ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 47

1996

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mishima, Sizuoka-ken, Japan
1997

Annual Reportof theNational Institute of Genetics
No. 47, 1996

(Computer Building)Published by

The National Institute of Genetics, Japan
1997

CONTENTS
General statement
Staff
Council and advisory committee
Research activities in 1996

A.

.
.
.
.

Department of Molecular Genetics
A-a. Division of Molecular Genetics
Molecular Anatomy of the Alpha Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Fine Mapping of the Contact Surface with DNA VP Elements and Class-I Transcription Factors. MURAKAMI, K., ARTSIMOVITCH, I., CHUGANI, S. A., BOUCHER, P. E., TANG, Y., GILADI, H., HOWE,
M. M., CHAKRABARTY, A. N., STIBITZ, S., DEHASETH, P. L.,
OPPENHEIM, A. B. and ISHIHAMA, A.
Molecular Anatomy of the Alpha Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Evidence for the Direct
Contact with DNA VP Elements and Class-I Transcription
Factors. MURAKAMI, K., OZOLINE, 0., NEGISHI, T., FUJITA, N., OWENS, J. T., MEARES, C. F. and ISHIHAMA, A.

1
3
8
10

10

12
Regulation of Intracellular Levels of Sigma Subunits in Escherichia
coli. JISHAGE, M., IWATA, A., VEDA, S. and ISHIHAMA, A.
13
Regulation of the Activity of Sigma Subunits from Escherichia coli.
KUSANO, S., KUNDU, T., TANAKA, K. and ISHIHAMA, A.
14
Cloning and Sequencing of the RNA Polymerase II Genes from
Schizosaccharomyces pombe. SAKURAI, H., MIYAO, T., MITSUZAWA, H., KIMURA, M. and ISHIHAMA A.
Subunit-subunit Contact Network within Schizosaccharomyces
pombe RNA Polymerase II. MIYAO, T., YASUI, K., ISHIGURO, A., IWATA, A., KIMURA, M., MITSUZAWA, H., VEDA, S.
and ISHIHAMA, A. .
.

15

16

ii

Transcription and Replication of Influenza Virus RNA: Evidence
for de novo Initiation of Replication. HONDA, A., MIZUMOTO, K. and ISHIHAMA, A.
The Molecular Anatomy of Influenza Virus RNA Polymerase.
ASANo, Y., HONDA, A., TOYODA, T. and ISHIHAMA, A.

17

18

A-b. Division of Mutagenesis
A Ubiquitin Pathway Essential for Onset of Anaphase in Mitosis.
SEINO, H., OSAKA, F. and YAMAO, F. .
.
CDC34 Dependent-Ubiquitin Pathway: Role of GRRI in the Ubiquitination of G 1 Cyclin Cln2. KISHI, T. and YAMAO, F.

21

22
A-c. Division of Nucleic Acid Chemistry
Host Proteins Involved in Transcription of The Sendai Virus.
Genome. MIZUMOTO, K. .
Crystal Structure of the Escherichia coli Replication TerminatorProtein Complexed with Ter DNA. KAMADA, K. and MORIKAWA, K
.
B.

23

24

Department of Cell Genetics
B-a. Division of Cytogenetics
Localization of Mouse Rad5l and Lim 15 on Meiotic Chromosomes
at Late Stages of Prophase 1. IKEYA, T. and OGAWA, T.

26
Characterization of DNA Ligase from the Fungus Coprinus Cinereus. MATSUDA, S. .
.
Genetic Characteristics of New recA Mutants of Escherichia coli
K-12. ALEXSEYEV, A.
Chromatin Structure of the Yeast URA3 Gene at High Resolution
Provides Insight into Structure and Positioning of Nucleosomes in the Chromosomal Context. TANAKA, S. .
.
B-b. Division of Microbial Genetics
Minus-Strand Origin of Filamentous Phage versus Transcriptional
Promoters in Recognition of RNA Polymerase. HIGASHITANI, A., HIGASHITANI, N. and HORIUCHI, K. .
.
The Active Center of the Initiator Protein of Filamentous Phage fl.
ASANo, S., HIGASHITANI, A. and HORIUCHI, K
.

26
27

28

29

30

CONTENTS

iii

A New Class of Mutations of Penicillin-Binding Protein 3 ofEscherichia coli. HARA, H. and HORIUCHI, K. .
.
The First Two Genes of the Cell Envelope Biosynthetic/Cell Division Gene Cluster in the Escherichia coli Chromosome 2Minute Region. HARA, H. and HORIUCHI, K. .
.
The spr Gene of Escherichia coli. OHFUCHI, H., NAKAKOUJI, M.,
ISHIHARA, M., NAKAZAwA, Y., NISHIMURA, Y. and HARA,
H.

Peptidoglycan Polymerase with no Crosslinking Activity in Escherichia coli. HARA, H.
Functional Dissection of Cell-Division Inhibitor SulA of Escherichia coli and Its Negative Regulation by Lon. HIGASHITANI,A., ISHII, Y., KATO, Y. and HORIUCHI, K. .
.
Search for Homologs of Human ATM Gene in C. elegans. HIGASHITANI, A., SATO, S. and ISHIHARA, T. .
.
B-c. Division of Cytoplasmic Genetics
Cell-cycle-dependent Expression of the STK-l Gene Encoding a
Novel Murine Putative Protein Kinase. NIWA, H., ABE, K.,
KUNISADA, T. and YAMAMURA, K. .
.
Endoderm-Specific Gene Expression in Embryonic Stem Cells
Differentiated to Embryoid Bodies. ABE, K., NIWA, H.,
IWASE, K., TAKIGUCHI, M., MORI, M., ABE, S., ABE, K. andYAMAMURA, K.
Nuclear Organization in Fission Yeast Meiosis. HIRAOKA, Y.

31

32

33
34

35
36

37

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38

Fluorescence Imaging of Living Mammalian Cells.

HIRAOKA, Y.

39

C.

Department of Ontogenetics
C-a. Division of Developmental Genetics
Control of Neuronal Identities by the Drosophila Nuclear Receptor,
SEVEN-UP. HIROMI, Y. and WEST, S. R.
. 41
biparous, a Novel bHLH Ggene Expressed in Neuronal and Glial
Precursors in Drosophila. BUSH, A., HIROMI, Y. and COLE,
M. 42
KLINGON, a New Member of the Drosophila Immunoglobulin
Superfamily, Participates in the Neuronal Differentiation of

iv

the R7 Photoreceptor Neuron. BUTLER, S. J., RAY, S. and
HIROMI, Y. .
Cell Lineage Analysis of the Mushroom Body. ITo, K., AWANO,
W., SUZUKI, K., HIROMI, Y. and YAMAMOTO, D.
o.
Hydra Peptide Project I. LWamides as Induces of Metamorphosisof Planula Larvae ofCnidarians. FUJISAWA, T., HATTA, M.,
SHIMIZU, H., IWAO, K., KOIZUMI, 0., KOBAYAKAWA, Y,
TAKAHASHI, T., MUNEOKA, Y. and SUGIYAMA, T
Hydra Peptide Project II. Two Peptides Involved in Regulation ofFoot Formation in Hydra. SHIMIZU, H., HATTA, M., TAKAHASHI, T., MUNEOKA, Y., SUGIYAMA, T. and FUJISAWA,
T. . o'o' o' o' o' o' o' o' o'C-b. Division of Gene Expression
Regulation of the EDG84A Gene by FTZ-Fl during Metamorphosis in Drosophila melanogaster. MURATA, T., KAGEYAMA,
Yo, HIROSE, S. and VEDA, H.
Transcriptional Activation through Interaction of MBF2 withTFIIA. LI, F.-Q., TAKEMARU, K., GOTO, M., VEDA, H.,
HANDA, H. and HIROSE, S.
Determination of Wing Cell Fate by the escargot and snail Genes in
Drosophila. FUSE, N., HIROSE, S. and HAYASHI, S.
On a Critically Sensitive Period for the Methoprene-induced Supernumeral Larval Ecdyses in the Mulberry Silkworm, Bombyx
mori (L.). MURAKAMI, A. and SHIMADA, J. .
o' o'Effects of Methoprene, a Juvenile Hormone Analogue, on the
Induction of Supernumeral Molting under the Influence ofBrain in the Bombyx Silkworm. SHIMADA, J. and MURAKAMI, A. .
On a Biological Clock in the Mulberry Silkworm, Bombyx mori(L.). MURAKAMI, A.
o'
Biological Meanings of Silk Production in the Mulberry Silkworm,
Bombyx Mori (L.). MURAKAMI, A. o'o'
Are Cultured Cells Situated between Vni- and Multicellular Organo'isms? IWABUCHI, K. and MURAKAMI, A. .
Origin of Holometabolous Metamorphosis in Insects. MINATO,
K.
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CONTENTS
C--c. Division of Physiological Genetics
A New Class of Transcription Factor DSIF, Which Regulates RNA
Polymerase II Processivity before Transcription Initiation.
WADA, T., TAKAGI, T., FERDOUS, A., YAMAGUCHI, Y. and
HANDA, H.
Analysis of the Mammalian Genome by Fluorescence in situ
Hybridization. OKUMURA, K. .
.
D.

Department of Population Genetics
D-a. Division of Population Genetics
Variation in Synonymous Substitution Rates Among Mammalian
Genes and the Correlation Between Synonymous and Nonsy.
nonymous Divergences. OHTA, T. and INA, Y. .
Population Biology of Antigen Presentation by MHC Class I Molecules. PARHAM, P. and OHTA, T. .
.
The Current Significance and Standing of Neutral and Nearly.
Neutral Theories. OHTA, T. .
Study of Gene Evolution as Observed as Interspecific Hybrid Anomaly in Drosophila. TAKANO, T. S. .
.
Lineage-dependent Protein-sequence Evolution of Drosophila
Genes. TAKANO, T. S.
Variance and Covariance of the Number of Amino Acid Substitutions Estimated by Kimura's Method. INA, Y. .
.
Correlation between Synonymous and Nonsynonymous Substitutions and Variation in Synonymous Substitution Numbers.
INA, Y.
Pattern of Synonymous and Nonsynonymous Substitutions: An
Indicator of Mechanisms of Molecular Evolution. INA, Y.

v

58
59

63
63
64
64
65

66

66

67

D-b. Division of Evolutionary Genetics
Replication Timing for the GC Content Transition Area between
Classes II and III in the Human MHC. TENZEN, T., WATANABE, Y., FUIIYAMA, A., OKUMURA, K., ANDO, A., INOKO,
H. and IKEMURA, T.
Human Pseudoautosomal Boundary-Like Sequences: Their Expression and Involvement in Evolutionary Formation of the Present-Day Pseudoautosomal Boundary of Human Sex Chromo-

68

vi

somes. FUKAGAWA, T., NAKAMURA, Y., OKUMURA, K.,
NOGAMI, M., ANDO, A., INOKO, H., SAITOU, N. and IKEMURA, T.
Chromosomal Localization of the Proteasome Z Subunit Gene
Reveals an Ancient Chromosomal Duplication Involving the
Major Histocompatibility Complex. KASAHARA, M., HAYASHI, M., TANAKA, K., INoKo, H., SUGAYA, K., IKEMURA, T.
and ISHIBASHI, T. .
.
Multivariate Diversity in Codon Usage in Escherichia coli Sequences Determined by Genome Projects. KANAYA, S.,
KUDO, Y., NAKAMURA, Y. and IKEMURA, T. .
.
cDNA Cloning of the Human Homologues of the Mouse Ke4 and
Ke6 Genes at the Centromeric End of the Human MHC
Region. ANDO, A., KIKUTI, Y. Y., SHIGENARI, A., KAWATA, H., OKAMOTO, N., SHIINA, T., CHEN, L., IKEMURA, T.,
ABE, K., KIMURA, M. and INOKO, H. .
.
Tenascin-X Is Essential for the Development of Epicardium andCoronary Vessels. MATSUMOTO, K., YAMAMOTO, H., TAKEDA, N., YOSHIDA, T., IMANAKA-YOSHIDA, K., SHIRAYOSHI,
Y., SAKAKURA, T., AIZAWA, S., IKEMURA, T. and NAKATSUJI, N.
Gene Organization of Human NOTCH4 and (CTG)n Polymorphism in This Human Counterpart Gene of Mouse Protooncogene Int3. SUGAYA, K., SASANUMA, S., NOHATA, J.,
KIMURA, T., FUKAGAWA, T., NAKAMURA, Y., ANDO, A.,
INOKO, H., IKEMURA, T. and MITA, K.
Reconstruction of Gene Trees from Sequence Data. SAITOU, N.

69

70

70

71

72

73
73

The Presence/Absence Polymorphism and Evolution of the p53
Pseudogene in the Genus Mus. OHTSUKA, H., OYANAGI, M.,
MAFUNE, Y., MIYASHITA, N., SHIROISHI, T., MORIWAKI, K.,
KOMINAMI, R. and SAITOU, N. .
.
Contrasting Gene Trees and Population Trees on the Evolution ofModern Humans. SAITOU, N. .
.
Extensive Polymorphism of ABO Blood Group Gene: Three Major
Lineages of the Alleles for the Common ABO Phenotypes.
OGASAWARA, K., SAITOU, N. and TOKUNAGA, K
.

74
75

75

CONTENTS
Molecular Genetic Analysis of Variant Phenotypes of the ABO
Blood Group System. OGASAWARA, K., SAITOU, N. and
TOKUNAGA, K. .
.
Evolution of Class III POU Genes: Amplification of Characteristic
Amino Acid Residues by G+C Pressure in Mammals. SUMIYAMA, K., SAITOU, N. and UEDA, S.
D-c. Division of Theoretical Genetics
Current Topics on Molecular Evolution. NEI, M. and T AKAHATA, N.
Neutral Theory of Molecular Evolution. TAKAHATA, N.

vii

76

77

80
80

Multiregional or Uniregional Origin of Modern Humans: What Do
Genetic Data Indicate? TAKAHATA, N. and KLEIN, J.
81
Evolution of the Primate Lineage Leading to Modern Humans:
Phylogenetic and Demographic Inferences from DNA Se.
quences. TAKAHATA, N. and SATTA, Y
Studies on the Structure of Visual Pigments Responsible for the
Function of Photoreceptor Cells. KOJIMA, D., OURA, T.,
SHICHIDA, Y., FUKADA, Y., HISATOMI, 0., TOKUNAGA, F.
and YOSHIZAWA, T.
Functional Analysis of Lipid Modifications on Photoreceptor
G-Protein. FUKADA, Y, MATsuDA, T., SHOUNO, 0., ARAKI, M., TAKAO, T., SHIMONISHI, Y., TANAKA, H. and MIKI,
N.
Functional Studies on the Ca2+ -Binding Protein in Photoreceptor
Cells. FUKADA, Y., SANADA, K., SHIMIZU, F., KAMEYAMA,
K., HAGA, K. and HAGA, T. .
.
Studies on Photoreception in the Brain. FUKADA, Y, OKANO, T.,
SANADA, K., YOSHIKAWA, T. and TAKANAKA, Y
.

E.

81

82

83

84
85

Department of Integrated Genetics
E-a. Division of Human Genetics
Isolation of Transcribed Sequences in Cloned Genomic DNA with
Known Location on the Human Chromosome 18. IMAMURA, T., SAKAI, M., INABA, R. and NAKASHIMA, H.
87

viii

Molecular Definition of Maternally Imprinted Genes on Chromosome 15 and the Development of Prader-Willi Syndrome.
IMAMURA, T., SAKAI, M., INABA, R. and HASEGAWA, T.
88Interleukin-4 Induces Association of the c-fes Proto-oncogene Product with Phosphatidylinositol-3-kinase and Activates Two Dis. 89
tinct Signal Transduction Pathways. IZUHARA, K
Mitochondrial DNA Polymorphism in East Asian Populations,with Special Reference to the Peopling of Japan. HORAI, S.,
FUCHAROEN, G., HARIHARA, S., PARK, K. S., OMOTO, K.
and PAN, I-H. 90
The Origin and Dispersal of Modern Humans as Viewed from
Mitochondrial DNA. HORAI, S., ODANI, S., NAKAZAwA,
M. and OHTSUKA, R. 91
Molecular Phylogeny of Macaques: Implications of Nucleotide
Sequences from an 896 Base Pair Region of MitochondrialDNA. HAYASAKA, K., FUJII, K. and HORAI, S. .
. 92
Detection of DNA Fragments Encompassing the Deletion Junctionof Mitochondrial Genome. GOTO, Y., NIsHINo, I., HORAI,
S. and NONAKA, I.
. 93
Thr
A Novel Mutation in the Mitochondrial tRNA Gene Associatedwith Mitochondrial Encephalomyopathy. NISHINO, I., TAKESHITA, K., HORAI, S., NONAKA, I. and GOTO, Y. . ..... 94
The 3260 Mutation in Mitochondrial DNA Can Cause Mitochondrial Myopathy Encephalopathy Lactic Acidosis and Stroke-like
Episodes (MELAS). NISHINO, I., HORAI, S., NONAKA, I.
and GOTO, Y. 95
E-b. Division of Agricultural Genetics
Inbreeding Depression, Outbreeding Depression and Hybrid Vigor
with Special Reference to the Mating System in Rice Species.
MORISHIMA, H. .
. 97
Analysis of Quantitative Trait Loci Underlying Domestication in. 98
Rice. CAl, H.-W. and MORISHIMA, H
Geographical Differentiation Found in Annual Types of Oryza
rufipogon. CAl, H.-W. and MORISHIMA, H. .
. 99
Basic Study of Genetic Erosion of Wild Rice (Oryza rufipogon) in
Thailand. AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA,CONTENTS

ix

H
. 100
Inter- and Intra-Specific Differentiation of Oryza glumaepatula
Found in Central and South America. AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA, H. .
. 101
E--c. Division of Applied Genetics
Mitochondrial Genome Polymorphisms in Lolium perenne. SHIMAMOTO, Y. 102
The Extent of Natural Cross Pollination in Wild Soybean (Glycinesoja). SHIMAMOTO, Y. 103
Molecular Genetic Studies of Genomic Imprinting. SASAKI, H.

103
F.

Genetic Stock Research Center
F-a. Mammalian Genetics Laboratory
Geological Distribution of a Y chromosome Specific Sequence
DYH1 in Wild Mice. UEDA, Y., SAGAI, T., BALL, S. T.,
MORIWAKI, K., SHIROISHI, T. 106
Molecular Characterization of Ph Hotspot in the ClassII of Mouse
MHC. ISOBE, T., YOSHINO, M., FISCHER LINDAHL, K.,
SAGAI, T., MIZUNO, K., KOIDE, T., SHIROISHI, T. .
. 107
Genetic Analysis of Coat-Color Mutation, rim2. III. SAGAI, T.,
KOIDE, T., TANOUE, K., ISHIGURO, S., YONEKAWA, H., MORIWAKI, K., WAKANA, S. and SHIROISHI, T. 108
Mapping and Histological Analysis of the Mouse Mutant (Rim3)
Affecting Skin and Eye. SATO, H., KOIDE, T., MASUYA, H.,
WAKANA, S., SAGAI, T., MORIWAKI, K. and SHIROISHI, T.

109
Organization of the Typel Keratin Genes (Krtl) in the Mouse
Genome. SATO, H., KOIDE, T. and SHIROISHI, T. 110
Gene Mapping of Rim4 Modifier. MASUYA, H., SAGAI, T.,
WAKANA, S. and SHIROISHI, T. .
. 111
Genetic Analysis of Hemimelic extra-toe (Hx) Mutant. SAGAI,
T., MASUYA, H. and SHIROISHI, T. 112
Positional Cloning of the Mouse Skeletal Mutant, Tail short (Ts).
SHIMIZU, K., KOIDE, T., MITA, A., UCHIDA, K., WAKANA,
S., KIKKAWA, Y., YONEKAWA, H. and SHIROISHI, T. 113
Morphological Characterization of the Mouse Mutant Tail-short:

xSkeletal Patterning Along the Anteroposterior Axis. ISHIJIMA, J., UCHIDA, K., MITA, A. and SHIROISHI, T. 114
Searching for Genes Regulating the Phenotype of Tail-short. ISHIJIMA, J., UCHIDA, K., MITA, A. and SHIROISHI, T. 114
A New Inbred Strain JFl from Japanese Fancy Mouse Carrying theClassic Piebald Allele for Use in Biomedical Research.
KOIDE, T., UCHIDA, K., MITA, A., SAGAI, T., SHIROISH.I, T.
and MORIWAKI, K. .
. 116
Establishment of Intersubspecific Consomic Mouse Strains II.
SHIROISHI, T., MITA, A., TAKADA, K., WAKANA, S., YONEKAWA, H. and MORIWAKI, K. 118
Genetic Analysis of Non-MHC Susceptibility Genes to IDDM in
Mice. WAKANA, S., SHIROISHI, T., ITo, M., SATO, T.,
HABU, S., MORIWAKI, K. and NOMURA, T. 119
F-b. Invertebrate Genetics Laboratory
Specification of Embryonic Limb Primordium by Graded Activityof Decapentaplegic. GOTO, S. and HAYASHI, S. .
. 120
Cadherin-mediated Cell Adhesion and Cell Motility in Drosophila
Trachea Regulated by Transcription Factor Escargot. TANAKA-MATAKATSU, M., UEMURA, T., ODA, H., TAKEICHI,
M., SHIGA, Y. and HAYASHI, S. 121
Role of the Drosophila EGF Receptor in Determination of Dorsoventral Domains of escargot Expression during Primary Neuro. 122
genesis. YAGI, Y. and HAYASHI, S. .
plexus, a Gene Required for Establishment of Adult Wing Vein
Pattern. MATAKATSU, H., GAMOU, S. and HAYASHI, S.

123
F-c. Plant Genetics Laboratory
Structural and Functional Analysis of the Genes Expressed in Early
Embryogenesis in Rice (Oryza sativa). ITO, Y., EIGUCHI, M.
and KURATA, N. .
. 124
Positional Cloning of a Segregation Distortion Gene Detected in
the Progenies of Crosses with Japonica and Indica Rices.
HARUSHIMA, Y. and KURATA, N. 124
Molecular Cytogenetic Analysis of Nuclear Organization of Rice
Chromosomes in Meiosis and Mitosis. NONOMURA, K. and
KURATA, N. 125

CONTENTS

xi

Generation of Enhancer and Gene Trap Lines in Rice. ITo, Y,
EIGUCHI, M., NONOMURA, K. and KURATA, N. 125
F-d. Microbial Genetics Laboratory
Molecular Mechanism that Determines the Timing of Cell Divisionin the Cell Cycle of Escherichia coli. NISHIMURA, A.
127
F-e. Genetic Resources Laboratory
Construction of Genetic Resources Database. YAMAZAKI, Y.
128
F-f. Mammalian Development Laboratory
Analysis of Cellular and Molecular Mechanisms in Development of
Mouse Fetal Germ Cells. NAKATSUJI, N. and SHIRAYOSHI,Y
129
Establishment of Neuronal Stem Cell Lines for Analysis of Cell
Lineages and Differentiation in Mouse Central Nervous
System Development. NAKATSUJI, N. and SHIRAYOSHI, Y.
131
Molecular Analysis of Cell Differentiation and Morphogenesis in
Postimplantation Mouse Embryos. SHIRAYOSHI, Y. and NAKATSUJI, N
. 132

G.

Structural Biology Center
G-a. Molecular Biomechanism Laboratory
Mechanism of Abortive Synthesis by E. coli RNA Polymerase.
KUBORI, T., NAGAI, H., SEN, R. and SHIMAMOTO, N.
133
Single-Molecule Dynamics of Transcription: Sliding of Proteins
along DNA. KABATA, H., TAKEUCHI, M., KINEBUCHI, T.,
MIYASHlTA, H. and SHIMAMOTO, N
134
Crystallographic Analysis of E. coli Replication Termination
Protein. KAMADA, K., MORIKAWA, K. and SHIMAMOTO,
N
135
G-b. Multicellular Organization Laboratory
Fluoride-resistant Mutants of the Nematode Caenorhabditis elegans. TAKE-UCHI, M., ISHIHARA, T. and KATSURA, L
136
A Gene That Controls Both Hatching and Cell Migration in C.

xiielegans.

HISHIDA, R., ISHIHARA, T. and KATSURA, I.

137
Analysis of the Head Neural Circuit of C. elegans as Studied byFormation of Dauer Larvae. SUZUKI, N., ISHIHARA, T. and
KATSURA, I.
. 139
Reverse Genetics of Neural Genes of C. elegans and the Analysis of
Their Expression Using Jellyfish Green Fluorescent Protein.
ISHIHARA, T., FUJIWARA, M. and KATSURA, I.
. 140
G-c. Biomolecular Structure Laboratory
Crystallographic Study of a3f33 Sub-complex of Fl-ATPase: Structural Analysis of Supramolecule. SHIRAKIHARA, Y. . ..... 141
Crystallographic Study of the Transcription Activator, PhoB.
AKIBA, T. and SHIRAKIHARA, Y. .
. 142
Crystallographic Study of the Transcription Repressor, CamR.
SHIRAKIHARA, Y. .
. 143
G-d. Gene Network Laboratory
Expression Pattern Map of the C. elegans Genome. KOHARA, Y.,
MOTOHASHI, T., TABARA, H., WATANABE, H., SANO, M.,
MIYATA, A., OHBA, T., MITANI, Y, IIDA, K. and UESUGI, H.

143
Pos-l, a Gene Which Shows Localization of Its mRNA to the P
Lineage during Early Cleavage of C. elegans Embryos.
TABARA, H., MELLO, C., HILL, R., PRIESS, J. and KOHARA,
Y

. 144

H. Center for Information Biology
H-a. Laboratory for DNA Data Analysis
Large-Scale Search for Genes on Which Positive Selection May
Operate and Analysis of Their Biological Meanings. ENDo,. 146
T. and GOJOBORI, T
Phylogenetic Analysis of Duplicated Regions within Human Chromosomes. ENDo, T., IMANISHI, T., GOJOBORI, T. and INOKO, H. 147
The Origin of Domestic Fowls. FUMIHITO, A., MIYAKE, T.,
TAKADA, M., SHINGU, R., ENDo, T., GOJOBORI, T., KONDO,
N. and OHNO, S. 148
The Molecular Mechanisms of Choosing Infected Clones in Mothe-

CONTENTS

xiii

r-To-Child Transmission of HIV-l. TAKAHASHI, K. and
GOJOBORI, T. 148
HLA Class II Alleles in Ainu Living in Hidaka District, Hokkaido,
Northern Japan. BANNAI, M., TOKUNAGA, K., IMANISHI,
T., HARIHARA, S., FunSAWA, K., Jun, T. and OMOTO, K.

149
Patterns of Amino Acid Substitutions in the V3 Region of HIV
Within Single Hosts. YAMAGUCHI, Y. and GOJOBORI, T.

149
An Analysis of Amino Acid Diversity at One Site in the Envelope
Glycoprotein of HIV-l. YAMAGUCHI, Y. and GOJOBORI, T.

150
Exceptionally Conserved Regions Found in Extensively Rearranged
Microbe Genomes. WATANABE, H., ITOH, T., MORI, H. and
GOJOBORI, T. 151
Characterization of the Methanococcusjannaschii Genome in Termsof Its ORF Composition and the Structures within and around
ORFs. WATANABE, H., GOJOBORI, T. and MIURA, K.

152
Implication about the Evolutionary Process of the Generation ofRegulator-Regulatee Relationships after Speciation between
Escherichia coli and Haemophilus injiuenzae. WATANABE,
H., GOTO, K. and GOJOBORI, T. 153
H-b. Laboratory for Gene-Product Informatics
Improvement of Protein Sequence-Structure Compatibility Methodby the Best-Five Test. OTA, M. and NISHIKAWA, K. 155
A Distant Evolutionary Relationship between Bacterial Sphingomyelinase and Mammalian DNase I. MATSUO, Y. and NISHIKAWA, K. 156
Comparison of Protein Structures Using 3D Profile Alignment.
SUYAMA, M. and NISHIKAWA, K. 156
H-c. Laboratory of Gene Function Research
DNA Database and Its Applications to the Study of Molecular. 157
Evolution. GOJOBORI, T. and TATENO, Y.
A Biological Taxonomy Database System on World-wide Web.
KITAKAMI, H., MORI, Y., TATENO, Y. and GOJOBORI, T.

158

xiv

Development of New DDBJ DNA Sequence Database with Data
Annotation Tool Yamato II. KOIKE, T., OKAYAMA, T.,
ISHII, J., MIZUNUMA, T., TAMURA, T., TATENO, Y., SUGAWARA, H., NISHIKAWA, K., IMANISHI, T., FUKAMI-KoBAYASHI, K., IKEo, K. and GOJOBORI, T
159
H-d. Molecular Classification Laboratory
Improvement of the Data Processing System for DDBJ. SUGAWARA, H., TATENO, Y., IKEO, K., SAlTOU, N., IMANISHI, T.,
FUKAMI-KoBAYASHI, K., MIYAZAKI, S., OTA, M., GOJOBORI, T. and IsoNo, K
160
Databases and Network Agents on Biological Data
160
Research and Development of Classification and Identification of
Biological Objects
162

I.

Radioisotope CenterTranscriptional Analysis of the Bacillus subtilis Sporulation Gene:
Promoter Selectivity of RNA Polymerase. FUJITA, M. andSADAIE, Y.
165
Nucleotide Sequence of the phoB-rrnE-groESL Region of the Bacillus subtilis Chromosome. SADAIE, Y., YATA, K., FUJITA,
M., SAGAI, H., ITAYA, M., KASAHARA, Y. and OGASAWARA,
N
166

J.

Experimental FarmDevelopment and Reevaluation of the Genetic Stocks of Rice.
NONoMuRA, K., EIGUCHI, M., MIYABAYASHI, T. and KURATA, N
168

Abstracts of diary for 1996
Foreign visitors in 1996 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Association for propagation of the knowledge of genetics
Author index

169
172
173
175

GENERAL STATEMENT
Our institute was established 47 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofresearch in genetics, in particular population genetics, plant genetics andmolecular genetics, by members of our institute have made it the premiercenter for genetic study in our country and a major institution with worldwide recognition. In 1984 the institute was reorganized into a National
Inter-university Research Institute to promote research activities throughcooperation with other institutions. Together with seven inter-university
research institutes, we formed the Graduate University for Advanced Studies, in 1988. Our institute admits graduate students to the Department of
Genetics of the Graduate School of Life Science. We have 27 such students
at present and in addition, about ten special research students from otheruniversities, including those from foreign countries. In addition, we havefour post-doctoral fellows of the center of excellence program. I consider itvital for our research activities to have a steady flow of young scientists.
Recent rapid progress in the field of genetics using newly developed
approaches has greatly shifted the course of study in biology. I consider itnatural that research in our institute has recently focused more on molecularand cellular studies of various aspects of genetics, without ignoring theimportance of more traditional approaches to genetics. Our institute is
uniquely suited for pursuing cooperative work with scientists of various
disciplines. Through interactions among various research disciplines, ourinstitute will flourish.
We have been carrying out several research related services. The DNA
Data Bank of Japan (DDBJ) is one of the three central banks in the worldthat gather, annotate, store and distribute information on DNA sequences.
Our institute also stores and distributes various organisms with geneticallycharacterized traits. Among them, services with mice, rice and Escherichia
coli are particularly significant. These service activities will continue todevelop in the coming years.
I hope that with guidance from people in and outside this institute andfurther support from governmental and private sources, I will be able to leadthe institute into a more successful future.
The Center for Information Biology was established by members from the2

DNA Research Center in 1995. This year the DNA Research Center was
reorganized to form the Structural Biology Center with four laboratories.
Nobuo Shimamoto, Molecular Biomechanism Laboratory, and Yuji Kohara,
Gene Network Laboratory, were promoted to professor.
Dr. Takeshi Seno, the Vice-Director and the head of the Division of
Mutagenesis, and Dr. Tsutomu Sugiyama, the Head of the Department of
Ontogenetics, retired at the end of March. The past year also saw a numberof changes in the staff of the institute. Promoted as associate professor,
Hiroyuki Hirano was transferred to the University of Tokyo. The following
twelve people joined us: as professor, Yasushi Hiromi in the Division of
Developmental Genetics, Hideaki Sugawara in the newly established Laboratory of Molecular Classification in the Center for Information Biology; asassociate professor, Nori Kurata in the Plant Genetics Laboratory. Hiroshi
Mitsuzawa and Makoto Kimura joined the Division of Molecular Genetics,
Tsutomu Ohta the Division of Cytogenetics, Hong-Wei Cai the Division ofAgricultural Genetics, Toshihiko Akiba the Biomolecular Structure Laboratory, Motonori Ota the Laboratory for Gene-Product Informatics, Kaoru
Fukami-Kobayashi the Gene Function Laboratory, and Satoru Miyazaki the
Molecular Classification Laboratory, and Ken-ichi Nonomura the Experimental Farm.

3

STAFF

(as of December 31, 1996)
Director-General

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

OKINO-MoRISHIMA, Hiroko, D. Ag.
Members

1. 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.
MITSUZAWA, Hiroshi, D. Sc.
KIMURA, Makoto, D. Sc.
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Associate professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry (Guest member)
MIZUMOTO, Kiyohisa, D. Sc., Professor
MORIKAWA, Kosuke, D. Pharm., Professor

2. Department of Cell Genetics
HORIUCHI, Kensuke, D. Sc., Head of the Department
Division of Cytogenetics
OGAWA, Tomoko, Ph. D., Professor
IMAI, Hirotami, T., D. Sc., Associate professor
TANAKA, Shigeo, D. Med.
OHTA, Tsutomu, D. Med.
GOTOH, 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)
YAMAMURA, Ken-ichi, D. Med., Professor
HIRAOKA, Yasushi, D. Sc., Associate professor
3. Department of Ontogenetics
HIROSE, Susumu, Ph. D., Head of the Department
Division of Developmental Genetics
HIROMI, Yasushi, D. Sc., 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., Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-aki, D. Sc.
VEDA, Hitoshi, D. Ag.
Division of Physiological Genetics (Guest member)
HANDA, HIROSHI, D. Med., Professor
OKUMURA, Katsuzumi, D. Ag., Associate professor
4. Department of Population Genetics
IKEMURA, Toshimichi, D. Sc., Head of the Department
Division of Population Genetics
HARADA-OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKANO, Toshiyuki, D. Sc.
INA, Yasuo, D. Sc.
Division of Evolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., D. Sc. Associate professor
MATSUMOTO, Ken-ichi, D. Ag.
TENZEN, Toyoaki, D. Ag.

STAFF

Division of Theoretical Genetics (Guest member)
TAKAHATA, Naoyuki, D. Sc., Professor
FUKADA, Yoshitaka, D. Sc., Professor
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
IZUHARA, Kenji, D. Med.
Division of Agricultural Genetics
OKINO-MoRISHIMA, Hiroko, D. Ag., Professor
CAl, Hong-Wei, D. Ag.
Division of Applied Genetics (Guest member)
SHIMAMOTO, Yoshiya, D. Ag., Professor
SASAKI, Hiroyuki, D. Med., Associate Professor
6. Genetic Stock Research Center
NAKATSUJI, Norio, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Associate professor
KOIDE, Tsuyoshi, D. Med.
Invertebrate Genetics Laboratory
HAYASHI, Shigeo, D. Sc., Associate professor
GOTO, Satoshi, D. Sc.
Plant Genetics Laboratory
KURATA, Nori, D. Ag., Associate professor
ITO, Yukihiro, D. Ag.
Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Associate professor
KANAMARU, Kengo, D. Ag.
Genetic Resources Laboratory
YAMAZAKI, Yukiko, D. Sc., Associate professor
FUJITA, Masaya, D. Eng.
Mammalian Development Laboratory
NAKATSUJI, Norio, D. Sc., Professor

5

6

SHIRAYOSHI, Yasuaki, D. SC.

7. Structural Biology Center
KATSURA, Isao, D. Sc., Head of the Center
Biological Macromolecules Laboratory
KATSURA, Isao, D. Sc., Head
Molecular Biomechanism Laboratory
SHIMAMOTO, Nobuo, D. Sc., Professor
NAGAI, Hiroki, D. Sc.
Multicellular Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate professor
Gene Network Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
8. Center for Information Biology
GOJOBORI, Takashi, D. Sc., Head of the Center
Laboratory for DNA Data Analysis
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
IMANISHI, Tadashi, D. Sc.
Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
OHTA, Motonori, M. Sc.
Gene Function Laboratory
TATENO, Yoshio, Ph. D., D. Sc., Professor
KOBAYASHI-FuKAMI, Kaoru, Ph. D.
Molecular Classification Laboratory
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, M. Sc.
9. Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate professor, Head of the Center

STAFF

10. Experimental Farm
OKINO-MoRISHIMA, Hiroko, D. Ag., Head of the FarmNONOMURA, Ken-Ichi, D. Ag.
11. Technical Section
MITA, Akihiko, Chief of the Section
12. Department of Administration
KURODA, Hideo, Head of the Department
TAlMA, Hitoshi, Chief of the General Affairs Section
TAMURA, Mituo, Chief of the Finance Section

7

8

COUNCIL(as of December 31, 1996)

Chairman
SUGIMURA, Takashi; President, Toho University
Vice-chairman
OSA W A, Shozo; Adviser, Biohistory Research Hall
Members (Alphabetical order)
HARA, Hiroko; Professor, Institute for Gender Studies, Ochanomizu
University
HI RASA, Takao; Presidenet, Osaka Prefectural University
HIROTA, Eiji; President, The Graduate University for Advanced Studies
IwATUSKI, Kunio; Professor, Rikkyo University
KATOH, Nobuo; President, Nagoya University
KYOGOKU, Yoshimasa; Professor, Institute for Protein Research, Osaka
University
MATSUBARA, Ken-ichi; Professor, Institute of Molecular and Cellular
Biology, Osaka University
MIURA, Kin-ichiro; Director, Institute for Biomolecular Science, Gakushuin University
MIYAMOTO, Misako; President, Nihon Women's University
MOHRI, Hideo; Director-General. National Institute for Basic Biology
NOJIMA, Shoshichi; Professor Emeritus, Tokyo University
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
SUGANO, Haruo; Director Emeritus, Cancer Institute
SUGIMURA, Takashi; President, Toho University
TAKANAMI, Mitsuru; Director, Kazusa DNA Research Institute
TAKEUCHI, Ikuo; President, Okazaki National Research Institutes
TANAKA, Ryuso; President, Hiroshima City University
TOYOSHIMA, Kumao; President, The Center for Adult Diseases, Osaka
YAMANOUTI, Kazuya; Senior Scientific Staff. Nippon Institute for Biological Sciense

9

ADVISORY COMMITTEE(as of December 31, 1996)

Chairman
MORISHIMA, Hiroko; Professor, Division of Agricultural Genetics
Vice-chairman
OKADA, Masukichi; Professor Emeritus, University of Tsukuba
Outside Members (Alphabetical order)
Go, Mitiko; Professor, Graduate School of Science, Nagoya University
HANAOKA, Fumio; Professor, Institute of Molecular and Cellular Biology, Osaka University
HINATA, Kokichi; Professor, Faculty of Agriculture, Tohoku University
HOTTA, Yoshiki; Professor, Graduate School of Sciences, The University of TokyoISONO, Katsumi; Professor, Faculty of Science, Kobe University
ITo, Koreaki; Professor, Institute for Virus Reserch, Kyoto University
KATSUKI, Motoya; Professor, The Insutitute of Medical Science, The
Univetsity of Tokyo
SEKIGUTI, Mutsuo; Professor, Fukuoka Dental College
T AJIMA, Fumio; Professor, Graduate School of science, The Univetsityof TokyoInside Members (Alphabetical order)
GOJOBORI, Takashi; Professor, Center for Information Biology
HIROSE, Susumu; Professor, Division of Gene Expression
HORIUCHI, Kensuke; Professor, Division of Microbial Genetics
IKEMURA, Toshimichi; Professor, Division of Evolutionary Genetics
IMAMURA, Takashi; Professor, Division of Human Genetics
ISHIHAMA, Akira; Professor, Division of Molecular Genetics
KATSURA, Isao; Professor, Structural Biology Center
NAKATSUJI, Norio; Professor, Genetic Stock Research Center
OGAWA, Tomoko; Professor, Division of Cytogenetics
OHTA, Tomoko; Professor, Division of Population Genetics

10

RESEARCH ACTIVITIES IN 1996

A.

DEPARTMENT OF MOLECULAR GENETICS
A-a.

(1)

Division of Molecular Genetics

Molecular Anatomy of the Alpha Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Fine Mapping of
the Contact Surface with DNA UP Elements
and Class-I Transcription Factors

Katsuhiko MURAKAMI, Irina ARTSIMOVITCH 1, Sudha A. CHUGANI 2, Philip
E. BoucHER3, Yang TANG4, Hilla GILADI 5, Martha M. HowE!, A. N.
CHAKRABARTy2, Scott STIBITZ3 , Pieter L. DEHASETH 4 , Amos B. OPPENHEIM 5, and Akira ISHIHAMA CUniv. Tennessee, Memphis, 2Univ. Illinois,
Chicago, 3 Center for Food and Drug Administration, Bethesda, 4 Case
Western Reserve University, Cleveland, 5 Hebrew University-Hadassha Medical School, Jerusalem)
The carboxy-terminal one-third of Escherichia coli RNA polymerase a
subunit plays a key role in transcription regulation by class-I transcription
factors and DNA enhancer (UP) elements. The roles of individual aminoacid residues within this regulatory domain of a subunit were examined after
systematic mutagenesis of the putative contact regions (residues 258 to 275,
and 297 to 298) for cAMP receptor protein (CRP). The reconstituted RNA
polymerases containing the mutant a subunits were examined for their
response to transcription activation by cAMP-CRP and the rrnBPI UP
element. Mutations affecting CRP responsiveness were located on thesurface of the putative CRP contact helix and most of these mutations also
influenced the response to the rrnB UP element. These observations raise apossibility that the CRP contact surface is also involved in contact with the
DNA UP element, although some amino acid residues within this region playdifferent roles in molecular communication with CRP and the UP element.
Among the amino acid residues constituting the contact surface, Arg265 was

A.

DEPARTMENT OF MOLECULAR GENETICS

11

found to play a major role in response to both CRP and the DNA UP
element. Judged by DNase I footprinting analysis, a mutants defective intranscription from the CRP-dependent lacPl promoter showed decreased
activity in the cooperative binding of CRP. Likewise, mutants defective in
rrnBPI transcription showed decreased binding to the UP element.
The same set of reconstituted mutant RNA polymerases containing Alasubstituted a subunit was used for mapping of the contact sites for phage
Mu-encoded Mor protein for middle transcription (ref. 1), Pseudomonas
CatR protein for transcriptin activation of the cat operon for catechol
catabolism (ref. 4) and Bordetella pertussis BvgA for transcription of thevirulence genes (ref. 3). Mor protein is a novel type of regulatory protein,which requires both a and 0 70 function (ref. 1). For contact with the asubunit, Asp258 plays a key role and residues Leu262, Arg265 and Asn268
indirectly affect Mor-RNA polymerase interaction by stabilizing the ternarycomplex via a-DNA contact. On the other hand, the possible contact site for
the CatR protein was identified to include Arg265 and Asn268 (ref. 4) andthat for the BvgA protein was also mapped at Arg265 and Asn268 (ref. 3).
An DNA UP element defines a supplementary promoter element locatedupstream of the promoter-35 signal and stimulates transcription by interacting with the C-terminal domain of RNA polymerase a subunit. Both P L andP RM promoters of phage it also carry UP elements even though the activities
are lower than the ribosomal RNA (rrnB) gene UP element (ref. 7 and 29).
Mutant a with Ala-substitution at Leu260, Leu262, Arg265 and Cys269 allgave severe effect on the P L UP element recognition (ref. 29), while theactivation ofP RM requires Leu260, Leu262, Arg265 and Asn268 (ref. 7). The
effects of Ala substitutions of aCTD on the phage it UP elements are
essentially the same as that on the rrnB promoter (ref. 24).

12

(2)

Molecular Anatomy of the Alpha Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Evidence forthe Direct Contact with DNA UP Elements
and Class-I Transcription Factors

Katsuhiko MURAKAMI, Olga OZOLINE, Tomofumi NEGISHI, Nobuyuki
FUJITA, Jeffrey T. OWENS!, Claude F. MEARES·, and Akira ISHIHAMAC Univ. California, Davis)
The reactivity of both isolated a-subunit and a-subunit assembled in the
RNA polymerase holoenzyme against a monomercury derivative of fluoresceine acetate (FMMA) was analyzed. In both cases, the largest reactivity was
identified to display Cys-269, positioned in the regulatory helix of C-terminal
domain (CTD), including the contact sites for both class-I transcription
factors and DNA UP elements. Substitution of Ala for both Cys269 and Cys
176 completely eliminates the reactivity of a-subunit against the fluorescentdye, supporting the prediction that another reactive amino acid is Cysl76
which is positioned within or near the region important for dimerization of
a-subunit and its binding of {3' subunit. Mutant a-subunits, bearing only one
cysteine residue at either 269 or 176, could be reconstituted into locallymodified and active enzymes which provide a tool suitable for studies of
intra- and intermolecular interactions of this subunit.
The modified enzyme was used to investigate RNA polymerase interaction
with different promoters, either containing or lacking an upstream enhancer
element. Using a single-round transcription assay, it was shown that thepresence of the label decreases the efficiency of RNA polymerase interaction
with promoters with UP element such as rrnBP 1 but not with many otherwithout the UP elements. Interaction with the rrnBP 1 UP element leads tosubstantial alterations in the spectral parameters of the reporter label, which
are different from those induced by interaction with promoters without UP
elements. A pronounced spectral blue shift suggests that the labeled surface
of aCTD closely approaches the charged UP DNA helix. Thus we proposethat Cys-269 is directly involved in the contact with the UP element ofpromoter DNA.
Taking advantage of the recently discovered polynucleotide and polypeptide hydrolysis reaction by iron chelete Fe-BABE [(p-bromoacetamidobenzyl)-EDTA· Fe], the chelate was tethered to the reactive Cys residue at

A. DEPARTMENT OF MOLECULAR GENETICS

13

position 269 and used to map its proximity to DNA UP elements. Furthermore, in order to identify possible differences in the functional roles of thetwo a subunits, we have developed a reconstitution method for hybrid RNA
polymerases containing two distinct a subunit derivatives in a defined orientation ("oriented a-heterodimer"). The binding sites of two a C-terminal
domains on the UP element DNA were determined by hydroxyl radical-based
DNA cleavage mediated by Fe· BABE which was bound at Cys-269 on the
UP-recognition surface of one or both a subunits (ref. 25). The results
clearly indicated that the two a subunits bind in tandem to two helix turns of
the rrnBP 1 UP element, and that the,8'-associated a subunit is bound to thepromoter-distal region.
(3)

Regulation of Intracellular Levels of Sigma
Subunits in Escherichia coli

Miki JISHAGE, Akira IWATA!, Susume UEDA l , and Akira ISHIHAMA C Nippon Institute of Biological Science, Ohme)
Using a quantitative Western immnoblot analysis, the intracellular levels of
two principal 0 subunits, 0 70 (aD, the rpoD gene product) and if8 (as, the rpoS
gene product), and of two minor 0 subunits, d 4 (aN, the rpoN gene product)and d 8 (uF, the rpoF gene product), were determined in two Escherichia coli
strains, W3110 and MC4100 (ref. 16). The results indicated that: the levelsof 0 54 and d 8 are maintained at 10 and 50% the level of 0 70 in both strains
growing at both exponential and stationary phases; but in agreement with ourprevious measurement for strain MC4100, the level of a3 8 was undetectable atthe exponential growth phase but increased to 30% the level of 0 70 at thestationary phase. Stress-coupled change in the intracellular level was observed for two 0 subunits: the increase in a3 8 level and the decrease in d 8 levelupon exposure to heat shock at the exponential phase; and the increase in if8
level under high osmolality conditions at both the exponential and thestationary phases.
In the course of these studies, we realized that heterogeneity exists among
the stock strains of Escherichia coli K 12 strain W3110 in Japan with respect
to two 0 subunits, d 8 (uF, the rpoF gene product) and a3 8 (05, the rpoS geneproduct) (ref. 17). Five different types of W3110 were identified: A-typelineages have both 0 subunits in intact forms; B-type lineages carry truncated14d 8 subunit and intact d'8 subunit; C-type lineages carry intact d'8 subunit but

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lack 8 subunit; D-type lineages have only 8 subunit without 8 subunit;
and E-type stocks lack both 0 subunits. All the lineages examined, however,contain the intact forms of 0 70 (aD, the rpoD gene product) and 05 4 (aN, therpoN gene product). As expected from the lack of 8 subunit, cells of D-type
and E-type lineages are nonmotile. The truncated form of d 8 subunit inB-type stocks carries two mutations near its N-terminus and lacks the
C-terminal proximal region 4 due to an amber mutation. The failure of Cand E-type W3110 cells to express 8 and that of D- and E-type cells to
express d'8 was found to be due to defects in transcription even though therespective 0 subunit genes remain intact. These findings emphasize theimportance of paying attention to possible variations in the genetic background between laboratory stocks originating from the same strain.

