ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 43

1992

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1993

Annual Reportof theNational Institute of Genetics
No. 43, 1992

Published by

The National Institute of Genetics, Japan
1993

CONTENTS
General Statement..........................................
Staff. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Council and Advisory committee..............................
Association for propagation of the knowledge of genetics
Projects of research for 1992
Research activities in 1992.............................

I.

1
3
6
8
10
15

Molecular Genetics
The promoter selectivity control of Escherichia coli RNA polymerase: Mapping of the contact sites on RNA polymerase withtranscription factors. ISHIHAMA, A., Zou, c., KUMAR, A.,
FUJITA, N., SAKURAI, H., MAKINO, K. and HAYWARD, R. S.
15
The promoter selectivity control of Escherichia coli RNA polymerase: Modulation of RNA polymerase at stationary growthphase. SAKURAI, H., TANAKA, K., FUJITA, N. and ISHIHAMA, A.
.
.
Starvation-coupled modulation of Escherichia coli ribosomes: Regulation of the rmf gene encoding the ribosome modulation
factor. YAMAGISHI, M., MATSUSHIMA, H., WADA, A. and
ISHIHAMA, A.
.
.
Molecular composition of fission yeast RNA polymerase II: Analysis of the S. pombe rpb2 gene for RNA polymerase subunit 2.
KAWAGISHI, M., Y AMAGISHI, M. and ISHIHAMA, A.
Molecular composition of fission yeast RNA polymerase II: Analysis of the S. pambe rpb3 gene for RNA polymerase subunit 3.
AZUMA, Y, Y AMAGISHI, M. and ISHIHAMA, A. .
.
Molecular anatomy of influenza virus RNA polymerase: in vitro
reconstitution studies. KOBAYASHI, M., ASANO, Y, MIZUMOTO, K. and ISHIHAMA, A. .
.
Molecular anatomy of influenza virus RNA polymerase: in vitro
replication studies.
TOYODA, T., NAKADA, S. and ISHIHAMA, A. .
.

17

18

18

20

21

22

ii

Regulatory role of the matrix protein on virus growth. YASUDA,
J. and ISHIHAMA, A.
.
.
Molecular assembly of influenza virus: Association of the NS2protein with virion matrix. YASUDA, J., NAKADA, S., KATO,
A., TOYODA, T and ISHIHAMA, A.
.
.
Structure and function of mouse Mx 1 protein: Inhibition mechanism of influenza virus growth. ASANO, Y., TOYODA, T,
SATO, S. and ISHIHAMA, A.
.
.
Ambisense coding strategy of rice stripe virus. HAMAMATSU, c.,
KOBAYASHI, M., TOYODA, T, TORIYAMA, S. and ISHIHAMA,A.
Regulatory elements located at the 5' end of the first intron of thehuman thymidylate synthase gene. KANEDA, S. and SENO, T.

22

23

24

25

25
Cell cycle control by ubiquitin system in mammalian cells; Is theubiquitin-activating enzyme, El, a substrate of cdc2 kinase?
NAGAI, Y, KANEDA, S., SENO, T. and YAMAO, F.
Cell cycle control by ubiquitin system in mammalian cells; Ubiquitin conjugating enzymes specific for DNA synthesis.
KANEDA, S., NAGAI, Y., SENO, T. and YAMAO, F.
Pulverization of chromosomal NORs in the mouse cell mutant of
temperature-sensitive ubiquitin-activating enzyme with thearrest point at the G2 phase of the cell-cycle. SUDHA, T.,
KANEDA, S., NAGAI, Y., Y AMAO, F. and SENO, T.
Mechanism of mRNA capping reaction. MIZUMOTO, K. . .....
Transcription of Sendai virus (HVJ) genome. MIZUMOTO, K.

26

27

28
29
30

Kinetic study on tanscription using immobilized operons: Early
events in transcription elongation. KUBORI, T. and SHIMAMOTO, N.
.
.
Single-molecule dynamics of transcription using immobilized operons: Sliding of RNA polymerase on DNA. KABATA, H.
and SHIMAMOTO, N.
.
.
Functional and structural study of E. coli single-stranded DNA
binding protein. KAMADA, K., NAGAI, H. and SHIMAMOTO,N.
The tissue specific expression of the wx + gene in transgenic rice and

31

31

32

CONTENTS
petunia. HIRANO, H.-Y., MATSUMURA, T, TABAYASHI, N.,
TAN IDA, M., KOMEDA, Y. and SANO, Y.
II.

iii

33

Microbial Genetics
Recognition of DNA replication ongm of bacteriophage fl by
RNA polymerase. HIGASHITANI, N., HIGASHITANI, A. and
HORIUCHI, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Requirement of the correct phasing within the replication enhancerof phage fl. ASANO, S., HIGASHITANI, A. and HORIUCHI,
K.
.
Conformational changes induced in a rolling circle replication
origin by intiator protein. HIGASHITANI, A., HIROKAWA, H.
and HORIUCHI, K.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Stabilization of replication initiator protein DnaA of Escherichia
coli by DnaK chaperone. YASUDA, S., HIGASHITANI, A.,
HORIUCHI, K. and SAKAKIBARA, Y.
A 1.9-kb upstream sequence is required for expression of the fts!
gene coding for penicillin-binding protein 3 of Escherichia coli.
HARA, H., HORIUCHI, K. and PARK, J. T.
A chain-forming strain, PM61, of Escherichia coli contains, inaddition to the envC mutation, an IS4 insertion in the prc genewhich causes accumulation of the precursor form of penicillinbinding protein 3. YAMAMOTO, Y., HARA, H., KARIBIAN,
D., HORIUCHI, K., PARK, J. T. and NISHIMURA, Y.
Osmoregulation of the fatty acid receptor gene fadL through
OmpR protein in Escherichia coli. HIGASHITANI, A., NISHIMURA, Y., HARA, H., AlBA, H., MIZUNO, T and HORIUCHI,
K.
.
Molecular mechanism in determination of timing of cell division in
Escherichia coli K-12(I). NISHIMURA, A.
Molecular mechansim in determination of timing of cell division in
Escherichia coli K-12(II). UKAI, H. and NISHIMURA, A.

34

35

36

38

39

40

41
42

43
III.

Mammalian Genetics

High-order chromatin structure of the recombinational hotspot in
the mouse MHC. MIZUNO, K-I., SAGAI, T, SHIROISHI, T.

iv

and MORIWAKI, K.
Functional analysis of the Lmp-2 gene encoding a subunit of themouse proteasome. MIZUNO, K-I., SAGAI, T., SHIROISHI, T.
and MORIWAKI, K.
Establishment of embryonic stem cell lines from BlO.H-2 congenic
strains II. SAGAI, T., NAKATSUJI, N., SHIROISHI, T. and
MORIWAKI, K.
Genetic control of male-specific suppression of meiotic recombination. SHIROISHI, T., SAGAI, T. and MORIWAKI, K.
Establishment of new congenic strain for insulin-dependent diabetes
mellitus (IDDM) susceptibility gene, Idd-4. WAKANA, S.,SHIROISHI, T. and MORIWAKI, K.
Gene dosage effects of recombinational hotspots in the mouse
MHC. YOSHINO, M., SAGAI, T., FISCHER-LINDAHL, K.,
SHIROISHI, T. and MORIWAKI, K.
Mutation rate of intra-MHC recombinant mice. YOSHINO, M.,
SAGAI, T., SHIROISHI, T. and MORIWAKI, K.
Comparison of the DNA sequence in the D-Ioop region of mousemitochondrial DNA. III. Close relatedness between Japanese
northern population and the castaneus population in the Southeastern Asia. YONEKAWA, H., WATANABE, S., MIYASHITA,
N. and MORIWAKI, K.
A new Pas genes derived from Asian wild mouse. MIYASHITA, N.,
SHIROISHI, T., WAKANA, S., YONEKAWA, H. and MORIWAKI,
K.
.

IV.

44

44

45
46

47

47
48

49

50

Developmental and Somatic Cell Genetics

Formation of a new nerve network in the nerve-free tissue ofepithelial hydra through reintroduction of interstitial cells.
SUGIYAMA, T., MINOBE, S. and KOIZUMI, O.
A simplified method for detecting cell type specific eDNA clones
from Hydra. OHKAWA, T., SHIOKAWA, K. and FUJISAWA,
T.
Protease sensitivities of Hydra cell-cell adhesion. HATTA, M. and
SUGIYAMA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
DNA supercoiling facilitates the assembly of transcriptionallyactive chromatin on the adenovirus major late promoter.

52

53
54

CONTENTS

v

OHBA, R., T ABUCHI, H. and HIROSE, S. .. . . . . . . . . . . . . . .
Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-FI, a member of the steroid receptor superfamily.
TSUKIYAMA, T., VEDA, H., HIROSE, S. and NIWA, O.
A novel DNA-binding motif abuts the zinc finger domain of insect
nuclear hormone receptor FTZ-Fl and mouse embryonal longterminal repeat-binding protein.
VEDA, H., SUN, G.-c.,
MURATA, T. and HIROSE, S.
Production of antibodies specific for double stranded antigen DNA
clones from immune complexes in plasma of a SLE patient.
TERADA, K., HIROSE, S. and OKUHARA, E.
. . . . . . . . . . ..
Permeability of dechorionated eggs and developmental anomalies in
Drosophila. MINATO, K.
. . . . . . . . . . . . . . . . . . . . . . . . . . ..
Control of imaginal cell development by the escargot gene of
Drosophila. HAYASHI, S., HIROSE, S., METCALFE, T. and
SHIRRAS, A.
Role of the escargot gene during Drosophila development. FUSE,
N., HIROSE, S. and HAYASHI, S.
. . . . . . . . . . . . . . . . . . . . ..
Regulation of Drosophila neural development by a putative secreted
protein. OKANO, H., HAYASHI, S., TANIMURA, T., SAWAMOTa, K., YOSHIKAWA, S., WATANABE, J., IWASAKI, M.,
HIROSE, S., MIKOSHIBA, K. and MaNTELL, C. . . . . . . . . ..
Genetics of fluoride-resistance of the nematode Caenorhabditis elegans. KAWAKAMI, M., ISHIHARA, T. and KATSURA, I.
Caenorhabditis elegans larval lethal mutations that cause grossmorphological changes. KATSURA, I., HISHIDA, R. and ISHIHARA, T.
C. elegans mutants abnormal in movement or shape of the head.
ISHIHARA, T. and KATSURA, I.
Construction of lacZ markers for various nerve cells in the head.
ISHIHARA, T. and KATSURA, I.
Systematic analysis of C. elegans eDNA. KOHARA, Y., MITSUKI,
H., NISHIGAKI, A., MOTOHASHI, T. and SUGIMOTO, A.
Tel insertional mutant bank of C. elegans. ANDACHI, Y. and
KOHARA, Y.
Search for localized maternal mRNA in early embryos of C.
elegans. TABARA, H. and KOHARA, Y.

55

55

56

56
57

57
58

59
60

61
62
62
63
64
65

VI

Analysis of morphogenesis in postimplantation mouse embryos.
NAKATSUJI, N. and SHIRAYOSHI, Y.
Manipulation of embryogenesis using mammalian embryonic cells.
NAKATSUJI, N. and SHIRAYOSHI, Y.
Molecular analysis of cell differentiation in postimplantation mouseembryos. SHIRAYOSHI, Y. and NAKATSUJI, N.

V.

66
66
67

Population Genetics
The meaning of natural selection revisited at the molecular level.
OHTA, T.
Theoretical study of near neutrality. II. Effect of subdivided population structure with local extinction and recolonization.
OHTA, T.
Simulation study of a multigene family, with special reference
to the evolution of compensatory advantageous mutations.
BASTEN, C. J. and OHTA, T.
Gene conversion generates hypervariability at the variable regionsof kallikreins and their inhibitors. OHTA, T. and BASTEN, C.
J.
Statistical method for estimating the effective population size in
Pacific salmon. TAJIMA, F. . . . . . . . . . . . . . . . . . . . . . . . . ..
Statistical method for estimating the standard errors of branch
lengths in a phylogenetic tree reconstructed without assuming
equal rates of nucleotide substitution among different lineages.
TAJIMA, F.
Evolutionary genetics of human paleo-populations. TAKAHATA,
N.
.
Some comments on calibration of molecular evolutionary rates.
TAKAHATA, N. and SATTA, Y.
..
.
..
Trans-species polymorphism of the major histocompatibility complex (Mhc) loci. TAKAHATA, N., SATTA, Y. and KLEIN, J.

68

68

69

69
70

70
70
71

72

Polymorphism and balancing selection at major histocompatibility
complex loci. TAKAHATA, N., SATTA, Y. and KLEIN, J. . .
Evolution of the mouse t haplotype: Recent and worldwide introgression to Mus musculus.
MORITA, T., KUBOTA, H.,
MURATA, K., NOZAKI, M., DELARBRE, C. WILLISON, K.,

73

CONTENTS
SATTA, Y., SAKAIZUMI, M., TAKAHATA, N., GACHELIN, G.
and MATSUSHIRO, A. .
.
Evolution of the primate DRB region. KLEIN, J., SATTA, Y.,O'HVIGIN, c., MAYER, W. E. and TAKAHATA, N
.

VI.

VII

74
74

Evolutionary Genetics

Long-range G + C% mosaic structures of the human genome and aborder of the mosaic domains; new genes found in the MHC
class III region. SUGAYA, K., WADA, K., MATSUMOTO, K.
I. and IKEMURA, T. .
.
Analyses on codon usage. IKEMURA, T. .
.
Evolutionary rate of insertions and deletions in nucleotide sequences. SAITOU, N. .
.
Concerted evolution of the primate immunoglobulin alpha genethrough gene conversion. KAWAMURA, S., SAITOU, N. and
VEDA, S.
.
.
Gen~tic affinities of human populations.
SAITOU, N., TOKUNAGA,
K. and OMOTO, K. .
.
Construction of the MHC database and its utilization to anthropological studies. GOJOBORI, T.
.
.
Rates of synonymous substitution and base composition for nuclear
genes in Drosophila. MORIYAMA, E. N. and GOJOBORI, T.

76
76
78

78
78

80

80
Patterns of nucleotide substitution inferred from the phylogenies of
the class I major histocompatibility complex genes. IMANISHI,
T. and GOJOBORI, T.
.
Evolutionary origin of a Kunitz-type trypsin inhibitor domain
inserted in the amyloid /3 precursor protein of Alzheimer's
disease. IKEO, K., TAKAHASHI, K. and GOJOBORI, T.
Allele and haplotype frequencies for HLA and complement loci invarious ethnic groups. IMANISHI, T., AKAzA, T., KIMURA,
A., TOKUNAGA, K. and GOJOBORI, T. .
.
Six strains of human immunodeficiency virus type-l isolated inJapan and their molecular phylogeny. SHIMIZU, N., TAKEUCHI, Y., NARUSE, T., INAGAKI, M., MORIYAMA, E. N.,
GOJOBORI, T. and HOSHINO, H.
.
.
Complement studies of the eleventh international histocompatibility

81

82

82

83

Viii

workshop. TOKUNAGA, K., TANAKA, H., MAuFF, G.,
SUZUKI, K., NISHI MUKAI, H., HAUPTMANN, G., FINLAY, J.,
IMANISHI, T., GOJOBORI, T., AKAZA, T., Jun, T. and DAwKINS, R. L.
VII.

Human Genetics

A simple method for generating sequence tagged sites adjacent to
the human telomere associated repeat sequence. IMAMURA,
T., NAKASHIMA, H., SAKAI, M. and INABA, R.
Mapping of 60 cosmid clones isolated from a human 18 chromosome library by fluorescent in situ hybridization. NAKASHIMA, H., SAKAI, M., INABA, R. and IMAMURA, T.
Human chromosome 18-specific microsatellite polymorphic markers spatially resolvable on the map by fluorescent in situ
hybridization. NAKASHIMA, H., SAKAI, M., INABA, R. and
IMAMURA, T.
......................................
Man's place in Hominoidea revealed by mitochondrial DNA genealogy. HORAI, S., SATTA, Y, HAYASAKA, K., KONDO, R.,
INOUE, T., ISHIDA, T., HAYASHI, S. and TAKAHATA, N.
Mitochondrial DNA mutation and Leigh's syndrome. SAKUTA,
R, GOTO, Y, HORAI, S., OGINO, T., YOSHINAGA, H., OHTAHARA, S. and NONAKA, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Is Parkinson's disease a mitochondrial disorder? NAKAGAWAHATTORI, Y., YOSHINO, H., KONDO, T., MIZUNO, Y andHORAI, S.
Mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episodes (MELAS): A correlative study of theclinical features and mitochondrial DNA mutation. GOTO,
Y., HORAI, S., MATSUOKA, T., KOGA, Y., NlHEI, K. and
NONAKA, I.
"
A novel point mutation in the mitochondrial tRNA Leu(UURj gene ina family with mitochondrial myopathy. GOTO, Y, ToJO, M.,
TOHYAMA, J., HORAI, S. and NONAKA, I. . .... .... . ....
VIII.

84

Applied Genetics

Polymorphism of bacterial blight resistance in natural populationsof wild and cultivated rice. MORISHIMA, H. and MIYABA-

85

86

88

89

89

90

91

91

CONTENTS
YASHI, T.
'" .. .
..
. . . .. . .
. . ..
The birth ofjaponica rice in East China. SATO, Y. I., TANG, S. X.,
YANG, L. U. and TANG, L. H. . . . . . . . . . . . . . . . . . . . . . . ..
Sampling strategy in constructing a core collection from an entire
collection of germplasm. YONEZAWA, K., NOMURA, T. and
MORISHIMA, H.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Recognition of the water surface by a deepwater-tolerance gene(dW3) in rice. EIGUCHI, M., HIRANO, H.-Y., MORISHIMA,
H. and SANO, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Genetic comparisons of chromosome 6 between wild and cultivatedrice. SANO, Y.
Is an egg-killer present in rice? SANO, Y. . . . . . . . . . . . . . . . . ..

IX.

IX

93
94

95

96
97
98

Database

DNA database release 11 from DNA Data Bank of Japan.
UGAWA, Y., IKEO, K., YAMAZAKI, Y., HORIE, M., IWASE,
M., SAITO, M., SATO, Y., SUZUKI, S., HAWAGAWA, Y., HASEGAWA, A., HATTORI, Y., SHIMOYAMA, M., SUZUKI, R.,
UCHIDA, R., SHIDAHARA, Y., MATSUSHIMA, Y., WATANABE,
A., UEDA, Y., KAWAMOTO, T., KITAKAMI, H., SAITOU, N.,
GOJOBORI, T. and TATENO, Y.
100
Integration and search system for a large-scale DNA database.
KITAKAMI, H. and YAMAZAKI, Y.
. . . . . . . . . . . . . . . . . . .. 100
Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Abstracts of diary for 1992
Foreign visitors in 1992 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Author index

102
114
117
120

GENERAL STATEMENT
Our institute was established 43 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofresearch on genetics, in particular population genetics, plant genetics andmolecular biology, by members of our institute have made it the center ofgenetic study in our country and a major institution with world-wide recognition. In 1984 the institute was reorganized into a National Inter-university
Research Institute to promote research activities through cooperation withother institutions. In addition, together with seven inter-university research
institutes, we formed the Graduate University for Advanced Studies, in 1988.
Our institute has admitted graduate students in the Department of Geneticsof the Graduate School of Life Science. We have now about 30 such students
and thus maintaining a steady flow of young scientists that is vital for our
research activities.
Recent rapid progress in the field of genetics with newly developed
approaches has greatly shifted the course of study in biology. Accordingly,
researches in our institute are focused more on molecular studies on various
aspects of genetics. This is not to ignore the importance of more traditional
approaches on genetics of various organisms. Our institute is uniquely
suitable to pursue cooperative works by scientists of various disciplines.
Through interactions between these lines of researches, our institute will
flourish.
We have been carrying out several research related services. We house the
DNA Data Bank of Japan (DDBJ) that gather, annotate, store and distribute information on DNA sequences. As one of the three banks in the world,
DDBJ will continue to serve the world-wide community of biological scienceand technology. Our institute also stores and distributes various organisms
with genetically characterized traits. Among them services on mice, rice andEscherichia coli are particularly significant. These service activities will beadvanced. However, as the responsible manager of these activities, I honestly
feel pain in running service operations in the conditions of understaffing,underpaying and underfunding. Nation-wide recognition of importance of
these activities and appropriate actions to amend these conditions are essential.

2

I hope that with guidance from people in and outside this institute andfurther supports from governmental and private sources, I could lead theinstitute into a more successful future.
In the past year, Associate Prof. S. Hirose was promoted to the professorof the Laboratory of Phenogenetics and Dr. F. Tajima (Laboratory of
Population Genetics) was promoted to the rank of associate professor.
Associate Prof. T. K. Watanabe was transferred to Kyoto Institute ofTechnology, Associate Prof. N. Takahata to the Graduate University for
Advanced Studies both as professor, Dr. H. Tezuka to Yamanashi University
School of Medicine and Dr. H. Tachida to Kyushu University both asassociate professor. Dr. T. Toyoda joined us in the Laboratory of Molecular
Biology and Dr. M. Hatta in the Laboratory of Developmental Genetics.
The DNA Research Center gained the following four as research members:
Drs. H. Nagai, T. Ishihara, Y. Andachi and K. Ikeo.
It is a pleasure to note that Dr. M. Kimura, Emeritus Professor, washonored by the Darwin Medal by the British Royal Society for his work on
the role of stochastic events in determining the rate of molecular evolution,
and Dr. T. Shiroishi the Encouragement Award of the Genetics Society ofJapan for his study on recombinational hotspots of the mouse major histocompatibility complex.

STAFF
Director-General

TOMIZAWA, Jun-ichi, Ph. D.
Vice-Director

MORIWAKI, Kazuo, D. Sc.
Members1.

Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Division of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
YAMAGISHI, Masahiro, D. Sc.
TOYODA, Tetsuya, D. Med.
Division of Mutagenesis
SENO, Takeshi, D. Sc., Professor
YAMAO, Fumiaki, D. Sc., Associate professor
KANEDA, Sumiko, D. Pharm.
Division of Nucleic Acid Chemistry (Guest member)
MIZUMOTO, Kiyohisa, D. Sc., Professor
YASUDA, Hideyo, D. Pharm., Associate professor

2.

Department of Cell Genetics
MORIWAKI, Kazuo, D. Sc., Head of the Department
Division of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami T., D. Sc., Associate professor
SHIROISHI, Toshihiko, D. Sc.
GOTO, Hideo, D. Ag.

4Division of Microbial Genetics
HORIUCHI, Kensuke, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Division of Cytoplasmic Genetics (Guest member)
OHTSUBO, Eiichi, D. Sc., Professor
YONEKAWA, Hiromichi, D. Sc., Associate professor
3.

Department of Ontogenetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
Division of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
HATTA, Masayuki, D. Sc.
Division of Phenogenetics
HIROSE, Susumu, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Division of Physiological Genetics (Guest member)
KOIZUMI, Osamu, D. Sc., Professor

4.

Department of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Head of the Department
Division of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAJIMA, Fumio, Ph. D., Associate professor
Division of Evolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate professor
MATsuMoTo, Ken-ichi, D. Ag.
MORIYAMA, Etsuko, Ph. D.
Division of Theoretical Genetics (Guest member)
TAKAHATA, Naoyuki, D. Sc., Professor
YASUNAGA, Teruo, Ph. D., Associate professor

STAFF

5.

Department of Integrated Genetics
IMAMURA, Takashi, D. Med., Head of the Department
Division of Human Genetics
IMAMURA, Takashi, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate professor
HORAI, Satoshi, D. Med., Associate professor
NAKASHIMA, Hitoshi, D. Med.
Division of Agricultural Genetics
MORISHIMA, Hiroko, D. Ag., Professor
SANO, Yoshio, D. Ag., Associate professor
SATO, Yoichiro, D. Ag.
HIRANO, Hiro-Yuki, D. Ag.
Division of Applied Genetics (Guest member)
WATANABE, Takeshi, D. Med., Professor
YONEZAWA, Katsuei, D. Ag., Professor

6.

Research Facilities
Genetic Stock Research Center
MORISHIMA, Hiroko, D. Ag., Head of the Center
NAKATSUJI, Norio, D. Sc., Professor
MIYASHITA, Nobumoto, D. Med.
SHIRAYOSHI, Yasuaki, D. Sc.
UEDA, Hitoshi, D. Ag.
NISHIMURA, Akiko, D. Ag., Associate professor
TATENO, Yoshio, Ph. D., D. Sc., Associate professor
DNA Research Center
SENO, Takeshi, D. Sc., Head of the Center
SHIMAMOTO, Nobuo, D. Sc., Associate professor
NAGAI, Hiroki, D. Sc.
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
HAYASHI, Shigeo, D. Sc.
GOJOBORI, Takashi, D. Sc., Professor
UGAWA, Yoshihiro, D. Sc.
IKEO, Kazuho, D. Sc.
KOHARA, Yuji, D. Sc., Associate professor
ANDACHI, Yoshiki, D. Sc.

5

6

KITAKAMI, Hajime, D. Eng., Associate professor
YAMAZAKI, Yukiko, D. Sc.
Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate professor, Head of the Center
Experimental Farm
SANO, Yoshio, D. Ag., Head of the Farm
NAKAMURA, Ikuo, D. Ag.

7.

Technical Section
MITA, Akihiko, Chief of the Section

8.

Department of Administration
EBIHARA, Akira, Head of the Department
NINOMIYA, Shigeo, Chief of the General Affairs Section
MABUCHI, Kenji, Chief of the Finance Section

COUNCIL
Chairman
NAGAKURA, Saburo; President, Graduate University for Advanced
StudiesVice-chairman
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
Members (Alphabetical order)
EBASHI, Setsuro; President, Okazaki National Research Institutes
HAMAl, Osamu; Dean, Faculty of Letters, University of TokyoICHIKA W A, Atsunobu; Deputy Director, the National Institute forEnvironmental Sciences
IINO, Tetsuo; Professor, Faculty of Human Science, Waseda University
KIMURA, Motoo; Professor emeritus, National Institute of Genetics
NOJIMA, Shoshichi; Dean, Faculty of Pharmaceutical Science, Teikyo
University
ONOE, Hisao; President, Shiga University
Om, Tatsuo; Professor, Faculty of Home Economics, Kyoto Women's
University
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
SANO, Hirotoshi; President, Tokyo Metropolitan University

ADVISORY COMMITTEE

7

SUGANO, Haruo; Director, Cancer Institute
SUGIMURA, Takashi; President emeritus, National Cancer Center
T AKANAMI, Mitsuru; Professor, Institute for Chemical Research, Kyoto
University
TAKEUCHI, Ikuo; Director-General, Institute for Basic Biology
TANAKA, Ryuso; President, Hiroshima University
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University
YOSHIOKA, Morimasa; President, Tokyo Women's Medical College
YURA, Takashi; Director, Institute for Virus Research, Kyoto University

ADVISORY COMMITTEE
ChairmanMORIWAKI, Kazuo; Professor, Division of Cytogenetics
Vice-chairman
OSAWA, Shozo; Adviser Planning Department, Japan Tabacco Inc.
Members (Alphabetical order)
FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
HINATA, Kokichi; Professor, Faculty of Agriculture, Tohoku University
ISHIWA, Sadao; Professor, Faculty of Science, Ochanomizu Women's
University
MIURA, Kin-ichiro; Professor, Faculty of Science, Gakushuin University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
T AKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto University
TAKEUCHI, Takuji; Professor, Faculty of Science, Tohoku University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YOSHIKAWA, Hiroshi; Professor, Faculty of Medicine, Osaka University
GOJOBORI, Takashi; Professor, DNA Resarch Center
HORIUCHI, Kensuke; Professor, Division of Microbial Genetics
IKEMURA, Toshimichi; Professor, Division of Evolutionary Genetics

8

IMAMURA, Takashi; Professor, Division of Human Genetics
ISHIHAMA, Akira; Professor, Division of Molecular Genetics
MORISHIMA, Hiroko; Professor, Division of Agricultural Genetics
NAKATUJI, Norio; Professor, Genetic Stock Research Center
OHTA, Tomoko; Professor, Division of Population Genetics
SENO, Takeshi; Professor, Division of Mutagenesis
SUGIYAMA, Tsutomu; Professor, Division of Developmental Genetics

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

ADVISORY COMMITTEE

9

MORISHIMA, Hiroko; Professor, National Institute of Genetics

Adviser
MORIWAKI, Daigoro; Honorary member, National Institute of Genetics

PROJECTS OF RESEARCH FOR 1992
1.

DEPARTMENT OF MOLECULAR GENETICS
Division of Molecular Genetics

Regulatory mechanisms of gene transcription in prokaryotes (lSHIHAMA,
FUJITA and YAMAGISHI)
Molecular architecture of transcription apparatus from eukaryotes (lSHIHAMA and YAMAGISHI)
Molecular mechanisms of transcription and replication of animal and plantviruses (IsHIHAMA, TOYODA and NAKAMURA)
Division of Mutagenesis

Molecular and genetic studies of cell-cycle associated genes (SENO, YAMAO
and KANEDA)
Gt:ndil.: l.:onsequences of thymidylate stress in mammalian cells (SENO andYAMAO)
Radiation sensitivity in mammals (TEZUKA)
Division of Nucleic Acid Chemistry

Mechanism of mRNA capping (Mizumoto)Transcription of the Sendai virus genome (Mizumoto)
Cell-cycle regulation by cyclin and CDC2 kinase (YASUDA)
2.

DEPARTMENT OF CELL GENETICS
Division of Cytogenetics

Genetic differentiation of mouse species (MORIWAKI, SHIROISHI and MIYASHITA*)* Genetic stock research center.

PROJECTS OF RESEARCH FOR 1992

11

Recombinational hotspots in the mouse MHC (SHIROISHI and MORIWAKI)
Theoretical and experimental bases for karyotype evolution (IMAi)
Study of adrenal function in mouse development by transgene (GOTOH,SHIROISHI and MORIWAKI)
Division of Microbial Genetics

DNA replication in E. coli (YASUDA, HIGASHITANI and HORIUCHI)
Cell cycle regulation in E. coli (HARA, HIGASHITANI and HORIUCHi)
Division of Cytoplasmic Genetics

Studies on cytoplasmic genes during subspecies differentiation of house
mouse Mus musculus (YONEKAWA)
Studies on bacterial plasmids and transposons (OHTSUBO)
3.

DEPARTMENT OF ONTOGENETICS
Division of Developmental Genetics

Genetic and molecular analysis of developmental mechanisms III hydra(SUGIYAMA, FUJISAWA, SHIMIZU and HATTA)
Division of Phenogenetics

Gene expression in eukaryotes (HIROSE, VEDA and HAYASHI)
Genetic studies on the life history characters in Bombyx (MURAKAMI)
Genetic studies on the nerve system characters in Bombyx (MURAKAMI)
Genetic studies on development and growth of insect (YAMADA andMINATO)
Division of Physiological Genetics

Nerve net formation in Hydra (KOIZUMI)

12

4.

DEPARTMENT OF POPULATION GENETICS
Division of Population Genetics

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

Studies on codon usage (IKEMURA)
Studies on chromosome band structures at the DNA sequence level (lKEMURA and MATSUMOTO)
Studies on genes in HLA locus (MATSUMOTO and IKEMURA)
Molecular evolutionary analysis of nucleotide sequence data (SAITOU)
Studies on the genetic affinity of human populations (SAITOU)
Studies on molecular evolution on viruses (MORIYAMA)
Studies on molecular evolution of Drosophila genes (MORIYAMA)
Division of Theoretical Genetics

Population immunogenetics (TAKAHATA)
Molecular anthropology (TAKAHATA)
Computer analysis of genetic information (YASUNAGA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Division of Human Genetics

Genetic and physical mapping of human genome (IMAMURA and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA and NAKASHIMA)
Molecular biology of oncogenes (FUJIYAMA)PROJECTS OF RESEARCH FOR 1992

13

Studies on DNA polymorphisms in human populations (HORAI)
Division of Agricultural Genetics

Evolutionary and ecological genetics in wild and cultivated nce species(MORISHIMA and SATO)
Genetic studies of speciation in rice (SANO and HIRANO)
Studies on plant gene expression (HIRANO and SANO)
Bio-archaeological analysis of plant remains (SATO and NAKAMURA)
Division of Applied Genetics

Molecular genetics of human immune mechanisms (WATANABE)
Theoretical studies on conservation biology (YONEZA W A)
6.

RESEARCH FACILITIESGenetic Stock Research Center

Analysis of morphogenesis in postimplantation mouse embryos (NAKATSUJI
and SHIRAYOSHI)
Manipulation of embryogenesis using mammalian embryonic cells (NAKATSUJI and SHIRAYOSHI)
Molecular analysis of cell differentiation in postimplantation mouse embryos(SHIRAYOSHI and NAKATSUJI)
Genetic mechanisms for regulating tumor development in the laboratory andwild mice (MIYASHITA and MORIWAKI)
Evolutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA and HIROSE)
Molecular mechanism of cell division in E. coli (NISHIMURA)
Theoretical studies in molecular phylogeny (TATENO)14

DNA Research Center

Interaction between proteins and nucleic acids (SHIMAMOTO and NAGAI)
Developmental and behavioral genetics of C. elegans (KATSURA and ISHIHARA)
Computer analysis of DNA sequences (GOJOBORI and UGAWA)
Construction of DNA sequence database (GOJOBORI, UGAWA, IKEO andTATENO)
Molecular evolution of DNA sequences (lKEO and GOJOBORI)
Molecular genetics of Drosophila development (HAYASHI and HIROSE)
Molecular biology of C. elegans development (KOHARA and ANDACHI)
Genome analysis of C. elegans (KOHARA)
Large-scale genetic information analysis project (KITAKAMI and YAMAZAKI)Radioisotope Center

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

Molecular genetics of morphological mutants in rice (NAKAMURA)
DNA fingerprinting of plant species (NAKAMURA)RESEARCH ACTIVITIES IN 1992

I.

