ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT1994

No. 45

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mishima, Sizuoka-ken, Japan
1995

Annual Reportof theNational Institute of Genetics
No. 45, 1994

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Published by

The National Institute of Genetics, Japan
1995

CONTENTS
General statement
Staff
Council and advisory committee
Association for propagation of the knowledge of genetics
Projects of research for 1994
Research activities in 1994
1.

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Molecular Genetics

Molecular anatomy of RNA polymerase alpha subunit from Escherichia coil: functinal map of the amino-terminal assembly
domain. KIMURA, M. and ISHIHAMA, A. .. . . . . . . . . . . . .
Molecular anatomy of RNA polymerase alpha subunit: analysis of
the carboxy-terminal activation domain by class-I factors andupstream enhancer elements. MURAKAMI, K., FUJITA, N.,
TAO, K., HEYDUK, T. and ISHIHANA, A.
Molecular anatomy of RNA polymerase alpha subunit: structure of
the carboxy-terminal activation domain. NEGISHI, T.,
FUJITA, N., KYOGOKU, Y. and ISHIHAMA, A.
Promoter selectivity control of Escherichai coil RNA polymerase:
intracellular levels of sigma subunits. JISHAGE, M., IWATA,
A. and ISHIHAMA, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Promoter selectivity control of Escherichia coil RNA polymerase:functional comparison of sigma subunits. KUSANO, S.,. . . .. ..... . . .... . . . . . ...
FUJITA, N. and ISHIHAMA, A.
Molecular anatomy of Escherichia coil RNA polymerase: modes of
RNA polymerase-transcription factor interaction (progress ininternational collaborative studies). ISHIHAMA, A.
Gene cloning and characterization of RNA polymerase II subunits
from the fission yeast Schizosaccharomyces pombe: cloning of
the genes encoding small subunits. SAKURAI, H., SUZUKI, H.
and ISHIHAMA, A.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Gene cloning and characterization of RNA polymerase II subunits
from the fission yeast Schizosaccharomyces pombe: thermolabile

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RNA polymerase II with mutations in subunit 3. Y ASUI, K.,AZUMA, Y., YAMAGISHI, M. and ISHIHAMA, A. .
.
Gene cloning and characterization of RNA polymerase II subunits
from the fission yeast Schizosaccharomyces pombe: structureand function of subunit 5. MIYAo, T., AZUMA, Y, SAKURAI, H., YAMAGISHI, M. and ISHIHAMA, A.
. ...
Molecular anatomy of influenza virus RNA polymerase: analysis ofsubunit functions. KOBAYASHI, M., TOYODA, T., NAKADA,
S., MlzuMoTo, Ko and ISHIHAMA, Ao
Molecular anatomy of influenza virus RNA polymerase: analysis ofsubunit-subunit contact sites. ADYSHEV, D., TOYODA, T.,
IWATA, A., VEDA, S. and ISHIHAMA, A. .
Molecular anatomy of influenza virus RNA polymerase: mappingof nucleotide-binding sites. . ASANO, Y, MlzuMoTo, K.,
MARUYAMA, T. and ISHIHAMA, A.
Molecular dissection of influenaza virus nucleoprotein (NP): identification of RNA-binding region. KOBAYASHI, M., ToyoDA, T. and ISHIHAMA, Ao..
Growth control of influenza virus by membrane protein (MI).
YASUDA, J. and ISHIHAMA, A. . .
Molecular dissection of mouse Mx 1 protein with anti-influenza
virus activity. TOYODA, T., ASANO, Y. and ISHIHAMA, A.
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DNA replication/repair is impaired in a temperature-sensitive ubiquitin-activating enzyme mutant of mouse cells,without theaccompanied enhanced mutation frequency-a possible involvement of deoxyribonucleotide metabolism. AOKI, F.,
WATAYA, Y. and SENO, T.
Perturbation of nucleolar dissolution in the G2-M transition in amouse cell mutant with temperature-sensitive ubiquitinactivating enzyme EI: possible involvement of HSP70 andproteasome. SUDHA To, SAMASHIMA, M., TSUJI, H.,
Y AMAO, F. and SENO, T.
..
Vbiquitin-activating Enzyme, El is phosphorylated in mammaliancells by the protein kinase CDC2. NAGAI, Y., KANEDA, S.,
SENO, To and YAMAO, F.
Screening for cDNAs encoding the ubiquitin-conjugating enzyme
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CONTENTS
in fission yeast, Schizosaccharomyces pombe. OSAKA, F.,
SEINO, H., SENO, T. and YAMAO, F. .
.
Regulation of G liS transition by the ubiquitin system in yeast
Saccharomyces cerevisiae. KISHI, T., VI, M. and SENO, T.

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Cell cycle regulation by the ubiquitin system in eukaryote. SEINO,
H., YAGURA, T., YAMAO, F. and SENO, T. .
.
Crystallographic studies of proteins involved in DNA repair.
MORIKAWA, K.
Kinetic study on transcription using immobilized operons: branched pathway in early elongation. KUBORI, T. and SHIMAMOTO, N.
.
.
Single-molecule dynamics of transcription using immobilized operons: sliding of P. putida CamR protein on DNA. KABATA,
H. and SHIMAMOTO, N.
Functional and structural study of E. coil single-stranded DNA
binding protein. NAGAI, H. and SHIMAMOTO, N.
In vitro transcriptional analysis of the Bacillus subtilis sporulation
gene. FUJITA, M. and SADAIE, Y.
.

II.

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Microbial Genetics
Recognition of the minus strand DNA replication origin of thefilamentous phage by E. coli RNA polymerase. GUAN, Z. W.,
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.
The psv gene coding for penicillin-binding protein 7 of Escherichia
coli. HARA, H., ABE, N., NAKAKOHJI, M. NISHIMURA, Y.
and HORIUCHI, K.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
A cell division inhibitor SulA of Escherichia coli directly interacts
with FtsZ through GTP hydrolysis. HIGASHITANI, A. and
HORIUCHI, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Isolation and characterization of mutations that suppress the effectof a cell-division inhibitor SulA in Escherichia coli. HIGASHITANI, A. and HORIUCHI, K.
..
.
Molecular mechanisms that determine the timing of cell division in
Escherichia coli K12. NISHIMURA, A.
A new gene which couples cell growth and cell division in Escherichia coli K-12. VKAI, H., WATANABE, N. and NISHIMURA,

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The role of the secA protein of Bacillus subtillis in sporulation.
SADAIE, Y. and FUJITA, M.
.
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III.

Mammalian Genetics

Genetical analysis of rim2 coat color mutaion. SAGAI, T.,W AKANA, S., MORIWAKI, K. and SHIROISHI, T. .
.
Meiotic recombination at the Lmp2 hotspot in the mouse major
histocompatibility complex is tolerant of substitutional basemismatches. YOSHINO, M., SAGAI, T and SSHIROISHI, T.

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Genetic analysis of mouse mutants affecting limb pattern formation.
MASUYA, H., SAGAI, T, MORIWAKI, K. and SHIRaIsHI, T.
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Linkage analysis of the skeleton mouse mutant, Tail short (Ts).
SHIROISHI, T, UCHIDA, K., KOOPMAN, P., MlTA, A.,
WAKANA, S. and MORIWAKI, K. .
.
Gene mapping toward positional cloning of the Rim3 gene.
MASUYA, H., WAKANA, S., SAGAI, T., MORIWAKI, K. and
SHIRaIsHI, T.
.
.
Meitotic recombination on DNase I-hypersensitive sites in themouse major histocompatibility complex. MIZUNO, K-I.,
SAGAI, T., MORIWAKI, K. and SHIROISHI, T. .
.

IV.

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Developmental Genetics

Sexual differentiation potency of multi-potent stem cells in hydra.
NISHIMIYA-FuJISAWA, C. and SUGIYAMA, T.
Hydra regeneration from recombined ectodermal and endodermal
tissue. I. Cell rearrangement. KISHI MOTa, Y, MURATE, M.
and SUGIYAMA, T.
.
.
Hydra regeneration from recombined ectodermal and endodermal
tissue. II. Electron microscopic study. MURATE, M., KISHIMOTa, Y., SUGIYAMA, T., TAKAHASHI-IWANAGA, H. and
IWANAGA, T.
Isolation of cytoskeletal molecules in hydra by epitope selection.
HATTA, M., SAKAGUCHI, M., KOBAYAKAWA, Y., KOIZUMI,O. and SUGIYAMA, T. .
.

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CONTENTSv

Disappearance and reappearance of mesoglea in hydra tissue during
regeneration after head removal. SHIMIZU, H. and SARRAS,
M. J. Jr.
Hydra peptide project. I. Isolation,structure determination andchemical synthesis. MUNEOKA, Yo, TAKAHASHI, T., FUJISAWA, T, SHIMIZU, H., HATTA, M., SUGIYAMA, T., KorZUMI, 0., BOSCH, T., DEHARO, M., LOHMANN, J., DAVID, C.
and BODE, H.
Hydra peptide project. II. Differential display-PCR assay for signal
molecule activity. FUJISAWA, T., SHIMIZU, H., HATTA, M.,
SUGIYAMA, T., BOSCH, T., DEHARO, M., LOHMANN, J.,
DAVID, C., TAKAHASHI, T., MUNEOKA, Y., KorZUMI, o. andBODE, H.
Hydra peptide project. III. Biological function assay. FUJISAWA,
T, SHIMIZU, H., HATTA, M., SUGIYAMA, T, KOIZUMI, 0.,
BODE, H, BOSCH, T., DEHARO, M., LOHMANN, J., DAVID, C.,
TAKAHASHI, T. and MUNEOKA, Y.
.
.
Intermittent expression of BmFTZ-Fl, a member of the nuclear
hormone receptor superfamily during development of the silkworm Bombyx mori. SUN, G.-C., HIROSE, S. and VEDA, H.
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Mediators of activation of jushi tarazu gene transcription by
BmFTZ-Fl. LI, F.-Q., VEDA, H. and HIROSE, S. .
Sequences of two cDNAs encoding silkworm homologues of Drosophila melanogaster squid gene. LI, F.-Q., SUN, G.-c.,
VEDA, H. and HIROSE, S.
.
The Drosophila nuclear receptors FTZ-Fla and FTZ-Fl,B compete
as monomers for binding to a site in the jushi tarazu gene.
OHNO, C. K., VEDA, H. and PETKOVICHI, M.
..
The Bombyx silkworm is a semslparous insect with a long life in the
post-reproductive period. MURAKAMI, A.
Homoeostasis in the process of growth and senility in the Bombyx
silkworm: the relationship between Larval growth period andtemperature. MURAKAMI, A.
Homoeostasis in growth and senility in the Bombyx silkworm: therelationship between adult lifespan and body-weight. MURAKAMI, A.
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Growth phenomena in Bombyx mori (L.) with a special reference togenetic factors responsible for growth acceleration and molutinism. MURAKAMI, A.
Climatic differential phenotypic expression of voltine genes inBombyx mori L. SUBRAMANYA, G. and MURAKAMI, A. ..
Isolation and characterization of enhancer trap lines expressed inovary in Drosophila melanogaster. YAMADA, M.-A.
The retinoblastoma binding factor 1(RBF-I) site in RB gene promoter binds preferentially E4TFI, a member of the Ets transcriprion factors family. SAVOYSKY, E., MIZUNO, T., SOWA,
Y., WATANABE, H., SAWADA, J.-I., NOMURA, H., OHSUGI,Y, HANDA, H. and SAKAI, T. . . . . . . . . . . . . . . . . . . . . . . ..
Transcriptional activation through the tetrameric complex formation of E4TFI subunits. SAWADA, J.-I., GOTO, M., SAWA,
c., WATANABE, H. and HANDA, H.
Formation of Drosophila wing and haltare imaginal disc depends
on two related zinc finger proteins Snail and Escargot. FUSE,
N. and HAYASHI, S.
Control of esg expression. YAGI, Y and HAYASHI, S.
Use of green fluorescent protein as a live cell marker and its
application to the study of tracheal morphogenesis. SHIGA,
Y. and HAYASHI, S.
Developmental mechanisms and manipulation of germ cells inmouse embryos. NAKATSUJI, N. and SHIRAYOSHI, Y. ....
Molecular analysis of cell differentiation and morphogenesis in
postimplantation mouse embryos. SHIRAYOSHI, Y and NAKATSUJI, N.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Cell differentiation and morphogenesis of the mouse central nervous. .... . ....
system. NAKATSUJI, N. and SHIRAYOSHI, Y.
Fluoride-resistant mutants of the nematode Caenorhabditis elegans.
KAWAKAMI, M., ISHIHARA, T., URASAKI, M. and KATSURA,I.
Caenorhabditis elegans larval lethal mutations that cause detachment of the outer surface of the intestine from the innersurface of the body wall. HISHIDA, R., ISHIHARA, T. and
KATSURA, I.
A gene that controls both hatching and cell migration in C. elegans.

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CONTENTSvii

HISHIDA, R., ISHIHARA, T. and KATSURA, I.
Construction of GFP markers for various nerve cells in C. elegans
and their use for mutant isolation. ISHIHARA, T., FUJIWARA,
M. and KATSURA, I.
..
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..
Analysis of the head neural system of C. elegans as studied byformation of dauer larvae. SUZUKI, N., URASAKI, M., ISHIHARA, T. and KATSURA, I.
The C. elegans cDNA project: towards an expression map of the
genome. KOHARA, Y., MOTOHASHI, T., TABARA, H., SUGIMOTO, A. and WATANABE, H.
. . . . . . . . . . . . . . . . . . . . . . ..
In situ hybridization of the multi-well format on whole mountembryos of C. elegans. TABARA, H., MOTOHASHI, T. and
KOHARA, Y.
Efficient isolation of TCI-insertion mutans from a mutant bankrepresenting 20,000 worms of C. elegans. ANDACHI, Y. and
KOHARA, Y.

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Cell Genetics
Fine analysis of DNA replication timing in the human MHC region
by fluorescence in situ hybridization: temporal order of DNA
replication closely correlates with G + C % gradient of genome
sequences. OKUMURA, K., FUKAGAWA, T., and IKEMURA,
T.

VI. Population Genetics
Interaction of selection and drift in molecular evolution.
T.

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OHTA,

.

On hypervariability at the reactive center of proteolytic enzymes
and their inhibitors. OHTA, T.
Further examples of evolution by gene duplication revealed throughDNA sequence comparisons. OHTA, T.
Statistical methods for estimating the effective number of alleles,
expected heterozygosity and genetic distance in self-incompatibility locus. TAJIMA, F., TOKUNAGA, T. and MIYASHITA, N. T.
Comments on the detection of reciprocal recombination or geneconversion. TAKAHATA, N.

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The neutral theory of molecular evolution. TAKAHATA, N. and
KIMURA, M.
Structure, function, and evolution of mouse TL genes, nonclassicalclass I genes of the major histocompatibility complex.
OBATA, Y., SATTA, Y., MORIWAKI, K., SHIROISHI, T., HASEGAWA, H., TAKAHASHI, T. and TAKAHATA, N.
Polymorphism at Mhc loci and isolation by the immune system invertebrates. TAKAHATA, N.
Intensity of natural selection at the major histocompatibility complex loci. SATTA, Y., O'HUIGIN, C., TAKAHATA N. and
KLEIN, J.
.
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Repeated failures that led to the eventual success in human evolution. TAKAHATA, N.
Population genetic studies on the evolution of SINE II. T ACHIDA,
H.
.
Genetic and geographical differentiation of subspecies groups of
Mus musculus in east Asia. MORIWAKI, K., MIYASHITA, N.,
TSUCHIYA, K., MATUSDA, Y., NATSUME-SAKAI, S., YONEKAWA, H., SUZUKI, H., WANG, C.-H., Wu, X.-L., JIN, M. L.,
LI, H., WANG, F., KRYUKOV, A. P., FRIESMAN, L. V. and
YAKIMENKO, L. V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
VII.

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Evolutionary Genetics

Early evolution of genes and genomes. OHTA, T.
Estimation of evolutionary distance for reconstructing molecularphylogenetic trees. TAJIMA, F. and TAKEZAKI, N.
Human pseudoautosomal boundary-like sequences (PABLs): coreand consensus sequence, expression, and involvement in evolutionary formation of the present day pseudoautosomal boundary of human sex chromosomes. FUKAGAWA, T., NAKAMURA, Y., OKUMURA, K., ANDO, A., INOKO, H., SAITOU, N.
and IKEMURA, T.
A Notch-family gene found in the human MHC class III region
near the junction with the class II: human counterpart ofmouse mammary tumor gene int-3. SUGAYA, K., ANDO, A.,
INOKO, H. and IKEMURA, T.
Estimation of protein-production levels for ORFs registered in E.

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CONTENTS

IX

coil genome projects, based on levels of "optimal codon" usage,
in connection with the feasibility of protein coding ability andwith assignment to foreign-type genes. NAKAMuRA, Y. and
IKEMURA, T. 100
Evolutionary rate of insertions and deletions in non-coding nucleo101
tide sequences of primates. SAITOU, N. and VEDA, S.
Genetic analysis and molecular phylogeny of simian T-celllymphotropic virus type I: evidence for independent virus evolution in
Asia and Africa. SONG, K.-J., SAITOU, N. and YANAGIHARA, R.
.
. 101
Population genetic study in Hainan Island, China, II. Genetic
affinity analysis. SAITOU, N., OMOTO, K., Du, C. and Du, R.
102
Molecular evolution of serine protease and its inhibitor with specialreference to domain evolution. GOJOBORI, T. and IKEO, K.
103
NRSub: a non-redundant data base for the Bacillus subtilis genome.
. 103
PERRIERE, G., GOUY, M. and GOJOBORI, T.
Confirmation by molecular evolutionary analysis of hepatitis C
virus transmission through needlestick accidents. SUZUKI,
K., MIZOKAMI, M., LAu, J. Y. N., MIZOGUCHI, N., KATO,
K., MIZUNO, Y, SODEYAMA, T., KIYOSAWA, K. and GoJOBORI, T. 104
Evolution of pathogenic viruses with special reference to the rates
of synonymous and nonsynonymous substitutions. GoJOBORI, T., YAMAGUCHI, Y, IKEO, K. and MIZOKAMI, M. .. 104
Reduction of synonymous substitutions in the core protein gene of
hapatitis C virus. INA, Y, MIZOKAMI, M., OHBA, K. and
GOJOBORI, T.
.
. 105
Phylogenetic subtypes of human T-Iymphotropic virus type I andtheir relations to the anthropological background. MIURA,. 106
T., INA, Y., GOJOBORI, T. and HAYAMI, M. .
Toward unification of taxonomy databases in a distributed computer environment. KITAKAMI, H., TATENO, Y. and GOJOBORI,
T.
.
. 106
Molecular evolutionary virology: its application to hepatitis C virus.
MIZOKAMI, M., GOJOBORI, T. and LAu, J. Y N. .
. 107

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Molecular evolution of the major epitopes of HIV within a single. 107
host. YAMAGUCHI, Y. and GOJOBORI, T. .
A large-scale search for genes on which positive selection may
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operate. ENDO, T, IKEO, K. and GOJOBORI, T. . ....
Gene conversion in the major histocompatibility complex genes.
IMANISHI, T. . ..
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Studies of genetic variability in human MHC genes. IMANISHI, T.
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Evolution of glutamine synthetase gene is in accordance with the110
neutral theory of molecular evolution. TATENO, Y
Yamato and Asuka: DNA database management system. KITAKAMI, H., SHIN-I, T, IKEO, Ko, UGAWA, Y., SAITOU, N.,
GOJOBORI, T and TATENO, Y
110
Building and search system for a large-scale DNA database. KITAKAMI, H., YAMAZAKI, Y., IKEO, K., UGAWA, Y., SHIN-I,
T., SAITOU, N., GOJOBORI, T. and TATENO, Y. .
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VIII. Human Genetics
Molecular definition of the prader-Willi syndrome chromosome
region and isolation of transcribed sequences by positional
cloning method. IMAMURA, T., SAKAI, M., INABA, R., IzuHARA, K. and HASEGAWA, T.
. .....
112
Isolation of candidate transcribed sequences in cloned genomic
DNA with known lacation on human chromosome 18. IMAMURA, T., SAKAI, M., INABA, R., and NAKASHIMA, H.
113
Abnormality of JAK-STAT pathway induced by interleukin-4 inpatients with X-linked severe combined immunodeficiency.
IZUHARA, K. HElKE, T., SAKAI, Mo, INABA, R., MAYUMI, M.,
IMAMURA, T. and HARADA, N
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A new point mutation at nucleotide pair 3291 of the mitochondrialtRNALeu(UUR) gene in a patient with mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke-like episodes(MELAS). GOTO, Y, TSUGANE, K., TANABE, Y, NoNAKA, I. and HORAI, S. . .....
115
Point mutations in mitochondrial tRNA genes: sequence analysis ofchronic progressive external ophthalmoplegia (CPEO).
HATTORI, Y., GOTO, Y., SAKUTA, R., NONAKA, I., MIZUNO,
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Y., and HORAI, S
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Mitochondrial myopathy with progressive decrease in mitochondrial tRNALeu(UUR) mutant genomes. KAWAKAMI, Y., SAKUTA, R., GOTO, Y., NONAKA, I. and HORAI, S.
.
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A caucasian family with the 3271 mutation in mitochondrial DNA.
MARIE, S. K. N., GOTO, Y., NONAKA, I. and HORAI, S.
118
A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. KADOWAKI, T., YAZAKI, Y., GOTO, Y.,NONAKA, I. and HORAI, S
118
Enzymatic and genetic adaptation of soleus muscle mitochondria tophysical training in rats. MURAKAMI, T., SHIMOMURA, Y.,
OZAWA, T. and HORAI, S. .
119
Polymorphisms of complement component I and CIR subcomponent of Cl in nine aboriginal Taiwanese populations.
UMETSU, K., YUASA, I., SUZUKI, T., SUN, c.-S., PAN, I.-H.,
ISHIDA, T., SAITOU, N. and HORAI, S
120
Construction and expression of chimeric antibodies by a simple
replacement of heavy and light chain V genes into a single
cassette vector. KANDA, H., MORI, K., TANIGUCHI, K. and
WATANABE, T
121

IX. Plant Genetics
Genetic diversity of Chinese wild-rice populations. CAl, H. W.
and MORISHIMA, H
122
Variation study of weedy rice by morpho-physiological characters,
isozyme and RAPD markers. SUH, H. S., SATO, Y.-I., andMORISHIMA, H.
.
123
Allozyme variation and genetic structure of Oryza glumaepatula
populations distributed in the Amazon basin. AKIMOTO, M.,
SHIMAMOTO, Y. and MORISHIMA, H. . . . . . . . . . . . . . . . . .. 124
Genetic mechanism of unidirectional cross-incompatibility in rice.
SANO, Y.
.
125
The expression of the rice Wx gene in response to sugars. KUNIEDA, M., SANO, Y. and HIRANO, H.-Y.
126
Genetic diversity of wild soybean populations in Japan. SHIMAMOTO, Y.
.
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Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Abstracts of diary for 1994
Foreign visitors in 1994
Author index

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GENERAL STATEMENT
Our institute was established 45 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofresearch on genetics, in particular population genetics, plant genetics andmolecular genetics, by members of our institute have made it the center ofgenetic study in our country and a major institution with world-wide recognition. In 1984 the institute was reorganized into a National Inter-university
Research Institute to promote research activities through cooperation withother institutions. 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 other universities, 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 banks in the world thatgather, annotate, store and distribute information on DNA sequences.
DDBJ will continue to be active as a regional center for such activities. Our
institute also stores and distributes various organisms with genetically characterized traits. Among them, services with mice, rice and Escherichia coli are
particularly significant. These service activities will continue to develop 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 successful future.
Dr. K. Moriwaki, the Deputy Director and the Head of Division of

2

Cytogenetics, retired at the end of March. I appreciate very much his
assistance in the administration of this institue. He continues his research on
mice at Fukuyama University. Dr. T. Seno succeeded in the role of Deputy
Director.
We mourn the sudden loss of our honorary and honored member, Dr. M.
Kimura, who died on the 13th of November at the age of 71. His works aremonumental as they lay the foundation for modern population genetics andevolution biology.
In the past year, we have had many personnel changes. The Gene
Function Research Laboratory was established in the DNA Research Centerand Dr. Y. Tateno was promoted to professor and was transferred to the newlaboratory from the Genetic Resources Laboratory, which Dr. M. Fujitajoined as a research member. Dr. Y. Sato was transferred to Shizuoka
University as associate professor and Dr. H. Nakashima to Kyushu University. Dr. S. Kaneda retired to work at the HSP Research Institute, Inc., andDr. E. Moriyama to study at Harvard University. In addition to Dr. Fujita
the following five people joined us as research members: Dr. H. Seino in the
Division of Mutagenesis, Dr. Y. Ina in the Division of Population Genetics,
Dr. T. Tenzen in the Division of Evolutionary Genetics, Dr. K. lzuhara in
the Division of Human Genetics and Dr. T. Imanishi in the DNA Data
Analysis Laboratory.
It is a pleasure to note that Dr. A. Ishihama was honored by the Cultural
Award of the Chunichi Press for his studies on the regulation of transcription
by RNA polymerase.

3

STAFF

(as of December 31, 1994)
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.
YAMAGISHI, Masahiro, D. Sc.
TOYODA, Tetsuya, D. Med.
Division of mutagenesis
SENO, Takeshi, D. Sc., Professor
YAMAO, Fumiaki, D. Sc., Associate professor
KISHI, Tsutomu, D. Eng.
SErNO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry (Guest member)
MIZUMOTO, Kiyohisa, D. Sc., Profesor
MORIKAWA, Kousuke, D. Sc., Profesor

2.

Department of Cell Genetics
HORIUCHI, Kensuke, D. Sc., Head of the Department
Division of Cytogenetics
IMAI, Hirotami T., D. Sc., Associate professor
GOTO, Hideo, D. Ag.

4Division of Microbial Genetics

HORIUCHI, Kensuke, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Division of Cytoplasmic Genetics (Guest member)
OHTSUBO, Eiichi, D. Sc., Professor
MORIWAKI, Kazuo, D. Sc., 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 profesor
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
OHTA, Tomoko, Ph. D., D. Sc., Head of the Department
Division of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Professor
T AJIMA, Fumio, Ph. D., Associate professor
T AKANO, 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
SANO, Yoshio, D. Ag., Associate 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
Plant Genetics Laboratory
Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Associate professor

5

6

KANAMARU, Kengo, D. Ag.
Genetic Resources Laboratory
FUJITA, Masaya, D. Eng.
Mammalian Development Laboratory
NAKATSUJI, Norio, D. Sc., Professor
SHIRAYOSHI, Yasuaki, D. Sc.

7.

DNA Research Center
GOJOBORI, Takashi, D. Sc., Head of the Center
DNA-Protein Inateraction Laboratory
SHIMAMOTO, Nobuo, D. Sc., Associate professor
NAGAI, Hiroki, D. Sc.
Recombinant DNA Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
DNA Synthesis Laboratory
DNA Data Analysis Laboratory
GOJOBORI, Takashi, D. Sc., Professor
IKEo, Kazuho, D. Sc.
IMANIsHI, Tadashi, D. Sc.
Gene Library Laboratory
KOHARA, Yuji, D. Sc., Associate professor
ANDACHI, Yoshiki, D. Sc.
Gene Function Laboratory
TATENO, Yoshio, Ph. D., D. Sc., Professor

8.

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

9.

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

10.

Technical Section
MITA, Akihiko, Chief of the Section

STAFF

11.

Department of Administration
KaNa, Kenji, head of the Department
MIY AJI, Hideo, Chief of the General Affairs Section
YOKI, Yoshihisa, Chief of the Finance Section

7

8

COUNCIL(as of December 31, 1994)

Chairman
NAGAKURA, Saburo; President,Graduate University for Advanced Studies
Vice-chairman
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
Members (Alphabetical order)
HAMAl, Osamu; Dean, Faculty of Letters, University of Tokyo
HI RASA, Takao; Presidenet, Osaka Prefectural University
INOKUCHI, Hiroo; President, Okazaki National Research Institutes
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
NOJIMA, Shoshichi; Dean, Faculty of Pharmaceutical Science, Teikyo
University
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
SUGANO, Haruo; Director emeritus, Cancer Institute
SUGIMURA, Takashi; President, Toho University
TAKANAMI, Mitsuru; Director, Kazusa DNA Research Institute
TAKEUCHI, Ikuo; Director-General, National Institute for Basic Biology
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 31,1994)

Chairman
SENO, Takeshi; Professor, Division of Mutagenesis
Vice-chairman
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
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, Institute of Molecular and Cellular Biosciences
TAKAGI, Nobuo; Professor, Graduate School of Environmental Science,
Hokkaido University
TAKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto University
GOJOBORI, Takashi; Professor, DNA Research Center
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
MORISHlMA, 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

ASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS
President
KONDO, Norio; Professor emeritus, Tokyo University of Agriculture
Managing Director
MORIWAKI, Kazuo; Professor emeritus, National Institute of Genetics
GOJOBORI, Takashi; Professor, National Institute of Genetics
Manager
TOMIZAWA, Jun-ichi; Director-General, National Institute of Genetics
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
YAMAGUCHI, Hikoyuki; Professor emeritus, University of Tokyo
MIURA, Kin-ichiro; Director, Institute for Biomolecular Science, Gakushuin University
KURODA, Yukiaki; Professor emeritus, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of Genetics
TrusteeT AZIMA, Yataro; Director, Honorary member, National Institute of
Genetics
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Professor, Faculty of Medicine, Teikyo University
SHIGETO, Gakuji; Managing Director, The Inouye Foundation for the
Promotion of Science
MATSUNAGA, Ei; Professor emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, Shokabo Publishing Co.
T AKAGAKI, Yoshio; Chairman of the Board of Directors, CSK Research Park
SENO, Takeshi; Professor, National Institute of Genetics
T ATENO, 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; Honorary member, National Institute of Genetics

11

PROJECTS OF RESEARCH FOR 1994
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 (lSHIHAMA and YAMAGISHI)
Molecular mechanisms of transcription and replication of animal and plantviruses (lSHIHAMA and TOYODA)
Division of Mutagenesis

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

Mechanism of mRNA capping (MIZUMOTO)
Transcription of the Sendai virus genome (MIZUMOTO)
Structural genetics: X-ray crystallography of proteins (MORIKAWA)
2.

DEPARTMENT OF CELL GENETICS
Division of Cytogenetics

Genetic differentiation of mouse species (MORIWAKI, SHIROISHI* and MIYASHITA*)
Theoretical and experimental bases for chromosome evolution (lMAI)
Study of adrenal function in mouse development by transgenesis (GOTOH,SHIROISHI* and MORIWAKI)* Genetic stock research center.

12

Division of Microbial Genetics

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

Studies on cytoplasmic genes during subspecies differentiation of house
mouse Mus musculus (YONEKAWA)
Cell-cycle regulation by cyclin and CDC2 kinase (YASUDA)
3.

DEPARTMENT OF ONTOGENETICS
Division of Developmental Genetics

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

Gene expression in eukaryotes (HIROSE and UEDA)
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)
4.

DEPARTMENT OF POPULATION GENETICS
Division of Population Genetics

Theoretical studies of population genetics (OHTA, TAJIMA and INA)PROJECTS OF RESEARCH FOR 1994

13

Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (TAJIMA and INA)
Statistics for DNA polymorphisms (TAJIMA and 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 (IKEMURA)
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)
Molecular biology of oncogenes (FuJIYAMA)
Studies on DNA polymorphisms in human populations (HORAI)14

Division of Agricultural Genetics

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

Molecular genetics of human genome imprinting mechanisms (SASAKI)
Domestication dynamics in (SHIMAMOTO)
6.

GENETIC STOCK RESEARCH CENTER
Mammalian Genetics Laboratory

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

Molecular genetics of Drosophila melanogaster (HAYAsHI)
Plant Genetics Laboratory

Microbial Genetics Laboratory

Timing of cell division in E. coli (NISHIMURA)
Division apparatus of E. coli (KANAMARU and NISHIMURA)PROJECTS OF RESEARCH FOR 1994

15

Genetic Resources Laboratory

Theoretical studies in molecular phylogeny (TATENO)
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

DNA Data Analysis Laboratory

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

16

Gene Library Laboratory

Molecular genetics of Caenorhabditics elegans development (KOHARA andANDACHI)Genome analysis of Caenorhabditis elegans (KOHARA)
Gene Function Laboratory

Molecular phylogenetic analysis of DNA and protein sequence data
(TATENO)
8. RADIOISOTOPE CENTER
Molecular mechanisms of sporulation in Bacillus subtilis (SADAIE andFUJITA)
9.

EXPERIMENTAL FARM

17

RESEARCH ACTIVITIES IN 1994

I.

MOLECULAR GENETICS

Molecular Anatomy of RNA Polymerase Alpha Subunit from
Escherichia coli: Functinal Map of the Amino-terminal
Assembly Domain
MakoID KIMURA and Akira ISHIHAMA

The a subunit of E. coli RNA polymerase consisting of 329 amino acidresidues plays a major role in assembly of the core enzyme. Analysis ofcarboxy-terminal deletion derivatives of a indicated that the amino-terminal
two-thirds of a, down to amino acid residue 235, is involved in this assembly(Igarashi, K. et al. (1991) J. Mol. Bioi. 218, 1-6; Hayward, R. S. et al. (1991)
J. Mol. BioI. 221, 23-29). Detailed analysis of the assembly domain bymaking sets of terminal and internal deletion a mutants indicated that theminimal fragment with the full activity of subunit assembly is a(21-235)(Kimura, M. et al. (1994) J. Mol. BioI. 242, 107-115). Insertion of Ala-Ser(AS) dipeptide made a inactive in dimerization, (3- and/or (3'-binding depending on the position of AS insertion (Kimura, M. and Ishihama, A. (1995) J.
Mol. Bioi. 248, 710-718). For fine mapping, we then made a set of mutants
with Ala substitution near the subunit-subunit contact regions. Results
altogether indicate that for a dimerization, multiple contact sites are involved, which cluster within the whole sequence of the amino-terminal
assembly domain, while the contact with the two large subunits, (3 and (3',includes each two sites (Kimura, M. and Ishihama, A. (1995) J. Mol. BioI.
254, in press).
To confirm the subunit-subunit contact sites on a thus determined using invitro reconstitution system, we made the expression plasmids for mutant awith His-tag and analyzed subunit complexes isolated from cell extracts by
Ni + -affinity column chromatography (Kimura, M. and Ishihama, A., submitted for publication). Results of the in vivo analysis agreed well with theconclusion obtained from the in vitro studies.

18

Molecular Anatomy of RNA Polymerase Alpha Subunit: Analysis of the
Carboxy-terminal Activation Domain by Class-I Factors
and Upstream Enhancer Elements
Katsuhiko MURAKAMI, Nobuyuki FUJITA, Kazuyuki TAO*,
Tomasz HEYDUK** and Akira ISHIHAMA

The carboxy-terminal one-third of RNA polymerase a subunit plays anessential role in transcription activation by class-I transcription factors [weproposed to classify transcription factors on the basis of the contact site on
RNA polymerase]. Mutant RNA polymerases containing carboxy-terminal
truncated a carry the basic function of RNA synthesis but are inactive incAMP receptor protein (CRP)-dependent transcription of lac (Igarashi, K.
and Ishihama, A. (1991) Cel/65, 1015-1022). A number of class-I transcription factors have since been identified, which all require the contact site I on
the carboxy-terminal domain of a (reviewed in Ishihama, A. (1993) J.
Bacteriol. 175, 2483-2489). Later, this region was found to playa role inrecognition of promoter upstream (UP) elements with enhancing activity oftranscription (Ross, W. et a/. (1993) Science 262, 1407-1413). Amino acidsubstitution mutations of rpoA affecting the response to cAMP receptor
protein (CRP) are mapped mostly within a narrow region between aminoacid residues 265 and 270 [and a region between 298-300 for some mutations
with weak effects] (Zou, C. et a/. (1992) Mol. Microbiol. 6, 2599-2605).
Likewise, mutations affecting the response to OxyR, another class-I factor
involved in transcription activation of the oxidative stress-response genes,
were mapped in the same two regions in the vicinity of CRP site [although
the mutations at 293-300 region influenced as much as the mutations at 265270 region] (Tao, K. et a/. (1993) Mol. Microbiol. 7, 859-864; Tao, K. et a/.
(1995) J. Bacteriol., in press).
For detailed analysis of the individual amino acids in this region, we haveconstructed two sets of mutant a, i.e., the first-set mutants carrying a single
Trp substitution at all the residues in the major CRP contact site region
between residues 260-270, and the second-set mutant a carrying the substitution of Arg265 for all kinds of other amino acids. In vitro transcription assay
using the reconstituted RNA polymerases from overexpressed and purified***

Radioisotope Center, University of Tokyo, Tokyo.
Department of Biochemistry and Molecular Biology, University of saint Louis, St.
Louis.

I.

MOLECULAR GENETICS

19

mutant a indicated that this region is involved in not only CRP contact but
also rrnB UP element recognition, but the roles of individual amino acidresidues in these two functions are different because some mutations affected
only transcription activation by the UP element but not by CRP (Murakami,
K. et al., in preparation). Likewise, mutations affecting the response to
DNA UP element do not always influence OxyR response (Tao, K. et al.
(1995) J. Bacterial., in press). DNase I footprinting analysis showedsignificant decrease in the cooperative promoter binding of the mutant
holoenzymes with CRP.
Molecular Anatomy of RNA Polymerase Alpha Subunit:
Structure of the Carboxy-terminal Activation Domain
Tomofumi NEGISHI, Nobuyuki FUJITA, Yoshimasa KVOGOKU*
and Akira ISHIHAMA

The a subunit of E. coli RNA polymerase is composed of two major
structural domains, i.e., the amino-terminal domain upstream from Arg235
and the carboxy-terminal domain downstream of Glu245, both being connected by a protease-sensitive inter-domain linker (Negishi, T. et al. (1995)
J. Mol. Bioi. 248, 723-728). This structural organization is in good agreement with the functional map of this subunit, consisting of the aminoterminal assembly domain and the carboxy-terminal transcription activation
domain by class-I transcrition factors (reviewed in Ishihama, A. (1993) J.
Bacterial. 175, 2483-2489) and DNA UP elements (Ross, W. et al. (1993)
Science 262, 1407-1413).
Upon prolonged incubation with proteases, the amino-terminal assembly
domain is cleaved between Arg45 and Glu68 into two sub-domains Na and
Nb. The subdomain Nb of 168 amino acids in length is then cleaved between
Arg 150 and Gly 151 into two segments; the amino-terminal proximal segmentof 11 kDa containing the (3 subunit-binding site (Kimura, M. and Ishihama,A. (1995) J. Mol. Bioi. 254, in press) was resistant to further digestion withtrypsin, while the carboxy-terminal segment containing the (3' subunitbinding site was digested rapidly upon continued proteolysis.
The carboxy-terminal domain for transcription activation is highly resistant to proteolysis, indicating that this fragment forms a highly structured*

Institute for Protein Research, Osaka University, Suita.

20

domain. We expressed and purified this domain and subjected to NMR
spectoscopic analysis by means of 15N-single and lSNj13C-double labelingtechniques. The three dimensional structure predicted from the NMR
analysis is in good agreement with the results obtained from mutant studies
and limited proteolysis (Jeon, Y. O. et al. (1995) Science, in press).

