ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT1995

No. 46

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mishima, Sizuoka-ken, Japan
1996

Annual Reportof theNational Institute of Genetics
No. 46, 1995

(Laboratory Building)Published by

The National Institute of Genetics, Japan
1996

CONTENTS
General statement
Staff
Council and advisory committee
Projects of research for 1995. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Research activities in 1995. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A.

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Department of Molecular Genetics
A-a. Division of Molecular Genetics
Functional Map of RNA Polymerase Alpha Subunit from Escherichia coli: Subunit-Subunit Contact Sites. KIMURA, M. and
ISHIHAMA, A.
Functional Map of RNA Polymerase Alpha Subunit from Escherichia coli: Transcription Factor- and DNA VP ElementContact Sites. MURAKAMI, K., NEGISHI, T., FUJITA, N. and
ISHIHAMA, A.
Structural Map of RNA Polymerase Alpha Subunit from Escherichia coli. NEGISHI, T., JEON, Y. H., SHIRAKAWA, M., YAMAZAKI, T., MURAKAMI, K., FUJITA, N., OZOLINE, 0., KYoGOKU, Y. and ISHIHAMA, A... .
...
...
Regulation and Specificity of RNA Polymerase Sigma Subunits
from Escherichia coli. Intracellular Levels of Various Sigma
Subunits. JISHAGE, M., KUSANO, S., IWATA, A., VEDA, S.
and ISHIHAMA, A.
Regulation and Specificity of RNA Polymerase Sigma Subunits
from Escherichia coli: Promoter Selectivity of Various Sigma
Subunits. KUSANO, S., KUNDU, T. K., DING, Q., TANAKA,
K., KOLB, A., FUJITA, N. and ISHIHAMA, A.
Subunit Composition of RNA Polymerase II from the Fission yeast
Schizosaccharomyces pombe: Subunit Composition. SAKURAI, H., MIYAO, T., SUZUKI, H., YAMAGISHI, M. and ISHIHAMA, A.............................................
Subunit Function of RNA Polymerase II from the Fission Yeast

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Schizosaccharornyces pornbe: Subunit 2, 3 and 5. MIYAO, T.,
YASUI, K., KAWAGISHI, M., AZUMA, Y., YAMAGISHI, M. and
ISHIHAMA, A. .
Subunit Function of Influenza Virus RNA Polymerase: Crosslinking Studies of Nucleotides. ASANO, Yo, MARUYAMA, T.
and ISHIHAMA, A. . ....
Subunit Function of Influenza Virus RNA Polymerase: RNA Synthesis by Subunits and Subassemblies. TOYODA, T., KOBAYASHI, Mo and ISHIHAMA, A. . ..
Subunit Function of Influenza Virus RNA Polymerase: Mapping of
Subunit-subunit Contact Sites. ADYSHEV, D., TOYODA, T.
and ISHIHAMA, A. . ..
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Division of Mutagenesis

Abnormal Integrity of Nucleolus Associated with Cell Cycle Arrest
Owing to the Temperature-Sensitive Vbiquitin-Activating
Enzyme El. SUDHA, T., AOKI, F., SAMESHIMA, M.,
KANEDA, S., YAMAO, F. and SENO, To .
Decrease in Intracellular Deoxyribonucleoside Triphosphate Pools
Associated with Arrest in DNA Replication and Defective
DNA Repair in a Mouse Cell Mutant tsFS20 with Thermolabile Vbiquitin-Activating Enzyme El. AOKI, F., WATAYA, Y,
KANKAWA, S., AYUSAWA, D., KANEDA, S., YAMAO, F. and
SENO, T
Cell Cycle-Dependent Phosphorylation and Nuclear Targeting of
Vbiquitin-Activating Enzyme, EI, in Mammalian Cells.
SEINO, H., NAGAI, Y., SENO, T. and YAMAO, F ....
Screening for cDNAs Encoding the Vbiquitin-Conjugating Enzyme
in Fission Yeast, Schizosaccharornyces pornbe. OSAKA, F.,
SEINO, H., SENO, To and YAMAO, F.
Regulation of G l/S transition by the Vbiquitin System in Yeast
Saccharamyces cerevisiae. KISHI, T., VI, M. and SENO, T.
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Human Vbiquitin Pathway Homologous to that Mediated by Vbc
P4 in Fission Yeast. SEINO, H., OHSAKA, F., SENO, T. and
YAMAO, F .....
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CONTENTS
A-c.

Division of Nucleic Acid Chemistry

Crystal Structure Determination of the Escherichia coli Replication
Terminator Protein (Tus) Complexed with Ter DNA. MoRIKAWA, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yeast mRNA Capping Enzyme Mutants: Isolation of TemperatureSensitive Mutations and Characterization of the Altered
Enzymes. YAMAGISHI, M., MIZUMOTO, K. and ISHIHAMA,
A

B.

iii

Department of Cell Genetics
B-a. Division of Cytogenetics
A Species-specific Interaction of Rad5l and Rad52 proteins III
Eukaryotes. OGAWA, T., SHINOHARA, A. and IKEYA, T.
Homologous Recombination and The Roles of Double-strand..
Breaks. SHINOHARA, A. and OGAWA, T.
Localization of RecA-like Recombination Protein on Chromosomes of The Lily at Various Meiotic Stages. TERASAWA,
M., HINOHARA, A. and OGAWA, T.
Localization of Mouse Rad51 and Lim 15 on Meiotic Chromosomesin Late Stages of Prophase 1. IKEYA, T., SHINOHARA, A. and
OGAWA, T.
Roles of the Rad52 Protein of Saccharomyces cerevisiae in Homologous Recombination. SHINOHARA, A., SHINOHARA, M.
and OGAWA, T.
B-b. Division of Microbial Genetics
The Initiator Protein of Filamentous Phage Forms a Covalent
Complex with the Origin DNA. ASANO, S., HIGASHITANI,
A. and HORIUCHI, K.
Mutational Analysis of the Minus Strand Replication Origin ofFilamentous Bacteriophage. HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.
.
A Promoter at the Beginning of the Cell Envelope BiosyntheticjCell
Division Gene Cluster in the Escherichia coli Chromosome 2Minute Region is Required for Expression of Nine Genes up to
its W. HARA, H. and HORIUCHI, K.
Functional Penicillin-Binding Protein 7 is a Multicopy Suppressor

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IV

of Thermosensitive Growth Defect at Low Osmolarity Due toan spr Mutation of Escherichia coli. HARA, H. and HORIUCHI, K.
C-terminal Region of an Escherichia coli Cell-Division Inhibitor
SulA is Involved in Negative Regulation of the SOS Response.
HIGASHITANI, A., ISHII, Y., KATO, Y. and HORIUCHI, K ...
B-c.

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Division of Cytoplasmic Genetics

Identification and Characterization of the Linear IS3 Molecules
Generated by Staggered Breaks. OHTSUBO, E.
Characterization of TraI helicase-endonuclease which is involved in
the initiation and termination steps of DNA transfer. OHTSUBO, E.
A 6-kb Upstream Region of the Human Transthyretin Gene Can
Direct Developmental, Tissue-Specific and Quantitatively
Normal Expression in Transgenic Mouse. NAGATA, Y,
TASHIRO, F., VI, S., MURAKAMI, T., MAEDA, S., T AKAHASHI, K., SHIMADA, K., OKAMURA, H. and Y AMAMURA, K.

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Cell-Cycle-Dependent Expression of the STK-I Gene Encoding a
Novel Murine Putative Protein Kinase. NIWA, H., ABE, K.,
KUNISADA, T. and Y AMAMURA, K. .
.

C.

50

Department of Ontogenetics
C-a.

Division of Developmental Genetics

Nerve Cell Differentiation in Epithelial Hydra: Epithelial Tissue
Provides Cues for Region-Specific Differentiation. MINOBE,
S., KOIZUMI, O. and SUGIYAMA, T. .
.
Hydra Peptide Project II. Biological Function Assay. FUJISAWA,
T., SHIMIZU, H., HATTA, M., KOIZUMI, 0., BODE, H., BOSCH,
T., deHARO, M., LOHMANN, J., DAVID, C., TAKAHASHI, T.
and MUNEOKA, Y.
Disappearance and Reappearance of Mesoglea in Hydra Tissue
During Head Regeneration. SHIMIZU, H. and SARRAS, M. P.
Jr.
.
Analysis of Hydra Cell Adhesion Molecules. HATTA, M., HOBMAYER, E. and SUGIYAMA, T
.

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CONTENTSv

Evolutionary Study of Reef-Building Corals, Genus Acropora.
FUKAMI, H., WANG, W., OHMORI, M., HAYASHIBARA, T,
SHIMOIKE, K., HATTA, M. and SUGIYAMA, T.
Cloning of Female Germ Stem Cells from a Male Strain of Hydra
magnipapillata. NISHIMIYA-FuJISAWA, C. and SUGIYAMA,

56

T
C-b.

.

56

Division of Phenogenetics

Cloning of a cDNA for DNA Supercoiling Factor Reveals aDistinctive Ca2+ -Binding Protein. OHTA, T., KOBAYASHI,
M. and HIROSE, S.
Molecular Cloning of a cDNA for Transcriptional Positive Cofactor MBF2. LI, F.-Q., TAKEMARU, K., VEDA, H. and
HIROSE, S.
Molecular Cloning of a cDNA Encoding Transcriptional Mediator
MBFl. TAKEMARU, K., LI, F.-Q, VEDA, H. and HIROSE, S.

58

58

59
Consideration of the Hormonal Function of Aging Cells and Organisms in Insects. IWABUCHI, K. and MURAKAMI, A .....
Neuro-Endocrinological Studies on a Mutant sdi (Short Durationof Imaginal Lifespan) in the Bombyx Silkworm. SHIMADA,
J., KANBARA, M. and MURAKAMI, A.
.
Linkage Analysis of the sdi (short duration of imaginal lifespan)
Gene. FUJII, H., DOIRA, H. and MURAKAMI. A. .
.
Genetic Studies on Chromosomal Translocation in Bombyx Mori
(L.) (a) Construction of a New Reciprocal Translocation.
MURAKAMI, A. .
.
Genetic Studies on Chromosomal Translocation in Bombyx Mori
(L.) (b) Genetic Function of the Y-Chromosome. MURAKAMI, A.
Origin of Holometabolous Metamorphosis in Insects. MINATO,
K
.
C-c.

Division of Physiological Genetics

Hydra Peptide Project I. Isolation, Structure Determination andChemical Synthesis. MUNEOKA, Y, TAKAHASHI, T, FUJISAWA, T, SHIMIZU, H., HATTA, M., SUGIYAMA, T, KOIZUMI, 0., BOSCH, T, deHARO, M., LOHMANN, J., Charles

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63

64

67
68

VI

DAVID, C. and BODE, H.
Analyses of Mammalian Genome by Fluorescence in situ Hybridization. OKUMURA, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

D.

69
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Department of Population Genetics
D-a.

Division of Population Genetics

Synonymous and Nonsynonymous Substitutions in Mammalian
Genes and the Nearly Neutral Theory. OHTA, T.
Gene Conversion vs Point Mutation in Generating Variability at
the Antigen Recognition Site of Major Histocompatibility.
Complex Loci. OHTA. T................. .
Genetic study of Species Differences as Observed as Loss of Bristles
in Interspecific Hybrids of Drosophila. TAKANO, T.
New methods for Estimating the Numbers of Synonymous andNonsynonymous Substitutions. INA, Y
D-b.

73

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Division of Evolutionary Genetics

Studies of Human Pseudoautosomal Boundary-Like Sequences(PABLs). FUKAGAWA, T, OHONO, M., NAKAMURA, Y,
OKUMURA, K., ANDo, A., INOKO, H., SAITOU, N. and IKEMURA, T.
75
Studies of Notch-Related MHC-Class III Gene INT3: Structure of
the Human Counterpart of Mouse Mammary Tumor Gene int3 and (CTG)n Polymorphism in the 5'-Upstream Region.
SUGAYA, K. MITA, K., ANDO, A., INOKO, H. and IKEMURA,
T
77
Precise Replication Timing around a Boundary of Long-Range G +C% Mosaic Domains in the Human MHC Region. TENZEN,
T, FUJIYAMA, A., OKUMURA, K., ANDo, A., INOKO, H.,
GOJOBORI, T. and IKEMURA, T.
77
Detection of Genes in Escherichia coli Sequences Determined by
Genome Projects and Prediction of Protein-Production Levels,
Based on Multivariate Diversity in Codon Usage. KANAYA,
S., KUDO, Y, NAKAMURA, Y and IKEMURA, T. . . . . . . . . .. 78
Tenascin-X Expression in Tumor Cells and Fibroblast in Culture.
SAKAI, T., FURUKAWA, Y, CHIQUET-EHRISMAN, R., IKEMURA, T. and MATSUMOTO, K.
79

CONTENTS

vii

Codon Usage Tabulated from the International DNA Sequence
Databases. NAKAMuRA, Y., GOJOBORI, T. and IKEMURA,T

A Genetic Affinity Analysis of Human Populations.

.

79

SAITOU, N.
80

Methods for Building Phylogenetic Trees of Genes and Species.
SAITOU, N.
Application of a Parallel Logic Programming for Reconstruction of
Molecular Phylogenetic Trees Using the Maximum Likelihood
Method. OOTA, S., SAITOU, N., and KUNIFUJI, S.
Mitochondrial DNA Sequences of Various Species of the Genus
Equus with a Special Reference to the Phylogenetic Relationship between Przewalskii's Wild Horse and Domestic Horse.
ISHIDA, N., OYUNSUREN, T., MASHlMA, S., MUKOYAMA, H.
and SAITOU N. .
.
A Genetic Study of 2,OOO-Year-Old Human Remains from Japan
Using Mitochondrial DNA Sequences. OOTA, H., SAITOU,
N., MATSUSHITA, T. and UEDA, S
.
D-c.

80

81

81

82

Division of Theoretical Genetics

A Genetic Perspective on the Origin and History of Humans.
TAKAHATA, N.
MHC Diversity and Selection. TAKAHATA, N. .
.
Divergence Time and Population Size in the Lineage Leading toModern Humans. TAKAHATA, N., SATTA, N. and KLEIN, J.

84
84

85
A Population Genetic Study on the Evolution of SINEs. II. Sequence Evolution under the Master Copy Model. T ACHIDA,

H

.

86

Molecular Evolution of the 5'-flanking Regions of Duplicated Amy
Genes in the Drosophila melanogaster Species Subgroup.
OKUYAMA, E., SHIBATA, H., TACHIDA, H. and YAMAZAKI,

T
E.

.

Department of Integrated Genetics
E~a.
Division of Human Genetics
Isolation of Transcribed Sequences in Cloned Genomic DNA with

86

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Known Location on the Human Chromosome 18. IMAMURA, T., SAKAI, M., INABA, R. and NAKASHIMA, H.
88
Isolation of Transcribed Sequences by Kinetic Enrichment ofcDNA Fragments Derived from Chromosome 15 and Molecular Definition of the Prader-Willi Syndrome. IMAMURA, T,
SAKAI, M., INABA, R. and HASEGAWA, T.
89
A Signal Transduction Pathway ofInterieukin-4 and Interleukin-13
in Human B Cells Derived from X-linked Severe Combined
Immunodeficiency Patients. IZUHARA, K., IMAMURA, T.,
91
HElKE, T., NIHO, Y. and HARADA, N.
A Synthetic Peptide Corresponding to a Critical Intracellular Signalling Region of the Human IL-4 Receptor Inhibits IL-4Dependent Proliferation. IZUHARA, K., SAKAI, M., INABA,
R., IMAMURA, T. and HARADA, N.
91
Recent African Origin of Modern Humans Revealed by CompleteSequences of Hominoid Mitochondrial DNAs. HORAI, S.,
HAYASAKA, K., KONDO, R., TSUGANE, K. and TAKAHATA,
N
92
Different Waves of Migration to the New World: Implications ofMitochondrial DNA Polymorphism in Native Americans.
HORAI, S............................................. 93
Y Chromosomal DNA Variation and the Peopling of Japan.
HAMMER, M. F. and HORAI, S.
94
Molecular Phylogeny of Gibbons Inferred from Mitochondrial
DNA Sequences: Preliminary Report. HAYASHI, S., HAYASAKA, K., TAKENAKA, O. and HORAI, S.
95
Orosomucoid Phenotyping with Monoclonal Antibodies: Polymorphic Occurrence of ORMl*QO in Aboriginal Taiwanese Populations. UMETSU, K., YUASA, I., HARADA, A., SUZUKI, T,
PAN, I.-H., ISHIDA, T., SAITOU, S. and HORAI, S.
96
Genetic Polymorphism of ADA in Taiwan Aboriginal Populations:
New ADA Variants Detected by Isoelectric Focusing Method.
JIN, F., SAITOU, N., ISHIDA, T, SUN, C-H., PAN, I-H.,OMOTO, K. and HORAI, S.
97
E-b.

Division of Agricultural Genetics

Isozyme Variation in Asian Common Wild Rice Oryza rufipogon.

CONTENTS

ix

CAl, H. W., WANG, X. K. and MORISHIMA, H.
99
QTL Analysis of Annual/Perennial Life-History Variation in Oryza
rufipogon. KOHN, J. R. and MORISHIMA, H
101
Ecological Genetic Studies on American Wild Rice (Oryza glumaepatula Steud.) Distributed around the Amazon Basin. AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA, H. . . . . . . . . .. 102
Gene Regulation of Two Alleles at the wx Locus of Rice.
103
HIRANO, H.-Y., EIGUCHI, M. and SANO, Y.
Genetic Analysis of Chromosome 7 in an Annual Type Wild Rice.
104
EIGUCHI, M. and SANO, Y.
E-c.

Division of Applied Genetics

Mitochondrial DNA Analysis Reveals Cytoplasmic Variation
within a Single Cultivar of Perennial Ryegrass (Lolium per106
enne L.). SHIMAMOTO, Y.
Genetic Differentiation between Summer and Autumn Maturing
Cultivars of Soybean (Glycine max L.) in the Kyushu districtof Japan. SHIMAMOTO, Y.
106
Molecular Genetic Studies of Genomic Imprinting. SASAKI, H.,
HATANO, N., ZUBAIR, M. and KATO, R.
107

F.

Genetic Stock Research Center
F-a. Mammalian Genetics Laboratory
Establishment of System for Tetrad Analysis in Mouse Female
Meiosis II. MIZUNO, K-I., SAGAI, T., MORIWAKI, K. and
SHIROISHI, T.
109
Fine Structure of the Ph Hotspot in the Mouse MHC class II
Region. ISOBE, T., YOSHINO, M., MIZUNO, K-I., KOIDE, T.,
MORIWAKI,. K. and SHIROISHI, T
111
Genetic Analysis of Coat-Color Mutation, rim2 . II. SAGAI, T.,
KOIDE, T., ENDO, M., TANOUE, K., YONEKAWA, H., MORIWAKI, K. and SHIROISHI, T
112
Linkage Analysis of the Mouse Mutant, Rim3, Affecting Skin andEye. SATO, H., MASUYA, H., WAKANA, S., SAGAI, T., MoRIWAKI, K. and SHIROISHI, T
113
Multigenic Control of Localization of the Zone of Polarizing Activity in Mice Limb Morphogenesis. MASUYA, H., SAGAI, T.,

x

WAKANA, S., MORIWAKI, K. and SHIROISHI, T. 114
Searching for Modifier Genes for the Phenotype of Mutation Tailshort. ISHIJIMA, J., UCHIDA, K., MITA A., KOIDE, T. and
SHIRaISHI, T. 116
Morphological Characterization of the Mouse Mutant Tail-short:
Skeletal Patterning Along the Anteroposterior Axis. ISHIJIMA, J., UCHIDA, K., MITA, A., KOIDE, T. and SHIROISHI, T.
116
Fine Gene Mapping of the Skeleton Mouse Mutant, Tail short (Ts),
Toward Positional Cloning. KOIDE, T., UCHIDA, K., MITA,
A., WAKANA, S., KIKKAWA, Y., YONEKAWA, H., MORIWAKI,
K. and SHIRaISHI, T. 117
Establishment of Intersubspecific Consomic Mouse Strains. SHIRaISHI, T., MITA, A., TAKADA, K., WAKANA, S., YONEKAWA, H. and MORIWAKI, K
. 118
F-b.

Invertebrate Genetics Laboratory

Determination of wing cell fate by the escargot and snail genes in
Drosophila. FUSE, N., HIROSE, S. and HAYASHI, S. . ..... 119
A Cdc2 dependent checkpoint maintains diploidy in Drosophila.
HAYASHI, S. .
. 120
Roles of Escargot and DE-Cadherin in the Formation of Drosophila
Tracheal Network. TANAKA-MATAKATsu, M., UEMURA,
T., aDA, H., TAKEICHI, M. and HAYASHI, S. 121
The Early Development of Drosophila Imaginal Discs. GOTO, S.
and HAYASHI, S. 122
A Nuclear GFPI,B-Galactosidase Fusion Protein as a Marker for
Morphogenesis in Living Drosophila. SHIGA, Y., T ANAKAMATAKASTU, M. and HAYASHI, S. 122
Control of esg Expression. YAGI, Y. and HAYASHI, S. .
. 123
argos is Reqired for Projection of Photoreceptor Axons During
Optic Lobe Development in Drosophila. SAWAMOTO, K.,
OKABE, M., TANIMURA, T., HAYASHI, S., MIKOSIBA, K. and
OKANO, H
. 123
F-c.

Plant Genetics Laboratory

Structural and functional analysis of the genes expressed during
early embryogenesis in rice. ITo, Y.
. 124

CONTENTS
F-d.

xi

Microbial Genetics Laboratory

A New Gene Determining the Frequency of Cell Division in E. coli.
TADENUMA, M. and NISHIMURA, A
125
Control of Cell Division at Low pH. UKAI, H., WATANABE, N.,SUZUKI, K. and NISHIMURA, A.
125
The Instability of Membranes Affect the Transcription of Cell
Division Gene, ftsZ. NISHIMORI, K. and NISHIMURA, A. .. 126
sdiA and rpoS-Mediated Dual Transcriptional Regulation of 7aHSDH Gene, hdhA, in Escherichia coli. KANAMARU, K. and
KANAMARU. K.
127
A Novel Member of the cspA Family of Genes, cspG, which is
Induced Upon Cold-shock in Escherichia coli. KANAMARU
K. and KANAMARU, K.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 127
F-e.

Genetic Resources Laboratory

Compilation of Genetic Resources Information.
YAMAZAKI, Y.
F-f.

SAITO, M. and128

Mammalian Development Laboratory

Analysis of Cellular and Molecular Mechanisms in Development of
Mouse Fetal Germ Cells. NAKATSUJI, N. and SHIRAYOSHI,Y
130
Molecular Analysis of Cell Differentiation and Morphogenesis in
Postimplantation Mouse Embryos. SHIRAYOSHI, Y. and
NAKATSUJI, N.
131
Establishment of Neuronal Stem Cell Lines for Analysis of Cell
Lineages and Differentiation in the Mouse Central Nervous
System Development. NAKATSUJI, N., SHOKUNBI, M. T. and
SHIRAYOSHI, Y
132
Analysis of the Migration Pattern of N euroblasts and Morphogenesis in Development of the Mouse Central Nervous System.
NAKATSUJI, N. and SHOKUNBI, M. T.
133
G.

DNA Research Center
G-a.

DNA-Protein Interaction Laboratory

Kinetic Study on Transcription by E. coli RNA polymerase: 1.
Irreversible Trapping in Abortive Cycle. KUBORI, T. and
SHIMAMOTO, N
135

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Single-Molecule Dynamics of Transcription Using Immobilized
Operons: Sliding of P. putida CamR protein on DNA.
KABATA, H. and SHIMAMOTO, N.
136
Protein Footprint analysis of Transcription Complexes. NAGAI,
H., T AKAGISHI, T. and SHIMAMOTO, N.
136
G-b.

Recombinant DNA Laboratory

Fluoride-resistant Mutants of the Nematode Caenorhabditis elegans. KAWAKAMI, M., TAKE-ueHI, M., URASAKI, M., ISHIHARA, T. and KATSURA, I.
137
A Gene that Controls Both Hatching and Cell Migration in C.
elegans. HISHIDA, R., ISHIHARA, T. and KATSURA, I. .... 139
Analysis of the Head Neural Circuit of C. elegans as Studied byFormation of Dauer Larvae. SUZUKI, N., URASAKI, M.,
ISHIHARA, T. and KATSURA, I.
140
Construction of GFP Markers for Various Neurons in C. elegans
and Their Use for Mutant Isolation. ISHIHARA, T., FUJIWARA, M. and KATSURA, I.
141
G-c.

DNA Synthesis Laboratory

Crystallographic Study of af33 Sub-complex of F1-ATPase: Structural Analysis of Supramolecule. SHiRAKIHARA, Y
142
Crystallographic Study of Transcription Factors, PhoB and CamR.
SHIRAKIHARA, Y.
143
G-d.

Gene Library Laboratory

Expression map of the C. elegans genome. KOHARA, Y, MOTOHASHI, T., TABARA, H., SUGIMOTO, A., WATANABE, H.,
SANO, M. and MIYATA, A
144
A Multi-well Version of in situ Hybridization on Whole Mount
Embryos of C. elegans. TABARA, H., MOTOHASHI, T. and
KOHARA, Y.
145
Pos-I, a Gene Which Shows Localization of Its mRNA to the P
Lineage During Early Cleavage of C. elegans Embryos.
TABARA, H., MOTOHASHI, T., MELLO, C., PRIESS, J. and
KOHARA, Y.
145

CONTENTS

xiii

H. Center for Information Biology
H-a. DNA Data Analysis Laboratory
Domain Evolution of Serine Protease and Its Inhibitor Genes.
GOJOBORI, T., ENDo, T. and IKEO, K. 148
Sequence Divergence Estimation. GOJOBORI, T. and IKEo, K . 148
Different Evolutionary Histories of Kringle and Protease Domains
in Serine Proteases: A Typical Example of Domain Evolution.
IKEO, K., TAKAHASHI, K. and GOJOBORI, To
149
Evolution of Genes and The DNA Database-Implications for Bio149
information. GOJOBORI, T, ENDo, T and IKEO, K. o'
Large-Scale Search for Genes on Which Positive Selection May
Operate. ENDo, T., IKEO, K. and GOJOBORI, T. .
. 150
Large-Scale Search for Within-Gene Regions Where Positive Selection May Operate. ENDo, T., IKEO, K. and GOJOBORI, T.
0

0

"

0

•••

150
Evolutionary Relationship of Hepatitis C, Pesti-, Flavi-, Plantviruses, and Newly Discovered GB Hepatitis Agents. OHBA,
K., MIZOKAMI, M., LAU, J. Y. N., ORITO, Eo, IKEo, K. and
GOJOBORI, T
151
Classification of Hepatitis C Virus into Major Types and Subtypes
Based on Molecular Evolutionary Analysis. OHBA, K., MlzoKAMI, Mo, OHNO, T, SUZUKI, K., ORITO, E., INA, Y., GOJOBORI, T. and LAU, J. Y. N. .
152
Patterns of Amino Acid Substitutions in the Third Variable Envelope Region of HIV Within Single Hosts. YAMAGUCHI, Y.
and GOJOBORI, T. .
152
Molecular Epidemiology of Human Immunodeficiency Virus Type
1. YAMAGUCHI, Y. and GOJOBORI, T. 153
Molecular Evolution of HIV-l and its Implication to Mother-ToChild Transmission. TAKAHASHI, K. and GOJOBORI, T . . . 153
Molecular Evolution of Marburg and Ebola Viruses. SUZUKI, Y.
and GOJOBORI, T. .
. 154
Molecular Phylogenetic Trees Based on Minimum Description
Length Principle. REN, F., TANAKA, H., FUKUDA, N. and
GOJOBORI, T. 154
NRSub: a Non-Redundant Database for Bacillus subtilis. PERRI0

0

••••

0

••••••••••••••••••••••••••••••••••••

0••••••••••••••••••••••

xiv

ERE, G., MOSZER, I. and GOJOBORI, T
155
Analysis of the Structure of the Escherichia coli Genome by Seeing
the Distance between Homologous Genes. WATANABE, H.
and GOJOBORI, T.
156
Analyses of the Structures of Microbe Genomes on the Basis of the
Homologous Relationships between Genes. WATANABE, H.,
MORI, H. and GOJOBORI, T. .
156
Monophyletic Origin and Unique Dispersal Patterns of Domestic
Fowls. AKISHINONOMIYA, F., MIYAKE, T., TAKADA, M.,
SHINGU, R., ENDo, T., GOJOBORI, T., KONDO, N. and OHNO,S
157
Isolation and Charactarization of cDNA Clones for Japanese Quail(Coturnix Japonica) Major Histocompatibility Complex(MhcCoja) Class I Molecules. SHIINA, T., ANDo, A., IMANISHI, T., KAWATA, H., HANAZAWA, K., GOJOBORI, T.,
INOKO, H. and WATANABE, S. . . . . . . . . . . . . . . . . . . . . . . . . .. 158
Determination of HLA Class II Alleles by Genotyping in a Manchu
Population in the Northern Part of China and Its Relationshipwith Han and Japanese Populations. GENG, L., IMANISHI,
T., TOKUNAGA, K., ZHU, D., MIZUKI, N., Xu, S., GENG, Z.,
GOJOBORI, T., TSUJI, K. and INOKO, H. .
159
Distribution of Shared Polymorphisms and I ts Relation to Gene
Conversion in the Major Histocompatibility Complex Genes.
IMANISHI, T.
160
Y AMATO AND ASUKA: DNA Database Management System.
KITAKAMI, H., SHIN-I, T., IKEO, K., UGAWA, Y., SAITOU, N.,
GOJOBORI, T. and TATENO, Y.
161
H-b.

Laboratory of Gene Function Research

Molecular Evolution of a Protein Seemingly Responsible for Epoxidation in Eukaryote. TATENO, Y.
162
H-c. Laboratory for Gene-Product Informatics
Prediction of Protein Three-Dimensional Structures by A 3D-1D
Compatibility Method. NISHIKAWA, K. .
164
Protein Stability Change Caused by Single Amino Acid Substitutions: Estimation by The 3D-Profile Method. NISHIKAWA,
K
165

CONTENTS

I.

xv

Radioisotope CenterTranscriptional Analysis of the Bacillus subtilis Sporulation Gene.
FUJITA, M. and SADAIE, Y.
166
Abstracts of diary for 1995
Foreign visitors in 1995 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Association for propagation of the knowledge of genetics
Author index

167
172
174
176

GENERAL STATEMENT
Our institute was established 46 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 32 such students
at present and in addition, about ten special research students from otheruniversities, including those from foreign countries. I consider it vital 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 molecularstudies of various aspects of genetics, without ignoring the importance ofmore traditional approaches to genetics. Our institute is uniquely suited for
pursuing cooperative work with scientists of various disciplines. Throughinteractions among various research disciplines, our institute 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.
In the past year, we have had an important shift in the organizationaccording to the demands of the times. Separated from the DNA Research2Center, laboratories concerned with information biology were set up in a newCenter for Information Biology which consists of four laboratories. The past
year also saw a number of changes in the staff of the institute. Promoted as
professor, Yoshio Sano was transferred to the Hokkaido University, Fumio
Tajima to the University of Tokyo, and Tetsuya Toyoda to the Kurume
University, respectively, and Masahiro Yamagishi to the Aichi Cancer Center
Research Institute as senior researcher. The following eight people joined us:
as professor, Tomoko Ogawa in the Division of Cytogenetics, Ken Nishikawa
in the newly established Laboratory for Gene-Product Informatics; as associate professor, Yukiko Yamazaki in the Genetic Resources Laboratory, Yasuo
Shirakihara in the DNA Synthesis Laboratory. Shigeo Tanaka joined theDivision of Cytogenetics, Tsuyoshi Koide the Mammalian Genetics Laboratory, Satoshi Goto the Invertebrate Genetics Laboratory, Yukihiro Ito the
Plant Genetics Laboratory.
It is a pleasure to note that the following three were honored with awards:
Takashi Gojobori by the Kihara Memorial Yokohama Foundation for the
Advancement of Life Sciences, Naruya Saitou by the Genetics Society ofJapan and Nobuo Shimamoto by the Industry Applications Society of the
U.S.A. Jun-ichi Tomizawa was elected a foreign associate of the National
Academy of Sciences of the U.S.A.

3

STAFF

(as of December 31, 1995)

Director-General

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

SENO, Takeshi, D. Sc.
Members1.

Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Division of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
TOYODA, Tetsuya, D. Med.
Division of Mutagenesis
SENO TAKESHI, D. Sc., Professor
YAMAO, Fumiaki, D. Sc., Associate professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry (Guest member)
MORIKAWA, Kosuki, D. Sc., Professor
YAMAGISHI, Masahiro, D. Sc.

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.

4

GOTO, Hideo, D. Ag.
Division of Microbial Genetics
HORIUCHI, Kensuke, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Division of Cytoplasmic Genetics (Guest member)
OHTSUBO, Eiichi, D. Sc., Professor
YAMAMURA, Ken-ichi, D. Med., Professor

3.

Department of Ontogenetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
Division of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
HATTA, Masayuki, D. Sc.
Division of Phenogenetics
HIROSE, Susumu, D. Sc., Professor
MURAKAMI, Akio, D. Ag., Sc., Associate professor
VEDA, Hitoshi, D. Ag.

YAMADA, Masa-aki, D. Sc.
MINATO, Kiyoshi, M. Sc.
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
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKANO, Toshiyuki, D. Sc.
INA, Yasuo, D. Sc.

STAFF

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.
Division of Theoretical Genetics (Guest member)
TAKAHATA, Naoyuki, D. Sc., Professor
TACHIDA, Hidenori, D. Sc., Associate 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
MORISHIMA, Hiroko, D. Ag., Professor
HIRANO, Hiro-Yuki, D. Ag.
Division of Applied Genetics (Guest member)
WATANABE, Takeshi, D. Med., Professor
SHIMAMOTO, Yoshiya, D. Ag., Professor

6.

Genetic Stock Research Center
NAKATSUJI, Norio, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Associate professor
Invertebrate Genetics Laboratory
HAYASHI, Shigeo, D. Sc., Associate professor
GOTO, Satoshi, D. Sc.
Plant Genetics Laboratory
ITo, Yukihiro, D. Ag.

5

6

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
SHIRAYOSHI, Yasuaki, D. Sc.

7.

DNA Research Center
KATSURA, Isao, D. Sc., Head of the Center
DNA-Protein Interaction Laboratory
SHIMAMOTO, Nabuo, D. Sc., Associate professor
NAGAI, Hiraki, D. Sc.
Recombinant DNA Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
DNA Synthesis Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate professor
Gene Library Laboratory
KOHARA, Yuji, D. Sc., Associate professor
ANDACHI, Yoshiki, D. Sc.

8.

Center for Information Biology
GOJOBORI, Takashi, D. Sc., Head of the Center
DNA Data Analysis Laboratory
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
IMANISHI, Tadashi, D. Sc.
Laboratory of Gene Function Research
TATENO, Yoshio, Ph. D., D. Sc., Professor

STAFF

7

Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
9.

Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate professor, Head of the Center

10.

Experimental Farm
MORISHIMA, Hiroko, D. Ag., Head of the Farm

11.

Technical Section
MITA, Akihiko, Chief of the Section

12.

Department of Administration
KONO, Kenji, Head of the Department
SATO, Shigeru, Chief of the General Affairs Section
TAMURA, Mituo, Chief of the Finance Section

8

COUNCIL(as of December 31, 1995)

Chairman
NAGAKURA, Saburo; Director, Kanagawa Academy of Science andTechnology FoundationVice-chairman
SUGANO, Haruo; Director emeritus, Cancer InstituteMembers (Alphabetical order)
HAMAl, Osamu; Professor, Graduate School of Humanities and Sociology. The University of Tokyo
HI RASA, Takao; President, Osaka Prefectural University
KATO, Nobuo; President, Nagoya University
KYOGOKU, Yoshimasa; Professor, Institute for Protein Research, Osaka
University
MATSUBARA, Kenichi; Director, Institute for 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
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
OSAWA, Shozo; Adviser, Biohistory Research Hall
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
YAMAGATA, Hirotada; Professor, Faculty of Bioscience and Technology, Kinki University

9

ADVISORY COMMITTEE(as of December 31, 1995)

Chairman
SENO, Takeshi; Professor, Division of Mutagenesis
Vice-chairman
OKADA, Masukichi; Professor emeritus, University of Tsukuba
Outside Members (Alphabetical order)
Go, Mitiko; Professor, Faculty of Science, Nagoya University
HANAOKA, Fumio; Professor, Institute for Molecular and Cellular
Biology, Osaka University
HINATA, Kokichi; Professor, Faculty of Agriculture, Tohoku University
HOTTA, Yasuo; Professor, Graduate School of Biological Sciences, Nara
Institute of Science and Technology
ISHIWA, Sadao; Professor, Faculty of Science, Ochanomizu Women's
University
IsoNo, Katsumi; Professor, Faculty of Science, Kobe University
OISHI, Michio; Director-General, National Institute of Bioscience andHuman-Technology
TAKAGI, Nobuo; Professor, Graduate School of Environmental Science,
Hokkaido University
TAKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto University
Inside Members (Alphabetical order)
GOJOBORI, Takashi; Professor, Center for Information Biology
HIROSE, Susumu; Professor, Division of Phenogenetics
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
MORISHIMA, Hiroko; Professor, Division of Agricultural Genetics
NAKATSUJI, Norio; Professor, Genetic Stock Research Center
OHTA, Tomoko; Professor, Division of Population Genetics
SUGIYAMA, Tsutomu; Professor, Division of Developmental Genetics

10

PROJECTS OF RESEARCH FOR 1995
1.

DEPARTMENT OF MOLECULAR GENETICS
Division of Molecular Genetics

Regulatory mechanisms of gene transcription in prokaryotes (ISHIHAMA andFUJITA)

Molecular architecture of transcription apparatus from eukaryotes (ISHIHAMA)
Molecular mechanisms of transcription and replication of animal and plantviruses (ISHIHAMA and TOYODA)
Division of Mutagenesis

Multiple role of ubiquitin system in the cell-cycle regulation (SENO, YAMAO,KANEDA, KISHI and SEINO)Division of Nucleic Acid Chemistry

Structural genetics: X-ray crystallography of proteins (MORIKAWA)
Isolation and characterization of yeast mRNA capping enzyme mutants(YAMAGISHI)
2.

DEPARTMENT OF CELL GENETICS
Division of Cytogenetics

Mechanisms of genetic recombination (OGAWA)
Theoretical and experimental bases for chromosome evolution (IMAI)
Chromatin structure involved in recombination (TANAKA and OGAWA)PROJECTS OF RESEARCH FOR 1995

11

Division of Microbial Genetics

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

Studies on bacterial plasmids and transposons (OHTSUBO)
Molecular mechanisms of mammalian development and diseases (YAMAMURA)
3.

DEPARTMENT OF ONTOGENETICS
Division of Developmental Genetics

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

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

Studies on DNA replication timing (OKUMURA)
Systematic studies on neuropeptides (MUNEOKA)

12

4.

DEPARTMENT OF POPULATION GENETICS
Division of Population Genetics

Theoretical studies of population genetics (OHTA and INA)
Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (INA)
Statistics for DNA polymorphisms (INA)
Experimental population genetics on DNA polymorphism and evolution in
Drosophila (TAKANO)
Genetic studies on interspecific variation in Drosophila (TAKANO)
Division of Evolutionary Genetics

Studies on codon usage (lKEMURA)
Studies on chromosome band structures at the DNA sequence level (lKEMURA and TENZEN)
Studies on genes in the MHC region (IKEMURA and MATSUMOTO)
Studies on functions of extracellular matrix proteins (MATSUMOTO andIKEMURA)Studies on DNA replication of human genome (TENZEN and IKEMURA)
Molecular evolutionary analysis of nucleotide sequence data (SAITOU)
Studies on the genetic affinity of human populations (SAITOU)
Division of Theoretical Genetics

Population immunogenetics (TAKAHATA)
Molecular anthropology (TAKAHATA)
Theoretical studies of population genetics (TACHIDA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Division of Human Genetics

Genetic and physical mapping of human genome (IMAMURA and IZUHARA)
Molecular genetics of human metabolic disorders (IMAMURA and IZUHARA)PROJECTS OF RESEARCH FOR 1995

13

Molecular biology of oncogenes (FUJIYAMA)
Studies on DNA polymorphisms in human populations (HORAI)
Division of Agricultural Genetics

Evolutionary and ecological genetics in wild and cultivated nce species(MORISHIMA)
Studies on plant gene expression (HIRANO and SANO)
Division of Applied Genetics

Molecular genetics of human genome imprinting mechanisms (SASAKI)
Preservation mechanisms of genetic diversity in plant populations (SHIMAMOTO)
6.

GENETIC STOCK RESEARCH CENTER
Mammalian Genetics Laboratory

Recombinational hotspots in the mouse MHC (SHIROISHI and MORIWAKI)
Genetic Mechanisms for regulating tumor development in the laboratory andwild mice (MIYASHITA and MORIWAKI)
Invertebrate Genetics Laboratory

Molecular genetics of Drosophila melanogaster (HAYASHI and GOTO)
Plant Genetics Laboratory

Molecular genetics of rice (ITO)
Microbial Genetics Laboratory

Timing of cell division in E. coli (NISHIMURA)
Division apparatus of E. coli (NISHIMURA)
Environmental adaptation in E. coli (KANAMARU)

14

Genetic Resources Laboratory

Compilation of genetic resources information (SAITO and YAMAZAKI)
Mammalian Development Laboratory

Developmental mechanisms and manipulation of germ cells in mouse embryos (NAKATSUJI and SHIRAYOSHI)
Molecular analysis of cell differentiation and morphogenesis in postimplantation mouse embryos (SHIRAYOSHI and NAKATSUJI)
Cell differentiation and morphogenesis of the mouse central nervous system(NAKATSUJI and SHIRAYOSHI)
7.

DNA RESEARCH CENTER

DNA-Protein Inateraction Laboratory

Nanobiology of DNA-protein interaction (SHIMAMOTO and NAGAI)
Recombinant DNA Laboratory

Genetics and molecular genetics of development and behavior of Caenorhabditis elegans (KATSURA and ISHIHARA)DNA Synthesis Laboratory

Crystallographic study of supramolecules and transcription factors (SHIRAKIHARA)
Gene Library Laboratory

Molecular genetics of Caenorhabditics elegans development (KOHARA andANDACHI)Genome analysis of Caenorhabditis elegans (KOHARA)PROJECTS OF RESEARCH FOR 1995
8.

15

CENTER FOR INFORMATION BIOLOGYDNA Data Analysis Laboratory

Molecular evolution of pathogenic viruses (GOJOBORI and IKEo)
Molecular evolution of mosaic proteins (lKEO and GOJOBORI)
Human evolution based on polymorphisms in MHC genes (lMANISHI andGOJOBORI)
Genetic information analysis by using DNA databases (GOJOBORI, IKEO andIMANISHI)
Laboratory of Gene Function Research

Molecular phylogenetic analysis of DNA and protein sequence data
(TATENO)
Laboratory for Gene-Product Informatics

Computational analysis and prediction of protein three-dimensional structure(NISHIKAWA)
9. RADIOISOTOPE CENTER
Molecular mechanisms of sporulation in Bacillus subtilis (SADAIE andFUJITA)

10.

EXPERIMENTAL FARM

16

RESEARCH ACTIVITIES IN 1995

A.

DEPARTMENT OF MOLECULAR GENETICS
A-a.

(1)

Division of Molecular Genetics

Functional Map of RNA Polymerase Alpha Subunit from
Escherichia coli: Subunit-Subunit Contact Sites

Makoto KIMURA l and Akira ISHIHAMA CPresent address: Rockefeller
University, New York)The RNA polymerase core enzyme of Escherichia coli with a subunit
composition of a-j3{3' is assembled in the sequence: a~a2~a-j3~a-j3{3'.
Dimerization of the a subunit is the first step in this assembly pathway andthus the a subunit plays a key role in RNA polymerase formation. The a
subunit also plays an important role in molecular interactions with both agroup of positive factors, which we defined as class I factors, and DNA UP(or upstream activation) elements.
Using the in vitro reconstitution system of RNA polymerase from deletionmutant a, we identified that the minimum fragment of a with the enzyme
assembly activity is a(21-235) consisting of a region from residues 21 to 235[wild-type a subunit contains 329 residues], while a carboxy-terminal regionof a up to amino acid 235 is involved in the transcription activation. For finemapping of the subunit-subunit contact sites, we made eleven species of
a-insertion mutants, each containing an extra alanine-serine (AS) dipeptide
at 20 amino acid intervals within this domain. From the results of subunit
assembly in vitro for these a mutants (Kimura and Ishihama, 1995a), we
proposed the functional map of subunit assembly domain on a as follows: aa contact involves multiple sites; a-{3 contact site is located near residue 80;
and (3' assembly involves the site(s) near residues 80 and 180-200.
For detailed mapping, Ala was substituted for 26 highly conserved aminoacids around residues 40, 80 and 170 to 210. The a-point mutants were also
analyzed in vitro for their abilities to form dimers and to assemble {3{3'A.