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(4)

Regulation of the Activity of Sigma Subunits from Escherichia coli

Shuichi KUSANO, Tapas KUNDU, Kan TANAKA l , and Akira ISHIHAMA
CUniv. Tokyo, Inst. Cell. Mol. BioI.)
Minor 0 subunits are induced under various stress conditions, including thestationary growth phase. Upon exposure to such environmental conditions,the intracellular conditions are also altered even though details are not known
yet. In the stationary phase cells, for instance, there are accumulations oftrehalose, glycine betaine, glycogen, polyphosphate and other components incytoplasm. In addition, the structure of nucleoids changes as to reduce
superhelicity.
The effect of trehalose on transcription in vitro was compared between two
holoenzymes, Ea70 and Ed8, which were reconstituted from purified core
enzyme and either 0 70 (the major 0 at exponentially growing phase) or 8
(the essential 0 at stationary growth phase), respectively. The optimum
trehalose concentration giving maximum transcription by Ed8 was higher
than that by Ea70 , in good agreement with the increase in intracellulartrehalose concentration under certain stress conditions where d 8 is produced.
Transcription activation by trehalose was attributed to both enhanced 8binding to the core enzyme and increased transcription initiation by Ed 8
8-dependent promoters. The activation of Ed8 by
holoenzyme fromtrehalose was additive with the transcription enhancement by decreased

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A. DEPARTMENT OF MOLECULAR GENETICS

15

superhelicity of template DNA prepared from stationary-phase cells. We
thus propose that the selective activation of transcription by £d8 holoenzyme
takes place in the presence of specific condition(s) and factor(s) present
under stress conditions.
The rpoF gene of E. coli codes for d' (or 8 ) subunit of RNA polymerase
which is involved in transcription of the flagellar class-III operons and thechemotaxis genes. (d' was overexpressed, purified to homogeneity, andcompared with a70 , the major a in exponentially growing cells, with respect tothe activity, specificity and response to changes in reaction conditions. The
affinity of d' to core RNA polymerase (E) is higher than that of d'. In an invitro transcription system, the holoenzyme containing d' (Ed') selectivelytranscribed the flagellar class-III and chemotaxis genes, all of which cannot
be transcribed by Ea70 • This strict promoter recognition property is similar to
those of other stress-response minor a subunits such as 03 2 but different fromthose of the principal a subunits, a 70 and 03 8 • The d'-dependent transcription
was highly sensitive to the species and concentration of salts: the level ofsingle-round transcription was maximum at 50 mM KCI and then decreased
to negligible level at 300 mM; while the effect was quite contrasting in thecase of K-acetate and K-glutamate, giving the maximum transcription level
between 200-300 mM. The activity of Ed' was more thermolabile than thatof Ea70 , being inhibited at high temperature (42°C). DNase I foot printing ofthefiiC andfiiD promoters indicated that d' alone cannot bind to DNA but
Ed' specifically recognizes the promoter -10 and -35 regions. Possible
alteration of the promoter structure after binding of Ed' was suggested.

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(5)

Cloning and Sequencing of the RNA Polymerase II
Genes from Schizosaccharomyces pombe

Hitomi SAKURAI, Takenori MIYAO,KIMURA, and Akira ISHIHAMA

Hiroshi

MITSUZAW A,

MakotoThe subunit composition of RNA polymerase II (pol II) was compared
between the budding yeast Saccharomyces cerevisiae and the fission yeast
Schizosaccharomyces pombe. For this purpose, we partially purified the
enzyme from S. pombe. Judging from the co-elution profiles in column
chromatographies of both the RNA polymerase activity and the two large
subunit polypeptides [subunit 1 (prokaryotic f3' homologue) and subunit 2 (f316homologue)], the mInimum number of S. pombe pol II-associated polypeptides was estimated to be lO, less than the proposed subunit number of theS. cerevisiae enzyme (ref. 28). These lO putative subunits of S. pombe pol II
correpond to subunits 1, 2, 3, 5, 6, 7, 8, lO, 11 and 12 of the S. cerevisiaecounterparts.
RNA polymerase II purified from the fission yeast Schizosaccharomyces
pombe contains ten different species of polypeptides. Previously, we cloned
and sequenced both cDNA and the genes encoding the three large subunits,
Rpbl, Rpb2 and Rpb3. Later the rpb5+ gene encoding subunit 5 of RNApolymerase II was cloned and sequenced (ref. 23). The polypeptide predictedfrom DNA sequence of the rpb5+ gene consists of 2lO amino acids with acalculated molecular weight of 23,914. The homology of the amino acidsequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human
hRPB25, respectively. Recently, other groups isolated the genes for Rpb6
and Rpb12 and cDNA for RpblO. This year we cloned both cDNA and thegenes encoding four small subunits, Rpb7, Rpb8, RpblO and Rpbll. From
the nucleotide sequences, Rpb7, Rpb8, RpblO and Rpbll were found to
consist of 172 (Mr 19,081), 125 (Mr 14,284), 71 (Mr 8,266) and 123 (Mr14, Ill) amino acid residues, respectively. All these four subunits arehomologous to the corresponding subunits of S. cerevisiae RNA polymerase
II. The rpb7 gene contains one intron while the rpb8, rpblO and rpbll genes
contain two introns. Taken altogether the gene organization and the predicted protein sequence have been determined for all ten subunits of the S.
pombe RNA polymerase II.
(6)

Subunit-subunit Contact Network within Schizosaccharomyces
pombe RNA Polymerase II

Takenori MIYAO, Kiyoshi YASUI, Akira ISHIGURO, Akira IWATA 1, Makoto
KIMURA, Hiroshi MITSUZAWA, Susumu VEDA l , and Akira ISHIHAMAC Nippon Institute for Biological Science, Ohme)
Eukaryotic RNA polymerase II is composed of more than lO polypeptide
chains. The minimum and essential subunits for RNA synthesis have not yet
been identified. Toward this ultimate goal, we analyzed the topologicalarrangement of the putative subunits by analyzing the subunit-subunit contact network withinS. pombe RNA polymerase II. Far-Western blot analysis

A. DEPARTMENT OF MOLECULAR GENETICS

17

of S. pombe RNA polymerase II using 32P-Iabeled recombinant Rpb3 or Rpb
S fused to glutathione S-transferase (GST) as a probe, indicated that Rpb3
binds to membrane-immobilized Rpbl, Rpb2, RpbS and Rpbll, and RpbS
binds strongly to Rpbl, Rpb2 and Rpb3 and weakly to RpbS and a lS-kDa
subunit (Rpb8 or Rpbll) (ref. 23). The existence of RpbS-Rpb3 contact was
supported by detection of complexes formed between these two proteins
synthesized in vitro using protein-immobilized beads. The contact sites on
Rpb 1 and Rpb2 with Rpb3 and RpbS are being analyzed in details using
fragments of these two large subunits expressed in E. coli.
The same line of studies is being carried out using 32P-Iabeled Rpb6, Rpb7,
Rpb8, RpblO, Rpbll and Rpbl2 as far-Western probes.
(7)

Transcription and Replication of Influenza Virus RNA:
Evidence for de novo Initiation of Replication

Ayae HONDA, Kiyohisa MIZUMOT0 1, and Akira ISHIHAMA CKitasato
Univ., Fac. Pharm.)
Influenza virus genome is composed of eight RNA segments of negative
polarity. Transcription of the influenza virus genome RNAs (vRNAs) by thevirus-associated RNA-dependent RNA polymerase is initiated by using hostcell capped RNAs as primers, while the mechanism of replication initiation
remained unsolved. De novo initiation for the RNA replication was suggested
because the majority of all eight vRNA segments was found to carry
triphosphate at their S' termini based on the observations: (i) S' phosphorylation of vRNAs by T4 polynucleotide kinase takes place only after phosphatase treatment; (ii) capping of vRNAs can be observed with the intact yeast
capping enzyme consisting of a (RNA guanylyltransferase) and /3 (RNA S'triphosphatase) subunits; but (iii) the a subunit, which exhibits guanylyltransferase activity by itself but lacks RNA S'-triphosphatase activity, is unable
to cap vRNAs.

18

(8)

The Molecular Anatomy of Influenza Virus RNA Polymerase

Yukiyasu ASAN0 1, Ayae HONDA, Tetsuya TOYODA2 , and Akira ISHIHAMA
CPresent address: Sanwa Kagaku Research Inst., 2 Kurume University Med.
Sch.)
Influenza virus RNA polymerase is a multifunctional and multisubunit
enzyme, consisting of three viral P proteins, PB I, PB2 and PA. Several linesof reserach have been carried out for mapping the functional sites on each P
protein subunit. Radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB I subunit. Specific crosslinking of PB I was
confirmed using oxidized GTP. The GTP analogue-binding domains wereidentified after proteolytic cleavage of the crosslinked PB I with V8 protease.
The cleavage pattern of PBI was determined by analysis of the aminoterminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. Results indicate two GTP-bindingsites in the PBI subunit, i.e., the amino terminal-proximal site I and on the
carboxy terminal-proximal site II. The location of GTP-binding site I and II
is close to that of sequence motif A and motif D, respectively, conserved
among RNA-dependent RNA polymerases. On the other hand, radio-labeled
capped RNA was UV cross-linked to the PB2 subunit. Proteolytic cleavage
experiments suggested that capped RNA was cross-linked to two segments.
By transfection of two P protein genes, one intact gene for one P protein
and the other trauncated gene for another P protein, and immunoprecipitation of binary complexes, we determined the subunit-subunit contact sites
among three P proteins. The PBI is the core subunit of RNA polymerase, towhich both PB2 and PA bind independently (ref. 30).
Publications
1.

Artsimovitch, I., Murakami, K., Ishihama, A., and Howe, M. M.: Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal region of both a and700subunits of Escherichia coli RNA polymerase. J. BioI. Chern., 271, 32343-32348,

2.

Belyaeva, T., Bown, J., Fujita, N., Ishihama, A., and Busby, S.: Location of the
C-terminal domain of the RNA polymerase alpha subunit in different open complexes at
the Escherichia coli galactose operon regulatory region. Nucleic Acids Res., 24, 2243-

1996.

2251, 1996.

3.

Boucher, P. E., Murakami, K., Ishihama, A., and Stibitz, S.: Mapping of the contact sites

A.

4.

5.

6.

7.

8.

9.

10.

11.
12.

13.

14.

15.

16.

DEPARTMENT OF MOLECULAR GENETICS

19

on RNA polymerase for BvgA phosphate on thejha promoter. J. Bacteriol., 179, 17551763, 1997.
Chugani, S. A., Parsek, M. R., Douglas, Hershberger, C. D., Murakami, K., Ishihama,A., and Chakrabarty, A. M.: Activation of the catBCA promoter: Probing the interactionof CatR and RNA polymerase through in vitro transcription. J. Bacteriol., 179, 22212227, 1997.
Eichenberger, P., Dethiollaz, S., Fujita, N., Ishihama, A., and Geiselmann, J.: The
influence of the location of the activator binding site on the geometry of a transcriptional
activation complex in Escherichia coli. Bichemistry, 35, 15302-15312, 1996.
Fujita, N. and Ishihama, A.: Reconstitution of RNA polymerase. In "Meth. Enzymol."
273, RNA Polymerase and Associated Factors, Part A, (Adhya, S., ed.), pp. 121-130,
Academic Press, New York, 1996.
Giladi, H., Murakami, K., Ishihama, A., and Oppenheim, A. B.: Identification of an UP
element within the IHF binding site at the Pd-P L 2 tandem promoters of bacteriophage A.
J. Mol. BioI., 260, 484-491, 1996.
Greiner, D. P., Miyake, R., Moran, J. K., Jones, A. D., Negishi, T., Ishihama, A., andMeares, C. F.: Synthesis of the protein cutting reagent iron (S)-I-(p-bromoacetamidebenzym)ethylenediaminetetraacetate and conjucation to cysteine side chains. Bioconjugate Chemistry, 8, 44-48, 1997.
Imamura, R., Yamanaka, K., Ogura, T., Hiraga, S., Fujita, N., Ishihama, A., and Niki,
H.: Identification of the cdpA gene encoding cyclic 3' ,5'-adenosine phosphophate phosphodiesterase in Escherchia coli. J. BioI. Chern., 271, 25423-25429, 1996.
Ishihama, A.: Promoter selectivity control of Escherichia coli RNA polymerase. Nucleic
Acids & Molecular Biology, Vol. 11, Mechanism of Transcription, Eds. F. Eckstein and D.
Lilley, Springer-Verlag, Heidelberg, pp. 53-70, 1997.
Ishihama, A.: A multi-functional enzyme with RNA polymerase and Rnase activities:
Molecular anatomy of influenza virus RNA polymerase. Biochimie, in press.
Ishihama, A., Asano, Y., Honda, A., Toyoda, T., Mizumoto, K., and Nakada, S.:
Structure, function and formation of influenza virus RNA polymerase. In: Options for
the Control of Influenza, (Brown, L. E., Hampson, A. W. and Webster, R. G., eds.),
Elsevier Sci. B. V., Amsterdam, pp. 372-380, 1996.
Jair, K.-W., Fawcett, W. P., Fujita, N., Ishihama, A. and Wolf, Jr., R. E.: Ambidextrous
transcription activation by SoxS: Requirement for the C-terminal domain of the RNA
polymerase alpha subunit at a subset of superoxide inducible genes. Mol. Microbiol., 19,
307-314, 1996.
Jair, K.-W., Yu, X., Skarstad, K., Thony, B., Fujita, N., Ishihama A., and Wolf, Jr., R.
E.: Transcription activation of promoters of the superoxide and multiple antibiotic
resistance regulation by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. J. Bacteriol., 178, 2507-2513, 1996.
Jeon, Y. H., Yamazaki, T., Otomo, T., Ishihama, A., and Kyogoku, Y: Flexible linker inthe RNA polymerase a subunit facilitates the independent motion of the C-terminal
activator contact domain. J. Mol. BioI., 267, 953-962, 1997.
Jishage, M. and Ishihama, A.: Variation in RNA polymerase sigma subunit composition
within different stocks of Escherichia coli strain W311O. J. Bacteriol., 179,959-963,

20

17.

18.

19.

20.
21.

22.

23.

24.

25.

26.

27.

28.
29.

30.

1997.
Jishage, M., Iwata, A., Ueda, S., and Ishihama, A.: Regulation of RNA polymerase sigma
subunit synthesis in Escherichia coli: Intracellular levels of four species of sigma subunit
under various growth conditions. J. Bacteriol., 178, 5447-5451, 1996.
Kawagishi-Kobayashi, M., Yamamoto, M., and Ishihama, A.: Mutational analysis of the
RNase-like domain in subunit 2 of fission yeast RNA polymerase II. Mol. Gen. Genet.,
205, 1-5, 1996.
Kimura, M. and Ishihama, A.: Subunit assembly in vivo of Escherichia coli RNA
polymerase: Role of the amino-terminal assembly domain of alpha subunit. Genes To
Cells, 1, 517-528, 1996.
Kobayashi, M., Toyoda, T., and Ishihama, A.: Influenza virus PBl protein is the minimaland essential subunit of RNA polymerase. Arch. Virol., 141, 525-533, 1996.
Kusano, S., Ding, Q., Fujita, N., and Ishihama, A.: Promoter selectivity of Escherichia
coli RNA Polymerase Ea70 and Eo" holoenzymes: Effect of DNA supercoiling. J. BioI.
Chern., 271, 1998-2004, 1996.
Luo, J., Sharif, K. A., Jin, R., Fujita, N., Ishihama, A., and Krakow, J. S.: Molecular
anatomy of the {3' subunit of the E. coli RNA polymerase: Identification of regions
involved in polymerase assembly. Genes to Cells, 1, 819-828, 1996.
Miyao, T., Yasui, K., Sakurai, H., Yamagishi, M., and Ishihama, A.: Molecular assemblyof RNA polymerase II from the fission yeast Schizosacharomyces pombe: Subunit-subunit
contact network involving Rpb5. Genes to Cells, 1, 843-855, 1996.
Murakami, K., Fujita, N., and Ishihama, A.: Transcription factor recognition surface on
the RNA polymerase a subunit is involved in contact with the DNA enhancer element.
EMBO J., 15,4358-4367, 1996.
Murakami, K., Kimura, M., Owens, J. T., Meares, C. F. and Ishihama, A.: The two a
subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact
different halves of the DNA upstream element. Proc. Natl. Acad. Sci. USA, 94, 17091714, 1997.
Nagai, S., Nagasawa, Y., Yagihashi, T., and Ishihama, A.: The cyclic AMP receptor
protein (CRP) gene function is required for expression of a (cell-bound)-type hemolysis
in an avian pathogenic Escherichia coli strain. Infect. Immun., in press.
Rajkumari, K., Kusano, S., Ishihama, A., T. Mizuno and Gowrishankar, J.: Effects of
H-NS and potassium glutamate on
and o7°-directed transcription in vitro fromosmotically regulated PI and P2 promoters of proU in Eschercihia coli. J. Bacteriol., 178,
4176--4181,1996.
Sakurai, H., Miyao, T., and Ishihama, A.: Subunit composition of RNA polymerase IIfrom the fission yeast Schizosaccharomyces pombe. Gene, 180, 63-67, 1996.
Tang, Y., Murakami, K., Ishihama, A., and deHaseth, P. L.: Upstream interactions at thelambda P RM promoter are sequence nonspecific and activate the promoter to a lesser
extent than an introduced UP element on rRNA promoter. J. Bacteriol., 178, 6945-6951,
1996.
Toyoda, T., Adyshev, D. M., Kobayashi, M., Iwata, A., and Ishihama, A.: Molecularassembly of influenza virus RNA polymerase: Identification of the subunit-subunit
contact sites on each P protein subunit. J. Gen. Virol., 77, 2149-2157, 1996.

as-A. DEPARTMENT OF MOLECULAR GENETICS

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Toyoda, T., Adyshev, D. M., Kobayashi, M., Iwata, A., Ueda, S., and Ishihama, A.: The
subunit-subunit contact sites of the influenza virus RNA polymerase. In: Options forControl of Influenza (Brown, L. E., Hampson, A. W. and Webster, R. G., eds.), Elsevier
Sci. B. V., Amsterdam, pp. 419-426, 1996.
32. Toyoda, T., Kobayashi, M., Nakada, S., and Ishihama, A.: Molecular dissection of
influenza virus RNA polymerase: PBI subunit alone is able to catalyze RNA synthesis.
Virus Genes, 12, 155-161, 1996.

31.

A-b.
(1)

Division of Mutagenesis

A Ubiquitin Pathway Essential for Onset of Anaphase in Mitosis

Hiroaki SEINO, Fumio OSAKA, and Fumiaki YAMAO
A cDNA encoding a novel ubiquitin-conjugating enzyme designated as
UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed
that it was essential for cell growth. In vivo depletion of the UbcP4 protein
demonstrated that it was necessary for cell cyele progression at two phases,
G2/M and metaphase/anaphase transitions. The G2-arrest of UbcP4depleted cells was dependent upon chkl which mediates checkpoint pathway.
UbcP4-depleted cells arrested at metaphase had duplicated and condensed
chromosomes but were defective in separation. However, septum formationand cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9'S cells at a
restrictive temperature. cut9 encodes a component of the anaphase promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyelin-specific ubiquitin ligase. Cdc 13, a
mitotic cyelin in fission yeast, was accumulated in the UbcP4-depleted cells.
These results strongly suggested that UbcP4 is a ubiquitin-conjugating
enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset ofanaphase and possibly of mitotic cyelin at the exit of mitosis.

22

(2)

CDC34 Dependent-Ubiquitin Pathway: Role of GRRI in
the Ubiquitination of G1 Cyelin Cln2

Tsutomu KISHI and Fumiaki Y AMAO
In S. cerevisiae, Cdc34, Cdc4 and Cdc53 form a complex, and are required
for the G 1-S transition. CDC34 encodes a ubiquitin-conjugating enzyme,
suggesting that Cdc34-dependent proteolysis is necessary for the cell cycle
progression. Sic1, an S-phase CDK inhibitor, has been identified as an
essential target of this pathway. Other essential target proteins could be
elucidated since a cdc34 sid double mutant still arrests in G2.
We isolated extragenic suppressors of a cdc34-1 sid mutant. One group of
suppressors was recessive, and was found to be allelic to GRR1, a gene shown
to be involved in degradation of Gl cyclins (Clnl and Cln2) as well as
glucose repression. Conversely, overproduction of Grrl in cdc34 sid cellsoimpaired colony formation at 30 e. We isolated MGOl (Multicopy suppressor of Grrl Overproduction defect) which suppressed this growth defectwhen introduced on a multicopy vector. Sequence analysis revealed that
MGOl is identical to SKP1, which has been shown to be required for theubiquitin-mediated proteolysis of Cln2 and Sicl. Using GST pull-down
assay, we found that in vitro translated Grrl bound to both bacterially
produced Gst-Skp I and Gst-Cln2. Furthermore, in vitro translated Cln2
bound to Gst-Skpl, depending upon the presence of Grrl. Association of
Grrl and Gst-Cln2 did not require the carboxyl-terminal PEST sequences.
These data suggest that Grrl serves as a bridge between ubiquitination
machinery and Cln2.
Publications
Yamaguchi, T., Kim, N., Sekine, S., Seino, H., Osaka, F., Yamao, F., and Kato, S.:
Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme
similar to the Drosophila bendless gene product. J. Biochem., 120, 494-497, 1996.
2. Nakajima, T., Morita, K., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., Osaka, F., Yamao,
F., and Oda, K.: Degradation of topoisomerase IIa during adenovirus EIA-induced
apoptosis is mediated by the activation of the ubiquitin proteolysis system. J. BioI.
Chern., 271, 24842-24849, 1996.
3. Azuma, Y., Seino, H., Seki, T., Uzawa, S., Kleb, C., Ohba, T., Wittinghufer, A., Hayashi,N., and Nishimoto, T.: Conserved Histidine Residues of RCCI Are Essential for
Nucleotide Exchange on Ran. J. Biochem., 120, 82-91, 1996.
1.

A.
4.

5.

DEPARTMENT OF MOLECULAR GENETICS

23

Hayashi, N., Seino, H., Ide, K., Watanabe, M., Clark, K. L., Matsumoto, K., andNishimoto, T.: Genetic Interaction of DED I Encoding a Putative ATP-dependent RNA
Helicase with SRMI Encoding a Mammalian RCCI Homolog in Saccharomyces cerevisiae. Mol. gen. Genet., 253, 149-156, 1996.
Ikehata, H., Kaneda, S., Yamao, F., Seno, T., Ono, T., and Hanaoka, F.: Mouse cells
expressing temperature-sensitive ubiquitin-activating enzyme El mutant show ultravioletlight sensitivity and deficiency in induced mutagenesis. Molecular & Cellular Biology, 17,1484-1489, 1997.

A-c.

(1)

Division of Nucleic Acid Chemistry

Host Proteins Involved in Transcription of
The Sendai Virus Genome

Kiyohisa MIZUMOTO (School of Pharmaceutical Sciences, Kitazato University)
The Sendai virus, a member of the Paramyxoviridae, contains a single,
nonsegmented RNA genome of 15 kb long with negative polarity, which
encodes six viral proteins, nucleocapsid protein (NP), phosphoprotein (P),matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN),and large protein (L). The NP, P, and L proteins are associated with the
genome to form a nucleocapsid. The genetic information of this RNA
genome is expressed through at least six monocistronic mRNA species
transcribed by a virion-associated RNA-dependent RNA polymerase, whichis thought to be composed of the P and L proteins.
In vitro mRNA synthesis by The Sendai virus is almost entirely dependent
on the addition of cellular proteins (positive host factors), one of which could
be tubulin. We investigated the distribution of host factors in various rat
organs. Extracts from the brain, thymus, heart, lung, testis, ovary and uterus
all supported in vitro Sendai virus transcription, among which the highest
activity was obtained with the brain extract. On the other hand, little or no
activity was detected in the liver, spleen, and kidney extracts. An inverse
correlation between the apparent host factor activity to stimulate mRNA
synthesis and RNase activity that hydrolyzes Sendai virus mRNAs wasfound, except in the liver extract. However, when a transcription initiation
complex was isolated and subjected to RNA chain elongation reaction, all of
the extracts including those from liver, spleen and kidney, were active.

24

Immunoblotting showed that tubulin molecules were integrated in these
initiation complexes, supporting the notion that tubulin is involved in initiation complex formation. We alsoidentified transcription inhibitory activity
without any detectable RNase activity in the liver extract. This negative hostfactor seemed to act on RNA chain elongation. It is likely that Sendai virustranscription is regulated by both positive and negative regulatory factors.
(For details, see Ref. 1.)(2)

Crystal Structure of the Escherichia coli Replication
Terminator Protein Complexed with Ter DNA

Katsuhiko KAMADA and Kosuke MORIKAWA (Biomolecular Engineering
Research Institute)
Replication is terminated at defined sequences of DNA in prokaryotic
chromosomes. In Escherichia coli, this termination is mediated by thesite-specific DNA-binding protein designated as Tus. The Tus protein (mw:
36 kDa) is a monomeric molecule that binds six specific sequences (Ter)within the replication terminus region, and the Tus-Ter complex prevents thepassage of DnaB helicase, which is a constituent of replication machinery.
The three-dimensional (3D) structure at the atomic level is required to gain
insight into the mechanism by which the Tus protein recognizes the Ter sites
and blocks the replication fork in a polar manner.
The most suitable crystals for X-ray analysis were grown from a mixture of
the Tus protein and a 16 base long Ter DNA using the microdialysis method
in PEG solution (ref. 3). The crystal structure was resolved at 2.7 A
resolution and refined (ref. 4). The Tus protein shows a very uniquestructure as compared with other DNA binding proteins. The protein
embraces the Ter element from both sides of the DNA with two protruded
a-helical regions. The most remarkable feature of the complex is an extensive
protein-DNA interface which involves many direct and indirect polar interactions. Two .a-sheets are responsible for the recognition of the DNA in the
major groove.
A genetic approach allowed the isolation of the Tus mutant. Most of thesesingle mutation sites are mapped on the .a-sheet region in the 3D-structure.
These mutants that affect the efficiency of replication arrest, partially orcompletely impair the binding of the Tus protein with DNA. This suggests

A.

DEPARTMENT OF MOLECULAR GENETICS

25

that replication arrest and Tus-DNA binding might be inseparable.
The overall structural feature of the complex also implies that the a-helicalregions on the fork-blocking side act as a directional DNA clump and preventthe passage of the DnaB helicase. Presumably, a mechanism that enables thereplication fork from the opposite side to pass through, would rely upon the
structural disturbance of the ,B-sheet region induced by the unwinding motionof DnaB. This interpretation agrees with the physical collision model thatshows the intrinsic structure of the complex dominating the directional arrestof the replication fork.
Publications
1.

2.
3.

4.

Takagi, T., Iwama, M., Seta, K., Kanda, T., Tsukamoto, T., Tominaga, S., and Mizumoto, K.: Positive and negative host factors for Sendai virus transcription and their organ
distribution in rat. Arch. Virol., 141, 1623-1635, 1996.
Watanabe, K., Handa, H., Mizumoto, K., and Nagata, K.: Mechanism for inhibition of
influenza virus RNA polymerase activity by matrix protein. J. Virol., 70, 241-247, 1996.
Kamada, K., Ohsumi, K., Horiuch, T., Shimamoto, N., and Morikawa, K.: Crystallization and Preliminary X-ray Analysis of the Escherichia coli Replication TerminatorProtein Complexed with DNA. PROTEINS: Structure, Function, and Genetics, 24,
402-403, 1996.
Kamada, K., Horiuch, T., Ohsumi, K., Shimamoto, N., and Morikawa, K.: Structure ofa replication-terminator protein complexed with DNA. Nature, 383, 598-603, 1996.

26

B.

DEPARTMENT OF CELL GENETICS
B-a.

(1)

Division of Cytogenetics

Localization of Mouse Rad51 and Lim15 on Meiotic
Chromosomes at Late Stages of Prophase 1

Tomoatsu IKEYA and Tomoko OGAWA
Studies on the localization of Rad5 I and Lim I5 proteins on meiotic
chromosomes of lily showed that these proteins participate in searching forhomologous chromosomes, which is initiated on the leptotene stage and leads
to pairing in the zygotene stage. However, the roles of these proteins inchiasma formation and chromosomal exchanges are not known. Taking
advantage of the much shorter lengths of mouse chromosomes which facilitates the examination of on an entire chromosome with a chiasma region
through optical methods, we studied the localization of Rad5 I and Lim I 5
homologues on the mouse chromosomes.
In the chromosomes of mouse testis during the late stages of meiotic
prophase I, the Rad51 and Liml5 proteins were present in the core regions of
the chromosomes which remained after removal of the chromatin by DNase
II. Rad5 I was located along the core of the synaptonemal complexes (SC) in
the pachytene stage and in the chiasma regions in the diplotene stage. The
protein was not present on separated homologous cores in the diplotene stage.
On the other hand, the Liml5 protein was present almost exclusively at bothends of the core of chromosomes from the pachytene to diplotene stages andeven in diakinesis. Differences in the roles of these proteins in the late stageof recombination were evident from these findings. Ref. I(2)Characterization of DNA Ligase from the Fungus
Coprinus Cinereus

Shimako MATSUDA
DNA ligase was highly purified from the fungus Coprinus cinereus at the

B.

DEPARTMENT OF CELL GENETICS

27

meiotic recombination stage, pachytene. The pachytene DNA ligase showed
three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimatedby a-[ 32 P]AMP-Iabe1ing assay. These three polypeptides were susceptible toreaction with mAb against a 16-amino-acid sequence in human DNA ligase
I, which is conserved in the C-terminal regions of mammalian, vaccinia virus
and yeast DNA ligases. Since rapidly purified preparations from freshpachytene cells exhibited a single polypeptide of DNA ligase with a molecularmass of 88 kDa, the smaller polypeptides seemed to be limited-degradation
products of 88-kDa polypeptide during the isolation and purification procedures. Km values for ATP and (dTho hybridized with (dA)n were 1.5 mM
and 90 nM, respectively. This enzyme was capable of joining (dTho' (rA)nand (rA)12-18,(dT)n as well as (dTho'(dA)n, and able to ligate blunt-end
DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also
partially purified from zygotene cells at the meiotic pairing stage and mitotic
mycelium cells. In their molecular mass, immuno-reactivity, Km value andsubstrate specificity, they were indistinguishable from pachytene DNA ligase.
These results suggest that the fungus C. cinereus in the pachytene stage
contains DNA ligase with a molecular mass of 88 kDa as a main or a single
species, which is quite similar to DNA ligases from the zygotene andmycelium cells in molecular and catalytic properties. Ref. 2(3)

Genetic Characteristics of New recA Mutants ofEscherichia coli K-12

Andrei ALEXSEYEV (ALEXEEV)To search for functionally thermosensitive (FT) recA mutations, as well as
mutations which differently affect RecA protein functions, seven new recA
mutations in three different regions of the RecA protein structure proposedby Story et al. were constructed. Additionally, the recA2283 allele responsible for the FT phenotype of the recA200 mutant was sequenced. Five single
mutations (recA2277, recA2278, recA2283 , recA2283E and recA2284) and one
double mutation (recA2278-5) generated, respectively, the amino acid substitutions L-277";,N, G-278";,P, L-283";,P, L-283";,E, 1-284";,D, and G-278";,
T plus V-275";,F in the alpha-helix H-beta-strand 9 region of the C-terminal
domain of the RecA protein structure. According to recombination, repair,and SOS-inducible characteristics, these six mutations fall into four phenoty-28

pic classes: (i) an FT class, with either inhibition of all three analyzed
functions at 42 degrees C (recA2283), preferable inhibition at 42 degrees C ofrecombination and the SOS response (recA2278), or inhibition at 42 degrees
C of recombination only (recA2278-5); (ii) a moderately deficient class(recA2277); (iii) a nondeficient class (recA2283E); and (iv) a mutation witha null phenotype (recA2284). The recA2223 mutation generates an L-223--;>M substitution in beta-strand 6 in the central domain of the RecA structure.
This FT mutation shows preferable inhibition of the SOS response at 42
degrees C. The recA2183 mutation produces a K-183;>M substitution inalpha-helix F of the same domain. The Lys-183 position in the Escherichia
coli RecA protein was found among positions which are important forinterfilament interaction. Ref. 3(4)

Chromatin Structure of the Yeast URA3 Gene at High Resolution
Provides Insight into Structure and Positioning of
Nucleosomes in the Chromosomal Context

Shigeo TANAKA
To characterize nucleosome structure and positioning in the chromosomal
context, the chromatin structure of the whole VRA3 gene was studied in the
genome and in a minichromosome by testing the accessibility of DNA tomicrococcal nuclease and DNase I. The cutting patterns and hence thechromatin structures were almost indistinguishable in the genome and in theminichromosomes. The only notable exception was enhanced cutting between nucleosomes U3/U4 and U4/U5 in the minichromosomes. The results
demonstrate that there is no severe constraint acting from outside the URA
3 gene in chromosomes and minichromosomes. While low-resolution mapping showed six regions with a positioned nucleosome (VI to V6), each region
resolved in a complex pattern consistent with multiple overlapping positions.
Some regions (VI, U4, U5 and V6) showed multiple positions with adominant rotational setting (DNase I pattern), while U2 showed positioning
within 10 bp but with no defined rotational setting, demonstrating that
nucleosome positions were not in phase and not coordinately regulated.
Reduced DNase I cutting from about 50 bp form the 5' end towards 3' end
was common to all nuc1eosome regions. This polarity has been observed on
isolated core particles. The results demonstrate that the DNase I pattern

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DEPARTMENT OF CELL GENETICS

29

observed in vitro indeed reflects a structural property of nuc1eosomes in thechromosomal context. It is emphasized that despite the local heterogeneity
revealed by high-resolution mapping, the low-resolution map is a reasonably
accurate representation of the chromatin structure. Ref. 4
Publications
1.

2.
3.

4.

Ikeya, T., Shinohara, A., Sato, S., Tabata, S., and Ogawa, T.: Localization of mouse
Rad51 and Lim 15 on meiotic chromosomes at late stages of prophase 1. Genes to Cells,
1,379-389, 1996.
Matsuda, S., Sakaguchi, K., Tsukada, K., and Teraoka, H.: Characterization of DNA
ligase from the fungus Coprinus cinereus. European J. Biochem., 237, 691-697, 1996.
Alexseyev, A. A., Bakhlanova, I. V., Zaltsev, E. N., and Lanzov, V. A.: Genetic characterization of new recA mutants of Escherichia coli K12. J. BacteriD/., 178, 2018-2024,
1996
Tanaka, S., Livingstone-Zatchej, M., and Thoma, F.: Chromatin structure of the yeast
URA3 gene at high resolution provides insight into structure and positioning of nucleosomes in the chromosomal context. J. Mol. BioI., 257, 919-934, 1996.

Oral presentaions
1.

2.
3.

4.

Ogawa, T., Ikeya, T., Shinohara, M., Shinohara, A., and Terasawa, M.: Roles of Rad51,
Rad52 and Lim15 proteins in meiotic recombination. Keyston meeting: Replication andRecombination, USA, FEb.
Ogawa, T., Ikeya, T., and Terasawa, M.: Dual nature of Rad51 and Lim15 proteins inmeiotic recombination. EMBO Meeting: Recombination, France, May.
Ikeya, T., Sato, S., Tabata, S., and Ogawa, T.: Localization of mouse Rad51 and Lim15
proteins on meiotic chromosomes at late stage of prophase 1. Gordon Research
Conference: Meiosis, USA, June.
Ogawa, T. and Shinohara, A. Biochemical activities of Rad52 protein in homologous
recombination. Gordon Research Conference: Meiosis, USA, June.

B-b.

(1)

Division of Microbial Genetics

Minus-Strand Origin of Filamentous Phage versus Transcriptional
Promoters in Recognition of RNA Polymerase

Atsushi HIGASHITANI, Nahoko HIGASHITANI, and Kensuke HORIUCHI
Replication of complementary strand DNA in filamentous phages is lll-

30

itiated by a primer RNA, which is synthesized at the minus-strand origin on
the viral single-stranded DNA by E. coli RNA polymerase holoenzyme
containing the 0 70 subunit. We demonstrated that the affinity of RNApolymerase in vitro to the origin was about 16-fold higher than that to thelacUV5 promoter. We also showed that the temperature dependence of theprimer RNA synthesis was much lower than that of lacUV5 transcription.
The high affinity of RNA polymerase to the origin depended on the singlestrandedness of the "-10 region". The nucleotide sequence of the nontemplate strand in the "-10 region" was found to be important for thefunction, while that of the template strand was not. These observations
suggest that the 0 70 subunit directly interacts with the single-stranded nontemplate strand containing adenine residue(s) at the -10 region of thepromoter. For details, see Ref. 1.
(2)

The Active Center of the Initiator Protein ofFilamentous Phage fl

Satoshi ASANO, Atsushi HIGASHITANI, and Kensuke HORIUCHI
The gene II protein (gpII) of filamentous phages (fl, fd, M13) is amultifuctional protein that plays several key roles in phage plus-strand DNA
replication. First, it binds to the plus-strand origin and introduces a specificnick in the plus-strand of supercoiled replicative form DNA (RF I). The 3'hydroxyl end of the nick serves as the primer for the rolling-circle typereplication. Binding of gpII induces DNA bending and duplex melting
around the nicking site. The melting requires the negative superhe1icity ofDNA, and the nicking reaction requires the Mg2+ ion. Upon completion ofa round of replication, gpII functions in termination by cleaving and circularizing the displaced single strand.
We showed that a small fraction of the nicked DNA product was covalently attached at the 5' end to gpII. The covalent bond was extremely
unstable in the presence of active gpII. The residue that bridged gpII to the
5' end of DNA was phospho-tyrosine.
There are 15 tyrosine residues in gpII. We therefore constructed 15
mutant gpII, in each of which a tyrosine residue was replaced by phenylalanine. Complementation tests of a gene II amber phage were carried out using
these mutant proteins provided in trans from plasmids. Three mutants only

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DEPARTMENT OF CELL GENETICS

31

weekly complemented the amber phage. Three other mutants were temperature sensitive. Only one mutant(gpII Y197F) was totally incapable ofcomplementing the amber mutant. The remaining mutants strongly complemented the amber phage. We purified gpII Y 197F gpII and tested fornicking activity and sequence-specific DNA binding activity for the origin.
The results indicated that gpII Y197F could specifically bind to the origin andbend DNA normally. However, it could not introduce the nick.
In order to directly determine the tyrosyl residue of gpII to which thenicked DNA product is covalently attached at the 5' end, the covalent
complex was digested with trypsin, and the peptide-DNA complex was
isolated by ion-exchange chromatography and gel filtration. The results of
peptide sequencing of the isolated peptide-DNA complex indicated that thetyrosine residue at position 197 from the N-terminus was covalently attachedto the DNA.
We concluded that the tyrosine residue at position 197 is involved in thenicking reaction as the active center.
In contrast to the case of the cisA protein of lytic phage phiXl74, thecovalent bond between gpII and the 5' end of DNA was quickly cleaved by
gpII itself. The instability of the covalent complex may be related to theunique mode of DNA replication in filamentous phages which allows coordinated growth of the phage and the host bacteria.
(3)

A New Class of Mutations of Penicillin-Binding
Protein 3 of Escherichia coli

Hiroshi HARA and Kensuke HORIUCHI
Penicillin-binding protein 3 (PBP 3) of E. coli is a peptidoglycan-crosslinking enzyme essential for formation of the division septum. The crosslinking activity is carried by the C-terminal penicillin-binding domain. We
suspected, from the following findings, that PBP 3 acted as an oligomer or acomplex with other cell division proteins and that the function for theoligomer/complex formation was carried by the N-terminal region: (i) A
mutant PBP 3 with an altered active site for the crosslinking activity (S307A)
showed a dominant lethal effect, and the effect was reversed by replacementof the N-terminal 40 residues with an uncleavable signal peptide. (ii)Overproduction of N-terminal fragments caused a dominant negative effect.

32

We mutagenized the N-terminal region and selected mutations that reversed the dominant lethal effect by S307A. Such intragenic suppressors in the
N-terminal region were then combined with the wild-type crosslinking activity domain in the C-terminal region. Among them we found PBP 3 mutants
that were non-functional in septum formation. One of these mutants wasfound to have a deletion of the 4 residues Arg-Val-Ala-Trp, at the 41st-44th
positions (R41-W44) from the N terminus.
In a separate experiment to exchange the N-terminal region of PBP 3 withother sequences, we inserted a synthetic 12-base oligonucleotide containing
an EcoRI-recognizing sequence into the Mlul site within the PBP 3 gene.
The resultant mutant PBP 3, which had a 4-residue insertion of Asn-Ser-ArgVal after V42, was also found non-functional in septum formation. When
combined with the S307A C-terminal domain, it suppressed the dominantlethal effect.
These mutants (i) retained penicillin-binding ability, indicating normal
crosslinking activity, and (ii) were C-terminally processed by a periplasmic
protease Prc as was wild-type PBP 3, indicating normal translocation acrossthe cytoplasmic membrane. In contrast to crosslinking-defective mutations
isolated so far by monitoring penicillin-binding ability, these mutations are of
a novel class, probably defective in the oligomer/complex formation. The
deletion and insertion were within the N-terminal hydrophobic sequence F24148. We converted R41, the sole charged residue in the sequence, to 19 otherkinds of amino acid residues, but the septum-forming ability was not lost. A
residue(s) within V42-W44 or the length of the hydrophobic sequence may be
important for PBP 3 function.
(4)

The First Two Genes of the Cell Envelope Biosynthetic/Cell
Division Gene Cluster in the Escherichia coli
Chromosome 2-Minute Region

Hiroshi HARA and Kensuke HORIUCHI
In the 2-min region of the E. coli chromosome, cell envelope biosynthetic
and cell division genes are tightly clustered in the same orientation. A
promoter, P mra, at the beginning of this mra cluster is required for expressionof the first nine genes, up to Its w. We concluded this because a P mra :: P1ac
strain in which the promoter was displaced by the lac promoter grew in the

B.

DEPARTMENT OF CELL GENETICS

33

absence of a lac inducer IPTG, only if it harbored a plasmid containing achromosomal fragment ranging from P mra to fts W or further downstream.
The first two genes of the cluster, not known for their function, have beensuspected to be involved in cell division, because their homologs are wellconserved in the homologous clusters of many other bacterial species including Micoplasma genitalium, a peptidoglycan-free bacterium. However, whena plasmid expressing seven genes ranging from ftsL, the third gene of thecluster, tofts W under a promoter in the vector region was introduced into theP mra :: Plac strain, the strain grew normally in the absence of IPTG. The thirdand fourth genes,ftsL andftsI, have been proven essential for cell division,and a plasmid expressing five genes ranging from murE, the fifth gene, toftsW did not support the growth of the Pmra :: Plac strain in the absence of
IPTG. When the first two genes on the chromosome were destroyed bydisplacing a Pvull fragment covering most parts of them with an antibiotic
resistance gene, the resultant strain showed no defect in growth or division.
We concluded that these two genes are dispensable at least under laboratory
conditions.
(5)

The spr Gene of Escherichia coli

Hiroko OHFUCHI 1, Masayo NAKAKOUJI!, Mitsuaki ISHIHARA!, Yasuo
NAKAZAWA!, Yukinobu NISHIMURA!, and Hiroshi HARA C Department of
Biomolecular Sciences, Faculty of Science, Toho University)
Escherichia coli !:¥Jrc mutants lacking Pre, a periplasmic proteolytic
enzyme involved in the C-terminal processing of penicillin-binding protein(PBP) 3, show thermosensitive growth at low osmolarity. Suppressor
mutations that reversed the thermosensitivity were all mapped at 47 minuteon the chromosome map. These spr mutations caused thermosensitivity at
low osmolarity in the prc + background.
From the Kohara it phage genomic library we found a clone that could
transduce spr into an spr- recipient expressing the cI repressor from aplasmid. We subcloned the spr gene and determined its nucleotide sequence.
The predicted product was a 21-kDa lipoprotein. When the spr gene wasoverexpressed, a 21-kDa protein was detected in the envelope fraction. This
product seemed to be degraded by Pre protease, because the protein band on
SDS-gel was more intense in a !:¥Jrc mutant than in the wild type. Primer

34

extension experiments revealed two promoters for the spr gene, the upstream
one being stronger than the other. Two spr mutations were analyzed for their
nucleotide sequences: one had an insertion of lSI between the two promoter
sequences, reducing the expression a great deal; and the other had a one-baseinsertion, truncating the product to about one third of the wild-type product.
Hyperexpression of spr using the A PL promoter caused cell lysis. A !1prc
mutant, in which the Spr protein is not degraded by the protease, lysed fromsimultaneous thermal and osmotic stresses. Such stresses caused spr mutants
to cease growing. Hyperproduction of Prc protease, which would reduce theamount of intact Spr, also caused growth cessation. Thermosensitivity on a
low osmolarity medium of an spr mutant was suppressed by overproductionof PBP 7, a peptidoglycan DD-endopeptidase. This multicopy suppression
was not observed by mutant PBP 7 lacking the Do-endopeptidase activity.
The deduced amino acid sequence of Spr was found to show significant
homology to peptidoglycan endopeptidase II of Bacillus sphaericus and to the
C-terminal region of p60 proteins of many Listeria species, which seems
involved in cell separation after division. Thus we suppose that Spr may
function in cell wall formation as a peptidoglycan-hydrolyzing enzyme.
(6)

Peptidoglycan Polymerase with no Crosslinking
Activity in Escherichia coli

Hiroshi HARA
Penicillin-binding proteins (PBPs) la and lb of E. coli are bifunctional
peptidoglycan-synthesizing enzymes with a glycan-polymerizing domain in
their N-terminal halves and a peptide-crosslinking domain in their C-terminal
halves. Their activity is essential for growth: a mutant lacking both PBP laand Ibis not viable, although a mutant lacking eitherof them is viable. Morethan 10 years ago we discovered a monofunctional enzyme that possess
glycan-polymerizing, but no crosslinking activity, and purified and characterized it. It was to be shown responsible for a considerable part of the total
in vitro glycan-polymerizing activity measured.
Recent progress in the genome project led to identification of a gene, at 72
minute on the chromosome map, whose predicted product shows homology
to the N-terminal glycan-polymerizing domain of the bifunctional PBPs, butlacks a region corresponding to the C-terminal domain. A Swiss group

B.

DEPARTMENT OF CELL GENETICS

35

cloned this mgt gene, and showed a significant increase in in vitro peptidoglycan synthesis with overexpression of the gene. I disrupted this gene byinserting an antibiotic resistance gene, and found such a mutant strain grewnormally. This insertion mutation could be transduced into mutants defective in either PBP la or 1b without causing any apparent growth defect.
These results indicate that mgt is not an essential gene.
(7)

Functional Dissection of Cell-Division Inhibitor SulA of
Escherichia coli and Its Negative Regulation by Lon

Atsushi HIGASHITANI, Yoshiyuki ISHII!, Yasuhiko KATO!, and Kensuke
HORIUCHI C Department of Applied Chemistry, Faculty of Engineering,
Kyusyu Institute of Technology)

E. coli SulA is induced by SOS response and inhibits cell division throughinteraction with FtsZ. To determine which region of SulA is essential for thefunction of cell division inhibition, we constructed a series of N-terminal andC-terminal deletions of SulA and a series of alanine substitution mutants.
Arg 62, Leu 67, Trp 77, and Lys 87 in the central region of SulA were foundessential for the inhibitory activity. N-terminal residues of SulA ranging
from the 3rd to the 27th amino acid and C-terminal 21 residues weredispensable for the activity. The mutant protein lacking N-terminal residues
from the 3rd to 47th was inactive as was that lacking C-terminal 34 residues.
C-terminal deletions of 8 or 21 residues increased the growth-inhibiting
activity in Ion + cells, but not in Ion - cells. The wild-type and mutant SulA
proteins were isolated in a form fused to E. coli maltose-binding protein(MBP), and tested in vitro for sensitivity to Lon protease. Lon degradedwild-type SulA and its deletion mutant lacking N-terminal 93 amino-acids,
but did not degrade it if 21 residues at the C-terminus were deleted.
Futhermore, the wild-type SulA and the N-terminus-deleted mutant formed
a stable complex with Lon, while the C-terminus-deleted mutant did not.
Only 20 C-terminal residues fused to MBP formed a stable complex with, butwere not degraded by, Lon. The LacZ protein that was fused at its
C-termnus to 8 or 20 amino-acid residues from the C-terminus of SulA wasstable in Ion + cells. These results indicate that the C-terminal 20 residues of
SulA are sufficient for recognition by, and for complex formation with, Lon.
They are necessary, but not sufficient, for degradation of SulA by Lon. For

36details, see Ref. 3.
(8)

Search for Homologs of Human ATM Gene in C. elegans

Atsushi HIGASHITANI, Shusei SATOI, and Takeshi ISHIHARA 2 C Kazusa
DNA Research Institute, 2 Multicellular Organization Laboratory, NIG)
Weare interested in check point controls after meiotic recombination andmitotic DNA damage. In the last year the human ataxia telangiectasia
mutated (ATM) gene was isolated and characterized by Savitsky, K. et al.
(Science, 268, 1749-1753, 1995). The ATM protein contains a PI-3 kinasedomain in its C-terminal region. This feature is shared with a group ofproteins including rad3 in S. pombe, MECI (ESRl) and TELl in S. cerevisiae,
MEI-4l in D. melanogaster, and human DNA-dependent protein kinase.
From a blast search of a C. elegans database (ACEDB), a homolog of
ATM was identified in cosmid T06E4.3. We named the gene atl-l (ATM like
1). The primary structure of the cDNA was determined by sequencing the
RT-PCR products of C. elegans mRNA and the yk76g7 cDNA clone. The
total length of the cDN A is 7711 bp and the putative gene product consists of2514 amino acid residues. Comparison of amino-acid sequences of the PI-3
kinase motif indicated that the ATL-l sequence is much closer to MECI
(ESRl), TELl, MEI-4l, than to bovine plIO which is the catalytic subunitof PI-3 kinase. By using RT-PCR, it was found that the level of mRNA of
atl-l was increased about 3-5 fold in the adult stage, while it was expressed inall stages. We have carried out injection experiments of atl-l anti-sense
RNA, whose length was about 2 kb starting from the 3' poly A terminus, into
the N2 wild-type worm. Surprisingly, the phenotype of the fl progenies was
extremely pleiotrophic. We could classify the phenotypes into various
categories such as dead egg, Ll lethal (irregular Ll), protruding vulva,
abnormal gonad, irregular oocyte, sterile, dpy or small size, burst from vulva,
abnormal intestine, unc, and high incidence of males. These results may
suggest that atl-l is involved in a check point control of mitosis and meiosis,
as ATM is. The defect of the gene function seems to result in premature
segregation of meiotic chromosomes and/or in increase of mutations inseveral different cells.
We thank Shigemi Sasamoto and Kaoru Naruo for technical assistance,
Kensuke Horiuchi, Ryuichi Hishida, Norio Suzuki, Isao Katura, Hiroaki

B. DEPARTMENT OF CELL GENETICS

37

Tabara and Yoshito Sadaie for helpful comments, Yuji Kohara for cDNA
clones and discussions, and Tim Schedl for very helpful suggestions.
Publications

Higashitani, A., Higashitani, N., and Horiuchi, K.: Minus-strand origin of filamentous
phage versus transcriptional promoters in recognition of RNA polymerase. Proc. Natl.
Acad. Sci. USA, 94, 2909-2914, 1997.
2. Higashitani, N., Higashitani, A., Guan, Z. W., and Horiuchi, K.: Recognition mechanisms of the minus-strand origin of phage fl by Escherichia coli RNA polymerase. Genes
to Cells, 1, 829-841, 1996.
3. Higashitani, A., Ishii, Y., Kato, Y., and Horiuchi, K.: Functional Dissection of aCell-Division Inhibitor SulA of Escherichia coli and its negative regulation by Lon. Mol.
Gen. Genet, 254, 351-357, 1997.
I.