Promoter Selectivity Control of Escherichai coli RNA Polymerase:
Intracellular Levels of Sigma Subunits
Miki JISHAGE, Akira IWATA* and Akira ISHIHAMA

Bacterial cells have the capacity to respond rapidly to eventual environmental changes by turning off and on the expression of sets of genes.
For the changes in global gene expression pattern, the RNA polymerase isconsidered to playa key role by rapidly modulating its promoter selectivity(Ishihama, A. (1988)Trends Genet. 4, 282-286). One major mechanism of
the promoter selectivity control of RNA polymerase is the replacement of 0
subunit, the promoter recognition factor, on core enzyme with the catalytic
function of RNA synthesis. Up to now, six different molecular species of 0
subunit have been identified in Escherichia coli (reviewed in Ishihama, A.
(1988) Trends Genet. 4, 282-286). However, little is known about the
intracellular level of each 0 subunit under various growth conditions of E.
coli.
In order to determine the levels and control of individual 0 subunits, we
produced mono-specific polyclonal antibodies against each 0 subunit andmeasured the concentrations of 0 subunits in cell extracts by quantitative
Western blot analysis. Results indicated that: i) OD (or 0 7°), the princial 0
subunit in exponentially growing cells, is maintained at a constant level
throughout the growth change from log to stationary phase; ii) in contrast,
the level of oS (or 0 38 ), the stationary-specific 0 subunit, increases when cell
growth enters into the stationary phase; iii) the level of oS increases furthermore by exposure of the stationary-phase cells to osmotic stress; and iv) evenin the middle of exponentially growing phase, the level of oS transiently
increases when cells are exposed to heat shock (Jishage, M. and Ishihama, A.
(1995) J. Bacterial., in press). The intracellular localization of oS, i.e. free
dissociated form or core enzyme-associated form, is being examined by*

Nippon Institute of Biological Science, Ohme, Tokyo.

1.

MOLECULAR GENETICS

21

glycerol gradient centrifugation.
Promoter Selectivity Control of Escherichia coli RNA Polymerase:
Functional Comparison of Sigma Subunits
Shuichi KUSANO, Nobuyuki FUJITA and Akira ISHIHAMARNA polymerase core enzyme of Escherichia coli with the subunit composition of a2/3/3' is functionally differentiated into different forms of holoenzyme by binding one of the multiple molecular species of a subunit, thepromoter recognition subunit (Ishihama, A. (1988) Trends Genet. 4, 282~
286). The switching of gene expression pattern is considered to be controlled
by the replacement of core enzyme-associated a subunit. Until now, six
different species of a subunit have been identified in E. coli. In order tounderstand the molecular mechanism of a replacement, we have performed asystematic comparison of the activities between different a subunits.
First, the level of a subunits required for the maximum transcription in thepresence of a fixed amount of core enzyme was compared between various a
subunits. Results indicated that the core-binding affinity of oS (or a 38 ; theprincipal a in statinary-phase cells) is 4- to 7-fold weaker than aD (or a 70 ; theprincipal a in exponentially growing cells), a H (or a 32 ; the heat-shock genespecific a subunit) and a F (or a2 8; the flagella gene-specific a subunit)(Kusano, S. et al. (1995) J. Bioi. Chern., in press). To confirm this prediction, we then measured directly the core-binding affinity. For this purpose,
mixtures of a fixed amount of core enzyme and various amounts of a subunits
were subjected to gel-filtration HPLC and the core enzyme-bound a subunits
were determined. The core enzyme was saturated with aD by adding 2-foldmolar excess over the core enzyme but to achieve the same extent of corebinding, at least 6-fold molar excess of oS was needed, indicating that the core
enzyme-binding affinity of oS is at least three fold weaker than aD. Thesefindings are rather unexpected because the intracellular concentration of oS is
not higher than that of aD even in the stationary-phase E. coli (Jishage, M.
and Ishihama, A. (1995) J. Bacterial., in press). Thus, it appears that astationary-specific factor(s) or condition(s) is involved in either efficientreplacement of a subunit from aD to oS or efficient utilization of EoS
holoenzyme for transcription of stationary-phase specific genes.
As an attempt to examine these possibilities, we analyzed the effect of DNA

22superhelicity on the promoter selectivity by £aD and £dl holoenzymes.
Results indicated that the optimum superhelical density for maximum transcription of lacUV5 and osmY promoters is lower for £dl than EaD . This isin good agreement with the decrease in superhelicity of the chromosomal
DNA in stationary-phase E. coli.
Molecular Anatomy of Escherichia coli RNA polymerase: Modes of
RNA Polymerase-Transcription Factor Interaction(Progress in International Collaborative Studies)
Akira ISHIHAMA

A number of transcription factors have been identified in E. coli, which
activate transcription by modulating the promoter selectivity of RNA polymerase via direct protein-protein contacts (reviewed in Ishihama, A. (1992)
Mol. Microbial. 6, 3283-3288; Ishihama, A. (1993) J. Bacterial. 175, 24832489). In collaboration with more than 50 laboratories from 18 countries, asystematic mapping of the domain(s) on RNA polymerase subunits for
activation by each of these transcription factors is being carried out. Basedon the results of our collaborative studies, we have proposed a classification
system of the transcription factors. This new system is based on the locationof the activation domain on RNA polymerase subunits. Table 1 shows a listof the up-to-date classification. Among these transcrption factors, finemapping of the contact sites on RNA polymerase has been done extensively
for the class-I factors. Analysis of the protein structure is also being focussed
on the C-terminal region of a subunit.
Beside transcription activation by the protein factors, upstream (UP)enhancer DNA elements make direct contacts with RNA polymerase andactivates transcription. Thus, the C-terminal domain of a subunit is involved
in recognition of not only class-I transcription factors but also DNA UP
elements (Ross, W. et al. (1993) Science 262, 1407-1413). DNA-a subunit
interaction is now recognized as one of the attractive research subjects.
Followings are the major progress in this year's collaborations:
CRP (in collaboration with S. Busby and colleagues, School of Biochemistry, Univerisity of Birmingham, Birmingham): cAMP receptor protein(CRP) functions as both class-I and class-I transcription factors dependingon the DNA site of CRP binding relative to promoter (Igarashi, K. andI.

Table I.
Class

Location of theactivation domaina subunit
II
III
IV

a subunit(3 subunit(3' subunit

MOLECULAR GENETICS

23

Classification of E. coli transcription factors
Transcription factor*
Ada!, AraC 2 , CRP(lac)3/4, CysB, O(P4), FlhDfFlhC', Fnr,
IHF6, MelR, Ogr(P2), OmpR 7, OxyR 8, SoXS(zwf)9, TyrR IO
CRP(gal)3/II, cI(A)IJ, Fis, MerR IJ , NtrC 14, PhoB'" SoxS(sodA)9
DnaA'6, ppG pp 17----NusA, Rho* Underlined, factors analyzed in this laboratory in collaboration with: 'K. Sakumi and M.
Sekiguchi, Kyushu Univerisity School of Medicine, Fukuoka; 2 R. Schleif and colleagues, John
Hopkins Univeristy, Baltimore; 3 Hiroji Aiba and colleagues, Nagoya University, Faculty of
Science, Nagoya; 4 A. Kolb and H. Buc, Pasteur Institute; 'P. Matsumura and colleagues,
University of Illinois, Chicago; 6A. Oppenheim and colleagues, Hebrew Univerisity School of
Medicine, Jerusalem; 7 Hirofumi Aiba and T. Mizuno, Nagoya Univerisity, Faculty ofAgriculture, Nagoya; 8 K. Tao, University of Tokyo, Radioisotope Center, Tokyo; 9R. E.
Wolf, JT. and colleagues, University of Maryland Baltimore County, Baltimore; 10 B. Lawly
and A. J. Pittard, University of Melbourne, Melbourne; II S. Busby and colleagues, Univ.
Birmingham, Birmingham; 12G. N. Gussin, Univ. Iowa, Iowa City; IJ A. Ansari and T.
O'Halloran, Northwestern Univ.; 14S. Kustu and colleagues, University of California,
Berkeley; F. G. Hansen and T. Atlung, Department of Microbiology, Technical Universityof Denmark, Lyngby; 17 D. Chatterji and colleagues, Centre for Cellular and Molecular
Biology, Hyderabad.
Ishihama, A. (1991) Cel/65, 1015-1022; Igarashi, K. et al. (1991) Proc. Natl.
Acad. Sci. USA 88,8958-8962). In the case of transcription activation of lac,
one molecule of the CRP dimer interacts with the C-terminal region of one
molecule of the a subunit dimer [contact site I]. On the other hand, one
molecule of the CRP dimer interacts with the C-terminal region of a subunit
for activation of gal transcription (Kumar, A. et al. (1994) J. Mol. BioI. 235,405-413). We found, however, that the other molecule of the CRP dimer
bound to the gal promoter interacts with the C-terminal region of a (Attey,
A. et al. (1995) Ncleic Acids Res. 22,4375-4380). This CRP-a interaction
appears to shift the a contact site on the gal promoter to a putative UP
element, ultimately leading to enhanced transcription.
FlhD/FlhC (in collaboration with P. Matsumura and colleagues, Department of Microbiology and Immunology, University of Illinois, Chicago):
Transcription of the flagellar operons is ordered in a hierarchy, allowing
coordinated expression of flagellar components in the order of asssembly. In
the early step of flagellar operon transcription, the FlhD/FlhC complex plays
a key role as a master regulatory factor which activates transcription of the24genes expressed in the late stage of flagellar formation. In vitro transcription
studies using three FlhDjFlhC-dependent promoters, fliA, flhB and fliL,indicated that the mutant RNA polymerases containing C-terminal truncateda subunits were not activated by FlhDjFlhC complex (Liu, X. et al. (1995)
J. Bacteriol. 177, 5186-5188). Thus, we concluded that the FlhDjFlhC
complex is a class-I transcription factor.
SoxS (in collaboration with R. E. Wolf, Jr. and colleagues, Departmentof Biological Sciences, University of Maryland Baltimore County, Baltimore): In response to the oxidative stress, E. coli activates the SoxRS
regulon, which is comprised of at least 10 genes. For this coordinate
expression, SoxS, a XylSjAraC family protein, plays as a transcription
activator of the member genes. SoxS binds to "soxbox" DNA located near
the promoter - 35 sequence and is considered to make direct contact with the
C-terminal domain of a subunit, because the mutant RNA polymerases
consisting of C-terminal truncated a subunit were unable to transcribe SoxSdependent zwf (glucose-6-phosphate dehydrogenase) andfpr (NADPH: ferredoxin oxidoreductase) (lair, K.-W. et al. (1995) Mol. Microbiol., in press).
However, SoxS-dependent transcription of fume (oxygen stable fumarase),
micF (antisense regulator RNA of ompF) , nfo (endonuclease IV) and sodA
(manganese superoxide dismutase) was indifferent to the truncation of a
subunit C-terminus. Thus, like the cAMP receptor protein (CRP or CAP),
SoxS is an "ambidextrous" activator.
Gene Cloning and Characterization of RNA Polymerase II Subunits
from the Fission Yeast Schizosaccharomyces pombe:
Cloning of the Genes Encoding Small Subunits
Hitomi SAKURAI, Hisako SUZUKI and Akira ISHIHAMA

Knowledge of the structure and function of RNA polymerase II is essential
for understanding the molecular mechanisms of transcription regulation in
eukaryotes. Purified RNA polymerase II from the fission yeast Schizosaccharomyces pombe contains more than 10 polypeptides as analyzed by SDS-gel
electrophoresis. Up to the present time, we have cloned the genes coding forthe subunits 1 (RPBI), 2 (RPB2), 3 (RPB3) and 5 (RPB5) (Azuma, Y. et
al. (1991) Nucleic Acids Res. 19, 461-468; Kawagishi, M. et al. (1993)
Nucleic Acids Res. 21, 469-473; Azuma, Y. et al. (1993) Nucleic Acids Res.

I.

MOLECULAR GENETICS

25

21, 3749-3754; Miyao, T. et al., in preparation). The rpb] and rpb2 genes
were isolated using the corresponding genes from the budding yeast Saccharomyces cerevisiae as the hybridization probes. For cloning of the rpb3 andrpb5 genes, we determined amino acid sequences for parts of the isolated
subunit proteins. The genes were PCR-amplified using primers designed
based on the protein sequences determined in this study or the publishedamino acid sequences of the corresponsind genes from the budding yeast
Saccharomyces cerevisiae or human. In addition to these four subunits, wehave determined partial amino acid sequences for subunits 6, 7, 10 and 11.
Results showed sequence similarity to the corresponding subunits of Saccharomyces cerevisiae RNA polymerase II. The knowledge of amino acid sequences can now be used for cloning of the genes coding for these proteins.
Gene Cloning and Characterization of RNA Polymerase II Subunits
from the Fission Yeast Schizosaccharomyces pombe:
Thermolabile RNA Polymerase II with
Mutations in Subunit 3
Kiyoshi YASUI, Yoshinao AZUMA *, Masahiro YAMAGISHI
and Akira ISHIHAMA

For analysis of the structure-function relationship of RNA polymerase II
subunits from S. pombe, we constructed a number of temperature-sensitive
mutants with mutations in the rpb3 gene encoding subunit 3. For thispurpose, we first PCR-amplified the rpb3 gene in the presence of manganese
ion to induce inaccurate DNA synthesis. The mutagenized rpb3 gene wastransformed into S. pombe and the recombinants were screened for temperature-sensitive cell growth. The ts mutants were classified into two groups:
group 1 mutants stopped cell growth immediately after temperature up-shift,while group 2 mutants exhibited delayed response to the growth arrest at hightemperatures. RNA polymersae II partially purified from Ts54, one of thegroup 2 mutants, was thermo-labile in vitro as measured by non-specifictranscription assay (Azuma, Y. et al. (1995) J. Biochem. 118, 216-220).
These results suggested that RPB3 plays roles not only in enzyme assembly
but also in transcription regulation. The RNA polymerase II mutants
isolated in this study can be used as reference strains for in vivo studies oftranscription regulation.

* Present address: University of California, Irvine.

26

Gene Cloning and Characterization of RNA Polymerase II Subunits
from the Fission Yeast Schizosaccharomyces pombe:
Structure and Function of Subunit 5 (RPBS)
Takenori MIYAO, Yoshinao AZUMA*, Hitomi SAKURAI,
Masahiro YAMAGISHI and Akira ISHIHAMA

The rpb5 gene coding for subunit 5 of S. pombe RNA polymerase II was
amplified by PCR using primers degenerated from the knowledge of aminoacid sequences of the subunit 5 from S. cerevisiae and human (Miyao, T. etal., in preparation). In parallel, we determined the amino acid sequence ofparts of the isolated RPB5 polypeptide. The amino acid sequence of thepolypeptide predicated from the cloned gene agreed well with the sequence of
RPB5. In addition, the predicted amino acid sequence is 56 and 44%
identical with that of the RNA polymerase subunit 5 from budding yeast andhuman, respectively. In particular, the homology is high at the carboxyterminal proximal region. We then concluded that the cloned gene represents
the S. pombe rpb5 gene encoding RNA polymerase II subunit 5.
Recently, it was found that the trans-acting factor from hepatitis B virus
directly interacts with the subunit 5 of human RNA polymerase II (Murakami, S. et al. (1995) EMBO J. 14, 143-150). This finding raises a possibility
that cellular factors with the regulatory functions of transcription also bindto and form complexes with this subunit. To test this possibility, we aretrying to identify S. pombe proteins specifically associating with RPB5.
Molecular Anatomy of Influenza Virus RNA Polymerase:
Analysis of Subunit Functions
Makoto KOBAYASHI**, Tetsuya TOYODA, Susumu NAKADA***,Kiyohisa MIZUMOTO**** and Akira ISHIHAMARNA polymerase of influenza virus is composed of three viral coded P
proteins, PBl, PB2 and PA, and is involved in all step reactions in bothtranscription and replication (reviewed in Ishihama, A. and Nagata, K.
(1989) CRC Crit. Rev. Biochem. 23, 27-76; Ishihama, A. and Barbier, P.
*
**
***
****

Present address: University of California, Irvine.
Present address: Jichi Medical School, Department of Biology, Tochigi.
Science University of Tokyo, Noda, and Yamanouchi Pharm. Co., Ltd., Tsukuba.
Kitasato University, School of Phemaceutical Sciences, Tokyo.

I.

MOLECULAR GENETICS

27(1993) Arch. Virol. 134, 235-258). The subunit composition was first
determined by purification of the RNA polymerase from virus particles(Honda, A. et al. (1989) J. Biochem. 107, 1021-1026) and later confirmed byreconstitution of the enzyme from three P protein subunits which were
purified from individually expressed in insect cells after infection of therecombinant baculoviruses carrying the cloned cDNAs (Kobayashi, M. et al.
(1992) Virus Res. 22,235-245).
In order to determine the function(s) of each subunit, we examinedpossible activities of viral RNA synthesis in nuclear extracts prepared from
insect cells infected with various combinations of the recombinant baculoviruses. RNA synthesis activity was measured using 84mer short model RNA
templates, each carrying 5'- and 3'-terminal conserved sequences of the
genome RNA segment 8. Results indicated that: i) nuclear extracts expressing only PB 1 subunit was able to catalyze viral RNA synthesis in the presenceof ApG primer, but the activity was very low in its absence; ii) the nuclear
extracts containing both PBl and PB2 exhibited the 84mer model RNAdirected globin mRNA-primed RNA synthesis, producing transcripts ofabout 100 nucleotides in length; and iii) the nuclear extract containing all
three P proteins exhibited the activity of complete replication, i.e., minusstrand model RNA-dependent plus-strand RNA synthesis and plus-strand
RNA-dependent minus-strand RNA synthesis in the absence of primers(Kobayashi, M. et al., submitted for publication). These observations
altogether indicate that: PB 1 is a catalytic subunit of the influenza virus RNA
polymerase; PB2 is required for mRNA-primed transcription; but the complete set of P proteins is required for the complete cycle of replication.
Essentially the same results were obtained when recombinant vaccinia viruses
carrying the cDNA for influenza virus P proteins were infected into HeLa
cells (Toyoda, T. et al., submitted for publication).

28

Molecular Anatomy of Influenza Virus RNA Polymerase:
Analysis of Subunit-Subunit Contact Sites
Djanybeck ADYSHEV*, Tetsuya TOYODA, Akira IWATA**,
Susumu UEDA** and Akira ISHIHAMA

Influenza virus RNA polymerase is composed of three viral coded proteins,
PB I, PB2 and PA, each playing a different role( s) in transcription andreplication. For the expression of intrinsic subunit activities in vitro, theassembly of all three P proteins is required (Kobayashi, M. et al. (1992)
Virus Res. 22, 235-245), while in vivo, individual subunits and binary
complexes consisting of two P proteins exhibit low levels of the activities (seeabove). Thus, subunit-subunit contacts are required for the expression of
intrinsic subunit functions or at least for the enhancement of the activities.
In order to understand the mechanism of subunit assembly and thefunctinal and structural changes of individual subunits during the assembly,
attempts were made this year to determine the subunit-subunit contact sites
between the three P proteins. For this purpose, we prepared sets of aminoterminal and carboxy-terminal deletion mutants for each P protein. eDNAof the mutant P proteins were expressed in various combination. In order toexamine binary complex formation, two kinds of the eDNA clones, one
deletion mutant P protein and the other intact P protein were simultaneouslytransfected, and expressed P proteins were immuno-precipitated using monospecific anti-P protein antibodies. Results indicated that both PB2 and PA
can bind to PBI, and that the PB2-binding site is located at the carboxyterminal region of PBI while PBI-binding site is located at the aminoterminal region of PB2.
Molecular Anatomy of Influenza Virus RNA Polymerase:
Mapping of Nucleotide-Binding Sites
Yukiyasu ASANo, Koyohisa MIZUMOTO***, Tokumi MARUYAMA****and Akira ISHIHAMA

8-Azido GTP (8-N 3 GTP) was demonstrated to be polymerized into RNA
*
**
***
****

On leave of absence from Institute of Biochemistry and Physiology, Kyrghyz
Academy of Sciences, Bishkek, Kyrghysztan.
Nippon Institute of Biological Science, Ohme, Tokyo.
Kitasato University, School of Pharmaceutical Sciences, Tokyo.
Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Tokushima.

I.

MOLECULAR GENETICS

29

by influenza virus-associated RNA polymerase at about one tenth the rate of
GTP incorporation. The K m value for the azido analogue of GTP inprimer-dependent RNA synthesis (transcription) was 9411M whereas K m forthe natural substrate, GTP, was 6.7 11M. Upon exposure of a mixture of 8-N 3[a_ 32 P]GTP and influenza virus RNP to UV light, the PB 1 subunit was
selectively radio-labeled. The photo-labeling of PB 1 was strongly competedby GTP and to lesser extents by other nucleoside 5'-triphosphates. These
results altogether support the prediction that the substrate-binding site (Ssite) is located on the PB1 subunit. In the presence of ApG primer, the 8-N 3
GTP binding was reduced to about 40% level, suggesting that the GTP
analogue can bind not only to the S site but also the primer- and productbinding site (P site).
Molecular Dissection of Influenaza Virus Nucleoprotein (NP):
Identification of RNA-binding Region
Makoto KOBAYASHI*, Tetsuya TOYODA and Akira ISHIHAMA

Influenza virus nucleoprotein (NP) is associated with the genome RNA,
forming ribonucleoptorein (RNP) cores. Isolated NP binds to viral RNA at
an interval of about 20 nucleotides in a cooperative manner. To identify theamino acid sequence on NP involved in this RNA binding, we performed
Northwestern blot analysis using a set of amino- and carboxy-terminal
deletion mutants of NP produced in Escherichia coli. The RNA-binding
region was mapped between amino acid residues 91 and 188, a stretch ofresidues that contains a sequence highly conserved among A-, B- and C-type
influenza viruses (Kobayashi, M. et a!' (1994) J. Viral. 68, 8433-8436). A
similar sequence was found in the RNA-binding domain of a plant virus
movement protein and of ribosomal protein S9.
Growth Control of Influenza Virus by Membrane Protein (Ml)Jim YASUDA** and Akira ISHIHAMA

Analysis of fast-growing reassortants (AWM viruses) of A-type influenza
virus produced by mixed infection of a fast-growing WSN and a slowly***

Present address: Jichi Medical School, Department of Biology, Tochigi.
Present adress: University of Alabama, Birmingham.

30

growing Aichi strain indicated that RNA segment 7 including the M1 andM2 genes plays a role in the control of influenza virus growth at an early stepof infection (Yasuda, J. et al. (1993) Arch. Virol. 68,8141-8146). In orderto determine which of the two M genes, M1 or M2, encoded by RNA
segment 7 is resonsible for the growth rate control, one recombinant WSN
virus (CWA) clone possessing a chimeric M gene (WSN M 1-Aichi M2) was
generated by using an improved reverse genetics and transfection system.
The recombinant CWA virus retained the phenotype of both large plaqueformation and early onset of virus growth (Yasuda, J. et al. (1994) J. Virol.
68, 8141-8146). This indicates that the WSN M1 protein is responsible for
rapid virus growth.

Molecular Dissection of Mouse Mxl Protein with
Anti-Influenza Virus Activity
Testuya TOYODA, Yukiyasu ASANO and Akira ISHIHAMA

Mouse Mx 1 protein is an interferon-inducible nuclear protein, which
renders the expressing cells resistant to influenza virus infection. Previously
we identified that Mx1 protein is a GTP-binding protein with GTPase
activity and forms self-asseblies (Nakayama, M. et al. (1991) J. Bioi. Chern.
266,21404-21408; Nakayama, M. et al. (1992) Virus Res. 22,227-234). The
motifs for the GTPase and self-assembly have been mapped using a set ofdeletion and amino acid substitution Mx1 mutants (Nakayama, M. et al.
(1993) J. BioI. Chern. 268, 15033-15038).
In order to address possible relationship between these biochemical activities and the antiviral activity, we constructed cell lines expessing intact ormutant Mx 1 proteins and examined for the growth of influenza virus intransfected cells. Results indicate that: i) one mutant Mx 1 (S50I) with Ser
to He substitution at residue 50 within the GTP-binding motif showed a low
activity of GTP hydrolysis in vitro, formed apparently linear large aggregate
bundles in expressed cell nuclei, and lost the anti-viral activity; and ii)
another mutant Mx1 (C71S) with Cys to Ser substitution at residue 71 within
the self-assembly domain, did not form self-assemblies in vitro, showed
uniform diffuse distribution in expressed nuclei, but retained the anti-viral
activity. Thus, we concluded that the GTPase activity is essential for the

I.

MOLECULAR GENETICS

31

anti-influenza acitivity (Toyoda, T. et al. (1995) J. Gen. Virol. 76, 18671869).
DNA Replication/Repair is Impaired in a Temperature-Sensitive
Ubiquitin-Activating Enzyme Mutant of Mouse Cells,
without the Accompanied Enhanced Mutation
Frequency- A Possible Involvement of
Deoxyribonucleotide Metabolism
Fumiko AOKI, Yusuke WATAYA* and Takeshi SENO

A mouse cell mutant, tsFS20 with the temperature-sensitive ubiquitinactivating enzyme £1, was arrested in a S-phase at the nonpermissive
temperature (39°C) (Ayusawa D. et al. (1992) Cell Struct. Funct. 17, 113~122), where cellular DNA synthesis was inhibited, but not total RNA andprotein synthesis. DNA synthetic activity decreased to 30% after 4 h
incubation at 39°C and was near 0% of the initial level in the mutant cells
after 16 h. When the DNA synthetic activity in vitro was measured after the
4 h-arrested cells were permeabilized by lysolecithin, the activity had recovered to a normal level. It also recovered to near normal level in the 16 harrested cells. Thus, the inhibition of DNA replication in this mutant seems
to be reversible. DNA synthesis in the permeabilized cells was also nottemperature-sensitive. To explain these results, the possibility has been raisedthat the enzyme system involving a reduction of ribonucleotides is affected inthis mutant. In support of this supposition, the mutant showed increased
sensitivity to hydroxyurea, a specific inhibitor of ribonucleotide reductase.
Also, the mutant showed a significant decrease in intracellular pools of dTTP
and dCTP when cells were cultured at a nonpermissive temperature. Thisdecrease in the nuclotide pool seemed to be specific for the mutation in theubiquitin-activating enzyme, since a mouse cell mutant tsFT20 with temperature-sensitive DNA polymerase a, arrested in the S-phase at a nonpermissive
temperature, and wild-type cells arrested in the S-phase in the presence of a
DNA synthesis inhibitor, aphidicolin, did not show a decrease in the deoxyribonucleotide pool. Hence, targeted ubiquitination in relation to the mutant
phenotype may not be involved on component(s) of DNA replication machinery, but rather on protein(s) involving reduction of nucleotides as* Faculty of Pharmaceutical Sciences, Okayama University.

32

suggested above.
tsFS20 cells showed an increased sensitivity to the alkylating agent
MNNG. Interestingly, the increased sensitivity to MNNG was not accompanied by an increased mutation frequency as measured at the HPRT
locus. These results suggest that the ubiquitin system is involved in the DNA
repair mechanisms and more specifically in the induced mutation fixation
processes. This phenotype has not been reported for mammalian mutants,
but is reminiscent of the S. cerevisiae rad6 mutant in which a mutation in theubiquitin-conjugating enzyme E2/UBC2 has been identified. Attempt tocomplement the mutant phenotype by transfection of cloned cDNA encoding
the human homologue of RAD6 have failed so far.
Perturbation of Nucleolar Dissolution in the G2-M Transition in a
Mouse Cell Mutant with Temperature-Sensitive Ubiquitin-Activating
Enzyme El: Possible Involvement of HSP70 and Proteasome
T. SUDHA,* Masazumi SAMASHIMA, Hideo TSUJI,
Fumiaki YAMAO and Takeshi SENO

Ubiquitin stress created by a shift-up of temperature in a mouse cellmutant, ts85 with the temperature-sensitive ubiquitin-activating enzyme El,
brings about cell cycle arrest at the G2 phase accompanied by abnormal
integrity of the nucleolus represented by a U-shaped or ring-shaped arrangement of nucleolar lobes. More severe fragmentation of the lobes wasobserved when the mutant cells were synchronized at the G2 phase before thetemperature shift-up. A cloud of unidentified electron-dense particles (70 nm
in diameter) was observed in regions surrounded by the nucleolar lobes(Sudha, T. (1995) Chromosome Res. 3, 115-123). The particles grew innumber when cells in the G2-phase were subjected to a temperature-shiftup.
Also, among several proteins tested, immuno-histochemical signals for inducible type HSP70 and proteasome were concentrated and colocalized in theregion. It should be mentioned that such local concentrations of HSP70 andproteasome in nucleoli were also observed for canonically heat-shockedwild-type cells. Following the release of the arrested mutant cells into the Mphase, nucleolar materials persisted in prophase and prometaphase chromosomes which brought about a peculiar bouquet-like figure of metaphase*

The Tokyo Metropolitan Institute of Medical Science, Tokyo.

I.

MOLECULAR GENETICS

33

chromosomes. In such odd chromosomal figures, condensation and separation of nucleolar organizer regions seemed to be incomplete as shown by
FISH with rDNA and NOR-Ag staining. The mutant phenotype wascomplemented by transfection with a cloned cDNA for the El enzyme (Imai,
N. et al. (1992) Gene 118, 279-282). Hence, the ubiquitin system inassociation with HSPs seems to playa key role in the dissociation processesof the nucleolus during G2-M transition.
Ubiquitin-activating Enzyme, El Is Phosphorylated in Mammalian
Cells by the Protein Kinase CDC2
Yukiko NAGAI, Sumiko KANEDA, Takeshi SENO and Fumiaki YAMAO

The ubiquitin-activating enzyme (El) is the first enzyme in the pathway
leading to the formation of ubiquitin-protein conjugates. E 1 was found to bephosphorylated in cells of a mouse mammary carcinoma cell line, FM3A.
Peptide mapping of trypsin digests of labeled El indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were alsolabeled in vitro with Cdc2 kinase-mediated phosphorylation of El, affinitypurified from the same cell line. Cdc2 kinase is a key enzyme playing apivotal role in G2/M transition in the cell cycle. The phosphorylation of oneof the two oligopeptides was prominent 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. These findings suggestthat El is a target of Cdc2 kinase in the cell, implying that the ubiquitin
system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.
Screening for cDNAs Encoding the Ubiquitin-Conjugating Enzyme
in Fission Yeast, Schizosaccharomyces pombe
Fumio OSAKA, Hiroaki SEINO, Takeshi SENO and Fumiaki YAMAO

By screening the cDNA gene products expressed in E. coli cells with theirability to bind activated ubiquitin, we have isolated cDNAs for a family ofubiquitin-conjugating enzymes (UBC) of S. pambe. Among more than34

20,000 clones, twenty were found to encode protein with UBC-like activity,and subsequent sequence analysis identified four types of genes. We concluded that these four were ubc genes and designated them, ubcPI, ubcP2,
ubcP3 and ubcP4. Their gene products form a thiolester bond with activatedubiquitin in vitro in the presence of the ubiquitin-activating enzyme. A UBC
domain structure which included cysteine residue forming a thiolester bondwith ubiquitin was found in their sequences and is characteristic to all known
UBC's. ubcP I and ubcP2 which were predominantly isolated in the screening were found to encode the homologue of Ubc4 and Ubc2/Rad6 in budding
yeast, Saccharomyces cerevisiae, respectively. The other two, ubcP3 andubcP4 were novel ubc genes. ubcP4 was found to be essential for growth, andwas arrested at the G2 period of the cell cycle when its expression was
abolished. The terminal phenotypes of the cells in which the ubcP4 gene wasrepressed under the control of the nmt promoter were not unique but very
close to those found when there is overexpression of the dominant negative
cdc2 kinase mutant. The genetic and biochemical analysis of the gene and its
product, including the identification of the target protein of this ubiquitin
pathway are now underway.
Regulation of Gl/S transition by the Ubiquitin System in
Yeast Saccharamyces cerevisiae
Tsutomu KISHI, Motoyasu UI* and Takeshi SENO

When GI yeast cells reach a critical size, they not only initiate DNA
replication but they also form buds and duplicate their spindle pole bodies.
Three genes (CDC4, CDC34 and CDC53) are specifically needed to enter S
phase. Mutants defective in each of these three genes showed three characteristics; they failed to initiate DNA replication, fail to separate their spindle
pole body, and they budded repeatedly.
CDC34 has already been shown to code for ubiquitin conjugating enzyme
E2 (Ubc3). Therefore, degradation of some specific target proteins has beensuggested to be essential for the initiation of DNA replication. One of thetarget proteins was shown to be Sic1 which functions as an inhibitor of B-type
cyclin dependent kinase activity. Deletion of SICl in cdc34 ts strain allowed
the cells to enter the S phase at its restriction temperature. However, they*

Graduate School of the University of Tokyo.

I.

MOLECULAR GENETICS

35

failed to undergo nuclear division and accumulated as large budded cells with
2N DNA content. These evidences suggested that Cdc34 regulated not only
initiation of DNA replication by promoting the degradation of Sic I, but also
nuclear division. However, the function of Cdc34 on nuclear division is notknown. We are trying to identify the target proteins to be ubiquitinated.
( I) Detection of target proteins of Cdc34 by in vitro system
Cdc34 was expressed and purified from E. coli. First, the protein was
mixed with labeled ubiquitin and crude yeast extract prepared from the G I
phase of the cell cycle. However, detection of ubiquitinated protein by Cdc
34 was not possible. Then, we prepared the extract from yeast nuclei. Using
this nuclear extract, we detected one major signal (ubiquitinated histone) and
one faint signal (a protein about 65 kDa). As histone is not an in vivo
substrate, we are trying to deplete histone from the nuclear extract.
(2) Isolation of suppressor sensitive mutants of the cdc34 strain
We deleted SICI from cdc34ls strain. As reported, this strain failed toundergo nuclear division, and were arrested with a large bud. Several
suppressor mutants were isolated and cloning of the revertant genes is under
way.
Cell Cycle Regulation by the Ubiquitin System in Eukaryote
Hiroaki SEINO, Tatsuo YAGURA *, Fumiaki YAMAO and Takeshi SENO

Ubiquitin is activated by the ubiquitin-activating enzyme, EI, and ubiquitin is transferred from the E I enzyme to one member of a family of isozymes
called ubiquitin-conjugating enzyme E2, which catalyzes the conjugation ofubiquitin to specific target proteins. There is increasing evidence that each E
2 enzyme has a specific target protein(s). Detailed analysis of pleiotropic
phenotypes of mutant strains of mammalian cells which have a mutation on
the structure gene coding the EI enzyme suggest that the ubiquitin system isinvolved not only in G2jM phase regulation, but also in G I and S phaseregulation. To identify the ubiquitin pathway(s) involved in each phase of
the cell cycle, we examined ubiquitin transfer from EI to E2 in vitro using
extracts of various temperature-sensitive EI mutants isolated from the mouse
cell line, FM3A. Ubiquitin transfer to an E2 protein of approximately 35
kDa was found to be reduced specifically in the extract of the mutant cell line,* Department of Biology, Faculty of Science, Kansei Gakuin University, Nishinomiya.

36

tsFS20, which was predominantly arrested in the S phase at a restrictive
temperature (39°C). Furthermore, the activity of this £2 protein was foundto increase during the S phase in the extract of wild-type cells. These results
suggest that this 35 kDa £2 protein is involved in progression through the Sphase. We purified this protein from cell extract by using affinity column
chromatography and deduced a partial amino acid sequence. Cloning of acDNA encoding this protein and characterization of its properties are underway.
Crystallographic Studies of Proteins Involved in DNA Repair
Kousuke MORIKAWA

In view of short term survival, DNA must be chemically stable in order tomaintain a genetic record, even though the evolution of organisms requires
some alteration of genes. Adequate stability of genetic records on DNA ismaintained not only through an extremely accurate mechanism for copying
DNA sequences but also through precise and sophisticated mechanisms forrepairing the many accidental lesions in DNA that occur spontaneously
within cells.
By making a rough classification, the mechanisms of DNA repair can bedivided into two groups, recombinational repairs and excision repairs. We
have determined the crystal structures of two key enzymes which are involved in these two DNA repair mechanisms. The structure of the enzyme
involved in recombinational repair has been determined in a DNA-free state,while that for excision repair has been analyzed in a complex state with a
DNA substrate duplex containing a pyrimidine dimer (PD). These structures, both refined to atomic resolution, reveal how the respective enzymes
interact with a key intermediate in recombinational repair and with damaged
bases to catalyze DNA excision repair.
1. Holliday junction resolvase from E. coli (RuvC protein).
In all organisms, homologous recombination is a crucial process not only
for generation of genetic diversity but also for repair of damaged chromosomes. The Holliday junction is a universal intermediate produced in theprocess of homologous DNA recombination. We elucidated the crystal
structure of the RuvC Holliday junction resolvase (19 kd) from E. coli.
Together with results from extensive mutational analyses, the refined struc-

I.

MOLECULAR GENETICS

37

ture at 2.5 A reveals that the catalytic center, comprising four acidic residues,
lies at the bottom of a cleft that nicely fits a DNA duplex (Ariyoshi, M. et al.
(1994) Cell 78, 1063-1072). The structural features of the dimeric protein,with a 30 A spacing between the two catalytic centers, provide a substantiallydefined image of the Holliday junction architecture. Thus, based on thedocking examination of the RuvC protein with DNA, a new model of theHolliday junction was proposed. This model, which consists of two antiparallel quasicontinuous DNA duplexes, is similar to the stacked X structure(vin Kitzing, E. et al. (1990) Nucl. Acids Res. 18,2671-2673) but has longer
extended linkers with at least two nucleotides. The folding topology in thevicinity of the catalytic site exhibits a striking similarity to that of RNaseHI
from E. coli. More recently, the RNaseH-like fold also has been found in
HIV integrase (Dyda, F. et al. (1994) Science 266, 1981-1986), suggestingthat it forms a ubiquitous and important super family (Yang, W. and Steitz,
T. A. (1995) Structure 3, 131-134).
2. DNA excision repair enzyme in a complex with a DNA substrate
T4 endonuclease V (16kd) is a pyrimidine dimer specific excision repair
enzyme encoded by the bacteriophage T4 gene, and it catalyzes the firstreaction step in the excision repair pathway for a pyrimidine dimer. This
enzyme possesses two distinct catalytic activities: a pyrimidine dimer (PD)glycosylase and an apurinic-apyrimidinic lyase.
We determined the crystal structure of an active site mutant enzyme in acomplex with a DNA substrate containing a thymidine dimer at 2.75 A
resolution. This enzyme contains GIn in place of Glu23, which is a catalytic
residue for glycosylase reactions (Morikawa, K. et al. (1992) Science 256,523-526). The abolishment of glycosylase activity in crystallization drops
allowed us to obtain crystals of the enzyme complexed with the substrate.
The atomic model of the complex has revealed the detailed catalytic mechanism and the major factors, by which the enzyme recognizes PD specifically.
The bound DNA adopts a unique structure which is sharply kinked at thecentral dimer position with a 60° inclination. Thus, the DNA duplex isdivided to two regions, both of which retain the DNA-B form. The adenine
base complementary to the 5' side of the dimer, is completely flipped out fromthe B-DNA duplex and trapped into a cavity on the protein surface. This
striking local distortion disrupts the base pair, whereas the adjacent base pairwithin the dimer can be retained in spite of its substantial deformation. A
shortened phosphodiester bond distance at the dimer site is recognized by38four basic residues. These structural features implicate a general mechanismof how damaged DNA is recognized by repair enzymes.
Kinetic Study on Transcription Using Immobilized Operons~
Branched pathway in early elongation
Tomoko KUBORI and Nobuo SHIMAMOTO

In transcription experiments in vitro a template DNA fragment was fixed toacrylamide beads at the tight binding between avidin and biotin. The
immobilized DNA was a good template in transcription by E. coli RNA
polymerase. This technique enabled us to separate transcription complexes
from free components with brief centrifugation. In addition, dead-end
complexes which lost elongation activity can be assayed by rapidly diluting areaction mixture with a new substrate solution. We studied the relationships
between active transcription complexes, dead end complexes, and abortive
release.
In the E. coli transcription system, we found abortive release is greatly
activated in the presence of polymerase in excess of a promoter. Whentranslocation of a polymerase on DNA is inhibited by another preceding
molecule stuck at 32 nt from a promoter, a misincorporation at 6 nt was
found in addition to further enhancement of abortive release. The misincorporated transcripts were never elongated into long RNA even when thestacked molecule was removed. Thus misincorporation was checked in theearly steps in elongation.
The immobilized DNA was preincubated with RNA polymerase holoenzyme and packed in a 5 f11 column. Reactions were started by eluting thecolumn with a buffer containing substrates. The kinetics of releases ofabortive transcripts and the sigma factor were thus studied.
Single-Molecule Dynamics of Transcription Using Immobilized Operons:
Sliding of P. putida CamR Protein on DNA
Hiroyuki KABATA and Nobuo SHIMAMOTO

The sliding motion of RNA polymerase along DNA during promoter
search has proved by direct visualization of single molecules of the enzyme.
To address whether sliding motion is common to other DNA binding

I.