MOLECULAR GENETICS

The Promoter Selectivity Control of Escherichia coli RNA Polymerase:
Mapping of the Contact Sites on RNA Polymerase withTranscription Factors
Akira ISHIHAMA, Chao Zou, Ashok KUMAR*, Nobuyuki FUJITA,
Hitomi SAKURAI, Kozo MAKINO** and Richard S. Hayward*

The promoter selectivity control of RNA polymerase plays a key role inglobal regulation of gene transcription in prokaryotes. A number of accessory transcription factors have been identified in Escherichia coli, which interact
with RNA polymerase and modulate its promoter selection pattern. One
group factors form RNA polymerase complexes in the absence of DNA,
while the other group factors bind to DNA and interect with RNA polymerase when both are aligned along the same DNA strand (Ishihama, A.
(1988) Trends Genet. 4, 482-486). Regardless of apparent difference in theorder of molecular assembly, ternary complexes are formed between RNA
polymerase, transcription factors and DNA signals (promoter and enhancer)
prior to transcription initiation.
Since the finding of the presence of at least two contact sites on E. coli
RNA polymerase for CRP (cAMP receptor protein) (Igarashi, A. and
Ishihama, A. (1991) Cell 65, 1015-1022; Igarashi et al. (1991) Proc. Natl.
Acad. Sci. USA 88, 8958-8962), our effort has been focussed towards theclassification of various transcription factors on the basis of the contact site
selection on RNA polymerase. In collaboration with a large number oflaboratories, various transcription factors have been examined as to theirabilities to activate mutant RNA polymerases contsisting of C-terminal
truncated a subunits which lack the contact site I for class-I factors (review***

On leave of absence from Institute of Cell and Molecular Biology, University of
Edinburgh, Edinburgh, UK.
Institute of Microbial Diseases, Osaka University, Suita, Osaka, Japan.

16
Table I.
FactorContact site*

Ada
CRP

I
I/II

IHF
itd/clI
NtrC
OmpR
OxyR
PhoB

I
II
II
I
I
II

Classification of transcription factors
Publications

Sakumi, K. et al. (1993) J. Bacterial. 175, 1162-1167.
Igarashi, K. and Ishihama, A. (1991) Ce1/65, 1015-1022.
Igarashi, K. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8958-8962.
Kolb, A. et al. (1993) Nucleic Acids Res. 21, 319-326.
Zou, C. et al. (1992) Mol. Microbial. 6,2599-2605.
Giladi, H. et al. (1992) J. Mol. Bioi. 227,985-990.
Gussin, G. N. et al. (1992) J. Bacterial. 174,5156-5160.
Lee, H.-S. et al. (1993) J. Bacterial. 175, 1568-1572.
Igarashi, K. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8958-8962.
Tao, K. et al. (1993) Mol. Microbial. 7, 859-864.
Igarashi, K. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8958-8962.

* Contact site I=a subunit; contact site II=a subunit

ed in Ishihama, A. (1992) Mol. Microbial. 6,3283-3288; Ishihama, A. (1993)
J. Bacterial. 175, 2483-2489). Some of the results have been published aslisted in Table 1.
Fine mapping of the contact site I region was carried out for CRP (Zou etal., 1992) and OxyR (Tao, K., unpublished). Taken together with resultsfrom other laboratories, it turned clear that multiple contact sites exist within
the contact site-I region in the C-terminal one-third of RNA polymerase a
subunit, each interacting with a different set of transcription factors and eachconsisting of a structure formed by a short peptide of about 10 amino acidresidues (reviewed in Ishihama, A. (1992) Mol. Microbial. 6, 3283-3288;
Ishihama, A. (1993) J. Bacterial. 175, 2483-2489).
Recently two trascription factors, PhoB (in the case of pstS transcription)
and CRP (in the case of galPl transcription), were found to make contact
with 0 70 subunit at upstream from the region 4 (the recognition domain ofpromoter -35) (Makino, K. et al. (1993) Genes Devel. 7, 149-169; Ashok,
K. et al. (1993) J. Mol. Bioi., in press). Analysis of C-terminal deletion of the70
0
subunit indicated that these two class-II transcription factors interact atdifferent sites within this contact site-II region (Ashok, K. et al. (1993) J.
Mol. Bioi., in press).

1.

MOLECULAR GENETICS

17

The Promoter Selectivity Control of Escherichia coli RNA Polymerase:
Modulation of RNA Polymerase at Stationary Growth-Phase
Hitomi SAKURAI, Kan TANAKA*, Nobuyuki FUITA and Akira ISHIHAMA

Under starved environmental conditions, some bacterial species such as B.
subtilis maintain cell viability by forming dormant spores. Even in nondifferentiating bacteria such as E. coli, however, genetic programs operate as
to establish a metabolically less active and more stress-resistant state. Resultsof our previous studies led us to postulate that the modulations of both RNA
polymerase and ribosomes play major roles in this switch of the genetic
program (Ozaki, M. et al. (1991) Mol. Gen. Genet. 230, 17-23; Ozaki, M. etal. (1992) Nucleic Acids Res. 20,257-261; Wada, A. et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 2657-2661). The marked decrease in the net charge forthe stationary-specific core enzymes itself indicated protein modification withmoieties of highly negative charge. Preliminary experiments indicated theattachment of phosphate moieties in this modification. The chemical natureof this modification is being analyzed, by making a large amount of stationary-specific RNA polymerase at different phases of cell growth.
In addition to the core enzyme modification, replacement of the a subunit
has been proposed. In collaboration with Dr. K. Tanaka et al. (Univ.
Tokyo), we succeded the detection in vitro of sigma activity for expressed andpurified d 8 (or ~; the rpoS(katF) gene product) when bound to the core
enzyme isolated from exponentially growing cells (Tanaka, K. et al. (1993)
Proc. Natl. Acad. Sci. USA, 90,3511-3518). A systematic comparison of thepromoter selectivity between Ea70 and Ed 8 holoenzymes indicated that Ea70
recognizes a set of promoters associated with highly expressed genes in
exponentially growing E. coli while Ea38 initiates transcription from promoters of stationary phase-specific genes. However, a group of promoters arerecognized by both holoenzymes, indicating cross-talk between two sigma
factors.

*

Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan.

18

Starvation-Coupled Modulation of Escherichia coli Ribosomes:
Regulation of the rmf Gene Encoding the
Ribosome Modulation Factor
Masahiro YAMAGISHI, Hiroshi MATSUSHIMA*, Akira WADA** and Akira ISHlHAMA

Ribosome modulation factor (RMF) is a protein specifically associatedwith WOS ribosome dimers which start to accumulate in Escherichia coli upon
growth transition from exponential to stationary phase (Wada, A. et a/.
(1990) Proc. Nat/. A cad. Sci. USA 87,2657-2661). The gene, rmf, encoding
RMF was cloned from 21. 8 min region of the E. coli genome, and sequence
analysis indicated that rmf codes for a small basic protein of 55 amino acidresidues (Yamagishi, M. et al. (1993) EMBO J. 12,625-630). By Northernblot analysis, it was found that rmf was silent in rapidly growing cells at
exponential phase, but became highly expressed concomitantly with thegrowth transition to stationary phase.
Under slow growth conditions, however, rmf was expressed even at theexponential growth phase, and an inverse relationship was observed between
the expression of rmf and the cell growth rate. Thus, the expression profile
of rmf is contrary to that of the genes for components of translational
appratus such as ribosomal proteins and ribosome-associated proteins. Disruption of rmf resulted in loss of ribosome dimers and reduction of cell
viability during stationary phase.
Molecular Composition of Fission Yeast RNA Polymerase II:
Analysis of the S. pombe rpb2 Gene for RNA
Polymerase Subunit 2
Makiko KAWAGISHI, Masahiro YAMAGISHI and Akira ISHIHAMARNA polymerase plays a central role in gene expression. Analysis of thestructure and function of RNA polymerase is essential to understand themolecular mechanisms of transcriptional regulation. In eukaryotes, threeclasses of nuclear RNA polymerases, I, II and III have been identified, each
responsible for transcription of large ribosomal RNA, mRNA, and small
ribosomal RNA and tRNA, respectively. Each class RNA polymerase is* On leave of absence from Health Science Research Institute, Yokohama, Japan.
**

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

I.

MOLECULAR GENETICS

19

composed of two large polypeptides and about 10 smaller subunits. However,
the minimum essential subunits have not been identified for anyone of thethree RNA polymerases. In order to extend our knowledge of the subunit
composition of RNA polymerase II and the role of each subunit in transcription, we started biochemical and genetical studies of RNA polymerase IIfrom the fission yeast, Schizosaccharomyces pombe. Last year, we cloned andsequenced the rpbI gene for the largest subunit (subunit 1) using thecorresponding gene (RPBl) of the budding yeast, Saccharomyces cerevisiae,
as the hybridization probe (Azuma, Y. et al. (1991) Nucleic Acids Res. 19,
461-468).
This year we cloned the S. pombe rpb2 gene by cross-hybridization with theS. cerevisiae RPB2 gene. The rpb2 gene encodes the subunit 2 (the second
largest subunit) of RNA polymerase II with 1210 amino acid residues and acalculated molecular weight of 138 kDa (Kawagishi, M. et al. (1993) Nucleic
Acids Res. 21,469-473). From the result of gene disruption experiment, weconcluded that the rpb2 gene is essential for cell viability.
Because of a local sequence similarity between a part of the subunit 2 of
RNA polymerase II and bacterial RNase such as barnase, we tried to test
whether this RNase-like region of the subunit 2 carries RNase activity or not.
For this purpose, we expressed in E. coli a GST-fusion protein including thisRNase-like region of the subunit 2. In a preliminary experiment, the partiallypurified GST-fused protein showed RNase activity in vitro, even though its
activity was much weaker than the authentic barnase. As an attempt toconfirm this observation, we introduced a mutation at an essential site for thecatalytic activity of barnase. The GST-fusion protein containing this mutant
rpb2 segment showed weaker activity of RNA hydrolysis than the unmodified
fusion protein. Further confirmatory experiments are being carried out using
a highly purified preparation of the fusion proteins. The specificity of thissubunit 2-associated RNase, and the biological role(s) of this RNase-likeregion all remain unsolved.

20

Molecular Composition of Fission Yeast RNA Polymerase II:
Analysis of the S. pombe rpb3 Gene for RNA
Polymerase Subunit 3
Yoshinao AZUMA, Masahiro Y AMAGISHI and Akira ISHIHAMAIn order to improve our understanding of the structure-function relationship of RNA polymerase II, we continued the cloning, sequencing andcharacterization of the genes encoding proteins identified in a highly purified
RNA polymerase II fraction from the fission yeast S. pombe. Up to thepresent time, we have isolated the genes, rpbI and rpb2, coding for the largest(subunit I) and the second-largest (subunit 2) subunits, respectively, of
RNA polymerase II (Azuma, Y. et al. (1991) Nucleic Acids Res. 19,461-468;
Kawagishi, M. et al. (1993) Nucleic Acids Res. 21, 469-473). This year wecloned and sequenced the gene, rpb3, coding for the third-largest subunit(subunit 3).
Since the RPB3 gene for the RNA polymerase II subunit 3 of the budding
yeast Saccharomyces cerevisiae did not cross-hybridize with S. pombe DNA,
we tried to clone the subunit 3 gene using synthetic probes designed based on
the knowledge of amino acid sequence. For this purpose, we first purified
RNA polymerase II from S. pombe. By SDS-gel electrophoresis, a highly
purified RNA polymerase II fraction gave at least 11 bands. The sizes of twolarge subunits were estimated to be about 210 and 150 kDa, respectively,
close to those calculated from the nucleotide sequences of the cloned rpbI andrpb2 genes and similar to those of subunits 1 and 2 of S. cerevisiae RNA
polymerase II. We isolated the third-largest subunit with approximate
molecular weight of 40 kDa and subjected it to amino acid microsequencing.
With use of three different oligonucleotide primers designed from the partial
amino acid sequences, we cloned the gene encoding the putative subunit 3
polypeptide. Using the genomic clone as a probe, we also cloned cDNA for
this polypeptide. Sequence determination indicated that this gene, interrupted by two introns, codes for a polypeptide of 307 amino acid residues
with a calculated molecular weight of 34 kDa. This protein contains fiveconserved structural domains of the third largest subunits from other eukaryotic RNA polymerase II. Thus, we concluded that the gene we cloned is S.
pombe rpb3 encoding the subunit 3 of RNA polymerase II.

I.

MOLECULAR GENETICS

21

Molecular Anatomy of Influenza Virus RNA Polymerase:in vitro Reconstitution Studies
Makoto KOBAYASHI, Yukiyasu ASANO, Kiyohisa MIZUMOTO* and Akira ISHIHAMA

Influenza virus RNA polymerase catalyzes multiple step reactions intranscription and replication of the genome RNA. The core enzyme iscomposed of each one of the three P proteins, PB I, PB2 and PA (Honda, A.
et al. (1990) J. Biochem. 107, 624~628). Subunit PB1 is involved in RNA
polymerization, while PB2 is involved in capped RNA recognition. The
function of PA protein is totally unknown. For detailed analysis of the roleof each P protein and functional domains on each P polypeptide, we
expressed individual P proteins in insect cells infected with recombinantbaculoviruses carrying cDNA for each P protein (Kobayashi, M. et. al.
(1992) Virus Res. 22, 235-245). RNA polymerase reconstituted by mixing
three purified P proteins was able to transcribe short model RNA templates
containing the conserved terminal sequences of influenza virus RNA segments. The mixture of any two of the three P proteins did not exhibittranscription activity, confirming that the RNA polymerase is composed of
the three P proteins. We started to make a set of mutant RNA polymerases
containing mutant P proteins. For this purpose, we established expression
systems of each P protein in Escherichia coli using T7 RNA polymerase!
promoter system. In parallel, we started to identify functional sites on each
P protein subunit by chemical cross-linking with analogues of substrates,
primers, templates and products. As expected, a GTP analogue was crosslinked to the PB 1 protein.
In addition to the three P proteins, NP protein is required for efficient
elongation of RNA chains (Honda et al. (1988) J. Biochem. 104, 10211026). In order to construct in vitro transcription system of long RNA
templates, the NP protein was prepared from E. coli expressing cDNA for the
NP gene. A set of deletion mutants of the NP protein was made in order to
identify the functional domain required for RNA elongation.

*

Division of Nucleic Acid Chemistry, Department of Molecular Genetics.

22

Molecular Anatomy of Influenza Virus RNA Polymerase:in vitro Replication Studies
Tetsuya TOYODA, Susumu NAKADA* and Akira ISHIHAMA

RNA-dependent RNA polymerase associated with influenza vIrIons iscomposed of each one of the three P proteins (PBl, PB2 and PA) (Honda,
A. et al. (1990) J. Bichem. 107,624-628; Kobayashi et al. (1992) Virus Res.
22, 235-245). In influenza virus-infected cells, the RNA polymerase is
invloved in both transciption and replication of eight genomic RNAs(vRNAs). In order to reveal the difference in the molecular architecture
between the two RNA polymerase complexes, i.e., transcriptase and replicase, we prepared nuclear extracts from influenza virus AjPR8-infected
MDCK and HeLa cells. Both extracts catalyzed the synthesis of not onlymRNA but also cRNA (the first step reaction of replication). We also
detected these two activities for cell extracts of the clone 76 cell line, which
expresses influenza virus three P and NP proteins (Nakamura et al. (1991) J.
Biochem. 110, 395-401). Theses observations indicates that three P proteins
and NP are enough for transcription and replication.
We reconstituted an in vitro system of primer-independent RNA synthesis
using high salt-treated RNP from virus-infected cells and nuclear extracts of
uninfected cells. Identification of the host factor( s) required for replicationof the influenza virus genome is in progress.
Regulatory Role of the Matrix Protein on Virus Growth
Jiro YASUDA and Akira ISHIHAMA

The genome of influenza A virus is composed of eight RNA segments ofnegative polarity. RNA segment 7 encodes two molecular species of the Mprotein. The Ml protein forms matrix (or membrane) of virions, while M2
forms an ion channel in virus-infected cell membranes. M 1 is an amphiphilicprotein, interacting with both RNP cores at its hydrophilic inside surface andviral envelope at the hydrophobic outside surface. The activity of RNApolymerase associated with RNP cores is repressed by the M 1 protein(Hankins, R. W. et al. (1989) Virus Genes 3, 111-126; Hankins, R. W. et al.
(1990) Res. Virol. 141,305-314).
*

Science University of Tokyo, Noda, Chiba, Japan.

I.

MOLECULAR GENETICS

23

Influenza virus A/WSN/33 forms large plaques (> 3mm diameter) on
MDCK cells whereas A/Aichi/2/68 forms only small plaques « lmm diameter). Fast" growing reassortants (AWM), isolated by mixed infection of
MDCK cells with these two virus strains in the presence of anti-WSN
antibodies, all carried the M gene from WSN (Yasuda, J. et al., (1993) Arch.
Viral., in press). Upon sinlge-round infection, these reassortants showed
rapid appearance and high yield of progeny viruses. The fast-growing
reassortants overcame the growth inhibitory effect of lignins. Pulse-labelingexperiments at various times after virus infection showed that the reassortant
AWM started to synthesize viral proteins earlier than Aichi. Taken together,
we conclude that upon infecting MDCK cells, the reassortant viruses advance rapidly into the growth cycle, thereby leading to elevated levels of viralgene expression and progeny viruses in the early period of infection.
Molecular Assembly of Influenza Virus: Association of the NS2
Protein with Virion Matrix
Jira YASUDA, Susumu NAKADA*, Atsushi KATO**,
Tetsuya TOYODA and Akira ISHIHAMA

The genome of influenza A virus is composed of eight RNA segments ofnegative polarity, which altogether encode ten different viral proteins. RNA
segment 8 (vRNA 8) codes for two non-structural proteins, NSI and NS2.
NSI is translated from a colinear transcript (mRNA 8) of vRNA 8, while
NS2 is a translation product of an RNA generated from mRNA 8 after
splicing. NSI seems to be invloved in control of this splicing. Nothing is
known on the function of NS2.
The NS2 protein was found to exist in virus particles (Yasuda, J. et al.,(1993) J. Viral. 196, 249-255). By immunochemical method, the average
number of NS2 in a virus particle was estimated to be 130-200 molecules.
After solubilization of the viral envelope, NS2 was still associated withribonucleoprotein (RNP) cores, but was later dissociated upon removal of
the M 1 protein.
A filter binding assay in vitro indicated direct protein-protein contact
between MI and NS2. Following chemical cleavage of the MI protein, NS2***

Science University of Tokyo, Noda, Chiba, Japan.
Nippon Institute for Biological Science, Ohme, Tokyo, Japan.

24bound only a C-terminal fragment of M 1. By immunoprecipitation, NS2-M 1
complexes were also detected in virus-infected cell lysates. These observations altogether indicate specific molecular association between M 1 and NS2.
Possible involvement of NS2 in the control of M1 functions is being tested.
Structure and Function of Mouse Mxl Protein: Inhibition
Mechanism of Influenza Virus Growth
Yukiyasu ASANO*, Tetsuya TOYODA, Satoshi SATO** and Akira ISHIHAMA

The mouse Mx1 protein, a host factor invloved in resistance against
influenza virus infection is an interferon-inducible nuclear protein. The Mx 1
protein does not affect virus adsorpotion, penetration, uncoating, and transport of RNP complexes into nuclei, but appears to block primary transcription of the parental influenza viral genome. Previously, we found that the Mx
1 protein was a GTP-binding protein with GTPase activity (Nakayama, M.,
et al. (1991) J. Bioi. Chern. 266,21404-21408; Nakayama, M., et al. (1992)
Virus Res. 22,227-234). By electron microscopic observation, Mx1 formed
self-assembled polymers consisting of more than 30 monomers (Nakayama,
M. et al. (1993) J. BioI. Chern., 268, 15033-15038). Deletion analysis
revealed that the main motif for self-assembly was mapped between aminoacid residues 51-99 of the Mx 1 protein.
To address questions concerning these biochemical activities and anti-viralfunction of the Mxl protein, we constructed mutand Mxl proteins carrying
mutations in the GTPase and self-assembly domains and compared theirbiochemical activities and anti-viral function. In parrallel, we attemped toconstruct in vitro inhibition system of viral transcription by the Mx 1 protein.
For this pourpose, we prepared a 0.42 M NaCl extract of nuclei containing
the Mx1 protein from interferon-treated A2G mouse embryonic cells. Western blot analysis using polyclonal antibodies against the Mx1 protein indicated that 20-30% of the Mx 1 protein was extracted. Preliminary results
indicate that this nuclear extract containing the Mx 1 protein had an inhibitory activity of viral transcription in vitro.

***

On leave of absence from Rational Drug Design Lab., Fukushima, Japn.
Faculty of Science, Kyoto University, Kyoto, Japan.

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25

Ambisense Coding Strategy of Rice Stripe Virus
Chika HAMAMATSU, Makoto KOBAYASHI, Tetsuya TOYODA,
Shigemitsu TORIYAMA* and Akira ISHIHAMA

Rice Stripe Virus (RSV), the prototype member of the tenuiviruses, hasfour-RNA segments of single-stranded RNA as the genome. Sequence
analyses of the two small RNA segments revealed that both carry large open
reading frames at 5'-proximal region of both genome-sense and anti-genomesense RNAs. This year the sequence of RNA segment 3 was determined,
which again suggested the ambisense coding strategy (Takahashi, M. et al.
(1993) J. Gen. Virol. 74,769-773). To examine this ambisense nature of thegeome, we synthesized in vitro RNAs of both porality by transcribing thecDNA clones for three RNA segments (RNA 2, 3 and 4) using T7 RNApolymerase and translated in vitro each RNA using two translation systems,
reticulocyte lysate and wheat germ extract. Translation products wereanalyzed by SDS-gel electrophoresis and autoradiography. We detected all
the proteins expected from the open reading frames on RNAs of bothpolarities (Hamamatsu, C. et al. (1993) J. Gen. Viral. 74, 1125-1131).
By immunoprecipitation experiments, the product of anti-sense RNA of
segment 3 was identified as the coat protein of RSV, while that of the genome
sense RNA of segment 4 was the major non-structural protein (NS4). We
also translated in vitro total virus RNA and detected the proteins generated
from the open reading frames located in the 5'regions of vRNAs. The overall
results are consistent with the prediction that RSV RNAs, at least up to thesegment 2, are ambisense in coding strategy.
Regulatory Elements Located at the 5' end of the First Intronof the Human Thymidylate Synthase Gene
Sumiko KANEDA and Takeshi SENO

Thymidylate synthase(TS) plays an essential role in regulating the balanced supply of the four DNA precursors for normal DNA replication. We
isolated A phage clones containing the functionally active human TS gene,
determining the complete nucleotide sequence and the major transcriptional
start sites. The biologically active unit spans about 16 kilo bases and is*

National Institute of Agro-Environmental Sciences, Tsukuba, Japan.

26

composed of seven exons and six introns. There is no typical promoter//
sequence. A human TS minigene containing 5 - and 3 -flanking sequences, all
the exons and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes inwhich intron 1 was replaced by intron 2 or deleted in the above construct,
showed only less percent of the above frequency. Introduction of intron 1
into the above intronless or intron 2-minigene restored the transforming
activities regardless of its position and orientation. Deletion analysis revealed
two positive and one negative regulatory sequences in the 5'-end of intron 1,each of which seemed to bind specific proteins as shown by gel shift analysis.
Intron 1 also stimulated expression of a TS promoter-CAT gene construct but
not that of "an SV40 promoter-CAT gene construct. These results clearly
indicate that the intron 1 regulatory sequence cluster is a promoter specificenhancer which is basically different from known transcriptional enhancers.
Intron 1 was also shown to be a major determinant of the cell cycledependent fluctuation of TS mRNA at the posttranscriptional step. Therefore, we postulated that there is a link between transcription and theprocessing of the precursor mRNA in the expression of human TS minigenes.
Cell Cycle Control by Ubiquitin System in Mammalian Cells;
Is the Ubiquitin-activating Enzyme, EI,
a Substrate of cdc2 Kinase?
Yukiko NAGAI, Sumiko KANEDA, Takeshi SENO and Fumiaki Y AMAO

The ubiquitin system is essential for cell growth since the mutational blockof one of the ubiquitin-conjugation steps is lethal to cells, arresting them atspecific stages of mitotic cell cycle. Historically, ts85, a temperature-sensitive(ts) mutant of the mouse mammary carcinoma cell line FM3A, has beenshown to be defective in the ubiquitin-activating enzyme and arrested at thelate S to G2 period of the cell cycle under restrictive temperature. Recently,
other types of £1 mutants from the same cell line were also reported; someare arrested predominantly in the S phase while others are arrested at allphases of G 1, Sand G2/M, implying that multiple steps during the cell cycle
are differentially regulated by the ubiquitin system. In addition, a series ofkey proteins regulating the cell cycle were shown to be ubiquitinated. Cyclin
B, a regulatory subunit of the mitosis promoting factor, has been shown to be

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MOLECULAR GENETICS

27

degraded by the ubiquitin-dependent proteolytic pathway. p53 and c-Mos,have also been reported to be ubiquitinated at least in vivo. Thus, theconjugation of ubiquitin to the target molecules is associated with various
functions of the cell cycle progression, presumably by way of the proteolytic
breakdown of the proteins involved in the essential function of the processes.
It has been well established that the transitions of the critical points in the
eukaryotic cell cycle is controlled by a series of cyclin-dependent protein
kinases. A variety of mammalian cyclins and kinases associated with them
have been identified. Their functions, however, are largely unknown except
for cyclin B/p34cdc2 . Which cyclin associates with which kinases, the timingof the associations, and the in vivo substrates of the kinases are a matter ofgreat interest. To investigate the coupling of the ubiquitin system and thecyclin dependent kinases during cell cycle control, we examined cdc2 kinase
mediated phosphorylation of the ubiquitin activating enzyme El in mouse
cells. In the amino acid sequence of mouse El enzyme deduced from its
eDNA were found at least five possible target sites of phosphorylation by the
cdc2 kinase. The El enzyme from mouse cells was proved to be phosphorylated in vivo. It was also phosphorylated at least in vitro by the immunopurified cdc2 kinase. These results strongly suggest that the E 1 enzyme is a
substrate of cdc2 kinase in the cell.
Cell Cycle Control by Ubiquitin System in Mammalian Cells;
Ubiquitin Conjugating Enzymes Specificfor DNA Synthesis
Sumiko KANEDA, Yukiko NAGAI, Takeshi SENO and Fumiaki Y AMAO

Ubiquitin activated by the ubiquitin-activating enzyme, E 1, and ATP istransferred from the El enzyme to one of a family of isoenzymes calledubiquitin conjugating enzymes (E2's) which catalyze conjugation of ubiquitin to specific target proteins. There is increasing evidence that each E2
enzyme has a preferred target protein (s). Detailed analysis of pleiotropic
phenotypes of mutations of the ubiquitin-activating enzyme (E 1) showedthat the inhibition of the ubiquitin system caused cell cycle arrest not only atphase G2/M but also at Gland S in mammalian cells. To identify theubiquitin pathway conferring the progression of each phase of the cell cycle,
we examined in vitro ubiquitin transfer from El enzyme to E2 conjugating

28

enzymes in the extract of the various ts E 1 mutants of the mouse cell line, FM
3A. Ubiquitin transfer to an E2 protein of approximately 35 kDa was foundto be reduced specifically in lysates of mutant cell lines which were predominantly blocked in the S phase at restrictive temperature. From such S
phase-specific El mutants, we isolated partial revertants by selecting clones
that could grow at the semi-restrictive temperature, 37°C, but still could not
at 39°C. The arresting points during the mitotic cell cycle of these revertants
at the restrictive temperature were Gland G2/M phases, and specificallysuppressed the defect in S-phase progression of the parental mutant cell lines.
In the extract of the partial revertants of the El mutants, the reduced
ubiquitin transfer to the particular E2 was restored. Thus, E2 (35 kDa)
appears to be involved specifically in DNA synthesis.
Pulverization of Chromosomal NORs in the Mouse Cell Mutant
of Temperature-sensitive Ubiquitin-activating Enzyme with
the Arrest Point at the G2 Phase of the Cell-cycle
Thangirala SUDHA, Sumiko KANEDA, Yukiko NAGAI,
Fumiaki Y AMAO and Takeshi SENO

The temperature-sensitive mutation of the ubiquitin-activating enzyme El
in mammalian cells has given rise to unique phenotypes in which somemutants are arrested at the S phase of the cell-cycle but some others arearrested at the G2 phase. We found that mutant ts85 with the arrest point at
the G2 phase shows a peculiar chromosomal aberration which is not observedin mutant tsFS20 which has the arrest point at the S phase. When ts85 cells
were cultured at nonpermissive temperature for the indicated time, followedby culturing at permissive temperature in the presence of colcemid, we
observed an interesting metaphase configuration, in which specific chromosomes arrayed radially together with pulverization shown only near thecentromeric region of the chromosomes. This confuguration was observed ata frequency of 15-20%. We speculated that the configuration could be theresult of interference in the dissociation of the interphase nucleolus. In fact,
fluorescent signals of in situ hybridization using a segment of mouse rDNA
gene as probe as well as silver staining specific for the nucleolar organizer
region(NOR) coincided with the pulverized region. In addition, in situ
hybridization using a mouse centromeric satellite DNA as probe demonstra-

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MOLECULAR GENETICS

29

ted that the chromosomes arrayed with centromeres towards the center. To
identify the role of a particular ubiquitin pathway involved in the dissociation
process of the nucleolus further analysis of the nucleolus in the arrested cells
is essential. We collaborated with Dr. Hideo Tsuji and Dr. Yoichi Matsuda(the National Institute of Radiological Sciences, Chiba) in the latter part ofthis work. T. Sudha is a visiting scientist and on leave of absence from the
Down's Research Society (India).
Mechanism of mRNA Capping Reaction
Kiyohisa MIZUMOTO/
Most of the cellular as well as viral mRNAs in eukaryotes contain a 5 terminal cap structure, m 7GpppN. The cap structure is required for efficient
initiation of translation and for pre-mRNA splicing. It has also beensuggested that the synthesis of the methylated cap structure may play animportant role in transcription itself. The cap structure is synthesized at theinitial stage of mRNA synthesis and conserved at the 5' -termini of RNAs
while they are processed in the nucleus and transported to the cytoplasm.
Elucidation of the mechamism of the cap structure is thus important forunderstanding the molecular mechamism of eukaryotic gene expression. We
have been studying mRNA capping mechanism with purified capping enzymes from various eukaryotic cells (Prog. Nucl. Acid Res. Mol. BioI. 34, 1,
1987).
The highly purified yeast mRNA capping enzyme is composed of two
separate chains of 52 (a) and 80 kDa (/3), responsible for the activities ofmRNA guanylyltransferase (ppN-RNA +pppG~GpppN-RNA+ PPi) andRNA 5 -triphosphatase (pppN-RNA ~ ppN-RNA + Pi), respectively (J.
BioI. Chern. 262, 1989, 1987). The gene encoding the a subunit, CEGI, hasbeen isolated by immunological screening of a yeast genomic expression
library with anti-capping enzyme antibody (J. BioI. Chern. 267,9521, 1992).
The identity of CEG1 was comfirmed by expressing the gene in E. coli to givea catalytically active guanylyltransferase. Gene disruption experiment indicated that the CEGI gene is essential for the growth of yeast. The a subunit
protein synthesized in E. coli catalyzed the overall capping reaction with
ppN-RNA as well as the formation of enzyme-GMP (E-pG) covalent
reaction intermediate in the absence of the /3 subunit. The GMP was foundI30

to be linked to the E-amino group of a lysine residue of the enzyme. Locationof the active site for E-pG formation was sudied by isolating a 32P_guanyly_
lated peptide after trypsin digestion of E_[ 32 p]pG.
Transcription of Sendai Virus (HV J) Genome
Kiyohisa MIZUMOTO

Sendai virus (HVJ), a member of the paramyxovirus group, contains asingle nonsegmented negative strand RNA genome of approximately 15 kblong, encoding at least seven proteins, NP, P, C, M, F, HN and L. The
genetic information of this negative strand RNA is expressed through monocistronic mRNAs that are transcribed by a virion-associated RNA-dependent
RNA polymerase composed of P and L proteins.
We have developed a faithful in vitro transcription system with purified
Sendai virus particles, in which RNA synthesis is almost entirely dependent
on host cell proteins (host factors). The host factor activity was partiallypurified from bovine brain, and was shown to be separated into at least twocomplementary fractions. One of them could be replaced by highly purified
tubulin.
Sendai virus particles were incubated with bovine brain extract in theabsence of 4NTPs, and the viral nucleocapsid (RNP) was isolated bycentrifugation through a glycerol cushion. The RNP complex thus obtained
catalyzed the synthesis of viral mRNAs upon the addition of 4NTPs without
any supply of host proteins, indicating that an active transcription initiation
complex is formed. On the other hand, RNP isolated from Sendai virus notincubated with cell extract was completely inert.
When the active RNP was subjected to SDS-polyacrylamide gel electrophoresis, a single extra protein band, which was similar in size to tubulin, wasobserved, in addition to the protein components of viral nucleocapsid. The
identity of this band as tubulin was confirmed by Western blot analysis.
Highly purified tubulin from bovine brain also supported initiation complex
formation. These rusults suggest that tubulin molecules are integrated into
the initiation complex and may function in transcription initiation and/or
elongation processes.