B-c.

(1)

Division of Cytoplasmic Genetics

Cell-cycle-dependent Expression of the STK-l Gene Encodinga Novel Murine Putative Protein Kinase

Hitoshi NIWA, Kuniya ABE, Takahiro KUNISADA, and Ken-ichi YAMAMURA
We cloned a novel putative serine/threonine kinase-encoding gene, designated STK-l, from murine embryonic stem (ES) cell and testis cDNA
libraries. The kinase most closely related to STK-1 is Xenopus laevis XLP46
protein kinase which shows 71 % amino acid identity to STK-l between theirkinase domains. Nevertheless, STK-l is conserved throughout the phylogeny
with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast. STK-l mRNA is detected in testis, intestine andspleen, tissues that contain a large number of proliferating cells, but not inother tissues. All cell lines tested expressed STK-I mRNA with levels being
dependent upon proliferation rates. In NIH 3T3 cells, STK-l is expressed ina cell-cycle-dependent fashion. These findings suggest a role for STK-l in cell
growth. For details, see Ref. 1.

38

(2)

Endoderm-Specific Gene Expression in Embryonic Stem Cells
Differentiated to Embryoid Bodies

Koichiro ABE, Hitoshi NlwA, Katsuro IwASE, Masaki TAKIGUCHI, Masataka MORI, Shinichi ABE, Kuniya ABE, and Ken-ichi YAMAMURA
Mouse embryonic stem (ES) cells can differentiate into various cell types
within cell aggregates called embryoid bodies (EBs). This structure consistsof ectodermal, mesodermal and endodermal tissues, and resembles embryosof the egg-cylinder stage. After 8-10 days in culture, about half of the EBs
expand into large cystic structures homologous to the visceral yolk sac of
postimplantation embryos. To study endoderm differentiation at the molecular level, we examined expression of endoderm marker genes during theprocess of embryoid body (EB) development. a-fetoprotein (AFP) andtransthyretin (TTR) transcripts increased at the stage when embryoid bodies
began to form yolk sac like structures and were expressed strongly thereafter.
Expression of HNF4 (HNF; hepatocyte nuclear factor ), a variant form of
HNFI (vHNFI, also called HNFl,8), and HNF3,8 started before the onset of
AFP and TTR expression. HNFI (also called HNFla) expression began a
few days after the onset of the expression of the transcription factors
described above. The serum albumin (ALB) transcript was only found inlate large cystic EBs. Also AFP gene expression preceded ALB gene
expression. These results suggest that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo, and EB formation may serve as an in vitro system for studying
these differentiation processes. For the details, see Ref. 2.
(3)

Nuclear Organization in Fission Yeast Meiosis

Yasushi HIRAOKA
We found that in fission yeast meiotic prophase, telomeres are clusterednear the spindle pole body (SPB; a centrosome-equivalent structure in fungi)
and take the leading position in chromosome movement while centromeres
are separated from the SPB. The movement of chromosomes that preludes
meiosis was observed in living cells of fission yeast by fluorescence microscopy. Further analysis by in situ hybridization revealed that the telomeres
remain clustered at the leading end of premeiotic chromosome movement,

B.

DEPARTMENT OF CELL GENETICS

39

unlike mitotic chromosome movement in which the centromere leads. While
it is generally believed that the centromere is a unique chromosomal site that
leads the chromosome movement, our finding demonstrates a striking example in which telomeres can lead chromosome movement.
The meiotic telomere position contrasts with mitotic nuclear organizationin which centromeres remain clustered near the SPB and lead chromosome
movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. We analyzed thenuclear location of centromeres and telomeres in genetically wellcharacterized meiotic mutant strains. An intermediate structure for thetelomere-centromere switching was observed in haploid cells induced tomeiosis by a synthetic mating pheromone; fluorescence in situ hybridization
revealed that in these cells, both telomeres and centromeres were clusterednear the SPB. Further analyses in a series of mutants showed that thetelomere-centromere switching takes place in two steps; first, association of
telomeres with the SPB and second, dissociation of centromeres from the
SPB. The first step can occur during the haploid state in response to a mating
pheromone, but the second step does not take place in haploid cells andprobably depends on conjugation-related events. In addition, a linear minichromosome was also colocalized with authentic telomeres instead of centromeres, suggesting that telomere clustering plays a role in organizing
chromosomes within a meiotic prophase nucleus. (Chikashige et al., EMBO
J., 16, 193-202, 1997)(4)

Fluorescence Imaging of Living Mammalian Cells

Yasushi HIRAOKA
Cellular events are accomplished by a coordinated interaction of molecularcomponents within a cell. Continuous observation of such interaction inliving cells can be essential to the understanding of the temporal and spatial
relationships of those molecular components. With its molecular selectivity
in imaging, fluorescence microscopy has become of increasing importance formolecular cell biology, especially since the advent of green fluorescentprotein. Toward this end, we have developed fluorescence microscopy
techniques to examine the dynamics of specific molecules in living mammalian cells. Multple-wavelength imaging in living cells enabled visualiztion of

40

dynamic interactions of cellular structures during mitosis. Continuous
observations provide a unique oppotunity to detect transient, ephemeral
events which can not be detected in fixed specimens. (Hiraoka and Haraguchi, Chromosome Res., 4, 173-176, 1996)
Publications
1.

2.

3.
4.

5.

Niwa, H., Abe, K., Kunisada, T., and Yamamura, K.: Cell-cycle-dependent expression of
the STK-I gene encoding a novel murine putative protein kinase. Gene, 169, 197-201,
1996.
Abe, K., Niwa, H., Iwase, K., Takiguchi, M., Mori, M., Abe, S., Abe, K., andYamamura, K.: Endoderm-specific gene expression in embryonic stem cells differentiatedto embryoid bodies. Exp. Cell Res., 229, 27-34, 1996.
Hiraoka, Y. and Haraguchi, T.: Fluorescence imaging of mammalian living cells. Chromosome Res., 4, 173-176, 1996.
Kikuchi, S., Sonobe, K., Shinohara, D., Ohyama, N., Mashiko, S., and Hiraoka, Y.: A
double-axis microscope; and its three-dimensional image position adjustment based on an
optical marker method. Optics Commun., 129, 237-244, 1996.
Chikashige, Y., Ding, D.-Q., Imai, Y., Yamamoto, M., Haraguchi, T., and Hiraoka, Y.:
Meiotic nuclear reorganization: switching the position of centromeres and telomeres infission yeast Schizosaccharomyces pombe. EMBO J., 16, 193-202, 1997.

41

(1)

C.

DEPARTMENT OF ONTOGENETICS

C-a.

Division of Developmental Genetics

Control of Neuronal Identities by the Drosophila
Nuclear Receptor, SEVEN-UP

Yasushi HIROMI and Steven R. WEST! C Department of Molecular Biology,
Princeton University)
Our major research goal is to understand the molecular mechanism bywhich neuronal diversity is generated. While much has been learned about
how a cell becomes a neuron, little is known about how each neuron adopts
its identity as a specific neuron. We have previously shown that the seven-up
gene controls photoreceptor neuronal identities in the Drosophila retina; lossof seven-up function results in the transformation of one neuronal type toanother. This is one of the clearest examples of genes that control thedecision between different neuronal types. SEVEN-UP is a member of thenuclear receptor family and is likely to act by regulating transcription of its
target genes. Expression of seven-up is confined to the neurons whose fate isaffected in seven-up mutants, thus SEVEN-UP is likely to be an autonomous
factor that specifies the fate of individual neurons. SEVEN-UP is highly
conserved throughout evolution. The human and Drosophila homologs of
SEVEN-UP exhibit more than 90% amino acid identity in both the DNA
binding domain and the ligand binding domain. Vertebrate homologs of
SEVEN-UP are also expressed in a spatially restricted pattern during nervous
system development, suggesting that they may also playa role in neuronal
specification.
Through a series of misexpression studies, we showed that expression of
SEVEN-UP in any cell type within the ommatidium that normally does not
express SEVEN-UP results in an alteration of its cell fate. Each cell has aunique response and unique responsive period, suggesting that misexpressionof SEVEN-UP interferes with a molecular machinery that is used in manycell fate decisions. Surprisingly, generation of these phenotypes does not
require the presence of the DNA binding domain of SEVEN-UP; expression

42of the chimeric molecules containing a heterologous DNA binding domain
and the SEVEN-UP ligand binding domain causes many of the same phenotypes in the eye observed following misexpression of the intact SEVEN-UP
protein. The presence of a conserved DNA binding domain in SEVEN-UP
implies that the endogenous protein acts by binding to its target DNA. On
the other hand, production of similar phenotypes by misexpressing the intact
protein and the protein lacking its DNA binding domain suggests that themechanism by which misexpressed SEVEN-UP acts is by titrating anothermolecule, presumably a protein (which we call protein X). We expect
protein X to be an evolutionarily conserved molecule that is involved in manycell fate decisions. Currently we are conducting genetic and molecular
screens to identify protein X.

(2)biparous, a Novel bHLH Ggene Expressed in Neuronaland Glial Precursors in DrosophilaAndrew BUSH!, Yasushi HIROMI, and Michael COLE!
Molecular Biology, Princeton University)

C Department of

Genetic studies have uncovered many genes that are involved in the firststeps of neuronal development in Drosophila. Less is known about theintermediate steps during which individual precursor cells follow either theneuronal pathway or the glial pathway. We have identified a novel bHLH
gene, biparous, that is expressed in neuronal and glial precursors in the
Drosophila embryo. Unlike most bHLH genes whose expression is confinedto early neurogenesis, biparous expression continues towards the final stagesof embryonic development. Expression of biparous is not observed in
postmitotic neurons and precedes the expression of repo, a gene activated inlater stages of glial differentiation. The bHLH domain is sufficiently different
from previously described bHLH domains to imply a novel function. See
ref. 1.

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(3)

DEPARTMENT OF ONTOGENETICS

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KLINGON, a New Member of the Drosophila Immunoglobulin
Superfamily, Participates in the Neuronal Differentiationof the R7 Photoreceptor Neuron

Samantha J. BUTLER!, Sandip RAY!, and Yasushi HIROMI C Department of
Molecular Biology, Princeton University)
Of the eight photoreceptor neurons of the Drosophila compound eye, theR7 neuron is the only UV-sensitive neuron and also synapses in the layer ofthe optic lobe that is different from all other photoreceptor neurons. Startingfrom an enhancer trap line, we identified a transcription unit, klingon, that isexpressed in a restricted pattern of neurons during embryonic neurogenesis
and in the R 7 photoreceptor precursor throughout its development. klingon
is a member of the Immunoglobulin superfamily and encodes a putative
protein of 528 amino acids and contains three C2-type Immunoglobulin-likedomains followed by one fibronectin type III repeat. When KLINGON isexpressed in S2 tissue culture cells, it is associated with the cell membrane bya glycosyl-phosphatidylinositollinkage and can mediate homophilic adhesion.
Genetic analysis has revealed that klingon is an essential gene that participates in the development of the R 7 neuron. Ectopic expression of klingon in
all neurons in a genetic background that lacks the R 7 neuron causes therhabdomere of the R8 neuron to project more apically, suggesting that
KLINGON may be involved in positioning of the photoreceptor rhabdomere. See ref. 2.
(4)

Cell Lineage Analysis of the Mushroom Body

Kei ITO!, Wakae AWANO!, Kazumi SUZUKI!, Yasushi HIROMI, and Daisuke
YAMAMOTO! C ERATO at Mitsubishi Kasei Institute of Life Sciences)
During neurogenesis, a large number of neuron and glia are generated
from neuroblasts, bipotential precursors for both cell types. We developed amethod to express any UAS-linked reporter gene in clonal progenies ofneuroblasts that express GAL4 under a constitutive promoter. Using thismethod we performed cell lineage analysis in the adult Drosophila brain, toanalyze the development and organization of the mushroom body (MB),which is an important center for higher-order sensory integration and learning. We showed that the four mushroom body neuroblasts (MBNbs) give

44

birth exclusively to the neurons and glial cells of the MB, and that each of thefour MBNb clones contributes to the entire MB structure. The expression
patterns of 19 GAL4 enhancer-trap strains that mark various subsets of MB
cells revealed overlapping cell types in all four of the MBNb lineages. Partial
ablation of MBNbs using hydroxyurea showed that each of the four neuroblasts autonomously generates the entire repertoire of the known MB substructures. See ref. 3.
(5)

Hydra Peptide Project I. LWamides as Induces ofMetamorphosis of Planula Larvae of Cnidarians

Toshitaka FUJISAWA, Masayuki HATTA, Hiroshi SHIMIZU, Kenji IWAOl,
Osamu KOIZUMI 2 , Yoshihisa KOBAYAKAWA 3 , Toshio TAKAHASHI4, Yojiro
MUNEOKA4, and Tsutomu SUGIYAMA5 C Akajima Marine Science Laboratory, 2 Fukuoka Women's University, 3 Kyushu University, 4 Hiroshima University, 5 Ishinomaki-Senshu University)
Using the approach described previously (Ann. Report No. 46, 1995), we
isolated 338 peptides from Hydra magnipapiIlata, determined the structure of
222 peptides and chemically synthesized 30 peptides. We selected two groupsof peptides which are involved in morphogenesis of hydra as well as othercnidarians for further analyses.
We isolated 7 LWamides which have a consensus sequence of GLWamide
in their C-termini. A similar peptide, metamorphosin A (pEQPGLWamide)
was isolated from sea-anemone and shown to induce metamorphosis ofplanula larvae of Hydractinia echinata. We also showed that the Hydra
peptides induced metamophosis of planula larvae of Hydractinia serrata andthat the effective domain of the peptides for metamorphosis is G LWamide attheir C-termini (Takahashi et al., 1997). We further examined whether
LWamides are general inducers of metamorphosis of cnidarian planulae. The
phylum cnidaria is composed of 3 classes of animals: hydrozoa, anthozoa andschyphozoa. We chose one or two representatives in each class. Hydractinia
is the representative of hydrozoa, reef-building coral, Acropora of anthozoa
and Cassiopea and Aurelia of schyphozoa. Although preliminary, one of the
LWamides, Hym-54 (GPMTGLWamide) was shown to be effective ininducing metamorphosis of Acropora planulae. Metamorphoses of schyphozoans are currently under examination. These results show that LWamides

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are general inducers of metamorphosis in at least two of the 3 classes ofcnidarians.
(6)

Hydra Peptide Project II. Two Peptides Involved inRegulation of Foot Formation in Hydra

Hiroshi SHIMIZU, Masayuki HATTA, Toshio TAKAHASHI!, Yojiro MUNEOKA!, Tsutomu SUGIYAMA2 and Toshitaka FUJISAWA C Hiroshima University, 2Ishinomaki-Senshu University)
We screened 30 synthetic peptides for their ability to enhance foot regeneration of hydra. Animals were treated with a peptide for 24 h and the upper
foot region was isolated and allowed to regenerate for another 24 h in theabsence of the peptide. At the end of regeneration, the pieces from treatedand untreated control animals, as well, were fixed for immunostaining of footspecific monoclonal antibody (AE-03, a gift of Dr. Kobayakawa of Kyushu
Univ.). Two peptides, Hym-323 and Hym-346 ,were identified as giving a
positive signal in about 20% of regenerating tissues, while control tissues
gave essentially no positive signal. Hym-323, composed of 16 amino acids,
has some homology to head activator (Schaller and Bodenmueller, 1989) at
its C-terminal half. Head activator was originally identified as a head specificmorphogen but later it was shown to be a mitogen and inducer of neurons.
Hym-323 has no effect on head formation. Hym-346, composed of 20 aminoacids, is identical to a recetnly reported foot specific morphogenetic factor,
pedibin purified from H. vulgaris. (Hoffmeister, 1996) except that Hym-323
lacks the C-terminal residue, glutamic acid, of pedibin.
Since the pretreatment of peptides resulted in the enhancement of footregeneration, we assumed that the peptides were involved in chaging positional information along the body axis rather than in speeding up the regeneration process. To confirm this, lateral tissue transplantation experiments were carried out. A small tissue was excised from the mid-position
between the head and the budding region of hydra which had been treated forseveral days and was transplanted to the same position of untreated hosthydra. The frequencies of ectopic foot formation for Hym-323 and Hym-346
were 15% and 17%, respectively. In control transplants, where no peptide
treatment was involved, the frequency of ectopic foot formation was 5%.
These differences were statistically significant. The results strongly suggest

46
that these peptides are involved in determining axial positional values involved in foot formation.

Publications
1.
2.

3.

4.

5.

6.

7.

Bush, A., Hiromi, Y., and Cole, M.: biparous: a novel bHLH gene expressed in glial andneuronal precursors in Drosophila. Dev. BioI., 180, 759-772, 1996.
Butler, S. J., Ray, S., and Hiromi, Y.: klingon, a new member of the Drosophila
immunoglobulin superfamily, is required for the development of the R7 photoreceptor
neuron. Development, 124, 781-792, 1997.
Ito, K., Awano, W., Suzuki, K., Hiromi, Y., and Yamamoto, D.: The Drosophila
mushroom body is a quadruple structure of clonal units each of which contains a virtually
identical set of neurons and glial cells. Development, 124, 761-771, 1997.
Hobmayer, B., Hatta, M., Fujisawa, T., Holstein, T., and Sugiyama, T.: Identification ofa Hydra homologue of the b-catenin/placoglobin/armadillo gene famiily. Gene, 172,155-159.
Takahashi, T., Muneoka, Y., Lohmann, J., Lopez de Haro, Bosch, T. C. G., David, C.
N., Bode, H. R., Koizumi, 0., Shimizu, H., Hatta, M., Fujisawa, T., and Sugiyama, T.:
Systematic isolation of peptide signal molecules regulating development in Hydra: I.
LWamide and PW families. Proc. Natl. Acad. Sci., U.S.A., 94, 1241-1246, 1997.
Takahashi, T., Ohtani, M., Muneoka, Y, Arimoto, S., Hatta, M., Shimizu, H., Fujisawa,T., Sugiyama, T., and Koizumi, O. Structure-activity relation of LWamide peptides
synthesized with a multipeptide synthesizer. Peptide Chemistry, 1996, pp. 193-196, 1997.
Murate, M., Kishimoto, Y., Sugiyama, T., Fujisawa, T., Takahashi-Iwanaga, H., andIwanaga, T. Hydra regeneration from recombined ectodermal and endodermal tissue. II.
Differential stability in the ectodermal and endodermal epithelial organization. J. Cell
Sci., 110, 1919-1934, 1977.

C-b.
(I)

Division of Gene Expression

Regulation of the EDG84A Gene by FTZ-FI during
Metamorphosis in Drosophila melanogaster

Takehide MURATA, Yuji KAGEYAMA, Susumu HIROSE, and Hitoshi VEDA
Transcription factor FTZ-FI is a member of the nuclear hormone receptor
superfamily and is transiently expressed during the mid- and late prepupal
periods in Drosophila melanogaster. A putative pupal cuticle gene, EDG84A,
is expressed slightly following FTZ-FI expression during the prepupal period
and carries a strong FTZ-FI binding site between bases 100 and 92 upstream

C. DEPARTMENT OF ONTOGENETICS

47

of its transcription start site. In this study, EDG84A mRNA was found to beprematurely expressed upon heat induction of FTZ-F1 in prepupae carrying
the heat shock promoter-FTZ-F1 cDNA fusion gene construct. Transgenic
fly lines which had the 0.8-kb region of the EDG84A promoter fused to lacZ
expressed the reporter gene in a tissue- and stage-specific manner. Basesubstitutions in the FTZ-F1 binding site within the 0.8-kb promoter abolishedexpression of lacZ. These results strongly suggest that the EDG84A gene isa direct target ofFTZ-F1. Deletion studies of the cis-regulatory region of the
EDG84A gene revealed that space-specific expression in imaginal disc-derived
epidermis is controlled by the region -408 and -104 bp from the transcription
start site. The region between bp-408 and -194 is necessary to repress
expression in a posterior part of the body, while the region between bp-193
and -104 carries a positive element for activation in an anterior part of the
body. These results suggest that FTZ-F1 governs expression of the EDG84A
gene in conjunction with putative tissue-specific regulators. For details, see
Ref. 1.
(2)

Transcriptional Activation through Interaction of
MBF2 with TFIIA

Feng-Qian LI, Ken-ichi TAKEMARU, Masahide GOTOl, Hitoshi VEDA,
Hiroshi HANDA l , and Susumu HIROSE C Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama)Transcriptional activation of the Drosophila melanogaster fushi tarazu gene
by FTZ-F1 or its silkworm counterpart BmFTZ-F1 requires two cofactors,
MBF1 and MBF2, which do not directly bind to DNA. MBF1 is a bridging
molecule that connects FTZ-F1 (or BmFTZ- F1), MBF2 and the TATA
binding protein TBP. MBF2 is a positive cofactor that activates transcription(Li, F.-Q., Veda, H., and Hirose, S., Mol. Cell. Biol., 14, 3014-3021, 1994).
To elucidate the mechanism of transcriptional activation by MBF2, we
isolated the cDNA coding for the factor. Northern blot analyses showedtemporally restricted expression of MBF2 mRNA similar to that of BmFTZF1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas
natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acidsequence of the factor showed no homology with proteins in the databases.
Farwestern analyses and glutathione S-transferase interaction assays demon-

48

strated that MBF2 makes direct contact with the ,B-subunit of TFIIA. In a
HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activatedtranscription from various promoters as natural MBF2 did. This activation
requires the MBF2-TFIIA interaction. When recombinant MBF2 was added
to the HeLa cell nuclear extract in the presence of MBFl and FTZ622
bearing the DNA-binding region of FTZ-FI, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the
MBF2-TFIIA interaction. From these results, we propose a model in which
FTZ-FI (or BmFTZ-Fl) and MBFl recruit MBF2 to a promoter carrying
the FTZ-FI site and MBF2 activates transcription through its interaction
with TFIIA to allow selective activation in a FTZ-FI site dependent manner.
For details, see Ref. 2.
(3)

Determination of Wing Cell Fate by the escargot
and snail Genes in Drosophila

Naoyuki FUSE l , Susumu HIROSE, and Shigeo HAYASHI lInvertebrate Genetics)

eLaboratory of

Insect appendages such as the wing and the leg are formed in response to
inductive signals in the embryonic field. In Drosophila, cells receiving suchsignals initiate developmental programs which allow them to become imaginal discs. Subsequently, these discs autonomously organize patterns specificfor each appendage. We here report that two related transcription factors,
Escargot and Snail, expressed in the embryonic wing disc, function as
intrinsic determinants of the wing cell fate. In escargot or snail mutant
embryos, wing-specific expression of Snail, Vestigial and ,B-galactosidase
regulated by the escargot enhancer were found as well as in wild-type
embryos. However, in escargot snail double mutant embryos, wing development proceeded until stage 13, but the marker expression was not maintainedin later stages, and the invagination of the primordium was absent. Fromsuch analyses, it was concluded that Escargot and Snail expression in thewing disc are maintained by auto- and crossactivation. Ubiquitous escargot
or snail expression induced from the hsp70 promoter rescued the escargot
snail double mutant phenotype with the effects confined to the prospective
wing cells. The similar DNA binding specificities of Escargot and Snailsuggest that they control the same set of genes required for wing develop-

C. DEPARTMENT OF ONTOGENETICS

49

ment. We thus propose the following scenario for early wing disc development. Prospective wing cells respond to induction by turning on escargot andsnail transcription, and become competent for regulation by Escargot andSnail. Such cells initiate auto- and crossregulatory circuits of escargot andsnail. The sustained Escargot and Snail expression then activates vestigial
and other target genes that are essential for wing development. This
maintains commitment to wing cell fate and induces wing-specific cell shapechange. For details, see Ref. 3.
(4)

On a Critically Sensitive Period for the Methoprene-induced
Supernumeral Larval Ecdyses in the Mulberry
Silkworm, Bombyx mori (L.)

Akio MURAKAMI and Jun SHIMADA l
Agric. and Technology, Tokyo)

eFac. of Agric., Tokyo Univ. of

The process of metamorphosis from larval to pupal forms is principally
controlled by developmental mechanisms, but this is to some extent influenced by certain endocrine mechanisms. The mechanisms are also concerned in the areas of the central nervous system and brain, involved withperception, recognition, and integration of environmental information.
There is an accumulation of data on effects of juvenile hormone (JH) on
metamorphosis in this insect. The action of JH has effects on several insect
species in terms of larval maintainance or status co. However, the interaction
between JH and brain function has not been analyzed with the silkworm, sofar. In addition, the relationship between the supernumeral larval ecdysis
events and regenerate of the mid-gut has not been elucidated in the insect.
Therefore, we attempted to analyze the developmental processes of the JH
analogue, methoprene-induced supernumeral larval ecdyses in the silkworm.
In this series of experiments, the biological materials used in this study
were F 1 hybrid silkworms obtained from a cross between Jl06 females andDaizo males. The larvae were fed on mulberry leaves at 25°C under standard
conditions. Five different developmental groups, 48, 60, 72, 84 and 96 hr-old
larvae after the 4th ecdysis, were prepared and 50 larvae were assigned toeach group. The insects used in this experiment started spinning about 120
hr after the 4th ecdysis. Methoprene (GL Science, Tokyo) was used as a JH
analogue, JH(a), which has a powerful function similar to JH. The chemical,

50

which was dissolved in acetone solutions at the rate of 10 flg per 10 ml
acetone was applied to the larval body surface at 10 flg per body weight.
Acetone alone was spread on larvae used as controls.
The results of the experiment clearly indicated that larvae at 48 hrs are
indifferent to JH (a), but almost 50% of the treated larvae at 60 hr showedrepeated larval molting. 84 hr larvae showed the most sensitive activity. A
large number of supernumeral ecdysed larvae was detected among larvae
ranging from 60 to 84 hrs. Most larvae at 96 hrs were normally developed
into pupae, but less then 10% of the larvae were super molted again
suggesting that older larvae are irreversibly directed to the next form. Theseobservations showed that larvae ranging from 60 to 84 hrs are in a critical
period for JH(a)-induced supernumeral ecdytsial events.
JH(a) is highly toxic to larvae at 48 and 60 hrs but all surviveing larvae
were transformed to pupal forms, indicating that even the young last instar
larva have the potential to start to some extent for the next form. The
developmental process of the last larval stage before the critical period is acomplicated situation: the larvae have not yet perfectly attained the state ofmetamorphosis into pupae but have the potential to do so. In any case, thelast instar larvae are classified into three developmental steps: before andafter the critical period, which ranges from 72 to 84 hrs. The critical period
is the turning point for developmental direction, either normal pupal molting
or repeated larval molting in the silkworm. During this period larval molting
can occur again in contrast to the normal developmental process in thisspecies. This suggests that even certain genetically determined developmental
processes can be reversed by JH(a). This phenomenon seems to be exceptional, but the repeat of a small period of developmental growth in this insect may
provide a clue to rejuvenescence. The larvae at 96 hr-old did not respond tothe chemical, indicating that they had already irreversibly entered the metamorphosic process to the pupal form. The same view has been described by
Sakurai (1985).
It is a well-known fact that tetramolting larvae at 48 hr-old have asignificant amount of the JH titre, but not the ecdysone. No supernumeral
ecdysial event has occurred in such a hormonal situation. There is apossibility that a JH receptor cell is involved in this developmentally stage
specific phenomenon. It can be assumed that the receptor cell in young larvae
just after the 4th ecdysis is inactivated by certain regulators under the controlof the developmental mechanism. As will be made clear in the nextC.

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communication, the brain is a regulator of the JH receptor cell.
(5)Effects of Methoprene, a Juvenile Hormone Analogue,
on the Induction of Supernumeral Molting underthe Influence of Brain in the Bombyx Silkworm

June SHIMADA l and Akio MURAKAMI C Fac. of Agric., Tokyo Univ. ofAgric. and Technol. Tokyo)
This study was carried out to clarify the brain function on the methoprene
induced in supernumerally molting larvae. The experimental materials andmethods used in the research were the same as in the previous report except
for certain kinds of operations, the extirpation and implantation of the brain.
These operations were involved the ether anesthesia method at 20 e (Shimada, 1989). Furthermore, certain larvae, which their brains removed were
implanted with two brains per larva, obtained from two donors in the samedevelopmental stage. The larvae were further kept for 12 hrs at 20 e after
anesthesia until complete recuperation. The larvae, which had brains removed, could not feed on mulberry leaves and they showed a fasting
condition. Accordingly, the larvae on total abstinence from the leaves forseveral days were prepared as corresponding controls. Methoprene was also
spread to the larvae starved as occasion demands. An ointment of methoprene dissolved in acetone solution was spread on the body surface of brainlesslarvae.
The results of the experiments under fasting conditions clearly showed thatthe supernumerary ecdysed larvae ranging from 72 to 84 hr-old were highly
detected from 40 to 82% in the methoprene treated insects, but no supernumerary ecdysed larvae were observed in the sham operation as the control.
The event was induced in 8% of the subject at 96 hr-old larvae. This feature
was the same as the experimental group after treatment of larvae withmethoprene under feeding condition as described in the previous communication.
In the brain extirpation group, all surviving larvae developed and/or
metamorphosed to pupae, indicating that an effect of methoprene on the
induction of the supernumerally ecdysed larva was not observed without theexistence of the brain regardless of methoprene treatment.
In the next experiment, brain implantations were conducted to confirm the
0

0

52

experimental results. The experimental plane was limited to 20 larvae at 84
hr-old, because the operation is complicated and/or required much time. In
this experiment, following extirpation, implantation of brain and applicationof methoprene, no dead larvae were detected and only two larvae among 20
insects developed to pupae, but the remaining 18 larvae exhibited supernumeral molting, and once again larval ecdysed. Thus, it can be concluded
that in larvae treated with methoprene, the brain is absolutely required for
induction of supernumerally ecdysed larvae.
Supernumeral events were not detected in brainless larvae regardless oftreatment with methoprene. No supernumerally molt larvae at any stage
were detected, in brainless larvae, irrespective of the post-spreading of
methoprene in addition to a high frequency of death in larvae ranging from48 to 84 hr-old. In other words, the brain has a critical function in theexertion of the original function of JH(a) or the JH function is under thecontrol of the brain.
As is well-known, in the silkworm, JH titres of larvae during an early
developmental stage of the last instar are significant, but this event did nottake place. This contradiction can be explained on the basis of the hypothesisthat an additional step, activation of the JH(a) receptor cells, as has beenreported in numerous hormones, takes part in this biological event. At
present, it appears that some substance secreted from secretary cells in thebrain activates the receptor cell to receive JH(a) and consequently, thehormone becomes functional.
(6)

On a Biological Clock in the Mulberry Silkworm,
Bombyx mori (L.)

Akio MURAKAMIIn the silkworm, the daily rhythm of either hatching or eclosion is notdetected in the strict sense of the word, because the egg-hatching neveroccurs again and/or the adult eclosion never occurs again. Nevertheless,some sericulturists have reported that hatching and/or eclosion in this insect
is controlled by a circadian oscillator or biological clock system. Consideringsuch a situation, we have analyzed biological rhythms on the level of the
population.
We have been preserving several chronology mutants relative to both the

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behaviors in the insect species. In the silkworm, normal hatching (h +) andeclosion (e+) behaviors begin 15 to 30 min after sun-rise and reach the
maximum ca. 45 to 60 min after the start of these behaviors every day
throughout the year. After that the behaviors usually decrease and aresuspended about 1 hr later. The maximum frequency of these behaviors innormal stocks correctly changes with the seasons, indicating that both eventsare under the control of the movement of two heavenly bodies, the sun andthe earth. For reference, the hatching event appears to some extent earlier
than the eclosion. Accordingly, it seems unlikely to stand between certain
circadian pacemaker systems and these biological phenomena.
Some larvae of a mutant stock showed a serious delay in hatching times for
4 to 5 hrs as compared with the normal stock, and in addition the stock is alsoca. 15 min delayed for maximum time. A similar event is also observed in an
eclosion mutant e - . These phenomena seem to show a defect in time
regulation mechanism in the brain rather than what is called the biologicalclock. A stock of h tI (and e tI) showed earlier hatching (and eclosion) event
than the normal stock (h + and/or e+) and these stocks began their behavior
before sunrise, suggesting that the unique behaviors are controlled by theirown biological clock rather than the photoperiodic rhythm.
Accordingly, it is likely that these normal behaviors are under the control
of both the biological clock and the movement of the heavenly bodies: thebehaviors seemed to originally arise near sunrise.
When the embryos, at hatching time and/or near sunrise were kept in dark
conditions, hatching was significantly prolonged and the remainder hatched
the next day. This feature suggests the existence of a circadian oscillator.
The embryos at hatching time were lit up for a very short time in the earlymorning around 2-3 o'clock, most of them hatched. The behavior is thought
to be a reflex reaction to the flash light, since the behavior occurred a shorttime after illumination with the flash light, suggesting that these behaviors aremainly dependent on certain nervous systems. The same response seemed to
be operating on the eclosion. Therefore, these biological behaviors in thesilkworm are difficult to separate in term of the neurosecretory system andhormonal systems. Accordingly, it is unlikely that there is exclusive participation of the hormonal system in the behaviors. The instantaneous response
seems to be involved in certain neurotransmittants.
Furthermore, adult exclusion consists of two steps: the first emerged frompupal cuticles and from cocoons in the next step. The primary behavior

54

occurs during the developmental process or their metamorphosis whichslowly takes place during the latter half of the pupal stage within theircocoons. It is logical to assume that certain endocinological substances areconcerned in the process to some exent. The moths survive on certain protein
enzymes before emergence from their cocoon shells. This terminal event in
the pupae seems to be regulated by either the strength (or weakness) of thesunlight conditions or their own circadian pacemaker or by both. Accordingly, it is possible to say that the behavior involves enzymes rather thanhormonal substances. For reference, we are preserving a recessive mutant
stock which shows an incomplete pupal ecdysis for adults.
(7)

Biological Meanings of Silk Production in the Mulberry
Silkworm, Bombyx Mori (L.)

Akio MURAKAMI
All living things ingest food to maintain life as well as to preserve their
species. The silkworms use mulberry leaves to maintain their life, but why dothey produce silk and/or cocoons. The cocoon may function to protect pupafrom a various kinds of external hazardous factors. In fact, the silkworm
spins silk fibers. In this report, the author considers the biological meaning ofsilk production in this insect.
It is axiomatic that the silkworm produces the proteinous fiber for theirlife, not for human beings. This statement is logical, even though, people are
apt to think that the insect makes the silk for humans. This view is not
acceptable from a biological viewpoint. The silkworm is regarded as anoligophagous insect feeds on mulberry leaves as its natural food. The leaves
are complete in various kind of amino acids. The leaves are nutritional food
stuffs for the silkworm larvae, but at the same time they contain many more
non-essential amino acids. It is clear that the essential amino acids have ahalf of all kinds of fibroin and/or sericine, and contain large amounts ofalanine (Ala), glycine (Gly), serine (Ser) and tryptophane (Try), indicatingthat these amino acids are of a non-essential type for the Bombyx larvae. As
the larvae ingest a large quantity of both essential and non-essential aminoacids from the mulberry leaves, the latter amino acids need to be excreted
outsides of the body as polymeric silk proteins in neutralized forms. The
more the insect feeds on mulberry leaves, the more nitrogenous substances

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accumulate within the body. In general, an excess of nitrogenous substancesin the body is toxic to the insect as in other organisms. This point requires
attention in rearing the Bombyx silkworm as one step in detoxification of theunnecessary amino acids. Accordingly, the insect has to protect itself fromthe toxic substances. As is well known, the silk proteins are the product ofpolymerization of amino acids in silkglands. Polymerization of the aminoacids during neutralization seems to be a sophisticated detoxification mechanism. It is clear that the silkworm excretes an excess of the non-essential
amino acids for the larvae as the form of silk proteins rather than an objective
product, suggesting that the silk production is necessary for maintaining life.
In the silkworm, high productive lines often show physiological weakness,sensitivity to infection and so on. A mutant stock, Nd, is remarkably weakbecause this spineless line lacks the ability to spin the silk matter to the out
side of the body, so the matter remains in the pupal-body. In this insect, an
overfed larva is apt to have a shortened adult lifetime.
In domesticated animals, beef cattle, for example, the use of the entire or
partial body is the object of breeding them. As learned from the mechanismof silk making, the strategy of Bombyx breeding is essentially different fromthat for mammals. However, the current breeding methods for silkworms ismuch the same as that for animals. It seems to be to force the larva to growfat or to overfed it. In other words, it appears to force the insect to excrete
the disused amino acids. But the silkworm appears to defend itself against
from such rearing conditions. Consequently, it is necessary to improve themethods of rearing and/or breeding the domesticated silkworm in terms ofbetter balanced dietetic control.
(8)

Are Cultured Cells Situated between Uni- andMulticellular Organisms?

Kikuo IWABUCHI 1 and Akio MURAKAMI C Fac. of Agric., Tokyo Univ. ofAgric. and Technol. Fuchu, Tokyo)
Living organisms can be classified into unicellular and multicellular types
according to the nature of their cell organization. Both types live as
individual organisms. The latter type is composed of multiple cells, each ofwhich does not act individually. In this type of organism, the central nervous
system or brain maintains body function. Any cell of this class of organisms

56

has identical genetic information. However, each cell has a tissue specificcharacter as a result of cell differentiation. Any cultured cell line, which isderived from a multiple organism, has a large genomic size compared tounicellulars.
Cultured cells, which are derived from various kinds of tissues in multicellular organisms, are free from the brain-nerve system and its relatedorgans. Although cultured cells possess the same genome set as their original
tissues, but only express the characteristics for living singly in culture media.
Accordingly, nature may allow the cells to live a life of independence fromthe control of the brain-nerve system. It is likely that the primitive phase in
the course of the establishment of cell lines takes place in three steps: the firststep it becomes independent of cells from the original tissues, and the second
one is adaptation of the cells to the culture medium, which is a novel
environment for the cell line. In the adaptation of the cells, it should benecessary as a result of changes of gene expression in the new environmental
condition. Thus, the cell line can be established an unicellular organism inthe culture media independent of a subordinate state of the brain-nerve
system.
The body structure and its activity in insects are regulated under thecontrol of the brain-nerve and/or endocrine systems. In addition, it should
be noted that a certain cultured cell line in insects is sensitive to ecdysone, as
communicated in the previous report (lwabuchi and Murakami, 1996).
Certain ecdysone treated culture cells are transformed to the nerve cell type
(Courgeon, 1972). Beermann and Clever (1964) reported a certain puffing
on salivary gland chromosomes after the treatment of Chironomus tentans
with ecdysone, suggesting a direct binding of the hormone to DNA. The
response is considered to be the result of gene activation by ecdysone.
Ecdysone binds to the hormone receptor (Turberg et al., 1988) and finally to
DNA (Turberg et al., 1992) in the C. tentans cultured cell. From thisevidence, it is thought that ecdysone directly activates gene transcription in
cultured cells. It is also possible to say that certain cultured cell lines of theinsect possess the ability to accept endocrinal regulation as does the originalin vivo multicellular organism, suggesting that the culture cell may have an
intracellular regulato~. Consequently, it can be said that the cultured celllines are generally more closely related to the multicellular organisms than tounicellular organisms.

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DEPARTMENT OF ONTOGENETICS

57

Origin of Holometabolous Metamorphosis in Insects

Kiyoshi MINATO
The evolutional ongm of the holometabolous metamorphosis in endopterigote, which is characteristic to the specific developmental stage ofpupa and the unique larval forms which are relatively simple and dissimilar to
adult, which is one of the reasons insects have evolved so successfully, wasinvestigated and analyzed through available literature and observations of thelife styles of various insects in the fields.
Though the meaning of the pupal stage and how it originated in evolutionaly remain unclear, it seems now most reasonable that it corresponds to thelast larval instar stage in the exopterigote, and that it has evolved asintermediate in the large morphological gaps between the larval and adultstages (Hinton, H. E., 1963b). However, considering the hormonal conditions during these stages in which the ecdysis into pupa occurs without any
juvenile hormone, the pupal phase is likely to belong to the adult rather thanthe larval stage. The possibility that "the pupal stage is another one of thetwo adult stages (Poyarkeff, 1914; Hinton, 1948)" may remain to be reconsidered
The unique larval forms most different from those of adults in endopterygotes proved to have probably evolved in order to get actively the newstyles of habitation and feeding, climbed and sticked to the labile, complicated, and sometimes large structure of grasses and trees, and bored or dipped
into some three-dimensional structures such as wood, fruits, pasty-liquids,
and soils, the both circumstances of which must have appeared newly in theprocess of evolution, rather than in order to get the advantages of theunnecessary of the competition for the same habitat and food between thelarval and adult stages.
Publications
1.

2.
3.

Murata, T., Kageyama, Y., Hirose, S., and Veda, H.: Regulation of the EDG84A gene by
FTZ-FI during metamorphosis in Drosophila melanogaster. Mol. Cell. BioI., 16, 65096515, 1996.
Li, F.-Q., Takemaru, K., Goto, M., Veda, H., Handa, H., and Hirose, S.: Transcriptional
activation through interaction of MBF2 with TFIIA. Genes to Cells, 2, 143-153, 1997.
Fuse, N., Hirose, S., and Hayashi, S.: Determination of wing cell fate by the escargot and58

snail genes in Drosophila. Development, 122, 1059-1067, 1996.

C-c.
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Division of Physiological Genetics

A New Class of Transcription Factor DSIF, Which Regulates RNA
Polymerase II Processivity before Transcription Initiation

Tadashi W ADA, Toshiyuki TAKAGI, Anwarul FERDOUS, Yuki YAMAGUCHI,
and Hiroshi HANDA (Faculty of Bioscience and Biotechnology, Tokyo
Institute of Technology)
The purine nucleotide analog 5,6-dichloro-I-,B-O-ribofuranosylbenzimidazole (ORB) has been used to investigate the mechanism of transcription
regulation in vivo and in vitro. We investigated the mechanism of ORB byusing an in vitro reconstituted transcription system. We purified a ORB
sensitivity inducing factor (OSIF) from HeLa cell nuclear extracts. OSIF is
composed of at least two subunits with molecular masses of 160 kOa (pI60)
and 14 kOa (pI4) that confer ORB-mediated transcription inhibition(OMTI) of RNA polymerase II and act as ORB-mediated elongation
inhibitors. Biochemical studies on OMTI with a kinetically synchronizedtranscription reaction demonstratet that OSIF activity can be blocked by anunidentified protein phosphorylation which is inhibited by ORB and thisphosphorylation event takes place before transcription initiation, but affects
the processivity of RNA polymerase II during elongation. Isolation of acorresponding cONA identified OSIF p160 as a 1087 residue protein with anextremely acidic amino terminus and a novel six amino acid repeat at the
carboxyl terminus (consensus=K/R-T-P-A/M-Y-G). In a search of theavailable sequence databases it was found that, p160 has strong homology to
the yeast SPT5 and a Caenorhabditis elegans SPT5 homolog of unknown
function. Recombinant Supt4h protein which is encoded by a human homolog of yeast SPT4 gene was functionally equivalent to OSIF p14, suggestingthat OSIF comprises human homologs of SPT5 and SPT4. The convergenceof biochemical studies on OSIF and genetic analysis of SPT5 and SPT4
suggest that OSIF works as an elongation factor and regulates RNA polymerase II processivity.

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DEPARTMENT OF ONTOGENETICS

59

Analysis of the Mammalian Genome by Fluorescencein situ Hybridization

Katsuzumi OKUMURA (Faculty of Bioresources, Mie University)
We analyzed the structures and DNA replication in the human and mouse
genomes using fluorescence in situ hybridization (FISH). We assigned thechromosomal localization of various genes by using eDNA or genomic
clones. For details, see Ref. 19-26, 28, 29.
On the other hand, FISH was used to analyze how DNA replication timing
is related to genome structure and how the specific genome domains areorganized in the nucleus.
First, we determined the replication timing patterns of DNA segments
within imprinted gene regions, such as those for Igf2, Igf2r, and PraderWilli!Angelman Syndrome (PW/ AS). Asynchronous replication timing wasobserved in the large area of each domain. These regions may consist of
long-range replication domains which cover larger regions than those subjected to genomic imprinting. In particulor, we found that the PW/ AS
region consists of smaller replication timing domains in which either thepaternal or maternal allele replicates earlier than the other. To analyze theintranuclear organization of imprinted gene regions, mouse embryonic fibroblast cells were cultured on microscope slides, and fixed with paraformaldehyde to keep the three dimensional structure of the nuclei. The nuclei were
subjected to multicolor FISH using the three probes in the Igf2r domain. The
FISH signals of the early replicating allele were dispersed in the nuclei,
whereas those of the late replicating allele occupied a compact area, before
and after their DNA replication. The experiment using the probes in the
PW/ AS region showed the same results, strongly suggesting that the imprinted gene regions are regulated to form the allele specific genome organization in the nuclei. For details, see Ref. 31.
Other studies on DNA replication timing and the chromosome bands weredone in the human MHC region, as descriped in Ref. 17, 32.
We also studied biosynthesis and metabolism in NAD and its related
compounds. For the details, see Ref. 18, 27, 30.

60

Publications
1.

2.

3.
4.

5.

6.

7.

8.

9.

10.

11.

12.

Hayashida, N., Sumi, Y., Wada, T., Handa, H., and Shinozaki, K.: Construction of acDNA library for a specific region of chromosome by a novel cDNA selection method
using latex particles. Gene, 165, 155-161, 1995.
Tanaka, H., Makino, Y, Hiramoto, M., Handa, H., and Makino, I.: Potentiation of
glucocorticoid-mediated gene expression by the novel benzoquinone derivative (2E)-3-[5(2,3-dimethoxy-o-methyl-l,4-benzoquinoyl)]-2-propenoic acid (E3330). Eur. J. Pharm.,
291, 121-127, 1996.
Watanabe, K., Handa, H., Mizumoto, K., and Nagata, K.: Mechanism for inhibition of
influenza virus RNA polymerase activity by matrix protein. J. Virol., 70, 241-247, 1996.
Goto, M., Shimizu, T., Sawada, J-I., Sawa, C., Watanabe, H., Ichikawa, H., Ohira, M.,
Ohki, M., and Handa, H.: Assignment of the E4TFI-60 gene, an Ets-related transcription
factor, to human chromosome 21q21.2--q21.3. Gene, 166, 337-338, 1996.
Imai, T., Sumi, Y., Hatakeyama, M., Fujimoto, K., Kawaguchi, H., Hayashida, N.,
Shinozaki, K., Yajima, H., and Handa, H.: Selective. purification of DNA or RNA using
single-stranded DNA affinity latex particles. Colloid Interface Sci., 177, 245-249, 1996.
Hirano, F., Tanaka, H., Makino, Y., Okamoto, K., Hiramoto, M., Handa, H., andMakino, I.: Induction of the transcription factor AP-l in cultured human colon adenocarcinoma cells following exposure to bile acids. Carcinogenesis, 17,427-433, 1996.
Wada, T., Takagi, T., Yamaguchi, Y., Hiramoto, M., Ferdous, A., Takayama, M., Lee,K. A. W., Hurst, H. C., and Handa, H.: Copurification of casein kinase II with transcription factor ATF/E4TF3. Nucl. Acids Res., 24, 876-884, 1996.
Shiio, Y., Sawada, J-I., Handa, H., Yamamoto, T., and Inoue, J-I.: Activation of theretinoblastoma gene expression by BcI-3: implication for muscle cell differentiation.
Oncogene, 12, 1837-1845, 1996.
Shimada, H., Wada, T., Handa, H., Ohta, H., Mizuguchi, H., Nishimura, K., Matsuda,T., Shioi, Y., and Takamiya, K-I.: A transcription factor with a leucine-zipper motif
involved in light-dependent inhibition of expression of the puf operon in the photosynthetic bacterium phodobater sphaeroides. Plant Cell Physiol., 37, 515-522, 1996.
Hashimoto, M., Chayama, K., Tsubota, A., Kobayashi, M., Saito, S., Arase, Y., Ikeda,
K., Kobayashi, M., Nakano, A., Takagi, K., Koike, H., Okamoto, K., Handa, H., andKumada, H.: Typing six major hepatitis C virus genotypes by polymerase chain reaction
using promers derived from nucleotide sequences of the NS5 region. Int. Hepatol.
Commun., 4, 263-267, 1996.
Kosukegawa, A., Arisaka, F., Takayama, M., Yajima, H., Kaidow, A., and Handa, H.:
Purification and characteriztion of virus-like particles and pentamers produced by theexpression of SV40 capsid proteins in insect cells. Biochim. Biophys. Acta, 1290, 37-45,
1996.
Okazaki, T., Takita, J., Handa, H., and Yokota, J.: Detection of amplified genomic
sequences in human small-cell lung carcinoma cell lines by arbitrarily primed-PCR
genomic fingerprinting. Human Genomics, 98, 253-258, 1996.