DEPARTMENT OF MOLECULAR GENETICS

17

subunits (Kimura and Ishihama, 1995b). New types of assembly-defective
mutants were identified: a-R45A dimerized but did not assemble (3 (and (3/)
subunits; and a-L48A showed a decreased level of a2f3 subassembly formation, indicating that this region (residues 45 to 48) is responsible for (3binding. Isolation of two mutants, a-K86A and a-VI73A, both forming a2f3
but not a2f3(3 complex, confirmed our previous conclusion that two separated
regions participate in (3/ -binding.
This year, we started the analysis of the subunit assembly in vivo. At first,
we analyzed eleven species of AS (alanine-serine) dipeptide-insertion andfour species of amino-terminal deletion mutants of a. In order to detect thesubunit assemblies in vivo, we added hexa-histidine (H 6 ) tag to wild-type andmutant a, expressed H 6-tagged a, and isolated subunit complexes by Ni H affinity chromatography. The assembly properties of a mutants agreed wellwith those observed in vitro, except that some mutants which showed decreased assembly in vitro were assembled into core and holoenzymes in vivo,
suggesting that a specific condition(s) or factor(s) supports the efficient assembly in vivo.
These mutant a were also tested for their ability to complement two
temperature-sensitive (ts) alleles, i.e., assembly-defective rpoA 112 and function-defective rpoA 101. The in vivo results generally supported the functional
map of a amino-terminal assembly domain previously established based on
the in vitro studies. Most assembly-competent a mutants complemented twotemperature-sensitive mutant alleles of rpoA, but the mutants carrying mutations at the extreme amino-terminal region and the mutant with a mutation
at residue 60 failed to complement these ts mutants, suggesting that theseassembly-competent but complementation-negative a mutants lack as yet
unidentified function(s). With subunit complexes containing some assemblycompetent/complementation-negative mutants and all the assembly-defective
mutants was associated DnaK (hsp70) protein, suggesting that molecularchaperones are involved in RNA polymerase assembly.

18

(2)

Functional Map of RNA Polymerase Alpha Subunit from
Escherichia coli: Transcription Factor- andDNA UP Element-Contact Sites

Katsuhiko MURAKAMI, Tomofumi NEGISHI, Nobuyuki FUJITA and Akira
ISHIHAMA
Escherichia coli RNA polymerase holoenzyme is composed of four different
subunit (aiJ/3'a). The a subunit, consisting of 329 amino acid residues, plays
a key role in RNA polymerase assembly, of which as many as 94 carboxy(C)-terminal amino acid residues can be removed without preventing theformation of RNA polymerase complexes. On the other hand, the C-terminal
proximal region is needed for transcription regulation by interaction with not
only a group of transcription activators such as cyclic AMP receptor protein(CRP) (Murakami et af., 1996), OxyR (Tao et af., 1995), SoxS (lair et af.,
1995), MarR (lair et af., 1996a), Rob (lair et af., 1996b) and OmpR, butalso transcription repressor GalR (Choy et af., 1995) [transcription factors
which require the C-terminal domain of RNA polymerase a subunit, directly
or indirectly, for action are classified into class-I factors]. The C-terminal
proximal region also plays a role in the recognition of promoter upstream(UP) elements, which consist of (A +T)-rich sequences, with enhancing
activity of transcription. Mutational mapping revealed that the majorcontact site for CRP on a subunit (CRP contact site-I) is located within anarrow region between amino acid residues 258 to 270. This region is also
required for contact with OxyR (Tao et af., 1995).
In order to investigate the role of each amino acid around this region(residues 258 to 275) and the secondary OxyR-contact region (residues 297
to 298), we carried out systematic mutagenesis to make a derivatives, eachcarrying a single amino acid (either Trp or Ala) substitution from 258 to 275,
and from 297 to 298. The mutant a subunit proteins were overexpressed,
purified and reconstituted into mutant RNA polymerases. The abilities of themutant RNA polymerases to respond to CRP and the UP element were
examined in transcription reactions in vitro initiated at the cAMPjCRPdependent facPl and UP element-dependent rrnBPI promoters. The mutations affecting CRP response were located on the surface of contact helix andmoreover most of these mutatios also influenced the response to the rrnB UP
element. Since Arg-265 was found to playa major role in response to bothA. DEPARTMENT OF MOLECULAR GENETICS

19

CRP and UP, we also made another set of mutant RNA polymerases by
replacing Arg-265 with amino acids other than Trp and Ala, and examined
their responses to CRP and UP. From the location of mutations affecting
CRP and UP responses on the three dimensional structure of a subunit
C-terminal domain (Jeon et al., 1995), we propose that the CRP contact
surface is also involved in contact with the DNA UP element, although someamino acid residues within this region play different roles in molecularcommunication with CRP and the UP element.
The contact between CRP and aCTD was competitively inhibited byadding peptides with the sequence present on the CRP contact surface of a
(Negishi et al., in preparation). The competitive inhibition assay withsynthetic peptides would be a useful method for identification of the locationof transcription factor contact sites on RNA polymerase and the role of eachamino acid in the molecular communication. Judged by DNase I footprinting analysis, the a mutants defective in transcription from the CRPdependent lacPl promoter showed decreased activity in the cooperative
binding of CRP. Likewise, the mutants defective in rrnBPI transcription
showed decreased binding to the UP element. The amino acid residues
important for the protein and DNA contact are conserved in the a subunits
of not only bacterial but also chloroplast RNA polymerases.
(3)

Structural Map of RNA Polymerase Alpha Subunit from
Escherichia coli:

Tomofumi NEGISHI, Young Ho JEON I , Masahiro SHIRAKAWA 2 , Toshio
YAMAZAKI I , Katsuhiko MURAKAMI, Nobuyuki FUJITA, Olga OZOLINE 3 ,Yoshimasa KYOGOKU I and Akria ISHIHAMA COaka University, Institute forProtein Research, 2 Nara Institute of Science and Technology, Graduate
School of Bioscience, 3 On leave of absence from Institute of Cell Biophysics,
Pushchino, Russia)
RNA polymerase of E. coli alters its promoter selectivity after interaction
with transcription protein factors. One of the regions responsible fortranscription regulation by transcription factors has been localized to the
C-terminal domain of a subunit. This group of proteins requiring the
C-terminal domain of a is designated as class-I factors. Fine mapping of thecontact sites for CRP, OxyR and other c1ass-1 factors is being carried out by

20

using mutant a with single amino acid substitutions (Murakami et al., 1996;
Tao et al., 1995). Limited proteolysis suggested that both N-terminl andC-terminal regions form independent compact globular domains, each being
connected by a protease-sensitive and presumably fragile linker (Negishi etal., 1995).
The structure of the C-terminal domain of a (aCTD), as determined by
nuclear magnetic resonance (NMR) spectroscopy, is compactly folded andcontains four helices and two long arms enclosing its hydrophobic core (Jeonet al., 1995). Most of the mutations affecting activation by CRP and UP
element are located on the surface of helix 1, indicating that the hydrophilic
surface of helix 1 is involved in direct contact with the protein and DNA
factors. The folding topology is distinct from those of other DNA-binding
proteins. The UP element-biding sites as determined by monitoring theselective signal loose by NMR agreed well with the site of mutations affecting
UP element response. In order to directly observe the molecular interactionsof a with class-I transcription factors and DNA UP elements on this surface,
we succeded to set up a condition to modify a by binding a fluorescent dye
only at Cys269 within helix 1. The spectroscopic studies are being carried out
for detection of protein-protein or protein-DNA contacts using the modified
a subunits.
(4)

Regulation and Specificity of RNA Polymerase Sigma Subunits
from Escherichia coli: Intracellular Levels of
Various Sigma Subunits

Miki JrsHAGE, Shuichi KUSANO, Akira IWATA l , Susumu UEDA! and Akira
ISHIHAMA CNippon Institute of Biological Science, Ohme, Tokyo)
In order to adapt to such various environments as the mammalian gut,
fresh and sea water and soil, bacteria carry sophysticated systems sensing thechanges in the nutrient availability, osmolarity, temperature, and otherexternal factors, and responding by turning on and off the specific sets ofstress response genes. For the global changes in gene expression pattern, theRNA polymerase is considered to playa key role by rapidly modulating its
promoter selectivity. One major mechanism of the promoter selectivity
control of RNA polymerase is the replacement of a subunit [the promoter
recognition factor] on core enzyme, which alone is unable to initiate trans-

A.

DEPARTMENT OF MOLECULAR GENETICS

21

cription from promoter even though it carries the catalytic function of RNA
synthesis. Up to the present time, seven different molecular species of a
subunit have been identified in Escherichia coli. The model of a replacement
relies on the change in the intracellular concentration of individual a subunits, because the level of core enzyme stays almost at a constant level.
However, little is known about the intracellular level of each a subunit undervarious conditions. We then measured the intracellular levels of various a
subunits in E. coli at various growth phases. For this purpose, we preparedmono-specific antibodies against each a subunit purified from over-expressed
E. coli cells and determined the a subunits in two E. coli strains, MC4100 and
W311O, by a quantitative Western blot method.
The intracellular levels of the major a subunit, a 70 (aD, the rpoD geneproduct) in E. coli MC4100 is maintained at 5O-80fmol of per /Lg of total
protein throughout the growth transition from the exponential growth to thestationary phase, while the level of d 8 protein (oS, the rpoS gene product) is
below the detection level at the exponential growth phase but increases to
30% the level of a 70 when the cell growth stops to enter into the stationary
phase. Besides the stationary phase, the increase in a 38 level was observed in
two cases: exposure to heat shock at the exponential phase; and osmotic shockat the stationary phase.
(5)Regulation and Specificity of RNA Polymerase Sigma Subunits
from Escherichia coli: Promoter Selectivity of
Various Sigma Subunits

Shuichi KUSANO, Tapas Kumar KUNDU, Quingquam DING!, Kan TANAKA 2,
Annie KOLB 3, Nobuyuki FUJITA and Akira ISHIHAMA COn leave of absencefrom Wuhan Institute of Virology, Wuhan, China, 2University of Tokyo,
Institute of Molecular and Cellular Biology, 3 Pasteur Institut, Paris)
In E. coli, the total number of RNA polymerase core enzyme is fixed at alevel characteristic of the rate of cell growth, which ranges from 1,000 to
3,000 molecules per genome equivalent of DNA. The levels of individual a
subunits vary depending on the cell growth conditions, but the total numberof a subunits is less than that of core enzyme (Jishage and Ishihama, 1995).
On the other hand, the total number of genes on the E. coli genome isestimated to be about 4,000. Thus, competition must take place between

22

promoters for binding a small number of RNA polymerase. Among about
4,000 genes on the Escherichia coli genome, about 1,000 genes are expressedat various levels in exponentially growing cells under laboratory culture
conditions, i.e., at 37°C and with aeration. The rest of genes is considered to
be expressed under various stress conditions that E. coli meets in nature.
Transcription of at least some of these stationary-specific genes is catalyzed
by RNA polymerase holoenzyme containing 03 8 (05; the rpoS gene product).
Promoters from the stationary-specific genes, however, do not have a single
consensus sequence (Kolb et al., 1995; Tanaka et al., 1995). However, wefound that transcription of the o5-dependent genes by RNA polymerasecontaining 05 requires a specific condition(s) such as high concentrations ofpotassium glutamate for transcription of the osmo-regulated genes. Thisfinding indicates that each stationary-specific promoter carries a specificsequence which is recognized by EcT under a specific reaction condition andthat the promoter sequences recognized by EcT differ between gene groups
sharing the same requirements. Our effort has since been focussed to identify
specific conditions or factors required for transcription of each stationaryspecific gene by EcT holoenzyme and, as an extension along this line, tounderstand the molecular mechanism(s) of a replacement under various
growth conditions.
Along this line, we anlyzed the effect of trehalose on EcT and Ea70
holoenzymes. Trehalose is known to accumulate in stationary-phase cells of
various microoganisms. Results indicated that high concentrations of trehalose specifically stimulate transcription by EcT. Likewise, we analyzed theeffect of DNA superhelicity on the promoter recognition by Ea38 holoenzyme.
DNA superhelicity is known to change depending on the cell growth conditions. For instance, nutrient downshift and stationary growth-phase cause adecrease in the DNA superhelical density while high osmolarity leads to anincrease in the superhelicity. The selectivity for stationary phase-specificpromoters by ~8 increases concomitantly with the decrease in DNA superhelicity, and that the effects of decreased DNA superhelicity and highpotassium glutamate concentrations are additive in enhancing the selectivity
for ~8. The transcription by Ea38 was, however, enhanced with the use of
templates with low superhelical density, in good agreement with the decrease
in DNA superhelicity in the stationary growth phase (Kusano and Ishihama,1996). We thus propose that the selective transcription of stationary-specificgenes by ~8 holoenzyme requires either a specific reaction condition(s) orA. DEPARTMENT OF MOLECULAR GENETICS

23

a specific factor(s). The same line of studies is being carried out for otherholoenzymes containing other a subunits.
(6)

Subunit Composition of RNA Polymerase II from the Fission Yeast
Schizosaccharomyces pombe: Subunit Composition

Hitomi SAKURAI, Takanori MIYAO, Hisako SUZUKI, Masahiro YAMAGISHI 1
and Akira ISHIHAMA
Present address: Aichi Cancer Center ResearchInstitute, Nagoya)eKnowledge of the structure and function of RNA polymerase II is essential
for understanding the molecular mechanism of transcription regulation in
eukaryotes. At present, however, little is known about the moleculararchitecture of RNA polymerase II. Purified RNA polymerase II fromyeasts, animals and plants all contain more than 10 polypeptides, but it has
not been determined yet whether or not all these polypeptides are essential for
RNA polymerase functions. One way to address the question whether allthese subunits are necessary for the RNA polymerase functions is to develop
an in vitro reconstitution system of RNA polymerase from isolated individual
subunits and to analyze the functions of complexes reconstituted fromdifferent combinations of isolated subunits. For this ultimate goal, we started
the cloning and sequencing of the Schizosaccharomyces pombe genes coding
for the proteins associated with purified RNA polymerase II. Judging fromthe co-elution profiles in column chromatographies along the RNA polymerase activity and the two large subunits [subunit 1 (prokaryotic /3'
homologue) and subunit 2 (/3 homologue)], the minimum number of S.
pombe RNA polymerase II-associated components was estimated to be 10,less than the subunit number (12) of Saccharomyces cerevisiae enzyme.
Among these 10 putative subunits, the S. cerevisiae counterparts have been
identified for nine. Up to the present time, we cloned and sequenced thegenes for subunits 1, 2, 3 and 5.

24

(7)

Subunit Function of RNA Polymerase II from the Fission Yeast
Schizosaccharomyces pombe: Subunit 2, 3 and 5

Takanori MIYAO, Kiyoshi YASUI, Makiko KAWAGISHI 1, Yoshinao AzuMA 2,
Masahiro YAMAGISHI 3 and Akira ISHIHAMA CPresent address: National
Institutes of Health, Bethesda, USA, 2 University of California, Irvine, 3 Aichi
Cancer Center Research Institute, Nagoya)The genes coding for the two large subunits of RNA polymerase II havebeen cloned from a number of organisms. Sequence analysis indicated that
subunits 1 and 2 exhibit notable homology with the (3/ and (3 subunits,
respectively, of prokaryotic RNA polymerases. The functional roles of these
two large subunits of eukaryotic RNA polymerases can now be predictedfrom the knowledge of the subunit functions of the well-studied Escherichia
coli RNA polymerase: the catalytic site is located on (3, while (3/ exhibits
non-specific DNA binding activity. Within the subunit 2 sequence, there is aconserved sequence for barnase, an extracelluar ribonuclease from grampositive bacteria. In order to identify possible function(s) associated withthis domain, we expressed a short polypeptide including this barnase domain
and examined activities for degrading RNA. So far, however, we failed todetect activities of RNA degradation, suggesting that this domain does notcarry the RNase activity (Kawagishi-Kobayashi et al., 1995).
On the other hand, the gene for subunit 3 has been cloned from Saccharomyces cerevisiae, Tetrahymena thermophila, and human. We also cloned thesubunit 3 gene from Schizosaccharomyces pombe. Sequence analysis indicatedthat subunit 3 is a eukaryotic homologue of the prokaryotic RNA polymerase
a subunit, of which the amino-terminal proximal domain plays a key role in
RNA polymerase assembly by providing the protein-protein contact surfaces
for a dimerization and for interaction with the two large subunits, (3 and (3/.
On other hand, the carboxy-terminal region of the prokaryotic a subunit
provides the contact surfaces for class-I transcription factors, and DNA UP
elements or prokaryotic enhancers. As judged from the sequence comparison, the subunit 3 of eukaryotic RNA polymerase II appears to lack thistranscription activation domain. For the identification of subunit 3 function(s), we started to isolate temperature-sensitive mutants with mutations in therpb3 gene encoding subunit 3. We analyzed in details thermolabile RNA
polymerase II from temperature-sensitive mutants of S. pombe with muta-

A. DEPARTMENT OF MOLECULAR GENETICS

25

tions in the rpb3 gene. Subunit a of prokaryotic RNA polymerases plays key
roles in protein-protein contacts for both subunit assembly and transcription
activation. To gain an insight into the roles of subunit 3, the eukaryotic
homologue of a, temperature-sensitive mutants of S. pombe have been
isolated after transformation of the mutagenized rpb3 gene (Yasui et al.,1995). A total of 68 ts mutants were classified into two groups: mutants
comprising one group ceased growing immediately after a temperature
up-shift, while mutants comprising the other group exhibited delayed growtharrest at high temperatures. RNA polymerase II partially purified from Ts54,
one of the group 2 mutants, was thermolabile in vitro, as measured by anon-specific transcription assay. This mutant carries double mutations indomain A of subunit 3, and thus can be used as a reference mutant of RNApolymerase II.
(8)

Subunit Function of Influenza Virus RNA Polymerase:
Cross-linking Studies of Nucleotides

Yasuyuki ASAN0 1, Tokumi MARUYAMA 2 and Akira ISHIHAMA COn leaveof absence from Sanwa Kagaku Research Institute, 2 Tokushima Bunri
University, Tokushima)
Influenza virus RNA-dependent RNA polymerase plays an essential role inboth transcription and replication of the viral genome. In transcription, theRNA polymerase cleaves cap-l (m 7GpppNm)-containing RNA of host cells
at 10-13 nucleotides from 5' cap structure, transcribes viral RNA (vRNA)
by elongating the resulting capped fragments until nucleotides 17 to 22 from
5' end of vRNA, and there adds multiple A residues to nascent RNA chains,altogether leading to formation of viral mRNA with both 5'-cap structure/and 3 -poly(A) tail. The RNA polymerase, in conjunction with an yet
unidentified host factor(s), is also involved in two step reactions of RNA
replication, i.e., the synthesis of full-sized complementary RNA (cRNA)
without these modifications and the cRNA-directed synthesis of vRNA.
The influenza virus RNA polymerase consists of three virus-encoded
proteins, PBI, PB2 and PA. Knowledge of the function of each subunit isstill limited. Ultraviolet (UV) light-induced crosslinking studies indicatedthat PB2 is the cap I-recognition protein, while PBI can be UV-crosslinked
to 3' terminal nucleotides of elongating mRNA chains, indicating that PB 1 is

26

the subunit involved in the catalytic activity of nucleotide polymerization.
Both PBI and PB2 can be crosslinked to synthetic RNA with the 3/ terminal
sequence of vRNA. Thus, these two basic subunits may also be involved inrecognition of transcription promoter and/or replication origin on templatevRNA. In contrast, no specific function has been identified for PA protein.
In order to get insight into the function of each subunit of influenza virus
RNA polymerase and to make the functional map of each subunit polypeptide, we are trying to set up an efficient reconstitution system of RNApolymerase from isolated individual P proteins and to analyze functions ofreconstituted mutant RNA polymerases.
In parallel, we carried out photoaffinity labeling of nucleotide-binding sites
on influenza virus RNA polymerase with azido-substituted nucleotides. 8Azidopurines are widely used for monitoring nucleotide-binding sites onproteins at the level of primary sequence. Upon exposure to a low dose of UV
light, the azide moiety of these nucleotide analogues is converted to a highly
reactive nitrene. If the analogue is associated at or near the active site of an
enzyme at the time of UV exposure, the nitrene forms a covalent bond withmost amino acids within the crosslinking distance. Once crosslinked thesereagents can be used for isolation of active site peptides. Thus the site
specificity imparted by the nucleotide, coupled with indiscriminate bondformation of the nitrene, permits tagging of the enzyme active site withregard to which amino acids are involved in forming the nucleotide-binding
domain. This year, we used 8-azido guanosine 5/ -triphosphate (8-N 3 GTP),an azido analogue of GTP, because GTP is the first substrate that is added tocapped RNA primers in transcription initiation by the influenza virus RNA
polymerase. Results demonstrated that: i) 8-N3 GTP serves as a substrate for
RNA synthesis by the influenza virus RNA polymerase; ii) the K m value forthe azido analogue of GTP in primer-dependent RNA synthesis was 94,uM
whereasK m for the natural substrate, GTP, was 6.7 ,uM; iii) 8-azido GTP (8N 3 GTP) can be polymerized into RNA at about one tenth the rate of GTP
incorporation; iv) 8-azido GTP is specifically photo-crosslinked to the PBI
subunit. These biochemical data provide a conclusive evidence supporting
the prediction that the nucleotide binding site is located on the PB I protein.
In the presence of ApG primer, the 8-N) GTP binding was reduced to about
40% level, suggesting that the GTP analogue can bind not only to thesubstrate site (S site) but also to the primer- and product-binding site (Psite).

A. DEPARTMENT OF MOLECULAR GENETICS(9)27

Subunit Function of Influenza Virus RNA Polymerase:
RNA Synthesis by Subunits and Subassemblies

Tetsuya TOYODAl, Makoto KOBAYASHI 2 and Akira ISHIHAMA CPresent
address: Kurume University School of Medicine, Kurume, 2 Jichi Medical
School, Department of Biology, Tochigi)
Influenza virus contains eight single-stranded RNA segments of negative
polarity as the genome and an RNA-dependent RNA polymerase as a virion
component. After virus infection, the RNA polymerase participates in bothtranscription [the synthesis of plus-strand mRNA containing host cell/
derived cap structure at 5'-terminus and poly(A) tail at 3 -terminus) andreplication [the synthesis of full-length plus-strand complementary RNA(cRNA) and the cRNA-dependent synthesis of minus-strand viral RNA(vRNA»). The viral RNA polymerase is composed of one molecule each of
the three viral coded P proteins, PB I, PB2 and PA. Genetic and biochemical
studies suggested that: PBI is involved in polymerization of RNA chains; andPB2 is involved in recognition of host cell capped RNA. The role of PA is
not clear yet, but temperature-sensitive mutations in the PA gene affect only
vRNA synthesis, but not mRNA synthesis. In order to identify the minimaland essential viral component(s) required for transcription and replication,
we analyzed in vitro activities of model template-directed RNA synthesis
associated with nuclear extracts from cells infected with each of the recombinant baculoviruses or various combinations of these viruses (Kobayashi etal., 1995). The nuclear extract of cells expressing all three P proteinscatalyzed model template-directed RNA synthesis in the absence of primers(an indication of RNA replication), supporting the notion that the complete
set of three P proteins is required for RNA replication. All the nuclear
extracts containing the PBI subunit, including the extract containing PBI
alone, were able to catalyze model template-directed dinucleotide-primed
RNA synthesis (an indication of transcription).
In parallel, we also examined the transcription and replication activities ofnuclear extracts of HeLa cells infected with one of the recombinant vaccinia
viruses, each carrying cDNA for one of the three P proteins, or in various
combinations of these viruses (Toyoda et al., 1996). Results demonstratethat the complete replication, i.e., vRNA~cRNA~vRNA, takes place in theextract containing the complete set of RNA polymerase subunits. Again, the28

nuclear extract containing PBl alone was found to catalyze primerdependent RNA synthesis. Taken together, we conclude that PBl is thecatalytic subunit of RNA polymerase and propose that under certain conditions, PB 1 alone is able to express the catalytic activity in vitro.
(10)

Subunit Function of Influenza Virus RNA Polymerase:
Mapping of Subunit-subunit Contact Sites

Djanybek AOYSHEv!, Tetsuya TOYOOA 2 and Akira ISHIHAMA COn leave ofabsence from Institute of Cell Biology, Kyrgyz Academy of Science, 2 Present
address: Kurume University School of Medicine, Kurume)
Influenza virus RNA polyfmerase is composed of three subunits, PBl, PB2
and PA, all encoded by the viral genome. The P protein complex is able tocatalyze transcription, as defined by the requirement of primers and vRNA
templates (but not cRNA templates). For replication, an additional
factor(s) present in infected cells is needed. Transfection experiments byrecombinant viruses carrying one of the three P subunits indicated that thenuclear extracts containing PB 1 alone is able to catalyze transcription(Kobayashi et al., 1995; Toyoda et al., 1996). However, reconstitution
experiments indicate that none of the P proteins alone is able to catalyzetranscription in vitro but that active enzymes are formed only by mixing all
three P proteins. Moreover, the efficiency of reconstitution from isolated
individual subunits is still at low level. In order to clarify the subunit
assembly mechanism of influenza virus RNA polymerase, we made a series ofdeletion mutants for each P protein. Three combinations of P proteins, i.e.,
PBl-PB2, PBl-PA and PB2-PA, were expressed simultaneouly in the same
cells. Binary P protein complexes were immunoprecipitated with use ofmono-specific antibodies again each P protein. Results indicated that thebinary complexes are formed between PBI-PB2 and PBl-PA but not PB2PA. Thus, PBl is the core subunit for RNA polymerase assembly.
Using N-terminal and C-terminal deletion mutants of each P protein, we
further identified the subunit-subunit contact sites on each P protein. Results
indicate that the C-terminal 158 amino acids of PBI associates with the
N-terminal249 stretch of PB2, while the N-terminal 140 amino acids of PBI
binds to the C-terminal region of PA.

A.

DEPARTMENT OF MOLECULAR GENETICS

29

Publications
1.

2.

3.

4.

5.

6.
7.

8.

9.

10.

II.
12.
13.

ASANO, Y., MIZUMOTO, K., MARUYAMA, T. and ISHIHAMA, A.: Molecular anatomy ofinfluenza virus RNA polymerase: Photoaffinity labeling of PBI subunit with 8-azido GTP.
J. Biochem., 117(3), 677-682, 1995.
AZUMA, Y., YASUI, K., YAMAGISHI, M. and ISHIHAMA, A.: Isolation of thermolabile
mutant RNA polymerase II from fission yeast Schizosaccharomyces pombe with mutalions
in the subunit 3 gene. J. Biochem., 118(7), 216-220, 1995.
CHOY, H., PARK, S. W., AKI, T., PARRACK, P., FUJITA, N., ISHIHAMA, A. and ADHYA,
S.: Repression and activation of transcription by Gal and Lac repressors: Involvement ofalpha-subunit of RNA polymerase. EMBO J., 14(18),4523-4529, 1995.
DING, Q., KUSANO, S. VILLAREJO, M. and ISHIHAMA, A.: Promoter selectivity ofosmoregulated promoters between ED and E S holoenzymes. Mol. Microbiol., 16, 649-656,
1995.
FUKUCHI, J., KASHIWAGI, K., YAMAGISHI, M., ISHIHAMA, A. and IGARASHI, K.:
Decrease in cell viability due to the accumulation of spermidine in spermidine in spermidine
acetyltransferease-deficient mutant of Escherichia coli. J. BioI. Chern., 270(32), 1883118835, 1995.
ISHIHAMA, A.: Genetic strategies of bacteria for stationary-phase survival. Actinomycetologia, 9, 236-243, 1995.
JAIR, K.-W., MARTIN, R. G., ROSNER, J. L., FUJITA, N., ISHIHAMA, A. and WOLF, Jr.,R. E.: Purification and regulatory properties of MarA protein: a tarnscriptional activatorof Escherichia coli multiple antibiotic and superoxide resistance promoters. J. Bacteriol.,
177,7100-7104, 1995.
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(2), 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(9), 2507-2513, 1996.
JEON, Y. H., NEGISHI, T., SHIRAKAWA, M., YAMAZAKI, M., FUJITA, N., ISHIHAMA, A.
and KYOGOKU, Y.: Solution structure of the C-terminal domain of RNA polymerase a
subunit responsible for the contact with trans-acting transcription factors and cis-acting UP
element of promoter. Science, 270, 1495-1997, 1995.
JISHAGE, M. and ISHIHAMA, A.: Regulation of RNA polymerase sigma subunit synthesis
in Escherichia coli: Intracellular levels of 0 70 and 0'8. J. Bacteriol., 177, 6832-6835, 1995.
KATO, A. FUJINO, M., NAKAMURA, T., ISHIHAMA, A. and OTAKI, Y.: Gene organizationof chicken anemia virus. Virology, 209( I), 480-488, 1995.
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.,

30
250(1 ), 1-6, 1996.
KIMURA, M. and ISHIHAMA, A.: Functional map of the alpha subunit of Escherichia coli
RNA polymerase: Insertion analysis of the amino-terminal assembly domain. J. Mol. BioI.,
248(2), 756-767, 1995a.
15. KIMURA, M. and ISHIHAMA, A.: Functional map of the alpha subunit of Escherichia coli
RNA polymerase: Amino acid substitutution within the amino-terminal assembly domain.
J. Mol. BioI., 254(3), 342-349, 1995b.
16. KOBAYASHI, M., TOYODA, T. and ISHIHAMA, A.: Influenza virus PBI protein is theminimal and essential subunit of RNA polymerase. Arch. Virol., 141, 525-539, 1996.
17. KOLB, A., KOTLARZ, D., KUSANO, S. and ISHIHAMA, A.: Selectivity of the Escherichia coli
RNA polymerase Ea" for overlapping promoters and ability to support CRP activation.
Nucleic Acids Res., 23(5), 819-826, 1995.
18. KUMAR, A., WILLIAMSON, H. E., FUJITA, N., ISHIHAMA, A. and HAYWARD, R. S.: A
partially functional 245 amino acid internal deletion derivative of Escherichia coli sigma-70.
J. Bacteriol., 177(17), 5193-5196,1995.
19. KusANo, S., DING, Q., FUJITA, N. and ISHIHAMA, A.: Promoter selectivity of Escherichia
coli RNA Polymerase Ea70 and Ed' holoenzymes: Effect of DNA supercoiling. J. BioI.
Chern. 271(4), 1998-2004, 1996.
20. LIU, X., FUJITA, N., ISHIHAMA, A. and MATSUMURA, P.: The C-terminal region of thea subunit of Escherichia coli RNA polymerase is required for transcription activation of theflagellar level II operons by the FlhD/FlhC complex. J. Bacteriol., 177(17), 5186-5188,
1995.
21. NAKAGAWA, Y., KIMURA, N., TOYODA, T., MIZUMOTO, K., ISHIHAMA, A., ODA, K. and
NAKADA, S.: RNA polymerase PB2 subunit is not required for replication of the influenza
virus genome, but is involved in capped RNA synthesis. J. Virol., 69, 728-733, 1995.
22. NEGISHI, T., FUJITA, N. and ISHIHAMA, A.: Structural map of the alpha subunit ofEscherichia coli RNA polymerase: Structural domains identified by proteolytic cleavage. J.
Mol. BioI., 248(2),723-728, 1995.
23. SHINKAWA, H., FUJITA, N., SHIINA, T., TANAKA, K., TAKAHASHI, H., ISHIHAMA, A. and
NIIMI, 0.: Purification and characterization of RNA polymerase holoenzyme (Ea") fromvegetative-phase mycelia of Streptomyce griseus. J. Biochem., 118(3),488-493, 1995.
24. TANAKA, K., KUSANO, S., FUJITA, N., ISHIHAMA, A. and TAKAHASHI, H.: Promoter
determinants for Escherichia coli RNA polymerase holoenzyme containing a" (the rpoS
gene product). Nucleic Acids Res., 23, 827-834, 1995.
25. TAO, K. ZOU, C. FUJITA, N. and ISHIHAMA, A.: Mapping of the OxyR protein contact site
in the C-terminal region of RNA polymerase a subunit. J. Bacteriol., 177, 6740-6744,
1995.
26. TOYODA, T., ASANo, Y. and ISHIHAMA, A.: Role of GTPase activity of Mxl protein in
nuclear localization and antiviral activity. J. Gen. Virol., 76, 1867-1869, 1995.
27. 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, in press.
28. WADA, A., IGARASHI, K., YOSHIMURA, S., AIMOTO, S. and ISHIHAMA, A.: Ribosome
modulation factor: Stationary growth phase-specific inhibitor of ribosome functions from14.

A. DEPARTMENT OF MOLECULAR GENETICS

31

Escherichia coli. Biochem. Biophys. Res. Commun., 214(2),410-417, 1995.
29. XUAN, X., NAKAMURA, T., IHARA, T., SATO, 1., TUCHIYA, K., NOSETTO, E., ISHIHAMA,
A. and UEDA, S.: Characterization of pseudorabies virus glycroprotein gIl expressed byrecombinant bacu1ovirus. Virus Res., 36, 151-161, 1995.
30. YAMAGISHI, M., MIZUMOTO, K. and ISHIHAMA, A.: Isolation of temperature-sensitive
mutants of mRNA capping enzyme in Saccharomyces cerevisiae. Mol. Gen. Genet., 249,
147-154, 1995.

A-b.
(1)

Division of Mutagenesis

Abnormal Integrity of Nucleolus Associated with Cell
Cycle Arrest Owing to the Temperature-Sensitive
Ubiquitin-Activating Enzyme El

Thangirala SUDHA, Fumiko AOKI, Masazumi SAMESHIMAl, Sumiko
KANEDA, Fumiaki Y AMAO and Takeshi SENO (' Metropol. Inst. Med. Sci.,
Tokyo)A mouse cell mutant, ts85 with the temperature-sensitive ubiquitinactivating enzyme El, was arrested mainly at the G2 phase at a nonpermissive temperature. In the arrested cells, Azure C, a nucleolar specificstain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes
with an unstained region in the center. Silver staining of the nucleolarorganizer region (NOR) and fluorescence in situ hybridization (FISH) with
rDNA, both gave signals in azure C-positive regions. The extent of nucleolar
abnormality was more severe when the mutant cells were synchronized in theG2 phase before the temperature shift-up. The Azure C-negative region ofabnormal nucleoli coincided with a cloud of unidentified electron-dense
particles (diameter, 70nm) which grew in number following the temperature
shift-up. The region also coincided with strong fluorescence signals detectedby immuno-histochemical staining for polyubiquitin, proteasomes and inducible HSP70, but not HSP90, DNA topoisomerase I and so on. Using
immuno-electron microscopy with anti-polyubiquitin, the gold particles werecoincided with the 70 nm particles. However, it was not clear whether thesignals represented a free form of polyubiquitin or ubiquitinated protein.
Under the canonical heat-shock conditions, such a coincidence of signals forproteasomes and HSP70 in nucleoli was also detected for wild-type cells.
When the arrested ts85 cells were released into the M phase, we observed a32

frequent occurrence of persistent nucleolar materials in prophase and prometaphase chromosomes that resulted in a peculiar bouquet-like figure of
metaphase-chromosomes in which regions of the nucleolar organizer seemed
to have failed in condensation and separation as supported by FISH with
rDNA and NOR-silver staining. Stable transformation of ts85 by a cloned
mouse El cDNA recovered the mutant phenotypes. It appears the ubiquitin
system plays a role in the dissolution processes of the nucleolus during G2M transition, possibly involving a specific protein(s) that is degraded. For
details, see ref. 1.
(2)

Decrease in Intracellular Deoxyribonucleoside Triphosphate Pools
Associated with Arrest in DNA Replication and Defective DNA
Repair in a Mouse Cell Mutant tsFS20 with Thermolabile
Ubiquitin-Activating Enzyme El

Fumiko AOKI, Yusuke WATAYA 1, Shiro KANKAWA', Dai AYUSAWA 2,
Sumiko KANEDA, Fumiaki YAMAO and Takeshi SENO Okayama Univ.,
Okayama, 2 Kihara Inst. BioI. Res., Yokohama City Univ., Yokohama)eA mouse cell mutant, tsFS20, with the thermolabile ubiquitin-activating
enzyme El is arrested in the S-phase of the cell-cycle with concomitant lossof survival when cultured at non-permissive temperature. The arrest was
accompanied by inhibition of DNA replication, but not in total RNA andprotein synthesis. When the arrested cells were permeabilized and incubatedwith an extracellular supply of dNTPs, DNA synthesis occurred normally.
We found that the intracellular dTTP pool was specifically decreased in thearrested mutant cells. They showed increased sensitivity to hydroxyurea, aspecific inhibitor of ribonucleotide reductase. tsFS20 cells also showed anincreased sensitivity to cell kill by N-methyl-N'-nitro-N-nitrosoguanidine.
However, this defect in DNA repair was not accompanied by increase in an
induced mutation, the phenotype being reminiscent of Saccharomyces cerevisiae rad6, a mutant of the ubiquitin-conjugating enzyme UBC2. Hence, in
addition to the regulation of DNA replication at the level of ribonucleotide
reduction, DNA repair and induced mutation fixation may also be controlled
by the ubiquitin system in this mammalian cell mutant. All of the phenotypes
were corrected concomitantly by stable introduction of mouse El cDNA to
the mutant. (Paper in preparation).

A. DEPARTMENT OF MOLECULAR GENETICS

(3)

33

Cell Cycle-Dependent Phosphorylation and Nuclear Targeting of
Ubiquitin-Activating Enzyme, El, in Mammalian Cells

Hiroaki SEINO, Yukio NAGAI, Takeshi SENO and Fumiaki YAMAO
The ubiquitin-activating enzyme El was found to be phosphorylated incells of a mouse mammary carcinoma cell line, FM3A(2). Peptide mappingof trypsin digests of labeled El indicated that two oligopeptides were mainly
phosphorylated in vivo. The same oligopeptides were also labeled in vitro on
Cdc2 kinase-mediated phosphorylation of El which was affinity-purified fromthe same cell line. The phosphorylation of one of the two oligopeptides wasprominent at the G2/M phase of the cell cycle, and dependent upon Cdc2
kinase activity in vivo since it was significantly reduced in tsFT21O, a mutant
cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine
residue at the fourth position of the El enzyme was a phosphorylation site of
Cdc2 kinase. A tentative nuclear targeting signal, KKRR, was found to linkwith the Cdc2 kinase target site, which implied the coupled function of Cdc2mediated phosphorylation and the KKRR signal to localize El in the nucleus
in a cell cycle-dependent fashion. In fact, the truncated El protein which wastagged with a FLAG sequence and transiently expressed in Cos7 cells, wasfound, by indirect immunofluorescent method with anti-FLAG, to be localized in nucleus, while the Ala4 mutant derivative, an unphosphorylatable
form, was not. These are now being confirmed using synchronized cells thatwas stably transformed with El derivatives.

(4)

Screening for cDNAs Encoding the Ubiquitin-Conjugating Enzyme
in Fission Yeast, Schizosaccharomyces pombe

Fumio OSAKA, Hiroaki SEINO, Takeshi SENO and Fumiaki YAMAO
We isolated cDNAs for ubiquitin-conjugating enzymes (Ubc) of S. pombe.
Among more than 20000 clones, twenty were found to encode proteins with
Ubc-like activity, and subsequent sequence analysis identified four types ofgenes. The bacterially expressed proteins formed thiolester bonds withactivated ubiquitin in vitro in the presence of EI enzyme. An Ubc domain
structure was found in their sequences, including a cysteine residue forminga thiolester bond with ubiquitin. Thus, we concluded that these four were
ubc genes and designated them, ubcP1, ubcP2, ubcP3 and ubcP4. ubcP1 and34

ubcP2, predominantly isolated by the screening, were found to encode thehomologue of Ubc4 and Ubc2/Rad6, respectively, in budding yeast, Saccharomyces cerevisiae. The other two, ubcP3 and ubcP4, were novel ubc genes.
ubcP4 was found to be essential for growth, and arrested at two points, G2
and metaphase, of the cell cycle when its expression was abolished. The
metaphase arrest resembled to that by cut9 mutant cells. cut9 has been shownto encode a subunit of ubiquitin-ligase complex (designated as the anaphasepromoting complex, APC, or cyclosome) which is required for cyclin B
degradation during the exit from mitosis. This strongly suggested that the
VbcP4 mediated ubiquitin pathway specifically required at the onset ofanaphase for ubiquitin-mediated degradation of both cyclin B and an as yet
unknown protein necessary for sister chromatid pairing during metaphase.

(5)Regulation of Gl/S transition by the Ubiquitin System in Yeast
Saccharamyces cerevisiae

Tsutomu KISHI, Motoyasu VI and Takeshi SENO
The phenotypes of various cell division cycle mutants of Saccharomyces
cerevisiae suggest a complex array of interactions among the corresponding
gene products leading to DNA replication and other early functions of thecell cycle. Cells bearing cdc4, cdc34 and cdc53 mutations execute the Startfunction, which initiates the processes that lead ultimately to cell division, but
fail to enter the S phase. Various specific functions including bud emergenceand spindle pole body duplication take place in these mutants, but fail toproceed to bud maturation, formation of bipolar spindle or DNA replication.
Molecular characterization of CDC34 has revealed that it codes for aubiquitin conjugating enzyme, which catalyzes the addition of ubiquitin to
the substrates and promotes degradation via a ubiquitin dependent proteolysis pathway. One of the substrates was suggested as the B-type cyclin
dependent kinase inhibitor Sicl, since deletion of SICI allowed cdc34 cells toperform DNA replication at restriction temperatures. However, it did not
suppress the lethality of the cdc34 mutation, suggesting that Cdc34 may havean other essential function in addition to promoting Sic! degradation. We
wished to identify mutations that rescue the temperature sensitivity of the cdc
34L1sicl strain, in the hope of identifying genes acting downstream of the
CDC34 (containing genes of proteins to be ubiquitinated by Cdc34). Twen-A. DEPARTMENT OF MOLECULAR GENETICS

35

ty-one mutants that were viable at restriction temperatures in the cdc34L1 sic 1
background were isolated. They were composed of three complementationgroups. One group showed cold-sensitivity and the suppressor gene wascloned by complementation. DNA sequence analysis revealed that the
suppressor gene was identical to GRRI. GRRI is implicated in the regulation of glucose uptake, glucose repression, divalent cation transport, and theturn-over of G I cyclins. We are now comparing the stability of G 1 cyclins
between the cdc34L1sicl strain and its suppressor strain.

(6)

Human Ubiquitin Pathway Homologous to that Mediated by
Ubc P4 in Fission Yeast

Hiroaki SEINO, Fumio OHSAKA, Takesi SENO, Fumiaki Y AMAO
Ubc P4, an £2 family gene isolated from fission yeast is involved in theregulation of G2jM and metaphase anaphase transition in the cell cycle(Ohsaka et ai., in preparation). We isolated the eDNA homologous to thisgene in human and designated it hubcP4. The predicted amino acid sequenceof hubcP4 was highly homologous to fission yeast UbcP4 (identity 50%,
similarity 80%). This suggested that the biological function of the ubiquitin
pathway mediated by UbcP4 is conserved between yeast and human. Now,
we are examining whether hUbcP4 eDNA is functional in fission yeast cells.
Furthermore, we will investigate the biochemical function of the hUbcP4 ina mammalian culture cell line or Xenopus egg extract which are more suitablefor biochemical experiments.
Publications

I. SUDHA, T., TSUJI, H., SAMESHIMA, M., MATSUDA, Y, KANEDA, S., NAGAI, Y, YAMAO,
F. and SENO, T.: Abnormal integrity of nucleolus associated with cell cycle arrest owing
to the temperature-sensitive ubiquitin-activating enzyme EI. Chromosome Res., 3, 115123, 1995.
2. NAGAI, Y., KANEDA, S., NOMURA, K., YASUDA, H., SENO, T. and YAMAO, F.: Ubiquitin-activating enzyme, EI, is phosphorylated in mammalian cells by the protein kinase
CDC2. J. Cell ScL, 108(6), 2145-2152, 1995.
3. INOKUCHI, H. and YAMAO, F.: Structure and expression of prokaryotic tRNA genes. In
tRNA: Structure, Biosynthesis and Function, D. Soli and U. L. RajBhandary, eds.
(Washington D.C.: American Society for Microbiology (ASM», pp. 17-30, 1995.

36

A-c.
(1)

Division of Nucleic Acid Chemistry

Crystal Structure Determination of the Escherichia coli Replication
Terminator Protein (Tus) Complexed with Ter DNA

Kosuke MORIKAWA l CBiomolecular Engineering Research Institute)
Replication is terminated at defined sequences in the DNA of prokaryotic
chromosomes. In Escherichia coli, this termination is mediated by a sitespecific DNA-binding protein designated as Tus. The Tus protein (row: 36kDa) is a monomeric molecule that binds six specific sequences (Ter) within
the replication terminus region, and it prevents the passage of DnaB helicase,which is a constituent of replication machinery. Knowledge of the threedimensional (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 crystal structure has been determined at 2.7 A resolution by themultiple isomorphous replacement method and anomalous scattering(MIRAS), and has been refined to provide R-factor= 17.0%.
The Tus protein has a very unique structure as compared with otherproteins. The protein embraces the Ter element from both sides of the DNA
with two protruded a-helical domains. The most remarkable feature of thecomplex is an extensive protein-DNA interface which involves strikingly
many direct and indirect polar interactions. Two )3-sheets are responsible forthe recognition of the DNA in the major groove. This structure motif issomewhat similar to the complexes of MetJ and Arc, although the )3-sheets
lies flat against the groove. Except for a slight bend and unwinding of the
DNA in the vicinity of this)3 sheets region, the Ter site DNA in the complex
deviates to a small but significant extent from the canonical B-form DNA.
A genetic approach allowed the isolation of the mutant Tus proteins. Mostof these single mutants are mapped on the )3-sheet region in the 3D-structure.
These mutants that affect the efficiency of replication arrest probably impair
the binding of the Tus protein with DNA. This suggests that replicationarrest and Tus-DNA binding might be inseparable. This interpretation
appears to be consistent with the collision action of Tus against DNA-

A. DEPARTMENT OF MOLECULAR GENETICS

37

translocating and unwinding proteins.
(2) Yeast mRNA Capping Enzyme Mutants: Isolation of
Temperature-Sensitive Mutations and Characterizationof the Altered Enzymes
Masahiro YAMAGISHI, Kiyohisa MIZUMOT0 1 and Akira ISHIHAMA CKitasato University)
Eukaryotic mRNA is capped at the 5' end by the addition of a guanylyl
residue via a 5'-5' triphosphate bridge followed by methylation to form an
m 7G(5')ppp(5')N structure. The cap structure is required for several
important steps of gene expression; i.e., mRNA stabilization, splicing,mRNA export from the nucleus, and initiation of translation. The mechanisms by which the cap structure functions in these steps have not yet been
well characterized. One of the important features of capping is that them 7G(5')ppp(5')N end formation in the nucleus is specific to RNA transcribed by RNA polymerase II, although isolated capping enzyme is capableof adding caps to other RNA species in vitro. The mechanism that underlies
the highly specific and efficient capping of polymerase II-transcribed RNA isalso unknown.
Genetic and biochemical analyses of yeast capping enzyme will provide
insights not only into the catalytic and structural properties of the enzyme
but also into the regulation of mRNA capping and molecular mechanisms
involved in the functions of the cap structure. The yeast capping enzyme iscomposed of a and (3 subunits possessing guanylyltransferase and triphoshatase activities, respectively. In this study, we isolated 10 recessive
temperature-sensitive mutations of the CEGI gene encoding the a subunit
and determined the mutation sites. Nine of the mutations (cegl-l to cegl9) were isolated on a single-copy plasmid and the remaining one (cegl-lO) ona multi-copy plasmid. The presence of cegl-lO in multiple copies is essential
for the viability of cells carrying the mutation, and a shift to a restrictive
temperature resulted in quick growth arrest of cegl-lO cells while othermutants decreased growth rates gradually upon the temperature up-shift.
Intragenic complementation was not observed for pairwise combinations of
the mutations. Guanylyltransferase activity was examined for each mutant
protein by assaying a covalent Ceglp-GMP complex formation. While most38of the mutant proteins showed clear heat-lability, Cegl-8p maintained substantial activity during incubation at a high temperature. Detailed characterization of the cegl mutant cells, including investigation of synthesis, accumulation and subcellular localization of mRNA, remains to be done. In
addition, isolation and analysis of extragenic suppressors will help us understand the regulation of mRNA capping and the functions of the cap structure
in gene expression pathway, from transcription in the nucleus to translationin the cytoplasm.
Publication
1.