MOLECULAR GENETICS

39

proteins, we applied similar single molecule dynamics to a bacterial repressor
protein, P. putida CamR, which is a homodimer molecule unable to relocateby the intersegment transfer mechanism, another relocation mechanism
requiring two DNA binding sites. The protein was observed to slide along
DNA, and the movement is common to the proteins so far examined. In theabsence of its inducer, d-camphor, CamR molecules were trapped at threepositions on a A DNA, one was its cognate operator cloned on A DNA, andthe other two were likely to be regions homologous to the operator. The
homologous regions turned out to be 400 and 4,000 times weaker bindingsites than the cognate fragments by a gel-shift assay. But CamR stayed onthem as long as the cognate site by the direct visualization. Another binding
assay using an affinity column immobilizing CamR protein showed a result
similar to the direct visualization, indicating the limitation of the gel-shift
assay.
Addition of the inducer, d-camphor, significantly decreased trapped molecules, but the frequency of sliding increased. The result showed that theinducer destabilized specific interactions but not those for sliding. Theinteraction in the specific complex is different from that during sliding.
Functional and Structural Study of E. coli Single-Stranded
DNA Binding Protein
Hiroki NAGAI, and Nobuo SHIMAMOTO

E. coli Single-Stranded DNA Binding Protein (SSB) is a 19 kda protein
and essential to replication and recombination. In physiological conditions,
it exists as a tetramer and binds to both single-stranded DNA and agroup ofmRNAs which have a homologous RNA element, termed SSB box. Both of
the bindings are cooperative and the protein molecules form clusters on such
nucleic acid molecules. These bindings suggest a coupling between replication and translation through the exchange of SSB between replication forks
and mRNAs. Because the translation initiation region of the ssb genecontains three putative SSB boxes, it is possible that the expression of ssb geneis regulated autogenously. We investigated the expression from ssb-IacZ
fusion genes in cells overproducing or depleting SSB protein. The results
showed, however, that cellular amounts of SSB protein hardly affect theexpression of ssb-IacZ fusion genes, suggesting that autogenous regulation of

40

the ssb gene expression through putative SSB boxes is unlikely.
In vitro Transcriptional Analysis of the Bacillus subtilis Sporulation Gene
Masaya FUJITA and Yoshito SADAIEInterconversion of transcription apparatus with different promoter selectivity among RNA polymerases of a single species is considered to be themajor mechanism of global control of transcription in bacteria. At least fivesporulation specific sigma factors have been identified in Bacillus subtilis atpresent. Replacement of the sigma subunit leading to the generation ofmultiple forms of the holo enzyme and reversible interaction of the RNA
polymerase with one of the transcription factors are the main determinant ofregulation of sporulation gene expression.
In this study, we constructed in vitro transcription system with EaA, EaE,and EaH. Under our standard reaction conditions, the order of promoter
strength of sporulation genes was determined. Several sporulation genes wereanalyzed with this system in the presence or absence of some transcription
factors, such as SpolIID or SpoOA. For example, the abrB gene encoding atranscriptional regulator was transcribed with EaA and repressed by SpoOA.
Transcription of the spa VE gene was initiated with EaE and repressed by
SpolIID. The SpoIIID binding site was located around the - 10 region of
the spoVE promoter by DNase I footprint analysis.

41

II.

MICROBIAL GENETICS

Recognition of the Minus Strand DNA Replication Origin of theFilamentous Phage by E. coli RNA Polymerase
Zhi Wen GUAN, Nahoko HIGASHITANI, Atsushi HIGASHITANI,
and Kensuke HORIUCHI

Synthesis of the minus strand DNA of filamentous phage f1 is initiated bya RNA primer, which is synthesized by the host RNA polymerase holoenzyme. The minus strand origin contains two hairpin structures called [B]
and [C]. Footprinting and mutational analyses have indicated that theregion containing the two hairpins is specifically recognized by RNA polymerase in the presence of SSB (single-stranded DNA binding protein).
We previously constructed a number of deletion and insertion mutations ineach of the hairpins and base substitution mutations in hairpin [B], andanalyzed them for the origin function (Higashitani, N. et al. (1990) annual
rep. 41, 31-32, and Higashitani, N. et al. (1992) annual rep. 43, 34-35). In
the present study, we constructed and tested base substitutions in hairpin[C]. From all the results obtained, it appears that the origin DNA may takea secondary structure as shown in Fig. 1 and may function through amechanism similar to transcriptional initiation.
For most regions within the stem of hairpin [B], double-strandedness isrequired for active origin function. A specific nucleotide sequence is not
required except for a short region, which is about 35 bases away from thestart site of the primer RNA when the secondary structure of the origin is as
depicted in Fig. 1. Thus, this particular region may correspond to the - 35
region of transcriptional promoters. The base sequence of this region
includes 5'-TTTACG which can be found in lists of -35 regions of weakpromoters of E. coli. In hairpin [C], we constructed base substitutions in aregion that corresponds to the -10 region in relation to the primer RNA
start site. In the wild type origin, this region carries a sequence, 5'AAAAAA, in the upper strand as shown in Fig. 1, and 3' -TTTTT in thelower strand. When the 3'-TTTTT was replaced with 3'-GGGGG, the origin
was active. When 5'-AAAAAA was replaced with 5'-CCCCCC, the origin
was inactive. When both of the substitutions were introduced into a single

42base sequence:base-pairing :

-I

+
+

+

1-11-11-1

I-I__ "\ TT TT

IS]

TT

-110

1--1
(57 00)
1~1

H T~:~~~~~;~;~C:~~CACCTCGAccc?~~~~~3)(5650)

cT(5620)'T

Ie]

'AG'

c-<

C

T

C

C

T-<
T-<

G

Fig. 1.

(5750) G

I

I

5

3'

A possible secondary structure of hairpins [B] and [C] at the minus-strand
origin. The long arrow indicates primer RNA. The shaded nucleotides and thearrowheads represent regions that are protected from nuclease digestion andregions that become hypersensitive to nucleases, respectively, upon binding of the
RNA polymerase holoenzyme. Nucleotide numbers are shown in a manner
analogous to that generally used for transcriptional promoters. The Requirementof the DNA sequence and that of the base-paired structure for the originfunction are indicated by +, while - indicates the absence of the requirement.

origin, it was inactive. Thus, it is specific bases and not paired strands thatare required in this region for the origin function. In addition, it has beenreported that the nucleotide sequence at the region around - 10 to + 1 of atranscriptional promoter could be replaced by unpaired sequences that lead tothe formation of bubble-like structures without loss of RNA synthesizing
activity (Tripatara and deHaseth (1993) J. Mol. Bioi. 233,349-358).
These results suggest structural and functional similarity between theminus strand origin and transcriptional promoters. The recognition mechanism of the RNA polymerase holoenzyme at the minus strand origin may berelated to that in transcriptional promoters. Then, the recognition mechanism of the ~ 10 region by sigma (a) factor may involve a single-stranded
DNA structure with specific base sequences.

II.

MICROBIAL GENETICS43

The psv Gene Coding for Penicillin-Binding Protein 7 of Escherichia coli
Hiroshi HARA, Noriko ABE*, Masayo NAKAKOHJI*,
Yukinobu NISHIMURA*, and Kensuke HORIUCHI

We identified the gene encoding penicillin-binding protein (PBP) 7 of E,
coli, a peptidoglycan endopeptidase, in the course of our study on periplasmic
protease Pre, which was originally found to cleave the C-terminal II residues
from the precursor form of PBP3. A tiprc mutant lacking this proteaseshows thermosensitive growth at low osmolarity. Its suppressor (spr) mutations also caused thermosensitivity at low osmolarity when placed in the prc +background. They were all located around fruA at 48 min on the chromosome map, not in the gene for PBP3. A gene that could correct thethermosensitive defect of an spr mutant was cloned from the ordered cosmid
library, and the gene turned out to be a multicopy suppressor of spr. The
cloned gene and the spr gene were mapped about I min apart from each other.
Analysis of the sequence of this multicopy suppressor gene revealed theexistence of three motifs conserved among PBPs and ,B-lactamases in the geneproduct. [14C]penicillin-binding experiments showed it was a PBP of about
31 kDa that had high affinity to N-formimidoylthienamycin and was converted to a PBP of about 30 kDa by protease OmpT. The product was thus
identified as PBP7, and the gene designated as psv. PBP7 seemed to be
degraded by protease Pre, because the band of [14C]penicilloyl-PBP7 wasmore intense in a tiprc mutant than in the wild type. Disruption of the psv
gene on the chromosome through the insertion of a drug resistance genecaused no apparent defect in cell division and growth.
A Cell Division Inhibitor SulA of Escherichia coli Directly Interacts
with FtsZ through GTP Hydrolysis
Atsushi HIGASHITANI and Kensuke HORIUCHI

The FtsZ protein plays a central role in bacterial cell division and is one of
the earliest factors involved in septum synthesis (Lutkenhaus, J. (1990)
Trends Genet. 6, 22-25). It forms a ring-like structure at the division site
prior to invagination of the cell wall (Bi, E. and Lutkenhaus, J. (1991)
Nature 354, 161-164). The purified FtsZ protein displays binding activities*

Department of Biomolecular Sciences, Faculty of Science, Toho University, Funabashi.

44

for GTP and GDP, and GTPase activity (RayChaudhuri, D. and Park, J. T.
(1992) Nature 359, 251-254). The putative binding domain for GTP
contains an amino acid sequence that is highly conserved among eukaryotic
tubulins (de Boer, P. et al. (1992) Nature 359, 254-256). Like tubulins, FtsZ
assembles upon incubation with GTP in vitro into a filamentous structure(Mukherjee, A. and Lutkenhaus, J. (1994) J. Bacterial. 176,2754-2758) that
may be comparable to the ring-like structure formed in vivo.
DNA damage in bacteria results in induction of at least 15 genes named
SOS genes, which include those involved in DNA repair (Walker, G. C.
(1987) in E. coli and S. typhimurium, ASM 1346-1357). One of these genes,
sulA, is a cell-division inhibitor and prevents premature segregation of thedamaged DNA into daughter cells during the DNA repair process (Gottesman, S. et al. (1981) J. Bacterial. 148,265-273). Genetic evidence suggeststhat the target of the SulA protein is FtsZ, because a class of mutations in
ftsZ (Rsa, sulB, or sfiB) which confer resistance to overproduction of SulA
has been isolated (Bi, E. and Lutkenhaus, J. (1990) J. Bacterial. 172, 56025610). In a recent study, induction of SulA blocked the formation of thering-like structure of FtsZ (Bi, E. and Lutkenhaus, J. (1993) J. Bacterial.
175,1118-1125). On the other hand, it has not been determined whether
FtsZ and SulA interact directly with each other, and if they do, whether theinteraction requires any other factor(s). In this study, we show directinteraction between purified FtsZ and SulA in vitro.
Direct interaction was examined in vitro using SuIA, which was fused tothe maltose binding protein (MBP-SuIA; Sonezaki, S. et al. (1995) Appl.
Microbial. Biotechnol. 43, 304-309), and purified FtsZ. Since MBP-SuIA
binds to amylose resin and is eluted from it with 10 mM maltose, it isexpected that FtsZ can be co-eluted with MBP-SuIA if a stable complex isformed between the two proteins prior to application to the resin. During
incubation at 37°C for 30min, FtsZ did form a complex with MBP-SuIA.
When MBP-SuIA was omitted, or MBP-SuIA was replaced by MBP, FtsZ
was not detected in the eluate. These results indicate that a complex wasformed by direct interaction of FtsZ and SuIA.
In order to determine if any co-factor(s) are required for complex formation, the effects of Mg and Ca ions, and various nucleotides on complex
formation were tested. Complex formation required both GTP and Mg ions.
Ca ions could not be substituted for Mg ions. Various nucleotides, ATP,
CTP, UTP, ADP, GOP, and GTPrS all failed as substitutes for GTP.

II.

MICROBIAL GENETICS

45

Complex formation proceeded progressively with time until the reaction wascompleted after 20 min of incubation at 37°C. At 20°C for 20 min, complex
formation was about 13 % complete, and less than 3 % complete at 4°C. A
good correlation between complex formation and GTP hydrolysis in each
rection was observed. These results suggest that the complex formation of
FtsZ and SulA requires GTP hydrolysis.
GTPase activity was only dependent upon FtsZ, and not on SuIA. The
GTPase activity of FtsZ was not affected by the addition of MBP-SuIA.
Furthermore, specific GTPase activity of the complex that was eluted fromamylose resin was identical to that of FtsZ alone. These results indicate that
the GTPase activity of FtsZ is not affected by formation of the complex withSuIA.
The amount of FtsZ found in the complex was proportional to the amountof FtsZ added to the reaction, and was saturated when the molar ratio of
FtsZ to MBP-SuIA reached about one to one. This suggests that the complex
was formed in a molar ratio of approximately one to one of the two proteins.
It is likely that the complex formation we reported here reflects the in vivo
mechanism by which SulA inhibits cell division.
Isolation and Characterization of Mutations that Suppress the Effectof a Cell-Division Inhibitor SulA in Escherichia coli
Atsushi HIGASHITANI and Kensuke HORIUCHI

The sulA gene of Escherichia coli codes for a cell division inhibitor whichis induced upon SOS response. In order to determine the interaction between
SulA and other proteins, we isolated suppressor mutants that survive overproduction of SulA (sso phenotype). We have constructed a plasmid, pSulA
5, which carries the wild-type sulA gene under lacUV5 promoter control(Higashitani, A et al. (1995) Biochem. Biophys. Res. Comm. 209, 198-204).
E. coli cells harboring this plasmid fail to grow in the presence of IPTG.
Dominant mutants that were able to grow on IPTG-containing plates arose
spontaneously at a frequency of about 1O~6. Twelve such mutants wereindependently isolated and named sso mutants. The chromosomal positionsof the mutations were mapped by a method previously reported (Higashitani,
A. et al. (1994) Nucl. Acids Res. 22, 2426-2427), and the results indicatedthat they could be classified into two alleles,pcnB (class I) andftsZ (class II).

46

The pcnB gene has been reported to be involved in the copy number control
of ColE I-related plasmids (Liu, J. and Parkinson, J. S. (1989) J. Bacterial.
171, 1254-1261). In the class I sso mutants, the copy number of plasmid
pSulA5 was about 10% of that of wild-type cells. Thus, the level of the SulA
protein in the presence of IPTG may not be so high in these mutants, and thismay explain why the mutants can grow on IPTG plates. TheftsZ gene playsa central role in cell division (for review, Lutkenhaus, J. (1990) Trends
Genet. 6, 22-25), and the class II mutants are probably similar to previously
isolated mutants in ftsZ (Rsa, sulB, or sfiB) which confer resistance tooverproduction of SulA. In our recent studies, direct interaction between the
SulA protein and the FtsZ protein was found in vitro. These results suggestthat FtsZ may be the only target of SulA, and we may not be able to obtainother suppressor mutants that would bypass the loss of function of FtsZ.
Molecular Mechanisms that Determine the Timing of Cell
Division in Escherichia coli K12
Akiko NISHIMURA

To elucidate the molecular mechanisms of the timing of cell division in E.
coli K 12, we isolated novel mutants, cfcA, B, C, D, E, and F, (Nishimura, A.
(1989) Mol. Gen. Genet. 215, 286-293) in which DNA replication and cell
division are uncoupled. These mutations partially suppressed both divisionarrest and lethality induced by treatments that inhibit DNA replication.
Under permissive conditions for DNA replication, the cfc mutants divide 1.3
to 1.5 times more frequently per round of DNA replication. Complementation tests and sequencing analyses showed that the cfcA gene encodes the asubunit of glycine tRNA synthetase. From genetic and physiologic analysesof the cfcA] mutant, the cfcA gene was found to be specifically involved in theregulation of the cell cycle by a pathway different from stringent response or
SOS induction. The locus of the cfcB] mutation was found in the apaH gene
mapped at 1 min of the E. coli genetic map. The ApaH gene encodes
diadenosine tetraphosphate (AppppA) hydrolase, and apaH mutants
showed a :::0; 16 fold increase in the cellular level of AppppA. AppppA may be
a pleiotropic signal for cell growth and DNA replication (review: Zamecnik,
P. (1983) Anal. Biochem. 134, 1-10). AppppA is formed in vitro byaminoacyl tRNA synthetases in a reaction in which an enzyme-boundII.

MICROBIAL GENETICS

47

aminoacyladenylate intermediate donates AMP to ATP (Goerlich, O. et. at.
(1982) Eur. J. Biochem. 126, 135-142). However, so far, neither aminoacyltRNA synthetase mutants, inhibitory treatments of aminoacylation, nor
structural mutants of tRNA has been known to overproduce AppppA in vivo.
The cfcAllglyS mutation was suppressed by a high copy number plasmidcarrying the apaH+ gene. HPLC analyses showed that the intracellular levelof AppppA in cfcAl and cfcBl cells was about lOO-times higher than in cfc+
cells. This suggests that cfcAl, the mutant of glycine tRNA synthetase, could
synthesize AppppA in vivo and that the AppppA nucleotide is involved in thedetermination of the timing of cell division in a manner that couples cell
division to DNA replication. We are currently analyzing other cfc mutants
to search for targets of AppppA.
A New Gene Which Couples Cell Growth and Cell Division in
Escherichia coli K-12
Hideki UKAI, Naoko WATANABE, and Akiko NISHIMURA

Our research focused on the regulatory mechanisms of cell division in E.
coli. We hypothesized that a cell has mechanisms to coordinate cell growthwith cell division. Recently we identified a novel gene, sun U, which uncouples cell division and cell growth. Multiple copies of the sun U gene partiallysuppressed the decline of cell growth, but not the defect of cell division, of
ftsE, cpcA58, andftsU349 mutants. FtsE gene has the concensus sequence for
the ABC-transporter. The cpcA58 mutation stopped cell division at 42°C in
media of pH 5.8 containing cAMP. The phenotype was more marked in thepresence of CaH . The cpcA58 mutant which harbors the tetracycline
resistance gene shows less resistance than cpcA + mutants. This indicates thatthe cpcA58 mutation causes a defect in the antitransport system of tetracycline resistance. The FtsU gene has a 'gearbox' promoter, and the mutation
stopped cell growth and cell division at 42°C showing high ploidy. A possible
explanation is that all three mutations may be related to an ion pump, and thesun U gene may couple the ion pump and cell division.

48

The Role of the SecA protein of Bacillus subtilis in Sporulation
Yoshito SADAIE and Masaya FUJITA

We investigated the expression and role of the secA gene of Bacillus subtilis
during early stages of sporulation as it plays a crucial role in sporulation andsporulation associated events as well as in protein secretion and cell septation.
Its RNA transcript and protein product were measured with Sl nucleasemapping and immunoblotting, while a temperature sensitive secA mutation
was used to examine the role of the SecA in the expression of the component
genes of phosphorelay and sigma cascade. The start site of the synthesis ofmRNA was 110 bases upstream of the initiation codon for the secA gene,preceeded by - 10 and - 35 sequences atypical of if' promoters. The amountof secA mRNA decreased upon sporulation, while a constant amount of SecA
protein was recovered throughout early sporulation stages after a temporaldecrease around TO.5 (30 min after the end of the logarithmic phase of
growth). A temperature shift experiment revealed an arrest of sporulation by
the temperature-sensitive secA341 (ts) mutation at 37°C up to the T3 stage,
and that the transcriptional expression of abrB, spoOH, kinA, spoOA, spoIIG,
and spoIIIG genes monitored with lacZ activity was blocked by the secA341
mutation at 37°C except for abrB and spoOH genes. Since these blocked
genes have promoters depending on a H and/or phosphorylated SpoOA, theseresults suggest the SecA protein is necessary for the functioning of the
product of the spoOH gene.

49

III.

MAMMALIAN GENETICS

Genetical Analysis of rim2 Coat Color Mutation
Tomoko SAGAI, Sigeharu WAKANA*I, Kazuo MORIWAKI
and Toshihiko SHIROISHI

rim2 is a single recessive mutant that arose spontaneously in one of therecombinants, BlO.A(R201) strain, which has a recombinational breakpointat the LMP-2 hotspot. The mutant is characterized by diluted pigmentationof the entire body hair, ears and tail. In particular, the amount of pigment in
the eyes at birth is markedly reduced from that of the original wild typestrain. In addition to hypo-pigmentation, eye defects such as cataracts,
eyelessness and small eyes are frequently observed. To search for the rim2
mutant gene, we carried out chromosomal mapping using micro-satellite
markers. Backcross mating with MSM strain revealed that the rim2 locus
was tightly linked with several microsatellite markers located on the chromosome 13.
At the near side of the rim2 loci, an old coat color mutation, pearl, has been
mapped. A mating cross of rim2 and pearl mutants showed that they areallelic. It is known that the pearl and many other hypo-pigmented mousemutants cause a platelet storage pool deficiency (SPD). In humans, SPD isa recessive autosomal disease associated with hemorrhagic symptoms andphenotypically heterogeneous syndromes. Chediak-Higashi Syndrome(CHS)and Hermansky-Pudlak Syndrome(HPS) are the most intensely studied ofthese diseases. The rim2 and the hypopigment mutants are expected to serve
as models for heterogeneous human SPDs.
On the basis of a linkage analysis of 1550 backcross mice, we obtained
several YAC clones including the marker sequences tightly linked to the rim
2 mutation. Construction of YAC contig is in progress.

*'

Central Institute for Experimental Animal, Kawasaki.

50

Meiotic Recombination at the Lmp2 Hotspot in the Mouse
Major Histocompatibility Complex Is Tolerant of
Substitutional Base Mismatches
Masayasu YOSHINO, Tomoko SAGAI and Toshihiko SHIROISHI

Recombination is widely considered to linearly depend on the length of thehomologous sequences. It was reported that II % mismatch decreased therate of phage-plasmid recombination 240-fold. It was found that two single
nucleotide mismatches that reduce the longest uninterrupted stretch ofhomology from 232 bp to 134 bp resulted in a 20-fold reduction in geneconversion of plasmid DNA in mouse L cells.
In this study we were interested in determining whether or not a very highdegree of sequence identity between homologous chromosomes enhances
recombinational events even in mouse meiosis. We made a cross between B
lO.A(R209) strain with the MHC haplotype of Kwm 7-Ab k--cyp21 d_D d and B
lO.BR strain with the MHC haplotype of K k-Ab k--cyp21 L Dk, both of whichhave the serologically identical k haplotype from the Lmp2 hotspot to therecombinational breakpoint of the a haplotype located between the Int3 andCyp21 genes in the major histocompatibility complex (MHC) region on
mouse chromosome 17. The physical length of this serologically identicalsegment is roughly estimated to be about 300kb. We screened the K-D
recombinants by cytotoxicity test using anti_Kwm7 and anti-D d antibodies. The
resultant K-D recombinants were genotyped by RFLP analysis using theLmp2 hotspot DNA as a probe and the Cyp21 probe and the recombinationalfrequency around the hotspot was estimated.
We obtained twenty-two K-D recombinants from 749 offspring and eighteen breakpoints of these were confined to the chromosomal segment spanning from the polymorphic DraI site which is located 1.2 kb proximal to the
MT-consensus sequence at the Lmp2 hotspot to the polymorphic EcoR V site
detected by the Cyp21 probe. Nine of the 18 Lmp2 hotspot-Cyp21 recombinational breakpoints were proximal to the polymorphic MspI site at the 1174
nucleotide position and they were confined to the 2.4 kb DNA segment
overlapping the Lmp2 hotspot. The remaining nine of the 18 Lmp2 hotspotCyp21 recombinational breakpoints were distal to the polymorphic MspI siteat the 1174 and they were confined to the 300 kb serologically identical region
spanning from the hotspot. Since there was no polymorphic restriction site in
the serologically identical region from the breakpoint at the Lmp2 hotspot toIII.

MAMMALIAN GENETICS51

the breakpoint at the Int3--cyp21 interval, the distal end of the breakpoints
could not be determined in this chromosomal segment. The remaining four
breakpoints of the 22 K-D recombinants were located in the Cyp21-Tnfa
interval.
Previous fine mapping of the recombinational breakpoints at the Lmp2
hotspot from the crosses of BlO.A (MHC type KLAb k--cyp21 c D d) andBlO.MOL-SGR (MHC type Kwm7-Abwm7--cyp21 wm7_Dwm7), and BlO.BR(MHC typeKLAbLcyp21 L D k ) and BlO.A(R218) (MHC type Kwm7_Ab wmL
Cyp21awILDd) revealed that the breakpoints were equally distributed inregions both proximal and distal to the MspI site (Shiroishi et a/., 1991). In
the cross of BlO.BR (K k-AbLcyp21 L D k ) and BlO.A(R209) (Kwm7_Ab kCyp21d_D d), the breakpoints were again equally divided into the two segments. The five sites of single base nucleotide mismatch were located in the(BlO.AXBlO.MOL-SGR)F 1 at the 1243,1261,1377,1388 and 1649 nucleotide positions, while these sites are matched and identity spans along 300 kb
distal to the hotspot in a cross of BlO.BR and BlO.A(R209). Therefore,
these results indicate that the distribution of the breakpoints was not biased
toward the more homologous region distal to the MspI site in the middle ofthe hotspot and that recombination was not elevated in the same region.
The recombinational frequency in the Lmp2 hotspot and Cyp21 interval in
the [B 1O.BR X B1O.A(R209)] F I female mice in which there is identity over
300 kb around the Lmp2 hotspot was 3.2% and this is almost equal to the
2.8% frequency at the Lmp2 hotspot in the [BlO X BlO.A(R209) ]F 1 female
mice. In males, the frequency in the Lmp2 hotspot and Cyp21 interval was
1.5% in the [BlO.BR X BlO.A(R209)]F 1 and this is almost equal to the 2.2%
frequency at the Lmp2 hotspot in the [BlOXBlO.A(R209)]F 1• The resultswith both sexes indicate that the long identity of the chromosomal segment
did not enhance recombination around the Lmp2 hotspot during meiosis.
The rate of mitotic recombination in mammalian cells has been thought toprimarily depend on the amount of uninterrupted homology. In the present
study, no enhancing effect on meiotic recombination around the hotspot wasobserved in crosses, in which two homologous chromosomes have longidentity spanning 300 kb, suggesting that meiotic recombination around thehotspot in the mouse MHC is tolerant of substitutional base mismatches.
Since mitotic and meiotic recombination have been thought to be mechanistically distinct in yeast, the present results may reflect some differences
between mitosis and meiosis.

52

Genetic Analysis of Mouse Mutants Affecting Limb Pattern Formation
Hiroshi MASUYA, Tomoko SAGAI, Kazuo MORIWAKI and Toshihiko SHIROISHI

Positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at theposterior margin. It is postulated that Sonic hedghog (Shh), a homologue ofthe segment polarity gene hedgehog in Drosophila, mediates ZPA activity.
The downstream activity of the ZPA is thought to be performed by genes
such as Hoxd and bone morphogenic protein-2 (Bmp-2), which may direct
positional specification during pattern formation in vertebrate limbs. It isalso known that some members of the FGF family expressed in the AER canfunctionally replace the AER. It has been evident for a long time that ZPA
stimulates signaling of the AER, while the posterior portion of the AER
maintains functional ZPA in the posterior mesoderm. In vertebrate tetrapods, there are many congenital abnormalities affecting the pattern formationof limbs. Preaxial polydactyly which causes extra digit(s) on the side of digit
I is one of the major abnormalities.
In this study, we carried out a morphological analysis of mouse mutants
with preaxial polydactyly. It revealed that a new mouse mutant, Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes(Hx) and Extra toes (Xt) , exhibit mirror-image duplication of the skeletal
pattern of the digits. In situ hybridization of the embryos of these mutants
indicated ectopic expression of Shh and fibroblast growth factor-4 (Fgf-4)
genes at the anterior margin of limb buds. The new mutation, Rim4, wasmapped to chromosome 6 with linkage to HoxA but was segregated from
HoxA. No linkage to other known polydactylous mutations was detected. In
this mutant, ectopic expression of Hoxd-ll gene which is thought to be thedownstream of ZPA was also observed at the anterior margin of the limb
buds. All results demonstrate the presence of an additional ZPA at theanterior margin of limb buds of these mutants. Thus, it appeared that there
are multiple endogenous genes that regulate the spatial localization of the
ZPA in the developing mouse limb bud.
When C57BL/lOJ-Rim4/+ mice were crossed with other laboratory
strains, the frequency of mice with the Rim4 phenotype was variable,
depending on the strain with which the Rim4 was crossed. The penetrance
was incomplete in heterozygotes, ranging from 0% in crosses with MSM
strain to almost 100% in crosses with NZB strain. The difference inIII.

MAMMALIAN GENETICS

53

penetrance indicates that the Rim4 phenotype is controlled by allelic forms of
the Rim4 locus or other genes, which are not linked to the Rim4.
Linkage Analysis of the Skeleton Mouse Mutant, Tail short (Ts)Toshihiko SHIROISHI, Kikue UCHIDA, Peter KOOPMAN*l, Akihiko MITA,
Sigeharu WAKANA*2 and Kazuo MORIWAKI

Tail short (Ts) is an old skeletal mutant (Morgan, 1950). Heterozygotes
for Ts have shorter kinked tails and smaller bodies than normal mice. Otherskeletal abnormalities of various kinds scattered over the whole body werealso observed. Homozygous embryos die at about the time implantation of
their litter mates occurs. Abnormalities in homozygous mice can be traced
back to 3 1/2 days post coitum (p.c.) through several criteria including low cellnumber and absence of cavitation. All these indicated the indispensable
function of the Ts gene not only in skeletal development but also in the early
development of mice. The Ts gene was mapped to the teromeric region ofchromosome 11. It was reported that mutant mice fell short of the Mendelian expectation by 30-40% in Ts/ + X +/+ mating. This indicated thatsome fraction of mice heterozygous for Ts were lethal at the time of birth,
rendering genetic analysis difficult. We have started a fine linkage analysis ofTs, toward positional cloning of this gene. We conducted backcross mating
between Ts/+ and MSM inbred strain derived from Japanese wild mouse.
Mice heterozygous for the Ts from this cross were less defective andlitter-size was relatively large. Furthermore, penetrance of the Ts phenotype
was almost complete in this cross. In the last year, two candidate genes for
the Ts mutation were cloned and mapped to the syntenic region of humanchromosome 17q24.3-q25.1. Sox9, a Sry-related gene, was identified as a
candidate gene for human campomelic dysplasia (CMPDI) displaying features similar to Ts (Foster et ai., 1994). The M33 gene, a human homologue
for a polycomb gene which modifies expression of Hox genes, was mapped tothe same region of the human chromosome. To elucidate whether either
Sox9 or M33 is a candidate for the Ts mutant gene, we analyzed linkage of Ts
phenotype and these two genes on the Ts/MSM backcross panel. It appearedthat Ts was segregated from both Sox9 and M33, excluding these two genes*1*2

The University of Queensland, Australia.
Central Institute for Experimental Animal, Kawasaki.

54

as candidates for the Ts mutant gene. The order of the genes is; centromere-sox9-(3.8cM)-Ts-(l7.0cM)-M33. We are continuing to make a fine
linkage map around the Ts for positional cloning of this gene.
Gene Mapping Toward Positional Cloning of the Rim3 Gene
Hiroshi MASUYA, Shigeharu WAKANA*t, Tomoko SAGAI,
Kazuo MORIWAKI and Toshihiko SHIROISHI

Rim3 is a hairless mutant derived from an intra-MHC recombinant, whichshows hyperkeratinozation in the cornea and probably throughout the wholebody. In the present study, we extended gene mapping of the Rim3, using
crosses between C57BL/1OJ-Rim3 and MSM strains. As a result, the Rim3
gene was tightly linked to a microsatellite marker DII Mitl4. on chromosome
11. The Krt-l (Keratin typel) locus has been mapped to the same position.
It is known that Krt-l is a multi gene family consisting of more than 10 genes
related to each other. It is likely that one of the members of Krt-l, which isexpressed in the cornea, is affected by the Rim3 mutation. We carried out
PCR-screening of YAC library provided from Research Genetics, Alabama,
USA, using DllMitl4 primers, and identified a single positive clone. A
search for a candidate gene for Rim3 is now in progress using this YAC
clone.
Meiotic Recombination on DNase I-hypersensitive Sites in the
Mouse Major Histocompatibility Complex
Ken-ichi MIZUNO, Tomoko SAGAI, Kazuo MORIWAKI
and Toshihiko SHIROISHIIn murine major histocompatibility complex (MHC), meiotic recombination does not occur at random in an array of DNA. We can serologicallydetect meiotic recombinations in the MHC using antibodies for H2K and
H2D marker loci, which are physically separated by more than 1Mb of
distance. Almost all breakpoints of the recombinants are clustered within
limited regions. At present, four clusters of recombinational breakpoints in
MHC class II are known and the clusters are called hotspots. One of them
was identified at the downstream of the Lmp2 gene and is designated the*'

Cenlral Institute for Experimental Animal, Kawasaki.

III.

MAMMALIAN GENETICS

55

Lmp2 hotspot. The Lmp2 hotspot was mapped within a 2 kb segment of
DNA and meiotic recombination occurs at an extremely high frequency,
about two percent, in this hotspot. However the molecular nature which
determines the location of recombinational hotspots is not well understood.
It is also well known that hotspots exist during meiotic recombinations in
the lower eukaryote Saccharomyces cerevisiae and, moreover that transient
double-strand DNA breaks (DSBs) occur at these hotspots, leading to theinitiation of recombination. Detailed analysis of chromatin structure aroundthe hotspots using endonucleases, such as DNase I, indicated that the
distribution of DSBs overlaps with that of constitutive DNase I-hypersensitive sites (DHSSs). It seems likely that features of the chromatin structure,
which is established prior to meiosis, playa key role in determining the sites
at which recombination is initiated. It is conceivable that DSBs occur at sites
where nucleosomes are disrupted and the DNA is accessible to the enzymes
involved in the recombination. In this context, we have analyzed thechromatin structure around the hotspots in the mouse MHC. The results ofthe study indicated an absence of DHSSs within the hotspot. There wereclear DHSSs in a region proximal to the hotspot instead. Although we haveobtained the data indicating that features of the chromatin structure during
mouse meiosis do not playa critical role in determining the sites of recombinational hotspots, the contribution of the chromatin structure to meiotic
recombination is still controversial in the mouse.
If DHSSs determine the initiation sites of recombination but not theresolution sites of recombinations around the hotspots, it is possible that there
are gene conversions and recombinations in the vicinity of the DHSSs
observed in the region proximal to the hotspot. We attempted to examine
whether these events occur at the DHSSs around the hotspot in the mouse
MHC. BlO.A(R209) strain shows recombination at the hotspot during malemeiosis and BlO.MOL-SGR dose not. These strains were crossed with
C57BLjlOJ strains and the FI progeny were backcrossed with C57BLjlOJ
strain. Genomic DNA were prepared from the tails of the backcrossed
progeny (BC2) and were examined by Southern analysis and PCR. Now weare typing the mice for five loci (H2K, H2D, Lmp2 hotspot and, the proximaland distal flanking sites of a mouse meiosis specific DHSS located proximal to
the Lmp2 hotspot).

56

IV.

DEVELOPMENTAL GENETICS

Sexual Differentiation Potency of Multi-Potent Stem Cells in Hydra
Chiemi NISHIMIYA-FuJISAWA and Tsutomu SUGIYAMAInterstitial stem cells of hydra consist of at least two subpopulations. One
is the germline-restricted stem cells differentiating only into sperm or eggs
(Littlefield, 1985). The other is the multi-potent stem cells capable ofdifferentiating into both somatic (nerve, nematocyte, gland cell) and gametic
cells (probably through germline-restricted cells) (Bosch and David, 1987).
"Pseudo-epithelial hydra" contain only the former but not the latter cells.
The stem cells in these animals are all localized only in and above, but not
below the budding zone. Evidence exists which suggests that germlinerestricted cells are also absent below the budding zone in normal hydra.
Therefore, the foot tissue in normal hydra probably contains only multipotent but no germline-restricted cells. Under the assumption that this viewis correct, sexual differentiation potency of the stem cells present in the foottissue of normal hydra was examined.
The experiment was done using 5 male (nem-l, SSCom, nB-2, ms-l andSSE) and 3 female (nem-Iof-l, sse and SSB) strains of Hydra magnipapillala. Nem-l (male) had been female in the past. Strain nem-Iof-l (female)arose from nem-l (male) and SSeom (male) from sse (female) throughspontaneous sex reversal. Foot tissue was cut out from the 8 strains, allowed
to regenerate, and the regenerates were induced to differentiate sexually. Sex
phenotype was identical between the regenerates and their corresponding
parents for the 3 female and 2 of the 5 male strains (ms-l and SSE).
However, it was not identical for the remaining 3 male strains. The footregenerates of the 3 strains (nem-l, sse m and nB-2) all produced eggs
instead of sperm.
The results obtained suggest that multi-potent stem cells present in the foottissue of the 3 strains (nem-l, sse m and nB-2) differentiate into eggs(presumably via egg-restricted stem cells), although sexual phenotype is malein these strains. This observation can be interpreted as follows:
The 3 strains were originally females, and their multi-potent stem cells
differentiated into eggs via egg-restricted stem cells. In the past, sex reversal
0

0

IV.