C.
13.

14.

15.

16.
17.

18.

19.

20.

21.

22.

23.

24.

DEPARTMENT OF ONTOGENETICS

61

Yajima, H., Kosukegawa, A., Hoque, M. M., Shimojima, T., Takayama, M., Sasaki, Y.,
Sakai, H., Ohtsuka, M., Inokuchi, S., and Handa, H.: Construction and characterizationof a recombinat adenovirus vector carrying the human preproinsulin gene under thecontrol of the methallothionein gene promoter. Biochem. Biophys. Res. Commun., 229,
778-787, 1996.
Sawa, C., Goto, M., Suzuki, F., Watanabe, H., Sawada, J-I., and Handa, H.: Functional
domains of transcription factor hGABPbetallE4TFI-53 required for nuclear localization and transcription activation. Nuc\. Acids Res., 24, 4954-4961, 1996.
Nabeshima, R., Aizawa, S., Nakano, M., Toyama, K., Sugimoto, K., Kaidow, A., Imai,T., and Handa, H.: Effects of vesnarinone on the bone marrow stromal cell-dependent
proliferation and differentiation of HL60 cells in vitro. Exp. Hemato\., in press.
Aoki. H., Seki, T., Sawada, J-I., Handa, H., and Adachi, K.: The promoter of theandrogen dependent gene in the flank organ of hamsters. J. Dermatol. Sci., in press.
Fukagawa, T., Nakamura, Y., Okumura, K., Nogami, M., Ando, A., Inoko, H., Saitou,N., and Ikemura, T.: Human Pseudoautosomal Boundary-Like Sequences (PABLs):
Expression and a Model of Formation of Present-day Pseudoautosomal Boundary of Sex
Chromosomes. Hum. Mol. Genet., 5, 23-32, 1996.
Nishitani, H., Nishitsuji, K., Okumura, K., and Taguchi, H.: Proton Pump Activation
and Growth Promotion by Cinchomeronic Acid in Radish. Phytochemistry, 42, 1-6,
1996.
Mizuki, N., Kimura, M., Ohno, S., Miyata, S., Sato, M., Ando, H., Ishihara, M., Goto,
K., Watanabe, S., Yamazaki, M., Ono, A., Taguchi, S., Okumura, K., Nogami, M.,
Taguchi, H., Ando, A., and Inoko, H.: Isolation of cDNA and Genomic Clones for a
Human Ras-related GTP-Binding Protein and Its Chromosomal Localization to the Long
Arm of Chromosome 7, 7q36. Genomics, 34, 114-118, 1996.
Makino, Y., Ohga, T., Toh, S., Koike, K., Okumura, K., Wada, M., Kuwano, M., andKohno, K.: Structural and Functional Analysis of the Human Y-Box Binding Protein(YB-l) Gene Promoter. Nucl. Acid Res., 24, 1873-1878, 1996.
Taniguchi, K., Wada, M., Kohno, K., Nakamura, T., Kawakami, M., Kagotani, K.,
Okumura, K., Akiyama, S., and Kuwano, M.: A Human Canalicular MultispecificOrganic Anion Transporter (cMOAT) Gene Is Overexpressed in Cisplatin-resistant
Human Cancer Cell Lines with Decreased Drug Accumulation. Cancer Res., 56, 41244129, 1996.
Eki, T., Okumura, K., Amin, A., Ishiai, M., Abe, M., Nogami, M., Taguchi, H., Hurwitz,
J., Murakami, Y, and Hanaoka, F.: Mapping of the Human Homologue (ORCIL) of the
Yeast Origin Recognition Complex Subunit 1 Gene to chromosome Band Ip32.2-p32.3.
Genomics, 36, 559-561, 1996.
Matsuzaka, Y., Kagotani, K., Okumura, K., Kimura, M., Inoko, H., and Taniguchi, Y.:
Isolation and Structural Analysis of the Mouse Nat (Ring3) gene in the MHC Class II
Region. Nuc\. Acids Symp. Ser., 35, 225-226, 1996.
Eki, T., Okumura, K., Shiratori, A., Abe, M., Nogami, M., Taguchi, H., Shibata, T.,
Murakami, Y., and Hanaoka, F.: Assignment of the Closest Human Homologue (DNA2L; KIAAOO83) of the Yeast Dna2 Helicase Gene to Chromosome Band lOq21.3--q22.1.
Genomics, 37, 408-410, 1996.

62
25.

26.

27.

28.

29.

30.

31.

Taniguchi, Y., Katsumata, Y, Koido, S., Suemizu, H., Yoshimura, S., Moriuchi, T.,
Okumura, K., Kagotani, K., Taguchi, H., Imanishi, T., Gojobori, T., and Inoko, H.:
Cloning, Sequencing, and Chromosomal Localization of Two Tandemly Arranged
Human Pseudogenes for the Proliferating Cell Nuclear Antigen (PCNA). Mammalian
Genome, in press.
Tran, P. T., Hori, H., Hori, Y., Okumura, K., Kagotani, K., and Taguchi, Y.: Molecular
Cloning of the Human Methylthioadenosine Phosphorylase Processed Pseudogene andLocalization to 3q28. Gene, in press.
Oshima, N., Nishitani, H., Nishikawa, S., Okumura, K., and Taguchi, H.: 1-0Nicotinoyl-,B-D-glucopyranose Found in Cultured Tobacco Cells as a New Niacin Metabolite. Phytochemistry, in press.
Hata, M., Okumura, K., Seto, M., and Ohtuka, K.: Genomic Cloning of a Human Heat
Shock Protein (Hsp40) Gene and Its Chromosomal Localization to 19p13.2. Genomics,
in press.
Fujiwara, Y., Miwa, M., Nogami, M., Okumura, K., Nobori, T., Suzuki, T., and Veda,
M.: Genomic Organization and Chromosomal Localization of the Human Casein Gene
Family. Hum. Genet., in press.
Taguchi, H., Sasatani, K., Nishitani, H., and Okumura, K.: Finding of VDP-Glucose:
Nicotinic Acid-N-glucosyltransferase Activity in Cultured Tobacco Cells and Its Properties. Biosci. Biotech. Biochem., in press.
Okumura, K.: Recent Progress in the Analysis of the Intranuclear Organization of theAnimal Cell Genome by FISH. Seikagaku (in Japanese), in press.

International Meeting
32.

Delineation of DNA Replication Timing in The Human MHC and Intranuclear Arrangement of Genome Domains by FISH: Okumura, K., Nogami, M., Hishida, K., Kagotani,K., Taguchi, H., Ando, A., Inoko, H., Fukagawa, T., Sugaya, K., Matsumoto, K., andIkemura, T.: Human Genome Meeting '96, Heidelberg, Germany, 1996.3.

63

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DEPARTMENT OF POPULATION GENETICS

D-a.

(1)

Division of Population Genetics

Variation in Synonymous Substitution Rates Among Mammalian
Genes and the Correlation Between Synonymous andNonsynonymous Divergences

Tomoko OHTA and Yasuo INA
Using mammalian gene sequences, the variances in the numbers of synonymous and nonsynonymous substitutions among genes were estimated together with the correlation coefficient between the two. The expected
correlation coefficient can be obtained under the neutral theory using theseestimated values of the variances. The expected coefficient is found to often
be one-half to two-thirds of the observed value. Possible causes for thedisagreement were discussed, such as correlated selective constraints on the
two types of substitutions and excess doublet mutations. The variance ofmutation rate and that of selective constraint were also estimated. The
results show that the coefficient of variation of the former is 0.2-D.3, whereasthat of the latter is 0.7--0.9. For details, see Ref. 1.

(2)

Population Biology of Antigen Presentation by
MHC Class I MoleculesPeter PARHAM and Tomoko OHTA
In principle, the function of major histocompatibility complex (MHC)
molecules is simple: to bind a peptide and engage a T cell. In practice, placing
this function within the context of the immune response begs questions of
population biology: How does the immune response emerge from the interactions among populations of peptides, T cells, and MHC molecules? Within apopulation of vertebrates, how does MHC polymorphism stamp individuality
on the response? Does polymorphism confer differential advantages in
responding to parasites? How are the pressures on the MHC reflected in64turnover of alleles? Examination of MHC molecules of North and SouthAmerican Indians showed that allelic turnover occurred only in South
populations. The roles of mutation, recombination, selection, and drift in thegeneration and maintenance of MHC class I polymorphism were discussed.
For details, see Ref. 2.
(3)

The Current Significance and Standing of Neutral andNearly Neutral Theories

Tomoko OHTA
Comparative studies of DNA sequences provide opportunities for testing
the neutral and the selection theories of molecular evolution. In particular,
the separate estimation of the numbers of synonymous and nonsynonymous
substitutions is a powerful tool for detecting selection of the latter. The
difference in the patterns of these two types of substitutions of mammaliangenes turned out to be in accord with the slightly deleterious or nearly neutral
mutation theory for nonsynonymous changes. Interaction systems at theamino acid level were suggested to be responsible for such nearly neutral, orvery weak selection. Synonymous substitutions are not strictly neutral, but
because of their minute effect, random drift predominates such that the rateof substitution is only slightly less than the completely neutral prediction. It
was concluded that the strictly neutral theory has not held up as well as thenearly neutral theory, yet remains invaluable as a null hypothesis for detecting selection. On the other hand, the main difference between the nearly
neutral and the traditional selection theories is that the former predicts rapid
evolution in small populations, whereas the latter predicts rapid evolution inlarge populations. For details, see Ref. 4.
(4)Study of Gene Evolution as Observed as InterspecificHybrid Anomaly in Drosophila

Toshiyuki S. TAKANO
Loss of macrochaetes of the notum is one of the anomalies seen ininterspecific hybrids between D. melanogaster and its closely related species.
The emergence and divisions of sensory mother cells was monitored by using

D. DEPARTMENT OF POPULATION GENETICS

65

a transformant line, AlOl and a rabbit anti-asense antibody. There was noclear anomaly in late third instar larvae nor in pupae up to I hr after
puparium formation (APF). Immunostaining using the mouse antibody
22ClO detected no neurons at many sites in the hybrid pupae of 25 hr APF,
which means no double neuron phenotype. It was also found that hybrid
pupae of 15 hr APF had no or very reduced levels of staining with theanti-cut antibody at the significant number of sites. These results suggest thatthe defect does not lie in the cell fate decisions during the development ofbristles, but in the maintenance of neural fate and/or differentiation of thedescendants of SMCs. Interspecific hybrids between a line of D. melanogasterand D. simulans iso-female lines exhibited a wide range in the number ofmissing bristles on the thorax. On the contrary, D. mauritiana and D.
seehellia lines showed almost no reduction in bristle number in hybrids with
D. melanogaster. This suggests that at least one of mutations occurred
recently along the D. simulans lineage. Genetic analysis clarified a significant
and partially recessive effect of the X chromosome of D. simulans, althoughgene(s) on the autosome(s) should interact with the gene(s) on the X
chromosome. In attempts to isolate genes responsible for the bristle anomaly,
deficiency screening was performed. QTL mapping based on the withinspecies variation of D. simulans was done as well by using nine length and
SSCP polymorphism on the X chromosome. These screenings resulted in theidentification of three candidate regions.
(5)

Lineage-dependent Protein-sequence Evolution of
Drosophila Genes

Toshiyuki S. TAKANO
The aehaete-seute complex consists of the aehaete (ac), seute (sc) andlethal of seute (l'se) proneural genes, and the neural precursor gene asense(ase). All of these genes encode basic helix-loop-helix (bHLH)-type transcription factors. The DNA sequence of these four genes from D. yakuba
were determined in order to compare their evolutionary rate. While there
was no significant variation in synonymous substitution rate among the fourgenes, the ac gene accumulated significantly larger number of replacement
substitutions than the other three genes based on the expectation from theestimates of the number of synonymous substitutions. This is mostly due to66

a high replacement substitution rate along the lineage leading to D. yakuba.
When the sequences were compared between D. melanogaster and its close
relative, D. simulans, the ratio of replacement to synonymous substitutions
for the ase gene was found to be significantly higher along the lines leading tothese two species than along the D. yakuba lineage. In sum, these datasuggests a significant variation in protein sequence evolution among the four
AS-C genes, depending On lineage.
(6)

Variance and Covariance of the Number of Amino Acid
Substitutions Estimated by Kimura's Method

Yasuo INA
Since the process of amino acid substitution is stochastic, it is of great
importance to consider the variance and covariance of the number of aminoacid substitutions. Formulae for the variance and covariance of the numberof amino acid substitutions estimated by Kimura's method are presented.
The bootstrap method shows that the formulae are accurate. The formulae
are useful for studies of molecular evolution, e.g., reconstruction of aminimum-evolution tree and the relative-rate test. For details, see Ref. 6.
(7)

Correlation between Synonymous and Nonsynonymous
Substitutions and Variation in Synonymous
Substitution Numbers

Yasuo INA
To study the correlation between synonymous and nonsynonymous substitutions, two models of nucleotide substitution were considered. It was
assumed that mutation rate and functional constraints on nucleotide changes
are mutually independent in the first model, whereas these parameters arecorrelated with each other in the second model. Using an analytical method
and computer simulations, the correlation coefficient between the numbers ofsynonymous and nonsynonymous substitutions under these two models wasinvestigated. A correlation between functional constraint on synonymoussites and that on nonsynonymous sites does not strongly affect the correlation
coefficient between the estimated numbers of synonymous and nonsynony-

D.

DEPARTMENT OF POPULATION GENETICS

67

mous substitutions, unless estimation errors are small. On the other hand,variation in mutation rate has a strong effect on increasing the correlation
coefficient. For details, see Ref. 7.
(8)

Pattern of Synonymous and Nonsynonymous Substitutions:
An Indicator of Mechanisms of Molecular Evolution

Yasuo INA
Comparison of numbers of synonymous and nonsynonymous substitutions
is useful for understanding mechanisms of molecular evolution. In thispaper, I examine the statistical properties of six methods for estimating
numbers of synonymous and nonsynonymous substitutions. The six methods
are Miyata and Yasunaga's (MY) method; Nei and Gojobori's (NG)method; Li, Wu, and Luo's (LWL) method; Pamilo, Bianchi, and Li's (PBL)
method; and Ina's (Ina) two methods. When the transition/transversion bias
at the mutation level is strong, the numbers of synonymous and nonsynonymous substitutions are estimated more accurately by the PBL and Ina
methods than by the NG, MY, and LWL methods. When the nucleotidefrequency bias is strong and distantly related sequences are compared, all thesix methods give underestimates of the number of synonymous substitutions.
The concept of synonymous and nonsynonymous categories is also useful for
analysis of DNA polymorphism data. For details, see Ref. 8.
Publications
1.

2.
3.
4.
5.

Ohta, T. and Ina Y.: Variation in synonymous substitution rates among mammalian genes
and the correlation between synonymous and nonsynonymous divergences. J. Mol. Evol.,41,717-720, 1995.
Parham, P. and Ohta, T.: Population biology of antigen presentation by MHC class I
molecules. Science, 272, 67-74, 1996.
Ohta, T. and Gillespie, J. H.: Development of neutral and nearly neutral theories. Theor.
Popul. BioI., 49, 128-142, 1996.
Ohta, T.: The current significance and standing of neutral and nearly neutral theories.
BioEssays, 18, 673-677, 1996.
Takano, T. S.: Genetic study of species differences as observed as loss of bristles ininterspecific hybrids of Drosophila. In "Current topics on molecular evolution" (Nei, M.
and Takahata, N. eds.), pp. 255-260, Institute of Molecular Evolutionary Genetics, The
Pennsylvania State University, USA, and The Graduate School for Advanced Studies,

68

Hayama, Japan, 1996.
Ina, Y.: Variance and covariance of the number of amino acid substitutions estimated by
Kimura's method. Genes Genet. Syst., 71, 43-46, 1996.
7. Ina, Y.: Correlation between synonymous and nonsynonymous substitutions and variation in synonymous substitution numbers. In "Current topics of molecular evolution"(Nei, M. and Takahata, N. eds.), pp. 105-113, Institute of Molecular Evolutionary
Genetics, The Pennsylvania State University, Pennsylvania, USA, and The Graduate
University for Advanced Studies, Hayama, Japan, 1996.
8. Ina, Y.: Pattern of synonymous and nonsynonymous substitutions: An indicator ofmechanisms of molecular evolution. J. Genet., 75, 91-115, 1996.
6.

D-b.

(1)

Division of Evolutionary Genetics

Replication Timing for the GC Content Transition Area between
Classes II and III in the Human MHC

Toyoaki TENZEN, Yoshihisa WATANABE, Asao FUJIYAMA, Katsuzumi
OKUMURA, Asako AND0 1, Hidetoshi INOK0 1, and Toshimichi IKEMURA
CSchool of Medicine, Tokai University)
The human genome is composed of long-range G+C% (GC%) mosaic
structures thought to be related to chromosome bands. We previously
reported an example of a boundary of megabase (Mb)-sized GC% mosaic
domains at the junction area between major histocompatibility complex(MHC) classes II and III, proposing it as a possible chromosome bandboundary. DNA replication timing during S phase is known to be correlated
cytogenetically with chromosome band zones, and thus the band boundaries
have been predicted to contain a switch point for DNA replication timing. In
this study, to identify at the nucleotide sequence level the replication switch
point during S phase, we determined the precise DNA replication timing for
MHC classes II and III, focusing on the junction area. To do this we used
PCR-based quantitation of nascent DNA obtained from synchronizedhuman myeloid leukemia HL60 cells. The replication timing changed
precisely in the boundary region with a 2-hour difference between the twosides, supporting the prediction that this region may be a chromosome bandboundary. We supposed that replication fork movement terminates (pauses)
or significantly slows down in the switch region, which contains dense Aluclusters, polypurine/polypyrimidine tracts, di-, tri- or tetranucleotide repeats,

D. DEPARTMENT OF POPULATION GENETICS

69

and medium reiteration frequency sequences (MERs). As the nascent DNA
in the switch region was recovered at low efficiency, we investigated whether
this region is associated with the nuclear scaffold and found three scaffoldassociated regions (SARs) in and around the switch region. We are also
determining the replication timing for the region containing pseudoautosomal boundary (PAB) of human sex chromosomes in order to assign theboundary of the X inactivation domain. For details, see Mol. Cell. BioI., 17,4043-4050.
(2) Human Pseudoautosomal Boundary-Like Sequences:
Their Expression and Involvement in Evolutionary
Formation of the Present-Day Pseudoautosomal
Boundary of Human Sex Chromosomes
Tatsuo FUKAGAWA, Yasukazu NAKAMURA, Katsuzumi OKUMURA, Masahiro NOGAMI, Asako AND0 1, Hidetoshi INOKOt, Naruya SAITOU, and Toshimichi IKEMURA C School of Medicine, Tokai University)
The human genome is composed of long-range mosaic structures of
G+ C% (GC%), which are thought to be related to chromosome bands. We
previously identified a boundary of Mb-level domains of GC% mosaic
structures in the human major histocompatibility complex (MHC) and foundin the domain boundary, a sequence very similar to pseudoautosomal boundary (PAB) sequences of human sex chromosomes. We designated it "PABL"
and found many PABLs in the human genome. By analyzing six genomic
and six transcribed PABLs, a core and consensus sequence of about 650 nt
were defined; the 3'- and 5'-edges of the PABLs were strictly conserved.
Northern blot analysis showed sizes of PABL transcripts to be 5-10 kb inlength. Divergence time of PABLs was estimated to be 60-120 million years
ago from an analysis of human PABLs and PABXYI of seven primates, andthe evolutionary rates deduced showed PABLs to have been under selective
constraints. A model for evolutionary formation of the present pseudoautosomal boundary was proposed by postulation of illegitimate recombination
between two PABLs. For details, see Hum. Mol. Genet., 5, 23-32, 1996.

70

(3)

Chromosomal Localization of the Proteasome Z Subunit Gene
Reveals an Ancient Chromosomal Duplication Involving
the Major Histocompatibility Complex

Masanori KASAHARA!, Masaru HAYASHI!, Keiji TANAKA 2, Hidetoshi
INOK03, Kimihiko SUGAYA, Toshimichi IKEMURA, and Teruo ISHIBASHI 1
CSchool of Medicine, Hokkaido University, 2 Institute for Enzyme Research, The University of Tokushima, 3 School of Medicine, Tokai University)
Proteasomes are the multi-subunit protease thought to play a key role in
the generation of peptides presented by major histocompatibility complex(MHC) class I molecules. When cells are stimulated with interferon r, two
MHC-encoded subunits, low molecular mass polypeptide LMP2 and LMP7,
and the MECLI subunit encoded outside the MHC are incorporated into theproteasomal complex, presumably by displacing the housekeeping subunits
designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably byaltering the cleavage specificities of the proteasome.
Here we show that the mouse gene encoding the Z subunit (Psmb7) maps
to the paracentromeric region of chromosome 2. Inspection of the mouse lociadjacent to the Psmb7 locus provides evidence that the paracentromericregion of chromosome 2 and the MHC region on chromosome 17 most likely
arose as a result of a duplication that took place at an early stage of vertebrate
evolution. Traces of this duplication are also evident in the homologous
human chromosome regions (6p21.3 and 9q33-q34). These observations
have implications for understanding the genomic organization of the presentday MHC and offer insight into the origin of the MHC. For details, see Proc.
Nat!. Acad. Sci. (USA), 93, 9096-9101, 1996.
(4)

Multivariate Diversity in Codon Usage in Escherichia coli
Sequences Determined by Genome Projects

Shigehiko KANAYA 1, Yoshihiro KUDO!, Yasukazu NAKAMURA, and Toshimichi IKEMURA CFaculty of Engineering, Yamagata University)
We developed measures (called Z-parameters in this study) which reflect
the diversity of codon usage in Escherichia coli genes using principal compo-

D. DEPARTMENT OF POPULATION GENETICS

71

nent analysis. Protein-production levels for 1500 CDSs (protein-coding
sequences) proposed by genome projects in Japan and USA were estimatedby a correlation equation between Zj and cellular protein content, which was
obtained through analysis of the genes which were experimentally characterized. Through a profile analysis of Zl for E. coli sequences determined in theJapan genome project, we predicted additional 36 CDSs that had not yet beenannotated in the International DNA Databases. Thirty-two out of thethirty-six CDSs could be assigned to presumed protein genes also through a
BLASTX search of recent protein databases in the Genome Net in Japan.
Detailed examination of the ZI-parameter profile also led us to assess sequencing errors which cause frame-shift. For details, see CABIOS, 12, 213225, 1996.
(5)

cDNA Cloning of the Human Homologues of the Mouse Ke4 and
Ke6 Genes at the Centromeric End of the Human MHC Region

Asako AND0 1, Yara Yukie KIKUTI 1, Atsuko SHIGENARI!, Hisako KAWATA!,
Naoaki OKAMOTO!, Takashi SHIINA!, Lei CHEN!, Toshimichi IKEMURA,
Kuniya ABE!, Minoru KIMURA 1, and Hidetoshi INoKo 1 C School of Medicine, Tokai University)
eDNA clones corresponding to the HKE4 and HKE6 genes at the centromeric end of the HLA region on human chromosome 6p21.3 were isolated
and characterized. The predicted amino acid sequences of HKE4 and HKE
6 exhibited 81.5 and 85.6% identity to the mouse homologues, Ke4 and Ke
6, respectively. HKE4 may encode a membrane protein with histidine-richcharge clusters. HKE6 possesses remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function and also
shows significant homology with the two unique functional domains containing the nucleotide cofactor binding site and the consensus motif characteristic of the members of the superfamily of short-chain alcohol dehydrogenases
such as human and rat steroid and prostaglandin dehydrogenases. For
details, see Genomics, 35, 60D---602, 1996.

72

(6)

Tenascin-X Is Essential for the Development of
Epicardium and Coronary Vessels

Ken-ichi MATSUMOTO, Hiroshi YAMAMOTO, Naoki TAKEDA!, Toshimichi
YOSHlDA 2, Kyoko IMANAKA-YOSHIDA 2, Yasuaki SHIRAYOSHI, Teruyo
SAKAKURA2 , Shinichi AIZAWA!, Toshimichi IKEMURA, and Norio NAKATSUJI C Kumamoto University School of Medicine, 2 Mie University Schoolof Medicine)
Tenascin-X (TN-X), a new member of the tenascin family of extracellularmatrix proteins, is ubiquitously expressed, with the highest levels detected in
the heart and muscle. However almost nothing is known about the functionsof TN-X during embryo development. To investigate roles of this extracellular matrix in vivo, we attempted to generate TN-X-deficient mice bygene targeting in embryonic stem (ES) cells. But TN-X (+ / -) chimericmice with a high level of ES cell contribution were not obtained. To evaluate
embryonic viability in TN-X ( +/ - ) chimeras, embryos at various developmental stages were examined. The TN-X (+ / -) chimeric fetuses expresseda reduced amount of TN-X and showed two conspicuous defects, both ofwhich led to generalized edema and embryonic death. One defect occurred
during the development of the epicardium and coronary blood vessels in theheart. Congestion of coronary vessels in the epicardium and thickened
epicardium were observed. The other defect involved massive necrosis of thehepatocytes in the liver. The epicardium and coronary vessels are tissues
where TN-X is most highly expressed, and thus are assumed to be where its
important functions lie. Our results suggest possible roles of TN-X inepicardial cell migration and the development of coronary blood vessels in atight dose-dependent manner.

D. DEPARTMENT OF POPULATION GENETICS(7)

73

Gene Organization of Human NOTCH4 and (CTG)nPolymorphism in This Human Counterpart
Gene of Mouse Proto-oncogene Int3

Kimihiko SUGAYA', Shun-ich SASANUMA!, Junko NOHATA!, Terumi KIMURA', Tatsuo FUKAGAWA, Yasukazu NAKAMURA, Asako AND0 2, Hidetoshi
INOKd, Toshimichi IKEMURA, and Kazuei MITA 1 C National Institute ofRadiological Sciences, 2 School of Medicine, Tokai University)
The cDNA and genomic clones for the human counterpart of the mouse
mammary tumor gene Int3 were isolated and sequenced. We designated thishuman major histocompatibility complex (MHC) class III gene as NOTCH
4, since very recently, by sequencing cDNA clones, the complete form of themouse proto-oncogene Int3 was clarified and named Notch4. The present
human NOTCH4 sequence is the first example of the genomic sequence forthe extracellular portion of mammalian Notch4, and by comparing it with themouse Notch4 cDNA sequence, the exon/intron organization was clarified.
Comparison of the predicted amino acid sequence of human NOTCH4 withthose of other Notch homologues of a wide range of species revealed four
subfamilies for mammalian Notch. In the protein coding region of human
NOTCH4, we found (CTG)n repeats showing variable number tandem
repeat (VNTR) polymorphisms for different human leukocyte antigen(HLA) haplotypes.
Here we show also that ten genes mapped on 6p21.3, including NOTCH4,
have counterparts structurally and functionally similar to those mostly
mapped on 9q33-q34, indicating segmental chromosome duplication during
the course of evolution. Similarity of genes on chromosome 1, 6, 9 and 19 was
also discussed. For details, see Gene, 189, 235-244, 1997.
(8)

Reconstruction of Gene Trees from Sequence Data

Naruya SAITOU
Reconstruction of the phylogeny of genes is essential not only for study of
evolution but also for biology in general. This is because replication of
nucleotide sequences automatically produce bifurcating tree of genes. It
should be emphasized that the phylogenetic relationship of genes is different
from the mutation process. The former always exists, while mutations may

74

or may not happen within a certain time period and DNA region. Therefore,
even if several nucleotide sequences happen to be identical, there must be a
genealogical relationship for those sequences.
However, it is impossible to reconstruct the genealogical relationshipwithout occurrence of mutational events. In this respect, the extraction of
mutations from genes and their products is also important for reconstructing
phylogenetic trees of genes. The advancement of molecular biotechnology
has made it possible to routinely produce nucleotide sequences. We will
therefore focus on the analysis of nucleotide sequences. However, a substantial part of this chapter also applies to other molecular data. For details, see
Ref. 25.
(9)The Presence/Absence Polymorphism and Evolution of
the p53 Pseudogene in the Genus Mus

Hiroshi OHTSUKA 1, Mituru OYANAGI 1, Yoshio MAFUNE 1, Nobumoto MIYASHITA2 , Toshihiko SHIROIsHI 3 , Kazuo MORIWAKI4, Ryo KOMINAMI 1, andNaruya SAITOU CFirst Department of Biochemistry, Niigata University
School of Medicine, 2 Kagawa Medical School, 3 Mammalian Genetics Laboratory, 4 Graduate University for Advanced Studies)
Distribution of the p53 pseudogene within the house mouse species (genusMus) was studied with polymerase chain reaction for thirty seven individuals
that were caught at different localities. Pseudogene-specific fragments weredetected in some, but not all, individuals of Mus musculus subspecies
regardless of locality and type of subspecies. Besides, three of seven individuals belonging to different Mus species carried the pseudogene in theirgenomes. These results show an existence of an interspecific presence/
absence polymorphism of the p53 pseudogene in mice. Sequence analysis ofeleven amplified 0.3 kb fragments suggested that the pseudogene originated in
an ancestral mouse about 10 million years ago. Thus alleles with and without
the p53 pseudogene have persisted through the mice speciation. The evolutionary rate for the p53 functional gene was also estimated to be about 2.5 109 per nucleotide site per year. For details, see Ref. 26.

D. DEPARTMENT OF POPULATION GENETICS

75

Contrasting Gene Trees and Population Trees on
the Evolution of Modern Humans(10)

Naruya SAITOU
A gene tree is an essential descriptor of any evolutionary process, for thesemiconservative replication of DNA double helix automatically produces abifurcating gene tree. It should be emphasized that the genealogical relationship of genes is independent from the mutation process, especially when theneutral evolution is considered. The former is a direct product of DNA
replication, while the latter mayor may not happen within a certain time
period and DNA region. Therefore, even if many nucleotide sequences
happened to be identical, there must be a genealogical relationship for thosesequences. However, it is impossible to reconstruct the genealogical relationship without mutational events. In this respect, extraction of mutations fromgenes and their products is critical for reconstructing phylogenetic trees. We
first reviewed gene trees for HTLV-I DNA, then compared gene trees andpopulation trees for extant and ancient mitochondrial DNA. Our recent
studies on gene and population trees for nuclear DNAs were also reviewd.
Lastly, Importance of finiteness for evolutionary studies was discussed. For
details, see Ref. 28.
(11)

Extensive Polymorphism of ABO Blood Group Gene:Three Major Lineages of the Alleles for
the Common ABO Phenotypes

Kenichi OGASAWARA 1, Naruya SAITOU, and Katsushi TOKUNAGA 2 C Japanese Red Cross Tokyo Metropolitan Blood Center, 2 University of Tokyo
Medical School)
Polymorphism of the ABO blood group gene was investigated in 262
healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles wereidentified. The number of alleles identified in each group was 4 for Al(provisionally called ABO*AI0l, *A102, *A103 and *A104 according to theguidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and*B103), and 6 for 0 (ABO*01OI, *0102, *0103, *0201, *0202 and*0203). Nucleotide sequences of the amplified fragments with different

76

SSCP patterns were determined by direct sequencing. Phylogenetic network
analysis revealed that these alleles could be classified into three major
lineages, *A/*Ol, *B and *02. In Japanese, *A102 and *B101 were thepredominant alleles with frequencies of 83% and 97% in each group,
respectively, whereas in group 0, two common alleles, *0101 (43%) and*0201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be
advantageous indicators for population studies. For details, see Ref. 27.
(12)

Molecular Genetic Analysis of Variant Phenotypes of
the ABO Blood Group System

Kenichi OGASAWARA', Naruya SAITOU, and Katsushi TOKUNAGA 2 ( ' Japanese Red Cross Tokyo Metropolitan Blood Center, 2 Graduate School of
Medical Science, University of Tokyo)
ABO is clinically the most important blood group system in transfusion
medicine and includes many variant phenotypes. To understand the molecular genetic basis of this polymorphic system, we have analyzed genomic
DNAs obtained from Japanese individuals possessing variant ABO phenotypes including A2, Ax, Ael, cis-AB, Bx, and Bel. By polymerase chain
reaction-single-strand conformation polymorphism (SSCP) and nucleotide
sequence analyses, we identified 11 different alleles. These alleles had
nucleotide sequences different from those of the previously described 13
different alleles responsible for the common ABO phenotypes. Analysis of
the nucleotide sequences of the alleles responsible for those variant phenotypes showed that the amino acid residues at positions 266 and 268 may be
crucial for transferase specificity, whereas those at positions 214, 216, 223,
291, and 352 may be critical for the activity level. Nine of the 11 alleles,
responsible for the A2, Ax, Ael, cis-AB, Bx, and Bel phenotypes, were
presumed to be generated from common ABO alleles by single nucleotide
mutations such as nonsynonymous substitution, deletion, or insertion. Twoother alleles, responsible for the A2, and Ael phenotypes, may have originated by recombination, gene conversionlike events or accumulation of nucleotide substitutions. Our data indicate that different alleles could cause thesame ABO variant phenotypes, and that these alleles do not necessarily
belong to a single evolutionary lineage. For details, see Ref. 30.

D.

(13)

DEPARTMENT OF POPULATION GENETICS

77

Evolution of Class III POU Genes: Amplification ofCharacteristic Amino Acid Residues by
G + C Pressure in Mammals

Kenta SUMIYAMA, Naruya SAITOU, and Shintaroh UEDA!
School of Science, University of Tokyo)

C Graduate

The class III POU transcription factor genes play an important role innervous system. Comparison of their entire amino acid sequences disclosed
a remarkable feature of particular mammalian class III POU genes. Alanine-, glycine-, and proline-repeats were present in the mammalian Brain-lgene, whereas most part of these repeats were absent in the non-mammalian
homologue. The mammalian Brain-2 gene had alanine-, glycine-, proline-,and glutamine-repeats, which were missing in the non-mammalian homologue. The mammalian Scip gene had alanine-, glycine-, proline-, andhistidine-repeats, but the non-mammalian homologue completely lacked these
repeats. In contrast, the mammalian Brain-4 gene had no amino acid repeats
like its non-mammalian homologue. The mammalian genes containing thecharacteristic amino acid repeats had another feature, the higher GC content.
We found a positive correlation between the GC content and the amino acidrepeats ratio. Those amino acids were encoded by triplet codons withrelatively high GC content. These results suggest that the GC pressure has
facilitated generation of the homopolymeric amino acid repeats. For details,
see Ref. 29.
Publications
Tenzen, T., Yamagata, T., Fukagawa, T., Sugaya, K., Ando, A., Inoko, H., Gojobori, T.,
Fujiyama, A., Okumura, K., and Ikemura, T.: Precise switching of DNA replication
timing in the GC content transition area in the human MHC. Mol. Cell. BioI., 17, 40434050, 1977.
2. Fukagawa, T., Nakamura, Y., Okumura, K., Ando, A., Inoko, H., Saitou, N., andIkemura, T.: Human pseudoautosomal boundary-like sequences (PABLs): Core andconsensus sequence, expression, and involvement in formation of the present day pseudoautosomal boundary of short arm of human sex chromosomes. Hum. Mol. Genet., 5,1.

23-32, 1996.

3. Sugaya, K., Sasanuma, S., Nohata, J., Kimura, T., Fukagawa, T., Nakamura, Y., Ando,A., Inoko, H., Ikemura, T., and Mita, K.: Gene organization of human NOTCH4 and(CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.

78

4.

5.
6.

7.

8.

9.

10.

II.

12.

13.

14.

IS.

16.

Gene, 189, 235-244, 1997.
Kasahara, M., Hayashi, M., Tanaka, K., Inoko, H., Sugaya, K., Ikemura, T., andIshibashi, T.: Chromosomal localization of the proteasome Z subunit gene reveals anancient chromosomal duplication involving the major histocompatibility complex. Proc.
Natl. Acad. Sci. (USA), 93, 9096-9101, 1996.
Nakamura, Y., Gojobori, T., and Ikemura, T.: Codon usage tabulated from the international DNA sequence databases. Nuc!. Acids Res., 1997.
Kanaya, S., Kudo, Y., Nakamura, Y., and Ikemura, T.: Detection of genes in Escherichia
coil sequences determined by genome projects and prediction of protein production levels
based on multivariate diversity in codon usage. CABIOS, 12, 213-225, 1996.
Ando, A., Kikuti, Y., Shigenari, A., Kawata, H., Okamoto, N., Shiina, T., Chen, L.,
Ikemura, T., Abe, K., Kimura, M., and Inoko, H.: cDNA cloning of the humanhomologues of the mouse Ke4 and Ke6 genes at the centromeric end of the human MHC
region. Genomics, 35, 600-602, 1996.
Nakamura, Y., Wada, K., Wada, Y., Doi, H., Kanaya, S., Gojobori, T., and Ikemura, T.:
Codon usage tabulated from international DNA sequence databases. Nucl. Acids Res.,
24,214-215, 1996.
Kanaya, K., Suzuki, S., Kudo, Y., and Ikemura, T.: Systematization of species-specificdiversity of genes in codon usage: comparison of the diversity among bacteria andprediction of the protein production levels in cells. Genome Informatics, 1996, 61-71,
1996.
Pi, K., Angata, M., Ikemura, T., Yanagisawa, Y., and Tanaka, Y.: Analysis of a tRNA
gene-like sequence(t-element) with TTA at the anticodon position in mitochondorial
DNA of Dictyostelium discoideum. J. Plant Res., 109, 1-6, 1996.
Hasegawa, K., Yoshida, T., Matsumoto, K., Katsuta, K., Waga, S., and Sakakura, T.:
Differential expression of tenascin-C and tenascin-X in human astrocytomas. Acta
Neuropathol., in press.
Sakai, T., Furukawa, Y., Chiquet-Ehrismann, R., Nakamura, M., Kitagawa, S., Ikemura,T., and Matsumoto, K.: Tenascin-X expression in tumor cells and fibroblasts: glucocorticoids as negative regulators in fibloblasts. J. Cell Sci., 109, 2069-2077, 1996.
Kasahara, M., Hayashi, M., Tanaka, K., Inoko, H., Sugaya, K., Ikemura, T., andIshibashi, T.: Chromosomal localization of the proteasome Z subunit gene reveals anancient chromosomal duplication involving the major histocompatibiliy complex. Proc.
Natl. Acad. Sci. (USA), 93, 9096-9101, 1996.
Fukagawa, T., Nakamura, Y., Okumura, K., Ando, A., Inoko, H., Saitou, N., andIkemura, T.: Human pseudoautosomal boundary-like sequences (PABLs): Core andconsensus sequence, expression, and involvement in formation of the present day pseudoautosomal boundary of short arm of human sex chromosomes. Hum. Mol. Genet., 5,23-32, 1996.
Sugaya, K., Sasanuma, S., Nohata, J., Kimura, T., Fukagawa, T., Nakamura, Y., Ando,A., Inoko, H., Ikemura, T., and Mita, K.: Gene organization of human NOTCH4 and(CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.
Gene.
Tenzen, T., Yamagata, T., Fukagawa, T., Sugaya, K., Ando, A., Inoko, H., Gojobori, T.,

D.

17.
18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

DEPARTMENT OF POPULATION GENETICS

79

Fujiyama, A., Okumura, K., and Ikemura, T.: Precise switching of DNA replication
timing in the GC content transition area in the human MHC. Submitted.
Nakamura, Y., Gojobori, T., and Ikemura, T.: Codon usage tabulated from the international DNA sequence databases. Nucl. Acids Res., 1997.
Kanaya, S., Kudo, Y., Nakamura, Y, and Ikemura, T.: Detection of genes in Escherichia
coil sequences determined by genome projects and prediction of protein production levels
based on multivariate diversity in codon usage. CABlOS, 12, 213-225, 1996.
Ando, A., Kikuti, Y., Shigenari, A., Kawata, H., Okamoto, N., Shiina, T., Chen, L.,
Ikemura, T., Abe, K., Kimura, M., and Inoko, H.: cDNA cloning of the humanhomologues of the mouse Ke4 and Ke6 genes at the centromeric end of the human MHC
region. Genomics, 35, 600-602, 1996.
Nakamura, Y., Wada, K., Wada, Y., Doi, H., Kanaya, S., Gojobori, T., and Ikemura, T.:
Codon usage tabulated from international DNA sequence databases. Nucl. Acids Res.,
24,214-215, 1996.
Sakai, T., Furukawa, Y., Chiquet-Ehrismann, R., Nakamura, M., Kitagawa, S., Ikemura,T., and Matsumoto, K.: Tenascin-X expression in tumor cells and fibroblasts: glucocorticoids as negative regulators in fibloblasts. J. Cell Sci., 109,2069-2077, 1996.
Pi, K., Angata, M., Ikemura, T., Yanagisawa, Y., and Tanaka, Y.: Analysis of a tRNA
gene-like sequence (t-element) with TTA at the anticodon position in mitochondorial
DNA of Dictyostelium discoideum. J. Plant Res., 109, 1-6, 1996.
Hasegawa, K., Yoshida, T., Matsumoto, K., Katsuta, K., Waga, S., and Sakakura, T.:
Differential expression of tenascin-C and tenascin-X in human astrocytomas. Acta
Neuropathol., in press.
Omoto, K., Hirai, M., Harihara, S., Jin, F., Misawa, S., Washio, K., Tokunaga, K.,
Saitou, N., Yamazaki, K., Du, R, Hao, L., Yuan, Y., Luo, Z., Xu, J., Hu, J., Wei, X., Li,
S., Zhao, H., Zhang, Z., Niu, K., Du, C., and Lu, B. (1996) Population genetic studies
on national minorities in China. In Akazawa, T. and Szathmary, E. (eds.), Proceedingsof the First Conference on the Dispersal of Prehistoric Mongoloids, Oxford University
Press, Oxford, pp. 135-145.
Saitou, N. (1996) Methods for reconstructing phylogenetic relationships of genes. In
Doolittle R. (ed.), Methods in Enzymology, Vo!. 266, pp. 427-449 Academic Press, Inc.,
Orlando.
Ohtsuka, H., Oyanagi, M., Mafune, Y., Miyashita, N., Shiroishi, T., Moriwaki, K.,
Kominami, R., and Saitou, N.: The presence/absence polymorphism and evolution of p53
pseudogene within the genus Mus. Mol. Phylogenet. Evo!., 5, 548-556, 1996.
Ogasawara, K., Bannai, M., Saitou, N., Yabe, R., Nakata, K., Takenaka, M., Fujisawa,
K., Uchikawa, M., Ishikawa, Y., Juji, T., and Tokunaga, K.: Extensive polymorphism of
ABO blood group gene: 1. Three major lineages of the alleles for the common ABO
phenotypes. Human Genetics, 97, 777-783, 1996.
Saitou, N. (1996) Contrasting gene trees and population trees on the evolution ofmodern humans. In Boyce A. J. and Mascie-Taylor, C. G. N. (eds.), Society for the
Study of Human Biology Symposium Series: Molecular Biology and Human Diversity,
pp. 265-282, Cambridge University Press, Cambridge, pp. 265-282.
Sumiyama, K., Washio-Watanabe, K., Saitou, N., Hayakawa, T., and Ueda, S.: Evolu-

80

tion of class III POU genes: amplification of characteristic amino acid residues by G + C
pressure in mammals. J. Mol. Evol., 43, 170-178, 1996.
30. Ogasawara, K., Yabe, R., Uchikawa, M., Saitou, N., Bannai, M., Nakata, K., Takenaka,
M., Fujisawa, K., Juji, T., and Tokunaga, K.: Molecular genetic analysis of the variant
phenotypes of ABO blood group system. Blood, 88, 2732-2737, 1996.
D-c.
(1)

Division of Theoretical Genetics

Current Topics on Molecular Evolution

Masatoshi NEIl and Naoyuki TAKAHATA 2 (' The Pennsylvania State University, 2 The Graduate University for Advanced Studies)This volume is the outcome of the US-Japan Binational Workshop on
Molecular Evolution held at the Graduate University for Advanced Studies,
Hayama, Japan, August 25-27, 1995, in conjunction with the Third International Meeting of the Society for Molecular Biology and Evolution. The
purpose of the workshop was to promote collaborative research between
young investigators from the United States and Japan in the area of molecular evolutionary biology. Since studies on molecular evolutionary biology are
so dispersed, emphasis was given to the study of molecular phylogeny andevolution of adaptive characters. In each of these areas, seven representatives
each from the United States and Japan presented papers. The papers werethen exposed to critical comments by other participants. There was ample
time for the representatives from both countries to get acquainted and discusseach subject matter in detail. This volume includes short versions of thetwenty-eight papers presented at the workshop. They are divided into twoparts: (1) Molecular phylogeny and (2) Evolution of adaptive characters.
However, the subject matters discussed are quite broad, and the distinction
between the two parts is not always clear-cut. We hope this volume will givesome flavor of the enthusiastic workshop attended by young investigators.
For details, see refs. 9-11.

(2)

Neutral Theory of Molecular Evolution

Naoyuki TAKAHATA (The Graduate University for Advanced Studies)
DNA sequence data are generally interpreted as favouring Kimura's

D. DEPARTMENT OF POPULATION GENETICS

81

neutral theory but not without dissent and often with a great deal ofcontroversy. Here I review the current status of the neutral theory withspecial reference to molecular clocks, DNA polymorphism, adaptive evolution, and gene genealogy. While the theory serves as a guiding principle,
many issues concerning mutation, recombination, and selection remain unsettled. Of particular importance is the need for more knowledge about thefunction and structure of molecules. For details, see Ref. 12.
(3)

Multiregional or Uniregional Origin of Modern Humans:
What Do Genetic Data Indicate?

