ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No.50

1999

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mlshlma, Shizuoka-Ken, Japan

Annual Reportof theNational Institute of Genetics
No. 50, 1999

Published by

The National Institute of Genetics, Japan

CONTENTS
General statement
Staff
Council and advisory committee
Research activities in 1999
A.

.
.
..
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1
3

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Department of Molecular Genetics
A-a. Division of Molecular Genetics
Mapping of Protein'protein Contact Network Involving the {3 and{3' Subunits of Escherichia coli RNA Polymerase. NOMURA,
T., KATAYAMA, A, FUJITA, N. and ISHIHAMA, A. 10
Structural Organization of the a Subunit of Escherichia coli
RNA Polymerase. FUJITA, N., ENDO, S. and ISHIHAMA. A.. 11
Functional Organization of the a Subunit of Escherichia coli
RNA Polymerase. OZOUNE, O.N., FuJITA, N. and ISIDHAMA,A
13
Competition among Seven E. coli a Subunits: Intracellular
Concentrations and Binding Affinity to the Core Enzyme.
MAEDA, H., JISHAGE, M., NOMURA, T., KATAYAMA, A.,
FUJITA, N. and ISHIHAMA, A. 14
Control of the Activities of a Subunits by Anti, a Factors.
JISHAGE, M., DASGUPTA, D. and ISIDHAMA, A.. 16
Contact Site Mapping of a Subunits and Transcription Factors
on the RNA Polymerase Core Enzyme Subunits Using
FeBABE. WIGNESHWERARAJ, S.R., COLLAND, F., FUJITA,
N., MEARES, C.F. and ISHIHAMA, A.. 17
Growth Phase'dependent Variation in the Protein Compositionof Escherichia coli Nucleoid. TALUKDER, A.A., IWATA, A.,
UEDA, S. and ISHIHAMA, A. 18
Functional Organization of Two Large Subunits of the Fission
Yeast Schizosaccharomyces pombe RNA Polymerase II.

II

WLASOFF, W.A., KIMURA, M. and ISHIHAMA, A.
Twelve Subunits ofthe Fission Yeast RNA Polymerase II: Intra-

19

cellular Contents, Transcription Organization and mRNA
Levels. SAKURAI, H., KIMURA, M., IWATA, A., UEDA, S. and
ISHIHAMA, A.
20
Involvement of Multiple Subunit-Subunit Contacts in the As'
sembly of RNA Polymerase II. KIMURA, M., SUZUKI, H. and
ISHIHAMA, A.
Mutant Studies of the Schizosaccharomyces pombe RNA Polymerase II: Rpb3 and Rpb6 Mutants. MITOBE, J., ISHIGURO,
A., MITSUZAWA, H., NOG!, T. and ISHIHAMA, A.

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Two WD Repeat'Containing TAFs in Fission Yeast that Suppress
Cell Cycle Arrest in Mitosis. MITSUZAWA, H., SEINO, H.,
YAMAO, F. and ISHIHAMA, A.
Identification of the RNA Cap'Binding Site on Influenza Virus
RNA Polymerase.

25

HONDA, A., MIZUMOTO, K. and ISHIHAMA,A

26

Functional Modulation of Influenza Virus RNA Polymerase
by Viral RNA: Differential Roles of vRNA and cRNA.
HONDA, A., ENDO, A. and ISHIHAMA, A.
27
Expression of Functional Influenza Viral RNA Polymerasein the Methylotrophic Yeast Pichia pastoris.

HWANG, J. -S.,

YAMADA, K., HONDA, A., NAKADE, K. and ISHIHAMA, A. ......... 28
Search for Host Factors Interacting with Influenza Virus RNA
Polymerase. HONDA, A., OKAMOTO, T. and ISHIHAMA, A..... 30
A-b. Division of Mutagenesis
Ubiquitin-conjugating enzymes in fission yeast.
SEINO, H. and YAMAO, F.
Destruction of S-phase cyclin, Cig2, in fission yeast.

Park, J.H.,
34
SEINO, H.

and YAMAO, F.
Ubiquitin pathways functioning in mitotic regulatory mecha-

34

nism. SEINO, H. and YAMAO, F.
35
Characterization of SCFGrrl that ubiquitinates G 1 cyclins in

Saccharomyces cerevisiae. KISHI, T. and YAMAO, F.

36

CONTENTS
A-c. Division of Nucleic Acid ChemistryProteomic analysis of Escherichia coli proteins in the stationary
phase by the RFHR 2-D PAGE. WADA, A.

B. Department of Cell Genetics
B-a. Division of Cytogenetics
Roles of Mre 11 of Saccharomyces cerevisiae in Meiotic Recombination and Mitotic Repair Reactions. TATSUDA, D.,
OSHIUMI, H., OHTA, T., TOMIZAWA, J. and OGAWA, T.
Roles of Rad52, Rad55 and Rad57 Proteins in Rad51-mediatedHomologous Recombination in Saccharomyces cerevisiae.
WATANABE, K, MATSUDA, S., OHTA, T. and OGAWA, T.
B-b. Division of Microbial Genetics
Dpbll controls the association between DNA polymerases a and
E and the autonomously replicating sequence region of budding yeast. MAsUMOTO, H. and AR.AKI, H.
Functional characterization of Sld3 and Sld4 that geneticallyinteract with Dpbll. KAMIMURA, Y. and ARAKI, H.
Function of the Sld5/Psfl complex which interacts with DNA
polymerase E and Dpbll in Saccharomyces cerevisiae.
TAKAYAMA, Y., KAMIMURA, Y. and ARAKI, H.
Novel mutations of DPBll. MURAMATSU, S. and AR.AKI, H.
B·c. Division of Cytoplasmic Genetics
Studies on behavioral disorders of knockout mice. NIKI, H. .....
C. Department of Developmental Genetics
C·a. Division of Developmental Genetics
Regulation of neural induction by negative regulators of theraslMAPKsignalingpathway. IWANAMI, M., OKABE, M. and
HIROMI, Y.
Positional information required for sensory organ identity in the
peripheral nervous system. OKABE, M. and HIROMI, Y. ......
Spatio-temporal constraints of organogenesis in Drosophila
imaginal discs. NIWA, N., OKABE, M. and HIROMI, Y. .........
Mechanism of action of a nuclear receptor Seven-up.

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IV

MATSUNO, M., KOSE, H. and HIROMI, Y.
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Hydra Peptide Project: Epitheliopeptides which are involved in
Hydra morphogenesis. TAKAHASHI, T., HARAFUJI, N., YUM,
S., HATTA, M., SHIMIZU, H., KOIZUMI, 0., KOBAYAKAWA, Yand
FUJISAWA, T.

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Expression of nanos related genes in Hydra. MOCHIZUKI, K,
NISHIMIYA-FUJISAWA, C. and FUJISAWA, T.
54
Feeding and wounding responses of hydra: nervous system oftentacles show unidirectional conduction of stimulus.
SHIMIZU, H.
Evolution of reef-building corals. HATTA, M.
Differentiation mechanisms of neurons and glia.
UMESONO, Y., TAKIZAWA, K and HOTTA, Y.
Axon guidance mechanisms.

55
55
SASAMURA, T.,
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TAKIZAWA, K, HIRAMOTO, M. and

HOTTA, Y
Function of the gcm'related transcriptional regulators.

57
HOSOYA,T. and HOTTA, Y........................................................................

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C'b. Division of Gene Expression
Functional dissection of DNA supercoiling factor: EF-handdomains and C-terminal HDEF motif are essential for its
activity. KOBAYASHI, M. and HIROSE, S.

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The Role of Human MBF1 as a Transcriptional Coactivator.
KABE, Y, GoTO, M., SHIMA, D., IMAI, T., WADA, T., MOROHASHI,

K, SHIRAKAWA, M., HIROSE, S. and HANDA, H.
Identification of the core domain and the secondary structureof the transcriptional coactivator MBFl. OZAKI, J.,

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TAKEMARU, K, IKEGAMI, T., MISHIMA, M., UEDA, H., HIROSE,
S., KABE, Y., HANDA,H. and SHIRAKAWA, M
61
Upstream element ofthe sea urchin arylsulfatase gene serves
as an insulator. AKASAKA, K, NISHIMURA, A., TAKATA, K,
MITSUNAGA, K, MIBUKA, F., UEDA, H., HIROSE, S., TSUTSUI,
K. and SHIMADA, H.
63
C'c. Division of Early Embryogenesis
Mesoderm induction and its dorsoventral specification inCONTENTS
zebrafish. MIZUNO, T., YAMAHA, E. and TAKEDA, H.
Cell'cell interaction in zebrafish floor'plate development.
SHINYA, M. and TAKEDA, H.
Roles of mesp gene family in zebrafish segmentation. SAWADA,
A., FRITZ, A., JIANG, Y. 'J., SAGA, Y. and TAKEDA, H.
Cod. Division of Physiological Genetics
Codon usage diversity of bacterial genes: An application ofself-organization mapping for investigating origin of hori'zontal transfer genes. KANAYA, S. and IKEMURA, T.

D.

Department of Population Genetics
D·a. Division of Population Genetics
Causes ofthe irregular synonymous substitution rates in Drosophila lineages. TAKANO'SHIMIZU, T.
Study of species difference as observed as interspecific hybrid
anomaly in Drosophila: genetic screens for genes involved in
the bristle loss of hybrids. TAKANO-SHIMIZU, T.
D·b. Division of Evolutionary Genetics
Fine replication' timing map ofthe entire human chromosome
llq at a genome sequence level: genome-wide approach to
put landmarks with biological significance along the humangenome sequence by defining transition zones for the repli'
cation·timing during S phase and GC% level. WATANABE, Y.,
FUJIYAMA, A., HATTORI, M., SAKAKI, Y. and IKEMURA, T. .......
Triplex·forming sticky DNAs in human interphase nuclei; withspecial emphasis on trinucleotide repeat (GAA/TTC)n.
IKEMURA, T., OHNO, M. and FUKAGAWA, T.
Distribution of polypurine/polypyrimidine tract sequences in
the human MHC region and their possible functions.
KANAYA, S., FuKAGAWA, T., ANno, A., INOKO, H., KUDO, Y. and
IKEMURA, T.
Functional analyses of centromere proteins of higher vertebratecells. FUKAGAWA, T., OKAMURA, A. and IKEMURA, T.
Development of mammalian artificial chromosomes. FUKAGAWA,

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T. and IKEMURA, T.
Molecular genetic analysis of remains of a 2, QQQ-year-old humanpopulation in China'and its relevance for the origin of themodern Japanese population. OOTA, H., SAITOU, N. andDEDA, S.
Phylogenetic relationship of muscle tissues deduced from superimposition of gene trees. OOTA, S. and SAITOU, N.
Alpha 1, 3-fucosyltransferase IX (Fuc'TIX) is very highly conserved
between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX. KANEKO, M.,
NISHIHARA, S., INOKO, H., SAlTOU, N. and NARIMATSU, H.....
Population Genetic Studies on Nine Aboriginal Ethnic Groups of
Taiwan. I. Red Cell Enzyme Systems. JIN, F., SAITOU, N.,
ISHIDA, T., SUN, C.-S., PAN. I.-H. and HORAI, S.
Evolution ofRh Blood Group Genes Have Experienced Gene Conversions and Positive Selection. KITANO, T. and SAlTOU, N.
..................................................................................................
Phylogenetic Analysis of Pseudomonas syringae Pathovars Sug'
gests the Horizontal Gene Transfer of argK and the Evolu'
tionary Stability of hrp Gene Cluster. SAWADA, H. and
SAITOU, N.
D-c. Division of Theoretical Genetics
Effect of gene conversion on polymorphic patterns at major histo'
compatibility complex loci. OHTA, T.

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E. Department of Integrated Genetics
E·a. Division of Human Genetics
Cloning and structural analysis ofthe mouse 7F4/F5 imprinted
domain. SASAKI, H., KATO, R., SUDA, C., SHIROHZU, H.,
PURBOWASITO, W., YOKOMINE, T., MIZUNO, S., MUKAI, T.,
HATTORI, M. and SAKAKI, Y.
84
Regulation of imprinting of the mouse Igf2/H19 sub·domain.
SASAKI, H., ISHIHARA, K., KATO, R., FURUUMI, H., OHNO, M.,
HATANO, N. and IWAKI, T.
84

CONTENTS
Cloning of a family of novel mammalian methyltransferases.
SASAKI, H., CHIJIWA, T., TSUJIMOTO, N., MIZUNO, S. andTAJIMA, S.
Is the chicken genome imprinted? SASAKI, H., YOKOMINE, T.,
MATSUDA, Y. and TSUDUKI, M..................................................
Are the polycomb group genes involved in genomic imprinting?
SASAKI, H., TSUJIMOTO, N. and KOSEKI, H.
X-inactivation in the mouse embryo deficient for DNA
methyltransferase 1 (Dnmt1). SADO, T., FENNER, M.H.,
TAN, S.-S., TAM, P.P.L., SHIODA, T. and LI, K
Analysis of X-inactivation center (Xic) in mouse. SADO, T.,
MIURA, A., SASAKI, H. and LI, E.
Human Genome Resources and Their Application to the Completion of Human Chromosome 21 Sequencing. FUJIYAMA, A,
ENDO, A. and MOTOYAMA, A.
Whole Genome Analysis of Signal-Transduction Pathways in
Fission Yeast. BONG, Y. -S., DANJO, I., INOUYE, M. and
FUJIYAMA, A.
E-b. Division of Brain Function
Mechanisms of Collateral Branching and Target Invasion of
Mitral Cell Axons during Development. HIRATA, N.
E-c. Division of Applied Genetics
Characterization of 13 -amylase and its deficiency in various ricecultivars. YAMAGUCHI, J., ITOH, S.-I., SAITOH, T., IKEDA, A,
TASHIRO, T. and NAGATO, Y.
Differential in situ expression of three ABA-responsive genes ofrice, Rab16A, REG2 and OSBZ8, during seed development.
MIYOSHI, K., NAKATA, K, NAGATO, Y. and HATTORI, T...........
Initiation of shoot apical meristem in rice: characterization offour
SHOOTLESS genes. SATOH, N., HONG, S.-K., NISHIMURA, A,
MATSUOKA, M., KrTANO, H. and NAGATO, Y.
Population Genetic Studies on Nine Aboriginal Ethnic Groups of
Taiwan. I. Red Cell Enzyme Systems. JIN, F., SAITOU, N.,
ISHIDA, T., SUN, C-S., PAN, I-H., OMOTO, K. and HORAI, S. .....

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The presence of ancient human T'celllymphotropic virus type I
provirus DNA in an Andean mummy. LI, H-C., FUJIYOSHI, T.,
Lou, H., YASHIKI, S., SONODA, S., CARTIER, L., NUNEZ, L.,
MUNOZ, I., HORAI, S. and TAJIMA, K

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E Genetic Strains Research Center
F-a. Mammalian Genetics Laboratory
Phenotype characterization of male sterility of a consomic strain
B6.MSM-ChrX and fine mapping of the causative gene(s) in
the X chromosome. OKA, A., TAKAGI, N., MITA, A., MIZUSINA,
Y., SAKURAI, N. and SHIROISHI, T.
95
Phenotype characterization and fine mapping of a preaxial polydactylous mouse mutantion, X-linked polydactyly(XpJ).
YADA, Y., MASUYA, H., MAKINO, S. and SHIROISHI, T.............
97
Developmental genetics of a mouse mutant, mesenchymal dys'
plasia (mes). MAKINO, S., YADA, Y., MASUYA, H., ISHIJIMA,J., TAMURA, M. and SHIROISHI, T.
98
Positional cloning of the mouse skeletal mutation, Tail'short(Ts). SHIMIZU, K, KOIDE, T., MITA, A., UCHIDA, K, ISHIJIMA,
J., WAKANA, S., HIRASAWA, T., KrKKAWA, Y., YONEKAWA, H.,
SASAKI, H. and SHIROISHI. T.
99
Positional cloning ofthe preaxial polydactyly mutation, hemimelicextra toes (Hx). SAGAI, T., MASUYA, H., SHIMIZU, K and
SHIROISHI, T.
100
Gasdermin (Gsdm) localizing to mouse chromosome 11 is predominantly expressed in upper gastrointestinal tract but sig'
nificantly suppressed in human gastric cancer cells. SAEKI, N.,
KUWAHARA, Y., SASAKI, H., AOKI, A., MIZUSHINA, Y., KOSEKI,
H. and SHIROISHI, T.
101
Behavioral study of inbred strains established from wild mice.
III. Active avoidance test. KOIDE, T., MORIWAKI, K and
SHIROISHI, T.
102
F·b. Plant Genetics Laboratory
Analysis of genetic mechanisms controlling embryogenesis andregeneration ofrice (Oryza sativa)

CONTENTS(I)-a. Regulation of expression of KNI-type homeobox genes inrice. ITO, Y., NIWA, Y. and KURATA, N.
(I)-b. Isolation of a mutant for a KNI-like homeobox gene inrice. ITO, Y., RIROCHIKA, R. and KURATA. N.
(I)-c. Generation and screenig ofretrotransposon insertion lines inrice. KURATA, N., MIYOSHI, K, EIGUCHI, M., KITANO, R.,
MATSUOKA, M., NAGATO, Y. and IWASAKI, Y.
(I)-d. Isolation and structural analysis of OSLECI genes offunctional
importance in embryogenesis. MIYOSHI, K, and KURATA, N...
(I)-e. Isolation and structural analysis of genes for receptor-likekinases in rice. TAKAYA, K, ITO, Y. and KURATA, N.
Positional cloning of a heterochronic gene, PIal, regulating theplastochron and the duration of vegetative phase inrice. AHN, B.-O., MIYOSHI, K, ITOH, J-I., NAGATO, Y. and
KURATA, N.
Study for the Chromosome and Nucleus Organization in Rice(3)-a. Structural analyses for the centromeric region of rice chromosome 5 and construction of rice artificial chromosomes.
NONOMURA, K and KURATA, N.
(3)-b. Analysis of chromosome dynamics in the nucleus by using
GFP-tags. KURATA, N.
Analysis of Genetic Factors causing Segregation Distortion.
RARUSHIMA, Y. and KURATA, N.
Generation of enhancer trap lines of rice. ITO, Y., EIGUCHI, M.
and KURATA, N.
F-c. Microbial Genetics Laboratory
The kdsA mutations affect the FtsZ-ring formation in Escherichia
coli K12. NISHIMORI, K., TAKAGI, H., WACHI, M.,
FUJISHIMA, H., KAWABATA, T., NISHIKAWA, K. andNISHIMURA, A.
F-d. Invertebrate Genetics Laboratory
Proximal to distal cell communication in the Drosophila leg
provides a basis for an intercalary mechanism of limb
patterning. GOTO, S. and HAYASHI, S.

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Interplay of Notch and FGF signaling restricts cell fate and MAPK
activation in the Drosophila trachea.

IKEYA, T. and

HAYASHI, S.

115

Repression of the wing vein development in Drosophila by the
nuclear matrix protein Plexus.

MATAKATSU, H., TADOKORO,

R., GAMO, S. and HAYASHI, S. 116
Snail·type zinc finger proteins prevent neurogenesis in Scutoid
and transgenic animals of Drosophila.

FUSE, N.,MATAKATSU, H., TANIGUCHI, M. and HAYASHI, S

.

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.

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Enhancer trap screen for genes involved in pattern formation.
GOTO, S., TANIGUCHI, M., SADO, Y. and HAYASHI, S

G.

Center for Genetic Resource Information
G·a. Genetic Informatics Laboratory
ORYZABASE··INTEGRATED RICE SCIENCE DATABASE··YAMAKAWA, T., MITSUI, K., KURATA, N., YOSHIMURA, A.,
NAGATo, Y. and YAMAZAKI, Y..

PEC:Profiling of Escherichia coli Chromosome.

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YAMAZAKI, Y,

IKEGAMI, T., YAMAKAWA, T., MITSUI, K., KAWABATA, T.,NISHIKAWA, K, MORI, T., NISHIMURA, A. and KATO, J.