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31

Kinetic Study on Transcription Using Immobilized Operons:
Early Events in Transcription Elongation
Tomoko KUBORI and Nobuo SHIMAMOTO

A template DNA fragment was fixed to acrylamide beads by the tightbinding between avidin and biotin. The immobilized DNA was a template as
good as free DNA in transcription by E. coli, bacteriophage T7, T3, and SP
6 RNA polymerases. This technique enabled us to separate transcripts
retained in the transcription complexes from those released by brief centrifugation. In addition, dead end complexes which lost elongation activity canbe assayed by rapidly diluting a reaction mixture with a new optimal
substrate solution. We studied the relationships between active transcription
complexes, dead end complexes, and abortive release.
In a bacteriophage transcription system, we found a new kind of transcription complexes which intervenes the two complexes and abortive release, andnamed it the moribund complex. The moribund complex is formed from theactive complex an obligatory intermediate in the abortive pathway, andelongate their RNA 100-1,000 times as slow as the active complexes, and isslowly converted into dead end complexes.
In the E. coli transcription system, we found the abortive release is greatly
activated by the presence of polymerase in excess of the promoter. Whentranslocation of a polymerase on DNA is inhibited by another preceding
molecule stuck at 32nt from a promoter, a misincorporation at 5nt was foundin addition to a further enhancement of the abortive release. The misincorporated transcripts never elongated into long RNA even when thestacked molecule was removed. Thus misincorporation was checked in theearly steps in elongation.
Single-Molecule Dynamics of Transcription Using Immobilized Operons:
Sliding of RNA Polymerase on DNA
Hiroyuki KABATA and Nobuo SHIMAMOTO

The first step of transcription is the search for and binding of RNApolymerase to a promoter, and this promoter search is the major determinant
in selective expression of genes. The enzyme could directly bind to apromoter on a long DNA, or alternatively, first bind to DNA in a nonspecific32

manner and then form the promoter complex by one-dimensional diffusion
along the DNA chain. Unfortunately, kinetic evidence for the two conflicting models has been reported and further study should be based on more
confident evidence. The immobilized operon provides an excellent methodfor determining which case is true. Linear DNA immobilized on a slide glass
was prepared. Fluorescently labeled RNA polymerase was added to thelinearly fixed DNA, and the movement of a single molecule of RNApolymerase was followed through microscopy and recorded on a video-tape.
One-dimensional movement was detected and such movements were inhibited when the labeled polymerase was preincubated with heparin or free
DNA fragment bearing a strong promoter.
Functional and Structural Study of E. coli Single-Stranded
DNA Binding Protein
Katsuhiko KAMADA, Hiraki NAGAI and Nobuo SHIMAMOTO

E. coli Single-Stranded DNA Binding Protein (SSB) is a 19 kda protein
and essential to replication and recombination. In physiological conditions,
it exists as a tetramer and binds to both single-stranded DNA and a group ofmRNAs which have a homologous RNA element called SSB box. Both of
the bindings are cooperative and the protein molecules form clusters on such
nucleic acid molecules. These bindings suggest a coupling between replication and translation through the exchange of SSB between replication forks
and mRNAs. To substantiate this hypothesis, we investigated the expressionof the ssb gene under various conditions. The ssb-lacZ operon and protein
fusions were employed for determination of the expression of the ssb gene invivo. The results showed that neither overproduction of SSB protein nor
addition of SOS-inducing agents (e.g. Mitomycin C) affect the expression of
the ssb-IacZ fusion genes. The construction and examination of strains which
deplete functional SSB protein is now in progress.
Structure analysis of native SSB protein is in the finishing stages. Crystalsof the protein were reproducibly formed by a salting out with ammonium
sulfate and a larger crystal suitable for X-ray crystallography was obtained.
A diffraction pattern of 3-3.5 angstrom is now being analyzed. Isomorphous
replacement with uranium was successful and the location of the metal was
determined. We are continuing the effort to isolate ssDNA-protein co-

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33

crystals or soak ssDNA into native crystals.
The Tissue Specific Expression of the wx t Genein Transgenic Rice and Petunia
Hiro-Yuki HIRANO, Takeshi MATSUMURA*, Noriko TABAYASHI*, Masatoshi TANIDA*,
Yoshibumi KOMEDA** and Yoshio SANO

The wx locus of rice controls amylose synthesis in the endosperm. We havereported that the wx t gene expressed in a tissue-specific manner, namely, it isexpressed only in the endosperm and pollen (Hirano and Sano (1991) Plant
Cell Physiol. 32, 989~997).
In order to examine the promoter function for tissue specificity, we
introduced the chimeric DNA consisting of the 5' upstream region of the rice
wx' gene and bacterial P'-glucronidase (GUS) gene as a reporter into cells ofrice and petunia and regenerated whole plants from them. The functions of
the 5' upstream region were monitored by measuring the enzymatic activities
and/or histochemical analysis of the GUS gene in various tissues (organs) oftransgenic plants. In the seed of transgenic rice, GUS activity was located in
the endosperm and no activity was detected in the embryo. GUS activity was
also detected in the pollen but was not in the leaves. These results indicatedthat the 5' upstream region introduced was sufficient for tissue specificexpression in the endosperm and pollen in transgenic rice. On the other hand,in the transgenic plants of petunia, the same chimeric gene was expressed in
the pollen but was not properly expressed in the endosperm. These results
suggest that the cis-acting elements that drive the gene in the pollen arecommon to both monocotyledonous and dicotyledonous plants but those forendosperm specific expression in rice, did not function in dicotyledonous
plants.

***Hokkaido Green-Bio Institute, Sapporo.
Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo.

34

II.

MICROBIAL GENETICS

Recognition of DNA Replication Origin of Bacteriophage fl
by RNA Polymerase
Nahoko HIGA3HITANI, Atsushi HIGASHITANI and Kensuke HORIUCHI

Synthesis of the minus strand DNA of bacteriophage f1 is initiated by an
RNA primer, which is synthesized by the host RNA polymerase. The minus
strand origin contains two hairpin structures called [B] and [C] (Gray et al.
(1978) Proc. Natl. Acad. Sci. USA. 75,50-53). The mechanism by which theprimer RNA is synthesized at a specific site is not well understood. It has
been generally assumed that primer RNA synthesis is initiated by somemechanism which is entirely different from the initiation of general transcription.
Previously, we constructed a number of deletion and insertion mutants in
the hairpins [B] and [C], and analyzed them for the origin function(Higashitani et al. (1990) annual rep. 41, 31-32). Furthermore, we havedetermined the start site of the primer RNA synthesis by sequencing theprimer using chain-terminating ribonucleoside triphosphate analogues (3'deoxyNTP's) (Higashitani et al. (1993) J. viol. 67,2175-2181). The results
indicated that the size of the primer RNA was 20 nucleotides and that thesequence was complementary to the f1 DNA sequence of nucleotides 5736 to
5717. This meant that the primer synthesis started at a site 20 bases
downstream from the site previously reported by Geider et al. « 1978) Proc.
Natl. Acad. Sci. USA. 75, 645-649). Since the newly determined start site
was located within the hairpin [C], a possibility was raised that the primer
synthesis is initiated by a mechanism similar to that for general transcription.
To examine the detailed mechanisms of recognition by RNA polymerase,
we constructed a number of base substitution mutants in the hairpin [B].
These mutants were examined for the activity of the origin. The results
indicated that a specific sequence which is required for the origin function
exists within the stem of the hairpin [B]. In most of the other regions of thestem, double-strandedness, but not specific sequence, was required for theorigin function.
Results of footprinting experiments of E. coli RNA polymerase on the

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MICROBIAL GENETICS

35

minus strand origin indicated that the bottom half of the stem in hairpin [B]and the bottom half of the stem in hairpin [C] which contains the initiationsite of the primer RNA were protected by RNA polymerase from nuclease
digestion. The binding of RNA polymerase was dependent on the presenceof sigma (a) factor.
These results suggest structural and functional similarity between theminus strand origin and transcriptional promoter. The recognition mechanism of RNA polymerase at the minus strand origin may be related to themechanisms of transcriptional initiation. Indeed, the nucleotide sequence ofthe protected region showed weakly transcriptional promoter activity in vivo.
Although we don't find a very prominent promoter consensus sequence in thehairpins, further mutational analyses may shed light on the relationshipbetween the priming reaction and general transcription.
Requirement of the Correct Phasing within the Replication
Enhancer of Phage fl
Satoshi ASANO, Atushi HIGASHITANI and Kensuke HORIUCHI

The plus-strand replication of filamentous coliphage is initiated by gp2,
which is a phage-encoded initiator protein. gp2 binds to the plus-strand
origin and introduces a specific nick in the plus-strand. The 3'-hydroxyl endof the nick serves as the primer for plus-strand rolling-circle-type replication.
The replication origin consists of the core region and the enhancer region.
While the former is absolutely required for initiation and termination, thelatter, located immediately downstream of the core, enhances initiation of
replication. The enhancer region contains three binding sites (1, 2' and 2")
for integration host factor (IHF). IHF binds strongly to site 1, but only
weakly to sites 2' and 2". In vivo studies have revealed that either deletionof the enhancer region or lack of IHF reduces initiation of replication to
one-hundredth of its normal level.
In this study, we investigated the mechanism of action of the enhancer
region. The following results were obtained: 1) correct phasing between thecore and the enhancer regions is important for replication; 2) correct phasing
between IHF binding site 1 and site 2', 2" is important for replication; 3)
when IHF site 1 was replaced by a sequence that IHF could not bind to,initiation efficiency was reduced; 4) when IHF site 2', or both sites 2' and 2",

36

was replaced by a sequence that IHF could not bind to, initiation efficiency
was reduced; 5) when IHF site 2" was replaced by a sequence that IHF could
not bind to, initiation efficiency was only weakly influenced. Our current
working hypothesis is as follows: IHF binds to site 1 and introduces DNA
bending in such a way that gp2 bound to the core region interacts with a yetunidentified factor that is bound to a distal portion of the enhancer region
that includes sites 2' and 2".
Conformational Changes Induced in a Rolling Circle
Replication Origin by Intiator Protein
Atsushi HIGASHITANI, Hideo HIROKAWA* and Kensuke HORIUCHI

Gene II protein (gpll) of the filamentous phage f1 is a multifunctional
protein that participates in DNA replication. It introduces a single-strand
break at a specific site on the plus strand of the negatively supercoiled
replicative form (RF-I). The 3' hydroxyl end of the nick serves as the primer
for initiation of plus strand rolling circle replication. Gpll binds to the origin
and forms two complexes, I and II, that are separable by polyacrylamide gelelectrophoresis (Greenstein & Horiuchi (1987) J. Mol. BioI. 197, 157-174).
The complex I results from interaction of two gpll molecules with theminimal binding sequence containing the inverted repeat, /3 and r. In thesecond binding step, two additional gpll molecules bind to form complex II
which is the functional complex for the nicking of replication initiation(Greenstein & Horiuchi (1990) J. Mol. BioI. 211, 91-101). Bent DNA
structures in these complexes were inferred from the gel mobility of a seriesof circularly permuted DNA fragments that differ in the placement of the
gpII binding site (Greenstein & Horiuchi, unpublished result).
In this study, we characterized the bent structures of the complexes
through electron microscopy. A 838 base pair HpallHaeII restriction fragment containing the complete f1 plus strand origin was used. In the absenceof gpll, the DNA fragment appeared extended. When gpII was mixed with
the DNA in a molar ratio of 1: 1, 34% of the DNA molecules formed bent
structures, with a bending angle of about 90 0 • The bent occurred near thenicking site. Gel retardation assay indicated that approximately 30% of the
DNA molecules formed complex I and 2% formed complex II under the*Life Science Institute, Sophia University.

II.

MICROBIAL GENETICS

37

conditions used. When gpll was mixed with the DNA in a molar ratio of
2: 1, approximately 38% of the DNA formed complex I and 8% formed
complex II. Under electron microscope, 43% of the DNA molecules werebent by approximately 90° and 12% were bent by approximately 150°. Whenthe molar ratio of gpll to DNA was increased to 4: 1, 45% of the DNA
formed complex I and 22% formed complex II, and 41 % of the DNA
molecules were bent by 90° and 19% were bent by 150°. These results
suggest that binding of gpll induces bending of the origin DNA and that thebending angles in complex I and complex II are approximately 90° and 150°,
respectively.
We further investigated whether melting of the duplex structure is inducedin the origin region by the binding of gpll. The potassium permanganate(KMn0 4) assay (Vos et al. (1991) Cell 65, 105~ 113) was used to detect
unpaired regions of DNA. Negatively supercoiled and linear DNA, bothcarrying the fl origin, were incubated with gpll at 37°C in the presence orabsence of Mg 2 '. In the presence of Mg 2 ' , a half of the population ofsupercoiled DNA molecules was converted to the nicked form (RF-II), andthe other half was converted to the relaxed form (RF-IV). The samples were
subsequently treated with KMn0 4 to modify unpaired thymidine residues,
and were cleaved with piperidine. The sites cleaved were determined byprimer extension.
The results obtained indicated that, in supercoiled DNA, binding of gpll in
the absence of Mg 2i made certain thymidine residues within the origin
hyperreactive to KMn04' On the plus strand, the residues at positions - 2,
-1, and + 3 in reference to the nicking site were made hyperreactive and theone at - 3 became weakly reactive. On the minus strand, the thymidines at
positions + 1 and +2 became hyperreactive. In the reaction with the linear
DNA, no signals of DNA melting by gpll were observed. These results
indicate that a short region of duplex DNA around the nicking site becomessingle-stranded upon binding of gpll and that this melting occurs only insupercoiled DNA.
To study which sequence element(s) are required for the DNA melting by
gpll, we used deletion mutants L176 and d83. L176 lacks the a repeat
sequence in the plus strand origin, which is essential for termination but notfor initiation of DNA replication. L183 lacks the a sequence and additional 7
bases which include the gpll nicking site (Dotto et al. (1982) J. Mol. Bioi.
162, 335-343). Both the mutant origins can form complexes I and II. L176

38

origin is nicked specifically by gpII, but Li83 is not. Our results showed that
gpII binding caused DNA melting in the Li76 origin in the same way as in thewild-type origin. This indicates that the DNA melting by gpII does not
require formation of a hypothetical cruciform between the inverted repeats aand /3. No melting was observed in the Li83 origin.
In summary, we showed that DNA melting around the nicking site is
induced by gpII binding on negatively supercoiled DNA, and that thesequence around the nicking site, but not the a repeat sequence, is required
for the melting to occur.
Stabilization of Replication Initiator Protein DnaAof Escherichia coli by DnaK Chaperone
Seiichi YASUDA, Atsushi HIGASHITANI, Kensuke HORIUCHI
and Yoshimasa SAKAKIBARA*

Dnak protein is a major heat shock protein of Escherichia coli and thehomologue of eukaryotic hsp70. DnaK protein interacts with various
proteins and activates them. In vivo studies of a dnaK mutant have shownthat DnaK protein is involved in initiation of chromosome replication. In
vitro studies using oriC plasmid replication systems have suggested that
DnaK protein modulates the activity of DnaA protein which is essential for
initiation of DNA replication. In this study we showed that DnaK protein
stabilizes the heat-labile wild type DnaA protein so that it is fully active evenafter heating at 70°C for 10 min. DnaK protein cannot reactivate heatinactivated DnaA protein in the presence or absence of ATP. DnaK protein
can form a stable complex with DnaA protein; the complex consists of one or
two molecules of each protein. The complex formation as well as the
DnaK-dependent stabilization of DnaA protein is inhibited by ATP without
its hydrolysis. These findings suggest that the function of DnaK protein in
initiation of DNA replication is to stabilize DnaA protein by forming acomplex with it.

*

National Institute of Health, Japan.

II.

MICROBIAL GENETICS

39

A 1.9-kb Upstream Sequence Is Required for Expressionof the ItsI Gene Coding for Penicillin-Binding
Protein 3 of Escherichia coli
Hiroshi HARA, Kensuke HORIUCHI and James T. PARK*

The ftsI gene of Escherichia coli codes for penicillin-binding protein 3(PBP 3), a cytoplasmic membrane protein that functions as an enzyme forthe formation of a septum of the murein sacculus and is the principal lethal
target of $-lactam antibiotics. This gene is located at 2 min on the E. coli
genetic map, a little distal to leu, and is among a large cluster of mureinbiosynthetic genes (mur genes and mraY and ddl) and cell division genes (jtsgenes and envA).
In an attempt to generate and characterize variousftsI mutant alleles on aplasmid, we realized that it would be useful to have a null allele of this geneon the chromosome. We replaced the 1.5-kb MluI-NarI fragment within thecloned ftsI gene (nucleotides 124-1628 out of its 1764-nucleotide open
reading frame [ORF]) with a chloramphenicol resistance (cat) gene in thesame orientation as the ftsI gene, and exchanged it with a chromosomal
wild-type allele in a polA (Ts) strain. This null allele (LlftsI:: cat) could becotransduced with leu by PI phage only if a recipient strain carried additional
copies of wild-type ftsI on a plasmid. When the additional ftsI was undercontrol of the lac operator-promoter, the cells grew normally in the presenceof IPTG, but failed to divide and formed long filaments when incubatedwithout the inducer. These results provided final proofthatftsI is an essential
cell division gene.
It was unexpectedly found that a 2.6-kb Pvull fragment containingftsI and0.5 kb of its upstream region could not complement LlftsI ::cat when cloned
on a single-copy mini-F vector, although the fragment contains at least two
promoter-like sequences and was shown to complement anftsI(Ts) mutation
when cloned on a multicopy vector. A 7.3-kb HindIII-EcoRI fragment
containingftsI and 1.9 kb of its upstream cloned on a mini-F vector could notcomplement the null allele either, and the addition of a further upstream
1.8-kb HindIII fragment was necessary for complementation by a single copy
fts/. (Complementation tests of ftsI-carrying mini-F plasmids using anftsI(Ts) mutant did not give clear results because of thermosensitive mainte-*

Department of Molecular Biology and Microbiology, Tufts University School of
Medicine, Boston, U.S.A.

40

nance of the F replicon.) Disruption of the HindIII site 1.9-kb upstream of
fts!, by the filling reaction of T4 DNA polymerase, resulted in loss of thecomplementation ability. Since this site was intact on the chromosome in theLifts! ::cat strain, there seemed to be a sequence required in cis. A sequencewith homology to the consensus promoter sequence recognized by RNA
polymerase containing 0 70 was found overlapping the HindIII site. Thissequence was fused to a promoterless lacZ gene on a mini-F plasmid andshown to function as a promoter.
This promoter sequence lies near the 3' end of a 0.6-kb region whichcontains no ORF of significant length and is followed by murein biosynthetic
and cell division genes. These genes are tightly clustered in the sameorientation without a recognizable transcription terminator. It was reported
that a null allele of the ftsZ gene could not be complemented by a Atransducing phage containing ftsZ and the preceding 6 kb region (Dai and
Lutkenhaus (1991) J. Bacterial. 173,3500-3506), and the authors proposeda possibility of a long transcript being required for full expression of ftsZ.
Transcription from the promoter identified in the present study at 1.9-kb
upstream offts! might proceed for 18 kb (i.e. beyondftsZ) to the only known
terminator just downstream of envA.
A Chain-Forming Strain, PM61, of Escherichia coli Contains, in Addition
to the envC Mutation, an IS4 Insertion in the prc Gene
which Causes Accumulation of the Precursor
Form of Penicillin-Binding Protein 3
Yoshihiro YAMAMOTO*, Hiroshi HARA, Doris KARIBIAN**, Kensuke HORIUCHI,James T. PARK*** and Yukinobu NISHIMURA****

Penicillin-binding protein 3 (PBP 3) of Escherichia coli is an essential
enzyme involved in septum formation. Temperature-sensitive fts! mutants
that are defective in PBP 3 are incapable of cell division and grow asfilaments at the restrictive temperature. Another class of mutants defective in
*
**
***
****

Laboratory of Genetics, Hyogo College of Medicine, Nishinomiya.
Centre National de la Recherche Scientifique, Universite Paris-Sud, Orsay, France.
Department of Molecular Biology and Microbiology, Tufts University School of
Medicine, Boston, U.S.A.
Department of Biomolecular Sciences, Faculty of Science, Toho University,
Funabashi.

II.

MICROBIAL GENETICS

41

septation form chains of cells rather than filaments: septa are formed but thepoles of daughter cells remain fused to each other for a period of time. One
such mutant is an envC mutant PM6l (Rodolakis et al. (1973) J. Gen.
Microbiol. 75, 409-416). Since the defect was related to completion of
septum formation, we examined penicillin-binding proteins of PM6l andfound that it had PBP 3 of larger molecular weight than normal.
By PI transduction experiments and by transformation with plasmids
carrying relevant genes it was shown that the PBP 3 defect was not caused by
the envC mutation nor by an unidentified mutation inftsI, the structural genefor PBP 3, but by a mutation in the prc gene coding for a processing enzymethat cleaves a C-terminal peptide from the precursor form of PBP 3.
Southern hybridization experiments showed that an insertion was present in
the prc gene of PM61. We cloned and sequenced the gene and found an IS4
sequence in its midst. So the prc function is knocked out in PM6l, and thelarge-size PBP 3 in PM6l is an unprocessed precursor form. Since precursor
PBP 3 is apparently functional for septum formation (Hara et al. (1991) J.
Bacteriol. 173, 4779-4813), this alteration must have little to do with theincomplete septation. At the IS4 insertion point there was a duplication of
12-nucleotide sequence. Comparison of the target sequence for IS4 insertion
into prc with previously published ones suggested that considerable variations
are allowed for the target sequence.
Osmoregulation of the Fatty Acid Receptor Gene fadL
through OmpR Protein in Escherichia coli
Atsushi HIGASHITANI, Yukinobu NISHIMURA*, Hiroshi HARA, Hirofumi AIBA**.
Takeshi MIZUNO** and Kensuke HORIUCHI

ThefadL gene of Escherichia coli codes for an outer membrane protein thatis involved in the uptake of long-chain fatty acids. The uptake is regulated byenvironmental osmolarity, and decreases when the cells are under highosmotic conditions. A temperature-sensitive mutant that requires fatty acidfor growth at 42°C was unable to grow at the high temperature even in thepresence of fatty acid if the medium contained 10% sucrose. Promoter
activity of the fadL gene in vivo was repressed by high osmolarity in a FadR***

Faculty of Science, Toho University.
School of Agriculture, Nagoya University.

42

repressor null mutant. Furthermore, in vitro transcription of the fadL gene
was strongly repressed by the addition of OmpR and EnvZ proteins. The
results of gel retardation and DNaseI protection experiments indicated that
OmpR protein, only after incubation with the protein kinase, EnvZ, in thepresence of ATP, specifically binds to at least four sites around the fadL
promoter, two upstream and two downstream from the transcriptional startsite. These results suggest that transcription of the fadL gene is osmoticallyregulated by the OmpR-EnvZ two-component system.
Molecular Mechanism in Determination of Timing of Cell
Division in Escherichia coli K-12 (I)Akiko NISHIMURA

To elucidate the molecular mechanism of the timing of cell division in
Escherichia coli K-12, novel mutants which uncouple DNA replication andcell division were isolated and named cfcAI, cfcBI, cfcCI, and cfcDI. The
mutations partially suppressed division arrest and lethality induced by inhibition of DNA replication. Under permissive conditions for DNA replication,
the division rate of cfc mutants was about 1.5 times higher than that of cfc rcells. Complementation and sequencing analysis showed that the cfcA geneencoded the a subunit of glycil-tRNA synthetase. The mutated cfcAI generevealed substitution, l43A by C; this corresponds to the amino acid substitution of Glu48 by Ala. The cfcBI mutation was mapped at 1 min ,near ileS,on the E. coli genetic map. The EcoRl-PvuII DNA fragment (2Kb) which
can complement the cfcBI mutation was subcloned and is continuing to be sequenced. The cfcCI and cfcDI mutations were unstable and tend to revert to
the Cfc t phenotype with high frequency. These two genes might be involvedin an essential process for cell growth. From the genetic and physiologic
analysis of cfc mutants and double mutants with relAl, lon, sfiA, or min B,
C, D, it was concluded that the cfc genes certainly participate in thefrequency of cell division. A search to elucidate the global network or themutual recognition system between processes in the cell cycle such as protein
synthesis, DNA replication, cell division, and/or murein synthesis could solve
the problem of timing of cell division in the cell cycle.

II.

MICROBIAL GENETICS

43

Molecular Mechanism in Determination of Timing of cell
Division in Escherichia coli K-12 (II)
Hideki UKAI and Akiko NISHIMURA

We found many multicopy suppressor genes mapped in the ter region,
which suppressed the temperature sensitive mutation of cell division genes(its) mapped within 10 min from oriC. Sequencing analysis of the genes
showed primary structure interesting for the cell cycle. The ftsNI (Ts)mutation was mapped at 95 min, and was complemented by one of themulticopy suppressor genes, sunU. TheftsNI mutant showed high ploidy at
41°C. Cell division of theftsNI mutant was arrested gradually at 41°C, but
an in crease in viable count immediately ceased. The ftsN gene is presumed
to be a growth phase-regulated gene because sequence analysis showed thatthe deduced ftsN gene had a consensus sequence of gear box at the promoter
region. The sun U gene was predicted to encode a protein composed of 198
amino acids. The C-terminal region of the deduced Sun U protein showedsignificant sequence similarity to SulA, a division inhibiter involved in the
SOS mechanism. Deletion of 6 amino acids from the C-terminal caused
decreased complementation activity for the ftsN1 mutation and deletion of 19
amino acids caused complete loss of comple mentation activity even when it
was overproduced in the cell. Further characterization of these mutants
would elucidate the fine control mechanisms of the cell cycle.

44

III.

MAMMALIAN GENETICS

High-Order Chromatin Structure of the Recombinational
Hotspot in the Mouse MHC
Ken-ichi MIZUNO, Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKIIt has been established that reciprocal recombination occurs with highfrequency in the mouse major histocompatibility complex (MHC) whensome mouse strains are crossed. The breakpoints for recombination in the
MHC are clustered at specific sites termed hotspots. The wm7 MHC
haplotype derived from Japanese wild mouse enhances the recombination at
the hotspot located between the Pb and Ob genes (wm7 hotspot). The
breakpoints of the recombinants turned out to be confined to a DNA
fragment of 2 kb. The molecular mechanism that controls the site-specificityof the recombinational breakpoints is yet to be defined. One possibility is thatthe recombinational hotspot has an open chromatin structure leading to theenhanced accessibility of DNA to the recombination machinery. We attempted to examine this hypothesis with the wm7 hotspot. First, we fractionatedtesticular germ cells prepared from several different mouse strains by centrifugal elutriation after collagenase and trypsin treatment of testes, and enriched the pachytene cells at which meiotic recombination is thought tooccur. In order to analyze the high-order structure of chromatin around thehotspot, we tried to detect DNasel hypersensitivity sites (DHSS) in thechromosomal region surrounding the hotspot. Clear DHSS was not detectedin the 2 kb segment in the vicinity of the hotspot. Now we have extended thisanalysis to much broader chromosomal segments.

Functional Analysis of the LMP·2 Gene Encoding a Subunitof the Mouse Proteasome
Ken-ichi MIZUNO, Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Exogenous antigenic proteins, as viruses, are digested to small octomeric or
nanomeric peptides, before being presented in the context of the MHC class
I molecules to the T-cell receptors (TCR). A multisubunit protein complex

III.

MAMMALIAN GENETICS

45

termed proteasome, exerts this protein degradation in an energy-dependent
manner. In mouse, the LMP-2 gene encodes a subunit of proteasome, which
is mapped to a 9 kb distal to the recombinational hotspot which has been
identified in the wm7 and cas3 MHC haplotypes. It is still unclear whether
the LMP-2 gene plays a role in protein degradation for antigen presentationin immune recognition. We intended to elucidate the function of the LMP2 gene by destroying this gene through a gene targeting experiment. In
addition, we attempted to test the possibility of enhancing the efficiency ofhomologous recombination using a vector construction which contains therecombinational hotspot. In the beginning of the study, we analyzed the
genome structure of the LMP-2 gene and defined the intron-exon organization of this gene. We constructed two different vectors, pLMP2neo-Hot andpLMP2neo-Cold. Both have the Neomycin phosphotransferase (Neo) genein exon 1 as a positive selection marker and the Herpes simplex virus
thymidine kinase (tk) gene at the 3' end of the LMP-2 gene as a negative
selection marker. Only pLMP2neo-Hot has the recombinational hotspot in
the 3' region of the LMP-2 gene. We are planning to transfect embryonic celllines, Fl-l, with these two vector DNAs.
Establishment of Embryonic Stem Cell Linesfrom BlO.H-2 Congenic Strains II
Tomoko SAGAI, Norio NAKATSUJI, Toshihiko SHIROISHI and Kazuo MORIWAKI

We have continued to isolate ES cell lines from BlO.H-2 congenic strains,for studying the molecular mechanism of meiotic recombination in the H-2
region. Many ES cell lines have been isolated from various mouse strains, but
production of germ-line chimeras has been achieved with only limited strains,for example 129/Sv. As shown in the previous report, we isolated twenty ES
cell lines from one BlO.H-2 congenic strain, BlO.A(R209), which showed
high recombination frequency in the H-2 region during meiosis. Thus far,three of them have been tested for chimera formation and germ-line transmission through microinjection into 8 cell embryos of BALB/c, A/] and ICR
strains. As a result, we have obtained several chimera mice from them, but
no germ-line chimera yet. Several reports suggest that the combination of the
genotype of ES cells and host embryo is essential for germ-line transmissionof ES cells. Recently Tokunaga and Tsunoda established a new ES cell line,46

Fill, from a (B6xCBA)Fl mouse. They reported that chimeras obtained
from Fill cells by 8 cell injection produced exclusively ES-derived offsprings(Tokunaga, T. and Tsunoda, Y. (1992) Develop. Growth and Differ. 34, 561566). From the point of view that the two mouse strains, B6 and BIO, aregenetically very related, it is expected that the (BIO.A(R209) XCBA)Fl
mouse would yield ES cell lines with a potential for germ-line transmisson as
well. We isolated three ES strains from the (BIO.A(R209) X CBA)Fl
mouse. Now we have obtained several chimera mice from the new ES celllines and are testing for germ-line transmission of them.
Genetic Control of Male-specific Suppression of Meiotic Recomibnation
Toshihiko SHIROISHI, Tomoko SAGAI and Kazuo MORIWAKI

Sexual difference in the frequency of meiotic recombination is observed inhuman and mouse chromosomes. The difference is significant in certain
chromosomes or particular parts of some chromosomes. We have reported
previously that the wm7 MHC haplotype derived from Japanese wild mouseenhances meiotic recombination specifically in females at the recombinational
hotspot located in the proximal region of the MHC. Genetic analysis
demonstrated that the wm7 haplotype possesses a suppressive factor forrecombinations in male meiosis, which is located on the chromosomal segment distal to the hotspot. In the present study, we attempted to map the
suppressive factor more precisely. For this purpose, we carried out mating
experiments using two intra-MHC recombinants, BIO.A(R202) and BIO.A(R203), which were generated from the wm7 haplotype. Both recombinants
have a genotype of H_2Ka_H_2D wm7• The recombinational breakpoints arelocated between the Bfand H-2D genes in BIO.A(R202), and between the Ea
and Cyp21 genes in BIO.A(R203). We estimated the recombination frequency in crosses of BIO.A(209) and these two recombinants, between the H-2K
and H-2D marker loci during male and female meiosis. As a result, crosses
involving BIO.A(R202) yielded a high recombination frequency both in maleand female meiosis. By contrast, crosses involving BIO.A(R203) yielded ahigh recombination frequency only in females. The result clearly showed thatthe male suppressor was preserved in BIO.A(R203), but not in BIO.A(R202),
indicating that the male suppressor is located between the hotspot and Cyp21
genes, which span about 400 kb in length. The breakpoints of the recombina-

III.