Naoyuki TAKAHATA' and Jan KLEIN 2 CThe Graduate University for Advanced Studies, 2 Max-Planck-Institut flir Biologie, Abteilung Immungenetik
and Department of Microbiology and Immunology, University of Miami
School of Medicine)
We examined hypotheses on the origin of Homo sapiens in terms of genegenealogy and compared the theoretical results with available genetic data.
All data suggest (1) frequent gene exchange and/or extinction and recolonization oflocal populations; (2) a relatively short coalescence time of neutral
genes or a relatively small effective population size for humans throughout
the late Pleistocene; and (3) characteristic intra- and inter-populational
genetic diversity among Africans. Any hypothesis that regards human
evolution as static cannot explain these observations. The most importantfactor, but one that has been overlooked in the evolution of H. sapiens,
appears to be the frequent extinction and recolonization of local populations.
For details, see Ref. 13.
(4)

Evolution of the Primate Lineage Leading to Modern Humans:
Phylogenetic and Demographic Inferences fromDNA Sequences

Naoyuki TAKAHATA and Yoko SATTA (The Graduate University for Advanced Studies)
For the dating of major divergences that occurred in the primate lineage
leading to modern humans, and to infer a demographic parameter (effective

82

population size) of the ancestral lineage that existed prior to each divergence,a maximum likelihood (ML) method was applied to autosomal DNA sequence data currently available for pairs of orthologous genes between
human and chimpanzee, gorilla, Old World monkey (OWM) and New
World monkey (NWM). A statistical test was carried out to support theassumption that silent substitutions have accumulated in a clock-like fashion
over loci between primate taxa or even among sites within a locus. It was
shown that the human ancestral lineage became distinct from the NWM 57.5
Myr ago, the OWM 31 Myr ago, the gorilla 8.0 Myr ago, and the chimpanzee
4.5 Myr ago, and that the effective population size at these divergences was
generally much greater than that of modern humans. It was argued that thehuman ancestral lineage branched off from the NWM and OWM earlier thanonce thought and that significant demographic changes might have occurredat different evolutionary stages, particularly at the hominid stage. For
details, see Ref. 16.
(5)

Studies on the Structure of Visual Pigments Responsible for
the Function of Photoreceptor Cells

Daisuke KOJIMA l , Tomonori OURAl, Yoshinori SHICHIDAl, Yoshitaka
FUKADA, Osamu HISATOMI 2, Fumio TOKUNAGA2 , and T6ru YOSHIZAWA 3
C Department of Biophysics, Faculty of Science, Kyoto University, 2 Department of Earth and Space Science, Faculty of Science, Osaka University,
3 Department of Information Systems Engineering, Faculty of Engineering,Osaka Sangyo University)
In spite of the high similarity in amino acid sequence between rod visual
pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only
the spectral sensitivities but also the thermal decay rates of the meta 11- andIII-intermediates are noticeably different from one another. In order to
identify the protein region(s) that contain(s) key residues being responsible
for the functional difference, we constructed six chimerical mutants derived
from gecko blue and bovine rhodopsin, with the aid of protein production ina human embryonic kidney cell line (293S). While the absorption maximumof every mutant was located in between gecko blue (466 nm) and bovine
rhodopsin (500 nm), a large blue-shift (18 nm) was observed when helices 1III of rhodopsin were replaced with those of gecko blue. A time-resolved

D. DEPARTMENT OF POPULATION GENETICS

83

spectroscopic study demonstrated that this replacement also accelerated the
decay rate of the meta II-intermediate. The decay of the meta 111intermediate of the mutants became faster as the compartment of gecko blue
was increased. Thus, the faster decay of the meta II-intermediate of gecko
blue is largely attributed to residues within helices I-III, while the decay ofthe meta III-intermediate apparently depends on the overall structure of theprotein. For details, see Ref. 1.
(6)

Functional Analysis of Lipid Modifications on
Photoreceptor G-Protein

Yoshitaka FUKADA, Takahiko MATSUDA!, Osamu SHOUN0 1, Masasuke
ARAKI 2 , Toshifumi TAKA0 3, Yasutsugu SHIMONISHI 3, Hidekazu TANAKA4 ,and Naomasa MIKI 4 C Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2 Department of Biology,
Kyoto Prefectural University of Medicine, 3 Institute for Protein Research,
Osaka University, 4 Department of Pharmacology 1, Medical School, Osaka
University)
Photoreceptor G protein transducin [a- and.Br-subunits (Ta/T.Br)] playa
central role in the visual transduction process. The amino-terminus of bovine
Ta is modified by one of four distinct fatty acids-laurate (C12 : 0), myristate (C14 : 0), C14: 1 (5·ds), and Cl4 : 2 (5-eis, 8-eis)-but the biologicalsignificance and the localization of the four isoforms of Ta are poorly
understood. To investigate the cellular distribution of each isoform, weprepared monoclonal antibodies against a synthetic C12: 0-, C14: 0-,
C14: 1-, or C14 : 2-nonapeptide corresponding to the N-terminal region of
Ta. Among several types of antibodies isolated, only one type, represented
by LA4, reacted specifically with the C12 : O-peptide as well as purified Ta
but not with the other proteins in bovine retinal homogenate, including
recoverin, indicating that the epitope comprises both C12 : 0 and the Nterminal amino acids of Ta. Immunohistochemical analyses of bovine retinal
sections by LA4 showed the uniform distribution of Cl2 : O-Ta in almost all
the rod outer segments. Hence, it seemed unlikely that each isoform of Ta
was localized in specific cells. This observation, together with evidence for a
possible functional diversity among the isoforms, suggests that the four
isoforms of Ta in a single rod cell may contribute simultaneously to a fine

84

tuning of the photon-signal transduction process. For details, see Ref. 3.
On the other hand, hydrophobic modifications of Tr, such as farnesyl- andcarboxyl-methylation, are essential for the association of Tf3r with thephotoreceptor disc membrane, and MEKA/phosducin is known to inhibit theassociation. We examined the effect of MEKA on the hydrophobicity of Tf3r.
MEKA could bind to Tf3r without farnesyllcarboxyl-methyl moieties as wellas native Tf3r. In the triton X-114 phase separation assay, Tf3r-MEKA
complex was recovered in the aqueous phase, whereas Tf3r was recovered in
the detergent phase. The N-terminal portion of MEKA which includes
Tf3r-binding domain was not sufficient to reduce the hydrophobicity of Tf3r
or to dissociate Tf3r from the membrane. The data suggest that MEKA
attenuates the hydrophobicity of Tf3r for dissociating Tf3r from the membrane without directly binding to farnesyllcarboxyl-methyl moieties. For
details, see Ref. 5.
(7)Functional Studies on the CaH -Binding Protein in
Photoreceptor Cells

Yoshitaka FUKADA, Kamon SANADA 1, Fumiko SHIMIZU!, Kimihiko KAMEYAMA2, Kazuko HAGA 2 , and Tatsuya HAGA2 C Department of Biophysics
and Biochemistry, Graduate School of Science, The University of Tokyo,
2 Department of Biochemistry, Institute of Brain Research, Faculty of Medicine, The University of Tokyo)
Recoverin, a new member of the EF-hand superfamily, plays a critical rolein the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a CaH -dependent manner. In rods, CaH -bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation ofrhodopsin. However, the functional significance of CaH -induced membraneassociation of recoverin has not been fully evaluated. We found that
CaH -bound recoverin forms a complex with rhodopsin kinase preferentiallyat the membrane surface. The addition of increasing amounts of membranes
promoted the membrane association of recoverin, and remarkably suppressedrhodopsin kinase activity. It was concluded that the CaH -recoverinrhodopsin kinase complex is stabilized by membrane association, leading toeffective suppression of kinase activity. For details, see Ref. 4.

D.

DEPARTMENT OF POPULATION GENETICS(8)

Studies on Photoreception in the Brain.

85

Yoshitaka FUKADA, Toshiyuki OKANO 1, Kamon SANADA 1, Tomoko YOSHIKAWAt, and Yoko T AKANAKA 1

C Department of Biophysics and Biochemis-

try, Graduate School of Science, The University of Tokyo)
We are studying the molecular mechanism of the light-dependent phaseshift of the chicken pineal circadian clock system.

Investigation of thephoton-signalling pathway mediated by pinopsin will be one fruitful approachto the circadian oscillator.
Publications
Nei, M. and Takahata, N.: (eds.) "Current Topics on Molecular Evolution", The
Pennsylvania State University/The Graduate University for Advanced Studies, 1996.
2. Kondo, R., Takahata, N., and Horai, S.: Nucleotide substitutions in the hominoid
mitochondrial DNA. In "Current Topics on Molecular Evolution", (M. Nei and N.
Takahata eds.), pp. 45-51, The Pennsylvania State University/The Graduate University
for Advanced Studies, 1996.
3. Satta, Y., O'huigin, c., Takahata, N., and Klein, J.: Intensity of overdominant selection
at the major histocompatibility complex loci. In "Current Topics on Molecular Evolution", (M. Nei and N. Takahata eds.), pp. 179-187, The Pennsylvania State University/
The Graduate University for Advanced Studies, 1996.
4. Takahata, N.: Neutral theory of molecular evolution. Current Opinion in Genetics andDevelopment, 6, 767-772, 1996.
5. Takahata, N. and Klein, J.: Multiregional or uniregional origin of modern humans: Whatdo genetic data indicate? In "The Origins and Past of Modern Humans-Toward a
Reconciliation" (K. Omoto and K. Hanihara eds.), World Scientofic Publishing Company PTE LTD, Singapore, 1997, in press.
6. Vincek, V., O'huigin, C., Satta, Y., Takahata, N., Boag, P. T., Grant, P. R., Grant, B. R.,
and Klein, J.: How large was the founding populations of Darwin's finches? Proc. Roy.
Soc. 1997, in press.
7. Kasahara, M., Nakaya, J., Satta, Y., and Takahata, N.: 1997 Chromosomal duplicationand the emergence of the adaptive immune system. Trends in Genetics, 1997, in press.
8. Takahata, N. and Satta, Y: Evolution of the primate lineage leading to: Phylogenetic anddemographic inferences from primate DNA sequences. Proc. Nat!. Acad. Sci. USA.
1997, in press.
9. Kojima, D., Oura, T., Hisatomi, 0., Tokunaga, F., Fukada, Y., Yoshizawa, T., andShichida, Y: Molecular properties of chimerical mutants of Gecko blue and bovine
rhodopsin. Biochemistry, 35, 2625-2629, 1996.
10. Ohguro, H., Kitamura, K., Konari, K., Sohma, H., Fukada, Y., and Akino, T.: The
differences in the expression of visual pigments and transducin in photoreceptor cell
I.

86

11.

12.

13.

14.

15.
16.

differentiation. Tohoku J. Exp. Med., 178, 233-240, 1996.
Shouno, 0., Kokame, K., Araki, M., Takao, T., Shimonishi, Y., Murata, M., Yoshizawa,T., and Fukada, Y.: Preparation and characterization of monoclonal antibodies specificfor lauroylated isoform of bovine transducin a-subunit: Immunohistochemical analysis ofbovine retinas. J. Neurochem., 66, 2188-2196, 1996.
Sanada, K., Shimizu, F., Kameyama, K., Haga, K., Haga, T., and Fukada, Y.: Calciumbound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation. FEBS Lett., 384, 227-230, 1996.
Tanaka, H., Kuo, C.-H., Matsuda, T., Fukada, Y., Hayashi, F., Ding, Y., Irie, Y., andMiki, N.: MEKA/Phosducin attenuates hydrophobicity of transducin bg subunits without binding to farnesyl moiety. Biochem. Biophys. Res. Commun., 223, 587-591, 1996.
Nishimura, S., Sasaki, J., Kandori, H., Matsuda, T., Fukada, Y., and Maeda, A.:
Structural changes in the peptide backbone in complex formation between activatedrhodopsin and transducin studied by FTIR spectroscopy. Biochemistry, 35, 1326713271, 1996.
Okano, T., Yamazaki, K., Kasahara, T., and Fukada, Y.: Molecular cloning of heterotrimeric G-protein a-subunits in chicken pineal gland. J. Mol. Evol., in press.
Hirunagi, K., Ebihara, S., Okano, T., Takanaka, Y., and Fukada, Y.: Immuno-electron
microscopic investigation for the subcellular localization of pinopsin in the pineal organof the chicken. Cell Tissue Res., in press.

87

E.

DEPARTMENT OF INTEGRATED GENETICS
E-a.

(1)

Division of Human Genetics

Isolation of Transcribed Sequences in Cloned Genomic DNA
with Known Location on the Human Chromosome 18

Takashi IMAMURA, Masako SAKAI, Rie INABA, and Hitoshi NAKASHIMA l
CFirst Department of Medicine, Kyushu University)
Identification and recovery of transcribed sequences from cloned humangenomic DNA remain an important problem in isolating genes on the basis of
their chromosomal location. Tetrasomy 18p and trisomy 18 syndromes arecaused by an excessive number of genes on chromosome 18. To identify
genes involved in these disorders, construction of high-resolution expression
map of this chromosome is a rational strategy. We have developed a strategy
that facilitates the recovery of transcribed sequences from random pieces ofcloned genomic DNA. Thus, we isolated 600 cosmid clones of which 60 new
cosmids are mapped on the short and long arms of chromosome 18 either by
R or by DAPI banding and simultaneous fluorescence in situ hybridization.
Our approach to generate region-specific eDNA libraries used hybridizationof eDNA library inserts to genomic DNA from cosmids, and subsequentcloning of annealed cDNAs. In one application of the scheme, DNA
fragments of a digested cosmid were ligated to an oligonucleotide linker/
adaptor, which were hybridized with human fetal brain eDNA library boundto the surface of streptavidin-coated magnetic beads, and cDNAs thus
selected were amplified by PCR before cloning.
While considerable progress has been made in genetic and physical mapping of the human genome, approaches are now needed to identify the protein
coding sequences contained therein. Although most genes are composed ofmultiple exons, the identification of a gene requires the recovery of only asingle exon. These candidate exons are ideally suited for establishing thepresence of a gene in a cosmid insert and facilitating the subsequent isolationof this gene. Our current experience suggests that everyone or two cosmids
contain at least one transcribed sequence. Further screening of unchar-

88

acterized cosmids will determine the utility of transcribed sequence fragments of known chromosomal location, as identifiers of candidate coding
sequences. For the details, see reference 1.
(2)

Molecular Definition of Maternally Imprinted Genes on
Chromosome 15 and the Development of
Prader-Willi Syndrome

Takashi IMAMURA, Masako SAKAI, Rie INABA, and Tomoko HASEGAWA 1
C Shizuoka Children's Hospital)
The Prader-Willi syndrome (PWS) is characterized by generalized muscular hypotonia and severe feeding problems in early infancy, followed byhyperphagia and subsequent central obesity in childhood. In addition toshort statue and characteristic dysfunction causing hypogonadism, dysmorphic signs such as almond-shaped palpebral fissures, narrow bifrontal diameter, down-turned mouth and acromicria become evident. In time, patients
may manifest intellectual deficiency, behavioral abnormalities, and poor
articulations. Originally defined as a clinical entity, PWS was later found tobe associated with abnormalities of chromosome 15, in particular a smallinterstitial deletion of bands qll-q13 of the paternally derived chromosome
15. Non-deletion PWS patients exihits maternal disomy for chromosome 15,
further demonstrating that loss of the expressed paternal alleles of maternallyimprinted genes is responsible for the PWS phenotype. Thus, PWS is adistinct neurogenic disorder, caused by the loss of function of a gene orclosely linked genes on chromosome 15. Paternal deletion and maternal
disomy in PWS suggest that the PWS genes are transcribed from the paternal
chromosome only. A system was developed in which subtraction and kinetic
enrichment was used to purify restriction endonuclease fragments present ina population of cDNA fragments from the normal individual but not in theother from the patient. Application of this method to cDNA library of
reduced complexity resulted in isolation of more than 20 probes present assingle copies that are down-regulated for their expression in the PWS library.
These probes could detect difference of levels between patient's cells andthose of normal individuals.
A mechanism of imprinting may have evolved in mammals because of
conflicting interests of maternal and paternal genes in relation to the transfer

E.

DEPARTMENT OF INTEGRATED GENETICS89

of nutrients from the mother to her offspring. This hypothesis predicts mostimprinted genes will affect how much nourishment an offspring receives fromits mother at the expense of its siblings. Imprinted genes will include loci that
influence placental growth, neonatal behavior, suckling and appetite control,
nutrient metabolism, and postnatal neuronal growth. The mapping of genes
identified here to within the PWS critical region, together with the indications
that they function in the brain and may playa role in regulating neuronal
growth and feeding behavior as well, should prompt us further to predictthese genes as a candidate imprinting loci responsible for syndrome.
(3)

Interleukin-4 Induces Association of the c-fes Proto-oncogene
Product with Phosphatidylinositol-3-kinase and Activates
Two Distinct Signal Transduction Pathways

Kenji IZUHARA
Interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely
related to the c-fes proto-oncogene product (FES), and association of thisprotein with IL-4 receptor a chain (IL-4Ra). IL-4 is known to induce
association of phosphatidylinositol-3 (PI3) kinase with IL-4Ra. Since FES
contains the consensus motifs for binding to PI3, FES may associate with PI
3 kinase upon IL-4 stimulation. We demonstrated that IL-4 stimulationinduced in mouse T cell lines rapid association of FES (or a related protein)with PI3 kinase, and that human FES (hFES) associated with src homology
2 (SH2) domain of PI3 kinase in a COS? cell expression system. The in vitro
PI3 kinase assay using COS? cells suggestes that hFES partly contributes toan association between hIL-4Ra and PI3 kinase. We further identified theimportant region in the cytoplasmic domain of hIL-4Ra for the association oftyrosine-phosphorylated hFES with hIL-4Ra and SH2 domain of PI3 kinase
using a COS? cell expression system. These results suggest that the FES (ora related protein)-PI3 kinase pathway may playa role in the pleiotropic
effects of IL-4.
c-Fes protooncogene product (or a FES-related protein) associates with
IL-4Ra and PI3 kinase in mouse T cell lines. PI3 kinase has been reported
to associate with IL-4Ra through tyrosine-phosphorylated-insulin receptor
substrate (IRS-2) in other cell types. We analyzed association of PI3 kinasewith IRS-2, and tyrosine-phosphorylation of IRS-2 in a mouse pro-B cell line,

90

Ba/F3, and a mouse mast cell line, MC9. In both cell lines, IL-4 induced

tyrosine phosphorylation of IRS-2, association of PI3 kinase with IRS-2, FES
or a FES-related protein. These results indicate that IL-4 activates two
distinct PI3 kinase pathways in the same cells. We further identified thecritical region in the cytoplasmic domain of IL-4Ra for tyrosine phosphorylation of IRS-2. For the details, see Ref. 2 and 3.
(4)

Mitochondrial DNA Polymorphism in East Asian Populations,with Special Reference to the Peopling of Japan

Satoshi HORAI, Goonnapa FUCHAROEN 1, Shinji HARIHARA2 , Kyung Sook
PARK 3, Keiichi OMOT04, and I-Hung PANs C Khon Kaen University,
2 Department of Anthropology, Graduate School of Science, University ofTokyo, 3 Department of Biology, Sung-Shin Women's University, 4 International research Center for Japanese Studies,s National Taiwan University)
Nucleotide sequences of the major noncoding (D-loop) region of humanmitochondrial DNA from five East Asian populations including mainland
Japanese, Ainu, Ryukyuans, Koreans and Chinese were analyzed. Based on
a comparison of 482 base pair sequences in 293 East Asians, 207 different
sequence types were observed. Of these, 189 were unique to their respective
populations, whereas 18 were shared between two or three populations.
Among the shared types, eight were found in common between the mainland
Japanese and Koreans, which is the largest n~mber in the comparison. Theintergenic COII/tRNALys 9 base pair deletion was observed in every East
Asian population with varying frequencies. The D-loop sequence variationsuggests that the deletion event occurred only once in the ancestry of East
Asians. Phylogenetic analysis revealed that East Asian lineages were classified into at least 18 monophyletic clusters, though lineages from the five
populations were completely intermingled in the phylogenetic tree. However,
we assigned 14 out of the 18 clusters for their specificity based on the
population from which the maximum number of individuals in each cluster
was derived. Of note is the finding that 50% of the mainland Japanese had
continental specificity in which Chinese or Koreans were dominant, while lessthan 20% of either Ryukyuans or Ainu possessed continental specificity.
Phylogenetic analysis of the entire human population revealed the closest
genetic affinity between the mainland Japanese and Koreans. Thus, theE. DEPARTMENT OF INTEGRATED GENETICS

91

results of this study are compatible with the hybridization model on the originof modern Japanese. It is suggested that about 65% of the gene pool inmainland Japanese was derived from the continental gene flow after the
Yayoi Age. For details, see Ref. 4.
(5)

The Origin and Dispersal of Modern Humans as
Viewed from Mitochondrial DNA

Satoshi HORAI, Shingo ODANIt, Minato NAKAZAWA 1, and Ryutaro OHTSUKAt CDepartment of Human Ecology, School of Health Sciences, Universityof Tokyo)Modern Homo sapiens originated in Africa somewhere between 125,000
and 161,000 years ago. Since then, a number of migrations out of Africa
have taken place, one of the longest journeys being the peopling of the New
World. To investigate the peopling all over the world, we examined the
mtDNA sequence variations in the D-Ioop region. We have compared
nucleotide sequences of the D-Ioop region for 271 individuals from six major
geographic regions: the Americas, Africa, Europe, Southeast Asia, East Asia,
and Papua New Guinea. The nucleotide diversity is highest in Africans,which is estimated at 2.11 %, while the lowest value is observed in Europeans,
0.96%. Almost the same values are observed in four Mongoloid populations
such as Asians from two regions, Native Americans and Papuans, althoughsampling size and nature of the populations are different; these range from1.30 to 1.37%. On the basis of pairwise number of nucleotide substitutions,a phylogenetic tree was constructed for 271 humans. It is obvious that mostAfricans located near the root of the tree, while some appeared in various
parts of the tree. Most Europeans fell into a final cluster, which is far fromthe root. This agrees with the lowest value of nucleotide diversity observedin Europeans. About 50,000 years ago, a sea level was over 80 meters below
what it is today. The lower sea level connected the Australian continent andPapua New Guinea, and exposed Sahulland. The first appearance of humans
in this region took place around this time. The Gidra people in Papua New
Guinea are considered to be descendants of such early migrants, who speaka non-Austronesian language. Some Papuans appeared in early diverging
clusters near the root, while the rest fell into four other clusters in different
locations of the tree. East Asians such as Japanese, Koreans and mainland

92

Chinese are dispersed throughout the tree. The same applies to Southeast
Asians such as Thais, Malays and Indonesians. Although 21 individuals wereanalyzed, they are dispersedly located in the tree. Contrary to the Asian
populations, the majority of Native Americans fell into four distinct clusters.
Assuming the deepest root of human mitochondrial divergence as 143,000
years ago, we estimated coalescence times of each human population; thelongest coalescence time was observed in the African population, and theshortest in the European population. It is to be noted that Papuans also
showed a deeper coalescence time, which was estimated around 124,000 years
ago. This may indicate that ancestors of some Papuans were the earliest
migrants of Homo sapiens who went out of Africa. For details, see Ref. 5.
(6)

Molecular Phylogeny of Macaques: Implications of Nucleotide
Sequences from an 896 Base Pair Region of
Mitochondrial DNA

Kenji HAYASAKA, Kunihiko FUJII, and Satoshi HORAI
We determined the nucleotide sequences of an 896 base pair region of
mitochondrial DNA (mtDNA) from 20 primates representing 13 species ofmacaques, a baboon, and a patas. We compared these sequences and thehomologous sequences from four macaques and a human against each otherand deduced the phylogenetic relationships of macaques. The results fromthe phylogenetic analyses revealed five groups among the macaques: (1)Barbary macaque, (2) two species of Sulawesi macaques, (3) Japanese,rhesus, Taiwanese, crab-eating, and stump-tailed macaques, (4) toque, pigtailed, and lion-tailed macaques, and (5) Assamese and bonnet macaques.
The phylogenetic position of Tibetan macaque remains ambiguous as towhether it belongs to the fourth or fifth group. Phylogenetic trees revealed
that Barbary macaque diverged first from the other Asian macaques. Subsequently, the four groups of Asian macaques diverged from one another in a
relatively short period of time. Within each group, most of the species
diverged in a relatively short period of time following the divergence of thegroups. Assuming that the Asian macaques diverged from the outgroup
Barbary macaque three million years ago (MYA), the divergence times
among groups of Asian macaques were estimated at 2.1-2.5 MYA, andwithin groups at 1.4-2.2 MYA. The intraspecific nucleotide diversity ob-

E. DEPARTMENT OF INTEGRATED GENETICS

93

served among three rhesus macaques was so large that they did not form amonophyletic cluster in the phylogenetic trees. Instead, one of them formed
a cluster with Japanese and Taiwanese macaques, while the other two formed
a separate cluster. This implies that either polymorphisms of mtDNA
sequences which existed before the divergence of these three species (ca.
700,000 years ago) have been retained in rhesus macaques or introgression
has occurred among the three species. For details, see Ref. 6.
(7)

Detection of DNA Fragments Encompassing the Deletion
Junction of Mitochondrial Genome

Yu-ichi GOT0 1, Ichizo NISHIN0 1, Satoshi HORAI, and Ikuya NONAKA 1
C Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry)
Rearrangements of human mtDNA are associated with not only mitochondrial diseases, including CPEO and Pearson's marrow-pancreas syndrome, but also with the aging process in various tissues such as the brain.
Both Southern blot analysis and the PCR are usually applied to the detectionof deletions in mtDNA because these methods have their own advantages.
We here describe a combined method of a long PCR and digestion with three
restriction enzymes that simplifies the detection of the deletion site andenables us to determine the adjacent junctional sequences. The long PCR
method together with restriction enzyme digestion is also useful to map thedeletion site on mtDNA, and it is simple to estimate the deletion site. One of
the reasons why we chose Xba I, Hind III and Bam HI is that it is convenient
to use the three enzymes in a single reaction tube because the optimal
enzymatic conditions (e.g., salt concentration) are almost the same. Anotherreason is that polymorphisms associated with the three enzymes are uncommon. Among 120 Japanese, only one morph has been observed in XbaI and
BamHI, and the most frequent morph in Hind III is seen in 97.5%. The
above estimation immediately allows sequencing of the deletion junction.
The determination of junctional sequences is essential for considering theprobes within and without the deletion to detect a possible duplication on
Southern blot. Because duplicated genomes are sometimes transmitted
maternally, their presence offers an important information for genetic consultation in clinical practice. The disadvantages of the method described here94

are the inability to detect duplications and to determine the proportion ofmutants. These are the same disadvantages as those of the conventional PCR
method, so they can be complemented by Southern-blot as before. We againemphasize that the combined use of Southern blot and PCR methods arenecessary to determine the exact nature of mtDNA deletions. Nevertheless,the long PCR and three-enzyme digestion method is a very useful means forscreening mtDNA rearrangements. For details, see Ref. 7.
(8)

A Novel Mutation in the Mitochondrial tRNAThr Gene Associatedwith Mitochondrial Encephalomyopathy

Ichizo NISHINO!, Kenzo TAKESHITA 2 , Satoshi HORAI, Ikuya NONAKA 1, andYu-ichi GOT0 1 C Department of Ultrastructural Research, National Instituteof Neuroscience, 2 Department of Neuropediatrics, Tottori University Hospital)
A novel G-to-A transition at nucleotide 15915 in mtDNA is described.
The patient showed a combination of muscle weakness, hearing loss, mental
retardation, and seizures. Muscle biopsy showed RRFs and focal COX
deficiency. We sequenced all mtDNA, and found 5 novel nucleotide substitutions. Three of them were synonymous mutations, one was a missense
mutation in cytochrome b gene (A~G at nt 15422), and the last one was the15915 mutation in tRNAThr gene. We screened for the 15422 and the 15915
mutations with mismatch primers and found that one of 104 normal individuals carried the former one and none of 175 had the latter one. The 15422
mutation existed in homoplasmic states both in the patient and the normal
individual, suggesting that this is a polymorphism. In contrast the 15915
mutation resided in heteroplasmic states in muscle, skin fibroblast and blood.
The nucleotide substitution at nt 15915 disrupts a highly conserved base pairin anticodon stem of the tRN A Thr . Our data suggest that the 15915 mutation
is an additional mtDNA mutation responsible for mitochondrial encephalomyopathies. For details, see Ref. 8.

E. DEPARTMENT OF INTEGRATED GENETICS(9)95

The 3260 Mutation in Mitochondrial DNA Can Cause
Mitochondrial Myopathy Encephalopathy Lactic
Acidosis and Stroke-like Episodes (MELAS)

Ichizo NISHINO 1, Satoshi HORAI, Ikuya NONAKAl, and Yu-Ichi GOT0 1(' Department of Ultrastructural Research, National Institute of Neuroscience)
Maternally inherited myopathy and cardiomyopathy (MIMyCa) is one ofthe phenotypic subgroups of mitochondrial diseases. There are two reports ofEuropean families with MIMyCa associated with an A-to-G transition at
nucleotide (nt) 3260 in the mitochondrial tRNALeu(UUR) gene. We haveidentified the 3260 mutation in a Japanese family with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)
accompanied by myoclonic epilepsy. We directly sequenced a series ofpolymerase chain reaction (PCR)-amplified fragments encompassing all themitochondrial tRNA genomes. Thirty sequence primers were set for both L
and H strands. The data were compared with the reference sequence andonly one substitution was found at nt 3260. Restriction fragment length
polymorphism analysis using the previously reported mismatch primer thatcreates the Xmn I recognition site showed heteroplasmy not only in theproband but also in the mother and the siblings. To estimate the proportionof the mutant mitochondrial DNA, we performed a last cycle cold PCRI
RFLP method which allowed us to avoid errors due to heteroduplex formation during PCR. The amplified fragments were digested with Xmn I
over-night and analyzed in an automated sequencer. The proportions of themutant mtDNA were estimated as follows: the mother 29.4% in blood; theproband 87.1 % in muscle, 51.0%; the first brother 31.6%; and the second
brother 44.1 %. Is the MELAS phenotype of the 3260 mutation specific to
the Japanese? The mitochondrial tRNALeu(UUR) gene is a hot spot for thepathogenic mutations including the 3243, 3252, 3271, and 3291, all of whichhave been associated with MELAS. On the other hand, cardiomyopathy is arelatively frequent complication of mitochondrial encephalomyopathies including MELAS. Thus, we could consider both MELAS and MIMyCa aspart of a spectrum of phenotypic expressions of the 3260 mutation. Althoughfurther studies are needed to elucidate the mechanism for the variablephenotypic expression, our data confirm that the 3260 mutation is a genetic

96

cause of MELAS as well as MIMyCa.
Publications
Sakai, M., Inaba, R., and Imamura, T.: eDNA selection: Efficient PCR approach for theselection of cDNAs encoded in chromosome 18 DNA fragments (CIT: 763480). In:
Report of the Fourth International Workshop on Human Chromosome 18 Mapping
1996, G. A. Silverman, J. Overhauser, S. Gerken, and Ad Geurts van Kessel, Eds.,
Cytogenetics and Cell Genetics, 75, 111-131, 1996.
2. Izuhara, K., Feldman, R. A., Greer, P., and Harada, N.: Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase. Blood, 88,
391~3918, 1996.
3. Izuhara, K. and Harada, N.: Interleukin-4 activates two distinct pathways of phosphatidylinositol-3-kinase in the same cells. Biochem. Biophys. Res. Commun., in press.
4. Horai, S., Murayama, K., Hayasaka, K., Matsubayashi, S., Hattori, Y., Fucharoen, G.,
Harihara, S., Park, K-S., Omoto, K., and Pan, I-H.: mtDNA polymorphism in East
Asian populations, with special reference to the peopling of Japan. Am. J. Hum. Genet.,
59, 579-590, 1996.
5. Horai, S., Odani, S., Nakazawa, N. and Otsuka, R.: The origin and dispersal of modern
humans as viewed from mitochondrial DNA. In: Ethnoepidemiology on Cancer. K.
Tajima & S. Sonoda (eds.), Gann Monograph on Cancer Research No. 44, Japan
Scientific Societies Press, pp. 97-105, 1996.
6. Hayasaka, K., Fujii, K., and Horai, S.: Molecular phylogeny of macaques: implicationsof nucleotide sequences from an 896 base pair region of mitochondrial DNA. Mol. BioI.
Evol., 13, 1044-1053, 1996.
7. Goto, Y., Nishino, I., Horai, S., and Nonaka, I.: Detection of DNA fragments encompassing the deletion junction of mitochondrial genome. Biochem. Biophy. Res.
Commun., 222, 215-219, 1996.
8. Nishino, I., Seki, A., Maegaki, Y., Takeshita, K., Horai, S., Nonaka, I., and Goto, Y.: A
novel mutation in the mitochondrial tRNA Th ' gene associated with a mitochondrial
encephalomyopathy. Biochem. Biophy. Res. Commun., 225, 18~185, 1996.
9. Nishino, I., Komatsu, M., Kodama, S., Horai, S., Nonaka, I., and Goto, Y.: The 3260
mutation in mitochondrial DNA can cause mitochondrial myopathy, encephalopathy,
lactic acidosis, and stroke-like episodes (MELAS). Muscle & Nerve 19,1603-1604,1996.
10. Horai, S., Kondo, R., Sonoda, S., and Tajima, K.: The first Americans: Different wavesof migration to the New World inferred from mitochondrial DNA sequence polymorphisms. In: Prehistoric dispersal of Mongoloids. T. Akazawa & E. Szathmary (eds.),
Oxford University Press, pp. 27~283, 1996.
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Division of Agricultural Genetics

Inbreeding Depression, Outbreeding Depression andHybrid Vigor with Special Reference tothe Mating System in Rice Species

Hiroko MORISHIMA
The level of inbreeding depression and outbreeding depression is fundamental to the evolution of the mating system in plants. This problem wasinvestigated using wild-rice populations (Oryza rufipogon) having different
outcrossing rates.
I) Estimation of the level of inbreeding depression in natural populations: Relative fitness of selfed progeny (inbreeding depression) was indirectly estimated from inbreeding coefficients observed before and after natural
selection of the progeny generation (seed and adult), and selfing rate. These
parameters were estimated based on isozyme polymorphism data obtained
from four wild-rice populations collected in Thailand. Relative fitness was
0.28 and 0.09 for two predominantly selfing populations, and 0.42 and 0.68
for two partially outbreeding populations. The former values were significantly below the 0.5 threshhold thought to favor selfing.
2) Deleterious genes maintained in inbreeding and outbreeding populations: 25 first-generation individuals derived from the original seeds (collected in nature) of three wild-rice populations were selfed and crossed with eachother (incomplete diallele cross). Seeds of their selfed progeny showed a highgermination percentage in inbreeding annuals as well as in outbreeding
perennials, though the germination percentage of original seeds was high in
inbreeding annuals but low in partially outbreeding perennials. This indicatesthat outbreeding perennials harbor recessive deleterious alleles but they were
purged through seed propagation. The germination percentages of F 1 seeds
obtained from crosses between individuals belonging to an annual population
was significantly lower than those from crosses between different populations.
Histological observation of those ungerminated intra-populational F 1 seeds
showed abnormal development of embryos. Outbreeding depression or
hybrid invariability is usually considered a mechanism to separate species or98subspecies. The outbreeding depression within an inbreeding population
found in this experiment could be a genetic mechanism to favor selfing.
3) Variation in hybrid vigor found within wild rice population: Hybrid
vigor was quantified in terms of the F l/midparent of plant biomass using F1s
obtained from the above-mentioned diallel cross and a number of previous
crossing experiments. In hybrids between related species in rice, the values
usually exceed 1.0, showing hybrid vigor. Fts with wild rice generally showed
lower values. Within a partially outbreeding wild-rice population, the valuesof hybrid vigor ranged from 0.6 to 1.8. The coexistence of hybrid vigor andhybrid weakness within a population might contribute to the maintenance ofa mix-mating system in wild rice.
(2)

Analysis of Quantitative Trait Loci Underlying
Domestication in Rice

Hong-Wei CAl and Hiroko MORISHIMA
The essential differences between cultivated rice (Oryza. sativa L.) andwild rice (0. rufipogon Griff.) lie in self-propagating ability and seed productivity. The component traits underlying these characteristics are mostly
quantitative. Recent developments in the rice genome project enabled us tomap a number of loci controlling quantitative traits which could be analyzed
only by quantitative genetic methods in the past. The objective of the present
study is to map QTL responsible for rice domestication and elucidate their
network, if any, on the rice genome. An F 7 recombinant inbred populationconsisting of 118 lines derived from a cross between cultivated rice (l08;
Indica from Taiwan) and wild rice (W1944: O. rufipogon from China) wasused. Eighty-five RFLP and isozyme markers were examined. An RFLP
assay was carried out using the ECL non-radioisotope method.
A total of 29 characters were observed. They included seed dormancy,
seed shattering, regenerating ability and panicle exertion as self-propagating
ability, with panicle number, panicle length, seed fertility, etc. as seedproductivity. Cosegregation between markers and characters was analyzed
using qGENE software. In all the characters studied, two to 44 QTLs weredetected with single point analysis at a 5% significance level. The QTLs forrespective characters generally had small gene effects and proved to be widelydistributed on different chromosomes. This suggests that domestication of

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rice was a gradual process accumulating a number of mutations with small
effects. Further, we found that some developmentally correlated charactersshared common QTLs indicating pleiotropy. On the other hand, several
QTLs controlling coadapted characters tended to cluster on the particularregions of several chromosomes. This suggests that there may be an "adaptive gene block" promoting domestication.
(3)

Geographical Differentiation Found in
Annual Types of Oryza rufipogon

Hong-Wei CAl and Hiroko MORISHIMATo examine whether or not differentiation exists in annual-type O. rufipogon, a total of 73 annual strains collected in tropical continental areas, suchas Thailand, Laos, Cambodia, Burma, India, Bangladesh and Sri Lanka were
examined. Thirty-two perennial strains collected in the same regions wereused as controls. Polymorphisms at 20 polymorphic isozyme loci of 7
enzymes were analyzed.
The results showed that there are no clear differences in allele frequencies
between annual and perennial types except for a few loci such as Estl. The
alleles of Est13-l, Est9-l andAmpl-3 frequently detected in Chinese strains in
our previous study were rare in tropical strains. Strains collected on the west
coast of India showed high frequencies of alleles Est5-3, Sdhl-3, Pgil-3 andPgil-4 which were rarely detected in other areas. The results of factor
analysis demonstrated that the strains examined can be separated into two
major groups. One group consists of the strains collected mostly from theIndian west coast including both annual and perennial types, and anothergroup contains the strains from other areas. These results indicated annualtypes of O. rufipogon showed geographical differentiation at the isozyme levelin parallel with perennial types.
Judging from the results of our previous study (Cai et al., 1995) and thepresent study, we concluded that Asian common wild rice can be largely
divided into three geographical groups based on isozyme variation, i.e., 1)
Chinese strains carrying some Japonica-specific isozyme alleles, 2) Indian
west coast strains characterized by unique genotypes rarely found in otherareas, 3) strains distributed in the tropics excluding the Indian west coast.
The geographical differentiation thus revealed in Asian common wild-rice100seems to be independent of ecotypic differentiation into perennial and annualtypes. (Ref. 3)(4)

Basic Study of Genetic Erosion of Wild Rice(Oryza rufipogon) in Thailand

Masahiro AKIMOTOI, Yoshiya SHIMAMOTOI, and Hiroko MORISHIMA
CFaculty of Agriculture, Hokkaido University)
In recent years, many natural populations of wild rice (Oryza rufipogon)
have approached new extinction. The major factors of genetic erosion in wild
plants are destruction of their habitats by human activity and genetic
contamination by cultivated rice. To obtain basic information on the genetic
erosion of wild rice, we are investigating the influence of environmental
change on the genetic structure of wild-rice populations. The site we studied
is a road-side swamp inhabited by perennial or an annual/perennial intermediate type of 0. rufipogon, which is located in the suburb of Ayuthya city inthe central plane of Thailand, and adjacent to a cultivated rice field on one
side. We examined allozyme diversity at 15 loci of 10 enzymes by starch andpolyacrylamide gel electrophoresis using two sets of seed samples collectedfrom the same site in 1985 and 1994, respectively.
Based on allele frequencies, we estimated the gene diversity parameters.
Gene diversity of the overall population decreased from 0.278 to 0.249 during
the period between 1985 and 1994. Recently a gasoline station was constructed on the site and the wild-rice population was seriously damaged on
account of destruction of their habitat and water pollution. Apparently adrastic decline in plant number forced surviving plants to inbreed and thiscaused a decrease in gene diversity.
An allele Pgil-l, is known to be common among Indica rice varieties andso dominant in cultivars of this area, while wild rice in this area generally
carry another allele, Pgil-2. In this study-site, we found Pgil-l in somewild-rice plants growing near cultivated rice. Most probably, introgression
has occurred from cultivated to wild rice populations. The frequency ofplants having the Pgil-l allele was higher in 1994 than in 1985, indicatingthat the intrinsic features of wild rice have been continually replaced by thoseof cultivated rice.
We must recognize the present situation in which genetic erosion is rapidly

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progressing and conservation efforts for natural environments are urgently
needed. (Ref. 2)(5)Inter- and Intra-Specific Differentiation of Oryza glumaepatula
Found in Central and South America

Masahiro AKIMOT0 1, Yoshiya SHIMAMOT0 1, and Hiroko MORISHIMAC Faculty of Agriculture, Hokkaido University)

Oryza glumaepatula is a diploid wild rice species found in Central andSouth America. It shares the genome AA with Asian cultivated rice and isthought to be a useful genetic resource for rice breeding. However, we havelittle information on the life-history of this taxa and evolutionary relationships with other related species. We examined variation in ecological andmorphological characters, isozymes and mtDNA polymorphisms among 40
accessions of O. glumaepatula collected in Central and South America. In
addition, several accessions of Asian (0. rujipogon), and Australian rice (0.
meridionalis) , and two African (0. longistaminata and O. barthii) wild ricespecies were also examined for comparison. I) From a survey of character
variation, we could divide O. glumaepatula into three ecotypes. Type I;
found in Central America and the northern part of South America. Regenerating ability and anther length (thought be related to outcrossing rate)
were the same as those of perennial O. rujipogon. Type II; found in the
Amazon flood basin. Regenerating ability and anther length showed anintermediate value between annual (predominantly selfing) and perennial(partially outbreeding) types of O. rujipogon. In the flooded period theirculms are easily broken at the lower part and the upper parts of plant bodies
become free floating on the water surface. Type III; found in southern Brazil(Pantanal swamp). Regenerating ability and anther length showed values
similar to those of type II. Matured seeds (about 5 mm in length) of type III
occupy only the lower half of the hull (11-12 mm length) leaving an empty
space inside the hull.
2) Allozymes were not so variable within O. glumaepatula and noremarkable differentiation was recognized among the three ecotypes. It was
found, however, that O. glumaepatula has developed unique isozyme genotypes by which it is separated from other related taxa.
3) Based on the mtDNA haplotype polymorphisms, O. glumaepatula

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accessions were categorized into two groups. One group consisted of theaccessions belonging to ecotype I and III, and their mtDNA haplotypes weresimilar to that of O. longistaminata. Another group consisted of theaccessions belonging to ecotype II, and their mtDNA haplotypes were similar
to that of O. meridionalis.
The phylogenetic relationships between O. glumaepatula and related taxa
found on other continents remains unclear.
Publications
1.

2.
3.

4.

5.

6.
7.

8.

Akimoto, M., Shimamoto, Y., and Morishima, H.: Completely sterile populations of wild
rice found in the suburbs of Bangkok. RGN 13,62-65, 1996.
Akimoto, M., Shimamoto, Y., and Morishima, H.: A case study of genetic erosion of wild
rice in Thailand. RGN 13, 60-62, 1996.
Cai, H. W., Wang, X. K., and Morishima, H.: Geographical differentiation of annual
type of O. rujipogon. RGN 13, 67-69, 1996.
Hirano, H.-Y, Eiguchi, M., and Sano, Y: A point mutation, G to T, causes thedifferentiation of the Wx' allele from Wx" allele, which is specific to Japonica rice. RGN
13, 148-149, 1996.
Morishima, H. and Chitrakon, S.: Genetic erosion in rice species. In "Biodiversity inAsia"., Ministry of Sci., Tech. & Env., Thailand, pp. 113-116, 1996.
Sato, Y. I., Chitrakon, S., Charoendham, P., Shimamoto, Y., and Morishima, H.: Set-upof a permanent observation site of wild rice in Thailand. RGN 13, 65-67, 1996.
Suh, H. S., Cho, Y. C., Chung, T. Y., and Morishima, H.: Genetic basis of variation in
weedy rice collected from Republic of Korea. Int. Rice Res. Note, 21(1), 7-8, 1996.
YONEZAWA, K., ISHII, T., NOMURA, T., and MORISHIMA, H.: Effectiveness of somemanagement procedures for seed regeneration of plant genetic resources accessions.
Genetic Resources and Crop Evolution, 43, 517-524, 1996.

E-c.
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Division of Applied Genetics

Mitochondrial Genome Polymorphisms in Lolium perenne

Yoshiya SHIMAMOTO
The restriction fragment length polymorphisms (RFLPs) of mitochondrial
DNA (mtDNA) of perennial ryegrass (Loliumperenne L.) were investigatedto elucidate the genetic relatedness among 124 cultivars including diploid andtetraploid ones. Many patterns of RFLPs were observed and allowed toE.

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assign the cultivars into the main eight haplotypes of mitochondrial genome.
The American cultivars were classified to haplotype I and VIII which wereremote at mitochondrial genome relatedness each other, the European ones
were distributed thoroughly to all haplotypes and tetraploid ones were mostly
assigned into the haplotype V. In addition the two genes, coxlII and nad9, on
mitochondrial genome can be located closely each other. The assessment of
RFLPs upon mtDNA may be a valuable method in analyzing a cytoplasmic
differentiation among the perennial ryegrass cultivars. Further investigations
remained to elucidate the mtDNA diversity in relation with the maternal
effects on the agronomic traits of perennial ryegrass.
(2)

The Extent of Natural Cross Pollination in
Wild Soybean (Glycine soja)

Yoshiya SHIMAMOTO
To evaluate the extent of natural cross pollination in wild soybean (Glycinesoja), the genetic structures of four populations along the Omono River in
Akita Prefecture, Japan, were investigated by examining variations of several
allozymes. In addition, observations of pollinators were made in the natural
population. Although it was thought previously that G. soja is autogamous,
as is cultivated soybean (G. max), within-population genetic variation (H s =.315) were higher, and genetic divergence among population (G st = .087)
were lower than the values expected for selfing plant species. Based on allelicfrequencies of isozyme loci, the means ofmutilocus (tm) and single locus (ts)outcrossing rate estimates were 13% (9.3%-19%) and 8% (5%-14.7%)
among the four populations, respectively. These values are much higher thanthe outcrossing rates previously reported for both the wild and cultivatedsoybeans. This higher outcrossing rate was supported by observations of thefrequent visits of honey bees and hornets to the flowers. For details, see
Ref. 1.
(3)

Molecular Genetic Studies of Genomic Imprinting

Hiroyuki SASAKI!

eInstitute of Genetic Information, Kyushu University)

Genomic or parental imprinting denotes a biological process that modifies

104

the expression of the genome differently in male and female germline cells,
leading to differential activity of the parental genome in offspring. In orderto understand the molecular mechanisms involved in imprinting, we are
studying the distal portion of mouse chromosome 7, which contains at least
three imprinted genes, insulin 2 (Ins2) , insulin-like growth factor 2 (Igf2) ,and H19. The results obtained so far are as follows:(I) Based on findings obtained in human and mouse, we discussed thepossibility of imprinted genomic regions serving as a model to study thelong-range mechanisms that control gene expression (ref. 2). (2) We isolated
YAC clones that cover the imprinted Ins2/Igf2/H19 domain, and are making
a physical map of the region. (3) We reported the complete nucleotide
sequence of the 28-kb region containing the entire Igf2 (ref. 3). (4) We
cloned the L23mrp gene, which is located downstream of H19 and isbiallelically expressed. Furthermore, it was shown that L23mrp is functionally insulated from theH19 enhancers, suggesting that there may be a domainboundary between H19 and L23mrp (in preparation). (5) We have sequenced most of this putative boundary region, which is about 30 kb in size.
(6) We showed that Igf2, which has three different promoters, is controlled
mainly by a locus-wide mechanism (in preparation). (7) A convenientmethod for detection and mapping CpG islands has been devised (in preparation). This technique should be useful in identifying new imprinted genes.
(8) We determined the timing of the erasure and establishment of themethylation imprint which is present in the 5' flank of H19 (in preparation).
(9) In collaboration with Hayashizaki's group in RIKEN, we isolated a newpaternally expressed mouse gene, RAS-specific guanine nucleotide exchange
factor (Grfl) , on chromosome 9 (ref. 4). (10) We found; in cultured
embryonic fibroblast cells; an apparently new arrest-specific up-regulatory
sequence common to many imprinted genes, which may give us a clue to theevolution of the genomic imprinting mechanisms (submitted).

Publications
1.

2.
3.

Fujita, R., Ohara, M., Okazaki, K., and Shimamoto, Y.: The extent of natural crosspollination in wild soybean (Glycine soja). J. Heredity, 88, 74-78, 1997.
Nakao, M. and Sasaki, H.: Genomic imprinting: significance in development and diseases
and the molecular mechanisms (review). J. Biochem., 120, 467-473, 1996.
Sasaki, H., Shimozaki, K., Zubair, M., Aoki, N., Ohta, K., Hatano, N., Moore, T., Feil,

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R., Constancia, M., Reik, W., and Rotwein, P.: Nucleotide sequence of a 28-kb mouse
genomic region comprising the imprinted Igf2 gene. DNA Res., 3, 331-335, 1996.
Plass, C., Shibata, H., Kalcheva, I., Mullins, L., Kotelevtseva, N., Mullins, J., Kato, R.,
Sasaki, H., Hirotsune, S., Okazaki, Y., Held, W. A., Hayashizaki, Y., and Chapman, V.
M.: Identification of Grfl on mouse chromosome 9 as an imprinted gene by RLGS-M.
Nature Genet., 14, 106-109, 1996.

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GENETIC STOCK RESEARCH CENTER
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(I)

Mammalian Genetics Laboratory

Geological Distribution of a Y chromosome Specific
Sequence DYHI in Wild Mice

Yoshihiro UEDA 1,2, Tomoko SAGAI 1, S. T. BALL3, Kazuo MORIWAKI4, Toshihiko SHIROISHI 1 C Nat!. Inst. Genet., 2 Fukuyama Univ., 3 MRC Radiobiology Unit, UK, 4 Grad. Univ. for Adv. Sci.)
Polymorphic genes on the Y chromosome are unique markers for study ofgenetic differentiation in the wild mice population, because the Y chromosome is inherited from male and the genes on the Y chromosome have neverbeen shuffled by recombinational event except for the pseudautosomal region.
DYH I is a novel sequence specific to the Y chromosome, which has
homology with the sequence for the human proto-oncogene, KRAS2. We
investigated the geographical distribution of polymorphisms of the DYHI inwild mice populations by analyzing the PCR amplified sequences. As a result,
we found that DYHI is absent in populations of Mus musculus domesticus inwest-Europe and Mus musculus castaneus in southeast Asia. It was distributed in populations of Mus musculus musculus, from east Europe to east
Asia including Japan. This distribution pattern is unique when comparedwith those obtained from analyses with two other markers specific to the Y
chromosome, the Zinc-finger protein on Y-2 (Zfy-2) and the Sex determining
region-Y (Sry). Based on the geographical distribution pattern of these threedifferent markers specific to the Y chromosome, we intend to study theprocess of subspeciation of mouse.

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Molecular Characterization of Ph Hotspot in
the ClassII of Mouse MHC

Taku ISOBE, Masayasu YOSHINO l , Kirsten FISCHER LINDAHL l , Tomoko
SAGAI, Kenichi MIZUN0 2, Tsuyoshi KOIDE, Toshihiko SHIROISHI C Howard
Hughes Med. Inst., Univ. Texas, Dallas, 2 Grad. Univ. for Adv. Sci., Kanagawa)
The murine major histocompatibility complex (MHC) is an excellent
system for studying the molecular mechanisms of meiotic recombination,
where breakpoints of recombinations are systematically studied at the molecular level. It is well known that meiotic recombinations in this region do notoccur at random but are clustered in limited regions known as hotspots. Thusfar, four hotspots have been identified in this region. Three hotspots otherthan Pb have been well characterized at the molecular level. Namely, the finelocations of breakpoints of these three hotspots were analyzed and sequences .
around the Eb and Lmp2 hotspots were determined. Comparison of the isequence between the Eb and Lmp2 hotspots revealed several molecularmotifs commonly shared by the two hotspots. In order to elucidate the rolesof these motifs in recombination at the hotspots and to understand themechanism by which recombinations are restricted to hotspots, we need tocharacterize other hotspots at the molecular level.
Meiotic recombination takes place at a high frequency at a hotspot in thevicinity of the Pb gene, when the wild mouse-derived cas4 haplotype is usedin the genetic cross. Molecular characterization of this hotspot, has not yet
been done. So far, we have obtained six independent recombinants at the Pb
hotspot after screening six hundred mice generated from crosses between cas
4 and wm7 haplotypes. The breakpoints of these recombinants were localized
to a 15 kbp of DNA fragment in the vicinity of the Pb gene. In this study, we
attempted to make a more complete map of the breakpoints of the six cas4/
wm7 recombinants. First, we constructed the restriction map of the 15 kbp
of DNA fragment including the breakpoints. Subsequently, we determined
the parental origins of several short DNA segments. At a result, at least fiverecombinants were found to be confined to a 2.4 kbp of DNA segment
located proximal to the 3/ end of the Pb gene. Then we determined thesequence of this segment. The sequence of this Pb hotspot appeared to haveneither MT-middle repetitive family, the TCTG repeat or the solitary type108

LTR, indicating that these molecular motifs commonly shared by the Eb andLmp2 hotspots do not play key roles in determining the site specificity ofrecombinational breakpoints in the mouse MHC.
In addition to the cas4/wm7 recombinants, we obtained other recombinants generated from cas4/cas3 and cas4/b crosses. The breakpoints of theserecombinants were analyzed in the same way used for the cas4/wm7 recombinants. The results indicated that the breakpoints are located in a comparatively larger fragment at least 15 kbp in length, including the same 2.4 kbp
segment to which the breakpoints of the cas4/wm7 recombinants wereconfined.