DEVELOPMENTAL GENETICS

57

took place from female to male in these strains. This occurred at the level ofgermline-restricted cells through transdiffe~entiation from egg-restricted tosperm-restricted cells. This type of transdiff~rentiationwas observed to occurin pseudo-epithelial hydra containing the egg-restricted stem cells. Transdifferentiation in the germline-restricted cells reversed the sexual phenotypeof the 3 strains without altering the original potency of the multi-potent stem
cells to differentiate into eggs.
Hydra Regeneration from Recombined Ectodermal andEndodermal Tissue. I. Cell rearrangement
Yasuyuki KISHIMOTO, Motohide MURATE and Tsutomu SUGIYAMA

Hydra regeneration can occur from dissociated and reaggregated cells(Noda, 1971; Gierer et al. 1972). In the early stage of this process, a sphereis formed which consists of an ectodermal cell layer outside, an endodermal
cell layer inside, and an empty space in the center. This observation showsthat hydra ectodermal and endodermal epithelial cells together have theability to self-organize themselves into a two-layered epithelial sheet. In thisstudy, a new system was developed to examine the interaction and selforganization of the ectodermal and endodermal epithelial cells into the
two-layered epithelial sheet.
Procaine treatment (Epp et al. 1979) was used to separate the ectodermaland endodermal layers of Hydra magnipapillata (strain 105). In someexperiments the separated tissue was dissociated into single cells and thenreaggregated to produce ectodermal or endodermal cell aggregates. Whentwo pieces of tissue or aggregate derived from the two layers were placed indirect contact with each other, a firm adhesion was quickly establishedbetween them. This was followed by a rapid spreading of the ectodermal
epithelial cells as a thin layer over the endoderm as in "epiboly" in developing
embryos of fish, amphibians, etc. Spot-labeling with Dil was used to follow
cell rearrangement taking place in the epibolic process. On the contact
surface of the recombined ectoderm-endoderm, cells initially located insidemigrated to the surface to intercalate themselves among the cells originallypresent in the contact surface. Continuous cell intercalation occurring on
both sides of the contact surface produced a rapid increase of the contact
surface, eventually resulting in a complete covering of the entire endoderm by

58

a thin ectodermal layer in about a day. The structure produced in this way,
which closely resembled the sphere produced during regeneration from mixed
cell aggregate, later developed into a new hydra.
An extract prepared from hydra membrane fraction specifically inhibitedthe epiboly, but not the initial adhesion or later regeneration.
These observations suggest that the major driving force for the epiboly inhydra is the cell intercalation occurring at the contact surface.
The epiboly probably requires an exchange of signals between the ectodermal and endodermal cells. And the active factor in the membrane
extract may be involved in the signal exchange mechanism in some way.
Hydra Regeneration from Recombined Ectodermal andEndodermal Tissue. II. Electron Microscopic Study
Motohide MURATE, Yasuyuki KISHIMOTO, Tsutomu SUGIYAMA,
Hiromi TAKAHASHI-IwANAGA* and Toshihiko IWANAGA*

In the epibolic process in recombined hydra ectoderm-endoderm, extensive
cell rearrangement takes place near the contact surface of the two tissue(preceding abstract). Electron microscopy was used in this study to examine
structural changes that occur in cells and tissue at various stages of the
epiboly.
Soon after recombining, major structures of the ectoderm and endoderm
were similar to those originally present in normal hydra. One new feature,
however, was present at the contact surface between the two tissue. The
ectodermal epithelial cells extended onto the endodermal surface many
membranous or bulbous processes resembling lamellipodia in migrating fibroblast cells.
By 4-6 hours, some ectodermal epithelial cells moved onto, and formed athin layer on, the endoderm. At the advancing edge, these cells extended longprocesses which formed multi-layers on the endoderm. In the endoderm,many cells lost their typical epithelial characteristics, turning into a mass of
irregularly-shaped cells with large empty spaces between them. Large
vacuoles initially present were missing in these cells.
At 10-20 hours, the ectodermal and endodermal cells started to regaincuboidal or columnar shape. These cells, now containing large vacuoles,*

School of Medicine, Niigata University.

IV.

DEVELOPMENTAL GENETICS

59

were organized in layered structure, in characteristic 2 layers in some parts,
but also in multi-layers in others. The layer formation started at the initial
contact site, gradually moved to surrounding areas, and eventually covered
the entire tissue.
Studies reported in this and preceding abstracts have revealed two important differences in the behaviors of the ectodermal and endodermal epithelial
cells. One was flexibility of the overall tissue shape. The ectoderm was
flexible, changing from the initial small spherical shape to the final thin layer
covering the endoderm. In contrast, the endoderm made relatively muchsmaller change, from the initial rod-shape to a near sphere. This difference
may be produced by the differential adhesiveness of the two epithelial cell
types (Steinberg, 1970). The other was cell organization in the tissue. The
ectodermal cells maintained the epithelial sheet organization throughout. In
contrast, the organization was once lost and later produced again in theendodermal cells. Thus, the ectodermal cells are persistent in maintaining
tissue organization, but flexible in overall tissue shape. The endodermal cells
are opposite in both properties. These two contrasting characteristics in thetwo epithelia may play important roles in hydra morphogenesis.
Isolation of Cytoskeletal Molecules in Hydra by Epitope Selection
Masayuki HATTA, Masahiko SAGAGUCHl', Yoshihisa KOBAYAKAWA 2,
Osamu KOIZUMI 3 and Tsutomu SUGIYAMA

Marker molecules were isolated for fine description of hydra cell structuresand examination of their changes in morphogenesis, with emphasis on
isolating molecules associated with cell membrane or cytoskeletal structures.
Epitope selection was used for isolation. A cDNA expression library was
screened with antisera raised against an insoluble fraction of hydra homogenate. 200 clones were isolated. Each clone was grown individually on apetri-dish. The fusion-protein expressed by each clone was lifted onto amembrane, which was incubated with the antisera. The bound antibodies
were recovered from the membrane, and then used to examine localization of
the antigen in hydra cells and tissue using indirect FITC staining.
I2
3

Shinshu University.
Kyushu University.
Fukuoka Women's University.

60

Three clones were isolated. (l) Clone-4 is a homolog of ABP-280 (actin
binding protein, non-muscle filamin) initially found in erythrocytes. The
antibody stains cell-cell boundaries of the ectodermal cells, particularly in thearea of adherence and septate junctions. The stain disappears when cells are
dissociated, suggesting that the molecule is involved in maintaining thestability of cell adhesion apparatuses and epithelial integrity. (2) Clone-l isa myosin heavy chain homolog. The muscle processes of ectodermal epithelial cells are specifically stained. Stained fibers, relatively short in young
animals, become longer during growth. Staining becomes negative in regenerating tissue after head removal, and possibly also in presumptive bud tissue.
Fiber depolymerization (muscle process reorganization?) may occur in cells
ready to start morphogenesis. (3) The antibody for clone-95 specificallystains muscle processes of both ectodermal and endodermal epithelial cells.
Sequence analysis has not been completed for this clone.
(4) Another clone (clone-F2-1) was isolated using antisera raised against
small-size cells separated by elutriation. Its DNA sequence shows homology
to tropomyosin and myosin heavy chain. The antibody stains a fibrous
structure throughout the entire cytoplasm of developing stenotele and desmoneme nematoblasts. In maturation, the structure gradually moves to andgathers at the base of the nematocyst capsule, suggesting that this molecule
may play a role in the positioning and anchoring of the capsule within thenematocyte cytoplasm.
Screening is in progress to isolate more markers. Markers isolated will be
used for fine structural examination of hydra morphogenesis. Epitope
selection is a rapid and convenient approach for simultaneous isolation of
specific antibodies and cDNA clones for marker molecules.
Disappearance and Reappearance of Mesoglea in Hydra Tissue
During Regeneration after Head Removal
Hiroshi Shimizu and Michael J. Sarras, J r*

The mesoglea is a thick layer of extracellular matrix that exists between theectodermal and endodermal layers in hydra. Using monoclonal antibodies
raised against hydra mesoglea, changes occurring in the mesoglea in regenerating tissue after head removal were examined.
*1

University of Kansas Medical Center, KS, USA.

IV.

DEVELOPMENTAL GENETICS

61

A monoclonal antibody, mAb52, specifically recognizes the laminin BI
chain in hydra mesoglea. mAb39 specifically recognizes the central fibrouszone of mesoglea. Indirect FITC-staining using these 2 preparations was
employed to visualize the mesoglea in the regenerating tissue. Both antibodies gave similar results.
Soon after head removal, the mesoglea disappeared from the regenerating
tissue. One hour after amputation, no staining was observed in the tissue
near the amputation site using either of the two antibody preparations.
Mesoglea was present in the adjacent tissue. The border of the remaining
mesoglea was located 40-50 fJ.m from the amputation site. The staining
intensity was stronger at this site than in the rest of the tissue. Histologicalexamination by electron microscopy showed that the thickness of the mesoglea was approximately 5 fJ.m at the edge and 2-3 fJ.m in the rest of the tissue.
The mesoglea started to appear again in the regenerating tissue at about 24
hours after head removal. The staining intensity was weak initially, andbecame gradually stronger, recovering to almost the normal level in 4 days.
During this period, however, the distinct edge of high staining intensity
observed soon after amputation could still be recognized at the same position.
The mechanisms for the disappearance and reappearance of the mesoglea
in regenerating tissue are unclear at present. The disappearance of themesoglea can occur either through degradation at the site, or withdrawal ofthe mesoglea layer from the amputation site into a more basal part of thetissue. A strong staining intensity and thickening of the mesoglea at the edge
suggests that withdrawal may be responsible for the disappearance. The
reappearance of mesoglea could occur either through new synthesis orextension of the existing mesoglea. The former appears likely since strong
staining intensity remains at the same site during the recovery process.
Experiments are in progress to critically examine the mechanisms for disappearance and reappearance.

62

Hydra Peptide Project. I. Isolation, Structure Determination
and Chemical Synthesis
Yojiro MUNEOKAI, Toshio TAKAHASHI', Toshitaka Fujisawa, Hiroshi SHIMIZU,
Masayuki HATTA, Tsutomu SUGIYAMA, Osamu KOIZUMI 2, Thomas BOSCH 3,
Maria deHaro3, Jan LOHMANN3, Charles DAVID 3 and Hans BODE'

Morphogens are thought to play important roles in regulating development
in hydra. A few of them have been isolated and characterized in past studies.
In order to systematically isolate new hydra morphogens, a project based ona novel approach was started. It aims to isolate peptides which function as
signal molecules to regulate gene expression in hydra. The project consists ofthe following 5 parts: (I) Peptide isolation from hydra tissue, (2) selection of
peptides capable of altering gene expression in hydra, using Differential
Display PCR (Liang and Pardee, 1992), (3) structure determination of
peptides active in DD-PCR, (4) chemical synthesis of peptides, and (5)examination of biological functions of synthetic peptides. Part (1), (3) and(4) are reported here, the rest are reported in the following abstracts.
(1) Peptide isolation. Two batches of crude peptide preparations wereextracted from Hydra magnipapillata tissue, batch 1 (150 g tissue) using hot
acetic acid method and batch 2 (500 g), the cold acetone method. Each
extract was separated by large scale reverse-phase chromatography into 15
fractions. Fraction 8 of both batches were selected as pilot samples. Fromthese samples, a total of 188 peptides were isolated by 5-8 alternating steps ofreverse-phase and ion-exchange HPLC. (2) Amino acid sequence. Aminoacid sequence analysis was successfully performed for 97 peptides using an
automatic analyzer. It was, however, not successful for 39 samples (21%)
because of N-terminal blocking (or non-peptide). A homology searchsuggested that at least 37% and 2% in batch 1 and 2, respectively, arefragments of known proteins in the data base. (3) Chemical synthesis. 14
peptides were chemically synthesized and confirmed to be identical to native
peptides by HPLC. They include a family of 4 peptides having a common
sequence of G LWamide at the C-terminus (as in Metamorphosin-A, Leitz et
aI, 1994).
1
2

3

,

Hiroshima University.
Fukuoka Women's University.
University of Munich.
University of California at Irvine.

IV.

DEVELOPMENTAL GENETICS63

The results described so far suggest that the procedure developed in thisstudy is a powerful approach for isolating biologically active peptides. They
also show that batch 2 contains less protein fragments than batch 1. 500 or
more peptides are estimated to be present in the remaining 14 fractions inbatch 2. All these peptides will be processed by the procedures describedabove.
Hydra Peptide Project. II. Differential Display-PCR Assay For
Signal Molecule Activity
Toshitaka FUJISAWA, Hiroshi SHIMIZU, Masayuki HATTA, Tsutomu SUGIYAMA,
Thomas BOSCH', Maria DEHAR0 1, Jan LOHMANN!, Charles DAVID',
Toshio TAKAHASHI', Yojiro MUNEOKA', Osamu KOIZUMI 3
and Hans BODE'

Differential Display-PCR (Liang and Pardee, 1992) was adopted as thecritical step for detecting signal molecule activities of peptides isolated fromhydra tissue. The procedure was originally developed to display small
differences of gene expression present in two similar tissue samples. This
procedure was used to detect alterations of gene expression in hydra tissue
treated by isolated peptides.
A part of each isolated peptide was diluted in a small volume of hydra
culture solution, producing a peptide solution ranging from 0.5 to lOX 10 - 9
M in concentration. This solution was used to treat hydra in the presence orabsence of 2% DMSO during the first hour. DMSO was used to make hydra"permeable" to peptides. RNA was extracted from the treated and untreatedcontrol hydra, and used to examine and compare gene expression patterns
using DD-PCR. Peptides that alter gene expression in the treated animals
were selected by this assay.
46 peptides were examined. Using 2-4 primer sets, 9 (20%) produced
major changes and 4 (9%) produced minor changes in gene expression. The
percentage values should become higher when using more primer sets. Some
produced additional bands (up-regulation), some produced disappearing
bands (down-regulation), and some produced both in the display. 8 out of
1

,
3

,

University of Munich.
Hiroshima University.
Fukuoka Women's University.
University of California, Irvine.

64

the 9 peptides which produced strong effects required DMSO to be effective,
indicating that "permealization" is required for the uptake of most peptides.
Several cDNA clones were isolated for genes whose expression was affectedby the peptides.
DD-PCR was found to be effective in selecting peptides capable of affecting
gene expression in hydra. However, one unexpected feature was also found.
Gene expression was affected by the peptide treatment as distinct additionalor missing bands in DD-PCR. However, northern analysis carried out using
the isolated cDNA clones has revealed that actual differences in expression ofsuch genes were relatively small (2-3 X) in some cases. The reason for thisis unclear at present. The display results, therefore, need to be interpretedwith caution. The procedure, however, is highly powerful in examining
signal molecule activities using limited quantities of isolated peptides.
Hydra Peptide Project. III. Biological Function AssayToshitaka FUJISAWA, Hiroshi SHIMIZU, Masayuki HATTA, Tsutomu SUGIYAMA,
Osamu KOIZUMI', Hans BODE 2, Thomas BoscH3, Maria DEHAR0 3,
Jan LOHMANN3, Charles DA vm 3 , Toshio T AKAHASHI 4
and Yojiro MUNEOKA 4

A functional test of the synthetic peptides is the final crucial step of thepeptide project. Simple but reliable assay procedures are required to identify
peptides which play roles in regulating developmental processes. Foursynthetic peptides were selected as model peptides to establish such tests.
They were (I) Hyd-l, KPPRRCYLNGYCSP-amide, (2) Hyd-23,
KWVQGK~TQEVKQ!K!::, (3) Hym-33H, AALPW, and (4) Hym-54,
GPMTGLW-amide. Hyd-23 and Hym-54 have identical amino acid residues
to head-activator (Schaller and Bodenmtiller, 1981) and Metamophosin-A(Leitz et al. 1994), respectively, in the positions indicated by underlines.
In Hydractinia, the peptide effect was tested on metamorphosis fromplanula to polyp. In hydra, i~ was tested on budding under a variety ofconditions, head or foot regeneration and TSI9+ antigen expression afterhead or foot removal, expression patterns of Cnox-2 and other locally
,
2

3
4

Fukuoka Women's University.
University of California, Irvine.
University of Munich.
Hiroshima University.

IV.

DEVELOPMENTAL GENETICS

65

expressed genes, formation of RF+ neuronal network, cell ratios (i/epi,
nerve/epi, nerve/i, etc) in whole body column and different regions, and
BrdU-labeling index for various cell types in pulse and continuous BrdUlabeling. The following results were obtained.
In Hydractinia, (l) Hym-54 (10- 6 M) induced complete metamorphosis
from planula to polyp. (2) Hym-33H (10- 6 M) induced tissue contraction(first step of metamorphosis?), but not complete metamorphosis by itself.
However, it enhanced metamorphosis with marginal concentrations (10- 7M) of Hym-54.
In hydra, (3) Hym-54 (1O- 6 M, +2% DMSO) enlarged the zone of RF+
neuron distribution in the upper body column, and increased the nerve/epi
ratio (about 1.5 X) in the same region. (4) Hym-33H (10 6 M) contracted
the same zone of RF + neuron distribution.
The procedures developed in this project were found to be effective inisolating signal peptides involved in regulating various developmental processes. In the future, the project can be extended to isolate genes encoding
the peptides. A new way, then, will be open to elucidate the complete"molecular circuitry" of a specific developmental process that starts from amaster control gene, goes through signal pathways and early gene regulation,and finally ends in the expression and functioning of the realizator genes.
Intermittent Expression of BmFTZ-Fl, a Member of the Nuclear
Hormone Receptor Superfamily during Developmentof the Silkworm Bombyx mori
Guan-Cheng SUN, Susumu HIROSE and Hitoshi VEDA

BmFTZ-FI is a sequence-specific DNA-binding factor in the silkworm
Bombyx mori sharing similar biochemical characteristics with Drosophila
FTZ-FI, a member of the nuclear hormone receptor superfamily. Using
DNA sequence homology with FTZ-FI and information on the tryptic
peptide sequences of BmFTZ-FI, we isolated a cDNA encoding for
BmFTZ-F1. Amino acid sequences in the zinc finger DNA-binding region
and the putative ligand-binding domain of BmFTZ-FI showed strong similarity not only to FTZ-FI but also its mammalian homologues, LRH-I, ELP,
and Ad4BP, suggesting the importance of each region for the function ofthese proteins. Northern blot analyses of RNA isolated from the middle and66

posterior silk glands and fat bodies showed that a 6.1-kb BmFTZ-Fl mRNA
is present in all tissues so far examined. Expression of BmFTZ-FI mRNA isintermittent, being high during larval molting and both larval-pupal andpupal-adult transformations. Injection of 20-hydroxyecdysone on the third
day of the 5th instar larvae induced BmFTZ-Fl mRNA in the posterior silk
gland after 24 hr. When 5th instar silk glands were cultured in vitro,
BmFTZ-Fl mRNA was induced by a 6-hr exposure to 20-hydroxyecdysone
followed by 6 hr in hormone-free medium. These results suggest that
BmFTZ-Fl is inducible through a decline in the ecdysteroid titer and may
play an important role in the development of the silkworm as a transcription
factor. For details, see Dev. Bioi. 162, 426-437, 1994.
Mediators of Activation oflushi tarazu Gene Transcription
Activation by BmFTZ-Fl
Feng-Qian LI, Hitoshi VEDA and Susumu HIROSE

Transcriptional activation by many eukaryotic sequence-specific regulators
appears to be mediated through transcription factors which do not directlybind to DNA. BmFTZ-Fl is a silkworm counterpart of FTZ-F1, a sequence-specific activator of the fushi tarazu gene in Drosophila melanogaster.
We report here the isolation of 18- and 22-kDa polypeptides termed MBF1
and MBF2, respectively, that form a heterodimer and mediate activation by
BmFTZ-FI of in vitro transcription from thefushi tarazu promoter. Neither
MBF1, MBF2, nor a combination of them binds to DNA. MBFl interacts
with BmFTZ-Fl and stabilizes the BmFTZ-FI-DNA complex. MBFl also
makes direct contact with the TATA-binding protein (TBP). Both MBFl
and MBF2 are needed to form a complex between BmFTZ-Fl and TBP. We
propose a model in which MBF1 and MBF2 form a bridge between BmFTZF1 and TBP and mediate transactivation by stabilizing the protein-DNA
interactions. For details, see Moi. Cell. Bioi. 14, 3013-3021, 1994.
Sequences of Two cDNAs Encoding Silkworm Homologues ofDrosophila melanogaster squid Gene
Feng-Qian LI, Guan-Cheng SUN, Hitoshi VEDA and Susumu HIROSE

The squid (sqd) gene of Drosophila melanogaster encodes a few isoforms of

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DEVELOPMENTAL GENETICS

67

a heterogeneous nuclear (hn) RNA-binding protein. We isolated two typesof cDNAs coding for homologues of the Sqd protein from the silkworm
Bombyx mori. The two predicted amino acid sequences are identical up toamino acid 280 and then diverge. The silkworm and fruit fly proteins share
80% homology in the RNA-binding motif region. These cDNAs detect 2.0-,
1.8- and l-kb mRNAs in the middle and posterior silk glands. Thesetranscripts were most abundant at the molting stage. This may respond to adramatic change in RNA species synthesized in the silk glands during themolting stage. For details, see Gene 154, 295-296, 1995.
The Drosophila Nuclear Receptors FTZ-Fla and FTZ-F1,8 Compete as
Monomers for Binding to a Site in the fushi tarazu Gene
Carolyn K. OHNO*, Hitoshi VEDA and Martin PETKOVICHI*The striped pattern offushi tarazu (jtz) expression found in the blastodermof the Drosophila melanogaster embryo is generated largely through complex
interactions between multiple transcription factors that bind to the zebra
element of the ftz gene. A motif in the zebra element, the FTZ-Fl
recognition element (FIRE), has been shown to bind a transcription factor,
FTZ-Fl, which is a member of the nuclear receptor family. A second,
related member of this family, FTZ-Fl,8, which also binds to this motif has
been identified recently. To investigate the possibility that FTZ-Fla and
FTZ-Fl,8 coregulateftz transcription through the FIRE, we have studied the
DNA binding properties of FTZ-Fla and FTZ-Fl,8. We found thatrecombinant FTZ-Fla and FTZ-Fl,8 proteins produce similar in vitro
DNase I footprint patterns on a l4-nucleotide region of the zebra element
and bind to this site with similar affinities and sequence specificities. Usingwild-type and N-terminally truncated receptors, we have determined that
FTZ-Fla and FTZ-Fl,8 both bind as monomers to the 9-bp FIRE in thezebra element, as well as to an imperfect inverted FIRE repeat present in the
Drosophila alcohol dehydrogenase gene. A polyclonal antibody raised against
FTZ-Fl,8 identifies a predominant FIRE-binding component in embryonicnuclear extracts. Although FTZ-Fla is also present in these extracts, FTZFla and FTZ-Fl,8 do not appear to form heterodimers with each other.
*Cancer Research Laboratories and Departments of Biochemistry and Pathology,
Queen's University, Kingston, Ontario, Canada K7L 3N6.

68

Cotransfection assays in mammalian cell culture indicate that both receptors
contribute to the net transcriptional activity of a reporter gene through theirdirect interaction with the FIRE. These data suggest that FTZ-Fla and
FTZ~Fl,8 likely coregulate common target genes through competition forbinding to a 9-bp recognition element. For details, see Mol. Cell. BioI. 14,
3166-3175, 1994.
The Bombyx silkworm Is a Semelparous Insect with a Long Lifein the Most-reproductive Period
Akio MURAKAMI

There is a widely accepted view that semelparous organisms are quick
aging and have a short lifespan in the post-reproductive term as seen in ocean
salmon, trout and so on. The silkworm, Bombyx mori (L.), is a semelparous
insect. It is debatable whether aging in the post-reproductive term for
Bombyx follows the same pattern as in other semelparous animals. In
Bombyx after a 2 week embryo period, newly hatched larvae start feeding on
mulberry leaves. This period lasts about 3 weeks. However, female moths lay
offspring within half a day after eclosion. Accordingly, the entire postembryonic life of this insect, from egg hatching to moth eclosion, is calculated to be about 40 days in addition to several days of an adult period at thestandard breeding temperature of 23-25°C. But the popular view of theadult lifespan of Bombyx has long been accepted as several days. Results of
our observations indicate that the adult lifespan differs between the sexes:
female moths generally live 1.5-1.9 times longer than males: freshlyeclosed
moths live ca. 5-6 days in the case of males and ca. 9-10 days in the case of
females. It is worthwhile to note that some individuals, have had lifespan up
to ca. 23 days in the post-reproductive period, regardless of sex. Accordingly,
the lifespan of Bombyx is markedly longer than what is conventional viewaccepted: lasting about 45 days in males for the post-embryonic period andabout 50 days in females.
In this insect species, eclosion of moths of both sexes takes place at sunrise
and is completed by 8-9 a.m. at temperatures of 23-25°C throughout theyear. Thirty minutes after eclosion newly emerged moths begin to mate andthey continue copulation for 1-2 hrs. The separated female moth begins tolay eggs around sundown and continues for half the night. During this short

IV.

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69

term, female moths deposit almost all the eggs from their bodies. Accordingly, it can be said that the reproductive term of Bombyx silkworms is less thanhalf a day. The moth that develops seems useful only for reproductive
purposes. The female moth that has completed her duty of laying offspring
lives for a fairly long post-reproductive period. Generally, the male moth
lives a markedly shorter period than the female. In any case, it is clear that
Bombyx silkworms do no have a quick-aging or early death characteristic in
the post-reproductive period. Their situation seems to be analogous toiteroparous animals including human beings.
Non-mated female moths in Bombyx slowly deposit unfertilized eggs inaccordance with a clear-cut circadian rhythm for several days: they usually
lay eggs from sundown until sunrise of the next day, but aged moths lose
such the rhythm. It is of interest to note that there is no significant differencein the adult lifespan of mated moths from unmated ones so far as we haveobserved, suggesting that insemination stimulates only egg-laying. The
behavior of egg-laying of a wild type silkworm, B. mandarina (M.), is tosome extent different from the domesticated silkworm, B. mori. B. mandarina, a putative ancestor of B. mori, is a semelparous insect too, but has apeculiar habit of laying eggs on branches of the mulberry tree, bit by bit,extending over several days.
Thus, it can be inferred from the present observations and others that in B.
mori slow aging (and a short lifespan) in the post-reproductive phase is areflection of a genetic trait preserved in mandarina silkworms and semelparous animals can be grouped, at least, into two types with either a short orlong lifespan in the post-reproductive term. Briefly, it appears there is no
positive relationships between the lifespan of the post-reproductive phase andthe number of reproductions that occur in the life of a semelparous organism.
Homoeostasis in the Process of Growth and Senility in the Bombyx
Silkworm: the Relationship between Larval Growth
Period and Temperature
Akio MURAKAMI

The domesticated mulberry silkworm, Bombyx mori (L.), has widely beenbred throughout the Northern Hemisphere from the subtropics to the temperate zones and is a holometabolous insect passing through four different

70stages: embryo (or egg), larva, pupa, and adult. Larvae principally consume
mulberry leaves as a source of energy and during the other three stages use ismade preserved nutrients: embryos use vitellins, but both pupae and moths
utilize some nutrients remanufactured from several worn out tissues andorgans from each of the previous developmental forms as well as preserved
nutriments in adipose tissues. Bombyx larvae have a fairly narrow range of
permissible breeding temperatures in contrast to the other three life forms:
larvae are usually bred within a temperature ranging from 20 to 25°C in thetemperate zones including Japan, while it is cultured in some measure in hightemperatures in the subtropics. In general, the entire larval stage and firsthalf of embryogenesis (prior to the completion of organogenesis), are affected negatively by low (below 15°C) and high (above 35°C) temperatures. On
the other hand, the pupal, adult, and certain developed embryonic stages are
fairly resistant to a wide range of temperatures from near the freezing point
to around 35°C, except for certain developmental steps. This stage differential temperature sensitivity appears to be dependent upon the type of nutrients being used in each developmental stage. As a matter of course, embryosin organogenesis and pupae in the first half of metamorphosis as well as theentire larval stage are sensitive to extremely high and low temperatures aboveand below 19-25°C. Practically, this insect is bred around 2G-25°C in thetemperate zone, while in the tropics to some measure in temperatures higher
than the standard rearing temperature of the temperate zones (but notexceeding 30°C).
In the Bombyx silkworm, a number of breeding records on the larvalrearing period used with many tetramoulter stocks at various temperatures
have accumulated. Using data from Morohoshi (1972), an analysis was
carried out to obtain information on the relationship between larval growthterms and breeding temperatures at 19 and 24°C. As a result, it was foundthat there is no marked difference in the duration of the larval period between
the two breeding temperatures: it takes ca. 700 hrs at the two temperatures on
average among five pure stocks regardless of sex. However, for the pupal
period a great difference was found between the two temperatures: its took
about 300 hrs at 24°C for male pupae and 350 hrs for females, to go throughthe larval period, but ca. 550hrs at 19°C for both sexes. This suggests that
Bombyx larvae have the ability to stabilize the process of their growth incontrast to pupae. Such a large stage differential in temperature effect on
growth-rate between the larval and pupal stages may be a reflection ofIV.

DEVELOPMENTAL GENETICS

71

different physiological demands based on the nutrients utilized for each life
form.
Homoeostasis in Growth and Senility in the Bombyx Silkworm:
the Relationship between Adult Lifespan and Body-Weight
Akio MURAKAMI

Although the adult lifespan of Bombyx is known to be under the control ofa complex genetic system, a peculiar phenomenon in which the duration of
the moth stage is inversely proportional to the body-weight of adults (orpupae) has often been seen. As shown in Figure 1, the more body-weightgained above a population mean, the shorter the adult lifespan and covensely,
the lower the body-weight, the more extended adult lifespan. The phenomenon is observable with both sexes. In addition, some corpulent individuals,
whose weight is in excess of the mean population body-weight have atendency to be susceptible to several diseases and also to be physically

15

o

0
....... Female

~c

10

~
~

~

:g
-<

1.0

1.5

2.0

Pupal body-weigh (g)Fig. 1.

Relationship between adult lifespan and body weight of a certain F, hybridline in Bambyx mar; (L). [A stock used is an F, hybrid between Kinshu andShowa.]

72

inactive as adults as well as pupae. Observations showed that within the same
genetic population, the lifespan of adults is to some extent affected by theamount of food injested at the later larval phase, too. From the result of thepresent investigations, it is clear that lifespan of Bombyx adults depends ontheir body-weight, which goes up a larval feeding activity, and adults whichhave the average body-weight show their mean population lifespan for eachstock. The average body-weight as well as lifespan of adults is appeared to bea reflection of physiologically more stable state for each genetic stock own. It
can be said that this homeostatic state is peculiarly under the control of an
inherently fixed genomic framework to each stock.
Growth Phenomena in Bombyx mori (L.) with a Special Reference
to Genetic Factors Responsible for Growth
Acceleration and Molutinism
Akio MURAKAMI

The tropical race has a certain growth accelerating factor as a novel
biological trait, besides various peculiarities of multivoltinism, robustness(highly resistant to either high temperature or high humidity and some insect
diseases, etc. ) and a unique feel to the silk produced from this silkworm race.
The growth accelerating trait is recessive and seems to be comprised of theprecocity compound gene loci located on the X chromosome of Cambodge
stock. Each member is specially responsible for the growth acceleration foreach developmental stage, in addition to the wide range over the growthphase. Considering such a genetic feature of the compound loci, it can besaid that the growth phenomena in this insect species is under the control of
the Mendelian genetic system rather than the so-called polygenic system.
Phenotypic expression of the compound loci is independent of changes ofphotoperiod, light, temperature and other environmental variables unlike themoultinistic gene system.
For details, see Murakami (1994), Indian J. Sericulture 33, 12-14.

IV.

DEVELOPMENTAL GENETICS

73

Climatic Differential Phenotypic Expression of Voltine
Genes in Bombyx mori L.
G. SUBRAMANYA* and A. MURAKAMI

Voltinism was considered to be under the control of autosomes and sex
chromosomes in addition to autosomal modifiers. Earlier reports emphasized
this phenomenon as a maternal trait. A detailed investigation on the genetic
mechanism of voltinism resulted in the identification of a "npnd" stock, thegenes for which are located on the 'X' chromosomes. As a result, it is now
understood that a certain voltinism is a maternally inherited biological event
under the control of sex linked genes. The photoperiod plays an importantrole in this phenomenon. This knowledge of voltinism and 'X' chromosomes
is essential in silkworm breeding when studying combining ability or whenevolving robust races. Polygenic involvement in silkworm breeding is questionable since it has been shown that 'X' chromosomes carry specific genes
for various life history characters of Bombyx mori L. This new insight into
the biology of voltinism of tropical races may open up new avenues forstudying neurobiology, memory, learning, adaptation and evolution.
For details, see Subramanya and Murakami (1994), Indian J. Sericult. 33,
103-109.
Isolation and Characterization of Enhancer Trap Lines expressedin Ovary in Drosophila melanogaster
Masa-Aki YAMADAIn Drosophila, development of the oocyte is controlled by various genes
during oogenesis. In order to understand the mechanism of oogenesis,
isolation of P-element mediated enhancer trap lines was carried out using
histochemical detection of the ,,-galactosidase activity in the ovaries oftransformants.
Of the 260 transformants carrying P[lwB] on the X chromosome, 26 lines
expressed ,,-galactosidase specifically in the cells of the ovary. In 6 lines, theooplasm and germarium were labeled and four lines were expressed within
the germarium and all subsequent stages of the oogenesis. For three lines,,-galactosidase activity was detected during stage 8-10 in nurse cell nuclei*

Department of Sericulture, University of Mysore, Mysore -570 006, India.

74

and ooplasm. One line was labeled in the germarium, nurse cell nuclei andooplasm. In three lines, .a-galactosidase activity was detected throughout theovary and four lines were labeled in the columnar follicle cells and the border
cells.
These enhancer trap lines capable of being stained in specific cells and at
specific stages of the oogenesis provide useful markers for the recognition ofcell types in ovaries. Further analyses on these lines are in progress.
The Retinoblastoma Binding Factor 1 (RBF-l) Site in RB Gene
Promoter Binds Preferentially E4TFl, a Member of the
Ets Transcription Factors Family
Ericka SAVOYSKY*, Takakazu MIZUNO*, Yoshihiro SOWA**, Hajime WATANABE,
Jun-ichi SAWADA, Hitoshi NOMURA*, Yoshiyuki OHSUGI*,
Hiroshi HANDA and Toshiyuki SAKAI**

The tumor suppresser retinoblastoma gene product, pRB, is a well known
regulator of G liS cell cycle progression. Moreover, mutational inactivations
within the retinoblastoma gene (RB) are found in many human malignant
tumors, and thus, believed to be an essential step in tumor formation. The
human RB gene is considered a housekeeping gene with no characteristic
TATA or CAAT elements in its promoter region, but the sequence between
206 and 185 bases upstream of the initiation codon, essential for RB promoter
activity, contains putative Spl and ATF recognition sites. We previously
reported that point mutations in this region, causing low penetrance retinoblastomas, completely reduced RB promoter activity, and that a nuclear
factor, named RBF-l (retinoblastoma binding factor 1), could specificallybind to this sequence, overlapping the SP 1 recognition sequence. We show
here, that RBF-l can recognize a specific DNA sequence, 5'-GGCGGAAGT-3', overlapping the Spl and ATF sites and corresponding to the consensus DNA binding site for members of the Ets transcription factors family.
When the RBF-l site was used for sequence specific DNA affinity purification from erythroleukemia cells, reconstitution assays, immunoblotting analysis and peptide mapping showed that the two major co-purified proteins are***

Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135
Komakado, Gotemba, Shizuoka 412, Japan.
Department of Preventive Medicine, Kyoto Prefectural University of Medicine,
Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan.

IV.

DEVELOPMENTAL GENETICS

75

identical to human E4TFI-60 and -53 proteins. This reveals that E4TFI canbind to the RBF-l site of the RB gene promoter, which thus, constitutes a
new target member of the Ets transcription factors family. For details, see
Oncogene. 9, 1839-1846, 1994.
Transcriptional Activation through the Tetrameric Complex
Formation of E4TFI Subunits
Jun-ichi SAWADA, Masahide GOTO, Chika SAWA,
Hajime WATANABE and Hiroshi HANDA

Transcription factor E4FTI is involved in the regulation of the adenovirus
E4 gene. The factor is composed of two types of subunits, an ets-related
DNA binding protein, E4TFI-60, and its associated proteins with four
tandemly repeated Notch-ankyrin motifs, E4TFI-53 and E4TFI-47. To
determine the functional domains, we constructed various mutants of thesubunits. E4TFI-60 bound to DNA as a monomer. The ets domain and its
N-terminal flanking region were necessary to recognize the specific DNA
sequence. The 48 amino acids at the E4TFI-60 C-terminus were required forinteraction with the other type of subunit. E4TFI-53 and E4TFI-47 share
the N-terminal 332 amino acids but differ at the C-termini. They interactedwith E4TFI-60 through the N-terminal flanking region to form a heterodimer. E4TFI-53 dimerized with itself, whereas E4TFI-47 did not. The
C-terminal region specific for E4TFI-53 was required for the dimerization
Therefore, heterodimers composed ofE4TFI-53 and E4TFI-60 were further
dimerized, resulting in the formation of a tetrameric complex, which stimulated transcription in vitro. Heterodimers of E4TFI-47 and E4TFI-60
weakly stimulated transcription in vitro. The results indicated that the
tetrameric complex formation of E4TFI subunits was necessary to activatetranscription efficiently in vitro. For datails, see EMBO J., 13, 1396-1402,
1994.
Formation of Drosophila Wing and Haltare Imaginal Disc Depends on
Two Related Zinc Finger Proteins Snail and Escargot
Naoyuki FUSE and Shigeo HAYASHI

Drosophila escargot (esg) and snail (sna) genes encode closely related C2-

76

H2 type zinc finger transcription factors. esg is expressed in all imaginal discs
and histoblasts during embryogenesis and in the larva. In early embryo, sna
is transiently expressed in mesodermal primordia and in the nervous system.
During mid to late embryogenesis, expression of these two genes overlap inwing, haltare and genital imaginal discs. These imaginal discs do not develop
in embryos deficient for both sna and esg activity. The presence of one copyof either one of the genes was sufficient for the formation of these discs.
Furthermore, the ubiquitous expression of esg from a heat shock promoter
partially rescued the mutant phenotype. In the rescued embryo, partial wingor haltare discs were observed in the position where they are normallyformed, suggesting that esg and sna expression alone is not sufficient to induce
wing and haltare discs in the ectopic position. These results suggest that local
activation of esg and/or sna triggers differentiation of cells which werealready made competent through the action of segmentation and homeotic
genes.
Control of esg Expression
Yoshimasa YAGI* and Shigeo HAYASH)esg is expressed in a spatially and temporally complex pattern during
embryogenesis. Its expression in the imaginal cell primordia is thought to be
responsible for the initial formation of some imaginal discs and for maintenance of diploidy. Expression in embryonic gonads and testes at later stages
has a relevance for esg's role in spermatogenesis. In addition, esg is expressedin a subset of embryonic tracheal cells which play an important role intracheal morphogenesis. To elucidate the role of esg in those tissues, and its
relation to other known regulatory genes, we have been mapping cis-control
elements in the upstream region of the esg gene. So far we have mapped thetestis specific enhancer within a I kb fragment in the vicinity of the esg
promoter. This fragment can direct expression on heterologous genes in thetestis hub cell. Control elements for the nervous system, gut imaginal cells
and tracheal tip cells are scattered within a region of more than 20 kb
upstream from the esg promoter. These elements are candidates for thetranscriptional target of regulatory genes such as, wigless and dpp which are
involved in the morphogenesis of many tissues expressing esg.

* on a leave from Department of Biology, Faculty of Science, Nagoya University.

IV.