120

G·b. Genome Biology Laboratory
Analysis of gene expression patterns in Caenorhabditis

elegans. KOHARA, Y., SHIN·I, T., SUGIURA, 1., OBA, T., OBARA,
M., SUZUKI, T., KITAYAMA, S., MOTOHASHI, T., HIRONO, K,
NAGAOKA, T., ITO, M., MINAKUCHI, Y. and THIERRY'MIEG, J.
and D

.

Studies on translational regulation of maternal mRNA.
K. and KOHARA, Y
Application of cDNA array of G.elegans.

.

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MOCHII, M., YOSHIDA,

S., MORITA, K, DESUGI, H., KOHARA, Y. and DENO, N
Functional analysis of the G. elegansT'box gene tbx-9.

y.
H.

121

OGURA,

Structural Biology Center
H·a. Biological Macromolecules Laboratory.

123

ANDACHI,.

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CONTENTS
Single Molecule Imaging of Biological Functions.

xi

TOKUNAGA,

M.
Single molecule imaging of nuclear transport. lMAMOTO, N. and
TOKUNAGA, M.
Single molecule measurement of intermolecular and intramo'
lecular interactions using subpiconewton intermolecular force.
microscopy. HIROSHIMA, M. and TOKUNAGA, M
Single molecule measurement of protein folding using intermo'
lecular force microscopy. SAKANE, 1., KUWAJIMA, K and

TOKUNAGA, M.
H·b. Molecular Biomechanism Laboratory
The arrest of E. coli RNA polymerase at promoters, and theeffect of the Gre factors. SUSA, M., KUBORI, T., NAGAI, H.,
GAAL, T., MISKELISHIVILI, V.G. and SHIMAMOTO, N..
Role of w subunit of E. coli RNA polymerase. CHATTERJI, D.,
MUKHERJEE, K, NAGAI, H. and SHIMAMOTO, N.
Inactivation of a 70 by oligomerization and identification of the
NAGAI, H., KAUSCIUKOVIC, T.,..
Single'Molecule Dynamics of Transcription: Sliding of proteins
along DNA. KINEBUCHI, T., SOGAWA, K, KABATA, H.,

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127

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130

role of its spacer region.

HAYWARD, R.S., SATO, Y. and SHIMAMOTO, N

131

SHIMAMOTO, N., KUROSAWA, 0., ARAMAKI, H., WASHIZU,M..
131
H·c. Multicellular Organization Laboratory
Fluoride'resistant Mutants of the Nematode Caenorhabditis

elegans. OISHI, A, ISHIHARA, T. and KATSURA, 1.
.
Analysis of Synthetic Dauer'constitutive Mutations.
MIYAHARA, K, OHKURA, K, ISHIHARA, T. and KATSURA, 1. ....
Analysis of Mutants That Show Abnormality in the Selection
between Two Behaviors. ISHIHARA, T., IINO, Y. and KATSURA,

1

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135

.

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H·d. Biomolecular Structure Laboratory
Crystallographic Study of F1'ATPase:Structural Analysis ofSupramolecule. SHIRAKIHARA, Y., and SHIRATORI, A ........

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Crystallization of ATPsynthase. SHINDO, K and SHIRAKIHARA,y.
Crystallization of cytoplasmic domain of CD40 receptor in B-cell.
SHIRAKIHARA, Y.
Crystallization of D'aminoacylase from Alcaligenes.
SHIRAKIHARA, Y.
Crystallization of Na+·translocating ATPase. SHIRAKIHARA, Y.
..................................................................................................
I. Center for Information Biology
I-a. Laboratory for DNA Data Analysis
Reconstruction of the Ancestral Genome and Evolutionary Studyof Genome Structures by Comparison of Complete Genome
Sequences. ITOH, T. and GOJOBORI, T.
Different evolutionary histories in two subgenomic regions of the
major histocompatibility complex. GAUDIERI, S., KULSKI,
J.K, DAWKINS, R.L. and GOJYOBORI, T.
Identification of a ribonuclease R gene in both Mycoplasma
genitalium and Mycoplasma pneumoniae by a new method for
exhaustive identification of ORFs in the complete genome
sequences. BELLGARD, M.l. and GOJOBORI, T.
Significant differences between the G+C content of synonymouscodons in orthologous genes and the genomic G+C content.
BELLGARD, M.l. and GOJOBORI, T.
MITEs, interspersed nuclear elements, in the plant genomes andtheir significance in the plant evolution. MINETA, K, IKEo,
K, OGIHARA, Y. and GOJOBORI, T.
I-b. Laboratory for Gene-Product Informatics
Cooperative approach for the protein fold recognition. OTA, M.,
KAWABATA, T., KINJO, A.R. and NISHIKAWA, K
Strong hydrophobic nature of cysteine residues in proteins.
NAGANO, N., OTA, M. and NISHIKAWA, K
Design and synthesis of a globin fold. ISOGAI, Y., OTA, M.,
FUJISAWA, T., IZUNO, R., MUKAI, M., NAKAMURA, R., IIZUKA, T.
and NISHIKAWA, K.

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CONTENTS
Experimental verification of the 'stability profile of mutant
protein' (SPMP) data using mutant human lysozymes.
TAKANO, K, OTA, M., OGASAHARA, K, YAMAGATA, Y.,NISHIKAWA, K and YUTANI, K
I·c. Laboratory of Gene Function
DNA Data Bank of Japan dealing with large' scale datasubmission. SUGAWARA, R., MIYAZAKI, S., GOJOBORI, T.

151

and TATENO, Y.
A phosphate'induced gene which promotes Penicillium mediatedbioconversion of cis-propenylphosphonic acid to Fosfomycin.
Applied and Environmental Microbiology. WATANABE, M.,
SUMIDA, N., MURAKAMI, S., ANZAI, R., THOMPSON, C.J.,TATENO, Y. and MURAKAMI, T.
Genomic organization around the centromeric end of the RLA
class I region: Large'scale sequence analysis. YAMAZAKI, M.,

152

TATENO, Y. and INOKO, R.
Domain dislocation: a change of core structure in periplasmic
binding proteins in their evolutionary history.

154

FUKAMI'KoBAYASHI, K, TATENO, Y. and NISHIKAWA, K .......
Evolutionary pattern of influenza B viruses based on the HA and
NS genes during 1940 to 1999: origin ofthe NS genes after
1977. Luo, C., MORISHITA, T., SATOU, K, TATENO, Y.,

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NAKAJIMA, K, NOBUSAWA. K and E.
I -d. Laboratory for Molecular Classification
Data processing system for DDBJ. SUGAWARA, R. and

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MIYAZAKI, S.
WFCC·MIRCEN World Data Centre for Microorganisms(WDCM). SUGAWARA, R., MIYAZAKI, S. and FUJISAWA, Y...
Workbench for the microbial systematics. SUGAWARA, R.,

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MIYAZAKI, S. and OGAWA, R.
J.

xiii

Experimental FarmDevelopment, propagation and distribution of the genetic stocksof rice. NONOMURA, KI., EIGUCHI, M., MIYABAYASHI, T. and

153

157
157

xiv
KURATA, N

Abstracts of diary for 1999
Foreign visitors in 1999
Association for propagation of the knowledge of genetics
Author index

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..
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159
161
167
168
170

GENERAL STATEMENT

We had two special events this year. National Institute of Genetics (NIG)
was established 50 years ago, and we cerebrated the semi'centennial on June
1st by inviting many distinguished guests and our old friends. To commemorate
the anniversary, we started a "Virtual Museum of Genetics" by expanding ourhome page. We would like to make it as a useful and enjoyable site for citizens
and students. At present, it is still in its infancy, but please visit it and give us
your feedbacks.
We also had a special day on May 31"t, when Emperor and Empress visited
us and enjoyed watching our scientific activities. Emperor himself is a biologist
and asked many questions. We were very happy to see their relaxedatmosphere.
Major contributions ofNIG in the 50'year research in genetics, in particular,plant genetics, population genetics and molecular and developmental genetics,
have made it one of the most important centers with worldwide recognition. In
1984, NIG was reorganized into an Inter-university Research Institute topromote collaborative activities. Together with other inter-university research
institutes, we founded the Graduate University for Advanced Studies in 1988.
We are serving as Department of Genetics of the Graduate School of Life
Science. This year, we have 33 graduate students and 10 special research
students from other universities, including those from abroad. Nine students
obtained Ph.D. this year. In addition, we have the Center Of Excellence (COE)program with which 5 foreign and 7 Japanese postdoctoral fellows conducted
research.
We have been carrying out several research,related services. The DNA Data
Bank of Japan (DDBJ) is one of the three central data banks in the world thatgather, annotate, store and distribute DNA sequence information. It receives
data input not only from Japanese institutions but also from institutes inother Asian countries. Recent development of new technology using various

model organisms has enhanced the importance of genetics as the bases ofmany branches of biological studies. We have Genetic Strains Research Center
and Genetic Resource Information Center that are designed to organize andsupport the use of genetic strains and resources. Our institute also stores anddistributes various organisms with genetically characterized traits. Amongthem, services with Escherichia coli, mice and Drosophila are particularly
significant. These activities will continue to develop in the coming years. Our
institute is uniquely suited for pursuing cooperative works with scientists ofvarious disciplines by sharing the genetic resources.

Yoshiki Hotta, Director-General

3

STAFF

(as of December 31,1999)

Director-General
HOTTA, Yoshiki, D. Med.

Vi ce-Di rector
OGAWA, Tomoko, D. Pha.

Members
1. Department 01 Molecular Cenetics
ISHIHAMA, Akira, D. Sc., Head of the Department

Division ofMolecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
MITSUZAWA, Hiroshi, D. Sc.
KIMURA, Makoto, D. Sc.

Division ofMutagenesis
YAMAO, Fumiaki, D. Sc., Associate Professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.

Division ofNucleic Acid Chemistry
MIZUMOTO, Kiyohisa, D. Pha., Adjunct Professor
W ADA, Akira, D. Pha., Adjunct Professor
2. Department 01Cell Cenetics

ARAKI, Hiroyuki, D. Sc., Head of the Department

Division ofCytogenetics
OGAWA, Tomoko, D. Pha., Professor
IMAI, Hirotami T., D. Sc., Associate Professor
TANAKA, Shigeo, D. Med.
OHTA, Tsutomu, D. Med.

Division ofMicrobial Genetics
ARAKI, Hiroyuki, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate Professor

4

KAMIMURA, Yoichiro, D. Med.

Division o/Cytoplasmic Genetics
TOMIZAWA, Jun-ichi, D. Pha., Adjunct Professor
NIKI, Hiroaki, D. Med. Sci., Ph. D. Adjunct Professor
3. Department ofDevelopmental Genetics

HIROSE, Susumu, D. Sc., Head ofthe Department

Division o/Developmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
HATTA, Masayuki, D. Sc.
OKABE, Masataka, M. D., Ph. D.
HOSOYOA, Toshihiko, D. Sc.

Division o/Gene Expression
HIROSE, Susumu, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate Professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-aki, D. Ag.
UEDA, Hitoshi, D. Ag.
Division 0/Early Embryogenesis
TAKEDA, Hiroyuki, D. Sc., Professor
Division 0/Physiological Genetics
SHIRAKAWA, Masahiro, D. Sc., Adjunct Associate Professor
KANAYA, Shigehiko, D. Ind., Adjunct Associate Professor
4. Department ofPopulation Genetics

IKEMURA, Toshimichi, D. Sc_, Head of the Department

Division 0/Population Genetics
TAKANO, Toshiyuki, D. Sc.

Division 0/Evolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate Professor
TENZEN, Toyoaki, D. Ag.

STAFF
FUKAGAWA, Tatsuro, D. Sc.

Division ofTheoretical Genetics
HARADA-OHTA, Tomoko, Ph. D. D. Sc., Adjunct Professor
KITANO, Hiroaki, D. Eng., Adjunct Professor

5. Department ofIntegrated Genetics
SASAKI, Hiroyuki, D. Med., Head of the Department

Division ofHuman Genetics
SASAKI, Hiroyuki, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate Professor
SADO, Takashi, D. Sc.

Division ofAgricultural Genetics
Division ofBrain Function
HIRATA, Tatsumi, D. Med., Associate Professor
KAWASAKI, Takahiko, D. Sc.

Division ofApplied Genetics
NAGATa, Yasuo, D. Ag., Adjunct Professor
HORAI, Satoshi, D. Med., Adjunct Professor

6. Genetic Strains Research Center
SHIROISHI, Toshihiko, D. Sc., Head ofthe Center

Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Professor
KaInE, Tsuyoshi, D. Med.

Mammalian Development Laboratory
NAKATSUJI, Norio, D. Sc., Professor
SAITO, Tetsuichiro, D. Sc.
TADA, Takashi, D. Sc.

Plant Genetics Laboratory
KURATA, Nori, D. Ag., Associate Professor
ITO, Yukihiro, D. Ag.

Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Associate Professor

Invertebrate Genetics Laboratory

6

HAYASHI, Shigeo, D. Sci, Professor
GOTO, Satoshi, D. Sc.

7. Center for Cenetic Resource Information
KOHARA, Yuji, D. Sc., Head ofthe Center
Genetic Informatics Laboratory
YAMAZAKI, Yukiko, D. Sc., Associate Professor
FUJITA, Masaya, D. Eng.
Genome Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
8. StructuralBiolo!fY Center
KATSURA, Isao, D. Sc., Head of the Center

Biological Macromolecules Laboratory
TOKUNAGA, Makio, D. Sc., Associate Professor

Molecular Biomechanism Laboratory
SHIMAMOTO, Nobuo, D. Sc., Professor
NAGAI, Hiroki, D. Sc.

Multicelluar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.

Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor

Gene Network Laboratory
9. Center for Information Biolo!fY

GOJOBORI, Takashi, D. Sc., Head of the Center

Laboratory for DNA Data Analysis
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
IMANISHI, Tadashi, D. Sc.

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

STAFF

Laboratory for Gene Function
TATENO, Yoshio, Ph. D. D. Sc., Professor
FUKAMI"KOBAYASHI, Kaoru, Ph. D.

Laboratory for Molecular Classification
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, D. Sc.
10.

Radioisotope Center

ISHIHAMA, Akira, D. Sc., Professor, Head ofthe Center
11.

Experimental Farm

KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken'ichi, D. Ag.
12.

Teehnleal Seetlon

HOTTA, Yoshiki, D. Med., Chief ofthe Section
13.

Department ofAdministration

SUNADA, Hitoshi, Head of the Department
KOBAYASHI, Akira, Chief of the General Affairs Section
KOSEKI, Kenzo, Chief of the Finance Section

7

8

COUNCIL(as of December 31, 1999)

Chairman
SUGIMURA, Takashi; President, Toho University
Vice-chairman
OSAWA, Shozo; Adviser, Biohistory Research Hall
Members (Alphabetical order)
HIROTA, Eiji; President, The Graduate University for Advanced Studies
ISHII, Shiro; Professor, The International Research Center for JapaneseStudies
ITO, Mitsuo; President, Okazaki National Research Institutes
IWATSUKI, Kunio; Professor, College of Science Rikkyo University
KURODA, Reiko; Professor, Graduate School ofArts and Sciences, The University of Tokyo
KYOGOKU, Yoshimasa; Professor, Fukui University of Technology
MATSUBARA, Ken-ichi; Vice-Director, International Institute for Advanced
Studies
MATSUO, Minoru; President, Nagoya University
MIURA, Kin-ichiro; Director, Institute for Biomolecular Science, Gakushuin
University
MIYAMOTO, Misako; President, Nihon Women's University
MOHRI, Hideo; Director-General, National Institute for Basic Biology
OKADA, Masukichi; Professor Emeritus, University of Tsukuba
OSAKI, Hitoshi; Director-General, Center for National University Finance
OTSUKA, Eiko; Professor Emeritus, Hokkaido University
TANAKA, Ryuso; President, Hiroshima City University
TOYOSHIMA, Kumao; Director, Sumitomo Hospital
TSUNEWAKI, Koichiro; President, Fukui Prefectural University
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Science9

ADVISORY COMMITTEE(as of December 31,1999)

Chairman
OGAWA, Tomoko; Professor, National Institute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Professor Fukuoka Dental College
Outside Members (Alphabetical order)
Go, Michiko; Professor, Graduate School of Science, Nagoya University
HANAOKA, Fumio; Professor, Institute of Molecular and Cellular Biology,
Osaka University
HINATA, Kokichi; Representative Managing Director, Research Instituteof Seed Production Co., Ltd.
ISONO, Katsumi; Professor, Faculty of Science, Kobe University
ITO, Koreaki; Professor, Institute for Virus Research, Kyoto University
KATSUKI, Motoya; Professor, The Institute of Medical Science, The
University of Tokyo
MATUURA, Etsuko; Professor, Faculty of Science, Ochanomizu University
SASAZUKI, Takehiko; Professor, Medical Institute ofBioregulation, Kyusyu
University
TAJIMA, Fumio; Professor, Graduate School ofScience, The University oftokYo
Inside Members (Ajphabeticalorder)
ARAKI, Hiroyuki; Professor, National Institute of Genetics
GOJOBORI, Takashi; Professor, National Institute of Genetics
HIROMI, Yasushi; Professor, National Institute of Genetics
HIROSE, Susumu; Professor, National Institute of Genetics
IKEMURA, Toshimichi; Professor, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
KOHARA, Yuji; Professor, National Institute of Genetics
SASAKI, Hiroyuki; Professor, National Institute of Genetics
SHIROISHI, Toshihiko; Professor, National Institute of Genetics

10

RESEARCH ACTIVITIES IN 1999

A.

DEPARTMENT OF MOLECULAR GENETICS
A-a. Division of Molecular Genetics(1)

Mapping of Protein-protein Contact Network Involving the Ii and
Ii' Subunits of Escherichia colJ RNA Polymerase

Tasuku NOMURA, Akira KATAYAMA, Nobuyuki FUJITA andAkira ISHIHAMAThe RNA polymerase holoenzyme of Escherichia coli is composed of the core
enzyme with the subunit composition of 0: 2 {3 {3' and one of seven different
species of the a subunit. The core enzyme carries the catalytic activity of RNApolymerization, but the a subunit is required for the promoter recognition andtranscription initiation from the promoter. The core enzyme is assembled
sequentially both in vitro and in vivo under the order: 20:-+ 0: 2 -+ 0: 2 {3-+ 0: 2 {3 {3'(premature core) -+ E (active core). Previously we performed detailed map'ping of the subunit·subunit contact sites on the 0: subunit. Along this line, we
carried out the mapping of subunit'subunit contact sites on two large subunits, {3 and {3' subunits. For functional mapping the {3 subunit (1342 residues), we employed two approaches: (i) comparative analysis ofthe proteolytic
cleavage pattern of the unassembled free {3 subunit and the intermediate
subassembly 0: 2 {3 complex; and (ii) analysis of the complex formation between various {3 fragments and His 6 -tagged 0: subunit or between various
His6 -tagged {3 fragments and the intact 0: subunit. The results indicated thatthe primary and tight contact site on the {3 subunit with the 0: subunit islocated in the central portion of {3 polypeptide but in addition, the C·terminal
proximal region is needed as the secondary and probably regulatory site for
either efficient binding of the 0: subunit or stabilization ofthe 0:-{3 contact. All
the {3 fragment- 0: binary complexes isolated were able to bind {3' subunit

A.