(3)

Genetic Analysis of Coat-Color Mutation, rim2. III

Tomoko SAGAI, Tsuyoshi KOIDE, Kenjiro TANOUE!, Seiich ISHIGUR0 2 ,Hiromich YONEKAWA 1, Kazuo MORIWAKI 3 , Shigeharu WAKANA4, and Toshihiko SHIROISHI CThe Tokyo Metropolitan Institute for Medical science,
Tokyo, 2 Tohoku University, 3 The Graduate Universiy for Advanced Studies, Kanagawa, 4 Central institute for Experimental Animals, Kawasaki)
As previously reported, one of the RIMs (Recombination Induced Mutants), rim2, is a new allele of an old coat color mutation, pearl, and has
abnormalities similar to human hemorragic symptoms accompanied withalbinism, platelet storage pool deficiency (SPD). Platelet aggregation and
ATP release in platelets of the rim2 mouse using several agonists were clearly
reduced in comparison to those of wild type mouse. These results indicate thepossibility that rim2 could serve as a mouse model for human SPDs.
Prior to the positional cloning of the rim2 or pearl gene, we tried to makethe YAC contig covering the rim2 region. We screened YAC libraries withmicrosatellite markers tightly linked to the rim2 gene. First we obtained two
YAC clones including two marker sequences which perfectly cosegregatedwith the rim2 phenotype in 1570 back cross progeny. These clones were
nonchimeric, but were discontinuous with each other. Then we isolated the
STS end markers from the two clones and screened the YAC libraries again.
Finally we assembled a contiguous array of YAC clones spanning of rim2
region. The length of the entire contig was speculated to be a maximum of
1.5Mb. Now we are ready to carry out a rescue experiment via thetransgenesis of these YAC clones into the rim2 homozygotes to identify the

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GENETIC STOCK RESEARCH CENTER109

Y AC clone including the rim2 gene.

(4)

Mapping and Histological Analysis of the Mouse
Mutant (Rim3) Affecting Skin and Eye

Hajime SATO, Tsuyoshi KOIDE, Hiroshi MASUYA, Shigeharu WAKANA 1,
Tomoko SAGAI, Kazuo MORIWAKI 2, and Toshihiko SHIROISHI C Central
Institute for Experimental Animals, Kawasaki, 2 The Graduate University
for Advanced Studies, Kanagawa)Rim3 is a dominant mutant derived from an intra-MHC recombinant. It
is characterized by hairlessness and corneal opacity. In order to determine
the locus of the Rim3 gene, we carried out two kinds of crosses, cross I:(C57BL/lOSnJ-Rim3/ + X MSM)F] X C57BL/lOSnslc; cross II: (C57BL/
lOSnJ-Rim3/+ XJFl)F 1 XC57BL/lOSnslc. MSM is an inbred strain derived from Japanese wild mouse, while JFl is an inbred strain derived fromJapanese fancy mouse. These mice are suitable for linkage analysis because
their genetic backgrounds are far from those of laboratory mice. We finallyobtained 1545 progeny from cross I and 667 progeny from cross II. Linkage
analysis using microsatellite markers revealed that the Rim3 locus was tightly
linked to DllMitl45, 14, 124, 197 on Chromosome 11. Gene and microsatellite markers orders obtained from the two crosses are as follows, cross I:
centromere-Mit145-(0.26 cM)-Rim3-(0.06 cM)-Mit14, 124, 197; cross II:
centromere-Mit145-(0.30 cM)-Rim3, Mit14, 197-(0.15 cM)-Mitl24.
The Krt-1 (Keratin type 1) locus has been mapped near this position.
Keratins are a group of water-insoluble proteins that form lO-nm intermediate filaments in epithelial cells. These can be divided into acidic and basic
subfamilies according to their relative charge, immuno-reactivity, and sequence homologies. They are designated as type I and II keratins, respectively. Type I and II keratins contribute to cytoskeletal architecture as heterodimers. It is known that Krt-1 consists of at least 11 genes and its expression
patterns are tissue- and differentiation- specific. Therefore we intended to
examine whether Krt1-12 which is expressed in cornea and Krt1-10, 14, 16, 17
which are expressed in epidermis are possible candidates for the Rim3 gene.
At the present time, we have excluded Krtl-lO, 12, 14, 17 as candidate genes
for the Rim3 gene, because linkage analysis has indicated segregation of Rim
3 phenotype and these marker loci.

110

Several mouse mutants affecting the skin and cornea have also been
mapped to the region near the Rim3 locus. The bare skin (Bsk) mutation
causes baldness and corneal opacity. The denuded allele at the Rex locus(Re den ) has a similar phenotype. To characterize the corneal opacity andhairlessness in detail, we carried out histological analysis in Rim3 and Re den •We observed remarkable changes in the center of the cornea of Rim3
mutants. Keratin covered the surface of the cornea and the corneal epithelium were about three fold thicker than that of control mice. Keratinocytes
in the stromal layer proliferated and neutrophils infiltrated into the stromal
layer, which may have caused the thickness of the stromal layer. There werealso some vessels in this lesion. In contrast, we could not find any changes in
the periphery of the cornea of Rim3 mutants. Additionally, morphologicalalterations in the epidermis of Rim3 were observed. The epidermis of Rim3
mutants was much thicker than that of control mice and contained two to
three times the number of spinous layers. The number of the hair follicles of
Rim3 mutants were markedly reduced. Similar abnormalities could be seen in
Re den mutants.
We are making a physical map around the Rim3 locus by using YAC and
BAC sequences and searching for candidate genes which may play roles ingrowth and differentiation of epithelial cells.
(5)

Organization of the Typel Keratin Genes (Krtl) in
the Mouse Genome

Hajime SATO, Tsuyoshi KOIDE, and Toshihiko SHIROISHI
Mapping studies of keratin genes in the mouse genome have revealed thatmost of the type 1 keratin genes (krt1) are located in the region 58.0 eM fromthe centromere on Chromosome 11. This is a syntenic region l7q12-q2l in
the human Chromosome, where most of the human type 1 keratin genes are
clustered. In the human type 1 keratin genes, KRTlO, 13, 14, 15, 16, 17, 18,
19 have been already cloned, and the genes separated into two clusters. One
contains KRT15 and KRTl9, and the other cluster contains KRT14, KRT16
and KR T17. Recently, it was reported that the two clusters were very closely
located and confined to at most a 55-kb DNA fragment in the humangenome. The genes were organized in the following order: 5'-KRT19KRTl5-KRT17-KRT16-KRTl4-3' (Milisavljeic etal., 1996).

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In mouse, Krtl-1O, 12, 13, 15, 19, 14 (partial), 17 (partial) have been
already cloned, but their relative positions and the physical distance between
them have not been studied. In order to examine the organization of themouse Krtl gene cluster, we carried out fine linkage analysis using the panels
which were constructed for Rim3 mapping. Our results suggested that there
are at least two clusters in the mouse Krtl genes as well. One contains Krtl10 and Krtl-12, and the other cluster contains Krtl-15,19, 14 and 17. We are
now making a physical map using Y AC sequences containing these Krtl
genes and intend to resolve the organization of Krtl genes in mouse genome.
(6)

Gene Mapping of Rim4 Modifier

Hiroshi MASUYA, Tomoko SAGAI, Shigeharu WAKANAl, and Toshihiko
SHIROISHI (' Central Institute for Experimental Animals, Kawasaki)
A network of interactions among the products of numerous genes isprobably essential for morphogenesis in limb buds. An analysis of mutants inwhich such genes are affected is obviously critical if we are to identify the key
roles of the various genes. Mating experiments between different mutants
with similar phenotypes would provide information about the interactions
among genes that are involved in morphogenesis. Although such genetic
studies have been very successful in the fruitfly, fewer studies have been
performed in house mouse, Mus musculus. There are many mutants withpreaxial polydactyly in mice. We reported previously that disruption of theanteroposterior axis formation of limb bud induces the preaxial polydactyly
in six preaxial polydactyly mutants, namely, Recombination induced mutant
4 (Rim4), Hemimelic extra toes (Hx), extra-toes-Jackson (Xt') , Strong's
luxoid (1st), luxate (Ix), and X-linked polydactyly (Xpl). In these mutants,
region specific expression of Sonic hedgehog (Shh) which is mediator gene forthe anteroposterior axis formation was affected in early limb development.
It has been well documented that a mutant phenotype depends not only on
the genotype at the mutated locus but also on the genotypes at other lociunlinked to the mutant gene. This fact, known as the genetic backgroundeffect, indicates that there is a second gene, called a modifier, which interacts
with the first mutant gene and that such modifier genes are functionallypolymorphic among inbred strains. We have also reported that the appearance of the phenotype of the polydactyly mutants depended on the genetic

112

background of mouse strain. For example, polydactylous phenotype of Rim4
heterozygotes is dominantly and completely suppressed by the MSM background, which is derived from Japanese wild mouse and is dominantly
accelerated by NZB background.
We carried out the gene mapping of the modifier for the one of polydactyly
mutants, Rim4. We crossed C57BL/lOJ-Rim4/+ with the F 1 progeny of
MSM and NZB. We obtained 57 polydactylous progeny out of total 381
progeny generated from the above cross. Using the 57 polydactylous
progeny, we carried out a systematic search for loci which show significantly
biased frequency for one parental allele. As a result, two marker loci weredetected as the candidates for the modifier genes of Rim4. At D2Mit13, D2
Mit126 and D2Mit130 in chromosome 2, the frequency of the MSM derived
allele was 22.8% and the LOD score was 3.9. At D15Mit2, D15Mit31 and
D15Mit6 in chromosome 15, the LOD score was 1.6. When randomly
extracted progeny, irrespective of polydactylous or not, were used in the same
analysis, the frequency of MSM allele at the D2Mit13 and D15Mit2 wasalmost 50%. Thus, it revealed that the biased frequency in the polydactylous
progeny was not due to transmission ratio distortion (TRD).
Recently, it was reported that preaxial polydactyly mutant, 1st, was tightly
linked to D2Mit130. We previously reported that 1st exhibits a phenotype
similar to that of Rim4. It is possible that 1st per se is a downstream modifiergene for Rim4 in the genetic cascade regulating the anteroposterior axis
formation in mouse limb development.
(7)

Genetic Analysis of Hemimelic extra-toe (Hx) Mutant

Tomoko SAGAI, Hiroshi MASUYA, and Toshihiko SHIROISHI

Hemimelic extra-toe (Hx) is a limb morphological mutant which arose in
the BlO.D2 strain. Heterozygotes have preaxial polydactyly on all four feet.
Typical expression includes shortening of the radius, tibia, and talus withsupernumerary metacarpals, metatarsals, and digits. The supernumerary
digits are always preaxial. The fibula and ulna are normal size but often
bowed. The Hx gene is located on chromosome 5 and very closely linked to
the Hammer-toe (Hm) mutation which shows interdigital webbing regression. This region is homologous to a segment of human chromosome 7q36,
where congenital limb deformity (polysyndactyly) in a large family has been

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located. These data suggest that the homologous region of human and mouse
contain genes involved in limb pattern formation. Additionally ectopic
expression of sonic hedgehog (Shh) has been detected in several preaxial
polydactylous mutants including Hx (Masuya et al., Genes & Dev., 9, 16451653, 1995). Because Hx and Hm are mapped in a position close to the shh
locus, shh is one possible candidate gene for Hx and Hm. For the elucidationof genes concerning limb patterning through Hx mutation, we are carryingout fine linkage analysis of the Hx locus.

(8)

Positional Cloning of the Mouse Skeletal Mutant, Tail short (Ts)Kunihiko SHIMIZU, Tuyoshi KOIDE, Akihiko MITA, Kikue UCHIDA, Shigeharu WAKANAl, Yoshiaki KIKKAWA 2, Hiromichi YONEKAWA 2, and Toshihiko SHIROISHI C Central Institute for Experimental Animals, Kawasaki,
2 Department of Laboratory Experimental Animal Science, The Tokyo Metropolitan Institute for Medical Science, Tokyo)

Tail short (Ts) was initially found as a mutant which produces a shortened
kinked tail in heterozygotes. Ts gene was mapped to the teromeric region ofchromosome 11. It has been proposed that Ts is a animal model for a humanskeletal dysmorphology known as Meckel syndrome (MESl). MES has been
mapped to 17q21-24 in human chromosome and this region is homologous
and syntenic to the distal region of mouse chromosome 11.
To elucidate the function of the Ts gene in mouse embryogenesis, we aretrying to clone the gene using positional cloning. The Ts gene was mapped
within a region of 0.16 cM that is located between two microsatellite markers, DIIMit128 and DIIMit256. The distance between these two markers is
estimated to be approximately 320 kb from calculations based on the average
recombination frequency in the mouse genome. We screened the mouse
YAC library and obtained YAC clones which carry microsatellite markers
tightly linked to the Ts locus. A new YAC library constructed by Whitehead
Institute, MIT has been made available from Research Genetics Co. Ltd. We
obtained six additive YACs by screening this new library. In the present
study, we isolated terminal sequences of YAC clones. Further screening of
the mouse YAC and Bacterial Artificial Chromosome (BAC) library andchromosome walking is underway to make a contig between these twomarkers.

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(9)

Morphological Characterization of the Mouse Mutant Tail-short:
Skeletal Patterning Along the Anteroposterior Axis

Junko ISHIJIMA, Kikue UCHIDA, Akihiko MITA, and Toshihiko SHIROISHI
Mice heterozygous for Tail-short (Ts) exhibit shortened a kinked tail andnumerous skeletal abnormalities throughout the whole body and severe
defects in head structural development. These phenotypes differ depending
upon the genetic background in which the mutant gene is introduced. In
particular, Ts heterozygotes generated from the cross of (C57BL/6J- Ts/ +X MSM)F 1 X C57BL/6J show several homeotic transformations that arehighly reproducibe. Two most characteristic phenotypes of the mutant mice
were transformation of the cervical vertebrae to thoracic vertebrae and an
additional vertebrae in upper thorathic region. It is of interest to note thatthe cervical region transformation was observed asymmetrically, namely, left
side of the vertebra was preferentially transformed to a posterior structure.
Thus, it is possible that Ts gene plays some role in providing segmental
identity along anteroposterior and left-right axes in mouse embryogenesis.
The precise relationship between this mutant phenotype and a number ofgenes known to be involved in determination of segmental identity along twoaxes remains to be determined.
(10)

Searching for Genes Regulating the Phenotype of Tail-short

Junko ISHIJIMA, Kikue UCHIDA, Akihiko MITA, and Toshihiko SHIROISHI
The phenotype of Tail-short (Ts) mutant is varies according to different
genetic backgrounds as is seen in many other mouse mutants. This indicatesthat some unknown genes play critical roles in mouse embryogenesis incooperation with Ts gene. The genes which interact with the mutant geneand change the mutant phenotype are called modifier gene. The large
variation in Ts phenotype observed in different inbred strains indicates that
Ts modifier genes are functionally polymorphic among inbred strains. We
have carried out two experiments to reveal the nature of the modifier genes.
(I) Polymorphism of Ts modifier genes in mouse inbred strains. In order
to examine the effect of genetic background of different inbred strains on Ts
phenotype, Ts heterozygotes were crossed with several inbred strains, such as
A/J, C3HSW/SnJ, CBA/J, AKR/J, BALB/cAnN, C57BL/6J, DBAI2J

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and SJL/J. Microsatellite markers linked to Ts locus were used to determining the genotype of the resultant progeny. This mating experiment clearly
indicated that crosses with A/J, C3HSWISnJ, CBA/J and AKR/J mousestrains did not give rise to viable progeny which had Ts mutant allele. This
led us to the conclusion that the above four strains possess polymorphic gene(s) which causes prenatal lethality in cooperation with Ts mutant gene. We
intended to map the gene which causes the lethality. The above four inbred
strains were crossed with C57BL/6J strain, then F 1 mice were subsequently
crossed with Ts heterozygotes. The resultant progeny were subjected togenotyping through entire chromosomes. The result clearly demonstrated
that the progeny carrying alleles of A/J, C3HSWISnJ, CBA/J and AKR/J
only at the microsatellite loci linked to Ts were lethal. This indicated that theobjective gene is tightly linked to Ts locus and probably is Ts locus per se. It
is possible that the allelic forms of the four inbred strains at Ts locus
improperly interact with Ts allele which results in prenatal lethality. A/J,
C3HSWISnJ and CBA/J originated from a common stock and have diverged
into three inbred strains about seventy years ago. Detailed analysis of early
embryogenesis revealed that mutant embryos were mostly defective in theirdistal mesodermal region at early primitive streak stage, leading to lethality inembryos in utero around 12.5 dpc. Thus, the results indicate that Ts geneplays a crucial role in early embryonic stage and that there is polymorphism
at Ts locus among inbred strains. The functional differences of the polymorphisms become evident when they interact with Ts allele.
2) Searching for Modifier Genes for the Phenotype of Mutation Tailshort When (TSJ/Le-Tsl + X C57BL/6J)F 1 mice were backcrossed to
C57BL/6J, the frequency of the resultant progeny carrying the Ts allele isalmost 0%. Fetal death soon after birth is associated with a high incidenceof cranial neural tube defects including exencephaly, spinal bifida and cleft
palate. In turn, when the MSM inbred strain was used as a backcrosspartner, mice with the Ts allele were less defective and viable. The differencesin expressivity of the mutant phenotype indicates that there are non-allelicgenes which modify the severity of Ts phenotypes and are polymorphic
among C57BL/6J and MSM strains. We intend to map these genes andinvestigate the mechanisms of the neural tube defects.

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(11)

A New Inbred Strain JFl from Japanese Fancy Mouse
Carrying the Classic Piebald Allele for Use
in Biomedical Research

Tsuyoshi KOIDE, Kikue UCHIDA, Akihiko MITA, Tomoko SAGAI, Toshihiko
SHIROISHI, and Kazuo MORIWAKI 1 C The Graduate University for Advanced Studies, Kanagawa)
There is strong evidence that laboratory mice were established from theEuropean or North American wild mouse, M. m. domesticus. Analysis of themitochondrial genome, and many of the nuclear genes of the laboratory
mouse have revealed that it shares common polymorphisms with those of thewest European subspecies, M. m. domesticus. Most laboratory strains
probably originated from a small number of fancy mice kept by mouse
breeders in Europe and North America. It is known that Abbie E. C.
Lathrop maintained a number of fancy mice on her farm in Granby,
Massachusetts and also supplied her mice to scientists in the early 1900s. Old
laboratory strains such as C57BL/6, and C58 were established from her mice.
On the other hand, there have been many mouse fanciers in Japan and
China since the late 1700s. Varieties of mutant mice such as albino, pink-eye
dilution, dwarf, waltzer and piebald already existed in Japan and those mice
were described in the book titled "Chingan-sodategusa" published in 1787.
One of those mice, Japanese Waltzing Mouse, famous for its characteristic
behavior was introduced into Europe in the late l800s by Brehm. Historical
records indicate that Japanese fancy mice were crossed with European andNorth American mice and some genetic factors were introduced into them.
An analysis of genes on the Y chromosome like Zfy2 and Sry has shown that
the Y chromosome of most strains of laboratory mouse was derived from
Asian mice, probably the Japanese wild mouse, M. m. molossinus. Even
though M. m. molossinus contributed to the gene pool of many laboratory
mouse strains, morphological similarities are not apparent between M m.
molossinus and laboratory mice. Thus, most laboratory strains are genetically similar to M. m. domesticus and differences between laboratory strains
are quite small, even though there has been substantial contributions from M.
m. molossinus to the establishment of laboratory inbred strains. This implies
that if we use only current laboratory strains, there is a risk of ignoring many
unknown genetic factors which could be found only in strains genetically

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remote from laboratory strains. Such polymorphisms are precious and usefulfor much scientific research. In fact, a new inbred strain which is genetically
far different from old laboratory strains but easy for use in biomedical
research work in terms of breeding, handling and manipulation has beenrequired for a long time.
Although the original strains of the Japanese fancy mouse were believed tohave become extinct by the middle of the 1900s, we found a strain of fancy
mouse being maintained in Denmark with the name "Japanese Mouse" in
1987. We introduced this Japanese Mouse stock into the National Instituteof Genetics, Japan and have bred the stock through full-sib mating. Now, themouse has been established as an inbred strain and designated JFl. The
mouse is phenotypically similar to one of the mice reported in "Chingansodategusa". Morphological analysis of JFl showed that the mouse is highly
similar to the Japanese mouse M. m. molossinus. In addition to this, many
genetic features such as the polymorphism in Zfy2 on the Y chromosome, thegenotype for biochemical markers, and the haplotype of H-2Kf suggest thatJFl has the genetic profile peculiar to M. m. molossinus. Therefore weconcluded that JFl originated in Japanese wild mouse M. m. molossinus.
Linkage analysis for the spotting coat color in JFl showed that the mutation
is located on the piebald locus. Characterization and comparison of thecausative gene for piebald, endothelin receptor type B (ednrb), indicated thatthe allele of ednrb in JFl is the same as that of the classic piebald allele
suggesting that these two mutations have a common origin. Possibly, theclassic piebald mutation was introduced from a Japanese tame mouse which
was already reported at the end of the l700s.
JFl has the potential to provide a new source for genetic research. It
appeared from SSLP analysis that the microsatellite markers are highly
polymorphic between JFl and old laboratory strains. We observed that 80%of the microsatellite markers are polymorphic between JFl and laboratory
strains. These polymorphisms facilitate a high density mapping of any
objective genes. It is also noteworthy that both the males and females of theF, progeny between JFl and either C57BL/6 and DBA/2 are completely
fertile and have no difficulty when crossing to any of the parental strains.
This makes mapping experiments extremely easy. We found that F, progeny
from these crosses showed more increased activity than any of the parental
strains. This can be explained by heterosis. These F[ progeny are still easier
to handle than any strains established from wild mouse.

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Currently, we are investigating modifier effects on several morphologicalmutants and observed different phenotypes of mutations in the genetic
background of JFl. These modifier effects must be caused by polymorphic
genes in JFl, which functionally cooperate with the mutant genes. It istheoretically easier to detect modifier effects for a variety of mutations byintroducing mutant genes into a genetically different strain such as JFl rather
than into closely related strains.
JFl will also provide a new experimental system for studying mouse
behavior, because JFl has been highly domesticated and is obedient compared to the genetically related strain MSM which still maintains the character of wild mouse and is very active. The clarified genotypes of JFl are "aa
BB CC DD ss".

(12)

Establishment of Intersubspecific Consomic Mouse Strains II

Toshihiko SHIROISHI, Akihiko MITA, Kyoko T AKADA, Shigeharu W AKANA!, Hiromichi YONEKAWA 2 , and Kazuo MORIWAKI 3 C Central Institute forExperimental Animal, Kawasaki, 2 Deppartment of Laboratory Experimental Animal Science, Tokyo Metropolitan Institute for Medical Science,
Tokyo, 3 The Graduate University for Advanced Studies, Hayama, Kanagawa)By definition, two strains of mouse are referred to as consomic when theydiffer by one complete chromosome pair. Given that mouse has a karyotype
with 21 different chromosomes (19 autosomes + X + Y), 21 consomic mousestrains are required for full coverage of mouse chromosomes by donor
strains, from which one complete chromosome is introduced into thecommon genetic background referred to as the recipient. Chromosome
substitution is achieved by six to seven times backcrossing of individuals
identified as heterozygous for the selected chromosome to the recipient strain.
It has been well established that many pathological conditions and diseases
like cancer and diabetes are under multigenic control. They are generally
difficult to analyze when a great number of genes are involved and epistaticinteractions are present among the genes. In these cases, if a phenotype ofinterest appears to deviate in a consomic strain from the recipient strain, its
genetic control must be related to the chromosomal substitution concerned.
Further genetic analysis will then be considerably simplified.

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We have started to establish consomic strains, using the MSM strain,
which is derived from Japanese wild mouse, Mus musculus molossinus, as thedonor strain and, C57BL/6J laboratory strain as the recipient. These twostrains are genetically very remote from each other so that they differ in a
number of genetic traits. This genetic difference facilitates chromosomal
assignment of any phenotype to be examined and is of great use in linkage
analysis based on polymorphisms of DNA markers. We are using microsatellite DNA markers, at least five markers per chromosome, for selection ofindividuals in each step of the backcrossing. At present, we have obtained
third to fourth generations of the backcross for most of the chromosomes.
(13)

Genetic Analysis of Non-MHC Susceptibility
Genes to IDDM in Mice

Shigeharu WAKANA 1, Toshihiko SHIROISHI, Mamoru ITO!, Takehito SATOZ,
Sonoko HABU 2 , Kazuo MORIWAKI3, and Tatsuji NOMURA! Central Institute for Experimental Animals, Kawasaki, 2 Dept. of Immunology, Tokai
University, Isehara3, 3 The Graduate Univ. for Advanced Studies, Hayama)e

The NOD mouse is a good animal model for insulin-dependent diabetes
mellitus (IDDM). It is also used as a model for studying multigenic diseases,
because at least sixteen different Idd loci responsible for susceptibility to
IDDM have been reported. The first discovered locus Idd, is linked to the
MHC, and essential to the development of diabetes. However, the contributions of other Idd loci not linked to MHC, to IDDM remain unclear. To
investigate the functions of the Idd3 and Idd4 without the influence of othersusceptibility genes, we have established two congenic strains for Idd3 andIdd4 by introducing the chromosomal segments of a Japanese wild mousederived MSM strain into the NOD mouse. The NOD-Idd3 msm congenic mice
showed a reduced level of IDDM, but were not completely free from it. On
the other hand, the NOD-Idd4 msm congenic mice developed insulitis, although
the incidence of diabetes and insulitis in these congenic mice was not lower
than that in NOD mice. To elucidate the function of the Idd3 in detail, weconstructed a fine map for Idd3 using a cross between NOD_Idd4msm/msm and
NOD_Idd3nod/msm. A region responsible for severe insulitis could be localized
in the Idd3-region between D3MIT169 and D3MIIT181 including the IL2
gene. Moreover, the coding sequence of IL2 from NOD was different from120

that of MSM. The amount of IL2 secreted from anti-CD3 stimulated T cellsof NOD/shi and MSM was measured by ELISA. The level of IL2 in the
MSM was higher than that of NOD/shi. These results suggest that IL2 is a
plausible candidate for Idd3.
Publications
1.

2.

3.

4.

5.

6.

7.

Reuss, F. U., Frankel, W. N., Moriwaki, K., Shiroishi, T., and Coffin, J. M.: Genetics ofintracisternal-A-partcle-related envelope-encoding proviral elements in mice. J. Virol.,70, 6450-6454, 1996.
Ohtsuka, H., Oyanagi, M., Mafune, Y., Miyashita, N., Shiroishi, T., Moriwaki, K.,
Kominami, R., and Saitou, N.: The presence/absence polymorphism and evolution of thep53 pseudogene in the genus Mus. Phylogenet. Evol., 5, 548-556, 1996.
Kanai, Y., Kanai-Azuma, M., Noce, T., Saido, T. c., Shiroishi, T., Hayashi, Y., andYazaki, K.: Identification of two Soxl7 messenger RNA isoforms, with and without thehigh mobility group box region, and their differential expression in mouse spermatogenesis. J. Cell BioI., 133, 1-15, 1996.
Mizuno, K., Koide, T., Sagai, T., Moriwaki, K., and Shiroishi, T.: Molecular analysis ofrecombinational hotspot adjacent to Lmp2 gene in the mouse MHC: fine location andchromatin structure. Mammal. Genome, 7, 490-496, 1996.
Uchida, K., Koopman, P., Mita, A., Wakan, S., Wright, E., Kikkawa, Y., Yonekawa, H.
Moriwaki, K., and Shiroishi, T.: Exclusion of Sox9 as a candidate for the mouse mutant
Tail short. Mammal. Genome, 7, 481-485, 1996.
Kuroiwa, Y., Kaneko-Ishino, T., Kagitani, F., Kohda, T., Li, L.-L., Tada, M., Suzuki,
R., Yokoyama, M., Shiroishi, T., Wakana, S., Barton, S. c., Ishino, F., and Surani, A.:
Peg3 imprinted gene on proximal chromosome 7 encodes for a zink finger protein.
Nature Genetics, 12, 186-190, 1996.
Tani, M., Shindo-Okada, N., Hashimoto, Y., Shiroishi, T., Takenoshita, S., Nagamachi,Y., and Yokota, J.: Isolation of a novel Sry-related Gene that is expressed in highmetastatic K1735 murine melanoma cells. Genomics, 39, 30-37, 1997.

F-b.

(1)

Invertebrate Genetics Laboratory

Specification of Embryonic Limb Primordium by
Graded Activity of Decapentaplegic

Satoshi GOT a and Shigeo HAYASHI
Two thoracic limbs of Drosophila, the leg and the wing, originate from acommon cluster of cells including the sources of two secreted signaling

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molecules Decapentaplegic and Wingless. We showed that Wingless, but not
Decapentaplegic, is responsible for the initial specification of limb primordia
with a distal identity. Limb formation is restricted to the lateral position of
the embryo by negative control of the early function of Decapentaplegic andthe EGF receptor homolog that determines the global dorso-ventral pattern.
Late function of Decapentaplegic locally determines two additional cell
identities in a dosage dependent manner. Loss of Decapentaplegic activity
resulted in a deletion of the proximal structures of the limb, which is incontrast to the consequence of decapentaplegic mutations in the imaginal discthat cause a deletion of distal structures. The results indicate that the limb
pattern elements are added in a distal to proximal direction in the embryo,which is opposite to what is happens in the growing imaginal disc. We
propose that Wingless and Decapentaplegic act sequentially to initiate theproximodistal axis. For details, see Ref. 7.
(2)

Cadherin-mediated Cell Adhesion and Cell Motility in Drosophila
Trachea Regulated by Transcription Factor Escargot

Miho TANAKA-MATAKATSU, Tadashi UEMURA', Hiroki ODA', Masatoshi
TAKEICHI', Yasuhiro SHIGA2 , and Shigeo HAYASHI (' Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto 606, 2 Present address, Tokyo University of Pharmacy)
Coordination of cell motility and adhesion is essential for concerted
movement of tissues during animal morphogenesis. The Drosophila tracheal
network is formed by branching, migration and fusion of tubular ectodermal
epithelia. The tracheal tip cell is located at the end of each branch that isgoing to fuse. Tip cells extend filopodia to search for targets and later change
their cell shape to that of a seamless ring to allow passage of lumen. The celladhesion molecule DE-cadherin is required for tracheal formation (Ref. 2),
and accumulates at the site of contact to form a ring that marks the site oflumen entry, and is essential for fusion. DE-cadherin expression in tip cellsof a subset of branches is dependent on escargot, a zinc finger gene expressedin all tip cells. Such escargot mutant tip cells failed to adhere to each otherand continued to search for alternative targets by extending long filopodia.
We present evidence indicating escargot positively regulates transcription of
the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescued the122defect in one of the fusion points in escargot mutants, demonstrating theessential role of DE-cadherin in target recognition and identifying escargot asa key regulator of cell adhesion and motility in tracheal morphogenesis. For
details, see Ref. 6.
These studies have proven that the tracheal system is ideal for the study ofcell motility and adhesion during morphogenesis. To facilitate the study oftracheal formation in live animals, we developed a gfp expression vector thatenabled us to observe tracheal cell migration in living embryos. For details,
see Ref. 4.
(3)

Role of the Drosophila EGF Receptor in Determination of
Dorsoventral Domains of escargot Expression
during Primary Neurogenesis

Yoshimasa Y AmI and Shigeo HAYASH! CDivision of Biological Science,
Graduate School of Science, Nagoya University, Nagoya 464-{)1, Japan)
Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains on each side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot(esg), whereas the intermediate column does not. We studied esg expression
as a probe to investigate the mechanism of neuroectoderm patterning. The
effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist were found to repress esg expression outside of theneuroectoderm. The expression of escargot in the intermediate column isnormally repressed, but repression was eliminated when the EGF receptor
homologue (DER) activity was either elevated or reduced. A neurogenic
enhancer of escargot was identified, and was shown to be separable into a
distal region that promotes ubiquitous expression in the neuroectoderm anda proximal region that represses the intermediate expression. It was concluded that decapentaplegic, snail, twist and an activator act through the
distal region to initiate transcription of escargot in the neuroectoderm. We
propose that the combination of opposing gradients of DER and its ligand
creates a peak of DER activity in the intermediate column where DER
represses escargot transcription through the proximal repressor region. These
two kinds of regulation establish the early escargot expression that prefigures

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the neuroectoderm patterning. For details, see Ref. 8.

(4)plexus, a Gene Required for Establishment of
Adult Wing Vein Pattern

Hitoshi MATAKATSU!, Sumiko GAMOU!, and Shigeo HAYASHI COsaka
Prefectural University)
Veins on the adult wing are good landmarks for anteroposterior anddorsoventral positional information. They also provide a stage where the
EGF receptor, Decapentaplegic and Notch/Delta signaling pathways interact. The gene plexus is required for supression of extra veins in specificintervein positions (between vein two and three, and between vein four andfive). The study of plexus may provide a clue to the mechanism thatorganizes the global pattern of the wing as well as vein differentiation. To
this end, we are cloning the plexus gene.
Publications
1.

2.

3.
4.

5.
6.

7.
8.

Sawamoto, K., Okabe, M., Tanimura, T., Hayashi, S., Mikoshiba, K., and Okano, H.:argos is required for projection of photoreceptor axons during optic lobe development in
Drosophila. Developmental Dynamics, 205, 162-171, 1996.
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kataoka, Y., and Takeichi, M.: Zygotic
DE-cadherin expression is required for processes of dynamic epithelial cell rearrangementin the Drosophila embryo. Genes Dev., 10, 659-671, 1996.
Hayashi, S.: A Cdc2 dependent checkpoint maintains diploidy in Drosophila. Development,122, 1051-1058, 1996.
Shiga, Y., Tanaka-Matakatsu, M. and Hayashi, S.: A nuclear GFP/tl-galactosidase fusion
protein as a marker for morphogenesis in living Drosophila. Development, Growth andDifferentiation, 38, 99-106, 1996.
Fuse, N., Hirose, S., and Hayashi, S.: Determination of wing cell fate by the escargot andsnail genes in Drosophila. Development, 122, 1059-1067, 1996.
Tanaka-Matakatsu, M., Uemura, T., Oda H., Takeichi, M., and Hayashi, S.: Cadherinmediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot. Development, 122, 3697-3705, 1996.
Goto, S. and Hayashi, S.: Specification of the embryonic limb primordium by graded
activity of Decapentaplegic. Development, 124, 125-132, 1997.
Yagi, Y. and Hayashi, S.: Role of the Drosophila EGF receptor in determination of thedorsoventral domains of escargot expression during primary neurogenesis. Genes to
Cells, 2, 41-53, 1997.

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F-c.
(1)

Plant Genetics Laboratory

Structural and Functional Analysis of the Genes Expressedin Early Embryogenesis in Rice (Oryza sativa)

Yukihiro ITO, Mitsugu EIGUCHI, and Nori KURATA
To elucidate the gene expression network essential for early embryogenesis
in rice, we cloned and conducted a functional analysis of transcription
activating factor genes. We constructed cDNA libraries of embryos at I, 2,
3,4 and 5 days post anthesis (DPA) and selected 33 cDNA clones probed byhomeo-domain-containing PCR fragments isolated originally from the 3
DPA embryo. These clones were categorized into 8 kinds of clones; HaS 9,
13, 16, 24, 58, 59 and 66, through sequence analysis. In addition, we cloned
another cDNA coding zinc-finger protein probed by a maize homologue anddesignated it HAZ 1. To detect the expression specificity of these genes,
RT-PCR analysis was carried out using fixed-cDNA libraries of 0, 1,2,3,4,
5, 6 and 22 DPA embryos. Three HaS clones, HaS 13, 16 and 24, and the
HAZ 1 clone showed stage specific expression patterns in the embryo. We
next analysed the spacial expression patterns of these clones by in situ
hybridization on sections of 3, 4 and 5 DPA embryos. The results showedthat Has 24 expresses on the shoot/radicle primordium at 3~4 DPA andHAZ 1 expresses on the surface cell layer of globular shape embryo at 3~4
DPA. These genes appear to have important roles in early rice embryogenesis. The analysis will be followed by searches for genes operating in up- anddown-stream flows of gene expression in embryogenesis as well as throughfurther functional analysis through generations of transgenic rice.
(2)

Positional Cloning of a Segregation Distortion Gene
Detected in the Progenies of Crosses
with Japonica and Indica Rices

Yoshiaki HARUSHIMA and Nori KURATA
Site specific reduced gene transmission, which is called segregation distortion, has been detected in several chromosomal regions in F 2 progenies of

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crosses betweenjaponica and indica rices (ref.s 1,2 and 6). To detect the finepositions and degree of causative genetic factors for the segregation distortion, each of 200 reciprocal backcrossed lines derived from either a backcrossof the japonica rice, Nipponbare, or that of the indica rice, Kasalath, to an F 1
plant of Nipponbare crossed with Kasalath were planted and leaf samples
were harvested. Fine mapping and positional cloning of these genetic factors
will be continued in 1997.
(3)

Molecular Cytogenetic Analysis of Nuclear Organization of
Rice Chromosomes in Meiosis and Mitosis

Kenichi NONOMURA and Nori KURATA
This study focused on unravelling the mechanisms of spacial and functional organization of chromosomes in the nucleus. Before screening of mutants
affecting both chromosome arrangement and function in the nucleus, we havebegun with the following two approaches starting in October, 1996. We are
now in the initial stage of the first important step of figuring out the basic andnormal chromosome architecture in the nucleus by reconstructing threedimensional images of in situ hybridization using position-known probes.
Another approach is to investigate the effect of mismatch repair genes inrelation to homologous and/or homoeologous chromosome interraction andrecombination in rice. We have cloned a candidate partial fragment of a rice
mismatch repair gene by PCR amplification using an adult leaf cDNA
library.
(4)

Generation of Enhancer and Gene Trap Lines in RiceYukihiro ITO, Mitsugu EIGUCHI, Kenichi NONOMURA, and Nori KURATA
To establish and to screen a lot of mutants affecting the steps of early
embryogenesis and of chromosome organization in meiotic and mitotic stages
in rice nuclei, we have started to produce gene and enhancer trap lines formost rice genes. For efficient generation of trap lines, we decided to use an
Ac/Ds transposon system of maize, GUS or GFP genes as reporters and the
T-DNA mediated gene transfer system of Agrobacterium. We chose useful
constructs already used in the Arabidopsis gene/enhancer trapping system

126

and replaced some components with fragments suitable for rice transformation system. Weare beginning to transform rice calli with either the vector
carrying Ac-transposase or that carrying Os + GUS gene and intend to crossthese two Ac- and Os-GUS transformed plants to obtain a lot of Os-GUS
transposed gene/enhancer trapped progenies.
Publications(I) Related papers and reviews published in the prvious institute.
I. Harushima, Y., Kurata, N., Yano, M., Nagamura, Y., Sasaki, T., Minobe, Y., andNakagahra, M.: Detection of segregation distortions in an indica-japonica rice cross using
a high-resolution molecular map. Theoretical and Applied Genetics, 92, 145-150, 1996.
2. Tsunematsu, H., Yoshimura, A., Harushima, Y., Nagamura, Y., Kurata, N., Yano, M.,
Sasaki, T., and Iwata, N.: RFLP Framework Map Using Recombinant Inbred Lines in
Rice. Breeding Science, 46, 279-284, 1996.
3. Hashizume, T., Shimamoto, I., Harushima, Y., Yui, M., Sato, T., Imai, T., and Hirai, M.:
Construction of a linkage map for watermelon (Citrullus lanatus (Thumb.) Matsum &Nakai) using random amplified polymorphic DNA (RAPD). Euphytica, 90, 265-273,
1996.
4. Yoshimura, S., Umehara, Y., Kurata, N., Nagamura, Y., Sasaki, T., Minobe, Y., andIwata, N.: Specifying a YAC clone which carries Xa-l, the bacterial blight resistance gene
locus in rice. Theoretical and Applied Genetics, 93, 117-122, 1996.
5. Umehara, Y., Tanoue, H., Kurata, N., Ashikawa, I., Minobe, Y., and Sasaki, T.: An
ordered yeast artificial chromosome library covering over half of rice chromosome 6.
Genome Res., 6, 935-942, 1996.
6. Antonio, B. A., Inoue, T., Kajiya, H., Nagamura, Y., Kurata, N., Minobe, Y., Yano, M.,
Nakagahra, M., and Sasaki, T.: Comparison of genetic distance and order of DNA
markers in five populations of rice. Genome, 39, 946-956, 1996.
7. Sasaki, T., Yano, M., Kurata, N., and Yamamoto, K.: The japanese rice genome research
program. Genome Res., 6, 661-666, 1996.
8. Wang, Z. X., Idonuma, A., Umehara, Y., Van Houten, W., Ashikawa, I., Minobe, Y.,
Kurata, N., and Sasaki, T.: Physical mapping of rice chromosome I with yeast artificial
chromosomes (YACs). DNA Res., 3, 291-296, 1996.
9. Saji, S., Umehara, Y., Kurata, N., Ashikawa, I., and Sasaki, T.: Construction of YAC
contigs on rice chromosome 5. DNA Res., 3, 297-302, 1996.
10. Yano, M., Nagamura, Y., Kurata, N., Sasaki, T., and Minobe, Y.: Toward the construction of an integrated genetic and physical map of rice. In: Genomes of Plants andanimals. Twenty-first Stadler Genetics Symposium. (ed. J. P. Gustafson and R. B.
Flavell), pp. 57-72, Plenum Publishing, New York (1996).
11. Miyao, A., Yamamoto, K., Havukkala, I., Yano, M., Kurata, N., Minobe, Y, and Sasaki,
T.: Rice as a model for genome analysis. In: ICRF Genome Analysis Handbook. pp. xxxx, Ed. N. K. Spurr., Blackwell Scientific Publications (in press).

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Kurata, N. and Sasaki, T.: Molecular analysis of the rice genome. In: DNA markers:
Protocols, Applications and Overviews. (ed. G. Caetano-Anolles and P. M. Gressholf),
pp. 271-282, John Wiley & Sons, Inc. NY (1997).
13. Van Houten, W., Kurata, N., Umehara, Y., Sasaki, T., and Minobe, Y. Generation of a
YAC contig encompassing the extra glume gene, eg, in rice. Genome, 39, 1072-1077,
12.

1996.
14.

Monna, L., Miyao, A., Zhong, H. S., Yano, M., Iwamoto, M., Umehara, Y., Kurata, N.,
Hayasaka, H., and Sasaki, T.: Saturation mapping with subc10nes of YACs: DNA marker
production targeting the rice disease resistance gene, Pi-b. Theor. Appl. Genet., 94, 170176, 1997.

F-d.
(1)

Microbial Genetics Laboratory

Molecular Mechanism that Determines the Timing of
Cell Division in the Cell Cycle of Escherichia coli

Akiko NISHIMURA
During the cell cycle of E. coli, several fundamental events take placethrough strictly periodic processes, though the synthetic pattern of DNA,
cytoplasm, and membrane in the cell cycle are completely different from eachother (Cooper, 1990), and two identical daughter cells are produced. The
cell must have a mechanism coordinating each event. Cell division, forexample, occurs after a constant period (20 min) and the coefficient ofvariation in cell size at the time of cell division is very small, about 10%(Schaechter et al., 1962). These facts suggest that E. coli must have amechanism that triggers the initiation of cell division at a specific cell length.
We isolated the novel mutants cfcA11 and cfcB1 which uncouple between
cell division and DNA replication in the normal cell cycle. The cfc mutations
reduced the period from nuclear division to cell division producing manysmall cells. The mutants grew exponentially, and the rates of macromolecular synthesis, DNA per mass ratio, elongation speed of DNA replication, andthe mass at the initiation of DNA replication were not affected significantly
by the mutations. Therefore, the cfc mutations affect only cell division, andthe time shortened by the early division might be compensated for by theextension of the period for cells staying mononuclear.
We also found that Ap4A accumulates in the mutant strain of the glyS
gene and overproduction of Ap4A causes early division in the cell cycle

128

uncoupling between cell division and DNA replication. It is known that Ap
4A affects many cellular processes (Zamecnik, 1983; Johnstone and Farr,
1991; Farr et al., 1989). The Ap4A nucleotide is synthesized by certain
aminoacyl-tRNA synthetases in a reaction in which an enzyme-bound aminoacyladenylate intermediate donates AMP to ATP (Zamecnik et al., 1966).
However, neither aminoacyl-tRNA synthetase mutants, inhibitory treatments of aminoacylation, nor structural mutants of tRNA are known to
produce Ap4A in vivo (Bochner et al., 1984). This is the first indication that
Ap4A is involved in the regulation of timing of cell division in E. coli.
F-e.

Genetic Resources Laboratory

Construction of Genetic Resources Database
Yukiko YAMAZAKI
The Genetic Resources Databank Project has entered its second trial
phase. Information on wheat, rice, and drosophila genetic resources maintained by several laboratories in Japan are provided by individual databases
via a WWW server (http://www.grs.nig.ac.jp) as well as local information
on mouse stocks and cloning vector collections. A unified database of wheat
and rice strains has also been constructed and has started to be used forexperimental purposes.
(1) Wheat germplasm database-KOMUGI version l.D--has been released. The database contains 13679 accessions of wheat stocks from 16
research laboratories in Japan. The data were classified into 8 groups
according to their genetic properties. Each record consists of 9 categories
and 63 items including genetic, biological and bibliographical information.
The server offers both global and targeted keyword searching of the database.
A data submission system, through which researchers can submit a newstrain,and add or update their data using a Web interface, has been developedand is ready for test use.
An improved format with additional information is scheduled to appear in
KOMUGI version 2.0. The construction of a barley germplasm database andwheat DNA clone databases have already been started with the collaborationof researchers.
(2) The first electronic database of rice genetic resources in Japan has

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been constructed and distributed this year. In addition to information on
11020 strains maintained by 33 laboratories in Japan, basic biological knowledge, such as anatomical data, gene designations and a morphological linkage
map, and about 900 citations are included. The data were divided into 10
groups and each group has individual items. Although the database is a trial
version and still contains known inconsistencies, this is the most substantial
collection among the currently accessible rice databases in the world. Release
1.0 will soon appear both on line and as a published catalog. Partnership withthe scientific community is necessary to enrich the database and improve thequality of it.
(3) A unified database which integrated wheat and rice strain data wasexperimentally constructed under Sybase DBMS. A database schema wasdesigned by which any syntactic inconsistencies between two plants were
accepted as long as they were semantically identical, allowing cross-species
search between them. The data were roughly divided into 9 semantic
categories and 90 items which cover all data compiled. Although a moresophisticated interface would be desirable to read browsing data, the method
allows quick integration of several different databases without changing any
entities. The next plan is an application of the idea to other species databases.
F-f.
(1)

Mammalian Development Laboratory

Analysis of Cellular and Molecular Mechanisms in
Development of Mouse Fetal Germ Cells

Norio NAKATSUJI and Yasuaki SHIRAYOSHI
Recent studies have shown that the stem cell factor (SCF), leukemia
inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and enhancement of the cAMP level increase proliferation and survival of mouse primordial germ cells (PGCs) in vitro. Even after the addition of these factors,
however, it is still not possible to obtain proliferation of PGCs at a rapid rate
similar to that in vivo, suggesting the presence of other growth factor(s) invivo. We found that tumor necrosis factor-a (TNF-a) stimulates proliferation of PGCs at earlier migration stages. We also analyzed the functionalrole of gp l30-mediated signaling in PGC growth in vitro (for details, see Ref.
1). A combination ofIL-6 and soluble IL-6 binding subunit (sIL-6R), which130

is known to activate intracellular signaling via gp130, fully reproduced the
LIF action on PGCs. The addition of a neutralizing antibody against gp130
in culture markedly blocked PGC survival. These results suggest the pivotal
role of gp130 in PGC development. We further demonstrated that a
combination of LIF with forskolin or retinoic acid caused continuation of
proliferation of PGCs, leading to propagation of embryonic stem cell-like EG
cells.
Then, we showed that use of SlIS14-m220 feeder cells and the addition of
medium conditioned with Buffalo rat liver cells (BRL-CM) and forskolin to
the culture medium stimulate PGCs obtained from 8.5 days post coitum(dpc) embryos to proliferate in culture at a rate comparable to that in vivo.
In this condition, their proliferation continued several days past the timing ofgrowth arrest, which we studied in detail by using the elonal culture method(for the details, see Ref. 2). Such proliferating PGCs continue to express
c-kit and Oct-3 proteins. The characteristics of the medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells.
For details, see Ref. 3.
Also, we evaluated electroporation, liposome-mediated transfection, andthe calcium phosphate (CaP04) co-precipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to theinvestigation of molecular mechanisms of germ cell development. We foundthat electroporation severely damaged PGCs, and the efficiency of liposomemediated transfection was very low. In contrast, using the CaP04 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs
obtained from embryonic gonads at 11.5 days post coitum (dpc) during 2days of culture after transfection. PGCs isolated from 11.5 dpc gonads
change from spread- to round-shape and exhibit growth arrest during a few
days of culture, and these rounded PGCs quickly disappear from the culture.
We found that the transfection and expression of Bel-XL or adenovirus type
2 EIB 19,000-molecular-weight protein (EIB 19K) significantly promoted
the survival of PGCs and retarded the disappearance of rounded PGCs fromthe culture system. These results suggest that Bel-XL or EIB 19K can
prevent the apoptosis of PGCs and inhibit cell death of rounded PGCs inculture. For details, see Ref. 4.