MAMMALIAN GENETICS

47

nts obtained in the present experiment were defined by restriction fragment
length polymorphism (RFLP) analysis. We found that all breakpoints
almost overlapped with the original hotspot identified in the cross between
the wm7 and laboratory MHC haplotypes.
Establishment of New Congenic Strain for Insulin-dependent
Diabetes Mellitus (lDDM) Susceptibility Gene, Idd-4
Shigeharu WAKANA, Toshihiko SHIROISHI and Kazuo MORIWAKIThe NOD (Nonobese diabetic) mouse is widely used as animal model ofinsulin-dependent diabetes mellitus (IDDM). Genetical analysis in breedingstudies between NOD and C57BL/lOJ mice has suggested that several genes
are involved in onset of diabetes and insulitis, a prediabetic condition
characterized by lymphocyte infiltration into the islets of Langerhans of thepancreas. At least four susceptibility genes for IDDM have been reported.
Among them, the Idd-I is linked to the MHC on chromosome 17 and Idd-4
is mapped in the region between the Acrb and Mpo genes on chromosome 11.
In order to investigate the function of the Idd-4 gene without influence ofother susceptibility genes for diabetes, we have tried to produce the congenic
mouse strain for the Idd-4 gene by introducing of the chromosomal segment
between the Acrb and Mpo gene from Japanese wild mouse-derived MSM
strain into the genetic background of the NOD mice. At present, thebackcross has reached to the 7th generations. Histopathological examination
revealed that the Idd-4 congenic mice, NOD.Idd_4 MSM , did not have insulitis
and showed reduced level of glucose content in peripheral blood. These
results confirmed the presence of a susceptibility gene for IDDM, which islocated between the Acrb and Mpo genes, and usefulness of the NOD.Idd4 MSM congenic strain to identify and clone the Idd-4 gene.
Gene Dosage Effects of Recombinational Hotspots in the Mouse MHC
Masayasu YOSHINO, Tomoko SAGAI, Kirsten FISCHER-LINDAHL*,
Toshihiko SHiROISHi and Kazuo MORIWAKIThe meiotic recombinations in the proximal region of the mouse major*

Howard Hughes Medical Institute, and Department of Microbiology and Biochemistry,
University of Texas Southwestern Medical Center, Dallas, TX 75235-9050, USA.

48

histocompatibility complex (MHC) are not random, but clustered at specificsites termed hotspots. Some of the MHC haplotypes derived from the Asian
wild mice, enhance recombination at the hotspots when they are heterozygous with standard laboratory haplotypes. The wm 7 and cas3 haplotypes
enhance recombination at the hotspot located between the Pb and Ob gene,and the cas4 at a different hotspot located at a distance of about 100 kb fromthe Pb-Ob hotspot. In order to test the effects of a double dosage of hotspots,
we estimated the rate of recombination frequency and determined the localizations of breakpoints in two different crosses, wm7/cas3 and wm7/cas4. In
total, we have screened 3570 of the backcross progeny from these two crosses
and have obtained 29 of new recombinants between the H-2K and Ab genes,
yielding a frequency of 0.81%. This rate was 40-fold higher than thestandard value estimated in crosses between laboratory haplotypes (0.02%),and very similar to those obtained in crosses between wild and laboratory
haplotypes. Thus, it appeared that a double dosage of hotspots has no
additive effect on meiotic recombination. Molecular mapping by RFLP
analysis revealed that the localizations of the recombinational breakpoints
were completely identical to those in crosses involving the wm 7, cas3 andcas4, indicating conservation of site-specificity in crosses between the two
haplotypes with the hotspot.
Mutation Rate of Intra-MHC Recombinant Mice
Masayasu YOSHINO, TOMOKO SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKIIn our laboratory, hundreds of intra-MHC recombinants have been established from thousands of progeny from crosses between a wild-derived MHC
congenic strain, BIO.MOL-SGR, and standard inbred strains. We found thatsome of these intra-MHC recombinants have spontaneously yielded multiple
visible mutations. The rate of recessive and dominant mutations per gamete
per locus was roughly estimated to be 2.8 X 10- 4 and 7.1 X 10- 4 , respectively.
These rates were much higher than the standard value, 3.8 X 10- 6 for recessive and 3.4 X 10- 7 for dominant mutations, which were estimated fromlaboratory mouse strains. In order to calculate the spontaneous and irradiation-induced mutation rates with the intra-MHC recombinants, we havestarted mutagenesis experiments. We used the PT/7af strain as a tester ingenetic crosses with the intra-MHC recombinants, as it carries mutated

III.

MAMMALIAN GENETICS

49

recessive alleles at seven specific loci. Besides these experiments, the dominant visible mutations were carefully observed as to whether irradiation
boosts the mutation rate. In practice, we irradiated the BIO.BR.(R228)
mouse strain, from which a dominant visible mutation of hairless phenotype(Rim3) was obtained, and mated those mice with testers of the PT strain. As
yet, we have detected neither recessive nor dominant mutation in 210
offspring from 5 Gy-irradiated females of BIO.BR.(R228). We intend tocontinue this experiment to screen at least 1000 offspring. The target of this
irradiation is the late stage of meiosis and a large lesion in the chromosome
is expected. A small lesion in the gene at the early stage of meiosis can be
induced by irradiating male mice with two fractions of 5 Gy separated by 24hours. We plan to set up another mutagenesis experiment using an intraMHC recombinant, BIO.(R233), which has spontaneously yielded a recessive
visible mutation of hairless phenotype (rim1) and a dominant visible mutation of yellow belly, (Rim5), to estimate the mutation rate after irradiation at
the early stage of meiosis in male mice.
Comparison of the DNA Sequence in the D-Ioop Region of Mouse
Mitochondrial DNA. III. Close Relatedness between
Japanese Northern Population and the castaneus
Population in the South-eastern Asia
Hiromichi YONEKAWA, Sumiyo WATANABE, Nobumoto MIYASHITA
and Kazuo MORIWAKI

Based on restriction analysis of mitochondrial DNA, we have proposed the
hypothesis that Japanese wild mouse "Mus musculus molossinus", is originated from a hybrid between ancestral colonies, possibly very small, of M. m.
musculus and of M. m. castaneus (Yonekawa et a/. (1988) Mol. Bio/. Evol. 5,63). To prove this hypothesis, we analyzed the mtDNA D-loop region fromthe mice in Japan as well as from those in its neighboring countries. Throughthis analysis we have previously showed the following lines of evidence: 1)
substituted nucleotides in the mtDNA D-loop region, like restriction sitevariation, can be used as a diagnostic marker for subspecies identification
when we construct phyo10genic trees using the unweighed pair-grope methodwith arithmetic mean (UPGMA) or the neighbor-joining (NJ) method. 2)
at least four different mtDNA types exist in the Southern population of

50

Japanese wild mice (Yonekawa et al. (1990) Ann. Rep. Nat!. Inst. Genetics.
Japan, 41, 46-47), although no restriction polymorphisms had been detectedin the population (Yonekawa et al. (1988) Mo!. Bio!. Eva!. 5, 63), 3) mtDNAof the Japanese southern population was clustered in that of Korean, northern-Chinese and eastern-Russian populations. Thus it is suggested that thesouthern population was originated from northern Far East and came fromthere through the Korean peninsula (Yonekawa et a!. (1991) Ann. Rep. Nat!.
Inst. Genetics. Japan, 42, 52-54).
Next question is what is the locality where the Japanese northern population with castaneus type mtDNA came from. To address this question, wesequenced mtDNA from 27 mice collected from the Southeastern Asia andsouthern China including 5 samples from Japanese mice. We found that 1)
castaneus type mtDNA showed at least three distinct clusters in the "castaneus" branch of phylogenetic tree, and 2) mtDNA of the Japanese northern
population was clustered in that of southeastern Asia, but not in southern
China. This suggests that the northern population was originated from the
population of southeastern Asia. It is thus concluded that there are two
possible routes through which the Japanese mice invade into Japan: one isfrom northern China through the Korean peninsula and the other is southeastern Asia possibly through islands in southern Pacific.
A New Pas Genes Derived from Asian wild Mouse
Nobumoto MIYASHITA, Toshihiko SHIROISHI, Shigeharu W AKANA*,
Hiromichi YONEKAWA** and Kazuo MORIWAKI

Rather classical mating experiments between a resistant strain C57BL anda susceptible strain A have demonstrated the possible presence of a single
recessive resistant gene in the susceptibility to urethane-induced pulmonary
adenoma in mice (Falconer and Bloom, 1964). This gene was designated"ptr" by them, but later "Pas-I" (Pulmonary Adenoma Susceptibility-I) by
Malkinson et al., 1984. Ryan et a!. (1984) and Gariboldi et a!. (1993)
demonstrated that the major gene for pulmonary adenoma susceptibility was
located on chromosome 6, though the relationship between Pas-I and Kras-2
was not necessarily clear, probably because of the additional effect of H-2***

Central Institute for Experimental Animals, Kawasaki.
Tokyo Metropolitan Institute for Medical Science, Tokyo.

III.

MAMMALIAN GENETICS

51

complex as shown by us (Miyashita and Moriwaki, 1987). We have achieved
mating experiments using A/Wyand BlO.A strains which have the same H2 haplotype and linkage analysis to make clear whether Pas-I is Kras-2 itself
or not. Two recombinants out of 68 N2 progeny were observed, indicatingthat Pas-I is a unique locus independent from Kras-2 on chromosome 6, buttightly linked to it.
BGR and MSM strains derived from Asian wild mice, Mus musculus
castaneus andM. m. molossinus, have shown a definitely dominant allele(s) intheir pulmonary adenoma susceptibility gene(s) which was strikingly different from the resistant strain such as C57BL. F2 progeny from A X BGRcross revealed that this resistant gene(s) is not Pas-I. This putative suppression gene is not linked with either Acrb (Trp53) on chromosome 11 or D14
Mit7 (Rb-l) on chromosome 14. This resistant allele(s) has possibly eliminated during the course of development of the present day's laboratory mousestrains.

52

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Formation of a New Nerve Network in the Nerve-free Tissue of
Epithelial Hydra through Reintroduction of Interstitial Cells
Tsutomu SUGIYAMA, Sumiko MINOBE and Osamu KOIZUMIFormation of a new nerve network in the nerve-free tissue of epithelial
hydra was examined through the reintroduction of interstitial cells intoepithelial hydra.
Hydra has a primitive nervous system known as a diffused nerve network.
It consists of a large number of nerve cells distributed throughout the tissue.
Neuron cell bodies extend one to several long processes into the tissue, andthese processes interconnect with each other to form an extensive meshlike
network throughout the entire body. Within the network, the neurons are
distributed not randomly, but in a highly region-specific manner. For
example, the neuron density is about 6 times higher in the head and about 3
times higher in the foot than in the central part of the body column. The
neurons are classified into several major types based on morphology, theneuropeptides they contain, or the specific antigens present in them andrecognized by various monoclonal antibodies. Distribution of the various
types is also highly region-dependent.
To examine the mechanisms responsible for determining the region-specificnerve cell distribution in the network, we employed epithelial hydra in thepresent study. The tissue of epithelial hydra consists of mostly epithelial cells
and is completely free from the interstitial cells and their differentiation
products, nerve cells and nematocytes. When the interstitial cells werereintroduced into epithelial hydra, these cells differentiated into nerve cells
rapidly, and established a new nerve network in the nerve-free tissue ofepithelial hydra within several days.
In the hypostome, the "ganglion" cells first appeared in the whole region at
day 1.5 after interstitial cell reintroduction. By day 4, a new network similar
to that present in the hypostome of intact hydra was established, with acluster of "sensory" cells at the hypostome tip and numerous ganglion cells in
the adjacent tissue. In the tentacles, nerve cells first appeared at day 1.5-2.5.
However, their distribution was initially restricted only to the base of theIV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

53

tentacles. The area of distribution then gradually expanded, reaching thetentacle tip in about 7 days. The rate of this expansion was about the sameas the speed of the epithelial tissue displacement from the base to the tip of
the tentacles.
These observations show that a new nerve network closely resembling thatin normal hydra can be established relatively quickly after reintroduction of
the interstitial cells into the nerve-free tissue of epithelial hydra. Thissuggests the the network formation is controlled primarily by the epithelial
tissue environment.
A Simplified Method for Detecting Cell TypeSpecific cDNA Clones from Hydra
Takeshi OHKAWA*, Koichiro SHIOKAWA* and Toshitaka FUJlSAWA

The cell type specific expression of a given cDNA is normally achieved
through in situ hybridization, which involves rather tedious procedures. Fora large scale screening of cDNA clones for any particular cell type, it isdesirable to have a more simplified and convenient method. For this purpose,
we established an efficient cell fractionation method utilizing elutriation.
About 1,000 hydra (10 8 cells) were treated with pronase (8 mg/ml of hypertonic cell dissociation medium) at room temperature for 1-2 h. By thetreatment, cell-cell adhesion molecules are digested and tissue is easily
dissociated into single cells. The resultant cell suspension was subjected toelutriation (constant flow rate of 15 ml/min and varying centrifugal speed
from 800 to 3,800 rpm) and 7 fractions were collected. An examination ofeach fraction under Nomarsky optics revealed that 7 basic cell types whichmake up hydra tissue were nicely separated according to their sizes.
Total RNA was extracted from each fraction, fractionated by agarose gelelectrophoresis and blotted onto nylon membranes. Using clones whose
expression with respect to cell types is already known, northern hybridization
was carried out. Signals were detected in fractions exactly as expected.
These results indicate that we have a significantly simplified method at handfor sorting cDNA clones according to their specific cell type expression.

*

Department of Zoology, Faculty of Science, Tokyo University, Tokyo.

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Protease Sensitivities of Hydra Cell-Cell Adhesion
Masayuki HATTA and Tsutomu SUGIYAMA

Morphogenesis of multicellular animals are mediated by harmony ofmultiple cellular movements such as folding, invagination or evagination ofepithelial cell sheets, transition of cells between epithelial and mesenchymal
states and segregation of different type of cells. In these cellular activities,
cell-cell adhesion mechanisms have been shown to play crucial roles. Hydrais an animal which has a simple body plan and a strong regenerative capacity,and morphogenesis during regeneration of Hydra has been investigated formany years. However cellular behavior or molecular mechanisms of theregeneration have been little investigated. We have started a project toexamine cellular and molecular aspects of Hydra regeneration processes,
focusing on cell-cell adhesion systems.
First, we developed solutions for protease treatment of Hydra tissue toobtain viable dissociated single cells and for reaggregation of the dissociatedcells. Three proteases were then used for digestion of cell surface molecules:
collagenase which has a high specificity on collagens, trypsin which has amoderate substrate specificity and pronase which has a wide spectrum of
substrate specificity. About 0.2 g wet weight of Hydra were treated with eachof the three proteases at 0.1 % concentration in dissociation solution for 30
minutes at 18°C, rotating at 80 rpm and pipetting every 10 minutes. Cells
were collected by centrifugation at 5000 rpm for 1 minute and resuspended inreaggregation solution. After removal of tissue fragments by decantation,
the suspension of cells were subjected to reaggegation assay in 24-wells
multiplates with rotation at 80 rpm for 30 minutes at 18°C. Cells treated withcollagenase and trypsin formed cell aggregates, but cells treated with pronase
did not. Most of the aggregates consisted of either ectodermal or endodermal
cells, resulting segregation of cell-type specific aggregates. Repeated experiments yielded reproducible results, except for trypsin treated cells whichsometimes failed to form aggregates.
These results indicate that Hydra cells have specific cell-cell adhesion
systems for ectoderm-ectoderm and endoderm~endodermadhesion, and thatthey are mediated by adhesion molecules which are resistant to collagenase,partially sensitive to trypsin and sensitive to pronase.
A project to identify and isolate these adhesion molecules is under way
presently.

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55

DNA Supercoiling Facilitates the Assembly of Transcriptionally
Active Chromatin on the Adenovirus Major Late Promoter
Reiko OHBA, Hisahiro TABUCHI and Susumu HIROSE

Assembly of nucleosomes on the adenovirus major late promoter blocked
initiation of transcription by RNA polymerase II. However, the formation oftranscription preinitiation complexes prevented subsequent assembly of promoter sequences into nucleosomes and allowed transcription on the chromatin templates. When the formation of preinitiation complexes was in competition with nucleosome assembly, transcription on linear or relaxed closed
circular DNA was inactivated by nucleosome assembly over the promoter
region. However, transcription on partially supercoiled DNA (mean superhelical density of -0.036) remained active because the rapid formation ofpreinitiation complexes prevented subsequent assembly of promoter sequences into nucleosomes. For details, see Biochem. Biophys. Res. Commun.
186, 963-969, 1992.
Embryonal Long Terminal Repeat-Binding Protein Is a Murine Homologof FTZ·Fl, a Member of the Steroid Receptor Superfamily
Toshio TSUKIYAMA*, Hitoshi UEDA, Susumu HIROSE and Ohtsura NIWA*

The embryonal long terminal repeat-binding protein, ELP, is present in
undifferentiated mouse embryonal carcinoma cells. It binds to and suppresses
transcription of the Moloney leukemia virus long terminal repeat in undifferentiated murine embryonal carcinoma cells. We found that ELP is amouse homolog of Drosophila FTZ-Fl, which positively regulates transcription of the fushi tarazu gene in blastoderm-stage embryos of the fly. As
members of the steroid receptor superfamily, ELP and FTZ-Fl have both
DNA binding and putative ligand binding domains which are well conserved
between the two. ELP and FTZ-Fl function in the extremely early stage ofdevelopment. A high degree of conservation between the two transcription
factors during the evolution of these species indicates the importance of theirfunctions in early-stage embryogenesis. In addition, the sequence elements
they recognize do not contain repeat units, in contrast to other steroid*

Department of Pathology, Research Institute for Nuclear Medicine and Biology,
Hiroshima University, Hiroshima.

56

receptors, which usually bind to either palindromic or direct repeat sequences. For details, see Mol. Cell. Bioi. 12, 1286-1291, 1992.
A Novel DNA-Binding Motif Abuts the Zinc Finger Domain of Insect
Nuclear Hormone Receptor FTZ-Fl and Mouse Embryonal
Long Terminal Repeat-Binding Protein
Hitoshi UEDA, Guan-Cheng SUN, Takehide MURATA and Susumu HIROSE

Fruit fly FTZ-Fl, silkworm BmFTZ-Fl, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor
superfamily, which recognizes the same sequence, 5'PyCAAGG PyCPu-3'.
Among these proteins, a 3D-amino-acid basic region abutting the C-terminalend of the zinc finger motif, designated the FTZ-Fl box, is conserved. Gel
mobility shift competition by various mutant peptides of the DNA-binding
region revealed that the FTZ-Fl box as well as the zinc finger motif is
involved in the high-affinity binding of FTZ-Fl to its target site. Using a gelmobility shift matrix competition assay, we demonstrated that the FTZ-Fl
box governs the recognition of the first three bases, while the zinc finger
region recognizes the remaining part of the binding sequence. We also
demonstrated that the DNA-binding region of FTZ-Fl recognizes and binds
to DNA as a monomer. The occurrence of the FTZ-F 1 box sequence in othermembers of the nuclear hormone receptor superfamily raises the possibility
that these receptors constitute a unique subfamily which binds to DNA as amonomer. For details, see Mol. Cell. Bioi. 12, 5667-5672, 1992.
Production of Antibodies Specific for Double Stranded Antigen DNA
Cloned from Immune Complexes in Plasma of a SLE Patient
Kunihiko TERADA*, Susumu HtROSE and Eiji OKUHARA*

The antigenicity of antigen DNA isolated from immune complexes in theplasma of an SLE patient was examined. DDY mice were immunized withthe cloned antigen DNA carrying a sequence homologous with a part ofbacteriophage fl (KS8 DNA) by the coupling method, and the antibody
response was estimated by radioimmuno assay. Antibodies specific to double
stranded DNA were elicited. Moreover, the antibodies showed preferential*

Department of Biochemistry, Akita University School of Medicine, Akita.

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binding to KS8 DNA than other DNA derived from Escherichia coli. These
results suggest that KS8 DNA has a significant antigenicity in mice. For
details, see Biochem. Biophys. Res. Commun. 183, 797-802, 1992
Permeability of Dechorionated Eggs and Developmental
Anomalies in Drosophila
Kiyoshi MINATO

In previous experiments, the freshly laid and dechorionated eggs of Drosophila melanogaster developed abnormally when incubated in aqueous solutions such as distilled water and 0.9% NaCI solution, whereas they developed
normally when incubated in a non-aqueous solution such as liquid paraffin.
Because this phenomenon was not found when the incubated eggs were at
more developed stages, it appeared to be due to the permeability of thevitelline membrane of the eggs to water or water-soluble substances, which isthought to remain for a while after the fertilization of eggs (Yoon and Fox
1965, Limboug and Zalokar 1973). When incubated in distilled water, mosteggs swelled gradually without developing any structure, while, when incubated in 0.9% NaCI solution, most eggs showed, interestingly, blastoderm
formation only in the posterior-half and resulting thereafter in embryos with
an amorphous bag-like structure herniated at the anterior half.
This time, the limiting time or the time, incubated after which, eggs candevelop normally was determined precisely. Consequently it proved to beabout 30 to 60 minutes after fertilization (at 25°C) during which nuclei are
still dividing actively in the inside embryos. This is much earlier than thetime of two hours, roughly determined previously, the time at which nuclei
had migrated to the surface of embryos to become syncythial blastoderm.
Control of Imaginal Cell Development by the escargot
Gene of Drosophila
Shigeo HAYASHI, Susumu HIROSE, Tony METCALFE and Allan SHiRRASDuring the larval period, larval cells undergo multiple rounds of DNA
replication without cell division and become giant polyploid cells. In contrast, imaginal cells remain diploid and retain the ability to divide. Mutationsin the escargot (esg) locus cause a variety of defects in adult structures

58

derived from imaginal discs and histoblasts, such as loss of the abdominal
cuticule and malformation of the wings and legs. The esg locus is more than
70 kb long and contains a small transcription unit of about 2.7 kb which
potentially encodes a zinc finger protein. esg RNA is expressed in wing,
haltare, leg and genital imaginal discs and in abdominal histoblast nests in theembryo. Expression in imaginal tissues is also found in third instar larvae. In
esg mutant larvae, normally diploid abdominal histoblasts replicate DNA
without cell division and become similar in appearance to the polyploid larval
epidermal cells. esg mutations cause an endo-replication in abdominal histoblasts during the larval period, and an increase in nuclear size and DNA
content. These apparently polyploid mutant histoblasts fail to proliferate
during pupariation. This leads to the observed deletion in the adult abdomen.
In contrast to the severe abdominal defects which are essentially due todeletion of histoblast-derived structures, the leg and wing defects in esg
mutants are limited to malformation. This difference in sensitivity to esg
mutations may reflect a different temporal pattern of cell division in imaginaldiscs and abdominal histoblasts. While imaginal disc cells are actively
dividing during the larval stages, abdominal histoblasts is arrested in G2
interphase. The histoblasts enter mitosis only after pupariation under the
influence of a change in hormonal environment. Cell cycle arrest of theabdominal histoblasts during the larval stage in addition to the reduction of
esg function may be a necessary condition for becoming polyploid. A similar
esg mutant phenotype was also found in some imaginal discs whose cell
division was blocked by the raf mutation. These results suggest that one of
the normal functions of esg is the maintenance of diploidy of imaginal cells.
Details of this work will be published (Development, in press).
Role of the escargot Gene during Drosophila Development
Naoyuki FUSE, Susumu HIROSE and Shigeo HAYASHI

To further investigate the role of the esg gene during embryonic andimaginal development, we studied the effects of ectopic esg expression. Flystrains containing the esg gene linked to a heat shock gene promoter havebeen constructed. The strains transiently expressed abundant esg proteinwith heat shock. However the animals tolerated this treatment and developed
to normal adults albeit with reduced fertility in some cases. As an alternative

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59

method of ectopic expression, strains carrying the esg gene placed downstream of the binding site for a yeast transcriptional activator GAL4 wereconstructed. When these strains were crossed to a strain carrying the GAL
4 gene under the control of salivary gland specific enhancer, the esg gene was
ectopically expressed in the salivary gland and great reduction of viability
was observed. In such larvae, endoreplication of salivary gland cells wasseverely inhibited and the size of salivary gland cells was reduced. Taken
together, these results suggest that, Drosophila can tolerate transient ectopic
esg expression but continuous expression is inhibitory to larval growth. These
results support our previous hypothesis that esg may playa positive role indistinguishing larval versus imaginal cell identity (S. Hayashi, S. Hirose, T.
Metcalfe, A. Shirras, Development in press).
Regulation of Drosophila Neural Development
by a Putative Secreted Protein
Hideyuki OKANO, Shigeo HAYASHI, Tei-ichi TANIMURA, Kazunobu SAWAMOTO,
Shingo YOSHIKAWA, Jira WATANABE, Masayuki IWASAKI, Susumu HIROSE,
Katsuhiko MIKOSHIBA and Craig MONOTELL

Drosophila strawberry (sty) was isolated as a novel visual system mutant
displaying a rough eye phenotype. Analyses of mutant phenotypes and theexpression pattern of the gene suggested that the sty gene has pleiotropic
functions. Mutations in the sty gene affect eye development, leading toirregular spacing of ommatidia, an increase in the number of photoreceptor
cells, and abnormal axonal projection and the disruption optic lobe structurein the adult fly. In addition to affecting the visual system, they cause
abnormal head involution, a change in the number of sensilla in the antennomaxillary complex in the embryonic stage, and abnormal morphogenesis of
maxillary palp and wings in later stages. We examined the expression of thesty gene during development in terms of lacZ expression from enhancer trap
elements inserted within the sty gene. During embryogenesis, expression oflacZ showed a segmental pattern in the ectoderm and in the nervous system.
In the eye imaginal discs, lacZ began to be expressed in photoreceptor cells,a few rows posterior to the morphogenetic furrow. It was also expressed in
the wing disc. In the adult, it was expressed in the retina and lamina. We
cloned the sty gene by P-element tagging. On the basis of the phenotype ofloss of function, its expression pattern, and the predicted structure of its

60

product, a secreted peptide with a putative epidermal growth factor (EGF)
motif, it appears that sty encodes a diffusible protein with pleiotropic functions acting as a signal involved in lateral inhibition within the developing
nervous system and also as a factor involved in axonal guidance. Details of
this work have been published (Okano et al., Differentiation 52, 1-11, 1992).
Genetics of Fluoride-resistance of the Nematode Caenorhabditis elegans
Minoru KAWAKAMI, Takeshi ISHIHARA and Isao KATSlJRA

Sodium fluoride (NaF) is toxic to almost all organisms. Biochemical
reactions in vitro suggest that it may interfere with interesting regulatory
mechanisms, because it depletes Ca t t , inhibits phosphatases, and activates
G-proteins. We are trying to discover and elucidate such regulatory mechanisms through the isolation and characterization of C. elegans mutants
resistant to NaF.
We have isolated 13 recessive fluoride-resistant mutants that map in 5 newgenes,jlr-lX,jlr-2 V,jlr-3 IV,jlr-4 X andjlr-5 V. They are grouped into two
categories, class 1 and class 2. Class 1 mutants (flr-l, jlr-3, and jlr-4) areresistant to 10 mM NaF, but they grow twice as slowly as wild type worms
and have a small brood size even in the absence of NaF. In contrast, class 2
mutants (flr-2 andjlr-5) are not completely resistant to lOmM NaF. They
are almost normal in growth rate and brood size in the absence of NaF.
Interestingly, class 2 mutations suppress the slow growth and small broodsize, but not the strong fluoride-resistance of class 1 mutations. Namely, class
2 mutations are epistatic to class 1 mutations concerning growth rate andbrood size, but hypostatic concerning the degree of fluoride-resistance. We
interpret this relationship by assuming that the jlr gene products form ametabolic, transport or signal transduction pathway that confers fluoride
sensitivity on C. elegans. Class 1 and class 2 gene products form the lower
and upper part of the pathway, respectively, and between the two parts there
is an intermediate that delays the growth of C. elegans when accumulated.
Mutations in class 1 genes cause fluoride-resistance by blocking the pathway.
Moreover, they cause accumulation of the intermediate and decrease of thegrowth rate. Class 2 mutations prevent the accumulation of the intermediate
and restore the growth rate. There is probably a narrow bypass to theintermediate, because class 2 mutants are only weakly resistant to fluoride

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61

ion.
To understand the biochemical reactions involved in fluoride-sensitivity wecloned some of theflr genes by the transposon-tagging method. For theflr-I
gene we first cloned a 2.8 kbBamHI genomic DNA fragment containing
transposon Tel (1.6kb) from a Tel-insertion mutantflr-I (utll) and then a
3.7 kb HindIII fragment from wild-type worms, using the DNA fragment
flanking the Tc 1 as the probe. For the flr-3 gene we cloned a 3.6 kb EcoRI
fragment containing Tel from a Tel-insertion mutantflr-3(ut9). In addition, we have isolated eDNA clones of flr-I and flr-3 using their genomic
DNA fragments as the probes. Sequencing of the genomic and eDNA clones
is in progress.
Caenorhabditis elegans Larval Lethal Mutations
That Cause Gross Morphological Changes
Isao KATSURA, Ryuichi HISHIDA and Takeshi ISHIHARA

We have been analyzing mutants that die as larvae with abnormal shapes,
in order to study essential functions in late embryonic and post-embryonicdevelopment (See the Annual Report of 1991 for details). Among thosemutants, we concentrated our study on those in which the outer surface of
the intestine is detached from the inner surface of the body wall. We callthem clr-I-like mutants, since the phenotype resembles that of clr-I among
known mutations. Ten such mutants have been isolated and mapped to 8
genes. Three of them map in known genes that are thought to act in signaltransduction: let-23 (receptor tyrosine kinase that acts in vulval induction),
let-34I (another gene that acts in vulval induction), and clr-I (lethal mutation in this gene is suppressed by mutations in sem-5, which codes for a
growth factor receptor binding protein). We therefore expect that most of
the clr-I-like mutations are related to signal transduction that acts in an
unidentified function essential to larvae as well as in vulval induction or sex
myoblast migration in some cases. Of the other seven mutants that we haveisolated, two were sent to J. Kimble's laboratory (University of Wisconsin)
and found to contain mutations deleting the lag-2 gene, which acts in various
cell-cell interactions. The remaining five mutants seem to define four new
genes, which we are trying to clone. In addition to the mutants mentioned
above we have isolated and are analyzing four new mutants with the clr-I-like

62

phenotype.

C. elegans Mutants Abnormal in Movement
or Shape of the HeadTakeshi ISHIHARA and Isao KATSURA

In relation to the studies mentioned above we isolated seven dr-I-likemutants of low penetrance, i.e., mutants of which some worms die as larvaethat have a space between the intestine and the body wall, while others growto adulthood and multiply. The adult worms are abnormal in the shape of thehead in two of the mutants, and in the movement of the head in four. These
results are important in the following aspects: first it shows that the dr-I-likephenotype may be related to defects in formation of an unidentified organ!
tissue in the head, and second it provides a method for selecting for mutantsin head movement, which are difficult to recognize. We are now mapping
those mutations and analyzing their phenotypes. We are also isolating
mutants in head movement by direct inspection, using those mutants aspositive controls.
Construction of lacZ Markers for Various Nerve Cells in the HeadTakeshi ISHIHARA and Isao KATSURA

In the head of C. elegans, there are circuits of many interneurons, which
process information from the head sensory organs and control motor neurons. To elucidate the mechanisms of formation and function of the neuralcircuits, we are genetically analyzing the nervous system of the head in C.
elegans. As a first step we are making markers for various nerve cells in thehead using the "promoter trapping" method (Hope I., 1991, Development
113, 399-408). Namely, we ligate random genomic DNA fragments of C.
elegans to the upstream of a cloned lacZ gene, transfer the DNA constructs
into C. elegans by microinjection and look for worms in which a defined setof nerve cells are stained by Xgal. We expect that such markers will be usefulin analyzing structural changes of the neural system in variQus mutants suchas those mentioned above and in isolating mutants abnormal in neuralmorphology.