DEVELOPMENTAL GENETICS77

Use of Green Fluorescent Protein As a Live Cell Marker and
Its Application to the Study of Tracheal Morphogenesis
Yasuhiro SHIGA* and Shigeo HAYASHIThe morphogenesis of embryonic trachea involves initial determination ofprimordium from ectoderm, invagination, extension and final joining of
precursors from each hemisegment to form a tubular network. During thefinal joining process, a set of marker genes are expressed in a single cell at the
tip of each growing tracheal branch. These cells contact each other andjoining occurs. esg is expressed in these tracheal tip cells and is required forfusion to occur. To elucidate the role of the tip cell, we are using two
approaches. i) Investigate the role of esg in the tip cell. We are currently
studying phenotypes of esg mutant tip cells using various markers. ii) Direct
ablation of the tip cell and real time observation may be an effective way toaccess the role of the tip cell. We are using green fluorescent protein (GFP)
to visualize tracheal cells in live embryo. A fusion gene, (nuc-GFP-lacZ)with the GFP coding sequence linked to the nuclear localization signal andbacterial ,B-galactosidase gene was expressed in embryo and in larva. The
green fluorescence of nuc-GFP-lacZ was detected in both fixed and live
embryo. This marker gene should allow us to follow the sequence of tracheal
migration and to label a subset of tracheal cells for ablation experiments.
Developmental Mechanisms and Manipulation of Germ
Cells in Mouse Embryos
Norio NAKATSUJI and Yasuaki SHIRAYOSHI

The postimplantation period of mammalian embryogenesis includes many
important events, such as formation of the primitive streak, central nervous
system (CNS) and primordial germ cells (PGCs). PGCs migrate to fetal
gonads, become the gonia cells, and take different courses of differentiation inmale and female embryos. We are studying the cellular and molecularmechanisms of such developmental processes in germ cells of mouse embryos.
There are several growth factors, such as the stem cell factor and leukemia
inhibitory factor, which are known to affect proliferation of PGCs in culture.
We found that another factor, tumor necrosis factor (TNF) -a, stimulates*

on a leave from Faculty of Agriculture, Nagoya University.

78

proliferation of PGCs at early migration stages when added to the culture
medium (Kawase, E. et al. (1994) Deve!. Bio!. 161,91-95). For the PGCs atlater stages, after arrival at fetal gonads, we found that retinoic acid acts asa potent mitogen (Koshimizu, U. et al. (1985) Devel. Bioi. 168, 683-685).
We are currently studying the differentiation of PGCs after they havesettled in gonads. After having developed culture conditions to allow
survival and differentiation of PGCs at these stages, we are now analyzing
molecular and cellular changes of germ cells and their interactions withgonadal somatic cells. Our aim is to develop experimental methods to study
developmental processes including meiosis and gamete formation in culture,and to enable the manipulation of germ cells, such as gene transfection, atvarious developmental stages to facilitate the molecular analysis of germ celldifferentiation.
Molecular Analysis of Cell Differentiation and Morphogenesis in
Postimplantation Mouse Embryos
Yasuaki SHlRAYOSHI 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, to clone 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 (eNS) during thepostimplantation period. We have constructed a cDNA library from 6.5~8.5
days 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 (acheatescute complex) 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 int3 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 embryos. The expression pattern of int-3 coincided with theIV.

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79

expression of receptor tyrosine kinase Flk-l, which is regarded as a major
regulator of vasculogenesis and angiogenesis. These results suggested that
int-3 might be involved in the formation of blood vessels in early mouseembryos. We will study its roles in blood vessel construction either by ectopic
expression or by altering the gene through the gene targeting method using
ES cells and homologous recombination. Various ES cell lines we obtained(Kawase et a/. (1994) int. J. Devel. Bioi. 38,385-390) may be useful for suchstudies. Another area of interest is the molecular mechanism of sex
differentiation in fetal gonads. We are currently trying to find genes whichshow specific expression patterns in male or female gonads.
Cell Differentiation and Morphogenesis of the Mouse
Central Nervous System
Norio NAKATsuJI and Yasuaki SHIRAYOSHI

In collaboration with another laboratory (Dr. I. Nagata at Tokyo Metropolitan Institute for Neuroscience), we are studying migration patterns ofneuroblasts 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 (Nagata, I. and Nakatsuji, N. (1994) Deve/. Growth Differ. 36,
19-27), and the ultrastructure of the neuroblasts showing perpendicular
contact guidance (Ono, K. et al. (1994) Deve/. Growth Differ. 36,29-38). We
are now analyzing the role of perpendicular contact guidance in the actual
morphogenesis of brain cortices using various histological and morphologicalmethods.
We are also studying the cellular and molecular mechanisms of celldifferentiation in CNS development. We have established several cell lines
from early CNS neuroepithelium dissected from embryos of a transgenic
mouse strain harboring a temperature-sensitive immortalizing gene. We are
currently studying the differentiation potency of these cells in vitro and also invivo after injecting them into embryonic brains.

80

Fluoride-resistant Mutants of the Nematode Caenorhabditis elegans
Minoru KAWAKAMI, Takeshi ISHIHARA, Misako URASAKI
and Isao KATSURA

Sodium fluoride (NaF) may interfere with a signal transduction system,
because it depletes Ca + +, inhibits phosphatases, and activates trimeric Gproteins. Weare trying to elucidate such a system through the isolation andcharacterization of C. elegans mutants resistant to NaF.
We have isolated 13 recessive fluoride-resistant mutants that map in 5 newgenes,flr-l X,flr-2 V,flr-3 IV,flr-4 X andflr-5 V. They are grouped into two
categories, class 1 and class 2. Class I mutants (jir-l,flr-3, andflr-4) areresistant to 10 mM NaF, but they grow slowly and have a small brood size
even in the absence of NaF. In contrast, class 2 mutants (jir-2 andflr-5) are
not completely resistant to 10 mM NaF, and they are almost normal in
growth rate and brood size in the absence of NaF. Interestingly, class 2
mutations suppress the slow growth and small brood size but not strong
fluoride-resistance of class I mutations. (Katsura, I. et al. (1994) Genetics
136, 145-154)
To understand the biochemical reactions in fluoride-sensitivity we cloned
flr-l andflr-3 genes from transposon-insertion mutants, using the transposon
Tcl as the probe. Then we cloned cDNAs of these genes from a C. elegans
cDNA library, using fragments of the genomic clones as the probes. So far
only a partial-length cDNA offlr-l has been obtained. The predicted aminoacid sequence shows weak homology to the MEC-4 and DEG-l ion channelsof C. elegans and amiloride-sensitive ion channels of mammals. Since thesimilarity is relatively high at the functionally important membrane-spanning
region, we thinkflr-l also codes for an ion channel. For flr-3 we obtained afull-length cDNA, which has the same length (2 kb) as the mRNA as
determined by Northern analysis. The predicted FLR-3 protein consists of
525 (or 589) amino acid residues. It contains the subdomains II to XI of theprotein kinase consensus sequences (Hanks, S. K. et al. (1988) Science 241,
42-52) but lacks the consensus sequence of subdomain I, the ATP-bindingsite. Such examples have been found among protein kinases and kinase-likeproteins.
During the course of the study it has become clear that the flr mutations
affect neuronal functions. They cause worms to grow to dauer larvae, if theworms also have a certain neuronal mutation such as unc-3, unc-31 or osm-

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81

I (See section 2b below). Furthermore, J. H. Thomas (University ofWashington) and E. Jorgensen (University of Utah) found that the class Iftr
mutants have shorter defecation periods than wild type worms, which show
a defecation every 45 seconds. We found also that some, if not all, ftr
mutants are abnormal in chemotaxis. We are trying to elucidate the functionof the ftr gene products by studying the time and site of gene expression as
well as interactions between the gene products.

Caenorhabditis elegans Larval Lethal Mutations That Cause
Detachment of the Outer Surface of the Intestinefrom the Inner Surface of the Body Wall
Ryuichi HISHIDA, Takeshi ISHIHARA and Isao KATSDRA

We have been analyzing mutants in which the outer surface of the intestineis detached from the inner surface of the body wall and which die as larvae(Katsura, I. (1993) Genetica 88, 137-146). We call them elr-l-Iike mutants,
since the phenotype resembles that of elr-I among known mutations. Tensuch mutants were isolated and mapped either to known genes that act insignal transduction (let-23, let-34I, lag-2 and elr-I) or to unknown genes. We
think the latter genes may also act in signal transduction or in cellularfunctions regulated by signal transduction.
We cloned one of the unknown genes, let(ut40, ut102), by the transposontagging method. Using a genomic fragment as the probe, we screened a C.
elegans cDNA library and found three types of clones overlapping each otherbut differing in the positions of the 5' and 3' ends. The difference in the 3'
ends is probably caused by two closely located poly-A addition sites. The
difference in the 5' ends may be due to multiple promoters, or it may result
from trans-splicing, since the shorter cDNA has the SLI splice-leader sequence. The predicted amino acid sequence of let (ut40, ut102) has no clear
homology to any known protein. To obtain further information we aredetermining the sites of gene expression in wild type worms and in signaltransduction mutants.

82

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 are defective both in hatching and in migrationof a neuroblast (Hedgecock, E. M. et al. (1987) Development 100, 365-382).
They cannot digest protein components of the eggshell, and a neuroblast
called QL cell moves anteriorly instead of posteriorly during larval development. Since we isolated a transposon-insertion mutant in hch-l (unpublishedresults by K. Kondo (Soka University) and I.K.), we have cloned the gene by
the transposon-tagging method and the cDNA by using the genomic fragment as the probe. The cloned cDNA has the same length (3 kb) as themRNA as detected by Northern analysis. The predicted amino acid sequence
contains a consensus of metal proteinases. It is intriguing to know the
relation between this enzyme and the hatching enzymes of fish and sea
urchin.
Construction of GFP Markers for Various Nerve Cells in C. elegans
and Their Use for Mutant Isolation
Takeshi ISHIHARA, Manabi FUJIWARA and Isao KATSURA

To analyze formation and function of the neural circuit in C. elegans, wemade 8 types of worms in which specific sets of neurons emit fluorescence.
They were prepared by integration (into the worm genome) of the DNA
constructs, in which the cDNA of GFP (jellyfish green fluorescent protein;
Chalfie, M. et al. (1994) Science 263, 802-805) was fused with various C.
elegans neural promoters. The promoters were selected by the "promoter
trapping" method (Hope I. (1991) Development 113, 399-408) or by the
DNA sequence as determined by the C. elegans genome project. The
following are the neurons that express GFP (and results of a homology
search of the genes that seem to be controlled in vivo by the promoters) for
the 8 types of worms: (1) all or almost all neurons (no homology), (2) a pairof head neurons called AFD cells (no homology), (3) G2 cell and its
descendants (no homology), (4) all or almost all neurons (unc-31 geneitself), (5) 8 neurons in the head and PVC cells in the tail (AMPA-typeglutamate receptor), (6) CEP, ADE and PDE neurons (dopamine transporter), (7) several head neurons including ASJ, ASI and URX as well as theIV.

DEVELOPMENTAL GENETICS83

PQR neuron in the tail (cyclic nucleotide-gated channel), and (8) a small
number of head and tail neurons including ASG, DVA, PDA and ALN as
well as the excretory cell (prospero homolog). We confirmed that at least forthe promoter (l), fluorescence is localized to cell nuclei and to processes andcell bodies, if the SV40 nuclear localization signal and a C. elegans unc-76
eDNA fragment, respectively, are inserted into the DNA construct.
We plan to use these worms to isolate mutants with structural abnormalities in the nervous system. As a first step we mutagenized the worms (I)containing SV40 NLS using EMS (ethyl methanesulfonate) and screened
1000 independent FI worms for those producing mutants abnormal in thenumber and/or location of neuronal nuclei in the tail. Mutants having
different numbers of neurons in the tail were found and are being analyzed.
Analysis of the Head Neural System of C. elegans as Studied byFormation 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
limited food supply, they deviate from the normal life cycle and develop toenduring, non-feeding larvae called dauer larvae or dauers. The developmental decision to become dauers seems to be regulated by the head neuralcircuit, with the smell of bacteria and the pheromone as inputs, which aresensed by amphids, a pair of sensory organs in the head. Since the assay ofdauer formation is much less time-consuming than behavioral assays such as
those of chemotaxis, we are analyzing the head neural circuit by detecting
dauer formation as the output.
To identify genes required for the neural circuit, we looked for combinations of known mutations that cause worms to grow to dauers irrespective of
the environment, only if two of them are present in the worm genome as
homozygous double mutations. We found more than 40 known mutations
have such a synthetic dauer-constitutive phenotype. They include the following: (I) most sensory mutations that cause structural abnormalities inamphids (che-2, che-3, che-ll, che-13, daf-lO, osm-1, osm-3, osm-5, osm-6,
che-lO, che-14, daf-6, mec-l) , (2) most chemotactic mutations that do notseem to affect amphid structures (che-1, che-6, che-7, tax-2, tax-4, tax-6, odr1, odr-2, odr-4, odr-5), (3) some mutations giving rise to abnormality in84

movement (unc-3, unc-31, unc-64, unc-101 , unc-104), (4) a few mutations in
the neuronal cell lineage (lin-32, vah-3), (4) some egg-laying mutations (egl4, egl-32), (5) a mutation resulting in abnormality in defecation (aex-3), (6)fluoride-resistant mutations (jlr-1 ,flr-2,flr-3 ,flr-4,flr-5), (7) some mutations
affecting body-size (sma-2, sma-4), (8) a mutation causing decrease in theamount of serotonin and dopamine (cat-1). Since cat-1 is included in the list,
we tested dauer formation of some single mutants in the presence of various
neurotransmitters, agonists and antagonists. Of 11 drugs that we tested,ketanserin, a serotonin antagonist, produced a dauer-constitutive phenotype
in mutants of unc-3, unc-31, osm-1 and egl-4 but not in wild type worms.
In this study it become clear that many genes affect dauer formation in
combination but not by themselves. The reason is probably that there areparallel pathways for the signals regulating dauer-formation. The pattern of
combinations of mutations that have the dauer-constitutive phenotype showthat there are more than three parallel pathways. Another peculiar aspect isthat many mutations listed under (1) have a dauer-defective rather thandauer-constitutive phenotype by themselves. This means that one of thesignals in the parallel pathway is unaffected by sensory inputs and always
inhibits dauer formation. We are especially interested in the nature of thissignal, which requires the function of unc-3, unc-31, unc-64 and unc-104
genes.
To look for new genes required for one of the parallel pathways andpossibly for the formation or function of the neural system, we isolated 44
new mutations that have the dauer-constitutive phenotype in combination
with unc-31 mutation but not by themselves. Although we have mapped only
about half of them, some of them are located evidently in new genes. We also
tested all but two of the mutants for structural abnormality in amphid
neurons by the dye-filling method (Hedgecock, E. M. et al. (1985) Dev. Bioi.
111, 158-170). Six of them were clearly abnormal, another six were weakly
abnormal, and 30 seemed to be normal in this respect.

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85

The C. Elegans cDNA Project: Towards an Expression
Map of the Genome
Yuji KOHARA, Tomoko MOTOHASHI, Hiroaki TABARA,
Akiko SUGIMOTO and Hisako WATANABE

Aiming to ultimately understand the network of gene expression in development of the worm, we are constructing an expression map of the 100 Mb
genome by identifying and characterizing all of its cDNA species, whose
number is estimated to be around 15,000. 36,000 cDNA clones were picked
up randomly from 3 different cDNA libraries (from size-fractionated (> 2
Kb) and unfractionated cDNA from a mixed-stage population, and fromembryos) and were subjected to (l) tag-sequencing from both 5'- and 3'ends, (2) mapping onto the genome, and (3) analysis of expression patterns.
After analysis of every some 4,000 clones, cDNA species which were represented by more than 4 clones in the previous analysis were removed tominimize redundancy in analyses.
(1) Tag sequencing: The 3'-tag sequences were compared by FASTA toclassify the cDNA clones into unique groups (genes). Thus far, 8,351 clean
3'-tag sequences have been obtained, and these were classified into 3,518
unique cDNA groups (assigned CELKOOOOI through CELK03521). Also
detected were many pairs of clones which appeared to be generated byalternative splicing or differential poly-A addition. In a BLASTX search of
the 5'-tags, 36% of the cDNA groups showed significant similarities (blastx
score> 100).
(2) Mapping onto the genome: Using YAC polytene filters, 1,416 cDNA
groups were mapped onto the genome. Mapping in silica using the cosmid
sequences available at the Sanger Centre was also effective.
(3) Analysis of expression patterns: We have developed a method of in situ
hybridization on whole mount embryos of the multi-well format, and we areapplying the method to the set of representative clones of the identified
cDNA groups.
In Situ Hybridization of the Multi-Well Format on
Whole Mount Embryos of C. Elegans
Hiroaki T ABARA, Tomoko MOTOHASHI and Yuji KOHARA

In order to perform a large scale screening of the expression patterns of86

worm genes during embryogenesis, we have adapted our method of in situ
hybridization on whole mount embryos to the standard 96-well format using
96-well dot-blot apparatuses. The results of model experiments using stagespecific gene probes (unc-54, myo-2, glp-l, clb-2, pepsinogen gene, etc.)verified the effectiveness of this procedure. Currently, 32 different probes canbe assayed on one apparatus at one time. We are applying the procedure toclassified cDNA groups produced by the cDNA project in this lab. Thus far,an approximate estimation is that 1/20 of the cDNA groups shows specificpatterns of expression during embryogenesis.
In the course of the in situ screening, we found that a cDNA clone,
yk6lhll, showed asymmetrical distribution of the mRNA in the 2-cell stageembryo. Closer examination showed that the mRNA (1) was detected firstat the corner of the gonad, (2) was distributed evenly in oocytes, (3) was
segregated posterially during psuedo-cleavage and first cleavage and (4)remained only in P4. This gene, namedpos-l (posterior), was mapped on LG
5 using a YAC polytene filter. The cDNA sequence showed strong homology
to the zinc finger region of the Tisll family which are known to betranscription factors induced by stimulation of FGF and TPA etc. Another
cDNA species having homology with Tisll was obtained in our cDNA
project, but the distribution of the mRNA turned out to be ubiquitous during
embryogenesis. Weare planning gene disruption experiments on the gene
pos-l.
Efficient Isolation of Tel-Insertion Mutants from a Mutant
Bank Representing 20,000 Worms of C. elegans
Yoshiki ANDACHI and Yuji KOHARA

Aiming for efficient gene disruption, we have devised a system for isolating
mutants of Tcl insertion in desired genes from a bank of frozen mutator
worms. The main point of the system is a pooling strategy. Empirically, atleast one Tc I insertion mutant in a given gene is expected in a population of
105 or fewer mutator worms. Thus, as a first step, 192 pools of about 100
worms (a total of about 20,000 worms) of a mutator strain, MT3126 (mut2), were fed to Fl progeny and a part of the Fl worms of each pool wasstored frozen to make a mutant bank. Genomic DNA were extracted fromthe remaining part of each pool. Parts of the genomic DNA of every 12 pools

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DEVELOPMENTAL GENETICS

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were further pooled to make 16 larger pools. The larger pools were examined
for Tcl insertion in a target gene by PCR, using primers from the target geneand Tcl. When a positive one was detected, its 12 pools were assayedindividually by the same PCR to detect a positive pool. Finally, the frozen
worms (~200) corresponding to the positive pool were cultured separately
and assayed by the PCR to identify a strain having the Tc 1 insertion.
Thus far, we have obtained insertion mutants for 9 out of 10 genes forwhich the bank was screened. Although the insertions in many cases were
within introns, deletion mutants have been successfully isolated from some of
the insertion mutants by a sib-selection procedure. Therefore, we think thatthe bank is sufficient for producing disruption of any of most genes of thisorganism.

88v.

CELL GENETICS

Fine Analysis of DNA Replication Timing in the Human MHC Region
by Fluorescence in situ Hybridization: Temporal Order of
DNA Replication Closely Correlates with G + C%
Gradient of Genome Sequences
Katsuzumi OKUMURA 1, Tatsuo FUKAGAWA 2 , and Toshimichi IKEMURA 2

We have used fluorescence in situ hybridization (FISH) on HL60 cells forstructural analyses and determination of replication timing patterns of DNA
segments over 2 Mb within the human MHC region. This region is composedof long-range G+C% (GC) mosaic structures related to chromosome bands,
and a boundary of GC mosaic domains exists between MHC classes II and
III. FISH with elongated chromatin fibers from the interphase nuclei of HL
60 cells showed that the flanking genome structures of the boundary arestrictly consistent with previously reported mapping data. This confirmedthat HL60 cells may not have large structural abnormalities in this region.
Multi-color FISH with interphase nuclei of HL60 cells was useful in comparing the temporal replication timings of two DNA fragments which arelocated in adjacent genomic positions and which replicate at a similar time
during the S phase in the cell cycle. Using this procedure it was possible todetermine precisely the relative time of replication of each DNA clone which
was randomly selected from the MHC region. The clones at the boundary
region were found to replicate the latest among all the clones examined.
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 the genome mosaic structure at thelevel of genome sequences as well as in chromosome bands (manuscript in
preparation) .

1
2

Division of Physiological Genetics
Division of Evolutionary Genetics, National Institute of Genetics, and The Graduate
University for Advanced Studies

89

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POPULATION GENETICS

Interaction of Selection and Drift in Molecular Evolution
Tomoko OHTA

The nearly neutral theory proposes that interaction of selection and drift isimportant in molecular evolution. The theory predicts that evolutionary rateis negatively correlated with the species population size, for those genes
whose function has been fixed long time ago. The prediction is tested throughanalysis of sequence data of 17 mammalian genes by estimating divergenceamong genes separately for synonymous substitutions and nonsynonymous
substitutions. Star phylogenies composed of rodentia, artiodactyla andprimates are examined. The generation-time effect is found to be more
conspicuous for synonymous substitutions than for nonsynonymous substitutions. This result supports the nearly neutral theory. For details, see Jpn. J.
Genet. 68, 529-537.
On Hypervariability at the Reactive Center of Proteolytic
Enzymes and Their Inhibitors
Tomoko OHTA

The pattern of synonymous and nonsynonymous substitutions at thereactive center of proteases (kallikrein) and their inhibitors (ai-antitrypsin
and serpin) was examined. In the case of ai-antitrypsin, the proportion ofdifferent nonsynonymous sites exceeds that of different synonymous sites at
the reactive center for sequence pairs of recent duplication. The result
indicates that the positive selection has operated after duplication to increasefunctional diversity. In the cases of kallikrein, serpin, and remote sequencepairs of ai-antitrypsin, the proportion of different synonymous sites at thereactive center exceeds that of different synonymous sites at the remaining
region. The result indicates that gene conversion followed by natural
selection is working. On the whole, it is concluded that hypervariability ofamino acids at the reactive center is generated by an interaction among
natural selection, random genetic drift, point mutation, and gene conversion.

90

Gene duplication may provide potential for them to interact. For details, see
J. Mol. Evol. 39, 614-619.

Further Examples of Evolution by Gene Duplication Revealed ThroughDNA Sequence Comparisons
Tomoko OHTA

To test the theory that evolution by gene duplication occurs as a result of
positive Darwinian selection that accompanies the acceleration of mutant
substitutions, DNA sequences of recent duplication were analyzed by estimating the numbers of synonymous and nonsynonymous substitutions. For
the troponin C family, at the period of differentiation of the fast and slow
isoforms, amino acid substitutions were shown to have been accelerated
relative to synonymous substitutions. Comparison of the first exon of a-actin
genes revealed that amino acid substitutions were accelerated when thesmooth muscle, skeletal and cardiac isoforms differentiated. Analysis ofmembers of the heat shock protein 70 gene family of mammals indicates thatheat shock responsive genes including duplicated copies are evolving rapidly,
contrary to the cognitive genes which have been evolutionarily conservative.
For the aI-antitrypsin reactive center, the acceleration of amino acid substitution has been found for gene pairs of recent duplication. For the details,
see Genetics 138, 1331-1337.
Statistical Methods for Estimating the Effective Number of Alleles,
Expected Heterozygosity and Genetic Distance in
Self-incompatibility Locus
Fumio TAJIMA, Tohru TOKUNAGA* and Naohiko T. MIYASHITA**

In order to understand the evolutionary process of self-incompatibility, wemust know the genetic variability of the self-incompatibility genes within andbetween populations. Statistical methods for estimating the effective numberof alleles, expected heterozygosity and genetic distance from pollination
experiments were developed, which can be applied to both gametophytic andsporophytic self-incompatibility systems. In these methods, bud-pollination,***

Kurume University Fusetsu High-School, Kurume, Fukuoka.
Laboratory of genetics, Faculty of Agriculture, Kyoto University, Kyoto.

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91

which is necessary for obtaining homozygotes, is not required. Since budpollination, which is time-consuming, is not required in the present methods,
they might be useful. For details, see Jpn. J. Genet. 69, 287-295, 1994.
Comments on the Detection of Reciprocal Recombination orGene Conversion
Naoyuki TAKAHATA

Reciprocal recombination and gene conversion (in short, exchanges) haveundoubtedly been playing important roles in molecular evolutionary processes. Such exchanges may be revealed by comparing DNA sequences.
However, it is not easy to obtain clear-cut evidence for the occurrence of
exchanges between orthologous regions of DNA, because alleles at a functionallocus generally differ very little within species. Demonstrable instances
often involve exchanges between paralogous loci. Several statistical tests for
detecting sequence exchanges are available, some of which examine whether
or not nucleotide substitutions are randomly distributed along the DNA
sequences compared. For instance, Satta (1992) considers two different
regions separately, measures their relative sizes p and q (p +q = 1), andcounts the total number of substitutions (M). If both regions had evolved
without exchanges, the number of substitutions in each region is expected tofollow the binomial distribution with parameters p and M. Other tests rely on
phylogenetic trees constructed by the maximum parsimony or maximum
likelihood methods. Discordant phylogenetic trees between different regions,if found, are taken as evidence for exchanges. In this communication, I
consider the statistical strength of the tests developed by Stephens (1985) and
Sawyer (1989) in comparison with that of the Runs Test. All these tests areapplied to Mhc DNA sequences. For details, see Immunogenetics 39, 146149, 1994.
The Neutral Theory of Molecular Evolution
Naoyuki TAKAHATA and Motoo KIMURA*

There are a variety of molecular mechanisms that are responsible forchanging the genome. However, molecular evolution has not been driven* deceased.

92

solely by these mechanisms. Mechanisms acting at the individual andpopulation levels have played an equally important role in determining theprocess of molecular evolution. Of particular importance are natural selection and random genetic drift. The neutral mutation and random genetic
drift theory of molecular evolution, the neutral theory in short, claims thatthe overwhelming majority of evolutionary changes at the molecular level arecaused by random fixation of selectively neutral (i.e., selectively equivalent)mutants. The theory also asserts that most of the protein or DNA variation
within species is maintained by the balance between mutational input andrandom extinction. In this Chapter, we overview molecular evolutionary
mechanisms, and describe the neutral theory and its predictions in the light ofrecent observations. For details, see pp. 205-234 in Principles of Medical
Biology (E. E. Bittar and N. Bittar eds.), JAI PRESS London 1994.
Structure, Function, and Evolution of Mouse TL Genes, NonclassicalClass I Genes of the Major Histocompatibility Complex
Yuichi OBATA, Yoko SATTA, Kazuo MORIWAKI, Toshihiko SHIROISHI,
Hitomi HASEGAWA, Toshitada TAKAHASHI and Naoyuki TAKAHATA

In contrast to well-studied "classical" class I genes of the major histocompatibility complex (MHC) , the biology of nonclassical class I genes remains
largely unexamined. The mouse TL genes constitute one of the best definedsystems among nonclassical class I genes in the T regiotl of the MHC. To
elucidate the function and the evolution of TL genes and their relationship toclassical class I genes, seven TL DNA sequences, including one from aJapanese wild mouse, were examined and compared with those of several
mouse and human classical class I genes. The TL genes differ from eitherclassical class I genes or pseudogenes in the extent and pattern of nucleotide
substitutions. Natural selection appears to have operated so as to preserve
the function of TL, which might have been acquired in an early stage of its
evolution. In a putative peptide-binding region encoded by TL genes, the rateof nonsynonymous (amino acid replacing) substitution is considerably lower
than that of synonymous substitution. This conservation is completely
opposite that in classical class I genes, in which the peptide-binding region has
evolved to diversify amino acid sequences so as to recognize a variety ofantigens. Thus, it is suggested that the function of TL antigens is distinct

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93

from that of classical class I antigens and is related to the recognition of a
relatively restricted repertoire of antigens and their presentation to T-cell
receptors. For details, see Proc. Nat/. Acad. Sci. USA 91, 6589-6593, 1994.
Polymorphism at Mhc Loci and Isolation by the Immune
System in Vertebrates
Naoyuki TAKAHATA

The major histocompatibility complex (Mhc) contains a family of genes
whose products play crucial roles in self and nonself discrimination. Self isthe mature T cell repertoire that is restricted by Mhc molecules, whereas
nonself is processed peptides of foreign antigens that can be presented in thecontext of Mhc. Because the T cell repertoire and the complex of Mhcpeptides provide molecular basis of self defense mechanism and both of them
depend critically on Mhc genotypes, the genes must be subjected to natural
selection. A model of individual fitness is formulated in terms of the numberof functional Mhc loci, the number of heterozygous loci, the size of T cell
repertoire, and the size of Mhc peptide repertoire. Available data delimit therange of values of these parameters in the model. The mode of natural
selection on Mhc loci depends primarily on the T cell repertoire shaped bydifferent gene products. If each Mhc gene product deletes a substantial
portion of T cells, balancing selection cannot operate simultaneously on alarge number of loci. Only when the number of functional Mhc loci islimited, natural selection acts so as to produce the unusual polymorphism. If
individuals are heterozygous at all these loci or possess haplotypes being
different in the constellation of Mhc loci, their fitness may be reduced. The
reduction may happen in hybrids between antigenically diverged populations,in which case the immune system acts as a reproductive isolation mechanism.
For details, see pp. 233-246 in Non-Neutral Evolution (B. Golding ed.),
Chapman & Hall, New York, 1994.
Intensity of Natural Selection at the Major Histocompatibility Complex Loci
Yoko SATTA, Colm O'HUIGIN, Naoyuki TAKAHATA and Jan KLEINLong persistence of allelic lineages, prevalence of nonsynonymous oversynonymous substitution in the peptide-binding region (PBR), and deviation

94

from neutrality of the expected gene identity parameter F all indicate
indirectly that balancing selection is operating at functional major histocompatibility complex (MHC) loci. Direct demonstrations of the existence ofbalancing selection at MHC loci are, however, either lacking or not fully
convincing. To define the conditions under which balancing selection could
be demonstrated, we estimated its intensity, from the mean number of
nonsynonymous substitutions, K B , at the PBR and the mutation rate fJ.. We
compared the five available methods for estimating K B by computer simulation and chose the most reliable ones for estimation of selection intensity.
For the human MHC, the selection coefficients of the HLA-A, -B, -C, -DRBI,-DQBI, -DQAI, and -DPBI loci are O.Q1S, 0.042, 0.0026. 0.019, 0.0085,
0.0028, and 0.0007, respectively. This low selection intensity places severe
restrictions on the possibility of measuring selection directly in vertebrate
populations. For details, see Proc. Nat!' Acad. Sci. USA 91, 7184-7188, 1994.
Repeated Failures That Led to the Eventual Successin Human Evolution
Naoyuki TAKAHATA

The effective population number estimated in humans is about 10,000.
This rather small number may contradict the fact that humans had been
occupying vast areas in Africa and Eurasia for a long time since Homo erectus
first migrated out of Africa 1-2 Mya. One possible cause is a recent
bottleneck, or a sudden reduction in population size, but a severe bottleneck
is incompatible with the unusual polymorphism at the major histocompatibility complex (Mhc) loci. In this letter, I argue that a more likely cause isfrequent extinction and recolonization (turnover) of subpopulations in thelineage leading to modern humans. For details, see Mo!' Bio!' Eva!' 11, 803805, 1994.
Population Genetic Studies on the Evolution of SINE II
Hidenori T ACHIDA

In eukaryote genomes, there are many dispersed repetitive elements called
retroposon which amplify themselves via RNA intermediates. In order toinvestigate how these amplifications occur, three models of retroposons wereVI.

POPULATION GENETICS

95

developed and analyzed. In the transposon model, all elements can duplicate
themselves. In the master copy model, only one element per haploid genome
has the ability of duplication. Intermediate between the two extremes is thethird model in which some elements have ability of duplication but others do
not have such ability. Variances of consensus gene frequencies in the threemodels were computed. The variances were shown to be different among thethree models if the amplification period is not short. One subfamily of thehuman Alu family of retroposon was analyzed by computing the variances of
the consensus frequencies and it was found that the data is compatible withany of the three model if we assume that the amplification period is short.
However, mutations were found to have occurred on duplicative elements inseveral sites and the probability of obtaining this pattern would be small if the
duplication period is short and the number of duplicative elements is small.
From these considerations, it was inferred that this subfamily of the Alu
element amplified in a short period and there were multiple duplicative
elements.
Genetic and Geographical Differentiation of Subspecies Groups of
Mus musculus in East Asia
Kazuo MORIWAKI, Nobumoto MIYASHITA*t, Kimiyuki TSUCHIYA*2, Yoichi MATSUDA*3,
Shunnosuke NATSUME-SAKAI*4, Hiromichi YONEKAWA*s, Hitoshi SUZUKI*6,
Cheng-huai W ANG*', Xiang-lin wu*', Mei-lei JIN*', Hai Li*9,
Fengshan WANG*", Alexei P. KRYUKOY*IO, Lubov V. FRIESMAN*IO
and Ludmila V. YAKIMENKO*IO

Since 1983, we have surveyed genetic variations among natural populationsof house mouse, Mus musculus, in vavious areas of China, the area west ofLanzhou, the southern area including Tibet, the central area, the northernarea including Mongolia and the northeast area of China. Molecular
*'
*2
*3
*4

*s
*6
*'
*'
*"

Kagawa Medical College, Takamatsu.
Miyazaki Medical College, Miyazaki.
National Institute of Radiological Science, Chiba.
Kanazawa University Medical School, Kanazawa.
Tokyo Metropolitan Institute of Medical Science, Tokyo.
Jikeikai Medical University, Tokyo.
Lanzhou Institute of Biological Products, Lanzhou.
Shanghai Laboratory Animal Center, Shanghai.
Institute of Biology and Pedology, Vladiovostok.

96

analyses of genetic polymorphisms in mitochondrial DNAs, ribosomal
DNAs, SRY DNAs on the Y-chromosome, hemoglobin-beta (Hbb) andothers, have revealed the presence of two major subspecies groups of thehouse mouse in these areas which are the castaneus group and the musculus
group. The former inhabits the area south of the Yangtze river and the latter
inhabits the area north of the river including the northeast, Korean peninsula
and Japan. More detailed analyses demonstrated a further differentiation of
the subspecies groups. In the musculus subspecies group, the western China
populations which were classified as the gansuensis subspecies based onmorphology could also be characterized by a new Hbb-wl. The central
populations, the homourus subspecies, were characterized by Hbb-p and thenorthern populations including the northeast, Korea and Japan, the molossinus subspecies, were separated by their characteristic SRYmo. In the
castaneus subspecies group, the southern China population and southeast
Asian population could be clearly discriminated by D-Ioop analysis in mitochondrial DNAs. It should be noted that the northern Japanese populations
show the latter type mt-DNA. This is probably related to the histoucal
human movement from southeast Asia to Japan.

97

VII.

EVOLUTIONARY GENETICS

Early Evolution of Genes and Genomes
Tomoko OHTA

In the early evolution of gens and genomes, ribozymes and reverse tarnscriptase probably played important roles. In such a system, reverse flow ofgenetic information may have been more frequent than in the present DNA
world. The interaction between natural selection and reverse flow of genetic
information might have provided an efficient environmental feedback to
genes and genomes. For details, see In Early Life on Earth (Bengtson, S. ed.)pp. 135-142, Columbia Univ. Press.
Estimation of Evolutionary Distance for Reconstructing
Molecular Phylogenetic Trees
Fumio T AJIMA and Naoko T AKEZAKI*The most commonly used measure of evolutionary distance in molecularphylogenetics is the number of nucleotide substitutions per site. However,this number is not necessarily most efficient for reconstructing a phylogenetic
tree. In order to evaluate the accuracy of evolutionary distance, D(t), forobtaining the correct tree topology, an accuracy index, A (t), was proposed.
This index is defined asD'(t)f!V[D(t)], whereD'(t) is the first derivative ofD(t) with respect to evolutionary time and V[D(t)] is the sampling varianceof evolutionary distance. Using A (t), namely, finding the condition underwhich A (t) gives the maximum value, we can obtain an evolutionary distancethat is efficient for obtaining the correct topology. Under the assumption thatthe transversional changes do not occur as frequently as the transitionalchanges, we obtained the evolutionary distances which are expected to givethe correct topology more often than are the other distances. For details, see
Mol. BioI. Eva!. 11, 278-286, 1994.

* Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania
State University, University Park, Pennsylvania, U.S.A.

98

Human Pseudoautosomal Boundary-Like Sequences (PABLs): Core andConsensus Sequence, Expression, and Involvement in Evolutionary
Formation of the Present Day Pseudoautosomal Boundaryof Human Sex Chromosomes
Tatsuo FUKAGAWA 1, Yasukazu NAKAMURA!, Katsuzumi OKUMURA 2,
Asako ANDO', Hidetoshi INOKo" Naruya SAITOU I
and Toshimichi IKEMURA 1

The human genome, like those of warm-blooded vertebrates in general, has
long-range mosaic structures of G+C% (GC%), which are thought to berelated to chromosome bands. We previously disclosed a boundary for
Mbp-Ievel domains of G+C% mosaic structures in the human MHC andfound in the domain boundary, a sequence highly homologous with thepseudoautosomal boundary (PAB) of human sex chromosomes. We designated the sequence "PABL" and found many PABL-type sequences in thehuman genome. Expression of the PABL sequences was also disclosed. In
analyzing a total of twelve PABLs (six genomic and six cDNA sequences), aca. 650 nt of the core and consensus sequence could be defined; a strict
conservation of the 3' and 5' edges of the PABLs including sex-chromosome
PABs was found. Northern blot analysis showed the sizes of PABL transcripts to be 5-10 kb.
The divergence time of PABLs was estimated to be 60-120 million years
ago after analyzing five human PABLs and sex-chromosome PABs of sevenprimates. The evolutionary rates deduced showed PABLs to have been undera selective constraint. A model of evolutionary formation of the present-day
pseudoautosomal boundary was proposed by postulating an illegitimate recombination between two PABLs. For details, see Genomics 25, 184-191(1995) & Human Molecular Genetics (in press).

I

2

,

Division of Evolutionary Genetics, National Institute of Genetics, and The Craduate
University for Advanced Studies.
Division of Physiological Genetics.
School of Medicine, Tokai University.

VII.

EVOLUTIONARY GENETICS

99

A Notch-family Gene Found in the Human MHC Class III Region
Near the Junction with the Class II: Human Counterpartof Mouse Mammary Tumor Gene int-3
Kimihiko SUGAYA 1, Asako AND02, Hidetoshi INOK0 2
and Toshimichi IKEMURA 1

The genomes of higher vertebrates are composed of long-range mosaic
structures of G+C content (GC%), which are related to chromosome
bands. During the processes to characterize the border of the Mb-level GC%
mosaic domains previously located in the human MHC, we found the humancounterpart of the mouse mammary tumor gene int-3 near the junction of the
MHC class III with the class II. Integration of the mouse mammary tumor
virus (MMTV) into the int-3 locus promotes the transcription of the flanking
mouse cellular int-3 sequence which shares significant homology with theintracellular domain of the Drosophila neurogenic Notch gene. The humangene found in the present work contained not only the intracellular domainpart present in the int-3 sequence but also the extracellular part present in
typical Notch-family genes, showing it to correspond to the human counterpart of an uninterrupted form of the transmembrane protein gene predicted
at the mouse int-3 locus. Human cDNA clones of this NOTCH-related gene
were isolated and sequenced, clarifying its gene organization. In the 5'
upstream region, there was a trinucleotide-repeat cluster, which shows evident polymorphism among human populations and thus is a useful marker instudying the correlation of this gene with the diseases suspected in therespective region, as well as in searching for other disease-related genes. By
constructing phylogenetic trees based on all available sequences of Notchfamily genes including the present gene, four subfamilies for mammalian
Notch were found (manuscript in preparation).