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Establishment of Neuronal Stem Cell Lines for Analysis of
Cell Lineages and Differentiation in Mouse Central
Nervous System Development

Norio NAKATSUJI and Yasuaki SHIRAYOSHI
We established a large number of immortalized cell lines from earlyneuroepithelium through isolation of the forebrain region at E8.5 or E9.5
from mouse embryos harboring transgene tsA58, the temperature sensitive
SV40 T-antigen gene (T-Ag) driven by the H2-Kb promoter. Expression of
the oncogene product can be controlled by temperature and also by thepresence or absence of interferon in cell culture. All of the NES8 lines
obtained from such embryos at E8.5 expressed marker proteins of the neuralprecursor cells, but they could not be induced to differentiate into neurons or
glia in various culture conditions. When injected into the ventricle of fetal
brains at E14.5, however, NES8 cells showed engrafting into brain tissues
and produced typical neurons in the cerebral cortical plate. Thus, NES8 celllines seem to represent the undifferentiated neuroepithelial cells at the earliest
stage. Half of the NES9 lines established from embryos at E9.5 were similar
to the NES8 lines, but the remaining half expressed neuronal or glial
differentiation marker proteins dependent on culture conditions. Thus, they
seemed to contain cell lines at various stages in their determination anddifferentiation. These results suggest that the early neuroepithelium at E8.5
is made of homogeneous cell population of the early stem cells, and there isa beginning of cell determination and differentiation at E9.5 producing
various types of multipotent stem cells and neuronal progenitor cells. In
addition, the engrafting of some of NES lines into fetal brains through theventricular zone indicates an important possibility that they may be used asa vehicle to investigate roles of various genes during CNS development by
gene transfection of a NES line and examination of effects of the expressedgene on fates of the engrafted cells and also on the surrounding nervous
system.

132(3)

Molecular Analysis of Cell Differentiation and Morphogenesis
in Postimplantation Mouse Embryos

Yasuaki SHIRAYOSHI and Norio NAKATSDJI
We cloned one Notch related gene "int-3" from the 8.5 day cDNA library.
The entire structure of the int-3 gene resembles that of Notch or vertebrate
Notch homologues, and the int-3 gene showed 50-60% identity to the related
Notch homologues in its amino acid sequence. We consider int-3 to be oneof the mouse homologues of Notch. RT-PCR and in situ hybridization
analysis revealed that int-3 was expressed in blood vessels in 9.5-10.5 day oldembryos. The expression pattern of int-3 coincided with the expression ofreceptor tyrosine kinase Flk-l, which is regarded as a major regulator ofvasculogenesis and angiogenesis. These results suggested that int-3 might beinvolved in the formation of blood vessels in early mouse embryos. For
details, see Ref. 5.
Publications
1.

2.

3.

4.

5.

Koshimizu, D., Taga, T., Watanabe, M., Saito, M., Shirayoshi, Y., Kishimoto, T., andNakatsuji, N.: Functional requirement of gp130-mediated signaling for growth andsurvival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG)cells. Development, 122, 1235-1242, 1996.
Ohkubo, Y., Shirayoshi, Y., and Nakatsuji, N.: Autonomous regulation of proliferationand growth arrest in mouse primordial germ cells studied by mixed and clonal cultures.
Exp. Cell Res., 222, 291-297, 1996.
Kawase, E., Shirayoshi, Y., Hashimoto, K., and Nakatsuji, N.: A combination of buffalo
rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor
stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that invivo. Devel. Growth Differ., 38, 315-322, 1996.
Watanabe, M., Shirayoshi, Y., Koshimizu, D., Hashimoto, S., Yonehara, S., Eguchi, Y.,
Tsujimoto, Y., and Nakatsuji, N.: Gene transfection of mouse primordial germ cells in
vitro and analysis of their survival and growth control. Exp. Cell Res., 230, 76-83, 1997.
Shirayoshi, Y., Yuasa, Y., Suzuki, T., Sugaya, K., Kawase., Ikemura, T., and Nakatsuji,
N.: Proto-oncogene int-3, a mouse Notch homologue, is expressed in endothelial cells
during early embryogenesis. Genes to Cells, 2, 213-224, 1997.

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G-a.

Molecular Biomechanism Laboratory

Mechanism of Abortive Synthesis by E. coli RNA Polymerase

Tomoko KUBORI, Hiroki NAGAI, Ranjan SEN, and Nobuo SHIMAMOTO
Transcription is regulated at initiation, and there are both positive andnegative controls. The regulation is afforded either by changing the rate of
the rate-determining step (r. d. s.), or by changing the active fraction of thetranscription complex or RNA polymerase. The latter mechanism works
irrespective of the position of r. d. s., and seems to be more general from akinetic viewpoint. The former mechanism, however, is conventionally supposed to be the case of most regulatory mechanism of transcription initiation.
Transcription of full-length products is always accompanied with abortivesynthesis in vitro; iterative synthesis and release of 2-14 nucleotide transcripts. The physiological significance of abortive synthesis is still unknown,
and its existence in a cell is technically difficult to prove. We speculated thatabortive synthesis may relate to the regulatory mechanism classified in thelatter mechanism.
In transcription from the APR promoter, we found a new transcription
complex which produces only abortive transcripts (see Ref. 1). The complex,
named moribund complex, cannot convert into the normal productive complex producing full-length transcript unless moribund complex is dissociatedfrom DNA. The complex releases abortive transcripts with a time constantof 2.5 min, and iteratively synthesizes abortive transcripts (see Ref. 2). It
also converts into a complex unable to elongate transcripts in 20 to 30 min.
RNA polymerase prepared from the productive complex can convert into
moribund complex, showing that productive and moribund complexes are
generated from the same molecule of RNA polymerase.
Some mutations in the 0 70 subunit decrease the synthesis of abortivetranscripts. We compared transcription from several promoters by using
RNA polymerases containing the wild-type and mutant 0 70 subunit. Kinetic
analysis showed that decrease of abortive synthesis is due to the destabiliza-

134tion of binary complexes of polymerase and a promoter, rather than adecrease of moribund complex. A part of the dissociated binary moribund
complex generates a binary productive complex upon rebinding to a promoter, resulting in indirect conversion from moribund complex into productive
complex. Weare accumulating evidence for difference between protein
conformation of moribund complex and that of productive complex using
protein and DNA footprinting techniques.
(2)

Single-Molecule Dynamics of Transcription:
Sliding of Proteins along DNA

Hiroyuki KABATA, Minoru TAKEUCHI, Takashi KINEBUCHI, Hisashi MIYASHITA, and Nobuo SHIMAMOTO
We previously showed the existence of a sliding motion of protein along
DNA through direct visualization of single molecules of E. coli RNA
polymerase. To check the generality of sliding, we applied similar single
molecule dynamics to a bacterial repressor protein, P. putida CamR, which
was observed to slide along DNA. In the absence of its inducer, d-camphor,
CamR molecules were trapped at three positions on a A DNA, one was its
cognate operator cloned in A DNA, and the other two are likely to behomologous to the operator. All trapping occurred at specific sites, and only
sliding complexes were observed at non-specific sites. This observation
indicates that the non-specific complex is the sliding complex itself.
Based on this observation, we developed a general theory of sliding. The
theory predicts that sliding does not always cause an enhancement of specificbinding. Sliding can accelerate not only association to a specific site but also
dissociation from it. Under certain conditions sliding enhances specificbinding, and the enhancement depends on the length of DNA. In this casenon-specific sites close to a specific site, work as antenna to harvest protein
molecules, but distant sites are competitors for specific sites. The criticaldistance distinguishing these two classes of non-specific sites is also derived
from the theory. This theory solves many seemingly conflicting results
previously obtained in kinetic assays of sliding.
We have been developing manipulating techniques for single molecules(see Ref. 3). As a fluorescent technique to detect single molecules, we havebeen improving theA. victoria Green Fluorescent Protein. The following two

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awards were given for these lines of work.
James Melcher Prize Paper Award of IEEE/IA Trans, 1996
Saitou Award, 1996
(3)

Crystallographic Analysis of E. coli Replication
Termination Protein

Katsuhiko KAMADA, Kosuke MORIKAWA, and Nobuo SHIMAMOTO
Genome replication is terminated at specific sites, and a protein-DNA
complex is reqired for this function. In E. coli, the Tus protein binds to one
of Tar sites, and blocks replisome which is progressing in one direction, but
the Tus-Tar complex does not block the one progressing in the otherdirection. This polar block is required for full replication of the genome. The
mechanism of this polar block is studied by clarifying the structure of theTus-Tar complex.
Among the 50 kinds of oligo DNA synthesized and tried, two DNA
formed protein-DNA cocrystals suitable for X-ray diffraction study. The
obtained structure with 2.7 angstrom resolution showed detailed features of
Tus-Ter interactions (see Ref. 4). In the direction of blocking, replisome
seems to collide with two alpha helices surrounding DNA, breaking no
Tus-Ter interactions on the other side of the helices. In the other direction
replisome collides with flat beta-sheets interacting specific bases of Ter DNA,and appears to break the interactions one by one, explaining the polar blockby the complex.
This work was done under collaboration with Takashi Horiuchi, andKatsufumi Ohsumi (National Institute of Basic Research).
Publications
1.

2.
3.
4.

Kubori, T. and Shimamoto, N.: A branched pathway in the early stage of transcription
by Escherichia coli RNA polymerase. J. Mol. BioI., 256, 449-457, 1996.
Kubori, T. and Shimamoto, N.: Kinetics of transcription in a minute column. Nuc1.
Acids Res., 24, 138D--138 I, 1996.
Washizu, M., Kurosawa, 0., Suzuki, S., and Shimamoto, N.: Space-resolved manipulation of biological macromolecules. SPIE,2716, 133-142, 1996.
Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto, N., and Morikawa, K.: Structure ofa replication terminator protein complexed with DNA. Nature, 383, 598-603, 1996.

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G-b.
(1)

Multicellular Organization Laboratory

Fluoride-resistant Mutants of the Nematode
Caenorhabditis elegans

Masaya TAKE-vem, Takeshi ISHIHARA, and Isao KATSVRA
We are isolating and characterizing fluoride-resistant mutants of C. elegans
to discover a new regulatory system. These mutants are recessive and map infive new genes,jIr-1 tojIr-5. They are grouped into two categories: strongly
resistant, slowly growing mutants (jIr-1,jIr-3 andjIr-4) and weakly resistant,normally growing mutants (jIr-2 and jIr-5). Interestingly, the latter mutations suppress the slow growth of the former (Katsura, I. et al., Genetics,
136, 145-154, 1994). All those mutations seem to affect the nervous system,
because the slowly growing mutants have short defecation-cycle periods (Dr.
J. H. Thomas, University of Washington), because all except thejIr-2 mutations have synthetic dauer-constitutive phenotypes (See section (3», andbecause some of them are defective in chemotaxis.
To learn what molecular mechanism is involved in the action of jIr genes,
we cloned the genes and cDNAs ofjIr-1 ,jIr-3, andjIr-4. The DNA sequencessuggest that jIr-1 encodes an ion channel related to amiloride-sensitive ionchannels of mammals. jIr-3 and jIr-4 code for a kinase-like molecule and anovel protein kinase, respectively, both of which have a hydrophobic domainon the carboxyl-terminal side of the kinase domain. This year we madeseveral advances in our research on jIr-1 and jIr-4. We obtained theirfull-length cDNA and determined the genomic DNA fragments that rescue
the mutant phenotypes by microinjection. Two mutations each injIr-1 andjIr-4 were identified by DNA sequencing. Both of the jIr-1 mutations were
missense mutations near membrane-spanning regions, while the jIr-4 mutations were a splice-acceptor mutation in the kinase domain and a missense
mutation in the C-terminal hydrophobic domain. This shows that thehydrophobic domain, in addition to the kinase domain, is essential for thefunction of the FLR-4 protein. We noticed a strange phenomenon onjIr-3.
Although we detected a single band of 2 kb in the Northern analysis, an 11
kb genomic fragment containing all of the 2 kb cDNA sequence did not

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rescue any of the flr-3 mutant phenotypes. However, a fragment 3.1 kb
longer on the 3' side did rescue all of the mutant phenotypes. We think there
is an essential mRNA inflr-3 that was not detected by the Northern analysis
and that extends to this downstream region. We actually detected a candidate for such an mRNA by RT-PCR.
It is known from our previous study that a flr-3-lacZ fusion gene isexpressed in most intestinal cells, beginning just before body elongation in the
mid-embryonic stage and ending at the end of the Ll or the beginning of theL2 larval stage. This year we investigated the expression of GFP fusion genesof flr-l and flr-4. Both of them were expressed in the intestinal cells frommiddle-stage embryos to adults. Furthermore, the flr-4-GFP fusion was
expressed also in a pair of head neurons from Ll larvae to adults. Thusflr1 andflr-3, and possibly alsoflr-4, may act in the intestine and affect neuralfunctions from outside the neurons.
We plan to continue the analysis of theflr genes to elucidate the molecularmechanisms of their action regulating various neural functions as well as
fluoride-sensitivity and growth-rate.
(2)

A Gene That Controls Both Hatching andCell Migration in C. elegans

Ryuichi HISHIDA, Takeshi ISHIHARA, and Isao KATSURA
Mutants in the hch-l gene have abnormalities both in hatching and in cell
migration. They cannot digest protein components of the eggshell, and aneuroblast called QL and its descendants move anteriorly instead of posteriorly during larval development (Hedgecock, E. M. et al., Development,
100, 365-382, 1987). Having isolated a transposon-insertion mutant in hch1 (K. Kondo (Soka University) and I. Katsura, unpublished results), wecloned the gene by the transposon-tagging method and the cDNA by using agenomic fragment as the probe. The cloned cDNA encoded a protein thatconsisted of a signal peptide, a Zn protease domain, an EGF domain and a
CUB domain, and that resembled the Drosophila TOLLOID and the mammalian BPM-l, both of which act in cell-differentiation. By whole-mount in situ
hybridization of embryos, we found that expression starts just before bodyelongation, continues through the comma stage and ends at the 1.5-fold or 2fold stage, while no expression was detected at later embryonic stages. The

138

hybridization signal was found on the dorsal and lateral sides of the middle
and posterior part of embryos before elongation, but was limited to the lateralsides (probably seam cells) at the 1.5-fold stage.
This year we investigated five hch-l alleles for their DNA sequence, thehatching defects, and the cell-migration abnormality. These alleles consistedof one nonsense and two missense mutations in the protease domain, atransposon-insertion mutation in the CUB domain, and a missense mutationin the Cys-rich region near the carboxyl terminal. While the former four
mutations were recessive and had low penetrance in the cell-migration
abnormality, the last allele was semi-dominant and had high penetrance,
which shows the importance of the C-terminal part of the molecule for thefunction. Although most mutants showed an average delay of several hours
in hatching and hatched completely in the end, the transposon-insertion
mutation had a delay of more than 10 hours, and about half of its embryos
did not ever hatch. Thus there was almost no correlation between theabnormality of hatching and that of the cell-migration.
We found that hch-l mutants show abnormality, though at much lower
penetrance, in the direction (normally anterior) of migration of the QR
neuroblast and its descendants, as well as in the direction (normally posterior) of migration of the QL neuroblast and its descendants. It is known thatboth of them migrate anteriorly in a loss-of-function mutant of the homeotic
gene mab-5, and posteriorly in a gain-of-function mutant. We constructeddouble mutants between each of the five hch-l alleles and both types of mab5 mutants. None of the double-mutants showed the same cell-migration
phenotype as either of the single-mutants, i.e., hch-l was neither epistatic nor
hypostatic to mab-5. Thus it is unlikely that hch-l gene acts simply in the
upstream or downstream of mab-5 in the regulatory cascade. Proteaseactivity in the hatching fluid was assayed in vitro with the soft eggshells of
hch-l embryos as the substrate. Activity in the hatching fluid of a hch-l nullmutant was not detectable, whereas a transformant embryo having many
copies of the wild-type hch-l gene exhibited about 10 times greater proteaseactivity than wild-type embryos. Thus, hch-l gene seems to encode thehatching enzyme itself or another factor that determines the level of hatching
enzyme activity.

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139

Analysis of the Head Neural Circuit of C. elegans
as Studied by Formation of Dauer Larvae

Norio SUZUKI, Takeshi ISHIHARA, and Isao KATSDRA
If newly hatched larvae of C. elegans are in a crowded state and given alimited food supply, they deviate from the normal life cycle and develop toenduring, non-feeding larvae called dauer larvae. The developmental decision to become dauer larvae is regulated by the neural circuit in a pair ofsensory organs called amphids, with the dauer pheromone and food as the
input. Since the assay of dauer formation is much less time-consuming than
behavioral assays such as chemotaxis assays, we are analyzing the head neuralcircuit by detecting dauer formation as the output. We found that more than
50 genes are involved in the synthetic dauer-constitutive (syn Daf-c) phenotype, i.e., the phenotype in which combinations of two mutations cause
worms to develop to dauer larvae irrespective of environmental conditions.
The synthetic nature of this phenotype, we think, is based on the structure of
the neural circuit, in which many neurons are positioned in parallel or in a
network, only a part of which can be blocked by a single mutation. If this is
the case, the specific combinations of mutations for the phenotype should
contain information on the structure of the functional neural network on
which the dauer regulatory signal is transmitted and processed.
We found that the pattern of combinations for the syn Daf-c phenotype isvery specific and can be explained by a model assuming three parallel
pathways, if we excludeflr mutations. However, a more complicated modelis necessary to explain the suppression of the syn Daf-c phenotype of various
double-mutants by known dauer-defective mutations.
We isolated and mapped 44 new mutations that show the syn Daf-c
phenotype in combination with the unc-31 (e169) mutation. Eight of themwere alleles of 4 known genes (tax-2, osm-6, che-ll, and aex-3), but most of
the remaining 36 mutations, which map in at least 18 genes, seem to be allelesof novel genes. We also examined the penetration of the fluorescent dye diD
into amphids and phasmids (sensory organs in the tail) to investigate possible
structural defects in those organs. Six mutants, of which four were alleles of
osm-6 and che-ll, showed clear abnormalities in dye-filling. We also foundthat one of the mutants, which is normal in dye-filling, escapes from benzaldehyde and isoamyl alcohol, which attract wild-type worms. These results

140

show that the syn Daf-c phenotype is useful for the isolation of new neuralmutations. It is known that wild-type worms become dauer-constitutive if
three types of amphid sensory neurons (ADF, ASI, ASG) are killed, while
the killing of only the ASI neurons is necessary to make the unc-31 mutant
dauer-constitutive. This suggests that many, if not all, of the above mutants
have defects in ASI. We plan to characterize them further to elucidate theirroles in sensory reception, transduction, or processing.
(4)

Reverse Genetics of Neural Genes of C. elegans andthe Analysis of Their Expression Using
Jellyfish Green Fluorescent Protein

Takeshi ISHIHARA, Manabi FUJIWARA, and Isao KATSURA
To analyze the formation and function of the neural circuit in C. elegans,
we made about 20 types of worms in which specific sets of neurons emit
fluorescence. They were prepared by integrative transformation of worms by
DNA constructs in which the eDNA of GFP Uellyfish green fluorescentprotein; Chalfie, M. et al., Science, 263,802-805, 1994) is fused with various
C. elegans neural promoters. Three of the promoters were selected by thepromoter trapping method (Hope, I., Development, 113, 399-408, 1991),
and others by looking at the genome DNA sequences. Most of the cells that
express GFP were identified for each promoter.
Of the promoters obtained by promoter trapping, H20 gave expression in
all neurons, H13 only in AFD neurons, and 185 in the post-embryonic blast
cell G2 and its descendants, including RMF neurons. The gene for H13
encoded a putative transcriptional factor possessinging Zn fingers.
Of the genes selected by looking at DNA sequences, we investigated threeglycine/GABA A receptor homologs and two metabotropic glutamate receptor homologs in more detail. We made their eDNA by RT-PCR, 5' -RACE,/
and 3 -RACE. We also isolated their deletion mutants by first selecting
transposon-insertion mutants and then selecting deletion mutants made byimprecise excision of the inserted transposon. The behavioral defects of thosemutants are now under study. We hope to elucidate the roles of interneurons
by studying the functions of those receptors, which are expressed in someinterneurons as well as other cells. More specifically, we are interested inmetabotropic glutamate receptors because they play a role in learning in

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mammals. Glycine/GABA A receptors are ligand-gated chloride channels
that repress the activity of neurons. Hence, the disruption of their genes may
be a useful means of producing the effects opposite to those from the killingof the neurons expressing them.
Publications

1. Hishida R., Ishihara T., Kondo K., and Katsura I.: hch-l, a gene required for normal
hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to
TOLLOID and BMP-1. EMBO J., 15,4111-4122,1996.
G-c.
(1)

Biomolecular Structure Laboratory

Crystallographic Study of a3fJ3 Sub-complex of FI-ATPase:
Structural Analysis of Supramolecule

Yasuo SHIRAKIHARA
FI-ATPase is a catalytic sector of the membrane bound ATP synthasewhich plays a central role in energy conversion in mitochondria, chloroplasts
and bacteria, generating ATP from ADP and inorganic phosphate using
energy derived from a trans-membrane electro-chemical potential. The a3{33
sub-assembly of FI-ATPase is an active complex with 20-25% of the ATPase
activity of intact FI, and has similar catalytic properties to those of Fl. Both
FI-ATPase and the a3{33 sub-assembly have been challenging targets forcrystallographic study because of their large sizes e.~. 380 kDa for FI and
320 kDa for the a3{33 sub-assembly.
The nucleotide-free form of a3{33 sub-assembly from Bacillus PS3 FI
formed the crystals which diffracted to 3.2A resolution. We solved thestructure of the sub complex by the molecular replacement technique using
bovine FI structure as a search model, and have finished refinement of thestructure this year (manuscript in preparation). The final model has R factorof 20.5% (free R factor 29.5%) and has a reasonable stereochemistry e.g. theroot mean square deviations from ideal values of o.Ol2A for bond lengths,
and 1.6° for bond angles. The structure solution was done in collaboration
with Andrew Leslie, Jan Pieter Abrahams and John Walker at MRC Laboratory of Molecular Biology, Cambridge, UK.

142

The following structural features have been established after finishing therefinement. In the absence of nucleotides and the single copy subunits, thea3/33 sub-assembly adopts a strictly symmetrical structure: the three a andthree /3 subunits have identical conformations. This is in contrast to theasymmetrical crystal structure of nucleotide-bound bovine mitochondrial Fl.
The bacterial /3 subunits adopt a conformation essentially identical to that of
the nucleotide-free /3 subunit in mitochondrial Fl. In contrast, the a subunits
have a similar conformation in both structures. When compared to themitochondrial enzyme, the major differences in structure are localised inmembrane-proximal half of the enzyme, while the remainder of the structureis highly conserved.
(2)

Crystallographic Study of the Transcription Activator, PhoB

Toshihiko AKIBA and Yasuo SHIRAKIHARA
PhoB Protein is a positive transcriptional activator for the genes in thephosphate (Pho) regulon of E. coli, such as phoA and pstS, that are inducedby phosphate starvation. PhoB acts by binding to the phD box in the
promoter region, which is the consensus sequence shared by the regulatory
regions of phoA, phoB, phoE and PstS. The activity of PhoB is regulated byphosphorylation by PhoR. The N-terminal domain ofPhoB is responsible forthis regulatory role, whereas the C-terminal domain has a DNA binding
ability.
The DNA-binding C-terminal domain, spanning from a residue 125 to the
C-terminus, of the protein has been crystallized in phosphate solutions by the
microdialysis method. Diffraction data were collected using synchrotron
radiation and the Weissenberg camera installed at the beam line BL18B of the
Photon Factory in KEK, Tsukuba. Crystals diffracted to 2.oA resolution andshowed good stability against X-ray irradiation at an ambient temperature.
The crystals belong to the space group P2 t with unit cell dimensions a = 30.74
A, b= 105.91A, and c=30.90A, /3= 110.27°. Using the collected data set, a
structural analysis has been attempted employing the molecular replacement
method with the structural model of OmpR, which has become available
recently. This work has been done in collaboration with Kozo Makino,
Research Institute for Microbial Diseases, Osaka University.

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143

Crystallographic Study of the Transcription Repressor, CamR

Yasuo SHIRAKIHARA
CamR protein is a repressor that regulates transcription of the cytochrome
P-450cam hydroxylase operon of Pseudomonas putida. Expression of the
camDCAB operon and the camR gene is regulated through interaction of the
CamR protein with the single operator located in the overlapping promoter
region between the camDCAB operon and the camR gene. D-camphor is aninducer which allows those genes to be transcribed. CamR is a homodimer
with a molecular mass of 40 kDa. Crystals of CamR with satisfactory size
was previously obtained from polyethyleneglycol solution in the presence orabsence of D-camphor. However, recent crystallization experiments havefailed to grow such crystals, probably due to variability of protein preparations. While trying to improve the quality of the CamR protein preparation,
we searched for new crystallization conditions and found that Na-Kphosphate gave crystals with the similar size in the presence of D-camphor.
These crystals diffract to at least 2.8A resolution, but are temperaturesensitive: the crystals grown at a low temperature got cracked at room
temperature where a diffraction study was made. Effort is being made to
overcome the problem. This work has been done in collaboration with
Hironori Aramaki, Daiichi College of Pharmaceutical Sciences.
G-d.
(1)

Gene Network Laboratory

Expression Pattern Map of the C. elegans Genome

Yuji KOHARA, Tomoko MOTOHASHI, Hiroaki TABARA, Hisako WATANABE, Masako SANO, Akiko MIYATA, Tokie OHBA, Yuko MITANI, Kouichi
IIDA, and Hiroko DEsum
Aiming to ultimately understand the network of gene expression in thedevelopment of the nematode C. elegans, we are constructing an expression
pattern map of the 100 Mb genome by identifying and characterizing its
cDNA species, whose total number is estimated to be around 15,000.
More than 100,000 cDNA clones were picked up randomly from different
cDNA libraries, stored and gridded. cDNA clones derived from already144

analyzed abundant cDNA species were identified by probing the high density
clone grids and removed to avoid unnecessary redundancy of analysis. The
remaining clones were subjected to analyses of (1) tag-sequencing from both1
51 -ends (with vector primers) and 3 -ends (with anchored oligo-dT primers),(2) mapping onto the genome, and (3) analysis of expression patterns. Thusfar, 32,000 clones have been processed, and 21,500 clean 3 /-tag sequenceswere obtained, which were classified into >6,000 unique cDNA species(nearly half of the total genes) by comparing the 3 -tags. About 70% ofthem were located on the genomic sequence determined by the Sanger Ctr/
WASH-U sequencing consortium. We are analyzing the expression patterns
during development of the classified cDNA species using the newly developed
method of in situ hybridization on whole mount specimens of embryos, larvae
and adults in the standard multi-well format. Thus far, about 30% of them
have shown specific expression patterns. The cDNA sequence and mapping
information has been made available at our WWW site (http://www.ddbj.
nig.ac.jp/htmls/c-elegans/htmllCE_INDEX.html) in collaboration with
DDBJ staff and we are preparing to make the expression patterns open at the
WWW site.
1(2)

Pos-I, a Gene Which Shows Localization of Its mRNA to the P
Lineage during Early Cleavage of C. elegans Embryos

Hiroaki TABARA, Craig MELL0 1, Russell HILL 2, Jim PRIESS!, and Yuji
KOHARA C Cancer Center, University of Massachusetts, Worcester, MA,
USA, 2 Fred Hutchinson Cancer Resear~h Center, Seattle, WA, USA)
Taking advantage of our cDNA project, we have identified a lot ofmaternal genes which showed interesting patterns of expression. One ofthem, the cDNA group CELK01662 (=YKI662 in ACEDB), showed an
asymmetric distribution of its mRNA in 2-cell stage embryos. Closer
examinations of the behavior of the mRNA in gonads and embryos showedthat (1) the mRNA started to appear at the turn of gonad, (2) it was
distributed evenly in oocytes, (3) it was segregated posteriorly as the firstcleavage proceeded, (4) the mRNA remained in P4 but disappeared in othersomatic blastomeres (perhaps through a mechanism of degradation). Thus,
we named this gene pos-l (posterior segregation).
Sequencing analysis showed that pos-l had strong homology to the zinc-

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145

finger region of the mammalian Tisll growth factor inducible genes. Thiszinc-finger motif is also found in a maternal gene pie-I. Interestingly, thephysical map position ofpos-I turned out to be very close to the genetic map
position of another maternal gene, skn-2. The skn-2 gene like pie-I, isrequired for proper development of the P lineage and skn-2 and pie-I mutants
exhibit dominant genetic interactions. Based on the following evidence weconcluded that skn-2 is a mutation of the pos-I gene; (1) injection experiments using pos-I antisense RNA caused phenocopies of skn-2, and (2)missense or deletion mutations were found in the zinc finger region of pos-I
in five skn-2 mutant alleles.
The overall phenotype of skn-2 mutants is the lack of germ line development. Although skn-2 embryos are lethal, some somatic lineages seem todifferentiate properly; we found the differentiation of hypodermis, gut andpharynx, but not body-wall muscle, in skn-2 embryos. It was shown that thepie-I gene, a genetically related gene to skn-2, repressed the transcription ofsomatic genes in the germ line. In situ hybridization experiments, however,showed that this was not the case in the skn-2 mutants. Antibodies against
the N-terminal part of the pos-I protein were raised and the distribution of
the pos-I protein was examined. The expression of the protein was coincident
with the pattern of the localized mRNA. Essentially no expression of theprotein was detected in I-cell embryos or AB blastomeres in 2-cell embryos.
The expression of the protein was detected in PI, P2, P3 and P4. Interestingly, the expression of the POS-l protein was cytoplasmic in contrast toother genes containing the Tis 11 zinc finger motif.
Based on these facts and preliminary observations of other genes containing the Tisll zinc finger motif, we hypothesize that the function of the POS1 protein is to stabilize or activate the translation of mRNA of other factors
which are necessary for establishing the fate of the germ line.
Publication
1.

Tabara, H., Motohashi, T., and Kohara, Y: A multi-well version of in situ hybridization
on whole mount embryos of Caenorhabditis elegans. Nucleic Acids Res., 24, 2119-2124,
1996.

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H.

CENTER FOR INFORMATION BIOLOGY

H-a.
(1)

Laboratory for DNA Data Analysis

Large-Scale Search for Genes on Which Positive Selection May
Operate and Analysis of Their Biological Meanings

Toshinori ENDO" 2 and Takashi GOJOBORI l ( ' National Institute of Genetics,
2 New Energy and Industrial Technology Development Organization)
With the aim of revealing the role of positive selection in gene evolution,
we have developed a method for searching and identifying the genes on which
positive selection may operate by comparing the numbers (dS and dN) of
synonymous and nonsynonymous substitutions of homologous DNA sequences. By searching through 3,595 homologous gene groups representing
all the coding sequences in the DDBJ database, we obtained 17 gene groups
as the candidates. We published the result in Mol. BioI. Evol., 13, 685-690,
1996. This method, however, failed to detect the MHC and Abalone sperm
lysin genes on which positive selection was shown to have operated previously. We found that the problem came from the comparison between dS anddN for whole coding regions. To resolve this problem, we developed a new
method for detecting positive selection within a small region of coding genes.
We named this method as Window Search. As a result, we could identify 192
candidate gene groups on which positive selection may operate. They
included genes that are reported to be under positive selection. We examined
genes for Abalone sperm lysin and Rhodobacteria photosynthetic reaction
center H subunit whose tertiary structure had been resolved, to study the
relationships between the positive selection and tertiary structure of the geneproduct. In the former case, the region under positive selection has a shapesimilar to scissors. This region is considered to have interaction to egg
surface molecules and it may be important for the egg to identify the spermsof the same species among many sperms in the ocean. Thus, positive selection
might help develop distinguishable genes for lysin molecules. In the latter
case, the gene product is a component of multisubunit protein. It is suggested
that the gene in question corresponds to the protein that interact with

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CENTER FOR INFORMATION BIOLOGY147

photoharvesting protein, which might vary depending on the wavelength ofabsorbing light. We reported these results at the 19th Annual Meeting of theJapan Society of Molecular Biology in Sapporo.
(2)Phylogenetic Analysis of Duplicated Regions within
Human Chromosomes

Toshinori END0 1,2, Tadashi IMANISHI 1, Takashi GOJOBORI!, and Hidetoshi
INoKo 3 C National Institute of Genetics, 2 New Energy and Industrial Technology Development Organization, 3 Tokai University School of Medicine)
We examined the evolution of duplicated band regions in human chromosomes on 6p21.3 and 9q33-34. The band 6p21.3 is well known as the major
histocompatibility complex (MHC) region and is well characterized. The
two regions contained more than 11 pairs of paralogous genes. We examined
the phylogenetic relationships between each pair of genes in the two regions,
together with the homologous genes from other organisms including miceand Drosophila. We concluded that those genes in the two regions diverged
around the time when vertebrates emerged, with some exceptions. The geneorder is conserved as well, though we need to consider some duplication andrearrangement events of some chromosomal segments. It is possible that the
duplication event might have triggered the emergence of vertebrates. Inspired by the finding above, we attempted to identify such duplicated regions byconducting homology searches among genes whose chromosomal locations
are already known. As a result, we found that at least 200 chromosomal
bands carry genes that have homologous genes on different chromosomal
bands. However, we could not identify a large-scale duplicated tract extending over several bands except for the chromosome-long homology foundbetween chromosomes 22 and X. Thus we concluded that duplication andrearrangement of chromosomes may be common events. We reported theresult above at International Symposium on Molecular Evolution at Guana
Caste in Costa Rica 1997.

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(3)

The Origin of Domestic Fowls

Akishinonomiya FUMIHITO" Tetsuo MIYAKE 2 , Masaru TAKADA3, Ryosuke
SHINGU4, Toshinori ENDo 5,6, Takashi GOJOBORI 5, Norio KOND03, andSusumu OHN06 CYamashina Institute for Ornithology, 2 Wakunaga Pharmaceutical Company Central Laboratories, 3 The Research Institute of
Evolutionary Biology, 4 Osaka University Medical School, 5 National
Institute of Genetics, 6 New Energy and Industrial Technology Development
Organization, 6 Beckman Research Institute of the City of Hope)
We attempted to identify the origin of domestic fowls by molecularphylogenetic analysis of mitochondrial DNA. We concluded that all thedomestic fowls have a single origin and their most closely related species
might be the red junglefowl in the Indochinese Peninsula. We published theresult in Proc. Nat!. Acad. Sci., 93, 6792-6795, 1996.
(4)

The Molecular Mechanisms of Choosing Infected Clones inMother-To-Child Transmission of HIV-l

Kazunari TAKAHASHI and Takashi GOJOBORI
Mother-to-child transmission of human immunodeficiency virus (HIV-I)
is one of the major paths of AIDS infection. With the aim of understanding
which virus clones are transmitted from mother to child, we extracted
nucleotide sequences of viruses in mothers and their children from DDBJ(DNA Data Bank of Japan) or journals and investigated the patterns ofmutations in the V3 amino acid sequences of the env gene product soon after
the birth of an infected child. By comparing the sequences between mothers
and children, we obtained the characteristic features of sequence differences
between transmitted and nontransmitted viruses. We also estimated theamino acid sites of the V3 region which may play an important role duringmother-to-child transmission.

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149

HLA Class II Alleles in Ainu Living in Hidaka District,
Hokkaido, Northern Japan

Makoto BANNAI!, Katsushi TOKUNAGA!, Tadashi IMANISHI, Shinji HARIHARA 3, Kiyoshi FUJISAWA\ Takeo JUJI 4 and Keiichi OMOTd C Departmentof Research, Japanese Red Cross Tokyo Metropolitan Blood Center,2 Department of Human Genetics, School of International Health, The
University of Tokyo, 3 Department of Biological Sciences, Graduate Schoolof Science, The University of Tokyo, 4 Japanese Red Cross Central Blood
Center, 5 Division of Research, International Research Center for JapaneseStudies)
The Ainu people are considered to be the descendants of preagricultural
native populations of northern Japan, while the majority of the population ofcontemporary Japan (Wajin) is descended mainly from postneolithic migrants. Polymorphisms of the HLA-DRB1, DRB3, and DQBl alleles were
investigated in DNA samples of 50 Ainu living in Hidaka district, Hokkaido.
Unique features of the Ainu in this study were high incidences of DRB1*
1401, DRB1*1406, and a newly described allele, DRB1*l106 (20%, 17%,and 5%, respectively). On the other hand, several common alleles in Wajin(DRB1*1502, 1302,0803, and 1502) were found at relatively low frequencies(1-2%) in Ainu. Previously DRBI *1406 was described as a characteristic
allele of some Native American or northeast Asian ethnic groups, andDRBI *1106 had been found in only two Singapore Chinese and one Korean.
Principal component analysis of various populations based on HLA class II
allele frequencies places the Ainu population midway between other east
Asian populations, including Wajin, and Native Americans. These observations support the hypothesis that the Ainu people are the descendants of someUpper Paleolithic populations of northeast Asia from which Native Americans are also descended. See Ref. 5 for details.
(6)

Patterns of Amino Acid Substitutions in the V3 Region of
HIV Within Single Hosts

Yumi YAMAGUCHI and Takashi GOJOBORIThe third variable envelope region (V3) is one of the major epitopes ofHIV. We intended to elucidate the evolutionary mechanism of molecular

150

evolution of the V3 region within a human body. We collected the nucleotide
sequence data of HIV clones which were isolated from single hosts at several
time points after infection. From the comparisons between the rates ofsynonymous and nonsynonymous substitutions, we previously showed that
there were the particular periods when positive selection was operating in theV3 region of HIV within single hosts. In this study, we attempted tocharacterize patterns of amino acid substitutions. For this purpose, weestimated frequencies and types of amino acid substitutions that occurred inthe V3 region. We also compared the substitution pattern obtained with that
expected by the assumption of the JTT model (Jones et al., 1992). We thenfound that the proportion of amino acid substitutions altering their electriccharge was significantly high in the V3 region compared with the expectationfrom the JTT model. Our results indicate that the V3 region may havecharacteristic patterns of amino acid substitution due to positive selection
which favors amino acid substitutions causing antigenic or phenotypic variations.
(7)

An Analysis of Amino Acid Diversity at One Site in
the Envelope Glycoprotein of HIV-l

Yumi Y AMAGUCHl and Takashi GOJOBORIIn protein evolution, the variability of amino acids at each site in proteins
may be affected by the level of functional constraints or selective advantages
for amino acid changes. We intended to elucidate the evolutionary mechanism of the protein at one amino acid site level by examining the correspondence of the variability of amino acids at one site to its biological function.
We developed a new method to estimate variability at each amino acid siteby extending the idea of the "nucleotide diversity." Our method uses aligned
amino acid sequence data. The amino acid diversity at a given site is definedas the proportion of pairs of two sequences whose amino acids on the site aredifferent. This quantity is estimated from data of frequencies of amino acids
or gaps at each amino acid site. In this method, the amino acid diversity canbe divided into several components of interest. For example, the whole
diversity including gaps can be divided into diversity by gaps and that bydifferent amino acids. We applied our new method to the envelope glycoprotein of HIV-1 to elucidate the relationships between the diversity of amino

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151

acids generating selective advantages and the functional constraints of theprotein that are important for the viral replication.
We analyzed 67 nucleotide sequences of gp120 (surface glycoprotein) of
HIV-1 (subtype B). Our findings are as follows: 1) insertions or deletions areconcentrated on six regions, 2) the V3 region (one of the major epitopes of
HIV-I) is one of the most variable regions in gp120, 3) there are several
highly variable regions with variability on electric charge of amino acids asmuch as the V3 region in gp120, 4) the amino acid sites that are responsible
for binding to CD4 locate on relatively conservative region.
(8)

Exceptionally Conserved Regions Found in Extensively
Rearranged Microbe Genomes

Hidemi WATANABE, Takeshi ITOH', Hirotada MORI', and Takashi GOJOBORI CNara Institute of Science and Technology, Japan)
To investigate the evolutionary process of genome structures in microbes,
completely or nearly completely sequenced genomes of Haemophilus influenzae Rd, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis
sp. strain PCC6803, Methanococcus jannaschii, Saccharomyces cerevisiae,
Escherichia coli and Bacillus subtilis were compared to each other using their
DNA sequences. The comparisons were carried out by examining thelocations of orthologous genes in different genomes. As a result, we foundthat the genomes of these microbes must have undergone frequent rearrangements except for a few extremely conserved gene clusters. Here, orthologous
genes are defined as those which originated from a single gene in the lastcommon ancestral genome. The orthologous gene pairs were identified
according to the following four criteria: (I) Each of an orthologous pair ofgenes should be more similar to each other than to any other genes it can bepaired with; (2) Each orthologous gene pair should show similarity ofstatistical significance; (3) All the possible pairs of the genes comprising an
orthologous gene group should satisfy the above-mentioned two criteria; (4)Gene phylogeny based on similarities of genes should be consistent with thephylogenetic relationships among those species. Through these analyses, werevealed that the order of orthologous genes identified between different
genomes is so unstable that almost all operons were broken, although operons
have been believed to be very conservative structural units. This indicates152

that dynamic rearrangements have frequently occurred in those microbe
genomes during their evolution. In the extensively rearranged microbe
genomes, however, we found several exceptionally conserved regions such as
ribosomal protein gene clusters, the RNA polymerase operon, the ATPase
operon, and a cluster composed of genes for cell wall synthesis and cell
division. This suggests that the gene order in these conserved regions must be
essential for survival of those organisms, implying the existence of special
structural constraints on the regions. Thus, this kind of study will provide
significant insights into the ancestral genome structures and their relationships to gene functions. This study was presented in the International
Symposium on Network and Evolution of Molecular Information, The 1996
Cold Spring Harbor Meeting on Molecular Genetics of Bacteria and Phages,
and J. Mol Evol., 44(Suppl. 1), S57-S64.
(9)

Characterization of the Methanococcus jannaschii Genome inTerms of Its ORF Composition and the Structures
within and around ORFs

Hidemi WATANABE, Takashi GOJOBORI, and Kinichiro MIURA 1 CInstitutefor Biomolecular Science, Gakushuin University)
As a result of genome projects, the sequence of the whole genome of anarchaeon, Methanococcus jannaschii, was recently presented as well as othercomplete sequences of bacterial and eucaryal genomes. Although previous
investigations mostly using translation-related genes showed that Archaea
were closely related to Eucarya rather than Bacteria, a classification of theentire genes of M. jannaschii on the basis of the sequence similarity clearlyshows that this archaeon had chimeric sets of bacterial-type (70%) andeucaryal-type ( < 30%) genes. Most of the minor eucaryal genes are involvedin the translation and transcription pathway. However, a few genes such as
the 16S ribosomal RNA and an EF-Tu-like gene, are bacterial. To understand the discrepancy between the phylogenetic place and the gene composition of this archaeon, we investigated whether the genome structure of M.
jannaschii is eucaryal or bacterial in terms of the base compositional patternaround and within ORFs. In this investigation, a statistical method, G-test,
was applied to evaluate the base biases around the boundaries of ORFs.
Then, we found that M. jannaschii possesses some bacterial features in the

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base biases around and even within ORFs. This indicates that the exceptional
bacterial genes for translation play crucial roles in the translation pathway.
We dare to hypothesize that this archaeon is more closely related to Bacteria
than Eucarya on the whole. This study was presented in the International
Workshop on Recent Advance in Genome Biology of Micro-organisms andthe Meeting of the International Society of Molecular Evolution "JUNK
DNA: The Role and the Evolution of Non-coding Sequences," and also isgoing to be published in a special issue of GENE.
(10)

Implication about the Evolutionary Process of the Generation ofRegulator-Regulatee Relationships after Speciation between
Escherichia coli and Haemophilus influenzae

Hidemi WATANABE, Kousuke GOT0 1, and Takashi GOJOBORI
systems technology Co., Ltd.)C Teijin

The transcriptional regulation relationships among genes in Escherichia
coli have been extensively studied. However, little about the generation
process of the relationships has been investigated. To get insight into theevolutionary processes of the generation of regulatory relationships, we
examined which regulators and regulatees in Escherichia coli were conservedin Haemophilus influenzae. For this examination, we first compiled the dataon transcriptional regulation relationships among the E. coli genes fromliteral sources: ninety-seven regulons in which 262 regulated operons (455
genes) are regulated. By identifying the orthologues of these E. coli genes inthe H. influenzae genome, it was revealed that only twenty-seven orthologous
regulators (28%) and 151 orthologous regulated genes (33%) had beenretained in E. coli and H. influenzae since the beginning of speciation, andthat among the ninety-seven operons each of which were composed of two or
more genes in E. coli, only fifteen operons were completely conserved in H.
influenzae. These findings strongly suggest that regulation systems andregulation relationships among genes in these organisms have been frequently
reconstituted after speciation. By searching for the origins of regulatory
regions, we are currently investigating the evolutionary events for the neogenesis of the regulatory regions of the genes and operons in H. influenzae
whose orthologous genes or operons together constitute a single operon in E.
coli.

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Publications
I.

2.

3.

4.

5.

6.

7.
8.

9.

10.
II.

12.
13.

Shiga, H., Shioda, T., Tomiyama, H., Takamiya, Y., Oka, S., Kimura, S., Yamaguchi, Y.,
Gojobori, T., Rammensee, H.-G., Miwa, K., and Takiguchi, M.: Identification ofmultiple HIV-I cytotoxic T-cell epitopes presented by human leukocyte antigen B35
molecules. AIDS, 10, 1075-1083, 1996.
Tokunaga, K., Imanishi, T., Takahashi, K., and Juji, T.: On the origin and dispersal of
East Asian populations as viewed from HLA haplotypes. In Prehistoric Mongoloid
Dispersals (Akazawa, T. and Szathmry, E. J. E. eds.), 187-197, Oxford Univ. Press, 1996.
Lin, L., Tokunaga, K., Tanaka, H., Nakajima, F., Imanishi, T., Kashiwase, K., Bannai,M., Mizuno, S., Akaza, T., Tadokoro, K., Shibata, Y., and Juji, T.: Further moleculardiversity in the HLA-BI5 group. Tisue Antigens, 47, 265-274, 1996.
Imanishi, T.: DNA polymorphisms shared among different loci of the major histocompatibility complex genes. In Current Topics on Molecular Evolution, Proceedings of the
U.S.-Japan Workshop (Nei, M. and Takahata, N. eds.), pp. 89-95, Inst. MEG, Penn.
State Univ., 1996.
Bannai, M., Tokunaga, K., Imanishi, T., Harihara, S., Fujisawa, K., Juji, T., and Omoto,K.: HLA class II alleles in Ainu living in Hidaka district, Hokkaido, northern Japan.
Am. J. Phys. Anthropo!., 101, 1-9, 1996.
Taniguchi, Y., Katsumata, Y., Koido, S., Suemizu, H., Yoshimura, S., Moriuchi, T.,
Okumura, K., Kagotani, K., Taguchi, H., Imanishi, T., Gojobori, T., and Inoko, H.:
Cloning, sequencing, and chromosomal localization of two tandemly arranged humanpseudogenes for the proliferating cell nuclear antigen (PCNA). Mammalian Genome 7,
906-908, 1996.
Endo, T., Ikeo, K., and Gojobori, T.: Large-scale search for genes on which positive
selection may operate. Mol. BioI. Evol., 13, 685-690, 1996.
Fumihito, A., Miyake, T., Takada, M., Shingu, R., Endo, T., Gojobori, T., Ohno, S., andKondo, N.: Monophyletic origin and unique dispersal patterns of domestic fowls. Proc.
Natl. Acad. Sci. USA, 93, 6792-6795, 1996.
Otsuka, J., Watanabe, H., and Mori, K.: Evolution of transcriptional regulation systemthrough promiscuous coupling of regulatory proteins with operons; suggestion fromprotein sequence similarities in Escherichia coli. J. Theor. Bio!., 178, 183-204, 1996.
Perriere, G., Moszer, I., and Gojobori, T.: NR Sub: a non-redundant database for
Bacillus subtilis. Nuc!. Acids Res. Oxford University Press, 24(1), 41-45, 1996.
Ohba, K., Mizokami, M., Lau, J. Y. N., Orito, E., Ikeo, K., and Gojobori, T.: Evolutionary relationship of hepatitis C, pesti-, flavi-, plantviruses, and newly discovered GB
hepatitis agents. FEBS Lett., 378, 232-234, 1996.
Gojobori, T., and Tateno, Y.: DNA database and its applications to the study ofmolecular evolution. In: "Bio Japan '96," Symposium Proceedings, 353-359, 1996.
Imanishi, T., and Gojobori, T.: Diversity in human MHC genes among ethnic groups
worldwide. In "Ethnoepidemiology of Cancer" (Eds., K. Tajima and S. Sonoda), 89-96,
Gann Monograph on Cancer Research No. 44, Japan Scientific Societies Press, Tokyo!