2.

KAMADA, K., OHSUMI, K., HORIUCHI, T., SHIMAMOTO, N., and MORIKAWA, K.:
Crystallization and Preliminary X-ray Analysis of the Eschericha coli Replication Terminator Protein Complexed with DNA. PROTEINS: Struc. Func. and Gen., in press.
YAMAGISHI, M., MIZUMOTO, K. and ISHIHAMA, A.: Isolation of temperature-sensitive
mutants for mRNA capping enzyme in Saccharomyces cerevisiae. Mol. Gen. Genet., 249,
147-154, 1995.

39

B.

DEPARTMENT OF CELL GENETICS
B-a.

(1)

Division of Cytogenetics

A Species-specific Interaction of Rad51 and Rad52 proteinsin Eukaryotes

Tomoko OGAWA, Akira SHINOHARA and Tomoatsu IKEYAThe properties of the rad51 and rad52 mutants in Saccharomyces cerevisiae
are very similar to the recA mutant in Escherichia coli. The wide distributionof the recA-like RAD51 and RAD52 homologs in a variety of organisms
suggests the presence of similar fundamental mechanisms of recombination in
all living cells. Although the protein structure of Rad51 homologs are highly
conserved (about 80%), the human RAD51 gene did not complement therad51 deletion mutant of S. cerevisiae. In the case of Rad52 homologs, only
one third of the N-terminal region was conserved. We found that the
N-terminal region of the Rad51 protein interacts with the C-terminal regionof the Rad52 protein and the interaction is species-specific. This speciesspecificity probably plays an important role in the functioning of these
proteins in meiotic recombination and repair of damaged DNA. Ref. 1.
(2)

Homologous Recombination and The Roles of Double-strand Breaks

Akira SHINOHARA and Tomoko OGAWA
Double-strand breaks (DSBs) and single-strand gaps in damaged DNA are
efficiently repaired by mechanisms associated with recombination. Recombination is a series of complex biological reactions, requiring at least 20 geneproducts, even in Escherichia coli. Genes homologous to bacterial and yeast
recombination genes have been cloned in higher eukaryotes, suggesting there
might be a common fundamental mechanism of recombination among a wide
variety of species. In eukaryotes, protein-protein interactions play importantroles in recombination: by interacting with a specific protein(s), the complex
involved in repair of DSBs is modified to carry out specialized cellular40functions, such as meiotic recombination and switching of mating types inyeast. Ref. 2.
(3)

Localization of RecA-like Recombination Protein on Chromosomesof The Lily at Various Meiotic Stages

Masahiro TERAsAwA, Akira SHINOHARA, Tomoko OGAWA
The Rad51 and Lim IS proteins of lily, which are homologs of the bacterial
RecA protein, were found on chromosomes in various stages of meiotic
prophase I. The presence of both Rad51 and Lim IS proteins as discrete focion leptotene and zygotene chromosomes and their colocalization suggest thatmeiotic recombination begins at the leptotene stage with the cooperation ofthese proteins and continues in the zygotene stage. Localization of the focion or adjacent to the chromosomes suggests that these proteins bind to thechromatin loops that extend from the axial cores. The protein in these focimay participate in the searching and pairing of homologous DNA sequences,
as the RecA protein does. The different pattern of localization of the Rad51
protein between the leptotene and pachytene stages and the absence of the
Lim IS protein in the pachytene stage suggest that the Rad51 protein plays
different roles in these stages. Ref. 3.
(4)

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

Tomoatsu IKEYA, Akira SHINOHARA, and Tomoko OGAWA
Studies on the localization of Rad51 and Lim IS proteins on meIOtic
chromosomes of lily showed that these proteins participate in searching forhomologous chromosomes, which is initiated in the leptotene stage and leadto 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 length of mouse chromosomes which facilitates the examination of an entire chromosome with a chiasma region
through optical methods, we studied the localization of Rad51 and Lim IS
homologues on mouse chromosomes.
In the chromosomes of mouse testis, during the late stages of meiotic

B.

DEPARTMENT OF CELL GENETICS

41

prophase 1, the Rad51 and Lim 15 proteins were present in the core regions of
the chromosomes which remained after removal of the chromatin by DNase
II. Rad51 located along the core of the synaptonemal complexes (SC) in thepachytene 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 Lim15 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. 4.

(5)Roles of the Rad52 Protein of Saccharomyces cerevisiae in
Homologous Recombination

Akira SHINOHARA, Miki SHINOHARA and Tomoko OGAWA

It has been reported that the RAD52 gene of Saccharomyces cerevisiae plays
crucial roles in several pathways of homologous recombination. The Rad52
protein is a single-stranded (ss) DNA binding protein which catalyzes
annealing of complementary ssDNAs. The protein interacts with a yeast
ssDNA binding protein, RPA. The RPA protein stimulates Rad52 activities
by recruiting Rad52 and forming a Rad52-RPA complex on ssDNA. The
Rad52 protein interacts directly with the Rad51 protein and the incorporation of the Rad52 protein stabilizes the complex formed by Rad51 on ssDNA.
The Rad52 protein appears to act by promoting the assembly of proteins
involved in recombination machinery for pairing of homologous sequences in
Rad51-dependent pathways, and also by directly promoting homology searches and homologous pairing in Rad51-independent recombination by virtue
of its annealing activity.
Publications
OGAWA, T., SHINOHARA, A. and IKEYA, T.: A species-specific interaction of Rad51 andRad52 proteins. Adv. Biophysics, 31, 93-100, 1995.
2. TERASAWA, M., SHINOHARA, A., HOTTA, Y., OGAWA, H. and OGAWA, T.: Localizationof RecA-like recombination proteins on chromosomes of the lily at various meiotic stages.
Genes & Dev., 9, 925-934, 1995.
3. SHiNOHARA, A. and OGAWA, T.: Homologous recombination: Role of double-strand
breaks, TiBS, 20, "DNA Repair" A special issue, 387-391, 1995.
1.

42
4.

IKEYA, T., SHINOHARA, A., SATO, S., TABATA, S. and OGAWA, T.: Localization of Mouse
Rad51 and Lim IS Proteins on Meiotic Chromosomes at Late Stages of Prophase 1. Genes
to Cells, I, 379-389, 1996.
B-b.
(1)

Division of Microbial Genetics

The Initiator Protein of Filamentous Phage Forms a Covalent
Complex with the Origin DNA

Satoshi ASANO, Atsushi HIGASHITANI and Kensuke HORIUCHI
The plus-strand replication of filamentous coliphage is initiated by gpII,
which is a phage-encoded initiator protein. gpII binds to the plus-strand
origin and introduces a specific nick in the plus-strand. The 3'-hydroxyl endof the nick serves as the primer for plus-strand rolling-circle-type replication.
We showed that DNA bending and duplex melting around the nicking site
are induced by gpII binding. The melting required negative superhelicity ofDNA, and the nicking reaction required the Mg+ + ion. gpII showedsingle-stranded DNA-specific and sequence-specific endnuclease activity. A
nicking substrate was prepared through hybridization of 39mer and 25mer
deoxyoligonucleotides. It contained a 25-nucleotide dsDNA domain for gpII
binding and a 9-nucleotide ssDNA domain around the nicking site, and wascompletely cleaved by gpII in less than 5 min. When this substrate was used
for nicking reactions, a small fraction of the nicked DNA product was foundcovalently attached at the 5' end to gpII. This covalent bond was extremely
unstable in the presence of active gpII.
G73A, a mutant gpII, formed the covalent complex more efficiently thanwild type gpII. To identify the amino acid residue that forms the covalentbond, we used this mutant gpII. A substrate DNA carrying 32p internally at
the nicking site was nicked with G73A gpII to yield a 32P-Iabelled gpII-DNA
complex. The result of hydrolysis of the complex in 6N HCI followed by highvoltage paper electrophoresis indicated that it is a phospho-tyrosine thatbridges the 5' end of the DNA and gpII.

B.

(2)

DEPARTMENT OF CELL GENETICS

43

Mutational Analysis of the Minus Strand Replication Origin ofFilamentous Bacteriophage

Nahoko HIGASHITANI, Atsushi HIGASHITANI and Kensuke HORIUCHI
The single-stranded viral DNA of F-specific filamentous bactriophages (fl,M13, and fd) is converted upon infection to the double-stranded replicative
form through the synthesis of the minus strand, which is initiated by a RNA
primer. The RNA primer is synthesized by the host RNA polymerase on theviral DNA template in the presence of the E. coli SSB protein, and isextended by the DNA polymerase III holoenzyme (Geider, K. et al. (1974)
J. BioI. Chern. 249, 3999-4005). The minus-strand origin contains two
inverted repeats that can form a structure containing two hairpins named [B]
and [C] (Gray, C. P. et al. (1978) Proc. Natl. Acad. Sci. USA. 75, 50-53).
The mechanism of recognition of the minus-strand origin by RNA polymerase is not well understood. The specificity of the priming reaction
depends on the 0 70 subunit of RNA polymerase (Kaguni, J. M. et al. (1982)
J. BioI. Chern. 257, 5437-5443). We previously determined the nucleotide
sequence of the primer RNA, and this prompted us to search for structuresthat are recognized by RNA polymerase in the priming reaction by taking
into account the analogy to the promoter recognition mechanism. By
depicting the two hairpins [B] and [C] horizontally, and by assuming thatthey correspond to a stretch of a double-stranded molecule, it was possible todraw the origin schematically as if it was comparable to a transcriptional
promoter (Higashitani, N. et al. (1993) J. Virol. 67, 2175-2181).
We carried out footprinting experiments with E. coli RNA polymerase on
the minus strand origin. The results indicated that the bottom half of thestem in hairpins [B] and [C] was protected by RNA polymerase and that thefootprint was dependent on the presence of the 0 70 factor. The protected
portions of DNA corresponded to the - 35 region and a region ranging from-10 to + 5, respectively, in the schematic presentation of the origin. We
constructed and analyzed a number of base substitution mutants on thehairpin [B]. The results showed that a specific sequence which is required forthe origin function exists within the stem of hairpin [B], located about 35
nucleotides upstream of the primer RNA start site in the scheme of the origin
described above.
We then constructed mutations in the region about 10 nucleotides up-

44

stream of the primer RNA start site in the scheme. The dAS sequence on theupper strand (non-template strand) was replaced by deS or dTS. Thesemutant origins were inactive. When the dTS sequence on the lower strand(template strand) was replaced by dGS, the origin was active. Furthermore,
we replaced six nucleotides on the non-template strand of this region by[TATAA T], the consensus sequence for the promoter - 10 region. Thismutant was active as the origin. These results indicated that in this region thesequence of the non-template strand was important for the origin activity
while that of the template strand was not.
We further constructed a set of mutants in which the region ranging from- 12 to - 1 was replaced by the sequence of the lacUVS promoter. When thesequences of both strands were replaced by those of lacUVS, the origin wasinactive. Starting from this mutant, replacement of the template strand withanother sequence to destroy the base pairing in this region produced a fully
active origin. On the other hand, replacement of the non-template strand byanother sequence to destroy the base pairing produced a totally inactive
origin. The results indicated that in the region ranging from - 12 to - 1 in
the origin, there are specific bases in the non-template strand that are required
for the origin recognition, but not in the template strand, and that the DNA
in this region must be in a single-stranded form for the origin to be active.
We also constructed mutants by changing the sequence of the region
ranging from + 2 to + 10 of the origin. The result clearly indicated that
double-strandedness of this region was vital for the origin activity and thatthe nucleotide sequence of either strand was irrelevant.
Altogether, the results obtained suggest that the interaction of RNApolymerase with the minus-strand origin shares some common features with
the interaction with transcriptional promoters, in spite of the fact that asignificant portion of the minus-strand origin is single-stranded.
In addition, we examined the role of SSB in the recognition of the origin.
When the viral single-stranded DNA of 6.4 kb was used as the template,
RNA polymerase produced a large amount of non-specific RNA in theabsence of SSE. In the presence of more than one molecule of SSB per S
nucleotides of the template DNA, synthesis of non-specific RNA was abolished, and synthesis of primer RNA was observed. Primer RNA was notsynthesized in the absence of SSE. To test if this was due to sequestration of
RNA polymerase by the non-specific single-stranded region of the DNA, a140-nucleotide fragment of the viral DNA containing the origin was pre-B.

DEPARTMENT OF CELL GENETICS

45

pared, and used as the template. An equal amount of primer RNA of normal
size (20 nucleotides) was produced no matter whether SSB was present or
not. This suggests that the function of SSB in primer synthesis is to block
non-specific RNA synthesis rather than to be directly involved in a specificinteraction between RNA polymerase and origin DNA.
(3)

A Promoter at the Beginning of the Cell Envelope Biosyntheticj
Cell Division Gene Cluster in the Escherichia coli Chromosome
2-Minute Region is Required for Expression ofNine Genes up to its W

Hiroshi HARA and Kensuke HORIUCHI
A promoter required for expression of the ftsI gene encoding penicillinbinding protein 3, a membrane-bound enzyme essential for cell division, ofEscherichia coli is located upstream of the gene by 3 open reading frames. It
was identified by analyzing chromosomal fragments necessary to complement
a chromosomal null allele of the gene (ftsID.::cat) at a single copy. In this nullallele an internal fragment of ftsI was replaced with the chloramphenicol
resistance gene (cat) of the same orientation. An attempt to construct a
similar chromosomal null allele but with cat of the opposite orientation was
unsuccessful in the presence of a plasmid carrying a chromosomal fragmentfrom the promoter to murF, the second downstream gene of ftsI, which
complementedftsID.::cat. The promoter (Pmra) is at the 5' end of the mra(murein biosynthetic gene cluster A) region at the map position of about 2
min, in which cell envelope biosynthetic and cell division genes are tightly
clustered in the same orientation with the only known terminator at the 3'
end. We suspected that Pmra might be responsible for expression of thegenes further downstream of murF. To examine the function of thispromoter we disrupted it on the chromosome by inserting the lac promoter ofthe same orientation between the - 35 and - 10 sequences of Pmra. The
resultant strain did not grow at all in the absence of a lac inducer IPTG.
Removal of IPTG from the culture medium caused cell lysis, indicating
defective expression of a cell wall synthetic gene(s). Complementation ofsuch a defect required a chromosomal fragment from Pmra tofts W that is thefifth downstream gene offtsI and is essential for cell division. With a plasmidcarrying genes up to murD, the fourth downstream gene, cell division was

46

blocked upon the removal of IPTG, and this defect was corrected with theadditional introduction of a plasmid carryingftsW: Thus we concluded that
the Pmra promoter is required for expression of the first nine genes, up to
ftsW, of the mra cluster.
(4)

Functional Penicillin-Binding Protein 7 is a Multicopy Suppressorof Thermosensitive Growth Defect at Low Osmolarity
Due to an spr Mutation of Escherichia coli

Hiroshi HARA and Kensuke HORIUCHI
Escherichia coli tlprc 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. An extragenic
suppressor mutation (spr) that reversed the thermosensitivity was isolated
and found to cause thermosensitivity at low osmolarity in the prc + background. Thus pre and spr are mutual suppressors. In the course of the study
on spr, the gene for PBP7, a DD-endopeptidase that hydrolyzes peptide
crossbridges of macromolecular murein sacculi, was identified as a multicopy
suppressor of spr. PBP7 seemed to be degraded by Pre, and we suspected thatthe multicopy suppression might be due to overproduction of the protease
substrate mimicking a Pre-deficient state. We converted the serine residue atthe 42nd position from the N terminus of the mature form of PBP7, which
was predicted to be the active site by its occurrence in the SXXK motif
conserved among PBPs and ,B-lactamases, to alanine by oligonucleotidedirected mutagenesis. This resulted in the loss of penicillin-binding ability,
indicating the serine residue is actually the active site. Overproduction of thisactive-site mutant protein did not suppress the spr growth defect. The
presence of the gene product was demonstrated by a maxicell experiment.
The multicopy suppression of spr did not seem simply due to an excess
amount of the Pre substrate, but the DD-endopeptidase activity of PBP7 wasnecessary. Spr protein might also function in murein metabolism. For
details, see Ref. 1.

B.

(5)

DEPARTMENT OF CELL GENETICS

47

C-terminal Region of an Escherichia coli Cell-Division Inhibitor
SulA is Involved in Negative Regulation of the SOS Response

Atsushi HIGASHITANI, Yasuyuki ISHII!, Yasuhiko KAT0 1 and Kensuke
HORIUCHI CDepartment of Applied Chemistry, Faculty of Engineering,
Kyushu Institute of Technology)
The suJA gene of Escherichia coli codes for a cell-division inhibitor that isexpressed in the bacterial SOS response to prevent premature segregation of
the damaged DNA into daughter cells during the DNA repair process. The
intracellular concentration of SulA is well regulated so that the divisioninhibition in SOS response is transient. suJA mutants were first isolated as
suppressors of Jon, which codes for an ATP-dependent protease. The SulA
protein is rapidly degraded by Lon protease. The average half-life of SulA
molecules is 1.2 min in wild-type cells and 19min in Jon- cells. In this study,
we constructed a series of deletion mutants of suJA gene and identified thenegative regulatory region of suJA for the degradation by Lon protease.
From deletion analyses, the N-terminal amino acid residues from the 3rd
position to the 27th position were found to be dispensable for the activity of
the cell-division inhibitor. Likewise, the 21 C-terminal residues were dispensable. We found that the inhibitory activity of the C-terminal deletions was
greater than that of the wild-type SulA in Jon + cells, but not in Jon - cells. In
vitro experiments using SulA fused to the maltose binding protein (MBPSuIA) as a substrate for Lon protease indicated that wild-type SulA was
specifically cleaved by Lon at two sites located within hydrophobic domains
at about the 50th and 110th codons. On the other hand, SulA lacking 21
C-terminal residues which was similarly fused to MBP, (MBP-SuIACI48)
was not cleaved by Lon. Thus, several C-terminal residues of SulA are likely
to have an important role in regulating cell division via the stability of theprotein, in the presence of ATP-dependent protease Lon, during the SOS
response. The C-terminal region of SulA contains a sequence similar to thatfound in the .Ie phage N protein which is also cleaved by Lon protease (4amino acids residues identity in 9 amino acids). This amino acid sequence
may be recognized by Lon. However, it is not enough by itself to make theprotein sensitive to Lon. The LacZ protein that was fused at its C-terminus
to the 8 or 20 amino acid residues of the SulA C-terminus was stable in the
Jon + cells. Lon is a heat shock protein whose function appears to be the

48

degradation of heat-denatured proteins. In vitro analysis has indicated that
heat-denatured proteins are good substrates for Lon, while their native forms
are not. The SulA and N protein, natural substrates of Lon protease, may
harbor a unique structure in common for recognition and cleavage by Lon.
Publications
1.

2.

3.

HARA, H., ABE, N., NAKAKOHJI, M., NISHIMURA, Y. and HORIUCHI, K.: Overproduction of penicillin-binding protein 7 suppresses thermosensitive growth defect at low
osmolarity due to an spr mutation of Escherichia coli. Microbial Drug Resistance, 2, 6372, 1996.
HIGASHITANI, A., HIGASHITANI, N. and HORIUCHI, K.: A cell division inhibitor SulA of
Escherichia coli directly interacts with FtsZ through GTP hydrolysis. Biochem. Biophys.
Res. Commun., 209, 198-204, 1995.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.: SOS induction in Escherichia coli
by single-stranded DNA of mutant filamentous phage: Monitoring by cleavage of LexA
repressor. J. Bacteriol., 177, 3610-3612, 1995.

B-c.

(1)

Division of Cytoplasmic Genetics

Identification and Characterization of the Linear IS3
Molecules Generated by Staggered Breaks

Eiichi OHTSUBO
Transposable elements are characterized by their ability to transpose. IS3(1258 bp in length) is an insertion element present in the Escherichia coli
chromosome and in plasmid F. IS3 is supposed to transpose in a nonreplicative manner. This element encodes two, out-of-phase, overlapping
open reading frames, orfA and orfB. The OrfAB transframe protein, that is
IS3 transposase, is produced by -1 translational frameshifting between orfA
and orfB. Efficient production of the IS3 transposase in cells harboring theIS3-carrying plasmid has been shown to generate miniplasmids as well as
characteristic minicircles, called IS3 circles, consisting of the entire IS3
sequence and one of the 3-bp sequences flanking IS3 in the parental plasmid.
We showed that IS3 transposase also generates the linear molecules of IS3
with 3-nt overhangs at the 5' ends. The nucleotide sequences of theoverhangs are the same as for those flanking IS3 in the parental plasmid,

B.

DEPARTMENT OF CELL GENETICS

49

suggesting that the linear IS3 molecules are generated from the parental
plasmid DNA through staggered double-strand breaks at the end regions of
IS3. We pointed out that the transposition reaction in IS3 is similar to thetransposition reaction in other transposons, such as TnlD and Tn7, and evenmore similar to the integration reaction in retroviruses, which generate linear
DNA molecules as the early intermediates in insertion.
(2)

Characterization of TraI Helicase-Endonuclease Which is Involvedin the Initiation and Termination Steps of DNA Transfer

Eiichi OHTSUBO
In conjugal DNA transfer promoted by bacterial sex factor plasmids, suchas RlOO and F, one of the initial events is supposed to be strand- andsite-specific nicking at the origin of transfer, oriT, by the plasmid-specified
endonuclease. Unwinding from the nick subsequently occurs to provide
single-stranded DNA, which is known to be transferred to a recipient cell.
The TraI protein of RlOO specifies both DNA he1icase activity and site- andstrand-specific nickase activity.
We have recently shown that the TraI
protein specifies a new activity in addition to the helicase-nickase activity,that is the single-strand DNA specific endonuclease-rejoining activity at oriT,
where nicking occurs. The TraI protein consequently generates one complete
unit of the single-stranded plasmid DNA, which is supposed to be transferred
into a recipient cell at the final stage of conjugation. We have also obtained
results indicating that in the nicking reaction, the TraI protein recognizes andbinds to a specific single-stranded DNA sequence around oriT, which appears
due to local melting of the double-stranded DNA molecule, and it cleaves thesequence at oriT.

B.

50

DEPARTMENT OF CELL GENETICS(3) A 6-kb Upstream Region of the Human Transthyretin GeneCan Direct Developmental, Tissue-Specific and Quantitatively
Normal Expression in Transgenic Mouse
Yasushi NAGATA, Fumi TASHIRO, Shigehiro VI, Tatsuhumi MURAKAMI,
Shuichiro MAEDA, Kiyoshi, TAKAHASHI, Kazunori SHIMADA, Hitoshi,
OKAMURA and Ken-ichi YAMAMURA
To ascertain whether a 6-kb upstream region of the human transthyretin(TTR) gene contains the cis-element(s) required for proper specificity andlevel of expression, transgenic mice carrying the human mutant TTR genecontaining either 6-kb (6.0-hMet30) or O.6-kb (O.6-hMet30) of the upstream
region were produced and studied. The 6.0-hMet30 gene was expressed in theyolk sac, liver and choroid plexus, where the mouse endogenous TTR gene isalso expressed. In contrast, expression of the O.6-hMet30 gene was restricted
to the yolk sac and liver. The expression levels of the 6.0-hMet30 gene in theliver and serum were similar to those of the mouse TTR gene, and about 10fold those of the O.6-hMet30 gene. Before birth, the developmental profiles ofthe expression of both transgenes in each tissue were similar to those of themouse TTR gene. However, the expression levels of the 6.0-hMet30 gene in
the liver and serum increased after birth to reach adult levels at an age of 4
weeks, while expression of the O.6-hMet30 gene remained at a low level after
birth. These results suggest that the 6-kb upstream sequence contains thecis-elements required for developmental, tissue-specific and quantitatively
normal expression. For the details, see Ref. 7.
(4)

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 have cloned a novel putative serine/threonine kinase-encoding gene,designated STK-I, from murine embryonic stem (ES) cell and testis cDNA
libraries. The kinase most closely related to STK-l is Xenopus laevis XLP46
protein kinase which shows 71% amino acid identity to STK-I between theirkinase domains. Nevertheless, STK-l is conserved throughout phylogeny
with hybridizing sequences being detected in DNA from mammals, amphi-

B.

DEPARTMENT OF CELL GENETICS

51

bians, 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-l 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 the details, see Ref. 8.
Publications
1.

2.

3.

4.
5.

6.
7.

8.

SEKINO, N., SEKINE, Y and OHTSUBO, E.: lSI-encoded proteins, InsA and the InsA-B'InsB transframe protein (transposase): Functions deduced from their DNA-binding ability. Adv. Biophys., 31, 209-222, 1995.
ABO, T. and OHTSUBO, E.: Characterization of the functional sites in the oriT region
involved in DNA transfer promoted by sex factor plasmid RIOO. J. Bacteriol., 177,43504355, 1995.
FUKUDA, H. and OHTSUBO, E.: Large scale purification and characterization of Tralendonuclease encoded by sex factor plasmid RIOO. J. BioI. Chern., 270, 21319-21325,
1995.
BAUER, W. B., OHTSUBO, H., OHTSUBO, E. and BENHAM, C. J.: Energetics of coupled
twist and writhe changes in closed circular pSMI DNA. J. Mol. BioI., 253, 438-452, 1995.
OHTSUBO, E., and SEKINE, Y.: Bacterial insertion sequences. Current topics in Microbiology and Immunology. Saedler, H. and Gierl, A. (Eds) 204: 1-26. Springer-Verlag,
Berlin-Heiderberg-New York-Tokyo, 1996.
SEKINE, Y., EISAKI, N. and OHTSUBO, E.: Identification and characterization of the linear
IS3 molecules generated by staggered breaks. J. BioI. Chern., 271, 197-202, 1996.
NAGATA, Y, TASHIRO, F, YI, S., MURAKAMI, T., MAEDA, S., TAKAHASHI, K., SHIMADA, K., OKAMURA, H. and YAMAMURA, K.: A 6-kb upstream region of the humantransthyretin gene van direct developmental, tissue-specific, and quantitatively normal
expression in transgenic mouse. J. Biochem. 117, 169-175, 1995.
NIWA, H., ABE, K., KUNISADA, T. and YAMAMURA, K.: Cell-cycle-dependent expressionof the STK-l gene encoding a novel murine putative protein kinase. Gene., 169, 197-201,
1996.

52

C.

DEPARTMENT OF ONTOGENETICS

C-a. Division of Developmental Genetics(1)

Nerve Cell Differentiation in Epithelial Hydra: Epithelial Tissue
Provides Cues for Region-Specific Differentiation

Sumiko MINOBE 1, Osamu KOIZUMI 1, and Tsutomu SUGIYAMA CFukuoka
Women's University)
Nerve precursor cells were introduced into nerve-free epithelial hydra bygrafting of normal hydra tissue, and their differentiation into nerve cells was
examined and compared in tentacles and hypostome of epithelial hydra host.
In tentacles, ganglion cells first appeared in tentacle base at 1.5 days aftergrafting. A boundary was present between tentacle base tissue containing
nerve cells and more distal tissue without them. The boundary moved
gradually, reaching tentacle tip in 7-8 days. Sensory cells also appeared intentacle base, but about I day later than ganglion cells. The boundary for thesensory cells also moved distally. The rates of boundary movement for bothnerve cell types were similar to the rate of displacement of carbon-marked
epithelial tissue from base to tip of tentacles.
In hypostome, a small number of ganglion cells first appeared in the apex
at 1.5 days. More nerve cells appeared during following days, eventuallyforming a cluster of sensory cells at the apex surrounded by numerous
ganglion cells in adjacent tissue as in the hypostome of normal hydra.
These results show that nerve cell differentiation in the epithelial hydra
host does not occur randomly. Instead, it occurs in a strongly region-specificmanner as in normal hydra. This observation suggests that epithelial cells in
different regions provide different cues or signals for region-specific nerve celldifferentiation.

C. DEPARTMENT OF ONTOGENETICS

(2)

53

Hydra Peptide Project II. Biological Function AssayToshitaka FUJISAWA, Hiroshi SHIMIZU, Masayuk~ HATTA, Osamu
KorZUMI 1, Hans BODE 2, Thomas BOSCH3, Maria deHARo 3 , Jan LOHMANN 3 ,Charles DAvm3, Toshio TAKAHASHI 4 and Yojiro MUNEOKA4 ) CFukuoka
Women's University, 2 University of California at Irvine, 3 University of
Munich, 4 Hiroshima University)
Using the approach described in Hydra Peptide Project I, we have isolated
286 hydra peptides, determined the structure of 112 peptides and synthesized
19 peptides. We selected 2 families of peptides (GLWamide family and PW
family) to examine their biological functions.
1) GL Wamide family. Six peptides belong to this family. They consistof 7 or 8 amino acids and all have a consensus sequence of GLWamide at the
C-terminus (e.g. Hym-54 GPMTGLWamide). Similar peptide, Metamorphosin-A (pEQPGLWamide), is isolated from sea-anemone, which can
induce metamorphosis of planula larvae of a marine hydrozoan, Hydractinia(Leitz et al., 1994). All 6 hydra GLWamide peptides can also induce
metamorphosis of Hydractinia planulae. In hydra they induce the contraction of ectodermal sphincter muscles which in turn promotes detachment ofbuds from the parental body. They also induce contraction of parietal
muscles of the sea anemone body wall.
2) PWfamily. Four peptides belong to this family. They are 5-8 aminoacids long and have PW in their C-termini (e.g. Hym-33H AALPW). All of
the PW peptides specifically inhibits neuron differentiation in hydra.
All of the members of each family affect the expression of the sametranscripts when analyzed in DD-PCR. The results suggest that they haveredundant functions being good agreement with the results of biological
function analysis. Further characterization of these peptide families as wellas other synthetic peptides are currently in progress.

(3)

Disappearance and Reappearance of Mesoglea in Hydra Tissue
During Head Regeneration

Hiroshi SHIMIZU and Michael P. SARRAS, Jr l CUniversity of Kansas Medical Center)
Mesoglea is a thin (2-3,um) layer of extracellular matrix (ECM) that

54

intervenes the ectodermal and endodermal epithelial layers in hydra. As an
attempt to elucidate the involvement of ECM in hydra morphogenesis,changes that occur in the mesoglea during head regeneration were examined.
Immunoflourescence staining using a monoclonal antibody m52 which specifically recognizes laminin B I chain in hydra mesoglea was employed tovisualize the mesoglea as a marker.
Immediately after head amputation, the apical edge of mesoglea was
located basal to that of epithelial tissue with a distance of about 50 flm. The
tissue at the site of amputation was thus free of mesoglea. The edge ofmesoglea was a ragged line with very high staining intensity. It was found bysectioning tissue that this bright staining represents a notable thickening of
mesoglea there up to 20-30 flm compared to 2-3 flm of intact mesoglea.
At the site of regeneration, no staining was seen until 18 hr after amputation, followed by a gradual and overall increase of staining intensity. Formation of tentacles began morphologically at 30-48 hr. There tentacles emergedin the area of tissue which was free of mesoglea.
The notable thickening of mesoglea at the amputation site strongly suggests that mesoglea retracted away the site thereby making the tissue there
free of mesoglea. The gradual and overall increase of staining intensity after
18 hr suggests that new synthesis of mesoglea occured at the amputation site
to fill the gap of mesoglea. The close correlation between the site ofretraction of mesoglea and the site of tentacle formation suggests that theretraction of mesoglea is involved somehow in determining the site of tentacle
formation. Two ways of involvement is possible. First, the direct contact of
ectodermal and endodermal tissue layers enabled by the retraction of mesoglea may give rise to massive interaction of the two layers resulting incommitment of tentacle formation at the spot. Second, the intact mesoglea
might work as a mechanical barrier for tentacle formation because of its
possible elasticity or rigidity. The long duration of time when there is muchless mesoglea than normal might make tentacle formation mechanicallyeasier at the spot.

C.

(4)

DEPARTMENT OF ONTOGENETICS

55

Analysis of Hydra Cell Adhesion Molecules

Masayuki HATTA, Engelbert HOBMAYER 1 and Tsutomu SUGIYAMA
CFrankfurt University)
Hydra can regenerate even if it is dissociated to single cells and allowed toform reaggregated cell mass. This process gives a good model system toanalyze principles of morphogenesis of multicellular animals. During regeneration from reaggregates, cell to cell recognition/adhesion and cell movement are thought to play key roles for morphogenesis giving rise to cellsorting and epithelium formation.
We investigated cell adhesion properties of dissociated hydra cells inrotation culture and detected two kinds of activities. In the early stage ofrotation culture, ectodermal and endodermal cells sorted out making ectodermal or endodermal homotypic small cell clumps. Once homotypic cells
adhered, they altered cell adhesion specificities quickly to accept the othertype of cells. This alteration resulted to make large mixed cell masses as thelater phase of reaggregation in rotation culture. When antiserum against
hydra cell membrane was added to the culture, the both type of the cells
mixed making small cell clumps and never grew to lager cell mass. These
results suggest that the quick alteration of cell adhesion specificities is one ofmajor driving force for epithelium formation and that this adhesion specificities are performed by molecules recognized by the antiserum.
Another approach on cell adhesion is identification of homologues ofknown cell adhesion molecules. We isolated cDNAs for ,B-catenin (ref.) andintegrin ,B-chain, and identified ABP-280 as a component of septate junctionthat was first identified to crosslink actin fibers and membrane glycoproteinsin mammalian blood cells. Both description of cell adhesion activities andidentification of cell adhesion molecules will complement each other to figureout molecular bases of epithelium formation.

56

(5)

Evolutionary Study of Reef-Building Corals, Genus Acropora

Hironobu FUKAMI!, Wenqiao WANG!, Makoto OHMORI 1, Takeshi HAYASHIBARA2, Kazuyuki SHIMOIKE 2, Masayuki HATTA and Tsutomu SUGIYAMA
CTokyo University of Fishery, 2 Akajima Marine Science Laboratory)

GenusAcropora shares dominant species in reef-building corals. Dozens ofAcropora species spawn at the same time in the small areas. This massspawning gives a problem how they could speciate without geological isolation. We tried to find out the evidences for the coral evolution by molecularphylogenetic analysis and experimental cross.
First we isolated the gene for mini-collagen encoding a major componentof nematocyst capsule from A. donei (ref.) and A. nasuta, and designed
primers for amplification of a fragment of the gene by PCR. DNA wasextracted from sperms used for cross experiments and subjected to PCR.
DNA sequences obtained were analyzed by molecular phylogenetic methods
to estimate genetic distances and variation, and compared with the results ofcross experiments. Five conclusions were obtained; (I) two species which do
not join mass-spawning are genetically distant from the others, (2) DNA
polymorphism are higher in two similar corymbose shape species than theothers, (3) intraspecific polymorphisms and interspecific divergence wereindistinguishable in the two corymbose species and interspecific cross wereobserved between the two, (4) one major staghorn shape species crossfertilized with one of the corymbose shape species at high frequency, (5) the
major table shape species revealed low frequency of intraspecific fertilizationsuggesting it is a sibling species.
It is suggested that Acropora is evolving through repeating speciation by
rapid establishment of reproductive isolation and gene introgression or
species fusion by interspecific fertilization, giving rise to variety of morphology.
(6)

Cloning of Female Germ Stem Cells from a Male Strain of
Hydra magnipapillata

Chiemi NISHIMIYA-FuJISAWA and Tsutomu SUGIYAMA
Three types of interstitial stem cell subpopulations were isolated from
Hydra magnipapillata, and their roles in sex determination were examined.

C.

DEPARTMENT OF ONTOGENETICS

57

A subpopulation of interstitial stem cells restricted to the sperm differentiation pathway was isolated previously from strain nem-l (male). Anothersubpopulation restricted to the egg differentiation pathway was also isolated
from the same strain. Hydroxyurea treatment was used for isolation in bothcases.
"Pseudo-epithelial hydra" containing only sperm- or egg-restricted stem
cells but no other interstitial stem cell types were maintained by force-feedingfor 2 years. Sex reversal from egg-restricted to sperm-restricted stem cells
occurred 3 times during this period. Both of these two stem cell types are
numerous in the central gastric region of the pseudo-epithelial hydra, butabsent in the foot region below the budding zone. Foot tissue was cut out
from normal nem-l polyps (male), and allowed to regenerate. The regenerates produced eggs but no sperm upon sex induction. These and other results
suggest that the foot tissue contains multi-potent stem cells capable ofdifferentiating into eggs during sexual differentiation.
These observations suggest that strain nem-l (male) contains 3 types ofinterstitial stem cell subpopulations: (l) sperm-restricted stem cells, (2)egg-restricted stem cells, and (3) multi-potent stem cells capable of differentiating into nerve cells, nematocytes and eggs. Upon sex induction, however,differentiation of eggs by the latter two types is suppressed, and only sperm
are produced by the sperm-restricted stem cells. Evidence is presented whichsuggests that similar "phenotypic males", which normally only produce
sperm but contain the stem cell types capable of differentiating into eggs,
occur widely in Hydra magnipapillata. A possible relationship between
phenotypic male hydra and hermaphroditic hydra is discussed.
Publications
1.

2.
3.

4.

MINOBE, S., KOIZUMI, O. and SUGIYAMA, T.: Nerve cell differentiation in nerve-freetissue of epithelial hydra from precursor cells introduced by grafting. I. Tentacles andHypostome. Dev. BioI., 172, 170-181, 1995.
SHIMIZU, H. and BODE, H. R.: Nematocyte differentiation in hydra: commitment tonematocyte type occurs at the beginning of the pathway. Dev. BioI., 169,136-150,1995.
SHIMIZU, H., BODE, P. M. and BODE, H. R.: Patterns of oriented cell division during thesteady state morphogenesis of the body column in hydra. Dev. Dynamics, 204, 349-357,
1995.
NISHIMIYA-FuJISAWA, C. and SUGIYAMA, T.: Genetic analysis of Developmental mechanisms in hydra. XXll. Two types of female germ stem cells are present in a male strain of

58

Hydra magnipapillata. Dev. BioI., 172, 324-336, 1995.
5. WANG, W., OHMORI, M., HAYASHIBARA, T., SHIMOIKE, HATTA, M.,et al.: Isolation andcharactrerization of a mini-collagen gene encoding a nematocyst capsule protein from areef-building coral, Acropora donei. Gene, 152, 195-200, 1995.
6. HOBMAYER, R, HATTA, M., FUJISAWA, T., HOLSTEIN, T. and SUGIYAMA, T.: Identification of a Hydra homologue of the b-cateninjplacoglobinjarma-dillo gene famiily. Gene, In
press.
7. KISHIMOTO, Y., MURATE, M. and SUGIYAMA, T.: Hydra regeneration from recombined
ectodermal and endodermal tissue. I. Epibolic ectodermal spreading is driven by cellintercalation.: J. Cell Sci., in press.

C-b.
(1)

Division of Phenogenetics

Cloning of a cDNA for DNA Supercoiling Factor Reveals
a Distinctive CaH -Binding Protein

Tsutomu OHTA, Masatomo KOBAYASHI and Susumu HIROSEThe DNA supercoiling factor is a protein capable of generating negative
supercoils into a relaxed DNA in conjunction with eukaryotic DNA topoisomerase II. A cDNA clone encoding the Bombyx mori supercoiling factor has
been isolated and characterized. The cDNA sequence contains an open
reading frame of 322 amino acids rich in acidic residues. A local region of
the predicted polypeptide shares a weak homology with the central portion of
the bacterial DNA gyrase A subunit. The cDNA sequence also predicts two
direct repeats within the factor. Each repeat unit consists of a potential
EF-hand domain. The factor contains three other potential EF-hand domains and actually binds Ca2+. Moreover, the supercoiling reaction has beenshown to be activated by Ca2+. These results implicate Ca2+ in the control ofthe superhelical state of DNA. For details, see Ref. 1.

(2)

Molecular Cloning of a cDNA for Transcriptional
Positive Cofactor MBF2

Feng-Qian LI, Ken-ichi TAKEMARU, Hitoshi VEDA and Susumu HIROSE
Transcriptional activation of the Drosophila melanogaster fushi tarazu gene
by FTZ-FI or its silkworm counterpart BmFTZ-Fl, requires two additional
factors MBFI and MBF2. Neither of these factors directly binds to DNA.

C. DEPARTMENT OF ONTOGENETICS

59

MBF1 serves as a bridging molecule that connects BmFTZ-F1 (or FTZ-F1),
MBF2 and the TATA binding protein TBP (F.-Q. Li, H. Veda, and S.
Hirose, Mol. Cell. BioI. 14, 3013-3021, 1994). To clarify the role of MBF2
in transcriptional activation, we isolated a cDNA coding for the factor fromthe silkworm Bombyx morL The deduced amino acid sequence of the factor
showed no homology with proteins in the databases. Farwestern analyses
demonstrated that MBF2 makes direct contact with the J3-subunit of TFIIA.
In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2
activated transcription from various promoters as natural MBF2 did. When
recombinant MBF2 was added to the HeLa cell nuclear extract in thepresence of MBF1 and BmFTZ-FI (or FTZ-F1), it selectively activatedtranscription of the fushi tarazu gene. We propose that MBF2 activatestranscription through its interaction with TFIIA to allow selective activation
in a FTZ-F1 site dependent manner.

(3)

Molecular Cloning of a cDNA Encoding Transcriptional
Mediator MBFl

Kin-ichi TAKEMARU, Feng-Qian LI, Hitoshi VEDA and Susumu HIROSE
MBF1 is a transcriptional mediator that connects the TATA e1ementbinding protein TBP and the Drosophila melanogaster nuclear hormone
receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1 (F.-Q. Li, H.
Veda, and S. Hirose, Mol. Cell. BioI. 14, 3013-3021, 1994). A cDNA clone
coding for MBF1 has been isolated from the silkworm Bombyx morL The
cDNA sequence encodes a basic protein consisting of 180 amino acids.
Homology search in the databases revealed that the deduced amino acidseguence of the factor is conserved across species from yeast to human.
Bacterially expressed recombinant MBF1 is functional in interactions with
TBP and a positive cofactor MBF2. Recombinant MBF1 also makes directcontact with FTZ-F1 and stimulates the FTZ-F1 binding to its recognitionsite. When recombinant MBF1 was added to a HeLa cell nuclear extract in
the presence of MBF2 and BmFTZ-F1, it supported selective transcriptional
activation of the fushi tarazu gene as natural MBF1 did.

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(4)

Consideration of the Hormonal Function of Aging Cells
and Organisms in Insects

Kikuo IWABUCHI 1 and Akio MURAKAMI CFac of Agric., Tokyo Univ. ofAgriculture and Technology, Fuchu, Tokyo)
The scenario for the developmental process of organisms might already bedescribed as a grand design in the genome. The life history of insects iscomprised of processes taking the organisms from through embryonic, larval,
pupal and adult forms: senescence begins after the reproductive phase in thelast form and is followed by the death event. As is well-known, theprothoracic gland hormone (ecdysone) and corpus allatum (juvenile) hormone (JH) take part in various processes in holometabola insects, among
other things, the larval growth period. Ecdysone plays a role in each larvalmolt-the periodic shedding of the larval cuticle in insects-and in each stepof metamorphosis from embryo to larva, larva to pupa, and pupa to theimaginal form in Bombyx, for example. As has widely been accepted, JH hasa juvenilizing function in larval stages (Wigglesworth, 1934). The relative
concentrations of ecdysone and JH determine whether juvenile or matureforms will follow a given molt. Thus, the function of ecdysone is to set thestage for a more mature form. In other words, the hormone pushes
organisms to the approach of death by opening several gates in developmental
steps.
The central nervous system is essential to the life of organisms. Accordingly, the neurons which comprise the neural networks in the system affect thelife span of individual organisms. Of note is another function of ecdysone inorganisms, as an activator in the cell nucleus inducing cell transformation
and/or differentiation. At a cellular level, ecdysone changes aspects of cellproliferation and differentiation leading to cell death. The fate of eachdifferentiated cell in tissue at a different developmental step is geneticallydetermined. The cellular mechanisms at each developmental step has a
special function to change cells from dividing to non-dividing types. It has
been observed in the polyembryonic wasp that JH induces a vegetable cell
propagation in the polymorula formation without differentiation (Iwabuchi,
1995). From such an observation, it can be imagined that a similar event may
occur in cultured cells of the insect: when Drosophila Kc cells were treatedwith ecdysone, it revealed a cell transformation from the dividing to non-C.