I

2

Division of Evolutionary Genetics, National Institute of Genetics, and The Graduate
University for Advanced Studies.
School of Medicine, Tokai University.

100

Estimation of Protein-Production Levels for ORFs Registered in E. coli
Genome Projects, Based on Levels of "Optimal Codon" Usage,in Connection with the Feasibility of Protein Coding Ability
and with Assignment to Foreign-Type Genes
Yasukazu NAKAMURA! and Toshimichi IKEMURA!

The choice among synonymous codons is clearly non-random, although it
does not affect the nature of proteins synthesized. Among the genes of each
unicellular organism, there is a clear similarity of codon choice patterns,
regardless of gene function; i.e., the "codon dialect" of the organism. We
previously found the extent of codon bias (accent of the codon dialect) isrelated to the protein production level. Codon usage in genes encoding
abundant proteins is always much more dependent on tRNA content (strong
accent) than that in moderately or poorly expressed genes (moderate accent).
It is also known that foreign-type genes (e.g., transposons, plasmids and virus
genes) often have quite different codon patterns from the host dialect. To
examine these features quantitatively, the frequency of use of "optimal
codons" was previously defined; the "optimal codon" is the codon translatedby the most abundant isoacceptor. The cellular content of many E. coliproteins was measured by Neidhardt and his colleagues allowing us to study
the correlation between frequency of optimal codon use (Fop) and gene
expressivity. A definite correlation between Fop and protein content was
revealed; Fop for the highly expressed genes was almost always high, but low
for weakly expressed genes. The Fop of foreign-type genes was significantly
lower than that of intrinsic genes. We calculated the Fop of 603 ORFs
registered in E. coli genome projects of Japan and USA. Based on the Foplevels, we could classify these ORFs into highly-, moderately- or weaklyexpressed genes, as well as into foreign-type genes. Among those registeredby the USA group, they often included ORFs that are shorter than those in
the Japanese project. The feasibility of individual short ORFs as proteincoding regions was proposed. For details, see Genome Informatics Workshop Series No.5, pp. 188-189 (1994) & Nucleic Acids Research (in press).

I

Division of Evolutionary Genetics, National Institute of Genetics, and The Graduate
University of Advanced Studies.

VII.

EVOLUTIONARY GENETICS

101

Evolutionary Rate of Insertions and Deletions in Non-Coding
Nucleotide Sequences of Primates
N. SAITOU and S. UEDA*

Insertions and deletions are responsible for gaps in aligned nucleotide
sequences, but they have been usually ignored when the number of nucleotide
substitutions was estimated. We compared six sets of nuclear and mitochondrial non-coding DNA sequences of primates, and obtained the estimates ofthe evolutionary rate of insertions and deletions. The maximum parsimony
principle was applied to locate insertions and deletions on a given phylogenetic tree. Deletions were about twice as frequent as insertions for nuclear
DNA, and single nucleotide insertions/deletions were the most frequent in all
events. The rate of insertions/deletions was found to be rather constantamong branches of the phylogenetic tree, and the rate (ca. 2.0 per kb per
million years) for mitochondrial DNA was found to be much higher thanthat (ca. 0.2 per kb per million years) for nuclear DNA. The rates ofnucleotide substitutions were about ten times higher than those of insertions/deletions both for nuclear and mitochondrial DNAs. For details, see Mol.
Bioi. Evol. 11, 504-512 (Saitou and Veda, 1994).
Genetic Analysis and Molecular Phylogeny of Simian T-cell
Lymphotropic Virus Type I: Evidence for Independent
Virus Evolution in Asia and Africa
K.-J. SONG**, N. SAITOU and R. YANAGIHARA**

Type C retroviruses, designated simian T-cell lymphotropic virus type I(STLV-I), have been isolated from several genera of Old World monkeys andapes, but not from New World monkeys and prosimians. To determine thegenomic diversity and molecular evolution of STLV-I and to clarify their
genetic relationship to human T-celllymphotoropic virus type I (HTLV-I),
we enzymatically amplified, then directly sequenced selected regions of thegag, pol, env, andpX genes ofSTLV-1 strains from Asia and Africa. STLV-I
strains Si-2, Matsu, and JM86 from Japanese macaques, which exhibitedsequence similarities ranging from 98.5 to 99.8% among themselves, diverged***

Graduate School of Science, University of Tokyo.
National Institute of Neurological Disorders and Stroke, NIH, U.S.A.

102

by 12.9 to 13.3% from STLV-I strain MM39-83 from a naturally infectedrhesus macaque, by 9.7 to 11.2% from STLV-I strains from Africa, and by
8.8 to 11.2% from HTLV-I strains originating in Japan, India, Africa, theCaribbean, the Americas, Polynesia, and Melanesia. By contrast, the interspecies nucleotide sequence similarity among African STLV-I strains fromgreen monkey, yellow baboon, sooty mangabey, and common chimpanzee
was remarkably high, ranging from 96.9 to 97.4%, and these STLV-I strains
diverged by only 2.2 to 2.8% from HTLV-I strain EL from equatorial Zaire.
Phylogenetic trees constructed by using the neighbor-joining and maximum
parsimony methods indicated that the Asian STLV-I strains diverged fromthe common ancestral virus prior to African STLV-I and cosmopolitan andMelanesian HTLV-I strains. Thus, our data are consistent with an archaicpresence of STLV-I in Asia, probably predating macaque speciation, withsubsequent independent virus evolution in Asia and Africa. For details, see
Virology 199, 56-66 (Song et al. 1994).
Population Genetic Study in "ainan Island, China.
II. Genetic Affinity Analysis
N. SAITOU, K. OMOTO*, C. Du** and R. Du***A population genetic study was carried out on six populations (two Li
populations, two Miao populations, one Hui population, and one Han population) in Hainan Island, Southern China. Allele frequency data for these six
populations and those for some other Asian populations were used to estimate
genetic distances between populations, and genetic affinity dendrograms andnetworks were constructed by using UPGMA and the neighbor-joining
method, respectively. The two Li populations showed a close relationship,
while the two Miao populations were relatively distant from each other.
These six populations of Hainan Island showed a close affinity with Zhuangof Southern China, Thailanders, and Javanese, but they are distant from
populations around northern China (Japanese, Mongolian, and Korean).
Divergence time between the Li-Miao group and Japanese was estimated to
be roughly 19,000-26,000 years. For details, see Anthropological Science
102, 129-147 (Saitou et al. 1994).
*
**
***

International Research Center for Japanese Studies.
Sun Yazt-sen University of Medical Sciences, China.
Institute of Genetics, Academica Sinica, China.

VII.

EVOLUTIONARY GENETICS

103

Molecular Evolution of Serine Protease and Its Inhibitor with
Special Reference to Domain Evolution
Takashi GOJOBORI and Kazuho IKEo

The evolution of serine protease and its inhibitor are 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 are made up of various functional
domains, these proteins are typical examples of the so-called mosaic proteins.
When Kringle domains in serine proteases and a Kunitz-type proteaseinhibitor domain in the amyloid b precursor protein in Alzheimer's disease
patients were examined by 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 evolutionarily movable. Thus, it was concluded that various functional
domains evolved independently of each other and that they have been shuffledto create the existent mosaic proteins. This conclusion leads us to thereasonable speculation that those functional domains must have been minigenes possibly at the time of primordial life or the origin of life. We call theseminigenes 'ancestral minigenes'. Every effort should be made to answer
questions about the minimum set of ancestral minigenes that must haveexisted and must have been needed for maintaining life forms. The DNA
sequence database is useful for making attempts to answer such difficult but
significant questions. For details, see Phil. Trans. Royal Soc. Land. B344,
411-415.
NRSub: A Non-redundant Data Base for the Bacillus subtilis Genome
Guy PERRIERE, Manolo GOUY and Takashi GOJOBORI

We have organized the DNA sequences of Bacillus subtilis from the EMBL
collection to build the NRSub data base. This data base is free from
duplications and all detected overlapping sequences were merged into contigs. Data on gene mapping and codon usage are also included. NRSub is
publically available through anonymous FTP in flat file format or structured
on the form of an ACNUC data base. Under this format, it is possible to use
NRSub with the retrieval program Query win. This program integrates a

104graphical interface and may be installed on any kind of UNIX computerunder X Window and on which the Vibrant and Motif libraries are available.
For details, see Nucl. Acids Res. 25, 5525-5529
Confirmation by Molecular Evolutionary Analysis of Hepatitis C
Virus Transmission through Needlestick Accidents
Kaoru SUZUKI, Masashi MIZOKAMI, Johnson Y. N. LAu, Naoto MIZOGUCHI,
Koji KATO, Yoshiki MIZUNO, Takeshi SODEYAMA,
Kendo KIYOSAWA and Takashi GOJOBORI

To document the transmission of hepatitis C virus (HCV) through needlestick accidents, 3 health workers who acquired HCV through such accidents
and their HCV donor patients were studied using molecular evolutionary
analysis based on the HCV E2 region. At least six clones were sequenced
from each subject. Nucleotide substitutions were estimated by the sixparameter method, and a phylogenetic tree was constructed by the neighborjoining method. HCV isolates from the donor patient and the recipient were
nested in one monophyletic cluster; this clustering was confirmed to bestatistically significant by bootstrap analysis. The nucleotide divergenceamong the isolates from the recipient was always smaller than that from thedonor, supporting the notion that the direction of transmission was from thedonor to the recipient. These findings provide evidence, at a molecular
evolutionary level, that HCV was transmitted through needle-stick accidents.
For details, see J. Infectious Diseases 170, 1575-1578.
Evolution of Pathogenic Viruses with Special Reference to theRates of Synonymous and Nonsynonymous Substitutions
Takashi GOJOBORI, Yumi YAMAGUCHI, Kazuho {KEOand Masashi MIZOKAMI

For pathogenic viruses such as human immunodeficiency virus (HIV),hepatitis B virus (HBV), hepatitis C virus (HCV), human influenza A virus,
and human T-cell leukemia virus type I (HTLV-I), evolutionary features
were briefly reviewed with special reference to the rates of synonymous andnonsynonymous substitutions. In particular, these rates were discussed inconnection with the neutral theory of molecular evolution. Common to all

VII.

EVOLUTIONARY GENETICS

lOS

five pathogenic viruses was the fact that the rate of synonymous substitution
was higher than that of nonsynonymous substitution particularly when entire
gene regions were compared between different isolates. This suggests thatviral proteins are quite conservative in regards to functional and structuralchanges even though most of these viral genomes are evolving at a speed
much higher than that of their host genomes. Thus, this feature is consistent
with the neutral theory. However, it can also be pointed out that positive
selection may be operating on some specific sites such as antigenic sites inorder for the pathogenic viruses to escape from the host immune system. For
details, see Jpn. J. Genet. 69, 481-488.
Reduction of Synonymous Substitutions in the Core Protein
Gene of Hepatitis C Virus
Yasuo INA, Masashi MIZOKAMI, Kenichi OHBA and Takashi GOJOBORI

Molecular evolutionary analyses were carried out to elucidate the phylogenetic relationships, evolutionary rate, and divergence times of hepatitis C
viruses. Using the nucleotide sequences of viruses isolated from various
locations in the world, we constructed phylogenetic trees. The trees showedthat strains isolated from a single location were not necessarily clustered as agroup. This suggests that the viruses may be transferred with blood on aworldwide scale. We estimated the evolutionary rates at synonymous andnonsynonymous sites for all genes in the viral genome. We then found thatthe rate (1.35 X 10- 3 per site per year) at synonymous sites for the C gene wasmuch smaller than those for the other genes (e.g., 6.29 X 10- 3 per site per
year for the E gene). This indicates that a special type of functional
constraint on synonymous substitutions may exist in the C gene. Because wefound an open reading frame (ORF) in the C gene region, the possibility
exists that synonymous substitutions for the C gene are constrained by theoverlapping ORF whose reading frame is different from that of the C gene.
Applying the evolutionary rates to the trees, we also suggest that majorgroups of hepatitis C viruses diverged from their common ancestor several
hundred years ago. For details, see J. Mol. Evol. 38, 50-56.

106Phylogenetic Subtypes of Human T-Iymphotropic Virus Type I and
Their Relations to the Anthropological Background
Tomoyuki MIURA, Yasilo INA, Takashi GOJOBORI and Masanori HAYAMI

Isolates of human T-Iymphotropic virus type I (HTLV-I) from native
inhabitants in India and South America (Colombia and Chile) and from the
Ainu were phylogenetically analyzed. Their genomes were partially sequenced together with isolates from Gabon in central Africa and from Ghana inWest Africa. A phylogenetic tree was constructed from the sequence dataobtained and those of previously reported HTLV-I isolates and simian
T-Iymphotropic virus type I (STLV-I) isolates. The heterogeneity of
HTLV-I was recently recognized, and one major type, generally called the"cosmopolitan" type, contained Japanese, Caribbean, and West Africa isolates. The phylogenetic tree constructed in the present study showed that thiscosmopolitan type can be further separated into three lineages (subtypes A,
B, and C). Subtype A consists of some Caribbean, two South America, andsome Japanese isolates, including that from the Ainu, in addition to an India
isolate. Subtype B consists of other Japanese isolates in addition to anotherIndian isolate, suggesting that there might be at least two ancestral lineages of
the Japanese HTLV-I. Subtype C consists of the West African and other
Caribbean isolates, indicating that not all parts of the Caribbean strains
directly originated from West Africa. The tree also has showed that the
HTLV-I isolate from Gabon in central Africa forms a cluster with STLV-I
from a chimpanzee, suggesting a possible interspecies transmission between
man and the chimpanzee in the past. For details, see Proc. Nat/. Acad. Sci.
USA 91, 1124-1127.
Toward Unification of Taxonomy Databases in a Distributed
Computer Environment
Hajime KITAKAMI, Yoshio TATENO and Takashi GOJOBORIAll the taxonomy databases constructed with the DNA databases of theinternational DNA data banks are powerful electronic dictionaries which aid
in biological research by computer. The taxonomy databases are, however,not consistently unified with a relational format. If we can achieve consistentunification of the taxonomy databases, it will be useful in comparing many

VII.

EVOLUTIONARY GENETICS

107

research results, and investigating future research directions from existentresearch results. In particular, it will be useful in comparing relationships
between phylogenetic trees inferred from molecular data and those constructed from morphological data. The goal of the present study is to unify
the existent taxonomy databases and eliminate inconsistencies (errors) thatare present in them. Inconsistencies occur particularly in the restructuring of
the existent taxonomy databases, since classification rules for constructing
the taxonomy have rapidly changed with biological advancements. A repair
system is needed to remove inconsistencies in each data bank and mismatches
among data banks. This paper describes a new methodology for removing
both inconsistencies and mismatches from the databases on a distributed
computer environment. The methodology is implemented in a relational
database management system, SYBASE. For details, see ISMB94, pp. 227235, Stanford Univ. Press.
Molecular Evolutionary Virology: Its Application to Hepatitis C Virus
Masashi MIZOKAMl, Takashi GOJOBORI and Johnson Y. N. LAu

We described the importance and usefulness of molecular evolutionary
analysis in the study of pathogenic viruses. In particular, HCV (hepatitis C
virus) and HIV (human immunodeficiency virus) are known to have a rate ofnucleotide substitution million times greater than hosts such as humans. The
extremely high rate of nucleotide substitutions for these viruses enables us topredict future evolution and to test it a few years later. For example, thepatterns of nucleotide substitutions can be estimated by constructing phylogenetic trees. In particular, the direction from a particular nucleotide to othernucleotides can be identified if we can successfully infer the ancestral
nucleotide at each branching point from the phylogenetic tree. This will bevery useful for developing effective vaccines because the future direction ofsubstitutions can be predicted with certain probabilities. For details, see
Gastroenterology 107, 1181-1182.
Molecular Evolution of the Major Epitopes of HIV within a Single Host
Yumi YAMAGUCHI and Takashi GOJOBORIThe third variable envelope region (V3) is one of the major epitopes of108

HIV (human immunodeficiency virus). We intended to elucidate the evolutionary mechanisms of the V3 region of HIV within a single host. We
collected nucleotide sequence data of HIV clones isolated from a single hostat several time points after infection. We estimated the rates of synonymousand nonsynonymous substitutions for the V3 region of HIV within a single
host during each period of time. In some periods of time after infection, therate of nonsynonymous substitution for the V3 region was higher than that ofsynonymous substitution. We also estimated the number and type of aminoacid substitutions that occurred at each amino acid site in the V3 region. It
was found that amino acid substitutions dominantly occurred at several
particular amino acid sites where the substitutions are known to be responsible for the production of antigenic variation and determination of viral
phenotypes. These observations indicate a strong possibility that positive
selection is operating in the V3 region of HIV within a single host.
A Large-Scale Search for Genes on which Positive Selection May Operate
Toshinori ENDO, Kazuho IKEo and Takashi GOJOBORIThe neutral theory of molecular evolution predicts that most genes evolveby accumulation and fixation of neutral mutations. The theory, however,does not deny the possibility that positive selection operates on some genes.
The proportion of such genes among all the genes available has not yet been
estimated. Thus, we have searched for such genes from the three DNA
sequence databases of DDBJ, EMBL, and GenBank. By making a large
number of alignments for homologous DNA sequences, we then compared
the estimated numbers of synonymous substitutions with those of nonsynonymous substitutions. As a result, we found that in 3,595 homologous
gene groups, positive selection may operate on sixteen gene groups. Eight outof sixteen gene groups were surface proteins of parasites and viruses. We
then performed the newly developed window analysis method to find thewithin-gene region on which positive selection may operate. The window
analysis revealed that positive selection may operate on a within-gene region
where an antigenic epitope is located. We further searched the genes that
contained in within-gene regions where positive selection may operate. We
found that these 186 gene groups were regarded as candidate genes on which
positive selection may operate.

VII.

EVOLUTIONARY GENETICS

109

Gene Conversion in the Major Histocompatibility Complex Genes
Tadashi IMANISHI

Human major histocompatibility complex (MHC) genes comprise a multigene family, and many of them show high degrees of genetic polymorphisms.
One of the hypothesized mechanisms that generate polymorphism is geneconversion, by which allelic segments of dozens of nucleotides are copied toother alleles or alleles of other loci. In this study, I examined the possibilityof gene conversion in the MHC genes and its evolutionary significancethrough extensive comparisons of nucleotide sequence data. In particular,
distribution of locus-specific substitutions, nucleotides observed exclusively ina particular locus and not in other loci, and shared DNA polymorphism
between different loci, were examined. Results showed shared DNA polymorphism was significantly abundant in the hyper-variable exons of bothclass I (HLA-A, B, and C) and class II (HLA-DRB1, DQB1, and DPB1)
MHC loci. This observation can be accounted for, either by gene conversion
between different, duplicated loci or by natural selection favor~ng the same
sets of amino acid sequences.
Studies of Genetic Variability in Human MHC Genes
Tadashi IMANISHI

The genetic variability in human MHC genes (HLA) was investigated at
the DNA level and at the population level. New alleles of class I HLA were
found and their nucleotide sequences were determined. The molecular
evolution of the new alleles was examined by constructing a phylogenetic tree
(Ishikawa, Y. et al. (1994) Human Immunology 39, 220-224). Moreover,the frequencies of DNA-typed HLA alleles and haplotypes were estimatedfor the Japanese population (Hashimoto, M. et al. (1994) Tissue Antigens 44,
166-173; Akaza, T. et al. (1994) Transplantation Now 7: s87-s99 (inJapanese)). Furthermore, the gene frequencies and haplotype frequencies in
HLA were estimated for the Buryat population in northeast Asia andJamaican populations in South America. The phylogenetic relationships ofthese populations and the surrounding populations were analyzed by using
dendrograms (Tokunaga, K. et al. (1995) Tissue Antigens 45, 98-102; Blank,
M. et al. (1995) Tissue Antigens 45, 111-116). These studies revealed not110only the distribution of genetic variability in human MHC genes at the
population level and at the molecular level, but also the evolution of humanpopulations and MHC genes themselves.
Evolution of Glutamine Synthetase Gene Is in Accordance with the
Neutral Theory of Molecular Evolution
Yoshio TATE NO

The evolution of the glutamine synthetase gene was studied by analyzing
30 homologous DNA sequences of the gene. The sequences of variousorganisms spanning from prokaryotes to eukaryotes were collected from the
DDBJ database. They were translated first, aligned next, then evolutionary
distances were computed, and the molecular phylogeny was finally obtained.
The results of the alignment revealed that functionary important regions of
this enzyme have been evolutionary more conserved than the remaining
regions which are not directory related to its function. The evolutionary
distances computed show that the rate of synonymous substitutions is higher
than that of nonsynonymous substitutions. These results are in accordancewith the neutral theory of molecular evolution.
Moreover, the molecular phylogeny estimated indicates that the origin ofglutamine synthetase gene is much older than the divergence between eukaryotes and prokaryotes, suggesting that the gene is one of the oldest genes
functioning at present. (published in Jpn. J. Genet 69, 489-502, 1994)
Yamato and Asuka: DNA Database Management System
H. KITAKAMI, T. SHIN-I, K. IKEO, Y. UGAWA, N. SAITOU,
T. GOJOBORI and Y. TATENO

We recently developed a relational schema for effectively building, integrating, and searching the DNA database on the relational databasemanagement system, Sybase at DDBJ. The schema is called the DDBJ
schema. The schema allows us to implement a DNA database management
system with two types of window interfaces, Yamato and Asuka, to easily
build the DNA database. The first type is used by the data reviewers forprocessing a single entry of the data sent by a researcher. The second one isused by genome project groups for processing multiple entries. Both types

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have a window interface similar to that for the flat file format.
We have also developed two subsidiary systems for Yamato and Asuka.
One is a restructuring tool for converting the data in the GenBank schema
into those in the DDBJ schema. The other is a tree search tool needed forcorrecting errors in the taxonomy database. A structured SQL programming
method is proposed for implementing these subsidiary systems. The method
was developed by making use of the SQL expansion and the control flow
language of Sybase. (published in The Proceedings of the 28 HawaiiInternational Conference on System Sciences, pp. 72-80, IEEE Computer
Society, 1994)
Building and Search System for a Large-Scale DNA Database
H. KITAKAMI, Y. YAMAZAKI, K. IKEO, Y. UGAWA, T. SHIN-I,
N. SAITOU, T. GOJOBORI and Y. TATENO

We at DDBJ had used a flat-file system to operate our DNA databasesystem until two years ago. But the flat-file system was inadequate for
integrating and searching a large-scale database in which the number of
entries was increasing at an explosive rate. Then we adopted a relational
database management system, the GenBank Schema, from GenBank in the
United States. This system worked on Sybase. We also installed theannotator's work bench (AWB), which was a tool for building the DNA
database with the schema. AWB, however, did not have a simultaneous
processing function for large amounts of DNA data such as EST data.
Moreover, the integrating function was not operated, nor was the searching
function at DDBJ.
Thus we recently developed a hierarchical relational schema for effectively
building, integrating, and searching the DNA database on Sybase. The
schema is called the DDBJ schema. The schema allows us to implement awindow interface to easily build the DNA database. The database in the
GenBank schema can be converted to that in the DDBJ schema by use of a
restructuring tool we also developed. We have proposed a structured SQL
programming method to implement the restructuring tool. The method wasdeveloped by using the control flow language of Sybase. We have also
proposed two more methods by which to execute those tools on the UNIX
based workstation connected to the computer network. (published in Advances in Molecular Bioinformatics, pp. 123-138, lOS press, 1994)

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VIII.

HUMAN GENETICS

Molecular Definition of the Prader-Willi Syndrome Chromosome
Region and Isolation of Transcribed Sequences by
Positional Cloning Method
Takashi IMAMURA, Masako SAKAI, Rie INABA, Kenji IZUHARA
and Tomoko HASEGAWA

The prader-Willi syndrome (PWS) is characterized by psychomoter andgrowth retardation, infantile hypotonia, characteristic dysfunction causing
hypogonadism and hyperphagia with obesity. Originally defined as a clinicalentity, PWS was later found to be associated with abnormalities of chromosome 15, in particular a small interstitial deletion of bands q 11.1-q 13 thatinvolve the paternally derived chromosome 15. Non-deletion PWS patients
exihits maternal disomy for chromosome 15 further demonstrating that lossof the expressed paternal alleles of maternally imprinted genes is responsible
for the PWS phenotype. Conversely, maternally derived deletions of thisregion or paternal disomy are associated with the Angelman syndrome (AS)that is clinically quite distinct and characterized by more severe mental
retardation, absent speech, microcephaly, seizures, a movement disorder,
inappropriate laughter and typical facies. AS can also be caused by paternal
disomy or maternally inherited as yet unidentified submicroscopic mutations.
Thus, PWS and AS are distinct neurogenic disorders, which are caused by theloss of function of closely linked genes on chromosome 15. Paternal deletionand maternal disomy in PWS suggest that the PWS genes are transcribedfrom the paternal chromosome only. Likewise, maternal deletions andpaternal disomy in AS suggest that the AS genes are transcribed from thematernal 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
another from the patient. Application of this method to cDNA library of

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113

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.
To identify the locus from which these cDNAs were derived and to characterize the genomic structure of loci, we have carryed out quantitative
polymerase chain reaction amplification of human genomic DNA using
primers derived from the cDNA sequences. Since the patient with PWS are
identified to be hemizygous for the genes, the results indicate that thesetranscribed sequences appear to be deleted on the paternally derived chromosome 15, and that genes on the maternally derived chromosome 15 are
imprinted and completely suppressed.
Isolation of Candidate Transcribed Sequences in Cloned Genomic DNA
with Known Lacation on Human Chromosome 18
Takashi IMAMURA, Masako SAKAI, Rie INABA and Hitoshi NAKASHIMA

Identification and recovery of transcribed sequences from cloned mammlian genomic DNA remains an important problem in isolating genes on thebasis of their chromosomal location. We have developed a strategy thatfacilitates the recovery of exons from random pieces of cloned genomic
DNA. We have isolated 600 cosmid clones of which 60 new cosmids are
mapped on the short and long arms of chromosome 18 either by R or by
DAPI banding and simultaneous fluorescence in situ hybridization. In thefurther extension of this approach, cDNA library have been probed withpools of microclones from a cosmid clone. Our approach to generateregion-specific cDNA libraries used hybridization of cDNA library inserts togenomic DNA from cosmids, and subsequent cloning of annealed cDNAs.
In one application of the scheme, human fetal brain cDNA library was
hybridized to linker-adaptor ligated fragments of a digested cosmid, andselected cDNAs are amplified by PCR before cloning.
The approaches are designed to detect transcribed sequences from genomic

114

DNA of known location. A regional assignment of cosmids could be madeby fluorescence in situ hybridization to metaphase chromosomes. The
purpose of this work was to get a molecular approach for identifying genes
within the human genome. 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, itssequences and its pattern of expression. Since most genes are composed ofmultiple exons and the identification of a gene requires the recovery of onlya single exon, this consideration should not be a limiting factor. These
candidtate exons are ideally suited for establishing the presence of a gene ina cosmid insert and facilitating the subsequent isolation of this gene. Ourcurrent experience suggests that as many as 20 cosmids can be screened
concurrently in a 4 week period, and that everyone or two cosmids containat least one transcribed sequence. This screen of uncharacterized cosmids
will determine the utility of transcribed sequence fragments of known chromosomal location, as identifiers of candidate coding sequences.
Chromosome 18 is involved in tetrasomy 18p syndrome as well as intrisomy 18. 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.
Abnormality of JAK-STAT Pathway Induced by Interleukin-4 inPatients with X-linked Severe Combined Immunodeficiency
Kenji IZUHARA, T. HElKE, Masako SAKAI, Rie INABA, M. MAYUMI,
Takashi IMAMURA and Nobuyuki HARADAInterleukin-4 (lL-4) activates members of a few tyrosine kinase family,
JAK1 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 (rc)with IL-2, IL-7 and IL-9 receptor systems. Mutations of rc are known toresult in human X-linked severe combined immunodeficiency (XSCID).
Since JAK3 has been shown to associate with rc, we analysed activation of

VIII.

HUMAN GENETICS

liS

JAK-STAT pathway in EBV-transformed B cell lines established from two
XSCID patients (XSCID-B), in order to investigate the involvement of rc in
IL-4 mediated signal transduction. We detected no tyrosine phosphorylationof 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 activated byIL-4 in normal B cells, but not in XSCID-B cell lines. These results indicatethat JAK-STAT pathway in IL-4 signal transduction is impaired in XSCID,
further suggesting that rc plays a pivotal role in activation of JAK-STAT
pathway by IL-4.

A New Point Mutation at Nucleotide Pair 3291 of the Mitochondrial
tRNA1eu<UURl Gene in a Patient with Mitochondrial Myopathy,
Encephalopathy, Lactic Acidosis, and Stroke-Like
Episodes (MELAS)
Yu-ichi GOTO*, Kazuo TSUGANE, Yuzo T ANABE**,
Ikuya NONAKA* and Satoshi HORAI

Examples of the association between mitochondrial DNA (mtDNA) mutations and mitochondrial encephalomyopathies have followed in succession
since the first report by Holt and his colleagues. This genotype-phenotype
relationship includes, for instance, mtDNA deletion or duplication in chronic
progressive external ophthalmoplegia (CPEO), a point mutation at nucleotide pair 8344 in myoclonus epilepsy with ragged-red fibers (MERRF) and apoint mutation at 3243 in mitochondrial myopathy, encephalopathy, lactic
acidosis and stroke like episodes (MELAS). In MELAS, we have found asecond common mutation at nucleotide pair 3271 in Japanese patients. Both3243 and 3271 mutations are not limited to the Japanese but have been foundin other racial groups. We have also shown that a mutation at nucleotide pair11084 which has been proposed as a third one could be an mtDNA polymorphism prevalent in Japanese. A new point mutation at nucleotide pair 3291
in the mitochondrial tRNA Leu(UUR) gene was found in a Japanese MELAS
patient. The nucleotides at the mutated site were evolutionarily invariant
from humans through sea urchins. The mutant genomes were detected in a***

National Center of Neurology and Psychiatry.
Chiba Children's Hospital.

116

heteroplasmic fashion in muscle and blood cells of the proband by means of
PCR-RFLP. Among 46 MELAS, 5 MERRF, 23 CPEO and 55 normal
controls examined, this is the only patient with the mutation. This is the third
mutation associated with MELAS in addition to nucleotides at 3243 and3271. All three mutations occurred within the tRNA-Leu(UUR) region
indicating that the tRNA alteration is responsible for the MELAS phenotype.
For details, see Biochem. Biophys. Res. Comm. 202(3), 1624-1630 (1994).
Point Mutations in Mitochondrial tRNA Genes: Sequence Analysis of
Chronic Progressive External Ophthalmoplegia (CPEO)
Yuko HATTORI*, Yu-ichi GOTO**, Ryoichi SAKUTA**, Ikuya NONAKA**,
Yoshikuni MIZUNO* and Satoshi HORAI

We have sequenced all mitochondrial tRNA genes from 9 Japanese patients with chronic progressive external ophthalmoplegia (CPEO) who had
no detectable large mtDNA deletions nor mutations previously reported, andidentified 6 different base substitutions in 6 patients. Since 5 of the 6
substitutions were homoplasmic in distribution and recognizable in somenormal controls, they were thought to be polymorphisms in normal individuals. One mutation at nucleotide (nt) 12311 in the tRNALeu(CUN) gene was
not present in 90 normal controls nor in 103 patients with other mitochondrial myopathies. This mutation was in a heteroplasmic state, and the mutatedsite was conserved among other species during evolution, suggesting a
disease-related mutation. However, the significance of this mutation has tobe studied further. In Japanese CPEO patients without large deletions, apoint mutation in the mitochondrial tRNA gene is not likely to be a frequentcause. For details, see J. Neurol. Sci. 125, 50-55 (1994)***

Juntendo University School of Medicine.
National Center of Neurology and Psychiatry.

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117

Mitochondrial Myopathy with Progressive Decrease in MitochondrialtRNALeu(UUR) Mutant Genomes
Yasuhiko KAWAKAMI*. Ryoichi SAKUTA**. Yu-ichi GOTO**.
Ikuya NONAKA** and Satoshi HORAI

Since the first description of mitochondrial myopathy by Luft and colleagues in 1962, many mitochondrial disorders have been described in whichalmost 40 of patients have the following three major clinical forms: chronic
progressive external ophthalmoplegia (CPEO) including Kearns-Sayre syndrome (KSS); mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke like episodes (MELAS); and myoclonus epilepsy associated withragged-red fibers (MERRF). Each disorder has disease-specific mtDNA
abnormalities including large-scale deletions in CPEO and KSS, point mutations at nucleotide pairs (nt) 3243 and 3271 in MELAS and 8344 in MERRF.
We present a female patient who had an A-to-G substitution at nt 3243 in
mtDNA (3243 mutation), commonly seen in patients with MELAS but withthe unusual feature of improvement in her muscle weakness. While the 3243
mutation is thought to induce various clinical expressions from none totypical MELAS phenotype, this is the first report describing a patient who
had reversible muscle weakness and pathology associated with a change in the
mutant genome population. A female patient with mitochondrial myopathy
had a mitochondrial DNA mutation at nucleotide pair 3243, commonly seenin patients with MELAS but unlike MELAS patients, she had no central
nervous system symptoms. Muscle weakness, which was most severe when
she was 7 years old, improved gradually with age. Comparison of two muscle
biopsies obtained at an interval of 12.5 years (7 and 20 years of age,respectively), revealed that the number of ragged-red fibers was markedly
decreased and histochemical cytochrome c oxidase activity increased inparallel with the decrease in population of mutant genomes. For details, seeAnn. Neurol. 35, 370-373 (1994).

***

Nippon Medical School.
National Center of Neurology and Psychiatry.

118

A Caucasian Family with the 3271 Mutation in Mitochondrial DNA
Suely K. N. MARIE*. Yu-ichi GOTO**. Ikuya NONAKA**
and Satoshi HORAIMELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episodes) is a distinct clinical entity characterized by episodic
headache and vomiting, convulsions, transient hemiplegia, hemianopsia, andhigh lactate levels in serum and cerebrospinal fluid (CSF). Approximately
80% of the patients have an A-to-G transition mutation at nucleotide
position 3243 in the mitochondrial (mt) tRNALeu(UUR) gene (3243 mutation), while about 10% have a T-to-C transition in the same gene at
nucleotide position 3271 (3271 mutation). Patients with the 3243 mutation
have been found in several races, and biochemical and genetic studies haveshown that the mutation plays a substantial role in the pathological process in
MELAS. Since all the patients with the 3271 mutation reported so far are ofJapanese descent, it has been debated whether the mutation is pathognomonicof mitochondrial diseases. Here we describe a Brazilian family with the 3271
mutation, offering further evidence that the 3271 mutation is closely related
to mitochondrial diseases. This mutation was found in a Brazilian family of
Portuguese and Italian descent, indicating that this mutation also exists in arace other than Japanese. The propositus had mild clinical manifestations
atypical of MELAS, suggesting that patients with the 3271 mutation exhibit
heterogeneous phenotypic expression as seen in the 3243 mutation. For
details, see Biochem. Med. Metabo!' BiD!. 52, 136-139 (1994).
A Subtype of Diabetes Mellitus Associated with a Mutationof Mitochondrial DNA
Takashi KADOWAKl***. Yoshio YAZAKl***. Yu-ichi GOTO***.
Ikuya NONAKA*** and Satoshi HORAI

Several families have been described in which a mutation of mitochondrial
DNA, the substitution of guanine for adenine (A G) at position 3243 of
Lucite transfer RNA, is associated with diabetes mellitus and deafness. The
*
**
***
****

University of Sao Pauro.
National Center of Hospital for Mental. Nervous and Muscular Disorders.
University of Tokyo.
National Center of Neurology and Psychiatry.

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119

prevalence, clinical features, and pathophysiology of diabetes with this mutation are largely undefined. We studied 55 patients with insulin-dependent
diabetes mellitus (lDDM) and a family history of diabetes (group 1), 85
patients with IDDM and no family history of diabetes (group 2), 100 patientswith non-insulin-dependent diabetes mellitus (NIDDM) and a family historyof diabetes (group 3), and 5 patients with diabetes and deafness (group 4) forthe mutation. We also studied the prevalence and characteristics of diabetes
in 39 patients with a syndrome consisting of mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke-like episodes who were known tohave the mutation and 127 of their relatives (group 5). We identified 16
unrelated patients with diabetes associated with the A--G mutation: 3
patients from group 1 (6 percent), 2 patients from group 3 (2 percent), 3
patients from group 4 (60 percent), and 8 patients from group 5 (21 percent).
We also identified 16 additional subjects who had diabetes and the mutation
among 42 relatives of the patients with diabetes and the mutation in groups
1, 2, 3, and 4 and 20 affected subjects among the 127 relatives of the patientsin group 5. Diabetes cosegregated with the mutation in a fashion consistent
with maternal transmission, was frequently (in 61 percent of cases) associated with sensory hearing loss, and was generally accompanied by impaired
insulin secretion. Diabetes mellitus associated with the A--G mutation at
position 3243 of mitochondrial leucine transfer RNA represents a subtype of
diabetes found in both patients with IDDM and patients with NIDDM in
Japan. For details, see N. Eng/. J. Med. 330,962-968 (1994).
Enzymatic and Genetic Adaptation of Soleus Muscle Mitochondria to
Physical Training in Rats
Taro MURAKAMI*, Yoshiharu SHIMOMURA*. Takayuki OZAWA**and Satoshi HORAI

To evaluate the effects of physical training on mitochondrial gene expression and mitochondrial biogenesis in slow-twitch muscle, adult female
Sprague-Dawley rats were trained for 3, 6, and 12 wk by running on a
motor-driven treadmill (speed of 25 m/min and duration of 90 min/day, 5days/wk), and the activities of citrate synthase, ubiquinolcytochrome-c ox***

Nagoya Institute of Technology.
University of Nagoya.

120

idoreductase, cytochrome oxidase, mitochondrial cytochrome b mRNA (byNorthern blot analysis), and mitochondrial DNA (by slot-blot and Southernblot analyses) were measured in rat soleus muscle. A DNA probe fordetection of mitochondrial mRNA and DNA was prepared from a 1,500-bp
fragment of human mitochondrial DNA that included the coding region of
the cytochrome b gene. Training for 3, 6, and 12 wk significantly increased
the activities of citrate synthase (31, 28, and 47%, respectively), ubiquinolcytochrome-c oxidoreductase (61, 63, and 77%, respectively), and cytochromeoxidase (25, 26, and 32%, respectively) in muscle. The concentration ofcytochrome b mRNA in the muscle was proportionally elevated with the
enzyme activities. On the other hand, the mitochondrial DNA concentrationin the muscle was not altered by training for 3 or 6 wk but increased
significantly after training for 12 wk (35% in the slot-blot analysis and 31%
in the Southern blot analysis). These results suggest that an increase in theoxidative capacity of slow-twitch muscle by the relatively short-term training
is regulated at the pretranslational step in mitochondrial protein synthesis butthat the increase by the long-term training involves mitochondrial replication.
For details, seeAm. J. Physiol. 267 (Endocrinol. Metab. 30),388-395 (1994).
Polymorphisms of Complement Component I and CIR Subcomponent of
Cl in Nine Aboriginal Taiwanese Populations
Kazuo UMETSU*. Isao YUASA**. Tsuneo SUZUKI*. Cheih-Shan SUN***.
I-Hung PAN****. Takafumi ISHIDA*****, Naruya SAITOU
and Satoshi HORAI

Complement component I (IF) and CIR subcomponent of Cl (CIR) types
were determined by isoelectric focusing and subsequent immunoblotting
techniques for 658 individuals from nine aboriginal Taiwanese populations.
The frequency of the IF*A allele ranges from 0.075 (Bunun) to 0.430(Saisiat), and a new variant allele IF*B2 was found to have polymorphic
frequency in the Atayal. The frequency of the CIR 1 allele ranges from
0.410 (Yami) to 0.650 (Atayal), and the frequency of the CIR *2 allele** Yamagata University School of Medicine.
** Tottori University School of Medicine.
*** National Taitung Hospital.
**** National Taiwan University medical School.
***** University of Tokyo.