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63

Systematic Analysis of C. elegans cDNA
Yuji KOHARA, Hironobu MITSUKI, Akiko NISHIGAKI, Tomoko MOTOHASHI
and Akiko SUGIMOTO

Aiming to ultimately understand the network of gene expression in development of the worm, we are trying to identify and characterize all of its
cDNA species.
(I) cDNA libraries were made from mRNA of a mixed-stage population
using ).ZAPII as a vector. The cDNA were size-fractionated on agarose gel
before cloning. Firstly, 8,000 independent plaques were picked up from alibrary of cDNA longer than 1.5 Kb. Abundant cDNA clones were removedby hybridization screening to establish a set of about 4,600 less abundant orrare cDNA clones.
(2) cDNA inserts of individual clones were PCR-amplified directly fromphage suspension using vector primers, purified on S-400 micro spin columnof the 96 well format, analyzed for their size and yield on agarose gel, andsubjected to the following analyses.
(a) Tag sequencing: by single pass sequencing from 5 -end (using avector primer) and 3 -poly-A end (using anchored oligo (dT) primer) on a
ABI auto-sequencer. The tags were subjected to DNA and protein databasesearch. The 3 -tags, as ID sequences of individual genes, were also comparedwith each other for classification.
(b) Mapping on the genome: by hybridization to the YAC polytene filters
using the DIG-based non-RI system.
(c) Analysis of expression pattern: by hybridization to the membrane
strips on which all cDNA species from single embryos at various stages havebeen dot-blotted.
Thus far, about 900 clones have been processed for (a) and (b). Briefly,
4% of them matches C. elegans gene or cosmid sequences, 8% the Chris
Martin's cDNA clones, and 23% shows significant similarity in the databasesearch. To speed the tag sequencing, the clones will be shared by the genomic
sequencing consortium. After assessing the extent of saturation with respect
to cDNA species in the clone set, we are going to make a set of rarer clones.
1

1

1

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Tel Insertional Mutant Bank of C. elegans
Yoshiki ANDACHI and Yuji KOHARA

In order to device efficient ways to disrupt any genes of interest, we havemade a transposon Tcl insertional mutant bank and have developed astrategy to retrieve mutant strains.
A mutator strain RW7097 (mut-6) gives visible mutants by Tcl insertionof a given gene spontaneously at the frequency of 10 -4-10 5, meaning that
one can expect to get one insertional mutant of a given gene out of 104_10 5
independent worms of this strain. Thus, pools of the mutant strain RW7097,
each containing about 100 larvae (LIs), were fed in 6 cm NGM-agarose
plates to produce Fl progeny. Duplicate frozen stocks were made from apart of the Fl worms and genomic DNA were extracted from the anotherpart. About 100 pools have been processed at one time in this way, and wehave prepared 800 pools (altogether 80,000 independent worms) thus far.
Methods to retrieve insertional mutants are as follows. Aliquots of the
DNA prepared from 12 individual pools (corresponding to 1200 independent
worms) were mixed and subjected to PCR analysis using one primer fromTc-l and another from a target gene. Since a lot of non-specific PCR
products were generated in the reaction, we have developed the following
nested PCR strategy: For the first PCR, a target-gene specific primer was
biotinylated at the 5/-end to purify the product amplified from the target
region by using streptavidin-conjugated magnetic beads. The product boundto the beads were subjected to second PCR using a set of nested primers andsubjected to agarose gel electrophoresis. "Hot-start" of PCR by simply
putting a reaction plate on the block of a PCR apparatus pre-heated at 94 0 C
also turned out to contribute to reduction of the non-specific products. Whena mix of 12 pools gave a positive band, the DNA from individual pools wereanalyzed as above to select a positive pool. From the frozen stock of thepositive pool, about 200 worms were grown separately and the same PCR
analysis as above was performed to finally identify a mutant worm.
Insertion mutants have been searched for several known genes to test thequality of the bank and insertion sites have been analyzed. Although the
problem of false positive bands in our PCR assay are still open, we havesuccessfully isolated at least one insertional mutant for unc-22 gene out of 200
pools and each one for unc-54 and lin-12 genes out of 600 pools. Positions of
the Tcl insertion were determined by direct sequencing of positive PCRIV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

65

bands obtained in the mutant screening. In the unc-22 mutant, Tc 1 turned
out to be inserted in the fifth exon at 554 bp upstream the splice junction.
This mutant showed a typical unc-22 phenotype (twitching) and we haveconfirmed co-segregation between the insertion and appearance of the phenotype by crossing with wild type N2 strain. The unc-54 mutant did not show
any clear phenotype and we found that the insertion was in the first intron.
As to the lin-12 mutant, we have located the insertion in the ninth exon at 62
bp downstream the splice junction. Some defects of gonad and analysis of its
phenotype is underway.
Search for Localized Maternal mRNA in Early Embryos of C. elegans
Hiroaki TABARA and Yuji KOIIARA

A fertilized egg of C. elegans asymmetrically divides to give rise to thesomatic founder cell AB and the germ line founder cell Pl. The PI
blastomere divides 3 times in the stem cell fashion, producing 3 somatic
founder cells EMS, C, D and the germ line precursor cell P4. Although thefates of the blastomeres are thought to be determined by maternal elements(cytoplasmic determinants) as well as cell-cell interaction, the substances of
the cytoplasmic determinants are largely unknown. Candidates for thedeterminants could be some maternal mRNA and such determinants arehighly expected to be asymmetrically segregated during early cleavage. Thus,
we have began to search for localized mRNAs in early embryos.
Embryos were obtained by alkaline bleach treatment of adults and egg
shells were lysed by chitinase. An embryo at 2 cell stage was picked up, andthe blastomeres were separated physically using siliconized glass needles
attached to a micro-manipulator. Nucleic acids were extracted from theindividual blastomeres and 3'-proximal region of whole eDNA species were
amplified by a method developed in this laboratory. The amplified eDNA
were labeled and used for differential screening of a eDNA library made fromearly embryos (mostly earlier than the stage of gastrulation) using AZapII asa vector. As a first trial, we screened 5 X 104 plaques and obtained 8 positive
clones. These clones were classified into three AB specific and one P I specificclones. Analysis of these clones are in progress.

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Analsis of Morphogenesis in Postimplantation Mouse Embryos
Norio NAKATSUJI and Yasuaki SHIRAYOSHI

The postimplantation period of mammalian embryogenesis includes many
important events such as formation of the primitive streak, central nervous
system and primordial germ cells. Analysis of molecular and cellularmechanisms of this period has advanced through the use of various experimental methods including whole embryo culture and culture of embryonictissues and cells (Nakatsuji, N. (1992) Devel. Growth Differ. 34,489-499). In
our studies, particular attention is paid to the formation of the mesodermal
cell layer at the primitive streak, the appearance and migration of theprimordial germ cells, and differentiation of the central nervous system.
In collaboration with another laboratory (Dr. I. Nagata at Tokyo Metropolitan Institute for Neuroscience), we are also studying migration patternsof neuroblasts during histogenesis of the mammalian central nervous system(CNS). We found a new type of cell behavior, "perpendicular contact
guidance," which is exhibited by CNS neurons but not by the peripheral
nervous system neurons on aligned parallel bundles of neurites. Such aperpendicular pattern occurred on an artificially fabricated microstructure on
the quartz surface (Nagata, I. and Nakatsuji, N. (1993) Development 117,
401-408). We are now analyzing the roles of the perpendicular contact
guidance in the actual morphogenesis of brain cortices.
Manipulation of Embryogenesis Using Mammalian Embryonic Cells
Norio NAKATSUJI and Yasuaki SHIRAYOSIII

Isolation and manipulation of embryonic cells in culture and reincorporation of such manipulated cells into embryos would facilitate many experimental strategies in basic research on embryogenesis and also in more appliedresearch in the medical and agricultural sciences.
We used embryonic stem (ES) cell lines established from mouse blastocysts
for an analysis of embryogenesis. For example, we isolated a cell line which
expresses ,B-galactosidase, and analyzed chimaeric tissues using these labeledcells. Also, we are trying to expand such strategy into other mammalian
species (Kashiwazaki, N. et al. (1992) Vet. Rec. 130, 186-187).
We have been studying the primordial germ cells (PGCs) which may be

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67

used for manipulation of the mouse germ line. As a first step, we isolated
germ cells and somatic cells from fetal ovaries (12.5-14.5 days post coitum),cultured them as dispersed cells, and made reaggregates. Such reaggregates(reconstituted fetal ovaries) were transplanted into ovary capsules of adultfemale mice. Then, they gave birth to offspring from transplanted germ cells(oogonia or oocytes) (Hashimoto, K. et al. (1992) Devel. Growth Differ. 34,
233-238). We are now developing a new in vitro experimental systemthrough the culture of PGCs for analysis of proliferation and differentiationof germ cells (Kawase, E. et al. (1993), Devel. Bioi., in press).
Molecular Analysis of Cell Differentiationin Postimplantation Mouse Embryos
Yasuaki SHIRAYOSHI and Norio NAKATSUJI

Determination of cell fate and cell differentiation are crucial events in early
embryogenesis. In our laboratory, we have started experiments to approachsuch problems from their molecular aspects. One area of experimentation
involves identifying important genes in determination and differentiation of
the central nervous system during the postimplantation period. We haveconstructed a eDNA library from 7.5-8.5 days old mouse embryos, in which
the neural plate and neural tube formation is initiated, and screened forimportant genes relating to CNS development.

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V.

POPULATION GENETICS

The Meaning of Natural Selection Revisited at the Molecular Level
Tomoko OHTA

Various molecular interaction mechanisms cause biased transmISSIOn ofgenes. Meiotic drive is an example of strong bias, and compartmentalizationof mammalian chromosomes reflects weak bias. Such biases are the results ofinteraction between DNA and proteins, and should be distinguished fromnatural selection. Separating the effects of molecular interaction from thoseof natural selection, however, is often very difficult. Natural selection andmolecular mechanisms interact, and our understanding of how selection
works requires revision. For details, see Trends in Ecology and Evolution 7,
311-312.
Theoretical Study of Near Neutrality. II. Effect of Subdivided Population
Structure With Local Extinction and Recolonization
Tomoko OHTA

There are several unsolved problems concerning the model of nearly
neutral mutations. One is the interaction of subdivided population structure
and weak selection that spatially fluctuates. The model of nearly neutral
mutations whose selection coefficient spatially fluctuates has been studied byadopting the island model with periodic extinction-recolonization. Both thenumber of colonies and the migration rate play significant roles in determining mutants' behavior, and selection is ineffective when the extinctionrecolonization is frequent with low migration rate. In summary, the numberof mutant substitutions decreases and the polymorphism increases by increasing the total population size, and/or decreasing the extinction-recolonization
rate. However, by increasing the total size of the population, the mutant
substitution rate does not become as low when compared with that inpanmictic populations, because of the extinction-recolonization, especiallywhen the migration rate is limited. It is also found that the model satisfactorily explains the contrasting patterns of molecular polymorphisms observed

V.

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69

in sibling species of Drosophila, including heterozygosity, proportion ofpolymorphism and fixation index. For details, see Genetics 130, 917-923.
Simulation Study of a Multigene Family, With Special Reference
to the Evolution of Compensatory Advantageous Mutations
Christopher J. BASTEN and Tomoko OHTA

We investigate the evolution of a multigene family incorporating the forcesof drift, mutation, gene conversion, unequal crossing over and selection. The
use of simulation studies is required due to the complexity of the model.
Selection is modeled in two modes: positive selection as a function of thenumber of different beneficial alleles and negative selection against deleterious alleles. We assume that gene conversion is unbiased, and that all
mutations are initially deleterious. Compensation between mutants creates
beneficial and neutral alleles, and allowances are made for compensatory
mutations either within or between the members of a multigene family. We
find that gene conversion can enhance the rate of acquisition of compensatory
advantageous mutations when genes are redundant. For details, see Genetics
132, 247-252.
Gene Conversion Generates Hypervariability at the Variable
Regions of Kallikreins and Their Inhibitors
Tomoko OHTA and Christopher J. BASTEN

The two mechanisms for generating hypervariability at the reactive centerof serine proteases and their inhibitors are gene conversion followed bynatural selection and natural selection for point mutation. One way to clarify
the effects of these two mechanisms is to calculate separately the number of
nonsynonymous substitutions and that of synonymous substitutions at thevariable regions and at the conserved regions. Our data analysis shows that
not only the number of nonsynonymous substitutions but also the number ofsynonymous substitutions at the variable regions exceed the corresponding
numbers at the conserved regions. Thus gene conversion has provided
needed variability at the variable regions of serine proteases and theirinhibitors. Natural selection has helped perpetuate such variability. For
details, see Molecular Phylogenetics and Evolution 1, 87-90.

70

Statistical Method for Estimating the Effective
Population Size in Pacific Salmon
Fumio T AJIMA

The effective population size is one of the most important parameters that
determine the genetic variation in a population. Recently, a statisticalmethod for estimating the effective breeding size of Pacific salmon (Oncorhynchus spp.) which have unusual life history features, i.e., semelparity withoverlapping year classes, has been developed (Waples (1990) J. Hered. 81,
277-289). In this method, however, computer simulations are required. I
have developed a simple algorithm for estimating the effective population size
without computer simulation. This algorithm can be applied as long as the
distribution of age at which a spawner returns is known. For details, see J.
H ered. 83, 309-311.
Statistical Method for Estimating the Standard Errors of Branch Lengths
in a Phylogenetic Tree Reconstructed without Assuming Equal Rates
of Nucleotide Substitution among Different Lineages
Fumio T AJIMA

A statistical method was developed for estimating the standard errors ofbranch lengths in a phylogenetic tree reconstructed without assuming equal
rates of nucleotide substitution among different lineages. This method can beeasily used for testing whether the length of an interior branch in a reconstructed tree is positive or not, i.e., whether the topology of the tree is correct
or not. Computer simulations indicated that this method is appropriate for astatistical test. As an example, this method was applied to phylogenetic trees
reconstructed for the four hominoid species: human, chimpanzee, gorilla, andorangutan. The results obtained show that the present method provides a
powerful statistical test. For details, see Mol. BioI. Evol. 9, 168-181.
Evolutionary Genetics of Human Paleo-populations
Naoyuki TAKAHATA

Genetic variation revealed at the DNA level provides useful information
about not only the evolutionary mechanisms involved but also the history of

v. POPULATION GENETICS

71

organisms. As to the former, we have learned, at least in principle, how andwhat kind of genetic variation is generated at the molecular level and how it
is shaped by the mechanisms acting at the population level. An importanttask is to clarify outcomes of simultaneously interacting molecular andpopulation mechanisms and to assess their relative importance in actual
evolutionary processes. Another exciting finding emerging in current evolutionary biology is the possibility that genetic variation, together with appropriate theories, may shed new light on the population history of organisms
and population dynamics including speciation processes. In this pursuit, it isimportant to delimit the time scale in which genetic variants have persisted
and therefore are useful as a document of population histories. Equallyimportant, to interpret these data, is development of appropriate theories.
Focussing on these two points, I present some theoretical results and apply
them to the major histocompatibility complex loci in humans. I argue thatthe human lineage has never experienced severe bottlenecks and that theextent of panmixia of the population might have been low before humans
migrated out of Africa but later became high. The implication is related tothe origin of modern humans, subsequent racial differentiation and thefounder principle, a popular mechanism often invoked in speciation. See
Population Paleo-Genetics, pp. 1-21, N. Takahata (ed.), Japan Scientific
Society Press, 1992 for details.
Some Comments on Calibration of Molecular Evolutionary Rates
Naoyuki TAKAHATA and Yoko SATTA

Molecular evolutionary studies rely much on the existence of a molecular
clock, which allows one to date molecular events on the geological timescale.
Any method of calibrating the rate of molecular evolution begins withaligning two homologous DNA sequences that have been separated for aknown time period (t) and counting the number of nucleotide differences (D)in a specified region. The proportion of nucleotide differences per site p is
obtained by dividing D by the total number (n) of nucleotide sites compared.
The value of p is used to estimate the actual number of substitutions (K) thathave occurred in both sequences during t and the evolutionary rate which is
given by K/(2t). This final step requires a stochastic model which describes
the total number of substitutions per site per unit time and substitution

72

patterns among the four different nucleotides. Two basic assumptions arecommonly made. First, the sites in a specified region of DNA sequenceschange stochastically according to a common rule and the change follows the
Poisson process. These assumptions imply that there is no heterogeneity inintrinsic substitution rates within the region and that accumulation of nucleotide changes can be described by a stationary Markov process. Elsewhere,
Takahata questioned the validity of the second assumption for nucleotide
substitutions in the antigen recognition site (ARS) of the major histocompatibility complex (Mhc) genes. In this communication, we focus on the first
assumption, although our discussion is intimately related to the second. The
same problem was discussed in relation to amino acid sequence data, but it
seems more important for DNA sequence data because the number ofpossible states treated in a model is only four rather than 20. See Immunogenetics 36, 126-129, 1992 for details.
Trans-species Polymorphism of the Major Histocompatibility
Complex (Mhc) Loci
Naoyuki TAKAHATA, Yoko SATTA and Jan KLEIN

The functional major histocompatibility complex (Mhc) genes are highly
polymorphic. The Mhc polymorphism is unusual in many respects, among
which the trans-species mode of evolution is the most remarkable. It has
convincingly been shown that a large number of allelic lineages at functional
Mhc loci have persisted for tens of millions of years in various lines of species
that include humans. Because of the long persistence time, some human Mhc(HLA) allelic lineages are shared not only be African apes, but also by Old
World monkeys, and the divergence of alleles often predates species divergences to a great extent. This finding has raised many challenging problems
for evolutionary biologists. In this paper we present our analysis of thenucleotide substitution rates as well as the mode and intensity of natural
selection occurring at the primate functional Mhc genes. The implication of
the HLA polymorphism for human evolution is discussed. See Progress in
Immunology VIII (Proceedings of the 8th International Congress of Immunology Budapest), pp. 153-158, J. Gergely et al. (eds.), Springer Hungarica,
1992 for details.

V.

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73

Polymorphism and Balancing Selection at Major
Histocompatibility Complex Loci
Naoyuki TAKAHATA, Yoko SATTA and Jan KLEIN

Amino acid replacements in the peptide-binding region (PBR) of thefunctional major histocompatibility complex (Mhc) genes appear to be driven
by balancing selection. Of the various types of balancing selection, we haveexamined a model equivalent to overdominance that confers heterozygote
advantage. As discussed by A. Robertson, overdominance selection tends tomaintain aIleles that have more or less the same degree of heterozygote
advantage. Because of this symmetry, the model makes various testablepredictions about the genealogical relationships among different aIleles andprovides ways of analyzing DNA sequences of Mhc aIleles. In this paper, weanalyze DNA sequences of 85 aIleIes at the HLA-A, -B, -C, -DRBI and-DQBI loci with respect to the number of aIleles and extent of nucleotide
differences at the PBR, as weIl as at the synonymous (presumably neutral)sites. Theory suggests that the number of aIleles that differ at the sites
targeted by selection (presumably the nonsynonymous sites in the PBR)
should be equal to the mean number of nucleotide substitutions among pairsof aIleles. We also demonstrate that the nucleotide substitution rate at thetargeted sites relative to that of neutral sites may be much larger than 1. The
predictions of the presented model are in surprisingly good agreement withthe actual data and thus provide means for inferring certain population
parameters. For overdominance selection in a finite population at equilibrium, the product of selection intensity (s) against homozygotes and theeffective population size (N) is estimated to be 350-3000, being largest at the
B locus and smaIlest at the C locus. We argue that N is of the order of 105
and s is several percent at most, if the mutation rate per site per generation is
10 8 • See Genetics 130, 925-938, 1992 for details.

74

Evolution of the Mouse t Haplotype: Recent and Worldwide
Introgression to Mus Musculus
Takashi MORITA, Hiroshi KUBOTA, Keiko MURATA, Masami NOZAKI,
Christiane DELARBRE, Keith WILLISON, Yoko SATTA,
Mitsuru SAKAIZUMI, Naoyuki TAKAHATA,
Gabriel GACHELIN and Aizo MATSUSHIRO

Mouse t haplotypes are variants of chromosome 17, consisting of four
inversions. Oespite the homozygous lethality and pleiotropic effect onembryonic development, sperm production, and recombination, they havewidely spread in natural populations of the house mouse (lD-40% in frequency) because of the meiotic drive advantage. We sequenced 14 Tcp-1 (tcomplex polypeptide 1) genes from four t haplotypes, nine wild mice, and a
rat as a reference. From a comparison of intron sequences of 610 base pairs,
we dated the origin of t haplotypes to 2.9±0.7 million years ago, which
predates the splitting of Mus musculus subspecies (:::::: 1 million years ago).
However, the Tcp-1 intron sequences of t haplotypes from different M.
musculus subspecies from various parts of the world show no divergence,indicating the recent introgression (no earlier than 0.8 million years ago) ofa single ancestral type. Nucleotide changes in coding regions are also
consistent with this conclusion. Hence, polymorphisms among t haplotypes
including lethality factors have accumulated during this short time period
independently in each M. musculus subspecies. See Proc. Natl. Acad. Sci.
USA. 89, 6851~6855, 1992 for details.
Evolution of the Primate DRB Region
Jan KLEIN, Yoko SATTA, Colm O'HUIGIN, Werner E. MAYER
and Naoyuki TAKAHATA

There are eight ORB loci in the human population, ORBI through ORB8,
of which four at the most are present on anyone chromosome; some humanchromosomes carry only one, two, or three ORB loci. We discuss theevolutionary origin of the individual loci, taking into account all the available
information on the ORB region not only of humans, but of other primates aswell. This information includes exon 2 sequences of over 200 genes, entire
coding sequences of some 60 genes, exon and intron sequences of 10 genes
and pseudogenes, sequences of 11 Alu repeats inserted into ORB (pseudo)

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POPULATION GENETICS

75

genes, and the worked-out organization of five different human, two gorilla,
one chimpanzee, and one orang-utan DRB haplotypes. Our discussion isbased on the consideration of evolutionary trees constructed by both distanceand maximum parsimony methods from nucleotide sequence data, of diagnostic substitutions (i.e. nucleotide or amino acid substitutions characterizing
a particular group of genes and absent in all or almost all other genes), of thepresence or absence of particular inserts in the genes, and of the overall
organization of the DRB haplotypes. See HLA 1991, pp. 45-56, T. Kimiyoshi, M. Aizawa and T. Sasazuki (ed.), Oxford University Press, Oxford,
1992 for details.

76

VI.

EVOLUTIONARY GENETICS

Long-range G+C% Mosaic Structures of the Human Genome
and a Border of the Mosaic Domains; New Genes Foundin the MHC Class III Region
SUGAYA, K*, WADA, K.**, MATSUMOTO, K. and IKEMURA, T.

The genome of warm-blooded vertebrate is known to be a mosaic ofcompartments differing in G+C content, designated "isochores", which
seemed to relate to staining chromosomal bands. One approach to investigating the evolutional process in producing the mosaic structure and understanding its biological significance, is thought to be in the characterization of
the border of the mosaic domains on the sequence level. After surveying
human gene sequences in DNA databases, which were linked within several
hundred kb, two possible borders of long-range G + C% mosaics werepreviously assigned; one between X-linked G6PD and F8C, and the otherbetween HLA-DRA (in MHC Class 2) and CYP2l (in MHC Class 3) on
6p21.3. Recently we obtained cosmids and YAC contiguous clones, which
completely cover the latter border in the MHC locus. Near (or at ) the
border, a characteristic genome portion (at least 50 kb) that is mainlycomposed of Alu repetitive sequences was found. In its class III-side
neighboring region, we found four new genes, Notch-like gene (homolog ofmouse mammary tumor int-3 gene), PBX2 gene (a homeobox gene), RAGE
(receptor for advanced glycosylation end products of proteins) gene, andextracellular matrix tenascin-like gene, in order from centromere to telomere.
For details, see "HLA 1991" (Tsuji et al., eds.), Oxford Univ. Press, 2, 125128 and 185-190., and Immunogenetics, 36, 40Q-404, 1992.
Analyses on Codon Usage
T.IKEMURA

The cellular tRNA content of E. coli, Salmonella typhimurium, andSaccharomyces cerevisiae was measured about ten years ago by our group, and* Dept. of Genetics, The Graduate University for Advanced Studies.
** Chukyo University, School of Computer and Congnitive Science.

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77

organism-specific codon choice patterns of these microorganisms were attributed to the availability of tRNA isoacceptors within a cell. In those studies,the following four deductions were made, and this year, based on a vast
amout of codon usage data accumulated after publication, these were reexamined and confirmed.
I) There is a close correlation between tRNA content and codon usage inmost protein genes sequenced for these organisms. The exceptional cases arelargely confined to genes with low expression. 2) There are clear similarities
in codon choice among different genes of one microorganism, regardless ofgene function. We called this organism specific codon-choice the "codon
dialect" of the organism. The codon dialect is related to the specific tRNA
isoacceptor population of an organism. 3) The extent of bias in codon choice
is related to the protein production level of each gene. Codon usage in genes
for abundant protein molecules is always more dependent on tRNA content
than that in moderately or poorly expressed genes. 4) Foreign-type genes
such as those of transposons, plasmids, and viruses often have quite different
codon patterns from those of host organisms, and thus the above deductions
are not necessarily applicable to them.
Regarding codon-choice patterns, the distinction between unicellular microorganisms and multicellular higher eukaryotes, rather than the distinction
between prokaryotes and eukaryotes,was emphasized. In unicellular organisms, most, if not all, genes are expressed in individual cells. To maintainefficiency of translation processes (e.g., to save GTP energy used in the
proof-reading process), these genes have an analogous codon-choice pattern(codon dialect) that fits the tRNA population. In the case of higher
eukaryotes, each body is composed of an enormous number of cells. Many of
the cells are highly differentiated, and a restricted spectrum of genes isexpressed in individual cells. The target of Darwinian selection is a wholebody instead of individual cells. Thus, fitness change caused by a synonymous change in an ordinary gene should be much smaller than that forunicellular organisms. A synonymous change to a codon with higher tRNAavailability may save a certain amount of GTP energy and effective working
time of ribosomes in a restricted portion of cells. However, the contributionof this change in overall fitness of a body should be extremely small, so it
would presumably be counted as a neutral mutation. This contrasts with thecase of unicellular organisms where the cell itself is the direct target of
selection. Codon usage in ordinary genes of multicellular organisms is78

therefore thought to be less stringently constrained by tRNA availability thanthat in genes of unicellular organisms. When some constraints from factors
other than translation efficiency exist (e.g., long-range G + C% mosaic
structures of the genome), their codon choices presumably follow the constraints. The evident diversity of the third codon G + C% and correlation ofthis diversity with the G+C% mosaic structures may reflect the weakerconstraint imposed by tRNA content. For details, see CODON USAGE, in"Plant Molecular Biology LABFAX" (Broun, T. A., ed.), Blacwell Scientific
Publications, in press, and CORRELAnON BETWEEN CODON USAGE
AND tRNA CONTENT IN MICROORGANISMS, in "Transfer RNA in
Protein Synthesis" (Hatfield, D. L. et al., ed.) CRC Press (1992).
Evolutionary Rate of Insertions and Deletions in Nucleotide Sequences
Naruya SAITOU

Gaps created through alignment are routinely eliminated when we compare nucleotide sequences, and nucleotide substitutions have been extensively
studied. However, insertions and deletions that are responsible for gaps inaligned sequences seem to occur rather frequently, especially in non-coding
regions. We studied the rate of insertions and deletions in pseudogene
sequences by using (1) Tajima and Nei's evolutionary distance due toinsertions and deletions and (2) the maximum parsimony principle to locate
insertion/deletion events on a phylogenetic tree. The rate of insertion/deletion events was found to be rather constant. For details, see Molecular
Paleo-Population Biology: Proceedings of the Seventeenth Taniguchi Symposium (N. Takahata ed.), pp. 335-343, Japan Science Society Press, Tokyo,
1992.
Concerted Evolution of the Primate Immunoglobulin
Alpha Gene through Gene Conversion
Shohji KAWAMURA*, Naruya SAITOU and Shintaroh UEDA*

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (Ca) genes (chimpanzee Ca2, gorilla Ca2, and gibbon
Cal and Ca2 genes), and these new data made it possible to examine gene*

Department of Anthropology, Faculty of Science, The University of Tokyo.

VI.

EVOLUTIONARY GENETICS

79

conversions of all the hominoid Ca genes. The following three methods wereused to detect gene conversions: 1) phenetic tree construction, 2) detection of
the DNA segment which has extremely low variability between duplicated
Ca genes under the assumption of random distribution of varied and unvaried sites, and 3) site-by-site search of shared nucleotide changes between
duplicated Ca genes by comparing sequences among species on the basis of aparsimony principle. Results by using method 1 suggest the occurrence ofgene conversion at least in the human, chimpanzee, and gorilla lineages, while
those by using method 2 do in the human, gorilla, and gibbon Ca genes. Lackof a long segment of unvaried sites between the chimpanzee Cal and Ca2
genes might be because gene conversions were so old that mutations had
accumulated, or because detectably long conversions did not occur. By using
method 3 we identified clusters of shared nucleotide changes between duplicated Ca genes in any of human, chimpanzee, gorilla, gibbon, and their
hypothetical ancestors. These results indicate that gene conversions occurredrather frequently between duplicated Ca genes during hominoid evolution.
For details, see J. BioI. Chem. 267,7359-7367, 1992.
Genetic Affinities of Human Populations
Naruya SAITOU, Katsushi TOKUNAGA* and Keiichi OMOTO*

Differentiation of human populations does not necessarily follow a simple
model of population fission, and gene migrations may occur after a relatively
long isolation. Thus for describing genetic relationships among populations,a network (unrooted tree) of genetic affinity seems to be more appropriate
than a dendrogram (rooted tree), which is expected to estimate the phylogenetic tree of populations. The neighbor-joining method, in which theprinciple of minimum evolution is used for constructing networks, may besuitable for this purpose. We applied this method to three sets of genetic
distance data and compared the resulting affinity networks with those obtained by other methods. It was found that the genetic affinity networks
generally reflect the geographical isolation and migration of human populations. For details, see Society for the Study of Human Biology Symposium
Series 33: Isolation, Migration. and Health (R. F. Roberts, N. Fujiki and K.
Torizuka eds.), pp. 118-129, Cambridge University Press, Cambridge, 1992.
*

Department of Anthropology, Faculty of Science, The University of Tokyo.

80Construction of the MHC Database and its Utilization
to Anthropological Studies
Takashi GOJOBORI

With help of many researchers, we successfully compiled all the data thatwere sent to the Central Data Analysis Committee by the participants of theEleventh International Histocompatibility Workshop. The data compiled
were stored into the relational database called "MHC database." This
database was created by use of a commercial relational database management
software (RDBMS). Specifically, we used Informix-SQL version 2.10. This
database contains the HLA typing data for a total of 27,765 individuals
sampled from various ethnic groups in the world.
Using the MHC database, anthropological studies were performed byobtaining the following data: (1) frequencies of HLA antigens for eachethnic group, (2) haplotype frequencies and linkage disequilibria among the
HLA genes in each ethnic group, (3) genetic distances among the various
ethnic groups, (4) a phylogenetic tree based on the genetic distances, and (5)geographical distributions of each HLA allele. From these data, we evaluated the degrees of genetic variation at the HLA loci for each ethnic group.
For details, see HLA 1991 (Oxford University Press) I: 65, 66~67, 67~76,
623-626, and 627-632.
Rates of Synonymous Substitution and Base Composition
for Nuclear Genes in Drosophila
Etsuko N. MORIYAMA and Takashi GOJOBORI

We compared the rates of synonymous (silent) substitution among various
genes in a number of species of Drosophila. First, we found that even for aparticular gene, the rate of synonymous substitution varied considerably with
Drosophila lineages. Second, we showed a large amount of variation insynonymous substitution rates among nuclear genes in Drosophila. These
rates of synonymous substitution were correlated negatively with C content
and positively with A content at the third codon positions. Nucleotide
sequences were also compared between pseudogenes and their functional
homologs. The C content of the pseudogenes was lower than that of thefunctional genes and the A content of the former was higher than that of the

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81

latter. Because the synonymous substitution for functional genes and thenucleotide substitution for pseudogenes are exempted from any selective
constraint at the protein level, these observations could be explained by abiased pattern of mutation in the Drosophila nuclear genome. Such a bias in
the mutation pattern may affect the molecular clock (local clock) of eachnuclear gene of each species. Finally, we obtained the average rates ofsynonymous substitution for three gene groups in Drosophila; 11.0 X 10 9,17.5 X 10 9 and 27.1 X 10 9isiteiyear.
For details, see Genetics 130: 855-864.
Patterns of Nucleotide Substitution Inferred from the Phylogeniesof the Class I Major Histocompatibility Complex Genes
Tadashi IMANISHI and Takashi GOJOBORI

Patterns of nucleotide substitutions in human major histocompatibility
complex (MHC) class I genes were estimated by using phylogenetic tress inthe form of a set of 12 parameters, each of which represents the relative
frequency of substitutions from a particular nucleotide to another. The
pattern at the antigen recognition sites (ARS) in functional MHC genes was
remarkably different from that at the remaining coding region (non-ARS).
In particular, the proportion of transitions (P,) among all the nucleotide
substitutions was extremely low at the third codon positions of ARS. In the
HLA-A genes, Ps at the third codon positions was only 6% in ARS, whereas
it was 69% in non-ARS. In HLA-B, the corresponding values were 30% in
ARS and 80% in non-ARS, respectively. On the other hand, Ps in a class I
pseudogene (HLA-H) was 57%, which was in good agreement with P s inother pseudogenes. Because pseudogenes are selectively neutral, the patternin pseudogenes is regarded as the pattern of spontaneous substitution mutations. In general, the pattern in functional genes that are subject to selective
forces deviates from the pattern in pseudogenes. At the third codon positions
in coding regions, transitions scarcely cause amino acid replacements, whereas about half of transversions do cause replacements. Accordingly, P, at thethird codon positions decreases if amino acid replacements are accelerated bynatural selection but increases if amino acids are conserved by functional
constraint. Our observations imply that the ARS region is subject to natural
selection favoring amino acid replacement, whereas the non-ARS region is

82

subject to functional constraint.
For details, see J. Mol. Evol., 35: 196-204 and HLA 1991 (Oxford
University Press), 1: 357-359.
Evolutionary Origin of a Kunitz-Type Trypsin Inhibitor Domain Inserted
in the Amyloid j3 Precursor Protein of Alzheimer's Disease
Kazuho IKEO, Kei TAKAHASHI, and Takashi GOJOBORIThe Kunitz-type protease inhibitor is one of the serine protease inhibitors.
Recently, a
Kunitz-type sequence was found in the protein sequence of the amyloid j3
precursor protein (j3AAP). It is known that j3AAP accumulates in theneuritic plaques and cerebrovascular deposits of patients with Alzheimer's
disease. Collagen type VI in chicken also has an insertion of a Kunitz-typesequence. To elucidate the evolutionary origin of these insertion sequences,
we constructed a phylogenetic tree by use of all the available sequences ofKunitz-type inhibitors. The tree shows that the ancestral gene of theKunitz-type inhibitor appeared about 500 million years ago. Thereafter, thisgene duplicated itself many times, and some of the duplicates were inserted
into other protein-coding genes. During this process, the Kunitz-type sequence in the present j3APP gene diverged from its ancestral gene about 270
million years ago and was inserted into the gene soon after duplication.
Although the function of the insertion sequences is unknown, our molecular
evolutionary analysis shows that these insertion sequences in j3AAP have an
evolutionarily close relationship with the inter-a-trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membranebound serine
protease in human T4+ lymphocytes.
For details, see J. Mol. Evol. 34: 536-543.
It is found in blood, saliva, and all tissues in mammals.