DEPARTMENT OF ONTOGENETICS

61

dividing nerve cell type (Courgeon, 1972). Again, ecdysone is closely related
to the longevity in both cells. Thus, it is clear that in insects, the longevity ofcultured cells is analogous to that of individual organisms: the nerve cells
derived either in vivo (organism) or in vitro (transformed cell types) possess
the key function to control life span for both cell types. Cultured cell
populations possess an inherently neuron type nature. When such a transformation once begins, cell division ceases and finally results in death. For
reference, it is worthwhile to note that cell lines derived from imaginal discs
of insects maintain a more undifferentiated state than those of vertebrates,
and in consequence insect cells are apt to exhibit a nature as the non-dividing
nerve cells among cultured cell populations.
It can be inferred that in insects, ecdysone exhibits cell transforming
activity from undifferentiated to differentiated forms in cultured cells. It isalso possible to infer that the hormone pushes both insect cells and organisms
toward death.
(5)

Neuro-Endocrinological Studies on a Mutant sdi (Short Durationof Imaginal Lifespan) in the Bombyx Silkworm

elun SHIMADA!, Masako KANBARA 2 and Akio MURAKAMI Fac. of Agric.,
Tokyo Univ. of Agric. and Technology, Fuchu, Tokyo, 2 Fac. of Agric.,
Univ. of Tokyo, Hongo, Tokyo)

The continuation of each biological species through transmission of genetic
information to progeny, is ensured by sophisticated reproductive mechanisms. However, some hold the view that when the mechanisms of life
maintenance in the post-reproductive phase fail it results in a death events
through the process of senescence. But the senescence process, which starts
at the time of completion of the reproductive phase is also a life phenomenon
that is part of the genome design.
As is well-known, the larvae of the Bombyx silkworm feed on mulberryleaves, and both the pupa and moth use nutrients stored-up in the larval stage.
Accordingly, moths are required to make efficient use of the stores tomaintain the post-reproductive period as possible. To do this, the Bombyx
moth should have well-organized physiological functions to manage theconsumption of stored nutrients under control of the brain-nerve system, tomaintain homeostasis during the post-reproductive period of the insect.

62

A spontaneous autosomal recessive mutant, sdi, was detected and fixed by
Murakami and its genetic characteristics analyzed by him and his co-workers
since 1986. In the present study, the effect of brain removal and/or implantation of the moths of the sdi strain, those of the 11 06 strain having a wild typegenesdi (or +sdi), and F} moths obtained from a cross between sdi and J106
strains were used to investigate the brain function of moths in regard to theimaginal lifespan. The experiment was performed at a temperature of 25°C
and a humidity of 75% using moths from the above groups. Brain removal
and implantation were performed according to conventional methods, andmoths wounded by a sham operation used as the control.
Results indicated that certain endoneurosecretory substances from theimplanted brain did not change the imaginal lifespan in each line observed inthis experiment. Accordingly, it is likely that the short adult lifespan of thesdi mutant line is caused by an abnormal mechanism concerning intercellulartransmission of neural information among brain neurons rather than by a
substance secreted from the brain.
The typical death in the moth of the sdi strain, suddenly occurred after
completion of the reproductive phase (or two to three days) after emergence
without a marked reduction of their body-weight (Murakami et al., 1986,1989). Similarly, if pupal brains were removed in certain wild-type stocks (orlines) having an average adult lifespan a drastic shortening of the imaginal
lifespan was observed (Murakami and Shimada, 1988).
As is well-known, ecdysone is able to activate genes of Diptera, as observedin the polytene chromosome (Clever and Karlson, 1960) and this induces
adult transformation from pupal forms in various insect species. Furthermore, the hormone seems to have the ability to transform cells from undifferentiated type cells to differentiated ones as described in another report.
Accordingly, it is possible to infer that ecdysone makes renews and/ortransforms undifferentiated neurons into differentiated ones in the central
nervous system. In addition, the hormone acts as an inducer of transformation from larval to adult forms.
From these observations, it appears that the short adult lifespan, as a
typical feature of the sdi mutant line, may be caused by a marked disorder ofthe life sustaining mechanism or homeostasis at an early stage of the
post-reproductive phase. In this regard, the possibility cannot be excluded
that some functional disorder in the ecdysone receptor exists and that agenetically programmed cell replacement of used neurons with new ones

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63

occurs in the preceding developmental stage.
(6)

Linkage Analysis of the sdi (short duration of imaginal lifespan) Gene

Hirosi FUJII!, Hirosi DOIRA 1 and Akio MURAKAMI
Kyushu Univ., Fukuoka)

CFac. of Agric.,

In the Bombyx silkworm the growth period from embryo via larva to pupa
in the mutant line sdi, is no different than that in the normal line. However,
the adult lifespan in the sdi line is shorter than that in common silkworm
lines. The adult lifespan of this mutant lasts at most two to three days after
emergence from the pupal form. Murakami and his colleagues have analyzed
the genetic as well as biological characteristics of the mutant sdi since 1986
and their findings indicate that the mutant characteristic is controlled by asingle recessive gene located on some autosome. It is not yet known which
linkage group the gene sdi belongs to.
For linkage analysis, the marker genes used were 2(Y), 3(Ze), 4(L), 5( -),6(Ekp), 7(q), 8(st). 9(la), lO( ~), l1(K), 13(ch), l4(U), l5(bl), l6(cts), 18(min), 19(nb), 20( -), 2l( -), 22(mw), 23( -), 24(Sel), 25(Nd), 26( -),27(so) and 28(Etr). The numbers indicate linkage chromosome groups.
When the dominant marker was used for the analysis, the F 1 hybrid females
were mated with the test male (sdi and +Nd, a normal cocooning wild type),
and then BF] progenies were examined for longevity. If the recessive marker
was used, F 2 progenys were examined for longevity. The results showed thatthe gene sdi was an independent relation to the following linkage groups, 2,
3, 4, 6, 7, 8, 9, 11, 13, 14, 15, 16, 18, 19, 22, 24, 27 and 28. In analyses
conducted so far, when silkworms marked with genes, sdi and +Nd, werecrossed with moths having the gene Nd and +sdi and then F 1 female moths
showing the Nd characteristics were again mated with moths having thegenessdi and +Nd. The BF 1 progenys obtained were examined for longevity.
The results showed that all 216 + Nd type moths, of both sexes, had shortadult lifespans, while thirty-three moths of the Nd type also had a short adult
lifespan and died within 4 days after eclosion. But a hundred and two (102)
Nd type moths had a long lifespan, over 5 days after emergence. The
thirty-three moths seem have a normal lifespan (+sdi) (as a death symptomof the moths showed natural) and the observed segregation ratio was far
from expected in an independent relationship. If the genes sdi and Nd had an64

independent relationship when sdi and normal (+Nd) moths were crossedwith Nd +sdi moths and F 1 females showing the Nd characteristics werecrossed with +Nd sdi, moths the segregation of the BF] progeny would havethe ratio of 1 sdi +Nd, 1 +sdi Nd, 1 sdi Nd, 1 +sdi+Nd, but in the cross, only
33 sdi and Nd moths appeared, and moreover, no +sdi +Nd moths wereobserved. The sdi and Nd moths, which died soon after emergence, weredifferent from those having natural (senescence) death: they were not sdi
mutants. Hence, the segregation of the BF] progeny should be considered tohave a ratio of I +Nd sdi to 1 Nd +sdi. Accordingly, we concluded that thegene sdi is linked with Nd on the 25th linkage group of B. mori (L.).
Then, we carried out the following cross to determine the recombination
value between the gene sdi and Nd. The BF 1 male moths, sdi + I +Nd, weremated to females homozygous for sdi +Nd, the progenys were segregated into
Nd and +Nd type cocoons. Each type progeny was examined for longevity.
The resultant progenys were segregated in the ratio of 151 Nd +sdi: 52 Nd sdi:
252+ Nd sdi; 20+ Nd +sdi. Thus, the recombination value between sdi and Nd
was calculated to be 12.3%. But as already noted above, Nd moths die inconsequence of physiological disorders throughout the entire developmental
period from embryo to pupa, and among other things, at the larval-pupal
ecdysis event. Therefore, we can calculate the recombination value using+Nd sdi individuals exclusive of Nd ones. The value estimated was 7.3% andthe gene sdi was located ±7.3% units from the gene Nd on the 25th linkage
group in the Bombyx silkworm.
(7)

Genetic Studies on Chromosomal Translocation in Bombyx Mori (L.)(a) Construction of a New Reciprocal Translocation

Akio MURAKAMIIn Bombyx silkworms, several numbers of the Y-chromosome marked withvarious kinds of visible gene characters have been constructed for breeding
strategies, to discard female insects before they enter the actively growing
instar phase, since male silkworms more efficiently produce silk-proteins thanfemales. To construct a certain type of translocated chromosome, it isconvenient to make use a chromosome marked with visible gene characteristics. In this insect, there are known to be various kinds of mutant genes interms of egg and cocoon color, larval marking patterns, and others. The Y

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65

DEPARTMENT OF ONTOGENETICS

chromosome is known only as a female determining factor, but other genes
having a biological function have not been detected so far. At present,
several marked Y-chromosomes have been preserved in many biologicalinstitutions. However, neither their origins, attachment or translocation,
have been sufficiently analyzed.
Such being the case, we have purposely produced a certain reciprocaltranslocation between the Y and 5th chromosomes. Wild type stocks, Aojuku
X-rays

X-rays

ytL

re+

pe+j

I

5
5

X

I

+

pe+

-+- -lI

I

pe+

re+

re

pe

re+

I

X

I

pe

Treated choromosomesof femalesreTester chromosomes(males)Breakage and reunion type(translocation)

Normal type

(B)

(A)

Femelesl:wwwv

+

+

I

I
I
re

I

pe

Black eggs and zebra
larvae

pe+

Ze

re+

pe

re

+ '\f\/\J\IV'v IruI -lI
Black eggs and zebra
larvae
(D)

(C)

Males

+

+

I

I
I
re

I

pe

White eggs and usual
larvaeFig. I.

Zere+

Iru -l-

I

I

pe

re

White eggs and zebra
larvaeA schematic construction procedure of a reciprocal translocation between the Y
and 5th chromosomes in B. mori (L.).

66

and C 108, in the latter of which the Y chromosome is marked with the Zebra
gene, were adopted for use in this study. A dozen F 1 hybrid female pupae
obtained from crosses between stocks were treated with X-rays (ca. 1000 R)
to break the chromosomes. These females were mated with the marker
males, homozygouspe (5-0.0) and re (5-31.5), in expectation of breakage
events on the two chromosomes.
In consequence, we detected a small number of whole-body type mutant
eggs colored with either yellowish-white or red serosa cells and mosaic typemutants with black and either yellowish-white or red cells among many
normal black eggs. Theoretically, these mosaic mutants were considered toinclude chromosomal aberrations on the 5th chromosome concerned with
egg-color gene loci. The female moths that hatched from mosaic mutants,
were again crossed with pe: re tester males for progeny tests.
A female moth, which originated from the mosaic egg with yellowish-whiteand black cells, laid black ( +pe) and yellowish-white (pe) eggs in the ratio of
approximately 1 : 1. Egg-color mutant were separately cultured. The sex oflarvae derived from the yellowish-white eggs was male, but zebra and normal(p or p +) type larvae were also found in the ratio of 1 : 1 without exception,
while the sex of black eggs was female in the ratio 1 : 1 for normal and zebra
types. Viability of females with black colored eggs and zebra type larva was
100%, but that of males with yellowish-white eggs and zebra markers waspoor to some extent, indicating that the female did not suffer from a loss ofchromosomes (or genetic information) from a shift of the genes located on
the chromosomes. But the male was completely lacking in some genes on theproximal region of the 5th chromosome. From these analyses, the chromosomal aberration type mutant can be regarded as a reciprocaltranslocation
between the Y and 5th chromosomes, rcpT[tl(Y:5)pe+:h(5:Y)Ze]. The
reciprocal translocation line together with the egg-color pe and re genes, hasbeen preserved in our laboratory.
In this line, the sex-ratio of females to males is normal, suggesting that atranslocated part of the Y-chromosome marked with the Ze gene on the 5thchromosome in the male showed maleness. Males having new constitution in
the 5th chromosome received a certain portion of the Y-chromosome marked
with the zebra gene did not express femaleness and females, which had aY-chromosome suffering from some structural changes, an exchange between
the Y and 5th chromosomes, did not change because of the original gender.
Those observations suggest that a female determinant factor is not dispersed

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DEPARTMENT OF ONTOGENETICS

67

along the Y-chromosome, but is localized at a specific site on the chromosome. Furthermore, it suggests that the factor did not exist or a translocatedpart of the Y to the 5th chromosome did not exist, and female silkworms
can't exist (survive) without the factor on the Y chromosome.
(8)

Genetic Studies on Chromosomal Translocation in Bombyx Mori (L.)(b) Genetic Function of the V-Chromosome

Akio MURAKAMIIn the reciprocal translocation reported above, the sex-ratio of females tomales was normal, indicating that silkworms, with an exchange between the
5th chromosome and the Y chromosome marked with the zebra gene, were
all males, while silkworms, which have a Y chromosome with a proximal partof the 5th chromosome, are females. As is well-known, in Bombyx silkworms, only a female determinant factor has been recognized on the Y
chromosome, but no other genes relating to life activities of this insect. Thisshows the factor is localized on a specific site of the Y chromosome rather
than being dispersed along the chromosome. It is also possible to say that the
Y chromosome fragment translocated onto the 5th chromosome does not
only retain the factor, but the remainder of the Y chromosome still maintains
the factor. In any case, the Y chromosome of several Y chromosometranslocation lines established showed a perfect function in expressing the sexof females. Moreover, it is likely that a would-be female silkworm does notexist without the female determinant factor. Furthermore, it is possible todeduce that the Y chromosome fragment shifted onto the autosome does nothave a killing effect on males in the translocation line.
The V-chromosome marked with the zebra gene was constructed by
Hashimoto (1956). This chromosome has been considered to be a product ofa reciprocal translocation between the Y and 3rd chromosomes, T[tl(Y:3)
Ze:t2(3:Y)( - )]. There is a low viability of males in the line, but not a severe
lethality in either embryonic or young larval male. Hashimoto interpreted
this phenomenon as being caused by a structural disorder of the 3rd chromosome, which received a fragment of the Y chromosome autosome on theoccasion of chromosome breakage. However, it may be possible that as analternative explanation, the poor viability of males in Hashimato's translocation line depended on a large deficiency of the 3rd chromosome from the fern

68

locus (3--0.0) to the Ze locus (3-20.8). As discussed above, there was also
low male viability in the rcpT(Y:5) line, probably, becouse of a deletion of asmall portion of the 5th chromosome. In the sex chromosome constitution of
Bombyx silkworms, males are XX and females XY. Accordingly, there is nochange of genome constituents in spite of a drastic shift of chromosomal
structure owing to chromosomal exchanges in the females. While, in themales, there is a deficiency of genome composition in the nature of the sex
chromosome composition of males. Consequently, it can be said that the
deficiency of a certain region in the autosome seems to be reflected directly inlethality in the male. The differential male viability observed between
Hashimoto's and the present translocation lines is thought to be due to thesize of deletions on the autosomes concerned in each line.
(9)

Origin of Holometabolous Metamorphosis in Insects

Kiyoshi MINATO
Among the organisms, those in the insecta classes are most successful in
not only the total number but also the number of species. One of the reasonsis thought to be in the very skillful life styles through the holometabolous
metamorphosis or the complete metamorphosis seen in groups of the animals.
That is, the holometabolous metamorphosis is specific in their larval forms
relatively simple and dissimilar to adult ones, and the intermediate phase of
pupa lying between larva and adult. Since the origin of the holometabolous
metamorphosis, and hence the origin of the pupal stage in evolution of insects
have never been thoroughly classified, we focused on the matter by investigating the life styles in various insect and examining the theories seen in available
literature's.
The Berlese theory (1913) that the larval forms in Holometabola derived
from a precocious hatching of embryos owing to less yolk reserves is now
received by few authors from some negative evidences. And many think that
endopterous conditions in Holometabola larva derived from a similar process
seen in the earlier nymphal stages of some Heterometabola, such as Thysanopter and Homoptera in which wing anlages development internally.
Furthermore, the larval forms in Holometabola are though to develop to be
simplified and specialized more and more through the physiological andmorphological independency between larva and imago made by the metamor-

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DEPARTMENT OF ONTOGENETICS

69

phic phase of pupa.
However, the origin of other features seen in the larvae of Holometabola,
such as defects in compound eyes and less-developed thoracic legs, have notexplained satisfactorily by the preceding authors. The precocious hatch ofembryo in the Berlese theory may not be acceptable, it is likely that Holometabola utilized a part of embryonic forms in making their larval forms by
suppressing the morphogenesis of some tissues, such as compound eyes andthoracic legs appeared later in embryonic development.
Publications

OHTA, T., KOBAYASHI, M. and HIROSE, S.: Cloning of a cDNA for DNA supercoilingfactor reveals a distinctive Ca2+ -binding protein. J. BioI. Chern., 270, 15571-15575, 1995.
2. LI, F.-Q., TAKEMARU, K., GOTO, M., UEDA, H., HANDA, H. and HIROSE, S.: Molecularcloning of MBF2 reveals its contact with TFIIA that leads to transcriptional activation.
Mol. Cell. BioI. in press.
3. SANO, Y., EIGUCHI, M., HIRANO, H.-Y. and YAMADA, M.-A.: A nuclear gene modifying
instability of fertility restoration in cytoplasmic sterile rice. Genet. Res., in press.
4. UEDA, H., MURATA, T., SUN, G.-c. and HIROSE, S.: Role of transcription factor FTZ-FI
in the process of ecdysis and metamorphosis. In "Molecular mechanisms of insect
metamorphosis and diapause" (A. SUZUKI et al. eds.), pp. 299-307, Industrial Publishingof Consulting, Inc., 1995.
I.

C-c.

(1)

Division of Physiological Genetics

Hydra Peptide Project I. Isolation, Structure Determination andChemical Synthesis

Yojiro MUNEOKAl, Toshio TAKAHASHI I , Toshitaka FUJISAWA, Hiroshi SHIMIZU, Masayuki HATTA, Tsutomu SUGIYAMA, Osamu KOIZUMI 2, Thomas
BOSCH 3, Maria deHARo 3 , Jan LOHMANN 3 , Charles DAVID 3 and Hans BODE4
CHiroshima University, 2 Fukuoka Women's University, 3 University of
Munich, 4University of California at Irvine)
Peptide signaling molecules are widely found among animal tissues andbear important biological functions, as neurotransmitters, hormones, growth
factors etc. However, very little is known about their involvement indevelopment. In order to systematically isolate peptide signaling molecules

70

involved in hydra development, we initiated a new project based on a novel
approach (see Annual Report, 1994). The project consists of the following 5
parts: (1) Peptide isolation from hydra tissue, (2) selection of peptides which
are capable of altering gene expression in hydra, by using Differential Display
PCR (DD-PCR) (Liang and Pardee, 1992), (3) structure determination of
peptides active in DD-PCR, (4) chemical synthesis of active peptides, and(5) examination of biological functions of synthetic peptides. Part (l), (3)and (4) are reported here and the rest are reported in the abstract "Hydra
Peptide Project II".
(l) Peptide isolation. As we have reported previously (see Annual
Report, 1994), two batches of crude peptide preparations were made from
Hydra magnipapillata tissue. Batch 1 (150 g) using hot acetic acid extraction
method and batch 2 (500 g), the cold acetone method. Each extract was
separated by reverse-phase HPLC into 15 factions. Fraction 8 from batch 1
and fractions 8 and 9 were first selected and subjected to 8-9 alternating stepsof reverse-phase and ion-exchange HPLC to isolate peptides. No screening
was involved in these steps. So far, 286 peptides were isolated. The results
suggest that hydra tissue contains close to 1,000 peptides.
(2) Amino acid sequence. Out of 286 peptides 193 peptides were subjected to amino acid sequence analysis by using an automatic amino acidsequencer (and a mass spectrometer in a few cases) and 112 peptides weresuccessfully analyzed. However, it was not successful for 81 samples (42%)
because of N-terminal blocking (or non-peptides).
(3) Chemical synthesis. 19 peptides were chemically synthesized andconfirmed to be identical to the native peptide by HPLC. They include afamily of 6 peptides which have a common sequence of GLWamide at the
C-terminus (as in Metamorphosin-A, Leitz et al., 1994) and a family of 4
peptides which have PW at the C-terminus. PW peptides are not reported sofar. Isolation, structure determination and chemical synthesis of peptides
from the remaining fractions are currently underway.
(2)

Analyses of Mammalian Genome by Fluorescence in situ Hybridization

Katsuzumi OKUMURA
We analyzed the human and mouse genomes by fluorescence in situ
hybridization (FISH), including the mapping of several genes, judgment of

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DEPARTMENT OF ONTOGENETICS71

YAC clone chimerism, detection of a transgene and its amplified unit,
ordering of genomic clones. For details, see Ref. 1, 2, 6-8, 10.
On the other hand, FISH was used to analyze how DNA replication timing
is related to the genome structure and how the specific genome domains arearranged in the nucleus.
First, we determined the replication timing pattern of DNA segments over
2 Mb within the human MHC on HL60 cells. This region is composed oflong-range G+C% (GC) mosaic structures related to chromosome bands
and a boundary of the GC mosaic domains exists between MHC classes II
and III. FISH with elongated chromatin fibers showed that the flanking
genome structures of the boundary are strictly consistent with the reported
mapping data. This confirmed that HL60 cells may not have extensive
structural abnormality in this region. Multi-color FISH with interphasenuclei was useful in comparing the temporal replication timing of two DNA
fragments at adjacent genomic positions and which have similar replication
timing in the cell cycle. Using this procedure, it was possible to determine theprecise replication timing of each DNA fragment which was randomly
selected from the MHC region. The fragments at the boundary region were
found to relatively replicate latest among all examined. It was also suggested
that this region consists of at least two replicon clusters. Interestingly, there
is a close correlation between the temporal order of replication and the GC
gradient of genome sequences in human MHC on HL60 cells. These results
suggested the possibility that replication of DNA in the mammalian cell
genome reflects GC mosaic domains at the level of genome sequences as well
as chromosome bands.
Second, we used FISH to delineate the intranuclear arrangement of specificgenome domains by using 16 cosmids scattered all over the entire chromosome 12. Each set of two probes was hybridized to the HL60 nuclei fixedwith paraformaldehyde to keep the 3D structure and detected by different
fluorochromes. The analysis of the distribution of the nuclear periphery or
the interior on each probe suggested that chromosome 12 exhibits a distinctly
non-random arrangement in G 1 nuclei; centromeric and q21 flanking regions
are localized on the nuclear periphery, while the other domains are largely
localized in the interior. The relationship between DNA replication and theintranuclear arrangement of specific genome domains are also discussed. For
details, see Ref. 12.
We also studied biosynthesis and the metabolism of NAD and its related

72

compounds. For details, see Ref. 3-5, 9, 11.
Publications
1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

OKUMURA, K., NOGAMI, M., TAGUCHI, H., HISAMATSU, H. and TANAKA, K.: The
Genes for the a-Type HC3 (PSMA2) and tI-Type HC5 (PSMBI) Subunits of Human
Proteasomes Map to Chromosomes 6q27 and 7pI2-p13 by Fluorescence in Situ Hybridization. Genomics, 27, 377-379, 1995.
SHIRATORI, A., OKUMURA, K., NOGAMI, M., TAGUCHI, H., INOUE, T., ANDO, T., IZUMI,M., MIYAZAWA, H., HANAOKA, F., MURAKAMI, Y., and EKI, T.: Assignment of the 49kDa (RPIMI) and 58-kDa (PRIM2A and PRIM2B) Subunit Genes of the Human DNA
Primase to Chromosome Bands Iq44 and 6pI1.1-pI2. Genomics, 28, 350--353,1995.
NISHITANI, H., YAMADA, Y., OHSHIMA, N., OKUMURA, K. and TAGUCHI, H.: Identification of N-(beta-D-glucopyranosyl)nicotinic Acid as a Major Metabolite from Niacin in
Cultured Tobacco Cells. Biosci. Biotech. Biochem., 59(7), 1336-1338, 1995.
NISHITANI, H., KIKUCHI, S., OKUMURA, K. and TAGUCHI, H.: Finding of a HomarineSynthesizing Enzyme in Turban Shell and Some Properties of the Enzyme. Arch. Biochem.
Biophys., 322(2), 327-332, 1995.
NISHITANI, H., MORIMURA, N., HAMANO, Y., OKUMURA, K. and TAGUCHI, H.: Homarine Promotes the Growth of Duckweed and Radish Seedlings. Bull. Fac. Biores. Mie
Univ., 15, 27-31, 1995.
KUSABA, H., KOHNO, K., ASAKUNO, K., KUWANO, M., OKUMURA, K., GREEN, E. D.,
SCHLESSINGER, D. and WADA, M.: Functional Expression of Yeast Artificial Chromosome (YAC)-Human Multidrug Resistance Genes in Mouse Cells. Genome Research, inpress.
TORIGOE, K., SATO, S., KOHNO, K., KUWANO, M., OKUMURA, K., GREEN, E. D., TSUI,
L.-c., SCHERER, S. W., SCHLESSINGER, D. and W ADA, M.: A YAC-based contig of 1.5
Mb spanning the human multidrug resistance gene region and delineating the amplification
unit in two human multidrug resistant cell lines. Genome research, 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., in press.
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., ONO, A., TAGUCHI, S., YAMAZAKI, M., OKUMURA, K., NOGAMI, M.,
TAGUCHI, H., ANDO, H. 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, in press.

73

D.

DEPARTMENT OF POPULATION GENETICS
D-a.

(1)

Division of Population Genetics

Synonymous and Nonsynonymous Substitutions in Mammalian
Genes and the Nearly Neutral Theory

Tomoko OHTA
The nearly neutral theory of molecular evolution predicts larger generation-time effects for synonymous than for nonsynonymous substitutions.
This prediction is tested using the sequences of 49 single-copy genes by
calculating the average and variance of synonymous and nonsynonymous
substitutions in mammalian star phylogenies (rodentia, artiodactyla, andprimates). The average pattern of the 49 genes supports the prediction of thenearly neutral theory, with some notable exceptions.
The nearly neutral theory also predicts that the variance of the evolutionary rate is larger than the value predicted by the completely neutral theory.
This prediction is tested by examining the dispersion index (ratio of thevariance to the mean), which is positively correlated with the average
substitution number. After weighting by the lineage effects, this correlation
almost disappears for nonsynonymous substitutions, but not quite so forsynonymous substitutions. After weighting, the dispersion indices of bothsynonymous and nonsynonymous substitutions still exceed values expectedunder the simple Poisson process. The results indicate that both the systematic bias in evolutionary rate among the lineages and the episodic type of ratevariation are contributing to the large variance. The former is moresignificant to synonymous substitutions than to nonsynonymous substiutions.
Isochore evolution may be similar to synonymous substitutions. The rate andpattern found here are consistent with the nearly neutral theory, such that therelative contributions of drift and selection differ between the two types ofsubstitutions. The results are also consistent with Gillespie's episodic selection theory. For details, see Ref.!.

74

Gene Conversion vs Point Mutation in Generating Variability at the
Antigen Recognition Site of Major Histocompatibility Complex Loci

(2)

Tomoko OHTA
In order to assess the roles of gene conversion followed by natural selection
and balancing selection for point mutations in polymorphisms at major
histocompatibility complex (MHC) loci, DNA sequences of several mammalian taxa were analyzed. Synonymous and nonsynonymous diversities were
estimated separately for the antigen recognition site (ARS) and the remaining region of class I and class II genes. In some sequence pairs, the numberof nonsynonymous substitutions exceeds that of synonymous substitutions at
the ARS. This result indicates that some kind of balancing selection for point
mutation is operating. In other sequence pairs (particularly of bovine and of
rabbit), the number of synonymous substitutions at the ARS exceeds thesame number at the remaining region. This result indicates that geneconversion involving a short region followed by natural selection is important. In general, a combination of gene conversion, point mutation, natural
selection and random drift is thought to have contributed to polymorphisms.
For details, see Ref. 3.
(3)

Genetic Study of Species Differences as Observed as Loss ofBristles in Interspecific Hybrids of Drosophila

Toshiyuki TAKA NO
Species differences can be revealed through inviability, sterility and/or
morphological anomalies of interspecific hybrids even if there is remarkable
morphological similarity between species. An analysis of bristle number ininterspecific hybrids has been used to search for genes responsible for genetic
differences among D. melanogaster and three closely related species, D.
simulans, D. mauritiana, and D. sechellia. There are clear-cut genetic
differences between D. simulans, on the one hand, and D. mauritiana and D.
sechellia, on the other hand, in reduction of the bristle number of hybrids
with D. melanogaster. Interspecific hybrids between a line of D. melanogasterand D. simulans iso-female lines exhibited a wide range of the number ofmissing bristles on the thorax. On the contrary, D. mauritiana and D.
sechellia lines showed almost no reduction of the bristle number in hybrids

D. DEPARTMENT OF POPULATION GENETICS75

with D. melanogaster. Furthermore, the application of deficiency screening
identified two potential regions on the X chromosome.
(4)

New Methods for Estimating the Numbers of Synonymous andNonsynonymous Substitutions

Yasuo INA
I developed two new methods for estimating the numbers, d s and d N , ofsynonymous and nonsynonymous substitutions. Using computer simulations,
I evaluated the accuracies of the two new methods and of four previously
proposed methods: Nei and Gojobori's method (NG), Miyata andYasunaga's method (MY), Li, Wu, and Luo's method (LWL), and Pamilo,
Bianchi, and Li's method (PBL). The following results were obtained: (1)When there is a strong transition/transversion bias at the mutation level, theNG, MY, and LWL methods overestimate d s and underestimate dNo (2)Unless there is a strong nucleotide-frequency bias, the new methods and the
PBL method give good estimates of ds and dNo For details, see Ref. 2.
Publications
1.

2.
3.

OHTA, T.:

Synonymous and nonsynonymous substitutions in mammalian genes and thenearly neutral theory. J. Mo!. Evo!., 40, 56-63, 1995.
INA, Y.: New methods for estimating the numbers of synonymous and nonsynonymous
substitutions. J. Mo!. Evo!., 40, 190-226, 1995.
OHTA, T.: Gene conversion vs point mutation in generating variability at the antigen
recognition site of major histocompatibility complex loci. J. Mo!. Evo!., 41, 115-119, 1995.

D-b.
(1)

Division of Evolutionary Genetics

Studies of Human Pseudoautosomal Boundary-Like
Sequences (PABLs)

Tatsuo FUKAGAWA, Mizuki OHONO, Yasukazu NAKAMURA, Katsuzumi
OKUMURA, Asako ANDO l , Hidetoshi INOKOl, Naruya SAITOU and Toshimichi IKEMURA CSchool of Medicine, Tokai University, Isehara)
The human genome is composed of long-range mosaic structures of G +76

C% (GC%), which are thought to relate with chromosome bands. We
previously found a boundary of Mbp-level mosaic domains in the humanmajor histocompatibility complex (MHC) region disclosing a sharp GC%
transition. Near this boundary to differentiate the global GC% distribution
along the genome, a sequence highly homologous (ca. 80% nucleotide
identity) with the pseudoautosomal boundaries of short arms of human sex
chromosomes was found and designated "PABLl": the pseudoautosomal
boundaries, PABXI and PABYI, are unique in the human genome as thestrict physical site at which the unusually high rate of recombination in thepseudoautosomal region PARI (known to be 20-fold greater than the
genome average) terminates abruptly, differentiating human sex chromosomes into functionally distinct regions, i.e., 2.6 Mbp of PARI and sexspecific regions.
To investigate structures and functions of the highly homologous sequences both of which are found in the boundaries differentiating global
genome characteristics, other pseudoautosomal boundary-like sequences(PABLs) were cloned and characterized. Comparing ten genomic PABLs
situated on different chromosomes, a ca. 650 nt consensus sequence of the
PABL core could be defined. From six independent cDNA libraries, we
isolated cDNA clones having PABL and found fairly strict conservation of
the 3' or 5' edge of their PABL cores. Northern blot experiment showed
sizes of PABL transcripts to range 5-10 kb length, and FISH analysis using
the cDNAs as probes showed characteristic localization of these transcripts
in interphase nuclei.
Divergence time of PABLs was estimated to be 60-120 million years ago
and their evolutionary rates showed evolutionary (and thus most likely
functional) constrains on PABL sequences. Based on structures of pseudoautosomal boundaries of short and long arms of human sex chromosomes,
evolutionary process to form the present day pseudoautosomal boundary of
Y chromosome, PABYI, is proposed postulating an illegitimate recombination between two PABLs of the ancestral Y chromosome. For details, see
Ref. I.

D. DEPARTMENT OF POPULATION GENETICS(2)

77

Studies of Notch-Related MHC-Class III Gene INT3: Structure of the
Human Counterpart of Mouse Mammary Tumor Gene int-3 and(CTG)n Polymorphism in the 5' -Upstream Region

Kimihiko SUGAYA 1, Kazuei MITA 1, Asako AND0 2, Hidetoshi INoKo 2 andToshimichi IKEMURA CNational Institute of Radiological Sciences, Anagawa, 2 School of Medicine, Tokai University, Isehara)
The cDNA clones for the human counterpart of mouse mammary tumor
gene int-3, which is a human MHC class III gene and designated INT3, wereisolated and sequenced. Mouse int-3 has been thought a Notch-homologous
gene, but the extracellular portion of this predicted transmembrane gene has
not been clarified. The human INT3 sequence determined in this work
corresponds not only to the intracellular portion but also to the extracellular
portion, being composed of Notch/lin-12 and EGF-like repeats, which areessential domains in Notch-homologues but uncharacterized for mouse int-3.
A phylogenetic comparison of INT3 with other Notch homologues of a wide
range of species revealed four subfamilies for mammalian Notch. In the 5 /upstream region of human INT3, we found (CTG)n repeats showing VNTR
polymorphism for different HLA haplotypes.
Eight genes mapped on 6p21.3, including INT3, were found to havecounterparts structurally and functionally similar to those mostly mapped on
9q33-q34, indicating the segmental chromosome duplication during thecourse of evolution (submitted).
(3)

Precise Replication Timing around a Boundary of Long-Range
G+C% Mosaic Domains in the Human MHC Region

Toyoaki TENZEN, Asao FUJIYAMA, Katsuzumi OKUMURA, Asako AND0 1,
Hidetoshi INOK0 1, Takashi GOJOBORI and Toshimichi IKEMURA CSchool of
Medicine, Tokai University, Isehara)
The human genome is composed of long-range G+C% (GC%) mosaic
structures related to chromosome bands. The human MHC region has beenfound to have a boundary of megabase-level GC% mosaic domains in thejunction area between MHC classes II and III. We determined the precise
DNA replication timing for several loci in the classes II and III, especially
focusing the GC% boundary region. The replication timings could be78

determined at the kb-level resolution by quantifying pulsely labeled nascent
DNAs which were isolated from synchronized HL60 cells. The class III
region including TNX, AGER, and INT3 replicates in the second hour of S
phase whereas the class II region including HLA-DRA, PABLI, and dense
LINE-l cluster replicates in the fourth hour of S-phase, i.e., it makes the lagof two hours between the classes II and III. The replication timing changes
around the 20-kb dense Alu cluster where a sharp GC% transition point
exists, showing a good correlation between replication timing and GC%
distribution.
(4)

Detection of Genes in Escherichia coli Sequences Determined by
Genome Projects and Prediction of Protein-Production Levels,
Based on Multivariate Diversity in Codon Usage

Shigehiko KANAYAl, Yoshihiro KUDO l , Yasukazu NAKAMURA and Toshimichi IKEMURA CDepartment of Electric and Information Engineering,
Faculty of Engineering, Yamagata University, Yonezawa)
We developed measures (called Z-parameters in this study) which reflect
the diversity of codon usage in Escherichia coli genes using principal component analysis. Protein-production levels for 1500 CDSs (protein-coding
sequences) proposed by genome projects in Japan and USA were estimatedby a correlation equation between Zl and cellular protein content, which was
obtained through advance analysis of the genes which were experimentally
characterized. Through a profile analysis of Zl for E. coli sequences
determined in the Japan genome project, we predicted additional 36 CDSs
that had not yet been annotated in the International DNA Databases.
Thirty-two out of the thirty-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 Zl-parameter profile also
led us to assess sequencing errors which cause frame-shift. For details, see
CABIOS, in press.

D. DEPARTMENT OF POPULATION GENETICS(5)79

Tenascin-X Expression in Tumor Cells and Fibroblast in Culture

Takao SAKAI 1, Yusuke FURUKAWA 1, Ruth CHlQUET-EHRISMANN 2, Toshimichi IKEMURA and Ken-ichi MATSUMOTO CInstitute of Hematology, Jichi
Medical School, Tochigi, 2 Friedrich Miescher-Institut, Switzerland)
Tenascin-X, a novel member oftenascin family, has recently been shown tobe distinct and its distribution is often reciprocal to that of tenascin-C in thedeveloping mouse embryo. We have investigated the expression of tenascinX in fibroblasts and carcinoma cells. Tenascin-X protein was secreted in vitro
in the conditioned media at an apparent molecular weight of 450 kDa, andfibroblasts contained a major tenascin-X isoform of 220 kDa. On Northernblots, a single major transcript with a size of 13 kb was detected. Overexpression of tenascin-X protein was not found in primary fibroblasts of knockout
mice for tenascin-C gene. Steroid hormone glucocorticoids, were found todownregulate tenascin-X mRNA levels and protein synthesis in fibroblasts at
the physiological concentrations. On the other hand, neither growth factors
nor cytokines examined affected the expression level of tenascin-X.
As in vivo study, carcinoma cells were transplanted into nude mice. In
contrast to the distinct tissue distribution of tenascin-X in adult skin, expression of tenascin-X protein during tumorigenesis was found to be downregulated considerably not only in tumor cells themselves but also in tumor
stroma. These findings provide evidence that the expression of tenascin-X
can be influenced by stromal-epithelial interactions. We have identified
glucocorticoids as physiological inhibitors for tenascin-X and suggest that
glucocorticoids may in part participate in the downregulation of tenascin-X
in fibroblasts in vivo. For details, see Ref. 3.
(6)

Codon Usage Tabulated from the International DNA
Sequence Databases

Yasukazu NAKAMURA, Takashi GOJOBORI and Toshimichi IKEMURA
Codon usage in 87,602 genes was calculated using the nucleotide sequence
data obtained from the GenBank Genetic Sequence Data Bank (Release 90.0;
Sep., 1995). The database is called the CUTG Database. To reveal thecharacteristics of codon use of a wide range of organisms, as well as viruses
and organella, the frequency (per one thousand) of codon use in 461

80organisms for each of which more than 20 genes were available was calculated by summing up numbers of codons used. WWW clients, such as NCSA
Mosaic and Netscape, can be used to query this file. A user can display acodon usage table by clicking an anchor for selecting species or searchingwith species' name. Complete form of the database is available by anonymous ftp from DDBJ: ftp://ftp.nig.ac.jp/pub/db/codon/GB90. The frequencies of codon use in the 461 organisms can be accessed on the following
WWW server: http://tisun4a.lab.nig.ac.jp/codon/CUTG.html. For details, see
Ref. 2.
(7)

A Genetic Affinity Analysis of Human Populations

Naruya SAITOU
Genetic affinity of human populations based on allele frequency data wasstudied from two viewpoints. (I) The effect of the number of polymorphic
loci on the reconstruction of a phylogenetic tree of human populations was
empirically investigated. Affinity networks (unrooted trees) were constructed based on data for 1-12 polymorphic loci, by using the neighborjoining method. Geographical clustering of populations gradually appeared
while the number ofloci was increased. A new classification and terminologyof higher order human population clusters is proposed based on those andother studies. (2) A new method of estimating absolute divergence time of
two populations is proposed, which is based on a diffusion equation that
describes random genetic drift. For details, see Ref. 5.
(8)

Methods for Building Phylogenetic Trees of Genes and Species

Naruya SAITOUThis chapter deals with phylogenetic trees of genes and species, that are
fundamental not only for evolutionary studies but for molecular biology in
general. The mathematical properties of phylogenetic trees such as thedifference between rooted and unrooted trees and the number of possible tree
topologies are first explained. Then the biological properties of phylogenetic
trees in general is discussed with special reference to the difference between
gene trees and species trees. The next section gives description of various

D. DEPARTMENT OF POPULATION GENETICS

81

tree-building methods such as the unweighted pair group method witharithmetic mean (UPGMA), the neighbor-joining, the maximum parsimony,and the maximum likelihood methods with worked-out examples. Resultsfrom computer simulation studies and statistical tests of estimated phylogenetic trees follow. Introduction of various computer packages for tree-building
analyses and future trends are given at the end. For details, see Ref. 6.
(9) Application of a Parallel Logic Programming for Reconstruction of
Molecular Phylogenetic Trees Using the Maximum Likelihood Method
Satoshi OOTA!, Naruya SAITOU and Susumu KUNIFUJI 1
Institute of Science and Technology)

eJapan Advanced

With rapid increase of DNA sequence data, it is required to develop
reliable application programs to infer molecular phylogenetic trees in parallel
environment. To implement a practical maximum likelihood method inparallel environment, we have developed programs Contour!] and Traverse!3
to calculate likelihood values of arbitrary molecular phylogenetic trees.
KLIC/KLl, a parallel logic programming language, was used for those
programs. Contour!] generates log likelihood surfaces and we can investigate
the surface to reach the maximum likelihood point, while Traverse!3 searches
the maximum likelihood values by traversing branches. We propose ideas
how to implement the maximum likelihood method in a parallel environment.
For details, see Ref. 7.
(10) Mitochondrial DNA Sequences of Various Species of the Genus
Equus with a Special Reference to the Phylogenetic Relationshipbetween Przewalskii's Wild Horse and Domestic Horse
Nobushige ISHIDA 1, Tsendsuren OYUNSUREN 2 , Suguru MASHIMA 1, Harutaka MUKOYAMA 1 and Naruya SAITOU Equine Research Institute, Japan
Racing Association, Japan, 2 Department of Molecular Biology, Institute of
Biotechnology, Mongolia)e

The non-coding region between tRNA Pro and the large conserved sequence
block is the most variable region in mammalian mitochondrial DNA D-Ioop
region. This variable region (ca. 270bp) of four species of Equus, including

82

Mongolian and Japanese native domestic horses as well as Przewalskii's (orMongolian) wild horse, were sequenced. These data were compared with ourrecently published Thoroughbred horse mitochondrial DNA sequences. The
evolutionary rate of this region among the four species of Equus wasestimated to be 2-4 X 10- 8 per site per year. Phylogenetic trees of Equus
species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number
change (probably increase) of the Przewalskii's wild horse occurred rather
recently. For details, see Ref. 8.
(11)

A Genetic Study of 2,OOO·Year-Old Human Remains fromJapan Using Mitochondrial DNA Sequences

Hiroki OOTAl, Naruya SAITOU, Takayuki MATSUSHITA 2 and Shintaroh
Department of Biological Sciences, Graduate School of Science,
UEDA'
University of Tokyo, 2 Doigahama Anthropological Museum)e

We present nucleotide sequence data for mitochondrial DNA extracted
from ancient human skeletons of the Yayoi era (ca. 2,000 BP) excavatedfrom the Takuta-Nishibun site in northern Kyushu of Japan. Nucleotide
sequence diversity showed that the Yayoi people of the Tak excavated fromthe Takuta-Nishibun site in northern Kyushu of Japan. Nucleotide sequence
diversity showed that the Yayoi people of the Takuta-Nishibun site were nota genetically homogeneous population. This site shows a diversity in theburial style. Phylogenetic analysis indicated a statistically significant correlation between burial style and the genetic background of the Takuta-Nishibun
individuales, and revealed no discrete clustering patterns for the Yayoi
individuales, for early modern Ainu, or for the Jomon people. For details, see
Ref. 9.

Pu blications
1.

FUKAGAWA, T., NAKAMURA, Y., OKUMURA, K., ANDO, A., INOKO, H., SAITOU, N. and
IKEMURA, 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.
2.

NAKAMURA, Y., WADA, K., WADA, Y., DOl, H., KANAYA, S., GOJOBORI, T. and

D.

3.

4.

5.
6.

7.

8.

9.

10.

II.

12.

DEPARTMENT OF POPULATION GENETICS

83

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., in press.
FUKAGAWA, T, SUGAYA, K., MATSUMOTO, K., OKUMURA, K., ANDO, A., INoKo, H.
and IKEMURA, T: A boundary of long-range G+C% mosaic domains in the human MHC
locus; Pseudoautosomal boundary-like sequence exists near the boundary. Genomics, 25,
184-191, 1995.
SAITOU, N.: A genetic affinity analysis of human populations. Human Evolution, Vol. 10,
No. I, pp. )7-33, 1995.
SAITOU, N.: Methods for building phylogenetic trees of genes and species. In Griffin H.
and Griffin A. (eds.), Molecular Biology: Current Innovations and Future Trends, Horizon
Scientific Press, Wymondham, U.K., pp. 115-135, 1995.
OOTA, S., SAITOU, N. and KUNIFUJI, S.: Application ofa parallel logic programming forreconstruction of molecular phylogenetic trees using the maximum likelihood method. In
Chikawama T., Nakashima H. and Tick E. (eds.), ICLP'95 Workshop on Parallel Logic
Programming, Department of Electronic Engineering, University of Tokyo, pp. 61-72,
1995.
ISHIDA, N., OYUNSUREN, T., MASHIMA, S., MUKOYAMA, H. and SAITOU, N.: Mitochondrial DNA sequences of various species of the genus Equus with a special reference tothe phylogenetic relationship between Przewalskii's wild horse and domestic horse. Journalof Molecular Evolution, Vol. 41, pp. 18(}-188, 1995.
OOTA, H., SAITOU, N., MATSUSHITA, T. and UEDA, S.: A genetic study of 2,000-year-old
human remains from Japan using mitochondrial DNA sequences. American Journal of
Physical Anthropology, Vol. 98, pp. 133-145, 1995.
JIN, F., SAITOU, N., ISHIDA, T., SUN, c.-S., PAN, I.-H., OMOTO, K. and HORAI, S.:
Genetic polymorphism of ADA in Taiwan ethnic groups. Anthropological Science, Vol.
103, No.3, pp. 227-234, 1995.
UMETSU, K., YUASA, I., HARADA, A., SUZUKI, T., PAN, I-H., ISHIDA, T., SAITOU, N. and
HORAI, S.: Orosomucoid phenotyping with monoclonal antibodies: polymorphic occueernce of ORMI*QO in aboriginal Taiwaniese populations. Human Heredity, Vol. 45, pp.
181-185,1995.
KITAKAMI, H., SHIN-I, T., IKEo, K., UGAWA, Y., SAITOU, N., GOJOBORI, T. andTATENO, Y.: Yamato and Asuka: DNA database management system. Proceedings of the
28th Annual Hawaii International Conference on System Sciences-1995, pp. 72-80, 1995.

