) as research members.
It is a pleasure to note that Prof. A. Ishihama was honored by the Encouragement Award of the Association for the Promotion of Genetic

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Studies for his molecular studies on gene expression, and Dr. S. Horai
the Encouragement Award of the Japan Society of Human Genetics forhis human genetic and molecular evolutionary studies on mitochondrial
DNA.

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., Professcr
YASUDA, Seiichi, D. Sc., Associate professor
NISHIMURA, Yukinobu, D. Sc.
HARA, Hiroshi, D. Sc.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Laboratory of Cytoplasmic Genetics (Guest member)
UCHIDA, Hisao, D. Sc., Professor
YONEKAWA, Hiromichi, D. Sc., Associate professor

3. Department of Ontogenetics
KURODA, Yukiaki, D. Sc., Head of the Department
Laboratory of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
Laboratory of Phenogenetics
KURODA, Yukiaki, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Laboratory of Physiological Genetics (Guest member)
SHIMADA, YUTAKA, D. Med., Professor
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 Genetics
TUTIKAWA, Kiyosi, Associate professor
GOJOBORI, Takashi, D. Sc., Associate professor
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

STAFF

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

Research Facilities
Genetic Stock Research Center!YAMA, Shin-ya, D. Ag., Head of the Center
MIYASHITA, Nobumoto, D. Med.
WATANABE, Takao K., D. Sc., Associate professor
VEDA, Hitoshi, D. Ag.
HIRANO, Hiroyuki, D. Ag.
NISHIMURA, Akiko, D. Ag.
!YAMA, Shin-ya, D. Ag., Associate professor
TATENO, Yoshio, Ph. D., D. Sc.
DNA Research Center
SENO, Takeshi, D. Sc., Head of the Center
SHIMAMOTO, Nobuo, D. Sc., Associate professor!KEMURA, TOSHIMICHI, D. Sc., Associate professor
MATSUMOTO, Kenichi, D. Ag.
HIROSE, Susumu, D. Sc., Associate professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
HAYASHIDA, Hidenori
KOHARA, Yuji, D. Sc., Associate professor
Radioisotope Center
SADAIE y oshito, D. Sc., Head of the Center
Experimental Farm
MORISHIMA-OKINO, Hiroko, D. Ag., Head of the Farm
NAKAMURA, !kuo, D. Ag.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

7.

Technical Section
ONIMARU, Kimiharu, Chief of the Section

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

COUNCIL
Chairman
YAMAMURA, Yuichi; Emeritus professor, Osaka University
Vice-chairman
NAGAKURA, Saburo; President, Graduate University for Advanced
Studies
Members (Alphabetical order)
HAYAISHI, Osamu; Director, Osaka Bioscience Institute
ICHIKAWA, Atsunobu; Dean, Graduate School at Nagatsuta, Tokyo
Institute of Technology
lINO, Tetsuo; Professor, Faculty of Human Science, Waseda Universtiy
IMAHORI, Kozo; President, Naruto University of Education
KIMURA, Motoo; Emeritus professor, National Institute of Genetics
KUWAHARA, Shogo; Director, Toho University
NAKAI, Junnosuke; President, Hamamatsu University School of
Medicine
NOMURA, Tatsuji; Director, Central Institute for Experimental
Animals
OKADA, Tokindo S.; President Okazaki National Research Institutes
ONOE, Hisao; President, Shiga University
OZEKI, Haruo; Emeritus professor, Kyoto University
SAKAI, Fuminori; Director, Japan Society for the Promotion of
Science
SUGANO, Haruo; Director, Cancer Institute
TAKANAMI, Mitsuru; Director, Institute for Chemical Research, Kyoto
University

COUNCIL

7

TAKEUCHI, Ikuo; Director-General, Institute for Basic Biology
WATANABE, Itaru; Emeritus professor, Keio University
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University

ADVISORY COMMITTEE
Chairman
KURODA, Yukiaki; Professor, Laboratory of Phenogenetics
Vice-chairman
SASAKI, Motomichi; Professor, Faculty of Science, Hokkaido 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, Universityof Tokyo
NAKAJIMA, Tetsuo; Professor, Faculty of Agriculture, Tamagawa
University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
OSAWA, Shozo; Professor, Faculty of Science, Nagoya University
TAKEUCHI, Takuji; Professor, Faculty of Science, Tohoku University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto
University
YOSHIKAWA, Hiroshi; Professor, Faculty of Medicine, Osaka University

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

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

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

PROJECTS OF RESEARCH FOR 1989
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular Genetics

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

Molecular and genetic studies of cell-cycle associated genes (SENO andKANEDA)
Genetic consequences of nucleotide pool imbalance in mammalian cells(SENO, Y AMAO and TEZUKA)
Radiation sensitivity in mammals (TEZUK)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 ubiquitinactivating 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)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Genetic mechanism of meiotic recombination in mice (SHIROISHI, IMAI
and MORIWAKI)
Labaratory 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)
3.

DEPARTMENT OF ONTOGENETICS
Laboratory of Developmental Genetics

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

Studies on development any growth of insect (MINATO)
Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on animal cells in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA)
Genetic studies on the life history characters in Bombyx (MURAKAMI)
Genetic studies on the nerve system characters in Bombyx (MURAKAMI)
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

Electron microscopic studies on differentiation of animal cells (SHIMADA
and KURODA)
Theoretical models for pattern formation in hydra (SAWADA, SHIMIZU andPROJECTS OF RESEARCH FOR 1989

11

SUGIYAMA)
4.

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

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

Studies on molecular evolution on viruses (GOJOBORI and MORIYAMA)
Computer analysis of DNA sequences (GOJOBORI)
Radiation genetics in mice (TUTIKAWA)
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, FUJlYAMA
and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA and NAKASHIMA)
Molecular biology of oncogenes (FUJlYAMA)
Studies on DNA polymorphisms on human populations (HORAI)12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Laboratory of Agricultural Genetics
Evolutionary genetics in wild and cultivated rice species (MORISHIMA, SANO
and SATO)
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
Genetic studies of speciation in rice (SANO and HIRANO)
Studies on plant gene expression (HIRANO and SANO)
Theoretical studies on breeding techniques (IYAMA)
Documentation of genetic stocks in Japan (IYAMA)
Studies on the management system of genetic stocks information (IYAMA)
Theoretical studies on molecular phylogeny (TATENO)
Evolutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA)
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)
Sequence and structure analysis of DNA and proteins (MIYAZAWA and
HAYASHIDA)
Molecular genetics of insect development (VEDA and HIROSE)PROJECTS OF RESEARCH FOR 1989

Control of gene expression in eukaryotes (HIROSE and VEDA)
Radioisotope Center

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

Experimental Farm

Molecular genetics of plant development (NAKAMURA)
Genetical analysis of physiological traits in plants (NAKAMURA)RESEARCH ACTIVITIES IN 1989

I.

MOLECULAR GENETICS

Promoter Selectivity of Escherichia coli RNA Polymerase, I.
Effect of Base Substitutions in Promoter - 35 Region
on the Promoter Strength
Makoto KOBAYASHI, Kyosuke NAGATA and Akira ISHIHAMADNA sequence analyses of a wide variety of prokaryotic promoters
have indicated that the promoters for the major form of E. coli RNA polymerase (Ea 70 ) are composed of two conserved hexanucleotide sequences,
TTGACA and TATAAT, which are located at 35 and 10 base-pairs upstream of the transcription initiation site, respectively. Among natural
E. coli promoters, however, there are considerable sequence differences,which might be related to differences in promoter strength (for a review
see Ishihama, A. (1988) Trends Genet. 4, 282-288). At present, however,
little is known about the role of individual bases within these two regionsin promoter function.
For better understanding of the relationship between promoter strengthand sequence, we constructed a set of variant promoters derived from the
JacUV5 promoter, each carrying a single base substitution within the -35
region (nucleotide positions from -36 to -31 relative to the transcription
start site). Using this promoter collection and purified E. coli RNA
polymerase holoenzyme, in vitro mixed transcription assays were performed
to determine two parameters governing the promoter strength: binding
affinity to RNA polymerase (parameter I); and rate of open complex formation (parameter II). The following conclusions were drawn from the experimental results (for details see Kobayashi, M. et al., submitted for
publication): (1) Alteration in the promoter strength depends on boththe position and base species of substitutions; (2) the consensus sequence(TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position -34 cause marked effects on both parameters; (4) cytosine at nucleotide position -32 can not be replaced with otherRESEARCH ACTIVITIES IN 1989

15

nucleotide without significant reduction in promoter strength; and (5)base substitution at nucleotide position -31 has little effect on parameter
I. All these findings were confirmed by abortive initiation assays. The
same type of analyses on the -10 region will provide us with detailed knowledge on the function of each promoter element.
The Promoter Selectivity of Escherichia coli RNA Polymerase, II.
Random Screening of Promoters and Classification
Based on the Promoter Strength
Masayo KUBOTA, Yukiko YAMAZAKI and Akira ISHIHAMA

To reveal the moleclar basis of promoter strength differences among
natural E. coli promoters, we developed the in vitro mixed transcription
assay system and determined two parameters of the promoter strength
for a number of natural promoters which were isolated from known genes(for a review see Ishihama, A. (1988) Trends Genet. 4, 282-288). In parallelwith the strength measurement of known promoters, we constructed acollection of all kinds of natural promoters from the total chromosomal
DNA of E. coli, to classify these promoters based on their strength, andafterward to identify the genes carrying these promoters.
Five hundred and fifty DNA fragments of 100-500 base pairs in length
were cloned from total E. coli DNA, each capable of inducing the synthesisof p-Iactamase when inserted upstream of the ampC gene without its own
promoter in a promoter-probe plasmid (for details see Kubota, M. et ai.,
submitted for publication). These clones were classified, based on theputative promoter strength as measured by the level of resistance to ampicillin, which ranged from 10 to more than 5,000 ,ugjmI. Some of the highlevels of drug resistance (more than 1,000,ugjml) were due to not only theinsertion of strong promoters but also to an increase in plasmid copy
number.
Some of the DNA fragments were mapped on the E. coli chromosome byhybridization with the DNA membrane filter of Kohara's miniset clones(the gene mapping membrane was developed in this institute and is being
produced by Takara Shuzo). The DNA fragments which produced thehighest level of drug resistance were all mapped at 5.7 min on the E. coli
chromosome and shared the same sequence. In these fragments, an upstream AT-rich sequence was found, in addition to -35 and -10 signals

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

with a high similarity to the promoter consensus sequence. The genecarrying this strong promoter has not, however, been identified yet.
Promoter Selectivity of Escherichia coli RNA Polymerase, llI.
Structural and Functional Modulations of RNA Polymerase
during Growth Phase Transition
Miwako OZAKI, Nobuyuki FUJITA, Akira WADA* and Akira ISHIHAMAIn order to reveal the regulatory mechanisms of growth phase-dependent
gene expression in E. coli, we examined possible alterations in the promoter
selectivity of RNA polymerase during the growth phase transition. Previously we reported that, although core enzyme subunits, a, 13 and 13', ofRNA polymerase are synthesized coordinately during exponential growthphase, this coordination is disrupted during the growth transition fromexponential growth to stationary phase (Kawakami, K. et al. (1979) Mol.
Gen. Genet. 174, 107-116). Also, newly synthesized subunits in stationaryphase cells are degraded preferentially (Enami, M. and Ishihama, A. (1982)
Mol. Gen. Genet. 185, 373-378).

In addition to the alteration in the synthesis and assembly of RNA polymerase, we found that the preexisting RNA polymerase is converted
into at least three different forms during the growth phase transition fromexponential growth to stationary phase. All three novel forms of RNApolymerase were eluted from phosphocellulose columns at KCl concentrations lower than that required for the elution of exponential-phase RNA
polymerase (for details see Ozaki, M. et a/., submitted for publication). The
relative levels of these three enzyme forms varied depending on the stageof growth phase transition.
To reveal the altered functions of stationary-phase specific RNA polymerases, the promoter selectivity of each enzyme was tested using an invitro mixed transcription system, in which transcription was performed
with mixtures of various E. coli promoter DNA fragments. All three RNA
polymerases exhibited altered promoter selectivity. In order to identify
differences in structure between exponential-phase and stationary-phase
RNA polymerases, both enzymes were dissociated into (170 subunit and core
enzyme, and a holoenzyme reconstitution was carried out using isolated* Department of Physics, Faculty of Science, Kyoto Universty, Kyoto.

RESEARCH ACTIVITIES IN 1989

17

components. The hybrid holoenzyme from the exponential-phase (170 andthe stationary-phase core showed essentially the same promoter selection
pattern as that of the stationary-phase holoenzyme. The results indicatethat the altered function of stationary-phase RNA polymerase is due to analteration in the core enzyme. These results altogether suggest that themodulation of RNA polymerase might play a major role in the global
switch of transcription pattern during the growth transition to stationary
phase.
Structural Modulation of Ribosomes during Growth Phase Transitionof Escherichia coli
Akira WADA*, Yukiko YAMAZAKI, Nobuyuki FUJITA and Akira ISHIHAMA

The decrease in overall translation activity occuring concomitantly withthe transition from the exponential growth phase to the stationary phaseof Escherichia coli was found to be accompanied by the appearance oflOOS ribosome (dimer of 70S ribosome monomers). Analysis of ribosomal
proteins by the radical-free and highly reducing (RFHR) method of twodimensional gel electrophoresis indicated that a protein, previously designated protein E, was exclusively associated with 100S ribosome (Wada,
A. et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 2657-2661). From theresults, we proposed that protein E is a "ribosome modulation factor"(RMF), which associates with 70S ribosomes and converts them to a dimeric form. The hypothesis that lOOS ribosomes are non-functional
stored form of translational apparatus is being tested experimentally.
A homology search of the partial amino acid sequence of RMF using
the DNA sequence data bases revealed that the rmf gene, which encodes
RMF, is located next to thefahA gene at 21.8 min on the E. coli chromosome.
This was found to be the case by PCR amplification of this region and direct
sequencing of PCR products. Structural and functional modulations ofboth transcription and translation apparatuses might play a major role in
the global control of gene expression during growth phase transitions ofE. coli.

* Department of Physics, Faculty of Science, Kyoto University, Kyoto.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

The Molecular Anatomy of Escherichia coli RNA Polymerase:
Functional Mapping of the a Subunit
Kazuhiko IGARASHI, Richard S. HAYWARD·, Nobuyuki FUJITA,
Masahiro Y AMAGISHI and Akira ISHIHAMAThe DNA-dependent RNA polymerase is composed of at least four
different subunits, a, p, p' and (J, each having different functions in genetranscription. The intrinsic functions of individual subunits are, however,expressed only when they are assembled into a complete enzyme. It istherefore important to analyze the molecular interactions among the subunits.
The assembly takes place in a sequential manner in the order: a~az~
azp~azpp'~azpp'(J (for a review see Ishihama, A. (1986) Adv. Biophys.
21, 163-173). Previously, we carried out a molecular anatomy study ofthe p subunit and identified the region involved in the interaction withother subunits (for a review see Ishihama, A. (1988) Trends Genet. 4, 282288). Along this line, we have analyzed the region of a involved in subunit
assembly.
First, we determined the mutation sites of the RNA polymerase a subunit
gene in two temperature-sensitive E. coli strains carrying mutations, rpoAlOl
or rpoA112 (Ishihama, A. et al. (1980) J. Mol. Bioi. 137, 137-150), because
the assembly of RNA polymerase is defective in the rpoAl12 mutant (Kawakami, K. and Ishihama, A. (1980) Biochemistry 19, 3491-3494). Sequence
analysis of PCR-amplified DNA indicated that both of the mutant rpoA
genes have a single base transition, which leads to a substitution of Cys
for Arg at position 191 (rpoAlOl) or 45 (rpoA112), respectively. The
location of the rpoA112 mutation raised the possibility that the aminoterminal region of a plays an important role in subunit assembly.
To test this possibility, we constructed a set of deletion mutations of
the rpoA gene. When carboxyl-terminally truncated a polypeptides weresynthesized in vitro and examined for their assembly-competence using
an in vitro reconstitution system, N-terminal fragments of 296, 256, or 235
amino acid residues in length were found to retain the ability to form
pseudo-azp (azf3 complex composed of truncated a) and pseudo-azpp'
complexes, while those truncated at residues 176 or 150 did not form suchcomplexes.
•

On leave of absence from Department of Molecular Biology, University of Edinburgh, UK.

RESEARCH ACTIVITIES IN 1989

19

Studies of E. coli strains producing the same truncated a subunits showedthat the 296, 256, and 235 amino acid species can even form pseudo-holoenzyme in vivo. These results indicate that the aminoterminal region of
the a subunit plays an important role in RNA polymerase assembly. However, rpoAlOl and rpoA1l2 mutations could not be complemented by theexpression of a-296, a-256, or a-235. Instead, a-256 and a-235 showed
inhibitory effects on cell growth, suggesting that, although these truncateda polypeptides lack some essential function associated with RNA polymerase, they interfere with the assembly of intact a subunit.
Promoter Selectivity of Micrococcus luteus RNA Polymerase:
Identification and Characterization of the
Major Sigma Factor
Manabu NAKAYAMA, Nobuyuki FUJITA, Syozo OSAWA* and Akira ISHIHAMA

Promoters of Micrococcus luteus, a bacterium whose chromosomal
DNA has a high G+C content (74 %), differ from the consensus prokaryotic promoter in having GC-rich DNA sequences at less important positions(Nakayama, M. et al. (1989) Mol. Gen. Genet. 218, 384-389). In order tocompare the promoter selectivity of RNA polymerase between M. luteus
and E. coli, we purified the enzyme from both organisms. The sets ofpromoters recognized by the two RNA polymerases were found to overlap
partly (for details see Nakayama, M. et al., submitted for publication).
Some, but not all, E. coli promoters were found to be correctly transcribedin vitro by the M. luteus RNA polymerase as well as the E. coli enzyme.
For better understanding of the molecular basis of different promoter
selectivity, we attempted to isolate the a subunit from M. luteus and tocompare it with the E. coli a subunit. One molecular species of the M.
luteus a factor, with an apparent molecular weight of 60 kDa, was isolated
from purified RNA polymerase. By the addition of either M. luteus orE. coli core enzyme, it was reconstituted into an active holoenzyme. The
hybrid holoenzyme between the M. luteus a subunit and the E. coli core
enzyme was able to initiate transcription only from promoters which wererecognized by both of the native holoenzymes. These observations indicate
that, although the a subunit plays a major role in promoter recognition~* Department of Biology, Faculty of Science, Nagoya University, Nagoya.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

the core enzyme also participates in promoter selection. This suggestion
is consistent with our previous observation that the promoter selectivity
is altered by mutations not only in a but also in core enzyme subunit genes(Nomura, T. et al. (1976) Mol. Gen. Genet. 193, 8-16).
In addition to the major a factor, we found a minor species of M. luteus
a factor, which is different from the major a factor in both promoter selectivity and molecular size.
Molecular Mechanisms of Transcription and Replication of Phage Q ~:Cloning, Sequencing and Mapping of Host Factor
Masayuki KAJITANI and Akira ISHIHAMA

Bacteriophage Q fi contains a positive-strand RNA as the genome, which
encodes coat ptotein, maturation protein and RNA-dependent RNA polymerase. The RNA polymerase is composed of four subunits, one phagecoded protein (subunit 13) and three host-derived proteins (subunit a=ribosomal protein SI, subunit r=EF-Tu, subunit o=EF-Ts). Phage RNAdependent synthesis of RNA by purified RNA polymerase requires in addition a host protein factor (HF). The nature of this host factor, its role inphage RNA-dependent RNA synthesis, and the physiological role of thisprotein in uninfected E. coli cells all remained unclear.
For better understanding of these problems, we purified HF from uninfected E. coli W3350 as the activity stimulating Q~ RNA-directed RNA
synthesis with purified Qfi RNA polymerase, determined the amino acidsequence of several parts of purificed HF and, based on the sequence data,prepared probes for hybridization. Using the gene mapping membraneof Kohara's miniset library, we identified the gene encoding HF, cloned
the hfq gene encoding HF and mapped it on the E. coli chromosome (fordetails see Kajitani, M. et al., submitted for publication). Sequence analysis revealed that HF is a protein of 102 amino acid residues with Mr
11,200. The physiological role of HF is being examined by constructing
mutants defective in the hfq gene.

RESEARCH ACTIVITIES IN 1989

21

Molecular Mechanisms of Transcription and Replication of
Influenza Virus RNA, I. Molecular Structure ofVirus-Associated RNA Polymerase
Akira ISHlHAMA, Ayae HONDA*, Jun MUKAIGAWA**, Atsushi KATO***,
Kyosuke NAGATA, Debi NAYAK**, Susumu UEDA***
and Mark KRYSTAL****The RNA-dependent RNA polymerase of influenza virus A/PR/8 was
isolated from virus particles by stepwise centrifugation in cesium salts.
First, RNP (viral RNA-NP-P proteins) complexes were isolated throughglycerol gradient centrfugation of detergent-treated viruses and subsequently
NP was dissociated from RNP by cesium chloride gradient centrifugation.
The P-RNA (P proteins-viral RNA) complexes thus isolated were further
dissociated into P proteins and viral RNA by cesium trifluoroacetate(CsTFA) gradient centrifugation. The nature of dissociated P protein
complexes was further analyed by glycerol gradient centrifugation andimmunoblotting using monospecific antibodies against each P protein(Honda, A. et al. (1990) J. Biochem. 107, 624-628).
The three P proteins, PB1, PB2 and PA, sedimented together as fast as
the marker protein with a molecular weight of about 250,000 dalton. Upon
addition of the template vRNA, the RNA-free P protein complex exhibitedthe activities of capped RNA cleavage and limited RNA synthesis. For
the synthesis of template-sized RNA, however, NP might be needed aspreviously demonstrated, using native P protein-vRNA complexes and-isolated NP (Honda, A. et al. (1988) J. Biochem. 104, 1021-1026). Whena cell line stably expressing eDNA for three P proteins and NP protein
was examined, three P proteins were found to be co-precipitated by treatment with antibodies against the individual P proteins. These results
altogether indicate that the influenza virus RNA-dependent RNA polymerase
is a heterocomplex composed of one each of the three P proteins and that
the RNA-free RNA polymerase can be isolated in an active form fromvirus particles. Furthermore, P proteins form a complex in the absence
ofvRNA.
* Present address: Department of Microbiology, State University of New York,
Stony Brook.
** Department of Microbiology and Immunology, University of California at Los
Angeles, Los Angeles.
*** Laboratory of Biotechnology, Nippon Institute for Biology, Ohme.
**** Department of Microbiology, Mount Sinai Medical School, New York.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO.4,

Molecular Mechanisms of Transcription and Replication of Influenza
Virus RNA, II. Function of Influenza Viral Proteins,
PB2 and NP, in Viral RNA Transcription
Kunitoshi YAMANAKA, Naotake OGASAWARA*, Masashi UEDA**,
Hiroshi YOSHIKAWA*, Akira ISHIHAMA
and Kyosuke NAGATA

Ribonucleoprotein (RNP) cores (RNA polymerase-RNA-NP-complexes)
are capable of synthesizing template-sized RMA in vitro, and of producing
infectious viruses when transfected to permissive cells. RNA polymerase,composed of PBl, PB2 and PA, is essential for transcription initiation as
well as elongation, while NP is required for efficient elongation of RNA
chains. However, the functions and functional domains of each component
are not well understood yet.
PB2 has been thought to interact with the cap-l structure. By comparing
dinucleotide- and capped RNA-primed in vitro RNA synthesis activities
between a wild type and a ts mutant carrying mutations in the PB2 gene,it was concluded that PB2 is indeed involved in the recogition of the cap
structure. Nucleotide sequence analysis revealed that the amino acidnumber 417 is essential for the recognition of cap-l structure and/or theinteraction with the catalytic unit of the RNA polymerase (Yamanaka,
K. et al. (1990) Arch. Virol., in press).
On the other hand, the analysis of RNA binding mode indicated that
NP is an oligonucleotide « 15 bases) binding protein without any basespecificity. The RNA-NP complexes reconstituted under the optimized
conditions are structurally equivalent to native RNA cores (Yamanaka,
K. et al. (1990) J. BioI. Chern., 265,11151-11155).
Thus, protein-protein and protein-RNA interactions play importantroles in the regulation of influenza virus RNA transcription. By the same
type of genetic and biochemical analyses, we hope to define functions andfunctional domains of other proteins involved in viral RNA transcription.
For detailed analysis of the structure-function relationships of these proteins,
we are trying to develop a bacterial expression system for large scale production of each protein.

* Department of Genetics, School of Medicine, Osaka University, Osaka.
** Department of Microbiology, Institute of Public Health, Tokyo.

RESEARCH ACTIVITIES IN 1989

23

Molecular Mechanisms of Transcription and Replication of Influenza
Virus RNA, III. Analysis of Transcription Promoters andReplication Origins of Viral Genomes
Kunitoshi YAMANAKA, Naotake OGASAWARA*, Hiroshi YOSHIKAWA*,
Akira ISHIHAMA and Kyosuke NAGATA

In order to determine the RNA signal (promoter) for transcription by
influenza virus RNA polymerase, we performed in vitro transcription experiments using RNA polymerase, NP and various kinds of short model
template in collaboration with the Department of Microbiology of Mount
Sinal Medical School in New York. The results showed that dinucleotide
primer-dependent transcription is directed by the putative promoter locatedwithin the 15 nucleotide 3' terminus of the genome (Parvin, J. et al. (1989)
J. Virol. 63, 5142-5152). In order to clarify RNA signals for transcription
in vivo, we developed an infectious RNA-protein complex system.
First, eDNA for the RNA segment 8 of influenza virus A/PR/8/34 wascloned. The region coding for the NS protein was deleted and replacedby the chloramphenicol acetyltransferase (CAT) gene. The resulting
DNA sequence was deposited under control of the promoter of T7 RNApolymerase and the antisense RNA (vRNA-sense) to CAT mRNA wasproduced by transcrbing recombinant DNA with T7 RNA polymerase.
Transfection of cells with this antisense CAT RNA in the presence ofhelper RNP cores led to no significant production of CAT. In contrast,
when the RNA was mixed with purified NP prior to transfection, the efficient
expression of CAT activity was observed. Using this infectious RNA-NP
complex system, the essential structure of RNA signals required for transcription and replication is being studied.
Molecular Mechanisms of Transcription and Replication of Influenza
Virus, IV. Mechanism of Anti-influenza Virus by Mouse Mx Gene
Manabu NAKAYAMA, Kyosuke NAGATA, Akiko YOKOIYAMA,
Yoichiro IWAKURA**, Atsushi KATO***and Akira ISHIHAMA

The inbred mouse strain A2G is known to be resistant to infection by* Department of Genetics, School of Medicine, Osaka University, Osaka.
** Institute of Medical Science, Universityof Tokyo, Tokyo.
*"'* Nippon Institute of Biological Science, Ohme, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

influenza virus. This resistance has been shown to be determined by asingle dominant allele designated Mx, and both cDNA and genomic DNA
encoding the Mx protein have been cloned and sequenced. The resistancemechanism by the Mx protein is still unclear. By western blotting andimmunoprecipitation analyses using antibodies against synthetic peptides
corresponding to either the carboxyl terminus or a region near the amino
terminus of the mouse Mx protein, we confirmed that the Mx protein was
induced by interferon.
In order to reveal the action mechanism of the Mx protein, we examinedthe effect of the Mx protein, induced in primary cultures of A2G mouseembryos by interferon, on each step of the influenza viral infectious cycle.
It was found that the infection of influenza virus was not affected, whereas
viral transcription, viral translation and the production of infectious virus
were reduced about 30 fold under the conditions employed. These results
suggest that the interferon-induced Mx protein inhibits the synthesis of
influenza viral mRNAs or the transport of influenza viral RNA-protein
complexes from cytoplasm to the nucleus. However, interferons induce
the expression of multiple genes including the Mx gene, which together
repress the growth of viruses. Thus, the possibility is not excluded yetthat the inhibitory effect is not due to the Mx protein but other interferoninduced proteins. We are trying to establish cell lines which produce theMx protein in the absence of interferon.

Molecular Mechanisms of Transcription and Replication of Adenovirus:
Involvement of Nuclear Factor I in Transcription Regulation
Ken MATSUMOTO*, Fumio HANAOKA*"', Akira ISHIHAMA and Kyosuke NAGATA

Recently, evidence has been accumulating indicating DNA replication-origins contain transcriptional regulatory elements. Studies using several
DNA viruses reveal that trans-acting factors essential for DNA replication
are aslo involved in transcription regulation. The replication origin of
adenovirus (Ad) DNA consists of three functional domains. The minimum
origin is located at the end of the genome, containing an AT-rich region
conserved in all human adenoviruses. The nuclear factor I (NFl) binding* Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo.
** Radiation Biology Laboratory, Riken, Wakoh.

RESEARCH ACTIVITIES IN 1989

25

site (FIB site) adjacent to this AT-rich region and the NFIII binding site are
essential for Ad DNA replication. We examined the transcription promoter
activity within the Ad replication origin in a cell-free system using nuclear
extracts of HeLa cells (Matsumoto, K. et al. (1989) Biochem. Biophys. Res.
Commun. 164, 1212-1219).
Transcription from a cloned inverted terminal repeat of Ad type 5 DNA
was found to take place in the opposite direction with respect to that of
DNA replication. The major transcript was initiated outside the Ad
sequence about 30 nucleotides downstream from the AT-rich region. Competitive transcription experiments using oligonucleotides and isolated
NFl revealed that NFl and the FIB site were involved in negative regulationof this transcription. DNase I footprinting experiments indicated that
the TATA-box binding protein, TFIID, was capable of binding to the ATrich region at the minimum origin but not when NFl binds to the adjacent
FIB site.
The results of this study indicate that NFl, which is essential for Ad
replication and is involved in the positive regulation of transcription of avariety of genes, can also function as a transcriptional repressor possibly
dependent on the position of its binding site relative to the TATA-box.
Molecular Mechanisms of Transcription and Replicationof Rice Stripe Virus
Akira ISHIHAMA, Pascale BARBIER, Mami TAKAHASHI*
and Shigemitsu TORIYAMA*

Rice stripe virus (RSV) is a member of the newly recognized tenUlvlrus
group of plant viruses. These viruses are able to infect their insect vectors
as well as their plant hosts. Biochemical studies have indicated that thisgroup of viruses has a unique genome structure. Purified RSV was foundto contain one major (32 kDa coat protein) and one minor (230 kDa putative
RNA polymerase protein) as the protein components, and four segmentsof both single-strand (ss) and double-strand (ds) RNA as the genome.
In addition, RNA-dependent RNA polymerase activity was found to be
associated with virus particles. As an initial attempt to reveal whether
both ss- and ds-RNA are required for virus growth, we stared the purifica*

Division of Microbiology, National Institute of Agro-Environmental Sciences, Tsukuba.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

tion and characterization of RNA polymerase as well as the determinationof the sequence of each RNA segment.
The sequencing analysis of both termini of ss-RNA segments indicatedthat terminal sequences are well-conserved among the four RNA segments
and moreover the 3'- and 5'-terminal sequences are complementary toeach other (for details see Fukuhara, M. et al., submitted for publication).
These properties suggest the negative-strand nature of ss-RNA segments,
indicative of the presence of RNA polymerase in virus particles. To confirm this prediction, several approaches are being employed, including
determination of the nucleotide sequences for RNA segments, determinationof the amino acid sequence for RNA polymerase, characterization oftranscripts formed by RNA polymerase, and identification of RNA polymerase-binding sites on viral RNA segments. Initial attempts haveindicated that 230 kDa viral protein, the putative RNA polymerase, canbe dissociated from viruses and recovered from vRNA through CsCI centrifugation, the procedure being successfully employed in this laboratory
for the purification of virus-associated RNA polymerases. Effort is being
made to establish an in vitro transcription system using isolated vRNAand RNA polymerase.
Molecular Studies on Evolution, I. Annual and Perennial
Differentiation in Wild Rice Oryza rufipogon
Pascale BARBIER and Akira ISHIHAMAThe Asian wild rice species Oryza rufipogon is differentiated into 2 ecotypes: an annual type and a perennial type. In spite of a clear cut differentiation in phenotypic traits, little differentiation has been found inisozymic markers between these two ecotypes (Barbier, P. (1989) lpn. l.
Genet. 64, 259-271; ibid., 273-285).
In order to estimate the divergence time of the two ecotypes, an attempt
was made to estimate the phylogeny of eight annual and perennial strains
originating from various regions of Asia (India, Indonesia and Thailand),using the nucleotide sequence of three introns located in the phytochrome
gene (about 600 base-pairs of introns and as many as exon borders). The
related African perennial species Oryza Iongistaminata was also analyzed
as a control outgroup. The nucleotide sequence of each of the nine strains
was determined without cloning, using PCR amplification of target sequences

RESEARCH ACTIVITIES IN 198927

and direct submission of purified PCR products to sequencing reactions
with the dideoxy method using Taq polymerase.
The variation in the phytochrome introns revealed that: i) the difference
between o. longistaminata and O. rufipogon was about 0.042 substitutions
per site; but ii) within o. rufipogon, strains from geographical origins asremote as India, Indonesia and Thailand, the difference was within therange of 0.0017-0.0050 substitutions per site. These results indicate that
extant strains of Asian wild rice diverged as recently as a few hundred
thousand years ago, compared to 5-6 million years ago for the divergenceof O. logistaminata and o. rufipogon. Annuals and perennials from thesame geographic region (= sympatric strains) seemed to be a little moreclosely related than allopatric ones. Thus, a monophyletic origin of annuals
is excluded. These data also indicate that the annual-perennial differentiation may be even more recent than the geographic differentiation between
strains. However, the existence of a shared substitution between a strain
from India and a strain from Thailand could also indicate that long-distancemigration and introgression between strains cannot be excluded in theevolutionary history of wild rice.
Molecular Studies on Evolution, II. Variation in the Sequence of a
Storage Protein Gene Family in Wild Rice
Pascale BARBIER and Akira ISHIHAMA

Variation in the 10 kDa prolamin gene family was probed using PCRamplification and direct sequencing. Eight strains of Asian wild rice
(Oryza rufipogon) and one strain of African wild rice (Oryza longistaminata)were examined (for details see Barbier, P. and Ishihama, A. (1990) Plant
Mol. Bio/., 15, 191-195). As PCR allows the amplification in bulk of all
copies of a gene family, the extent of heterogeneity among copies may bealso investigated. The sequencing of artificial template mixtures showed thatthe presence of minor variants representing 20 %of all copies can be detectedby the method employed. The 10 kDa prolamin genes from all the test
strains were found to be about 400 bp long, without introns. Withinrufipogon strains, variants were only detected in two strains: a strain from
West India and a strain from Thailand (one and two base substitutions,
respectively). How these two strains escaped the homogenization process(concerted evolution) seen in other strains is unknown. Large differences

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

were found in O. longistaminata as compared to O. rufipogon, including
the presence of two additional codons. No detectable heterogeneity
among the 10 kDa prolamin gene copies was found for all the strains analyzed. The number of copies is being determined.
Molecular Studies on Evolution, III. Introgression of Mitochondrial
DNA in Land Snail Lllchuphaedusa (Ophaedusa) ophidoon
Rei UESHIMA and Akira ISHIHAMA

L. (0.) ophidoon is a clausilid land snail endemic to Shimokoshiki Island
in Kyushu. Studies on morphological and allozymical variation haveindicated that at least three genetically distinct taxa, southern large form
(LS), southern small form (SM) and northern large form (LN) exist, eachcarrying characteristic morphological traits and having a different geographical distribution (for details see Ueshima, R. et al., in preparation).
The two southern taxa, LS and SM, form a narrow hybrid zone in natural
habitats. In order to reveal the evolutionary history of these taxa, withparticular focus on gene flow across the hybrid zone, we analyzed variations in mitochondrial DNA (mtDNA) sequences.
We analyzed 87 restriction enzyme sites of mitochondrial (mt) DNA
from 236 LS individuals and 100 SM individuals collected from 37 localities.
Altogether 17 different haplotypes were identified, among which 16 haplotypes were specific to each taxon (13 to LS and 3 to SM) but one haplotype
was shared by both taxa. The phylogeny analysis revealed that these
haplotypes could be classified into two distinct mtDNA lineages, LS- andSM-lineages. Only a single haplotype among those present in SM taxon
was found to belong to the SM-type lineage. The LS-type mtDNA lineage
found in the SM taxon was considered to be introgressed from LS taxon
through hybridization. In contrast, the flow of nuclear genes across the
hybridization seems to be prevented due to strong post-mating isolation.
These observations indicate that mtDNA was transferred from LS to SMlong enough ago to accumulate mutations in the transferred mtDNA
genome and to spread into most of the SM individuals. To explain the
introgression pattern of mtDNA in the land snails on this small island.
we proposed a model of two cycles of range contraction and expansion.