DEPARTMENT OF MOLECULAR GENETICS

11

leading to form pseudo·core complexes, suggesting that the (3' subunit"con"tact site(s) on the {3 subunit is located near the a"contact sites. For detailed
mapping of the subunit·subunit contact sites on the {3'subunit (1407 resi"dues) with the 0 70 subunit and the a 2 {3 complex, we employed two approaches:(i) identification of {3 tryptic fragments capable of forming complexes withpreformed a 2 {3 complex or purified 0 70 subunit; and (ii) systemtaic analysis
of complex formation of various {3' fragments, with or without His 6 -tag, withthe a 2 {3 complex and the 0 70 subunit. The results indicated that: two reo
gions, one central region and the other C"terminal proximal region are in"volved in binding the a 2 {3 complex; and one N-terminal proximal region plays
a major role in binding the 0 70 subunit. However, both a 2 {3 -binding sites
have weak activity of the 0 70 subunit; likewise, the 0 70 subunit-contact surface has weak binding activity of the a 2 {3 complex. The sites involved in the
catalytic function of RNA polymerization are all located within two spacer
regions sandwiched between these three subunit-subunit contact surfaces.
As an extension of this line of studies, we searched for class· III and -IV transcription factors which interact with the {3 and/or {3'subunits, respectively. For
this purpose, we employed an affinity isolation of candidate proteins from wholecell extracts of E. coli expressiong a low level of {3 - or {3' -subunts fused withglutathione S-transferase (GST). After N·terminal amino acid microsequencing,
we identified 7 proteins as the candidates for class- III ( (3 -contact) and 9 pro·teins as the candidates for class·IV ( (3 '-contact) factors. The 110 kDa RapAJ
HepA protein was included in both group of proteins. For details see Refs. 13,
14, 15, 17 and 23.
I(2)Structural Organization of the ex Subunit ofEscherichia colJ RNA Polymerase

Nobuyuki FUJITA, Shizuko ENDO and Akira ISHIHAMA
The a subunit of Escherichia coli RNA polymerase is comprised of two independently folded domains. The N-terminal domain (NTD; residues 8 to 235)
is required and sufficient for the assembly of core enzyme with the composition of a 2 {3 {3', while the C-terminal domain (CTD; residues 249 to 329) plays

12

key roles in transcription activation by interacting with either a large group oftranscription factors (class I factors) such as cAMP receptor protein (CRP) andOmpR, or the upstream promoter element (up element) which is present in acertain class of strong promoters including ribosomal RNA promoters. Theintervening sequence connecting these two domains is easily accessible toproteases and has high motional flexibility as revealed by the measurement of
NMR relaxation and the comparison of NMR signals between o:CTD and
intact 0: subunit. The flexible nature of interdomain linker is also suggested
from functional studies. The o:CTD is able to interact with CRP bound at
widely different positions along the DNA while the RNA polymerase is boundat a fixed position of the promoter. Different surfaces of o:CTD can be utilized
for the interaction with upstream DNA in different initiation complexes. Threedimensional structures of o:CTD and o:NTD of E. coli RNA polymerase havebeen determined by NMR spectroscopy and X-ray crystallography,
respectively. The o:CTD has a compact globular structure consisting of four
helices, two helical turns and a long C-terminalloop with defined structure. The
dimer of o:NTD has an elongated flat structure. The two 0: CTDs are supposed to extrude out from the core part of RNA polymerase, probably toward
the upstream direction, through the long flexible linkers. Such a moleculararchitecture would allow o:CTDs to behave as independent motional units
and interact with a variety of DNA and protein signals while the main part ofRNA polymerase stays at the promoter site. Despite the possible importanceof the interdomain linker in transcription activation, none of the reported
structures contain the linker region.
To elucidate structural requirements, if any, for the interdomain linker, weconstructed a set of mutant E. coli RNA polymerases, each consisting ofmu'
tant 0: subunit with altered interdomain linker. Deletion of 3 amino acids
from the linker exhibited 50% inhibition of CRP'dependent lac PI
transcription. Deletion of 6 amino acids completely knocked out the activity.
Insertion of 3 amino acids did not affect the activity, whereas 40-60% inhibi'tion was observed after insertion of 1, 2, or 4 amino acids. Substitution of 10
consecutive glycine residues resulted in nearly 90% reduction of the CRPdependent activity, whereas 50% activity was retained after substitution of 10
proline residues or a sequence expected to form a strong 0: - helix. Similar

A.

DEPARTMENT OF MOLECULAR GENETICS

13

results were obtained with UP element-dependent rrnBP1 transcription. These
results altogether suggest that: (j) sufficient length of the interdomain linker
is required for transcription activation mediated by a C-terminal domain; (ii)the linker directs positioning ofthe C-terminal domain to a preferred orientation for the interaction with CRP and UP element; (iii) in addition to theoverall flexibility, the amino acid sequence context ofthe linker is importantfor full function of the C-terminal domain, probably through the formation ofloose helix-like structure. See Refs. 6 and 14.

(3)Functional Organization of the a Subunit ofEscherichia colJ RNA Polymerase

Olga N. OZOLINEl, Nobuyuki FUJITA and Akira ISHIHAMA (Ion leave of absencefrom Institute of Cell Physics, RAS, Puschino)
In transcription initiation by the E. coli RNA polymerase, exchangeable
a subunits are responsible for recognition of promoter DNA while the C-terminal domain (CTD) of a subunit makes additional contacts with regulatory
signals. For instance, the upstream sequence of rrnBP1 promoter, generally
designated as UP element, directly contacts the aCTD and supports enhanced
transcription ofrRNA. The aCTD is also responsible for interaction with a setof transcription factors, designated as class-I (or a "contact) factors, regulating transcription efficiency. The most studied factor is cAMP receptor protein(CRP), which regulates transcription of more than 80 genes. Molecular mechanism oftranscription regulation by CRP depends on the position of its bindingsite on the promoter sequence. Promoters which have the CRP-binding site
centered between "60 and -100 usually require aCTD for activation.
To get insight into the detailed mechanism of transcription activation by
aCTD, we tried to monitor conformational changes within the aCTD uponinteractions with DNA UP element or transcription factor CRP. For this purpose, a fluorescent dye, fluorescein mercuric acetate (FMMA), was conjugatedat various positions of the aCTD using a newly constructed collection of single
Cys"mutant a subunit, and spectral changes were measured after interaction
with DNA UP elements or CRP. When FMMA was conjugated at Cys located14in the a-subunit helix-I and the loop between helix-III and -IV, the spectralchanges typical for DNA interaction were observed for the RNA polymerasepromoter binary complex with UP element-dependent rrnBP1 and the ternarycomplex with CRP-dependent uxuAB promoter in the presence of cAMPCRP. In addition, we found conformational changes of aCTD upon formationof binary complex with the uxuAB (in the absence of CRP) and factor-independent T7D promoters. In these cases, the spectral changes suggested that the
C-terminal proximal helix-IV of aCTD approaches the negatively charged
phosphate moiety of DNA. This prediction was supported after analysis ofcontact-dependent DNA cleavage by the chemical nuclease, Fe-(pbromoacetamidobenzyD-EDTA (Fe-BABE), conjugated to Cys-309 located on
the helix-IV. We propose that the helix-IV of aCTD is involved in direct interaction with some promoters. For details see Refs. 13, 14, 24 and 25.

(4)Competition among Seven E. colJ (J Subunits: Intracellular
Concentrations and Binding Affinity to the Core Enzyme

Hiroto MAEDA l , Miki JISHAGE, Tasuku NOMURA, Akira KATAYAMA, Nobuyuki
FUJITA andAkira ISHIHAMA (Ion leave of absence from Kagoshima Univ., Facul.
Fisheries)
The RNA polymerase core enzyme with the subunit composition a21313' has
the ability to transcribe the genetic information on DNA into RNA, but for
initiation of transcription at specific promoter sites on DNA, an additional
component, a subunit, is required. Seven different molecular species of thea subunit have been identified in E. coli, and replacement of the a subunit on
the RNA polymerase is an efficient way for switching the transcription
pattern. In Escherichia coli, seven different species of the a subunit, a 70 , aN
(also called a 54 ), as (a 38 ), all (a 32 ), a F (a 28 ), a E (a 24 ) and a Fed , are known toexist, each directing transcription of a specific set of genes. Most of the growthrelated and house-keeping genes expressed at the exponential phase of cell
growth are transcribed by the holoenzyme containing a70 (the rpoD gene product), while the holoenzyme Eas is essential for transcription of some stationary-phase specific genes. The stress response genes are transcribed by RNA

A.

DEPARTMENT OF MOLECULAR GENETICS

15

polymerase holoenzymes containing the alternative minor a subunits. We
have purified all seven species of the E. coli a subunit and analyzed theirrecognition specificity of various E. coli promoters. Using specific antibodies
raised against the purified a proteins, we also measured the intracellularconcentrations of all seven a subunits for both exponential and stationary
phase cultures of E. coli W31l0(A). The order of intracellular level was:a 70 > a F > aN> a S, a fI, a E, a FecI in exponential phase; and a 70 > as> a F >aN> a fI, a E, a FecI in stationary phase.
The global pattern of gene transcription is believed to be determined throughcompetition between available a subunits, and if this is the case, the replacement of core enzyme-associated a subunit from one species to another shouldbe the major determinant in the switching of global transcription pattern. To
test the a competition model in the global regulation oftranscription among
about 4000 genes on the E. coli genome, all seven a subunits have been purified, and compared for the binding affiniy to the same core RNA polymerase(E). In the presence of a fixed amount of a 70 (the principal a for growth-related genes), the level ofE a 70 holoenzyme formation increased linearly with
the increase in core enzyme level, giving the apparent ~ for the core enzyme of
0.26 nM. Mixed reconstitution experiments in the presence of a fixed amountof core enzyme and increasing amounts of an equimolar mixture of all sevena subunits indicated that a 70 is the strongest in the core enzyme binding,followed by aN, aF, aEI a FecI , afl, and as in the decreasing order. This order ofcore-binding affinity was confirmed by measuring the replacement of one coreassociated a by another a subunit. Taken together with the intracellular alevels, we tried to estimate the number of each holoenzyme form in growing E.
coli cells. Based on the estimation, we propose that, for effective replacementof the core-bound a70 by other minor a for stress-response gene transcription,
it appears that specific intracellular conditions or additional factors are
required. See Refs. 13, 14, 15, 16 and 19.

16

(5)

Control of the Activities of0

Subunits by Anti- 0 Factors

Miki JrsHAGE, Dipak DASGUPTA I and Akira ISHIHAMA (Ion leave of absencefrom Saha Institute of Nuclear Physics, Calcutta)
The intracellular concentrations of the seven a subunits change depending
on cell growth conditions. In addition to the level control, the activity ofa subunit is controlled. For instance, the subunit a 28 ( aF) involved in transcription ofthe genes for the flagella formation becomes inactive when it forms
a binary complex with the flgM gene product. Likewise the activity of a E, amember of the ECF (extracytoplasmic function) family a subunits, is regulated by the rseA (regulator of sigma E or anti- aE factor) gene product which is
associated with the inner membrane and inhibits the activity of a E by directlyinteracting with a E •Recently we discovered a novel E. coli protein, referred to Rsd (regulator ofsigma D), which forms a complex with a 70 and prevents its function. Purified
Rsd protein formed complexes in vitro with a 70 but not with other a subunits,
and inhibited a 7°-dependent transcription in vitro to various extents depending on the promoters used. Since Rsd is induced in the stationary phase of cell
growth, unused excess a70 subunit, without being involved in transcription
cycle, should be trapped by Rsd. Thus, the possibility has arisen that Rsd isan anti- a factor for the major a70 subunit for its storage in stationary phase. In
order to clarify the in vivo function of Rsd, we analyzed the influence of both
depletion and over-production of Rsd on a 70 - and as-dependent transcription
in vivo. On the basis of the results herewith described we propose that Rsd isa regulator that facilitates the switching of a subunit on RNA polymerase
from a 70 to a S during the transition from exponential growth to stationary
phase. To understand the in vivo role of Rsd level in E. coli, we analyzed thetranscription organization of the rsd gene. Primer extension analysis indicated the presence of two promoters, a~dependent PI and a7~dependent P2
with the gearbox sequence. The expression of ladl reporter gene fused to either a 70 - or as-dependent promoter was analyzed in the absence of Rsd orpresence of over-expressed Rsd. In the rsdnull mutant, the a 70 • and as'de'pendent gene expression is increased or decreased, respectively. On the other

A.

DEPARTMENT OF MOLECULAR GENETICS

17

hand, the a 70 • and as-dependent transcription was reduced or enhanced, respectively, after over-expression of Rsd. The repression of the as-dependenttranscription in the rsd mutant is overcome by increased production of theas subunit. These observations altogether support the prediction that Rsd isinvolved in replacement of the RNA polymerase a subunit from a70 to as during the transition from exponential growth to stationary phase.
An an extension of this line of studies, we have searched for anti-sigma
factors candicates for other minor a subunts. Using the affinity purification
method, we identified, besides the core enzyme subunits, extra components
associated with the overexpressed tagged a subunits. Functional characterization of these a-associated proteins is in progress. See Refs. 12, 13, 15 and
16.

(6)

Contact Site Mapping of (J Subunits and Transcription Factors
on the RNA Polymerase Core Enzyme Subunits Using FeBABE

Siva Ramesh WIGNESHWERARAJ I, Frederic COLLAND 2, Nobuyuki FUJITA, Claude
F. MEARES 3 and Akira ISHIHAMA (Ion leave of absence from Imperial College of
Science, Technology and Medicine, London; 20n leave of absence from Pasteur
Institut, Paris; and 3University of California, Davis)
Previously we developed, in collaboration with Claude F. Meares and colleagues (Univ. California, Davis), a new method of the mapping of proteinnucleid acid and protein-protein contact sites using protein·conjugated (pbromoacetamideenzyl)EDTA-Fe (FeBABE). This useful tool has been successfully used for mapping of the contact sites of RNA polymerase a and 0: subunits on promoters and the mapping of 0: and a 70 subunits on the core enzyme. This
year we employed this technique for mapping of the contact sites of as and aNsubunits on the core enzyme subunits. For this purpose, we constructed setsof single Cys mutant a S and a Nsubunits at various regions along these asubunits. After conjugation of FeBABE at each Cys residue, holoenzymes werereconstituted by mixing with the core enzyme, and the cleavage sites on thecore enzyme subunits were determined upon addition of H 2 0 2 andascorbate. The protein cutting by a ~ and a ~tethered FeBABE was observed

18

in two regions ofthe {3 subunit, one including regions C and D, and the otherincluding the region G and the dispensable region 2 (DR2), and one region ofthe {3' subunit, including regions C and D. The cleavage patterns are similar
to that observed with a 7°-tethered FeBABE, but there were minor but significant differences in the cleavage sites among the three a subunits.
As an initial attempt for systematic mapping of the contact sites of varioustranscription factors on the RNA polymerase, we developed a modified methodof FeBABE conjugation to protein Lys residues by using 2-iminothiolane (2-IT)
as a linker. Using the modified method, we succeded to locate the contact sites
on the core enzyme subnits oftwo elongation factors, NusA and GreA, and an
RNA polymerase chaperone w. For details see Refs. 2, 3, 5, 14, 33 and 37.
(7)

Growth Phase-dependent Variation in the Protein Composition ofEscherichia colJ Nucleoid

Ali Azam TALUKDER, Akira IWATA 1, Susumu UEDA 1 and AKIRA ISHIHAMA(INippon Institute for Biological Science, Ohme)
The genome DNA of Escherichia coli forms nucleoprotein complexes, often
called nucleoid, together with about 10 major DNA-binding proteins, among
which Fis, H-NS, HU and IHF have been believed to be the major molecularspecies. Several lines of evidence indicate that the intracellular levels of thenucleoid proteins and their localization along the genome DNA influence not
only the conformation of nucleoid but also the functions of DNA such as replication, recombination, repair and transcription. In certain cases, the regulatory roles of nucleoid proteins in DNA functions are attributed to not only
modulation of the genome conformation as a whole but also more direct effecton the local conformation of specific DNA regions or even direct interaction
with protein components. Using a quantitative immunoblot method, we
perfomed for the first time a systematic determination of the intracellularconcentrations for twelve species of the nucleoid protein in E. coli W3110,
including CbpA (Curved DNA-Binding Protein A), CbpB (Curved DNA-Binding Protein B; or Rob, Right Origin Binding protein), DnaA (DNA-binding pro·teinA), Dps (DNA-binding Protein from Starved cells), Fis (Factor for Inver-

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sion Stimulation), Hfq (Host Factor for phage Q~), H-NS (Histone-like Nucleoid Structuring protein), HU (Heat-Unstable nucleoid protein), IciA (Inhibitorof Chromosome Initiation A), IHF (Integration Host Factor), Lrp (LeucineResponsive regulatory Protein) and StpA (Suppressor of td - Phenotype A).
The level is maximum at the growing phase for nine proteins, CbpB (Rob),
DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp and StpA, which may play regulatory
roles in DNA replication and/or transcription of the growth-related genes. The
order of accumulation level, calculated as monomers, in growing E. coli is:
Fis>Hfq>HU>StpA>H-NS>IHF*>CbpB(Rob» Dps*>Lrp>DnaA>IciA>CbpA*(stars represent the stationary-phase proteins).
The order of abundance in the early stationary-phase is:Dps*>IHF*>HU>Hfq>H-NS>StpA>CbpB(Rob»DnaA>Lrp>IciA>CbpA>Fis,
while that in the late stationary phase is:Dps*>IHF*>Hfq>HU>CbpA*>StpA>H-NS>CbpB(Rob»DnaA>Lrp>IciA>Fis.
Thus, the major protein components of nucleoid change from Fis and HU in thegrowing phase to Dps in the stationary phase. The curved DNA-binding pro"tein, CbpA, appears only in the late stationary phase. These changes in thecomposition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the DNA
functions. The intracellular localization of these DNA-binding proteins is be"
ing analyzed using indirect immunofluorescence staining. For details see Refs.
12, 13, 30, 31 and 32.
(8)Functional Organization of Two Large Subunits of the Fission
Yeast Sehlzosaeeharomyees pombe RNA Polymerase II

Wjatschesslaw A. WLASOFF 1, Makoto KIMURA and Akira ISHlHAMA (Ion leave ofabsence from Institute of Cytology and Genetics, RAS, Novosibirsk)
The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces
pombe is a multifunctional and multisubunit enzyme consisting of 12 putative subunits. In contrast to prokaryotic RNA polymerases, little is known
about the molecular architectures of eukaryotic counterparts except that: (i)the two largest subunits, homologous to bacterial 8 and 8' subunits, are20

involved in the binding of DNA template, the polymerization of RNA chains,
and the association of nascent RNA chains; and (ii) the subunits 3 and 11,
homologues of bacterial a subunit, play roles in the assembly of Pol
II. Knowledge of the structure and function of the individual subunits is essential for understanding of the molecular mechanisms of transcription andregulation of the protein -coding genes in eukaryotes. As an attempt toward
this ultimate goal, we tried to locate the active center for RNA polymerization
using an affinity labeling with photo-reactive nucleotide analogues.
The catalytically competent transcription complex of Pol II was affinitylabeled with photo-reactive nucleotide analogues incorporated at 3' termini of
nascent RNA chains. To locate the catalytic site for RNA polymerization, thelabeled subunits were separated by SDS- PAGE and subjected to partial
proteolysis. After microsequencing of proteolytic fragments, a complex
multidomain organization was indicated for both of the two large subunits,
Rpb1 and Rpb2, with the most available sites of proteolysis in junctions between the conserved sequences among RNA polymerase from both prokaryotes and eukaryotes. The cross-linking studies indicate that: (i) the 3' terminiof growing RNA chains are most extensively cross·linked to the second largest
subunit Rpb2 between amino acids 825 and 994; and in addition, (in theregions 298-535 of Rpb2 and 614-917 ofRpb1 are cross-linked to less extents,suggesting that these regions are situated in the vicinity of the catalytic
site. All these regions include the conserved sequences of RNA polymerases,
and the catalytic site of Rpb2 belongs to an NH 2 -terminal part of its conserved
sequence H. For details see Ref_ 37.