84

D-c.
(1)

Division of Theoretical Genetics

A Genetic Perspective on the Origin and History of Humans

Naoyuki TAKAHATA
Recent topics in molecular anthropology are reviewed with special reference to hominoid DNA sequences and population genetics theory. To covera wide range of demographic situations in the human lineage since the
Miocene, a model is introduced that allows temporal changes in population
structure and size. The coalescence process of neutral genes is formulated
and used to make quantitative inferences on the origin and history ofhumans. Nuclear DNA sequence data support the theory that humans andchimpanzees diverged from each other 4.6 million years (my) ago and thegorilla lineage branched off as early as 7.0-7.4 my ago. The same data
estimate the effective size of the Pliocene hominoid population as lOS, a figure
similar to that obtained independently from alleles that have persisted in thehuman population for more than 5 my. Hypotheses about the origin of Homosapiens, genetic differentiation among human populations, and changes in
population size are quantified. None of the hypotheses seems compatible withthe observed DNA variation. The effective size decreased to 104 in the
Pleistocene, suggesting an important role of extinction/restoration in H.
sapiens populations. Natural selection against protein variation might berelaxed in the Pleistocene. The history of H. sapiens appears to have been
more dynamical than is postulated by the hypotheses. For the details, see
Ref. 1.
(2)

MHC Diversity and Selection

Naoyuki TAKAHATA
The major histocompatibility complex (Mhc) contains a family of genes
whose products play crucial roles in self and nonself discrimination. Self is
one's own peptides that bind to Mhc molecules and restrict the T-cell
repertoire in the thymus, whereas nonself is processed peptides of foreign
antigens that can be presented in the context of Mhc molecules and trigger

D. DEPARTMENT OF POPULATION GENETICS85

the immune response. Since the immune system thus controlled by Mhc
molecules affects the survival of individuals, the dual function of Mhc itself
must be subjected to natural selection. The operation of natural selection
calls for the necessity of studying Mhc genes from a viewpoint of evolutionary population genetics. This paper is concerned with convergence insequence motifs in the peptide-binding region (PBR) and T-cell mediatedevolutionary forces for Mhc molecules. For the details, see Ref. 2.
(3)

Divergence Time and Population Size in the Lineage
Leading to Modern Humans

Naoyuki TAKAHATA, Yoko SATTA and Jan KLEIN
We have developed maximum likelihood (ML) methods for comparisonsof nucleotide sequences from unlinked genomic regions. In the case of asingle species, the ML method primarily estimates the effective population
size (Ne ) under both constant size and abrupt expansion conditions. In thecase of two or three species, the ML method simultaneously estimates the
species divergence time and the effective size of ancestral populations. This
allows us to trace the evolutionary history of the human population over the
past several million years (my). Available sequences at human autosomal
loci indicate N e = 10,000 in the Late Pleistocene, a figure concordant with theresults obtained from mitochondrial DNA sequence and allele-frequency
data analysis, and there is no indication of population expansion. The ML
analysis of two species shows that humans diverged from chimpanzees 4.6 myago and that the human and chimpanzee clade diverged from the gorilla 7.2
my ago. Furthermore, the effective population size of humans more than 4.6
my ago is nearly ten times larger than N e of modern humans. The effective
population size in the human lineage does not seem to have remained
constant over the past several million years. The ML model for three species
predicts slightly results different from, but consistent with those obtained by
the two-species analysis. For the details, see Ref. 3.

86

(4)

A Population Genetic Study on the Evolution of SINEs.
II. Sequence Evolution under the Master Copy Model

Hidenori TACHIDA
A transient population genetic model of SINE evolution which assumes themaster copy model was theoretically investigated. The means and variancesof consensus frequency, nucleotide homozygosity, and the number of shareddifferences considered to have been caused by mutations occurring in mastercopy lineages were computed. All quantities investigated were shown to be
monotone functions of the duration of the expansion period. Thus, they canbe used to estimate the expansion period although their sampling varianceswere generally large. Using the theoretical results, the Sb subfamily ofhuman Alu sequences was analyzed. First, the expansion period was estimated from the observed mean and variance of homozygosity. The
expansion period was shown to be short compared to the time since the endof the expansion of the subfamily. However, the observed number of shareddifferences was less than one half of that expected under the master copy
model with the estimated expansion period. Alternative models including one
with multiple master copy loci as explanation of this observation werediscussed.
(5) Molecular Evolution of the 5'-flanking Regions of Duplicated
Amy Genes in the Drosophila melanogaster Species Subgroup
Eisaku OKUYAMA, Hiroki SHIBATA, Hidenori TACHIDA and Tsuneyuki
YAMAZAKI
The nucleotide sequences of the 5 ' -flanking regions of duplicated Amy
genes in eight sibling species belonging to the melanogaster species subgroupwere analyzed. In Drosophila melanogaster, about 450bp immediately upstream of the translation initiation site of the two paralogous genes (theproximal and distal genes) have sequence similarities. However, we could
not detect any significant sequence similarity in the upstream region beyond~ 450. This result indicates that the coding regions of the ancestral Amy gene
were duplicated together with 450 bp of the 5 ' -flanking region as one unit.
Multiple alignment of these 450 bp sequences in the proximal and distal genesof all eight species revealed a mosaic pattern of highly conserved andD.

DEPARTMENT OF POPULATION GENETICS

87

divergent regions. The conserved regions included almost all the putative
regulatory elements identified in previous analyses of the sequences. A
phylogenetic analysis of the aligned sequences shows that these 450 bp
sequences are clustered into proximal and distal groups. As a whole, thedivergence between groups in this region is very large in contrast to that in
the coding regions. Based on the divergence between groups, the 450 bp
region is divided into two subregions. We found that ratios of the divergence
between groups to that within groups differ in the two subregions. Fromthese observations, we discuss the possibility of positive selection acting on
the subregion immediately upstream of the Amy coding region to diverge
regulatory elements of the paralogous genes.
Publications
1.

2.
3.
4.

TAKAHATA, N.: A genetic perspective on the origin and history of humans. Annual
Review of Ecology and Systematics, 26, 343-372, 1995.
TAKAHATA, N.: MHC diversity and selection. Immunological Review, 143, 221-247,
1995.
TAKAHATA, N., SATTA, Y. and KLEIN, J.: Divergence time and population size in thelineage leading to modem humans. Theor. Popul. BioI., 48, 198-221, 1995.
HORAI, S., HAYASAKA, K., KONDO, R., TSUGANE, K. and TAKAHATA, N.: The recent
African origin of modem humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc. Natl. Acad. Sci. USA, 92, 532-536, 1995.

88

E.

DEPARTMENT OF INTEGRATED GENETICS
E-a. Division of Human Genetics(1)

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 lFirst Department of Medicine, Kyushu University)e

Chromosome 18 is involved in tetrasomy 18p and in trisomy 18 syndromes.
The latter is the second most common abnormality after Down syndrome
which is caused by an excessive number of chromosome. To identify genes
involved in these disorders, construction of high-resolution expression map of
this chromosome is a rational strategy. Identification and recovery oftranscribed sequences from cloned mammalian genomic DNA remains animportant problem in isolating genes on the basis of their chromosomal
location. We have developed a strategy that facilitates the recovery oftranscribed sequences from random pieces of cloned 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 andsimultaneous fluorescence in situ hybridization. In the further extension ofthis approach, eDNA library has been probed with pools of microclones froma cosmid clone. Our approach to generate region-specific eDNA libraries
used hybridization of cDNA library inserts to genomic DNA from cosmids,
and subsequent cloning of annealed cDNAs. In one application of thescheme, human fetal brain cDNA library was hybridized to linker-adaptor
ligated fragments of a digested cosmid, and selected cDNAs are 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. The ultimate goal would be an integrated genetic, physical and transcriptional map of the genome, combining
information on the position of a gene, its sequences and its pattern ofexpression. Since most genes are composed of multiple exons and theE. DEPARTMENT OF INTEGRATED GENETICS

89

identification of a gene requires the recovery of only a single exon, thisconsideration should not be a limiting factor. These candidate exons are
ideally suited for establishing the presence of a gene in a cosmid insert andfacilitating the subsequent isolation of this gene. Our current experiencesuggests that as many as 20 cosmids can be screened concurrently in a 4 week
period, and that everyone or two cosmids contain at least one transcribedsequence. Screening of uncharacterized cosmids will determine the utility oftranscribed sequence fragments of known chromosomal location, as identifiers of candidate coding sequences. For the details, see reference 1.
(2) Isolation of Transcribed Sequences by Kinetic Enrichment ofcDNA Fragments Derived from Chromosome 15 and MolecularDefinition of the Prader-Willi Syndrome
Takashi IMAMURA, Masako SAKAI, Rie INABA and Tomoko HASEGAWA l
CShizuoka 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 q ll-q 13 that involve the paternally derived
chromosome 15. Non-deletion PWS patients exihits maternal disomy forchromosome 15 further demonstrating that loss of the expressed paternalalleles of maternally imprinted genes is responsible for the PWS phenotype.
Conversely, maternally derived deletions of this region or paternal disomy areassociated with the Angelman syndrome (AS). AS can also be caused bypaternal disomy or maternally inherited as yet unidentified submicroscopic
mutations. Thus, PWS and AS are distinct neurogenic disorders, caused by
the loss of function of closely linked genes on chromosome 15. Paternaldeletion and maternal disomy in PWS suggest that the PWS genes aretranscribed from the paternal chromosome only. Likewise, maternal dele-90

tions and paternal disomy in AS suggest that the AS genes are transcribedfrom the maternal chromosome only.
Lymphoblastoid cell lines from 5 PWS patients and one AS patient andthose from healthy individuals were made in this laboratory. Messenger
RNAs were prepared from the cells, from which the cDNA libraries wereconstructed. 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 as compared to the genomic DNAs resulted in theisolation of 13 probes present as single copies in the normal library. These
probes were 120--500 base pairs in length, which could detect difference
between transcribed sequences of patient's cells and those of normal individuals. In principle, this system was used for isolating probes linked tomaternally imprinted genes on the chromosome 15 PWS region. Results ofinitial analysis of mRNAs from lymphoblastoid cell lines implied that thesesequences are transcribed in the normal individuals but not in PWS patients.
A mechanism of imprinting may have evolved in mammals because of
conflicting interests of maternal and paternal genes in relation to the transfer
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, suckling, neonatal behavior, appetite control,
nutrient metabolism, and postnatal growth. Perhaps imprinted gene in PWS
region, and in Angelman region as well, is just such a gene. The mapping ofgenes identified here to within the PWS critical region, together with theindications that they function in the brain and may playa role in regulating
feeding behavior, should prompt us further to predict these genes as a
candidate imprinting loci responsible for syndrome.

E. DEPARTMENT OF INTEGRATED GENETICS(3)91

A Signal Transduction Pathway of Interleukin-4 and Interleukin-13
in Human B Cells Derived from X-linked Severe Combined
Immunodeficiency Patients

Kenji IZUHARA, Takashi IMAMURA, Toshio HElKE!, Yoshiyuki Nmo 2 andNobuyuki HARADA 3 CInstitute of Medical Science, Tokyo University, 2 First
Department of Medicine, Kyushu University, 3 Research Institute of Tuberculosis)
Interleukin-4 (IL-4) activates members of a few tyrosine kinase family,
JAKI and JAK3, and a cytoplasmic protein termed STAT (signal transducers and activators of transcription) by tyrosine phosphorylatioion. The IL-4
receptor has been demonstrated to share the common gamma chain (gc) with
IL-2, IL-7 and IL-9 receptor systems. Mutations of gc are known to result inhuman X-linked severe combined immunodeficiency (XSCID). Since JAK3
has been shown to associate with gc, we analysed activation of JAK~STAT
pathway in EBV-transformed B cells established from two XSCID patients(XSCID-B) upon IL-4 stimulation in order to investigate the involvement of
gc in IL-4 mediated signal transduction. We detected no tyrosine phosphorylation of JAK3 in two XSCID-B cell lines, whereas tyrosine phophorylation of JAK3 in EBV-transformed normal B cell lines was clearly detected.
Using electrophoretic mobility shift assay, we found that STAT was activatedby IL-4 in normal B cells, but not in XSCID-B cell lines. These results
indicate that JAK-STAT pathway in IL-4 signal transduction is impaired in
XSCID, further suggesting that gc plays a pivotal role in activation of JAKSTAT pathway by IL-4. For the details, see Ref. 1.
(4)

A Synthetic Peptide Corresponding to a Critical Intracellular
Signalling Region of the Human IL-4 Receptor Inhibits
IL-4-Dependent Proliferation

Kenji IZUHARA, M. SAKAI, R. INABA, T. IMAMURA and N. HARADA'
CResearch Institute of Tuberculosis)
We have identified the critical region for growth signal transduction in thecytoplasmic domain of the human IL-4 receptor (hIL-4R). IL-4R is likely to
associate with other signal transducing molecules through this critical cytoplasmic region. We tested how a synthetic peptide (SP-I), corresponding to92this critical cytoplasmic region interfered with IL-4-dependent proliferationby competition with the IL-4R in binding to intracellular signal transducing
molecules. Our results indicated that lOOmg/ml of SP-l peptide completely
inhibited hIL-4-induced proliferation of Ba/F3 cell transfectants expressing
the full-length hIL-4R. In contrast, an alternated synthetic peptide corresponding to the C-terminal region of the hIL-4R (SP-2) did not affect hIL-4induced proliferation of hIL-4R-Ba/F3 transfectants. The specificity of SP1 mediated inhibition of IL-4-induced proliferation was further supported by
the fact that SP-l peptide had no effect on IL-3-induced proliferation of thesame hIL-4-Ba/F3 transfectants. In addition, SP-l peptide did not affecteither IL-2-induced proliferation of Ba/F3 transfectants expressing thehuman IL-2 receptor b chain (hIL-2Rb), or hIL-4-induced proliferation of
Ba/F3 transfectants expressing a chimeric receptor consisting of a hIL-4R
extracellular domain and a hIL-2Rb cytoplasmic domain. For the details, see
Ref. 2.
(5) Recent African Origin of Modern Humans Revealed byComplete Sequences of Hominoid Mitochondrial DNAs
Satoshi HORAI, Kenji HAYASAKA, Rumi KONDO!, Kazuo TSUGANE andNaoyuki TAKAHATA 1 CThe Graduate University for Advanced Studies)
Extensive analyses of cleavage sites and sequences of mitochondrial DNA(mtDNA) have shown that African is most variable among various ethnic
groups, and have supported an earlier suggestion that the last common
ancestor (LCA) of contemporary human mtDNAs existed some 200,000
years ago. These results have been taken as evidence for the notion thatmodern humans originated in Africa and migrated to Eurasia, replacing
Homo erectus and the Neanderthals with no or little gene exchanges. However, this notion was strongly disputed by proponents of the multiregional
hypothesis, who claim that modern humans originated simultaneously invarious geographic regions and that regional continuity in human charactershas evolved over the past 1 million years (Myr). Under this hypothesis, theage of the LCA is unlikely to be as low as 200,000 years. The main
controversy, therefore, has centered around the estimated age of the LCA
and the reliability of the mitochondrial molecular clock (constancy of
nucleotide substitution rates) on which it is based. To resolve close relation-

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DEPARTMENT OF INTEGRATED GENETICS

93

ships among humans, recent studies have focused on sequence analysis in the
rapidly evolving D (displacement)-loop region of mtDNA. Sequence datafrom the D-loop region alone are insufficient for the accurate estimation ofsubstitution rates and divergence times of hominoid mtDNAs, and sequencesfrom coding regions are required. To this end, we have analyzed thecomplete mtDNA sequences of three humans (African, European and Japanese), three African apes (common and pygmy chimpanzees, and gorilla),
and one orangutan in an attempt to estimate most accurately the substitution
rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated with an approximately clock-like regularity. From these substitutions and under the assumption that the orangutan and African apes diverged 13 million years ago, we
obtained a divergence time for humans and chimpanzees of 4.9 million years.
This divergence time permitted calibration of the synonymous substitution
rate (3.89 X 1O-8!site!year). To obtain the substitution rate in the D-loop
region, we compared the three human mtDNAs and measured the relative
abundance of substitutions in the D-loop region and at synonymous sites.
The estimated substitution rate in the D-loop region was 7.00 X 1O-8!site!year.
Using both synonymous and D-loop substitutions, we inferred the age of thelast common ancestor of the human mtDNAs as 143,000± 18,000 years. The
shallow ancestry of human mtDNAs, together with the observation that theAfrican sequence is most diverged among humans, strongly supports therecent African origin of modern humans, Homo sapiens sapiens. For thedetails, see Refs. 15, 16 and 17.
(6)

Different Waves of Migration to the New World: Implications ofMitochondrial DNA Polymorphism in Native Americans

Satoshi HORAI
Mitochondrial DNA sequences of the major noncoding region (482 basepairs) were determined for 72 Native Americans from 16 different local
populations in the South, Central and North Americas. On the basis of asequence comparison, 43 different types were observed. Phylogenetic analysis
revealed that most Native American lineages are classified into four major
distinct clusters. Individuals belonging to each cluster share at least two
specific polymorphic sites that are nearly absent in other human populations,

94

indicating a unique phylogenetic position of Native Americans. Phylogenetic
analysis was extended with 193 individuals including Africans, Europeans,
Asians and Native Americans, indicating that the four Native American
clusters are distinct and dispersed in the tree. Although a majority ofmembers are Native Americans, there are some Asian individuals in eachcluster. For example, in cluster II, one Japanese appeared together with two
Native Americans in a small cluster which diverged at a final branching
point. This coalescence occurs at only 7% in length of the total way from thetips to the root of the tree. As the Japanese and the Native Americans resideon two different continents, ancestors of these Native Americans must havemigrated after the time of initial coalescence of the two lineages. Assumingthat the human mitochondrial lineage divergence took place 143,000-179,000
yr. RP. (Horai et al. 1995), the dates of this initial coalescence were
estimated as 10,000-12,500 yr. RP. The same magnitude of coalescence
between the Asians and Native Americans are also observed in other threeclusters. The phylogenetic tree also suggests that the four distinct clusters
containing Native Americans may represent different waves of migration to
the New World. The coalescence time within each cluster is less than
one-fourth of the coalescence time for the entire tree. However, the coalescence time of the four clusters is a little longer than half the latter coalescencetime. It appears more likely that individuals in the four clusters aredescendants of four different ancient populations, which had been wellisolated from each other for a relatively long period of time. I therefore
postulate that the four clusters represent different waves of migration to the
New World. Although it is difficult to determine the times of occurrence ofthe four waves of migration, they seem to have taken place 10,000-12,500 yr.
B.P. For the details, see Ref. 18.
(7)

Y Chromosomal DNA Variation and the Peopling of Japan

Michael F. HAMMER l and Satoshi HORAI

eArizona University)

Four loci mapping to the nonrecombining portion of the Y chromosome
were genotyped in three Japanese populations (Okinawa, Shizuoka and
Aomori) and Taiwanese. The Y Alu polymorphic (YAP) element is present
in 42% of the Japanese and absent in the Taiwanese, confirming the irregular
distribution of this polymorphism in Asia. Data from the four loci were used

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DEPARTMENT OF INTEGRATED GENETICS95

to determine genetic distances among populations, construct Y chromosome
haplotypes, and estimate the degree of genetic diversity in each population.
Evolutionary analysis of Y haplotypes suggests that polymorphisms at theYAP (DYS287) and DXYS5Y loci originated a single time, whereas restriction patterns at the DYS I locus and microsatellite alleles at the DYS 19 locus
arose more than once. Genetic distance analysis indicated that the Okinawans are differentiated from Japanese living on Honshu. The data support
the hypotheses that modern Japanese populations have resulted from distinctive genetic contributions involving the ancient Jomon people and Yayoi
immigrants from Korea or mainland China, with Okinawans experiencing
the least amount of admixture with the Yayoi. It is suggested that YAP( +)chromosomes migrated to Japan with the Jomon people> 10,000 years ago
and that a large infusion of YAP( -) chromosomes entered Japan with the
Yayoi migration starting 2,300 years ago. Different degrees of genetic
diversity carried by these two ancient lineages may be explained by thedifferent lifestyles of the migrant groups, the size of the founding populations,and the antiquities of the founding events. For the details, see Ref. 19.
(8)

Molecular Phylogeny of Gibbons Inferred from Mitochondrial
DNA Sequences: Preliminary Report

Seiji HAYASHI!, Kenji HAYASAKA, Osamu TAKENAKA 2 and Satoshi HORAI
CDepartment of Planetary and Earth Sciences, School of Science, Nagoya
University, 2 Primate Research Institute, Kyoto University)
A large amount of data have been accumulated to elucidate phylogenetic
relationships within Hominoidea (humans and apes). These studies revealed
that the closest relative to human are African apes, ultimately chimpanzees.
The phylogenetic position of gibbons (family Hylobatidae) as a sister group
to human and great apes was also supported by these studies. Gibbons, genus
Hylobates, inhabit in a wide range of Southeast Asia, from eastern India tosouthern China on the mainland, as well as Borneo, Java, Sumatra, and theMentawai islands. Nine nominal species are generally accepted. They are theconcolor gibbon (H. concolor, lives in Vietnam, Laos, Yunnan, and Hainan
Island), the siamang (H. syndactylus, in Sumatra and Malaya), the hoolock
gibbon (H. hoolock, in Assam, northern Burma, and Bangladesh), the Kloss's
gibbon (H. klossii, in the Mentawai Islands), the white-handed gibbon (H.

96lar, in Thailand, southeastern Burma, Malaya, and northern Sumatra), theagile gibbon (H. agilis, in Malaya, central and southern Sumatra, andsouthwestern Borneo), the pileated gibbon (H. pileatus, in southwestern
Thailand and Kampuchea), the silvery gibbon (H. moloch, in Java), andMuller's gibbon (H. muelleri, in Borneo). The former three species areplaced in monotypic subgenera Nomascus, Symphalangus and Bunopithecus,
respectively. The latter six species are classified into the subgenus Hylobates.
In the subgenus Hylobates, the latter five species, lar, agilis, pileatus, moloch,and muelleri, are called the lar-group or lar species complex, because theyshare derived morphological characters, such as very dense fur and largeears. klossii is excluded from lar-group due to lack of the synapomorphic
characteristic of the lar-group. We analyzed the 896 base-pair (bp) mitochondrial DNA fragment comprising partial sequences of ND4 and ND5(NADH dehydrogenase complex) genes and adjacent three complete tRNA
genes (tRNA Hi" tRNALeu(CUN), and tRNASer(AGY») from seven gibbons, representative of three out of four subgenera. A result from our molecularanalysis is consistent with the previous studies as to the monophyly ofsubgenus Hylobates species, yet the relationship among subgenera remains
slightly ambiguous. A striking result of the analysis is phylogenetic locationof the Kloss's gibbon (H. klossii). The Kloss's gibbon has been considered to
be an initial off-shoot of the subgenus Hylobates because of its morphologicalprimitiveness. However our molecular data strongly support the Kloss's
gibbon speciated most recently within the subgenus Hylobates. For thedetails, see Ref. 20.
(9)Orosomucoid Phenotyping with Monoclonal Antibodies:
Polymorphic Occurrence of ORMl*QO in Aboriginal
Taiwanese Populations

Kazuo UMETSU!, Isao YUASA 2 , Akira HARADA l , Tsuneo SUZUKI!, I-Hung
PAN 3, Takafumi ISHIDA4, Naruya SAITOU 5 and Satoshi HORAI CYamagata
University School of Medicine, 2 Tottori University School of Medicine,
3 National Taiwan University School, 4 University of Tokyo, 5 Division of
Evolutionary Genetics)
Human orosomucoid (ORM) or at-acid glycoprotein is a major acutephase plasma protein with a molecular mass of approximately 49 kD, of

E. DEPARTMENT OF INTEGRATED GENETICS

97

which 45 % is carbohydrate. ORM is predominantly produced in the liverand suppresses blastogenesis of lymphocytes and neutrophil activation.
ORM shows extensive genetic variation, and about 30 alleles have been
described at the two loci ORMl and ORM2. The ORMl and ORM2
alloproteins are encoded by two closely linked genes on the long arm ofchromosome 9 and are distinguishable from each other in band intensity. In
the course of ORM phenotyping, we have occasionally encountered somebands for which it was difficult to determine whether they belonged to the
ORMl or ORM2 product. To overcome such confusions, we attempted toprepare locus-specific monoclonal antibodies. Using polyclonal and monoclonal antibodies, we performed ORM phenotyping on native Taiwanese
populations and carried out ORM I QO typing.
Three monoclonal antibodies (OR35, OR40 and OR48) against orosomucoid (ORM) were prepared for the phenotyping of the human ORM system.
The OR35 and OR48 antibodies recognized ORMI and ORM2 products,
respectively. OR40 reacted strongly to the products of ORMI but poorly tothose of ORM2. With the help of these monoclonal antibodies, ORM
phenotyping was performed on 658 individuals from nine subpopulations ofaboriginal Taiwanese, with close attention to two individuals with an ORMI
QO homozygous phenotype. The ORMl*QO allele was found to be at apolymorphic frequency in eight of the nine subpopulations. For the details,
see Ref. 21.
(10)Genetic Polymorphism of ADA in Taiwan Aboriginal Populations:
New ADA Variants Detected by Isoelectric Focusing Method

Feng JINt, Naruya SAITOU 2 , Takafumi ISHIDAt, Cheih-Shan SUN 3 , I-Hung
PAN4, Keiichi OMOT0 1 and Satoshi HORAI CUniversity of Tokyo, 2 Divisionof Evolutionary Genetics, 3 Taidong Hospital, Taiwan, 4 National Taiwan
University, Taiwan)
Human adenosine deaminase (ADA) catalyzes the conversion of adenosine to inosine with the release of ammonia. Genetic polymorphism of ADA
has been recognized by Spencer et aJ. (1968) by means of starch-gel electrophoresis. In the following two decades, several new genetic variants of ADA
were recognized by the same method. In the past, the investigation of the
ADA polymorphism has been carried out in most human population studies

98

using starch gel electrophoresis. In the present study, we improved themethod in typing of ADA by employing isoelectric focusing (lEF). Nine
aboriginal populations (Gaoshan) of Taiwan including 654 individuals were
investigated. The improved method led us to find two new variants, whichwere tentatively namedADA*Taiwanl andADA*Taiwan2. It was also foundthat one of the common alleles ADA*2, had relatively high frequencies insome (the Paiwans and the Amis) of the Taiwan aboriginal populations. For
the details, see Ref. 22.
Publications
1.

2.

3.

4.

5.

6.

7.
8.
9.

10.

II.

NAKASHIMA, H., HASEGAWA, T., SAKAI, M., INABA, R. and IMAMURA, T.: Identificationof iso( 18p) marker chromosome by fluorescence in situ hybridization with single-copy
DNA probe. Jpn. J. Human Genet., 40, 185-188, 1995.
IZUHARA, K., HElKE, T., OTSUKA, T., YAMAOKA, K., IMAMURA, T., MAYUMI, M.,
NIHO, Y. and HARADA, N.: Signal transduction pathway of interleukin-4 and interleukin13 in human B cells derived from X-linked severe combined immunodeficiency patients. J.
BioI. Chern., 271, 619-622, 1996.
IZUHARA, K., SAKAI, M., INABA, R., IMAMURA, T., HOWARD, M. and HARADA, N.: A
synthetic peptide corresponding to a critical intracellular signalling region of the human IL4 receptor inhibits IL-4-dependent proliferation. Cell. Immunol., 163, 254-259, 1995.
IMAMURA, T: Mutation at the DNA level and molecular pathology of genetic disorders(in Japanese). In: Human Genetics-Principles and Practice, 2nd Ed., ed. by Yanase, T.,
Kanehara & Co., Ltd., Tokyo, pp. 283-294, 1995.
IMAMURA, T: Prophylaxis and therapy of genetic diseases and congenital disorders (inJapanese). In: Human Genetics-Principles and Practics, 2nd Ed., ed. by Yanase, T,
Kanehara & Co., Ltd., Tokyo, pp. 408-417, 1995.
IMAMURA, T.: Hematological and neoplastic diseases-thalassemia syndrome (in Japanese). In: Practical Guide for Genetic Diagnosis, ed. by Nakai, T., et al., Chugai Igakusha,
Tokyo, pp. 239-244, 1995.
IMAMURA, T.: Genetic diagnosis, present and future (in Japanese). Igakuno Ayumi,
174(5), 357-362, 1995.
IMAMURA, T.: Genetic diagnosis of thalassemia (in Japanese). Rinsho Kensa, 397, 775780, 1995.
HARADA, N. and IZUHARA, K.: IL-4 receptors (CDwI24): structure and function. In:
Leucocyte Typing Y, ed. by Schlossman S. F., et aI., Oxford University Press, pp. 18701873, 1995.
SCHULTZ, C., IZUHARA, K., COFFMAN, R. and HARADA, N.: The critical region in thecytoplasmic domain of human IL-4 receptor for induction of IgE synthesis. Immunol.
Lett., 46, 215-219,1995.
IZUHARA, K., IDEGUCHI, H., OHTSUKA, T., OKUBO, K., OGO, T., HAMASAKI, N. and

E.

12.
13.
14.
15.

16.

17.

18.

19.
20.

21.

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NIHO, Y.: High incidence of a polymorphic variant of erythrocyte membrane protein, band
3-Memphis, on a Japanese western island. Jpn. J. Human. Genet., 40, 265-270, 1995.
IZUHARA, K.: A transcriptional factor induced by IL-4; IL-4 STAT (in Japanese). Rinsho
Meneki, 27, 809-813, 1995.
IZUHARA, K. and HARADA, N.: The signal transduction mechanism of IL-4. Igakuno
Ayumi, 174, 1019-1022, 1995. (in Japanese)
IZUHARA, K.: Signal transductions through IL-4 and IL-7 receptors (in Japanese). Saibo
Kogaku, 14, 1153-1157, 1995.
HORAI, S., HAYASAKA, K., KONDO, R., TSUGANE, K. and TAKAHATA, N.: Recent
African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc. Natl. Acad. Sci. USA. 92, 532-536, 1995.
HORAI, S.: Origin of Homo sapiens inferred from the age of the common ancestral humanmitochondrial DNA. In: The origin and past of modern humans as viewed from DNA. S.
Brenner and K. Hanihara (eds.), World Scientific, pp. 171-185, 1995.
HORAI, S.: Evolution and origins of man: clues from complete sequences of hominoid
mitochondrial DNA. Southeast Asian J. Trop. Med. Public Health 26(Sup. I), 146-154,
1995.
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 and E. Szathmary (eds.),
Oxford University Press, pp. 270--283, 1996.
HAMMER, M. and HORAI, S.: Y chromosomal DNA variation and the peopling of Japan.
Am. J. Hum. Genet. 56, 951-962, 1995.
HAYASHI, S., HAYASAKA, K., TAKENAKA, O. and HORAI, S.: Molecular phylogeny ofgibbons inferred from mitochondrial DNA sequences: Preliminary report. J. Mol. Evol. 41(3),359-365, 1995.
UMETSU, K., YUASA, I., HARADA, A., SUZUKI, T., ISHIDA, T., SAITOU, N. and HORAI, S.:
Orosomucoid phenotyping with monoclonal antibodies: Polymorphic occurrence of
ORM1*QO in Aboriginal Taiwanese populations. Hum. Hered. 45(4),181-185,1995.
JIN, F., SAITOU, N., ISHIDA, T., SUN, C-H., PAN, I-H., OMOTO, K. and HORAI, S.:
Genetic polymorphism of ADA in Taiwan aboriginal populations: New ADA variants
detected by isoelectric focusing method. Antropological Science 103(3), 227-234, 1995.

E-b.
(1)

Division of Agricultural Genetics

Isozyme Variation in Asian Common Wild Rice Oryza rufipogon

Hong-Wei CAl, Xang-Kun WANG l and Hiroko MORISHIMA

CBeijing Agri-cultural University, China)
A total of 106 strains of Asian wild rice, Oryza rufipogon, collected from 13
countries were examined for isozyme variation. Polymorphism at 21 loci was

100

detected using starch and poly-acrylamide gel systems.
The geographical distribution area was divided into four regions, China,
Indochina, the Indian subcontinent and the Malayan Peninsula. Strains from
China were found to carry some alleles at high frequencies which were rarely
found in other regions. Of them, Cat-I 2 , Est-l3 I and Est-l2° are known to be
specific to Japonica cultivars. Genetic distances between the four regions andaverage gene diversities within region were estimated. Genetic distances
ranged from 0.085 (Indian subcontinent and Indochina) to 0.254 (China andthe Malayan Peninsula).
To examine overall variation patterns, data for 21 loci was analyzed byfactor analysis. Variation explained by the first three factors was only 24% of
the total variation, indicating that differentiation within O. rufipogon is not so
distinct at the isozyme level. The first factor extracted a variation characterized by the alleles at Cat-I and Est-l3, which are differentially distributed in
the wild rices of China and other regions and are also diagnostic in classifying
Indica and Japonica cultivars. The second factor distinguished a few strains
carrying a rare allele, Est-Yo The third factor extracted a variation characterized by the alleles at Acp-I and Pox-2, which are also Indica-Japonica
diagnostic loci. A scatter diagram of 106 strains plotted by the first and third
factor scores showed that Chinese strains and the strains from other regions
are separated. Strains from the Indian subcontinent tend to be located in
between the Chinese and Indochina/Malayan Peninsula strains. Perennial andannual types, which are the two major ecotypes, differentiated within thisspecies were not detected at the isozyme variation level.
In order to examine to what extent alleles at different loci are nonrandomly
associated in Asian wild rice, corresponding to Indica-Japonica differentiation in cultivars, we calculated the R 2 (squared correlation coefficient ofallelic frequencies) between 8 Japonica-Indica diagnostic loci. Several combinations showed significant nonrandom associations. But generally the R 2
values were much lower (R2 =0.022) than those found in O. sativa (R 2 =0.382, Sano and Morishima 1992). This indicates that no clear JaponicaIndica differentiation occurs in O. rufipogon.
From the above results, it may be concluded that O. rufipogon has acontinuum at the isozyme level, but tends to be differentiated geograpically.
For the details, see Ref. 2.

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DEPARTMENT OF INTEGRATED GENETICS101

QTL Analysis of Annual/Perennial Life-History Variationin Oryza rujipogon

Joshua R. KOHN! and Hiroko MORISHIMA
Diego)

eUniversity of California at San

The use of molecular markers to identify quantitative trait loci (QTL) has
become a key approach in modern breeding programs. This approach,however, has been little used as a means to understand the genetic basis of
ecologically important adaptation. In this study, we use molecular markers
to map genomic regions controlling trait variation among annual and perennial ecotypes of wild rice, Oryza rujipogon. Annual types exhibit earlyheading, self-fertilizing flowers, high seed yield and low regenerating ability.
Perennial types exhibit late heading, mixed-mating flowers, low seed yieldand high vegetative reproduction.
One hundred and one F 2 offspring from a cross between annual andperennial strains were used. Each F 2 plant was split into two clones andgrown as replications. Various quantitative traits related to life history were
measured. The broad sense heritability of each trait and genetic correlations
among traits were calculated. Each plant was scored for 40 molecularmarkers (one isozyme locus and 39 genomic RFLP probes provided by the
Rice Genome Research Program, Japan and from Cornell University).
Single and multiple regression were used to estimate the proportion ofphenotypic and genotypic variation explained.
Genotype effects were significant for all traits and broad-sense heritabilities
ranged from 0.81 to 0.37. Traits expressed early in the life cycle (e.g. headingdate) or features of highly canalized floral structures (e.g. anther length) had
higher heritabilities than did traits expressed late in the life cycle, or composite traits such as the ratio of panicle number to tiller number. As expected,
many traits were correlated and negative genetic correlations were foundbetween most measures of fertility and vegetative persistence.
Single QTL explained up to 27% of the phenotypic variation in the traits
examined. Multiple regressions explained 7-38% of the phenotypic and 1862% of the genotypic variation in each trait examined. Some QTL appear to
explain significant proportions of variation in several different traits, suggesting the possibility of pleitropic effects. In particular, marker Gl184Ca
located on chromosome 1, explained significant variation in heading date,102height, panicle number/tiller number, panicle length, and post-harvest regeneration.
In many other studies, chromosomal regions controlling large fractions ofphenotypic variation have been found. In contrast, we found only one casein which greater than 25% of the phenotypic variation was explained by asingle marker. One reason was the moderate heritability of the traits understudy. Lack of marker saturation might also have reduced the explanatory
capacity of markers. Finally, ecological adjustments that contribute to
intraspecific annual/perennial differentiation may be more polygenic than thespecies-level differences examined in previous studies. Annual and perennial
ecotypic syndromes reflect differences in allocation to reproduction versus
survival and vegetative spread. Little is known about the genetics ofalternative allocation patterns and the results of this study should be ofinterest to evolutionary and developmental biologists as well as to those
involved in plant breeding.
(3)

Ecological Genetic Studies on American Wild Rice(Oryza glumaepatula Steud.) Distributed aroundthe Amazon Basin

Masahiro AKIMOTOl, Yoshiya SHIMAMOT0 1 and Hiroko MORISHIMA CFac.
Agr., Hokkaido University, Sapporo)

Oryza glumaepatula distributed in the tropical America is a wild-ricespecies having an AA genome in common with cultivated rice and their wild
relatives. Populations of this species growing in the flood plain of the
Amazon, where the annual oscillation of water level is about 10m, show
unique life-history traits. Most probably they propagate mainly by seeds. To
study the influence of life-history on the genetic population structure, we
examined several phenotypic characteristics supposedly related to life history
and allozyme variability of natural populations of O. glumaepatula collectedin the Amazon basin.
a) Germination tests indicated that the kernel dormancy (physiologicaldormancy) is not so strong. However, the seeds showed grain dormancy(physical dormancy) to some extent which restricted germination under deep
water.
b) They had high deep-water tolerance conferred with internode elonga-

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tion and culm brittleness. On the other hand, they seemed to have weak
deep-water resistance. Large amounts of resources are used to vegetative
organs even after heading. This characteristicis is assumed to be necessary
for surviving the flood conditions of the Amazon.
c) O. glumaepatula had well developed anthers and produced a large
amount of pollen, as much as the outbreeding ecotype of Asian O. rufipogon.
But the proportion of resource allocation to anther and stigma was not sohigh as expected from outbreeding plants.
d) Observed heterozygosities were much lower than expected and the
Hardy-Weinberg equilibrium did not hold in the populations. This suggeststhat Amazonian O. glumaepatula developed self-pollination system.
e) Allozymes were not so variable and overall gene diversity was low( = 0.113) as compared with those of Asian wild rice O. rufipogon. Presumably, a bottleneck caused by periodical extinction of populations, scarcenessof inter-populational hybridization owing to a self-pollinating nature and theabsence of related species growing sympatrically may hamper the development of allozyme variability.
f) It was found that gene diversities gradually increased going down fromthe upper to lower basin. This is probably because gene flow has proceeded
in a one-way direction from the upper to lower basin. A low degree ofinter-populational differentiation (D ST = 0.050 and H s = 0.068) was found,
contrary to the expectation that they would be predominantly selfing. The
plants appear to be able to migrate a long distance by seeds drifting on theriver, resulting in frequent inter-regional gene flow in the basin. For thedetails, see Ref. 1.
(4)

Gene Regulation of Two Alleles at the wx Locus of Rice

Hiro-Yuki HIRANO, Mitsugu EIGUCHI and Yoshio SANO
The rice wx locus controls amylose synthesis in the endosperm and has twofunctional alleles, Wx and Wx b , which were initially defined on the basis of
the amount of their gene product, the Wx protein. The Wx allele produces
about 10-fold higher levels of mRNA and protein than Wx b does. Wx b isfound in Oryza sativa Japonica, and Wx is found in O. sativa Indica andvarious wild rice species including o. rufipogon. Since both cultivated rices,
Japonica and Indica, evolved from a wild rice, 0. rufipogon, Wx b is thought
Q

Q

Q

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to have been derived from Wx a• What kind of mutation caused such a largechange in the level of Wx gene expression during the short period of theevolution of Japonica? We have great interest in the molecular mechanism(s) underlying the differences in expression levels of wx a and Wx b as
related to the evolution of gene regulation.
The rice Wx gene has a long intron ( > 1 kb) upstream of the ATG codon.
The DNA sequence (PI-DNA) that includes the promoter region (P) andthe first intron (I) was fused to the GUS gene and GUS activity analyzed ina transient assay system using rice protoplasts. The PI-DNA from wx ashowed about IOO-fold higher activity for driving the GUS gene than thatfrom Wx b• To identify the sequence responsible for the large difference in
expression level in the two alleles, various chimeric sequences of the PI-DNA
consisting of Wx a and Wx b were constructed and used for a transient assay.
The results indicated that the sequence responsible for the difference was notin the promoter region but in the downstream region (about 140bp) of theinitiation codon, where two sequence differences were detected between Wx aand Wx b• One difference was the number of CT repeats before the 1st intron.
Another difference was the splice donor site of the 1st intron. The donor
sequence of Wx b was TT, while that of Wx b was the normal sequence, GT.
The Northern blot analysis for the transcript from the seeds of Japonica
showed a larger band including the 1st intron, in addition to the maturetranscript. It seems likely that poor efficiency in splicing causes the lower
transcription of Wx b , or that quick degradation of the abnormal transcript
results in a reduced level of the mature transcript in Japonica. Alternatively,
the number of CT repeats may affect the transcription. Further analysis
involving site-directed mutagenesis at the splice donor site are in progress toascertain which mutation affects the level of transient expression of GUS
derived by PI-DNA.
(5)

Genetic Analysis of Chromosome 7 in an Annual Type Wild Rice

Mitsugu EIGUCHI and Yoshio SANO
In order to look into the genetic divergence of wild and cultivated rice, asegment of chromosome 7 was examined after introducing it into a cultivatedform from a common wild rice through successive backcrossings. An annual
type wild rice from India (W107) was crossed to a near-isogenic line of

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Taichung 65 (Japonica type from Taiwan) having g (long empty glume) and19 (liguleless) which was used as the reccurent parent (T65glg). W107, likemost common wild rice shows red pericarps (Rc) and short empty glumes(g+) and T65glg shows white pericarps and long empty glumes. The twodominant genes were used to transfer the segment with Rc and g+. The
genetic divergence on the segment linked to Rc needs to be studied since
weedy forms frequently show red pericarps even after introgression, suggesting the adaptive significance of the segment under natural conditions.
Two dominant genes were detected on the segment. One was responsible
for short stature only in the vegetative phase (tentatively called Ssv) while nodifference in culm length was observed at the flowering stage. Dominant
dwarfism expressed at the vegetative phase has not been reported so far. The
other gene was responsible for late heading in Mishima (tentatively calledLh) while plants with Lh headed earlier than T65glg in a short-day field,
which indicated that the dominant gene may be a photosensitivity gene. A
ratio of 3 : 1 was repeatedly found from BC 3F 2 to BC sF 2 showing that shortstature and late heading both are controlled by a single dominant gene in thegenetic background. Allelism tests between Lh and other known photosensitivity genes remain to be carried out.
Segregation in B sF 2 showed that these genes were linked as g-Ssv-Rc-Lh on
chromosome 7. Distorted segregations were detected for Rc and Lh, showing
significant values in X2• Although the mechanism for the distortion is
unknown, the frequency of phenotypes with Rc decreased more sharply in
BsF 2 than expected (60.8%, significant at 1%) , suggesting that the introduced segment is quickly eliminated when backcrossed with T65glg.
Publications
1.

2.
3.
4.

AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA, H.: Gene flow and population structureof Oryza glumaepatula distributed in the Amazon basin. Rice Genet. Newslett., 12, 177180, 1995.
CAl, H. W., WANG, X. K. and MORISHIMA, H.: Isozyme variation in Asian common wild
rice, Oryza rufipogon. Rice Genet. Newslett., 12, 174-17S, 1995.
EIGUCHI, M. and SANa, Y.: Evolutionary significance of chromosome 7 in an annual typeof wild rice. Rice Genet. Newslett., 12, 18S-187, 1995.
HIRANO, H.-Y., TABAYASHI, N., MATSUMURA, T., TAN IDA, M., KOMEDA, Y. and SANa,
Y.: Tissue-dependent expression of the rice wx + gene promoter in transgenic rice andpetunia. Plant Cell Physiol. 36, 37-44, 1995.

106
YONEZAWA, K., NOMURA, T. and MORISHIMA, H.:
Sampling strategies for use instratified germplasm collections. In Core Collections of Plant Genetic Resources (T.
Hodgkin et al. eds.). pp. 35-53, Wiley-Sayee Pub!., 1995.

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Division of Applied Genetics

Mitochondrial DNA Analysis Reveals Cytoplasmic Variation withina Single Cultivar of Perennial Ryegrass (Lolium perenne L.)