RESEARCH ACTIVITIES IN 1989

29

Structural and Functional Analysis of the Human
Thymidylate Sythase Gene
Sumiko KANEDA, Josephine NALBANTOGLu*, Dai AYUSAWA, Keiichi TAKEISHI**,
Kimiko SHIMIZU***, Osamu GOTOH*** and Takeshi SENO

The complete nucleotide sequence of the human thymidylate synthase(TS) gene was determined. Genomic DNA fragments for human TS
were cloned using a DNA mediated transformation of mouse mutant
cells deficient in TS. The biologically active unit spans about 16 kilobases (kb) and is composed of seven exons ranging in size from 72 basepairs (bp) to 250bp, and six introns ranging in size from 507bp to 6271bp.
The nucleotide sequence of the exons was in agreement with that reported
for eDNA (Takeishi et al., 1985, Nucl. Acids Res. 13, 2035). Comparisonof the positions of the exons in human and mouse TS genes (Deng et al.•1986, J. BioI. Chern. 261, 16000) showed that their splice junctions are
identical; each exon is interrupted at the same amino acid position and eachcodon is interruted in the same phase in the two TS genes. The gene is G+
C-rich and has a high concentration of CpG in the 5' flanking region, exon
I and intron I. The promoter region and the major transcriptional startsites were located within about 400bp and 160-180 bp, respectively, upstream
from the ATG initiation codon. The 5' upstream region lacks the threecanonical transcriptional signals, the TATA box, CAAT box, and GC box(GGGCGG), whereas GC boxes are present in the corresponding regionof the mouse gene. Instead, there are three GC box esquences in intron
I and all of them seem to be functional, as determined by gel retardation
assay. A new enhancer-like sequence is also present in intron 1 (Kaneda
et al., in preparation). There are two polyadenylation signals 288bp
apart around the 3' region, corresponding to the polyadenylation site of
the eDNA. In both cases, the polyadenylation signal of AATAAA is approximately 20bp upstream from the sequence CAPyTG which contains the
dinucleotide CA, the preferred sites of polyadenylation in eukaryotic genes.
The second polyadenylation signal seems to be used in normal fibroblastic
cells transformed by SV40, as estimated by 3' mapping of 7 different TS
eDNA clones. In the mouse TS mRNA, the 3' untranslated region is* INRS-Sante, Universite du Quebec, Pointe-Claire, Canada.
** School of Food and Nutritional Science, University of Shizuoka, Shizuoka.
*** Saitama Cancer Center Res. Inst., Ina-machi, Saitama-ken.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

absent, that is, poly (A) is added to the G of the stop codon VAG. Thus,
polyadenylation sites are movable and the untranslated region of TS mRNA
may have no essential and rigid function.
Functional Analysis of the Human Thymidylate Synthase
Gene by Moleucular Dissection
Atsushi TAKAYANAGI, Sumiko KANEDA, Dai AYUSAWA and Takeshi SENO

We reported previously that cell cycle-dependent expression of humanthymidylate synthase (TS) is largely regulated posttranscriptionally, thatis, in normal human diploid cells the amount of TS mRNA increases about
14 fold from the GO/Gl phase to the S phase, while the initial velocity oftranscription in the TS gene is almost the same between the two phases
as measured by "nuclear run-off" (Ayusawa et aI., 1986, J. Mol. BioI.
190, 559). In order to locate the regulatory sequences responsible forthe posttranscriptional regulation within the gene, we constructed a minigene of the human TS and related chimeric genes partially replaced bycorresponding SV40 promoter sequences or 3' regulatory sequences in the
Okayama-Berg expression vector and introduced them into a TS-negative
mutant of rat 3Yl cells to establish stable transformant lines. The minigene
was composed of about 4kb of a 5' flanking sequence, a coding sequencewith or without interruption by intron 1 and about 1.7kb of a 3' flanking
sequence.
Each transformant line was synchronized in the GO/G I phase by serum
starvation, and then stimulated to traverse into the S phase through theaddition of serum. The amount of TS mRNAs was assayed between cells
in GO/Gl and S phases by Northern hybridization analysis with a fragmentof human TS cDNA clone as a probe. The results showed that the above
minigene gave an increment of mRNA in the S phase and showed a repressionof it in the GO/Gl phase, whereas the eDNA in the SV40 promoter-driven
vector produced an abundant amount of mRNA equally in the two phases.
The same minigene, but without an interruption by intron 1, showed theS phase-specific expression pattern but to a lesser extent. These results
suggested that the 5' flanking and/or 3' flanking genomic sequences in theminigene are responsible for the S phase-specific increase of mRNA. Furthermore it was shown that the presence of the genomic 5' flanking sequence,but not the 3' flanking sequence is sufficient for the S phase-specific increment

RESEARCH ACTIVITIES IN 198931

of mRNA and subsequent repression in the GO/G 1 phase by using chimericgenes of genomic and viral sequences. The role of intron 1 in the S phasespecific expression is under investigation. Elucidation of the differential
role of the 5' flanking sequence and intron 1 in the S phase-specific expressionof the TS gene is of central interst in understanding the posttranscriptional
regulatory mechanism.
Enhancement by I-Leucovorin of Thymidylate Stress-Induced
DNA Double Strand Breaks in Mouse Cells
Shugo AKAzAwA"', Dai AYUSAWA, Yusuke WATAYA"'''',
Hideo TEZUKA and Takeshi SENO

We previously demonstrated that thymidylate stress (thymine nucleotide
starvation) in cultured mammalian cells results in cell death with concomitant DNA double strand breaks associated with ongoing DNA replication, resulting in the accumulation of DNA fragments of a particularsize (5O-150kb) as measured by pulsed-field gel electrophoresis (Ayusawa,
D. et al., 1988, Mutat. Res. 200, 221). These results are consistent withthe known effectiveness of fluorinated derivatives of pyrimidine in cancer
chemotherapy. Since inhibition of TS is a cause of thymidylate stress,
one may expect a positive correlation between the extent of inhibition of
TS and that of DNA double strand breaks. In this report we treated mouse
mammary FM3A cells with the TS specific inhibitor 5-FdUrd or 5-fluoro2'-deoxy-3'-O-0Ieoyluridine-5'-(2-trimethylammonioethy1) phosphate (T506)
in combination with I-leucovorin which forms a covalent bonded ternarycomplex with the TS enzyme, and derived 5-FdUMP. The results showedthat (1) 5-FdUrd and T506, but not 5-FUra produced DNA double strand
breaks as detected by pulsed-field gel electrophoresis; 5-FUra produced
DNA strand breaks only at high concentrations, (2) the extent of the DNA
double strand breaks increased with time in the drug-treatment, (3) 1leucovorin enhanced both cytotoxicity and DNA double strand breaks
brought about by 5-FdUrd or T506 treatment, and (4) a clinical test showedthat treatment of a patient with 5-FdUrd or T506 produced in the bloodfor a reasonable period of time, a range of 5-FdUrd concentration comparable
to that which brought about DNA double strand breaks in the above in'" Saitama Cancer Center Hospital, Ina-machi, Saitama-ken.
"'''' Faculty of Pharmaceutical Sciences, Okayama University, Okayama

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

vitro experiment. In the case of 5-FUra, its concentration in the blooddecreased easily to below the level necessary for DNA strand breaks to be
induced. Theses results suggest that the presently used combination therapyof 5-FUra and leucovorin should be replaced by that ofT506 and leucovorin.
Genetic Analysis of Mammalian Cell Cycle Mutants
in a Unique Complementation GroupDai AYUSAWA, Sumiko KANEDA and Takeshi SENO

So far, a wide variety of conditional cell cycle mutants and non-cellcyle mutants have been isolated from various mammalian cells. Curiously
enough, a large number of such mutants fell into a single complementationgroup based on cell hybrid formation. Some of these mutants are shown tohave the temperature-sensitive ubiquitin activating enzyme El.
We purified a human gene which rescued temperature-sensitive traits inseveral mouse cell mutants through two-rounds of DNA-mediated genetransfer with human genomic DNA. From the DNA library of a secondarytransformant, genomic DNA segments derived from the human gene werecloned in a A phage vector with an Alu sequence as a probe. Then, usinga DNA segment which detects 3.6 kb mRNA expressed in transformant
and human cells, we cloned full-length cDNAs for the mRNA from ahuman cDNA library. These cDNAs were ligated to a mammalian expression vector and found to have the ability to rescue genetic defects inthe temperature-sensitive mouse cell mutants.
Now we can employ a genetic analysis to reveal the mechanism underlying
phenotypic diversity in the mutants. The functions of the EI enzyme
central to various cellular processes will be addressed taking advantageof the use of such mutants and the cloned genes.

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

E. coli single-stranded DNA binding protein (SSB) specifically binds
to a group of mRNA, including its own mRNA, in a cooperative manner.
Three RNA segments responsible for the specific and cooperative binding

RESEARCH ACTIVITIES IN 1989

33

with homologous 12 base sequences, named SSB box, are located on ssb
mRNA outside of the coding regions. Genes alkA and sec Y mRNA havesuch sequences. The following evidence implicates a regulatory role ofthe binding of SSB to mRNA. The binding takes place at physiologicalconcentrations, and a significant amount of SSB was found complexedwith RNA in cell lysate. The binding inactivates template activity of such
a mRNA in a cell-free translation system in vitro. The translation of recA
and nusA, with no such sequences, was not affected by SSB. Thus thebinding protein may be a pleiotropic translation factor presumble SSB
translational regulons. From these lines of evidence the following picture
emerges. When cells grow in a favorable environment, replication forks
increases, catching more binding protein. Thus more mRNA is exposed
and used in translation. The state of replication can be thus transmitted
to a translation system.
To check this model, the intracellular concentration of SSB was changedby overproducing SSB or RNA containing SSB box. The overproductionof either SSB protein or SSB box RNA was lethal. The overproductionof both, however, was not harmful. These results suggest that the amountof uncomplexed SSB is strictly regulated and that the binding of overproduced SSB to DNA and mRNA is toxic. Consistently, several proteins
were underexpressed upon the overproduction of SSB. These candidatesof SSB translational regulon are now being sequenced.
Kinetic Study on Transcription by Immobilized Operons:
Requirement for p, r-Phosphodiester Bond of
ATP in Initiation
Nobuo SHIMAMOTO and Miki FUJIOKA

The requirements for the /3, r-phosphodiester bond of ATP in transcription initiation by E. coli RNA polymerase has long been questioned. We
devised a novel method for studing these requirements and this method
enabled us to substitute substrates during elongation without changing
the ionic 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 RNA polymerase. If necessary, transcription
was interrupted by rapidly diluting substrates to 0.1 pM and resumed by

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

adding new substrates. Using ATP analogues as substrates, we clarified
that the (3, r-phosphodiester bond of ATP is required for effective transcription in the early stage of elongation. When transcription was started fromA P R promoter by adding four natural substrates, and eloncated 20 sec toform an elongation complex, this complex resumed elongation upon addition of a substrate set containing a (3, r-unhydrolyzable analogue of ATP.
The same substrate set, however, could not support the initiation. The
results suggest the presence of a new initiation complex which requires
ATP until elongation is established.
Kinetic Study on Transcription by Immobilized Operons:
Sliding of RNA Polymerase on DNA
Nobuo SHIMAMOTO

The first step of transcription is the binding of RNA polymerase to apromoter. The enzyme could directly bind to a promoter on a long DNA,or alternatively, first bind to DNA in a nonspecfic manner and then form
the promoter complex by one-dimensional diffusion along the DNA chain.
Unfortunately, kinetic evidence for the two conflicting models has beenreported and further study should be based on more confildent evidence.
The immobilized operon provides a critical method for determining whichcase is true. A fragment of DNA with one end immobilized on a slide
glass was extended by electric field of 30-100 Vjcm and the other end wasphotocrosslinked on the same slide glass. Fluorescently labeled RNA
polymerase was added to the linearly fixed DNA thus prepared and the
movement of a single molecule of RNA polymerase was followed throughmicroscopy. The one-dimensional movement was detected and its contribution to the promoter binding is now being evaluated.
Giant G+ C % Mosaic Structures of Human Genome Foundat a Level of DNA Sequences
Toshimichi IKEMURA, Ken-nosuke WADA, Shin-ichi AOTA,
and Ken-ichi MASTUMOTO

To determine the overall variation of G+C % distribution over longranges of the human genome, DNA sequences of human genes, whichwere closely linked genetically or physically, were surveyed in the GenBank

RESEARCH ACTIVITIES IN 1989

35

Data Base. A total of 72 sequences longer than 2 kb, which were mutually
linked within 500 kb, were identified. The sequences belonged to 17 linkage
groups and were ordered in each group according to their genetic positions.
Analyses of G+C % distribution along the ordered sequences showed thatsequences within each group almost always had similar G+C % levels, but
those belonging to different groups often had different levels. Similar
analyses of more distantly linked sequences (e. g., > ten megabases) showedmosaic structures of G+C % distribution. These findings are consistent
with predictions made from the 'isochore' structures found by Bernardi
and his colleagues using CsCl equilibrium centrifugation, in that structures
having a homogeneous base composition stretched over at least several
hundred kilobases. Two possible boundaries of the giant G+C % mosaic
structures were identified between classes II and III in the HLA locus andbetween X-linked G6PD and F8C. We are now attempting to clone theentire junction of the classes II and III in the HLA in order to clarify thecharacterisis of the boundaries. For details, see Ikemura et al., Genomics, 8(in press, 1990).
Codon Usage Tabulated from GenBank Genetic Sequence Data
Ken-nosuke WADA, Shin-ichi AOTA*, Rye TSUCHIYA,
Fumie ISHIBASHI, Takashi GOJOBORI
and Toshimichi IKEMURA

In 1986 and 1988, we reported codon usages in a total of 1638 and 3681
protein genes, respectively analyzing all the data available in those days.
Now codon usages in 11415 genes can be analyzed using nucleotide sequence
data obtained from the GenBank Genetic Sequence Data Bank (Release
62.0, Dec., 1989). Because of the growing size of the database, this year
a part of the data is listed in Nucl. Acids Res. (1990). [We will send uponrequest, a magnetic tape, or a hard copy listing codon usages in 11415
genes, to any Institutes of any countries.] To reveal the characteristics
of codon use in a wide range of organisms, as well as viruses and organella,
the frequency of codon use in each organism for which more than 20 genes
were available was calculated by summing up numbers of codon use. Confirming the "genome hypothesis" of Grantham et al., among taxonomically*

Present address:
Health, USA.

Department of Health & Human Services, National Institute of

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

related organisms (e. g. among mammals) codon-choice patterns resemble
each other but they differ between distant organisms. Synonymous codonchoice patterns in different genes of a single unicellular organism are known
to be usually similar to each other regardless of gene functions (dialectal
codon-choice pattern found for individual unicellular organisms as pointed out previously). However, codon-choice patterns in one higher vertebrate often differ significantly between different genes: we previously
pointed out the diverse codon-choice patterns found among genes of asingle higher vertebrate in connection with the evident diversity in the
G+C % at the codon third position among the genes. It should be stressedthat the characteristic pattern for the mammals mentioned above is obtained
only after summing up the genes with varying functions. When codon usages of approximately 10 or more genes with varying functions were summed
up for each mammal, they usually resulted in a very similar pattern, regardless of differences in the genes used for the summation. The fact that thepattern common among mammals does not depend on the genes used forthe summation, shows that this relates to the general characteristics oftheir genomes; 1) deficiency of CpG and TpA dinucleotides; 2) paucity ofgenes in the A+ T-rich genome portion, i. e. paucity of genes in A+ T-rich
isochores and in G/Q bands (thus A- and T-ending codons are less frequent);3) gross similarity of tRNA populations between different higher vertebrates(our preliminary data); 4) gross similarity of amino acid composition between different proteins, as well as between different mammals. For details,
see Wada, K., Aota, S., Tsuchiya, R., Ishibashi, F., Gojobori. T., andIkemura, T. (1990) Nucl. Acids Res., 18, Supplement 2367-2411.
G+ C % Mosaic Structures of the Higher Vertebrate Genome andDistribution of Dinucleotide Frequencies
Ken-nosuke WADA*, Isamu WATANABE**, Rye TSUCHIYA
and Toshimichi IKEMURA

It has become clear that the genome of higher vertebrates is a mosaic
of very long DNA sequences, each of which is fairly homogeneous in its* International Institute for Advanced Study of Social Information Science, Fujitsu
Limited.
** Present Address: School of Informaion and Cognitive Sciences, Chukyo University, Toyota, Aichi, Japan.

RESEARCH ACTIVITIES IN 1989

37

G+C content with several different levels. We attempted to characterize
the distribution of dinucleotide frequencies in different domains of the
G+C % mosaic structures of the human genome by comparing it with the
distribution along invertebrate (Drosophila and nematode) genome sequences. As a sensitive tool for visualizing both long-range and the shortrange features of G+C % variation along genome sequences, we proposed
the analysis of the frequency distribution of dinucleotides that are composed
only of G and/or C, or of A and/or T, as well as the distribution of theirdifferences. For details, see Wada, K., Watanabe, I., Tsuchiya, R., andIkemura, T. in New Aspects of Genetics in Molecular Evolution (editted
by N. Takahara) 1990, Japan Sci. Soc. Press.
Effect of DNA Supercoiling on in vitro Transcription from the
Adenovirus Early Region 4
Hiroshi HANDA*, Hajime WATANABE*, Yoshiaki SUZUKI**
and Susumu HIROSE

Recent studies have indicated that a specific DNA topology is required
for the expression of certain eukaryotic genes (see Weintraub, H., 1985,
Cell 42: 705-711 for review). To investigate the possibility that superhelical tension of DNA plays a direct role in the regulation of transcription,a cell-free transcription system that maintains superhelical DNA templates
has been developed using a Bombyx mori silk gland extract (Hirose, S.
et al., 1985, J. BioI. Chern. 260: 10557-10562). Using this in vitro system,
we examined the effect of DNA topology on the regulation of transcription
from the early region 4 (£4) of adenovirus type 5. The supercoiled DNAs
yield several-fold higher levels of £4 transcripts and more faithfully reflect
the regulation of in vivo transcription than linear DNAs do. For details,
see FEBS Letters, 249: 17-20, 1989.

* Department of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo.
** Department of Developmental Biology, National Institute for Basic Biology,
Okazaki.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Supercoiling of DNA by DNA Topoisomerase II and a Supercoiling
Factor Purified from the Posterior Silk Glandof Bomhyx mori
Tsutomu OHTA and Susumu HIROSE

A protein factor with an estimated molecular mass of 50 kDa was purified
to homogeneity from the silk gland of Bombyx mori. In the presence of
molar excess of this factor and eukaryotic DNA topoisomerase II, relaxed
circular DNA was converted to the negatively supercoiled form. Eukaryotic
DNA topoisomerase I could not substitute for eukaryotic DNA topoisomerase II in the supercoiling reaction. The reaction was dependent on
ATP and was inhibited by VP-16, a specific inhibitor of eukaryotic DNA
topoisomerase II. When DNA topoisomerase I was subsequently added
to the supercoiling reaction mixture, the supercoiled DNA became relaxed.
These results suggest that when both the 50 kDa protein and eukaryotic
DNA topoisomerase II are present in excess, unconstrained negative supercoils are introduced into DNA. For details, see Proc. Natl. Acad.
Sci. USA, 87: 5307-5311, 1990.
A Sequence-Specific DNA-Binding Protein that Activateslushi tarazu Segmentation Gene Expression
Hitoshi UEDA*, Sandra SONODA**, J. Lesley BROWN***,
Matthew P. SCOTT** and Carl Wu***

The Drosophila segmentation gene fushi tarazu (ftz) is expressed at thecellular blastoderm stage in a pattern of seven transverse stripes; the stripes
lie out of register with the segmental primordia, spanning alternate segmental
boundaries. The zebra element, a 740-bp DNA sequence upstream of
the ftz translational start, directs striped expression of lacZ when introduced
into the fly genome. We purified to homogeneity a sequence-specific DNAbinding factor, FTZ-Fl, which binds to two sites located within the zebra
element and to two sites within the ftz protein-coding sequence. FTZ-Fl* National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan.
** Laboratory of Biochemistry, National Cancer Institute, National Institute of
Health, Bethesda, Maryland 20892 USA.
*** Department of Molecular, Cellular, and Developmental Biology, University ofColorado, Boulder, Colorado 80309 USA.

RESEARCH ACTIVITIES IN 198939

DNA-binding activity is first detected in extracts of 1.5- to 4-hr embryos,
coincident with the time of ftz expression in stripes; the activity then diminishes before reappearing during the late embryo, larval, and adultstages. When one of the FTZ-F1-binding sequences in the zebra element
is mutated by 2- or 4-base substitutions, the binding to FTZ-F1 is disrupted
in vitro, and the intensity of lacZ expression is reduced in transformed
embryos, especially in stripes 1, 2, 3, and 6. These results suggest that
FTZ-F1 is a transcriptional activator necessary for the proper expressionof the ftz gene.
Cloning and Structural Analysis of the waxy (wx) Locus
Gene of Rice (Oryza sativa)
Hiro-Yuki HIRANO and Yoshio SANO

The wx locus controls amylose synthesis in rice seeds. Endosperms
of deficient mutants of the locus contain starch only composed of amylopectin. In wild-type rice, the amylose content (relative to total starch)varies among varieties or species in the Oryza genus and is genetically controlled in each strain. For example, in the Japonica-type rice of Oryza
sativa, the amylose content is 14-18 %, while in Indica-type rice it is 2027 %. In earlier studies, we found the wx locus product (Wx protein)through electrophoretic analysis of protein extracts from starch granules.
There is also a difference in the amount of Wx protein between the twovarietal groups; Indica rice has Wx protein about lO-times as much as
Japonica rice. In addition, we indicated that there is a positive correlation
between the amount of Wx protein and amylose content, suggesting thatthe extent of synthesis and accumulation of amylose is controlled by theamount of Wx protein. The molecular mechanism of the quantitative
gene regulation of the wx gene is of great interest for research. Since
amylose content is a major determinant of rice quality for eating or cooking,studies on the wx gene expression are not only attractive in terms of understanding gene regulation but also important for applied uses, such as
molecular breeding.
To approach the above problems, we tried to clone the wx locus gene ofrice using the wx locus gene of maize as a probe. We obtained DNA
clones from both Japonica- and Indica-type rice. Since Northern analysis
indicated that RNA, homologous to the cloned rice DNA segment, is

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

expressed in wild-type rice strains and not in mutants, this strongly suggeststhat cloned DNA is derived from the wx locus of rice. Southern blotting
analysis showed that each varieties of rice contains a single copy of thegene. Previous genetic studies support this result. The fact the gene to
be examined is single copy in the genome is a great advantage for genetic
and molecular biological studies. A cloned 4.5 kb DNA segment, which
may correspond to the wx locus of Japonica, has been completely sequenced.
The wx locus gene of rice is composed of many exons and introns asthat of maize is. A long open reading frame indicates that the wx geneproducts are about 59 kb polypeptides made up of 532 amino acids. In
comparison to the amino acid sequence of the N-terminal region of themature Wx protein, the precursor protein was shown to have an additional
polypeptide of 77 amino acids, which may be a transit peptide necessary
for importing the protein into amyloplasts. The size of the predictedpolypeptide from the nucleotide sequence is consistent with that of theWx protein as determined by polyacrylamide gel electrophoresis. The
deduced amino acid sequence of the rice wx locus gene has 88 % homology
with the maize gene. Since the maize wx locus is a gene for an enzyme,
UDP-glucose starch glycosyl transferase, the wx locus of rice may also
encode the same enzyme, which catalyzes the last step of amylose synthesis.
We purified the rice Wx protein and prepared the anti-sera for it. In
immunoblotting analysis of total protein extracts, the quantitative differencein Wx protein expression between Japonica and Indica was also shown as
in earlier experiments. These result eliminated the possibility that thedifference in amount of Wx protein in previous electrophoretic analysis
was due to the reduced affinity of the Wx protein to starch granules andloss during fractionation in Japonica rice. Northern blotting analysis
clearly indicated that the diffierences in quantitative expression of the wx
locus in the two varietal groups is regulated at the transcriptional level.
Studies on the Mechanism of mRNA Capping and the Process ofTranscription Initiation by RNA Polymerase II
Kiyohisa MIZUMOTO

Most of the cellular as well as viral mRNA in eukaryotes contain a 5'terminal cap structure, m7 GpppN. This cap structure is required for theefficient initiation of translation and for RNA splicing. It has also been

RESEARCH ACTIVITIES IN 1989

41

suggested that the synthesis of the methylated cap structure may play animportant role in transcription itself. The cap structure is synthesized
at the initial stage of mRNA synthesis and conserved at the 5'-termini ofRNAs while they are processed in the nucleus and transported to the cytoplasm. Consequently, elucidation of the mechanism of the cap structure
is important for understanding the molecular mechanism of eukaryotic
gene expression.
We characterized the reactions catalyzed by the mRNA capping enzyme(mRNA guanyltransferase), the key enzyme in the cap formation, fromvarious eukaryotic cells. The capping enzyme from animal cells consistsof a single polypeptide chain with an approximate Mr of 70,000 containing
two catalytic domains for each of the initial two consecutive reactions involved in the cap formation, i. e., those catalyzed by mRNA guanylyltransferase and RNA 5'-triphosphatase. In contrast, the higWy purified
yeast capping enzyme is composed of two separate chains of 52 kDa (a)and 80 kDa (~), responsible for the activities of mRNA guanylyltransferase
and RNA 5'-triphosphatase, respectively (Mizumoto, K. and Kaziro, Y.
(1987) Prog. Nucl. Acid Res. Mol. BioI., 34, 1).
The gene encoding yeast a subunit has been isolated by immunologicalscreening of a yeast genomic expression library in Agtll with antibodies
against the yeast capping enzyme. The idetity of the gene was confirmedby expressing it in E. coli to produce catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, andencodes a protein of 459 amino acid residues. Gene disruption experiments
showed that the a gene is essential for growth (Mizumoto, K. et al. (1990)
in Nucleic Acid Methylation (Eds: G. Clawson, D. Willis, A. Weissbach
and P. Jones), Alan R. Liss, Inc., NY, pp. 45-56).
We characterized an RNA polymerase II transcription initiation complex
formed with HeLa cell extracts and a DNA fragment containing the adenovirus major late promoter in the presence of ATP. An initiation complex
with an approximate size of 50S could be isolated by glycerol gradientcentrifugation free from the bulk of RNA polymerase II and the capping
enzyme. Specific transcription was detected with this complex when
supplemented with the remaining nucleoside triphosphates. Analyses of
the 5' -terminal structure of the transcript revealed the presence of a cap
structure m 7 GpppA in more than 50% of the RNA chains. These results
indicate that at least two cap-related enzymes, mRNA guanylyltransferase

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

and mRNA (guanine-7-) methyltransferase, are specifically associatedwith the complexes. The requirement of ATP hydrolysis for the initiation
complex formation is also being studied.
Transcription of Sendai Virus (HV J) Genome
Kiyohisa MrZUMOTO

Sendai virus (HVJ), a member of the paramyxovirus group, contains asingle nonsegmented negative strand RNA genome of approximately 15 kb
long, encoding at least seven proteins, NP, P, C, M, F, HN and L. The
genetic information of this negative strand RNA is expressed throughmonocistronic mRNAs that are transcribed by a virion associated RNAdependent RNA polymerase composed of P and L proteins.
We developed an efficient and faithful in vitro transcription system using
purified virus particles, and found that the HVJ mRNA synthesis is almost
entirely dependent on the presence of a host factor (s). The host factor
essential for the in vitro transcription was partially purified from mammaliancells and was shown to be separated into at least two complementary fractions, one of which, surprisingly, could be replaced by highly purified
tubulin from bovine brain. Furthermore, it was suggested that tubulin
is involved in the formation of transcription initiation complex by viralRNA-dependent RNA polymerase.
The analysis of the 5'-terminal sequences of in vitro-synthesized poly(A)+RNA demonstrated that almost all RNA molecules terminate inm7 GpppAmpGpGp-. mRNA was synthesized in vitro in the presence of[a- or ~_32P] ATP or GTP as the labeled substrates. The a and ~ phosphatesof GTP and the a phosphate of ATP, but not the ~ phosphate of ATP,were incorporated into the cap structure. These 32P-Iabeling data wereconsistent with a unique mechanism of cap formation by condensation of
GDP residue from GTP with pA- at the 5' temini of HVJ mRNAs.

RESEARCH ACTIVITIES IN 1989

II.