Allele and Haplotype Frequencies for HLA and Complement Lociin Various Ethnic Groups
Tadashi IMANISHI, Tatsuya AKAZA, Akinori KIMURA,
Katsushi TOKUNAGA, and Takashi GOJOBORI

Frequencies of alleles and haplotypes for HLA and complement loci were
estimated from the typing data in the MHC database compiled at the

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83

Eleventh International Histocompatibility Workshop. All the frequencies
were estimated by the maximum likelihood method assuming the HardyWeinberg law. We used the data only for the ethnic groups whose number ofsampled individuals exceeded 50. As the results, we found that there were
vast amounts of heterogeneities not only within a single ethnic group but also
among different ethnic groups. This confirms that the HLA loci are genetically very heterogeneous.
We also computed the values of linkage disequilibria for two, three, four,and five loci. Our results show that some loci are strongly linked.
For details, see HLA 1991 (Oxford University Press) 1: 76-79 and 10651220.
Six Strains of Human Immunodeficiency Virus Type-! Isolated
in Japan and their Molecular Phylogeny
Nobuaki SHIMIZU, Yasuhito TAKEUCHI, Takaji NARUSE, Minoru INAGAKI,
Etsuko N. MORIYAMA, Takashi GOJOBORI, and Hiroo HOSHINOFive strains of human immunodeficiency virus type 1 (HIV-1) wereisolated from five Japanese hemophilia patients. Two isolates, HIV-l [GUN1] and HIV-l [GUN-2], were from brother patients with hemophilia B. The
other three isolates, HIV-l [GUN-3], HIV-l [GUN-4], and HIV-l [GUNS], were from homophilia A patients. Another HIV-l strain, HIV-l [GUN6], was isolated from a Canadian male homosexual with AIDS. The
restriction endonuclease cleavage maps of the proviral genomes of these six
HIV-1 strains revealed that they were apparently different from each other.
The phylogenetic trees constructed using restriction maps and nucleotide
sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses.
Phylogenetic analyses showed that they were more closely related to HIV-ls
which had been reported to be isolated from homosexual patients in the
United States than those isolated from African patients. In particular, GUN1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2,
while GUN-5 and GUN-6 isolates were on the branch of HTLV-IIIB-related
isolates from New York.
For details, see J. Mol. Evol. 35: 329-336.

84

Complement Studies of the Eleventh International
Histocompatibility Workshop
Katsushi TOKUNAGA, Hidenori TANAKA, Gottfried MAUFF, Koichi SUZUKI,
Hiroaki NISHI MUKAI, Georges HAUPTMANN, Julie FINLAY, Tadashi IMANISHI,
Takashi GOJOBORI, Tatsuya AKAZA, Takeo JUJI, and Roger L. DAWKINSIn this study, we had three main purposes: (l) to see the associations
between complement and HLA alleles in various ethnic groups, (2) to
identify common MHC haplotypes in different ethnic groups, and (3) toexamine the association of complement alleles with certain diseases. In orderto achieve these purposes, we used complement data from as many individuals as possible.
We successfully identified a number of 'new' variants when a large numberof samples from various ethnic groups were examined. In particular, two
new and until-now-not-described variants of C2 were isolated from European
Caucasoids, and at least 10 new variants of C4 were observed in various
ethnic groups.
A total of 5 I ethnic groups with approximately 4500 individuals were
examined for the complement allotypes. All gene frequencies of Bf, C4A,
and C4B, and haplotype frequencies of Bf-C4A-C4B, were estimated for eachethnic group. Five-locus complement-HLA haplotype frequencies with
HLA-A and HLA-B alleles or with HLA-B and HLA-DR alleles were also
calculated. We found that there were apparently two major types of MHC
haplotype patterns. First, many of MHC haplotypes showed a rather
restricted distribution of geography. Thus, these multilocus haplotypes
should be excellent ancestral markers for the characterization of various
human populations. Second, some MHC haplotypes showed a very wide
distribution beyond ethnic boundaries. At present, we are still examining theassociation of complement alleles with certain diseases.
For details, see HLA 1991 (Oxford University Press) 1: 947-954.

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VII.

HUMAN GENETICSA Simple Method for Generating Sequence Tagged Sites Adjacentto the Human Telomere Associated Repeat Sequence
Takashi IMAMURA, Hitoshi NAKASHIMA, Masako SAKAI and Rie INABA

Tandemly repetitive DNA sequences are thought to have the potential toexpress the locus-specific organization of their folding structure in the
nucleus. Because of the inherent possibilities of rapid evolutionary sequence
alterations, including a tolerance for mutation events, repetitive sequence
units are predestined to develop and balance locus-specific repetitve higher
order structures. This potential may create a specific chromatin folding
structure whenever there is selection force at the position of this repetitive
DNA sequence in the genome. Human tandemly repetitive sequences are ofinterest for two reasons. Firstly, they define the boundaries or the signpostsof the physical and genetic maps of human chromosomes and thus are
particularly important in the mapping of the human genome. Secondly, someof them are essential for human chromosome function.
This study attempted to explore the principal organization of humantelomere-associated (or proterminal) DNA at 48 distinct telomeres. We
devised a conventional ligation mediated PCR protocol employing magnetic
bead- immobilized primer to create relatively short restriction enzyme fragments from human telomere-associated regions. The general scheme to
producing proterminal sequences involves, as a first step, PCR with one
telomere repeat primer biotinylated. Genomic DNAs were restriction
enzyme digested and were ligated to a unidirectional linker primer, or apUCl9 polylinker site which was cleaved and dephosphorylated. The PCR
amplified products were immobilized on streptavidin coated magnetic beads,
which were substrates for the second round PCR. Using three different kindsof enzymes, the 48 telomere-associated regions have been cloned. The
sequence studies will yield new STS markers. The primer extension techniqueprovides a powerful tool for preferentially cloning sequences adjacent toblocks of the telomeric repeat. The cloning strategy is dependent on a given
restriction site present within reach of the PCR amplification. The small
fragment sizes of the insert might contribute to their stability in the plasmid

86

vector and facilitate their rapid sequencing. This way long, the specifictelomeric sequence can be coupled to a solid matrix that might be useful inaffinity isolation of overlapping sequences for walking towards centromere.
The cloning of the subtelomeric sequences devoid of the terminal repeats also
facilitates molecular analysis of specific chromosomal localization by hybridization in situ to metaphase chromosomes. This work is in progress.
The preliminary sequencing results suggest that subtelomeric regions areorganized as a string of distinct short sequence elements several kilobases
throughout their length, comprising several multiple, different, but related
tandem motifs. The chromosomal distribution of these sequences could behighly polymorphic, which might constitute an apparently new repeat sequence family. The data thus identify a new source of human genetic
variation on the human chromosome ends.
Mapping of 60 Cosmid Clones Isolated from a Human 18 Chromosome
Library by Fluorescent in situ Hybridization
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA and Takashi IMAMURA

Sixty cosmids have been mapped to metaphase chromosomes by fluorescentin situ hybridization under conditions that suppress signals from repetitive
DNA sequences. The cosmid clones were isolated from a human chromosome 18 specific library prepared from the several human-mouse hybrid celllines, each containing, as the only human chromosome component, either one
whole chromosome 18 or its short arm and the centromere portion. The longarm of chromosome 18 appeared to be deleted entirely in the latter cells.
These hybrid cell lines were established by fusion of the human-mouse hybrid
cells (NAllOlO) that retained human chromosomes 9 and 18 (ATCC,
Rockville, MD) with the rodent cells (THY-M), kindly provided by Dr. S.
Kaneda, National Institute of Genetics, which were selected in culture by thethymidylate synthetase activity. The human chromosome 18-specific hybrid
cell lines, 126-16 and 126-21, etc. were finally established by these proceduresin this laboratory. The cosmid library, prepared using pSuperCosl vector(Strategene, Inc., La Jolla, CA), was plated at low density on LB agar plates
containing ampicillin and grown overnight. Single colonies were picked up
and inoculated individually in 96-well microtiter plates containing LB/amp
medium. Colonies from 16 each of the 96-well microtiter plate were gridded

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87

onto a nylon membrane using a 96-well replicator. These membranes were
hybridized with total human DNA radiolabeled by nick-translation. Colony
selection was performed in duplicate and only those colonies that gave intense
autoradiographic signals were grown overnight. Pure cosmid DNA wereprepared by plasmid SELECT-MP Miniprep DNA purification system (3'5', Inc., Boulder, CO). Cosmid contaning large inserts (3D-45kb) wereidentified by restiriction digestion analysis.
To map single-copy genes in the human genome we have used fluorescentin situ hybridization (FISH) of nonisotopically labeled probes to human
metaphase chromosomes by a modification of a published procedure (Pinkel,
1986). The fluorescent images were recorded using the CCD camera andmicroscopic image prosessing system (Astromed Ltd., UK). Of the 100
cosmids thus far analysed, 60 were mapped on the specific chromosome band
region of chromosome 18, which include both telomeric and pericentromericregions of the short and long arms. None of the cosmids was mapped
fortuitously to the other autosomes, suggesting that the library contained
human DNA fragments only from chromosome 18. For construction of thereference map by in situ hybridization, 200 cosmid clones were randomly
chosen from the library of short arm, and 400 clones from the library ofwhole chromosome 18, which may be comprising about 80% (or 20%) asmuch DNA of the short arm (or whole regions) of human chromosome 18.
The resources of human chromosome cosmid libraries have recently been
available for mapping, but the localization in situ of a large number ofcosmids to this chromosome has not been reported. The technique of FISH
has proven to be a very efficient and accurate methodology for gene mappingand has been used to verify mapping data generated by somatic hybrid panels.
Cosmid localization to specific bands of chromosome 18 in our studies willcontribute to the physical and genetic mapping of this chromosome in generaland may have some potential for their application in clinical cytogenetics aswell. It is especially interesting to reveal that initially, the majority of cosmid
DNA markers mapped to human chromosome 18 appeared to be on the
R-banded negative regions. Such a nonrandom distribution pattern, causedby either cloning or mapping techniques as suggested by previous reports, has
not been obvious. We selected recently a number of cosmids that gave apositive fluorochrome signals on the supposedly R-band positive regions of
DAPI stained chromosomes, but not on the R-banded metaphase chromosome preparations. It is likely that these clones contain DNA derived from

88

the R-band positive regions. Further characterization of these clones may
provide more understanding of the structure and organization of GC- or
AT-rich sequences, and the possible function of the human chromosome
relative to the G- or R-banding patterns.
Human Chromosome IS-specific Microsatellite Polymorphic Markers
Spatially Resolvable on the Map by Fluorescentin situ Hybridization
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABE and Takashi IMAMURAAn area of increasing importance in human genetics research is thestrategy of reverse genetics or positional cloning, whereby a gene associatedwith a genetic disease is isolated on the basis of its approximate chromosomal
position. Genetic analyses of the type require polymorphic markers that are
informative, i.e. heterozygous, in the relevant individuals. In this study, we
have isolated 12 cosmid clones mapped to the R-banded positive or negative
region of the short arm of chromosome 18, which contained (CA)n dinucleotide repeat sequence. We also isolated several other clones positive for (CA)n repeat sequence out of 50 clones mapped to specific band region of the longarm of chromosome 18. Since polymorphisms of this type are generally
detected by peR amplification using primers homologous to unique seqences
flanking the repeats, and could be effective single locus probes, we are
analysing the sequences of subclones containing microsatellite and the flanking region. These markers isolated in this study will be useful for the genetic
linkage analysis.
The past decade has seen great advances in the field of human gene
mapping. In particular, it has become possible to isolate genes mutated incertain human diseases. The first step towards isolation of a disease gene inthis way is its localization to a specific chromosome region. Except for
disorders associated with cytognetically detectabe chromosomal rearrangements, chromosomal localization of the gene involved will usually rely on
linkage mapping. This process has been revolutionalized by the ability todetect variation between individuals at the DNA level, in the form of
restriction fragement length polymorphisms (RFLPs). Probes that detectsuch variation, and are of known chromosomal localization, can be used to
study cosegregation of an RFLP with the disease phenotypes in affectedVII.

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89

families. More recently, polymorphisms in the length of tandemly repeatedsimple sequences such as (CA)n (microsatellites) have proven to be a sourceof easily detectable highly polymorphic markers for genetic analyses. The
high frequency of simple repeats and their apparently random distribution
throughtout the genome means that markers from virtually any genomic
region can be made informative for genetic analysis.
Man's Place in Hominoidea Revealed by Mitochondrial DNA Genealogy
Satoshi HORAI, Yoko SATTA, Kenji HAYASAKA, Rumi KONDO, Tadashi INOUE,
Takafumi ISHIDA, Seiji HAYASHI and Naoyuki T AKAHATA

Molecular biology has resurrected C. Darwin and T. H. Huxley's question
about the origin of man, but the precise branching pattern and dating remain
controversial. To settle this issue, a large amount of sequence information isrequired. We determined mitochondrial (mt) DNA sequences for fivehominoids; pygmy and common chimpanzees, gorilla, orangutan, and siamango The common region compared with the known human sequence is4759 base pairs (bp) long, encompassing genes for 11 transfer RNAs and 6
proteins. Because of the high substitution rates in mammalian mtDNA andan unprecedentedly large region compared, the sequence differences clearly
indicate that the closest relatives to human are chimpanzees rather thangorilla. For dating the divergences of human, chimpanzee and gorilla, weused only unsaturated parts of sequence differences in which the mtDNA
genealogy is not obscured by multiple substitutions. The result suggests thatgorilla branched off7.7±0.7 million years (Myr) ago (0.59±0.05 relative tothe divergence time between orangutan and the African apes) and human did
4.7±0.5 Myr ago (0.36±0.04), the time difference between these divergences
being as long as 3 Myr (0.23). For details, seeJ. Mol. Eval. 35,32-43 (1992).
Mitochondrial DNA Mutation and Leigh's Syndrome
Ryoichi SAKUTA, Yu-ichi GOTO, Satoshi HORAI, Tatsuya OGtNO,
Harumi YOSHINAGA, Shunsuke OHTAHARA and Ikuya NONAKA

Leigh's syndrome, a progressive neurodegenerative disorder with preferential involvement of the basal ganglia, is a group of heterogeneous metabolic
disorders.

90

After Tatuch and coworkers reported mtDNA mutation from T to G at
position 8993 within the ATPase 6 gene in a female infant with Leigh's
syndrome and her mother, we examined mtDNA in muscle biopsy specimentsfrom 23 patients with the clinical characteristics of Leigh's syndrome todetermine whether this mutation is a common abnormality in this disorder.
MtDNA in the region from positions 8838 to 9017 within the ATPase 6
gene was amplified by polymerase chain reaction and digested with therestriction enzyme Hpall. Since the 8993 mutation created a Hpall recognition site (CCTG to CCGG). With this procedure, we detected the mutationin 1 of 23 patients in heteroplasmic fashion. In a previous study on mtDNA
restriction analysis, there was no recognition site of Hpall at position 8993 in
116 normal Japanese subjects.
This point mutation leads to an amino acid change from a highly conserved
leucine to arginine in ATPase subunit 6, thus probably causing failure in ATP
synthesis. Because the mutation is detectable in mtDNA from blood samples
by using a simple PCR method, more patients with identical mtDNA
mutation may be identified in Leigh's syndrome and other neurologicaldisorders. For details, see Ann. Neural. 32, 597-598 (1992).
Is Parkinson's Disease a Mitochondrial Disorder?
Yuko NAKAGAWA-HATTORI, Hiroya YOSHINO, Tomoyoshi KONDO,
Yoshikuni MIZUNO and Satoshi HORAI

Parkinson's disease (PD) is a common degenerative disease, but its etiology is still unknown. However, since the discovery of MPTP, many investigators have been interested in the mitochondrial function in PD. We investigated mitochondrial functions in PD patients using the methods whichhave successfully been applied to mitochondrial myopathies (MM), i.e. assay
of lactate and pyruvate, measurement of muscle mitochondrial respiratory
enzyme activities and Southern blot analysis of muscle mitochondrial DNA.
Parkinson's disease patients did not differ from controls in the mean bloodand CSF (cerebrospinal fluid) lactate and pyruvate levels at the basal restingstate or during an aerobic exercise. But mitochondrial complex I activity ofthe skeletal muscle was significantly decreased in PD. In the Southern blot
analysis, we could not find major deletions or insertions of mitochondrial
DNA in PD. Our studies disclosed a differential mitochondrial impairment

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91

between PD and MM. We discuss the implication of our observation. For
details, see J. Neural. Sci. 107,29-33 (1992).
Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, andStroke-like Episodes (MELAS): A Correlative Study of theClinical Features and Mitochondrial DNA Mutation
Yu-ichi GOTO, Satoshi HORAI, Taro MATSUOKA, Yasutomi KOGA,
Kenji NIHEI and Ikuya NONAKA

We studied 40 MELAS patients (21 male and 19 female) to characterize
the clinical features and biochemical and muscle biopsy findings related to the
mtDNA mutation at the nucleotide position of 3,243, the most common
genetic defect in MELAS. The most frequent symptom was episodic sudden
headache with vomiting and convulsions, which commonly affected patients
aged 5 to 15 years (80%). Biochemical defects in the muscle were variable;
13 patients had complex I, seven complex IV, and four complexes I + IV
deficiencies. In four muscle biopsies without ragged-red fibers or any enzyme
defect, we based the diagnosis on the identification of strongly SDH-reactive
blood vessels, which occurred in 87.5% of the biopsies. The mtDNA
mutation was present in 32 of 40 patients (80%). We conclude that there are
no clinical and pathologic differences between the patients with and without
this mtDNA mutation. For details, see Neurology 42, 545-550 (1992).
A Novel Point Mutation in the Mitochondrial tRNA Leu(UUR)
Gene in a Family with Mitochondrial Myopathy
Yu-ichi GOTO, Megumu ToJO, Jun TOHYAMA, Satoshi HORAI and Ikuya NONAKA

A T-to-C transition mutation at nucleotide position 3,250 in the mitochondrial tRNA Leu(UUR) gene was present in a family with mitochondrial myopathy. Two of three muscle biopsies examined had complex I (NADHubiquinone oxidoreductase) deficiency. Heteroplasmy of wild and mutant
mitochondrial DNA was detected by Nae I digestion of the polymerase chain
reaction products with a modified primer. This was found in blood or muscle
samples or both from all seven members examined. Similar to the 3,243
mutation in most patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), the new mutation site

92

was located in the dihydrouridine loop and embedded in the binding region of
mitochondrial transcription termination factor. Elucidation of the effects ofthis mutation may help clarify the role of mitochondrial tRNAs and transcription termination. For details, see Ann. Neural. 31, 672-675 (1992).

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VIII.

APPLIED GENETICS

Polymorphism of Bacterial Blight Resistance in Natural Populationsof Wild and Cultivated Rice
Hiroko MORISHIMA and Toshie MIY ABA YASHIThis study was initiated to investigate the evolutionary dynamics of aplant-pathogen system in natural and agricultural ecosystems. We havealready demonstrated that land-races of rice and their wild progenitors arepolymorphic within populations in resistance to a bacterial blight diseasecaused by Xanthomonous oryzae pv. oryzae (Xoo). The following is ourrecent experimental result which suggests possible mechanisms for maintaining resistance polymorphism in natural populations. Bacterial blight resistance in rice plants was assessed by innoculation of four representative Xooraces using the clipping method.
(I) The effect of habitat on resistance variations
In wild rice, perennial ecotypes adapted to deepwater conditions consist
mostly of resistant plants, while annual ecotypes adapted to drier sites tend to
be polymorphic, preserving a high frequency of susceptible plants. A similartendency was found in Bangladesh cultivars: floating rice grown in a deepwater area were generally resistant, while cultivars in non-deepwater areas
and those in the dry season were either resistant or susceptible. It wasinferred from the above results that deepwater condition which favours thegrowth and development of pathogen Xoo has produces selection pressure
resulting in a high frequency of resistant plants. On the other hand, in dry
condition which is unfavorable to Xoo, selection for resistance must havebeen relaxed, resulting in polymorphism.
(2) Resistance variations on a fine scale in local populations
Two wild rice populations of the annual type in a suburb of Bangkok,
which were located I km apart from each other (NE3 and NE4), and samples
taken along a transect line connecting the above two populations were
examined regarding the frequency of resistant plants.
NE4 showed a higher frequency of resistant plants (resistant to all four
races: 58%) than NE3 (6%). This tendency was consistently observed in thesamples collected in different years. Populations sampled on a transect line

94

showed a cline of decreasing frequency from NE4 to NE3 sites. Similar
geographical clines between these two populations were found for somephenotypic characteristics such as plant height and flowering time, but nosuch trend was found in the allelic frequency of polymorphic isozyme loci.
The habitat of NE3 is a roadside grass land which is completely parchedduring the dry season; typical of an annual type habitat, while, NE4 is an
exceptional annual population which inhabits a swampy place adjacent to apond which retains water throughout the year. This geographical cline inbacterial blight resistance observed on a local scale could be explained by thedifferences in water conditions between NE3 and NE4. Namely, selection forresistant plants must have operated more strongly in NE4 in which theenvironment favours the pathogen than in NE3.
(3) Is there a resistance cost?
Frequency dependent selection between genetically heterogeneous pathogens and different host genotypes is one possible mechanism for maintaining
resistance polymorphism. The cost of resistance is often argued as anothermechanism for resistance polymorphism. We found a negative correlation
between the degree of resistance and fitness character in a disease-freeenvironment among lines derived from NE4. If this is the case, resistant
plants are less fit than susceptible ones in a disease-free environment, andpolymorphism can occur due to a balance of opposing selective forces.
The birth of japonica Rice in East China
Yo-Ichiro SATO, S. X. TANG*, L. U. YANG** and L. H. TANG***To inquire into the geographical origin of cultivated rice, spikelets excavated from archaeological sites at Homedu (as old as 7,000 years), were
examined to determine whether they contained spikelets of wild rice. Out ofeighty-one spikelets examined, five showed characteristics of wild rice undera scanning electron microscope (SEM), although the previous observation
had not revealed their existence. Other spikelets showed characteristics lying
between those of wild and cultivated rice, suggesting the occurrence of
natural hybridization between wild and cultivated rice, or the existence of an
*
**
***

China National Rice Research Institute, Hangzhou, China.
Zhejiang Museum, Hangzhou, China.
Jiangsu Academy of Agricultural Sciences, Nangjing, China.

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95

intermediate type. This indicates that a primitive stage of domestication had
developed there 7,000 years ago. Recent archaeological data suggests theexistence of an older site with proof of rice cultivation in the middle basin of
the Yangtze River. Molecular genetic analysis indicated that the progenitorsof indica andjaponica inhabited the tropics and China, respectively. It may
be inferred that the middle and lower basins of the Yangtze river including
the Homedu area are the places of origin of Japonica rice.
Sampling Strategy in Constructing a Core Collection
from on Entire Collection of Germplasms
Katsuei YONEZAWA, Tetsuro NOMuRA* and Hiroko MORISHIMA

A core collection is composed of representative sample entries taken froman entire germplasm collection. Sample size and stratification strategy forsampling core entries from a structured germplasm collection were investigated, based on a theoretical model which takes account of both theamount of genetic diversity to be retained and its maintenance. It wasderived that the optimum sample fraction could be defined but not be
uniquely specified. It depends on various genetic and resource parameters,
primarily on the degree of genetic redundancy in the entire collection, and theamount of resources available for maintenance (quantified by the number ofplants treated for rejuvenation of core entries). The optimum sample
fraction is larger with a lower redundancy among accessions, and/or with alower allelic diversity within accessions, indicating that the sample fraction
should be larger in species with a higher selfing rate. A larger sample fraction
will be optimum with a smaller total amount of available resources and/orwith a longer duration of maintenance of the core collection (defined by the
number of generations for the maintenance). A sample fraction in the rangeof 20 to 30 percent may be taken as appropriate in a situation where
accessions in the entire collection are neither very heavily nor very lightly
redundant (0.90> Dr >0.2, in terms of the degree of redundancy as definedby the authors), and the core entries are maintained for ten to twenty cycles
of rejuvenation with plants of the order of 10 3 being treated for one cycle ofrejuvenation.
Five stratified sampling strategies were compared by analyzing fourteen* Kyoto Sangyo University.

96

hypothetical and four real germplasm collections composed of a number ofgroups. It was concluded that a proportional strategy, where the sample size
is stratified in proportion to the number of accessions in each group, is themost reliable, while, of course, a strategy where the stratification is made inproportion to the range of genetic diversity is the best when the genetic
diversity is known in advance. Sampling procedures for germplasm collections where accessions are hierarchically structured from the level of groups
or single accessions were discussed. In the aim of drawing both the pattern
and range of genetic diversity in the germplasm collection, procedures using
the uniqueness value of Crozier (1992) were developed.
Recognition of the Water Surface by a Deepwater-Tolerance
Gene (dW3) in Rice
Mitsugu EIGUCHI, Hiro-Yuki HIRANO, Hiroko MORISHIMA and Yoshio SANO

Phenotypic plasticity which is characterized by changes in the morphology
and physiology of a plant associated with changes in the environmental
conditions is particularly important in plants due to their sessile life style.
Deepwater and floating rice (Oryza sativa) in which internodal elongation
starts with an increase in water depth in order to overcome the stressassociated with submergence and the lack of internodal elongation in airgrown deepwater rice or in non-deepwater rice are examples of plant plasticity. In previous studies the physiological basis for tolerance to deepwater was
ascribed to the enhancement of internodal elongation resulting from theaction of the plant hormones, ethylene and gibberellin. Recently, we haveobserved that the gene dW3 harboured by a floating type of wild rice (Oryza
rufipogon) induces internodal elongation in response to submergence and is
responsible for the survival of the plant at a depth of 1m (Eiguchi et at.
(1993) J. Heredity 84, 201-205). In addition, the response induced by dw]
was markedly attenuated after floral initiation, indicating that the genic
expression was regulated during the development of the plant. This observation prompted us to try to determine whether dw] was responsible only for thetiming of induction of internodal elongation under varying water regimes.
We report here that in rice plants harbouring the dW3 gene, stem elongationand heading time undergo considerable changes in response to variations inwater depth.

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97

To analyse the genic expression of dWj, which is responsible for the
induction of internodal elongation in response to submergence, phenotypicchanges were examined under varying water regimes (water depths ranging
from 0 m to 1.5 m) by using a rice cultivar (T65) susceptible to submergencein deepwater and its near-isogenic line carrying the gene dWj (T65dw]). T65
dWj survived after being submerged in water at various depths while T65 diedwhen submerged in water at 1. 5 m- and 1.0 m-depths. The plant height of
T65dwj markedly changed depending on the water depth. An increase in thenumber of elongated internodes and a delay in heading time were manifested
after submergence in deepwater. These observations indicate that T65dw]
recognized the water surface and adjusted the number of elongated internodes to complete its life cycle.
Deepwater rice cultivars are mostly photoperiod sensitive. Practically,
photoperiod sensitivity may be essential for deepwater rice when flood
hazards occur over a long period of time and the optimum harvest date ispredicted for safety. Information on the plasticity of floral induction affectedby deepwater is limited. The present study confirmed that the timing of floral
induction in T65dwj changed substantially in response to variations in thewater depth in spite of the fact that is almost neutral in regards to photoperiod. For details see lpn. l. Breed. 43, 135-139, 1993.
Genetic Comparisons of Chromosome 6 between Wild
and Cultivated Rice
Yoshio SANG

The objective of this study was to detect genetic differentiation between
wild and cultivated rice. Recent works on RFLP mapping encourage
identification of agronomically important genes which have not yet been
mapped on chromosomes. This implies that conventional techniques are notsufficient for identification of a complex nature of genes. Genomic differentiation is so complicated that it is hard to choose chromosomal regions to bestudied. I have chosen chromosome 6 in this experiment because of thefollowing points; 1) in rice there are lots of conventional markers for genetic
analysis on it, 2) accumulated evidence suggests significant genetic divergences between and within species. To study the genetic differentiation on
chromosome 6 between wild and cultivated rice, recombinant inbred lines

98

were made in the present experiment. The construction of recombinant
inbred lines was confirmed to be a powerful method for detecting genes
otherwise difficult to detect.
A large chromosomal segment of chromosome 6 was introduced into a
genetic tester from the wild progenitor (Oryza rufipogon) by successive
backcrossing, which was readily carried out because of the presence of agamete eliminator S6 on the segment. After introducing the segment,
recombinants with various lengths of the segment were obtained. This madeit possible to identify genetic factors by comparing the various segments inthe same genetic background. One of the genes detected is an enhancer forphotoperiod-sensitivity whose cooperative interaction with Se J leads to sensitivity as strong as that in wild rice. The other is a low crossability gene which
produces unidirectional incompatibility. It was surprising that the three
genes contribute to sexual affinity, resulting in a cluster of genes for pre- andpost-fertilization barriers. Efforts are now under way to locate other genes
for plant height, plant and seed morphologies, and heading date on thesegment.
Comparisons of the same region of chromosome 6 between the two
cultivated rice species revealed that another gamete eliminator, its modifiers
and recombination restriction occur on it. Similar abnormalities have beenreported in fruit fly and mice in relation to meiotic drive, although they havebeen found to occur within a population. It is interesting to note that similarsystems appear in the mechanism of reproductive isolation among rice taxa.
Hopefully, further studies will be readily extended by using various molecular markers in the near future, after elucidating the complex nature of thechromosomal segment. The importance of efforts to detect unidentified genes
with biological significance must be recognized. Most genes are waiting to beanalyzed in various ways. For details see Jpn. J. Breed. 42, 561-572, 1992.

Is an Egg-Killer Present in Rice?
Yoshio SANO

Hybrid sterility due to allelic interaction was first reported in Nicotiana.
An alien gene (pollen killer) in Nicotiana plumbaginifolia caused the degeneration of pollen not carrying it, when the chromosome on which it occurred
was added to the Nicotiana tabacum complement. The genetic basis forVIII.

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99

hybrid sterility between species seems to be complex. Accumulated evidence,
however, suggests that such genes causing gametic abortion are of wide
spread between and within plant species. Most of such genes detected so far
were of the gametophytic type and they were classified into three types,
gamete eliminator, pollen-killer and egg-killer, depending on the gametes
affected. The former two were frequently detected in plants. The only
example of an egg-killer was reported in a maize-Tripsacum hybrid. An extra
chromosome derived from Tripsacum was transmitted to a high proportion of
the progeny through the egg. This was explained by assuming the existanceof an egg-killer on the extra chromosome, which induces abortion of megaspores not carrying it. Data however were limited. The present experiment
was carried out to confirm the presence of an egg-killer recently proposed as
the genetic mechanism of hybrid sterility observed in Asian rice cultivars(Oryza sativa).
Hybrid sterility due to allelic interaction at a single locus was also reported
in some varietal crosses within O. sativa. Recent progress included thedetection of the wide compatibility gene, Sjn, which was recognized as a genethat greatly reduces seed infertility in the Japonica-Indica hybrid. It was
assumed that there are two additional alleles at the Sj locus and that an Sj'
allele from the Indica parent induces abortion of megaspores carrying theother allele (SJ from Japonica parent) in the heterozygote (Sj'jSj) , suggesting that Sj' may act as an egg-killer against Sj. The locus attracted rice
breeders since the hybrid sterility gene might prevail among commonly used
breeding materials.
The present study was carried out in order to confirm that the proposed Sj'
allele acts as an egg-killer against the opposite SJ allele in the Indica-Japonica
hybrid. A conspicuous feature of an egg-killer is a high rate of its transmission into the progeny through the egg. Backcrossing experiments were madeby using the Indica-Japonica hybrid in which Sj' and Sj had been assumed to
be involved. Although an egg-killer was easily identified through backcrossexperiments, the present study failed to detect any egg-killer in the IndicaJaponica hybrid, providing evidence counter to the proposed genetic mechanism for hybrid sterility in Asian rice. This study will appear in Theor. Appl.
Genet. 1993.

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IX.

DATABASE

DNA Database Release 11 from DNA Data Bank of Japan
Yoshihiro UGAWA, Kazuho IKEO, Yukiko YAMAZAKI, Motono HORIE,
Masako IWASE, Mari SAITO, Yumiko SATO, Shigeko SUZUKI,
Yuko HAWAGAWA, Asako HASEGAWA, Yoshie HATTORI, Mary SHIMOYAMA,
Rikiko SUZUKI, Reiko UCHIDA, Yoko SHIDAHARA, Yoko MATSUSHIMA,
Akino WATANABE, Yoko UEDA, Tatsuko KAWAMOTO, Hajime KITAKAMI,
Naruya SAITOU, Takashi GOJOBORI and Yoshio TATENOThe DNA database Release 11 includes 65,693 entries which correspond to
84,839,075 bases. This release contains the newest data prepared by DDBJ,
GenBank and the EMBL Data Library as of June 1993. Thanks tointernational collaboration between the three data banks, the International
Nucleotide Sequence Database has been organized as a unified database.
All the entries designated by the accession numbers with "D's" have beencollected and processed by DDBJ, and the rest have been prepared by
GenBank and the EMBL Data Library. Because the release contains unified
data, all the entries have been annotated with common feature keys. The
present release was prepared by processing the data on the relational database management system (Sybase). It does not include amino acid sequencedata, because the genetic code system is known to be no longer uniform
among the species and organella, and DDBJ is not yet prepared for this.
Integration and Search System for a Large-Scale DNA Database
Hajime KITAKAMI and Yukiko YAMAZAKI

A flat-file system is inadequate for building, integrating and searching alarge-scale DNA database whose entries are continuously increasing in an
explosive manner. GenBank helped DDBJ to convert flat-file data constructed by DDBJ to a relational format and trained DDBJ staff to use theAnnotator's Workbench, A WB, which is a tool for building the DNA
database based on the relational database management system, SyBase.
However, integration and search functions are still performed utilizing theflat-file system at DDBJ. We are developing a new intelligent system, AlifeIX.