Yoshiya SHIMAMOTO
The extent of mitochondrial DNA diversity among individual plant samples of perennial ryegrass was determined by using RFLP. Polymorphisms
can be observed among the cultivars, showing that the cultivars were classified into the three major groups by similarity of fragment length. Three
cultivars revealed polymorphisms among individual plants, whereas most of
the cultivars were monomorphic in fragment length. Two of the threecultivars showed several different haplotypes of the mitochondria genome,
respectively, and had a common haplotype. These cultivars were the ecotypes
based on a number of selected plants which were collected from the oldsward. It seems most likely that mitochondrial genome diversity amongindividual plants within the cultivar has resulted from non-homogeneousancestor cytoplasms in perennial ryegrass. For the details, see Ref. 1.
(2)

Genetic Differentiation between Summer and Autumn Maturing
Cultivars of Soybean (Glycine max L.) in the
Kyushu district of Japan

Yoshiya SHIMAMOTO
Soybean landraces and pure line selections collected from the Kyushu
district of Japan were assayed for isozyme and seed protein loci in order todetermine the genetic structure of the groups of summer and autumn
maturing cultivars. Four of the 16 loci tested, Dial, Enp, Estl, and Ti,
exhibited a marked difference in allelic frequency between the two groups.
The summer and autumn cultivar groups had different predominant alleles atthese four loci. The analysis of multi-locus genotypes revealed that both

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groups mostly consisted of different allele-combinations at four loci. Two
major genotypes, Dia1-b, Enp-b, Estl-a, Ti-b and Dia1-a, Enp-a, Estl-b,
Ti-a, were unique to the summer and autumn groups, respectively. The
former type has not been observed in China or Korea. These results
indicated that both groups were appreciably differentiated from each otherand the summer group was originally established in the Kyushu district ofJapan. For the details, see Ref. 2.
(3)

Molecular Genetic Studies of Genomic Imprinting

Hiroyuki SASAKI, Naoya HATANOI, Mohamad ZUBAIR 1 and Reiko KATO'
CInstitute of Genetic Information, Kyushu University)
In order to understand the molecular mechanisms involved in the phenomenon of genomic imprinting in mammals, we are characterizing an imprinted
DNA region on mouse chromosome 7. This region contains three imprinted
genes, i.e. 1ns2, 19f2 and H19, and has an approximate size of 100kb. Both1ns2 and 19f2 are only expressed from the paternally derived copy and H19from the maternal copy. One mechanism that has been shown to be involved
in imprinting is DNA methylation. Our previous studies showed that theinactive, paternally derived H19 copy is highly methylated at its promoter.
We have now shown that this methylation occurs around the time of
implantation during early development. However, the inactivation of thepaternal copy preceded this methylation. Thus the promoter methylation isprobably a stabilizing mechanism rather than the primary inactivation mechanism. Because some sites further upstream were more methylated on thepaternal copy than on the maternal copy even at these early stages ofdevelopment, these sites could serve as targets for primary imprinting.
The recent study has shown that a 13 kb deletion, including the H19 geneand its upstream region, causes loss of imprinting of both 1ns2 and 19f2,
which are located more than 90 kb upstream of H 19. This imprinted region
proposes a excellent model to study gene regulation at the chromosomal
domain level. To identify regulatory elements such as enhancers, silencers,
insulators and imprinting elements, in this domain, we are now carrying out
a systematic screening for DNaseI hypersensitive sites, which could serve asa marker for such regulatory elements. So far, three novel clusters ofhypersensitive sites were identified. We found that some of them were108

constitutively present and others were embryonic stage-specific. These findings were reported in meetings and symposiums. We are currently studying
the rest of the domain and will set out to assay the functions of these
hypersensitive sites. For the details, see Ref. 3.
Publications
1.

2.

3.

4.

SATO, M., KATO, S., MIKAMI, T. and SHIMAMOTO, Y: Mitochondrial DNA analysis
reveals cytoplasmic variation within a single cultivar of perennial ryegrass (Lolium perenneL.). Euphytica 83,205-208, 1995.
HIRATA, T., KANEKO, M., ABE, J. and SHIMAMOTO, Y.: Genetic differentiation between
summer and autumn maturing cultivars of soybean (Glycine max (L.) Merr.) in Kyushu
district of Japan. Euphytica, 1995. (in press)
SASAKI, H., FERGUSON-SMITH, A. C, SHUM, A. S. W., BARTON, S. C and SURANI, M.
A.: Temporal and spatial regulation of Hl9 imprinting in normal and uniparental embryos.
Development, 121, 4195-4202, 1995.
SURANI, M. A., FERGUSON-SMITH, A. C., SASAKI, H. and BARTON, S. C: Imprinting of
H19 and Xis! in uniparental embryos. In: Parental imprinting: causes and consequences(ed. by Ohlsson, R., Hall, K. & Ritzen, M., Cambridge University Press, Cambridge, 1995,
pp. 142-156.

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Mammalian Genetics Laboratory

Establishment of System for Tetrad Analysis in Mouse
Female Meiosis II

Ken'ichi MIZUNO, Tomoko SAGAI, Kazuo MORIWAKI 1 and Toshihiko SHIROISHI CThe Graduate University for advanced Studies, Kanagawa)
Recombination occurs in two distinct ways, crossover and gene conversion. Single crossover displays reciprocal segregation of parental alleles at a
locus. On the other hand, gene conversion exhibits non-reciprocal segregation. In the meiosis of lower eukaryotes such as yeast, tetrad analysis allows
us to find out which type of recombination, crossover or gene conversion,
occurred at allelic loci. This tetrad analysis and assignment of recombinational breakpoints have given clues to understand recombinational processes.
In higher eukaryotes including mammals, tetrad analysis has never been
established, because four gametes derived from a single oogonium eventuallydispersed and it is by no means easy to trace all four gametes. Hence, themechanism of meiotic recombination in mammals has been poorly understood.
The meiotic process in female mice is relatively synchronized. Female
germ cells that migrate into prospective ovaries cease proliferation and enter
meiosis at 13.5 d.p.c. In postnatal ovary, oocytes approach in diplotene, thelate stage of meiotic prophase I, and then meiotic division is arrested until
sexual maturation. In this stage, there are four chromatids in a single nucleus
and they contain four times the haploid amount of DNA (4C). Under the
influence of sex hormones, oocytes complete meiotic division I, release asmall polar body and proceed to division II of meiosis. A egg and a polar
body derived from a oocyte are enveloped in single zona pellucida. Finally,
oocytes are arrested at meiotic metaphase II until fertilization occurs. Since
there are two chromatids in a single nucleus in an oocyte of sexually matured
female mouse, meiotic recombination should have finished. Therefore, we
can analyze the segregation of the alleles of four chromatids from a single

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matured mouse oocyte.
In the mouse major histocompatibility complex (MHC) class II region, thebreakpoints of meiotic recombination are not distributed at random, but are
clustered in specific sites, called hotspots. One of the hotspots is locateddownstream of the Lmp2 gene in the MHC class II, when wm 7 and cas3
MHC haplotypes derived from Asian wild mice are crossed with laboratory
standard haplotypes. Within 2 kb of the DNA segment of this Lmp2 hotspot,
meiotic recombination occurs at a 2% frequency, the highest value observed
among hotspots in the mouse MHC until now. Since this hotspot could be ofgreat use in analyzing the segregation of the alleles flanking the recombinational breakpoints, we decided to establish the first experimental system fortetrad analysis in mammalian meiosis.
We used a polymerase chain reaction (PCR) to amplify the recombinational breakpoints and each allele of the flanking regions from a single oocyte.
For this purpose, we established a method for preparing a single mature
oocyte. Ovaries were removed from sexually mature female mice, andmechanically picked with a needle in medium containing a chelate agent.
Mature oocytes could be distinguished by cell size, and easily isolated with acapillary. A single oocyte was purified by washing several times andtransferred into newly prepared excessive medium with a capillary. Second
we set primers across the Lmp2 hotspot and tried to select conditions for
DNA amplification from a small amount of purified genomic DNA corresponding to a single haploid ( 1C) and found a suitable one. Based on this, we
tried to amplify the DNA segment of the Lmp2 hotspot from a single oocyte.
After lysis of the oocyte, we could amplify 1.3 kb of DNA from a single
oocyte by PCR. To establish a system for tetrad analysis in mammals, weneed to establish the conditions for comparative-quantitative PCR fromsingle cells in order to detect two types of parental DNA molecules and othertwo recombinant molecules, if present. Once the whole system is established,
we can analyze rate, fashion and timing of meiotic recombination. The same
system might be applicable to analyzing the timing of DNA replication insingle cells.

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Fine Structure of the Ph Hotspot in the Mouse MHC class II Region

Taku ISOBE, Masayasu YOSHINO, Ken'ichi MIZUNO, Tsuyoshi KOIDE,
Kazuo MORIWAKI and Toshihiko SHIROISHI
In mammals, the murine major histocompatibility complex (MHC) is the
only region where breakpoints of meiotic recombination are systematicallystudied at the molecular level. In this region, meiotic recombinations do notoccur at random but are clustered in limited regions known as hotspots. Thusfar, four hotspots have been identified in this region. The presence or absenceof a hotspot on a certain DNA segment depends on the MHC haplotypes
involved in genetic crosses. For example, the Eb hotspot is observed incrosses between standard laboratory haplotypes. In genetic crosses including
cas3 and wm 7 haplotypes which were derived from Asian wild mice, most of
the recombinations occurred at the Lmp2 hotspot. Lmp2 and Eb hotspots
have been well characterized at the molecular level. Sequences around these
two hotspots were determined and the fine locations of the breakpoints wereanalyzed. Comparison of the sequences between Lmp2 and Eb hotspots
revealed several molecular motifs commonly shared by the two hotspots. In
order to elucidate the roles of these motifs in recombinations at the hotspots
and to understand the mechanism by which recombinations are restricted tohotspots, we need to characterize 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, designatedPb 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 cas4 and wm 7 haplotypes. The breakpoints of theserecombinants were localized to a 15 kb 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 recombinants. First, we constructed the restriction
map of the 15 kb of DNA fragment including the breakpoints. Subsequently,
we determined the parental origins of several short DNA segments consistingof the 15 kb of DNA fragment in the six recombinants, by examining
polymorphisms between parental DNA segments through PCR-SSCP analysis. At a result, at least five recombinations were found to be confined to a 5
kb of DNA segment located proximal to the 3'end of the Pb gene.

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Genetic Analysis of Coat-Color Mutation, rim2. II

Tomoko SAGAI, Tsuyoshi KOIDE, Mari ENDO,l Kenjiro TANOUE, 1 Hiromichi YONEKAWA2 , Kazuo MORIWAKI 3 and Toshihiko SHIROISHI CDepartment of Cardiovascular Research, The Tokyo Metropolitan Institute for
Medical Science, Tokyo, 2 Deppartment of Laboratory Experimental Animal
Science, The Tokyo Metropolitan Institute for Medical Science, Tokyo, 3 The
Graduate University for Advanced Studies, Kanagawa)
An MHC haplotype wm7, which was derived from Japanese wild mouse,enhances recombination specifically during female meiosis at the Lmp2
hotspot. The mouse mutation, rim2, is one of a series of spontaneous
mutations which arose in intra-MHC recombinants between wm 7 and laboratory haplotypes. As described in the previous report, rim2 is a single recessivecoat-color mutation and is allelic to the old mutation pearl, pe, mapped on
chromosome 13. It is known that many coat-color mutants in mouse haveabnormalities similar to human hemorrhagic symptoms accompanied withalbinism, platelet storage pool deficiency (SPD). Recently, it was shown thatplatelet aggregation and ATP release in platelets of the rim2 mutant mouseusing several agonists were clearly reduced in comparison to those of wild
type mouse. These results supported the possibility that rim2 may serve as
a mouse model for heterogeneous human SPDs.
On the basis of linkage analysis with 1570 backcross progeny, we identified
two tightly linked microsatellite markers on chromosome 13, which did notsegregate from rim2. The screening of the YAC libraries gave two YAC
clones positive for each microsatellite marker and FISH analysis confirmedthat these clones were not chimaeric. Construction of YAC contig between
these markers is now in progress. Weare planning to make transgenic micewith the DNA prepared from the YAC clones positive for these tightly linked
markers and rescue the rim2 phenotype in order to identify the clone which
includes rim2 gene.

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Linkage Analysis of the Mouse Mutant, Rim3, Affecting Skin and Eye

Hajime SATO, Hiroshi MASUYA, Shigeharu WAKANA,1 Tomoko SAGAI,
Kazuo MORIWAKI 2 and Toshihiko SHIROISHI CCentral Institute for Experimental Animals, Kawasaki, 2 The Graduate University for Advanced Studies, Kanagawa)Rim3 is a dominant mutation derived from an intra-MHC recombinant
between wild mouse-derived wm 7 and laboratory haplotypes. It is characterized by hairless and corneal opacity. Based on linkage analysis with backcrosses between C57BL/lOJ-Rim3/+ and an MSM inbred strain derived fromJapanese wild mouse, the Rim3 locus appeared to be tightly linked to three
microsatellite markers, Dll MitI4, 124 and 197, on chromosome 11. A
Keratin type 1 (Krt-I) locus has been mapped to the same position. Keratinsare a group of water-insoluble proteins that form lO-nm intermediate filaments in epithelial cells. These can be divided into two groups, type I andtype II. Type I keratins are relatively acidic and type II basic-neutral. The
two types differ in immunoreactivity and genes have higher sequence homology within each type. It is known that the Krt-I region consists of a clusterof more than 10 genes and each gene has, to some extent, tissue-specificexpression patterns in normal epithelia. Since keratin-lO, 12, 14, 16 and 17
genes are expressed in epidermis, hair follicle and cornea, they seemed to be
candidates for genes affected in Rim3. At present, it appears that keratin-lO
is not a candidate because it was genetically segregated from Rim3. Keratin
12 was also excluded from being a candidate, because we could not find any
alteration of sequence in the Rim3 mutant gene. Several mouse mutations
affecting the skin and cornea have also been mapped to the region close to the
Rim3 locus. The mutation Bare skin (Bsk) causes baldness and corneal
opacity. Another mutation Rex denuded (Rex den ) exhibits a similar phenotype. To characterize corneal opacity in more detail, we carried out ahistological analysis of the cornea of Rim3 and Rex den• In both mutants, thecorneal epithelium is hyperstratified at the center of the cornea and a clusterof unidentified cells are accumulated in that subepithelial region. In addition,
keratin-like substances are observed on the surface of hyperstratified epithelium. Now we intend to carry out a immunohistochemistrical analysis forthese two mutants and Bsk.

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Multigenic Control of Localization of the Zone of Polarizing
Activity in Mice Limb Morphogenesis

Hiroshi MASUYA, Tomoko SAGAI, Shigeharu WAKANA 1, Kazuo MORIwAKI 2 and Toshihiko SHIROISHI CCentral Institute for Experimental
Animal, Kawasaki, 2 The Graduate University for Advanced Studies, Kanagawa)In vertebrate limb development, the pattern formation along the anteroposterior and proximodistal axes is controlled by two signaling centers.
Polarizing activity along the anteroposterior axis of limb morphogenesis isprovided from the Zone of Polarizing Activity (ZPA) located at the posterior
margin of the limb bud mesoderm. Now it is accepted that Sonic hedgehog(Shh), a mouse homolog of the segment polarity gene, Hedgehog, in Drosophila, mediates the key activity of the ZPA. Expression of Shh in theposterior mesenchyme of limb bud, and fibroblast growth factor-4 (Fgf-4) inthe posterior portion of apical ectodermal ridge (AER), coordinately regulate pattern formation along the anteroposterior axis. We demonstrated theectopic expression of Shh and Fgf-4 at the anterior margin of limb buds inthree preaxial polydactyly mutants, Recombination induced mutant 4 (Rim4), Hemimelic extra toes (Hx) and Extra toes-J (Xt'), indicating that mirrorimage duplications of the skeletal pattern of the digits could be caused by
duplication of ZPA at the anterior margin oflimb buds. For details, see Ref.
1.
One of the above mutations, Xt', is thought to be a loss of function type,because the affected gene, Gli3, which encodes a transcription factor has along deletion including a Zinc-finger DNA binding domain in this mutation.
Our finding of the duplication of ZPA in Xt' mice suggests that there is a
genetic control that suppresses ZPA activity at the anterior margin of thelimb bud, and Gli3 is involved in this cascade. To explore the gene functionof Gli3 in limb morphogenesis, we examined the localization of the Gli3transcript during limb development by whole-mount in situ hybridization. As
a result, Gli3 was expressed in the limb bud mesenchyme except the posterior
margin which overlaps the location of ZPA. This expression pattern, whichis complementary to that of Shh, shows that GLI3 functions in the anterior
portion of the limb mesoderm to suppress Shh expression.
Three other mouse mutants, luxate (Ix), Strong's luxoid (1st), and X-linked

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polydactyly (Xpl), exhibit a mirror-image duplication similar to those of, Hx
Rim4 and xtl showing hyperphalangy and reduction of tibia, except that Ix
and Xpl show polydactyly only in the hindlimbs. To examine whether the
preaxial polydactylies of lx, 1st, and Xpl, are caused by ectopic ZPA activity
at the anterior region of the limb buds, we carried out whole-mount in situ
hybridization of 12.5 days embryos using riboprobes of the Shh, and Fgf-4
genes. Ectopic expression of Shh and Fgf-4 were observed in the limbs of all
mutants. These results in conjunction with previous ones from our study on
Rim4, Hx and xtl suggested that most preaxial polydactyly mutations withmirror-image duplication are caused by ectopic formation of ZPA at theanterior margin of limb buds. In the Ix mutant, ectopic expression of Fgf-4
was found in the AER in the forelimb buds, but no ectopic expression of Shh
was detected in the mesoderm. Hence, the anterior margin of AER was
posteriorized in the limb buds of this mutant, but not its mesodermal
mesenchyme. Thus, Ix seems to be a candidate gene which functions tosuppress Shh via the signaling pathway in AER.
When mice heterozygous for the Rim4 mutant were crossed with MSM, aninbred strain derived from Japanese wild mouse, Mus musculus molossinus,
the appearance of the polydactylous phenotype was reduced to 0%, though
half of the Fl hybrids carried the Rim4 mutant gene (data not shown).
When (C57BL/lOJ~Rim4/+ and MSM) Fl mice heterozygous for Rim4
were backcrossed to C57BL/lOJ, 14.5% of the progeny exhibited polydactyly.
These findings indicate that the polydactylous phenotype of Rim4 wascompletely rescued by genes of the MSM strain in the Fl hybrids, and thegenes responsible for the rescue of the polydactylous phenotype are not manyin number. To investigate whether the same genes are able to rescue themutant phenotypes of other polydactylous mutations, we crossed the MSM
strain and heterozygotes of other polydactylous mutants. The polydactylous
phenotype of Rim4, Hx, and 1st, was ameliorated in crosses with the MSM
strain, but the phenotype of Xpl did not change at all. These mating
experiments indicate the possibility that Rim4, Hx, Xt, and 1st genes playfunctions in upstream of the rescue gene in the MSM strain, and the Xpl geneplays function in downstream of the rescue genes. Normal functions of allthese genes are likely to be involved in the down regulation of ZPA activity
at the anterior margin of limb buds.

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(6)

Searching for Modifier Genes for the Phenotype of Mutation Tail-short

Junko ISHIJIMA, Kikue UCHIDA, Akihiko MITA, Tsuyoshi KOIDE andToshihiko SHIROISHI
Mice homozygous for Tail-short, Ts, exhibit abnormalities in early development and embryonic lethality at about the time of implantation. On the other
hand, heterozygotes have various kinds of skeletal abnormalities throughout
the whole body and severe defects in head structural development. Thesephenotypes differ depending upon the genetic background in which the
mutant gene is introduced. For example, when (TSJjLe-Tsj+ XC57BLj6J)
F I mice were backcrossed to C57BLj6J, the viability of the resultant progeny
carrying the Ts allele is almost 0%. In turn, when the MSM inbred strain
derived from Japanese wild mouse, Mus musculus molossinus, was used as a
backcross partner, mice with the Ts allele were less defective and viability
increased to 40%. The difference in expressivity of the mutant phenotype
indicates that there is a gene(s) which modifies the expressivity of Ts
phenotypes and is polymorphic between the C57BLj6J and MSM strains. We
intend to map these modifier gene(s) using backcross progeny from (TSJjLeTsj + X MSM)FI X C57BLj6J. At present, no major gene has been detected
as a candidate for the modifier, but it is becoming more apparent that strain
differences in expressivity of the mutant phenotype is under the control of anumber of minor genes.
(7)

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

Junko ISHIJIMA, Kikue UCHIDA, Akihiko MITA, Tsuyoshi KOIDE andToshihiko SHIROISHI
We explored the characterization of the morphological phenotype of themouse mutant Tail-short in embryonic development and skeletal patterning.
Detailed analysis of the skeleton of Tsj + mice revealed that a number ofhomeotic transformations occur along the anteroposterior axis. In particular, transformation of the seventh cervical vertebrae to the first thoracic
vertebrae was highly reproducible in Ts heterozygotes generated from thecross of (C57BLj6J-Tsj + X MSM)FI X C57BLj6J. It is of interest to notethat this transformation was observed asymmetrically, namely, the left side ofF.

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vertebrae was predominantly transformed. Besides the above skeletal anomalies, a neural tube defect in the rhombomere region was frequently observed.
Thus it is possible that the Ts gene plays some roles providing segmental
identity in mouse embryogenesis. More detailed analysis of the mutant
phenotype is underway to characterize the primary defect in the Ts mutation,using in situ hybridization with a number of probes for genes whose expression patterns seems to be related to the defect in the Ts mutation.
(8)

Fine Gene Mapping of the Skeleton Mouse Mutant, Tail short (Ts),
Toward Positional Cloning

Tsuyoshi KOIDE, Kikue UCHIDA, Akihiko MITA, Shigeharu WAKANA 1,
Yoshiakira KIKKAWA 2, Hiromichi YONEKAWA 2, Kazuo MORIWAKI 3 andToshihiko SHIROISHI CCentral Institute for Experimental Animals, Kawasaki, 2 Department of Laboratory Experimental Animal Science, The Tokyo
Metropolitan Institute for Medical Science, Tokyo, 3 The Graduate University for Advanced Studies, Kanagawa)

Tail short (Ts) was initially found as a mutant which exhibits shortened
kinked tail in heterozygotes. They also have numerous skeletal abnormalities
throughout the entire body. Homozygous embryos die at around the time of
implantation. This is probably due to a retardation in forming morulae
because pale-staining embryos have been found at this stage in homozygotes.
These various phenotypes indicate that the gene for Ts plays a crucial role
throughout development including the early embryonic stage and skeletal
formation. To elucidate the function of the Ts gene, we are trying to clone
the gene by the method of positional cloning. We have conducted backcrossmating between Tsl + and the MSM inbred strain derived from Japanese wild
mouse. The Ts gene was mapped within a region of 0.2 cM that locatedbetween two microsatellite markers, DllMit128 and DllMit214. Calculations estimate the distance between the two markers as approximately 400 kb.
We screened the mouse YAC library and obtained one YAC clone which
carries D11 Mit214. Further screening of the mouse genomic library isunderway to make a contig between the two markers.

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(9)

Establishment of Intersubspecific Consomic Mouse Strains

Toshihiko SHIROISHI, Akihiko MITA, Kyoko TAKADA, Shigeharu WAKANA1, Hiromichi YONEKAWA 2 and Kazuo MORIWAKI 3 Central Institute forExperimental Animal, Kawasaki, 2 Deppartment of Laboratory Experimental
Animal Science, The Tokyo Metropolitan Institute for Medical Science,
Tokyo, 3 The Graduate University for Advanced Studies, Hayama, Kanagawa)e

Two strains of mouse are said to be consomic when they differ by one
complete chromosome pair. The prefix "con" means that any two consomic
strains, in spite of specific differences, have a similar genetic background andthe suffix "somic" refers to a complete chromosomal element. Given thatmouse has a karyotype with 21 different chromosomes (19 autosomes + X +Y), 21 consomic mouse strains are required for full coverage of mouse
chromosomes by donor strains, from which one complete chromosome isintroduced into the common genetic background referred to as recipient.
Chromosome substitution is achieved by six to seven times backcrossing ofindividuals identified as heterozygous for the selected chromosome to therecipient strain.
Consomic strains can facilitate the genetic analysis of penetrance andexpressivity. Variation in expressivity depends upon both genetic and non
genetic determinism, and it is clear that a couple of backcrossings to three orfour different consomic strains may help in detecting genes likely to be
involved in the phenomenon of expressivity. Consomic strains also help in
the genetic analysis of phenotypes with multigenic determinism. It has been
well established that many pathological conditions and diseases like cancer
and diabetes are under multigenic control. They are generally difficult toanalyze when a great number of genes are involved and epistatic interactions
are present among the genes. In these cases, if a phenotype of interest
appears to deviate in a consomic strain from what is known in the genetic
background strain, its genetic control must be related to chromosomal
substitution. Further genetic analysis will then be considerably simplified. In
addition, consomic strains can facilitate chromosomal assignment of cloned
DNAs.
Considering all these benefits from using consomic strains, we have started
to establish consomic strains. We chose the MSM strain, which is derived

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from Japanese wild mouse, Mus musculus molossinus, as donor strain and, a
C57BL/6J laboratory strain as recipient. These two strains are geneticallyvery remote from each other so that they differ in a number of genetic traits.
This genetic difference facilitates in chromosomal assignment of any phenotype to be examined and is of great use in linkage analysis based onpolymorphism of DNA markers. We are using microsatellite DNA markers,
at least five markers per chromosome, for selection of individuals in each stepof backcrossing. At present, we have obtained second generations of the
backcross for substitution of most chromosomes.
Publications
I.

2.

3.

4.

TANOOKA, H., OOTSYAMA, A., SHIROISHI, T and MORIWAKI, K.: Distribution of the p53
psudogene among mouse species and subspecies. Mammalian Genome, 6, 360--362, 1995.
YOSHINO, M., SAGAI, T, FISCHER LINDAHL, K., TOYODA, Y, MORIWAKI, K. and
SHIROISHI, T: Allele-dependent recombination frequency: Homology requirement inmeiotic recombination at the hotspot in the mouse major histocompatibility complex.
Genomics, 27, 298-305, 1995.
MASUYA, H., SAGAI, T., WAKANA, S., MORIWAKI, K. and SHIROISHI, T.: A duplicatedzone of polarizing activity (ZPA) in polydactylous mouse mutants. Genes & Dev., 9,1645-1653, 1995.
YOSHINO, M., SAGAI, T., MORIWAKI, K. and SHIROISHI, T.: Meiotic recombination at the
Lmp2 hotspot tolerates minor sequence divergence between homologous chromosomes.
Immunogenet. in press.

F-b.
(1)

Invertebrate Genetics Laboratory

Determination of wing cell fate by the escargot andsnail genes in Drosophila

Naoyuki FUSE, Susumu HIROSE l and Shigeo HAYASHI
genetics)

eDivision of Pheno-

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 have found that two related transcription factors,

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Escargot and Snail that are 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 LacZ regulated byescargot enhancer were found as well as in wild type embryos. However, inescargot snail double mutant embryos, wing development proceeded until
stage 13, but the marker expression was not maintained in later stages, andthe invagination of the primordium was absent. From such analyses, it was
concluded that Escargot and Snail expression in the wing disc are maintainedby their auto- and cross-activation. 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. Similar
DNA binding specificities of Escargot and Snail suggest that they control thesame set of genes required for wing development. We thus propose thefollowing scenario for early wing disc development. Prospective wing cells
respond to the induction by turning on escargot and snail transcription, andbecome competent for regulation by Escargot and Snail. Such cells initiate
auto- and cross-regulatory circuits of escargot and snail. The sustained
Escargot and Snail expression then activates vestigial and other target genesthat are essential for wing development. This maintains the commitment to
the wing cell fate and induces wing specific cell shape change. For the details,
see Ref. 4.
(2)

A Cdc2 dependent checkpoint maintains diploidy in Drosophila

Shigeo HAYASHI
DNA replication in G2 does not normally occur due to the checkpoint
control. To elucidate its mechanism, functions of the escargot and Dmcdc2
genes of Drosophila were studied. When escargot function was eliminated,diploid imaginal cells that were arrested in G2 lost Cyclin A, a regulatory
subunit of G2/M cdk, and entered endocycle. escargot genetically interactedwith Dmcdc2 that encodes a catalytic subunit of G2/M cdk. The mutant
phenotypes of Dmcdc2 itself was similar to those of escargot; many of diploidcells in imaginal discs, salivary glands and the central nervous system enteredendocycle and sometimes formed polytene chromosomes. Since mitoticallyquiescent abdominal histoblasts still required Dmcdc2 to remain diploid, theinhibitory activity of G2/M cdk on DNA replication appeared to be separable

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from its activity as the mitosis promoting factor. These results suggest thatin G2, escargot is required to maintain a high level of G2jM cdk that actively
inhibit the S phase entry. For the details, see Ref. 3.
(3)

Roles of Escargot and DE-Cadherin in the Formation of
Drosophila Tracheal Network

Miho TANAKA-MATAKATSU, Tadashi UEMURA 1, Hiroki aDA!, Masatoshi
TAKEICHI 1 and Shigeo HAYASHI CKyoto University, Faculty of Science)
The Drosophila tracheal system is a network of tubular epithelium. It isderived from ten pairs of precursors that invaginate from ectoderm in thethoracic and abdominal segments. The precursors extend branches which
migrate toward branches from other segment and fuse to form continuous
tubes. At the site of fusion, a specialized cell called tip cell was identified.
When thin branches of the dorsal branch and lateral trunk fuse, tip cells
search their target by extrending filopodia which make a first contact. The
homophilic cell adhesion molecule DE-Cadherin plays an essential role in thefusion (Ref. 5). Among several cell adhesion molecules examined, DECadherin is the first molecule to be accumulated at the contact interface.
Other cell surface molecules such as Fasciculin III and Crumbs were detectedin later stages. Changes in the level and spacial pattern of DE-Cadherin
accumulation closely corelated with the dynamic process of epitherial rearrangement during the fusion. DE-Cadherin is essential for the tracheal
fusion, since tracheal branches failed to fuse in embryos mutant for DECadherin gene shotgun. The tracheal tip cells specifically express the transcriptional regulator Escargot (Esg). In esg mutant, a subset of tracheal
branch failed to express DE-Cadherin and did not fuse. In addition, tip cells
in these branches extended unusually long cytoplasmic processes which
migrated to search an alternative fusion partner. Such phenotypes were
rescued by an esg transgene. Furthermore, heat inducible DE-Cadherintransgene also rescued the tracheal defects in esg mutant. These analyses
demonstrated the essential role of DE-Cadherin in the tracheal fusion andidentifyesg as a regulator of cell adhesion and motility.

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(4)

The Early Development of Drosophila Imaginal Discs

Satoshi GOTO and Shigeo HAYASHIThe embryonic development of Drosophila imaginal discs provides animportant experimental system to study pattern formation. The developmentof wing/leg primordia is thought to be composed of three steps. The first stepis the detemination of the leg and wing primordia as a common precursor
expressing the homeodomain protein Distal-less. This step requires thesignaling molecule wingless. The second step is the segregation of wing
primordium from the leg primordium. The wing primordium migrates
dorsally within ectoderm expressing dpp-IacZ reporter, suggesting that decapentaplegic (dpp) might be involved in this step. The third step is themaintenance of the imaginal fates. We found that both the wing and leg
primordia failed to form in the absense of Dpp receptor encoded by thick veinand punt. In less severe tkv and punt mutant background, only the wing
primordium was affected, suggesting dpp signaling is required for the first two
steps.
(5)

A Nuclear GFP/fi-Galactosidase Fusion Protein as a Marker for
Morphogenesis in Living Drosophila

Yasuhiro SHIGA l , Miho TANAKA-MATAKATsu and Shigeo HAYASHI
CPresent Address: Tokyo University of Pharmacy and Life Science)
A general, non-invasive method to trace morphogenesis in living Drosophila was developed. To label specific cells, green fluorescence protein(GFP) of jellyfish Aequorea victoria was expressed by the GaI4-UAS system.
Green fluorescence from GFP fused to the nuclear localization signal was
detectable in polytene larval tissue, but not in diploid tissue. Further fusion
to bacterial,B-galactosidase produced GFPN-IacZ, which fluoresced brightly
in several diploid larval and embryonic tissues. GFPN-IacZ was used to
trace dynamic cell movement during the formation of embryonic tracheal
system. These results indicate that GFPN-IacZ can be used to mark specificcells to study cell movement and gene expression in living animal. For thedetail, see Ref. 2.

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123

Control of esg Expression

Yoshimasa YAGI 2 and Shigeo HAYASHI
Science, Nagoya University)

eDepartment of Biology, Faculty of

esg is expressed in spatially and temporally complex pattern during embryogenesis. To elucidate the regulatory mechanism of esg expression, we
have been mapping cis-control elements in the upstream of esg gene. We havemapped testis specific enhancer whithin a I kb fragment in the vicinity of esg
promoter. Control elements for expression in the nervous systems, gut
imaginal cells, the leg and genital imaginal discs and the tracheal tip cells
scatter within more than 20 kb region in the upstream from the esg promoter.
These elements are candidates for targets of regulatory genes such as wiglessand dpp which are involved in morphogenesis of many tissues expressing esg.
(7)argos is Reqired for Projection of Photoreceptor Axons During
Optic Lobe Development in Drosophila

Kazunobu SAWAMOT0 1, Masataka OKABE 1, Teiichi TANIMURA 2 , Shigeo
HAYASHI, Katsuhiko MIKOSIBA 3 and Hideyuki OKANO' (' University ofTsukuba, 2 Kyushu University, 3 University of Tokyo)
The Drosophila argos gene encodes a secreted protein with an EGF motif,
which acts as an inhibitor of cell recruitment in the developing eye and wing.
We have analyzed the role of argos during optic lobe development. argos
expression was observed in the optic lobes throughout the developmental
stages. In argos mutants, neuropiles failed to develop normally, and photoreceptor axons did not project propery into the lamina. Ubiquitous expressionof argos, under control of the hsp70 promoter, rescued the defects in optic
lobes. We have found that glial cells failed to differentiate in the larval optic
lobes of argos mutants. Correspondingly, in loss of function repo mutants,
whose glial cells also failed to differentiate, photoreceptor axons showed
impared projection pattern similar to the argos phenotype. These results
suggest that glial cells playa role for guidance of photoreceptor axons. The
loss of function Star mutation dominantly supressed the defect in the argos
optic lobes, suggesting that these two genes act in an antagonistic fashion
during optic lobe development. For the detail, see Ref. 1.

124

Publications
1.

and H.
argos is required for projection of photoreceptor axons during optic lobe
development in Drosophila. Developmental Dynamics, 205, 162-171.
SHIGA, Y, M. TANAKA-MATAKATSU and S. HAYASHI (1996)
A nuclear GFP/j3galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Development, Growth and Differentiation, 38, 99-106.
HA YASHI, S. (1996) A Cdc2 dependent checkpoint maintains diploidy in Drosophila.
Development, 122, 1051-1058.
FUSE, N., HIROSE, S. and HAYASHI, S. (1996) Determination of wing cell fate by theescargot and snail genes in Drosophila. Development, 122, 1059-1067.
UEMURA, T., ODA, H., KRAUT, R., HAYASHI, H., KATAOKA, Y and TAKEICHI, M.
(1996) Zygotic Drosophila E-cadherin expression is required for processes of dynmic
epitherial cell rearrangement in the Drosophila embryo. Genes and Development, 10, 659671.
SAWAMOTO, K., OKABE, M., TANIMURA, T., HAYASHI, S., MIKOSHIBA, K.
OKANO (1996)

2.

3.
4.
5.

F-c.
(1)

Plant Genetics Laboratory

Structural and Functional Analysis of the Genes Expressed
During Early Embryogenesis in RiceYukihiro I TO
As the first step in understanding the molecular mechanism which regulates embryogenesis in rice, I isolated several cDNA clones and PCR fragments from cDNA libraries, which were constructed from mRNAs of riceembryos three days after fertilization using three different methods. The
cDNA libraries constructed were the general lambda phage library, thelambda phage library in which cDNAs were amplified by PCR, and thesolid-phase cDNA library in which cDNAs were fixed to a metallic support.
I isolated several cDNA clones using cDNAs of homeobox genes of rice,maize, and Arabidopsis as probes from two lambda phage cDNA libraries,and several PCR fragments amplified from a solid-phase cDNA library using
degenerated oligonucleotide primers corresponding to the conserved aminoacid sequences of homeodomains. These results suggest that homeobox genes
are probably involved in early embryogenesis in rice.

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Publications
USAMI S., BANNO H., ITO Y., NISHIHAMA R., and MACHIDA Y.: Cutting activates a 46ki10dalton protein kinase in plants. Proc. Natl. Acad. Sci. USA 92, 8660-8664, 1995.
2. SHIBATA W., BANNO H., ITo Y, HIRANO K., IRIE K., USAMI S., MACHIDA c., and
MACHIDA Y.: A tobacco protein kinase, NPK2, has a domain homologous to a domain
found in activators of mitogen-activated protein kinases (MAPKKs). Mol. Gen. Genet.
246,401-410, 1995.
1.

F-d.
(1)

Microbial Genetics Laboratory

A New Gene Determining the Frequency of Cell Division in E. coli

Maki T ADENUMA and Akiko NISHIMURA
We have isolated a novel mutant of E. coli, MT1, and found it to have thefollowing characteristics. The MTI had two mutations, cfcM] and X-. The
cfcM] gene mapped within several minutes of the oriC gene. The cfcM]
mutation caused excess division per round of DNA replication at 41 Dc. As
a result, a lot of mini cells with DNA and short filamentous cells with highploidy were produced. However, the colony forming ability of the single
mutant strain which carried the cfcM] or the X- was normal at 41 Dc.
Temperature sensitivity for growth was only shown for the double mutant,cfcM] and X-. The cfcM] mutant continued to divide even when DNA
replication was inhibited in the presence of nalidixic acid (7.5,ug/ml). We
have subcloned the DNA region, which complemented the cfcM] mutation,
into the miniF plasmid but could not do so into pSClOl. Over production of
CfcM is assumed to be toxic for cell.

(2)

Control of Cell Division at Low pH

Hideki UKAI, Nahoko WATANABE, Keiko SUZUKI and Akiko NISHIMURA
We isolated a novel mutant, cpcA58 (Control by pH and cAMP). The
cpcA58 mutant strain stopped cell division when cultured in a low pHDmedium containing cAMP at 43 C. The cpcA58 mutant strain carrying thetetracycline (Tc) resistance gene did not show Tc resistance. Lack of Tc

126

resistance was shown even at pH 7.0 and was more marked in the presence ofcAMP. Tc resistance is known to be acquired through efflux of the drag outof the cells by antiporter, TetA, coupled with H+ influx in cells. The
multicopy suppressor gene of the cpcA58 mutation, sun U, was also isolated.
The plasmid carrying the sun U+ gene complemented the growth deficiency of
the acrA strain cultured in the presence of the H+ uncoupler. These results
suggest that the cpcA58 mutant might be defective in the H+ efflux systems
and .dpH might be decreased in the cpcA58 strain. Under these conditions,a low pH is assumed to lower the proton motive force. As a result,localization of some protein(s) required for cell division might be inhibited.
(3)

The Instability of Membranes Affect the Transcription of
Cell Division Gene, ftsZ

Kana NISHIMORI and Akiko NISHIMURA
We analyzed the properties of temperature sensitive cell division mutantsof E. coli,ftsK830 andftsKl167. They were alleles of the kdsA gene which is
involved in the synthesis of lipopolyshaccharide (LPS). LPS is a major
component of the outer membrane. Accoordingly, the barrier property of theouter membrane is supposed to be reduced in these ftsK mutants. Actually,
sensitivity for some hydrophobic agents such as novobiocin was increased in
the ftsK830 mutants. However unexpectedly, some other agents such as
methylene blue accelerated the growth of theftsK830 andftsKl167 mutants
at which concentration the parent strain was sensitive. These results suggested that the instability of the membrane in the ftsK mutants might affect theexpression of many genes rather than just the lack of protein localization into
the outer membrane. Preliminary experiments showed that theftsZ-mRNAin theftsK mutant was half that of the parent when cultured at nonpermissible temperatures for 50 min.

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sdiA and rpaS-Mediated Dual Transcriptional Regulation of
7a-HSDH Gene, hdhA, in Escherichia cali

Kengo KANAMARU and Kyoko KANAMARU 1 CDeep-Sea Microorganisms
Research Group, JAMSTEC)
To investigate the cell-cell communication of Escherichia coli, we focusedin the physiological function of a positive transcriptional activator of theftsQAZ operon, SdiA. At least on the level of amino acids sequence, SdiA
belongs to the LuxR family of regulators of cell-density dependent gene
expression in several other gram-negative bacteria. We have found thatsuper-multicopying of the sdiA gene causes the intracellular accumulation of
7a-hydroxysteroid dehydrogenase (7a-HSDH), encoded by the hdhA gene,which catalyzes the dehydration of primary bile acids to secondary bile acids.
Interestingly, the transcription of hdhA was controlled by both sdiA and rpaS,
the latter of which encodes a stationary phase-specific sigma factor. The
trancriptional regulation of hdhA was very similar to the regulation of theftsQAZ operon by these transcriptional factors. A more than 20-fold increase
in total hdhA-mRNA level was observed in the cells of early stationary phasewhen compared with those of early log phase. sdiA-dependent transcription
kept increasing in the log phase but decreased immediately before entering
the stationary phase. Marked activation of rpaS-dependent transcription
occurred only during early stationary phase. Deletion of sdiA did not affectrpaS-dependent transcription of hdhA, whereas, in contrast, deletion of rpaS
resulted in a significant stimulation of sdiA-dependent transcription in the log
phase without any change in transcription of sdiA. These results imply a link
between the expressions of the 7a-HSDH and the cell division machinery
encoded by ftsQAZ, and therefore an involvement of the E. coli adaptive
response system to the intestinal environment.
(5)

A Novel Member of the cspA Family of Genes, cspG, which is
Induced Upon Cold-shock in Escherichia coli

Kyoko KANAMARU 1 and Kengo KANAMARU CDeep-Sea Microorganisms
Research Group, JAMSTEC)

Escherichia coli contains a major cold-shock protein, named CspA (or CS
7.4), whose production is predominantly induced at low temperatures. This

128

bacterium is known to possess five additional genes each encoding a protein
highly homologous to CspA (referred as the CspA family). We haveidentified a new gene encoding a cold-shock inducible analogue of CspAj
CspB. This newly cloned cspG gene is located at 22 min on the E. coli genetic
map, at a distance from the other cspA family genes. Its gene-product (70amino acids) is 73% and 77% identical to CspA (70 amino acids) and CspB
(71 amino acids), respectively. Analyses of the cspG-IacZ transcriptional
fusion gene and Northern hybridization revealed that cspG is a low temperature responsive gene. Its low temperature inducible promoter elements were
determined, and the results indicate that the cspG sequence is highly similar
to both the cspA and cspB sequences not only in the coding-regions but also
in the 5'-upstream non-coding regions surrounding their own promoters.
Publication
None

F-e.

Genetic Resources Laboratory

Compilation of Genetic Resources Information

Mari SAITO and Yukiko YAMAZAKI
The first trial phase of the Genetic Resources Databank Project has been
initiated in this laboratory this year. The goal of this project is the collection,
designation, construction, and online distribution of an Integrated Database,which contains genetic resources information and the relevant objects ofdifferent organisms, under a single logical data model.
Starting from local stock information such as mouse stocks maintained by
the Mammalian Genetics Laboratory (T. Shiroishi) and drosophila stocks
generated by the Invertebrate Genetics Laboratory (S. Hayashi), we have
implemented the following databases available via the World Wide Web(WWW). At present we have compiled drosophila stocks of individuallaboratories in Japan, local wheat germline stocks, and the cloning vector
collection in cooperation with Yamamoto Y. (Kyoto Industrial University),
H. Tsujimoto (Kihara Institute for Biological Research), T. Kawahara(Kyoto Germline Center) and S. Yasuda (Division of Microbial Genetics).

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GENETIC STOCK RESEARCH CENTER129

The data formats of these databases are minimally processed so that eachdatabase has its own format.
We also collaborated with the Wheat Networking Group, which is supported by National Science Foundation B, with aim of constructing an
integrated wheat database called "KOMUGI-Wheat Network of Japan". We
developed a data submission tool to collect data from each stock center anddesigned the database schema. The database, which will appear on theinternet by the end of the fiscal year, is composed of nearly 50 entities
including the biological and molecular biological features of each strain, as
well as bibliographical information. The working group plans to continue thework on the database in several directions, such as incorporation of the wheat
DNA repository database and of image data and cross referencing withrelated databases. The development of a data management system throughwhich each researcher can constantly update their own data by connecting to
the remote computer running the databases is also an ongoing project.
Rice Stocks Data maintained by several laboratories in Japan have been
already compiled by T. Kinoshita (Hokkaido University) and H. Morishima(Division of Agricultural Genetics) over a period of several years and stored
as hardcopy. Processing the hardcopy data to produce an electronic databaseis under way and the online database will soon be made accessible.
The next step will be providing a common model of these databases inorder to accomplish inter-species searching. Although our current databases
are organized as a relational database under Sybase DBMS, we plan to pursue
the possibility of a new system like the unified relational and object-oriented
system in the future.
Publication
None

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F-f.
(1)

Mammalian Development Laboratory

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

Norio NAKATSUJI and Yasuaki SHIRAYOSHI

Autonomous regulation of proliferation and growth arrest. Primordial
germ cells (PGCs) isolated from mouse embryos show proliferation andgrowth arrest in culture, with a time course that is similar to the events invivo. Such a growth pattern could be regulated autonomously or by somatic
cells, which always co-exist with PGCs in culture. Mixed culture experiments
using PGCs from 8.5 and 11.5 dpc embryos indicated no detectable interaction between PGCs and somatic cells at the two stages. We then carried out
clonal cultures of PGCs, and examined the proliferation of and morphological changes in individual clones. Such clonal cultures did not reveal any
subpopulations of PGCs in terms of growth rate and morphological changes.
We concluded that there is an autonomous regulation of growth and cell
shape change in PGCs, which occur as stochastical events but not strictly
timed by the number of cell divisions. For details, see Ref. 3.
Roles of the gp 130 signal-transduction. The leukemia inhibitory factor(LIF) is a cytokine known to influence proliferation and/or survival of mouse
PGCs in culture. We analyzed the functional role of gp l30-mediatedsignaling in PGC growth in vitro. A combination of IL-6 and soluble IL-6
binding subunit (sIL-6R), which is known to activate intracellular signaling
via gp 130, fully reproduced the LIF action on PGCs. The addition of aneutralizing antibody against gp 130 in culture markedly blocked PGC
survival. These results suggest the pivotal role of gp 130 in PGC development. We further demonstrated that a combination of LIF with forskolin or
retinoic acid, a potent mitogen for PGCs (for the details, see Ref. 1), caused
continuation of proliferation of PGCs, leading to propagation of embryonicstem cell-like EG cells. For details, see Ref. 4.
Gene transfection ofPGCs in vitro. We tested the transfection methods ofelectroporation, liposome-mediated transfection and the calcium phosphate(CaP04) co-precipitation method. When PGCs were transfected withF.