43

MICROBIAL GENETICS

Replication of Filamentous Phage fl: Enhancer-independent
Mutation of an Initiator Protein (gpII)
Atsushi HIGASHITANI, Nahoko HIGASHITANI and Kensuke HORIUCHI

The plus-strand replication origin of the filamentous bacteriophage f1
has a bipartite structure consisting of a required core origin (domain A)
and an adjacent A+ T-rich enhancer sequence (domain B). The core
origin sequence (about 45 bp) is absolutely required for replication andcontains three repeats «(3, rand 0), which serve as the binding site for thephage-encoded initiator protein (gpII: Horiuchi, K. 1986, J. Mol. Bioi.
188: 215-223). gpII binds the core origin in two steps, forming a binding
intermediate (complex I) and a functional complex for nicking (complex
II). Greenstein, D. and Horiuchi, K. (1990. J. Mol. Bioi. 211: 91-101)
have indicated that complex I contains two gpII molecules per origin, andcomplex II contains four molecules per origin. The replication enhancer
stimulates replication approximately 100-fold (Dotto, G. P. et al., 1984,
J. Mol. Bioi. 172: 507-512; Johnston, S. and Ray, D. S., 1984, J. Mol.
Bioi. 177: 686-700) and contains three binding sites (1, 2' and 2") for the
E. coli integration host factor (IHF: Greenstein, D. et al., 1988, PNAS.
85: 6262-6266). Two kinds of enhancer independent mutations in gpII
were isolated, which allow wild-type levels of replication in the absenceof either the enhancer sequence or IHF (Dotto, G. P. and Zinder, N. D.,
1984, Nature 311: 279-280; Kim, M. H. and Ray, D. S., 1985, J. Virol.
53: 871-878; Horiuchi, K., unpublished). One is the mpl mutation in
gpII (Met 40->Ile) which increases the co-operativity with which the protein
binds to form complex II (Greenstein, D. and Horiuchi, K., 1990, J. Mol.
Bioi. 211: 91-101). To elucidate the molecular mechanism of enhancerindependent replication by the other gpII mutant, which was termed "HII",
we determined the mutation site and characterized the Hll mutant
protein.
The sequence data indicated that the Hll mutation was caused by a singleamino acid change, Gly 73->Ala in gpII. An overproduction system
under the Tac promotor of the Hll mutant protein was constructed, and44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

this mutant protein was purified in mg order per litre culture. We examined the binding properties of the purified Hll mutant protein to thereplication origin in vitro. The Hll mutation in gpII increases the cooperativity with which the protein binds to form complex II, but to a lessextent than the mp1 protein. In addition, the Hll mutant gpII forms bothcomplexes I and II with a DNA fragment containing only two (f3-r) of thethree repeats from the core origin sequence as the mp1 mutant does, while
the wild-type protein forms only complex I with this fragment. These
results suggest that both mpl (Met 40....Ile) and Hll (Gly 73 ....A1a) mutations increase the co-operativity of protein-protein interactions to form afunctional initiation complex (complex II), and stimulate DNA replicationin the absence of the enhancer domain.
Multicopy Suppression in E. coli: Cloning of sufB, a Dosagedependent Suppressor of a Thermosensitive
Cell-division Mutation, ftsB
Atsushi HIGASHITANI*, Yoshihiro YAMAMOTO" and Yukinobu NISHIMURA

The suppression of a mutation in one gene by amplified copies of a different wild-type gene has been termed 'multicopy suppression' (Berg et al.,
1988, Gene 65: 195). A thermosensitive cell-division mutation, ftsB84(Ricard and Hirota, 1973, J. Bacteriol. 116: 314), is allelic to nrdB (ribonucleotide reductase B2 subunit gene) which maps at min 48.5 (Higashitani
and Nishimura, unpublished work: Kren et al. 1987, J. Bacterio!. 169:
14; Taschner et al., 1987, ibid. 169: 19). The growth thermosensitivity ofthis mutant can be corrected by a multicopy plasmid, pLCl-41, whichbears the purM region around min 54 (Yamada and Hirota. 1981. thisAnn. Rep. 31: 33). We have named the suppressor sufB (suppressor ofof ftsB) for the dosage-dependent suppressor of ftsB84.
To learn more about the structure and function of the multicopy suppressor, we subcloned the sufB gene from pLCl-41. The sufB gene waswithin ca. 3 kb fragment obtained by PstI digestion (Fig. 1). Nucleotide
sequencing of this DNA fragment is now in progress.
Fontecave et al. (1989, PNAS 86: 2147) have found the suppression of
nrdA, nrdB and also ftsB84 mutations under anaerobic conditions, and* Nagoya University.
.. Hyogo College of Medicine.

RESEARCH ACTIVITIES IN 1989";:lji ', , l

45
i

2650

sufB purMN gua
I .... '

'"

l;
~; ;
'"
.
!:;
,,:

Co1E1

pLCl-41:i

pAH844
pAH843
pAH843Cm

1

i!
,I

I

-

it:'
,
,

-----.:,

..

~
Km" .
pACYCl77

~I

; ".

+

,

pAH846

I Ii

Suppressionof ftsB84

pLCl-41t>E

pACYC184i"~pLCl-41t>P

pAH845(5).::~:: :KmTc++

7iem Km
Fig. 1.

Table 1.

Multicopy suppression in E. coli

Mutation

Suppressor

ilvE
Ion
topA
cheA
dnaA
dnaB
ftsB (nrdB)
ftsH
ftsM(serU)
ftsI
secA, secY
htrA (degP)

avtA, aspC
alp,ompT
toecheY
groE, sudA
dnaC
sufB
ftsI
ftsZ (suIB)
sufI
groE
sohA

Reference

Gene 65: 195 '88
J. Bact. 171: 3348 '89
Mol. Microbiol. 3: 531 '89
PNAS 81: 5056 '84
MGG 202: 435 & 446 '86
J. Bact. 146: 418 '81
Ann. Rep. NIG 31: 33 '81
J. Bact. 169: 5776 '87
J.Bact 171:2090'89
J. Bact. 170: 3967 '88
Nature 342: 451 '89
J. Bact. 172: 1587 '90

identified a novel ribonucleotide reductase (Nrd) that specifically functions
under anaerobic conditions. We speculate that the sufB gene product be

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

this particular Nrd. Tests for this possibility are under way.
Diverse examples of multicopy suppression have been reported in E.
coli (Table 1) as well as other procaryotes such as S. typhimurium (Wasserman et al. 1983. J. Bacteriol. 153: 1439; van Dyk et al., 1989, Nature
342: 451) and Xanthomonas (Thome et al., 1987, J. Bacteriol. 169: 3593)
or eucaryotes such as yeast (Hinnebusch and Fink, 1983, PNAS 80: 5374)
and nematode C. elegans (Maruyama et al., 1989, MGG 219: 113).
It has often been suggested that gene amplification plays an importantrole in evolution since gene duplication may represent the initial event in
the evolution of new function and in the formation of multigene families.
Following this initial step, one member could change by mutation or geneconversion or by exon shuffling (Gilbert 1978, Nature 271: 501) to produce
new functional genes. In view of this, we believe that it is important toelucidate the molecular mechanism of the multicopy suppression in E.
coli as a model system.
A Novel DNA Topoisomerase Essential for the E. coli
Chromosome Partition
Jun-ichi KATO*, Yukinobu NISHIMURA, Sota HIRAGA**, and Hideho SUZUKI***

Twin chromosomes produced upon completion of one round of replication
are likely to be linked with each other in a catenane form, as previously
demonstrated for small replicons (Sakakibara et al. 1976. JMB 108: 569).
The primary event in chromosome partitioning should involve the topological resolution of the catenated chromosomes through the decatenase
activity of a DNA topoisomerase. DNA gyrase participates in the resolution of the catenated E. coli chromosomes (Steck and Drlica, 1984, Cell
36: 1081). Three kinds of DNA topoisomerases have so far been foundin E. coli: Topoisomerase I (Topo I), II (gyrase), and III (Topo III) (Wang,
1985, Ann. Rev. Biochem. 54: 665; DiGate and Marians, 1988, JEC 263:
13366; and see Fig. 1). Two par mutant phenotypes described as ParA
and ParD have been ascribed to gyrE and gyrA, respectively (Kato et al.,
1989, MGG 217: 178; Hussain et al., 1987, Mol. Microbiol. 1: 259). The* Dept of Bacteriology, NIH, Yokyo.
** Dept of Molecular Genetics, Institute for Medical Genetics, Kumamoto University
Medical School. Kumamoto.
*** Lab of Genetics, Dept of Biology, Faculty of Science, University of Tokyo, Tokyo.

RESEARCH ACTIVITIES IN 1989

47

topS

t

1

Topolll Topoll

Topom Topol
Fig. 1.

parC mutation is not related to gyr (Kato et al., 1988, J. Bacterial. 170:
3967).
The nucleotide sequence of the parC gene has now been determined.
The deduced amino acid sequence was homologous to that of the A subunitof gyrase (Fig. 2). The homology was particularly remarkable around thetyrosine residue in the active site for gyrase activity. The parC protein
was shown to bind DNA.
Another new gene was identified in the upstream region of the parC
gene. The nucleotide sequence indicated that the product of that gene
was homologous to the gyrase B subunit. Four mutants were found in
the gene by surveying our E. coli mutant bank. They showed the typicalPar phenotype, and we named this gene parE.
A Topo I defect was compensated for by introducting simultaneously
two multicopy plasmids, one with the parC gene and the other with theparE gene. Thus, toe, a suppressor of tapA (Dorman et al., 1989, Mol.
Microbial. 3: 531), is most probably parC plus parE. We now believethat parC and parE genes code for a novel topoisomerase, Topo IV, whichis essential for chromosome partition (Fig. 1). For details, see Kato et al.,Cell in press.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40.

MYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNAParC I'" .. "".
GyrA
1"
MSEQNTPQVRBINISQBMRTSFLDYAMSVIVSBALEDVR]gLlifvHRRILYAMNDLGMTS~.~ParC 35' SAKFKKSARTVGDVLGKYHPHGOSACYBAMVLMAQPFSYRYPLVOGQGNWGAPOOPKSFA• • • t • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . It
GyrA 61" OKPYKKSARIVGBVIGKYHP!gDSAVYBSMVRMaQOFNYRYM~VOGHgN!gSVDGDSA-A
ParC gS' AM!BSRLSKYSBLLLSBLOQOTAOWVPNFDOTLQBPKMLPARLPNILLNGTTGIAVGM• • • • • • • • • • • • • • • • • • • If
• • • • • • 1' •••.•.•••••••.•••••••
GyrA 120" AM_ BARMSKISMBILRDITKOTIOYQONYDOSBREEVVMPSRFENL&V~2AAg!AVgM
ParC ISS' ATDIPPHNLRBVAQAAIALIOQPKTTLDQLLDIVQOPDYPTBABIITSRABIRKIYBNGR•••••• ~ •• If • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •GyrA ISO" AINIPPHQLOBIIDGVLAVSBNPDITIPBLMBVIPGPDFPTAOQIL-GRSOIRKAYESGRParC US' GSVRMRAVWKKBO-----OAVVISALPHQVSOARVLBQlAAQMRNKKLPMVOOLROESOH'1, .1'. It......••.••.•...•.••..•.••...•••••••.
GyrA 23g" OSITIRAKABIEQTSSGKERIIVT!LEYQVNKAKLIEKIAOLVROKKIBOITOLROESDRPare 270' BNPTRLVIVPRSNRVOMOQVMNHLFATTOLBKSYRINLNMIOLOGRPAVKNLLBILSEWL'. I.
•.• •• • .•••••• .••• ' . • • • •• I ••••• ' ••.•.•
GyrA 299" TO-MRIVIBIRRO-ANANVILNNLYKQTALQTSFGINL-LALVOOQPKVLTLKQCLEHYL
ParC 330' VFRRDTVRRRLNYRLBKVLKRLHILBGLLVAFLNIDBVIBIIRNEDBP---KPALMSRFG.. ., • • • • ' • "• • • • • • • " . • ••• I I . • •••. . . •• .. ' •.. ' .
GyrA 356" DHQKVVIRRBTAYELRKABARAHILBGLRVALDHLDAVISLIRNSQTAEIARTGLIEQFS
parC 387' LTBTQABAILBLKLRHLAKLBBMKIRGBQSBLBKBROQLQGILASBRKMNNLLKKBLQAO~.E2.lltt••••••.•••.•.•..••

II

II

fa

••

•••••••••

fa ••••••••••• It•..•.•••.

•... ..

GyrA 416" £,TBKQAQAILDMRLQRLTGLBRBKI!iBEYQSLVKLIABLKOILANBYKVLEIIRBBbTEIParC 447' AQAYGDORRSPLQBRBBAKAMSBHDMLPSBPVTIVLSQ--------MGWVRSAKGHDIDA...•.••...........•... 1.......
••.•
GyrA 476" KBRFNDERRTEIVT-SGLBTIBDBOLIERBNIVVTLTHNGYVKRLPASTYRSQKRGGKGVParC 499' PGLNYKAGDSFKAAVKOKSNQPVVFVDSTGRSYAIDPITLPS-AR-GQOBPLTGKLTLPP. . .
. .'. .' .. ''GyrA 535" QGMGTNEDDFVBHLISTSTHDTILFFSNKOKVYRAKGYBIPBYORTAKGIPIINLLBVBKParC 557' OATVDHML--MB-SDDQKLLMASDAGYGFVCTFNDLVARNRAGKALITLPENAHVMPPVV'. .. ., '. . ' ... " '.
...
''GyrA 595" GEWINAIIPVTBFNABLYLFFTTKHOVSKRTSLSQFANIRNNOLIALSLRBDDBLMOVRLParC 614' IBOASDMLLAITQAORMLMFPVSOLPQLSK-OKONKIINIPSABAAROBOGLAQLYVLPP••••••••••••• a ••.• a •••••.••• a •••••••••••• a.•
GyrA 655" TOOTKQIIIG-TKNOLLIRFPBTDVRBMGRTAAOVKGITLTOOOVVVGMBILBBBSHVLIParC 673' QSTLTIHVGKRKIKLRPEBLQKVTOERGRRGTLMRGLQRIORVEIOSPRRASSOOSBEXGyrA 714" VTEKGYGKRTPABEYRTQSRGGKGLKTAKITBNNGQLVAVKATKGEEDLMI ITASGVLIRGyrA 774" MO I NO I SI TGRVTQOVRL I RMAEBBHVATVALVBKNBBOBNEBBQBBV.•.. ...•..

.. ... ..
.

Fig. 2.

...

.... ...• . ....

Amino acid sequence homology of E. coli ParC with B. Subtilis GyrA (Moriya et al., 1985, Nucleic Acids Res. 13: 2251). Identities are represented byasterisks and conservative substitutions by dots. Active site Tyr is boxed.

49

RESEARCH ACTIVITIES IN 1989

Identification and Cellular Localization of the Product of prc,
a Gene Involved in the C-Terminal Processing of
Penicillin-Binding Protein 3 of Escherichia coli
Hiroshi HARA and Hideho SUZUKI·

The maturation of penicillin-binding protein 3 (PBP 3), an enzyme essential
for cell division in Escherichia coli, involves the proteolytic cleavage of the
C-terminal part of the precursor (Hara et al., 1989, J. Bacteriol. 171: 58825889). We have found a prc mutant defective in this unique processing
reaction (Hara et al., 1988, Natl. [nst. Genet. Ann. Rep. 38: 48-49), andhave cloned the prc gene (Hara and Suzuki, 1989, Natl. [nst. Genet. Ann.
Rep. 39: 52-54). The recombinant plasmids pHR53 and pHR61 carrying
the prc gene are shown in Fig. 1.
To identify the prc gene product, proteins synthesized in maxicells andin an in vitro coupled trancription-translation system under the directionof pHR53 and pHR61 were analyzed by sodium dodecylsulfate (SDS)
gel electrophoresis. A faint band of a protein migrating at the positionof about 80 kilodalton (kDa) was detected for pHR6I, but not for pHR53.
It was previously inferred that prc was transcribed in the direction fromleft to right in Fig. 1, and thus, both in pHR53 and in pHR6I, prc would beplaced next to the promoter of the drug resistance genes of the vectors in
the opposite orientation. We recloned the 2.6 kb EcoRI-PstI fragment,1 kb,E

P BH PE

!

I

!I

P B
!

Vector

!

!

.....

catp pACYC184! ~blap!

tetRp. 'pHR53

pBR32261

pBR322140~
Fig. 1. The plasmids used for the identification of the pre gene product. Only thecloned chromosomal regions are shown. Wedges denote the direction of
the promoters on vectors, and an arrow denotes the inferred location andorientation of the pre gene. B, BamHI; E, EeoRI; H, HindUI; and P, Pst!'•

Laboratory of Genetics, Department of Biology, Faculty of Science, University ofTokyo. Present address: Department of Biological Sciences, Kanagawa University.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40<;ontaining pre into pBR322 to construct pHRl40 (Fig. 1), in which thedirection of transcription from the promoter originally for a tet repressor
gene matched the inferred direction of pre. A remarkable overproductionof the 80 kDa protein was observed with pHR140. Insertion of a synthetic translation terminator (Maruyama et al., 1989, Gene Anal. Teehn.
6: 57-61) into the PstI site within pre eliminated the 80 kDa protein. Whenthe terminator was inserted into the BamHI site, which was also withinpre, a product of almost normal size was observed. These results indicatethat the 80 kDa protein is the pre product and that the direction of transcription is from left to right in Fig. 1 as inferred. Plasmid pHR53 complemented the pre mutation but did not direct the synthesis of the 80 kDa
product to a detectable level, probably owing to a weak promoter of the
pre gene, or to the transcription from the cat promoter counteracting thetranscription of pre, or to both. The 80 kDa product was detected with
pHR61 although the bla promoter could counteract the pre transcription.
Transcription from somewhere in the 9.5 kb PstI-EcoRI fragment upstream
to pre might proceed beyond the EeoRI site and contribute to the higher
pre expression. Weak expression of pre in pHR53 was enough to complement the pre mutation. Hyperexpression of pre by a strong promoter
was rather deleterious to cell growth.
The location of the pre product in a cell was investigated in maxicells
harboring pHRl64 which carried the genes pre, bla, and tet. When the
maxicells were disintegrated by sonication and fractionated by centrifugation, the pre product was observed both in the soluble fraction and in theinsoluble membrane fraction. The membrane fraction was further treatedwith a detergent, Sarkosyl, and the pre product was found in the solubilized<;ytoplasmic membrane fraction. The bla and tet products were recovered,as expected, in the soluble fraction and in the cytoplasmic membrane fraction, respectively. When the maxicells were converted to spheroplasts
by treatment with lysozyme and centrifuged, the pre product was fractionatedboth to the supernatant periplasmic fraction and to the pelleted spheroplasts.
These results may indicate that the pre product is distributed both in the
periplasm and in the cytoplasmic membrane. However, it does not appear
that any major processing occurs in the pre product, because no differencein electrophoretic mobility was observed between the product synthesizedin vitro and that in maxicells, when tested in SDS gels of several polyacrylamide concetrations. When the maxicells were fractionated by the

RESEARCH ACTIVITIES IN 1989

51

method of Russel and Model, which consists of treatment with 0.1 N N aOH
followed by centrifugation and precipitates membrane proteins (Russel
and Model, 1982, Cell 28: 177-184), the prc product partitioned into the
NaOH pellet together with the tet product, leaving the bla product in the
supernatant. It seems probable that the prc product is loosely associatedwith the periplasmic side of the cytopolasmic membrane and easily released
into soluble fractions during fractionation procedures. It should be notedthat overproduction of the product in maxicells may affect its distribution.
Localization of the prc product outside the cytoplasmic membrane is consistent with the notion that the C-terminal processing probably occurs
after PBP 3 is translocated across the cytoplasmic membrane (Hara andSuzuki, 1988, Natl. Inst. Genet. Ann. Rep. 39: 56-57).
Nucleotide Sequence of the prc Gene of Escherichia coli
Yoshihiro YAMAMOTO·, Yukinobu NISHIMURA and Hiroshi HARA

The prc gene of Escherichia coli is involved in the unique processing
reaction which cleaves the C-terminal part of the precursor form of penicillinbinding protein 3, an enzyme essential for septum formation (Hara et al.,
1989, J. Bacteriol. 171: 5882-5589). The gene was located in a 2.6 kilobase(kb) EcoRI-PstI fragment at min 40.3 on the E. coli chromosome (Haraand Suzuki, 1989, Natl. [nst. Genet. Ann. Rep. 39: 52-54; Hara et al., 1989,
ibid. 55), and the product was identified as a protein of about 80 kilodalton(kDa) as described in the accompanying report (p. 49-51).
We determined the nucleotide sequence of a 3178 base-pair (bp) chromosomal fragment, the 2.9 kb EcoRI fragment cloned in pHR53, plus
220 bp in the contiguous 9.5 kb PstI-EcoRI fragment in pHR61 (see Fig.
1 on p.49). Only one major open reading frame (ORF) of significant
length, starting with an AUG initiation codon either at nucleotide (nt)
287 or at nt 293 and ending with a UAA termination codon at nt 2330,
was identified. Its location and direction were consistent with our previous
results. We do not know at present which of the two AUG codons is usedas the initiator. The ORF could code for a polypeptide of 682 or 680
amino acids with a predicted molecular weight of 76,677 or 76,432, in good
agreement with the size of the gene product estimated previously. The•

Laboratory of Genetics, Hyogo College of Medicine.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

product, as deduced from the identified ORF, showed a relatively low
polarity. Several hydrophobic regions were found near the N terminus
and towards the middle of the polypeptide. The N-terminal hydrophobic
sequence might serve as a signal directing the product to the outside of thecytoplasmic membrane, but it does not appear to be cleaved, as describedin the accompanying report (p. 49-51).
When the prc gene was placed under the control of the A PL promoter
and the temperature-sensitive cI857 repressor, production of a large amountof the pre product was induced upon temperature upshift to 42°C. Under
these conditions the rate of processing of PBP 3 was increased. The prc
product is possibly a processing enzyme, although we cannot completely
eliminate other possibilities, e. g. it may be an activator of the processing
enzyme. The deduced amino acid sequence exhibited no significant
homology to any proteases, peptidases, or other proteins in the European
Molecular Biology Laboratory protein database.
A Mutant Producing Temperature-Sensitive Farnesyl
Diphosphate Synthase of Escherichia coli
Shingo FUJISAKI*, Tokuzo NXSHlNO**, Hirohiko KATSUKI***,
Hiroshi HARA and Yukinobu NISHIMURA

A reaction utilizing isopentenyl diphosphate (IPP; C~) and farnesyl
diphosphate (FPP; Cl~) is a branch point in the synthetic pathway of various
isoprenoids. Escherichia coli provides an excellent model system for the
study of non-sterol isoprenoid synthesis because it is much simpler in theisoprenoid composition than eukaryotic cells. We screened Hirota's
temperature-sensitive (ts) mutant stock of E. coli for mutants defective in
isoprenoid synthesis, and found a mutant, JE 11046, having a ts defect in
FPP synthase activity. FPP synthase condenses IPP with dimethylallyl
diphosphate (C~) or geranly diphosphate (C10) to yield FPP. The mutation
for this defect was named ispA. Results of Hfr crosses and F'- and Plmediated transductions indicated that the map location of ispA was about
min 10 and that the gene order was tsx-ispA-lon. The ispA mutation* Department of Chemistry, Faculty of General Education, Gifu University.

** Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku
University, Sendai.
*** College of Liberal Arts, Kinki University, Higashi-Osaka.

RESEARCH ACTIVITIES IN 198953

was recessive to the wild-type allele.
An isogenic pair of ispA- and ispA+ strains was constructed and theirproperties were examined. Unexpectedly, the ispA mutant showed no
growth defect compared to the wild type both at 30°C and at 42°C, although
the in vitro activity of partially purified FPP synthase was significantly
lower for the mutant than for the wild type (21 % at 30°C and 5 % at 42°C).
The amounts of major isoprenoids in the mutant cells were almost thesame as those in the wild-type cells at 30°C and at 42°C except that thelevel of ubiquinone was moderately lower (66 %) in the mutant cells at 42°C.
FPP synthase may not be rate-limiting in general isoprenoid synthesis, orthe mutant FPP synthase may be stabilized in vivo. The possibility cannot
be excluded that there may be another short-chain prenyl diphosphatesynthase which can supply a priming substrate for synthesis of long-chain
prenyl diphosphate. The is growth of JEl1046 appears to be due to somemutation(s) other than ispA.
We suggest that ispA represents a structural gene for FPP synthase,because FPP synthase partially purified from the ispA mutant was ts andwild-type FPP synthase was not inhibited by the addition of the enzyme
fraction of the mutant. For details, see Fujisaki et aI., 1989, J. Bacteriol.
171: 5654--5658.
Mapping of a Whole Set of Cell Division Genes in
Escherichia coli K-12
Akiko NISHIMURA, Reiko IROBE, Keiko SUZUKI and KiyokoUEYAMAOne of the possible approaches to elucidate the cell division mechanism
in E. coli is to identify the whole set of genes involved in cell division, andto analyze their functions and relations among their expression. Such an
appeoach would also make possible to find key genes and key reactionsin the cell division cycle.
Up to now only a few scores of cell division genes have been identified.
However, E. coli is expected to have more than one hundred cell division
genes as judged from statistical analyses on the occurrence of known mutations (Maruyama, personal communication). We have developed a systemof procedures to identify a whole set of cell division genes in a short timeby use of three different types of E. coli culture bank.
The strategy is as follows: i). Screening mutants from the "Hirota's

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

TS mutant bank", which form filamentous cells at 40°C without immediatearrest of DNA synthesis and of cell mass increase. ii). Screening plasmids
from the "Clarke & Carbon's pLC-plasmid collection", complementing
TS defects of these mutants, which represent putative cell division genes.
iii). Identification of the map position of the insert in each pLC-plasmidby plaque hybridization to "Kohara's clone bank". The system provides
a convenient screening procedure for identifying plasmids harboring acell division gene, and its mutant strain at the same time, and for carryingout structural analyses of these genes. The 282 of cell division mutants
were mapped on the physical map of E. coli. For details, see "Control of
Cell Growth and Division" 1991, H. Yoshikawa and A. Ishihama (eds.).
The present study was focused on the analysis of cell division genes.
However, we notice that our physical mapping of pLC-plasmids would
provide by itself a useful tool for genetic analyses and gene cloning in E.
coli. (in preparation).
The secA Gene of Bacillus subtilis
Yoshito SADAIE, Hiromu TAKAMATsu*, Kouji NAKAMURA*
and Kunio YAMANE*

We determined the nucleotide sequence of the 4.1 kb efr13I chromosomal
fragment from pll-div-341+ DNA which carries the wild type div-341+
gene of Bacillus subtilis. Three open reading frames were found in thesame orientation. The biggest frame consisted of 841 codons and codes
for a protein with a molecular weight of 95,529. The protein shared a
50 % identity in amino acid sequence with the secA protein of Escherichia
coli as shown in Fig. 1. The two genes shared a 47.2 % identity in their
nucleotide sequences.
The wild type div-341 + gene of B. subtilis is required for sporulation,
secretion of extracellular enzymes, autolysis, competence development,
and spore outgrowth as well as for cell division. By analogy with thefunction of the E. coli secA gene, the wild type B. subtilis div-341+ gene is
responsible for the secretion of certain extracellular enzymes and translocation of certain proteins on the cell surface which are involved in cell division,
sporulation, autolysis and uptake of exogenous transforming DNA. (Mol.
Gen. Genet. 190 (1983) 176-178; J. Bacteriol. 163 (1985) 648-653; Jpn.
* Tsukuba University.

RESEARCH ACTIVITIES IN 1989

55

ML-giLnKmF-dptkRTLnRyeKiaNdldAirgdyEnLSDdaLKhKTiEF
keRLEKGattddLlvEAFAVVREASrRVtGMfpFkVQLmGGvaLhdgnlA
EMkTGEGKTLTsTLPvYLNALTGKGVHVVTVNeYLAsRDAEqmgkiFEFL
GLTVG1NLnsMskdeKREAYAADITYsTNNE1GFDYLRDNMvlykEqmVQ
RpLHfAviDEVDSILIDEARTPLIISGqAakStklYvqaNafvr----------TlkaEkdytyDiKtkaVqLTEeGmtkaEkaf---GI----dnLfd
vkhvaLnHHinqALkAHvamqkDVDYvVeDGqVvlVDsfTGR1MkGRRyS
eGLHQAiEAKEGlelQNEsmTLAtITFQNYFRmYEKLAGMTGTAkTEeeE
FrnlYnmqvVtiPTNRPvvRdDrPDLiYrTmegKfkAvaEDvaqRymtGQ
PVLVGTvavEtSELiSklLknkGlpHqVLNAKnHerEAqlieeAGqkgAV
TIATNMAGRGTDlkLGeG------------------------------Vk
E1GGLavvGTERHESRRIDNQLRGRSGRQGDpGitqFYLSMEDeLMRrFg
aeRtmaMldrfGMddstplqskmVsrAvessQkrVEgnNFDsRKQLLqYD
DVlrqQRevlYkQRfEviDsenlrEivenmikssleralaAYtPreeLpE
eWkldGLvdlinttyldegaLeksdifgKEP----demlellmdriltkY
neKEEqfGkEqMReFEKvivLravDSkWmdHidAMDqLRQGIHLRaYAQt
nPlrEYqmEgFaMFehMiESiedEVakfvmKaei-----ennLEreevvq
gqttAh----qpQegDdnkkAkkApvrkvvdiGRNaPChCGSGKKYKnCc
GRte
Fig. 1. The amino acid sequence of the Bacillus subtilis div-341+ gene. Capital'
letters indicate common amino acids with E. coli secA protein, which consists.
of 901 amino acids, 60 amino acids longer than the protein from the wild typeB. subtilis div-341 + gene.

J. Genet. 64 (1989) 111-119; Gene in press.)

:56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

III.

IMMUNOGENETICS

Fine Mapping of Recombinational Hotspots in the Mouse MHC
by Direct Sequencing of PCR Amplified DNA
Toshihiko SHIROISHI, Tomoko SAGAI, Hideo GOTOH
and Kazuo MORIWAKI

Meiotic recombinations within the major histocompatibility complex(MHC) of the mouse occur in clusters at restricted sites, the so-called,
recombinational hotspots. So far four hotspots have been identified in
the proximal region encompassing 400 kb of DNA in the mouse MHC.
Japanese wild mouse derived haplotype, wm7, exibits enhanced recombination
frequency in this region in genetic crosses with standard laboratory strains.
Recombinational breakpoints were confined to one of the hotspots whichis 1 kb of DNA between the Afi3 and Afi2 genes. Molecular characterization of this hotspot and previously identified hotspot located in the E figene revealed that two independent hotspots share a very similar molecularorganization, consisting of two molecular elements, mouse middle repetitive
MT-family and tetramer repeated sequences (Shiroishi et al., Immunogenetics 31, 79-88, 1990).
In order to elucidate the spatial relationship between the location of therecombinational breakpoints and these two elements, we attempted tomake a more fine mapping of recombinational breakpoints. For thispurpose, DNA segments including the hotspots were amplified by thepolymerase chain reaction (PCR) method from twelve independent re·combinants. After further amplification using one of two primers toyield the single stranded DNAs, approximately 1 kb of amplified DNAs
jncluding the breakpoints were directly sequenced. Comparison of thesesequences and those of parental strains made it possible to assign the finelocation of the breakpoints. All recombinants from the cross of wm7
and b MHC haplotypes have breakpoints in approximately 300 bp of
DNA located upstream from the tetramer repeated sequence, while mostrecombinants from the cross of wm7 and k haplotypes placed their breakpoints in 500 bp of DNA located downstream from the tetramer repeatedsequence. Thus, all recombinational breakpoints are clustered aroundRESEARCH ACTIVITIES IN 198957

the tetramer repeated sequence. The significance of this association of
breakpoints with the tetramer sequence remains to be determined.
Characterization of Asian Specific H-2Ku Class I Antigen
Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Among the polymorphic H-2K class I antignes, H-2Kf and Kd distributeat high frequencies (Kf; 29%, Kd; 31 %) in Asia and have been observed
at cerain frequencies in Europe (Kf; 6.5 %, Kd; 15 %) as well. A detailed
characterization of the H-2Kf antigens identified in both populations,revealed that they include many minor variants related to each other. Nevertheless, an indistinguishable Kf related molecule was detected in European(M. m. domesticus) and Asian (M. m. musculus, M. m. castaneus andM. m. molossinus) mouse populations, although they are genetically separated to large extent. The geographical survey of the minor variants
suggested that the various H-2Kf-related molecules have been generated
before and after M. musculus subspeciation and coexisted in wild mouse
populations during their long evolutionary process. These results support
a positive selection hypothesis for increasing the number of MHC alleles.
Another H-2 class I antigen, H-2KU showed a high frequency (22%)
in Asia, but it has been hardly detected in Europe (0.3 %). The geographical distribution of H-2KU almost overlaps the habitat of M. m. castaneusand M. m. molossinus which is considered to be a hybrid of musculus andcastaneus subspecies. A characterization of H-2KU antigens using monoclonal antibodies and a DNA probe showed that those observed in Japan,
Taiwan and China are very similar to each other and are clearly distinguishable from other H-2 haplotypes. In contrast to H-2Kf molecules, the
H-2KU molecules seemed to share a framework of molecular structurewith those of other antigen molecules, such as Kq and KS. These results
suggest that the ancestor of the H-2Ku antigen molecules was generated in
Asian population after the subspeciation of M. musculus.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Studies on Freeze Preservation of Embryos of
Drosophila melanogasterYukiaki KURODA and Yuko TAKADA

The freeze-preservation of Drosophila embryos is a useful procedure formaintaining many wild-type and mutant stocks of Drosophila which saves
enormous cost, labor and time spent for maintaining them at 20-25°C inordinary procedures. There are, however, some barriers to be cleared
up to accompolish freeze-preservation. One such barrier may be the introduction of freeze-protective agents, glycerol and dimethyl sulfoxide(DMSO), into embryos. The vitelline membrane of embryos prevents
the agent from entering embryos. Many attempts have been made toabolish the non-permissible property of the vitelline membrane. One
procedure is to use a laser microbeam to perforate the vitelline menbrane.
The procedure proved partially successful in obtaining living larvae andadults after freeze-preservation at a very low frequency.
The other procedure is chemical treatment and this might be accompolished with some enzyme treatment. The enzymes used were proteases
such as dispase (Boehringer Manheim), pancreatin (Difco), pectinase (Sigma),neuraminidase (Sigma) j3-galactosidase (Sigma) and cellulase (Sigma).
Eggs were dechorionated by treatment with 3 % sodium hypochloride
solution for 6 minutes and then treated with these enzymes at various
concentrations for 1-3 hours. After treatment with enzymes, eggs were
examined for the entrance of neutral red and their hatchability after incubation at 25°C.
j3-galactosidase and pancreatin had almost no effect on the permeabilityof the vitelline membrane. Neuraminidase, pectinase and cellulase enhanced the permeability of the vitelline membrane. The treatment ofeggs with 1 % cellulase for 3 hours was especially effective in enhancing
permeability and had no significant effect on the hatchability of eggs. Moreeffective and useful treatments with these enzymes are under investigation.