(9)

Twelve Subunits of the Fission Yeast RNA Polymerase II:
Intracellular Contents, Transcription Organizationand mRNA Levels

Hitomi SAKURAI, Makoto KIMURA, Akira IWATA l , Susumu VEDA l and Akira
ISHIHAMA (INippon Institute for Biological Science, Ohme, Tokyo)
The RNA polymerase II (Pol II) core enzyme of the fission yeast

Schizosaccharomyces pombe is composed of twelve polypeptides, Rpb 1 to

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Rpbl2, corresponding to RPBl to RPB12 of the budding yeast Saccharomyces
cerevisiae. The subunits, Rpbl, Rpb2, Rpb3 and Rpb11, have the sequencesimilarity with the subunits, {3', {3, a and a, respectively, of the prokaryotic
RNA polymerase core enzyme, and are considered to be the essential for the
Pol II assembly and the catalytic function of RNA polymerization. The pathway of subunit assembly also seems to be similar, at least in part, to that ofEscherichia coli RNA polymerase. The assembly core with the structure Rpb2Rpb3-Rpb 11 corresponds to the a 2 {3 complex of E. coli. In addition, a larger
complex consisting of 7 subunits, Rpbl, Rpb2, Rpb3, Rpb5, Rpb7, Rpb8 and
Rpb11 is formed in vivo supposedly as an assembly intermediate of the S.
pombe Pol II. Among the twelve Rpb subunits, five, Rpb5, Rpb6, Rpb8, RpblOand Rpbl2, are assembled in Pol I (for rRNA synthesis) and Pol III (for tRNA
synthesis) as well as Pol II, and thus the processes ofrRNA, mRNA and tRNA
synthesis should be tightly interrelated through sharing the common
subunits. At present, however, little is known on the regulation of synthesis
and assembly of the RNA polymerases in eukaryotes.
As an initial attempt to get insight into the regulation of the synthesis andassembly ofthe Pol II, we have performed a systematic measurement of theintracellular levels of all twelve subunits of the Pol II in S. pombe using
quantitative immunoblotting. The levels ofthree subunits, Rpbl, Rpb2 andRpb3, were found to be low, compared with those of small-sized subunits,
which range 2- to 15-fold higher than the levels of the three core subunits.
Glycerol gradient centrifugation of S. pombe extracts indicated the presence ofconsiderable pools of free unassembled forms (and subassemblies) for thesesmall-sized Rpb subunits. Based on the intracellular concentration of theleast abundant Rpb3 subunit, the total number of Pol II in a growing S. pombecell was estimated to be about 10,000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of
the green fluorescent protein (GFP)·fused Rpb proteins.
We also analyzed the transcription organization of the genes for all twelve
subunits of the S. pombe Pol II using the oligo capping method. Transcriptionof one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7and rpblOis initio
ated mainly at a single site, while that of the other group of genes for rpbl,
rpb2, rpbB, rpb9, rpbll and rpb12is initiated at multiple sites. The promot-

22ers of the first group genes contain the TATA box sequence between -26 to -62,
while the second group genes carry TATA-Iess promoters. Several common
sequence segments, tentatively designated "Rpb motifs", were identified in
the promoter regions of the rpb genes. As an extension, we determined themRNA levels of all twelve rpb genes using competitive peR method. The results indicate that the mRNA levels do not necessarily correspond to those ofprotein products. mRNAs for Rpbl, Rpb3, Rpb7 and Rpb9 were among thegroup oflow abundance, while the levels of Rpb6, Rpb7 and RpblO mRNAs
were about 5-fold and that of Rpb2 mRNA was about 40-fold higher than the
Rpb3 mRNA level. After comparison between the protein and mRNA levels,
we predict that the efficiency oftranslation is maximal for mRNA species with
5' untranslated region of about 100 nucleotides in length, but becomes low for
mRNAs with shorter or longer untranslated regions. The translation efficiency
is low for the rpbl, rpb2, rpb3 and rpbll genes, encoding the homologues ofsubunits {3: {3, a and a, respectively, of prokaryotic RNA polymerase core
enzyme, supposedly because the transcription initiation sites of these genes
are all located close to the translation initiation codons. See Refs. 18,27, 28
and 37.

(10)

Involvement of Multiple Subunit-Subunit Contacts
in the Assembly of RNA Polymerase II

Makoto KIMURA, Hisako SUZUKI and Akira ISHIHAMARNA polymerase II (Pol II) purified from the fission yeast Schizosaccharomyces
pombe consists of twelve species of subunits designated as Rpbl to Rpb12.
Among them the largest and the second largest subunits, Rpbl and Rpb2,
have sequence similarities with the {3' and the {3 subunits of prokaryotic RNA
polymerase, respectively. The Rpb3 and the Rpbll show limited similarities
with the N-terminal domain of a subunit. The four subunits, Rpbl, Rpb2,
Rpb3 and Rpbll, are therefore, supposed to form an enzyme core which corresponds to the core enzyme, a2 {3 {3', of the prokaryotic RNA polymerase. In thepresence of 6 M urea, the purified S pombe Pol II dissociates forming a Rpb2Rpb3-Rpbll complex, which is equivalent with the assembly intermediate, a 2 {3, of

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DEPARTMENT OF MOLECULAR GENETICS

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prokaryotic RNA polymerases.
Function and structure of other eukaryotic Pol II subunits have been studied
mainly using the enzyme from Saccharomyces cerevisiae. Except for the fragmentary knowledge of the functions of each subunit, the structure-function
organization within Pol II complex still remains unclear. To elucidate the func'
tionalorganization, we have analyzed the subunit· subunit interactions within
the S. pombe Pol II by chemical crosslinking and Far'western blotting. We
also mapped the RpbS'contact site on the Rpb1 and the Rpb3'contact site on
the Rpb2 using yeast two hybrid system. The subunit-subunit interactions
were limited in a sense that they only showed interactions between two subunits, except in the case with RpbS, which stimulates the Rpb3-Rpbll
heterodimer formation. Here we present the formation of multi-protein complexes of Pol II subunits in insect cells expressing the recombinant subunit
proteins. We expressed the eleven subunits, except Rpb4, simultaneously incultured insect cells with baculovirus expression vectors. For the isolation ofsubunit complexes formed in the virus'infected cells, a glutathione S-transferase sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion
protein and a decahistidine-tag sequence was inserted into the rpbl cDNA toproduce Rpb1H protein. After successive affinity chromatography on glu'
tathione and Ni 2+ columns, complexes consisting of the seven subunits, Rpb1H,
Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of
the GST-Rpb3 expression resulted in reduced assembly of the Rpbll into thecomplex. Direct interaction between the Rpb3 and other six subunits was
detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb 11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb 1'Rpb3 interaction to someextent. We propose a mechanism, in which the assembly of RNA polymerase II
is stabilized through multiple subunit"subunit contacts. See Refs. 18 and 27.
(U)

Mutant Studies of the Schlzosaccharomyces pombe RNAPolymerase II: Rpb3 and Rpb6 Mutants

Jiro MITOBE, Akira ISHIGURO, Hiroshi MITSUZAWA, Toshihisa Nom' and Akira
ISHlHAMA <'Saitama Medical College, Department of Biochemistry, Saitama)

24
The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces
pombe is composed of twelve subunits and plays a key role in transcription ofprotein"coding genes. Two large subunits, Rpb1 and Rpb2, have notable se"quence homology with the 13' and 13 subunit, respectively, of the prokaryotic
RNA polymerases. By catalytic crosslinking, we found that the catalytic sitefor RNA polymerization is formed by these two subunits. On the other hand,
two subunits, Rpb3 and Rpbll, have sequence homology with the amino (N)'terminal domain of prokaryotic RNA polymerase a subunit (aNTD). As ex"pected from the sequence similarity, we found that both Rpb3 and Rpbll
subunits are included in the core assemblies of Pol II.
In order to get insight into in vivo roles of individual subunits and further"more to identify accessory factors which interact each of the twelve subunits,
we carried out systematic studies of the S. pombe Pol II mutants with muta"
tions in one of the twelve subunits. Up to now, we have isolated temperature"sensitive (Ts) or cold"sensitive (Cs) S pombe mutants with mutations in therpb2, rpb3, rpb5, rpb6, rpb7and rpbll genes. This year systematic studies
have been carried out for the rpb3and rpb6mutants. All the rpb3mutants
showed various degrees ofthe assembly defect of Pol II. For functional charac"terization of the rpb3mutants, we established an improved system ofGAL4'
VP16 activator' dependent in vitro transcription by S. pombe cell extracts.
Cell extracts from the S pombemutants carrying mutations in the eukaryote'specific conserved regions Band C of Rpb3 showed lower activities of theactivated transcription compared with other mutants carrying mutations in
the regions A and D, implying that the regions Band C are involved in tran"
scription factor"dependent transcription. Since the cell extracts from all the
S pombe carrying rpb3region A or D mutations showed more reduced thermo"
stability than others, the regiond A and D may play more essential roles in theassembly.
This year we also performed genetic analyses of the Rpb6 subunit of S
pombe Pol II. Rpb6 is one of the five common subunits shared among threeforms of eukaryotic RNA polymerase. Deletion and truncation analyses of therpb6 gene in the S pombe indicated that Rpb6 consisting of 142 amino acidresidues is an essential protein for cell viability, and the essential region is
located in the C"terminal proximal half between residues 61"139. After ran'

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DEPARTMENT OF MOLECULAR GENETICS

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dom mutagenesis, a total of 14 temperature-sensitive (Ts) mutants were isolated, each carrying a single (or double in three cases and triple in one case)mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (BAD) that inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AD sensitivity
and Ts phenotypes ofthese rpb6mutants were suppressed by over-expression
ofTFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed
reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS- Pol II
complexes using a fusion form of TFIIS with glutathione S-transferase(GST). Moreover the direct interaction between TFIIS and Pol II was competed by the addition of Rpb6. Taken together we propose that Rpb6 plays arole in the assembly between the Pol II and the transcription elongation factor
TFIIS. For details see Refs. 10, 22, 27 and 28.
(12)

Two WD Repeat-Containing TAFs in Fission Yeast that
Suppress Cell Cycle Arrest in Mitosis

Hiroshi MITSUZAWA, Hiroaki SEINO!, Fumiaki YAMA0 1 and Akira ISHIHAMA(IDivision of Mutagenesis, NIG)
The general transcription factor TFIID plays a critical role in transcription
initiation of protein-coding genes by RNA polymerase II (Pol II). TFIID is amultiprotein complex comprising the TATA-binding protein (TBP) and TBPassociated factors (TAFs), both of which have been well conserved from yeast
to humans. TBP specifically recognizes TATA elements, while certain TAFs
directly interact with initiator or downstream promoter elements. In addition
to a role in core promoter recognition, TAFs have been proposed to function astargets of activators. Subsets of TAFs have also been found in histone acetylase complexes distinct from TFIID.
Ubiquitin-dependent proteolysis has been shown to playa key role in progression through the cell cycle. A ubiquitin-protein ligase complex known as
the anaphase-promoting complex or cyclosome (APC/C) promotes themetaphase-to-anaphase transition and the exit from mitosis by mediating

26
ubiquitination of anaphase inhibitors and mitotic cyclins, leading to theirdestruction by the 268 proteasome. In the fission yeast Schizosaccharomyces
pombe, the ubiquitin -conjugating enzyme UbcP4 seems to be involved in
APC/C-mediated proteolysis. We have isolated two related TAF genes, taf72+
and taf73+, from S. pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4+. The ubcP4ts
mutation causes cell cycle arrest in mitosis, probably due to defects inubiquitination mediated by the APC/C. TAF72 and TAF73 proteins have homology to WD repeat-containing TAFs such as hTAFIOO, dTAF80/85, andyTAF90. We found that both TAF72 and TAF73 are associated with TBP andother TAFs. We also show that overexpression ofTAF72 or TAF73 suppresses
cell cycle arrest in mitosis caused by a mutation in the APC/C subunit gene
cut9+. These results suggest that TAF72 and TAF73 may regulate the expres·sion of genes involved in ubiquitin -dependent proteolysis during mitosis. Our
study thus provides genetic evidence for a possible role ofWD repeat-containing TAFs in the expression of genes involved in progression through the Mphase of the cell cycle.
(13)

Identification of the RNA Cap-Binding Site
on Influenza Virus RNA Polymerase

Ayae HONDA, Kiyoshi MrZUMOTO I and Akira ISHIHAMA (lDivision of Nucleic Acid
Chemistry)
The genome of influenza virus is composed of eight RNA segments of negative polarity, which are transcribed after infection into positive-strand viralmRNAs for viral protein synthesis. The RNA polymerase responsible for the
primary transcription is associated, in virions, with each RNA segment at thepromoter formed by terminal sequences conserved among eight RNA segments.
Transcription is initiated by using 11-13 nucleotide-long capped RNAs which
are generated after cleavage of host cell mRNA by the RNA polymerase-asso·
ciated endonuclease. The RNA polymerase is involved in not only transcription but also replication of the genome RNA. Replication is, however, initiated de novo without using primers. Previously we demonstrated that the

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DEPARTMENT OF MOLECULAR GENETICS27

RNA polymerase is composed of one molecule each of three viral proteins, PB 1,
PB2 and PA. All three P proteins are required for the complete cycle of transcription and replication. Since the catalytic site of RNA polymerization is
located in the PBI subunit and since both the PB2 and PA subunits bind to
PBI in the P complex formation, the PBI is the core subunit in the influenza
virus RNA polymerase and plays key roles in both transcription and replication. Genetic and biochemical studies indicated that PB2 is involved in recognition and cleavage of capped RNA for generation of transcription primers.
This year we tried to identify the cap I-binding site(s) on the PB2 protein.
For this purpose, ribonucleoprotein (RNP) cores were purified from virus particles and subjected to UV-crosslinking to RNA with 32p only at the 5'-terminal
cap-I structure. The PB2 protein cross-linked with the radioactive cap structure was isolated by SDS- PAGE, mixed with unlabeled PB2, and digested
with V8 in SDS-polyacrylamide gels. Amino acid sequencing of the proteolytic
fragments indicated that two regions ofPB2 are involved in cap 1 binding, one(N-site) at the N-terminal proximal region between residues 200 and 300, andthe other (C-site) at the C-terminal proximal region between residues 520 and
580, suggesting the presence of two capped RNA-binding sites on the PB2
protein. To confirm the results, we expressed two kinds of PB2 fragments,
each carrying either the N-site or C-site, in Escherichia coli, and tested thecapped RNA-binding activity for the PB2 fragments. The results support theconclusion that two capped RNA-binding sites exist on the PB2 protein. See
Ref. 8.
(14)

Funetional Modulation of Influenza Virus RNA Polymerase
by Viral RNA: Differential Roles of vRNA and eRNA

Ayae HONDA, Atsushi END0 1 and Akira ISHIHAMA (IDaiichi PharmaceuticalCo., Exploratory Res. Lab., Tokyo)
The genome of influenza virus is composed of eight negative-strand viral
RNA (vRNA) segments which are transcribed, in virus-infected cell nuclei, intomRNA by virion-associated RNA-dependent RNA polymerase. The influenza
virus RNA polymerase is involved in both transcription <vRNA-directed mRNA

28
synthesis) and replication (vRNA-directed synthesis of complementary RNA(cRNA) and cRNA-directed vRNA synthesis). In transcription, the RNA polymerase catalyzes not only RNA synthesis but also the cleavage of capped hostRNA to generate capped RNA primers and polyadenylation at the 3'-termini ofmRNA. The RNA polymerase also carries an apparent proof-reading activity
for nascent RNA chains.
The RNA polymerase is composed of one molecule each of PB 1, PB2 and PA.
PBl plays a central role in RNA polymerase assembly by providing the contact
surfaces for both PB2 and PA. In the virus particles, this RNA polymerase isbound to a double-stranded region of viral RNA (vRNA) formed by base-pairing between its 5'- and 3'-termini. Since the RNA polymerase is tightly associated with vRNA, no purification method yielding large amounts of functional
RNA polymerase has been established. For detailed analysis of the structure-function relationship of each P protein, large amounts of functional RNA
polymerase are required in a template-free form. This year we succeded topurify the RNA polymerase from insect cells, in which three P proteins were
simultaneously expressed using recombinant baculoviruses. For purification
purpose of the 3P complex, the PA protein was expressed as a fusion with His6
tag added at its N-terminus. The affinity-purified 3P complex showed theactivities of capped RNA binding, capped RNA cleavage, v- and c-sense modelRNA binding, model vRNA-directed and ApG- or mRNA-primed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of influenza virus RNA polymerase with the catalytic specificity oftranscriptase is formed in baculovirus-infected insect cells. Moreover, capped
RNA cleavage was observed in the presence ofvRNA but not of cRNA, indicating that vRNA functions as a regulatory factor for the specificity control ofviral RNA polymerase as well as a template for transcription.

(15)

Expression of Functional Influenza Viral RNA Polymerasein the Methylotrophic Yeast Plcbla pastorIs

Jung-Shan HWANG, Kazunori YAMADA 1, Ayae HONDA, Kohji NAKADE 1 andAkira
ISHIHAMA (IMitsubishi Chemical Co., Yokohama Res. Cent., Yokohama)

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DEPARTMENT OF MOLECULAR GENETICS

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Influenza virus RNA polymerase with the subunit composition PB 1-PB2-PA
is a multi-functional enzyme with the activities of both synthesis and cleavageof RNA, and is involved in both transcription and replication of the viralgenome. In order to meet the demand for a large amount of the functional
influenza virus RNA polymerase in template"free form, an alternative ap"
proach has been employed, where all three P proteins were expressed in thesame cells, transiently after infection of recombinant baculoviruses(see above). In order to improve the expression levels, we employed the
methylotrophic yeast Pichia pastoris system as well as the recombinantbaculovirus system. Here we describe the first successful expression of nega"tive"strand viral RNA polymerase in yeast. The P. pastoris yeast is able toutilize methanol as its sole carbon source and has been developed as a host for
the expression of heterologous proteins. The major advantages of this expres"sion system include: (i) a strong, tightly"regulated alcohol oxidase promoter,
5'AOX1, is available; (in large scale protein production can be achieved in alarge volume fermentor culture; (iii) a secretary pathway allows the productsecreted into the medium, separating the foreign protein from most of the hostproteins; (iv) the expression system can be easily set up; and (v) the cost is asequally low as the Escherichia coli expression system. The results indicate
that: (i) an expression system of reasonable amounts ofthree P proteins in P.
pastoris was established, after searching for the optimum induction times
and the optimum concentration of methanol to give the maximum level of
induction; (ii) by adding a histidine"tag to the PB2 protein, the 3P protein
complex was isolated from the celllysates after Ni2+"nitrilotriacetic acid (NTA)
agarose affinity chromatography; (iii) by quantitative Western blotting, allthree P proteins in the isolated 3P complex were detected in stoichiometricmolar ratios; (iv) the catalytic activity of RNA synthesis was detected for the
3P complex only when model vRNA or cRNA templates with the conserved
terminal sequences were added; and (v) both synthetic dinucleotides and globin
mRNA served as primers, but the 3P protein complex was virtually inactive in
the absence of primers. The template recognition specificity was also exam"
ined using various kinds of model vRNA or cRNA with or without the terminal
conserved sequences. Taken all the results together we conclude that the functional influenza virus RNA polymerase can be produced in the methylotrophic

30

yeast P. pastoris, in amounts sufficient for detailed functional analysis andstructural studies. See Ref. 9.
(16)

Search for Host Factors Interacting with Influenza Virus
RNA Polymerase

Ayae HONDA, Takuto OKAMOTO! and Akira ISHIHAMA (lUniversity ofShizuoka,
Graduate School ofPharmacentical Sciences)
Influenza virus RNA polymerase consisting ofthree viral P proteins (PBI,PB2 and PA) carries two functions, one for transcription ofvRNAs to produce
vival mRNAs and the other for replicatin ofvRNAs to produce progeny vRNAs
via cRNA templates. Transcription of the vRNAs by the viral RNA polymerase
is initiated by using host cell capped RNAs as primers. Analysis of the 5'terminal structure of virus· associated vRNAs indicated that RNA synthesis
for replication is initiated de novo without using primers. Both purified andreconstituted RNA polymerases require primers for function, while the RNA
polymerase in either virus-infected cell extracts or lysates of cells expressing
three viral P proteins can catalyze RNA synthesis in the absence ofprimers. We
then proposed that an as yet unidentified host factor(s) is involved in thefunctional conversion of the RNA polymerase from transcriptase to replicase.
Attempts have been made to identify host proteins, which interact witheach of the P proteins, using yeast two hybrid screening system. Several positive clones have been isolated for each P protein, and complete cDNAs for someof these putative PBI-, PB2- and PA-interacting host factors have been iso'lated. After overexpression in E. coli, the purified proteins were used to raise
antibodies in rabbits, and the anti-host factors were used for detection ofprotein complexes in both uninfected and virus-infected cell extracts. The
overexpressed and purified host proteins were also tested for the activity ofbinding to the respective P proteins. Effects of these putative host factors on
the functions of influenza virus RNA polymerase are being examined.