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plasmid pSV-LT by the CaP04 co-precipitation method, transient expressionof simian virus 40 large-T antigen was detected in an average of 18% of
PGCs transfected. However, we could not detect any effects on the morphology or proliferation of PGCs. We also succeeded in detecting transient
expression of the adenovirus 2 E1B 19kDa protein, which is known tosuppress cell apoptosis, in about 15% of PGCs transfected with plasmidpEF-E1B. In this case, its expression significantly retarded the disappearance of PGCs from the culture system and increased the number of rounded
PGCs, which probably represent PGCs at more advanced stages of development and which can not survive in the presently available culture conditions.
(2)

Molecular Analysis of Cell Differentiation and Morphogenesis in
Postimplantation Mouse Embryos

Yasuaki SHIRAYOSHI and Norio NAKATSUJI
Determination of cell fate and cell differentiation are crucial events inmorphogenesis and embryogenesis. We are trying to approach such problems
from the molecular aspect, for example, by cloning mouse homologues ofDrosophila genes which could play important roles in cell fate decision or celldifferentiation. One area involves identifying important genes in the determination and differentiation of the central nervous system (CNS) during thepostimplantation period. We constructed a cDNA library from 6.5-8.5 day
old mouse embryos, in which neural plate and neural tube formation has been
initiated. We screened this mouse cDNA library for important genes relating
to CNS development such as the Notch neurogenic or ASC (acheate-scutecomplex) proneural genes in Drosophila.
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 the Notch orvertebrate 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 one of the mouse homologues of Notch. RT-PCR
and in situ hybridization analysis revealed that int-3 was expressed in the
blood vessels in 9.5-10.5 day old embryos. The expression pattern of int-3
coincided with the expression of receptor tyrosine kinase Flk-l, which isregarded as a major regulator of vasculogenesis and angiogenesis. These
results suggested that int-3 might be involved in the formation of blood

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vessels in early mouse embryos. We will study its roles in blood vessel
construction either through ectopic expression or by altering the gene through the gene targeting method using ES cells and homologous recombination. Another area of interest is the molecular mechanism of sex differentiation in fetal gonads. We are currently trying to find genes which show
specific expression patterns in male or female gonads.
(3)

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

Norio NAKATSUJI, M. T. SHOKUNBI and Yasuaki SHIRAYOSHI
We dissected out the neural plate from mouse embryos at 8.0-9.5 dpc,
which were transgenic for the temperature sensitive simian virus 40 (SY40)
large tumor antigen (TAg) gene, tsA58, driven by the mouse H-2kb genepromoter (Immorto Mouse, Charles River Lab., USA). We cultured thesecells at 33°C in a medium containing interferon-r. We characterized many
sublines after cell cloning of such established cell lines. Among them, a large
number of cloned lines showed similar characteristics suggesting very primitive neuroepithelial cells, one of which was named the NES-8 cell line. Other
cloned lines consisted of those at more advanced stages of development
because they were more easily induced to differentiate into neurons in vitro
than the NES-8 cells. We obtained only the NES-8 cell type from the neuralplate at 8.0-8.5 dpc, but both the NES-8 and more advanced types from theneuroepithelium at 9.0-9.5 dpc.
While growing under TAg permissive conditions, NES-8 cells expressed
nestin and both the RCI and A2B5 antigens, which are markers of theneuronal stem or progenitor cells. Under TAg impermissive conditions(39°C without interferon-r), NES-8 cells showed morphological changes andproduced both bipolar and flattened cells. Such bipolar cells continued toexpress nestin and the RCl antigen but lost A2B5 antigen expression. The
flattened cells seemed to lose all three marker expressions. However, wefailed to demonstrate expression of differentiation markers such as theneurofilament protein (NF) and the glial fibrillary acidic protein (GFAP)
even after various trials to induce differentiation with aggregation culture,use of the poly-lysine coated culture dishes, deprivation of serum, andF.

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addition of bFGF or PDGF.
In contrast, approximately half of the cell lines derived from the neuroepithelium at 9.D-9.5 dpc showed transformation into neuron-like cells withlong processes, with concomitant expression of NF when transferred into
TAg impermissive conditions. Therefore, we concluded that early neuronalstem cells in the neuroepithelium undergo an important step toward commitment for neuronal differentiation at 8.5-9.0 dpc, and such cellular characteristics can be studied by using cell lines established from these cells at theearliest stages of CNS development. We are currently carrying out transplantation experiments of NES-8 cells into embryonic brains to test the
hypotheses that they are the earliest neuronal stem cells, and that they can be
induced to produce many cell types of CNS when transferred into theenvironment of embryonic brains.
(4)

Analysis of the Migration Pattern of Neuroblasts and Morphogenesis
in Development of the Mouse Central Nervous System

Norio NAKATSDJI and M. T. SHOKDNBI
In collaboration with Dr. I. Nagata at Tokyo Metropolitan Institute for
Neuroscience and Dr. K. Ono at Okayama University Medical School(present affiliation, Shimane Medical College), we are studying migration
patterns of neuroblasts during histogenesis of the mammalian central nervous
system (CNS). We found a new type of cell behavior, "perpendicular contact
guidance", which is exhibited by CNS neurons, but not by the peripheral
nervous system neurons on aligned parallel bundles of neurites. We studied
migration patterns of neurons in explants of brain cortices, using fluorescence
cell labeling. We are now analyzing the role of perpendicular contact
guidance in the actual morphogenesis of brain cortices using various histological and morphological methods.
Publications
1.
2.

KOSHIMIZU, D., WATANABE, M. and NAKATSUJI, N.: Retinoic acid is a potent growth
activator of mouse primordial germ cells in vitro. Devel. BioI., 168, 683-685, 1995.
ILIe, D., FURUTA, Y, KANAZAWA, S., TAKEDA, N., SOBUE, K., NAKATSUJI, N.,
NOMURA, S., FUJIMOTO, J., OKADA, M., YAMAMOTO, T. and AIZAWA, S.: Reduced cell
motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.

134

3.

4.

Nature, 377, 539-544, 1995.
OHKUBO, Y., SHIRAYOSHI, Y. and NAKATSUJI, N.: Autonomous regulation of proliferation and growth arrest in mouse primordial germ cells studied by mixed and clonal cultures.
Exp. Cell Res., 222, 291-297, 1996.
KOSHIMIZU, D., TAGA, T., WATANABE, M., SAITO, M., SHIRAYOSHI, Y., KISHIMOTO, T.
NAKATSUJI, N.: Functional requirement of gp 130-mediated signaling for growth andsurvival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG)cells. Development, 122, 1235-1242, 1996.

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G. DNA RESEARCH CENTER
G-a.
(1)

DNA-Protein Inateraction Laboratory

Kinetic Study on Transcription by E. coli RNA polymerase:
1. Irreversible Trapping in Abortive Cycle

Tomoko KUBORI and Nobuo SHIMAMOTO
The time course of synthesis of long and short (abortive) transcripts byEscherichia coli RNA polymerase was investigated under single round conditions in vitro. The synthesis of long RNA initiated at the APR (with an alteredleader sequence) or at the lacUV5 promoter was completed within 5 min, butabortive transcripts were continuously synthesized for at least 20 min. The
results indicate the presence of transcription complexes that are capable onlyof abortive synthesis, and not of productive elongation. Consistently, only
one in four of the RNA polymerase molecules that initially associated with
the APR promoter synthesized long RNA. The enzyme reisolated fromproductive complexes synthesized both long and abortive transcripts, behaving just like original enzyme. This suggests that RNA polymerase is homogeneous with respect to its ability to synthesize the two types of product.
Overall, the results indicate that many transcription complexes can be
irreversibly trapped in vitro in an abortive cycle (Ref. I).
We found abortive release is greatly activated in the presence of polymerase in excess of a promoter. When translocation of a polymerase on
DNA is inhibited by another polymerase molecule that stacked in front at 32
nt from a promoter, a misincorporation at 6 nt and abortive release of 4-5 nt
were greatly enhanced. The misincorporated transcripts were never elongated into long RNA even when the stacked molecule was removed.

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(2)

Single-Molecule Dynamics of Transcription Using Immobilized
Operons: Sliding of P. pUlida CamR protein on DNA

Hiroyuki KABATA and Nobuo SHIMAMOTO
The sliding motion of RNA polymerase along DNA during promoter
search has been proved by direct visualization of single molecules of the
enzyme. To check the generality of sliding, we applied similar single
molecule dynamics to a bacterial repressor protein, P. pUlida CamR, which isa homodimer molecule unable to relocate by the intersegment transfer
mechanism, another relocation mechanism requiring two DNA binding sites.
The protein was observed to slide along DNA. In the absence of its inducer,
d-camphor, CamR molecules were trapped at three positions on a DNA, one
was its cognate operator cloned in DNA, and the other two were likely to behomologous to the operator. The homologous regions turned out to be 400
and 4,000 times weaker binding sites than the cognate fragments by a gel-shift
assay. But CamR stayed on them as long as the cognate site by the direct
visualization. Immunoprecipitation assay showed a result similar to thedirect visualization, indicating the limitation of the gel-shift assay.
In the presence of the inducer molecules trapped at the strong binding sites
were significantly decreased, but the frequency of sliding increased. The
result showed that the inducer destabilized only specific interactions but notsliding complexes. This shows difference in interactions maintaining specificcomplex and sliding complexes.
(3)

Protein Footprint analysis of Transcription Complexes

Hiroki NAGAI, Tadashi T AKAGISHI and Nobuo SHIMAMOTO
Structural informations of engaged transcription complexes have been
mainly obtained from DNA footprinting/Exonuclease III protection approaches. This fact means that the obtained information is based on the
projected views of RNA polymerase onto DNA. Recently a novel and direct
approach, analogous to DNA footprinting, for detecting inter-protein/protein-nucleic acid interactions is developed. We used this method, protein
footprinting, to analyze structure of transcription complexes from Echerichia
coli. We constructed and purified C-terminal HMK-tagged sigma and j3
subunits of RNA polymerase. HMK-tagged portions of these subunits wereG.

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end-labeled with 32P by heart muscle kinase in vitro. Reconstituted RNA
polymerases were incubated with DNA template containing T7AI promoter
to form open complex, which were challenged by hydroxyl radical, and thencleaved sites were mapped by SDS-PAGE analysis. Results obtained so far
indicated that a portion of sigma subunit (mapped to conserved subregion3.2) in open complex was more protected from hydroxyl radical attack thanin holoenzyme. Detailed analysis is now in progress.

Publications
I.

2.

3.

4.
5.

KUBORI, T. and SHiMAMOTO, N.: A branched pathway in the early stage of transcription
by Echerichia coli. RNA polymerase. J. Mol. BioI. in press.
ARAMAKI, H., KABATA, H., SHiMAMOTO, N., SAGARA, Y. and HORIUCHI, T.: Purification and characterization of a cam repressor (CamR) for the cytochrome P450camhydroxylase operon on the Pseudomonas putida CAM plasmid. J. Bacteriol. 177, 31203127, 1995.
WASHIZU, M., KUROSAWA, 0., ARAI, I., SUZUKI, S. and SHiMAMOTO, N.: Applicationsof Electrostatic Stretch-and-Positioning of DNA. IEEE Trans. on Indst. Appl. 31, 447456, 1995.
SHIMAMOTO, N.: Complexity and multiplicity in life viewed from molecular biology. J.
Plasma and Fusion Res. (in Japanese) 71, 842-846, 1995.
KABATA, H. and SHIMAMOTO, N.: Single-molecule dynamics of proteins on DNA.
Biophysics (in Japanese) 35, 43-45, 1995.

G-b.
(1)

Recombinant DNA Laboratory

Fluoride-resistant Mutants of the Nematode Caenorhabditis elegans

Minoru KAWAKAMI, Masaya TAKE-UCHI, Misako URASAKI, Takeshi ISHIHARA and Isao KATSURA
The fluoride ion is known to interact in vitro with components of signaltransduction systems such as Ca2+, protein phosphatases and trimeric Gproteins. We are isolating and characterizing fluoride-resistant mutants of C.
elegans to discover a new signal transduction system.
Thirteen fluoride-resistant mutants that we isolated are recessive and map
in five new genes, fir-l to fir-5. They are grouped into two categories:
strongly resistant, slow-growing mutants (flr-l, fir-3 and fir-4) and weakly
resistant, normally growing mutants (flr-2 andfir-5). Interestingly, the latter

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mutations suppress the slow-growing phenotype of the former (Katsura, I. etal. (1994) Genetics 136, 145-154). All those mutations seem to affect thenervous system, because some of them are defective in chemotaxis (Seebelow), the slow-growing mutants have short defecation-cycle periods (Dr. J.
H. Thomas, University of Washington), and all except thefir-2 mutants havesynthetic dauer-constitutive phenotypes (See section (3».
To learn the molecular mechanism of the action offir genes, we cloned thegenes and cDNAs offir-l andfir-3. The amino acid sequence deduced froma partial-length cDNA offir-l has weak homology to that of the MEC-4 andDEG-l ion channels of C. elegans and amiloride-sensitive ion channels of
mammals. We obtained a full-length cDNA offir-3, which seems to encodea kinase-like molecule that has all the protein kinase consensus sequences
except that of subdomain I, the ATP-binding site.
By comparing the physical map ("contigs") with the genetic map, we
found that the positions of fir-4 mutations are confined to a genomic regionof several hundred kb. Injection of cosmid clones in this region into fir-4
mutants revealed a clone that can rescue the phenotypes of slow growth andshort defecation-cycle period offir-4. We are now subcloning it and trying todefine the minimal DNA region that can rescue the phenotypes.
We investigated the expression pattern of fir-3, using a fir-3-lacZ fusion
gene that contained the upstream 3.65 kb and downstream 2.75 kb of thetranscriptional start point offir-3. Expression was detected in most intestinal
cells, beginning just before body elongation in the mid-embryonic stage andending at the end of the L1 or the beginning of the L2 larval stage.
To study the effect of fir mutations on the nervous system, chemotaxis
assays were performed for the reference alleles of all the fir genes. It was
found that thefir-2 mutant was abnormal in chemotaxis to many volatile andnon-volatile compounds, fir-5 to lysine, cAMP (attractive) and l-octanol(repulsive), andfir-4 to lysine. Thefir-l mutant occasionally showed weakabnormality, but no abnormality was detected for fir-3.
We plan to continue the analysis of the fir genes to elucidate the molecularmechanisms of fluoride-sensitivity, growth-rate regulation and neural functions.

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DNA RESEARCH CENTER139

A Gene that Controls Both Hatching and Cell 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 (Hedgecock, E. M. et al. (1987) Development 100, 365-382).
They cannot digest protein components of the eggshell, and a neuroblast
called QL moves anteriorly instead of posteriorly during larval development.
After we isolated a transposon-insertion mutant in hch-l (K. Kondo (Soka
University) and I. Katsura, unpublished results), we cloned the gene by thetransposon-tagging method and the cDNA by using the genomic fragment as
the probe. The cloned cDNA had the same length (3 kb) as the mRNA as
detected by Northern analysis. It encoded a protein that consisted 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-I, both ofwhich act in cell-differentiation. However, the protein most closely related to
hch-l is the sea urchin BPlO/SpAN, which is reported to act in morphogenesis but not in hatching. We performed in situ hybridization of hch-l toembryos to detect expression before hatching. The results showed that
expression starts just before body-elongation, continues through the comma
stage and ends at the 1.5-fold or 2-fold stage, while no expression was
detected at later embryonic stages. The 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 lateral sides (probably seam cells) at the
1.5-fold stage. Both the morphogenetic protein BPlO/SpAN and hatching
enzymes (sea urchin HE6/SpHE and teleost HCE/LCE) were reported to be
expressed in the middle of embryogenesis, considerably before hatching.
Hence hch-l may be a hatching enzyme, or may act in the processing/secretion of hatching enzymes or in the differentiation of the cells that produce
hatching enzymes.
We are going to analyze the role of each domain of hch-l and to investigate
whether hch-l acts upstream or downstream to the homeotic gene mab-5,
which also affects the direction of QL migration.

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(3)

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

Norio SUZUKI, Misako URASAKI, Takeshi ISHIHARA and Isao KATSURA
If larvae of C. elegans just after hatching are in a crowded state and given
a limited 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 head neural circuit, with thesmell of bacteria and the pheromone as the inputs, which are sensed byamphids, a pair of sensory organs in the head. Since the assay of dauer
formation is much less time-consuming than behavioral assays such as
chemotaxis assays, we are analyzing the head neural circuit by detecting
dauer formation as the output. We found that more than 40 genes are
involved in the synthetic dauer-constitutive (syn Daf-c) phenotype, i.e., thephenotype 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 neuralcircuit, 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, thespecific combinations of mutations for the phenotype should contain information for 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 assuming three parallel pathways, if we
excludeflr mutations. We plan to obtain further information by investigating
whether known dauer-defective mutations can suppress the syn Daf-c phenotype of various double-mutations.
We isolated 44 new mutations that show the syn Daf-c phenotype incombination with the unc-31 (e169) mutation, which causes abnormalities inbody movement, pharyngeal pumping, mechanosensation and egg-laying.
We mapped them by sequence-tagged site markers and are performing
complementation tests to learn whether they are alleles of known genes. We
also examined the penetration of the fluorescent dye diO into amphids andphasmids (sensory organs in the tail) to investigate possible structural defects
in those organs. Thirty-three mutants were normal, five slightly abnormal
and six clearly abnormal. The results together with the results of mapping

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show that a majority of the new mutations are located in unknown genes andthat this method is useful for the isolation of new neural mutations. For
further characterization of the mutants we plan to look for behavioral
abnormalities and, with the help of markers described in (4) below, abnormalities in the structure of the neural system and differentiation ofneurons.
(4)

Construction of GFP Markers for Various Neurons in C. elegans
and Their Use for Mutant Isolation

Takeshi ISHIHARA, Manabi FUJIWARA and Isao KATSURA
To analyze the formation and function of the neural circuit in C. elegans,
we made 15 types of worms in which specific sets of neurons emit fluorescence. They were prepared by integration (into the worm genome) of DNA
constructs in which the eDNA of GFP (jellyfish green fluorescent protein;
Chalfie, M. et al. (1994) Science 263, 802-805) is fused with various C.
elegans neural promoters. Three of the promoters were selected by the"promoter trapping" method (Hope I. (1991) Development 113, 399-408),
and twelve by looking at the genome DNA sequence. The number of cells
that express GFP ranged from only one pair to a great majority of theneurons. The fluorescence was localized to cell nuclei and to processes andcell bodies, if the SV40 nuclear localization signal and a C. elegans unc-76
genomic fragment, respectively, were inserted into the DNA constructs.
Six of the 15 promoters showed expression in interneurons. We are
studying functions of four of the six genes, which code for three putative
glycine (or GABA) receptors and a metabotropic glutamate receptor. Gene
disruption experiments are continuing.
To learn the functions of specific neurons we plan to kill them by introducing DNA constructs in which those promoters are connected to cell-deathgenes (mec-4(d), ced-3 or ced-4). We also plan to isolate mutants abnormal
in neural morphology using the GFP markers. Using a marker that isexpressed in most neurons, we have already performed such experiments andobtained three mutants in neural cell lineage or cell migration.

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Publications
None

G-c.
(1)

DNA Synthesis Laboratory

Crystallographic Study of aJ33 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 a133
sub-assembly of FI-ATPase is an active complex with 20-25% of theATPase activity of intact F 1, and has similar catalytic properties to those of
F1. Both FI-ATPase and the a133 sub-assembly have been challenging
targets for crystallographic study because of their large sizes e.g. 380 kDa for
Fl and 320 kDa for the a133 sub-assembly.
The nucleotide-free a133 sub-assembly from Bacillus PS3 Fl formed the
crystals which diffracted to 3.2 A resolution. We solved the structure of thesubcomplex by the molecular replacement technique using bovine Fl structure as a search model (manuscript in preparation). The current model has
R factor of 16.2% (free R factor 32.0%) and has a reasonable stereochemistrye.g. the root mean square deviations from ideal values of 0.0 1 A for bondlengths, and 1.5° for bond angles. The structure was solved in collaboration
with Andrew Leslie, Jan Pieter Abrahmas and John Walker at MRC Laboratory of Molecular Biology, Cambridge, UK.
The crystal structure of the a133 sub-assembly was symmetrical: that is, thethree a and three f3 subunits have identical conformations. This was incontrast to the asymmetry observed in the nucleotide-bound bovine Fl
structure. In PS3 structure the f3 subunits adopted a conformation essentially
identical to that of the nucleotide-free f3 subunit in bovine F1. This suggeststhat in Fl the nucleotide occupancy as well as the interactions with the r
subunit determine the conformation of the nucleotide-free f3 subunit. In spite

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of the difference in nucleotide ligation, the a subunit had a similar conformation in both structures.
(2)

Crystallographic Study of Transcription Factors, PhoB and CamR

Yasuo SHIRAKIHARA
PhoB Protein is a transcriptional activator for the genes in the phosphate
regulon of E. coli, such as phoA and pstS, that are induced by phosphate
deprivation. PhoB acts by binding to the pho 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 by phosphorylation
by PhoR. The C terminal domain of PhoB has a DNA binding ability, while
the N terminal domain has a regulatory role governed by phosporylation by
PhoR protein.
We previously crystallized the C terminal domain of PhoB. Crystals
diffracted to at least 2.4 A resolution, but had a minor disorder in the
crystalline lattice. Using a new C terminal fragment which is shorter by 8
amino acid residues than the fragment above, we got crystals with noappreciable disorder. Also we got crystals of the intact PhoB protein. This
work has been done with Kozo Makino, Research Institute for Microbial
Diseases, Osaka University.
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.
CamR was crystallized from polyethyleneglycol solution with a typical sizeof 0.5mmX0.4mmXO.3mm in the presence or absence of D-camphor.
However, recent crystallization experiments have failed to grow such large
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. In stead of polyethyleneglycol, Na-K-phosphate
was found to give crystals of the similar sizes in the presence of D-camphor.
This work has been done with Hironori Aramaki, Daiichi College of Pharm-

144

aceutical Sciences.
Publications
None

G-d.
(1)

Gene Library Laboratory

Expression map of the C. elegans genome

Yuji KOHARA, Tomoko MOTOHASHI, Hiroaki TABARA, Akiko SUGIMOTO,
Hisako WATANABE, Masako SANO and Akiko MIYATA
Aiming to ultimately understand the network of gene expression in thedevelopment of the nematode C. elegans, we are trying to construct anexpression map of the 100Mb genome by identifying and characterizing all of
its cDNA species, whose number is estimated to be around 13,000.
Altogether 38,000 cDNA clones were picked up randomly from 3 different
cDNA libraries (from a mixed-stage population, from size-fractionated ( > 2Kb) cDNA of a mixed stage population and from embryos), stored andgridded. To avoid unnecessary redundancy of analyses, cDNA clones thatbelong to previously identified abundant cDNA species were screened byprobing the cDNA grids and removed. cDNA inserts of individual clones
were PCR-amplified using vector primers and then subjected to the following
analyses.
(1) Tag sequencing: Single reads were made from both 5'-ends (using
vector primers) and 3'-ends (using anchored oligo-dT primers in order tominimize the effect of a long poly-A stretch) on ABI sequencers. 3'-tagsequences were compared with each other using FASTA to classify the
cDNA clones into unique groups (genes). Thus far, about 16,000 clones gave
11,104 clean 3'-tag sequences, which were classified into 4,279 unique cDNA
species (assigned group names CELKOOOOI through CELK04279). This
analysis also detected many pairs of clones which appeared to be generated byalternative splicing or differential poly-A addition. BLASTX search showedthat 44% of the cDNA groups gave significant similarities (blastx score> 100).
(2) Mapping onto the genome: Using the ordered YAC filters, 1,416

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cDNA groups have been mapped onto the genome; central regions ofautosomes turned out to be much denser for cDNA than other regions.
Using the genomic sequences kindly provided by the Sanger Centre (1,001
cosmids including 172 cosmids at a shotgun phase), 1,341 cDNA species outof the 4279 species were mapped ("mapping in silico").
(3) Analysis of expression pattern: We are interested in gene expression
patterns particularly during embryogenesis. For this purpose we havedeveloped a method of in situ hybridization of the standard mult-well format
on whole mount embryos, and we have started a systematic in situ analysis
using the set of representative clones of the identified cDNA groups. Thusfar, 594 cDNA species were analyzed in this way, providing a specific patternof expression at a frequency of 1/5.
(2)

A Multi-well Version of in situ Hybridization on Whole Mount
Embryos of C. elegans

Hiroaki TABARA, Tomoko MOTOHASHI and Yuji KOHARA
We have developed an efficient procedure for in situ hybridization of amulti-well format on C. elegans embryos for large scale screening of gene
expression patterns in this organism. Each hybridization well contains
embryos at various stages throughout embryogenesis. The validity of themethod was confirmed by results with control genes whose expression patterns had been reported; glp-l in very early embryos, myo-2 in pharyngeal
muscle and unc-54 in body-wall muscle. Several collagen genes and a
pepsinogen gene were also examined to establish a set of lineage specificmarkers.
(3)

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

Hiroaki T ABARA, Tomoko MOTOHASHI, Craig MELLO l , Jim PRIESS 2 andYuji KOHARA CCancer Center, University of Massachusetts, Worcester,MA, USA, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA)We have long searched for localized maternal mRNA in early embryos of
the worm using a range of approaches to differential screening. In the course

146

of the systematic analysis of in situ hybridization with a set of classified
cDNA clones which have been generated by our cDNA project, we foundthat the clone, yk61hl, of the cDNA group CELKOl662 (=YKI662 in
ACEDB) showed an asymmetric distribution of its mRNA in 2-cell stageembryos. Closer examinations on the behavior of the mRNA in gonads andembryos using in situ hybridization showed that (l) the mRNA started toappear at the turn of gonad, (2) it was distributed evenly in oocytes, (3) it
was segregated posteriorly as the first cleavage proceeded, (4) the mRNA
remained in P4 but disappeared in other somatic blastmeres (perhaps througha mechanism of degradation). In situ analysis using nop-I embryos which
lacked pseudo-cleavage suggested that the segregation of the mRNA occurred during the first cleavage. Northern hybridization showed that the mRNA
(1.3 Kb) was detected in embryos and adults but not in Ll larva. We named
this gene pos-I (posterior segregation), and have analyzed it further.
Full sequencing of the cDNA clone showed that the pos-I mRNA has thetrans-spliced leader SLI and a strong homology to the zinc-finger region of
the mammalian Tsi 11 growth factor inducible genes. This zinc-finger motifis also found in the maternally expressed C. elegans gene pie-I. Interestingly,
the position of pos-I turned out to be very close to a second maternal gene,skn-2. The skn-2 gene, like pie-I, is required for proper development of the
P lineage, and skn-2 and pie-I mutants exhibit dominant genetic interactions.
These genetic interactions and the physical and genetic proximity of pos-I
andskn-2 led us to invetigate the possibility thatpos-I =skn-2. The following
evidence supports this hypothesis: (1) injection experiments using pos-I
anitsense RNA caused phenocopies of skn-2, and (2) a missense mutation
was found in the zinc finger region of pos-I in a skn-2 mutant (zuI48).
The phenotypes of skn-2 mutants were examined closely. The lack of germline was confirmed by the fact that anti-P granules antibodies did not stain
late embryos of skn-2; it stained two germ line precursor cells Z2 and Z3 inwild type embryos. Although skn-2 embryos are lethal, some somatic
lineages seem to differentiate properly; we found the differentiation of hypodermis, gut and pharynx in skn-2 embryos. As to the function of pie-I, a
genetically related gene to skn-2, it was hypothesized that the pie-I protein
repressed the transcription of somatic genes in the germ line, based on theobservation that the mRNA of some zygotic genes, e.g. pes-ZO, which was
detected in all somatic cells but not in the germ line, was detected also in thegerm line of pie-I embryos. We perfomed in situ experiments with such

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zygotic genes including pes-lO and 4-3 on skn-2 embyros. However, themRNA of 4-3 was not detected in the germ line of skn-2 embryos.
Antibodies against the N-terminal part ofthepos-l protein were raised andthe distribution of the pos-l protein was examined. The expression of theprotein was coincident with the pattern of the localized mRNA. Essentiallyno expression of the protein was detected in I-cell embryos or AB blastmeresin 2-cell embryos. The expression of the protein was detected in PI, P2, P3
and P4. Very interestingly, the expression of the pos-l protein was cytoplasmic in contrast to other genes conatining the Tisll zinc finger motif.
Based on these facts and preliminary observations about other genes
containg the Tisll zinc finger motif, we hypothesize that the function of the
pos-l proteins is to stabilize or activate the translation of mRNA of otherfactors which are necessary for establishing the fate of the germ line.
Publication
1.

BARNES, T., KOHARA, Y., COULSON, A. and HEKIMI, S.:
Meiotic recombination,
noncoding DNA and genomic organization in Caenorhabditis elegans. Genetics, 141, 159179, 1995.

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(1)

DNA Data Analysis Laboratory

Domain Evolution of Serine Protease and Its Inhibitor Genes

Takashi GOJOBORI, Toshinori ENDO and Kazuho IKEo
The evolution of serine protease and its inhibitor was discussed with specialreference to domain evolution. It is now known that most proteins arecomposed of more than one functional domain. Because serine proteases
such as urokinase and plasminogen and some of their protease inhibitors are
made of various functional domains, these proteins are typical examples ofthe so-called mosaic proteins.
When kringle domains in serine proteases and a Kunitz-type proteaseinhibitor domain in the amyloid f3 precursor protein in Alzheimer's disease
patients were examined by the molecular evolutionary analysis, the phylogenetic trees constructed showed that these functional domains had undergone dynamic changes in the evolutionary process. In particular, these
domains are considered to be evolutionarily movable.
It was concluded that these functional domains evolved independently ofeach other and that they have been shuffled to create the existent mosaic
proteins. This conclusion leads us to the reasonable speculation that thesefunctional domains must have been minigenes, possibly at the time ofprimordial life or the origin of life. We may call these minigenes "ancestralminigenes". For more details, see Ref. 7.
(2)

Sequence Divergence Estimation

Takashi GOJOBORI and Kazuho IKEo
Methods for estimating sequence divergence are crucial for studies ofmolecular evolution. In particular, the number of nucleotide (or amino acid)substitutions is a good quantity to use in representing the degree of sequence
divergence when two homologous sequences are being compared. Thus,

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knowledge of the number of nucleotide (or amino acid) substitutions isimportant for computing evolutionary rate and for constructing phylogenetic
trees at the DNA level. We reviewed statistical methods for estimating thenumber of nucleotide and amino acid substitutions, putting particular emphasis on their practical usage and usefulness. For more details, see Ref. 10.
(3)

Different Evolutionary Histories of Kringle and Protease Domains
in Serine Proteases: A Typical Example of Domain Evolution

Kazuho IKEO, Kei TAKAHASHI l and Takashi GOJOBORI
Physiology, Shimane Medical University, Isumo)

eDepartment ofWith the aim of elucidating the evolutionary processes of the kringle andprotease domains in serine proteases which are involved with the system ofblood coagulation and fibrinolysis, we constructed phylogenetic trees for the
kringle and protease domains, separately, by use of amino acid sequence data.
The phylogenetic trees constructed clearly showed that the topologies weredifferent between the kringle and protease domains. Because both domains
are coded by single peptides of serine proteases, this strongly suggests that the
kringle and protease domains must have undergone different evolutionary
processes. Thus, these observations imply that serine proteases evolve in away such that each domain is a unit of evolution, exemplifying a typical modeof domain evolution. A possible relationship between the domain evolution
and the exon shuffling theory were also discussed from the viewpoint of gene
evolution. For more details, see Ref. 8.
(4)

Evolution of Genes and The DNA Database-Implications
for Bioinformation

Takashi GOJOBORI, Toshinori EN DO and Kazuho IKEO
Needless to say, the DNA sequence database has tremendous value inmany contexts. Data retrieval has been very useful not only in academic
circles but also in bioindustries. The DNA database can also be used to study
the molecular evolution of genes. We conducted a large-scale analysis ofmolecular evolution, using the DDBJ DNA sequence database. The results
obtained showed that homologous domains exist among remotely related

150

gene groups. This suggests that present genes may have emerged as "mosaic
genes" from different domains of their ancestral gene by shuffling of homologous domains during evolution. This concept of "mosaic genes" is very
useful and important not only for biotechnology but also for bioremediation.
For more details, see Ref. 11.
(5)

Large-Scale Search for Genes on Which
Positive Selection May Operate

Toshinori ENDO, Kazuho IKEo and Takashi GOJOBORI
We conducted systematic search for the candidate genes on which positive
selection may operate, on the premise that for such genes the number of
nonsynonymous substitution is expected to be larger than that of synonymous
substitutions when the nucleotide sequences of the genes under investigation
are compared with each other. By obtaining 3,595 groups of homologous
sequences from the DDBJ, EMBL, and GenBank DNA sequence databases,
we found that seventeen gene groups can be the candidates for the genes on
which positive selection may operate. Thus, such genes are found to occupy
only about 0.5% of the vast number of gene groups so far available.
Interestingly enough, nine out of the seventeen gene groups were the surface
antigens of parasites or viruses. This work is described in more details in
Molecular Biology and Evolution. For more details, see Ref. 13.
(6)

Large-Scale Search for Within-Gene Regions Where
Positive Selection May Operate

Toshinori ENDO, Kazuho IKEO and Takashi GOJOBORITo determine what is the proportion of genes on which positive selection
operates, we searched for genes on which positive selection may operate froma large number of DNA sequences in DDBJ, EMBL and GenBank DNA
databases. We also attempted to identify the within-gene regions which
responsible for positive selection. For this purpose we developed a window
search method which quickly identifies the within-gene regions possibly
responsible for positive selection on the basis of estimation and comparison of
the numbers of nonsynonymous substitution with that of nonsynonymous

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substitutions for each window in a homologous DNA alignment. From 3,595
homologous gene groups which we previously obtained using homology
search, we found that 192 genes (5.3%) of the gene groups examined werethe candidates on which positive selection may operate. Among these genes,
the most major category was the gene group for the surface proteins ofparasites and viruses. The candidates also contained the gene groups for
secretory proteins, various kinds of enzymes, toxins and so on. These results
suggest that there may be various reasons for positive selection. These results
were presented at the Society of Molecular Biology and Evolution Meeting
1995 at Hayama, the meeting of Genetics Society of Japan at Okayama andthe annual meeting of the Molecular Biology Society of Japan at Nagoya.
(7) Evolutionary Relationship of Hepatitis C, Pesti-, Flavi-,
Plantviruses, and Newly Discovered GB Hepatitis Agents
Ken-ichi OHBA 1, Masashi MIzOKAMIl, Johnson Y. N. LAu 2, Etsuro ORIT0 1,
Kazuho IKEo and Takashi GOJOBORI CSecond Department of Medicine,
Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya,
2Section of Hepatobiliary, Department of Medicine, University of Florida,
Gainesville, FL, USA)
Two flavivirus-like viruses, GB virus-A (GBV-A) and GB virus-B(GBV-B), were recently identified in the GB hepatitis agent, and are known
to be distinct from the hepatitis A to E viruses. The putative helicase domain
of GBV-A and GBV-B was found to have amino acid sequence homology
with that of hepatitis C virus (HCV), and to be distantly related to pestiviruses, flaviviruses, and plant viruses. A phylogenetic tree constructed showedthat GBVs and HCV were closely related to each other and they wereclustered with pestiviruses, flaviviruses and plant viruses in that order. Thisfinding may lead us to an interesting speculation that HCV and GBV may
have originated from some of insect and plant viruses. For more details, see
Ref. 15.

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(8)

Classification of Hepatitis C Virus into Major Types andSubtypes Based on Molecular Evolutionary Analysis

Ken-ichi OHBA 1, Masashi MIZOKAMI 1, Tomoyoshi OHNOl, Kaoru SUZUKI I,
Etsuro ORITOl, Yasuo INA, Takashi GOJOBORI and Johnson Y. N. LAU 2
Second Department of Internal Medicine, Nagoya City University Medical
School, Kawasumi, Mizuho, Nagoya, 2 Section of Hepatobiliary Diseases,
Department of Medicine, University of Florida, Gainesville, FL, USA)e

Molecular evolutionary analysis was applied to determine the number ofhepatitis C virus (HCV) types and subtypes based on all the HCV nucleotide
sequences available from the DNA data banks (DDBJ, GenBank (NCBI),
EMBL) and the literature. There was an excellent concordance among thetypes and subtypes assigned based on different HCV genomic regions. Onlyone HCV isolate was assigned to different HCV types based on the 5'
non-coding (NC) and envelope I (El) regions. The 5' NC region was wellconserved and could be used to assign only types and not subtypes. From thesequence data available, there were 13 subtypes based on the core region and
14 subtypes based on the El and non-structural protein 5 (NS5) regions. For
more details, see Ref. 12.
(9)

Patterns of Amino Acid Substitutions in the Third Variable
Envelope 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
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. We then estimated the rates of synonymous andnonsynonymous substitutions for the V3 region of HIV within single hosts ateach period of time. In some periods of time since infection, the rate ofnonsynonymous substitution was significantly higher than that of synonymous substitution. We also estimated frequencies and types of amino acidsubstitutions that occurred at each amino acid site in the V3 region. As aresult, we found that amino acid substitutions dominantly occurred at severalamino acid sites where the substitutions are known to be responsible forH. CENTER FOR INFORMATION BIOLOGY

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production of antigenic variation and determination of the viral phenotypes.
These observations indicate a strong possibility that positive selection is
operating in the V3 region of HIV within single hosts by changing aminoacids at particular sites.
(10)

Molecular Epidemiology of Human Immunodeficiency Virus Type 1

Yumi YAMAGUCHI and Takashi GOJOBORI
Human immunodeficiency virus type 1 (HIV-I) that possibly came fromAfrica is now distributed all over the world. Previous phylogenetic analyses
showed that almost all HIV-I isolates from developed countries and Haiti
form a distinct cluster known as "subtype B". Those studies suggested that
one type of HIV-I variants in Africa was introduced to developed countries
and Haiti. We intended to elucidate the course of transmission of HIV-I to
all over the world by phylogenetic analysis. We collected nucleotide sequences of HIV-I from international DNA databases. We constructed
phylogenetic trees of HIV-I using amino acid sequences in a part of theenvelope glycoprotein including the third variable envelope (V3) region. We
found that HIV-I isolates from Russia and Romania form each new cluster.
Our results suggest the possibility that course of transmission of HIV-I to
Russia and Romania might differ from that to most developed countries. Our
results also support the possibility that HIV-I might be introduced to India
and Thailand directly from Africa.
(11)

Molecular Evolution of HIV·1 and its Implication toMother-To-Child Transmission

Kazunari TAKAHAsHI and Takashi GOJOBORI
Transmission of human immunodeficiency virus type 1 (HIV-I) takes
place, with a high frequency, among drug users, sexual partners, and motherto-child pairs. Our study was conducted with the aim of elucidating theevolutionary modes of HIV-I at the time when it is transmitted to a new host.
We used the nucleotide sequences of HIV clones isolated at the time of themother-to-child transmission. These data were obtained from the three DNA
databases of DDBJ, EMBL, and GenBank. We then constructed phylogene-

154

tic trees by the neighbor-joining method. The phylogenetic tree constructed
clearly showed how particular HIV clones in a mother were transmitted toher child.
(12)

Molecular Evolution of Marburg and Ebola Viruses

Yoshiyuki SUZUKI and Takashi GOJOBORI
Marburg and Ebola viruses, members of the nonsegmented negativestranded RNA viruses, constitute the genus Filovirus, the sole member of thefamily Filoviridae which is one of the constituents of the order Mononegavirales. These viruses are known to be the etiological agents of the haemorrhagic fever with high mortality rates and have been grouped into the"biosafety level 4" agents. From the molecular evolutionary points of view,
it is of importance to elucidate the origin and evolutionary mode of theseviruses. In this study, the rate of nonsynonymous substitutions for Marburg
virus was estimated to be 4.3 X 10- 4 per nonsynonymous nucleotide site per
year, which was of almost the same order of magnitude with other RNA
viruses. When this rate was applied to the degrees of sequence divergences
the divergence time between Marburg and Ebola viruses was estimated to be
more than 300 years ago, possibly several thousand years ago. Moreover, thepattern of nucleotide substitutions for Marburg virus indicated that the
purifying selection has operated on the evolution of this virus. The results forthe rate of nonsynonymous substitutions and the divergence time between
Marburg and Ebola viruses were presented on the 43rd Annual Meeting of
the Society of Japanese Virologists at Okayama, Japan in October, 1995.
(13)

Molecular Phylogenetic Trees Based on Minimum
Description Length Principle

Fengrong RENt, Hiroshi TANAKA l , Norio FUKUDA l and Takashi GOJOBORI
CTokyo Medical and Dental University, Tokyo)
Ever since the discovery of a molecular clock, many methods have beendeveloped to reconstruct the molecular phylogenetic trees. In this study, we
dealt with this problem from the viewpoint of an inductive inference andapply Rissanen's minimum description length principle to extract the mini-H.

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mum complexity phylogenetic tree. Our method describes the complexity ofmolecular phylogenetic tree by three terms which are related to the tree
topology, the sum of branch lengths, and the difference between the modeland the data measured by logarithmic likelihood. Five mitochondrial DNA
sequences from human, common chimpanzee, pygmy chimpanzee, gorillaand orangutan were used for investigating the validity of this method. It issuggested that this method is superior to the traditional method in that it stillshows the good accuracy even near the root of phylogenetic trees. For moredetails, see Refs. 1 and 2.
(14)

NRSub: a Non-Redundant Database for Bacillus subtilis

Guy PERRIERE 1, Ivan MOSZER 2 and Takashi GOJOBORI CLaboratoire de
Biometrie, Genetique et Biologie des Populations, Universite Clause BernardLyon 1, France, 2 Unite de Regulation de I'Expression Genetique, Institut
Pasteur, 28, France
In the context of the international project aimed at sequencing the wholegenome of Bacillus subtilis, we have developed a non-redundant, fully annotated database of sequences from this organism. Starting from the B. subtilissequences available in the EMBL, GenBank and DDBJ collections, we haveremoved all encountered duplications and then added extra annotations tothe sequences (e.g. accession numbers for the genes, locations on the genetic
map, codon usage, etc.). We have also added cross-references to the EMBL,MEDLINE, SWISS-PROT and ENZYME data banks. The present system
results from merging of the NRSub and SubtiList databases and the sequence
contigs used in the two systems are identical. NRSub is distributed as aflatfile in EMBL format (which is supported by most sequence analysis soft
ware packages) and as an ACNUC database, while SubtiList is distributed asa relational database under 4th Dimension. It is possible to access the data
through two dedicated World Wide Web servers located in France andJapan. For more details, see Ref. 14.

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(15)

Analysis of the Structure of the Escherichia coli Genome by
Seeing the Distance between Homologous Genes

Hidemi WATANABE and Takashi GOJOBORl
A hypothesis has been proposed for the evolution of the Escherichia coligenome that the genome has increased in size not by gradual addition but
through at least two times genome doublings. Although this hypothesis was
originally presented on the basis of the regular distribution of functionallyrelated homologous genes in the genome, it seems rather unconvincing
because the number of the genes used was very small and the regularity was
not evaluated by any statistical method. To date, the genome of E. coli has
been sequenced around three quarters. Thus we tried to test the hypothesis
with all the genes already sequenced. The frequency distribution of thephysical distances between all homologous gene pairs was computed. In thiscomputation, the orientation of the genes of each pair in the genome wasconsidered, and thus two distributions were obtained for the pairs of genes inthe same orientation and those in different orientations. Several characteristic features are found in these distributions: Two dominant peaks are detectedin the distribution of the pairs in the same orientation whereas no obvious
peak is not found in the distribution of those in different orientations. One of
the two peaks shows that tandem repeats of homologous genes in the sameorientation are predominant among all the homologous gene pairs. The otherpeak is found around 18 min distance. In both of the two distributions, nopeak is found around 25 or 50 min distance. Therefore, it is concluded that
gradual additions and duplications of local regions rather than recent two
times genome doublings have been contributed to the evolution of genome
size in E. coli.
(16)

Analyses of the Structures of Microbe Genomes on the Basisof the Homologous Relationships between Genes

Hidemi WATANABE, Hirotada MORl and Takashi GOJOBORl
To test two different hypotheses on the evolution of microbe genomes thatmicrobe genomes are evolutionarily stable and that the variety of their sizes
is mainly brought by genome doubling during evolution, the genome structures of Haemophilus influenzae, Mycoplasma genitalium, Escherichia coli andH. CENTER FOR INFORMATION BIOLOGY

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Bacillus subtilis were compared by use of the DNA sequences of theirgenomes. In these comparisons, the locations of orthologous genes were
examined between different genomes. Comparisons of orthologous genes
guarantee that the differences revealed by the comparisons mostly reflect thechanges in genome structure after the speciation of the species compared. We
invented two new measures for the degree of conservation of relative genelocations and for the difference in gene number. The comparisons by the newmeasures primarily revealed striking evidence that genome rearrangements
have frequently occurred in microbe genomes during their evolution evenafter the relatively recent speciation between E. coli and H. infiuenzae. Quite
exceptionally are only a few regions conserved, e.g. the S10 region, the clusterof genes for cell division, and the ATP synthase operon. This indicates thateven the clusters consisting of genes functionally related to each other, e.g.
operons, are not preserved through evolution. An additional analysis led to
us to suggest that the common ancestral genome of E. coli and H. infiuenzae,
having genomes of 4.7 and 1.8 megabases, respectively, must have been as
large as the E. coli genome, and then the genome of H. infiuenzae has been
shrunk.