RESEARCH ACTIVITIES IN 1989

59

Detection of SR Spiroplasms in Embryonic Tissues of Drosophilamelanogaster by an Electron Microscope
Yukiaki KURODA, Kugao OISHI* and Yutaka SHIMADAi

In the previous study, it was found that sex-ratio (SR) spiroplasms might
produce some brown necrotic changes in cultured epithelial cells of embryosof Drosophila melanogaster, when they infected embryonic cells in vitro.
Under an electron microscope, embryonic cells infected with SR spiroplasms
contained many spiroplasm-like structures in intercellular spaces and thecytoplasm of necrotic cells.
In the present experiment, whether SR spiroplasms might selectively
infect any specific type of embryonic cells in normal development was
examined. Eggs were collected 3.5, 7.5, 12 and 13 hours after oviposition
from adult flies of the Oregon-R strain infected with SR spiroplasms.
They were pre-fixed in glutaraldehyde and paraformaldehyde and fixed in
I % osmic acid. Epon embeded eggs were sectioned and observed under
an electron microscope.
The results indicated that some necrotic cells were found in the brain
area and in some neuroblast cells under the hypoderm cells of embryos.
In embryos at 20 hours after oviposion, some structures which might be
SR spiroplasms were detected in intercellular spaces. These findings
suggest that SR spiroplasms may have a specific affinity for the brain andnervous tissues and produce necrotic changes in them.
Developmental Genetics of E-Pseudoallelic Domain in Bombyx
1. Genetic characteristics
Akio MURAKAMI

It was nearly half a century ago when the suggestion was made that Epseudoallelic gene loci (6-0.0) in Bombyx could be used as important experimental material for studies concerning the basic body structure of embryo.
Receiving impetus from recent studies on the homeotic gene in D. melanogaster, the analysis of Bombyx E-pseudoalleles has been started again indevelopmental genetics, using molecular biological techniques. A totalof about 30 mutant members which comprise the E-allele have been found,including spontaneously induced members and artificially induced ones

* Department of Science, Kobe University.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

created through the use of ionizing radiation etc. The main genetic characteristics of these members are their dominant characteristic and thefact most of them are embryonic lethals. They are also characterized bya versatile expression of various visible and invisible characteristics fromthe embryonic stage to the larval stage; including fewer abdominal legs,
larval pattern marking, morphological abnormality of gonad and malformation of larval epidermis. In addition, the allelic group can be induced
through ionizing radiation which has a chromosome-breaking ability as
described above, and recombination can be observed clearly in females of
the allelic group, though the recombination frequency is low compared tomales (crossing over occurs generally only in males in the case of Bombyx).
On the basis of these characteristics, it seems logical to assume that thehereditary nature of the E-allelic mutation originated from chromosome
aberration rather than gene mutation. Also, the size of the E-domain is
fairly large and random recombination of chromosomal DNA (unequalcrossing over) occurs at chromosome brekage (or easily breakable) areas
independent of sex, thus causing the creation of various types of E-member
group constituents.
Developmental Genetics of E-pseudoaUelic Domain in Bomhyx
2. Developmental Characteristics
Akio MURAKAMI

The expression of a supranumeral leg is a degeneration of phylogenesis:
There are many reports on the relationships among embryonic lethality,
abnormal numbers of larval abdominal legs and abnomality of larval bodymarking pattern in E-pseudoallelic domain mutants, but the mechanism
remains unclear. Such a subject is interesting also in terms of its relationship to the determination of the structural plan of Bombyx. Results ofrearrangement and analysis of observation data so far accumulated on therelationship between visible character and embryonic lethal have indicatedthe possibility that an excess (or reduced) number of abdominal legs has astrong relationship to embryonic lethality on the basis of the existing patterns of crescent marking and star spot marking.
In normally developed early stage embryos, appendices for the development of future larval organs, such as abdominal and thoracic legs, exist
in all of the 18 segments except for the extreme tip segment which becomes a

RESEARCH ACTIVITIES IN 1989

61

caudal leg in the future. These appendices, excluding those which become
head organs, thoracic legs and abdominal legs of Bombyx larvae in the
future, disappear just after the so-called "blastokinesis" when organogenesis
is almost completed. Among E-allelic mutants, however, a mutant having
the ability to express supranumeral abdominal leg exists as it is (sometimes
in an incomplete form) and in particular has an excess of larval legs. A
normal developmental programme results in a silkworm larval body having
three pairs of abdominal legs. It can be concluded that a mutant of theE-pseudoallelic group shows a morphology similar to that of Myriapoda
which is a progenic type of insect, because of the abnormal developmental
programme caused by structural abnormality (probaly a deficiency) in
its E-domain chromosome as descrived elsewhere (Murakami, 1989). It
seems that the high frequency of embryonic lethality is caused not only
by direct chromosomal abnomality and indirect abnormality of the structural plan, but also by functional abnormality. In conclusion, it can besaid that the supranumeral leg in the E-allelic mutant is a type of embryological degeneration.
Structural plan of Bombyx leg deduced from the distribution of supranumerallegs: As described in the preceding section, supranumeral abdominal
legs of a larva appear in the anterior abdominal part of abdominal segments (A l ,2,oo'7,s) but not in the posterior part of abdominal segments(Ag,lo,u). Naturally, such legs do not appear in the thoracic part (T l ,2,a)or the head which is comprises of four segments. The frequency of supranumeral abdominal legs at two abdominal segments (A7 and As) is quite
low (below 1 %).
In embryogenesis of Bombyx, similar to that of other insects, the headpart is formed from the first to fourth embryonic s(fgments and the following three segments form the thoracic part (5th, 6th and 7th segments).
The remaining 11 segments form the abdominal part. The formation of
the abdominal part starts almost at the same time as head formation. The
head formed by the fusion of the first to fourth segments (or ganglia).
Interestingly, the formation of the Alo _7 segments (or ganglia) of the abdominal part as well as the caudal leg (A ls ) are accomplished in a similar way
through the repeated fusion of two segments (or ganglia) until the A 7segment
os reached. On the other hand, the As-As segments of the abdominal
section are formed smoothly through the fusion of segments and ganglia.
Therefore, in normal embryogenesis, the abdominal part (abdominal com-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

plex: ABC) is divided into anterior ABC (ABca) comprising the 8th to13th segments and posterior ABC (ABO) which comprises the 14th to18th segments. When the condition of supranumeral legs is taken into
consideration, ABca can be divided further into a part which includes the10th to 13th embryonic segments (ABCa- z) where the appearance (decrease)of normal abdominal legs is observed and a second part comprising the
8th and 9th segments (ABCa- 1) where supranumeral abdominal legs appear
at a high frequency in E-allelic mutants. In the same manner, it is possible
to classify ABCP into a segment (ABCP-l) where supranumerallegs appear,though at a low frequency and another part comprising the 16th to 18th
segments (ABO-Z) where supranumerallegs do not appear.
Genetic Studies on the Diapause Phenomenon in Bombyx
1. Seasonal Adaptation of the Tropical Multivoltine
Race in the Temperate Zone
Akio MURAKAMIIn Bombyx mori L., univoltine races never change their genetically determined voltinism in the climate of Japan, while bivoltine races easilychange their inherent voltinism(s) between bi- and univoltine types. It
is of interest to note that tropical multivoltine races have in general an
unusually high adaptability: when these races are introduced to the temperate zone including Japan, from their native tropical or subtropical zones,
they easily acquire a high embryonic diapause state for only one or two
generations after introduction from their native places to the temperatezone. This short term of adaptability of seasonal changes seems to be
not in accordance with general adaptation theory. However, this unusual
phenomenon can be explained by the detection of a gene (or system) whichis responsible for the abnormal phenomenon. Tropical multivoltine races
including strain Cambodge, however, change remarkably their generation
times in a year from the multivoltine type (7-8 times) to the univoltine one(a single time) in Japan. In fact, the strain Cambodge is highly adaptable
to changes in climatic conditions in Japan and is sensitive to changes ofseasons and/or photoperiods (Murakami, 1988, '89, '90). Having foreknowledge of unfavorable environmental conditions to come in the near
future, the moths of multivoltine races raised in Japan tend to lay diapausing(and/or hibernating) eggs for life existence and/or species maintenance.

RESEARCH ACTIVITIES IN 1989

63

The embryonic diapause phenomenon of the multivoltine race in the temperate zone seems to be one of certain motive powers for expanding theirecological niche. It was revealed by author that these biological phenomena
are under the control of a sex-linked gene npnd (non-pigmented and nondiapausing egg) detected in the strain Cambodge. Of course, the geneitself never changes but only changse its phenotypic expression depending
on climatic conditions.
About twenty years ago, an autosomal recessive multivoltine gene pnd(pigmented but nondiapause egg) was isolated from the strain Cambodge
by Katsumata (1968) and its linkage was analyzed (Yamamoto et al., 1972).
The insect having the gene pnd produces only nondiapause eggs throughout
the year regardless of change in environmental conditions. According toour genetic analyses of interactions between these two multivoltine genes,
the gene npnd is epistatic to pnd, indicating that the phenotype of egg statesin silkworm lines having both genes is absolutely under the control of thenpnd gene.
In short, the npnd gene (system) controls the nature of multivoltinism
and is also highly responsible for seasonal adaptations. A number of
researchers have done extensive investigate to clarfy the diapause phenomenon and consequently they have accumulated much valuable informationon this subject. Among other things, the photoperiod or daylength has
been shown to be the most prominent factor in various physical variables
as an indicator of seasonal change. Accordingly, it seems more likelythat a function of the gene npnd takes part in a series of integrations ofvarious environmental information including the photoperiod. The
present interpretation appears to be consistent with some reports whichhave been shown a connection between brain and the determination of
egg-states (Fukuda, 1951; Morophoshi, 1972, '76).
Genetic Studies on the Diapause Phenomenon in Bombyx
2. A Possible Role of the Central Nerve
Center System on Adaptation
Akio MURAKAMI

The photoperiod changes more drastically in the temperate zone thanin the tropics. Daylength increases gradually from the winter solsticeto summer solstice in the temperate zone, while the situation is reversed

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

from the summer solstice to the winter solstice. At both vernal and autumnal equinoxes, daylength becomes 12 hours. In short, the photoperiod
is slightly changing day by day.
Considering this, it can be assumed that embryos of Bombyx recongnize
such a trend: embryos have some function for precisely measuring timedifferences creating a biological (or circadian) clock. It seems likely thatthey may store the received information as changes of environmental conditions for about 24 hours in their cerevral nerve center system (CNCS) or
brain, to infer time differences in daylength for two consecutive days. Surprisingly enough, in Bombyx, for the egg-state conversion of the tropicalmultivoltine races from the nondiapause type to a diapause one in the
climate in Japan, it takes about 1.5 to 2.0 months from the input of theenvironmental information to the outcome or secretion of diapause factor(DF) in the pupal body cavity, so that it is necessary for long term information preservation. It is also reasonable to say that the Bombyx CNCS
may have the ability to integrate various kinds of external information or
physical stimulations, and store and transmit the adjusted information
through the larval period to the mid-stage pupae. Some instructions fromthe pupal CNCS are dispatched to the SG which synthesizes and secrets
DF into the body cavity. DF is synthesized in all silkworm races. Thus,
the embryo CNCS which has already received information differ according
to the quality of photoperiod, temperature, etc., and transmits the integrated
information through the larval CNCS to the mid-stage pupal CNCS which
gives some instructions to SG to secret DF which decides finally the fateof the egg-state: during embryonic development some CNCS which havereceived information on short daylength and/or low temperature or theboth promote the secretion of DF, while those which have received longdaylength and/or high temperature information, inhibit the secretion.
Of course, timing of the endocrinological event is absolutely under thecontrol of the inherent developmental programme of Bombyx. In any
case, the time course in a series of neural transmission passways of information appears to require a remarkably long duration, suggesting that thisprocess comprises a certain type of mechanism for information storage, along term memory. It is of interest to understand how the operation ofsuch a storage system in Bombyx. It is well known that in Bombyx themost popular egg incubation method for obtaining a completely converted
egg-state with a voltinism opposite to the originally determined one (fromRESEARCH ACTIVITIES IN 1989

65

diapause to nondiapause type, and vice versa) is treatment of the egg bykeeping for several days under relevant incubation conditions. Thissuggests that the modification of voltinisms may take part in certain confirmational events.
To sum up, the conversion process of egg-state for the voltinism takes
place in three steps: first is the reception of environmental information incertain sensory organs, second is information integration, storage andtransmission in CNCS, and third is the endocrinological performance in
pupal SG, as the result of orders of environmental information or stimulation.
Genetic Studies on the Diapause Phenomenon in Bombyx
3. Artificial Modification of the Diapause State inTemperate Races to Nondiapause Type
Akoi MURAKAMIIn the preceding section, it was stated that egg-states in the tropicalmultivoltine races convert easily from the genetically determined nondiapause type to a diapause type in the process of seasons under natural climaticconditions in Japan. One of the Chinese bivoltine silkworm races, theDaizo strains (this is regarded as multivoltine races in India) and a few
other strains of either Chinese or Japanese races partly resume a 2nd
generation after the first spring generation in Japan. However, in general,most of the bivoltine races do not enter a 2nd generation during the year.
Univoltine strains do not repeat a summer generation after the spring
generation as far we know. Thus, it becomes clear that the conversion
from a nondiapause state to a diapuse one in tropical mUltivoltine races is
produced during the process of seasonal change, while the temperate univoltine as well as bivoltine races, except in a few cases, never change their
genetically determined diapause state to a nondiapause one under natural
conditions. Accordingly, it seems more likely that the former diapause
state is a facultative type and the latter an obligatory type, suggesting that
there is different fundamental mechanism for the induction of diapause
states between the tropical and temperate races.
It is of interest to note that as is well known in Bombyx, the diapause
state in the temperate uni- and bivoltine races can be highly changed after
artificial treatment of eggs (or embryos) under short daylength or complete

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darkness and low temperature incubation regimes for several days during
the latter half of embryonic development, contrary to environmental (orphotoperiodic) conditions which spontaneously induce diapause eggs from
nondiapause ones during seasomal changes in Japan. This fact clearly
indicates the basic mechanism concerned in the temperate races is unlikethat for tropical races. In this respect, egg incubation conditions of shortdaylength and low temerature or a combination of both are regarded asdiapause preventing stimuli by Lees (1955). It is necessary to select biological materials (or strains) for studies on the diapause phenomenon as
an arresting agent or factor of embryogenesis in Bombyx.
An example of chemical modifications in the diapausing state of thetemperate race compared to nondiapausing ones is that when one day old
eggs are immersed in acidious solutions including hot dilute HCl for a few
minutes, they turn into almost complete nondiapausing embryos. Also,
dechorination of diapausing eggs alters their original state to that of nondiapausing eggs and embryonic development resumes immediately to someextent in in vitro artificial culture media as well as saline (e. g., Ohtsuki
et al., 1975). A similar phenomenon is also observed in spontaneously
occurring-parthenogenetic diapausing eggs (Murakami et al., 1987). In
addition, various methods including electricity spark, picking, etc., arealso to some extent effective in breaking the diapausing state and resumptionof embryonic development. From this evidence, it can be said that a slight
modulation of a certain controlling principle(s) for the determination of
diapause state is enough to completely change eggs state to an opposite
one.
In short, the nature of already genetically determined egg-state or voltinism
in any voltinistic races of the Bombyx silkworm is easily convertible to the
opposite egg-state regardless of in both natural and artificial conditions.
The Mutagenic Activity of a Carcinostatic Drug, Neocarzinostatin*,in Germ Cells of Bombyx Mor; L.
Akoi MURAKAMI

A carcinostatic agent, Neocarcinostatine (NC) [C462H7osN130015S54;
m.w. 10707.29] is a product of Streptomyces carzinostaticus var F-41 andis composed of 109 amino acids and two -5-5- bonds. This acid polypeptide* This drug was purchased from Yamanouchi Pharmaceutical Co., Tokyo.

RESEARCH ACTIVITIES IN 1989

67

drug with a high molecular weight induces gene mutation or DNA damage
in certain stocks of E. coli (WP2' etc.) and an unscheduled DNA synthesis
in human leucocytes. There has been no report as to mutagenic effectsof NC on germ cells of higher organisms. A preliminary investigation
was carried out to determine whether NC has genetic toxicity for germ
cells of treated silkworm (B. mori). Three different doses, 2, 4 and 6 ng per
capita of NC dissolved in 0.025 ml saline were injected into the body cavityof 8-day-old F 1 hybrid pupae from a cross between wild-type strain CI08
females and Aojuku males. The germ cells at this stage of Bombyx pupae
are mature sperm and prophase I oocytes for males and females, respectively.
The incidence of mutations was determined by the specfic locus method
using egg-color genes, pe (5-0.0) and re (5-31.7).
The results obtained indicate that in pupal mature sperm none of dosesof NC induced specific locus mutation, either whole-body (complete) or
fractional-body (mosaic) type, while in prophase I oocytes similar dosesof NC were found to induce a significant oncrease in the number of mutantsin relationship to dosage. Most mutants detected were fractional-body
types, while the incidence of whole-body type was somewhat higher thanthat of the corresponding control.
The mutagenic effect of NC in Bombyx pupae appears to be the same asthat of acridine compounds (Murakami, 1972, '73), suggesting that themechanism by which acridine compounds induce recessive visible mutations
could be, in part, applied to that of NC: both prophase I oocytes and mitotic
cleavage nuclei are susceptible to NC mutagenesis, but mature sperm withcondensed and/or paracrystallized genetic materials are not. This cellulardifference in NC mutagenesis may be due to differential DNA conformations in germ cells rather tahn sex differential metabolic activity in the
pupal body including gonad, between sexes. Nevertheless, it should berecognized that pupal prophase I oocytes in vitellogenesis prior to thecompletion of chorion formation are apt to incorporate the drug into thecells in contrast to mature sperm, regardless of metabolic activity.
The preponderance of mosaic mutations in NC-treated female pupae
may be interpreted as an indication that the accumulated NC in oocytes.
may act as a mutagen on mitotic cleavage (DNA replication) nuclei as
observed in mutagenesis with acridine compounds. A possible explanation
for the occurrence of a small number of complete mutations in female
pupae is that this type of mutant may be caused by two independent mu-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

tagenic events of NC on the zygotic cell as well as a dingle event on theovum. Of course, other mechanisms (such as an induced recombinationlike event between paired homologous DNA) cannot be excluded as possibilities.
In the present experiment, high molecular weight NC or its metabolite(s)were only mutagenic for oocytes treated during vitellogenesis before thecompletion of chorion formation in the female pupae. Hence it islikely that a certain physical interaction with DNA bases such as an intercalation is a necessary, but not a sufficient condition, for NC mutagenesis.
At any rate, there is a possibility that a carcinostatic drug, NC, is able toinduce recessive visible mutations in Bombyx germ cells.
Egg Permeability and Development of Embryo in Drosophila
Kiyoshi MINATO

In general, it has been shown that insect eggs, even when dechorionated,
are impermeable to aqueous solutions. Limbourg and Zalokar ('73)
reported the existence to some extent of permeability (uptake of uridine)
only in freshly laid eggs (within 2hr at 25°C) in Drosophila, and made theeggs at various stages permeable to aqueous solution with octane treatment.
Those eggs became very sensitive to the quality of incubation media, andtherefore died early or developed abonrmally when incubated in inadequately prepared aqueous media. However, we found that freshly laid eggs
themselves are also permeable enough without treatment and die or develop
abnormally when incubated in aqueous media.
In preliminary experiments in which we searched for better incubation
conditions for microscopical observation of eggs in Drosophila melanogaster,
we sometimes got many un-hatched eggs when eggs were dechorionatedand incubated in aqueous media. But if these eggs were incubated in
non-aqueous media such as paraffin oil, this situtation was never seen.
Moreover, in aqueous incubation, it is found that the younger the used
eggs were, the more un-hatched eggs were produced. When the eggs older
than the syncytial blastoderm stage at the start of incubation were used,
they developed normally and few un-hatched eggs were seen.
The development of freshly laid eggs incubated in aqueous media wereobserved. Eggs incubated in distilled water soon became opaque, showing
no signs of development such as nuclear cleavage or migration to the egg

RESEARCH ACTIVITIES IN 1989

69

surface. After several hours there were swelling possibly due to the hypotonicity of the medium. Incubated in 0.9 % NaCI, eggs developed toa later stage and formed cellular blastoderms in limited areas of the eggs
(mostly at the posterior third to half), which, after several hours, caused
teratogenic embryos an opaque and amorphous area in the anterior anda more transparent bag-like structure made of cell layers in the posterior.
These embryos somewhat shrank and hence detached from the vitelline
membrane at the anterior and posterior ends. When incubated in 0.75 %NaCl, embryos showed the least degree of developmental anomaly: eggs
sometimes developed nearly to the sac-like midgut stage though they had
poorly developed organs, and they moved under the vitelline membrane
though they were rather small compared to the normal embryos and neverhatched.
Thus, the extent and features of developmental abnormalities observed
above appeared to depend upon the tonicity of the media used (hence, 0.75%
NaCI seems to be nearly isotonic). This relationship suggests that freshlylaid eggs of Drosophila are permeable enough at least for water to die earlyor develop abormally when incubated in aqueous media except in carefully
prepared media.
Axial and Lateral Head Formation in Hydra
Hiroshi SHIMIZU, Tsutomu SUGIYAMA and Yasuji SAWADA

Axial and lateral head formation in hydra was investigated. Hydra
produces two basic types of head-like structures. One is regeneration ofa new head after removal of the original head. This type of head formation
occurs at the most apical end and in the same direction as the apico-basal
axis of the original animal. The other is bud formation in an asexually
growing animal. This type of head formation occurs in the budding zone
and in a direction which is perpendicular to the axis of the parental polyp.
The relatonships btween these two types of head structure formation were
examined using the "mirror-image" grafting procedure described in thisreport last year.
The grafting was done as follows; Head was removed from polyps by
amputation with a sharp surgical knife. A nylon fishline was then pushed
through the gastric cavity of two decapitated polyps in such a manner as
to make the wound opening of the two polyps face and cover each other.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

The fisWine was removed after 3-5 hours, and the two polyps grafted together
symmetrically in a mirror-image fashion by this operation were then kept
under normal culture conditions for seven days.
Grafted polyps produced two different types of structures at the graftjunction. One was a ring of tentacles which formed around the circumference of the graft junction. These grafted polyps later separated fromeach other in some cases, producing two independent normal regenerates
eventually. Thus, tentacle ring formation can be regarded as a variationof normal head regeneration caused by the grafting.
The other type of structure formed was a "lateral head". Initially asmall protrusion appeared at one (occasionally two) point of the circumference of the graft junction. It grew in size gradually, and formed ahypostome and tentacles around it at the tip. This process was similar
to normal budding, except for one important aspect. A normal bud forms
a foot and detaches from the parent, whereas a lateral head forms no footand remains attached to the graft.
The ratio of grafts forming a tentacle ring and a lateral head varied
greatly depending on the sites of amputation and grafting on the original
polyps. As the site moved from a more apical to a more distal position,
the ratio of tentacle ring formation decreased, whereas the ration of lateralhead formation increased.
These observations show that the capacity to form a tentacle ring (headregeneration) forms a gradient from head to foot, whereas the capacity toform a lateral head (budding) forms a gradient in the opposite direction.
This suggests that the two head-type structures (head regeneration andbudding) are regulated by two different, but presumably closely related,mechanisms.
Attempts are currently being made to formulate a new theoretical modelfor hydra pattern formation. In the new model, nonlinear elements ofchemical reaction-diffusion (Gierer and Meinhardt, 1972) and non-linear
elements of "active boundary" produced by injury effect (MacWilliams,
1982) will be integrated to simultaneously account for axial and lateralhead formation in hydra.

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71

Two Way Controls of Interstitial Stem Cell
Proliferation in Hydra
Toshitaka FUJISAWA

Interstitial cells (I-cells) in hydra are undifferentiated multipotent stem
cells which proliferate to maintain their own population as well as differentiate 2 types of nerve cells and 4 types of nematocytes under normal growth
conditions. When the I-cell level is lowered artificially (e. g. by hydroxyurea treatment), I-cells grow faster at the beginning by increasing the selfrenewal probability but later grow slowly, eventually attaining the normal
growth rate (Bode, et al., 1976). These results indicate that there is ahomeostatic control mechanism for maintaining the I-cell level.
In the present study, I examined control mechanisms involved in the
homeostasis of hydra stem cells. The methods employed were to introduce
a small number of I-cells into an epithelial host which lacked all the cell
types in an I-cell lineage (stem cells, their differentiation intermediates andproducts; nerves and nematocytes). First, the distal half of an epithelial
polyp and the proximal half of a normal polyp were axially grafted allowing
I-cells to migrate from proximal to distal regions. After 24 hr, the distal
half was isolated to regenerate. During the first 3 days of regeneration,
essentially no migrated I-cells increased in number. Instead they produceda large number of nerve cells. The number of I-cells started to increase
rapidly thereafter. When epithelial and normal tissues were kept in contact,.
I-cells increased continuously in number (note that I-cell migration occurs
only during the first 24 hr and then ceases). These results suggest thatsome component necessary for I-cell growth is missing in epithelial tissue
and that the component is a product of nerve cells which either directly
affect I-cell proliferation or negatively control nerve cell differentiation,
thus indirectly affecting the rate of I-cell proliferation. Second, a small
piece of normal tissue was laterally transplanted to an epithelial polyp.
By doing so, an animal with a small number of I-cells and nerve cells (andsome other cell types as well) was created. Under these conditions, I-cellsgrew faster during the first 10 days by increasing the self-renewal probability,
but later the growth rate leveled off, reaching a normal rate. These results
together with those described above indicate that two different mechanismsare involved in the homeostatic control of I-cells. (1) When enough nerve
cells are present, the rate of I-cell growth depends on the I-cell density.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Namely, a negative feedback signal emanates from I-cells themselves tocontrol the growth rate of I-cells. (2) In the absence of nerve cells, theself-renewal probability of I-cells decreases, thus favoring nerve cells differentiation, since the signal necessary for I-cell growth or a negative feedback signal for nerve cell differentiation is missing.
Purification and Structure Analysis of an Autocrine Growth Factor in
Conditioned Medium Obtained from Primary Cultures of Scleral
Fibroblasts of the Chick Embryo
Miki FUJIOKA, Yoshie OHIYA, Kazuo WATANABE
and Nobuo SHIMAMOTO

The time course of cell proliferation and cell differentiation may be
determined by environment 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 the firststep to check this hypothesis, we purified a related growth factor.
We found that the cells secrete autocrine growth factors. One factor
was purified from conditioned medium collected from growing-phasecultures by DEAE-Sepharose column chromatography and non-denaturing
polyacrylamide gel electrophoresis. The specific activity was increased
1,100-fold through purification. The purificed growth factor was heatstable, and resistant to acidic or basic treatment. Removal of the glycosidechain did not alter activity but reduction by dithiothreitol or digestion
by trysin hampered activity. These characteristics are similar to those of
the plateletderived growth factor (PDGF) but its molecular weight, 32 kd,
is much larger than that of PDGF. Thus this factor might be form a new
family of growth factors. The primary sequence was determined for 20
amino acids at N terminal and the cDNA is now being screened.
Assembly of Connectin (Titin) in Relation to Myosin anda-Actinin in Cultured Cardiac Myocytes
Yutaka SHIMADA

By using polyclonal and monoclonal antibodies against connectin (titin)which stains the A-I junctional area and the A band domain (polyclonal

RESEARCH ACTIVITIES IN 1989

73

anti-connectin and monoclonal 4C9) and the I band domain (monoclonal
SM I), the developmental relationship of this elastic protein with sarcomeric
proteins, especially myosin and a-actinin, was examined in embryonicchick cardiac myocytes in vitro under fluorescence microscopy. During
premyofibril stages, I-Z-I proteins were detected first (a-actinin dots anddiffuse actin [phalloidin and anti-troponin CJ staining), and later in theseareas connectin and myosin dots appeared with nearly identical distribution.
Somewhat later, phalloidin-positive nonstriated fibrils were observed in astraight course. They were aleays reactive with antibodies against a-actinin
and troponin C, but unreactive or only weakly reactive with anti-connectin
and anti-myosin. Initally, a-actinin dots were aligned these fibrils but
did not form striations. As they aggregated to form Z bands, connectin
and myosin came to exhibit typical striations ("doublets" and A bands,
respectively). No difference in the staining pattern was observed with
two hinds of monoclonal antibodies against different domains of connectinfilaments (4C9 and SM1) at early phases. As myosin staining showed
clear A bands, connectin epitopes became arranged in polarized positions.
We conclude that primitive I-Z-I complexes appear prior to the assemblyof connectin and myosin filaments, and then connectin filaments developing
intimateyl and corrdinately with myosin become associated with the aactinin lines. Thus, it appears that the putative elastic protein connectin,
plays some role in integrating myosin filaments with the preexisting I-Z-I
brushes. The occasional absence of connectin and the absence of A bands
between the two Z bands, beyond both of which clear sarcomeres havebeen formed, indicate that connectin is not a preformed scaffold for myofibrils, at which sarcomeric proteins accumulate.
Translational Control of Troponin C Gene Expression in
Chicken Skeletal Muscles
Yutaka SHIMADA

The expression of the cardiac/slow troponin C (C/STnC) gene in developcardiac and skeletal muscles was analyzed with cDNA probes and an antibody specific for C/STnC. Northern blot, Sl nuclease protection assay
and Western blot analyses showed that both the C/STnC mRNA and the
C/STnC proteins were expressed in the ventriclar muscle of specimensfrom embryo to the adult. However, in the breast muscle, although C/

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

STnC mRNA and the C/STnC protein were present in the embryo, thesynthesis of the CjSTnC protein decreased as development proceeded. The
C/STnC protein was not found at detectable levels in the adult breast muscle
in spite of the presence of C/STnC mRNA. In vitro translation analysis
indicated that C/STnC mRNA transcribed in the adult breast muscle wastranslatable. These results suggest that functional C/STnC mRNA wastranslationally inactive in the adult breast muscle and that the expressionof CjSTnC gene was controlled at the level of translation in the adult breast
muscle. Thus, it appears that translational control plays an importantrole in tissue-specific and developmentally regulated isoform changes introponin C.

RESEARCH ACTIVITIES IN 1989

v.