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14.

15.

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CENTER FOR INFORMATION BIOLOGY155

CRC Press, Boca Raton, 1996.
Koike, T., Okayama, T., Ishii, J., Mizunuma, T., Tamura, T., Tateno, Y., Sugawara, H.,
Nishikawa, K., Imanishi, T., Fukami-Kobayashi, K., Ikeo, K., and Gojobori, T.: Development of new DDBJ DNA sequence database with data annotation tool Yamato II. In"Proceeding of the seventh workshop on Genome Informatics 1996," 157-165, Tokyo/
Universal Academy Press, Inc., Tokyo, 1996.
Nakamura, Y., Wada, K., Wada, Y., Doi, H., Kanaya, S., Gojobori, T., and Ikemura, T.:
Codon usage tabulated from international DNA sequence databases. Nuc!. Acids Res.,24, 214-215, 1996.
Kitakami, H., Mori, Y., Tateno Y., and Gojobori, T.: A biological taxonomy databasesystem on world-wide web. In "Proceedings of the German Conference on Bioinformatics (GCB '96)," 244~246, University of Leipzig, 1996.

H-b.
(1)

Laboratory for Gene-Product Informatics

Improvement of Protein Sequence-Structure Compatibility
Method by the Be·st-Five Test

Motonori OTA and Ken NISHIKAWA
We proposed a new assessment, called the best-five test, for the pseudoenergy potential empirically derived from the protein structural database.
The object of the test is the three-dimensional (3D) profiles of proteins,which are directly connected to pseudo-energy potentials. In the 3D profile,the fitness of each amino acid type is ranked at each residue site of a protein.
A site whose native residue type is ranked within the best-five out of 20 aminoacids is regarded as satisfactory and the ratio of satisfactory sites over all thesites of all proteins examined is indicative of the efficiency of the pseudoenergy potential employed. We applied the test to our potential functions, bysetting various kinds of definitions to constitute energy functions. Throughthis test, the validity of the minus average operation was confirmed, where
the energy level of potential functions is adjusted by referring to the randomenvironmental state of proteins. Failure without the operation was ascribedto bulky hydrophobic residues, which almost always occupy higher ranking
positions in the 3D profile table. A maximum success ratio of 55.6% wasattained with the final set of potentials. The efficiency of the final set was
further checked in the fold-recognition test for distantly related proteins.
The best-five test is a new use of the 3D profile table for assessing the ability

156

of pseudo-energy potentials. See Ref. I for details.
(2)

A Distant Evolutionary Relationship between Bacterial
Sphingomyelinase and Mammalian DNase I

Yo MATSUO! and Ken NISHIKAWA CInstitute for Social Information Science, Fujitsu Laboratories Ltd.)The three-dimensional structure of bacterial sphingomyelinase (SMase)
was predicted using a protein fold recognition method; the search of a libraryof known structures showed that the SMase sequence is highly compatible
with the mammalian DNase I structure, which suggested that SMase adopts
a structure similar to that of DNase I. The amino acid sequence alignment
based on the prediction revealed that, despite the lack of overall sequencesimilarity (less than 10% identity), those residues of DNase I that are
involved in the hydrolysis of the phosphodiester bond, including two histidine
residues (His 134 and His 252) of the active center, are conserved in SMase.
The functional roles of SMase residues identified by sequence comparison
were consistent with results from mutant studies. Two Bacillus cereus SMasemutants (H134A and H252A) were constructed by site-directed mutagenesis.
They completely abolished their catalytic activity (collaboration studies with
Prof. Hiroh Ikezawa of Nagoya City University). A model for the SMasesphingomyelin complex structure was built to investigate how SMase specifically recognizes its substrate. The model suggested that a set of residues
conserved among bacterial SMases, including Trp 28 and Phe 55, might beimportant in substrate recognition. The predicted structural similarity andthe conservation of functionally important residues strongly suggest a distantevolutionary relationship between bacterial SMase and mammalian DNase I.
See Ref. 2 for details.
(3)

Comparison of Protein Structures Using 3D Profile Alignment

Mikita SUYAMA! and Ken NISHIKAWA CBiomolecular Engineering Research Institute)
A novel method for protein structure comparison using 3D profile alignment was presented. The 3D profile is a position-dependent scoring matrix

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derived from three-dimensional structures, and is basically used to estimate
sequence-structure compatibility for prediction of protein structure. Ouridea was to compare two 3D profiles using a dynamic programming algorithm to obtain optimal alignment and a similarity score between them.
When the 3D profile of hemoglobin was compared with each of the profiles inthe structural library, all the profiles of other globins were detected withrelatively high scores, and proteins of the same structural class followed theglobins. Exhaustive comparison of 3D profiles in the library was also
performed to depict protein relatedness in the structure space. Using
multidimensional scaling, a planar projection of points in the protein structure space revealed an overall grouping in terms of structural classes, i.e.,
all-a, all-,8, a/,8, and a +,8. These results differ in implication from thoseobtained by the conventional structure-structure comparison method. Differences are discussed with respect to the structural divergence of proteins in thecourse of molecular evolution. See Ref. 3 for details.
Publications
1.

2.

3.

Ota, M. and Nishikawa, K.: Assessment of pseudo-energy potentials by the best-five test:
A new use of the three-dimensional profiles of proteins. Protein Engineer., 10, 339-351,
1997.
Matsuo, Y., Yamada, A., Tsukamoto, K., Tamura, H., Ikezawa, H., Nakamura, H., andNishikawa, K.: A distant evolutionary relationship between bacterial sphingomyelinaseand mammalian DNase I. Protein Sci., 5, 2459-2467, 1996.
Suyama, M., Matsuo, Y., and Nishikawa, K.: Comparison of protein structures using 3D
profile alignment. J. Mol. Evol., 44(Suppl I), SI63-S173, 1997.

H-c.

(1)

Laboratory of Gene Function Research

DNA Database and Its Applications to the Study ofMolecular Evolution

Takashi GOJOBORI and Yoshio TATENO
With the recent advancement of molecular biology and biotechnology, theDNA sequence data are accumulating with an enormous rate. In particular,
the rapid development of genome projects for various kinds of species
including humans spurs the increase rate of the DNA sequence data in

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collaboration with EBI (European Bioinformatics Institute) at EMBL (European Molecular Biology Laboratories) and NCBI (National Center for
Biotechnology Information) at NLM (National Library of Medicine)/NIH(National Institutes of Health). Moreover, these international DNA databanks also play roles in providing the data to researchers worldwide. In Ref.
1, we briefly overviewed the present activities of the international DNA databanks with special reference to DDBJ. Then, we presented how the DNA
sequence data are important for various research areas of life sciences. We
focused on the study of molecular evolution and showed some examples ofthe study of evolution of genes and genomes by use of the DNA sequence
database.
(2)

A Biological Taxonomy Database System on World-wide Web

Hajime KITAKAMIl, Yasuma MORI, Yoshio TATENo, and Takashi GOJOBORI C Hiroshima City University)
We newly developed an intelligent system which is needed to integrate andsearch across taxonomy databases constructed by the international DNA
data banks over international computer networks. Integration across any
two taxonomy databases raises complex problems. We tried to resolve theproblems by introducing the intelligent mechanisms to integrate across thetaxonomy databases with semantic heterogeneity. We also described intelligent tree search methods implemented in stored procedure and loosely
coupled interface between WWW and database servers. Existing databasesystems do not support such devices and easy interface to access the taxonomy databases over computer networks. For details, see Ref. 4.

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159

Development of New DDBJ DNA Sequence Database with
Data Annotation Tool Yamato II

Tomohiro KOIKE l , Toshitsugu OKAYAMA!, Jun ISHII!, Tadashi MIZUNUMAl, Takurou TAMURA 2, Yoshio TATENO, Hideaki SUGAWARA, Ken NISHIKAWA, Tadashi IMANISHI, Kaoru FUKAMI-KOBAYASHI, Kazuho IKEO, andTakashi GOJOBORI CHitachi Software Engineering Co., Ltd., 2 Association
for Propagation of the Knowledge of Genetics)
As the molecular biology has made a rapid progress, there has been a great
number of changes required for the methodology for maintaining and utilizing DNA sequence data. For example, annotation to sequences has becomecomplex and extensive. DDBJ which recognized the impending requirements
decided to develop a new DNA sequence database system in 1995. To copewith frequent changes of the data structures and significant increment of thedata in terms of quality and quantity, we designed a completely new database
schema. In the new system, physical changes of the data structure no longer
affect such applications as a tool for annotation. We also designed a new
annotation tool with object oriented concept that allows us to handle DNA
sequence data in computers as intuitively as in the real world. The annotation
tool is named as YAMATO II. We also take care of needs from DDBJ itself
in the new system. Data traffics and security in the database access areespecially analyzed and outside reviewers of data for DDBJ are now able toprocess the data safely and comfortably in the new system. The new system
also realizes more robust and effective data exchange with partners in theinternational nucleotide sequence banks, EBI and GenBank. For details, see
Ref. 5.
Pu bl ications
1.

2.
3.

4.

Gojobori, T. and Tateno, Y.: DNA database and its application to the study of molecular
evolution. In Symposium Proceedings of BioJapan '96, pp. 355-359, 1996, S. Fukui,Japan Bioindustry Association.
Tateno, Y.: The International DNA Data Banks and genome sequence data. Japanese
Scientific Monthly, 49, 932-936, 1996 (in Japanese).
Tateno, Y.: Evolution of DNA and protein structures. In Structural Biology, pp. 130143, 1996, K. Nagata, Y. Nakanishi and M. Shirakawa, eds., Kyouritsu Publishing
Company, Tokyo (in Japanese).
Kitakami, H., Mori, Y., Tateno, Y., and Gojobori, T.: A biological taxonomy database

160
system on World Wide Web, In Proceedings of the German Conference on Bioinformatics (GCB'96), pp. 244-246, 1996, Univ of Leipzig Press, Leipzig.
5. Koike, T., Okayama, T., Ishii, J., Mizunuma, T., Tamura, T., Tateno, Y., Sugawara, H.,
Nishikawa, K., Imanishi, T., Fukami-Kobayashi, K., Ikeo, K., and Gojobori, T.: Development of new DDBJ DNA sequence database with data annotation tool Yamato II. In
The Proceedings of the Seventh Workshop on Genome Informatics, T. Akutsu, K. Asai,
M. Hagiya, S. Kuhara, S. Miyano, and K. Nakai, eds., pp. 157-165, 1996, Universal
Academy Press, Tokyo.
6. Yamamoto, H., Tamura, T., Isono, K., Gojobori, T., Sugawara, H., Nishikawa, K.,
Saitou, N., Imanishi, T., Fukami-Kobayashi, K., Ikeo, K., and Tateno, Y.: SAKURA: A
new data submission system of DDBJ to meet users' needs in the age of mass productionof DNA sequences. In The Proceedings of the Seventh Workshop on Genome Informatics, T. Akutsu, K. Asai, M. Hagiya, S. Kuhara, S. Miyano, and K. Nakai, eds., pp. 204205, 1996, Universal Academy Press, Tokyo.

H-d.
(1)

Molecular Classification Laboratory

Improvement of the Data Processing System for DDBJ

Hideaki SUGAWARA, Yoshio TATENO, Kazuho IKEO, Naruya SAITOU, Tadashi IMANISHI, Kaoru FUKAMI-KoBAYASHI, Satoru MIYAZAKI, Motonori
OTA, Takashi GOJOBORI, and Katumi ISON0 1 C Kobe University)
We improved the data processing system for DDBJ which faces the
explosion of sequence data. The core system is the database management
system named Y AMATall that was designed based on object-oriented
concept and has three layers of application, access and client. The improved
system is flexible and expandable enough to accommodate massive andcomplicated sequence data in the future. We also implemented a new datasubmission system named SAKURA. It takes advantage of INTERNET andWorld Wide Web technology and easy to use. Therefore more than half ofthe submission has been already done through SAKURA and quality of thedata is higher than those submitted by other methods. For details, see Ref.
6 and Ref. 15.

(2)

Databases and Network Agents on Biological Data

INTERNET and distributed data management systems will be an indispensable information environment for biology. We surveyed, studied andH. CENTER FOR INFORMATION BIOLOGY

161

developed systems which are not model systems in laboratory but processactual data in the real world. They will be the elements or modules of theintegrated system for biology in the future. For details, see Ref. 8, 9, 11, 13
and 14.
a. Information retrieval from distributed data sources in INTERNET
Satoru MIYAZAKI, Junko SHIMURAl, Takashi NAKASEl, Hideaki SUGAWARA CThe Institute of Physical and Chemical Research)
We developed an agent that helps researchers retrieve reliable data fromservers which are distributed in INTERNET. The subject of the prototype ismicrobes such as bacteria, fungi and cell lines. For details, see Ref. 4 andRef. 12.
b. Utilization of Broad Band Network for Science and Technology
Satoru MIYAZAKI and Hideaki SUGAWARA
We participated G7 GIBN projects, in which we carried out on-line
demonstration of virtual laboratory, tele-education, video-on-demand andmulti-media database between Japan and US. We could show the value andfeasibility of ATM switch network, especially, the usefulness of the virtuallaboratory composed of a large database, a high performance computer andan experimental group which are physically distributed in Japan and US.
c.

Database on EPITOPE

L. LING!, Junko SHIMURA2 , Hideaki SUGAWARA and Akira TSUGITA 1

CScience University of Tokyo, 2 The Institute of Physical and Chemical
Research)
We developed a database on epitopes which are located in antigens andwhose sequences are determined. The database has links to PIR-International, DDBJ/EMBL/Genbank and Hybridoma Data Bank and is avaluable tool for the analysis of the nature of molecular interaction. For
details, see Ref. 3.
d.

Statistical Analysis and Database on Human Leukocyte Differentiation
Antigens (HLDA)
Hitoshi KIKUTANIl, Hideaki SUGAWARA, Satoru MIYAZAKI, Tadao OHN0 2,
Kaoru SIAJ0 2 , Junko SHIMURA2 COsaka University, 2 The Institute ofPhysical and Chemical Research)162

We developed and provided to the public the database on HLDA. It
includes the reaction of 333 antibodies which were tested in the occasion of
the 6th International Workshop of HLDA in 1996. Based on the result ofcluster analysis, the new CD numbers are assigned to the new antibodies.
(3)

Research and Development of Classification and
Identification of Biological Objects

Biological diversity will be one of major subjects of biology in 21st century
and systematics forms the common base of the relevant researches. The
systematics in the stage of the integration of conventional taxonomy based on
phenotypic data and the phylogenetic analysis based on molecular data.
Therefore, we have developed tools for poly-phasic analysis of biologicaldata.
a. Information-base for Systematics
Hideaki SUGAWARA, Satoru MIYAZAKI, Junko SHIMURA!, Yohihiro MAsuDA2 , and Yasuhiro ISHITOBI 2 (' The Institute of Physical and Chemical
Research, 2 Xerox Co.)
The information-base aims to provide an integrated information environment for taxonomists. The user is able to apply many kinds of analyses to thesame subset from the database and enjoy sophisticated graphical user interface (GUI). Thus he/she is able to compare the results to get a consistentview of the relationship among species and strains. It is proved by the
explosive increase of users of Web browsers that a good GUI is powerful,valuable and important. For details, see Ref. 2
b. Homology modeling of 3D structure of imunogloblin
Satoru MIYAZAKI, Junko SHIMURA 1, Hideaki SUGAWARA, Sachiko HIROSE 2 , Shunichi SHIRAI 2 (' The Institute of Physical and Chemical Research,
2 Juntendo University)
We investigated effects of somatic mutations in immunoglobulin variable
region genes on the affinity maturation of autoantibodies using single precursor B cell-derived anti-DNA monoclonal antibodies generated from an
autoimmune disease-prone NZB X NZW Fl mouse. Analyses of DNA
sequences, homology modeling on a graphic computer and molecular dynamics simulation of antigen-binding sites showed that any single site of mutation

H. CENTER FOR INFORMAnON BIOLOGY163

and changes in the electrostatic or hydrogen-bonding potential of the residues
and in the three dimensional structure could not solely explain the difference
in DNA-binding activities. However, a significant increase in the flexibilityof antigen-binding Fv loops was associated with affinity-maturated anti-DNA
antibodies. For details, see Ref. I
c. A New Measure for the Study of Molecular Evolution
Satoru MIYAZAKI, Hideaki SUGAWARA, and Masanori OHYA 1 CScience
Univeristy of Tokyo)
We examined the efficiency of the entropy evolution rate for the construction of phylogenetic trees with DNA sequences. Taking model trees with
branches of given evolutionary rates, we generated sequences with 1,000
nucleotides along the branches of each model. We repeated computer
simulation 100 times for each model and found that the entropy evolution
rate gives the correct topology of trees more often than the other methods
when the evolutionary rate of a lineage (branch) was higher than 0.5 per site.
For details, see Ref. 7.
d.

The Construction of Phylogenetic Tree from the Large Scale Data ofGene Sequences
Satoru MIYAZAKI and Hideaki SUGAwARA
The maximum likelihood method has several advantages to other methods
to construct phylogenetic trees. However, it is not practical in the case of alarge data sets because it requires a lot of computer resources. We implemented fastDNAml (a parallelized program of the maximum likelihood
algorithm) to VPP500 and evaluated the program. We constructed the tree
for full sequences of 16SrRNA and checked computational time changing thenumber of PEs (processors) from I to 40. The increase of the number of PEs
decrease the computation time linearly and we find a possibility to draw alarge phylogenetic tree by use of fastDNAml on VPP500. For details, see
Ref. 5 and Ref. 10
Publications
1.

Shimura, J., Miyazaki, S., Sugawara, H., Hirose, S., and Shirai, S.: Change of genes andaffinity maturation of immunogobulin in model mice of auto-immune diseases (inJapanese), Inflam. Immunol., 4, 26-32, 1996.

164

2.

3.

4.

5.

6.

7.
8.

9.
10.
11.
12.

13.
14.

15.

Sugawara, H., Shimura, J., Miyazaki, S., Masuda, Y, and Ishitobi, Y.: Application of a
New Data Model and Visualization to Systematics, In BIOCOMPUTING-Proceedingof the 1996 Pacific Symposium (eds. Lawrence Hunter & Teri E. Klein), pp. 746-747,
World Scientific, Singapore, 1996.
Ling, L., Shimura, J., Sugawara, H., and Tsugita, A.: Epitope data bank, The Information Revolution: Impact on Science and Technology (eds.) Dubois, J.-E. and Gershon,E., pp. 249, Springer, Berlin, 1996.
Miyazaki, S., Ichiyanagi, Y., Shimura, J. Nakase, T., and Sugawara, H.: Agent for
Hunting Microbial Information on INTERNET (AHMII), Microbiology and Culture
Collections, 12, 47-47, 1996.
Miyazaki, S. and Sugawara, H.: The construction of phylogenetic tree from the large
scale data of gene sequences, In High Performance Computing in RIKEN 1996 (Ebisuzaki, T. ed.), I, pp. 209, Universal Academy Press, Japan, 1996.
Koike, T., Okayama, T., Ishii, J., Mizunuma, T., Tamura, T., Tateno, Y., Sugawara, H.,
Nishikawa, K., Imanishi, T., Fukami-Kobayashi, K., Ikeo, K., and Gojobori, T.: Development of New DDBJ DNA Sequence Database with Data Annotation Tool Yamato II,
Genome Informatics 1996, Proceedings of the Seventh Workshop on Genome Informatics, (eds.) Akutu, T. etal., pp. 157-165, Universal Academy Press, Japan, 1996.
Miyazaki, S., Sugawara, and Ohya, M.: The efficiency of entropy evolution rate forconstruction of phylogenetic trees, Genes Genet. Syst., 71, 323-327, 1996.
Sugawara, H. (a contibutor): The resource base for biodiversity assessment, Global
Biodiversity Assessment, (Executive Editor) V. H. Heywood, pp. 545-605, Cambridge
University Press, New York, 1996.
Sugawara, H.: INTERNET and scientific information (in Japanese), J. of Pesticide
Science, 21, 105-110, 1996.
Nukada, Y., Sarai, A., and Sugawara, H.: High Performance Computing in Life Scineces(in Japanese), RISTNews, 21, 2-10, 1996.
Sugawara, H.: INTERNET is the information environment for biology (in Japanese),
KAGAKU-KOGYO, 47, 716-725, 1996.
Miyazaki, S. and Sugawara, H.: Integration of information resources of yeasts(inJapanese), IGEseries, Institute of Genetic Ecology, Tohoku University (Sendai), pp. 3348, 1996.
Sugawara, H.: Database on microbes and utilization of the data (in Japanese), NogyoGijutu, 51, 28-32, 1996.
Sugawara, H.: WDCM and worldwide information, Culture Collections to Improve theQuality of Life, Samson, R. A., Staplers, J. A., Mei, D. van der and Stouthamer, A. H.
(eds.), Centraalbureau voor Shimmelcultures, pp. 155-157, Baarn, The Netherland,
1996.
Yamamoto, H., Tamura, T., !sono, K., Gojobori, T., Sugawara, H., Nishikawa, K.,
Saitou, N., Imanishi, T., Fukami-Kobayashi, K., Ikeo, K., and Tateno, Y.: SAKURA: A
new data submission system of DDBJ to meet users' needs in the age of mass productionof DNA sequences, Genome Informatics 1996, Proceedings of the Seventh Workshop on
Genome Informatics, (eds.) Akutu, T. et al., pp. 204-205, Universal Academy Press,
Japan, 1996.

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I.
(1)

RADIOISOTOPE CENTER

Transcriptional Analysis of the Bacillus subtilis Sporulation Gene:
Promoter Selectivity of RNA Polymerase

Masaya FUJITA and Yoshito SADAIE
Nutrient deprivation in Bacillus subtilis leads to a process of differentiationthat results in endospore formation. The first critical event of sporulation is
an asymmetric cell division that produces two cell types of different size: alarger mother cell and a smaller forespore. More than fifty sporulation genes
are expressed sequentially during sporulation and their expressions are controlled differently in the mother cell and in the forespore. Differential gene
expression during sporulation is largely due to promoter selectivity by the five
sequentially activated RNA polymerase sigma factors. To understand themolecular mechanism of their promoter selectivity, we constructed an in vitrotranscription system with if'- and cfI-RNA polymerases. The first sporulation
specific cfI factor directs transcription of a large number of early sporulation
genes whereas the principal a factor, if', is essential for the transcription of
the genes for vegetative growth and early sporulation. The genes encoding if'
or cfI were cloned into an expression vector under the control of the T7
promoter. Both proteins were overproduced in Escherichia coli BL21 (DE3)and purified from inclusion bodies after solubilization with guanidine hydrochloride. Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins. Unlike the if' protein,
the cfI protein showed DNA-binding ability. To compare the promoter
selectivity of the if' protein with that of the cfI protein, transcription in vitroof 16 promoters was performed using RNA polymerase holoenzymes reconstituted from the purified core enzyme with either cfI or if'. These holoenzymes correctly recognized each of the cognate promoters; cfI-RNA polymerase recognized cfI promoters but not if' promoters, and vice versa. We
also determined the promoter strength, i.e., the rate of open complex formation between RNA polymerase and the promoter, and the saturation level ofthe open complex formation at equilibrium. a H bound to core RNA polymerase could be replaced effectively by if' in vitro; this was unexpected as the166replacement of if" in the if"-RNA polymerase holoenzyme with d' in the latelogarithmic growth phase is essential for entering the developmental processof sporulation. We infer from these results that growth phase-coupled
replacement of the a subunit in the holoenzyme from if" to d' may require an
additional factor, or the modification of the core enzyme or a factor.
(2)

Nucleotide Sequence of the phoB-rrnE-groESL Region of
the Bacillus subtiUs Chromosome

Yoshito SADAlE, Katsunori YATA, Masaya FuJITA, Hitoshi SAGAI1, Mitsuhiro ITAYA2 , Yasuhiro KASAHARA 3 , and Naotake OGASAWARA3 C Laboratory for Pharmacology, Asahi Chemical Industry Co., Ltd., 2 Mitsubishikasei Institute of Life Sciences, 3 Nara Advanced Institute of Science andTechnology)
Large-scale nucleotide sequencing eventually will reveal the structure andorganization of genes on the chromosomes of bacteria that have been
estimated by classical genetic methods in a limited number of organisms. A
cooperative effort, by many laboratories of different countries, to sequence
the whole genome of Bacillus subtilis is providing fundamental knowledge ofthe gene organization of the spore-forming bacterial cell.
We determined a 36 kb sequence of the phoB-rrnE-groESL region of the
Bacillus subtilis chromosome at around 55°. The sequenced region contained
thirty-six open reading frames including the phoB and groESL genes, and thewhole rrnE operon. The phoB gene is transcribed in the direction opposite tothat of chromosome replication while most ORFs, including groESL and therrnE operon, are transcribed in the same direction. Two newly identified
tRNA genes upstream of the rrnE operon were those for Arg-tRNA and
Gly-tRNA. The sequenced region contains an operon consisting of genes for
degradation and the uptake of mannan. The rrnE operon and its downstream
ORFs are well conserved among Mycoplasma genitalium, Haemophilus injiuenzae, Synechocystis sp, and Methanococcusjannaschii. Sigma H consensus
sequences are present in the promoter regions of three ORFs, including
groESL. (Microbiology, 143, 1861-1866, 1997).

I.

RADIOISOTOPE CENTER

167

Publications
1.

Asai, K., Kawamura, F., Sadaie, Y., and Takahashi, H.: Isolation and characterization ofa sporulation initiation mutation in the Bacillus subtilis secA gene. J. Bacteriol., 179,
544-547, 1997.

168

J.
(1)

EXPERIMENTAL FARM

Development and Reevaluation of the Genetic Stocks of Rice

Ken-Ichi NONoMuRA, Mitsugu EIGUCHI, Toshie MIYABAYASHI, and Nori
KURATAWe have conducted the reproduction and distribution of genetic stocks ofwild and cultivated rice. From October 1, 1997, we try to include a new
system of rice genetic stock generation and application. Additional resources
we will produce, utilize and distribute are the enhancer trap and gene trap
rice lines. Another trial program to develop and reevaluate interspecifichybrids of rice stocks in terms of the mechanism of reproductive isolation bychromosomal unpairing is also in progress. These projects are being conducted in cooperation with the plant genetics laboratory. For details, see thereports of the plant genetics lab.

169

ABSTRACTS OF DIARY FOR 1996

Biological Symposium
464th-Jan. 24
Cloning of Genes for Rifampicin Inactivation from
Mycobacteria and other Organisms (Eric Dabbs)
465th-Feb. 26
Cloning and Expression of the Ecdysone Receptor andUltrasperacle Homologs in Manduca sexta (Marek
Jindra)466th-Mar. 14
Molecular analysis of pattern formation in Hydra(Thomas C. G. Bosch)
467th-Mar. 28
Promoter Activation by E. coli RNA Polymerase: Alternative Programs (Olga N. Ozoline)
468th-Apr. 4
Regulation of rRNA Transcription (Richard L.
Gourse)469th-Apr. 4
The Role of Antisense RNA and a Pseudoknot inControlling Replication of B Group Plasmids (J. Pittard)
470th-Apr. 11
Transcription Activation by the Cyclic AMP Receptor
Protein (Susan Garges)
471st-Apr. 11
Structure and Function of E. coli RNA Polymerase:
The Translocation Process (Hermann Heumann)
472nd-May 13
ATP-sensitive K+ Channels and Insulin Secretion (JoeBryan)
473rd-June 7
Of mice and Meisl: the identification of a novel homeobox gene and its role in myeloid leukemia (Arthur
Buchberg)
474th-June 6
Mapping the Surface of RNA Polymerase with Metal
Ions (Claude Meares)
475th-June 24
Proteome Research: Complementarity and limitations
with respect to the DNA and RNA worlds (Ian Humphery-Smith)
Genetic and Immunological Dissection of the Beta476th-Sept. 12
Subunit of RNA Polymerase (Robert E. Glass)477th-Sept. 19
The Role of Pax Genes in Organogenesis (Rudolf Balling)
478th-Oct. 17
Wrestling with E. coli Sigma: Results of Kon-Basho170

479th-oct. 17
480th-oct. 21
481st-oct. 24
482nd-Nov. 11483rd-Dec. 3
484th-Dec.9

485th-Dec. 5
486th-Dec. 20
487th-Dec. 25(Richard S. Hayward)
Proteomics, towards a full description of the cell readout (Andrew Gooley)
IMAGE: Integrated Molecular Analysis of the Human
Genome and its Expression (Charles Auffray)
Function and Molecular Structure of Nematocytes. A
one vesicle system of exocytosis (Thomas W. Holstein)
Helical Filaments and Hexameric Rings in DNA Replication and Recombination: A Single Conserved Structure (Edward H. Egleman)
Site-specific recombinases: enzymes or building blocks
in morphogenesis? (Kiyoshi Mizuuchi)
LINEAGE COMMITMENT AND FATE OFNEURAL CREST-DERIVED SUBPOPULATIONS(James A. Weston)
TRANSFORMING MEN INTO MICE (Pavel Pevzner)
Evolution of Genome Composition: Effect of Mutation
Pressure and Selection (Noboru Sueoka)
The Evolution of Self-incompatibility in Natural Populations of Flowering Plants (Marcy Uyenoyama)

Mishima Geneticists' Club
482nd-Jan. 18
Information theory and its application to DNA sequence analysis (Satoru Miyazaki)
Molecular evolution of aldolase: how tissue specific483rd-Feb. 22
functions of isozymes were acquired in vertebrate (Katsuji Hori)
484th-Feb. 26
Molecular biology of retroposons and its applications(Norihiro Okada)
Molecular evolution: approach from informatics (Hi485th-Feb. 23
roshi Tanaka)
486th-Feb. 23
Genetic variation and differentiation of wild rice in Asia
(Cai Hong-Wei)
Molecular mechanism of neuronal determination in the
487th-Mar. 18
Drosophila eye (Yasushi Hiromi)
488th-Mar.21
Micronuclei formation and DM (double minute chro-ABSTRACTS OF DIARY FOR 1996

489th-Apr. 1

490th-Apr. 18
49 1st-Apr. 24
492nd-May 15
493rd-May 29

494th-June 20

495th-Aug. 1

496th-Sept. 17
497th-oct. 30
498th-Dec. 5499th-Dec. 18

500th-Dec. 19

171

mosome) capture (Noriaki Shimizu)
Regulatory mechanism of asymmetric cell division in
the nematode C. elegans-Role of Wnt and a receptorlike molecule (Hitoshi Sawa)
Protein Structure(3D)-Sequence( lD) compatibility
search (Motonori Ohta)
Genetics of Atopy (Taro Shirakawa)
How left and right of body axis is determined? (Naohiko Yokoyama)
Three-dimentional structure of bovine cytochrome bc 1
complex by electron cryomicroscopy and helical image
reconstruction (Toshihiko Akiba)
Imaging of intermolecular interactions of biologicalmolecules-development of an atomic-force microscopeFtsH, a membrane-bound, ATP-dependent metalloprotease in Escherichia coli: structure and function of the
AAA family proteins (Teru Ogura)
Making a gene by combining microgenes (Kiyotaka
Shiba)
Mechanisms of substrate recognition by eIF2 a kinase(Makiko Kobayashi-kawagishi)
Protein crystallography and structural biology (Kunio
Miki)
Color pattern formation on the wing of the butterfly:
Color determination and scale development (Eriko
Takayama)
The functional analysis of unc-51 and unc-14 genes
required for the formation of a neural network of anematode C. elegans (Kenichi Ogura)

172

FOREIGN VISITORS IN 1996

June 9, 1994March 31, 1996
Jan. 24, 1996
Feb. 26
Mar. 14Mar. 28
Apr. 4
Apr. 4
Apr. 11
Apr. 11
May 13
May 16
May 22-23
June 6
June 7
June 24
Sept. 12
Sept. 12
Sept. 12
Sept. 12
Sept. 12
Sept. 12
Sept. 12
Sept. 12

H. W. Cai, Beijing Agriculture University, China
Eric Dabbs, University of the Witwatersrand, South
AfricaMarek Jindra, University of Washington, U.S.A.
Thomas C. G. Bosch, University of Munich, Germany
Olga N. Ozoline, Institute of Cell Biophysics, Russia
Richard L. Gourse, University of Wisconsin, U.S.A.
J. Pittard, University of Melbourne, Australia
Susan Garges, NCI, National Institutes of Health,
U.S.A.
Hermann Heumann, Max-Planck-Institute fur Biochemie, Germany
Joe Bryan, Baylor College of Medicine, U.S.A.
Claire Stuart, Mayor of New Primouth, New Zealand
Marcy K. Uyenoyama, Duke University, U.S.A.
Claude Meares, University of California at Davis,
U.S.A.
Authur Buchberg, Jefferson Medical College, U.S.A.
Ian Humphery-Smith, University of Sydney, Australia
Robert E. Glass, Nottingham University, U.K.
Enrique Alberto Fugueroa, National Institute of Agriculture Technology, Argentina
Adel Mohamed Abd EI Razek Ghoneim, Rice Research
and Training Center, Egypt
Rapolu Mahender Kumar, Directorate of Rice Research, India
Daniel Atula Masatia, National Irrigation Board,
Ahero Irrigation Research Station, Kenya
Zulkifli Bin Romli, Muda Agricultural Development
Authority, MADA Headquarters, Malaysia
Muhammad Imtiaz Ali Asad, Rice Research Institute,
Pakistan
Maria Chona E. Maleza, Bohol Agricultural PromotionASSOC. FOR PROPAG. OF THE KNOWL. OF GENET.

Sept. 12

Sept. 19
Oct. 1-31
Oct. 17
Oct. 17
Oct. 21Oct. 23
Oct. 24
Nov. 11
Dec. 2-4
Dec. 5
Dec. 9Dec. 20
Dec. 25

173

Center, Department of Agricuture, Philippines
Julmanee Pithuncharunlap, Rice and Field Crops Promotion Division, Department of Agricultural Extension, Thailand
Rudolf Balling, Institut fur Saugetiergenetik, Germany
John H. Gillespie, University of California at Davis,
U.S.A.
Richard S. Hayward, University of Edinburgh, U.K.
Andrew Gooley, Macquarie University, Australia
Charles Auffray, CNRS Research Unit on Molecular
Genetics and Developmental Biology, France
Hidesaburo Hanafusa, Rockefeller Univesity, U.S.A.
Thomas W. Holstein, JW Goethe University, Germany
Edward H. Egleman, University of Minnesota, U.S.A.
Kiyoshi Mizuuchi, NIDDK, National Institutes of
Health, U.S.A.
Pavel Pevzner, University of Southern California,
U.S.A.
James A. Weston, University of Oregon, U.S.A.
Noboru Sueoka, University of Colorado at Boulder,
U.S.A.
Marcy Uenoyama, Duke University, U.S.A.
ASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS

President
KONDO, Norio; Director-General, The Research Institute of Evolutionary Biology
Managing Director
MORIWAKI, Kazuo; Vice-President, The Graduate University for Advanced Studies
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

174

MIURA, Kin-ichiro; Director, Institute for Biomolecular Science, Gakushuin University
KURODA, Yukiaki; Professor, College of Environmental Health, Azabu
University
ISHIHAMA, Akira; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of 'Genetics
Trustee
TAZIMA, Yataro; Director Emeritus, Institute of Silkworm Genetics andBreeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Director and Professor, Institute of Medical Mycology,
Teikyo University
SHIGETO, Gakuji; Managing Director, Inoue Foundation for Science
MATSUNAGA, Ei; Professor emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, Shokabo Publishing Co.
TAKAGAKI, Yoshio; Full-time Adviser, Chugai Pharmaceutical Co.
SENO, Takeshi; Professor, National Institute of Genetics
TATENO, Yoshio; Professor, National Institute of Genetics
Auditor
IMAMURA, Takashi; Professor, National Institute of Genetics
MORISHIMA, Hiroko; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
Adviser
MORIWAKI, Daigoro; Professor emeritus, Tokyo Metropolitan University

175

AUTHOR INDEX'38'37,38,71
ABE, S·38
AIZAW A, S.
.
·72
AKIBA, T·142
AKIMOTO, M'100, 101
AKISHINONOMIYA, F.·········148
ALEXSEYEV, A·27
ANDO, A.
.
'68,69,71,73
ABE, Ko.

.

ABE, Ku

ARAKI, M.························· ·83
ARTSIMOVITCH, 1.

·10·30
ASANO, Y·18
AWANO, W·43
BALL, S. T
·106
BANNAI, M·149
BOUCHER, P. E.'·10
BUSH, A . · · · · · · · · · · · · · · · · · · · · · · · · · · · ·42
BUTLER, S. J
·43CAl, H.-W'98,99CHAKRABARTY, A. N
·10CHEN, L.
·71
CHUGANI, S. A
10COLE, M
·42
ASANO, S

FUSE, N.··························· ·48
GAMOU, S.
GILADI, H························123
10

GOJOBORI, T.

. .. '146, 147, 148, 149, 150,
151, 152, 153, 157, 158, 159, 160
GOTO, K
·153
GOTO, M
·47
GOTO, S
·120
GOTO, Y.
93, 94, 95
HABU, S·119
HAGA, K·84
HAGA, T··84
HANDA, H'47,58
HARA, H. .
'31,32,33,34
HARIHARA, S'90, 149
HARUSHIMA, Y.
HASEGAWA, T.
HATTA, M
HAYASAKA, K.
HAYASHI, M

HIGASHITANI, N
HILL, R
HlRAOKA, Y.
HIROMI, Y

FUJIWARA, M.··140

FUJIYAMA, A
FUKADA, Y.

.

FUKAGAWA, T
FUKAMI-KoBA YASHI, K.

.

·68
82, 83, 84, 85'69,73'159, 160······················92·70

HIGASHITANI, A.

DEHASETH, P. L.

····················10'124, 125, 168
ENDO, T
'146, 147, 148
FERDOUS, A
58
FISCHER LINDAHL, K. .
107
FUCHAROEN, G. .
·90
FUJII, K.
.
·92
FUJISAWA, K·149
FUJISAW A, T.
'44,45
FUJITA, M'165, 166
FUJITA, N·12····················12488'44,45

HAYASHI, S

EIGUCHI, M.

.

.

HIROSE, S.····HISATOMI, O.

.

HISHIDA, R
HONDA, A
HORAI, S
HORIUCHI, K
HOWE, M. M.
IIDA, K.

.

IKEMURA, T
IKEO, K.

.

IKEYA, T
IMAMURA, T.

.

'48, 120, 121, 122, 123
29, 30, 35, 36·29·144
38, 39'41, 42, 43, 43'46,47,48·82·137'17, 18'90,91,92,93,94,95
29, 30, 31, 32, 35·10·143'68,69,70,71,72,73
159, 160·26
87, 88IMANAKA-YOSHIDA, K.
IMANISHI, T··············72'147, 149, 159, 160

176'63,66,67
87, 88INOKO, H.
68, 69, 70, 71, 73, 147
ISHIBASHI, T·70
ISHIGURO, A·16
ISHIGURO, S. .
·108
ISHIHAMA, A'10, 12, 13, 14, 15,
16, 17, 18
ISHIHARA, M.
. ••.....•.•....•..•.. ·33
ISHIHARA, T. .
36, 136, 137, 139, 140
ISHII, J
159
ISHII, Y·35
ISHIJIMA, J . . • . . . . . . . . . . . . . . . . . . . . . ·114
ISOBE, T·107
IsoNo, K . . . . . . . . . . . . . . . . . . . . . . . . • • . 160
ITAYA, M.
. ••..••••..••••••....•• ·166
ITO, K. . .........•..••••.•..••••.• ·43
ITO, M . • . . . . . . . . . . . . . . . . . . . . . . . . . . ·119
ITO, Y'124, 125
ITOH, T. .
·151
INA, Y.

INABA, R.

.
.

Iw ABU CHI, K. ······················55
IWAo, K . • • . • • . . . • . . . . . . . • . . . . . . . • ·44·81·44
KOHARA, Y. .
'143, 144
KOIDE, T. ····107, 108, 109, 110,113, 116
KOIKE, T.··
·159
KLEIN, J.

.

KOBAYAKAWA, Y.

KOIZUMI, O.

························44

KOJIMA, D.

························82·74

KOMINAMI, R
KONDO, N.
KUBORI, T.
KUDO, Y························148························133·70

KUNDU, T.························· ·14
KUNISADA, T.

······················37'124, 125, 168.
·14

KURATA, N.

.

KUSANO, S.

LI, F.-Q.··························· ·47·74· .. 109, 111, 112
MATAKATSU, H·123
MATSUDA, S·26
MATSUDA, T.·· .. ·· .. ············ .. · ·83

MAFUNE, Y

MASUYA, H.

·

MATSUMOTO, K.

IWASE, K .•••••..••••..•.••••••..•• ·38

MATSUO, Y'13, 16IZUHARA, K . . . . . . • . . • . • . . . . • . . • . . • • ·89
JISHAGE, M. .
13
JUJI, T
·149
KABATA, H.'·134
KAGEYAMA, Y·46
KAMADA, K . . . . . . . . . . . . . . . . • . . . . 24, 135
KAMEYAMA, K.
.
·84
KANAYA, S·70
KASAHARA, M·70
KASAHARA, Y.
.
·166
KATO, Y
·35
KATSURA, I.
'136, 137, 139, 140
KAWATA, H·71
KIKKAWA, Y·113
KIKUTI, Y. Y.
·71
KIMURA, M ..••...••••.••••.• '15,16,71
KIMURA, T·73
KINEBUCHI, T.
.
134
KISHI, T·22
KITAKAMI, H·158

MEARES, C. F.

IWATA, A.

.

.

MELLO, C.

.

.

····················72
·156
·12
·144

MIKI, N.··························· ·83
MINATO, K .•••••.••.••..•••.•..••• ·57"113, 114, 116, 118·73
MITANI, Y.
·143
MITSUZAWA, H"15, 16
MIURA, K. .
·152
MIYABAYASHI, T·168
MIYAKE, T·148
MIYAO, T. .
'15, 16MIYASHITA, H. .
·134
MIYASHITA, N·74
MIYATA, A·143
MIYAZAKI, S·160
MIZUMOTO, K. .
'17,23MIZUNO, K ..••..••.•..••••••.•••.. ·107
MIZUNUMA, T.
.
·159
MORI, H
·151
MORI, M
·38
MITA, A

MITA, K

AUTHOR INDEXMORI, YMORIKAWA,·158

K.················· '24, 135

MORISHIMA, H'97,98,99, 100, 101'74, 106, 108, 109, 116,
118, 119
MOTOHASHI, T. ····················143
MUNEOKA, Y'44,45
MURAKAMI, A. .
'49, 51, 52, 54, 55
MURAKAMI, K. .
10, 12
MURATA, T·46
NAGAI, H. .
·133
NAKAKOUJI, M.
.
·33
NAKAMURA, Y. .
'69,70,73
NAKASHIMA, H. ····················87
NAKATSUJI, N.········· '72,129,131,132
NAKAZAWA, M.
.
·91
NAKAZAWA, Y·33
NEGISHI, T. · · · · · · · · · · · · · · · · · · · · · · · · 1 2
NEI, M·80
MORIWAKI, K

NISHIKAWA, K
NISHIMURA, A.
NISHIMURA, YNISHINO, I.
NIWA,'155, 156, 159····················127·33'93,94,95

H.······················· '37,38························69.
·73
NOMURA, T.
.
119
NON AKA, I. .
93, 94, 95
NONOMURA, K'125, 168
ODA, H·121
ODANI, S . . . . . . . . . . . . . • . . . . • • . . • . • . ·91
OGASAWARA, K.
.
'75,76
OGASAWARA, N ....•.••••••..•••.•. ·166
NOGAMI, M.

NOHATA, J.

OGAWA, T.························· ·26
OHBA, T
·143
OHFUCHI, H·33

OHNO, S .•.•.••••••••.••••........ ·148
OHTA, TOHTSUKA, H
OHTSUKA, R
OKAMOTO, N.
OKANO, T
OKAYAMA, T
OKUMURA, K'63,64·74·91. ........•••••••••••• ·71·85·159
59, 68, 69

OMOTO, K.

177.

'90, 149
10
OSAKA, F·21
OTA, M. • ..••.•..•••••••••.•• '155, 160
OURA, T·82
OWENS, J. T
·12
OYANAGI, M.
• .••.•.•••••.•.•••••• ·74
OPPENHEIM, A. B.

························12·90PARHAM, P . • • • • . . . . . . . . . . . . . . . . . . . ·63

OZOLINE, O.
PAN, I-H. .

PARK, K. S.
PRIESS, J························90144·43'165, 166
166

RAY, SSADAIE, Y
SAGAI, H
SAGAI, T

SAITOU, N.
SAKAI, M.

'106, 107, 108, 109, 111,
112, 116• ••• 69, 73, 74, 75, 76, 77, 160. . . . . . . . . . . . • . . . . . . . . '87,88

SAKAKURA, T.

······················72·15SANADA, K
84, 85
SANO, M . • • • . . • . • • . • . . . . • • . . . . . . . . ·143
SASAKI, H. .
·103
SAKURAI, H······················73'109, 110
SATO, S····36
SATO, T
·119
SATTA, Y·81
SEINO, H·21
SEN, R·133
SHICHIDA, Y·82
SHIGA, Y·121
SHIGENARI, A·71
SHIINA, T·71
SHIMADA, J.
.
'49, 51SHIMAMOTO, N . • . . . . . . . . . . '133, 134, 135
SHIMAMOTO, Y'100, 101, 102, 103
SHIMIZU, F. ························84
SHIMIZU, H.····'44,45SHIMIZU, K
113
SHIMONISHI, Y·83
SHINGU, R·148
SHIRAKIHARA, Y'141, 142, 143
SASANUMA, S.

SATO, H178
• ••.•..•• ·121

SHIRAYOSHI, Y.

TANAKA-MATAKATSU, M.

SHIROISHI, T

TANG, Y. ··························10
T ANOUE, K
108'72, 129, 131, 132'74, 106, 107, 108, 109,
110, 111, 112, 113, 114, 116, 118, 119
SHOUNO, O. ························83
STIBITZ, S . . . . . . . . . . . . . . . . . . . . . . • . • • . 10
SUGAWARA, H'159, 160
SUGAYA, K
70, 73SUGIYAMA, T .•••.•....•......... '44,45
SUMIYAMA, K. ······················77
SUYAMA, M.
. . . . . . . . . . . . . . . . . • • • • ·156
SUZUKI, K·43·139
TABARA, H.····'143, 144
TAKADA, K.
.
·118
TAKADA, M. . ........••••••••.... ·148
TAKAGI, T.
• ..••.•............•••• ·58
TAKAHASHI, K·148
TAKAHASHI, T ....•.••••••••..•• '44,45
TAKAHATA, N'80,81SUZUKI, N

T AKANAKA, Y.

······················85
TAKANO, T. S.······'64, 65
TAKAO, T ............•••••••••••... ·83
TAKE-UCHI, M. • ••••.•••••••...... ·136
TAKEDA, N·72
TAKEICHI, M . • • • . . . . . . • . . . . . . . . . . . ·121
TAKEMARU, K·47
TAKESHITA, K·94
TAKEUCHI, M .•..••••••••.•.•...•... 134
T AKIGUCHI, M .•.•..•.••••••........ ·38
TAMURA, T . · · · · · · · · · · · · · · · · · · · · · · · ·159
TANAKA, H·83
TANAKA, K.
.
'14,70TANAKA, S. • .••.•.•••••••......... ·28'157, 158, 159, 160

TATENO, Y.

.

TENZEN, T.

. . . . . . . . . . • • • . . • • . . . . . . '68

TOKUNAGA, F.

······················82.
75, 76, 149
TOYODA, T. . . . . . . . . . . . . . . . . • . • • • . • '18
UCHIDA, K.·'113, 114, 116
UEDA, H'46,47
UEDA, S. .
'13, 16, 77
UEDA, Y . . . . . . . . . . . . . . . . • . • • • • . . • • ·106
UEMURA, T.·· ..•.•.•••..•.••••.... ·121
UESUGI, H. .
·143
WADA, T . • . . • • • . . . . . . . . . . . . . . . . . . . '58
TOKUNAGA, K.

WAKANA, S . . '108, 109, 111, 113, 118, 119
WATANABE, H'143, 151, 152, 153

WATANABE, Y .•.•••..••..........•. ·68
WEST, S. R.

.

·41

YAGI, Y . . • . . . . . . . . . . . . . . . . . . . • • • • ·122
YAMAGUCHI, Y.

. .......•••. 58, 149, 150

YAMAMOTO, D.'·43
72
Y AMAMURA, K
37, 38YAMAO, F'21,22YAMAZAKI, Y.· ......••..•.••••..••• 128
YASUI, K'16
YATA, K
·166
YAMAMOTO, H

YONEKAWA, H. ············108, 113, 118
YOSHIDA, T .........•.•••••••••..•. '72
YOSHIKAWA, T . · · · · · · · · · · · · · · · · · · · · · '85
YOSHINO, M.

. • • • . • . . . . . . . . . . . . . . . ·107

YOSHIZAWA, T . . . . . . . . . . . . . . . . . . . • . • '82

Annual Report of the National Institute of Genetics. No. 47
Printed on December 20th 1997
Issued on December 30th 1997,

Issued by Director-General: Jun-ichi Tomizawa, Ph. D.
National Institute of Genetics
Yata 1,111 Mishima, Shizuoka-Ken, 411 Japan
Tel. 0559-81-6718
Fax. 0559-81-6715
Edited by Tomoko Ogawa & Yukiko Yamazaki
Published by National Institute of Genetics
Printed by Kokusai Bunken Insatsusha Co., Ltd.

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