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DEPARTMENT OF MOLECULAR GENETICS

31

Pub I i ca t ions
1.

2.

3.

4.

APIRAKARAMWONG, A., KAsHIWAGI, K., RA.I, V.S., SAKATA, K., KAKINUMA, Y., ISHlHAMA, A.
and IGARASHI, K.: Involvement of ppGpp, ribosome modulation factor, and stationary
phase-specific sigma factor as in the decrease in cell viability caused by spermidine.
Biochem. Biophys. Res. Commun. 264, 643-647 (1999).
BOWN, J.A., KOLB, A., MEARES, C.F., ISHlHAMA, A., MINCHIN, S.D. and BUSBY, S.J.W.:
Positioning of region 4 of the Escherichia coli RNA polymerase a 70 subunit by atranscription activator. J. Bacteriol. 182, 2982-2984 (2000).
BOWN, J.A., OWENS, J.T., MEARES, C.F., FUJITA, N., ISHlHAMA, A., BUSBY, S.J. and MINCIIlN,
S.D.: Organisation of open complexes at Escherichia coli promoters: location of thepromoter DNA sites close to region 2.5 of the a 70 subunit of RNA polymerase. J. Biol.
Chem. 274, 2263-2270 (1999).
BURNS, H.D., ISHIHAMA, A. and MINCHIN, S.D.: Open complex formation during transcription initiatioin at the Escherichia coli galP1 promoter: the role of the RNA polymerase a subunit at promoters lacking an UP-element. Nucleic Acids Res. 27, 2051-

5.

2056 (1999).
COLLAND, F., FUJITA, N., KOTLARZ, D., BOWN, J.A., MEARES, C.F., ISHIHAMA, A. and KOLB,
A.: Positioning of a~ the stationary phase a factor, in Escherichia coli RNA polymerase-promoter open complexes. EMBO J. 18, 4049-4059 (1999).

6.

FUJITA, N., ENDO, S. and ISHlHAMA, A.: Structural requirements for the interdomain
linker of a subunit of Escherichia coli RNA polymerase. Biochemistry39,6243-6249(2000).

7.

HARADA, Y., FUNATSU, T., MURAKAMI, K., NONOYAMA, Y., ISHIHAMA, A. and YANAGIDA, T.:
Single molecule imaging of RNA polymerase- DNA interactions in real time. Biophys.
J. 76, 709-715 (1999).

8.

HONDA, A., MIZUMOTO, K. and ISHIHAMA, A.: Two separate sequences of PB2 subunit
constitute the RNA cap-binding site of influenza virus RNA polymerase. Genes Cells 4,475-485 (1999).
HWANG, J.-S., YAMADA, K., HONDA, A., NAKADE, K. and ISHIHAMA, A.: Expression of

9.

functional influenza viral RNA polymerase in the methylotrophic yeast Pichia pastoris.
J. Virol. 74, 4074-4084 (2000).
10. ISHIGURO, A., NOG!, Y., HISATAKE, K., MURAMATSU, M. and ISHIHAMA, A.: Ppb6 subunit of
the fission yeast RNA polymerase II is a contact target of the transcription elongation
factor TFIIS. Mol. Cell. Biol. 20, 1263-1270 (2000).
11. ISHIHAMA, A.: Stringent control. In: Encyclopedia ofMolecular Biology, T.E. Creighton,
Ed., John Wiley & Sons, Inc., New York, pp. 2451-2455 (1999).
12.

ISHlHAMA, A.: Functional modulation of Escherichia coli RNA polymerase. Ann. Rev.
Microbiol. 54, 499-518 (2000).

13.

ISHlHAMA, A.: Modulation of the nucleoid, the transcription apparatus, and the trans-

32

14.
15.

16.

17.

18.
19.

20.

lation machinery in bacteria for stationary phase survival. Genes Cells 4. 135-143(1999).
ISHIHAMA. A: Molecular anatomy of RNA polymerase using protein-conjugated metalprobes with nuclease and protease activities. Chem. Commun. %000. 1091-1094 (2000).
ISHIHAMA. A. Zou. C.• KOBAYASHI. M .• KUMAR. A. FUJITA. N .. SAKURAI. H. and HAYWARD.
RS.: Molecular recognition in gene transcription. In: Biomolecular Interactions in
DNA and Proteins. (Buck in. V. and Funck. T.. eds'>. Nova Sci. Publ.. in press.
JISHAGE. M. and ISHIHAMA. A: Transcriptional organization and in vivo role of the
Escherichia coli rsd gene. encoding the regulator of RNA polymerase sigma D. J
Bacteriol. 181, 3768-3776 (1999).
KATAYAMA. A.. FU.JITA. N. and ISHIHAMA. A: Mapping of subunit-subunit contact surfaces on the 13' subunit of Escherichia coli RNA polymerase. J Biol. Chem. %75. 35833592 (2000).
KIMURA. M. and ISHIHAMA. A: Involvement of multiple subunit-subunit contacts in theassembly of RNA polymerase II. Nucleic Acids Res. %8. 952-959 (2000).
MAEDA. H .. JISHAGE. M .. NOMURA. T.. FUJITA. N. and ISHIHAMA. A: Promoter selectivityof the RNA polymerase holoenzyme containing the extracytoplasmic function (ECF)
sigma subunit a E or a F.". J Bacteriol. 18%. 1181-1184 (2000).
MASUDA. H .. SUZUKI. T.• SUGIYAMA. Y. MURAKAMI. K.. MIYAMOTO. D.• Jwa HIDARI. K.I.-P..
ITO. T.• KIDA. M .• FUKUNAGA. K.. OBUCHI. M .• TOYODA. T.• ISHIHAMA. A. KAWAOKA. Y. and
SUZUKI. Y.: Substitution of amino acid residue in influenza A virus hemagglutinin
aggects recognition of glycosphingolipid containing N'glycolylneuraminic acid. FEBS
Lett. 464. 71-74 (1999).

21.

MASUNAGA. K.. MlZUMOTO. K.. KATO. H .. ISHIHAMA. A and TOYODA. T.: Molecular mapping of influenza virus RNA polymerase by site-specific antibodies. Virology %56. 130141 (1999).

22. MlTOBE. J .• MITSUZAWA. H .• YASUI. K. and ISHIHAMA. A: Isolation and characterization oftemperature-sensitive mutations in the gene (rpb3) for subunit 3 of RNA polymerase
II in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. %6%. 73-84(1999).
23. NOMURA. T.. FUJITA. N. and ISHIHAMA. A: Mapping of subunit-subunit contact sites on
the 13 subunit of Escherichia coli RNA polymerase. Biochemistry 38.1346-1355 (1999).
24. OZOLINE. O.N.• FUJITA. N. and ISHIHAMA. A: Functional topology of the bacterial RNA
polymerase. Multipoint monitoring by a fluorescent probe. In: SpectroscopyofBiological Molecules: New Directions (eds. J. Greve. G.J. Puppels. C. Otto). Kluwer Academic
Publ.. Dordrecht. Netherland. pp. 87-89 (1999).
25. OZOLINE. 0 .• FUJITA, N. and ISHIHAMA. A.: Transcription activation mediated by the
carboxy-terminal domain of RNA polymerase alpha-subunit: Multipoint monitoring
ssing fluorescent probe. J Biol. Chem. %75. 1119-1127 (2000).
26. PROST. J.-F.• NEGRE. D.• OUDOT. C .• MURAKAMI. K.. ISHIHAMA. A.. COZZONE. AJ. and
CORTAY. J.-C.: Cra-dependent transcriptional activation of the icd gene of Escherichia

A

DEPARTMENT OF MOLECULAR GENETICS

33

coli. J. Bacteriol. 181, 893-898 (1999).
27.

SAKURAI, H., MITSUZAWA, H., KIMURA, M. and ISHIHAMA, A: The RpM subunit of fission
yeast Schizasaccharomyces pambe RNA polymerase II is essential for cell viability andsimilar in structure to the corresponding subunits of higher eukaryotes. Mol. Cell.
BioI. 19, 7511-7518 (1999).

28.

SHPAKOVSKI, G.V., GADAL, 0., LABARRE-MARIOTTE, S., LEBEDENKO, E.N., MIKLOS, 1., SAKURAl,
H., PROSHKIN, S.A, VAN Mullem, v., ISHlHAMA, A and THURIAUX, P.: Functional conservation of RNA polymerase II in the fission and budding yeasts. J. Mol. Biol. 295, 11191127 (2000).

29.

30.

STEVENS, AM., FUJITA, N., ISHlHAMA, A. and GREENBERG, E.P.: Involvement of the RNA
polymerase a subunit C-terminal domain in LuxR-dependent activation of the Vibrio
ii'scheriluminescence genes. J. Bacteriol. 181, 4704-4707 (1999).
TALUKDER, AA and ISHIHAMA, A: '!\velve species of DNA-binding protein from Escheri-chia ca1J~ Sequence recognition specificity and DNA binding affinity. J. BioI. Chem.
274, 33105-33113 (1999).
31.

TALUKDER, AA., HIRAGA, S. and ISHIHAMA, A: '!\vo types oflocalization of the DNA-binding proteins within the Escherichia coli nucleoid. Genes Cells 5, 613-626 (2000).
32. TALUKDER, AA., IWATA, A, NISHIMURA, A, UEDA, A and ISHlHAMA, A: Growth phasedependent variation in the protein composition of Escherichia coli nucleoid. J. Bacterial.
181, 6361-6370 (1999).
33.

TRAVIGLIA, S.L., DATWYLER, S.A, YAN, D., ISHIHAMA, A. and MEARES, C.F.: Targeted
protein footprinting: Where different transcription factors bind to RNA polymerase.
Biochemistry, 38, 15774-15778 (1999).

34. WATANABE, T., HONDA, A, IWATA, A, UEDA, S., HIBI, T. and ISHIHAMA, A: Isolation fromtobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing 126K1183K protein heterodimer. J. Viral. 73, 26332640 (1999).
35. WIGNESHWERARAJ, S.R, FUJITA, N., ISHlHAMA, A and BUCK, M.: Conservation of sigmacore RNA polymerase proximity relationships between the enhancer independent andenhancer-dependent sigma classes. EMBO J. 19, 3038-3048 (2000).
36. WADA, A, MIKKOLA, R, KURLAND, C.G. and ISHIHAMA, A: Growth phase-coupled changesof the ribosome pattern in Escherichia coli. J. Bacterial. 182, 2893-2899 (2000).
37. WLASSOFF, W.A, KIMURA, M. and ISHIHAMA, A.: Functional organization of two large
subunits of the fission yeast Schizasaccharamyces pombe RNA polymerase II: Location of the catalytic sites. J. Bial. Chem. 274, 5104-5113 (1999).
38. YAMAMOTO, K., NAGURA, R, TANABE, H., FUJITA, N., ISHIHAMA, A and UTSUMI, R: Negative
regulation of the bolA1p of Escherichia caliK-12 by the transcription factor OmpR for
osmolarity response genes. FEMS Microbial. Lett. 186, 257-262 (2000).

34

A-b. Division of Mutagenesis(1)

Ublquitin-eonjugating enzymes In fission yeast

JOON"HYUM Park, Hiroaki SEINO and Fumiaki YAMAO
Selective protein degradation in eukaryotic cells is mainly carried out by theubiquitin system. The many short"lived proteins are subjected to be substrates
of ubiquitination. Ultimately causing the destruction of various regulatory
proteins, the ubiquitin system plays important roles in many cellular functions, including cell"cycle control, signal transduction, transcriptional regula"tion, the nuclear transport process, receptor control by endocytosis and soon. The diversity and specificity of ubiquitination are based on the molecular
heterogeneity of ubiquitin"conjugating enzymes (Ubc or E2) and ubiquitinligases (E3). To elicidate the functional spectrum of Ubc's, we surveyed Ubc's
in fission yeast. In our previous works, four Ubc's (UbcPl"P4) were identified
through screening by their enzymatic activities. Through homology search in
the fission yeast genome data base, other eight Ubc's (UbcP5"P12) have beenfound. The functional analyses of Ubc's are undergoing through disruption of
their genes, showing that some genes are found essential for the cell growth
while its ortholog in the budding yeast are not (i.e., UBClllubcP4, UBC4/
ubcPl). Phenotypic differences between the fission yeast Ubc's and their struc"tural homologues in the budding yeast have been found in many cases. Thus,
the whole spectrum of functional diversity in Ubc does not seem to be identicalbetween the two yeast lines, indicating the importance to investigating func"
tions of ubiquitin"conjugating enzymes in fission yeast.

(!)

Destruction of S-phase eyelln, Cig!, In fission yeast

Hiroaki SEINO and Fumiaki YAMAO
Ubiquitin"conjugating enzyme UbcP4 in fission yeast, working together with
anaphase promoting complex (ApC/C), is essential for mitotic progression. They
mediate ubiquitin"pathway forwarding Cut2 for destruction to initiate

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DEPARTMENT OF MOLECULAR GENETICS

35

anaphase in mitosis as well as Cdc13, a mitotic cyclin, to exit frommitosis. Another cyclin, Cig2, functions during S-phase in fission yeast, andis destroyed before mitosis. Since genetic interactions among cig2, ubcP4 andcut9 mutant genes were observed, we suggested that Cig2 protein was regu"
lated via proteolysis involving UbcP4 andAPC/C. The cyclin boxes ofCig2 andCdc13 were found interchangeable. The phase"dependent destruction of Cig2
and Cdc13, therefore, was evidently determined by their structures of N -terminus outside the cyclin box. Cig2 has a degradation signal (named destruc"tion box) in the N"terminal region. As expected, Cig2 degradation depended
on the destruction box. Since the destruction boxes of Cig2 and Cdc13 are very
similar, we are now identifying the determinant for their destruction timing inor near the destruction box. These results indicated that ubiquitin pathway
involving UbcP4/APC was active in both G2 phase and anaphase, and that amechanism for the phase" and substrate-specific regulation of this pathway
might exist.

(3)

Ubiquitln pathways functioning In
mitotic regulatory mechanism

Riroaki SEINO and Fumiaki YAMAO
Ubc specific for mitotic cyclin ubiquitination has been identified in fission
yeast (UbcP4), clam (Ubc"C), Xenopus (VBC"x) and human (UbcRIO), whichfunction together with APC/C. During biochemical analysis of M phase cyclin
ubiquitination using frog or clam egg extract, an additional ubiquitin conjugat"
ing enzyme, a Ubc4 homologue, was found to be capable of ubiquitinating M
phase cyclin under the function ofAPC/C. The functional differences, however,between the two Ubc's have not yet been clarified. We found both oftheir ortholog
in fission yeast, ubcP4 and ubcPI (ubc4), were essential for cell viability. UbcPI"
or UbcP4"depleted cells exhibited M phase defects, accumulating mitotic cyclin
in the cells. These results suggest, in accordance with the above observations inegg extract, that the two ubiquitin conjugating enzymes have different and es"
sential functions for ubiquitinating mitotic cyclin. Characterizations of the roles
ofthese ubiquitin pathways in the mitotic regulation are undergoing.

36

(4)

Characterization of SCFGrrl that ubiquitinates 61 eyelins
in Saccharomyces cerevlslae

Tsutomu KISHI and Fumiaki YAMAOSCF complexes, composed of Skp 1, Cdc53 and one of the F'box proteins,
have been implicated in the Cdc34-dependent ubiquitination in Saccharomyces cerevisiae. We have found that Grr1 required for degradation ofG1 cyclins,
Cln1 and Cln2, as well as for regulation of glucose repression, is an F-box
protein and interacts with Skp1 through the F-box motif. Furthermore, wehave found that Grr1 also interacts in vitro with phosphorylated Cln1 andCln2. From these data, we have proposed that Grrl is required for degradation of Cln2 through linking phosphorylated Cln2 to Skp 1 in SCFGrrl
complex. To isolate additional genes that are required for the degradation of
G1 cyclin Cln2, we isolated mutants that stabilize Cln2. We tentatively named
these mutants as dog (Degradation of G1 cyclin) mutants. Characterization ofthese mutants, and cloning of the genes are underway.

Publ ication
1.

JB Review: Ubiquitin System'Selectivity and Timing of Protein Destruction. J. Biochemistry 125, 223-229, 1999.

YAMAO, F.:A.

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A-c. Division of Nucleic Acid Chemistry(1)

Proteomic analysis of Escherlchla colJ proteins in thestationary phase by the RFHR 2-D PAGE

Akira WADA (Osaka Medical college, Dept. Physics)
The RFHR (radical'free and highly reducing) 2·D PAGE which we have de'veloped separates basic proteins in addition to neutral and acidic proteins
with high resolution. The more commonly used O'Farrell's method can not
resolve highly basic proteins, therefore our method is more suitable for com'prehensive proteomic analysis.
The total proteins of E. coh l.:ulLureu in the mddium E including 2%
polypeptone for 7 ± 1 days were separated on the 2'D gels of the RFHR 2'D
PAGE. As a result, 6 log'phase'specific proteins and 59 stationary'phase'
specific proteins were detected. Among the stationary'phase'specific proteins,
23 proteins were found during the entire stationary phase, while the other 36
proteins were detected only during limited short periods of the stationary
phase, respectively. These 36 proteins were classified to 10 groups according
to their detected periods. This shows that the E. coli cells are surviving for theentire stationary phase by continually changing their protein composition.
Among these stationary'phase'specific proteins, 31 proteins were identi'
fied by amino acid sequencing. The protein function distributions are as fol'lows: carbohydrate metabolism 1, amino acid metabolism 1, nitrate metabo'lism 1, protein folding 1, DNA binding 4, membrane transport 5, translation 4
and hypothetical ORF 14.

Pub I i ca t i cns
1.
2.

MAKI, Y., TANAKA, A. and WADA, A.:

Stoichiometric analysis of barley plastid ribosomal
proteins, Plant Cell Physiol. 41(3), 289-299 (2000),
WADA, A., MIKKOLA, R, KURLAND, C.G. and ISHIHAMA, A.: Growth phase'coupled changesof the ribosome profile in the natural isolates and laboratory strains of Escherichia
coli, J. Bacteriol. 18Z(10), 2893-2899 (2000).

38

B.