DATABASE

101(A large-scale database integration facility with e-mail server) to integrate
and search the DNA Database. The Alife system includes some functions
developed by GenBank for implementing an integrated international database on SyBase. We developed a relational schema, named ddbj-schema, as
well as an online and e-mail service system for database users. It is necessary
to convert the DNA database built with the GenBank schema using AWB,
into a relational format using the ddbj-schema. A conversion system wasdeveloped using DDBJ's virtual tables as a component of the Alife system.
The ddbj-schema will be utilized to integrate three databanks DDBJ,
NCBI including GenBank, and the EMBL Data Library. In addition, theddbj-schema will be utilized to update the ESTs (Expressed Sequence Tags)!
TSFs (Transcribed Sequence Fragments) database submitted by the Japanesehuman cDNA project at Prof. Matsubara's Laboratory, Osaka University
and other such projects.
We are investigating, with Prof. Makinouchi of Kyushu University, thepossibility of rewriting the Alife system implementing the relational databaseusing an object-oriented database system.

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PUBLICATIONSAYUSAWA, D., KANEDA, S., ITOH, Y., YASUDA, H., MURAKAMI, Y., SUGASAWA, K., HAN AOKA, F. and SENO, T.: Complementation by a cloned human ubiquitin-activating enzyme
EI of the S-phase arrested mouse FM3A cell mutant with thermolabile EJ. Cell Struct.
Funct., 17, 113-122, 1992.
BARBIER, P., TAKAHASHI, M., NAKAMURA, I., TORIYAMA, S. and ISHIHAMA, A.: Solubilization and promoter analysis of RNA polymerase from rice stripe virus. J. Virol., 66, 61716174,1992.
BASTEN, CHRISTOPHER J. and OHTA, T.: Simulation study of a multigene family, with specialreference to the evolution of compensatory advatageous mutations. Genetics, 132, 247-252,
1992.
CHIFU, Y., NAKASHIMA, H., YOKOTA, E. and IMAMURA, T.: ;3-Thalassemia major resulting
from a compound heterozygosity for the ;3-globin gene mutation: further evidence formultiple origin and migration of the thalassemia gene. Hum. Genet., 89, 343-346, 1992.
DAVID, C. N., FUJI SAW A, T. and BOSCH, T. C. G.: Interstitial stem cell proliferation in Hydra:
Evidence for strain-specific regulatory signals. Developmental Biology, 148, 501-507, 199J.
DODO, Y., ISHIDA, H. and SAITOU, N.: Population history of Japan: a cranial nonmetricapproach. In "The Evolution and Dispersal of Modern Humans in Asia" (Akazawa, T.,
Aoki, K. and Kimura, T., eds.), pp. 479-492, Hokusen-sha, Publ. Co., Tokyo, 1992.
FUJISAWA, T.: Model systems for stem cell research. A. Hydra. Hematopoietic Factor 3, 7176, 1992 (in Jap.).
FUJISAWA, T.: Morphogenetic factors in hydra and its related species. The Cell., 24, 14-18,
1992 (in Jap.).
FUJISAWA, T.: Homeostatic recovery of interstitial cell populations in Hydra. Developmental
Biology, 150, 185-192, 1992 (in Jap.).
GILADI, H., IGARASHI, K., ISHIHAMA, A. and OPPENHEIM, A. B.: Stimulation of the phage
ApL promoter by IHF requires the carboxy terminus of the a-subunit of RNA polymerase.
J. Mol. BioI., 227, 985-990, 1992.
GOJOBORI, T., IMANISHI, T. and INA, Y.: Statistics on the numbers of panels stored on the
MHC database. HLA 1991 (Oxford University Press), I, 67-76, 1992.
GOJOBORI, T., IMANISHI, T., INA, Y., SAITOU, N., TATENO, Y., SATO, K., TAKATA, H.,
KIMURA, A., NISHIMURA, Y., AKAZA, T., MIYOSHI, H., MURA, T., TOKUNAGA, K.,TANAKA, H., KONOEDA, Y., W AKISAKA, A., JUJI, T., SASAZUKI, T., TSUJI, K. and INOKO,
H.: Construction of the MHC database. Proceedings of the 11th MHC HLA 1991 (Oxford
University Press), I, 66-67, 1992.
GOTO, Y., HORAI, S., MATSUOKA, T., KOGA, Y., NIHEI, K., KOBAYASHI, M. and NONAKA, I.:
Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes(MELAS): A correlative study of the clinical features and mitochondrial DNA mutation.
Neurology., 42, 545-550, 1992.
GOTO, Y., ToJO, M., TOHYAMA, J., HORAI, S. and NONAKA, I.: A novel point mutation in thePUBLICAnONS

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mitochondrial tRNALeu (UUR) gene in a family with muscle form of mitochondrial
disorder. Ann. Neurol., 31, 672-675, 1992.
GUSSIN, G. N., OLSON, C, IGARASHI, K. and ISHIHAMA, A.: Activation defects caused bymutations"in E. coli rpoA are promoter-specific. J. Bacteriol., 174, 5156-5160, 1992.
HAMAMATSU, C, TORIYAMA, S., TOYODA, T. and ISHIHAMA, A.: Ambisense conding strategyof the rice stripe virus genome. J. Gen. Virol., in press.
HARA, H. and PARK, J. T.: A far upstream region is required for expression of theftsI genecoding for penicillin-binding protein 3 of Escherichia coli. In "Bacterial growth and lysis:
metabolism and structure of the bacterial sacculus." (M. A. de Pedro et al., eds.), Plenum,
New York, in press.
HASHIMOTO, K., NOGUCHI, M. and NAKATSUJI, N.: Mouse offspring derived from fetal ovaries
or reaggregates which were cultured and transplanted into adult females. Devel. GrowthDiffer., 34, 233-238, 1992.
HAYASHI, S., HIROSE, S., METCALFE, T. and SHIRRAS, A.: Control of imaginal cell development by the escargot gene of Drosophila. Development, in press.
HIGASHITANI, A., GREENSTEIN, D. and HORIUCHI, K.: A single amino acid substitution
reduces the superhelicity requirement of a replication initiator protein. Nucl. Acids Res.,20, 2685-2691, 1992.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.: Nucleotide sequence of the primer
RNA for DNA replication of filamentous bacteriophages. J. Virol., 67,2175-2181,1993.
HIGASHITANI, N., HIGASHITANI, A., ROTH, A. and HORIUCHI, K.: SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of thecomplementary strand DNA synthesis. J. Bacteriol., 174, 1612-1618, 1992.
HIROSE, S. and OHBA, R.: Supercoiling facilitates the assembly of active chromatin. In"Molecular Biology of DNA Toposisomerases and Its Application to Chemotherapy" (Andoh,T., ed.), pp. 85-92, CRC Press, Florida, 1992.
HIROSE, S., T ABU CHI, H. and MIZUTANI, M.: Supercoiling of DNA facilitates TFIID-promoter
interactions. In "Molecular Structure and Life" (Kyogoku, Y. and Nishimura, Y. eds.), pp.
229-235, CRC Press, Florida, 1992.
HONDA, R., OHBA, Y. and YASUDA, H.: The cell cycle reguator, human p50 w <el, is a tyrosine
kinase and not a serine/tyrosine kinase. Biochem. Biophys. Res. Commun., 186, 1333-1338,
1992.
HORAI, S.: Human mitochondrial DNA: a clue to the development and dispersion of Asian
populations. In "Japanese as a Member of the Asian and Pacific Populations." (Hanihara,
K. ed.), pp. 147-159, International Research Symposium No.4, International Research
center for Japanese Studies, 1992.
HORAI, S., SATTA, Y., HAYASAKA, K., KONDO, R., INOUE, T., ISHIDA, T., HAYASHI, S. and
TAKAHATA, N.: Man's place in Hominoidea revealed by mitochondrial DNA genealogy. J.
Mol. Evol., 35, 32-45, 1992.
IKEMURA, T.: CODON USAGE. In "Plant Molecular Biology LABFAX" (Brown, T. A., ed.),Blackwell Scientific Publications, in press.
IKEMURA, T.: Correlation between codon usage and tRNA content in microorganisms. In"Transfer RNA in Protein Synthesis" (Hatfield, D. L. et al., eds.), pp. 87-111, CRC Press,
Florida, USA, 1992.

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IKEMURA, T, MATSUMOTO, K., ISHIHARA, N., AN DO, A. and INOKO, H.: Giant G+C mosaic
structures in HLA locus and a border between the mosaic domains. In "HLA 1991" Vol 2,
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IKEO, K., TAKAHASHI, K. and GOJOBORI, T.: Evolutionary Origin of a Kunitz-type trypsin
inhibitor domain inserted in the amyloid tJ precursor protein of Alzheimer's disease. J. Mol.
Evol., 34, 536-543, 1992.
IMAI, H. T, HIRAI, H., SATTA, Y., SHIROISHI, T., YAMADA, M. and TAYLOR, R. W.: Phase
specific Ag-staning of nucleolar organizer regions (NORs) and kinetochores in the Australian ant Myrmecia croslandi. Jpn. J. Genet., 67, 473-447, 1992.
IMAI, N., KANEDA, S., NAGAI, Y., SENO, T, AYUSAWA, D., HANAOKA, F. and YAMAO, F.:
Cloning and sequence of a functionally active cDNA for mouse ubiquitin-activatingenzuyme E 1. Gene, 118, 279-282, 1992.
IMAMURA, T: Hemolobinopathies. In "Integrated Handbook of Internal Medicine, Vol. 18"(lmura, H., Ogata, E., Takaku, H., Tarui, S., ed.), pp. 347-363, Nakayama Shoten, Tokyo,
1992 (in Japanese).
IMAMURA, T: Molecular genetic methods for analysis of the etiology of diseases. Geriatric
Psychiatry, 3, 955-959, 1992 (in Japanese).
IMAMURA, T: Polymerase chain reaction amplification of genes and its application. In"Methods for Analysis of the Genetic Etiology of Chronic Diseases" (Suzuki, T., ed.), pp.
I-II, Ministry of Health and Welfare Study Group, Tokyo, 1993 (in Japanese).
IMANISHI, T, AKAZA, T., KIMURA, A., TOKUNAGA, K. and GOJOBORI, T: Allele andhaplotype frequencies for HLA and complement loci in various ethnic groups. HLA 1991
(Oxford Universty Press), 1, 1065-1220, 1992.
IMANISHI, T, AKAZA, T., KIMURA, A., TOKUNAGA, K. and GOJOBORI, T: Estimation of allele
and haplotype frequencies for HLA and complement loci. HLA 1991 HLA 1991 (Oxford
University Press), 1, 76-79, 1992.
IMANISHI, T, AKAZA, T, MURA, T., MIYOSHI, H., KASHIWASE, K., INA, Y., TANAKA, H.,
TOKUNAGA, K., GOJOBORI, T and JUJI, T: Allele and haplotype frequencies of MHC lociestimated from panel data of the 11th International Histocompatibility Workshop: A
preliminary analysis. Data Analysis Book II, 433-542, 1992.
IMANISHI, T. and GOJOBORI, T: Patterns of nucleotide substitution inferred from thephylogenis of the class I major histocompatibility complex genes. J. Mol. Evol., 35, 196204, 1992.
IMANISHI, T. and GOJOBORI, T.: Distinctive patterns of nucleotide substitution inferred fromthe phylogenies of class I MHC genes. HLA 1991 (Oxford University Press), 2, 357-359,
1992.
IMANISHI, T, WAKISAKA, A. and GOJOBORI, T: Genetic relationships among various humanpopulations indicated by MHC polymorphisms. HLA 1991 (Oxford University Press), 1,
627-632, 1992.
INOKO, H., SATO, K., TAKANO, H., IMANISHI, T., INA, Y., SAITOU, N., TATENO, Y, KIMURA,
A., NISHIMURA, Y., AKAZA, T, MIYOSHI, H., MURA, T, TOKUNAGA, K., TANAKA, H.,
KONOEDA, Y., WAKISAKA, A., JUJI, T, SASAZUKI, T, TSUJI, K. and GOJOBORI, T.: Data
flow from collection of raw data to construction of the MHC database. HLA 1991 (Oxford
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INOUE, Y. and WATANABE, T. K.: Chromomsomal polymorphism in isofemale lines and cage
populations of Drosphila melanogaster. Evolution, 36, 797-806, 1992.
ISHIHAMA, A.: Molecular communication between Escherichia coli RNA polymerase andtranscription factors. In "Molecular Structure and Life" (Y. Kyogoku and Y. Nishimura,eds.), pp. 195-205. Jpn. Sci. Soc. Press, Tokyo/CRC Press, Boca Raton, 1992.
ISHIHAMA, A.: Role of the RNA polymerase a subunit in transcription activation. Mol.
Microbiol. (MicroReview), 6, 3283-3288, 1992.
ISHIHAMA, A.: Protein-protein communication within the transcription apparatus. J. Bacteriol.,(Minireview), 175, 2483-2489, 1993.
ISHIHAMA, A., TOYODA, T., KOBAYASHI, M., YASUDA, J., ASANO, Y. and NAKAYAMA, M.:
Formation and control of influenza virus RNA polymerase. In "Options for the Control of
Influenza" (C Hannoun et al., eds.), pp. 289-297, EXCEPTA MEDICA, Amsterdam, 1992.
ISHIHAMA, A., Zou, C, KOBAYASHI, M., KUMAR, A., FUJITA, N., SAKURAI, H. and HAYWARD, R. S.: Molecular recognition in gene transcription. In "Molecular Recognition in
Biological Systems" (V. Buckin et al., eds.), in press.
ISHIHARA, T., SHIGEMOTO, R., MORI, K., TAKAHASHI, K. and NAGATA, S.: Functional
expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide.
Neuron, 8, 811-819, 1992.
IWATA, A., TSUCHIYA, T., VEDA, S., ISHIHAMA, A., ZHU, G.-S. and HIRAI, K.: Nucleotide
sequence and transcription organization of the tumor-associated region of Marek's diseasevirus. Proc. Internatl. Symp. Marek's Disease Virus, in press.
IWATA, A., VEDA, S., ISIIIHAMA, A. and HIRAI, K.: Sequence determination of cDNA clonesof transcripts from the tumor-associated region of Marek's disease virus genome. Virology,
187, 805-808, 1992.
KAMIJO, M., YASUDA, H. Peter, M., YAMASHITA, M., NAGAHAMA, Y. and OHBA, Y.:
Preference of human cdc2 kinase to various peptide substrate. Peptide Res., 5,281-285,
1992.
KANEDA, S., HORIE, N., TAKEISHI, K., TAKAYANAGI, A., SENO, T. and AYUSAWA, D.:
Regulatory sequences clustered at the 5' end of the first intron of the human thymidylate
synthase gene function in cooperation with the promoter region. Somat. Cell Mol. Genet.,
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KASHIWAZAKI, N., NAKAo, H., OHTANI, S. and NAKATSUJI, N.: Production of chimeric pigs
by the blastocyst injection method. Veterinary Record, 130, 186-187, 1992.
KATAYAMA, S., HIRANO, H.-Y., NAITO, S., TSUKAYA, Y. and KOMEDA, Y.: Analysis ofintergenic spacer in the nuclear rDNA of Pharbitis nil. Genome, 35, 92-97, 1992.
KATO, Y., MAEKAWA, M. and SANO, Y.: Effects of 2-amino-3-methylimidazo[4,5:f] quinoline(lQ) on somatic mutation in a soybean test system. Mutation Res., 279, 239-243, 1992.
KATSURA, I.: Towards biophysics in the future. Biophysics (Seibutsubutsuri), 32, 46-48, 1992
(in Jap.).
KATSURA, I.: Programmed and pathological cell deaths in the nematode C. elegans. Experimental Medicine 10, 2093-2095, 1992 (in Jap.).
KATSURA, I.: Replication and gene expression of lambda phage. Protein, Nucleic Acid andEnzyme, 37, 2580-2588, 1992 (in Jap.).
KAWAGISHI, M., YAMAGISHI, M. and ISHIHAMA, A.: Cloning and sequence determination of

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the Schizosaccharomyces pombe rpb2 gene encoding the subunit 2 of RNA polymerase II.
Nucleic Acids Res., 21,469-473, 1993.
KAWAMURA, S., SAITOU, N. and UEDA, S.: Concerted evolution of the primate immunoglobulin alpha gene through gene conversion. J. BioI. Chern., 276, 7359-7367.
KIMURA, N., NISHIDA, M., NAGATA, K., ISHIHAMA, A., ODA, K. and NAKADA, S.: Expression of a recombinant influenza viral RNA in cells permanently expressing the RNA
polymerase and NP genes ofinfluneza virus. J. Gen. Virol., 73,1321-1328,1992.
KITAKAMI, H., YAMAZAKI, Y, UGAWA, Y., IKEO, K., SAITOU, N., TATENO, Y. and GoJOBORI, T.: Building and searching biological information based on a relational detabasesystem. Bulletin of the Japan Federation for Culture Collections., 8,172-200,1992 (in Jap.)
KITAGAWA, M., SAITOH, S., OGINO, H., OKABE, T., MATSUMOTO, H., OKUMURA, A., TAMAI,
K., OHBA, Y., YASUDA, H., NISHIMURA, S. and TAYA, Y.: Cdc2-like kinase in associatedwith the retihnoblatoma protein. Oncogene, 7, 1067-1074, 1992.
KLEIN, J., SATTA, Y., O'HUIGIN, C. MAYER, W. E. and TAKAHATA, N.: Evolution of the
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KOBAYASHI, M., TSUCHIYA, K., NAGATA, K. and ISHIHAMA, A.: Reconstitution of influenza
virus RNA polymerase from three subunits expressed using recombinant baculovirus
system. Virus Res., 22, 235-245, 1992.
KOIDE, T., YOSHINO, M., NIWA, M., ISHIURA, M., SHIROISHI, T. and MORIWAKI, M.: The
amplified long genomic sequence (ALGS) located in the centromenric regions of mouse
chromosomes. Genomics, 13, 1186-1191, 1992.
KOLB, A., IGARASHI, K., ISHIHAMA, A., LAVIGNE, M., BUCKLE, M. and Buc, H.: E. coli RNA
polymerase, delected in the C-terminal part of its a-subunit, interacts differentially with thecAMP-CRP complex at the lac PI and the galPI promoter. Nucleic Acids Res., 21, 319-326,
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KOYAMA, H., SHIOMI, Y., ARATANI, Y, AYUSAWA, D. and SENO, T.: Gene correction byhomologous recombination at the deleted aprt locus in CHO cells. In "Molecular Approaches to the Study and Treatment of Human Diseases." (Yoshida, T. O. and Wilson, J.
M., eds), pp. 347-355, Elsevier Science Publishers B. V., Amsterdam, 1992.
KUBORI, T., SHIMAMOTO, N., YAMAGUCHI, S., NAMBA, K. and AIZAWA, S-I.: Molphologicalpathway of flagellar assembly in Salmonella typimurium. J. Mol. BioI., 226, 433-446, 1992.
KUMADA, Y., BENSON, D. R., HILLEMANN, D., HOSTED, T. J., ROCHEFORT, D. A., THOMPSON, C. J., WOHLLEBEN, W. and TATENO, Y: Evolution of the glutamine synthetase gene,one of the oldest existing and functioning genes. Proc. Natl. Acad. Sci. USA, 90, 30093013, 1993.
KUNIEDA, T., XIAN, M., KOBAYASHI, E., IMAMICHI, T., MORIWAKI, K. and TOYODA, Y.:
Sexing of muse preimplantation embryos by detection of Y chromosome-specific sequencesusing polymerase chain reaction. Biology of Reproduction, 46, 692-697, 1992.
KUSUBATA, M., TOKUI, T., MATSUOKA, Y., OKUMURA, E., TACHIBANA, K., HISAGANA, S.,
KISHIMOTO, T., YASUDA, H., KAMIJO, M., OHBA, Y., TSUJIMOTO, K., YATANI, R. and
INAGAKI, M.: p13'u<' suppresses the catalytic function of p34<d<' kinase for intermediatefilament proteins, in vitro. J. BioI. Chern., 267, 20937-20942, 1992.
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S.: The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiationof transcription at an "extended minus 10" promoter. J. Mol. BioI., 232, 406-418, 1993.
KURODA, Y., JAIN, A. K., TEZUKA, H. and KADA, T.: Antimutagenesity in cultured
mammalian cells. Mutation Res., 267, 201-209, 1992.
LEE, H.-S., ISHIHAMA, A. and KUTSlJ, S.: The C-terminus of the a-subunit of RNA polymerase
is not essential for transcription activation of o54-holoenzyme. J. Bacteriol., 175,2479-2484,
1993.
MAEKAwA, T, AMEMURA-MAEKAwA, J. and OHTSUBO, E.: DNA-binding domains in Tn3transposase. Mol. Gen. Genet., in press.
MARUYAMA, K. and TACH IDA, H.: Genetic variability and geographical structure in partiallyselfing populations. Jpn. J. Genet., 67, 39-51, 1992.
MAsuDA, Y., TSUCHIMOTO, K., NISHIMURA, A. and OHTSUBO, E.: Isolation of temperaturesensitive aminoacyl-tRNA synthetase mutants from an Escherichia coli strain haboring the
pemK pemK plasmid. Mol. Gen. Genet., in press.
MATSUMOTO, K., ARAI, M., ISHIHARA, N., ANDO, A., INoKo, H. and IKEMURA, T.: Cluster
offibronectin type III found in the human major histocompatibility complex class III region
shows the highest homology with the repeats in an extracellular matrix protein, tenascin.
Genomics, 12,485-491, 1992.
MATSUMOTO, K., ISHIHARA, N., ANDO, A., INoKo, H. and IKEMURA, T: Extracellular matrix
protein tenascin-like gene found in human MHC class III region. Immunogenetic, 36, 400403, 1992.
MATSUMOTO, K., ISHIHARA, N., ANDO, A., INOKO, H. and IKEMuRA, T: Clustered occurrence of fibronectin type-III repeats in the human major histocompatibility complex class
III region: a tenascin-like gene in the MHC locus. In "HLA 1991" (Tsuji el al., eds.),
Oxford Univ. Press, 2, pp. 185-190, 1992.
MATSUTANI, S. and OHTSUBO, E.: Distribution of the Shigella sonnei insertion elements inEnterobacteriaceae. Gene, in press.
MOCHIZUKI, K., UMEDA, M., OHTSUBO, H. and OHTSUBO, E.: Characterization of a plant
SINE, p-SINEI, in rice genomes. Jpn. J. Genet., 67, 155-166, 1992.
MORISHIMA, H., SANO, Y. and OKA, H. I.: Evolutionary studies in cultivated rice and its wild
relatives. Oxford Surveys in Evolutionary Biology, 8, pp. 135-184, Oxford, UK, 1992.
MORITA, T, KUBOTA, H., MURATA, K., DELARBRE, c., GACHALIN, G., WILLISON, K.,
SATTA, Y., TAKAHATA, N., SAKAIZUMI, M., NOZAKI, M. and MATSUSHIRO, A.: Evolution of Mouse I-Haplotype: Recent and World-wide Introgression to Mus musculus. Proc.
Natl. Acad. SCi. USA, 89, 6851-6855, 1992.
MORIYAMA, E. N. and GOJOBORI, T.: Rates of synonymous substitution and base compositionof nuclear genes in Drsophila. Genetics, 130, 855-864, 1992.
NAITO, M., KOHARA, Y. and KUROSAWA, Y.: Identification of a homeobox-containing genelocated between lin-45 and unc-24 on chromosome IV in the nematode Caenorhadilis
elegans. Nucl. Acids Res., 20, 2967-2969, 1992.
NAGAMINE, C. M., NISHIOKA, Y., Moriwaki, K., BOURSOT, P., Bonhomme, F. and LAu, YUN
FA! CHRIS: The musculus-type Y chromosome of the laboratory mouse is of Asian origin.
Mammalian Genenome, 3, 84-91, 1992.
NAKAGAWA-HATTORI, Y., YOSHINO, H., KONDO, T., MIZUNO, Y. and HORAI, S.: Is

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Parkinson's disease a mitochondrial disorder? J. Neurol. Sci., 107,29-33, 1992.
NAKAMURA, T., IHARA, T., NUNOYA, T., KUWAHARA, H., ISHIHAMA, A. and UEDA, S.: Roleof pseudorabies virus glycoprotein II in protection from lethal infection. Veterinary
Microbiol., 36, 83-90, 1993.
NAKAsu, S. and TOMIZAWA, J.: Structure of the ColEI DNA molecule before segregation todaughter molecules. Proc. Natl. Acad. Sci. USA, 89, 10139-10143, 1992.
NAKATSUJI, N.: Development of postimplantation mouse embryos: Unexplored field rich inunanswered questions. Devel. Growth Differ., 34, 489-499, 1992.
NAKAYAMA, M., NAGATA, K. and ISHIHAMA, A.: Enzymatic properties of mouse Mxl
protein-associated GTPase. Virus Res., 22, 227-234, 1992.
NAKAYAMA, M., YAZAKI, K., KUSANO, A., NAGATA, K., HANAI, N. and ISHIHAMA, A.:
Structure of mouse Mxl protein: Self-assembly and GTP-dependent conformationalchange. J. BioI. Chern., 268, 15033-15038, 1993.
NATSUME-SAKAI, S., HARADA, Y. and MORIWAKI, K.: Establishment ofa congenic strain withcomplement regulatory protein of factor H allotype derived from Mus musculus molossinus.
Exp. Anim., 41, 79-82 (Japanese), 1992.
NEVO, E., HONEYCUTT, R. L., YONEKAWA, H., NELSON, K. and HANZAWA, N.: Mitochondrial DNA polymorphisms in subterranean mole rats of the Spalax ehrenbergi superspecies
in Israel and its periferal isolates. Mol. BioI. Evol., in press.
NISHIMIY A-FUJI SAW A, C. and SUIGYAMA, T.: Genetic analysis of developmental Mechanisms
in hydra. XX. Cloning of interstitial stem cells restricted to the sperm differentiation
pathway in Hydra magnipapillate. Developmental Biology, in press.
NISHIMURA, A., AKIYAMA, K., KOHARA, Y. and HORIUCHI, K.: Correlation of a subset of thepLC plasmids to the physical map of Escherichia coli K-12. Microbiol. Rev., 56, 137-151,
1992.
NISHITANI, H., GOTO, H., KANEDA, S., YAMAO, F., SENO, T., HANDLEY, P., SCHWARTZ, A.
L. and NISHIMOTO, T.: The tsBN75 and tsBN423, temperature-sensitive X-linked mutantsof the BHK21 cell can be complemented by the ubiquitin-activating enzyme, EI eDNA.,
Biochem. Biophys. Res. Commun., 184, 1015-1021, 1992.
NON AKA, M., MATSUDA, Y., SHIROISHI, T., MORIWAKI, K., NONAKA, M. and NATSUUMESAKAI, S.: Molecular cloning of mouse b2-glycoprotein I and mapping of the gene tochromosome II. Genomics, 13, 1082-1087, 1992.
OHBA, R., TABUCHI, H. and HIROSE, S.: DNA supercoiling facilitates the assembly oftranscriptionally active chromatin on the adenovirus major late promoter. Biochem.
Biophys. Res. Commun., 186, 963-969, 1992.
OHTA, T.: Diversifying selection, gene conversion, and random drift: interactive effects onpolymorphism at MHC loci. In "HLA 1991", Vol. 2, pp. 20-27, (Tsuji et aI., eds.), Oxford
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OHTA, T.: Theoretical study of near neutrality. II. Effect of subdivided population structurewith local extinction and recolonization. Genetics, 130,917-923, 1992.
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OHTA, T. and BASTEN, CHRISTOPHER J.: Gene conversion generates hypervariability at thevariable regions of kallikreins and their inhibitors. Molecular Phylogenetics and Evolution,
1, 87-90, 1.992.
OHYA, Y., WATANABE, K., SHIMAMOTO, N. and AMANO, M.: Scleral fibroblasts of the Chickembrio can proliferate without transferrin in protein-free culture. Zoolog. Sci., 9, 749-755,
1992.
OKANO, H., HAYASHI, S., TANIMURA, T., SAWAMOTO, K., YOSHIKAWA, S., WATANABE, J.,IWASAKI, M., HIROSE, S., MIKOSHIBA, K. and MONTELL, c.: Regulation of Drosophila
neural development by a putative secreted protein. Differentiation, 52, I-II, 1992.
OKAZAKI, K., NISHIZAWA, M., FURUNO, N., YASUDA, H. and SAGATA, N.: Differential
occurence of CSF-like activity and transforming activity of Mos during the cell cycle infibroblasts. EMBO J., 11, 2447-2456, 1992.
OZAKI, M., FUJITA, N., WADA, A. and ISHIHAMA, A.: Promoter selectivity of stationary-phaseforms of Escherichia coli RNA polymerase and conversion in vitro of the S I from enzyme
into a log-phase enzyme-like form. Nucleic Acids Res., 20, 257-261, 1992.
PRAMOONJAGO, P., KANEKO, M., KINOSHITA, T., OHTSUBO, E., TAKEDA, J., HONG, K.,
INAGI, R. and INOUE, K.: Role of TraT protein, an anticomplementary protein producedin Escherichia coil by Rloo factor, in serum resistance. J. Immunol, 148, 827-836, 1992.
QADOTA, H., ISHII, 1., FUJI YAM A, A., OHYA, Y. and ANRAKU, Y.: RHO gene Prodicts,
Putative Small GT binding zproteins, are Important for Activation of the CALl/CDC43
Gene Product, a Protein Geranylgeranyltransferase in Saccharomyces cerevisiae. Yeast, 8,
735-741,1992.
SAITOU, N.: Evolutionary rate of insertions and deletions. In "Population Paleo-Genetics:
Proceedings of the Seventeenth Taniguchi International Symposium on Biophysics" (Takahata, N., ed.), pp. 335-349, Japan Scientific Societies Press, Tokyo, 1992.
SAITOU, N., TOKUNAGA, K. and OMOTO, K.: Genetic affinities of human populations. In"Monograph SSHB Series #30: Isolation and Migration" (Roberts, D. L., Fujiki, N. andTorizuka, K., eds.), pp. 118-129, Cambridge University Press, Cambridge, 1992.
SAKAGAMI, H., TAKEDA, M., KAWAZOE, Y., NAGATA, K., ISHIHAMA, A., UEDA, M. and
YAMAZAKI, S.: Anti-influenza virus activity of a lignin fraction from cone of Pinus
parviflora Sieb. et Zucco In Vivo, 6, 491-496, 1992.
SAKUMI, K., IGARASHI, K., SEKIGUCHI, M. and ISHIHAMA, A.: The Ada protein is a class-I
transcription factor of Escherichia coli. J. Bacteriol., 175, 1162-1167, 1993.
SAKUTA, R., GOTO, Y., HORAI, S., OGINO, T., YOSHINAGA, H., OHTAHARA, S. and NONAKA,
1.: Mitochondrial DNA mutation and Leigh's syndrome. Ann. Neurology., 32, 597-598,
1992.
SANO, R. and MORISHIMA, H.: Indica-Japonica differentiation of rice cultivars viewed fromvariations in key characters and isozyme, with special reference to land races from theHimalayan hilly areas. Theor Appl. Genet., 84, 266-274, 1992.
SA NO, Y.: Genetic comparisons of chromosome 6 between wild and cultivated rice. Japan. J.
Breed., 42, 561-572, 1992.
SANO, Y., EIGUCHI, M., HIRANO, H.-Y. and YAMADA, M.-A.: A nuclear gene modifying
instability of fertility restoration in cytoplasmic sterile rics. Genet. Res., 60, 195· 200(1992).