75

CYTOGENETICS

Effect of H-2 Complex on Morphological Abnormality of
Mouse Spermatozoa
Kazuo MORIWAKI and Akihiko MITA

Genes governing the morphological characteristics of sperm head seemto be located on several chromosomes including No. 17 and Y in mouse.
The possible relevance of the H-2 complex on chromosome 17 to the morphological abnormality of spermatozoa has already been demonstrated(Suh, Styrna & Moriwaki. Genet. Res. Camb., 53: 17, 1989). To confirm
the previous findings and to locate more precisely such a gene in the H-2
complex, the frequency of spermatozoa with abnormal head shape wasscorted in 43 BIO.H-2 congenic strains. The precentage of head abnormality ranges from 1% to 50 %. Thirty three strains including BIO, BIO.A,
BlO.A (2R), BIO.A (3R), BIO.A (4R), BIO.AKM, BlO.BR, BIO.D2, BIO.G,
BIO.HTT, BIO.PL (73NS), BIO.RIII (7INS), BIO.S, BIO.MOL-ANJ, BIO.
MOL-MSM, BIO.MOL-OHM, BIO.MOL-OKB, BIO.MOL-SGR, BIO.
MOL-TEN2, and BIO.M-YNG, exhibited the abnormality at frequencies
between 5 % and 30 %. Significantly higher frequencies in the abnormality
were observed in the following 3 strains: 30.3 % in BIO.A (5R), 50.5 % in
BIO.M and 32.2% in BIO.MOL-TEN!. On the other hand, lower frequencies ofless than 5 % were observed in BIO.DA (80NS), BIO.GD, BIO.Y,
BIO.HTG and BIO.Po.
As far as the present survey is concerned, those strains carrying the dor q haplotype in the H-2K gene show relatively lower frequency of sperm
abnormality. Both of the strains with H-2Kf exhibited considerably ahigher frequency of abnormal sperm. Results obtained from BIO.A (3R)
and BlO.A (5R) are something puzzling, since both strains have almost thesame genic constitution in the H-2 complex and in their genetic backgroundas well. Further analyses of the origin of their Y chromosomes and theBIO genetic background is necessary.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

A New Variant of the Mouse .a-globin Gene, Hbbq , inNorthwestern China
Tsuyoshi KAWASHIMA Nobumoto MIYASHITA, Toshihiko SHIROISHI,
Wang Chong YAN*, Wang Cheng HUAI* and Kazuo MORIWAKIWe studied the allelic frequency of the hemoglobin beta chain gene (Hbb)of wild mice, Mus musculus, mostly in Asia. The wild populations in thenorthern part of China, Korea and the central part of Japan exhibited analmost monomorphic distribution of Hbb p • The southern part of Asia
shows a lower frequency of Hbb p and a predominant distribution of Hbb d(Minezawa et al., 1979; Miyashita et al., 1985). In the process of the surveyof Hbb polymorphism in China, we found another allele of Hbb, named
Hbb q • It is distributed in a rather wider area in northwestern China.
From the electrophoretic pattern on cellulose acetate membrane, Hbb q
was assumed to be an intragenic recombinat between Hbb and Hbb p •But recent analysis on polyacrylamide gel electrophoresis suggests thepossibility that the Hbb q is a completely new allele at both the bI and b2
loci. Molecular analysis of bI and b2 genes in Hbb q is now in progressusing southernblotting with Hbb DNA probes.
B

In vitro Development of Preimplantation Embryos Derived fromInter Subspecific Hybrids between Mus museu/us m%ssinus
and M. m. domesticus
Michiko NIWA, Noboru WAKASUGI** and Kazuo MORIWAKI

Japanese wild mice, Mus musculus molossinus, are genetically remotefrom laboratory mice which are derived predominantly from European
wild mice M. m. domesticus. Developmental abnormality was observedin preimplantation embryos from F2 hybrids between the MOM strain(one of the inbred strain derived from Japanese wild mice) and the C57BL/6
strain (B6; an inbred strain of laboratory mice) (Niwa, M. and Wakasugi,
N. 1990, Zool. Sci. 7: 209). We examined the in vitro development ofpreimplantation embryos from hybrids between C3H/He (C3H) and M.
MOL-MSM (MSM) and between SK/Cam (SK) and MSM. C3H is alaboratory inbred strain. SK and MSM strains are inbred strains derived* Lanzhou Institute of Biological Products, China.

** Faculty of Agriculture, Nagoya University.

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Table 1. Developmental ability of embryos obtained on Day 1 of pregnancy
crosses~xc3'

C3HxC3H

No. ofNo. of embryos thatembryosa)
develoed tocollected
blastocysts during
examined (means± sem)
4 days in culture
No. of~~7

C3HxMSM

8

MSMxMSM

16

MSMxC3H(C3H x MSM)Fl x cm
(C3H x MSM)F2 x cm

1
1
18

C3H x (C3H x MSM)F2

9

SKxSK

6

MSMxMSM

16(SKxMSM)Fl xSK

8SK x MSM)Fl x MSM

7(SK x MSM)F2 x SK

14

(SK x MSM)F2 x MSM

20

BlO x (SK x MSM)F2

9(B6 x C3H)Fl x cm

9

(B6 x C3H)F2 x C3H

11(B6 x Cm)F2 x B61060(8.6±1.0)69(8.6±0.5)76(4.8±0.2)59124(6.9±0.5)89(9.9±0.9)45(9.8±0.7)76(4.8±0.2)41(5.1±0.5)30(4.3±0.7)75(5.4±0.6)114(5.7±0.4)78(8.7±0.4)89(9.9±0.9)
112(10.2±1.0)80(8.0±0.7)

57 ( 95.0%)
68 ( 98.6)
75 ( 98.7)
5 (100.0)
9 (100.0)
105 ( 84.7)
86 ( 96.9)
14 ( 31.1)
75 ( 98.7)
39 ( 95.1)
30 (100.0)
53 ( 70.7)
80 ( 70.2)
70 ( 89.7)
89 (100.0)111(99.1)
75 ( 93.8)a) Morphologically normal 2- to 6-cell embryos were counted

from M. m. domesticus wild mice and Japanese wild mice, respectively.
We also observed the embryos of hybrids between B6 and C3H as a control.
The results are shown in table 1. The SK strain showed a low level ofdevelopmental ability in preimplantation embryos. Fl hybrids from all
crosses showed a high embryonic survival rate. A very low level of abnormality was observed in embryos sired by F2 males. On the contrary, some

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

embryos from F2 females between C3H and MSM (15.3 %) and between
SK and MSM (29.8 %) died before the blastocyst stage. These F2 females
were divided into two types on the basis of the proportion of embryosthat developed to blastocysts: one type showing a higher rate of survival
embryos and the other a lower rate. Almost all embryos derived froin
F2 females between B6 and C3H developed to blastocysts. These results
suggest that embryonic mortality probably occurred during the preimplantation development of F2 females between M. m. molossinus and M. m. domesticus and that the abnormality rate depends on the strain combinationin genetic crosses.
Studies of Adrenal Function in Mouse Development by Transgene.
Hideo GOTOH Toshihiko SHIROISH and Kazuo MORIWAKIA chromosome of H-2 awl8 haplotype has a deletion in the H-2 class
III region spanning both the gene for fourth component of complement (C4)and the gene for the active form of steroid 21-hydroxylase (21-0Hase).
Because of the lack of 21-0Hase activity, awl8 homozygous mice are unable
to synthesize any adrenal steroids, neither glucocorticoids nor mineralocorticoids. So far, we have found several developmental abnormalities
associated with the awl8 haplotype.
(1) Neonatal lethality: Homozygotes for the awl8 haplotype die within
two weeks after birth. Administration of adrenal homogenate to supplement adrenal steroids rescued the awl8 homozygote. The cause of thelethality is assumed to be the defect in adrenal steroid synthesis.
(2) Adrenal organogenesis: Histological abnormalities were observed inwhole adrenal glands of awl8 homozygotes. These abnormalities are
known to arise at the mid-gestation period, when medurally cells migrate
into cortical anlage. The cell-to-cell interaction during adrenal organogenesis seems to be abnormal in these mice.
(3) Embryonic malformation: In fetuses from intercrosses of awl8/+
heterozygotes, several kinds of severe-type malformations were observedwith high frequency.
We are now introducing a genomic DNA clone of murine 21-0Hase
gene into the awl8 mice. If the exogenous gene is experssed in adrenal
glands in vivo, the awl8 homozygotes will be rescued. These transgenic
mice will become powerful tools for studying unknown functions of adrenal

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79

steroids in mouse development.
Antigenic Polymorphism of a Serum Protein Found in a Strain
Derived from Indonesian Wild Mice (Mus musculus castaneus)
Yoshi-nobu HARADA, Cece SUMANTRI·, Takeshi TOMITA·
and Kazuo MORIWAKI

M. Cas-Bgr 1 is a strain derived from Indonesian wild mice which havebeen classified into one subspecies, Mus musculus castaneus. Antigenic
specificities of the M. Cas-Bgr 1 strain were studied by gel precipitation
analyses with alloantisera produced by reciprocal alloimmunizations with
the BALB/c strain. A specific precipitation line, which migrated between
and regions of serum globulins, was observed by immunoelectrophoretic
analysis with BALB/c anti-M. Cas-Bgr 1 serum. This precipitation line
must be a serum protein whose antigenic polymorphism has not beenreported previously. Hereafter, the serum protein will be called alphaprotein-3A (phenotype symbol: APH-3A). None of the M. Cas-Bgr 1
mice immunized with BALB/c serum produced a specific antiserum thatgave clear precipitation lines on immunoelectrophoretic analysis. A putative counterpart for APH-3A was not observed in BALB/c serum.
A genetic analysis of APH-3A was carried out on F l , F 2 and backcrossprogeny produced by crosses of M. Cas-Bgr 1 and BALB/c strains. The
results suggested that APH-3A was controlled by an autosomal dominantgene. Therefore, the locus controlling the APH-3A was tentatively called
Table 1. List of mouse strains used for serological survey of
APH-3A in this study.
APH-3A-positive
APH-3A-negative•

M. Cas-Bgr 1
A/WySnJ, AKR/J, BALB/c, BALB/cAnN, CBA/Ms, CBA/J
CBA/CaHN, CE/J, C3H/He, C57BL/6J, C57BL/I0J,
C57L/J, C58L/J, CS, DBA/lJ, DBA/2J, DCR/c, DDK,
DDN, DM, DNI, HTG, HTH, HTI, HRS/J, ITES,
IIITES, IQI, IVC-E, IXBL, KR, MA/MyJ, NC, OZA,OZB, Ozc, Ozd, OZF, P/J, PONY, RFM, RIIIs/J,
SIIIB, SJL/J, SM/J, SWR/J, TF/GnLe, WB/ReJ-W, ZCA,
129/Sv, 129/J

Faculty of Agriculture, Nagoya University.

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alphaprotein-3 (gene symbol: Aph-3) and alleles for APH-3A-positive andAPH-3A-negative were named Aph-3a and Aph-3b, respectively.

A serological survey of APH-3A was conducted on 51 laboratory strains
but no strain including BALB/c had the APH-3A.
DNA Sequeuce Comparison in the D-Ioop Region of Mitochondrial DNA
within and Among Subspecies of the Species Mus musculus
Hiromichi YONEKAWA, 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 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, we carried out two lines of research;an extensive survey of wild mice in neighboring countries of Japan, especiallyChina, using restriction enzymes with which we could identify subspecies,
and the development of a new efficient method for base sequence comparison of intersting region(s) on mtDNA. The objective of the former
research was to clarify subspecies boundaries between M. m. musculus andM. m. castaneus in Japan and China. The latter's objective was to confirm
whether there are subspecies-specific base substitutions on the mtDNA
sequences. If there are, we can examine more precisely the molecular
evolution of mtDNA during the subspeciation of Mus musculus.
For the second objective, we sequenced four entire mtDNA by collaboration in Dr. K. Fischer Lindahl of Howard Hughes Medical Institute in
Dallas (Fischer Lindahl et al. (1989) Cold Spring Harbor Symp. Quant.
BioI. 54: 563). In comparing these four mtDNA sequences, we foundthat there are at least three highly polymorphic regions in mouse mtDNA;
in particular the replication origin of heavy stand (D-Ioop region) is thehighest. Based on this result, we selected this region as the first target ofsequence comparison, since we can expect the greatest opportunities offinding base substitutions in this region when we survey the mouse subspecies with monomorphic mtDNA. To do so, we introducted the polymerase chain reaction (PCR) method into the research. To carry out
DNA sequencing experiments efficiently, we made six sequencing primers
which have common sequences among Mus musculus, Rattus norvegicus
and Rattus rattus. We have finished seven sequences of the D-Ioop region

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81

so far and found there are two clusters of highly polymorphic sites in theregion. We also found that some substituted bases are shared by micebelonging to the same subspecies, and thus they look like subspecies-specificsubstitutions, suggesting that we can identify subspecies by checking thesesubstitutions. The D-Ioop region seems to have higher frequencies oftransversions and insertions/additions than other regions.

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VI.

MUTAGENESIS AND RADIATION GENETICS

Genetic Analysis of Pulmonary Adenoma Susceptibility (Pas) in Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIWe estimated the number of genes which affect urethane-induced pulmonary adenoma in crosses between adenoma-sensitive AjWySnJ andresistant BlO.AjSgSnJ strains of mice. As these strains carry an identical
H-2 haplotype (H-2 a ), the effect of the H-2 complex on induction of pulmonary adenoma can be disregarded. The mice were sacrificed in the 5thmonth after a single subcutaneous injection of 1.5 mgjg urethanejg bodyweight. After the lungs were fixed in ethanoljformaldehyde (9: 1), thenumber of adenoma foci was assessed.
The BlO.A strain developed a lower multiplicity of pulmonary adenomas,
1.70±O.23jmouse. In contrast, the AjWy strain developed a greater
multiplicity, 27.15±1.02jmouse. (AjWyx BIO.A)Fl mice exhibitedtumors similar to AjWy parents. The mean number of tumors averaged
25.23±O.79jmouse. In a backcross with BlO.A, a distinct bimodal distribution was shown and the numbers of animals in resistant (n=54) andsensitive (n=42) groups were nearly the same. This result shows that thedifference between those strains was due to a single locus (Pas-i) withsusceptibility dominant. We studied the linkage of this putative Pas-i
locus with 13 polymorphic loci between the parental strains. No significant
linkage was detected.

RESEARCH ACTIVITIES IN 1989

VII.

83

POPULATION GENETICS

Role of Gene Duplication in Evolution
Tomoko OHTA

It is now known that many multigene and supergene families exist in
eukaryote genomes: multigene families with uniform copy members, likegenes for ribosomal RNA, those with variable members like immunoglobulin
genes, and supergene families such as those for various growth factors andhormone receptors. Many such examples indicate that gene duplicationand subsequent differentiation are extremely important in organismal
evolution. In particular, gene duplication could well have been the primarymechanism for the evolution of complexity in higher organisms. Population genetic models for the origin of gene families with diverse functions.
are presented, in which natural selection favors those genomes with more
useful mutants in duplicated genes. Since any gene has a certain probabilityof degenerating through mutation, success versus failure in acquiring a
new gene by duplication may be expressed as the ratio of probabilities of
spreading of useful versus detrimental mutations in redundant gene copies.
Also examined are the effects of gene duplication on evolution by compensatory advantageous mutations. Results of the analyses show thatboth natural selection and random drift are important in the origin of genefamilies. In addition, interactions between molecular mechanisms suchas unequal crossingover and gene conversion, and selection or drift havebeen found to have a large effect on evolution by gene duplication. For
details, see Genome 31, 304-310.
The Mutational Load of a Multigene Family with
Uniform Members
Tomoko OHTA

The mutational load of a multigene family with uniform members wasstudied through computer simulations. Two models of selection, truncationand exponential fitness, were examined, using a simple model of gene conversion. It was found that the load is much smaller than the Haldane-

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Muller prediction under truncation selection, and that it becomes approximately equal to the value calculated by the formula, nv (l-q)/(m-nq), where
n is the copy number, v is the rate of detrimental mutation per gene copy,
m is the truncation point in terms of the number of detrimental genes eliminated, and q is the equilibrium frequency of detrimental mutation. However equilibrium frequency could not be analytically obtained. For the
exponential fitness model, the load is close to the Haldane-Muller value.
When there is no gene conversion, the load becomes larger than in caseswith conversion both for the truncation and the exponential fitness models.
Thus, gene conversion or other mechanisms that are responsible for thecontraction-expansion of mutants on chromosomes, help in eliminating
deleterious mutations occurring in multigene families. For details, see
Genet. Res. 53, 141-145.
Time for Spreading of Compensatory Mutations
under Gene Duplication
Tomoko OHTA

Evolution by compensatory mutations is accelerated by gene duplication
because selective constraint is relaxed by gene redundancy. A mutation
is called compensatory if it corrects the effect of an earlier deleterious
mutation. Without duplication, Kimura has shown that the time for
spreading of compensatory mutations is greatly reduced by the tight linkage
between the two chromosomal sites of mutations. In this report, the time
for spreading with gene duplication was studied using the diffusion equation
method of Kimura, together with computer simulations. It was shown
that, when 2Nv_ is much less than unity, the time for spreading is greatly
shortened by gene duplication as compared with the case in which there iscomplete linkage between the two sites of mutations, where 2N is the effective
population size (haploid) and v_ is the rate of compensatory mutations.
However, if 2Nv_ > 1, gene duplication is not effective for accelerating
evolution by such mutations. For details, see Genetics 123, 579-584.

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Gene Genealogy in Three Related Populations: Consistency
Probability between Gene and Population Trees
NaoyukiTAKAHATA

A genealogical relationship among genes at a locus (gene tree) sampledfrom three related populations was examined with special reference topopulation relatedness (population tree). A phylogenetically informative
event in a gene tree constructed from nucleotide differences consists ofinterspecific coalescences of genes in each of which two genes sampled fromdifferent populations are descended from a common ancestor. The consistency probability between gene and population trees in which they are
topologically identical was formulated in terms of interspecific coalescences.
It was found that the consistency probability thus derived substantially
increases as the sample size of genes increases, unless the divergence timeof populations is very long compared to population size. Hence, there arecases where large samples at a locus are very useful in inferring a population
tree. (See Genetics 122: 957-966, 1989 for details.)
Stochastic Models for Molecular Clocks
Naoyuki TAKAHATA

Compared with the rate of evolution at the phenotypic level, the rate ofmolecular evolution is fairly constant in time as well as among diverse
lineages. The most commonly used statistical model to describe such
constancy (molecular clock) is a simple Poisson process in which the varianceof the number of amino acid or nucleotide substitutions in a particulargene should be equal to the mean and henceforth the dispersion index, theratio of the variance to the mean, should be equal to one. Recent sequence
data have shown that the rate of molecular evolution is considerably overdispersed which has called into question the validity of using a simple
Poisson process. Several efforts have been made to develop more realistic
models of molecular evolution. Here I reviewed three such statisticalmodels based upon doubly stochastic, cluster, and renewal processes.
Although these models do not immediately specify what the mechanisms ofmolecular evolution might be, they do make qualitatively different predictions and give some insight into their inference. The main concern iswhether or not the neutral theory of molecular evolution can explain,

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without major modifications, highly overdispersed molecular clocks. (Seepp. 433-444 in Proceedings of the 47th Session, Vol L III (3) Bulletin of
the International Statistical Institute, Paris, 1989, FRANCE for details.)
A Population Genetic Model of Selection that Maintains
Specific Trinucleotides at a Specific Location
Hidenori T ACHIDA

The periodic appearance of specific trinucleotides along the DNA sequence
have been reported in chicken core DNA, and it has been suggested thatthe phenomenon is related to the supercoiling of DNA around nucleosomes.
A population genetic model was constructed in which selection is operating
to maintain specific trinucleotides at a specific location on the DNA sequence. Assuming low mutation rates, equilibrium probabilities of theappearances of respective trinucleotides were computed. Vague patterns
appeared if the product of the effective size and the selection coefficient
was 0.1-2.0. The genetic load and substitution rates were also computed.
When the model was applied to the chicken DNA data, the product of theeffective size and the selection coefficient was estimated to be 0.1-0.2. With
this intensity of selection, the substitution rate was hardly different fromthat in cases without selection. However, the genetic load became fairly
large. Considering the large number of times that DNA coils about nucleosomes, the number of trinucleotides sites must be very large, and thus
the total load might be too large. This suggests the existence of epistasis
among sites to reduce the total load if selection is responsible for this periodic
pattern observed in chicken core DNA. The details will be published in
J. Mol. Eval.
A Study on Fixation Probabilities in a Subdivided
Population with Varying Environments
Hidenori T ACHIDA

The probability with which a mutant gene appearing in a population
is ultimately fixed is an important quantity in evolutionary studies. Kimura
derived a general formula to compute the fixation probability in randommating populations (Kimura, M., 1964, J. Appl. Prabab. 1: 177-232).
This was extended by Maruyama to structured populations (Maruyama,

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87

T., 1970, Genet. Res. 15: 221-226). There it was shown that in haploidorganisms the fixation probability is the same as that in a panmictic population if selection is operating uniformly over subpopulations. In this study,
fixation probability was studied in a subdivided population in which selection
pressure was different from one subpopulation to another. A comparison
with the fixation probability in a corresponding panmictic population, in
which selection coefficients are the averages of those in subpopulations,was made. When selection was very weak, such that the product of theselection coefficient and the size of subpopulations were much less than one
or when the migration rate was very large, there was no difference between
the fixation probability in a subdivided population and that in a corresponding panmictic population. Next we considered the case in which the
migration rate is small and selection is not weak. In the case of two subpopulations, the fixation probability can be approximately computed if
the migration rate is very small. It was found that the fixation probability
in a subdivided population was larger than that in a corresponding panmictic
population if selection favored one allele in one subpopulation and favoredthe other in the other subpopulation. A simulation study is now in progress
to determine the validity of this approximation. Also cases with morethan two subpopulations will be studied using this approximation.
Theoretical Studies on DNA Polymorphism and Statistical
Method for Testing the Neutral Mutation Hypothesis
Fumio TAJIMA

A large amount of genetic variation at the DNA level is maintained innatural populations. The amount of genetic variation is determined bymutation, recombination, natural selection, random genetic drift, population
structure, and so on, and can be measured by the average number, 1<,of (pairwise) nucleotide differences and the number, S, of segregating sites
in a sample of DNA sequences.
When the amount of DNA polymorphism is determined solely by mutation and random genetic drift, the expectations of these numbers are givenby E(k)=M and E(S)=aM, where M=4Nv, N is the effective populationn-lsize, v is the mutation rate per generation per DNA sequence, a= .E (Iii),
i=land n is the sample size, so that we have the following relationship:

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E(k) = E(S)/a( 1)

When the population is panmictic and the population size is constant overa long period of time, we can test the neutral mutation hypothesis by examining whether or not Equation (1) holds. It can be shown that k tends
to be larger than S/a if natural selection which increases genetic variation,is operating, whereas k tends to be smaller than S/a if natural selection
which reduces genetic variation, is operating. I have developed a statisticalmethod for testing the neutral mutation hypothesis by using the relationshipbetween k and S. Applying this statistical method to DNA sequences sampled from natural populations of Drosophila melanogaster, it has beenshown that large deletions/insertions (longer than 100 pb), which may
be caused by transposable elements, are deleterious. For details, see
Genetics 123: 585-595.
When the population is not panmictic, the relationship between k and
S is quite complicated and Equation (1) does not necessarily hold. I
examined this relationship by using the two-subpopulation model andobtained the following results: (i) E(k) is independent of the migration
rate. (ii) E(S) is independent of the migration rate when two or three DNA
sequences are randomly sampled from the same subpopulation, but depends
on the migration rate when more than three DNA sequences are sampled.
(iii) The population subdivision can increase the amount of genetic variation
at the DNA level even in a subpopulation in some cases. For details, see
Genetics 123: 229-240.
The size of population often changes drastically, and the change in population size might affect the amount of genetic variation maintained in natural
populations. I studied the expected number, E(S), of segregating sites
and the expectation, E(k), of the average number of (pairwise) nucleotide
differences among DNA sequences sampled from a population which is
not in equilibrium. The results obtained indicate that, when the population
size has changed drastically, S is influenced by the size of the current population more strongly than k, while k is affected by the size of the original
population more sharply than S. The results also indicate that k is affectedby a population bottleneck more strongly than S. For details, see Genetics
121: 597-601.

RESEARCH ACTIVITIES IN 198989

The Neutral Theory of Molecular Evolution andthe World View of the Neutralists
Motoo KIMURA

The main tenet of the neutral theory is that the great majority of evolutionary changes at the molecular level are caused not by Darwinian
selection but by random fixation of selectively neutral (or very nearly neutral) alleles through random sampling drift under continued mutation pressure. The theory also asserts that the majority of protein and DNA
polymorphisms are selectively neutral, and that they are maintained in thespecies by mutational input balanced by random extinction rather thanby "balancing selection." The neutral theory is based on simple assumptions. This enabled us to develop mathematical theories (using the diffusion
equation method) that can treat these phenomena in quantitative terms
and that permit theory to be tested against actual observations. Although
the neutral theory has been severely criticized by the neo-Darwinian establishment, supporting evidence has accumulated over the last 20 years.
In particular, the recent burst of DNA sequence data has helped to strengthen the theory a great deal. I believe that the neutral theory triggered
reexamination of the traditional "synthetic theory of evolution."In this work, I review the present status of the neutral theory, including
discussions of such topics as "molecular evolutionary clock," very highevolutionary rates observed in RNA viruses, a deviant coding systemfound in Mycoplasm together, with the concept of mutation-driven neutral
evolution, and the origin of life. I also present a world view based on theconception of what I call "survival of the luckiest." For details, see Genome
31: 24-31.
A Shifting Balance Type Model for the Origin ofCultural Transmission
Kenichi AOKIMost selectively advantageous traits that are culturally transmitted
probably cannot be genetically determined. On the other hand, the capacity
for culture is undoubtedly a genetically determined trait, which may have
evolved because of associated advantages. Conditions for the spreadof a gene for cultural transmission based on a haploid sexual model of

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this hypothesis. Two genotypes, communicators and non-communicators,
were assumed, and communicators were further distinguished by thepresence or absence of the trait. Only cultural transmission from parent tochild was considered. In a large panmictic population, initial spread of thegene for cultural transmission is facilitated if both mother and father contribute to the education of their offspring. If only the mother or father
does, a two-fold advantage is required for the culturally transmitted trait.
Cultural transmission may also evolve without unduly strong selection ina subdivided population, like that of some primates, where a shifting balance
type process can occur. By assuming that the groups synchronously
undergo reduction to the same finite size at a point in the life cycle, it ispossible to define probability distribution of the numbers of the three types
among groups. Numerical results obtained by iterating this probability
distribution revealed a dependence on group size and migration rate thatcan be interpreted in terms of the three phases of the shifting balance process.
The independent rediscovery rate, defined in analogy with the mutation
rate, was also shown to be an important parameter.

RESEARCH ACTIVITIES IN 1989

VIII.

91

EVOLUTIONARY GENETICS

Pattern of Nucleotide Substitutions and Its Implications for the
Immunological Diversity of Human Immunodeficiency Virus
Takashi GOJOBORI, Etsuko N. MORIYAMA and Nobuaki SHIMIZU

Human immunodeficiency virus (HIV) exhibits immunological hypervariability, which has been an obstacle to the successful production ofeffective anti-HIV vaccines. With the aim of finding characteristics ofthe mechanism which generates the immunological diversity of the envelope
protein, we estimated patterns of nucleotide and amino acid substitutions in
the env gene of HIVs. The results obtained showed that nucleotide changes
between A and G are predominant compared to those between other nucleotides. Since this feature is consistent with the pattern of nucleotide
substitution of other retroviral genes but is quite different from those of
most eukaryotic genes, a high rate of nucleotide substitution between A
and G appears to be specific for retroviruses including HIVs. Moreover,the nucleotide substitutions between A and G at the first and second positions of a codon tend to cause changes in hydrophilic amino acids much
more frequently than in hydrophobic amino acids. Since epitopes on
the env protein of HIVs consist mainly of hydrophilic amino acids, a highrate of nucleotide substitution between A and G can markedly alter theantigenicities of epitopes on the env protein without affecting the distributionof electric charges. These specific patterns of nucleotide substitutions
may play an important role in generating the immunological hypervariabilityof the env protein in HIVs. For details, see FEBS Letters 250: 591-595,
1989.
Evolutionary Relationships between Human and Simian
Immunodeficiency Viruses
Takashi GOJOBORI, Etsuko N. MORIYAMA, Hajime TSUJIMOTO
and Masanori HAYAMI

To elucidate the evolutionary origin of human and simian immunodeficiency viruses (HIVs and SIVs), we determined the nucleotide sequence of92

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the complete genome of a novel simian lentivirus, SIVmnd, isolated froma wild-caught mandrill in Africa (Gabon). We compared the nucleotide
sequence of the pol region (the regions of reverse transcriptase and endonuclease) of SIVmnd with those of other primate and ungulate lentiviruses.
Phylogenetic trees constructed, showed that HIVs can be classified into
two groups, HIV-I and HIV-2 groups. It also showed that two types of
SIVs (SIVmac isolated from rhesus macaques and SIV. m isolated from asooty mangabey) were closely related to the HIV-2 group. However,
SIVal\'m isolated from an African green monkey and SIVmnd were distantly
related to HIVs and form the third and fourth groups. The results indicatethat these four viral groups might have diverged from a common ancestor
at almost the same time before the spread of AIDS in humans. For details,
see Nature 341: 539-541, 1989.
Rates of Nucleotide Substitution for Drosophila Species
Etsuko N. MORIYAMA and Takashi GOJOBORI

To examine the relationship between evolutionary rates and generation
time, we estimated the rates of nucleotide substitution for Drosophila
species. We computed the numbers of synonymous (silent) substitutions
between the species compared, using the nucleotide sequences of 14 functional genes and a pseudogene for a total of 13 Drosophila species. The
results obtained showed that the rate of synonymous substitution for Drosophila is two to five times higher than those for mammals and 1.5 to threetimes higher than those for rodents. Such a high rate for Drosophila canbe explained by the shorter generation time of this organism. Furthermore,
the rate of synonymous substitution for Drosophila is estimated to be(10.8-20.2) X 10- 9 jsitejyear, which varies with the gene examined. In
particular, Adh, Hsp82, Ubx and Rp49 have rather high rates, while Xdh,
pcp, Gart, en, per, 3'ORF of Adh and beta-2 tubulin have rather low ones.
On the other hand, the rate of nucleotide substitution for the pseudogeneof Adh was estimated to be about two times higher than that of synonymous
substitution for the functional Adh. Therefore, the variability of evolutionary rates for Drosophila genes may be caused by a difference in the
degrees of functional constraints against synonymous substitutions. For
details, see Genetics 122: s41, 1989 and J. Mol. Evol. 28: 391-397, 1989.

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Evolutionary Origin of Kringle Structure in Apolipoprotein (a)K.lKEo, K. TAKAHASHI and T. GOJOBORI

A group of serine proteases is involved in the system of blood coagulation and fibrinolysis. These serine proteases have "kringle" domains,
each of which represents a conspicuous secondary structure formed bythree pairs of intrachain disulfide bonds. The number of kringle domains
varies with the type of proteins. Both urokinase and coagulation factor
XII contain one kringle. Tissue-type plasminogen activator and prothrombin have two kringle domains. Plasminogen has five. Recently,
38 kringle structures have been found in human apolipoprotein (a) (apo(a)),
which is a LDL-like plasma protein. To estimate the evolutionary originof these kringle structures, we constructed a phylogenetic tree using of all
the available sequences of kringle domains. This tree showed that theplasminogen might originally have duplicated to become its present form
and is the ancestral gene of apolipoprotein (a). This ancestral moleculeof apolipoprotein (a) may have consisted of the fourth and fifth plasminogentype kringles and a protease domain. Thereafter, the fourth plasminogentype kringle underwent multiple duplications, resulting in 38 kringles inhuman apolipoprotein (a). In particular, the latest duplication of the
kringle domains is considered to have occurred several million years ago.
For details, see Thromb Haemostas 62: 345.

Molecular Evolution of Hepadnaviruses
Yasuo INA, Etsuro ORITO*, Masashi MIZOKAMI*,
Etsuko N. MORIYAMA and Takashi GOJOBORI

Hepatitis B virus (HBV) is the etiological agent of a hepatitis that affects
an estimated 200 million people worldwide. HBV is a member of thehepadnavirus family that includes woodchuck hepatitis virus (WHV),.
ground squirrel hepatitis virus (GSHV) and duck hepatitis B virus (DHBV).
To examine whether the evolution of the hepadnavirus family is hostdependent, we constructed phylogenetic trees using 18 nucleotide sequences.
The trees obtained show that all 12 strains of HBV can be classified into
four subgroups that are not compatible with conventional (serological)* Second Department of Internal Medicine, Nagoya City University Medical School,
Nagoya.