DEPARTMENT OF CELL GENETICS
B-a. Division of Cytogenetics(1)

Roles of Mre11 of Saccharomyces cerev}s}ae in Meiotic Recombination and Mitotic Repair Reactions

Daisuke TATSUDA, Hiroyuki OSHIUMI, Tsutomu OHTA, Jun-ichi TOMIZAWA andTomoko OGAWA
The genetic integrity of vegetative cells is maintained by various DNA repair systems. Among the damage, double strand breaks (DSBs) that were
induced by treatment with ionizing radiation or radiomimetic chemicals, suchas methyl-methane sulfonate (MMS), are repaired by the homologous recombination or by the non-homologous end-joining. On the other hand, genomic
diversity is provided by meiotic recombinatiion. Specific DSBs introduced in
the meiotic process are repaired by homologous recombination.
We studied the involvement ofthe MREll gene of S. cerevisiae in homologous recombination [1]. The gene was first identified by isolation of a mutant
that is defective in meiotic recombination and repair of DSBs induced by
MMS [2]. Mre 11 is involved in two temporally coupled processes in the earlyphase of meiotic recombination, formation ofDSBs and their processing. For
the DSB formation, Mre11 acts by forming the pre-DSB complex with at least
eight proteins including Rad50, Xrs2, Spo 11 and Mei4. For processing of the
DSB ends, the Mre11 forms the post-DSB complex in which Mre11 holds Rad50
and Xrs2 tightly as a binding core. Such a tight binding among these proteins
was not present in the pre-DSB complex. The C-terminal region of the Mre11
binds to four meiotic proteins at least. One of the proteins, Mei4, was identified (Ohta, T. unpublished results). The N-terminal region that contains aphosphodiesterase motif specifies ssDNA endonuclease, 3' to 5' ssDNA exonu'
clease and 3' to 5' dsDNA exonuclease activities that are collectively requiredfor processing of the DSBs.
Mre11 has two sites to bind to DNA. The central site (Site-A) is required for

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DEPARTMENT OF CELL GENETICS

39

the DSB processing, whereas the C-terminal site (Site-B) is involved in the
DSB formation in meiosis. Mre11 also binds to recombination hotspots on thechromosomal DNA (Ohta, T. unpublished results). The site-B is not necessary
for repair of the DNA damage caused by MMS. Mre 11 has two regions to bindto Rad50, one. at the N-terminal side and the other at the central region of theprotein. Because Rad50 has been shown to bind DNA, Mre11 could bind DNA
through Rad50. The presence oftwo binding sites, each for DNA and for Rad50,
permits the protein to bind to DNA differently for formation ofDSBs and theirprocessing in meiotic recombination and in repair reactions.
To elucidate further the mechanisms of joining of DSB ends, we studied
joining of blunt ended DNA, using the substrate that was made by cutting
pUCI8 plasmid once with SmaI enzyme. Mre11 protein catalized formationof linear oligomers, mainly dimmers, but not monomeric circular molecules.
The results suggested that the formation of oligomers requires homologous
sequences at the ends of dsDNA. The reaction depends on Mn++, but not on
Mg++ and ATP, and is enhanced by Rad50 protein. The joining is unstableagainst the heating and the alkaline treatment, indicating binding by hydrogen bonds. These results show that this end-joining activity depends on theunwinding of the ends of DNA and on annealing with the counter molecules.
The reaction is independent of the nuclease activities, because the mutant
protein that is defective in the nuclease activities can join the ends.
We then examined the unwinding activity ofMre11, using various substrates
that were made by annealing ofMI3 single-stranded circular or linear viral
DNA with various nucleotide sequences of oligonucleotides in order to create
ends with different structures. Mre11 released the oligonucleotide from thesubstrates that has blunt ends with 3' overhung and also that have no overhanging at the annealed oligonucleotides. On the other hand, both overhanging ends at the annealed oligonucleotides was not unwound. These results
suggested that, by Mre 11, the substrates that have blunt ends were
unwound_ The substrate with 5'overhung released the fragments, but thosewith 3' overhung were not, suggesting that 5' overhung enhances the release of
the fragment from the blunt ends, but 5' overhanging end could not be
unwound. Thus, the unwinding activity is dependent on the structures of theends of the substrates. The activity was dependent on Manganese, but not

40

ATP.
Mre11 also has an activity to anneal heat denatured linear plasmid DNA.
Therefore, homologous blunt ends by Mre11 must be carried out through unwinding of the ends followed by annealing of complementary ssDNA.
The ends oflinear DNA were joined covalently by T4 DNA ligase. We thenexamined whether the Mre11 enhances the joining. We used the condition
where a formation of oligomers and circular molecules from blunt ended linear
dsDNA was almost undetactable due to the very small concentration of theligase. However, when the Mre11 was also present, substantial amount oflinear oligomers were formed. For the reaction, homology of the ends of linear
dsDNA molecules was not required. The Rad50 enhanced the reaction. The
joint formed from DNA fragmetns that had 3' or 5' overhanging ends wascleaved by the restriction enzymes which were used for the preparation of thefragments, indicating that the joining is accurate. On the other hand, the joint
formed by blunt ended fragments by the wild-type Mre11 and Mre11'6 was
inaccurate. Because Mrel1-6 that has no nuclease activity joins the ends in'accurately, the inaccuracy by wild-type may results from processing ends andalso unwinding the ends.
These results showed that Mre11 participates in two types of end joining
reactions, homology'dependent end'joining and the enhancement of covalent
end'joining of non' homologous ends by DNA ligase. Mre 11 joins homologous
blunt ends through unwinding and annealing activities. It also facilitates co'valent joining of non'homologous ends by bringing the ends of two molecules
close and/or holding them together. Rad50 participates in the bindings ofMre 11
to DNA in this reaction. Because Mre11 uses different DNA binding sites fordifferent Mre11 reactions, the difference in the mode of binding of Mre11 to
DNA is critical for different roles of the protein in different reactions. Thus,
Mre11 plays a key role in selecting repair process that is suitable to repair
each particular structure at the DSB site.

B.

(!)

DEPARTMENT OF CELL GENETICS

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Roles of Rada!, Radaa and Rada7 Proteins in Radal-mediatedHomologous Recombination in Saccharomyces cereylslae

Koichi WATANABE, Shimako MATSUDA, Tsutomu OHTA and Tomoko OGAWA
RecA protein in Escherichia coli has been known to playa pivotal role insearching for homologous DNA sequences, their pairing and exchange of DNA
strands. The RecA forms a typical right-handed helical filament in the presence ofATP, in which the DNA strand is extended 1.5-1.6 times compared with
B-form DNA that is called a nucleoprotein filament or a pre-synaptic filament. The
structure is essential for recombination and repair of damaged DNA. We thensearched for mutants that show similar phenotypes as E. coli recA mutant
among radmutants of S. cerevisiae, which are defective in the repair of DNA
damage. The HAD51 and RAD52 genes were found. Mutants of either geneare defective in mitotic and meiotic recombination and repair of DNA damage
induced by MMS. The amino acid sequence ofRad51 protein has a weak similarity to the RecA protein [3] and found that Rad51 forms nucleoprotein filament [4]. On the other hand, Rad52 and RecA have no sequence homology [5]. Another recA homologue, DMC1 of S. cerevisiae was also identified [6]. Both
RAD51 and DMC1 genes are required for the meiotic recombination. Using
the homologous sequences of E. coli RecA and S. cerevisiae Rad51, RAD51
homologues from higher eukaryotes were isolated [7], suggesting that the recA
like genes are involved in homologous recombination in all living cells. Further
characterization of HAD51 homologues showed that several eukaryotic geneproducts, Rad52, Rad54, Rad55, Rad57, Dmcl and RPA, which is functional
homolog of SSB of E. coli, act for formation of the Rad51 filament [8, 9].
To understand the roles of various proteins that are involved in Rad51mediated strand exchange reaction, we analyzed the role of Rad52 for the
Rad51-mediated strand exchange reaction. We found that the Rad52 eliminates RPA from ssDNA [10] and thus facilitating formation ofRad51 filaments in both the strand-exchange and ATP hydrolysis reactions. We thenanalyzed the biochemical properties of a protein from rad52 mutant cells,
Rad52-1, the mutant strain shows severe defects in both mitotic repair reaction and homologous recombination. The wild-type of Rad52 protein binds to42both single and double stranded DNA, while the Rad52-1 protein has weakerbinding ability. Nevertheless, Rad52"1 can anneal complementary single"
stranded DNA similar to that of the wild-type. Although Rad52-1 can bind toboth Rad51 and RPA, Rad52-1 could not help the Rad51"mediated strand
exchange reaction in the presence of RPA. These results indicate that thebinding of Rad52 to ssDNA is more important than the binding of the protein
to RPA or Rad51 for the enhancement offormation ofRad51 filament.
Rad55 and Rad57 proteins form a complex and bind to ssDNA. A mutation in
the putative ATP binding consensus amino acid sequence of RAD55 gene inacti"
vated the function for repair of DNA damage induced by MMS and for meiotic
recombination. The mutant protein prevents the complex formation with Rad57,
whereas the DNA binding activity was not affected, showing that the complex
formation of Rad55 with Rad57 is important for these reactions. The analysis
ofthe rad55mutant in meiosis demonstrated that DSBs were introduced at thesame timing as the wild"type diploid, but that commitment to recombinationand completion of the formation of synaptonemal complexes delayed in the
mutant. These results suggest that Rad55"57 complex acts in formation of theRad51 filaments on exposed ssDNA tail at DSB ends and that the pairing withhomologous DNA molecules may stimulate formation of synaptonemal com"
plexes by stabilizing axial association between synaptic chromosomes.

References[I] USUl, T., OHTA, T., OSHIUMI, H., TOMIZAwA, J., OGAWA, H. and OGAWA, T: Complex formationand functional versatility ofMrell of budding yeast in recombination. Cell 95, 705-716(1998).
[2] AJIMURA, M., LEEM, SoH. and OGAWA, H.: Identification of New Genes Required for
Meiotic Recombination in Saccharomyces cerevisiae. Genetics 133:51-66 (1993).
[3] SHINOHARA, A., OGAWA, H. and OGAWA, T.: Rad51 protein involved in repair and recombi"
nation in S. cerevisiae is a RecA-like protein, Cell 69:457-470 (1992).
[4] OGAWA, T., Ru, X., SHINOHARA, A. and EGELMAN, E.H.: Similarity of the yeast Rad51
filament to the bacterial RecA filament. Science %59:1896-1899 (1993).
[5] ADZUMA, K., OGAWA, T. and OGAWA, H.: Primary structure of the RAD52gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2753-2744 (1984).
[6] BISHOP, D.K., PARK, D., Xu, L. and KLECKNER, N.: DMCl: a meiosis"specific yeast
homolog of recA required for meiotic recombination, synaptonemal complex formation,

B.

DEPARTMENT OF CELL GENETICS

43

and cell cycle progression. Cell 69:439-456 (1992).
[7] SHlNOHARA, A., OGAWA, H., MATSUDA, Y., USHIO, N., IKEO, K and OGAWA, T.: Cloning of
human, mouse and fission yeast recombination genes homologous to RAD51 and recA.
Nat. Genet. ,1:239-243 (1993).
[8] SHlNOHARA, A. and OGAWA, T.: Homologous recombination: Role of double-strand breaks.
TiBS 20: "DNA Repair" A special issue, 387- 391 (1995).
[9] SHINOHARA, A. and OGAWA, T.: Rad5l/RecA protein families and the associated proteinsin eukaryotes. Mutation Res. 435:13-21 (1999).
[10] SHlNOHARA, A. and OGAWA, T.: Stimulation of Rad51-mediated Recombination by Rad52
in Saccharomyces cerevisiae. Nature 391:404-407 (1998).

Publication
1. SHINOHARA, A. and OGAWA, T.: Rad5l/RecA protein families and the associated proteinsin eukaryotes. Mutation Res. 435:13-21 (1999).

Oral Presentations
1. OGAWA, T., OHTA, T., OSHIUMI, H., USUI, T. and OGAWA, H.: Versatility of function of yeast
Mrell in Recombination and Repair. Keystone Symposia on Molecular & Cellular
Biology Taos, USA February.
2. OGAWA, T., TATSUDA, D., OSHIUMI, H., KAWANE, K, OGAWA, H. and OHTA, T.: Homologydependent and -independent end-joining reactions by Mrel1. FASEB Summer Conference Snowmass, USA August.
3. OHTA, T., TATSUDA, D., OSHIUMI, H., TANAKA, S.and OGAWA,T.: Roles of Mrell protein inyeast recombination and repair. The 16th Radiation Biology Center International
Symposium Bioregulation of Radiation Response: Evolutional Dynamism of Damage
Tolerance Kyoto December.
4. OHTA, T., OSHIUMI, H. and OGAWA, T.: Roles of Mrell protein in yeast recombination
and repair. FASEB Summer Conference Snowmass USA August.
5. OSHIUMI, H., OGAWA, H. and OGAWA, T.: Functional domains of Mrell. FASEB Summer
Conference Snowmass USA August.
6. OHTA, T., TANAKA, S., and OGAWA, T.: Roles of MREll protein in yeast recombination
and repair. 3R Symposium Miki November.
7. TANAKA, S. and OGAWA, T.: Functional analysis of MREll on the telomere maintenance. 3R Symposium Miki November.
8. WATANABE, K, MATSUDA, S. and OGAWA, T.: Biochemical characterization of Rad55-57
complex. 3R Symposium Miki November.
9. TATSUDA, D., KAWANE, K, OSHlUMI, H., MATSUDA, S., OHTA, T., OGAWA, H. and OGAWA,

44
T.: Homology'dependent and independent end'joining reaction by MREll. 3R Sympo'
sium Miki November.
10. OSHIUMI. H .. TSUBOUCHI. H .. OGAWA. H. and OGAWA. T.: Functional domains of Mre11.
3R Symposium Miki November.
11. ALExEEv. A. and OGAWA. T.: Role ofthe CBF1 protein in RAD51 transcriptional regula'tion in response to the DNA damage. 3R Symposium Miki November.

B-b. Division of Microbial Genetics(1)

Dpbl1 controls the association between DNA polymerases IX and
E and the autonomously replicating sequence regionof budding yeast

Hiroshi MAsUMOTO and Hiroyuki ARAKI
Chromosomal DNA replication in eukaryotic cells initiates from multiple
origins which fire sequentially throughout the S phase; some fires early andothers late. The pre-replicative complex (pre-RC) starts to assemble at origins from late M phase and DNA polymerases are recruited onto origins toinitiate DNA synthesis during the S phase. The Dpb11 protein which geneti'
cally interacts with essential DNA polymerase E (Pol E), is required for DNA
replication and the S'phase checkpoint. The physical interaction between Pol E
and Dpb11 had not been detected. Using a cross-linker, we detected a complexof Pol E and Dpb 11 (Dpb 11'Pol E complex). During the S phase of the cell cycle,
Dpb 11 associated preferentially with DNA fragments containing autonomously
replicating sequences (ARSs), at the same time as Pol E associated with thesefragments. Association of Dpb11 and Pol E with these fragments was mutu'
ally dependent, suggesting that the Dpb 11'Pol E complex associates with the
ARS. Moreover, Dpb11 was required for the association ofthe Pol (¥ 'primasewith the fragments. Thus, it seems likely that association of the Dpb11'Pol E
complex with the ARS fragments is required for the association of the Pol (¥.
primase complex. Hydroxyurea inhibits late'origin firing in S. cerevisiae, andthe checkpoint genes, RAD53 and MEG], are involved in this inhibition. In thepresence of hydroxyurea at temperatures permissive for cell growth, Pol E in
dpbll-] cells associated with early' and late'origin fragments. In wild·type

B.

DEPARTMENT OF CELL GENETICS

45

cells, however, it only associated with early· origin fragments. This indicatesthat Dpb11 may also be involved in the regulation oflate·origin firing. Overall,these results suggest that Dpb11 controls the association between DNA poly·merase a and E, and the ARS (Masumoto et al., 2000).
(2)

Functional characterization of SId3 and SId4 thatgenetically Interact with Dpbl1

Yoichiro KAMIMURA and HiroyukiARAKI
To identify factors interacting with Dpb 11, we have isolated 10 sid (syn·thetic lethality with dpbll-l) mutations which fall into 6 complementaion
groups (sidl-6). So far, we have cloned SLDI-6and found that SLDI is identical to DPB3encoding the third largest subunit of Pol E , SLD4is identical to
CDC45 required for the initiation of chromosomal DNA replication and SLD6
is identical to .RAD53required for cell cycle checkpoints. The SLD2, -3and -S
genes were novel genes essential for the cell growth. We have shown that Sld2
forms complex with Dpb11 and this complex is involved in the early step of
DNA replication.
To know the function of Sld3, we have isolated a thermosensitive s1d3-S
mutation. S1d3-5 cells were defective in DNA replication at the restrictive
temperature. We further analyzed the DNA replication defect of sid3-S cellsby 2·D agarose gel electrophoresis. In s1d3-S cells, the bubble arc in ARS region
that is a signal indicative of the initiation of DNA synthesis, reduced quickly
after temperature shift-up, suggesting that Sld3 is required for the early stepof DNA replication. When sid3-S cells were arrested at permissive tempera·ture in S phase by hydroxyurea and were released at the restrictive tempera·ture, cells did not complete DNA replication, suggesting that Sld3 also func·
tions in the elongation step of DNA replication. Thermosensitive growth ofsid3-S mutant was suppressed by high copy CDC45/ SLD4. Moreover, 2-hybrid analysis showed an interaction between Sld3 and Cdc45. These results
suggest that Sld3 and Cdc45 form a complex.
We analyzed association of Sld3 and Cdc45 with ARS using chromatin immunoprecipitation assay. Both Sld3 and Cdc45 associated with early-firing

46

ARSs from G1 while their association with late-firing ARSs occurred in the Sphase. Their association with ARSs depended on McmS, a component of the
pre-RC (see section (1)) and was mutually dependent, consistent with complex
formation of Sld3 and Cdc4S. When the ARS is activated, it has been thoughtthat double· stranded DNA in the ARS is unwound to single-stranded DNA
that binds to single-stranded DNA binding protein, RPA. We, thus, examined
RPA association with ARSs in sld3-S cells at the restrictive temperature andfound that RPA does not bind ARSs. Therefore, Sld3 is required for RPA association and probably functions before unwinding ofARS region.

(3)

Function of the Sld5/Psft complex which interacts with DNA
polymerase & and Dpbl1 in Saccharomyces cereylslae

Yuko TAKAYAMA, Yoichiro KAMlMURA and Hiroyuki.ARAKIThe SLDS gene encodes an essential 34 kDa protein. To elucidate the function of SldS, we isolated the PSFl (Partner of SLD Five) gene as a multicopy
suppressor of the sldS-12mutation. PSFl encodes a 24 kDa protein essential
for cell growth. In two-hybrid analysis and co-immunoprecipitation experiment, SldS interacts with Psfl in yeast cells. The SldS-Psfl complex was
detected throughout the cell cycle. We also isolated a thermosensitive psfl-l
mutation by the plasmid shuffling method. Psfl-l mutant cells arrested witha dumbbell shape at the restrictive temperature as did sldS-12cells. We did
not detect the SldS/Psfl complex in psfl-l cells. By two hybrid assay, Psfl-l
was defective in an interaction with Dpbl1. These results suggest that the
SldS/Psfl complex, together with Dpb11, is required for the early step of DNA
replication.

(4)

Novel mutations of DPB1J

Sachiko MURAMATSU and Hiroyuki.ARAKI
Dpb 11 has four BRCT (BRCAI C-terminus) repeats that occur in several proteins involved in cell cycle checkpoints, and has been suggested to be a domain for

B.

DEPARTMENT OF CELL GENETICS

47

interaction between proteins. Since dpb11-1 is a nonsense mutation that occurs
at C-terminus of the fourth BRCT repeats, the function of each BRCT repeats is
obscure. We, therefore, isolated novel DPB11 mutations to know the function ofeach BRCT repeats. A mutagenized DPB11 gene library was created by random
PCR mutagenesis and was screened for Ts (thermosensitive), RU (hydroxyurea)sensitive or MM:S (methyl methanesulfonate)-sensitive alleles by using a plasmid shufl1ing strategy. So far, we have isolated eight Ts (dpbl1-3 - -9, -11), two
MMS-sensitive (dpb11-12, -13) and one RU, MMS'sensitive (dpbl1-14) alleles.
We first examined the Dpb11 protein level by tagging Dpb11 and western
blotting. The Dpb11 protein levels were reduced to 10 - 30 % of the wild typelevel in the Ts mutants except for two which have mutations in first BRCT
repeats (dpbl1-3, -4). Since most ofTs mutants grew normally at permissive
temperature, it was suggested that small amount of Dpb 11 is sufficient for thecell growth.
The novel Ts mutants lost viability quickly after temperature shift-up whenpopulation of cells having large bud increased during S-phase as dpb11-1 cells
do. Multicopy SLDs and PSF1 that were isolated as factors interacting with
Dpb11, were introduced to each dpb11 mutants. Multicopy SLD2, whose products form a complex with Dpb11, strongly suppressed the growth defects of theTs mutations occurring in C-terminal half, but not ofthe Ts mutations in Nterminal half. This result suggests that Sld2 interacts with C-terminal half
ofDpb11, and further suggests that all the novel mutations do not have thedefect in the same process although phenotypes are similar.
All the Ts mutants (dpbl1-3- -9, -11) were slightly sensitive to RU and MM:S,
while six Ts mutants (dpbl1-3- -6, -9, -11) were sensitive to IN and two mutants (dpbl1-7, °8) were not. Dpb11012and -13mutants showed semi-dominantphenotype in sensitivity to MMS, but not to RU and uv. Dpbl1-14 mutant wasslightly sensitive to RU and MMS but not to uv.
To know whether dpb11 mutants are defective in the checkpoint, we examined the Rad53 modification in response to damage. It was reported that
Rad53 modification in response to MMS treatment was reduced in dpbll-1
cells at semi-permissive temperature. The Rad53 modification after MMS
treatment, however, was not reduced in dpb11-1 cells at semi-permissive tem-

48

perature by our hands. Surprisingly Rad53 was modified in dpbll-l morethan WT in response to low level MMS at permissive temperature. This result suggests that Dpbll is not major determinant of Rad53 modification in
the DNA damage checkpoint pathway, and Dpbll may be involved in repair ofdamaged DNA.