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121

ranges from 0.265 (Atayal) to 0.586 (Saisiat). The CIR *5 allele was foundin five populations (Atayal, Bunun, Ami, Puyuma, Yami), and the CIR *9allele was found in two populations (Tsou, Puyuma). The results indicate aremarkable degree of genetic variability among these populations. The
variability may reflect long-term genetic and geographic isolation of each
population. For details, see Human Biology 66(2), 339-348 (1994).
Construction and Expression of Chimeric Antibodies by a Simple
Replacement of Heavy and Light Chain V Genes
into a Single Cassette Vector
Hidetoshi KANDA, Keiko MORI, Katsumi TANIGUCHI and Takeshi WATANABE

A novel vector pMH-gpt, which is proved to be useful for cloning mouse
immunoglobulin heavy and light chain V genes and for expression of mousehuman chimeric antibody, was constructed. The vector contains humangenomic Cr and CIf. genes, cloning sites for immunoglobulin V region genes,
murine Ig promoters, a human Ig heavy chain enhancer, and the selection
marker gene Eco-gpt. Because VH and VIf. genes can be cloned into a single
vector, a chimeric antibody gene is easily constructed by this simple insertion
procedure.
The usefulness of the vector was confirmed by construction of two mousehuman chimeric antibodies. Mouse monoclonal antibody (MAb) 196-14
recognizes the ovarian cancer-associated antigen (CA125), and MoAb 2-18
reacts with carcinoembryonic antigen (CEA). Mouse-human chimeric 19614 and 2-18 antibodies were readily constructed and efficiently produced in amouse myeloma cell line by utilizing the vector. Both chimeric antibodies
retained binding activity to their respective antigens. In biodistiribution andimmunoscintigraphy studies, specificity of radiolabeled chimeric 196-14 antibody was identical to that of its murine counterpart and significant accumulation at the tumor site was observed. The pMH-gpt vector is useful for
construction and production of mouse-human chimeric antibodies.

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IX.

PLANT GENETICS

Genetic Diversity of Chinese Wild-Rice Populations
Hong-Wei CAI* and Hiroko MORISHIMAThe Asian common wild rice (Oryza rufipogon), progenitor of cultivatedrice, is widely distributed in China. The genetic diversity of Chinese wild riceand phylogenetic relationships with other strains belonging to the same
species in tropical Asia have not been fully investigated. In the present study,
three Chinese population samples collected in their natural habitats, 1)
Dongxiang, Jiangxi Province (the northernmost among the known sites ofthis species), 2) Yuanjiang, Yunnan Province, and 3) Guigang, Guangxi
province, were examined regarding various phenotypic characters, 29 isozymes and nuclear genome RFLP. As controls, three perennial populations
collected in Thailand, India and Indonesia and one Thai annual population
were selected. Perennial and annual types represent two major ecotypes ofthis wild-rice species. A total of 292 plants were examined.
( 1) Chinese wild rice showed a perennial trait, but differed from otherperennial types collected from tropical countries in plant type, photoperiodic
response, cold tolerance, etc.
(2) The three Chinese populations studied were found to share somecommon molecular markers which are rarely found in the strains found inother countries.
(3) The Dongxiang and Yuanjiang populations which are completely
isolated from cultivated rice fields showed a lower degree of intrapopulational polymorphism than Guigang populations and other control
perennial populations. The Guigang population which proved to be highly
polymorphic most probably received repeated gene flow from neighboring
cultivated rice fields.
(4) Chinese wild rice populations contained "Japonica-specific alleles" athigher frequencies than the populations of other countries. At the same time,they carried "Indica-specific alleles" as well as alleles which are not foundeither in Japonica or in Indica cultivars.

* JSPS Postdoctoral Fellow.

IX.

PLANT GENETICS123

The above results suggest that Chinese wild rice has played an importantrole in the differentiation of Indica and Japonica types of Asian cultivatednce.
Variation Study of Weedy Rice by Morpho-Physiological Characters,
Isozyme and RAPD Markers
Hak Soo SUH*, Yo-Ichiro SATO** and Hiroko MORISHIMA

There are various weedy types of rice which are not intentionally cultivated
but which naturally grow in farmers' rice fields. Such weedy types is an
important genetic resource for evolutionary study and breeding in rice. The
variation patterns of 152 weedy rice strains collected from various regions in
the world were investigated to elucidate the genetic background of weedy
rice, based on morpho-physiological characters, isozymes and RAPD markers.
The weedy rice strains examined could be clearly classified into Indica andJaponica types, the two major varietal groups in Asian cultivated rice, by the
first vector of principal component analysis (PCA) based on allelic variation
at 14 isozyme loci. In the Indica group, strains from tropical regions wereseparated from those of temperate regions by the second vector of PCA. The
Japonica group, however, showed little variation. In the scatter diagramfrom the first and second vectors of factor analysis based on six characters(glume hair length, KCIO] resistance, score of the first vector of PCA basedon isozymes, IOO-grain weight, seed dormancy and seed shattering degree),
the weedy rice strains tested were classified into four groups. These groups
were designated as groups I, II, III and IV. Group I was Indica type withcultivar characters, group II was Indica type with wild-rice characters, group
III was Japonica type with cultivar characters and group IV was Japonica
type with wild-rice characters. Fifty-four strains were identified as belonging
to group I, 24 to group II, 63 to group III and 21 to group IV, respectively.
Variation in seven RAPD markers (six for nuclear and one for chloroplastDNA) and one isozyme locus, Est-lO, whose alleles were effectively used for
identification of Indica and Japonica cultivars, were examined in 28 weedy
rice strains. Six Japonica-type weedy strains showed no variation in the eight***

Yeungnan University, Korea.
Present address: Shizuoka University.

124

molecular markers tested. Among twenty weedy strains of Indica type,however, two from Korea and two from Japan showed Japonica-specific
RAPD markers at one or two loci, and two from Brazil and two from the
USA showed both Indica- and Japonica-specific markers in one or two loci.
Two strains from Brazil, one from the USA, one from China and one from
Nepal among the Indica type showed non-deletion type cpDNA which was
frequent in Japonica cultivars. One weedy strain from Bangladesh had allele
4 at Est-lO which was specific to wild rice. These results suggest that someweedy rices might have originated from the progenies of natural hybridization between Indica and Japonica types of cultivated rice or hybridization
between wild and cultivated rice. This is additional support for the hypothesis proposed by Oka (1988) and others concerning the origin of weedy
forms in crops.
Allozyme Variation and Genetic Structure of Oryza glumaepatula
Populations Distributed in the Amazon Basin
Masahiro AKIMOTO*. Yoshiya SHIMAMOTO* and Hiroko MORISHIMA

Oryza glumaepatula found in tropical America is a diploid wild ricespecies, closely related to Asian cultivated rice (0. sativa) and its wildprogenitor (0. rufipogon) sharing the AA genome. The plants of this species
were found in the flood plain of the Amazon, where the annual oscillation of
the water level is 10 to 20 m. Based on our field study and information from
Brasilian scientists, the following unique life history was revealed. Growth isinitiated on the parched riverbed in the late dry season mostly from seedlings
and their internodes elongate in response to the rapid increase of water levelto a certain extent. Then, the culms are easily broken at the lower internodes
and their bodies are released from the ground to the surface of the water.
Forming floating meadows, they develop many shoots and roots from eachnode and continue reproduction. These plants drift in the river according to
the wind and current, mostly downstream. This habit enables the plants of O.
glumaepatula to disperse their seeds over a long distance.
To study genetic variability and the inter- and intra-populational structureof Amazonian 0. glumaepatula, we assessed allozyme variability at 30
isozyme loci of 16 enzymes using about 700 plants of 62 populations collected*

Hokkaido University.

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125

in 1992 and 1993. Genetic structure was quantified by several parameterssuch as F-statistics and Nei's gene diversity. The main results obtained are as
follows.
1) Allozymes were not so variable in this Amazonian wild rice as in Asian
wild rice. Overall gene diversity had low values, though our collection sites
covered more than 30 myriad km 2• However, population genotypes tended tobe differentiated in tributaries isolated geographically.
2) Populations in the lower basin showed higher gene diversity than thosein the upper basin, suggesting that the gene flow is directed from upper tolower basin in the Amazon.
3) The observed frequency of heterozygotes and estimates of the inbreeding coefficient had about the same values as found in the annual populationsof O. rufipogon which are predominantly autogamous. This suggests that O.
glumaepatula has developed a selfing system to assure safe pollination against
the threat of decrease in population size.
4) Inter-populational gene diversity is relatively higher than intrapopulational gene diversity, indicating that gene flow among populations is
restricted.
Various wild rice species are distributed pantropically, and adapted to thediverse environments of their habitats. O. glumaepatula also has developed
its own adaptive strategy in the Amazon basin. The results of this study
demonstrate a pattern of gene flow and inter- and intra-population genetic
structure which reflects its unique life history.
Genetic Mechanism of Unidirectional Cross-Incompatibility in Rice
Yoshio SANO!)

Information on genetic bases for reduced crossability is limited in rice. A
dominant gene, Ler (low crossability), has been detected on chromosome 6 ofa wild strain (W593, O. rufipogon) and has been transferred into the genetic
background of Taichung 65 (T65, Japonica type). When the plant carrying
Ler was crossed with pollen of T65, the seed setting was markedly reduced
due to an occurrence of shrunken and ungerminable seeds while the reciprocal crosses produced only viable seeds, resulting in unidirectional crossincompatibility. Cyto-histological examinations revealed that no abnormalI)

Present address: Faculty of Agriculture, Hokkaido University.

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ltIes were observed in pollen tube growth and fertilization, and that theendosperm began to deteriorate about 2-5 days after fertilization. When T65
was crossed as the pollen parent, the hybrid embryo continued to develop
without endosperm, and the retardation of the endosperm caused defective
seeds, while shriveled hybrid seeds were not obtained because of abortion at
the early stage of development when crossed with Koshihikari. Theseobservations suggested that the reduced crossability is not due to crossincompatibility before fertilization but due to zygotic lethality. Genetic
experiments showed that the role of cytoplasm as well as gene dosage effect of
Lcr in the triploid endosperm was ruled out for explanations of the phenomenon.
When the plant carrying Lcr was crossed as the maternal parent, Japonica
type tended to show reduced seed setting while O. rufipogon and Indica type
tended to show a normal seed setting. A similar unidirectional crossincompatibility was recently reported in hybrids between IR and Japanesevarieties. IR36 and others showed a low rate of crossability when Japanesevarieties were used as the pollen parent. Genes which rescue zygotic lethality
were detected in W593 and an Indica type of O. sativa. The modifiers werealso transferred into the genetic background of T65 and one from W593 wasproved to be located near Se/ (photoperiod sensitivity) on Chromosome 6. A
modifier detected in W593 rescued defective seeds only when transmitted
through pollen. Thus, crossability observed in rice seemed to be associatedwith an epigenetic, gamete-of-origin dependent modification of the genome.
The Expression of the Rice Wx Gene in Response to Sugars
Motoji KUNI-EDA 1), Yoshio SANO and Hiro-Yuki HIRANO

The wx locus encodes an enzyme, ADPglucose starch glycosyl transferase,
which catalyzes amylose synthesis in the rice endosperm. Carbon fixedduring photosynthesis is transported as sucrose through phloem to seeds
during their maturation. Since rice seeds contain a large amount of starch,such as amylose, the gene for starch synthesis is expected to be controlled bysucrose, which is converted to the substrate for starch synthesis. In thisstudy, we analyzed the effect of sugars on the gene expression of the Wx gene.
The results indicated that the rice Wx gene is activated in seeds by supplying
I)

Meiji Univ.

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127

sugars endogeneously, such as sucrose, glucose and fructose, as comparedwith water. However, activation was not observed when mannitol orsorbitol, which is not metabolized in plant cells, were supplied. The extent of
the activation depended on the concentration of sucrose; the most effective
activation was observed at a concentration of lOOmM, which corresponds tothe concentration of sucrose in the phloem of rice. These results suggest that
the Wx gene is regulated by the sugars metabolized in the cell.
We developed a transient assay system using rice protoplast to analyze thepromoter function in response to sugars. The promoter of the rice Wx gene
was activated in the protoplast when the culture medium contained sugars to
be metabolized, as the gene was activated in the seed. Using this transient
assay system, we are trying to identify the cis-acting element responsive tosucrose.
Genetic Diversity of Wild Soybean Populations in Japan
Yoshiya SHIMAMOTO

The wild progenitor of crop species is a primary gene pool for the
improvement of respective crops and also gives valuable information about
the domestication process. Wild soybean (Glycine soja) is distributed
throughout Japan. Seeds from 447 collection sites were used to elucidate thedegree and characteristics of genetic diversity in Japanese wild soybean
populations. Electrophoretic assays of the germinated seeds were carried out
to determine the genotype of each plant at 17 isozyme loci. The results
showed that the number of alleles per polymorphic locus, frequency ofpolymorphic populations and the expected heterozygosity were 2.83, 82.4%
and 0.267, respectively. This level of genetic diversity was higher than thatfound in the landraces and economic cultivars of Japanese soybean (Hirata etal., in preparation).
The Hokkaido district showed remarkably low genetic diversity and especially the Hidaka area was completely monomorphic. Genetic diversity in
the Kanto district was the highest in Honshu and also higher than the
Shikoku and the Kyushu districts. One of the predominant alleles incultivated soybean, Ap-b, was not found in Hokkaido, but found at a low
frequency in Tohoku and increased proportionately with distance traveledsouth. Wild soybeans were found isolated from soybean fields in the Hidaka

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area, while adjacent to soybean fields in southern Japan. The vanatlOnpatterns mentioned above suggests that the alleles of cultivars have invaded
wild soybean populations. The Hokkaido populations were considered to begenetically differentiated from other Japanese populations judging from their
genetic distances and other information. Thus, the distribution pattern ofgenetic variation in wild soybean populations in Japan seemed to reflect theeffect of gene flow from the cultivated soybean.

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PUBLICATIONSABO, T. and OHTSUBO, E.: Characterization of the functional sites in the oriT region involved
in DNA transfer promoted by sex factor plasmid RlOO. J. Bacteriol., in press, 1995.
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ANDO, A., KIKUTI, Y. Y., KAWATA, H., OKAMOTO, N., IMAI, T., EKI, T., YOKOYAMA, K.,
SOEDA, E., IKEMURA, T., ABE, K. and INOKO, H.: Cloning of a new kinesin-related genelocated at the centromeric end of the human MHC region. Immunogenetics, 39, 194-200,
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ARAMAKI, H., FUJITA, M., SAGARA, Y., AMEMURA, A. and HORIUCHI, T.: Heterologous
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ARIYOSHI, M., YASSYLYEV, D. G., IWASAKI, H., NAKAMURA, H., SHiNAGAWA, H. and
MORIKAWA, K.: Atomic structure of the RuvC resolvase: a Holliday junction-specificendonuclease from E. coli. Cell, 78, 1063-1072, 1994.
ASANO, Y., MIZUMOTO, K., MARUYAMA, T. and ISHIHAMA, A.: Photoaffinity labeling of
influenza virus RNA polymerase PBI subunit with 8-azido GTP. J. Biochem., 117(3), 677682, 1995.
ATTEY, A., BELYAEVA, T., SAVERY, N., HOGGETT, J., FUJITA, N., ISHIHAMA, A. and BUSBY,
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BLANK, M., BLANK, A., KING, S., YASHIKI, S., KUWAYAMA, M., FUJIYAMA, C., GONGORA,
D., ZANINOVIC, Y., CRANSTON, B., HANCHARD, B., IMANISHI, T., MANNS, A., BLATTNER, W. A., TAJIMA, K., HAYAMI, M., FUJIYOSHI, T. and SONODA, S.: Distribution of
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BUSBY, S., WEST, D., LAWES, M., WEBSTER, c., ISHIHAMA, A. and KOLB, A.: Transcription
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CHIQUET-EHRISMANN, R., HAGIOS, C. and MATSUMOTO, K.: The tenascin-like gene family.
Perspect. Dev. Neurobiol., 18, 3-7, 1994.
DING, Q., KUSANO, S., YILLAREJO, M. and ISHIHAMA, A.: Promoter selectivity control ofEscherichia coli RNA polymerase: Differential recognition of osmoregulated promotersbetween Eo" and &r holoenzymes. Mol. Microbiol., 16(4), 649-656 (1995).
FRAIPONT, c., ADAM, M., NGUYEN-DISTECHE, M., KECK, W., VAN BEEUMEN, J., AYALA, J.
A., HARA H. and GHUYSEN J.-M.: Engineering and overexpression of periplasmic forms of
the penicillin-binding protein 3 of Escherichia coli. Biochem. J., 298, 189-195, 1994.
FUJITA, M., TANAKA, K., TAKAHASHI, H. and AMEMURA, A.: Transcription of the principal

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sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to thegrowth phase. Mol. Microbiol., 13, 1071-1077, 1994.
FUJITA, N., MORI, H., YURA, T. and ISHIHAMA, A.: Systematic sequencing of the Escherichia
coli genome: analysis of the 2.1-4.1 min (lIO,917-193,643bp) region. Nucleic Acids Res.,
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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
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FUSE, N., HIROSE, S. and HAYASHI, S.: Diploidy of Drosophila imaginal cells is maintained bya transcriptional repressor encoded by escargot. Genes Dev., 8, 2270-2281, 1994.
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GOJOBORI, T., YAMAGUCHI, Y., IKEO, K. and MIZOKAMI, M.: Evolution of pathogenic viruses
with special reference to the rates of synonymous and nonsynonymous substitutions. Jpn.
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GOTO, Y., TSUGANE, K., TANABE, Y., NONAKA, I. and HORAI, S.: A new point mutation at
nucleotide pair 3291 of the mitochondrial tRNALeu(UUR) gene in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS).
Biochem. Biophy. Res. Commun., 202(3), 1624-1630, 1994.
GOTOH, H., KUSAKABE, M., SHIROISHI, T. and MORIWAKI, K.: Survival of steroid 21hydroxylase-deficient mice without endogenous corticosteroids after neonatal treatment andgenetic rescue by transgenesis as model system for treatment of congenital adrenal hyperplasia in humans. Endocrinology, 135, 1470-1476, 1994.
GOTOH, H., SHIROISHI, T. and MORIWAKI, K.: Genetic Manupu1ation of a lethal mutation
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GREENSTEIN, D., HIRD, S., PLASTERK, R. H. A., ANDACHI, Y., KOHARA, Y., WANG, B.,
FINNEY, M. and RRVKUN, G.: Targetted mutations in the Caenorhabditis elegans POU
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HARADA, K., KINOSHITA, A., AB SHUKOR, N. A., TACHIDA, H. and YAMAZAKI, T.: Genetic
variation estimated in three Shorea species by the RAPD analysis. Jpn. J. Genet., 69, 713718, 1994.
HASHIMOTO, M., KINOSHITA, T., YAMASAKI, M., TANAKA, H., IMANISHI, T., IHARA, H.,
ICHIKAWA, Y. and FUKUNISHI, T.: Gene frequencies and haplotypic associations within
the HLA region in 916 unrelated Japanese individuals. Tissue Antigens, 44, 166-173, 1994.
HATTA, M. and TAKEICHI, M.: Complex Cell Type-Specific Transcriptional Regulation by thepromoter and an Intron of the Mouse P-Cadherin Gene. Develop. Growth and Differ., 36,
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HATTORI, Y., GOTO, Y., SAKUTA, R., NONAKA, I., MIZUNO, Y. and HORAI, S.: Point
mutations in mitochondrial tRNA genes: sequence analysis of chronic progressive external

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ophthalmoplegia (CPEO). J. Neurol. Sci., 125, 50-55, 1994.
HAYASHIZAKI, Y., SHIBATA, H., HIROTSUNE, S., SUGINO, H., OKAZAKI, Y., SASAKI, N.,
HIROSE, K., IMOTO, H., OKUIZUMI, H., MURAMATSU, M., KOMATSUBARA, M., SHIROISHI, T., MORIWAKI, K., KATSUKI, M., HATANO, N., SASAKI, H., UEDA, T., MISE, N.,
TAKAGI, N., PLASS, C. and CHAPMAN, V. M.: Identification of an imprinted U2afbinding
protein related sequence on mouse chromosome II using the RLGS method. Nature
Genet., 6, 33-40, 1994.
HIGASHITANI, A., GREENSTEIN, D., HIROKAWA, H., ASANO, S. and HORIUCHI, K.: Multiple
DNA conformational changes induced by an initiator protein precede the nicking reaction
in a rolling circle replication origin. J. Mol. BioI., 237, 388-400, 1994.
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., in press.
HIGASHITANI, A., HIGASHITANI, N., YASUDA, S. and HORIUCHI, K.: A general and fast
method for mapping mutations on the Escherichia coli chromosome. Nucleic Acids Res.,22, 2426~2427, 1994.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.: SOS Induction in Escherichia coli bySingle-Stranded DNA of Mutant Filamentous Phage: Monitoring by Cleavage of LexA
Repressor. J. Bacteriol, 177, 3610-3612, 1995.
HIRAI, H., YAMAMOTO, M.-T., OGURA, K., SATTA, Y., YAMADA, M., TAYLOR, R. W. andIMAI, H. T.: Multiplication of 28S rDNA and NOR activity in chromosome evolution
among ants of the Myrmecia pi/osula species complex. Chromosoma, 103, 171 ~ 178, 1994.
HIRANO, H.-Y., MOCHIZUKI, K., UMEDA, M., OHTSUBO, H., OHTSUBO, E. and SANO, Y.:
Retrotransposition of a plant SINE into the wx locus during evolution of rice. J. Mol. Evol.,38, 132~137, 1994.
HIRANO, H.-Y., TABAYASHI, N., MATSUMURA, T., TAN IDA, M., KOMEDA, Y. and SANO, Y.:
Tissue-dependent expression of the rice wx+ gene promoter in transgenic rice and petunia.
Plant Cell Physiol., 36, 37-44, 1995.
HONDA, M., SHIOSAKI, K., EDA, Y., TOKIYOSHI, S., YAMAMOTO, S., NAKASONE, T., IKEO, K.,
YAMAGUCHI, Y., GOJOBORI, T., KORBER, B., HACHIMORI, T. and YAMAZAKI, S.:
Father-to-mother-to-infant transmission of HIV-I: Clonally transmitted isolate of infant
mutates more rapidly than that of the mother and gradually loses reactivity with neutralizing antibody. J. Virology 1994 (in press).
IMAI, H. T., TAYLOR, R. W. and CROZIER, R. H.: Experimental bases for the minimum
interaction theory. I. Chromosome evolution in ants of the Myrmecia pi/osula species
complex (Hymenoptera: Formiciddae: Myrmiciinae). Jpn. J. Genet., 69, 137-182, 1994.
IMAI, S., OKUMOTO, M., IWAI, M., HAGA, S., MORI, N., MIYASHITA, N., MORIWAKI, K.,
HILGERS, J. and SARKAR, N. H.: Distribution of mouse mammary tumor virus in asianwild mice. J. Virol., 68, 3437~3442, 1994.
IMAMURA, T.: Haematologic disorders-Thalassemia syndromes and hemoglobinopathies.
Nihon-Rinshou, 1994 Special issue: 629~633, 1994. (in Japanese)
IMAMURA, T.: Moleuclar biology of the red cells-Genetic analysis of the thalassemia
syndrome. Hematology & Oncology, 28, 49-57, 1994. (in Japanese)
INA, Y.: ODEN: a program package for molecular evolutionary analysis and database search of

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DNA and amino acid sequences. Comput. Applic. Biosci., 10, 11-12, 1994.
INA, Y. and GOJOBORI, T.: Statistical analysis of nucleotide sequences of the hemagglutinin
gene of human influenza A viruses. Proc. Natl. Acad. Sci., U.S.A., 91, 8388-8392, 1994.
INA, Y., MIZOKAMI, M., OHBA, K. and GOJOBORI, T.: Reduction of synonymous substitutionsin the core protein gene of hepatitis C virus. J. Mol. Evol., 38, 5G-56, 1994.
INAMOTO, S., FUKUDA, H., ABO, T. and OHTSUBO, E.: Site- and strand-specific nicking at oriTof plasmid RIOO in a purified system: Enhancement of the nicking activity of TraI (Helicase
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ISHIBASHI, K., SASAKI, S., FUSHIMI, K., UCHIDA, S., KUWAHARA, M., SAITO, H., FURUKAWA,
T., NAKAJIMA, K., YAMAGUCHI, Y., GOJOBORI, T. and MARUMO, F.: Molecular cloning
and expression of a member of the aquaporin family with permeability to glycerol and urea
in addition to water expressed at the basolateral membrane of kidney collecting duct cells.
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ISHIHAMA, A. and Barbier, P.: Molecular anatomy of viral RNA-directed RNA polymerases.
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ISHIHAMA, A., Zou, c., KOBAYASHI, M., KUMAR, A., FUJITA, N., SAKURAI, H. and HAYWARD, R. S.: Molecular recognition in gene transcription. In: Molecular Recognition in
Biological Systems (Buckin, V. et al., eds.), in press.
ISHIKAWA, Y., TOKUNAGA, K., LIN, L., IMANISHI, T., SAITOU, S., KIMURA, A., KASHIWASE,
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IWATA, A., TSUCHIYA, T., UEDA, S., ISHIHAMA, A., ZHU, G.-S. and HIRAI, K.: Nucleotide
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IZUHARA, K., FELDMAN, R. A., GREER, P. and HARADA, N.: Interaction of the c-fes
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KABATA, H., NOMURA, A., SHIMAMOTO, N. and KUNUGI, S.: Effect of high pressure on EcoRI
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KADOWAKI, T., KADOWAKI, H., MORI, Y., TOBE, K., SAKUTA, R., SUZUKI, Y., TANABE, Y.,
SAKURA, H., AWATA, T., GOTO, Y., HAYAKAWA, T., MATSUOKA, K., KAWAMORI, R.,
KAMADA, T., HORAI, S., NONAKA, I., HAGURA, R., AKANUMA, Y. and YAZAKI, Y.: A
subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. New Eng.
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KAJITANI, M., KATO, A., WADA, A., INOKUCHI, Y. and ISHIHAMA, A.: Regulation of the
Escherichia coli hfq gene encoding the host factor for phage Qp. J. Bacteriol., 176(2), 531534, 1994.
KAMISUGI, Y., NAKAYAMA, S., NAKAJIMA, R., OHTSUBO, H., OTSUBO, E. and FUKUI, K.:
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KANAMARU, K., KASHIWAGI, S. and MIZUNO, T.: A copper-transporting P-type ATPase foundin the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942. Mol.
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KANDA, H., MORI, K., KOGA, H., TANIGUCHI, K., KOBAYASHI, H., SAKAHARA, H., KONISHI,
J., ENDO, K. and WATANABE, T.: Construction and expression of chimeric antibodies bya simple replacement of heavey and light chain V genes into a single cassette vector.
Hybridoma, 13, 359-366, 1994
KANO, R., MIZUTANI, Y., SAITOU, N., KOSEKI, H., MAKINO, Y., ADACHI, Y., AKASAKA, T.,
KANNO, M., SHIROISHI, T., MORIWAKI, K. and TANIGUCHI, M.: Evolution ofVal4 TCR
gene family in mice. In "Genetics in Wild Mice" (Moriwaki, K., Shiroishi, T. andYonekawa, H., eds.), pp. 193-202, Japan Sci. Soc. Press/ Karger, Tokyo/Basel, 1994.
KASHIWAGI, S., IRIE, J., KANAMARU, K. and MIZUNO, T.: Cloning and Sequencing of a
Synechococcus gene encoding a protein very similar to mammalian aldehyde dehydrogenase.
Biosci. Biotech. Biochem., 58, 2299-2300, 1994.
KASHIWAGI, S., KANAMARU, K. and MIZUNO, T.: A Synechococcus gene encoding a putative
pore-forming intrinsic membrane protein. Biochim. Biophys. Acta, 1237, 189-192, 1995.
KATO, A., FUJINO, M., NAKAMURA, T., ISHIHAMA, A. and OTAKI, Y.: Gene organization ofchicken anemia virus. Virology, 209, 480-488, 1995.
KATSURA, I., KONDO, K., AMANO, T., ISHIHARA, T. and KAWAKAMI, M.: Isolation,
characterization, and epistasis of fluoride-resistant mutants of Caenorhabditis elegans.
Genetics, 136, 145-154, 1994.
KAWAKAMI, Y., SAKUTA, R., HASHIMOTO, K., FUJINO, 0., FUJITA, T., HIDA, M., HORAI, S.,
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KAWASE, E., SUEMORI, H., TAKAHASHI, N., OKAZAKI, K., HASHIMOTO, K. and NAKATSUJI,
N.: Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int. J.
Devel. BioI., 38, 385-390, 1994.
KAWASE, E., YAMAMOTO, H., HASHIMOTO, K. and NAKATSUJI, N.: Tumor necrosis factor(TNF)-a stimulates proliferation of mouse primordial germ cells in culture. Devel. BioI.,
161, 91-95, 1994.
KIMURA, M., FUJITA, N. and ISHIHAMA, A.: Functional map of the alpha subunit ofEscherichia coli RNA polymerase: Deletion analysis of the amino-terminal assembly
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KIMURA, M. and ISHIHAMA, A.: Functional map of the alpha subunit of Escherichia coli RNA
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KITAKAMI, H., TATENO, Y. and GOJOBORI, T.: Toward unification of taxonomy databases ina distributed computer environment. "Proceedings of the Second International Conferenceon Intelligent System for Molecular Biology" (Altman, R., Brutlag, D., Karp, P., Lathrop,
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KITAKAMI, H., YAMAZAKI, Y., IKEO, K., UGAWA, Y., SHIN-I, T., SAITOU, N., GOJOBORI, T.
and TATENO, Y.: Building and search system for a large-scale DNA database. In"Advances in Molecular Bioinformatics" (S. Schulze-Kremer ed.), pp. 123-138, lOS Press,
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KOBAYASHI, M., TOYODA, T., ADYSHEV, D. M., AZUMA, Y. and ISHIHAMA, A.: Moleculardissection of influenza virus nucleoprotein: Deletion mapping of the RNA binding domain.
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KOLB, A., KOTLARZ, D., KUSANO, S. and ISHIHAMA, A.: Selectivity of the Escherichia coli
RNA polymerase Ed8 for overlapping promoters and ability to support CRP activation.
Nucleic Acids Res., 23(5), 819-826, 1995.
KOSHIMIZU, V., 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.
KUMAR, A., GRIMES, B., FUJITA, N., MAKINO, K., MALLOCH, R. A., HAYWARD, R. S. and
ISHIHAMA, A.: Role of the simga 70 subunit of Escherichia coli RNA polymerase intranscription activation. J. Mol. BioI., 235(2), 405-413, 1994.
LAWLEY, B., FUJITA, N., ISHIHAMA, A. and PITTARD, A. J.: The TyrR protein of Escherichia
coli is a class I transcription activator. J. Bacteriol. 177, 238-241, 1995.
LI, F. Q., SUN, G. c., VEDA, H. and HIROSE, S.: Sequences of two cDNAs encoding silkworm
homologues of Drosophila melanogaster squid gene. Gene, in press.
LI, F. Q., VEDA, H. and HIROSE, S.: Mediators of activation of fushi tarazu gene transcription
by BmFTZ-FI. Mol. Cell. BioI., 14, 3013-3021, 1994.
Luo, J., FUJITA, N., ISHIHAMA, A. and KRAKOW, J. S.: Molecular anatomy of the (3' subunitof E. coli RNA polymerase: Identification of the regions involved in assembly. Biochemistry, in press.
MAEKAWA, T. and OHTSUBO, E.: Identification of the region that determines the specificity ofbinding of the transposases encoded by TnJ and 70 to the terminal inverted repeat
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MARIE, S. K. N., Goto, Y., PAssos-BuENO, M. R., ZATX, M., CARVALHO, A. A. S.,CARVALHO, M., LEVY, J. A., PAULO, V. B., CAMPIOTTO, S., HORAI, S. and NONAKA, 1.:
A Caucasian family with 3271 mutation in mitochondrial DNA. Biochem. Med. Metabol.
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MASUDA, Y. and OHTSUBO, E.: Mapping and disruption of the chpB locus in Escherichia coli.
J. Bacteriol., 176, 5861-5863, 1994.
MATSUDA, Y., MORIWAKI, K., CHAPMAN, V. M., HOI-SEN, Y., AKBARZADEH, J. and SUZUKI,
H.: Chromosomal mapping of mouse 5S rRNA genes by direct R-banding fluorescence insitu hybridization. Cytogenet Cell Genet, 66, 246-249, 1994.
MATSUMOTO, K., SAGA, Y, IKEMURA, T., SAKAKURA, T., and EHRISMANN R. C.: The
distribution of tenascin-X is distinct and often reciprocal to that of tenascin-C. J. Cell BioI.,
125, 483-493, 1994.
MIURA, T., FUKUNAGA, T., IGARASHI, T., YAMASHITA, M., IDO, E., FUNAHASHI, S., ISHIDA,
T., WASHIO, K., VEDA, S., HASHIMOTO, K., YOSHIDA, M., OSAME, M., SINGHAL, B. S.,ZANINOVIC, V., CARTIER, L., SONODA, S., TAJIMA, K., INA, Y., GOJOBORI, T. and
HAYAMI, M.: Phylogenetic subtypes of humanT-Iymphotropic virus type I and theirrelations to the anthropological background. Proc. Natl. Acad. Sci. V.S.A, 91, 1124-1127,
1994.
MlzoKAMI, M., GOJOBORI, T. and LAU, J. Y N.: Molecular Evolutionary Virology: Its
Application to Hepatitis C. Virus. Gastroenterology, 107, 1181-1182, 1994.
MIZUMOTO, K., MUROYA, K., TAKAGI, T., OMATA-YAMADA, T., SHIBUTA, H. and IWASAKI,
K.: Protein factors required for in vitro transcription of Sendai virus genome. J. Biochem.
117, 527-534 1995.
MIZUTA, K., SUZUKI, H., INA, Y., YAZAKI, N., SAKAMOTO, M., KATSUSHlMA, N. andPUBLICATIONS135

NUMAZAKI, Y.: Six-year longitudinal analysis of adenovirus type 3 genome types isolatedin Yamagata, Japan. J. Med. Virol., 42, 198-202, 1994.
MORIKAWA, K., ARIYOSHl, M., VASSYLYEV, D. G., KATAYANAGI, K., NAKAMURA, H., DOl,
T., HORAI, N. and OHTSUKA, E.: Crystal structure of T4 endonuclease V. in "DNA
Damage" (Wallace, S. S., van Houten, B. and Kow, Y. W., ed.), pp. 198-207, The New
York Academy of Sciences, New York, 1994.
MORISHIMA, H.: Polymorphism of bacterial blight resistance in the populations of wild andcultivated rice: A lesson from natural-ecosystems. In "Toward Enhancement and Sustainable Agricultural Productivity in the 20000s: Breeding Research and Biotechnology. (Hsieh,
S. C., ed), pp. 139-144 SABRAO, Taipei, 1994.
MORISHIMA, H.: Observations at permanent study-sites in the suburb of Bangkok. Tropics 3,
227-234.
MORISHIMA, H.: Rice in the Mekong delta. Tropics 3,211-216, 1994.
MORIWAKI, K.: Introductory remarks: Wild mouse from a geneticist's viewpoint. In "Genetics
in Wild Mice" (Moriwaki, K., Shiroishi, T. and Yonekawa, H., eds.), pp. xiii-xxv, 1994.
MORIWAKI, K., SHIROISHI, T. and YONEKAWA, H. (Edt.): Genetics in Wild Mice, pp. 1-333,
Japan Sci. Soc. Press. Tokyo/S. Karger, Basel, 1994.
MURAKAMI, A.: Growth Phenomena in Bombyx mori (L.) with a special reference to genetic
factors responsible for growth acceleration and moulitinism (A review paper). Indian J.
Sericulture 33(1), 12-14, 1994.
MURAKAMI, T., SHIMOMURA, Y., FUJITSUKA, N., NAKAI, N., SUGIYAMA, S., OZAWA, T.,
SOKABA, M., HORAI, S., TOKUYAMA, K. and SUZUKI, M.: Enzymatic and genetic
adaptation of soleus muscle mitochondria to physical training in rats. Am. J. Physiol. 267(Endocrinol. Metab. 30), 388-395, 1994.
NAKAGAWA, Y., KIMURA, N., TOYODA, T., MIZUMOTO, K., ISHIHAMA, A., ODA, K. and
NAKADA, S.: The RNA polymerase PB2 subunit is not required for replication of theinfluenza virus genome but is involved in capped mRNA synthesis. J. Virol., 69, 728-733,
1995.
NAGAI, Y., KANEDA, S., NOMURA, K., YASUDA, H., SENO, T. and YAMAO, E: Ubiquitinactivating enzyme, EI, is phosphorylated in mammalian cells by the protein kinase CDC2.
J. Cell Sci., 108, 2145-2152, 1995.
NAGAI, S., YAGIHASHI, T. and ISHIHAMA, A.: DNA sequence analysis of an allelic variant of
the Actinobacillus pleuropneumoniae-RTX-toxin (ApxIA) from serotype 10. Microb.
Pathogenesis, 15, 485-495, 1994.
NAGAMINE, C. M., SHIROISHI, T., MIYASHITA, N., TSUCHIYA, K., IKEDA, H., TAKAO, N.,
Wu, X.-L., JIN, M.-L. WANG, F.-S., KRYUKOV, A. P., AKBARZADE, J. and MORIWAKI,
K.: Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice.
Mol. BioI. Evol. 11, 864-874, 1994.
NAGATA, I. and NAKATSUJI, N.: Migration behavior of granule cell neurons in cerebellar
cultures. I. A PKH26 labeling study in microexplant and organotypic cultures. Devel.
Growth Differ., 36, 19-27, 1994.
NAKANE, K., TAKAMATSU, H., OGURO, A., SADAIE, Y., NAKAMURA, K. and YAMANE, K.:
Acquisition of azide-resistence by elevated secA ATPase activity confers azide-resistanceupon cell growth and protein translocation in Bacillius subtilis. Microbiology 141, 113-121,

136
1994.
NAKASHIMA, H., SAKAI, M., INABA, R. and IMAMURA, T.: Dinucleotide repeat polymorphismat the DI8S336 locus. Hum. Mol. Genet., 3, 390, 1994.
NAKASHIMA, H., SAKAI, M., INABA, R. and IMAMURA, T.: Isolation and fluorescence in situ
hybridization mapping of 60 cosmid clones on human chromosome 18. Genomics, 19, 577580, 1994.
NEGISHI, T., FUJITA, N. and ISHIHAMA, A.: Structural map of the alpha subunit of Escherichia
coli RNA polymerase: Structural domains identified by proteolytic cleavage. J. Mol. BioI.,
248,723-728, 1995.
NEWFELD, S. J., TACHIDA, H. and YEDVOBNICK, R: Drive-selection equilibrium: Homopolymer evolution in the Drosophila gene mastermind. J. Mol. Evol., 38, 637-641, 1994.
NISHIMIYA-FuJiSAWA, C., and SUGIYAMA, T.: Genetic Analysis of Developmental Mechanisms in Hydra: XX. Cloning of Interstitial Stem Cells Restricted to the Sperm Differentiation Pathway in Hydra magnipapi/lata. Develop. BioI., 157, 1-9, 1993.
OBATA, Y., MORIWAKI, K., SHIROISHI, T., SATTA, Y., TAKAHATA, N., KATSUKI, M.,
HASEGAWA, H. and TAKAHASHI, T.: TL Genes in Asian wild mice: The evolutionary
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OBATA, Y., SATTA, Y., MORIWAKI, K., SHIROISHI, T., HASEGAWA, H., TAKAHASHI, Y. and
TAKAHATA, N.: The structure, function and evolution of mouse TL genes, nonclassicalclass I genes of the major histocompatibility complex. Proc. Natl. Acad. Sci. USA, 91,
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OHARA, M. and SHIMAMOTO, Y.: Some ecological and demographic characteristics of two
growth forms of wild soybean (Glycine soja Sieb. et Zucc). Can. J. Bot., 72,486-492, 1994.
OHKUBO, T., SAKASHITA, H., SAKUMA, T., KAINOSHO, M., SEKIGUCHI, M. and MORIKAWA,
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OHNO, C, UEDA, H. and PETKOVICH, M.: The Drosophila nuclear receptor FTZ-Fl a and
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OHTA, T.: Early evolution of genes and genomes. In "Early Life on Earth" (Bengtson, S. ed.),pp. 135-142, Columbia Univ. Press, 1994.
OHTA, T.: Further examples of evolution by gene duplication revealed through DNA sequencecomparisons. Genetics, 138, 1331-1337, 1994.
OHTA, T.: On hypervariability at the reactive center of proteolytic enzymes and their inhibitors.
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OHTSUBO, H. and OHTSUBO, E.: Involvement of transposition in dispersion of tandem repeat
sequences (TrsA) in rice genomes. Mol. Gen. Genet., 245, 449-455, 1994.
ONO, K., NAKATSUJI, N. and NAGATA, I.: Migration behavior of granule cell neurons in
cerebellar cultures. II. An electron microscopic study. Devel. Growth Differ., 36, 29-38,
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ONOZAWA, T., DANJO, I., and FUJIYAMA, A.: Biochemical Similarity of Schizosaccharomyces
pombe rasl Protein with RAS2 Protein of Saccharomyces cerevisiae. Yeast, in press.