110
SATO, Y. I.: Gametic selection in rice. In "Pol/en and Ovules in higher plants" (Mulcahy, D. L.
& Ottaviano, O. E., ed.), Elsevier. Amsterdam/New York (in press).
SEKINE, Y., NAGASAWA, H. and OHTSUBO, E.: Identification of the site of translationalframeshifting required for production of the transposase encoded by insertion sequence lSI.
Mol. Gen. Genet., 235, 317-324, 1992.
SEKINE, Y. and OHTSUBO, E.: DNA sequences required for translational frameshifting in
production of the transposase encoded by lSI. Mol. Gen. Genet., 235, 325-332, 1992.
SEONG, B. L., KOBAYASHI, M., NAGATA, K., BROWNLEE, G. G. and ISHIHAMA, A.: Comparison of two reconstitution systems for in vitro transcription and replication of influenza
virus. J. Biochem., 111,496-499, 1992.
SIIIBAGAKI, Y., IToH, N., YAMADA, H., NAGATA, S. and MIZUMOTO, K.: Messenger RNA
Capping Enzyme: Isolation and characterization of a gene encoding mRNA guanylyltransferase from Saccharomyces cerevisiae. J. BioI. Chern., 267, 9521-9528, 1992.
SHIMAMOTO, N., KABATA, H., KUROSAWA, O. and WASHIZU, M.: Visualization of single
moplecule of RNA polymerase and its application for detecting sliding motion on T7 DNA
dielectrophoretically manipulated, in "Structural Tools for the Analysis of Protein-Nucleci
Acid Complexes" (Lilley, D. M. J., Heumann, H. and Suck, D., eds.), pp. 241-253,
Birkhauser Verlag, Berlin, 1992.
SHIMIZU, H., SAWADA, Y. and SUGIYAMA, T.: Minimum tissue size required for hydra
regeneration. Developmental Biology, 155, 287-296, 1993.
SHIMIZU, N., TAKEUCHI, Y., NARUSE, T, INAGAKI, M., MORIYAMA, E. N., GOJOBORI, T. and
HOSHI NO, H.: Six strains of human immunodeficiency virus type I isolated in Japan andtheir molecular phylogeny. J. Mol. Evol., 35, 329-336, 1992.
SHIMOZAWA, N., TSUKAMOTO, T, SUZUKI, Y., ORII, T, SHIRAYOSHI, Y., MORI, T and
FUJIKI, Y: A human gene responsible for Zellweger syndrome that affects peroxisome
assembly. Science, 225, 1132-1134, 1992.
SMILLIE, D. A., HAYWARD, R. S., SUZUKI, T., FUJITA, N. and ISHIHAMA, A.: Location of alkylhydroperoxide reductase genes on the physical map of Escherichia coli. J. Bacterial., 174,3826-3827, 1992.
SPECTHRIE, L., BULLITT, E., HORIUCHI, K., MODEL, P., RUSSEL. M. and MAKOWSKI, L.:
Construction of a microphage variant of filamentous bacterophage. J. Mol. BioI., in press.
SUZUKI, H., SAKURAI, S., NISHIMURA, M., KOMINAKI, R. and MORIWAKI, K.: Compensatorychanges in silver-stainability of nucleolar organizer regions in mice. Jpn. J. Genet., 67, 217232, 1992.
TACH IDA, H.: Evolution of repeated sequences by replication slippage. In "Population
Paleo-Genetics: Proceedings of the Seventeenth Taniguchi International Symposium on
Biophysics" (Takahata, N., ed.), pp. 319-333, Japan Scientific Societies Press, Tokyo, 1992.
TACH IDA, H. and IIZUKA, M.: Persistence of repeated sequences that evolve by replication
slippage. Genetics, 131,471-478, 1992.
T AJIMA, F.: Measurement of DNA polymorphism. In "Population Paleo-Genetics: Proceedingsof the Seventeenth Taniguchi International Symposium on Biophysics" (Takahata, N., ed.),pp. 37-59, Japan Scientific Societies Press, Tokyo, 1992.
TAJIMA, F.: Statistical method for estimating the effective population size in Pacific salmon.
Journal of Heredity, 83, 309-311, 1992.

PUBLICAnONSIII

TAJIMA, F.: Statistical method for estimating the standard errors of branch lengths in aphylogenetics tree reconstructed without assuming equal rates of nucleotide substitution
among different lineages. Molecular Biology and Evolution, 9, 168-181, 1992.
TAKAHASHI, E.-i., AYUSAWA, D., KANEDA, S., ITOH, Y, SENO, T. and HORI, T.-a.: The
human ubiquitin-activating enzyme EI gene (UBEI) mapped to band Xpll.23-p. 11.3 byfluorescence in situ hybridization. Cytogenet. Cell Genet., 59, 268-269, 1992.
TAKAHASHI, K., HAU, C, KAWAAN, K., IKEo, K. and KOH, E.: Phosphorylation of a surface
receptor bound urokinase-type plasminogen activator in a human metastatic carcinomatous
cell line. Biochem. Biophys. Res. Commm., 182, 1466-1472, 1992.
TAKAHASHI, M., TORIYAMA, S., HAMAMATSU, C. and ISHIHAMA, A.: Nucleotide sequenceand possible ambisense coding strategy of rice stripe virus RNA aegment 2. J. Gen. Virol.,74,769-773, 1993.
TAKAHATA, N.: Evolutionary genetics of human paleo-populations. In: Molecular Paleopopulation Biology. (N. Takahata, ed.), Japan Scientific Society Press, 1992.
TAKAHATA, N.: Molecular Paleopopulation Biology, The Proceedings of the 17th TaniguchiInternational Symposium on Biophysics. (N. Takahata, ed.), Japan Scientific Society Press,
1992.
TAKAHATA, N. and SATTA, Y.: Some comments on molecular evolutionary rate. Immunogenetics, 36, 126-129, 1992.
TAKAHATA, N., SATTA, Y, and KLEIN, J.: Trans-species polymorphism of the major histocompatibility complex (Mhc) loci. In: Progress in Immunology VIII (Proceedings of the 8thInternational Congress of Immunology Budapest), pp. 153-158, (1. Gergely et al. eds.),
Springer Hungarica, 1992.
TAKAHATA, N., SATTA, Y. and KLEIN, J.: Polymorphism and balancing selection at major
histocompatibility complex loci. Genetics, 130, 925-938, 1992.
TAKAMATSU, H., FUMA, S.-I., NAKAMURA, K., SADAIE, Y., SINKAI, A., MATSUYAMA, S.-1.
MIZUSHIMA, S. and YAMANE, K.: In vivo and in vitro characterization of the secA geneproduct of Bacillus subtilis. J. Bacteriol., 174, 4308-4316, 1992.
TAKAYANAGI, A., KANEDA, S., AYUSAWA, D. and SENO, T.: Intron I and the 5'-flanking
region of the human thymidylate synthase gene as a regulatory determinant of growthdependent expression. Nucl. Acids Res., 20, 4021-4025, 1992.
TAKESIIITA, K., YAMAGISHI, I., SUGIMOTO, T., OTOMO., S. and MORIWAKI, K.: The hair
growing effect of minoxidil. The molecular and structural Biology of Hair, 642, 470-472,
1992.
TAMAI, K., TEzuKA, H. and KURODA, Y: Different modifications by vanillin in cytotoxicity
and genetic changes induced by EMS and H,O, in cultured Chinese hamster cells. MutationRes., 268, 231-237, 1992.
TANAKA, K., TAKAYANAGI, Y., FUJITA, N., ISHIHAMA, A. and TAKAHASHI, H.: Heterogeneity of the principal sigma factor in Escherichia coli: The rpoS gene product, d 8 , is a second
principal a factor of RNA polymerase in stationary-phase Escherichia coli. Proc. Natl.
Acad. Sci. USA, 90,3511-3518 (1993).
TAo, K., FUJITA, N. and ISHIHAMA, A.: Involvement of the RNA polymerase a-subunit
C-terminal region in cooperative interaction and transcription activation with OxyR protein. Mol. Microbiol., 7, 859-864, 1993.

112
TERADA, K., HIROSE, S. and OKUHARA, E.: Production of antibodies specific for double
stranded antigen DNA cloned from immune complexes in plasma of a SLE patient.
Biochem. Biophys. Res. Commun., 183, 797-802, 1992.
TOKUNAGA, K., TANAKA, H., MANFF, G., SUZUKI, K., NISHI MUKAI, H., HAUPTMANN, G.,
FEENEY., J., IMANISHI, T., GOJOBORI, T., JUJI, T. and DAWKINS, R. L.: Complement
studies of the Elventh International Histocompatibility Workshop. HLA 1991 (Oxford
University Press), 1,947-954, 1992.
TSUCHIMOTO, S. and OHTSUBO, E.: Cooperative binding of the PemI and PemK proteins to thepromoter region of the pem operon. Mol., Gen. Genet., in press.
TSUCHIMOTO, S., NISHIMURA, Y. and OHTSUBO, E.: The stable maintenance system pem ofPlasmid RlOO: Degradation of the PemI protein may allow the PemK proteins to inhibit cell
growth. J. Bacteriol., 174,4205-4211, 1992.
TSUCHIYA, K., MATSUURA, Y., KAWAI, A., ISHIHAMA, A. and UEDA, S.: Characterization ofrabies virus glycoprotein expressed by reocombinant baculovirus. Virus Res., 25, 1-13,
1992.
TSUKIYAMA, T., VEDA, H., HIROSE, S. and NIWA, 0.: Embryonal long terminal repeat-binding
protein is a murine himologue of FTZ-FI, a member of the steroid receptor superfamily.
Mol. Cell. BioI., 12, 1286-1291, 1992.
UEDA, H., SUN, G.-c. and HIROSE, S.: Cloning of BmFTZ-FI, a family of nuclear hormone
receptor superfamily in the silkworm, Bambyx mori and comparison with FTZ-FI, a
Drosophila homologue of BmFTZ-FI. J. Cell. Biochem., 16C, 31, 1992.
UEDA, H., SUN, G.-c., MURATA, T. and HIROSE, S.: A novel DNA-binding motif abuts thezinc finger domain of insect nuclear hormone receptor FTZ-FI and mouse embryonal longterminal repeat-binding protein. Mol. Cell. BioI., 12, 5667-5672, 1992.
UESHIMA, R., FUJITA, N. and ISHIHAMA, A.: Identification of Escherichia coli proteinscrossreating with antibodies against region 2.2 peptide of RNA polymerase sigma subunit.
Biochem. Biophys. Res. Commun., 184, 634-639, 1992.
WADA, K., WADA, Y., ISHIBASHI, F., GOJOBORI, T. and IKEMURA, T.: Codon usage tabulated
from the GenBank genetic sequence data. Nucl. Acids Res., 20, Supplement, 2111-2118,
1992.
WAKISAKA, A., IMANISHI, T., SAITOU, N., KONONEDA, Y. and GOJOBORI, T.: Introduction toand organization of the anthropology component. HLA 1991 (Oxford University Press), 1,
623-626, 1992.
Wu, X., WANG, c., KATOH, H., XING, R., ZHANG, X., YAO, G., ZHU, B. and MORIWAKI, K.:
Genetic profile of LIBP/l inbred strain derived from the Kunming outbred stock of themouse. Exp. Anim., 41,541-543, 1992 (Japanese).
XIAN, M., MORIWAKI, K. and TOYODA, Y.: Sex indentification and development of single
blastomeres from 2-cell mouse embryos. J. Rep. Dev., 38, 15-21, 1992.
YAMAGISHI, H., HOSSAIN, M. M. and YONEZAWA, K.: Production of somatic hybrids between
southern type Chinese cabbage "Kenshin" and cabbage "Yoshin" and their floweringcharacteristics. J. Jpn. Soc. Hort. Sci., 61,311-316, 1992.
YAMAGISHI, H. and YONEZAWA, K.: Genetic variations in flowering time, flower and siliquesizes of Suzukina strains (Brassica campestris L.). J. Jpn. Soc. Hort. Sci., 61, 353-357 (inJapanese), 1992.

PUBLICAnONS

113

YAMAGISHI, M., MATSUSHIMA, H., WADA, A., SAKAGAMI, M., FUJITA, N. and ISHIHAMA, A.:
Regulation of the Escherichia coli rmf gene encoding ribosome modulation factor (RMF):
Growth phase- and growth rate-dependent control. EMBO J., 12,6171-6174, 1993.
YAMAO, F., NAGAI, Y., KANEDA, S., YOSHIDA, S. and SENO, T: Conditional resistance tothymine-less death predominantly selects DNA synthesis-deficient mutants of mammaliancells. Mutation Res., 289, 83-89, 1993.
YAMASHITA, M., FUKUDA, S., YOSHIKllNI, M., BULET, P., HIRAI, T., YAMAGllCHI, A.,
YASUDA, H., OHBA, Y. and NAGAHAMA, Y.: M-phase-specific histone HI kinase from fish
oocytes: its purification, components and biochemical properties. Eurp. J. Biochem., 205,
537-543, 1992.
YASUDA, H., NAKATA, T., KAMIJO, M., HONDA, R., NAKAMURA, M., NINOMIYA-TSUJI, J.,
YAMASHITA, M., NAGAHAMA, Y. and OHBA, Y.: Cyclin dependent kinase (cdk2) in themurine cdc2 kinase ts mutant. Somatic Cell and Mol. Genet., 18, 403-408, 1992.
YONEZAWA, K.: On the factors and mechanism causing genetic differentiation in plant
populations. Acta Hum. & Sci., Kyoto Sangyo Univ., Nat. Sci. Series II, 22, No.2: 78-114(in Japanese), 1992.
YONEZAWA, K.: Genetic edge effect in plant populations. Recent Advance in Breeding 34 (inJapanese),1992.
YONEZAWA, K.: Core collection sampling strategy for promoting maintenance and utilizationof plant genetic resources accessions. Ann. Rept. Inst. Natl. Land Util. Develop., Kyoto
Sangyo Univ., 14, 89-113 (in Japanese), 1993.
YURA, T, MORI, H., NAGAI, H., NAGATA, T., ISHIHAMA, A., FUJITA, N., ISONO, K.,MIZOBUCHI, K. and NAKATA, A.: Systematic sequencing of the Escherichia coli genome:
Analysis of the G-2.4min region. Nucl. Acids Res., 20, 3305-3308, 1992.
YURA, T, NAGAI, H. and MORI, H.: Regulation of the heat shook response in bacteria. Ann.
Rev. Microbiol. Vol. 47, in press.
ZOll, C, FUJITA, N., IGARASHI, K. and ISHIHAMA, A.: Mapping the Escherichia coli RNA
polymerase contact site I for cAMP receptor protein. Mol. Microbiol., 6, 2599-2605, 1992.

114

ABSTRACTS OF DIARY FOR 1992

Biological Symposium
356th-Jan. 29
Processes to produce mosaic structures of GC content
of genome DNA (Gerald P. Holmquist)
357th-Feb. 3
Coat protein mediated resistance against plant virus intransgenic plants (R. N. Beachy)
358th-Mar. 18
Cultural revolution caused by organic evolution (William Provine)
359th-Mar. 21
1. Synthesis and degradation of polyphospahteRegulation of polyphosphate operon
2. Mutations occurred in the in vitro oriC replication
system (Masahiro Akiyama)
360th-Apr. 7
The house mouse as a ring species: radiation from thecradle and the evolution of coadapted gene system(Pierre Boursot)
36 1st-Apr. 10
The MARCKS protein is an actin filament and plasma
membrane crosslinking protein regulated by protein
kinase C and calmodulin (Angus C. Narin)
362nd-Apr. 22
Differentiation of rat neuronal stem cells to neuron andglia (Noboru Sueoka)
363rd-Apr. 23
Why is ethanolamine required for growth of animalcells? (Tamiko Sueoka)
364th-May 25
Functional role for intracellular protein transport ofmultiple small GTP-binding proteins (Dieter Gallwitz)
365th-June 5
Ubiquitin-dependent proteolysis-a genetic analysis(Stefan Jentsch)
366th-June 8
Molecular analysis of V(D)J recombination (Martin F.
Gellert)367th-June 9
Homologous recombination in mammalian cells (Maria
Jasin)
368th-June 29
The enigma of compatibility and h<;>st/symbiont specificity in hydra/algae symbioses (Menachem Rahat)
369th-July 2
Measurement of pH inside perialgal vacuoles in symbiotic Hydra (Vanda Reich)

ABSTRACTS OF DIARYFOR 1992

37Oth-July 4
37lst~July 9372nd-July 27
July 27
373rd-Aug. 26
374th~Nov. 10375th~Nov. 19

376th-Nov. 20
377th~Dec. 1

378th~Dec. 4379th~Dec. 11

380th~Dec. 11

115

The temporal control of gene activities by ecdysone(Michael Ashburner)
Molecular epidemiology of HIV in Europe and Africa:
virus variation and the age of the epidemic (Jaap Goudsmit)
Active site geometry of E. coli RNA polymerase (Dipankar Chatterji)
Selection of transcription initiation site by E. coli RNA
polymerase (Changwon Kang)
Nuclear DNA variation in Homo sapiens (Kenneth K.
Kidd)
Sequencing the C. elegans genome (Robert H. Waterston)
Molecular evolution of HLA class II genes (Susan W.
Serjeantson)
Organisation and expression of the globin gene domain
and the Unified Matrix Hypothesis (Klaus Scherrer)
Antigen presentation by MHC class I moleculesIdentification of a peptid recognized by alloreactive
cytotoxic T lymphocytes (Keiko Udaka)
Drosophila tumor suppressor genes and their humanhomologs (Peter J. Bryant)
New databases and software tools for molecular biology
at the National Center for Biotechnology Information(James Ostell)
Physical mapping and genome sequencing in the human(Maynard Olson)

Mishima Geneticists' Club
395th-Jan. 23
Molecular cloning and expression of the secretin and
VIP receptors (Takeshi Ishihara)
396th-Jan. 24
Regulatory mechanisms of the heat shock response in E.
coli (Hiroki Nagai)
397th-Feb. 24
Cell cycle-dependent assembly of chromatin-an approach from in vitro reconstitution system (Tatsuya
Hirano)398th-Apr. 23
Molecular dynamics of large proteins (Atsushi Ikai)

116

399th-July 20
40Oth-July 29
40Ist-Sept. 10
402nd-Dec. 3
403rd-Dec. 17

Factors that regulate proliferation of primordial germ
cells (Yasuhisa Matsui)
Genetic analysis of eNS development in zebrafish(Kohei Hatta)
Structure/function studies on sigma subunits of E. coli
RNA polymerase (Richard S. Hayward)
Mechanisms of germ line cell differentiation in Drosophila (Satoru Kobayashi)
Analysis of nucleosome positioning using yeast minichromosome (Shigeo Tanaka)

117

FOREIGN VISITORS IN 1992

Mar. 28, 1991Jan. 3Mar. 31, 1992
Jan. 28-30
Feb. 3-4
Mar. 5-29
Mar. 21
Apr. 7
Apr. 10
Apr. 13-June 30
Apr. 22-23
Apr. 22-23
May 25June 5June 7-9
June 9June 15-20
June 15-20
June 28-30
July 2-5
July 3-4
July 4-Aug. 10

Engelbert Hobmayer, University of Munich, Germany
Jianying Luo, Hunter ColIege of CUNY, U.S.A.
Gerald P. Holmquist, City of Hope Medical Center,
U.S.A.
Roger N. Beachy, The Scripps Research Institute,
U.S.A.
William Provine, CornelI University, U.S.A.
Masahiro Akiyama, Stanford University, U.S.A.
Pierre Boursot, University Monpellier, France.
Angus C. Nairn, The RockefelIer University, U.S.A.
Ashok Kumar, University of Edinburgh, U.K.
Noboru Sueoka, University of Colorado at Boulder,
U.S.A.
Tamiko Sueoka, University of Colorado at Boulder,
U.S.A.
Dieter GalIwitz, Max-Planck-Institute for Biophysical
Chemistry, Germany
Stefan Jentsch, Friedrich-Miescher-Laboratorium der
Max-Planck-Gesellschaft, Germany
Martin F. GelIert, National Institutes of Health, U.S.A.
Maria Jasin, Memorial Sloan-Kettering Cancer Center,
U.S.A.
Ok-Soon Heo, National Institute of Safety Research,
Ministry of Health and Social Affairs, Korea
Eui-Sik Han, National Institute of Health, Ministry of
Health and Social Affairs, Korea
Menachem Rahat, The Hebrew University of Jerusalem,
Israel
Yanda Reich, The Hebrew University of Jerusalem,
IsraelMichael Ashburner, University of Cambridge, U.K.
Flemming Hansen, Technical University of Denmark,118

July 4-Sept. 22
July 9July 21-22
July 21-22
July 21-25
July 24-25
July 25-27
July 25-28
July 24-26
July 25-Aug. 4
Aug. 15-21
Aug. 15-21
Aug. 15-21
Aug. 15-21
Aug. 15-21
Aug. 15-21
Aug. 24-27
Sept. 1~12
Sept. 27-30
Sept. 29-oct. 30
Sept. 16-Dec. 14

Denmark
Richard S. Hayward, University of Edinburgh, U.K.
Jaap Goudsmit, University of Amsterdam, The Netherlands
You-di Liao, Institute of Biomedical Science, Academia
Sinica, Taiwan, China
Soo-Chen Cheng, Institute of Molecular Biology,
Taiwan, China
Bei-Chang Yang, National Cheng-Kung University,
Korea
Younghoon Lee, Korea Advanced Institute of Scienceand Technology, Korea
Changwon Kang, Korea Advanced Institute of Scienceand Technology, Korea
Diopankar Chatterji, Centre for Cellular and Molecular
Biology, India
Samit Adhya, Indian Institute of Chemical Biology,
India
Tove Atlung, Technical University of Denmark, Denmark
Lobov V. Frisman, Institute of Biology and Pedology
Far East Branch, Russian Academy, Russia
Vladimir P. Korablev, Institute of Biology and Pedology Far East Branch, Russian Academy, Russia
Galina M. Chelomina, Institute of Biology and Pedology Far East Branch, Russian Academy, Russia
Lydumila Yakimenko, Institute of Biology and Pedology Far East Branch, Russian Academy, Russia
Alexei P. Kryukov, Institute of Biology and Pedology
Far East Branch, Russian Academy, Russia
Irina V. Kartavtseva, Institute of Biology and Pedology
Far East Branch, Russian Academy, Russia
Kenneth K. Kidd, Yale University, U.S.A.
Karin Petersen, University of Edinburgh, U.K.
O. G. Ward, Los Alamos National Laboratory, U.S.A.
Chitrakon Songkran, Rice Research Institute, Thailand
Toshie Kawano, LEZEP Instituto Butantan, Brazil

FOREIGIN VISITORS IN 1992

Nov. 9-16
Nov. 10
Nov. 19
Nov. 20-22
Nov. 30-Dec. 1
Dec. 3
Dec. 3-5
Dec. 11
Dec. 11119

Michael Griffiths, Royal Perth Hospital, Australia
Robert H. Waterston, Washington University School of
Medicine, U.S.A.
Susan W. Serjeantson, The Australian National University, Australia
Klaus Scherrer, Institut Jacques Monod,CNRS, France
Keiko Udaka, Max-Planck-Institute for Biology, Germany
Faith Keenan, Hearst Newspapers, Washington Bureau,
U.S.A.
Peter J. Bryant, University of California, U.S.A.
James Ostell, National Center for Biotechnology Information, U.S.A.
Maynard Olson, University of Washington, U.S.A.

120

AUTHOR INDEX.41
82, 84
ANDACHI, Y . . . . • . . . . . . . . • • . • . . . . . . . . 64
ASANO, S.
.
35
ASANO, Y.
. ..•...........•.••.•. 21,24
AZUMA, Y.
. ...•.••..........•.•.•. 20
BASTEN. C. J.
.
69
DAWKINS. R. L.
84
DELARBRE. C.
74
EIGUCHI. M. . •..•.........•.•.•.•... 96
FINLAY. J
84
FISCHER-LINDAHL. K. . .••..•.•...... .47
FUSISA W A. T.
.
53
FUJITA. N.
..
15. 17
FUSE. N.
.
58
GACHELIN, G.
. . . . . . . • . . • . . . . . . . . . . . 74
GOJOBORI, T
80.81.82.83,84.100
GOTO. Y . . . . . • • . . • . • . . . . . . . . . . . . . 89.91
HAMAMATSU. C.
.
25
HARA. H. . . • • . . . . . . . . . . . . . . . . 39. 40, 41
HASEGAWA. A.
. . . . . . . . . . . . . . . • . . • . 100
HATTA. M.
. . . . • . . • . . • . . . . . . . . . . . . • 54
HATTORI. Y. . ..••.••.•..•....... 90. 100
HAUPTMANN. G.
• •.•.............•.• 84
HAWAGAWA. Y. . .•.............•.•• 100
HA YASAKA. K. • •.•.............•.••. 89
HAYASHI. S.
.
57. 58. 59. 89
HAYWARD. R. S.
.
15
AlBA, H.

.

AKAZA, T.

• •.•. . 34.35.36.38.41

HIGASHITANI. A.
HIGASHITANI. N.

.

34

HIROKAWA. H . . . . . . . . . . . . . . • . . • . . . . . 36
. . . . . . . . . . . . • . • . . . 33,96

HIRANO. H.-Y.

55. 56. 57. 58, 59
61.89
HORAI. S
89.90.91
HORIE. M.
. .••..•.............•..• 100
HORIUCHI, K . • • . . 34. 35. 36. 38. 39. 40. 41
HOSHINO. H. .
83
IKEMURA, T. .
76
HIROSE. S.

HISHIDA, R.

.

.

'"

IKEO. K.

. . . . . . . . • • . • . • . . . . . . . . 82. 100

IMAMURA, T.

85. 86. 88
81.82.84
INABA. R. .
85. 86. 88
INAGAKI. M. . . . . . . . . • . • • . . . . . . . . . . . . 83
INOUE, T.
89
ISHIDA. T. .
89
ISHIHAMA. A.
. •.... 15. 17. 18.20,21.22.
23. 24, 25
ISHIHARA, T.
.
60.61,62
IWASAKI, M . • . • . . . . . . . . . . . . . . . . . . . • • 59
IWASE. M.
. . . . . . . . • . • . • . . . . . • . . . . . 100
JUJI. T.
.
84
KABATA, H.
. . . . . . . . . • . . • . • . . . . . . . . . 31
KAMADA. K. . ...•••.•.•............. 32
KANEDA, S.
.
25,26.27.28
KARIBIAN. D.
.
" . . . . . . • . . . . . 40
KATO. A.
• . . . . . • • • . . . . . . . . . . . . . . . . . 23
KATSURA, I.
60.61.62
KAW AGISHI, M.
. . . . . . . . . . . . . . . . . • • • 18
KAWAKAMI, M.
.
'" ....•.•.••• 60
KAWAMOTO. T.
.
100
KAWAMURA. S.
.
78
KIMURA. A.
• •.•••....•............. 82
KITAKAMI. H . • . • . . . . . • . . • . • . . . . . . . . 100
KLEIN. J.
.
72. 73, 74
KOBAYASHI. M. . . . . • . . . . . . . . . . . . . 21. 25
KOGA. Y.
91
KOHARA, Y.
63. 64. 65
KOIZUMI. O. .
52
KOMEDA. Y . • . • . . . . . . . . . . . . . . . . . . . . . 33
KONDO. R.
.
89
KONDO, T
90
KUBORI, T.
.
31
KUBOTA. H.
.
74
KUMAR. A.
. • . . • • . . . . . . . . . . • . . . . . . . 15
MAKINO. K . • • . • • • • . . . . . . . . . . . . . . . . . 15
MATSUMOTO. K. I.
76
MATSUMURA, T.
33
IMANISHI. T.

.
.

121

AUTHOR INDEX. . • . . • . • . . . . . . . . . . . . . • 91

MATSUOKA, T.
MATSUSHIRO, A.

• . • . . • . . . . . . . . . . . . . . 74

MATSUSHIMA, H . . . . . . . . • . . • . . . . . . . . . 18
MATSUSHIMA, Y.

100
84
MAYER, W. E.
. . . . • . . . . . . . . . . . . . . • . • 74
METCALFF, T.
57
MIKOSHIBA, K. .
59
MINATO, K.
.
57
MINOBE, S.
.
52
MITSUKI, H.
. .........•.•..•..•••..• 63
MIYABAYASHI, T ....•..•.•..•..•••.•• 93
MIYASHITA, N.
. . . . . . . . . . . . . . • . . . 49, 50
MIZUMOTO, K
21,29,30
MIZUNO, K-1.
.44
MIZUNO, T.
• ••.•....•..•.•........ .41
MIZUNO, Y.
.
90
MaNTELL, C.
59
MORISHIMA, H.
.
93, 95, 96
MORITA, T.
. . • . . . . • . • . . . . . . . . . . . . . . 74
MORIWAKI, K.
. .44,45,46,47,48,49, 50
MaRlY AMA, E. N. .
80, 83
MOTOHASHI, T.
. .........•...•...•• 63
MURATA, K. .
74
MURATA, T. . . . . . . . . . . . . . . . . . . . . . • . . 56
NAGAI, H . . . . • . . . . . . . . . . . . . . . . . . . • . . 32
NAGAI, Y. .
26, 27, 28
NAKADA, S.
. . . . • . . . . . . . . • . . . . . . 22, 23
NAKAGAWA-HATTORI, Y.
.
90
NAKASHIMA, H.
. .•........•.. 85, 86, 88
NAKATSUJI, N.
. .....•.•..•• .45, 66, 67
NARUSE, T.
.
83
NIHEI, K.
.
91
NISHIGAKI, A.
. .......•.•..•....•..• 63
NISHIMUKAI, H.
. . . • . . . . • . . . . . . . . . . . 84
NISHIMURA, A.
• . . . . . . . . . • . . . . . . .42, 43
NISHIMURA, Y.
.
.40, 41
NIWA, O.
. . . . . . . . . . . . . . . . . . . . . • • . . • 55
NOMURA, T. . . . . . . . . . . . . . . . . . . • . • . • • 95
NONAKA, I.
89, 91NOZAKI, M.
. . . . . . . . . . . . . . . . . . . • • . • • 74
OGINO, T.
89
OHBA, R.
. . . . . . • . . . . . . • . . . . . . . . . . . . 55
O'HUIGIN, C.
74
MAUFF, G.

.

53
68,69
OHTAHARA, S.
. • • . . . • . . . . . . . . . . . . . . . 89
OKANO, H.
59
OKUHARA, E.
• • • . . . . . . . . . . . . • . . . . . . 56
OMOTO, K.
.
79
PARK, J. T.
39,40
SAGAI, T. .
44,45,46,47,48
SAITO, M.
. • . • . . . . . . . . . . . . . . . . . . . . 100
SAITOU, N. • . . . • . . . . . . . . . . . . . 78, 79, 100
SAKAI, M. . . . . . . . . . . . . . . . . • • . . 85, 86, 88
SAKAIZUMI, M. . .•...•.....•..••....• 74
SAKAKIBARA, Y.
38
SAKURAI, H.
15, I7SAKUTA, R.
. . . . . . • • . . . . . . . . . . . . . . • . 89
SANa, Y.
33, 96, 97, 98
SATO, S . . . • . . . . . . . . . . . . . • . . . • . . . . . • . 24
SATO, Y.
.
100
SATO, Y.-1.
94
SATTA, Y.
71,72, 73, 74, 89
SAWAMaTO, K. .
59
SENO, T. .
25, 26, 27, 28
SHIDAHARA, Y.
.
100
SHIMA MOTa, N
31,32
SHIMOYAMA, M
100
SHIMAMOTO, Y.
.
31
SHIMIZU, N.
. • • • . . . . . . . . . . . • . . . . . . . . 83
SHIOKAW A, K.
.
53
SHIRAYOSHI, Y. .
66,67
SHIROISHI, T.
• ....• 44,45,46,47,48, 50
SHIRRAS, A.
. . . . . . . . . . . . . . . • • . . . . . • . 57
SUDHA, T . • . . • . . . . . . . . . . . . . • . . . . . . . . 28
SUGAVA, K.
.
76
SUGIMOTO, A.
. . . . . . . . . . . . . . • . . . . . . • 63
SUGIYAMA, T . . . . . . . . . . . . . . . . . . . . • 52,54
SUN, G.-c.
56
SUZUKI, K.
.
84
SUZUKI, R.
.
100
SUZUKI, S.
. . • . . . . . . . . . . . . . . . . . . . . . 100
T ABARA, H.
• . . . . . . . . . . . . . . . . . . . . . . • 65
TABUCHI, H . . . . . . • . . . . . . . . . . . . . . . . . . 55
T AJIMA, F.
.
70
TABAYASHI, N . . . . . . . . . . . . . . . . . . . . . . • 33
TAKAHASHI, K.
.
82
OHKAWA, T.

OHTA, T.

.

.

122
TAKAHATA N

70,71,72,73,74,89

TAKEUCHI, Y.

• . • . . • • . . . . . . . . . . . . . • • 83

TANAKA, H.

.

TANAKA, K.

.
.
.
.

84
17
TANG, L. H.
94
TANG, S. X.
94
T AN IDA, M.
33
TANIMURA, T.
59
TATENO, Y . . . . . . . . • . . . . . . . . . . . . . . . . 100
TERADA, K.
.
56
TOHYAMA, J
91
Tala, M.
.
91
TOKUNAGA, K.
.
79, 82, 84
TORIYAMA, S.
.
25
TOYODA, T.
22, 23, 24, 25
TSUKIYAMA, T.
55
UCHIDA, R. .
100
UEDA, H. .
55, 56
UEDA, S.
.
78
UEDA, Y . . . . . . . . . . . . . . . . . . . . . . . • • . . 100
UGAWA, Y. . . • . . . . . . . . . . . . . . . . . . • • . 100
UKAI, H.
.
.43

18
76
W AKANA, S.
.
.47, 50WATANABE, A.
.
100
WATANABE, J.
.
59
WATANABE, S.
.
.49
WILLISON, K.
.
74
Y AMAGISHI, M. .
18, 20
YAMAMOTO, Y.
40
YAMAO, F.
.
26,27,28
YANG, L. U. .
94
YAMAZAKI, Y.
. . . • . • . . . . • . . . . . . . . . 100
YASUDA, J. .
22, 23
YASUDA, S.
.
38
YONEKAWA, H.
.
.49,50YONEZAWA, K
95
YOSHIKAWA, S
59
YOSHINAGA, H.
.
89
YOSHINO, H. .
90
YOSHINO, M.
.
47,48
Zou, C.
15
WADA, A
WADA, K

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