Publications
1. KAMIMURA, Y, KAwASAKI, Y. OHARA, T. and SUGINO, A: DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w (HELl), highly unwinds covalently closed, circu'
lar DNA in the presence of a DNA topoisomerase and yRF-A J. Biochem., Z5, 236'244(1999).
2. MASUMOTO, R., SUGINO, A and ARAKI, R.: Dpbll controls the association between DNA
polymerases a and E and the autonomously replicating sequence region of budding
yeast. Mol. Cell. BioI., ZO, 2809'2817 (2000).

B-c. Division of Cytoplasmic Genetics(1)

Studies on behavioral disorders of knockout mice

Hiroaki NIKI (Lab. for Neurobiology of Emotion, Brain Science Institute,
RIKEN)The research is aimed at elucidating the behavioral abnormalities of Fyn'deficient mice and their pharmacological and physiological correlates. These
mice were more fearful in a variety of fear tests (light aversion test, novelty
preference test, elevated plus maze test, and passive avoidance test). Decreased
offensive aggression and increased defensive aggression were noted in thesemice.
We also found that these mice were more susceptible to seizures induced byintense sound and convulsive drugs. Related to these abnormalities of emo'
tional behavior and seizure susceptibility Fyn'deficient mice were found tohave a lower density of central benzodiazepine receptor binding sites in brain
tissues. We have recently found that these mice were hypersensitive to the
hypnotic effect of ethanol along with the loss of acute tolerance to ethanol

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DEPARTMENT OF CELL GENETICS

49

inhibition of NMDA receptor mediated EPSPs in the hippocampus slice
experiment. In wild-type mice administration of ethanol enhanced tyrosine
phosphorylation of the NMDA receptor in the hippocampus, but such a change
was not noted in Fyn-deficient mice. In 1999 we found a novel target of ethanol
in Xenopus oocyte protein expression system: ethanol activates G-protein activated inwardly rectifying potassium channel (GIRK channel). In addition,
physiological and behavioral analyses of weaver mutant mice having mutant
GIRK channel in the brain revealed that the activation of GIRK channel by
ethanol is important for ethanol-induced analgesia.

Publ ication
1.

KOBAYASHI, T., IKEDA, K., KOJIMA, H., NIKI, H., YANO, R., YOSHIOKA, T. and KUMANISHI, T.:
Ethanol opens G'protein'activated inwardly rectifying K+ channels. Nature Neurosci.
ll:1091-1097, 1999.

50

C.

DEPARTMENT OF DEVELOPMENTAL GENETICS
C-a. Division of Developmental Genetics(1)

Regulation of neural induction by negative regulators of theras/MAPK signaling pathway

Masaki IWANAMI, Masataka OKABE, Yasushi HIROMI
Induction is a fundamental strategy in cell fate decisions during develop'
ment, and is often used to generate cellular diversity. During neural develop'
ment, however, neurons or their precursors often induce their neighbors to takea fate similar to their own. This phenomenon, called "homeogenetic induction", occurs in at least two processes in Drosophila; photoreceptor neuron
development in the adult retina, and chordotonal organ formation in the em'bryonic peripheral nervous system. In both cases, the inducing factor is an
EGF-like molecule Spitz, which acts though EGF-receptor and raslMAPK signaling pathway to phosphorylate ETS transcription factor PointedP2. To prevent unlimited spread of the induction process, induction must be negatively
regulated. We showed that in both eye and chordotonal organs, three negative
regulatory system operate simultaneously to inhibit induction. Two of themproduces either cell autonomous or non-autonomous factors that inhibit ras/
MAPK signaling, in response to the inducing signal. While Argos had been
known to act non-autonomously to antagonize EGF-receptor function, we
showed that Sprouty acts autonomously to inhibit Spitz-mediatedinduction. Furthermore, we found a third mechanism; signal-dependent inhibition of inducing ability. PointedP2 activated by the inducing signal inhibits
the ability to induce, thereby ensuring that cells, once induced, does not act asa secondary source of induction. These negative regulatory systems are allnecessary, and none ofthe three is sufficient.Production of constant outcome
by induction must therefore involve interaction of these negative regulatory
systems. In fact, argos and sproutyexhibit genetic interactions. We are cur-

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

51

rently analyzing mechanisms of interaction between three negative regulatory
systems. For details, see ref. 1.
(!)

Positional information required for sensory organ identity in the
peripheral nervous system

Masataka OKABE and Yasushi HIROMIThe Drosophila larval peripheral nervous system consists of many types of
sensory organs, such as external sensory (Es) organ (mechanosensory organ) and
chordotonal (Ch) organ (stretch receptor). We are investigating how each organ
forms in a specific segment, at specific positions within each segment. Formationof sensory organs require activities of organ"specific proneural genes that en"code bHLH proteins; Es organ depends on proneural genes in the achaete'scutecomplex (AS-C), while Ch organ require atonal. A down stream target of AS-C
is cut, which encodes a homeobox transcription factor that acts as a genetic
switch between Es and Ch organs. Although the prevailing view is that organ"
specific bHLH transcriptional factors determine the distinction between Es and
Ch organs, we found that even in the absence of organ-specific bHLH proteins,correct organ formation and Es"specific cut expression can be achieved by anorgan'nonspecific bHLH protein Asense. This indicates that there must be PO"
sitional information that activates cut specifically in the Es organ precursors,
independently from organ specific bHLH proteins.
With the aim of identifying positional information that regulates cutexpression, we began analysis of cis 'elements of the cut gene, responsible for its ex'pression in Es organ precursors. We have identified a minimal enhancer of200bp
that directs Es organ specific expression. In near future, we hope to identify
factors that act through this element and regulate cut expression in Es organ
precursors.

52

(3)

Spatlo-temporal constraints of organogenesis in
Drosophila imaginal discs

Nao NIWA, Masataka OKABE and Yasushi HIROMIThe Drosophila eyeless (ey) gene has been identified as a master control genefor the compound eye development. Ey is required for eye formation and isexpressed in the entire eye imaginal disc. However, not all of eyexpressing cells
form the compound eye. For example, peripheral region of the eye disc form thehead cuticle surrounding compound eye. Furthermore, while eyis ubiquitously
expressed throughout the eye disc, retinal differentiation starts only from theposterior edge ofthe disc. Therefore, in addition to eyexpression, there must beother conditions required for the initiation of eye formation.
We are investigating the spatio·temporal constraints on eye formation us·
ing the ectopic eye formed by the ey gene expression as an experimental
paradigm. We found that the ectopic eye formation was most frequent wheneywas expressed at about 80 hours after egg laying (AEL), and the positions ofectopic eyes in all imaginal discs were restricted to several domains near theanterior-posterior compartment borders where decapentaplegiddpp) or wingless (wg) gene is expressed. To test whether dpp and wg have a regulatory rolein the responsiveness to ey, ey was expressed under the conditions where Dpp
or WG signaling is activated. Coexpression of eyand a constitutive active
form of a Dpp receptor could induce ectopic eyes where Dpp is normallyabsent. On the other hand, coexpression of wg with ey blocked ectopic eyeformation everywhere in the imaginal discs. These results suggest that the
Dpp signal is required for the ectopic eye formation, and that Wg expression
negatively regulates eye formation. In addition, we found that the period ofcompetence to eywas precisely the stage when endogenous retinal develop·
ment starts at the posterior edge of the eye imaginal disc. At this time, dppis
expressed at the posterior edge of the eye disc and is required for the initiationof retinal differentiation. These results indicate that spatial and temporalconditions for ectopic eye formation by eyare quite similar to those for theendogenous eye formation.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS(4)

53

Mechanism of action of a nuclear receptor Seven-up

Motomi MATSUNO, Hiroyuki KOSEl, Yasushi HIROMI (IUniversity of Tokushima,
School of Medicine)
Nuclear receptor Seven-up (SVP) is expressed in a subset of photoreceptor
neurons and functions as a genetic switch between photoreceptor subtypes. The
ligand binding domain (LBD) of SVP is extremely well conserved during evolution' suggesting that it functions by interacting with other molecules that arealso conserved through evolution. Using chimeric molecules of the GAL4 DNA
binding domain and the SVP LBD, we showed that SVP LBD has transcrip'
tional repression activity. To identify molecules that participate in SVP'me'
diated gene regulation, we searched for proteins that interact with SVP LBD
using the yeast two'hybrid screen. We identified two proteins that could act
in transcriptional regulation. One is a protein that contain the SAM domain
similar to the one found in a Polycomb group protein Polyhomeotic. Another isa p52 subunit of basal transcription factor TFIIH. An intriguing possibility isthat SVP exerts its repression function by interacting with the p52 subunit of
TFIIH. We have identified four strains that harbor nonsense mutation or deletion in the p52 ORF. Currently, we are studying the developmental role of p52
and the genetic/molecular interactions between p52 and SVP.
(5)

Hydra Peptide Project: Epitheliopeptides which are involved in
Hydra morphogenesis

Toshio TAKAHASHI, Naoe HARAFUJI, Seungshic YUM, Masayuki HATTA, Hiroshi
SHIMIZU, Osamu KOIZUMI l, Yoshitaka KOBAYAKAWA2 and Toshitaka FUJISAWA(IFukuoka Women's University, 2Faculty of Science, Kyushu University)
In our ongoing "Hydra Peptide Project" in which peptide signaling molecules
are systematically purified and identified, we found several peptides which
are produced and/or localized in epithelial cells. They were referred to asepitheliopeptides. Among them, two (Hym-323 and Hym-346) are involved infoot formation, one (Hym-301) in tentacle/head formation and another one

54(Hym-330) in budding. These peptides have entirely different amino acid sequences and are encoded in independent cDNAs. It has been shown that morphogenesis in hydra is primarily controlled by epithelial cells. This is particularly obvious by the fact that epithelial hydra which is composed of only epithelial cells still maintains its morphology and polarity, buds and regenerates
normally. Thus, important morphogenetic molecules are expected to be epithelial cell origin. We have shown that both Hym-323 and Hym-346 altered
the positional values along the body column when applied exogenously andenhanced foot regeneration. It is one of the important features attributed tomorphogens. Whether Hym-30l and Hym-330 affect positional values remains
to be seen. In the Project, we expect to find more peptides which are involved
in morphogenesis and hope to find true morphogens.

(6) Expression of nanos related genes in Hydra
Kazufumi MOCHIZUKI 1, Chiemi NISHIMIYA-FUJISAWA and Toshitaka FUJISAWA(IDepartment of Genetics, The Graduate University for Advanced Research,
Mishima)Hydra contains undifferentiated multipotent stem cells which give rise to
three somatic cell types as well as germ cells. In order to analyze germ celldifferentiation, the molecular markers specific to germline cells are
essential. For this reason, we cloned two nanos (nos) related genes, Cnnosl
and Cnnos2 from Hydra magnipapillata. Using whole mount in situ hybridization, expression of Cnnosl and Cnnos2 was examined. Cnnosl was weakly
expressed in multipotent stem cells and strongly expressed in germline cells
but not in somatic cells. Cnnos2 appears to be also expressed in germline
cells. However, it was expressed more conspicuously in the endoderm of thehypostome and suggested to be involved in head morphogenesis. In addition
to structural conservation in the zinc finger domain of nos related genes, functional conservation of Cnnosl was also shown by that a Cnnosl transgene canrescue, although partially, nosRC phenotype that is defective in the activity ofgermline stem cell maintenace in Drosophila. (Sano and Kobayashi, personal
commJ Thus, the function of nos related genes in germline appears to be well

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DEPARTMENT OF DEVELOPMENTAL GENETICS

55

conserved from primitive to highly evolved metazoans. Cnnos genes are thefirst molecular genes specific to germline cells in Hydra.'!

(7)

Feeding and wounding responses of hydra: nervous system oftentacles show unidirectional conduction of stimulus

Hiroshi SHIMIZU
Diffuse nervous system is primarily found in freshwater hydra as nerve net
which is dispersedly distributed throughout the animal. It has been assumed
that external stimulus is conducted in all directions through the nerve net,
which is in contrast with central nervous system where the stimulus is con"ducted unidirectionally into or out of brain. I examined feeding and wounding
responses of hydra and obtained evidence that hydra tentacle nervous system
responds in a polarized manner to feeding and wounding stimulus. Tissue
located proximal to the site of stimulus contracted enormously whereas not at
all or very little in the distal part of it. Further analyses using animals which
is devoid of nerve cells showed that this polarized response does not occur inthe tissue where there are no nerve cells thus suggesting that nervous system
is responsible for the response. This is so far the first demonstration thatpolarized nervous system as the most primitive form of central nervous sys"
tem was first established in hydra, the most primitive organism having nervous system.

(8)

Evolution of reef-building corals

Masayuki HATTA
Crossing experiments were carried out systematically using mass"spawn'
ing corals ofthe genus Acropora in Okinawa, Japan. Interspecific fertilization
was observed in specific combinations of species, even between species thathave very different morphologies. Molecular phylogenetic analysis ofthe mini,
collagen gene, rDNA and histone gene clusters suggested the species weredivided to three related groups. Within the groups, the genetic relationships

56of the species are intermixed and hybridization was observed. These
charcteristics are explained by interspecific gene introgression via the hybrids
produced by mass spawning, and are consistent with the concept of a species
complex under the processes of the fusion of multiple species.
Surveyrance of alternative reproductive behaviors and genetic relationships
suggested sympatric speciation by shifting spawning timing from mass·spawn·
ing as an alternative mechanism of speciation in sympatric Acropora corals(Hatta et al. 1999).
Phylogeny of the coral family Acroporidae based on mitochondrial genes
suggested that divergence of extant species in each genus is recent events after
extinction during palaeoclimatic changes. Mass·spawning appeared to be theancestral reproductive behavior (Fukami et al. in press).

(9)

Differentiation mechanisms of neurons and gila

Takeshi SASAMURA 1, Yoshihiko UMESON0 2, Kazunaga TAKIZAWA 1,2 and Yoshiki
HOTTA2,3 (IGraduate School of Science, University of Tokyo, 2CREST, JST, 3Na·
tional Insitute of Genetics)
The transcriptional regulation ofthe gcm gene was examined. In the periph·eral nervous system (PNS), dorsal bipolar dendritic (dbd) lineage is the most
simple lineage, in which the precursor once divides asymmetrically to give rise
to a neuron and a glia. gcm is transiently expressed in the glial precursor andit presence is required for induction of the glial fate, whereas its absence isrequired for the neuronal fate in the dbd lineage. Notch signaling pathway issufficient for the induction of gcm in this process. Loss of Notch function
results in the loss of glial differentiation as a consequence of no gcm expres·sion. Conversely, gain of Notch function leads to the glial transformation of
the neuron through the ectopic gcm expression. Notch signaling becomes actiovated only in the ~liallineage and its neuronal inactivation requires Numb.
Thus, we propose k simple mechanism that asymmetric activation of Notch
directs asymmetric expression of gcm to establish a binary decision of theneuron/glia fate in the dbd lineage.
Glial differentiation mechanisms were analyzed by investigating the func·

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DEPARTMENT OF DEVELOPMENTAL GENETICS

57

tions of genes expressed in glial cell lineage. Longitudinal glia (LG) are generated from a single precursor cell called longitudinal glioblast (LGB). LGB
generates 6 to 8 LGs, which migrate medially eventually to ensheath longitu·
dinal tract. We showed that gcm genetically upregulates some glial specificmakers. Investigation of glial morphology in these mutants showed that glial
differentiation proceeds in some different pathways such as migration, pro·
cess formation, induction of expression of neuronal antigen. Next we studied
the relationship between glial differentiation and axon pathfinding and findthat migration of glial cells are abnormal in the mutants that have defects inpioneer pathfinding.
To identify genes that regulate neural cell differentiation, we screened about
3,000 tau·lacZ enhancer trap lines. One line, Z477, whose embryos show CNS
axon defect, was selected for further analysis, and named as solo (snappedouter longitudinals). We cloned solo and showed that it encodes a transcrip·tion factor that has BTB/POZ domain and zinc finger motifs. The solo gene
was found to be most related to Drosophila tramtrack gene. The solo mRNAwas detected in all neurons, but not glial cells by in situ hybridization toembryos. We found that solo mutant embryos have defects in the formation of
one of the CNS axon fascicles and dendrites of the DBD neuron in the PNS.
These data suggest that solo is required for neural differentiation, especiallyfor axon guidance.
(to)Axon guidance mechanisms

Kazunaga TAKIZAWA1,2, Masaki HlRAMOT0 1, 2and Yoshiki HOTTA2,3 (1 Graduate
School of Science, University of Tokyo, 2CREST, JST, 3National Insitute of Ge·
netics)We investigated functions of glial cells in axonal pathfindings by examining
axonal behavior in the gcm mutants of Drosophila that lack glial cells in the
nervous system. In this study, we showed that the initial extension of pioneer
neurons and target recognition of motoneurons are normal in the absence of
glial cells. We also demonstrated that formation of axon bundles andpathfindings are disrupted at later stages in the absence of glial cells and58

abnormal migration of glial cells leads to misrouting ofaxons, indicating thatglial cells are required for correct pathfinding at later stages. From theseobservations, we propose that formation of the nervous system proceeds in
multisteps with respect to glial cell functions.
Netrin is a secreted protein that has properties of a chemotropic axon guidance molecule. In Drosophia, Frazzled is a member of the DCC class of Netrin
receptors, and its phenotype in the embryonic CNS is consistent with the ideathat it mediates Netrin signals. Here we demonstrate a novel form of Netrindependent axon patterning by Frazzled: Frazzled controls the spatial distribution of Netrin, 'presenting' this ligand molecule for recognition by otherreceptors. Moreover, Frazzled itself is actively relocalized within axons bymeans of its cytoplasmic domain, thereby precisely rearranging Netrin into aspatial pattern that is completely different from the pattern of Netrin source.
Finally, we show that Frazzled"dependence of one axon guidance decision, thelongitudinal turning of the pioneer neuron dMP2, can be accounted for solely
on the basis of this ability of Frazzled to redistribute Netrin and not by
Frazzled signaling. We propose a novel patterning mechanism in which controlled distribution of a receptor creates positional information for other reo
ceptors by rearranging secreted ligand molecules.

(11) Function of the gem-related transcriptional regulators
Toshihiko HOSOYA 1,2, Yoshiki HO'ITA 1,3 (lNationalInsitute of Genetics, 2PRESTO,
JST, 3CREST, JST)The gem protein is a member of a novel protein family, the gem-family. Members of this family share the gem-motif, a novel protein motif that is of about
150 amino acids in length. It has been shown that the gem-motif has sequencespecific DNA-binding activity and that the gem protein has transcriptional regulation activity. The gem"binding DNA sequences are found in tandem in the
upstream of a glia-specific protein whose expression is dependent on gem. Thus
it is strongly suggested that the gem protein is a novel transcriptional regulator
and that the gcm"family is a novel family of transcriptional regulators.
Drosophila has at least another gem family gene, gcm2, which is expressed

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DEPARTMENT OF DEVELOPMENTAL GENETICS

59

in a subset of glial cells and early hemocytes. gem is also expressed in hemocytes
and required for their differentiation. Two deficiency strains that lack both
gem and gem2 have smaller number of hemocytes than wild-type. These
hemocytes show defects in taking up corpses and in movement. Preliminary
experiments indicated that either misexpression of gem or gem2 can rescue
these phenotypes. Thus it was suggested that gem and gem2have overlapping
function in the hemocyte differentiation. Two gcm family genes have been
isolated from Mouse, which show tissue specific expression. 13 -gal knock-in
chimera mice have been generated.

Pub I i ca t ions
1.

KRAMER, S., OKABE, M., HACOHEN, N., KRASNOW, M.A and HIROMI, y.: Sprouty: a com'
mon antagonist of FGF and EGF signaling pathways in Drosophila. Development
126, 2515'2525 (1999).

2.