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PERRIERE, G., GOUY M. and GOJOBORI, T.: NRSub: a non-redundant data base for the
Bacillus subtilis genome. Nucleic Acids Research, 22(25), 5525-5529, 1994.
SABRAMANYA, G. and MURAKAMI, A.: Climatic differential phenotypic expression of voltine
genes in Bomyx mori L. Indian J. Sericulture, 33(2), 000-00o, 1994.
SAGAI, T., SAKAIZUMI, M., MIYASHITA, N., SHIROISHI, T. and MORIWAKI, K.: The Polymorphism of the H-2 class I antigen in Japanese Wild mouse. In "Genetics in Wild Mice"(Moriwaki, K., Shiroishi, T. and Yonekawa, H. eds.), pp. 1131-1140, Japan Sci. Soc.
Press/Karger, Tokyo/Basel, 1994.
SAITOU, N., OMOTO, K., Du, C and Du, R.: Population genetic study in Hainan Island, China.
II. Genetic affinity analyses. Anthropol. Sci., 102, 129-147, 1994.
SAITOU, N. and UEDA, S.: Evolutionary rate of insertions and deletions in non-coding
nucleotide sequences of primates. Mol. BioI. Evol., 11, 504-512, 1994.
SAKURAI, T, KATOH, H., MORIWAKI, K., NOGUCHI, T and NOGUCHI, M.: The ter primordial
germ cell deficiency mutation maps near Grl-I on mouse chromosome 18. Mammal.
Genome,S, 333-336, 1994.
SANO, Y., SANO, R., EIGUCHI, M. and HIRANO, H.-Y.: Gamete eliminator adjacent to the wx
locus as revealed by pollen analysis in rice. J. Hered. 85, 310-312, 1994.
SASSMUZ, E., NAKAMURA, T., XUAN, X., IHARA, T., ISHIHAMA, A. and UEDA, S.: T cell
epitope analysis of glycoprotein gill of Pseudorabies virus. J. Virol., in press.
SATTA, Y., O'HUIGIN, C, TAKAHATA, N. and KLEIN, J.: Intensity of natural selection at the
major histocompatibility complex. Proc. Natl. Acad. Sci. USA, 91, 7184-7188, 1994.
SAVOYSKY, E., MIZUNO, T., SOWA, Y., WATANABE, H., SAWADA, J-I., NORMURA, H.,
OHSUGI, Y., HANDA, H. and SAKAI, T.: The retinobrastoma binding factor I (RBF-I) sitein RB gene promoter binds preferentially E4TFI, a member of the Ets transcription facotrs
family. Oncogene, 9, 1839-1846, 1994.
SAWADA, J-I., GOTO, M., SAWA, C, WATANABE, H. and HANDA, H.: Transcriptional
activation through the tetrameric complex formation of E4TFI subunits. EMBO J., 13,1396-1400, 1994.
SAWAMOTO, K., OKANO, H., KOBAYAKAWA, Y., HAYASHI, S., MIKOSHIBA, K. and TANIMURA, T.: The function of argos in regulating cell fate decisions during the Drosophila eye
and wing vein development. Dev. BioI. 164,267-276, 1994.
SEKINE, Y., EISAKI, N. and OHTSUBO, E.: Translational control in production of transposase
and in transposition of insertion sequence IS3. J. Mol. BioI., 235, 1406-1420, 1994.
SEKINO, N., SEKINE, Y. and OHTSUBO, E.: lSI-encoded proteins, InsA and the InsA-B'-lnsB
transframe protein (transposase): Functions deduced from their DNA-binding ability.
Adv. Biophys., 31, 149-162, 1995.
SHIBATA, H., HIROTSUNE, S., OKAZAKI, Y., KOMATSUBARA, H., MURAMATSU, M., TAKAGI,N., UEDA, T, SHIROISHI, T., MORIWAKI, K., KATSUKI, M., CHAPMAN, V. M. and
HAYASHIZAKI, Y.: Genetic mapping and systematic screening of mouse endogenously
imprinted loci detected with restriction iandmark genome scanning method (RLGS).
Mammal. Genome, vol. 5,797-800, 1994.
SHIMIZU, H. and BODE, H. R.: Nematocyte Differentiation in Hydra: Commitment to
Nematocyte Type Occurs at the Beginning of the Pathway. Dev. BioI., 169, 136-150.
SHIMIZU, H., BODE, P. M. and BODE, H. R.: Patterns of oriented cell division show that

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epithelial cells rearrange continuously in both layers of adult hydra. Dev. Dynamics (inpress).
SHARIF, K. A., FUJITA, N., JIN, R., IGARASHI, K., ISHIHAMA, A. and KRAKOW, J. S.: Epitope
mapping and functional characterization of monoclonal antibodies specific for the a subunitof Escherichia coli RNA polymerase. J. BioI. Chern., 269, 23655-23660, 1994.
SHIN-I, T., IKEO, K., TATENO, Y. and GOJOBORI, T.: DNA Data Bank of Japan and its
bioinformatic activities. The Biochemist (in press)
SHIROISHI, T., KOIDE, T., YOSHINO, M., SAGAI, T. and MORIWAKI, K.: Hotspots ofhomologous recombination in mouse meiosis. Adv. Biophysics. vol. 31, pp. 119-131, Japansci. Soc. Press/Elsvier, Tokyo/Limerick, 1994.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.: Recombinational hotspots in H-2 haplotypes
derived from Asian wild mouse. In "Genetics in Wild Mice" (Moriwaki, K., Shiroishi, T.
and Yonekawa, H. eds.), pp. 141-152, Japan Sci. Soc. Press. Karger, Tokyo/Basel, 1994.
SONG, K.-J., NERURKAR, V. R., SAITOU, N., LAzo, A., BLAKESLEE, J. R., MIYOSHI, M. and
YANAGIHARA, R.: Genetic analysis and molecular phylogeny of simian T-celllymphotropic virus type I: evidence for independent virus evolution in Asia and Africa. Virology, 199,
56--66, 1994.
SUDA, T., OYANAGI, M., WAKANA, S., TAKAHASHI, Y., KANADA, H., YONEKAWA, H.,MIYASHITA, N., SHIROISHI, T., MORIWAKI, K. and KOMINAMI, R.: Novel mousemicrosatellites: Primer sequences and chromosomal location. DNA Res. vol. I, 169-174,
1994.
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 thetemperature-sensitive ubiquitin-activating enzyme EI. Chromosome Res., 3, 115-123,
1995.
SUGAYA, K., FUKAGAWA, T., MATSUMOTO, K., MITA, K., TAKAHASHI, E., ANDO, A., INOKO,
H. and IKEMURA, T.: Three genes in the human MHC class III region near the junction
with the class II: gene for receptor of advanced glycosylation end products, PBX2
homeobox gene and a Notch homolog, human counterpart of mouse mammary tumor gene,
int-3. Genomics, 23, 408-419, 1994.
SUGIYAMA, T.: Spontaneous Loss oflnterstitial Stem Cells from Hydra Tissue. Dev. BioI., 163,302-308, 1994.
SUGIYAMA, T. and WANEK, N.: Genetic Analysis of Developmental Mechanisms in Hydra:
XXI. Enhancement of Regeneration in a Regeneration-Deficient Mutant Strain by the
Elimination of Interstitial Cell Lineage. Dev. BioI., 160,64-72, 1993.
SUMI, Y., SHIROYA, T., FUJIMOTO, K., WADA, T., HANDA, H. and KAWAGUCHI, H.:
Application of cationic latex particles for protein purification. Collids and Surfaces B:
Biointerfaces, 18,419-427, 1994.
SUN, G. c., HIROSE, S. and VEDA, H.: Intermittent expression of BmFTZ-FI, a member of thenuclear hormone receptor superfamily during development of the silkworm Bombyx mori.
Dev. BioI., 162,426-437, 1994.
SUZUKI, K., MIZOKAMI, M, LAu, J. Y. N., MIZOGUCHI, N., KATO, K., MIZUNO, Y.,
SODEYAMA, T., KIYOSAWA, K. and GOJOBORI, T.: Confirmation of Hepatitis C Virus
Transmission through Needlestick Accidents by Molecular Evolutionary analysis. The

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Journal of Infectious Diseases, 170, 1575-1578, 1994.
TACH IDA, H.: Decay of linkage disequilibrium in a finite island model. Genet. Res., 64, 137144, 1994.
TAJIMA, F. and TAKEZAKI, N.: Estimation of evolutionary distance for reconstructing
molecular phylogenetic trees. Mol. BioI. Evol., 11, 278-286, 1994.
TAJIMA, F., TOKUNAGA, T. and MIYASHITA, N. T.: Statistical methods for estimating theeffective number of alleles, expected heterozygosity and genetic distance in self-incompatibility locus. Jpn. J. Genet., 69, 281-289, 1994.
TAKAHASHI, K., HATTORI, T., NAKANISHI, T., NOHNO, T., FUJITA, N., ISHIHAMA, A. and
TANIGUCHI, S.: Repression of in vitro transcription of the Escherichia coli Inr and narX
genes by FNR protein. FEBS Lett., 340, 59-64, 1994.
TAKAHATA, N.: Comments on the detection of reciprocal recombination or gene conversion.
Immunogenetics, 39, 146-149, 1994.
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, 225-247, 1995.
TAKAHATA, N. and KIMURA, M.: The Neutral Theory of Molecular Evolution. pp. 203-232.
In Principles of Medical Biology, vol. IB, Bioethics, eds by E. E. Bittar and N. Bittar. JAI
Press Inc. Connecticut, 1994.
TAKAHATA, N. (ed.): Selected Papers on Population Genetics and Molecular Evolution, Motoo
Kimura. The Chicago University Press, Chicago, 1994.
TAKAHATA, N.: Polymorphism at MHC loci and isolation by the immune system in vertebrates.
pp. 233-246. In Non-neutral Evolution: Theories and Molecular Data, ed. by B. Golding.
Chapman and Hall, New York, 1994.
TAKAHATA, N.: Repeated failures that led to the eventual success in human evolution. Mol.
BioI. Evol., 11, 803-805, 1994.
TAKAHATA, N., SATTA, Y. and KLEIN, J.: Divergence time and population size in the lineage
leading to modern humans. Theor. Popul. BioI., 48, 198-221, 1995.
TAKAMATSU, H., NAKANE, A., OGURO, A., SADAIE, Y., NAKAMURA, K. and YAMANE, K.: A
truncated Bacillus subtilis SecA protein consisting of the N-terminal 234 amino acidresidues forms a complex with Escherichia coli SecA51 (ts) protein and complements theprotein translocation defect of the secA51 mutant. J. Biochem., 116, 1287-1294, 1994.
TAKAMATSU, H., NAKANE, A., SADAIE, Y., NAKAMURA, K. and YAMANE, K.: The rapid
degradation of mutant SecA protein in the Bacillus subtilis secA341 (ts) mutant causes a
protein translocation defect in the cell. Biosci. Biotech. Biochem., 58, 1845-1850, 1994.
TANAKA, K., KUSANO, S., FUJITA, N., ISHIHAMA, A. and TAKAHASHI, H.: Promoter
determinants for Escherichia coli RNA polymerase holoenzyme containing 03 8 (the rpoS
gene product). Nucleic Acids Res., 23(5), 827-834, 1995.
TATENO, Y.: Evolution of glutamine synthetase genes is in accordance with the neutral theoryof molecular evolution. Jpn. J. Genet. 69, 489-502, 1994.
TENZEN, T., MATSUDA, Y., OHTSUBO, H. and OHTSUBO, E.: Transposition of Tnrl in ricegenomes to 5'-PuTAPy-Y sites, duplicating the TA sequence. Mol. Gen. Genet., 245, 441448, 1994.
TOKUNAGA, K., SIDELTSEVA, E. W., TANAKA, H., UCHIKAWA, c., NIEDA, M., SIDELTSEV, V.

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V., ZHURAVLEVA, E., IMANISHI, T., ITOH, K., AKAZA, T., TAKAHASHI, K., KHALTURIN,
V., ALEXEEV, L. P. and Jun, T: Distribution of HLA antigens and haplotypes in the
Buryat population of Siberia. Tissue Antigens, 45,98-102, 1995.
TORIYAMA, S., TAKAHASHI, M., SANO, Y., SHIMIZU, T. and ISHIHAMA, A.: Nucleotide
sequence of RNA I, the largest genomic segment of rice stripe virus, the prototype of thetenuiviruses. J. Gen. Viol., 75, 3569-3579, 1994.
TOYODA, T, ASANO, Y. and ISHIHAMA, A.: Role of GTPase activity of Mxl protein in nuclear
localization and antiviral activity. J. Gen. Virol., in press.
TOYODA, T, KOBAYASHI, M. and ISHIHAMA, A.: Replication in vitro of the influenza virus
genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein
complexes. Arch. Virol., 136(3/4), 269-286, 1994.
TOYODA, H., KOIDE, N., KAMIYAMA, M., TOBITA, K., MIZUMOTO, K. and IMURA, N.: Host
factors required for internal initiation of translation on poliuovirus RNA. Archiv. Virol.,138, 1-15, 1994.
UMETSU, K., YUASA, I., SUZUKI, T., SUN, C-SHAN, PAN, I-HUNG, ISHIDA, T., SAITOU, N. and
HORAI, S.: Polymorphisms of complement component I and CIR subcomponent of CI innine aboriginal Taiwanese populations. Human Biology, 66(2), 339-348, 1994.
WANG, W., OMORI, M., HAYASHIBARA, T, SHIMOIKI, K., HATTA, M., SUGIYAMA, T and
FUJISAWA, T.: Isolation and Characterization of a Mini-collagen Gene Encoding a
Nematocyst Capsule Protein from a Reef-building Coral, Acropora donei. Gene, 152, 195200, 1995.
Xu AN, X., NAKAMURA, T., IHARA, T., SATO, I., TUCHIYA, K., NOSETTO, E., ISHIHAMA, A.
and UEDA, S.: Characterization of pseudorabies virus glycoprotein gIl expressed byrecombinant baculovirus. Virus Res., 36, 151-161, 1995.
YAMAMOTO, H., MIURA, H., SATO, S., TANAKA, M., NAsu, K., IKEO, K., GOJOBORI, T and
TAKEUCHI, T: Regulatory regions of vertebrate tyrosinase genes: Divergence functional
conservation. Proc. Natl. Acad. Sci. U.S.A. 1994 (in press).
YASUDA, J., BUCHER, D. J. and ISHIHAMA, A.: Growth control of influenza A virus by MIprotein: Analysis of transfectant viruses carrying the chimeric M gene. J. Virol., 68(12),8141-8146, 1994.
YOSHINO, M., KANAI, Y., KUROHMARU, M., AZUMA, S., TOYODA, Y., GOTO, H., MORIWAKI,
K. and HAYASHI, Y.: Fetal ovotestes in XX <= >XY chimeric mice develop into testes inadults. J. Reprod. Develop., 40, 39-48, 1994.
YOSHINO, M., SAGAI, T., FISCHER LINDAHL, K., TOYODA, Y., SHIROISHI, T. and MORIWAKI,
K.: No dose effect of recombnational hotspots in the mouse MHC Immunogenet., 39, 381389, 1994.
YOSHINO, M., SAGAI, T., FISCHER LINHDAHL, K., MORIWAKI, K. and SHIROISHI, T.: Recombination in the class III region of the mouse major histocompatibility complex. Immunogenet., 40, 28()-286, 1994.
Zou, C, FUJITA, N. and ISHIHAMA, A.: Asymetric arrangement of two alpha subunits within
Escherichia coli RNA polymerase: Involvement of one alpha subunit in contact with cAMP
receptor protein. J. Mol. BioI., 236, 1283-1288, 1994.

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ABSTRACTS OF DIARY FOR 1994

Biological Symposium
1.
408th-Jan. 13

409th-Jan. 13
41Oth-Mar. 14

41lth-Mar. 16
412th-Feb. 28

413th-Mar. 1
414th-Mar. 14
415th-Mar. 17
416th-Mar. 28
417th-Apr. 13
418th-June 6419th-July 26

The transition from host to phage metabolism after
T4 infection (Elizabeth Kutter)
2. Cellular and population responses to starvation in
E. coli (Roberto Kolter)DNA domains: The role of transcriptional control, replication and recombination (G. P. Georgoev)
1. Ribosomal phenotype and bacterial growth (Ritta
Mikkola)2. Structure and regulation of eukaryotic translation
initiation factors (John Hershey)
Ribosomal pausing; some reasons and consequences(Leif Isaksson)
Recognizing distant and evolutionary relationships
amongst protein sequences with special application toviral capsids (Patrick Argos)
Prediction and recognition in signal transduction pathways (Geoff Barton)
HTLV-I sequence variability is associated with diseases(Charles R. M. Bangham)
Sporadic distribution of Fn3 domains in animal andbacterial proteins (Russel Doolittle)
Hydra: how does it maintain its nervous system as it
constantly produces and loses neurons? (Hans R. Bode)
Structure-Function relationships of Escherichia coli
RNA polymerase (Dipankar Chatterji)
Mx proteins: antiviral activity against tick-borne Thogoto and Dhori viruses (Otto Haller)
1. Micro- and macroevolutionary analysis of breeding
system diversity in the Pontederiaceae (Joshua R.
Kohn)
2. Evolution of mating incompatibilities (Marcy K.
Uenoyama)

142

420th-Aug. 5

42Ist~Aug. 31

422nd-Dct. 5423rd-Dct. 17
424th-Dct. 21
425th-Nov. 15426th-Dec. 7427th-Dec. 9428th-Dec. 9429th~Dec.

12

430th-Dec. 21
43 1st-Dec. 19

A telomeric-like sequence as a regulatory element at theorigin of replication of trypanosomal kinetoplast DNA(Joseph Shlomai)
Negative phototaxis by Escherichia coli bacteria andlOO-fold increased phtotaxis by a mutant (Julium
Adler)
Combination of data in phylogenetic analysis (JansHurgen Bandelt)
Mechanism of HIV DNA integration (Robert Craigie)
Biochemical and biophysical changes in the human eyelens with ageing and in cataract (D. Balasubramanian)
How spirochetes swim? (Nyles W. Charon)
The signal transduction mechanism of interleukin-4(Nobuyuki Harada)"The genomic TAG hypothesis; modern viruses as molecular fossils ancient strategies for genomic replication,and clues regarding the origin of protein synthesis" and"evolution of U2 RNA genes"
In vitro control of SV40 DNA replication (Jerard Hurwitz)
Early segmentation in flies, beetles and butterflies (Lisa
Nagy)
Transcriptional regulation of the N-ras oncogene: In
vitro analysis of a TATA-Iess promoter (Fujiko Watt)
I. Dual errors hypothesis of variation and heterogeneity of DNA base composition (Noboru Sueoka)
2. Isochore-Genome structure of higher organisms(Giorgio Bernardi)

Mishima Geneticists' Club
415th-Jan. 10
Phosphorylation of RNA polymerase IICTD: Its role in
the initiation of transcription and its regulation (Hiroaki Serizawa)
416th-Jan. 7
Progress in predicting DNA-binding specificity of proteins from amino-acid sequence (Masashi Suzuki)
417th~Jan. 31
Regulation of secondary metabolism by protein-phosphorylation in Streptomyces (Kouji Ishizuka)

ABSTRACTS OF DIARY FOR 1994

41 8th-Feb. 1
41 9th-Feb. 2420th-Feb. 15
42 1st-Feb. 15
422nd-Feb. 16
423rd-Mar. 1

424th-Feb. 21

425th-Mar. 23

426th-Apr. 19
427th-Apr. 20
428th-Apr. 27

429th-Apr. 28

430th-Apr. 28
43lst-May 6
432nd-May 24
433rd-Mar. 30
434th-June 7

143

The role of traM gene in initiation of DNA transfer inbacterial conjugation (Tatsuhiko Abo)
Regulation of heat-shock gene expression in Pseudomonas aeruginosa (Masaya Fujita)
Site-specific transposition ofIS630 and Tnrl to 5/-TA-3'(Toyoaki Tenzen)
Molecular orgnization of centromeric alphoid repeatson the human chromosome 21 (Masashi Ikeno)
Initiation factors of homologous recombination duringmeiosis of budding yeat (Katsuki Johzuka)
Function and developmental expression of a steroidgenic cell-specific transcription factor, Ad4BP (Kenichirou Morohashi)
Evolution of human populations based on polymorphism in the major histocompatibility complex genes(Tadashi Imanishi)
Identification of genes regulating neurogenesis of Drosophila and its application to study mammalian nervous
system (Hideyuki Okano)
Flightless mutations of Drosophila (Kaname Mogami)
Movile genetic elements and natural mutations of Drosophila (Kou Harada)
Trials for genetic analysis of relationship between
genome structure and chromosome function (Nori
Kurata)Molecule species and their differential function of phytochrome (photoreceptor protein in plants) (Akira
Nagatani)
Dynamic analysis of genome in higher plants (Yasunori
Ogiwara)The signal transduction mechanisms of interleukin-4(Kenji Izuhara)
Three-dimensional structure and function of RNase H(Kousuke Morikawa)Analysis of the functional domains of regulator of chromosome condensation, Rcc 1 protein (Hiroaki Seino)
Limb pattern formation and retinoic acid (Kouji

144

435th-June 27
436th-July 22
437th-July 27
438th-Nov. 2

439th-Nov. 24

44Oth-Dec. 20
44 Ist-Dec. 21

Tamura)Transmembrane signaling of the aspartate receptor(Ichiro Maruyama)
Estimation of the numbers of synonymous and nonsynonymous substitutions (Yasuo Ina)
Figuration and function in genetic research of plants(Ki-ichi Fukui)
Analysis of suppressors of programmed cell death, using
the nematode Caenorhabditis elegans as a model system(Asako Sugimoto)Three-dimensional structure and function of the E. coli
RuvC protein (Holliday junction resolvase) (Kosuke
Morikawa)
Role of dpp, a member of TGF-$ superfamily, in Drosophila pattern formation (Shigeru Morimura)
Structure and function of nuc1eosomes (Kiyoe Dra)

145

FOREIGN VISITORS IN 1994

Dec. 1, 1991Aug. 27, 1993July 5, 1994
Oct. 5, 1993Dec. 1, 1993Mar. 10, 1994
Dec. 1, 1993
Feb. 20, 1994
Dec. 3, 1993Apr. 15, 1994
Jan. 13, 1994
Jan. 13Jan. 13
Feb. 28
Mar. 1Mar. 10Mar. 14
Mar. 14Mar. 16Mar. 17Mar. 25-30
Apr. 1-28
Apr. 3-7
May 2-4
May 2-15
June 5-15
June 10-

Thangirala Sudha, Indian Association for Down's Syndrome, India
Ashok Kumar, University of Edinburgh, U.K.
Dzhanybek M. Adyshev, Institute of Biochemistry andPhysiology, Kyrgys Academy of Sciences, Kyrgyz
Ritta H. Mikkola, Uppsala University, Sweden
Stefanie A. Margarson, University of Nottingham Medical School, U.K
Dipankar Chatterji, Centre for Cellular and Molecular
Biology, India
Elizabeth Kutter, Harvard Medical School, U.S.A.
Roberto Kolter, Harvard Medical School, U.S.A.
G. P. Georgiev, Institute of Gene Biology, Russian
Academy of Sciences, Russia
Patrick Argos, European Molecular Biology Laboratory, Germany
Geoff Barton, University of Oxford, U.K.
Hak-Soo Suh, Yeungnam University, Korea
John Hershey, University of California at Davis, U.S.A.
Charles R. M. Bnagham, John Radcliffe Hospital, U.K.
Leif Isaksson, University of Stockholm, Sweden
Russel Doolittle, University of California, U.S.A.
Hans Bode, University of California at Irvine, U.S.A.
Guy B. Perriere, Centre National de la Recherche Scientifique, France
Ling Hua Tang, Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, China
V. Purushothaman, India
William B. Provine, Cornell University, U.S.A.
Otto Haller, University of Freiburg, Germany
Hong Wei Cai, Beijing Agricultural University, China

146

June 27-Sept. 30
June 29
June 30-July 30
July 5-27
July 8-Aug. 24
July 8-Aug. 29
July 8-Aug. 29
Aug. 5-6
Aug. 31
Sept. 1Sept. 21-Dct. 13
Sept. 23-Dct. 3
Sept. 24-Dct. 5
Sept. 24-Dct. 5
Sept. 25-Dct. 1
Sept. 25-Dct. 1
Oct. 3-29
Oct. 3-Nov. 29
Oct. 13-23
Oct. 17-18
Oct. 20-21
Nov. 8-Dec. 24
Nov. 8-Dec. 9
Nov. 9-11
Nov. 15
Nov. 22-Dec. 24

Joshua R. Kohn, University of California at San Diego,
U.S.A.
M. K. Tadjudin, University of Indonesia, Indonesia
Marcy K. Uyenoyama, Duke Univercity, U.S.A.
K. W. Jair, Maryland University, U.S.A.
S. Wedgewood, University of Edinburgh, U.K.
Konstanze Beck, University of Munich, Germany
Seielstad, Mark T., Harvard University, U.S.A.
Joseph Shlomai, Hebrew University-Hadassah Medical
School, Israel
Julius Adler, University of Wisconsin, U.S.A.
John R. Wakeley, University of California at Berkeley,
U.S.A.
Hans-Juergen Bandelt, University of Hamburg, Germany
Hans Bode, University of California at Irvine, U.S.A.
Charles David, University of Munich, Germany
Maria de Haro, University of Munich, Germany
Tomas Bosch, University of Munich, Germany
Jan Lohmann, University of Munich, Germany
Chitrakon Songkran, Pathumthani Rice ResearchCenter, Thailand
Goonnapa Fucharoen, Khon Kaen University, Thailand
Hans Bode, University of California at Irvine, U.S.A.
Robert Craigie, NIDDK, National Institutes of Health,
U.S.A.
D. Balasubramanian, Centre for Cellular and Molecular
Biology, India
L. W. Chuang, Singapore University of Technology,
Singapore
Y.E.L. Elgin, Singapore University of Technology, Singapore
Masashi Suzuki, MRC Laboratory of Molecular Biology, U.K.
Nyles W. Charon, West Virginia University, U.S.A.
Yap E. Lin, Singapore University of Technology, Singapore

FOREIGN VISITORS IN 1994

Dec. 7
Dec. 8-9
Dec. 9Dec. 12
Dec. 18-19
Dec. 18Dec. 19
Dec. 20-21

147

Nobuyuki Harada, DNAX Research Institute of Molecular and Cellular Biology, U.S.A.
Alan M. Weiner, Yale University School of Medicine,
U.S.A.
Jerard Hurwitz, Memorial Sloan-Kettering Cancer
Center, U.S.A.
Lisa Nagy, University of Wisconsin, U.S.A.
Noboru Sueoka and Tamiko Sueoka, University of Colorado at Boulder, U.S.A.
K. Sharif, New York City University, U.S.A.
Giorgio Bernardi, Institut Jacques Monod, France
Fujiko Watt, CSIRO, Australia

148

AUTHOR INDEX
ABE, N.

043.

• ........••.•.•..•.... 28

ADYSHEV, D.
AKIMOTO, M

124
86
ANDO, A.
. . . . . . . . . • . • • . . . . . . . . . 98, 99
AOKI, F . • . . . . . . . . . . . . • . . • • . . . . . . . . . . 31
ASANO, Y.
......•..•.•••........ 28, 30
AZUMA, Y. .
25,26
BODE, H.
• •.............•.•.• 62, 63, 64
BOSCH, T. .
62, 63, 64
122
CAl, H.-W
DAVID, C. .
62, 63, 64
DEHARO, M.
.
62, 63, 64
Du, C.
102
Du, R
102
ENDO, T108
FRIESMAN, L. V
95
FUJISAWA, T.
.
62,63,64
FUJITA, M. .
40, 48
FUJITA, N.
.
18, 19,21
FUJIWARA, M.
.
82
FUKAGAWA, T.
.
88, 98
FUSE, N.
.
75
GOJOBORI, T.
103, 104, 105, 106, 107,108, 110, III
GOTO, M.
.
75
GOTO, Y.
115, 116, 117, 118
GOUY, M.
.
103
GUAN, Z. W
41
HANDA, H. . . . . • . . • . . • . . . . . . . . . . • 74, 75
HARA, H.
. ..••..•..•.......••.•.•. 043
HARADA, N.
.
114
HASEGAWA, H. . ...•..••.•.•.......•. 92
HASEGAWA, T.
.
112
HATTA, M. .
59, 62, 63, 64
HATTORI, Y.
.
116
HA YAMI, M.
.
106
HA YASHI, S. .
75, 76, 77
HEIKI, T. .
115
ANDACHI, Y.

.

HEYDUK, T.

18

HIGASHITANI, A . . . . . . . . . . . . . • 041,43,45
HIGASHITANI, N.

.

HIRANO, H.-Y.

........••.•.•.•.••• 126

HIROSE, S.

65, 66
81,82
HORAI, S. . . . . 115, 116, 117, 118, 119, 120
HORIUCHI, K.
. • • . . . . . . . . . . . . AI, 43, 45
IKEMURA, T.
.
88, 98, 99, 100
IKEO, K. . . . . . . . . • . 103, 104, 108, 110, 111
IMAMURA, T. .
112, 113, 114
IMANISHI, T.
.
109
INA, Y. . • • . . . . . . . . . . . . . . . • . . . • . 105, 106
INABA, R.
.
112, 113, 114
INOKO, H.
. .•••.•..•.•.......... 98, 99
ISHIDA, T.
.
120
ISHIHAMA, A.
. .17, 18, 19,20,21,22,24,
25, 26, 28, 29, 30
ISHIHARA, T
80,81,82,83
IWANAGA, T.
58
IWATA, A.
20,28
IZUHARA, K.
. ...•.•.••........ 112, 114
liN, M. L.
95
lISHAGE, M.
.
20
KABATA, H.
• • . . . . . . . . . . . . . . . . • . . • . • 38
KADOWAKI, T.
.
118
KANDA, H. . . . • . . . . . . . . . . . . . . . . . . . . 121
KANEDA, S.
.
33
KATO, K . . . . . . . • • . • . • • . . • . . • • . . . . . . 104
KATSURA, I.
. .•..•.•...... 80,81,82,83
KAWAKAMI, M.
.
80
KAWAKAMI, Y.
.
117
KIMURA, M.
.
17,91
KISHI, T.
.
34
KISHIMOTO, Y.
.
57, 58KIT AKAMI, H.
..
106, 110, 111
KIYOSAWA, K.
• .•.••..•.•......... 104
KLEIN, l.
.
93
KOBAYAKAWA, Y.
......•.•.•..•.•.• 59
HISHIDA, R.

.

AI

.

149

AVTHORINDEX

26, 2985, 86KOIZUMI, O.
.
59, 62, 63, 64
KOOPMAN, P.
.
53
KRYUKOV, A. P.
.
95
KUBORI, T.
.
38
KUNI-EDA, M.
.
126
KUSANO, S.
.
21
KYOGOKU, Y.
.
19
LAU, 1. Y. N. .
104, 107
LI, F.-Q
66
LI, H
95
LOHMANN, 1.
.
62, 63, 64
MARIE, S. K. N
119
MARUYAMA, T.
.
28
MASUYA, H.
.
52, 54
MATSUDA, Y.
.
95
MAYUMI, M
114
MITA, A.
.
53
MIURA, T.
.
106
MIYAO. T
26
MIYASHITA, N
95
MIYASHITA, N. T.
.
90
MIZOGUCHI, N.
.
104
MIZOKAMI, M.
.
104, 105, 107
MIZUMOTO, K.
.
26. 28MIZUNO, K-1.
54
MIZUNO, T.
74
MIZUNO, Y. .
104
MIZUNO, Y. .
116
MORI, K121
MORIKAWA, K.
.
36
MORISHIMA, H. .
122, 123, 124
MORIWAKI, K. .
49, 52, 53. 54, 92, 95
MOTOHASHI, T.
.
85
MUNEOKA, Y.
•
62.63,64
MURAKAMI. A.
.
68.69,71.72,73
MURAKAMI, K.
.
18
MURAKAMI, T.
.
119
MURATE, M.
.
57,58
NAGAI, H . . . . . . . . . . . . . . . • . . . . . . . . . . . 39
NAGAI, Y . . . . . . . • . . . . . . . . . . . . . • • . . . . 33
NAKADA, S.
. . . • . . . . . . . . . . . . . . . . . . . . 26
NAKAKOHJI. M.
.
.43
KOBA YASHI, M.

KOHARA, Y.

.

98. 100
113
NAKATSUJI. N.
.
77. 78, 79
NATSUME-SAKAI, S
95
NEGISHI, T.
.
19
NISHIMIYA-FuJISAWA, C.
.
56
NISHIMURA, A.
.
.46, 47NISHIMURA, Y. .
.43
NOMURA, H. .
74
NONAKA, I.
115, 116, 117. 118
O'HVIGIN, C.
.
93
OBATA. Y
92
OHBA, K
105
OHNO, C. K. .
67
OHSUGI, Y.
.
74
OHTA, T.
.
89,90.97
OKUMURA. K.
.
88, 98OMOTO, K. .
102
OSAKA, F
33
OZAWA. T.
.
119
PAN, I.-H.
.
120
PERRIERE, G. .
103
PETKOVICHI, M.
.
67
SADAIE, Y. .
40,48
SAGAI, T.
.
.49, 50, 52. 54
SAITOU, N. .
98, 101, 102, 110, 111, 120
SAKAGUCHI, M.
.
59
SAKAI, M.
..
112. 113, 114
SAKAI, T.
.
74
SAKURAI, H.
.
24.26
SAKUTA, R
116.117
SAMASHIMA, M.
.
32
SANO, Y. .
125, 126
SARRAS, M. 1. lr.
60
SATO, Y.-1.
123
SATTA. Y.
.
92,93
SAVOYSKY, E.
.
74
SAWA. C.
.
75
SAWADA,I.-1.
74,75
SEINO. Ii.
.
33, 35
SENO, T.
.
31, 32, 33, 34. 35
SHIGA. Y.
.
77
SHIMAMOTO, N. .
38, 39SHIMAMOTO, Y. .
124, 128
NAKAMURA. Y.

NAKASHIMA, H.

.

150
SHIMIZU, H.

.

'"

60, 62, 63, 64
119SHIN-I, T.
.
110, IIISHIRAYOSHI, Y.
..
77, 78, 79
SHIROISHI, T.
.49, 50, 52, 53, 54, 92
SODEYAMA, T.
. . '"
104
SONG, K.-J. .
101
SOWA, Y.
.
74
SUBRAMANYA, G
73
SUDHA, T. .
32
SUGA YA, K.
.
99
SUGIMOTO, A.
.
85
SUGIYAMA, T.
.. 56, 57, 58, 59, 62, 63, 64
SUH, H. S.
.
123
SHIMOMURA, Y.

.

TSUGANE, K.
TSUJI, H.

.

.

VCHIDA, K.

.

VEDA, H.

.

VEDA, S.

.

VEDA, SVGAWA, Y.

VI, M.

..

.

VKAI, H.

.

VMETSU, K.

.

VRASAKI, M.

.

WAKANA, S
WANG, C.-H.
WANG, F.

.

.

SUN, C.-S.

.

120

WATANABE, H

SUN, G.-C.

.

65, 66
24
95

WATANABE, N

SUZUKI, H.

.

SUZUKI, H.

.

SUZUKI, K.

.

SUZUKI, N.

104.

SUZUKI, T.
T ABARA, H.

.

T ACHIDA, H.

.

TAJlMA, F.

.

TAKAHASHI, T.

.

TAKAHASHI, T
TAKAHASHI-IWANAGA, H.
TAKAHATA, N
TAKEZAKI, N.

.

TANABE, Y.
TANIGUCHI, K.

.

TAO, KTATENO, Y.

.

TOKUNAGA, T.
TOYODA, T.
TSUCHIYA, K.

.

.

83
120
85
94
90,97
62,63,64
92.
58
91,92,93,94
97
115
121
18
106, 110, 111
90
26, 28, 29, 30
95

WATANABE, H
WATANABE, T.

.

WATAYA, Y.

Wu, X. 1.
YAGI, Y.

..

YAGURA, T.

.

YAKIMENKO,1. V.
YAMADA, M.-A.
Y AMAGISHI, M.

.

YAMAGUCHI, Y.

.

Y AMAO, F.

.

YAMAZAKI, Y.

.

YANAGIHARA, R.
YASUDA, J.
YASUI, K.
Y AZAKI, Y.

.

.
.

YONEKAWA, H
YOSHINO, M
YUASA, 1.

.

.

.

115
32
53
65, 66, 67
28
101
110, 11134
47
120
80, 8349,53,54
95
95
74,75
85
.47
121
, .31
95
76
35
95
73
25, 26
104, 10832, 33, 35
111
101
29
25
118
95
50
120

Annual Report of the National Institute of Genetics. No. 45
Printed on December 20th 1995
Issued on December 26th 1995
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 Yoshio Tateno & Toshihiko Shiroishi
Published by National Institute of Genetics
Printed by Kokusai Bunken Insatsusha Co., Ltd.

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