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subtypes. We estimated the rate of synonymous (silent) substitution of
HBV for the P region to be 4.57 x 1O-~ per site per year. This value isabout 104 times higher than that of a host genome but 10- 2 that of theretroviral genes. Applying this rate to the phylogenetic tree, we estimatedthat DHBV diverged from a common ancestral virus about 30,000 years
ago, that WHY and aSHY diverged about 10,000 years ago, and that
HBV diverged within the last 3,000 years. Because these divergence timesof the viruses are much more recent than those of the host species, it suggeststhat the hepadnavirus family evolved independently of host-species divergence. For details, see Proc. Natl. Acad. Sci. USA 89: 7059-7062.
A Method for Molecular Phylogeny Construction by Direct Use of
Nucleotide Sequence Data
Yoshio TArENo

In estimating a molecular phylogenetic tree (or molecular tree), there
are several reasons for the view that nucleotide sequence and amino acidsequence data are more informative than a distance matrix which may
be obtained from the sequence data but not otherwise. First, consider acommon site in a set of nucleotide sequences of particular homologous
genes from various species in question. If the site is occupied by a seriesof nucleotides which consist of at least two different kinds with the sameone present at least two times, the site is called an "informative" site, whichmakes it possible to divide the series of nucleotides into two or more subsets.
In this sense, there are as many possible pieces of evolutionary information
as the number of the sites in a homologous gene. Second, a pattern ofevolutionary change of nucleotides can be hypothesized on the basis ofactual sequence data. For example, transition and transversion can be
distinguished from each other, and their ratio can be estimated. Third,such evolutionary changes as insertions and deletions can sometimes betaken into account when inferring the evolutionary relationships of species
or genes in question. Fourth, in the process of tree estimation, an evolutionary distance between a pair of nucleotide sequences can be estimatedby using any of the proposed methods whose basis for the nucleotide substitution pattern is in accordance with that of the pair. Those four points
also hold, to a great extent, for amino acid sequence data. One cannot
make use of any of the four significant features, if one is to estimate a mo-

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95

1ecu1ar tree from a distance matrix.
In 1981 Felsenstein developed the maximum likelihood (ML) method
to estimate a molecular tree from a set of nucleotide sequence data. Thismethod has become popular among molecular evolutionists, mainly because
its scientific background is endorsed by statistics. No other method hassuch a strong backbone. The ML method is, however, accompanied withat least four problems or drawbacks. First, there has been no guaranteethat a single maximal (not maximum) point uniquely exists in the parameter
range on the likelihood surface. If there are two or more maximal points
there, the ML method does not necessarily give the maximum likelihood
estimate. This problem has been partially solved by us (see our reportin the previous issue, pp. 95-96, No. 39, 1988). Second, as you might
think, "informative" sites sometimes conflict with one another, so thatthe true evolutionary relationships are smudged by the conflicting implications. This may be resolved by increasing the number of sites to be considered. Third, because the method depends heavily on a probabilistic
model, its performance may be too sensitive to make the method useful inpractice. The second and third problems will be discussed in the nextreport below. Fourth, the ML method takes uncompromisingly large
amounts of computer execution time until it produces the results. Thisis true even when only eight or so species or OTUs (operational taxonomic
units) are involved. There is so far no feasible way to relax this computational burden, which still restricts the method to a quite limited range
of uses.
It is known that the number of tree topologies increases steeply, as thenumber of OTUs gradually increases: the number is 34,459,425 for 10
OTUs and 654,729,075 for 11 OTUs, for example. If we consider this
together with the fact that nucleotide substitution in evolution is a stochastic
process, we realize that molecular tree estimation is a probabilistic work.
Thus it is not reasonable for most methods to give only one tree without any
statistical criterion.
Reflecting on such situations, I developed a molecular tree estimation
method. This method estimates a molecular tree and ancestral sequences
simultaneously from nucleotide sequence data on the basis of parsimony
and sequence homology. I named it the stepwise ancestral sequence(SAS) method. It produces plural trees for a set of nucleotide sequence
data in descending order of magnitude of parsimony. The final tree may

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be chosen from them by considering various lines of related evidence. I
think that biological evidence must take precedence over any others.
It is noted that there are two principles which govern molecular tree estimation; likelihood and parsimony. The two principles have been considered and treated to be mutually exclusive. Although a method developed onparsimony is statistically less endorsed than one on likelihood like Felsenstein's, the former takes much less execution time than the latter. This meansthat the former can estimate a molecular tree of 100 or more OTUs withina reasonable amount of computer time. The problem then is which is
better in estimating the correct tree in reality. Statistics does not necessarily
lead to reality or to the correct tree. This problem has not yet been solved
or worked out thoroughly, and remains to be studied in greater depth.
The properties and performance of the SAS method were then examinedthrough computer experiments, in which the evolution of a hypotheticalgene along a model molecular tree of six OTUs was simulated, and produced six descendant genes at the terminals of the tree. The SAS method
was then applied to the descendant genes to produce plural estimates tothe tree. This was repeated 100 times. The results show that the correcttree is found among the plural trees with a probability of about 90 %. It
is also shown that the method satisfactorily estimates the ancestral sequences.
Those results are of course model dependent, and interpreted as such.
For details, see J. Mol. Eval. 30: 85-93, 1990.
Robustness of Maximum Likelihood Tree Estimation
against Different Patterns of Base Substitutions
Kaoru FUKAMI* and Yoshio TATENO

As noted in the previous report above, Felsenstein's maximum likelihood(ML) method intrinsically depends on a probabilistic model of nucleotide(or base) substitutions, so that its performance may be quite sensitive tothe model. The model in this case is related to the transition versus transversion ratio and the relative contents of the four bases. It is also expected that the performance is affected by the conflict between "informative"
sites which is inevitable, as long as we assume that each nucleotide site
evolves independently. Note that the ML method does not work without•

Ochanomizu University.

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this assumption.
To study those problems, we first statistically evaluated the robustnessof the ML method in the estimation of molecular trees against different
base substitution patterns, including Jukes and Cantor's (1969), which
does not distinguish between transition and transversion, and is thesimples ever proposed. Namely, we conducted computer simulations, inwhich we could set up various evolutionary models of a hypothetical gene,and define a true tree to which an estimated tree from the ML method
was to be compared. In this case, two sizes of the gene were considered
to see the effect of the number of sites in a gene on performance; 300 and
1500 nucleotides were used. The rates of substitution per site for the twocases kept the same. The results show that topology estimation by the
ML method is considerably robust against different ratios of transition
to transversion and different GC contents, but branch length estimation
is not so. When the simplest model is used, the method is resistant to the
GC contents, but rather sensitive to the ratio of transition to transversion.
We then applied the ML method with different substitution patterns tosequence data on the tax gene from T-cell leukemia viruses, whose evolutionary process must have been more complicated than that of the hypothetical gene. The results indicate that the simplest model is asuseful as more complicated ones for making inferences about molecularphylogeny of the viruses.
On the other hand, comparison of the results between the 300 and 1500
nucleotides cases reveals that the performance is much better in the lattercase than in the former. This is mainly due to the fact that sampling
variance is reduced with enlarging gene size. (J. Mol. Evol. (in press)).
Publication from Genetic Resources Section,
Genetic Stocks Research Center
Shin-ya IYAMA and Yoshio TATENOThis section is in charge of collection, compilation, and publication of
information on experimental organisms preserved in universities and research institutes in Japan. In 1990 a catalog for laboratory rats was published and distributed to researchers. This was made possible in collaboration with a group of concerned researchers and the section. It is noteworthy that this catalog lists strain names under a new designation which

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

were discussed and formulated by both groups. The groups will proposethat the designation be used worldwide, because, unlike laboratory mice,no consistent designation has been established for laboratory rats.
We have cooperated with the Department of Cytogenetics and researchers
outside our institute to develop an image database system for ants. This
includes not only text information about their characteristics but also photoimage information on the organism taken by professionals. Thus thesystem gives both pictures and text on the display screen of a personal
computer as the results of retrieval. It is needless to say that pictures are
more convincing than text in a database for identification and classificationof organisms like the present one. For this reason, we anticipate thatimage databases like ours will become popular and have a large impact
on research and development in various areas. We have developed aprototype of the database system which includes the two types of information for 100 ant species.
This section is also in cooperation with the Rice Genetics Cooperative
for the publication of Rice Genetics Newsletter, which is an annually issued
international journal on rice genetics studies. The sixth volume was published and distributed worldwide in 1990.

RESEARCH ACTIVITIES IN 1989

IX.

99

HUMAN GENETICS

Characterization of the Telomeres and Sub-Telomeric DNA
Sequeuces of Human Chromosomes
Takashi IMAMURA, Hitoshi NAKASHIMA, Asao FUJIYAMA,
and Tomoko HASEGAWA

The chromosome termini, or telomeres, ofa variety of unicellular organisms
have been characterized in some detail. They consist of a series of simple
tandemly repeated sequeces that are G-rich on one strand, and C-rich on
the other. It has become clear over the past few years that unusual structural properties and modes of replication are associated with these terminal
repeats. These features may account for some of the special propertiesof telomeres, such as the ability to resist fusion to other telomeres and tochromosome breaks and random rearrangements.
Human telomeres are similar to those of simple organisms. It has beenshown that the telomere erpeat of (TTGGGG)n from Tetrahymena thermophila will cross hybridize with human telomeres, and that the cloned
Arabidopsis telomere repeat (TTAGGGG)n also recognized human telomeres. Human telomeres have repeating units of (TTAGGG), equivalent
to those of the trypanosome telomere. We have now used a series ofsynthetic oligonucleotides and restriction enzymes to clone directly thissequence from human chromosome DNA, mainly by using a polymerase
chain reaction. The present and the prvious reports suggest, though it
is not proven yet, that human telomeres are not uniform throughout theirlength, comprising some of multiple, different, but related tandem motifs.
In somatic tissue cells, the major, most distal, component consists of 5-10
kb of repeats that must be very similar to the sequence (TTAGGG)n. A(TTAGGG)4 probe can now be used to identify cloned telomeric DNA
fragments derived from both ends of each human chromosome. Study
is now being directed towards cloning and sequencing of the sub-telomeric
single copy fragments adjacent to these repeating units, which will prove
to be invaluable genetic markers for completing the linkage map of thehuman genome and for characterizing chromosome rearrangements involving telomeres, some of which may have been stabilized during evolution.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Genetic Polymorphisms of Gene Conversion within the
Duplicated Human a-Globin Loci
Hitoshi NAKASHIMA, Asao FUJIYAMA, Shunjiro KAGIYAMA
and Takashi IMAMURA

The identification of individuals possessing one or three adult a-globin
genes on a single chromosome, instead of the normal duplicated a-globin
genes, provides strong genetic evidence for intergenic recombination mechanisms, such as unequal crossover and gene conversion, in the evolutionof human multigene families. Additional evidence that this tandem arrangement of homologous a-globin gene sequences promotes unequal
recombination is the production of deletions, which are indistinguishable
from those found in human a-thalassemia genes, upon propagation of thecloned a-globin gene DNA in Escherichia coli. Although the duplicatedhuman a-globin genes encode identical polypeptides, previous studies haveestablished that the a 1 and a2 genes are not identical at the DNA level.
This finding could be inferred by the alignment of restriction maps of thea loci residing on the one-, two-, and three-gene chromosomes. We reportherein the first estimate by DNA analysis of the frequency of sequence
rearrangements within a-globin loci in Japan.
Of the 645 Japanese subjects studied, we identified 10 individuals heterozygous for a chromosome with the triplicated a-globin loci. The frequency of the triple a-loci was 0.008 in this population, while that of thesingle a-locus, i. e., a-thalassemia 2 gene, might be lower than 0.0008.
Analysis of haplotypes using particular RsaI site polymorphism in the
a-globin gene complex strongly suggests that the triple aaa loci may have
had multiple origins in this population. Of particular interest is that thefinding is strikingly different in Melanesia, where people are exposed todifferent malaria endemicities. The much higher prevalence of deletionala-thalassemia gene associated with endemicites of malaria in different partsof the Melenesian Islands, as compared to the triple aaa genotype, indicatesthat the former genotype confers an apparently selective advantage withrespect to malaria.
Duplication of a gene is the first step toward the evolution of the multigene
family. Although it seems clear that hemoglobin abnormalities will not
be a major cause of disease in Japan, the extensive survey in this country
could promise to provide an important insight into the origin, dissemination,

RESEARCH ACTIVITIES IN 1989

101

and persistence of mutations affecting a very important protein, a better
insight than can come from countries where selective factors may be coloring the picture much more strongly. Thus far, the situation in Japansuggests that genetic recombinations within the duplicated a-globin genes
responsible for the numerical variations of copies of the a-globin geneencountered here do not confer a marked selective advantage, but areapproximately neutral and owe their low frequency to a simple mutation
process and genetic drift that occurred in a relatively small number offounders in this population.
A Common Chinese fi- Thalassemia Mutation Found in a
Japanese Family
Yuji NARITOMI, Hitoshi NAKASHIMA, Masaaki KAGIMOTO,
Yasushi NAITO, Eisuke YOKOTA
and Takashi IMAMURA

We have identified the substitution of a thymine for a cytosine at nucleotide position 654 in the second intron of the j3-globin gene that causes
p-thalassemia in a Japanese family. This mutation has until now beenfound to occur rather frequently only in patients of Chinese origin but has
rarely been found in other ethnic groups. The molecular basis for thethalassemia syndrome of these patients can be explatined by aberrant mRNA splicing in IVS-2.
Beta-thalassemia is a heterogeneous inherited disorder of j3-globin gene
expression and one of the most common single gene defects. Althoughvarious mutations have been identified in defective j3-globin genes, a limited
number of specific mutation are prevalent in a given ethnic group in which
the thalassemia occurs rather frequently. The j3-thalassemia gene in thisJapanese family and those in Chinese may be of the same origin as they
are both framework I genes. The spread of the mutation to the presnt
day geographical pattern can be explained on the basis of population migration. Analysis of haplotypes of restriction enzyme site polymorphisms
supports this assumption.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Intraspecific Nucleotide Sequence Differences in the Major
Noncoding Region of Human Mitochondrial DNA
Satoshi HORAI and Kenji HAYASAKA

Nucleotide sequences in the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas were determined.
These sequences include a 482 base-pair (bp) long region encompassing
most of the D-loop forming region. Comparison of thses sequences withthose previously determined has revealed remarkable features of nucleotide
substitutions and insertion/deletion events. The average nucleotide diversity among the sequences is estimated as 1,45 %, which is three to fourfold higher than the corresponding value estimated from restriction-enzyme
analysis of the whole mtDNA genome. A hypervariable region was also
defined. In this 14 bp region, 17 different sequences were detected. Morethan 97 % of the base changes are transitions. A nonrandom distributionof nucleotide substitutions and sequence length variations was also noted.
Phylogenetic analysis indicates that diversity among Negroids is muchlarger than that among Caucasoids or Mongoloids. In fact, part of the
Negroids first diverged from the rest of the individuals in the phylogenetic
tree. A striking finding in the phylogenetic analysis was that Mongoloids
can be separated into two distinct groups. A divergence of part of theMongoloids follows the earliest divergence of part of the Negroids. The
remainder of the Mongoloids subsequently diverged together with the
Caucasoids. This observation confirmed our earlier study which clearly
demonstrated the existence, through restriction enzyme analysis, of two
distinct groups in the Japanese.
DNA Amplification from Ancient Human Skeletal Remains
and Their Sequence Analysis
Satoshi HORAI, Kenji HAYASAKA, Kumiko MURAYAMA,
Noriyuki WATE*, Hiroko KOIKE*
and Nobuyuki NAKAI**

The development of a polymerase chain reaction (PCR) makes it possible
to amplify the DNA of an aimed region from a very small amount. By* College of Liberal Arts, Saitama University.
** Faculty of Science, Nagoya University.

RESEARCH ACTIVITIES IN 1989

103

applying this technique, we tried to amplify mtDNA from 22 samples ofhuman bones derived from different eras. In the first PCR we were able
to amplify DNA only in the positive control. However, when we took a
portion of the first PCR product and performed a second 30-cycle PCR,.
we were able to amplify DNA in some samples of bone extracts. Although
we could amplify DNA in the second PCR for the majority of samples, it
was impossible to amplify DNA in some samples, which is probably due
to the condition of the preserved bones. However, we succeeded in amplifity mtDNA from the oldest one of them, a skull named Urawa-I, ex··cavated at a depth of five meters in Urawa city in 1988. Using collagen
which was simultaneously isolated from this skull, the absolute age of
Urawa-l was estimated at 5790+ 120 years B. P. through the direct detectionof 14C using accelerator mass spectrometry. This age is equivalent to thefirst term of the Jomon-period in Japanese prehistory.
We determined a 190-bp nucleotide sequences from the second PCR
product of Urawa-l by the direct sequence method. The nucleotide sequence from Urawa-l was compared with those from 101 contemporary
humans from three major racial groups and six additional sequences determined for individuals from Southeast Asia. In comparing the sequence
from Urawa-l with those from the 107 individuals, we found complete
identity in two Southeast Asians (a Malay and an Indonesian) out of the
16 non-Japanese Asians. However, we didn't observe an identical sequence
to Urawa-l among the 61 contemporary Japanese. Specifically, 14 Japanesediffered at one position and 8 Japanese differed at two positions. Furthermore, 38 Japanese differed at three to eight positions even in this shortregion. These observations indicate that this ancestor of Japanese who
presumably lived in the central part of Japan about 6000 years ago had acommon origin with some contemporary Southeast Asians.
Restriction enzyme analysis revealed that the Japanese population could
be separated into at least two distinct groups: the group with a smallerfrequency (Group I) first diverged from the other group with a larger frequency (Group II). This was also confirmed by our recent sequence analysis of the major noncoding region for Mongoloids including non-JapaneseAsians. In this connection the ancient Japanese speciman (Urawa-l)
belongs to one of the two groups (Group II) of modern Japanese. Partof the people who migrated from the continent during the period two to
three thousand years ago may be representative of the other group (Group

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

I) of modern Japanese, because the sequences from Group I individuals
showed three to eight nucleotide differences in the 190-bp region in comparison with that from Urawa-l. Although our findings were obtained
through the determination of nucleotide sequences from only one ancientindividual this archaeological and molecular genetical study gives a newsource of perspective on the evolutionary history of human populations.
For details, see Proc. Japan Acad. 65B: 229-233, 1989.
Heteroplasmies and Polymorphisms in the Major Noncoding Regionof Mitochondrial DNA in Japanese Monkeys Associated with
the Tandem Repeated Sequences
Kenji HAYASAKA, Takafumi ISHIDA* and Satoshi HORAI

We analyzed polymorphisms in the major noncoding region of mitochondrial DNAs (mtDNAs) in 100 Japanese monkeys derived from 12
local populations. Ninety of the 100 samples were analyzed using DNA
amplified by a polymerase chain reaction. We found individuals whichwere heteroplasmic for mtDNAs in two populations. These heteroplasmies were associated with length polymorphisms which resulted from thepresence of tandem repeated segments in the noncoding region. We
classified the mtDNA of the 100 Japanese monkeys into six types. One
type was observed in seven populations and another in two populations,while the remaining four types were observed only one population each.
In 11 out of the 12 populations, only one type of mtDNA was observed.
These findings support the maternal inheritance of mtDNAs and the life
history of Japanese monkeys.
Mitochondrial DNA Mutation in a Family with Leber's
Hereditary Optic Neuropathy
Makoto YONEDA**, Shoji TSUJI**, Toyoaki YAMAUCHI**,Takashi INuzuKA**, Tadashi MIYATAKE**,
Satoshi HORAI and Takayuki OZAWA***

Leber's hereditary optic neuropathy is characterized by optic neuropathy* Primate Research Institute, Kyoto University.
Brain Research Institute, Niigata University.
Faculty of Medicene, Nagoya University.

*****

RESEARCH ACTIVITIES IN 1989

105

and cardiac arrhythmia. The age of onset ranges from adolescence to lateadulthood. Maternal transmission in some pedigrees suggested a mutationin mitochondrial DNA (mtDNA). Sequencing of 85 % of the mtDNA
from a black patient with this disease revealed 25 base substitutions, compared with normal mtDNA (the Cambridge sequence). Of these substitutions one-a G-to-A transition at nucleotide position 11778 in the Cambridge sequence, converting the 340th arginine to histidine in NADH
dehydrogenase subunit 4-has been suggested as the mutation correlated
to Leber's hereditary optic neuropathy because the 340th arginine is highly
conserved among various species. The mutation removes an SfaNI restriction site, which has also been found in a few North American andEuropean pedigrees.
We studied leucocyte mtDNA SfaNI restriction fragment length polymorphisms (RFLP) in a Japanese family with Leber's hereditary optic
neuropathy and found the identical mutation only in maternal members,
including two affected patients. The loss of a 0.92 kbp fragment and a
0.68 kbp one, and the appearance of a 1.6 kbp fragment, were observed
only in the maternal members, including the patients. Restriction mappingwith other enzymes ruled out deletion or rearrangement of mtDNA.
Nucleotide sequence analysis of polymerase chain reaction products inpatients' mtDNA revealed a G-to-A conversion identical to that reported
by Wallace et at.
This mutation was found in two patients with the disease and was maternally inherited through three generations. It has been demonstrated
in patients of differrent ethnic origin and, taken together, these reports
strongly support the hypothesis that the G-to-A mutation at position 11778
is the cause of Leber's hereditary optic neuropathy. For details, see Lancet
i: 1076-1077, 1989.
Specific Amplification of Deleted Mitochondrial DNA from a
Myopathic Patient and Analysis of Deleted Region
with S, NucleaseTomoko TANAKA-YAMAMOTO*, Masashi TANAKA*, Kinji OHNo*,
Wataru SATO*, Satoshi HORA! and Takayuki OZAWA*

Heteroplasmy of the normal-sized and the deleted mitochondrial genome
has been observed in mitochondrial myopathy. The deleted region of the* Faculty of Medicine, University of Nagoya.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing
a combination of polymerase chain reaction and Sl nuclease digestion.
The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized thedeletion from at least position 9020 to 14955, but regions of uncertainty
of 1 kb remained on both ends of the deletion. Using the polymerase
chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two
fragments adjacent to the start and end of the deletion were amplified fromthe normal-sized genome. Sl nuclease analysis of the heteroduplexes
formed among these fragments demonstrated that the deletion extended
from position 8650+50 to 15660+60. This methods does not requireradioisotopes and, moreover, can determine the deleted region within
5 h, in contrast to the 2 days required by the conventional Southern blot
analysis. These results indicate that the novel method is faster and moreaccurate than the conventional method for determination of the deleted
region of the genome. For details, see BBA 1009: 151-155, 1989.
Increased Level of Mitochondrial Gene Expression in
Polyps of Familial Polyposis Coli Patients
Akiko YAMAMOTO*, Satoshi HORAI, and Yasuhito YUASA**

To elucidate the mechanisms of polyp formation in familial polyposis
coli patients, cDNA clones, expressed differently between normal colonicmucosae and polyps, were screened. Three polyp-derived cDNA clones
showed increased levels of transcription under neoplastic conditions.
Among them, clone P6 was determined to be derived from the mtDNA
encoding parts of subunit 1 of NADH dehydrogenase and l6S rRNA.
The numbers of copies and restriction enzyme patterns of the mtDNA
did not vary in either normal or neoplastic tissues, and a level of different
mitochondrial mRNA was increased in the same proportion, suggestinga change in the rate of synthesis and/or degradation of the mtRNA. No
cDNA clone which was expressed higher in normal colonic mucosae thanin polyps, was isolated from the cDNA library derived from normal mucosae.
For details, see Biochem. Biophy. Res. Commun. 159(3): 1100-1106, 1989.
* Department of Hygiene, Gunma University School of Medicine.
** Tokyo Medical and Dental University School of Medicine.

RESEARCH ACTIVITIES IN 1989

x.

107

APPLIED GENETICS

Association between Pox-l Variation and Seed Productivity
Potential in Wild Rice
Hiroko MORISHIMA

Alleles at an isozyme locus Pox-I in rice are differentially distributed in'Cultivated species and their wild relatives. Populations of seed propagatingtaxa (Oryza sativa, O. glaberrima, annual type O. rujipogon, O. breviligulata)
are nearly monomorphic with an allele called 2A, while those of vegetatively
propagating ones (perennial type O. rujipogon, O. longistaminata) carryanother allele called 4A at high frequency. This association between
allozyme variation and propagating system was consistently found overbroad areas at the species level as well as at the ecotype level.
Asian common wild rice O. rujipogon tends to be differentiated into two
ecotypes; perennial and annual types which differ not only in longevity but
also in many other life-history traits. A cross between an annual and aperennial strain of O. rujipogon which carry Pox_1 2A and Pox-1 4A in homozygous state, respectively, was extensively studied to elucidate the genetic
mechanism of association between Pox-I variation and life-history, 1)
In Fa, homozygotes with POX-FA showed higher reproductive allocation,shorter plant stature, earlier flowering and shorter anther, which are all'Characteristics of annual life history, than those with Pox_1 4A • 2) When the
hybrid population was selected for early flowering, allele 2A increased.
When it was naturally grown for three years under conditions favorable forvegetative propagation, allele 4A increased. 3) To check whether this association is due to pleiotropy of Pox-I, isogenic lines were made through selfing
for six generations holding the Pox-I locus heterozgous. In the isogenic
pairs thus prepared, the association between allozyme and charactersmostly disappeared.
The above results suggest that quantitative trait loci for these life-history
traits exist, and that some of them are clustered on a chromosome segment
marked by Pox-I, Their linkages, however, are not tight judging fromthe experiments with isogenic lines. There must be certain selective interactions between those genes including Pox-I to maintain this gene blockin nature.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Variation in Resistance to Bacterial Leaf Blight between
and within Wild Rice Populations
C. HAMAMATSU, Y. I. SATO and H. MORISHIMA

Host-pathogen interaction in a natural ecosystem, when compated withthat in an agroecosystem, provides a perspective on the evolutionary dynamics of plant-pathogen systems. The present study deals with interand intra-populaltional variability in response to bacterial leaf blight caused
by Xanthomonas campestris pv. oryzae in the wild rice, Oryza rufipogon.
Forty-two strains of wild rice (each representing a natural population)collected from various regions of Asia were examined regarding response
to infection by five pathogen races ioolated in Japan. Mulitvariate analysisof the data revealed variation in general resistance, and variation in racespecific resistance. Variation patterns obtained, however, did not show
distinct relationship to geographical distribution nor to ecotypic differentiation (perennial vs. annual type). Some Chinese strains showed race-specificresponses.
To assess intra-populational variation in respones to the pathogen, threewild rice populations from Thailand (one annual and two perennial types)
were studied together with two populations of Chinese primitive cultivars.
In each population, 30 to 40 lines derived from randomly chosen motherplants growing in their natural habitats were examined by injection method
and clip-inoculation using three Japanese pathogen races. Wild rices aswell as land races were found to preserve a large amount of intra-populational genetic variability in response to the pathogen races. To the pathogen
races used in the present study, wild rices were generally resistant thoughland races were susceptible. The amount of intra-populational variability
in resistance to this desease did not seem to be related to genic diversity
estimated from isozyme variations.
Responses to three different pathogen races were compared among lines
in three wild populations. In most cases, intensities of infection (lesion
length of leaves) were positively correlated between different races withinpopulation, indicating that race-nonspecific resistance possibly governed bypolygenes is mainly responsible for intensity of infection. In addition, theexistence of race-specific resistance governed by a major gene was suggested
in an annual population NE4, judging from discontinous variation in
response to race, T7l33. This was also confirmed by injection method.

RESEARCH ACTIVITIES IN 1989

109

Further, two populations NE4 and CP20, each consisting of 30 lines.
were compared as to genetic structure of population in resistance to a pathogen race, T7133. Analysis of variance indicated that between-line variation(heterogeneity in the field) was larger in the annual population NE4, while
within-line variation (heterozygosity in the field) was larger in the perennial
population CP20. This could be interpreted by differences in their breeding
systems.
To understand the complex pattern of host-pathogen interactions, useof more pathogen races, particularly those prevailing in areas from whichstudy-populations were sampled is needed.
Genetics of Life-history Trait Associations in Wild Rice from Thailand
P. BARBIER* and H. MORISHIMA

In natural populations of wild rice Oryza rufipogon, life-history traits(such as the age at flowering, the components of seed productivity, andinvestment in vegetative growth and asexual reproduction) as well as characteristics of the mating system, are found tightly intercorrelated. The
resulting trait-associations correspond to differentiation between annuals
and perennials occurring within this species. Intra-populational genetic
variability of most of these traits seems, however, to be low. In order todetermine which trait associations have a genetic basis, a series of crosses
were made between individuals issued from one annual and several perennial
populations located in Thailand. These perennial populations slightly differ
by their degree of relatedness to the annual population used, as judged fromthe genetic distances calculated from isozyme data and by their sequence
data at some nuclear genes.
F 2 and Fa progeny of four annual X perennial crosses with the plantsfrom the most closely related perennial population were grown in a glasshouse and various life-history traits were measured to examine their correlation pattern. In particular, the existence of negative correlations between
fitness components characterizing the annual vs. perennial habit, was tested.
In contrast to the situation in natural populations, only a few correlations between traits were found in the progeny of crosses. A negative
correlation between the number of days to flowering and the number of>I<

Faculty of Agriculture, Nagoya University.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

panicles (component of fecundity) was consistently found in F 2 and Fa.
This correlation, which probably has a genetic or developmental basis,
may have played a role in the evolution of early reproducing, high yielders(such as the annual type). However, only slight evidence could be found
for the existence of antagonistic pleiotropy between components of fecundity
and traits related to post-reproductive survival. F 2 progeny of othercrosses are under investigation.
A New Hypothesis on the Parentage and Disseminationof Japanese RiceYo-Ichiro SATO

The parentage of Japanese rice, and the origin of early-heading cultivars
distributed in the northeastern part of Japan were discussed from theviewpoint of genetics. Native cultivars in Japan and neighbouring countries were examined to survey the distribution of genes for hybrid weakness,
F 2 chlorosis and black hull-color which segregate only in crosses between
distantly-related cultivars. It was found that Japanese native cultivars
are polymorphic in the loci examined, suggesting that Japanese rice has
plural origins. The geographical distribution pattern of the gene for hybrid
weakness suggests two paths of dissemination; one was from mainland
China ("keng" type) and another was from tropical islands. Modern
Japanese rice is considered to be formed through a natural hybridization
between these two groups.
Early-heading progeny are expected to appear theoretically in hybrids
between two late-heading japonicas. In fact, part of F 1 plants derived
from a cross between "keng" and Philippino upland cultivar headed as
early as early-heading cultivars in the northeastern part of Japan. The
origin of early-heading cultivars can be well explained under the present
hypothesis. The previous hypothesis which assumed an early-heading
mutation, however, is not supported by recent finding that rice cultivation
had begun in the northern Tohoku area already in the early Yayoi period.
For details, see Archaeology and Natural Sciences Vol. 22.