(17)

Monophyletic Origin and Unique Dispersal Patterns ofDomestic Fowls

Akishinonomiya FUMIHITO', Tetsuo MIYAKE 2 , Masaru T AKADA, Ryosuke
SHINGU4, Toshinori ENDO, Takashi GOJOBORI, Norio KONDO) and Susumu
OHN0 5 (' Yamashina Institute for Ornithology, Chiba, 2 Wakunaga Pharmaceutical Company Central Laboratories, Hiroshima, ) The Research Instituteof Evolutionary Biology, Tokyo, 4 The 2nd Department of Internal Medicine,
Osaka University Medical School, Osaka, 5 Beckman Research Institute of
the City of Hope, USA)
With the aim of elucidating in more details the genealogical origin of thepresent domestic fowls of the world, we have determined mitochondrialDNA(mtDNA) sequences of the D-Ioop regions for a total of21 birds which
belong to red junglefowl (Gallus gallus) comprised of three subspecies (6Gallus gallus gallus, 3 Gallus gallus spadiceus and 3 Gallus gallus bankiva)
and 9 representing diverse domestic breeds (Gallus gallus domesticus). We
also sequenced 4 green junglefowl (Gallus varius), 2 Lafayatte's junglefowl

158(Gallus lafayettei) and 1 grey junglefowl (Gallus sonneratii). We thenconstructed a phylogenetic tree for these birds by the use of nucleotide
sequences, choosing the Japanese quail (Coturnix coturnix japonica) as an
outgroup. We found that a continental population of G. g. gallus was the realmatriarchic origin of all the domestic poultries examined in the present study.
It is also of particular interest to note that there were no discernible difference
among Gallus gallus subspecies; G. g. bankiva being a notable exception. Thiswas because G. g. spadiceus and a continental population of G. g. gallus
formed a single cluster in the phylogenetic tree. G. g. bankiva, on the other
hand, was a distinct entity, thus, deserving its subspecies status. It implies
that a continental population of G. g. gallus sufficed as the monophyleticancestor of all domestic breeds. We also discussed a possible significance of
the initial dispersal pattern of the present domestic fowls, utilizing thephylogenetic tree. For more details, see Ref. 16.

(18)

Isolation and Characterization of cDNA Clones for Japanese
Quail (Coturnix Japonica) Major Histocompatibility
Complex (MhcCoja) Class I Molecules

Takashi SHIINA!, Asako AND0 2, Tadashi IMANISHI, Hisako KAWATA 2, Kei
HANZAWA!, Takashi GOJOBORI, Hidetoshi INoKo 2 and Seiki WATANABE I(' Laboratory of Animal Physiology, Department Of Zootechnical Science,
Tokyo University of Agriculture, Tokyo, 2 Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Kanagawa)Two cDNA clones (QF41 and QF63) corresponding to the MHC class I
genes of the Japanese quail (Coturnixjaponica) were isolated by screening aliver cDNA library with the chicken class I cDNA (B-F) as a probe. The
overall amino acid sequence identity between the Japanese quail and chicken
MHC class I a chain was from 74% to 78%, with the most divergence in thetransmembrane (TM) and cytoplasmic region (CY). Between the QF41 and
QF63 cDNA clones, there was only 79% amino acid identity with the highest
homology (93%) in the functionally conserved a3 domain. PCR analysis
using genomic DNA as a template with the QF41- and QF63-specific primers
revealed the different and unique exon/intron organization around the al anda2 domain regions. Taken together, these results suggest that the JapaneseH.

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quail has at least two distinct expressed MHC class I loci, designated Coja-A
and Coja-B. For more details, see Ref. 6.
(19)

Determination of HLA Class II Alleles by Genotyping in a
Manchu Population in the Northern Part of China and
Its Relationship with Han and Japanese Populations

Liao GENGl, Tadashi IMANISHI, Katsushi TOKUNAGA 2, Dingliang ZHU 3,
Nobuhisa MIZUKI\ Shan Xu 5, Zhencheng GEN03, Takashi GOJOBORI, Kimiyoshi TSUJI! and Hidetoshi INoKo 5 CDepartment of Transplantation Immunology, Tokai University School of Medicine, Kanagawa, 2 Department ofResearch, Japanese Red Cross Central Blood Center, Tokyo, 3 Institute of
Genetics, Fudan University, China, 4 Department of Ophthalmology, Yokohama City University School of Medicine, Kanagawa, 5 Department of
Genetics Information, Division of Molecular Life Science, Tokai University
School of Medicine, Kanagawa)
The genetic polymorphism of the HLA class II loci was investigated in a
Manchu population resident in the northern part of China and comparedwith those of other Asian populations including Japanese and Han. In 8
DQA 1 alleles, the most frequent allele was DQA 1*03 with the gene frequency of 2S.S%. Of IS DQBl alleles tested, 11 were observed and the mostcommon allele was DQBl*0301 with the gene frequency of 24.S%. Among
19 DPBl alleles, 11 were detected and DPBl*OSOI (43.8%) was the mostfrequent allele as observed in other Asian populations such as Japanese,
Chinese and Korean. Of 43 DRB 1 alleles tested, 21 were detected and
DRBl*0901 (14.0%), *IS01 (11.0%), *1201 (11.0%), *07 (9.0%) and*1401 (9.0%) were highly predominant and account for the high frequencies
of DR9, DR2, DRS, DR7 and DR6. In the DRB3 gene (DRS2), DRB3*
0202 (18.0%) was the most frequent. With respect to the DRB4 gene
(DRS3), the gene frequency of DRB4*0101 was 3S.0%. Of 3 DRBS alleles
detected, DRBS*0101 (11.0%) was highly predominant. Comparison of
HLA class II allele frequencies in Manchu with those in Japanese and Han
Chinese populations (South & North) detected some significant differencesand genetic divergence between these Oriental populations. The dendrogram
constructed by the neighbor-joining (NJ) method based on the allele frequencies of DQAl, DQBl, DPBl and DRBI of 10 representative populations

160

over the world suggested that Manchu is the closest, but at the same genetic
distance to both Northern and Southern Han Chinese. For more details, see
Ref. 5.
(20)

Distribution of Shared Polymorphisms and Its Relation to Gene
Conversion in the Major Histocompatibility Complex Genes

Tadashi IMANISHI
Human major histocompatibility complex (MHC) genes are highly polymorphic, and numerous MHC alleles are thought to be maintained bybalancing selection. On the other hand, there seem to be three mechanisms
to produce new MHC alleles: point mutation, recombination, and geneconversion. However, importance of gene conversion has been controversial,
partly because its molecular mechanism is not yet clearly demonstrated. In
this study, I analyzed statistically the nucleotide sequence data of human
MHC loci to detect inter-locus gene conversion among MHC genes. By
comparing the consensus sequences of HLA-A, B, C, OPBI, OQBI, ORBI,
and ORB5, I found that a significantly large number of ONA polymorphisms
are shared between different, duplicated MHC loci at orthologous nucleotide
sites. The shared polymorphisms were observed in the hyper-variable exonsof both class I (HLA-A, B, and C) and class II (HLA-ORBI, OQBI, andOPBI) loci. The shared polymorphisms between MHC loci can be accounted for primarily by frequent inter-locus gene conversion of very short
gene fragments. Positive natural selection might have operated in order tomaintain the shared polymorphisms that were produced by gene conversion.
Moreover, it was suggested that the shared polymorphisms have ancientorigins. For example, the shared polymorphisms observed between ORB 1
and ORB5 implied that inter-locus gene conversion events took place immediately after the gene duplication of these loci. These results were reported at
the third international meeting of the Society for Molecular Biology and
Evolution and at the annual meeting of the Japanese Society of Molecular
Biology.

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161

YAMATO AND ASUKA: DNA Database Management System

Hajime KITAKAMI', Tadasu SHIN-I, Kazuho IKEo, Yoshihiro UGAWA 2,
Naruya SAITOU, Takashi GOJOBORI and Yoshio TATENO CHiroshima City
University, Hiroshima, 2 National Institute of Agrobiological Resources,
Ibaraki)
We have realized effective join processing with the newly developed
DDBJ-schema which is easy to use, because its structure looks like agrammatical structure of the flat-file format. The DDBJ-schema was successfully implemented as a relational schema with three parts. It is possible toprovide the SQL-service for on-line and E-mail users. Moreover, we showedthat the schema allowed us to implement two types of interfaces which werenamed YAMATO and ASUKA. We also implemented a tool to convert
EMBL and NCBI/GenBank-formats into the relational format with the
DDBJ-schema.
Finally, we proposed a structured SQL-programming
method. The restructuring and tree search were implemented in the programming method. We have succeeded in a trial restructuring data with the
GenBank-schema into those with the DDBJ-schema by use of the structuredSQL programming method. We are planning to move the current system tothe newly developed system at DDBJ. For details, see Ref. 9.
Publications
1.

2.
3.
4.
5.

REN, F., TANAKA, H., FUKUDA, N. and GOJOBORI, T.: Molecular Evolutionary Phylogenetic Trees Based on Minimum Description Length Principle. In "Proceedings of the 28th
Annual Hawaii Int'I Conf. on System Sciences" (L. Hunter and B. D. Shriver eds.), 5, 165173, IEEE Computer Society Press, Los Alamitos, California, 1995.
REN, F., TANAKA, H. and GOJOBORI, T.: Construction of Molecular Evolutionary
Phylogenetic Trees from DNA Sequences Based on Minimum Complexity Principle.
Computer Methods and Programs in Biomedicine, 46, 121-130, 1995.
GOJOBORI, T.: Genetic Diversity and Molecular Evolution of Pathogenic Viruses. Biology
International, Special Issue (Uniqueness & Universality) 33, 30, 1995.
KASHIWASE, K., TOKUNAGA, K., ISHIKAWA, Y., LIN, L., IMANISHI, T., AKAZA, T.,
TADOKORO, K. and JUJI, T.: A new HLA-A9 subtype lacking the Bw4 epitope: Ancestralor revertant allele? Human Immunology, 42, 221-226, 1995.
GENG, L., IMANISHI, T., TOKUNAGA, K., ZHU, D., MIZUKI, N., Xu, S., GENG, Z.,
GOJOBORI, T., TSUJI, K. and INOKO, H.: Determination of HLA class II alleles bygenotyping in a Manchu population in the northern part of China and its relationship with
Han and Japanese populations. Tissue Antigens, 46, 111-116, 1995.

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6.

7.

8.

9.

10.

II.

12.

13.
14.
15.

16.

SHIINA, T., ANDO, A., IMANISHI, T., KAWATA, H., HANZAWA, K., GOJOBORI, T., INOKO,
H. and WATANABE, S.: Isolation and characterization of eDNA clones for Japanese quail(Coturnix japonica) major histocompatibility complex (MhcCoja) class I molecules. Immunogenetics, 42, 213-216, 1995.
GOJOBORI, T., ENDo, T. and IKEO, K.: Domain evolution of serine protease and its
inhibitor genes. In "Tracing Biological Evolution in Protein and Gene Structures" (M. Go
and P. Schimmel eds.), pp. 249-260, Elsevier Science B. V., Amsterdam, 1995.
IKEO, K., TAKAHASHI, K., and GOJOBORI, T.: Different evolutionary histories ofkringle
and protease domains in serine proteases-A typical example of domain evolution. J. Mol.
Evol., 40, 331~336, 1995.
KITAKAMI, H., SHIN-I, T., IKEO, K., UGAWA, Y., SAITOU, N., GOJOBORI, T. andTATENO, Y.: YAMATO AND ASUKA: DNA database management system. In "Proccdings of the 28th Annual Hawaii Int'l Conf. on System Sciences." pp. 72-80, 1995.
GOJOBORI, T. and IKEO, K.: Sequence Divergence Estimation. In: Molecular Biology andBiotechnology. A Comprehensive Desk Reference. (R. A. Meyers ed.), VCH Publishers,
Inc. pp. 863~866, 1995.
GOJOBORI, T., ENDO, T. and IKEO, K.: Evolution of genes and the DNA databaseImplications for bioremediation-. Bioremediation: Tokyo '94 Workshop. OECD Documents, pp. 369-373, 1995.
OHBA, K., MIZOKAMI, M., OHNO, T., SUZUKI, K., ORITO, E., INA, Y., LAu, JYN. and
GOJOBORI, T.: Classification of hepatitis C virus into major types and subtypes based onmolecular evolutionary analysis. Virus Research, 36, 201~214, 1995.
ENDO, T., IKEO, K., GOJOBORI, T.: Large-Scale Search for Genes on Which Positive
Selection May Operate. Molecular Biology and Evolution, 13, 685-690, 1996.
PERRIERE, G., MoszER, I. and GOJOBORI, T.: NRSub: a non-redundant database for
Bacillus subtilis. Nucleic Acids Research, 24, 41-45.
OHBA, K., MIZOKAMI, M., LAu, JYN., ORITO, E., IKEO, K. and GOJOBORI, T.: Evolutionary relationship of hepatitis C, pesti-, ftavi-, plantviruses, and newly discovered GB
hepatitis agents. FEBS Letters, 378, 232-234, 1996.
FUMIHITO, A., MIYAKE, T., TAKADA M., SHINGU, R., ENDO, T., GOJOBORI, T., KONDO,
N. and OHNO, S.: Monophyletic Origin and Unique Dispersal Patterns of Domestic Fowls.
Proceedings of National Academy of Science, U.S.A., 93, 6792-6795, 1996.

H-b.

Laboratory of Gene Function Research

Molecular Evolution of a protein Seemingly Responsible for
Epoxidation in Eukaryote
Yoshio T A TENO
Fosfomycin (FOM) is an antibiotic discovered in cultures of Streptomyces. It has phosphite in the molecular structure and is characterized by a C-

H.

CENTER FOR INFORMATION BIOLOGY

163

P bond and an epoxy ring. FOM inhibits the initial reaction in biosynthesisof peptidoglycans in a broad spectrum of Gram-positive and Gram-negative
bacteria, and thus is used as an antibiotic. FOM is now chemically synthesized by epoxidation of cis-propenylphosphonic acid (cPA) and isolation of
the L-form compound in the chemical products. The isolation of the L-form,
however, is not necessary, if the epoxidation process is attained by microbialconversion which was devised 25 years ago. If a gene of an enzyme involvedin the microbial conversion is identified, the converting activity may bemarkedly increased by gene manipulation. The gene or the enzyme has notbeen identified yet.
The present study is to identify and search out the gene encoding epoxidase, which is to convert cPA into FOS in Penicillium decumbens. If the geneis available, the converting activity can be markedly increased by genemanipulation. We first screened Penicillium decumbens. The wild strains of
the species so far examined, however, showed low converting activities, andto increase the activity through mutation and selection was difficult without
identifying the responsible gene. The use of genetic engineering for enhancing the activity was another idea, but this again could not easily be realized
for the same reason.
Thus we took an approach to identifying the corresponding productinstead of the gene. By carrying out two-dimensional electrophoresis forcell-free extract of P. decumbens including cPA, we found possible proteins
with a molecular weight of 31 kD but different isoelectric points. On the basisof sequence information of the proteins, we could find a gene that enhanced
the converting activity when it was placed in a proper environment.
To confirm our observation further, we searched for the genes in the DDBJ
database which encode enzymes catalyzing similar activities as the epoxidation in question. We found five such genes in prokaryote, and aligned the
Penicillium gene with those sequences after translation. The alignment
showed that the six sequences shared several residues where tryptophan
resided. There were some more residues implying that the six sequences were
homologous.
(The present study is still underway in collaboration with Dr. H. Watababeat NIG, Dr. K. Murakami at Meiji Seika Co. Ltd., and his colleagues.)

164

Publications
1.

2.
3.

KITAKAMI, H., SHIN-I, T., IKEO, K., UGAWA, Y., SAITOU, N., GOJOBORI, T. andTATENO, Y.: YAMATO and ASUKA: DNA Database Management Systems. In Proceedings of the Twenty-Eighth Hawaii International Conference on System Sciences, pp. 72-80,
L. Hunter and B. D. Shriver, eds., IEEE Computer Society Press, Los Alamitos, California,
1995.
TATENO, Y.: Development of information biology. Protein, Nucleic Acid, and Enzyme
40, 102-108, 1995. (in Japanese)
TATENO, Y.: Gene. In Dictionary of Mathematics, Physics, and Information Science, pp.
39-42, M. Ohya, H. Imai, I. Kojima, Y. Nakamura, and M. Hirota, eds., Asakura
Publishing Company, Tokyo, 1995. (in Japanese)

H-c.
(1)

Laboratory for Gene-Product Informatics

Prediction of Protein Three-Dimensional Structures by
A 3D-ID Compatibility Method

Ken NISHIKAWA
After a number of unsuccessful trials over the years, the protein structure
prediction from the sequence information has been actualized by the structure (3D) and sequence (lD) compatibility approach. The prediction
program we developed, called COMPASS [Ref. 1], has been applied to anumber of proteins of unknown structure [Ref. 2]. We have obtained five
convincing predictions so far. Two of them, for spermidine/putrescinebinding protein and fJ subunit of F I-ATPase, were already verified incomparison with their X-ray structures later solved. Another application tosphingomyelinase (SMase) from Bacillus cereus was performed recently[Ref. 3]. Our COMPASS program detected its structural similarity withbovine DNase I with a high score, while the sequence identity between them
was around 10%, as low as the complete random matching. Apparently,
these two enzymes seem quite different, one for cleaving DNA and the otherfor phospholipid (sphingomyelin). However, they are common as phosphodiesterases, cleaving the phosphoester bond next to phosphorus atom. The
3D-ID alignment shows that several residues functionally important in
DNase I are also conserved in SMase. These results indicate that the 3D-ID

H.

CENTER FOR INFORMAnON BIOLOGY165

compatibility approach could successfully identify evolutionary distant relationships.
(2)

Protein Stability Change Caused by Single Amino Acid Substitutions:
Estimation by The 3D-Profile Method

Ken N ISHIKAWA
The 3D profile table, utilized in aligning the structure with any sequence inthe 3D-ID compatibility approach, has interesting features. The 3D profile
is expressed as a 20 X N table, where N is the number of residue positions ofa structure. Placing each of the 20 amino acids at each position, its
compatibility with the surroundings is quantitatively estimated and listed in
the table. A slight modification provides a rearranged profile table, which
represents the relative stability of all possible point mutations, all at once,
against the wild type protein. A comparison was made between stabilitychanges computed from the profile table and changes in the melting temperature experimentally obtained, for about 100 mutants of ribonuclease H. The
results showed a fairly good correlation between the computations andexperiments [Ref. 5], indicating that the 3D-lD compatibility function wedeveloped was realistic.
Pu bl ications
I.

2.

3.

4.

5.

MATSUO, Y., NAKAMURA, H. and NISHIKAWA, K.: Detection of protein 3D-ID
compatibility characterized by the evaluation of side-chain packing and electrostaticinteractions. J. Biochem., 118, 137-148, 1995.
MATSUO, Y. and NISHIKAWA, K.: Assessment of a protein fold recognition method thattakes into account four physicochemical properties: side-chain packing, solvation, hydrogen-bonding, and local conformation. Proteins: Str. Func. Genet., 23, 370-375, 1995.
TAMURA, H., TAMEISHI, K., YAMADA, A., TOMITA, M., MATSUO, Y., NISHIKAWA, K.
and IKEZAWA, H.: Mutation in aspartic acid residues modifies catalytic and haemolytic
activities of Bacillus cereus sphingomyelinase. Biochem. J., 309, 757-764, 1995.
SHIRAKI, K., NISHIKAWA, K. and GOTO, Y.: Trifluoroethanol-induced stabilization of thea-helical structure of ,B-Iactoglobulin: Implication for non-hierarchical protein folding. J.
Mol. BioI., 245, 180-194, 1995.
OTA, M., KANAYA, S. and NISHIKAWA, K.: Desk-top analysis of the structural stabilityof various point mutations introduced into ribonuclease H. J. Mol. BioI., 248, 733-738,
1995.

166

I.

Radioisotope CenterTranscriptional Analysis of the Bacillus subtilis Sporulation Gene
Masaya FUJITA and Yoshito SADAIE
Nutrient deprivation in Bacillus subtilis leads to a process of differentiationthat results in endspore formation. Sporulation takes place in a sporangium
consisting of two cellular compartments known as the forespore and themother cell. More than 50 specific genes are expressed sequentially in bothcompartments during sporulation. Their transcriptions are initiated by atleast five RNA polymerase sigma factors specific for sporulation, and enhanced or repressed by transcription factors which appear during sporulation. To understand the molecular mechanism of promoter selectivity withthese sigma factors as well as the interaction between RNA polymerase andthe sporulation specific transcription factors, we constructed an in vitrotranscription system with purified B. subtilis RNA polymerase whose components were overproduced in Escherichia coli. The genes for oA, oH, oE, of,oG, oK, /3, /3', and a were cloned into a T7 RNA polymerase expression
system. Over expressed proteins were purified and mixed to reconstitute an
active RNA polymerase holoenzyme. We also developed a simple, rapid
procedure for reconstitution of the B. subtilis RNA polymerase holoenzyme
from crude cell extract. A hexahistidine-tagged recombinant subunit protein
was purified from T7 expression system and incubated with crude B. subtilis
cell extract. The resulting reconstituted RNA polymerase was purified by
Ni-ion-affinity chromatography. Using this procedure, some proteins interacting with RNA polymerase were purified. We are now identifying these
proteins.
Publication
1.
2.

FUJITA, M., HANAURA, Y. and AMEMURA, A.: Analysis of the rpoD gene encoding theprincipal sigma factor of Pseudomonas putida. Gene, 167, 93-98, 1995.
NAKANE, A., TAKAMATSU, H., OGURO, A., SADAIE, Y., NAKAMURA, K. and YAMANE,
K.: Acquisition of azide-resistance by the elevated secA ATPase activity confers azideresistance upon cell growth and protein translocation in Bacillus subtilis. Microbiology. 141,113-121, 1995.

167

ABSTRACTS OF DIARY FOR 1995

Biological Symposium
432nd-Jan. 26
The molecular biology of rinderpest virus (Michael D.
Baron)A reanalysis of human mtDNA strongly supports the433rd-Feb. 16
out-of-Africa hypothesis (David Penny)
7
434th-Mar. 8
Roles of
0 °, N-HS and HU proteins in osmotic
regulation of proU transcription in E. coli (J. Gowrishankar)
435th-Mar. 24
Sox-9 and the molecular genetics of skeletal development (Peter Koopman)
436th-Mar. 27
An evaluation of genetic distances for use with microsatellite loci (Marcus W. Feldman)
437th-May 12
Function and regulation of sigma factors in Escherichia
coli (Carol A. Gross)438th-May 15
Crystal structure of T7 RNA polymerase (Bi-Cheng
Wang)
Combining protein secondary structure and evolution
439th-May 29(Jeffrey L. Thorne)
440th-July 19
Urokinase-type plasminogen activator gene regulationin kidney cells: coupling of hormonal regulation andcell-specific gene expression (Yoshikuni N agamine)
441st-Aug. 28
Location, structure and function of the target of atranscription activator protein (Richard H. Ebright)
442nd-Sept. 1
The elongin complex: A multi-subunit regulation ofelongation by RNA polymerase II (Joan W. Conaway)
443rd-Sept. 1
Mechanisms mediating transcription factor remodelingof chromatin structure (Jerry L. Workman)
444th-Aug. 28
Recent investigation on synonymous substitutions (Giorgio Bernardi)
28
Tetraploidorigin of mammals (Susumu Ohno)
445th-Aug.
446th-Aug. 28
Molecular evidence for human evolution (Simon Easteal)
Genetic variation of human Alu insert polymorphisms
447th-Aug. 28

cr,

168

448th-Aug. 28
449th-Aug. 28
450th-Sept. 20
45Ist-Sept. 18
452nd---Oct. 5453rd---Oct. 26
454th-Nov. 7455th-Nov. 17456th-Nov. 21

457th-Nov. 27
458th-Nov. 22459th-Dec. 1
460th-Dec. 11
46 1st-Dec. 8
462nd-Dec. 14
463rd-Dec. 15(Mark Stoneking)
Tracking male migrations in Asia (Michael Hammer)
New advances of genes mapping (Clay Stephens)
GATA transcription factor control of hemotopoietic
and neuronal cell fates (James Douglas Engel)
The mussel mtDNA: A new mode of cytoplasmic DNA
transmission and evolution (E. Zouros)
Negative control of transcription initiation in bacteria(Hyon Choy)
Towards sequencing the human MHC (Stephan Beck)
Hedgehog genes and the patterning of the vertebrate
embryo (Cliff Tabin)
The role of neutral mutations in evolution: Molecularmechanisms (Miroslav Radman)
Positional cloning and characterization of genes fordeafness in the mouse and human (Stephen D. M.
Brown)
Genetic analysis of transmission ratio distortion in interspecific cross (Xavier Montagutelli)
Molecular evolution of color vision in vertebrates(Syozo Yokoyama)
Searching mechanism for homologous DNA by RecA
protein (Masayuki Takahashi)
Structural aspects of phage M u transpososome (Kiyoshi
Mizuuchi)
New developments of biological utilization of the DNA
sequence database (David Lipman)Protein-directed molecular interactions during genetic
recombination (Stephen C. West)
From genome information to protein function andstructure (Chris Sander)

Mishima Geneticists' Club442nd-Jan. 11
Specific gene expression in embryonic cells (Ohtsura
Niwa)
443rd-Jan. 11
Developmental control of the DNA replication-related
genes in Drosophila (Akio Matsukage)ABSTRACTS OF DIARY FOR 1995

444th-Jan. 11445th-Jan. 19
446th-Jan. 19
447th-Jan. 30
448th-Jan. 25
449th-Jan. 26
450th-Jan. 26
451st-Feb. 13

452nd-Feb. 16

453rd-Feb. 28

454th-Mar. 1
455th-Mar. 2
456th-Mar. 15
457th-Apr. 3
458th-Mar. 27

459th-Apr. 5
460th-May 1

169

Genetic recombination-from molecular to chromosomal level (Tomoko Ogawa)Analyses of cell differentiation and pathways of signaltransduction in Drosophila (Leo Tsuda)
Pattern formation of Drosophila melanogaster-The imaginal discs and the nervous system (Satoshi Goto)
Structure and function of a tobacco protein kinase NPK
15 (Yukihiro Ito)
Map construction ofrice chromosome (Ken-Ichi Nonomura)Cis element controlling s-phase specific transcription ofplant histone genes (Norihiro Ohotsubo)
Acetyl CoA carboxylase in higher plants (Tomokazu
Konishi)
Processing of Sindbis virus non-structural polyproteins
and regulation of viral RNA replication (Yukio Shirako)
Regulation of DNA replication by the p21 inhibitor of
CDK-analysis of SV40 DNA replication in vitro(Shou Waga)
X protein of human hepatitis B virus: Molecular structure and mechanisms of trans-activation (Kiyoshi Murakami)
Target genes of mouse Hox gene products (Daihachiro
Tomotsune)
Genomic imprinting: What is the target for modification? (Tsuyoshi Koide)
Study of mouse P53 gene by two-steps gene targeting(Yoichi Gondo)
Cell migration and cell adhesion molecules in central
nervous system (Katsuhiko Ono)
Structure of a mini-collagen gene encoding a nematocyst capsule protein from a reef-building coral and its
use for phylogenetic analysis (Wenqiao Wang)
Approach on the biomolecule database construction
based on the experimental science (Yukiko Yamazaki)
X-ray crystallographic analysis of F1-ATPase from a

170

461st-Apr. 26
462nd-May 19

463rd-May 24
464th-June 1465th-June 8466th-June 12
467th-June 22

468th-June 27
469th-June 28
47Oth-June 30

471st-July 12
472nd-Aug. 7473rd-Aug. 15
474th-Sept. 6

475th-Dct. 4
476th-Dct. 18
477th-Dct. 25

thermophilic bacterium (Yasuo Shirakibara)
Prediction of protein three-dimensional structures basedon database (Ken Nishikawa)
NMR studies on the structure of transcription factors
and their interaction with nucleic acids (Yoshimasa
Kyogoku)
NMR studies of protein structure: SH2 domain and Trp
repressor (Toshio Yamazaki)
Structure and function of yeast chromatin as revealed
by in vivo foot printing (Shigeo Tanaka)
Dynamics of folding of DNA long chain (Kenichi Yoshikawa)
Molecular dissection of F-plasmid SopB protein (RyoHanai)
Function of the basement membrane component laminine during Drosophila development (Yasumitsu Takagi)
Ecology of influenza viruses (Hiroshi Kida)
Mouse mutation,jumonji, produced by gene-trap and its
function in embryogenesis (Takashi Takeuchi)
Construction of phylogenetic trees using a maximum
likelihood method and its speed-up through parallel
processing (Hideo Matsuda)
Function of cytokine common r chains (Masataka Nakamura)
From information biology to database (Hideaki Sugawara)
Solution structure of virus particles: study by UVresonance Raman (Shoji Kaminaka)
Residues of yeast farnesyltransferase that may be involved in protein substance recognition (Hiroshi Mitsuzawa)
Evolution of tertiary structures of proteins (Hideaki
Moriyama)
Structure and physiological functions of protocadherins(Shintaro Suzuki)
Evolution of RNA-binding domains (Kaoru Koba-

ABSTRACTS OF DIARY FOR 1995

478th-Nov. 10479th-Nov. 20
480th-Dec. 18
48 1st-Dec. 26

171

yashi)
Molecular phylogeny of HIV and HTLV (Tomoyuki
Miura)
Interaction between SWI proteins and TFIID (Tsutomu
Ohta)
Mechanism of transcriptional activation by heat shock
factor on reconstituted chromatin (Gaku Mizuguchi)
Study on rice chromosomes-Molecular and genetic analysis of fission yeast chromosomes as a model (Ken-Ichi
Nonomura)

172

FOREIGN VISITORS IN 1995

Dec. 1, 1991Feb. 28, 1995
Oct. 5, 1993Mar. 31, 1995
Mar. 10, 1994Mar. 30, 1995
Apr. 1, 1994Mar. 31, 1995
June 9, 1994Sept. 1, 1994Mar. 30, 1995
Jan. 26, 1995
Feb. 16
Feb. 16-Mar. 12
Mar. 18-21
Mar. 24-25
Mar. 26-27
Apr. 3-4
Mar 12-13
May 14-16
May 14-16
May 14-16
May 29June 21-July 14
June 28-30
June 29-30

Thangirala Sudha, Indian Association for Down's Syndrome, India
Dzhanybek M. Adyshev, Institute of Biochemistry andPhysiology, Kyrgys Academy of Sciences, Kyrgyz
Hak Soo Suh, Yeungnam Univerity, Korea
Perriere Guy Bernard, URA Centre National de laRecherche Scientifique, France
H. W. Cai. Beijing Agriculture Univcersity, China
John R. Wakeley, University of California at Berkeley,
U.S.A.
Michael D. Baron, Institute for Animal Health, U.K.
David Penny, Massey University, New Zealand
Jayaraman Gowrishankar, Centre for Cellular and Molecular Biology, India
Hans Bode, University of California at Irvine, U.S.A.
Peter Koopman, University of Queensland, Australia
Marcus W. Feldman, Stanford University, U.S.A.
Walter Doerfler, Institut fUr Genetik, Germany
Carol A. Gross, University of California at San Francisco, U.S.A.
Bi-Cheng Wang, University of Georgia, U.S.A.
Orna Amster-Choder, Hebrew University Hadassah
Medical School, Israel
Mordechai Choder, Hebrew University Hadassah Medical School, Israel
Jeffrey L. Thorne, North Carolina State University,
U.S.A.
Dipankar Chatterji, Centre for Cellular and Molecular
Biology, India
Casey Bergman, Cornell University, U.S.A.
Michael Brenner, National Institute of Neurological
Disorders and Stroke, NIH, U.S.A.

FOREIGN VISITORS IN 1995

July 19
Aug. 17-18
Aug. 17-21
Aug. 27-29
Aug. 27-29
Aug. 27-30
Aug. 28
Aug. 28
Aug. 28
Aug. 28
Aug. 28
Aug. 28
Aug. 28-29
Aug. 29-30
Aug. 31Sept. 1, 1995
Sept. 1

Sept. 17-19
Sept. 20-21
Oct. 5Oct. 8-16
Oct. 8-16
Oct. 26
Oct. 30-31
Nov. 7
Nov. 17
Nov. 21

173

Yoshikuni Nagamine, Friedrich Miescher Institute,
Switzerland
Noboru and Tamiko Sueoka, University of Colorado at
Boulder, U.S.A.
James F. Crow, University of Wisconsin, U.S.A.
Greg Gibson, University of Michigan, U.S.A.
Grey Gibson, Duke University, U.S.A.
Elizabeth Waters, University of Arizona, U.S.A.
Giorgio Bernardi, Institut Jacques Monod, France
Simon Easteal, The Australian National University,
Australia
Michael Hammer, University of Arizona, U.S.A.
Susumu Ohno, Beckman Research Institute of The Cityof Hope, U.S.A.
Mark Stoneking, Pennsylvania State University, U.S.A.
Clay Stephens, National Cancer Institute, NIH, U.S.A.
Richard H. Ebright, Rutgers University, U.S.A.
Spencer V. Muse, Pennsylvania State University, U.S.A.
Jerry L. Workman, Pennsylvania State University,
U.S.A.
Joan W. Conaway, Program in Molecular and Cellular
Biology, Oklahoma Medical Research Foundation,
U.S.A.
E. Zouros, Dalhousie University, Canada
James Douglas Engel, North-Western University,
U.S.A.
Hyon Choy, National Cancer Institute, NIH, U.S.A.
William B. Provine, Cornell University, U.S.A.
Tomoko Steen, Emory University, U.S.A.
Stephen Beck, DNA Sequencing Laboratory, Imperial
Cancer Research Fund, U.K.
Joe Inselburg, Dartmouth Medical School, U.S.A.
Cliff Tabin, Harvard Medical School, U.S.A.
Miroslav Radman, CNRS Institut Jacques Monod,
FranceStephen D.M. Brown, St. Mary's Hospital Medical
School, U.K.

174

Nov. 22
Nov. 27
Dec. 1
Dec. 8Dec. 9-12
Dec. 13-14
Dec. 15

Syozo Yokoyama, Syracuse University, U.S.A.
Xavier Montagutelli, Institut Pasteur, Fraance
Masayuki Takahashi, Institut Curie, Orsay, FranceDavid Lipman, National Center for Biotechnology Information, NIH, U.S.A.
Kiyoshi Mizuuchi, Laboratory of Molecular Biology,
NIDDK, NIH, U.S.A.
Stephen C. West, Clare Hall Laboratories, Imperial
Cancer Research Fund, U.K.
Chris Sander, EMBL-EBI, Cambridge, U.K.
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
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

ASSOC. FOR PROPAG. OF THE KNOWL. OF GENET.

175

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

176

AUTHOR INDEX
ABE, K50

HARA, H.

. ....•.•••.•.••••••.•. 28

HARADA, A.

102
AKISHINONOMIYA, F.
157
AN DO, A.
. . . . . . . • . . . . . . . . . . 75, 77, 158
AOKI, F.
• .•••..••...••..•.••.... 31,32
ASANO, S.
.
42
ASANO, Y.
25
AYUSAWA, D.
. . . . . . . . . . . . . . . • • . • . . • 32
AZUMA, Y.
. ......•..••.••.•••.•.•. 24
BODE, H. . . . • . . . . . . . . . . . . . . . . . . . . 53, 69
BOSCH, T.
.
53, 69CAl, H. W.
.
99
CHIQUET-EHRISMAN, R. .
79
DA VID, C.
53, 69deHARo, M.
.
53, 69
DING, Q.
.
21
DOIRA, H . . . . . . . • • . . . • . . • . . • . . . • . • • • 63
EIGUCHI, M.
.
103
ENDO, M112
ENDO, T.
00 00.00 00. 00148, 149, 150, 157
FUJII, H.
. ••••.••...••..•..•..•.••. 63
FUJISAWA, T
53,69
FUJITA, M.
.
166
FUJITA, N.
.
18, 19,21
FUJIWARA, M.
.
141
FUJlYAMA, A.
. . . . • . . . . . . . . . . . . . . . . . 77
FUKAGAWA, T
75
FUKAMI, H.
. ..........••.....••••.• 56
FUKUDA, N.
.
154
FURUKAWA, Y ..••..••...•.••.•••.•.• 79
FUSE, N.
.
119
GENG, L.
159
GENG, Z159
GOJOBORI, T.
..77,79, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 161
GOTO, S.
.
122
HAMMER, M. F.
.
94
HANAZAWA, K.
.
158

HARADA, N

ADYSHEV, D.
AKIMOTO, M.

.

• ...••.•....••........ 45, 46• •..••••.••••.•.....•... 96

91
89
HATANO, N.
.
107
HATTA, M
53,55,56,69
HA YASAKA, K.
.
92, 95
HAYASHI, S.
. .95,119,120,121,122,123
HAYASHIBARA, T.
.
56
HElKE, T.
.
91
HIGASHITANI, A. .
.42, 43, 47
HIGASHITANI, N. .
.43
HINOHARA, A. • •..•.••••.•.•.•....• .40
HIRANO, H.-Y.
• .••.••••.•.•.•.••.• 103
HIROSE, S.
.
58, 59, 119
HISHIDA, R.
.
139
HOBMA YER, E. • ••.•.•••••••..•...... 55
HORAI, S.
.
92, 93, 94, 95, 96, 97
HORIUCHI, K. .
42, 43, 45, 46, 47
IKEMURA, T. .
75, 77, 78, 79
IKEO, K
148, 149, 150, 151, 161
IKEYA, T.
39,40
IMAMURA, T.
.
88,89,91
IMANIsHI, T.
.
158, 159, 160
INA, Y.
• • . . . . . • . . • . . . . . . . . . . . . . 75, 152
INABA, R. .
88, 89, 91
INoKo, H.
.
75, 77, 158, 159
ISHIDA, N
81
ISHIDA, T.
.
96, 97
ISHIHAMA, A.
• •.... 16, 18, 19,20,21,23,
24, 25, 27, 28, 37
ISHIHARA, T. .
137, 139, 140, 141
ISHII, Y. • ..••••••...•••••......•... .47
ISHIJIMA, J. .
116
Iso BE, T. .
III
ITO, Y . . . . . . . . . . . . . . . . . . . . . . • . . . . . • 124
IWABUCHI, K.
.
60
IWATA, A
20
IZUHARA, K
91
HASEGA WA, T.

.

AUTHOR INDEXJEON, Y. H.
JIN, F.

.

.

JISHAGE, M.

.

KABATA, H.

.

KANAMARU, K.
KANAYA, S.

.

.

KANBARA, M.

.

KANEDA, S.

.

KANKAWA, S.

.

KATO, R
KATO, Y.

.

KATSURA, I.
KAWAGISHI, M.

.

KA WAKAMI, M.
KAWATA, H.

.

.

KIKKAWA, Y.

117

KIMURA, M.

.

KISHI, T.

.
KITAKAMI, H

KLEIN, J.

.

KOBAYASHI, M.
KOHARA, Y.

.

.

KOHN, J. R.

.

KOIDE, T
KOIZUMI, O.
KOLB, A.

.

.

KONDO, N.

.

KONDO, R.

.

KUBORI, T.

.

KUDO, Y.
KUNDU, T. K.
KUNIFUJI, S.

.

.

KUNISADA, T.

.

KUSANO, S
KYOGOKU, Y.

.

LAu, J. Y. N.
LI, F.-Q.

.

.

LOHMANN, J.

.

MAEDA, S
MARUYAMA, T.
MAS HI MA, S.
MASUYA, H.

19
97
20
136
127
78
61
31,32
32
107
47
137, 39, 40, 41
24
137
158

.

.

MATSUMOTO, K.

.

MATSUSHITA, T.

.

16
34
161
85
27,58
144, 145
101
111,112,116,117
52, 53, 69
21
157
92
135
78
21
81
50
20,21
19
151, 152
58, 59
53,69
50
25
81
113, 114
79
82

MELLO, C.

177

145
123
MINATO, K.
.
68
MINOBE, S.
.
52
MITA, A
116,117,118
MITA, K.
.
77
MIYAKE, T.
157
MIYAO, T.
.
23,24
MIYATA, A
144
MIZOKAMI, M.
.
151, 152
MIZUKI, N. .
159
MIZUMOTO, K
37
MIZUNO, K-I.
109, 111
MORI, H156
MORIKA WA, K.
.
36
MORISHIMA, H.
.
99, 101, 102
MORIWAKI, K. .
109, 111, 112, 113,
114,117,118
MOSZER, I.
155
MOTOHASHI, T. .
144, 145
MUKOYAMA, H.
.
81
MUNEOKA, Y.
53, 69MURAKAMI, A.
.
60,61, 63, 64, 67
MURAKAMI, K. ..
18, 19MURAKAMI, T. .
50
NAGAI, H.
.
136
NAGAI, Y
33
NAGATA, Y.
.
.48
NAKAMURA, Y.
75, 78, 79
NAKASHIMA, H.
.
88
NAKATSUJI, N.
.
130, 131, 132, 133
NEGISHI, T
18, 19
NIHO, Y.
.
91
NISHIKAWA, K. .
164, 165
NISHIMIYA-FuJiSAWA, C.
56
NISHIMORI, K.
.
126
NISHIMURA, A.
.
125, 126
NIWA, H.
.
50
ODA, H.
.
121
OGAWA, T.
.
39,40,41
OHBA, K
151, 152
OHMORI, M. .
56
OHNO, S. .
157
OHNO, T152
MIKOSIBA, K.

178
OHONO, M.

.

OHSAKA, F.

.

75
35
58,73,74.

.48

OHTA, T.

.

OHTSUBO, E.

123
OKAMURA, H.
.
50
OKANO, H.
.
123
OKUMURA, K. .
70, 75, 77
OKUYAMA, E.
.
86
OMOTO, K.
.
97
OOTA, H.
.
82
OOTA, S.
.
81
ORITO, E.
.
151, 152OSAKA, F33
OYUNSUREN, T.
.
81
OZOLINE, O. .
19
PAN, I.-H.
.
96,97
PERRIERE, G. .
155
PRIESS, J
145
REN, F.
. .•....................... 154
SADAIE, Y.
.
166
SAGAI, T.
.
109, 112, 113, 114
SAITO, M.
.
128
SAITOU, N. .
75, 80, 81, 82, 97, 161
SAITOU, S. .
96
SAKAI, M
88,89,91
SAKAI, T.
.
79
SAKURAI, H. .
23
SAMESHIMA, M.
.
31
SANO, M
144
SANO, Y.
103, 104
SARRAS, M. P. Jr.
53
SASAKI, H.
.
107
SATO, H113
SATTA, N
85
SAWAMOTO, K.
.
123
SEINO, H.
.
33, 35
SENO, T.
.
31, 32, 33, 34, 35
SHIBATA, H. .
86
SHIGA, Y
122
SHIMADA, K. .
50
SHIINA, T.
.
158
SHIMADA, J. .
61
SHIMAMOTO, N. .
135, 136
OKABE, M.

.

SHIMAMOTO, Y.

102, 10653, 69SHIMOIKE, K.
.
56
SHIN-I, T. .
161
SHINGU, R. .
157
SHINOHARA, A.
.
39,40,41
SHINOHARA, M.
.
41
SHIRAKAWA, M.
.
19
SHIRAKIHARA, Y.
.
142, 143
SHIRAYOSHI, Y. .
130, 131, 132
SHIROISHI, T.
.
109, 111, 112, 113, 114,
116, 117, 118
SHOKUNBI, M. T.
..
132, 133
SUDHA, T
31
SUGAYA, K.
.
77
SUGIMOTO, A. .
144
SUGIYAMA, T.
.
52, 55, 56, 69
SUN, CoHo97
SUZUKI, H.
.
23
SUZUKI, K. .
125, 152
SUZUKI, N.
.
140
SUZUKI, T. .
96
SUZUKI, Y.
.
154
TABARA, H.
.
144, 145
T ACHIDA, H. .
86
TADENUMA, M.
.
125
TAKADA, K.
.
118
T AKADA, M.
.
157
TAKAGISHI, T.
.
136
TAKAHASHI, K.
.
50,149,153
TAKAHASHI, T.
53, 69TAKAHATA, N
84,85,92
TAKANO, T.
.
74
TAKE-UCHI, M.
.
137
TAKEICHI, M. .
121
TAKEMARU, K.
.
58,59
T AKENAKA, O. .
95
TANAKA, H.
.
154
TANAKA, K. .
21
TANAKA-MATAKASTU, M.
.
121, 122
T ANIMURA, T.
.
123
TANOUE, K.
.
112
TASHIRO, F.
.
50
TATENO, Y.
161, 162
SHIMIZU, H.

.

AUTHOR INDEXTENZEN, T.

.

TERASAWA, M
TOKUNAGA, K.

.

TOYODA, T.
TSUGANE, K.

.

TSUJI, K.

.
UCHIDA, K.
UEDA, H. .
UEDA, S. .
UEMURA, T.
UGAWA, Y.

UI, M.

.

.

.

UKAI, HUMETSU, K.

.

URASAKI, M.

.

WAKANA, S
WANG, W
WANG, X. K.
WATANABE, H.

.
.

77
40
159
27,28
92
159
116, 117
58, 5920, 82
121
161
34
125
96
137, 140
113,114,117,118
56
99
144, 156

179

WATANABE, N.

.
.

WATANABE, S.
WATAYA, Y

Xu, S.

.

YAGI, Y.

.

Y AMAGISHI, M.

.

YAMAGUCHI, Y.

.

Y AMAMURA, K.

.

Y AMAO, F.
YAMAZAKI, T.
YAMAZAKI, Y.

.

YASUI, K
YI, S
YONEKAWA, H.
YOSHINO, M.
YUASA, I.
ZHU, D.

.

ZUBAIR, M.

.
.

.

125
158
32
159
123
23, 24, 37
152, 15350
31, 32, 33, 35
19, 86128
24
50
112, 117, 118
III
96
159
107

Annual Report of the National Institute of Genetics. No. 46
Printed on November 5th 1996
Issued on November 12th 1996
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 Hiroko Morishima & Naruya Saitou
Published by National Institute of Genetics
Printed by Kokusai Bunken Insatsusha Co.• Ltd.

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