RESEARCH ACTIVITIES IN 1989

111

Toward a Rationale of Genetic System in the Flowering
Response of the Rice Plant to Photoperiod
Yoshio SANO

Plants utilize a precise and predictable photoperiod for determining thetime of floral initiation so as to accomplish their life cycle during a favorable
period. Although one of the major genes controlling photoperiod sensitivity, Sel (or LmU ), has been genetically well characterized, our present
knowledge is still limited for concerning diversified flowering responses tophotoperiods in rice. A problem in genetic studies of rice often comesfrom distorted segregations which occur in crosses between ecotypes and/or
distantly related taxa. The author has worked on genetic differentiation
between distantly related taxa of rice and examined the genic differences
after introducing alien chromosomal segments carrying a given gene intoa genetic tester of rice. The Sel locus was also successfully introduced
from various accessions of rice including wild forms. During this procedure,
two additional genetic factors were found which drastically change theheading time in the presence of Sel' Their precise chromosomal locations
and their gene actions are under investigation and this information will
be published elsewhere. The discovery of these two genetic elements
might offer an opportunity for better understanding of the complex regulatory system in photoperiod sensitivity.
a) Suppressor gene for Sel
An Indica type, Oryza sativa, Peiku (AccI08) is photoperiod insensitiveand it comes to heading about a week earlier than T65 with Sel +/ Sel + .
The genotype of AcclO8 has been shown to be Sel/Sel since a large chromosomal segent from wx to Sel was successfully introduced into Taichung65 from Accl08Sl (g) utilizing a segment containing the Sl gene (hybrid
sterility from O. glaberrima) as a recombination suppressor around thewx locus. The resultant plant came to heading two to three weeks later
than T65 and as late as T65Se l (g). The F l plants between T65 and Acc
108Sl (g) suggested that AcclO8 possesses a dominant suppressor (s) for
Sel' To extract and examine the suppressor gene, early flowering segregants from selfed progenies were backrossed with T65Se l (g). BC lF 2 andBC 2 F 2 segregants gave a ratio of 1: 2: 1 regarding flowering time. The
Fa lines also showed that the early flowering plant is homozygous for thedominant supperssor. The suppressor gene was tentatively designated112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Su(t)-Se t .
b) Enhancer gene for Set
A wild progenitor (0. rufipogon) is strongly photoperiod sensitive andit produced no panicle during the summer season under natural conditionsin Mishima. The chromosomal segment from Wx to Set was also introduced from W593 (0. rufipogon from Malaysia) to T65wx (a near-isogenic
line of Taichung 65 with wx). The segment was successfully introduced
without the destruction of the segment since S6 (gamete eliminator) istightly liked with Set and most of the hybrid derivatives tend to carry Set.
Unexpectedly, the homozygote for Wx and Set which was derived from
BCrF 2 produced no panicle in Mishima while the homozygote for wx and
Set always headed as early as the T65Se t (g) strain. This suggests that
BCrF t is heterozygous for an enhancer gene which dramatically delayed
heading in the presence of Set and that the enhancer gene is tightly linked
to Wx. The data from BCsF t , BCrF 2 and BCrFa consistently supported
these assumptions. The enhancer gene was tentatively designated En(t)Set and the gene order was estimated to be wX-En(t)-Set-Set-S6 •The present results confirmed that there are at least two genetic elements
which negatively and positively regulate the experssion of Set. Although
the genic interaction remains to be studied, the present study revealed
that investigations of differential regulation at the Set might elucidate
further information about diversified photoperiod sensitivity in rice.
A Gene Complex Responsible for Seed Shedding and Spreading
Panicle Found in Wild Rice Species
Mitsugu EIGUCHI and Yoshio SANO

Generally, cultivated crops readily interbreed with their wild progenitors
when they coexist under natural conditions and produce fertile progenies,
revealing their close relationships. This holds true for Asian cultivatedand wild forms of rice, giving rise to difficulties in distinguishing them insome instances. The only reliable morphological trait which distinguishes
wild forms from cultivated forms is seed shedding. Wild rice forms show
uniformaly, a high degree of seed shedding, suggesting a strong natural
selection for seed shedding in wild species. On the other hand, cultivatedforms show a wide range of intensities in seed shedding but generally it isa weak degree of seed shedding, if any. This strongly suggests that the

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113

genetic changes which occurred during the formation of various rice cultivars were not simple. We decided to study on the genetics of seed shedding
in order to examine their genetic changes in relation to the evolution fromwild to cultivated rice forms.
The materials used were three strains of Oryza rufipogon (a wild progenitor
of O. sativa, W107 as an annual form and W149 and W573 as perennial
forms) and a strain of O. glumaepatula (W1192), which showed seed shedding. A genetic stock (Taichung 65 carrying genetic markers of g and19, designated T65g 19) was used as a cultivated form without seed shedding,in these crossing experiments. Degree of seed shedding as described heremeans seeds shedding naturally after ripening as observed in wild rice forms.
Such a high degree of seed shedding is never observed in cultivated forms.
In addition, spreading panicle which is characteristic of wild forms andseems to be important for seed dispersal under natural conditions, wasalso examined in segregating papulations.
The F 1 hybrids between T65g 19 and wild forms all showed seed shedding
and spreading panicle like wild forms, which suggested that the two traits
are controlled by dominant genes. The F 2 segregations between T65g
19 and O. rufipogon revealed that seed shedding is controlled by two
dominant genes and sperading panicle by one. The confirmation of thetwo dominant genes for seed shedding supports the previous finding by
Kadam (1936) that wild rice carries two dominant genes responsible forseed shedding. One of them is known to be loosely linked with la (lazyhabit, the 8th likage group). In addition, the other dominant gene
responsible for spreading panicle (designated Spra (t) tentatively) seems to
be linked with both 19 and Ph in the 2nd linkage group. The F 1 plantsof T65g IgxW107 and T65g IgxWl192 were backcrossed with T65wx inorder to introduce alien genes responsible for the traits into the sativa
genetic background. In each generation, plants showing seed shedding
and spreading panicle were used for further crossings. The high frequenciesof plants with both traits in each backcross generation suggested a tightlinkage between the genes controlling the two traits although such a linkage
was not detected in the F 2 generation.
The near-isogenic lines carrying two dominant genes, each of which
control seed shedding and spreading panicle, were selected from BCe (xW107) and BCs (xW1192) generations. F 2 and Fa data showed support
for the finding that each line carries two dominant genes and that the two

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

genes for seed shedding and spreading panicle were tightly linked with arecombination value of about 2 % in both cases. This indicates that wild
rice species generally carry a gene complex which is responsible for seeddispersal. The order of the two dominant genes from Wl192 was also
estimated as 19-5prs(t)-Shs-Ph, suggesting that the gene complex is preservedin distantly related wild rice taxa. Accordingly, two steps of mutation
are needed for the establishment of the gene complex found in cultivatedforms. At the time the gene complex was established during domestication,
it seems to have been readily maintained in cultivated forms by selecting
nonshattering and compact panicles, a typical syndrome resulting fromautomatic selection due to planting harvested seed.
Extraction and Amplification of a Chloroplast DNA Fragmentfrom a Single Ancient Rice Seed
Ikuo NAKAMURA and Yo-Ichiro SATO

If DNA extracted from ancient rice seeds can be analyzed, the scope of
the phylogenic view of rice would certainly be broadened. peR enables
us to amplify DNA from museum specimens and archaeological finds(Higuchi et al. 1984, Paabo 1985, Golenberg et al. 1990). Our purpose
was to investigate whether a chloroplast DNA fragment could be amplified
from a single ancient rice seed found in a Japanese archaeological excavation (1,200 years old). We were able to successfully extract 50--100 ng ofDNA from a single ancient rice seed. This was half the amount of DNA
extractable from a single living seed using the same extraction procedure.
The DNA fragments from the ancient seed were recovered as 15-20 kbp
fragments and were a little smaller than intact DNA. Since the ancientseeds were sunk in water-saturated soil, water must have prevented theancient DNA from their strand cut and modification by oxidation. This
is why the DNA was preserved in extremely good condition.
We were able to amplify the rice chloroplast sequence, a 1.3 kbp fragment
covering trnQ, psbK, psbI and trnS genes corresponding to 1 % of totalrice chloroplast DNA, from the ancient DNA through a polymerase chain
reaction. The 1.3 kbp chloroplast fragment of living leaves was hybridized
with 1.3 kbp fragments amplified from ancient seeds as well as from living
seeds. Restriction analysis showed that these 1.3 kbp fragments werecorrectly amplified from rice chloroplast DNA. The DNA analysis of

RESEARCH ACTIVITIES IN 1989

115

ancient rice seed described can greatly contribute to the investigation of
the origin and varietal diversification of rice cultivars.
Anomalous 3: 4: 1 Segregation of a- and .a-Subunits of Seed
Conglycinin in Mutant Soybean
Ikuo NAKAMURA, Hiroya ODANAKA and Norihiko KAIZUMA

A soybean mutant (dmJdm) lacks the a- and p-subunits of p-conglycinin
in seed storage protein and is a lethal during the early stage of germination.
This mutant was selected from progenies of a r-ray irradiated soybean
cultivar; Wase-suzunari (Odanaka and Kaizuma 1988). Seed protein&
were extracted from an anti-hilum portion of seed from progeny derived
from selfed heterozygous plants and were subjected to SDS-polyacrylamide
gel electrophoresis. These two subunits were simultaneously segregatedat the ratio of 3 (+J+): 4 (+Jdm): 1 (dm/dm). This segregation ratio
has not been observed in any higher animals and plants. As the a- andp-subunits are coded by at least two genes, respectively (Harada et al 1989}
and a weak genetic likage was reported between the a- and p-subunit gene&(Tsukada et al. 1986), a relatively large chromosomal region covering thesegenes might be lost in the mutant and such a deleterious chromosome
might be involved in the anomalous segregation.
Our hypothesis to explain this anomalous Mendelian segregation is thatsome marker gene aberrantly segregates at 3: 1 during the meiosis of female
or male gamatecytes while normal meiotic segregation (1: 1) occurs during
another gamategenesis in the same flower of the heterozygous plant. If
this were true, the resulting segregation of the marker gene would be 3: 4: 1
in the progeny. Many researchers reported aberrant 3: 1 (6: 2) segregationin yeasts and lower fungi known as gene conversion (Szostak et al 1983).
The frequencies of gene conversion in these organisms is quite low. However the deletion of the two subunits was stably inherited at a 3: 4: 1 ratio
in this soybean mutant. This suggests that the aberrant 3: 1 segregation
also stably occurs during miosis specific to only one-side gamategenesis.
We are now investigating this phenomena at the molecular level.

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

Construction of a Targeting Signal for Plant Mitochondria
Ikuo NAKAMURA, Junko KYOZUKA and Ko SHIMAMOTO

An artificial DNA sequence (306 bp) coding a targeting signal (97 aminoacids) for plant mitochondria was constructed. The ,a-subunit of F 1ATPase in Nicotiana plumbaginifolia. is encoded by a nuclear gene and islocalized in the mitochondria (Bountry and Chua 1985). Four pairs (A,B, C and D) of oligonucleotides were designed, based on the amino acidsequences, including the targeting signal of this protein, and unique restriction sites with unchanged amino acid sequences were arranged at bothends of each fragment to link these fragments. A total of eight oligonucleotides (76- to 89-mers) were synthesized by DNA synthesizer. Each pairof oligonucleotides was annealed and ligated into a pUC type vector. Then,the cloned fragments were sequenced and correctly synthesized fragments
were linked in turn (A-B-C-D). Some deletion clones were made by theexonuclease III digestion from the 3' end of this fragment, because thesignal processing site of F1-ATPase ,a-subunit was not known. These
synthesized and deleted DNA fragments were ligated with a reporter gene
(8-glucuronidase) and then truncated genes were integrated into a plant
expression vector. The targeting function of the synthesized signals forplant mitochondria is being assayed.
On the Conditions Causing the Periphery Effect in Genotype
Distribution within Plant Populations
Katsuei YONEZAWA

Plant individuals do not change their positions and tend to mate withneighbouring individuals because of limited dispersion of pollen. In
addition, many plant species do selfing with a fairly high rate. Genotypes
in a plant population therefore are expected to distribute very unevenly,
forming patches of various sizes and shapes. One type of unevenness ingenotype distribution, the difference of periphery area from central area,
i. e., periphery effect, has not been extensively investigated. A greater
understanding of periphery effect is necessary for accomplishing our knowledge on plant populations because every plant population in reality hasboundaries. This subject is important also for defining the optimum
mehtod of sampling from plant populations for both scientific surveys andRESEARCH ACTIVITIES IN 1989

117

practical purposes such as geneitc resource collection.
Genotype distribution in a population is determined by various breeding
and life-history characteristics (e. g., longevity, overlap of generations,flowering date and duration, mating system, distance and frequency ofdispersion of pollen and seeds, etc.), and heterogeneities in macro- andmicro-environmental conditions. Among these factors, dispersion ofpollen and seeds and the selfing rate of plants seem to be most fundamental,
and are now being investigated using Monte Carlo computer simulation.
The periphery effect (PE) was measured by a ratio, (Patchiness of peripheryarea- Patchiness of central area)-;-(Patchiness of central area). The computations obtained hitherto showed that the periphery effect occurs mostprominently not in the absence of but in the presence of some degree ofpollen dispersion, roughly, under a dispersion of 2-5 in units of distance
between adjacently neighbouring individuals. The contribution of seeddispersion could not be perceived as clearly, but a dispersion larger than
2 seemed to suppress development of the periphery effect. The influenceof selfing rate also remains to be confirmed through further computations,but a trend could be observed in which a more pronounced periphery effectoccurs with a lower rate of selfing. It may tentatively be suggested thatthe periphery effect best develops in the presence of a moderated rather
than highly limited dispersion of pollen. In plant populations with a strongly restricted gene flow, homozygosity and patchiness of homozygotes progress rapidly, but to the same extent in both central and boundary areas,
and as a result, no differences occur between the two areas.
Computer Simulated Evaluation of the Hybrid Weakness Gene System
as a Means of Preventing Genetic Contamination of Rice Cultivars
Katsuei YONEZAWA, Yo-Ichiro SATO, Tetsuro NOMURA'"
and Hiroko MORISHIMA

The effectiveness of hybrid-weakness caused by a complementary interaction of two dominant genes, Hwc-l and Hwc-2, in suppressing the
spread of the red-grain gene into ordinary white-grained rice cultivars wasinvestigated based on the Monte Carlo computer simulation method. It
was shown that the weakness genes, capable of reducing the fitness of the<.:arrier by 80 percent or more, work quite effectively to suppress the genetic'"

Faculty of Engineering, Kyoto Sangyo University.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

contamnination of white-grained cultivars due to the inflow of pollens from
red-grained cultivars. The complementary weakness genes, however, dolittle to restrain the spread of the red-grain gene once the gene has been
incorporated into the population in the form of red-grained progeny produced due to imperfect lethality of the initial hybrid between red- andwhite-grained cultivars. The weakness genes also have no or little effect,when contamination was initiated by the inflow of seeds or seedlings from
red-grained cultivars. In these situations, removal (selection) of redgrained plants is the only method for exterminating the red-grain gene.
The removal of red-grained plants needs not to be intensive if carried out
continually. The spread of the red-grain gene after initial contamination
is suppressed if the red-grain gene and weakness genes are linked. The
suppressing effect of the linkage, however, is not substantial when theoutcrossing rate is around or smaller than 0.01 as assumable in most ricecultivars. For details, see Plant Breeding 104: 241-247 (1990).

RESEARCH ACTIVITIES IN 1989

XI.

119

DATABASE

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

A primary task of the DDBJ is, of course, DNA sequence collection.
However, in addition to that, we have a wide range of activities; 1) DNA
data collection and data entry in collaboration with other databanks, 2)data distribution, including the secondary distribution of the GenBank
and EMBL databases in Japan, 3) development of research tools for sequence analysis, and 4) the regular publication of newsletters to inform
people of activities of the DDBJ. The DDBJ computer system is open
to researchers for data submission and for providing online access to DNA
and related databases. In the following, I will briefly report this year's
activities of the DDBJ. For details, see "DDBJ News Letter No.8, 1989"
and "No.9, 1990".
1) Data Entry and Management
Sanzo MIYAZAWA and Hidenori HAYASHIDA

Our data collection began in December, 1986 and is carried out in collaboration with the GenBank and the EMBL Data Library. The collaboration includes projects on designing a new feature table and rebuilding
a DNA database. DDBJ has charge of mainly scanning journals publishedin Japan, and before, data directly submitted by authors had been forwarded
to either GenBank or EMBL according to journal split. However, DDBJ
started locally processing all direct submissions in October 1989; Direct
data submissions which the EMBL Data Library is supposed to process
are still forwarded to the EMBL at this stage.
Since we released the first version of the DDBJ database in July, 1987.
our database has been released every half year; version 5, which included
679,378 bases in 395 entries, was released in July, 1989, and version 6 including 841,236 bases in 496 entries was released in January, 1990. The
DDBJ collected about 305 kb in the year from January 1989 to January
1990. This amount of data is almost equal to 1.25 times the data collectedlast year, but only 1/40 of the total DNA sequences collected in the world.

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

However, this may be reasonable, if the number of staff of the DDBJ is
compared with those of GenBank and the EMBL Data Library. Eachrelease included a coding sequence database and a peptide sequence databasethat were extracted and translated from the original DNA sequence database, and files of journal index, accession number index, short directory,
and data submission form.
2) FLAT Database and Sequence Analysis System for DNA and Proteins:
Release 1.2.
Sanzo MIYAZAWA

We have been developing a search and retrieval system for flat file databases in order to provide simple tools for using DNA and protein sequence
databases. This system called FLAT consists of primitives most of which
perform a single operation and work as filters in the UNIX system. Its
first version, 1.0.8, was released in 1988. The most recent version, 1.2,
was released in October, 1989. New features added to this release
include functions of (1) electronic mail database server and (2) automatic update of databases according to new data sent bye-mail. One
can search and retrieve DNA and protein sequence databases by sending search/retrieval commands by electronic mail to the specific address
through Junet, Usenet, Internet, and Bitnet. Available databases include
PIR, SwissProt and PRF as well as DDBJ, EMBL, and GenBank databases.
The EMBL and GenBank databases are updated twice a day by adding
new entries which are daily sent bye-mail from those databanks. The
DDBJ database is updated as well. Functions provided by the server
include keyword search on definition lines, search by author name, journalname and accession number, and entry retrieval by specifying entry names
or accession numbers. This FLAT search/retrieval system for sequence
databases is designed to be portable among UNIX systems which are available for a wide range of computers from super to micro computers.
For details, see "DNA Data Bank of Japan: present status and futureplans" by Sanzo Miyazawa in "Computers and DNA, SF! Studies in the
Sciences of Complexity, Eds. G. Bell and T. Marr (Reading MA: AddisonWesley), vol. VII, pp. 47-61, 1989".

RESEARCH ACTIVITIES IN 1989

121

Basigin, a New, Broadly Distributed Member of the Immunoglobulin
Superfamily, Has Strong Homology with Both the Immunoglobulin
V Domain and the ,a-Chain of Major Histocompatibility
Complex Class II Antigen
Teruo MIYAUCHI*, Takuro KANEKURA*, Akihiro YAMAOKA*,
Masayuki OZAWA*, Sanzo MIYAZAWA
and Takashi MURAMATSU*

Lotus tetragonolobus agglutinin (LTA) binds preferentially to early embryonic cells in the mouse. The affinity-purified antibody raised against
LTA receptors from embryonal carcinoma cells was used to screen a Agtll
expression library of F9 embryonal carcinoma cells, resulting in detectionof a eDNA clone specifying a new glycoprotein termed "basigin". The
glycoprotein has been suggested as being a transmembrane one, and wasfound to be a new member of the immunoglobulin (lg) superfamily. The
molecular weight of basigin was largely in the range between 43,000 and
66,000, while that of the peptide portion with a putative signal sequence
was inferred to be about 30,000. Significant levels of basigin mRNA weredetected not only in embryonal carcinoma cells, but also in mouse embryos
at 9-15 days of gestation and in various organs of the adult mouse. The
Ig-like domain of basigin is unique, since it has strong homology to boththe j3-chain of the major histocompatibility class II antigen and the Ig V
domain. The number of amino acids between the two conserved cysteine
residues is intermediate between those of the Ig V and C domains. Therefore, basigin is an interesting protein in connection with the molecular
evolution of the superfamily.
For details, see "J. Biochem. 107,316-323, 1990".

•

Faculty of Medicine, Kagoshima Unversity.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

PUBLICAnONS
ANDO, T., SAWADA, Y., SHIMIZU, H. and SUGIYAMA, T.: Pattern formation in hydra
tissue without developmental gradients. Dev. BioI. 133: 405-414, 1989.
AOIG, K.: A shifting balance type model for the origin of cultural transmission. In"Population Biology of Genes and Molecules" (N. Takahata and J. F Crow, eds.),
123-137, Baifukan, Tokyo, 1990.
ARAKI, T., HIRANO, H., NAITO, S. and KOMEDA, Y.: Introduction of foreign genes into
Pharbitis nil calli using a vector derived from Agrobacterium pTi. Plant Cel Rep. 8:259-262, 1989.
AYUSAWA, D., YAMAUCHI, M., SHIMIZU, K., SENO, T. and MATsuHAsm, M.: Two typesof mouse FM3A cel mutants deficient in 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and their transformants isolated by human chromosomemediated gene transfer. In "Purine and Pyrimidine Metabolism in Man VI, Part A:
Clinical and Molecular Biology" (Mikanagi, et aI., eds.), Plenum Press, New York andLondon, 537-542, 1989.
BARBIER, P.: Genetic variation and ecotypic differentiation in the wild rice species Oryza
ru/ipogon. I. Population differentiation in life-history traits and isozyme loci. Jpn.
J. Genet. 64: 259-217, 1989.
BABlER, P.: Genetic variation and ecotypic differentiation in the wild rice species Oryza
ru/ipogon. II. Inflnence of the mating system and life-history traits on the genetic
structure of populations. Jpn. J. Genet. 64: 273-285, 1989.
BARBIER, P. and ISHIHAMA, A.: Vairation in the nucleotide sequence of a prolamin genefamily in wild rice. Plant Mol. BioI., 15: 191-195, 1990.
BONHOMME, F., MIYASHITA, N., BOURSOT, P., CATALAN, J. and MORIWAKI, K.: Geneticalvariation and polyphyletic origin in Japanese Mus musculus. Heredity 63: 299-308,
1989.
BOURSOT, P., BONHOMME, F., CATALAN, J. and MORIWAKI, K.: Variations of a Y chromosome repeated sequence across subspecies of Mus musculus. Heredity 63: 289-297,
1989.
CHEN, Z.-L., NAITO, S., NAKAMURA, I. and Beachy, R. N.: Regulated expression of geneencoding soybean coglycinins in transgenic plants. Dev. Genet. 10: 112-122, 1989.
FISCHER Lindahl, K., HERMEL, E., E., LoVELAND, B. E., RICHARDS, S., WANG, C.-R. and
YONEKAWA, H.: Molecular definition of a mitochondrially encoded mouse minor
histocompatibility antigen. Cold Spring Harbor Symposium on Quantitative Biology.
54: 563-569, 1989.
FUJIOKA, M., SmMAMOTO, N., KAWAHARA, A. and WATANABE, K.: Purification of an
autocrine growth factor in conditioned medium obtained from primary cultures ofscleral fibroblasts of the chick embryo. Exp. Cell. Res. 181: 400-408, 1989.
FUJISAWA, T.: Role of intersitital cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra. Dev. BioI. 133: 77-82, 1989.

PUBLICAnONS123

FUJIYAMA, A. and TAMANOI, F.: RAS 2 protein of S. cerevisiae undergoes rein moval ofmethionine at N-terminus and removal of three amino acids at C-terminus. J.B.C.,
1989 (in press).
FunsAKI, S., NISHINO, T., KATSUKI, H., HARA, H., NISHIMURA, Y. and HIROTA, Y.: Isolation and characterization of an Escherichia coli mutant having temperature-sensitive
farnesyl diphosphate synthase. J. Bacteriol. 171: 5654-5658, 1989.
FUKAMI-KoBAYASm, K. and TATENO, Y.: Robustness of maximum likelihood tree estimation against different patterns of base substitutions. J. Mol. Evol. (in press).
GOJOBORI, T.: Comparative study of molecular evolution. In "Molecular Biosciences",
Vol. 3 (Kimura, M. and Osawa, S., eds.), Iwanami Publisher, 1989, (in Japanese).
GOJOBORI, T.: Genetics. In "Dictionary of Statistics", TKS Publisher, 1989, (in Japanese).
GOJOBORI, T., IKEO, K. and TAKAHASHI, K.: Molecular evolution of kringle domains
in apolipoprotein (a) and other serine proteases. Thromb Haemostas 62: 345, 1989.
GOJOBORI, T. and ISHIWA, S.: Population genetics-How the genes evolve?-NHK Publication Association, 1989, (in Japanese).
GOJOBORI, T., E. N. MORIYAMA: Molecular phylogeny of AIDS viruses and its application
to vaccine development. In "Population Biology of Genes nad Molecules" (N. Takahata and J. F. Crow, eds.), Baifukan, Tokyo, 1990.
GOJOBORI, T., E. N. MORIYAMA, and M. KIMURA: Statistical method for estimating
sequence divergence. In "Methods in Enzymology", (R. F. Doolittle ed.), Academic
Press, Inc., Orlando, Florida, (in press).
GOTOH, H., SHIODA, T., SAKAI, Y., MIZUMOTO, K., SHIBUTA, H.: Rescue of Sendai virus
from viralribonucleoprotein-transfected cells by infection with recombinant vaccinia
viruses carrying Sendai virus L and PIC genes. Virology 171: 434-443, 1989.
HANDA, H., WATANABE, H., SUZUKI, Y. and HIROSE, S.: Effect of DNA supecoiling on
in vitro transcription from the adenovirus early regon 4. FEBS Letters 249: 17-20,
1989.
HANKINS, R. W., NAGATA, K., BUCHER, D. J., POPPLE, S. and ISHIHAMA, A.: Monoclonal
antibody analysis of influenza virus matrix protein epitopes involved in transcription
inhibition. Virus Genes, 3(2): 111-126, 1989.
HANKINS, R. W., NAGATA, K., KATO, A. and ISHIHAMA, A.: Mechanism of influenza
virus matrix (M,) protein transcription inhibition. Res. Virol. 13: 11-22, 1990.
HARA, H., NISHIMURA, Y., KATO, J., SUZUKI, H., NAGASAWA, H., SUZUKI A. and HIROTA,
Y.: Gentic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli. J. Bacteriol. 171: 5882-5889, 1898.
HARADA, Y.-N., BONHOMME, F., NATSUUME-SAKAI, S., TOMITA, T. and MORIWAKI, K.:
Serological survey of complement factor H in common laboratory and wild mice:a new third allotype. Immunogenetics 29: 148-154, 1989.
HATADA, E., HASEGAWA, M., MUKAIGAWA, J., SHIMIZU, K. and FUKUDA, R.: Control
of influenza virus gene expression: Quantitative analysis of each viral RNA species
in infected cells. J. Biochem., 105(4), 537-546, 1989.
HAYAKAWA, T., MIZUKAMI, M., NAKAMURA, I. and SUZUKI, M.: Cloning and sequencing
of RNA-1 eDNA from cucumbermosaic virus strain O. Gene 85: 535-542, 1989.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40
HAYASHI, J.-I., YONEKAWA, H. and TAGASHIRA, Y.: Nuclear but not mitochondria!
genome involvement in 3-methyl cholatherene-induced expression of tumorigenicity
in mouse somatic cells. Cancer Res. 49: 4715~720, 1989.
HIRANO, H., KOMEDA, Y. and IINO, T.: Cloning and structural analysis of the snap-backDNA of Pharbitis nil. Plant Mol. BioI. 12: 235-244,1989.
HONDA, A., MUKAIGAWA, J., YOKOIYAMA, A., KATO, A., UEDA, S., NAGATA, K., KRYSTAL,
M., NAYAK, D. P. and ISHHAMA, A.: Purification and molecular structure of RNApolymerase from influenza virus A/PR8. J. Biochem. 107: 624-628, 1990.
HORAI, S., HAYASAKA, K., MURAYAMA, K. WATE, N., KOIKE, H. and NAKAI, N.: DNA
amplification from ancient human skeletal remains and their sequence analysis. Proc.
Japan Acad., 65B(l0): 229-233,1989.
HORIE, N., NALBANTOGLU, J., KANEDA, S. AYUSAWA, D., SENO, T. and TAKEISHI, K.:
Identification and characterization of an Ll family sequence with a very long open
reading frame in the third intron of the human thymidylate synthase gene. J. Biochem. 106: 1~, 1989.
IGARASHI, K., FUJITA, N. and ISHIHAMA, A.: Promoter selectivity of Escherichia coli RNA
polymerase: Omega factor is responsible for the ppGpp sensitivity. Nucleic Acids
Res., 17(21): 8755-8765, 1989.
IHARA, T., KATO, A., UEDA, S., ISHIHAMA, A. and HIRAI, K.: Comparison of the sequence of the secretary glycoprotein A (gA) gene between Md5 and BC-l strains of
Marek's disease virus type I. Virus Genes, 3(2): 127-140, 1989.
IKEMURA, T.: Purification of RNA molecules by gel techniques. In "Methods in Enzymology. Vol. 180 RNA Processing. Part A", 14-25, Academic Press Inc., 1989.
IKEMURA, T., WADA, K. and AOTA, S.: Giant G+C% mosaic structures of human genome
found by arrangement of GenBank human DNA sequences according to genetic
positions. Genomics (in press), 1990.
ISHIHAMA, A.: Molecular assembly and functional modulation of Escherichia coli RNA
polymerase. In "Adv Biophys.", vol. 26, pp. 19-31, 1990.
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" (Wu, F. Y.-H., ed.), pp. 145-152, Raven Press, New York,1990.
ISHIHAMA, A., FUJITA, N., IGARASHI, K., UESHIMA, R., NAKAYAMA, M. and YAMAZAKI,
Y.: Molecular mechanisms of transcription regulation: Promoter selectivity of
RNA polymerase. FAOB Congeress Preceedings (in press).
ISHIKAWA, R., MORISHIMA H., MORI, K. and KINOSHITA, T.: Chromosomal analysis of
isozyme loci and the allelic expression at cellualr level in rice. Genetical studies onrice plants, XCVII. J. Fac. Agr. Hokkaido Univ. 64(1): 85-98, 1989.
KANEDA, H., MAEDA, Y., MORIWAKI, K., SAKAIZUMI, M., TAYA, C. WATANABE, S. and
YONEKAWA, H.: Research News: 1. t-chromosomes found in East Asiatic wild mice,
M.m. molossinus and M.m. castaneus, etc. Mouse News Letter 84: 119-120, 1989.
KATO, A., SATO, I., IHARA, T., UEDA, S., ISHIHAMA, A. and HIRAI, K.: Homologies between herpesvirus of turkey and Marek's disease virus type-I DNAs within two
colinearly arranged open reading frames, one encoding glycoprotein A. Gene 84:

PVBLICATIONS

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399-405, 1989.
KATO, J., NISHIMURA, Y. and SUZUKI, H.: Escherichia coli par A is an allele of the gyrB
gene. Mol. Gen Genet. 217: 178-181,1989.
KIM, B. K., WATANABE, T. K. and KITAGAWA, 0.: Evolutionary genetics of the Drosophila
montium subgroup. I. Reproductive isolations and the phylogeny. Jpn. J. Genet.
64: 177-190,1989.
KIMURA, M.: Neutral theory. In "Evolution and Animal Breeding" (W. G. Hill and T.
F. C. Mackay, eds.), 13-16, C.A.B. International, Wallingford, 1989.
KIMURA, M.: The neutral theory of molecular evolution and the world view of the·neutralists. Genome 31: 24-31, 1989.
KLEIN, J. and TAKAHATA, N.: The major histocompatibi ity complex and the quest fororigins. Immunological Review 113: 5-25, 1990.
KOBATAKE, E. and SUGIYAMA, T.: Genetic analysis of developmental mechanisms in hydra.
XIX. Stimulation of regeneration by injury in the regeneration-deficient mutant
strain, reg-16. Development 105: 521-528, 1989.
KOHARA, Y.: Correlation between the physical and the genetic map of E. coli K-l!
chromosome. In "The Bacterial Chromosome" (M. Riley and K. Drilica, eds.), Am.
Soc. Microbiol., Washington D.C. (in press).
KOMIYAMA, M., TOYOTA, N. and SHIMADA, Y.: Morpholgenesis of myofibrils in culturesof cardiac myocytes. "Mechanobilogical Research on the Masticatory System"(Kubota, K., ed.), 183-187, VEB Verlage fur Medizin und Biologie, Berlin, 1989.
KURODA, Y.: Antimutagenesis studies in Japan. In "Antimutagenesis and Anticarcin-·ogenesis Mechanisms II" (Y. Kuroda, et al., eds.), 1-22, Plenum Press, New York andLondon, 1990.
KURODA, Y.: Antimutagenic activity of vitamins in cultured mammalian cells. In"Antimutagenesis and Anticarcinogenesis Mechanisms II" (Y. Kuroda, et al., eds.)~
233-256, 1990.
KURODA, Y., SHANKEL, D. M. and WATERS, M. D. (eds.): Antimutagenesis and Anticarcinogenesis Mechanisms II. 485 p, Plenum Press, New York and London, 1990.
KURODA, Y. and SHIMADA, Y.: Electron microscqpic studies on in vitro differentiated.
cel s from Drosophila embryos. In "Invertebrate Cell System Applications Vol. I."(J. Mitsuhashi, ed.), 77-89, CRC 77-89, CRC Press Inc., Boca Raton, Florida, 1989.
KURODA. Y., TAKADA, Y. and KASUYA, T.: Vse of the laser microbeam for preserving.
frozen Drosohila embryos. Zool. Sci. 6: 499-505, 1989.
MATSUMOTO, K., NAGATA, K., YAMANAKA, K., HANAOKA, F. and VI, M.: Nuclear
factor I represses the reverse-oriented transcripton from the adenovirus type 5 left
terminus. Biochem. Biophys. Res. Commun., 164: 1212-1219, 1989.
MATSUMOTO, K., NAGATA, K., HANAOKA F. and VI, M.: Tissue-specific DNA bindingof nuclear proteins that bind to the adenovirus inverted terminal repeat. J. Biochem.
105(6): 927-932, 1989.
MATSUNAGA, E.: Genetic epidemiology of retinoblastoma. In "Genetic Epidemiology
of Cancer" (H. T. Lynch & T. Hirayama, eds.), 119-132, CRC Press, Inc. Florida,
1989.
MATSUNAGA, E., MINODA, K. and SASAKI, M. S.: Parental age and seasonal variation

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40
in the births of children with sporadic retinoblastoma: A mutation-epidemiologic
study. Hum. Genet. 84: 155-158, 1990.
MIYASHITA, N., MORIWAKI, K. and MIGITA, S.: The H-2 class II genes and the susceptibility to the development of pulmonary adenoma in mice. Immunogenetics 29:
14-18, 1989.
MIYAZAWA, S.: DNA Data Bank of Japan: Present status and future plans. In "Computers and DNA: SFI Studies in the Sciences of Complexity. vol. VII", (G. Bell and
T. Marr, eds.), 47-61, Addison-Wesley, 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"(Clawson, G. et al., eds.), pp. 45-56, Alan R. Liss, Inc., New York, 1990.
MIZUMOTO, K., SHIBAGAKI, Y., ITOH, N., YAMADA, H., NAGATA, S. and KAZIRO, Y.:
Structure and function of yeast capping enzyme. J. Cell. Biochem. 130: 209, 1989.
MORISHIMA, H.: Intra-populational genetic diversity in landrace of rice. In "Breeding
Research: The key to the survival of the earth", Proc. 6h Int. Congr. SABRAO,
159-162, 1989.
MORIYAMA, E. N. and GOJOBORI, T.: Evolution of nested genes with special reference
to cuticle protein in Drosophila melanogaster. J. Mol. Evol. 28: 391-397, 1989.
MORIYAMA, E. N. and GOJOBORI, T.: High variability and molecular evolution of AIDS
viruses. In "89' Advanced Sciences-Molecular Biology" (Ozeki, H., ed.), 57-60,
Gijutsu Publisher, 1989, (in Japanese).
NAGASAWA, H., SAKAGAMI, Y., SUZUKI, A., SUZUKI, H., HARA, H. and HIROTA, Y.:
Determination of the cleavage site involved in C-terminal processing of penicillinbinding protein 3 of Escherichia coli. J. Bacteriol. 171: 5890-5893, 1989.
NAGATA, K., TAKEUCHI, K. and ISHIHAMA, A.: In vitro synthesis of influenza viral RNA:
Biochemical complementation assay of factors required for influenza virus replication.
J. Biochem. 106(2): 205-208, 1989.
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.: Size polymorphism of seed enzymes in the genus Glycine analyzed by
SDS-polyacrylamide gel electrophoresis. Proc. 6th Int. Congr. SABRAO, 361364, 1989.
NAKAMURA, K., HASHIMOTO, Y., MORIWAKI, K., YAMAKAWA, T. and SUZUKI, A.: Genetic regulation of GM4 (NeuAc) expression in mouse erythrocytes. J. Biochem.
107: 3-7, 1990.
NAKASHIMA, H., FUJIYAMA, A., KAGIYAMA, S. and Immamura, T.: Genetic polymorphisms of gene conversion within the duplicated human a-globin loci. Human Genet.
84: 568-570, 1990.
NAKAYAMA, M., FUJITA, N., OHAMA, T., OSAWA, S. and ISHIHAMA, A.: Micrococcus
luteus, a bacterium with a high G+C content, contains Escherichia coli-type promoters. Mol. Gen. Genet. 218(3): 384-389, 1989.
NISHIMURA, A.: A new gene controlling the frequency of cell division per round of DNA
replication in Escherichia coli. Mol. Gen. Genet. 215: 286-293, 1989

PUBLICATIONS127

NISHIMURA, A. and HIROTA, Y.: A cell division regulatory mechanism controls the flagellar regulon in Escherichia coli. Mol. Gen. Genet. 216: 340-346, 1989.
NISHIMURA, A., MORITA, M., NISHIMURA, Y., and SUGINO, Y.: A rapid and highly
efficient method for preparation of competent Escherichia coli cells. Nucl. Acids.
Res. 18/20, 1990 (in press).
NOBuHARA, H., KUIDA, K., FURUTANI, M., SHIROISHI, T., MORIWAKI, K., YANAGI, Y.
and TADA, T.: Polymorphism of T-cell receptor genes among laboratory and wild
mice: deverse origins of laboratory mice. Immunogenetics 30: 405-413, 1989.
OHTA, T.: Gene families. In "Evolution and Animal Breeding" (W. G. Hill and T. F. C.
Mackay, eds.) 61-65, C. A. B. International, Wallingford, 1989.
OHTA, T.: Some new aspects of population genetics arising from gene multiplicity.
In: "Population Biology of Genes and Molecules", (N. Takahata and J. F. Crow,eds.), 169-180, Baifukan, Tokyo, 1990.
OHTA, T.: Time for spreading of compensatory mutations under gene duplication.
Genetics 123: 579-584, 1989.
OHTA, T.: The mutational load of a multigene family with uniform members. Genet.
Res. 53: 141-145, 1989.
OHTA, T.: Role of gene duplication in evolution. Genome 31: 304-310, 1989.
OHTA, T. and Hirose, S.: Purification of DNA supercoiling factor from the posteriorsilk gland of Bombyx mori. Proc. Nat!. Acad. Sci. USA. 87: 5307-5311, 1990.
ORITO, E., MIZOKAMI, M., INA, Y., MORIYAMA, E. N., KAMESHIMA, N., YAMAMOTO, M.
and GOJOBORI, T.: Host-independent evolution and a genetic classification of thehepadnavirus family based on nucleotide sequences. Proc. Natl. Acad. Sci. USA,
86: 7059-7062, 1989.
PARVIN, J. D., PALESE, P., HONDA, A., ISHIHAMA, A. and KRYSTAL, M.: Promoter analysis of the influenza virus RNA polymerase. J. Viro1., 63(12), 5142-5152, 1989.
SADAIE, T. and SADAIE, Y.: Rad-2 dependent repair of radiation-induced chromosomal
aberrations in Caenorhabditis elegans. Mutation Res. 218: 25-31,1989.
SADAIE, Y.: Metabolic and cell growth regulation of B. subtilis. In "Bacillus subtilis:
Molecular Biology and industrial Application" (B. Maruo and H. Yoshikawa, eds.),
27-42, Kodansha-Elsevier, 1989.
SADAIE, Y.: Molecular cloning of a Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion. Jpn. J. Genet. 64: 111-119, 1989.
SAGAI, T., SAKAIZUMI, M., MIYASHITA, T. and MORIWAKI, K.: New evidence for transspecies evolution of the H-2 class I polymorphism. Immunogenetics 30: 89-98,
1989.
SANO, Y.: The direction of pollen flow between two co-occuring rice species, Oryza
sativa and O. glaberrina. Heredity 63: 353-357, 1989.
SANO, Y., YI, H. X., SHAO, Q. Q., and IYAMA, S.: Ribosomal DNA spacer-length variations in a wild rice population from Dongxiang, China. In "Breeding Research:
The Key to the Survival of the Earth" (S. Iyama and G. Takeda, eds.), 483-496,
Proc. of 6th Internat. Con. SABRAO, 1989.
SEKINE, M., SAKAIZUMI, M., MORIWAKI, K., YAMAKAWA, T. and SUZUKI, A.: Twogenes controlling the extended globoglycolipids in mouse kidney are closely linked

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40
to each other on chromosome 19. J. Biochem. 105: 680-683, 1989.
SHIMIZU, H.: The effect of injury on Hydra head regeneration. Forma, 4: 21-25, 1989.
SHIMIZU, N., MORIYAMA, E. N. and GOJOBORI,T.: The way to the successful developmentof HIV sythesized vaccines. Medical Immunology No.6, 625-640, 1989, (inJapanese).
SHIMIZU, N., OKAMOTO, T., MORIYAMA, E. N., TAKEUCHI, Y., GOJOBORI, T. and HOSHINO,
H.: Patterns of nucleotide substitutions and implications for the immunologicaldiversity of human immunodeficiency virus. FEBS Lett. 250: 591-595, 1989.
SHIROISHI, T., HANZAWA, N., SAGAI, T., ISHIURA, M., GOJOBORI, T., STEINMETZ, M. and
MORIWAKI, K.: Recombinational hotspot specific to female meiosis in the mouse
major histocompatibility complex. Immunogenet. 31: 79-88, 1990.
SHU, D. S., STYRNA, J. and MORIWAKI, K.: Effect of Y chromosome and H-2 complex
derived from Japanese wild mouse on sperm morphology. Genet. Res., Camb.
53: 17-19,1989.
TABATA, S., HIGASHITANI, A., TAKANAMI, M., AKIYAMA, K., KOHARA, Y., NISHIMURA.
Y., NISHIMURA, A., YASUDA, S. and HIROTA, Y.: Construction of an ordered cosmid
collection of the Escherichia coli K-12 W3110 chromosome. J. Bacteriol. 171:1214-1218, 1989.
TACHIDA, H. and COCKERHAM, C. c.: Evolution of neutral quantitative characters.
with gene interaction and mutation. In: "Population Biology ofGenes and Molecules",(N. Takahata and J. F. Crow, eds.), 233-249, Baifukan, Tokyo, 1990.
TACHIDA, H. and COCKERHAM, C. C.: A building block model for quantitative genetics.
Genetics 121: 839-844, 1989.
TACHIDA, H. and COCKERHAM, C. c.: Effects of identity disequilibrium and linkage on
quantitative variation in finite populations. Genet. Res. Camb. 53: 63-70, 1989.
TACHIDA, H., HARADA, K., LANGLEY, C. H. AQUADRO, C. F., YAMAZAKI, T., COCKERHAM,
C. C. and MUKAI, T.: Restriction map and a-amylase activity variation among
Drosophila mutation accumulation lines. Genet. Res. Camb. 54: 197-203, 1989.
TAJIMA, F.: DNA polymorphism in a subdivided population: the expected number ofsegregating sites in the two-subpopulation model. Genetics 123: 229-240, 1989.
TAJIMA, F.: Statistical method for testing the neutral mutation hypothesis by DNA
polymorphism. Genetics 123: 585-595, 1989.
TAJIMA, F.: The effect of change in population size on DNA polymorphism. Genetics
123: 597-601, 1989.
TAKAHATA, N.: Allelic genealogy and MHC polymorphisms. In: "Population Biologyof Genes and Molecules", (N. Takahata and J. F. Crow, eds.), pp. 267-286, Baifukan,
Tokyo, 1990.
TAKAHATA, N.: Stochasitic models for molecular clocks (with French summary). In"Proceedings for the 47th session of the International Statistical Institute", vol. LIlI:
433-444, Paris, France, 1989.
TAKAHATA, N.: Gene genealogy in three related populations: Consistency probability'
between gene and population trees. Genetics 122: 957-966, 1989.
TAKAHATA, N. and CROW, J. F. (eds.): "Population Biology of Genes and Molecules".
(Proceedings for the 4th International Prize for Biology). Baifukan, Tokyo, 1990.

PUBLICATIONS

129

TAKEISHI, K., KANEDA, S., AYUSAWA, D., SHIMIZU, K., GOTOH, O. and SENO, T.: Humanthymidylate synthase gene: Isolation of phage clones which cover a functionallyactive gene and structural analysis of the region upstream from the translation initiation codon. J. Biochem. 106: 575-583, 1989.
TAMANOI, F., CoBITZ, A., FUJlYAMA, A., GOODMAN, L. E. and PEROU, C.: Posttranslational modification of RAS proteins: Palmitoylation and phosphorylation of yeast
RAS proteins. In "RAS-oncogens", (D. Spandidas, ed.), Plenum Press, NY &London, 1989 (in press).
TANAKA, M., YONEDA, M., OHNO, K., SATO, W., YAMAMOTO, M., NONAKA, I., HORAI.
S. and OZAWA, T.: Differenctly deleted mitochondrial genomes in maternallyinherited chronic progressive external ophthalmoplegia. J. Inher. Metab. Dis.
12: 359-362, 1989.
TANAKA-YAMOMOTO, T., TANAKA, M., OHONO, K., SATO, W., HORAI, S. and OZAWA,
T. : Specific amplication of deleted mitochondrial DNA from a myopathic patientand analysis of deleted region with Sl nuclease. Biochim. Biophys. Acta, 1009(2):151-155, 1989.
TATENO, Y.: Is molecular evolution parsimonious? A theoretical approach to the
problem. In "Population Biology o/Genes and Molecules" (Takahata, N. and Crow,
J. F., eds.), 287-305, Baifukan, Tokyo Macmillan, London, 1990.
TATENO, Y.: A method for molecular phylogeny construction by direct use of nucleotide
sequence data. J. Mol. Evol. 30: 85-93, 1990.
TERAI, M., KOMIYAMA, M. and SHIMADA, Y.: Myofibril assembly in linked with vinculin,
a-actinin, and cell-substrate contacts in embryonic cardiac myocytes in vitro. Cell
MotH. Cytoskeleton 12: 185-194, 1989.
TEZUKA, H.: The mouse mutant, "wasted": tissue-specific radiation sensitivity andhematopoietic cell lineages. In "Antimutagenesis and Anticarcinogenesis MechanismsII" (Y. Kuroda, et al., eds.), Basic Life Sciences Vol. 52, 379-383, Plenum, New
York, (1990).
TSUJlMOTO, H., HASEGAWA, A., MAKI, N., FUKASAWA, M., MIURA, T., SPEIDEL, S., COOPER,
R. W., MORIYAMA, E. N., GOJOBORI, T. and HAYAMI, M.: Sequence of a novel
simian immunodeficiency virus from a wildcaught African mandrill. Nature 341:
539-541, 1989.
UEDA, S., WATANABE, Y., SAITOU, N., OMOTO, K., HAYASHIDA, H., MIYATA, T., HISAJIMA,
H. and HONJO, T.; Nucleotide sequences of immunoglobulinepsilon pseudogene
in man and apes and their phylogenetic relationships. J. Mol. BioI. 205: 85-90,
1989.
UESHIMA, R.; Generic position of Kaliella yaeyamensis pilsbry, 1901 (Pulmonata: Helicarionidae). Venus (Jpn. J. Malacology), 48(2): 85-95, 1989.
UESHIMA, R., FUJITA, N. and ISHIHAMA, A.: DNA supercoiling and temperature shiftaffect the promoter activity of the Escherichia coli rpoH gene encoding the heatshock sigma subunit of RNA polymerase. Mol. Gen. Genet. 215(2): 185-189, 1989.
WADA, A., YAMAZAKI, Y., FumA, N. and ISHIHAMA, A.: Structure and gene map of aribosome modulation factor associated with 100S ribosome in stationary-phase
Escherichia coli. Proc. Natl. Acad. Sci. USA. 87: 2657-2661, 1990.

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40
WADA, K., AOTA, S., TSUCHIYA, R., ISHIBASHI, F., GOJOBORI, T. and Ikemura, T.: Codon
usage tabulated from the GenBank Genetic Sequence Data. Nucl. Acids Res.
18 Supplement. 2367-2411, 1990.
WATANABE, T., MIYASHITA, N., NISHIMURA, M., SAITOU, N., HAYASHI, Y. and MORIWAKI,
K. : Evolutionary relationships between laboratory mice and subspecies of Mus
musculus based on the restriction fragment length variations of the chymotrypsin
gene at the Prt-210cus. Biochem. Genet. 27: 119-130, 1989.
YAMAMOTO, A., HORAI, S. and YUASA, Y.: Increased levels of mitochondrial gene expression in polyps of familial polyposis coli patients. Biochem. Biophys. Res.
Comm. 159(3): 1100-1106, 1989.
YAMAO, F., ANDACHI, Y., MUTO, A., IKEMURA, T. and OSAWA, S.: The tRNA levels inbacterial cells as affected by amino acid usage in proteins. Proc. Japan Acad. 65B:
73-75, 1989.
YAMAUCHI, M., AYUSAWA, D., SHIMIZU, K., SENO, T. and MATSUHASHI, M.: Two typesof mouse FM3A cell mutants deficient in 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and their transformants isolated by human chromosomemediated gene transfer. Somat. Cell Mol. Genet. 15: 39-48, 1989.
YONEDA, M., TANAKA, M., NISHIKIMI, M., SUZUKI, H., TANAKA, K., NISHIZAWA, M.,
ATSUMI, T., OHAMA, E., HORAI, S., IKuTA, F., MIYATAKE, T. and OZAWA, T.: Pleiotrophic molecular defects in energy-transducing complexes in mitochondrial encephalomyopathy (MELAS). J.Neurol. Sci. 92: 143-158, 1989.
YONEDA, M., TSUJI, S., YAMAUCHI, T., !NuZUKA, T., MIYATAKE, T., HORAI, S. and OZAWA,
T.: Mitochondrial DNA mutation in family with Leber's hereditary optic neuropathy. Lancet i: 1076-1077,1989.
YONEZAWA, K., SATO, Y. I., NOMURA, T. and MORISHIMA, H.: Computer simulated
evaluation of the hybrid weakness gene system as a means to prevent genetic contamination of rice cultivars. Plant Breed. 104: 241-247, 1989.
ZENG, Z-B., Tachida, H. and COCKERHAM, C. c.: Effects of mutation on selection limits
in finite populations with multiple alleles. Genetics 122: 977-984, 1989.

ABSTRACTS OF DIARY FOR 1989

131

ABSTRACTS OF DIARY FOR 1989
Biological Symposium
287th meeting Jan.

288th

289th
290th

291st
292nd

293rd
294th(A)294th(B)

295th
296th

5 Genome scanning method for cloning
uncharacterized genes: cloning and characterization of pvn from mutant mouse(Yoichi Gondo)
Jan. 12 1. Current genetic research in R. O. C.
Taiwan2. Computerized numerical assignment
for the nucleotide sequence of T24
human bladder carcinoma oncogene(I-Hung Pan)
Mar. 20 Physical mapping of the C. elegans genome(Alan Coulson)
Mar. 20 High resolution genetic maps of mouse
chromosomes 14 and 17 with special emphasis on the t complex and the disorganization mutation (Joe Nadeau)
Mar. 24 p elements in Drosophila (William R.
Engels)May 12 Genomic imprinting in the mouse andpossible example in man (Bruce M.
Cattanach)May 17 Evolutionary aspects of codon usage(Michael Bulmer)
June
Reverse transcriptase and retrovirus-likeelements in the prokaryotes (Masayori
Inoue)
June 1 M-protein of influenza virus: Antigenic
analysis and intracelluar localization withmonoclonal antibodies (Doris Bucher)Aug. 25 Analysis and prediction of protein structure (William R. Taylor)Aug. 28 Non-Mendelian inheritance and evolution

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

of maternal characters (Russell Lande)
2 G+C % mosaic structures of the genomesof higher vertebrates and plants (Giorgio
Bernardi)
Oct. 26 Eukaryotic transcription factors and mech298th
anisms (Robert Roeder)
299th
Nov. 20 Regulation of growth hormone gene expression (Christopher Lefever)Nov. 22 Studies of mechanisms of DNA transposi300th
tion reactions (Kiyoshi Mizuuchi)
Dec. 4 Microdissection and microcloning of human
301stbanded chromosome (Uwe Classen)
302ndDec. 8 Activation of a silent GC-rich promoter in
an intron caused by deletion of the original
promoter (Makoto Taketo)
Mishima Geneticists' Club
351st
meeting Feb.
A test of the neutral theory based on DNA
polymorphisms (Fumio Tajima)
Feb. 27 Physiological functions of the c-mos on352nd
cogene at the beginning of meiosis(Naoyuki Sagata)
May 2 Physiological roles of interferons: Analysis
353rdusing transgenic mice (Masahide Asano)May 24 What determines the composition and
354thlevels of anticodons in the cells? (Fumiaki
Yamao)
355th
June 2 Evolution of surface antigen genes underbalancing selection (Akira Sasaki)
Restriction map polymorphisms of X chromosome genes in Drosophila melanogaster(Naohiko Miyashita)
356th
June 5 Mouse gentic lethality caused by defect ofsteroid biosynthesis (Hideo Gotoh)
June 28 Control mechanisms of eukaryotic trans357th
cription by regulatory proteins (MasamiHorikoshi)
358thJuly 25 Regulation of expression of the Drosophila
297th

Oct.

ABSTRACTS OF DIARY FOR 1989

359th
360th

361st

362nd

363rd
364th

133

even skipped gene (Tada-atsu Goto)
Aug. 30 Future directions of the protein sequence
database (David G . George)
Sept. 7 Analysis of proteins involved in genetic
recombination in mouse spermatogonia(Atsushi Higashitani)
Function of C-terminal region of E. coli
recA protein (Satoshi Tateishi)
Sept. 9 Isozymes in fish (Yoshihisa Fujio)
Some topics concerned with chromosome
research (Junichi Furuyama)
Development of muscle cells (Yutaka
Shimada)Nov. 13 Plasticity of DNA conformation around the
Drosophila alchohol dehydrogenease gene
(Kaoru Miyahara)
Nov. 16 Genetics of chemosensory identification(Kunio Yamazaki)
Dec. 4 Regulation of gene expression by Drosophila Anthennapedia gene product (Shigeo
Hayashi)134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

FOREIGN VISITORS IN 1989
April 9, 1984-

Pascale Barbier, Universite des Sciences et Techniques
du Languedoc, Montpellier, France
August 3, 1987Ling Hua Tang, Institute of Food Crops, Jiangsu
March 31, 1989
Academy of Agricultural Sciences, China
December 21, 1987- Xiao Mei Wu, Lanzhou Institute of Biological
March 20, 1989
Product, Ministry of Public Health, China
December 21, 1987- He Zhao, Lanzhou Institute of Biological Product,
March 20, 1989
Ministry of Public Health, China
Septemer 1, 1988Guan Cheng Sun, Sericultural Research Institute,
Chinese Academy of Agricultural Sciences, China
January 7, 1989Yong Hong Wang, Lanzhou Institute of Biological
Product, Ministry of Public Health, China
I-Hung Pan, National Taiwan University, Taiwan
January 12
March 2Djoho T. Iskander, Bandung Institute of Technology,
May 30,1989
Indonesia
March 6-16
William B. Provine, Cornell University, U. S. A.
Joe Nadeau, The Jachson Laboratory, U. S. A.
March 20
March 20-22
Alan Coulson, Laboratory of Molecular Biology,
U.K.

March 20-22
March 21-28
March 23
April 7
April 10May 12

Dieter SoIl, Yale University, U. S. A.
William R. Engels, University of Wisconsin, U. S. A.
Cheng Huai Wang, Lanzhou Institute of Biological
Product, Ministry of Public Health, China
E. Liseecki, RERF, HiroshimaFengshan Wang, Tianjin Institute of Industrial
Hygiene and Occupational Diseases, China
Bruce M. Cattanach, MRC Radiobiology Unit,
U.K.

May 16-17
May 30-June 2June 1-2
June 15

Michael Bulmer, University of Oxford, U. K.
Doris Bucher, New York Medical College, U. S. A.
Masayori Inouye, University of Medicine & Dentistry of New Jersey, U. S. A.
A. T. Bachin, Cambridge, U. K.

FOREIGN VISITORS IN 1989

June 15
June 21-23
June 21-23
Jnue 21-23
June 21-23
Jnue 21-23
June 21-23
June 21-23
June 21-23
June 21-23
June 29
July 19
July 19
July 19
July 19
July 19
July 19
July 26
July 26
August 25-26
August 28-29
August 30
October 2
October 20
October 26
November 16

135

C. Queen, Palo Alto, CA, U. S. A.

Graham Cameron, EMBL Data Library, Heidelberg,
Germany
Patricia Kahn, EMBL Data Library, Heidelberg,.
GermanyDavid Hazledine, EMBL Data Library, Heidelberg,
Germany
Jane Peterson, GenBank, NIH, Bethesda, U. S. A.
Julie Ryals, GenBank, Mountain View, CA, U. S. A.
David Benton, GenBank, Mountain View, CA~
U.S.A.
Christian Burks, Los Alamos National Laboratory,
U.S.A.
Paul Gilna, GenBank, Los Alamos National Laboraatory, U. S. A.
Michael Cinkosky, GenBank, Los Alamos National
Laboratory, U. S. A.
Anne Androuais, CNRS, France
S. N. Raina, University of Delhi, India
A. V. Shurkhal, N. I. Vavilov Institute of General
Genetics, U. S. S. R.
Douglas Soltis, Washington State Universiry, U. S. A.
Pamela Soltis, Washington State Universiry, U. S. A.
Steven B. Browles, University of Georgia, U. S. A.
William F. Grant, McGill University, Canada
N. Schuppl, N. I. Vavilov Institure of General
Genetics, U. S. S. R. Academy of Sciences, U. S. S. R.
V. Kolubelov, Institute of Bioorganic Chemistry
U. S. S. R. Academy of Sciences, U. S. S. R.
William R. Taylor, National Institute for Medical
Research, U. K.
Russell Lande, University of Chicago, U. S. A.
David G. George, Georgetown University, U. S. A.
Giorgio Bernardi, Institut Jacques Monod, France
Hubert Hug, University of Zurich, Switzerland
Robert Roeder, The Rockefeller University, U. S. A.
Akkas Uddin Armed, Bangladesh Rice Research

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

November 16
November 16
November 16
November 16
November 16

November 16
November 16
November 16
November 20
November 22
December 4
December 8

Institute, BangladeshPeng Junhua, Crop Research Institute, Sichuan
Academy of Agricultural Sciences, China
Hamed E. EI-Hassawi, Seeds Production, Department, Behera Governorate, Egypt
Gogineni S. V. Prasad, Plant Breeding Division,
Directorate of Rice Research, India
Gholamabbas Kianoush, Manzandaran Agricultural
Research Center, IranMat H. B. Abdullah, Farmers Association, Bakat
Barn, Kemubu Agricultural Development Authority,
Malaysia
Rosa E. S. Infantes, Enterprise of Rice Commercialization, Peru
Primitivo M. Esteban, Jr., Cagayan State University,
Philippines
Sawat Somsa-ard, Department of Agriculture,ThailandChristopher Lefevere, Cedars-Sinai Medical Center,
U.S.A.
Kiyoshi Mizuuchi, National Institutes of Health,
U.S.A.
U. Claussen, University of Erlangen, Germany
Makoto Taketo, The Jackson Laboratory, U. S. A.

AUTHOR INDEX137

AUTHOR INDEXAKAZAWA, S

31

AOKI, K ••...•.•••••••••••..•.••••.• 89
AOTA, S

34,35
29, 30, 31, 32
BARBIER, P
25, 26, 27, 109
BROWN, J. L
38
EIGUCm, M. . •.•••••••.......•..... 112
FUJIOKA, M •.••••.....•.......... 33, 72
FUJISAKI, S
52
FUJISAWA, T. .
71
FUJITA, N
17, 18, 19
FUJIYAMA, A.
99,100
FUKAMI, K •..••••••••......•........ 96
GOJOBORI, T
35,91,92,93
GOTOH, H. • ..•••••.•.......•.... 56, 78
GOTOH, O
29
HAMAMATSU, C. .
108
HANAOKA, F •.•.•...••..••........•. 24
HANDA, H ..••.••••.••............•. 37
HARA, H •.•....••.•••••••... .49, 51, 52
HARADA, Y
79
HASEGAWA, T
99
HAYAMI, M. • ..••..••.........•..... 91
HAYASAKA, K • . • . . • . . . . . . . . . . . . 102, 104
HAYASHIDA, H •.•.....•.........•... 119
HAYWARD, R. S
18
HIGASHITANI, A. • •.•...•........ .43, 44
HIGASHITANI, N. .
.43
HIRAGA, S. .
.46
HIRANO, H. Y
39
HIROSE, S
37, 38, 39
HONDA, A ••.•••••..••.••.•••.•.•.•. 21
HORAI, S
102, 104, 105, 106
HORIUcm, K. . ••••..•.••••••....... .43
HUAI, W. C. .
76
IGARASHI, K. • .•.••••••••.••••••.... 18
IKEMURA, T
34,35,36
IKEo, K •.••.••••••.••.••••••.....•. 93
IMAMURA, T
99, 100, 101
INA, Y
93

AYUSAWA, D

INUZUKA, T

104
53
ISHIBASHI, F •••••••••..•.••••.•••••.. 35
ISHIDA, T
104
ISHIHAMA, A •.•••••• 14,15,16,17,18,19,
20, 21, 22, 23, 24, 25, 26, 27, 28
IWAKuRA, Y. .
23
IYAMA, S
97
KAGIMOTO, M. • ...•••.••.......•... 101
KAGIYAMA, S
100
KAIzUMA, N
115
KAJITANI, N. .
20
KANEDA, S
29,30,32
KANEKURA, T. .
121
KATO, A ...•..•............•••.•. 21, 23
KATO, J
.46
KATSUKI, H • . . . . . . . . . . • . . . . . . . . . . . . . 52
KAWASHIMA, T
76
KIMAURA, M. . ....•.••..........•... 89
KOBAYASHI, M. . . . . . . . • . . . . . . . . . • . . . 14
KOIKE, H ..•.•.•....•....•.•....... 102
KRYSTAL, M. . . . . . . • • . . . . . . . . . . . . . . . 21
KUBOTA, M. . ......••.•............. 15
KURODA, Y
58, 59KYOZUKA, J
116
MATSUMOTO, K ••...••.•.......... 24,34
MINATO, K. . •...••.••••....•....... 68
MITA, A •..........•.••••........... 75
MIYAUCHI, T
121
MIYASHITA, N. .
76, 80, 82
MIYATAKE, T
104
MIYAZAWA, S
119, 121MIZOKAMI, M •..•••••••••••.•....... 93
MrzUMOTO, K. . ...........•••.•• .40, 42
MORISHIMA, H. . ••.••.• 107, 108, 109, 117
MORIWAKI, K •••••• 56,57,75,76,78,79,
80, 82MORIYAMA, E. N
91,92,93
MUKAIGAWA, J.........••........... 21
MURAKAMI, A •••.••• 59, 60, 62, 63, 65, 66
IROBE, R

138

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 40

MURAMATSU, T ••••••••••••••••••••• 121
MURAYAMA,}C••••••••••••••••.•••• 102
NAGATA,}C.

• •••••••••• 14, 21, 22, 23, 24

NAITo, Y. . ............•........... 101
NAKAI, N ••••••.••••••••••.•••••••. 102
NAKAMURA, I.
114, 115, 116
NAKAMURA, }C••..•.••.•••••••.•••••. 54
NAKASHIMA, H
99, 100, 101
NAKAYAMA, M ...•••••••••••.•.•. 19, 23
NALBANTOGLU, J
29

SUGIYAMA, T •••.•••••••.•••••••••••• 69
SUMANTRI, C. • •••••••••••••••••••••• 79
SUZUKI, H.

.

.46, 49SUZUKI,}C. • •••.•••••••..••••••••.•. 53
SUZUKI, Y. • •••••••.•..•••••.•••••.• 37
TACHIDA, H. .

86

TAJIMA, F ••••••.•••••••...•••••••..• 87• ••••••.•••••••...•••••• 58

TAKADA, Y.

TAKAHASHI,}C••••••••••.•••.•.•••.•. 93
TAKAHASHI, M •••.•.•••••••.•.••••.. 25

NARITOMI, Y. . ...•••••....••••.•.•. 101

TAKAHATA, N ••..••••••.••••••••••.. 85

NAYAK, D

TAKAMATSU, H

21
NISHIMURA, A. • .••••....•••••...•.•. 53
NISHIMURA, Y ••...•...•••. 44, 46, 51, 52
NISHINO, T •.•...•...•.•••••.•...•.•• 52
NIWA, M •....•...•...•••....•..•••• 76
NOMURA, T ••.•......••••..•....... 117
ODANAKA, H. .
115
OGASAWARA, N •........•.•.•..... 22, 23
OHNO,}C. . ......•.•.........•...•. 105
OHOYA, Y .......•.•.......•.•.•••.•. 72
OHTA, T. . .......•.......•.•• 38, 83, 84
OISHI,}C. . ....•••.....•..••.....•... 59
ORITO, E ...•.........•••.......•••• 93
OSAWA, S •••...........••.......•••. 19
OZAKI, M ••.........•.•.....•.•.•... 16
OZAWA, M. '" •.......•... " ....••. 121
OZAWA, T •........•••.......•. 104, 105
SADAIE, Y •. " ..... '" .•••.•.....• " .54
SAGAI, T •.....•........•••...•.•••• 56
SANO, Y . . . . . . • • . • . . . . . . . . . 39,111,112
SATO, W .••••.•••.••.•.....•••••••. 105
SATO, Y. -I.
110,114,117
SAWADA, Y ......••.•.••..•.•••.•... 69
SCOTT, M. P
38
SENO, T ••••••••••••••••••• 29,30,31,32
SHIMADA, Y. . ••..•.•••••••••• 59, 72, 73
SHIMAMOTO, }C
116
SHIMAMOTO, N •..•••••..... 32, 33, 34, 72
SHIMIZU, H. .
69
SHlMIZU,}C. • .......••...•....•••... 29
SHIMIZU, N •..••.••.•.•••••••.•••••• 91
SHIROISHI, T •....•.......•. 56,57,76,78
SONODA, S. • •.....••.•....•••••....• 38

54

TAKAYANAGI, A •••..••••...••.••..•. 30
T AKEISHI,}C. • •...••••.....•.••••.... 29
TANAKA, M •..•••••.•..•••.•...•.•• 105
TANAKA-YAMAMOTO, T •.......•••••. 105
TATENO, Y.

• ...••••••......•• 94,96,97

TEZUKA, H

31

TOMITA, T •.••••••.•.••••••.•.•.•.•. 79
TORIYAMA, S.
TSUJI, S.

• •••••.......••.•...••• 25. ...•••••...•••.•••.....•. 104

TSUJIMOTO, H

91

TSUCHIYA, R •...••••...•.•••••••. 35, 36
UEDA, H38

UEDA, M ...••.••••••.•...•••••.•.•• 22
UEDA, S •.•••••••.....•.•••...••••.. 21
UESHIMA, R •.....•...••..•..•.••..•. 28
UEYAMA,}C ••••••.•.••.••••....••••. 53
WADA, A •.•...•••••••••••••••••• 16,17
W ADA, }C.....•.•••••..••••••• 34, 35, 36
WAKASUGI, N.
WATANABE, H. .•.•••••••.•.•••••••• 76

37
36
WATANABE,}C•••••••••••..•••••••••• 72
WATAYA, Y .•••..•••••••..•.•••••.•. 31
WATE, N •.•••••••...•••••••.•••••• 102
Wu, C38
Y AMAGISHI, M ....••••••...•.•••...•• 18
YAMAMOTO, A.
106
YAMAMOTO, Y .•..••••...••.•..••. 44, 51
YAMANAKA,}C. .
22, 23YAMANE,}C. . ........•........••.•.. 54
YAMAOKA, A. •121
YAMAUCHI, T •...••••..••••••..••..• 104
WATANABE, I.

AUTHOR INDEX
15,17
76
YOKOIYAMA, A. . ....•...............23
YOKOTA, E
101
YONEDA, M....................•... 104

139'

YAMAZAKI, Y

YONEKAWA, H •••.••••..•...•••.••••• 800

YAN, W. C

YONEZAWA,K.

116, 117
22, 23YUASA, Y •.•••••••••••••••••••••••• lOt>
YOSHIKAWA, H

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t=PJilU~

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T411.IlUJJliI,=.e,m~m 1111

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