HIROTA, Y., OKABE, M., IMAI, T., KURUSU, K., YAMAMOTO, A, MIYAO, S., NAKAMURA, M.,
SAWAMOTO, K. and OKANO, H.: Musashi and Seven in absentia downregulate Tramtrack
through distinct mechanisms in Drosophila eye development. Mech. Dev. 87,93-101(1999).

3.

FUJIMOTO, J., SAWAMOTO, K., OKABE, M., TAKAGI, Y., TEZUKA, T., YOSHIKAWA, S., Ryo, H.,
OKANO, H. and YAMAMOTO, T.: Cloning and characterization of Dfak56, a homolog of
focal adhesion kinase, in Drosophila melanogaster. J. BioI. Chern. 274, 29196-29201(1999).

4.

KUMPFMUELLER, G., RYBAKINE, Y, TAKAHASHI, T., FUJISAWA, T. and BOSCH, T.: Identificationof an astacin matrix metalloprotease as target gene for Hydra foot activator peptides.
Dev. Genes. Evol. 209: 601-607 (1999).

5.

GRENS, A, SHIMIZU, H., HOFFMEISTER, S., BODE, H.R. & FUJISAWA, T.: PedibinlHym'346
lowers positional value thereby enhancing foot formation in hydra. Development, 126,517-524 (1999).

6.

HATTA, M., FUKAMI, H., WANG, W., OMORI, M., SHIMOIKE, K., HAYASHIBARA, T., INA, Y. and
SUGIYAMA, T.: Reproductive and genetic evidence for a reticulate evolutionary history of
mass'spawning corals. Mol. BioI. Evol., 16, 1607-1613 (1999).

7.

HATTA, M., SAKAGUCHI, M., KOBAYAKAWA, Y., KISHIMOTO, Y. and KOIZUMI, 0.: Identificationof a homolog of actin' binding protein, ABP-280, localized at epithelial cell-cell bound'aries in hydra. Zool. Sci., 16, 439-443 (1999).

8.

AKIYAMA'ODA, Y., HOSOYA, T., HOTTA, Y.: Asymmetric cell division ofthoracic neuroblast
6'4 to bifurcate glial and neuronal lineage in Drosophila. Development 1999 May;
126(9): 1967-74.

60
9.

KOBAYASHI, M., TAKEZAWA, S., HARA, K, Yu, RT., UMESONO, Y., AGATA, K, TANIWAKI, M.,
YASUDA, K, UMESONO, K: Identification of a photoreceptor cell"specific nuclear recep'
tor. Proc Natl Acad Sci U.S.A. 96, 4814-9 (1999).

C-b. Division of Gene Expression(1) Functional dissection of DNA supercolling factor:
EF-hand domains and C-terminal HDEF motif are
essential for its activity
Masatomo KOBAYASHI and Susumu HIROSE
DNA supercoiling factor (SCF) was first identified in the silkworm as aprotein capable of generating negative supercoils into a relaxed DNA in conjunction with eukaryotic topoisomerase II. Drosophila melanogaster SCF 10'
calizes to puffs on polytene chromosomes, implicating its role in gene
expression. The factor is a Ca 2+'binding protein with four EF-hand domains
and possesses a tetrapeptide sequence HDEF at its C-terminus.
To clarify the roles of the domains of SCF, we carried out a functional dissec'
tion of the factor. A glutamic acid to glutamine substitution at the end of theloop in EF-hand domain II or III reduced both the Ca 2+-binding and supercoil'ing activities; simultaneous substitutions at both sites abolished these
activities. During native polyacrylamide gel electrophoresis, SCF migrated
more rapidly in the presence of Ca2+than in the presence ofMg2+ or EGTA. SCF
binds directly to topoisomerase II. Deletion ofthe C-terminal HDEF sequence
destroyed the binding and supercoiling activity.
From these results, we conclude that two regions of SCF play critical roles inthe supercoiling activity. The C-terminal HDEF is essential for the factor
binding to topoisomerase II. The EF-hand domains II and III are functional for
the Ca 2+ binding that induces a mobility change in the factor upon gel
electrophoresis. For details, see Ref. 1.

(2) The Role of Human MUF1 as a Transcriptional Coactivator
Yasuaki KABEl, Masahide GOTO I , Daisuke SHIMA I , Takeshi IMAI I , Tadashi

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

61

WADA 1, Ken-ichirou MOROHASHI 2, Masahiro SHIRAKAWA3, Susumu HIROSE andHiroshi HANDA4 (IFaculty of Bioscience and Biotechnology, Tokyo Institute ofTechnology, Midori-ku, Yokohama 226-8501, Japan, 2National Institute for
Basic Biology, Okazaki, Aichi 444-8585, Japan, 3Graduate School of Biological
Sciences, Nara Instiute of Science and Technology, Ikoma, Nara 630-0101,
Japan, and 4Frontier Collaborative Research Center, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan)
Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA
element-binding protein and gene-specific activators such as the Drosophila
nuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The
human homolog ofMBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning andfunctional analysis of hMBF1. Two isoforms, which we term hMBF1 a andhMBF1 {3, have been identified. hMBF1 a mRNA was detected in a number oftissues, whereas hMBF1 {3 exhibited tissue-specific expression. Both isoforms
bound to TBP and Ad4BP/SF-1, a mammalian counterpart ofFTZ-F1, andmediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1
was detected in the cytoplasm by immunostaining, coexpression ofthe nuclear
protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the
nucleus, suggesting that hMBF1 is localized in the nucleus through its binding
to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine
zipper protein family, and mediated its activity as a transcriptional
activator. These data establish that the coactivator MBF1 is functionallyconserved in eukaryotes. For details, see Ref. 2.
(3)

Identification of the core domain and the secondary structureof the transcriptional coactlvator MUF1

Jun OZAKI 1, Ken-ichi TAKEMARU, Takahisa IKEGAMI 1, Masaki MISHIMA1, Hitoshi
DEDA, Susumu HIROSE, Yasuaki KABE 2, Hiroshi HANDA 3 and Masahiro
SHIRAKAWA1 (1 Graduate School of Biological Sciences, Nara Institute of Scienceand Technology, Ikoma, Nara 630-0101, Japan, 2Faculty of Bioscience and Bio-

62
technology, and 3Frontier Collaborative Research Center, Tokyo Institute ofTechnology, Midori-ku, Yokohama 226-8501, Japan)
Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, suchas FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activa"
tors and the TATA"box binding protein (TBP), suggesting that MBF1 functions
by recruiting TBP to promoters where the activators are bound. In addition,
MBF1 stimulates DNA binding activities of the activators to their recognition
sites. To date, little is known about structures of coactivators that bind to
TBP.
The two"dimensional (2D) IH_15N correlation spectrum of 15N labeled MBF1
indicated that MBF1 consists of both flexible and well structured
parts. Limited digestion of MBF1 by 0: -chymotrypsin yielded a "" 9 kDa
fragment. N"terminal sequence analysis and NMR measurements revealed
that this fragment originates from the C"terminal80 residues of MBF1 andform a well structured C"terminal domain ofMBF1, MBF1 cTD ' As previous
deletion analyses have shown that MBF1 CTD is capable of binding to TBP, it issuggested that MBF1 cTD is the TBP binding domain ofMBF1. Sequential
assignments have been obtained by means ofthree"dimensional (3D) and four
dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure ofMBF1 CTD was determined. As a result, MBF1 CTD was shownto contain four amphipathic helices and a conserved C-terminal region. Asp 106
which is assumed to be responsible for the binding to TBP is located at thehydrophilic side of the third helix.
From these results we conclude that MBF1 consists of two structurally
different domains. A N-terminal region is indispensable for the binding toactivators, and does not form a well defined structure. In contrast, the Cterminal 80 residues, which is capable of binding to TBP by itself, form a wellstructured domain, MBF1 CTD . MBF1 CTD is made up of four amphipathic heli"ces and a conserved C"terminal tail. A putative TBP binding residue is locatedon the hydrophilic surface of the third helix. For details, see Ref. 3.

C.

(4)

DEPARTMENT OF DEVELOPMENTAL GENETICS

63

Upstream element of the sea urchin aryisulfatase geneserves as an insulator

Koji AKAsAKAI, Atuko NISHIMURAI, Kazuko TAKATA 1, Keiko MITSUNAGA 1, Fusako
MIBUKA 1, Hitoshi UEDA, Susumu HIROSE, Ken TsuTsur 2 and Hiraku SHIMADA 1(IGraduate Department of Gene Science, Faculty of Science, Hiroshima Uni'
versity, Kagamiyama, Higashi-Hiroshima, 739-8526, Japan and 2Departmentof Molecular Biology, Okayama University Medical School, 2-5-1 Shikatacho,
Okayama 700-8558, Japan)
Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we reportthat a DNA fragment located in the upstream region of sea urchin, H.
pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars
enhancer when positioned between the enhancer and the target promoter, inan orientation dependent manner. The Arsinsulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation
mechanism operates also unidirectionally in Drosophila. We also show thatthe efficiency of transformation of HeLa cells is enhanced when the integrated
gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These
results demonstrate that the mechanism of action of the insulator has beenconserved throughout evolution. For details, see Ref. 4.

Publications
1.

KOBAYASHI, M. and HIROSE, S.: Functional dissection of DNA supercoiling factor: EF·
hand domains and C-terminal HDEF motif are essential for its activity. Genes Cells

4, 33'40, 1999.
2. KABE, Y., GOTO, M., SHIMA, D., IMAI, T., WADA, T., MOROHASHI, K., SHIRAKAWA, M., HIROSE,
S. and HANDA, H,: The role of human MBFl as a transcriptional coactivator. J. Bioi.
Chem. %74, 34196-34202, 1999.
3.

OZAKI, J., TAKEMURA, K., IKEGAMI, T., MISHIMA, M., UEDA, H., HIROSE, S., KABE, Y., HANDA,

64
H. and SHlRAKAWA, M.: Identification of the core domain and the secondary structure of
the transcriptional coactivator MBFl. Genes Cells 4, 415'424, 1999.
4.

AKASAKA, K, NISHIMURA, A, TAKATA, K, MITSUNAGA, K, MIBUKA, F., UEDA, H., HIROSE, S.,
TSUTSUI, K and SHIMADA, H.: Upstream element ofthe sea urchim arylsulfatase geneserves as an insulator. Cell. Mol. BioI. 45, 555-565, 1999.

5.

MISHIMA, M., OZAKI, J., IKEGAMI, T., KABE, Y., GOTO, M., UEDA, H. HIROSE, S., HANDA, H.
and SHlRAKAWA, M.: Resonance assignments, secondary structure and ION relaxation
data of the human transcriptional coactivator hMBFl (57-148). J. Biomol NMR, 14,373-376, 1999.

C-c. Division of Early Embryogenesis(1)

Mesoderm induction and its dorsoventral specification in zebrafish

Toshiro MIZUN0 1, Etsuro YAMAHA2 and Hiroyuki TAKEDA (ISchool of Science,
Kagoshima University, 2Faculty of Fisheries, Hokkaido University)
In the previous study, we have demonstrated that zebrafish yolk cell is responsible for induction and dorsoventral patterning of the mesoderm (Mizuno
et aI., Nature383: 131, 1996). In order to examine the presence and localiza'tion of cytoplasmic determinants for axis formation in zebrafish, we havedeveloped a method with which we removed vegetal yolk hemisphere of earlyfertilized eggs (vegetal'removed embryos). When the vegetal half of the yolk
mass was removed at l'cell stage, the embryos showed severe dorsal deficiencies and bilaterally symmetrical morphology in later development. Furthermore,
vegetal'removed embryos examined at early gastrula stage (50%'epiboly) did
not express a dorsal marker, goosecoid (gsc), while the expression of a ventral
marker, evel, was expanded dorsally. These results suggest that the vegetal
yolk cell mass contains the cytoplasmic determinants responsible for dorsal
specification (dorsal determinants) of the zebrafish embryos. Furthermore,
we found that the frequency of abnormalities decreased as the age at which thevegetal yolk hemisphere was removed increased: The embryos operated before
I'cell stage showed the highest frequency of abnormalities while most of theembryos operated after 8'cell stage developed normally. This result suggests
a translocation of the dorsal determinants from the vegetal pole toward the

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DEPARTMENT OF DEVELOPMENTAL GENETICS

65

animal pole during early cleavage stages in zebrafish.
To determine which tissue, the yolk cell or blastomere, requires the dorsal
determinants for development of dorsal fate, recombination experiments be'
tween normal and vegetal-removed embryos were carried out. Normal yolkcell, when transplanted in the animal-pole region of intact host embryo, in'duced ectopic expression of gsc in the host blastoderms. By contrast, the yolkcell obtained from vegetal' removed embryo lost the ability to induce gsc in
normal blastomeres. Furthermore, the blastomeres of vegetal'removed embryo expressed gscwhen combined with normal yolk cell. These results indio
cate that the dorsal inducing ability of the yolk cell is highly dependent on thedorsal determinants and that the blastomeres without receiving these determinants are competent to express the dorsal specific gene in response to thesignals from the yolk cell.
(!)

Cell-cell interaction in zebraflsh floor-plate development

Minori SHINYA and Hiroyuki TAKEDAThe floor plate is located at the ventral midline of the neural tube invertebrates. The floor-plate development is severely impaired in zebrafish
one'eyedpinhead (oep) mutants. oep encodes a membrane'bound protein witha EGF motif and functions autonomously in floor-plate precursors. To under'
stand the cell behavior and cell'cell interaction during floor plate develop'
ment, we examined the distribution and gene expression of wild,type and oep
mutant cells in genetic mosaics. When mutant shield cells were transplanted
into a wild·type host, an ectopic neural tube with a floor plate was induced.
However, the floor plate of the secondary axis was consistently devoid ofmu'
tant cells while its notochord was composed entirely of mutant cells. This
indicates that oep shield cells adopt only a notochord fate in a wild·typeenvironment. In reciprocal transplants (wild .> oep), however, grafted shieldcells frequently contributed to part of the floor'plate region of the secondaryneural tube and expressed floor-plate markers. Careful examination of serial
sections revealed that a mutant neural cell, when located next to the wild·typecells at the ventral midline, inhibited floor'plate differentiation of the adja'

66

cent wild-type cells. This inhibition was effective over an area only one or twocells wide along the anteroposterior axis. Since the cells located at the ventral
midline of the oep neural tube are thought to possess a neural character, simi'
lar to those located on either side of the floor plate in a wild-type embryo, thisinhibition may play an important role during normal development in restricting the floor-plate region into the ventral-most midline by antagonizing
homeogenetic signals from the floor-plate cells.
(3)

Roles of mesp gene family in zebrafish segmentation

Atsushi SAWADA, Andreas FRITZI, Yun·Jin JIANG~, Yumiko SAGA] and Hiroyuki
TAKEDA (I Department of Biology, Emory University, ~Vertebrate Development
Laboratory, Imperial Cancer Research Fund, :lCellular and Molecular Toxicol'
ogy, National Institute of Health Sciences)
Segmentation of a vertebrate embryo begins with the subdivision of theparaxial mesoderm into somites through a not'well'understood process. Recent
studies provided evidence that the Notch'Delta and the FGFR (fibroblast growth
factor receptor) signalling pathways are required for segmentation. In addi'tion, the Mesp family of bHLH transcription factors have been implicated inestablishing a segmental prepattern in the presomitic mesoderm (PSM). In
this study, we have characterized zebrafish mesp-a and mesp'b genes that areclosely related to Mesp family genes in other vertebrates. During gastrula'tion, only mesp'a is expressed in the paraxial mesoderm at the blastoderm
margin. During the segmentation period, both genes are segmentally expressedwith one to three stripes in the anterior parts of somite primordia. In fused
somites ([ss) embryos, in which all early somite boundary formation is blocked,
initial mesp'a expression at the gastrula stage remains intact, but the ex'pression of mesp'a and mesp-b is not detected during the segmentation
period. This suggests that these genes are downstream targets of fss at thesegmentation stage.
Ectopic expression ofMesp'b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These
injected embryos show a reduction in expression of the posterior genes, myoD

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DEPARTMENT OF DEVELOPMENTAL GENETICS

67

and noteh5, with uniform expression in the anterior genes, FGFRl, pape andnoteh6. These observations suggest that zebrafish mesp genes are involved inanteroposterior specification within the presumptive somites, by regulating
the essential signalling pathways mediated by Notch-Delta and FGFR.

Publ icatians
1.

2.

3.

4.

5.

6.

SHINYA, M., FURUTANI-SEIKI, M., KUROIWA, A. & TAKEDA, H.: Mosaic analysis with oep
mutant reveals a repressive interaction between floor-plate and non-floor plate mu'tant cells in the zebrafish neural tube. Dev. Growth & Differ. 41: 135-142 (1999).
MIZUNO, T., YAMAHA, E., KUROIWA, A. & TAKEDA, H.: Removal of vegetal yolk causes
dorsal deficiencies and impairs dorsal inducing ability of the yolk cell in zebrafish.
Mech. Dev. 81: 51-63 (1999).
RODAWAY, A., TAKEDA, H., KOSHlDA, S., BROADBENT, J., PRICE, B., SMITH, J. C., PATIENT, R.
& HOLDER, N.: Induction ofthe mesendoderm in the zebrafish germ ring by yolk celldrived TGF-b family signals and dicreimination of mesoderm and endoderm by FGF.
Development lZ6: 3067-3078 (1999).
NIKAlDO, M., TADA, M., TAKEDA, H., KUROIWA, A. & DENO, N.: In vivo analysis using
variants of zebrafish BMPR-IA: range of action and involvement of BMP in ectoderm
patterning. Development lZ6: 181-190 (1999).
AMANUMA, K., TAKEDA, H., AMANUMA, H. & AOKI, Y.: Transgenic zebrafish for thedetection of mutations caused by compounds in aquatic environments. Nature Biotechnology 18: 62-65 (2000).
MIZUMO, T., SHINYA, M. & TAKEDA, H.: Cell and tissue transplantation in zebrafish
embryos. In Molecular Methods in Developmental Biology - Xenopus and ZebrafJsh.
Ed. M. Guille, Humana Press Inc. p.15-28 (1999).

68

C-d. Division of Physiological Genetics(1)

Codon usage diversity of bacterial genes: An application of self-organization mapping for investigating origin of horizontal transfer genes

Shigehiko KANAYAand Toshimichi IKEMURA 1 (lDivision of Evolutionary Genet"
ics)
Multivariate analysis such as factor corresponding analysis and principal
component analysis (PCA) have been used to study heterogeneous codon usage
systematically in various species. To characterize the species"specific hetero"geneity of genes in codon usage, we developed previously a measure denoted byZl based on the widest range of axis obtained by PCA of codon frequency
patterns of genes analyzing a wide range of bacterial species. Extent of codon
bias for each gene was found to be associated with the level of protein produc"tion in the respective bacterial species.
In the present study, we attempted to comprehend species'specific codon
usage of genes in intra" and inter"species by self"organizing mapping. This
neural networks have been proposed to visualize high"dimensional systems.
Using the modern multivariate analysis, we analyzed codon usage heteroge"
neities for intra" and inter-bacterial species with special emphasis on distinct
codon usage of horizontal transfer genes involving bacteriophages, restriction
enzymes, and transposons, and searched for the original species from which
the transferred genes were derived.

Pub Ii ca t ions!{ANAYA S., FUKAGAWA T., ANDO A., INoKo H., KUDO Y. and IKEMURA T.: Distribution ofpolypurine/polypyrimidine tract sequences in the human MHC region and their pos'sible functions. In: Major Histocompatibility Complex, Springer"Verlag Berlin I Heidel"
berg I New York I Tokyo, 131-145, 2000.
2. !{ANAYA S., YAMADA Y., KUDO Y. and IKEMuRA T.: Studies of codon usage and tRNAgenesof 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expres"sion level and species"specific diversity of codon usage based on multivariate analy"
sis. Gene, Z88, 143'155, 1999.
1.

69

D.

DEPARTMENT OF POPULATION GENETICS
D-a. Division of Population Genetics(1)

Causes of the irregular synonymous substitution rates in
Drosophila lineages

Toshiyuki TAKANO·SHIMIZU
Significant locus·lineage interaction exists in the synonymous substitution
rates among Drosophila lineages (Takano 1998; Zeng et al. 1998). I have stud·
ied its causes by using four species of the D. melanogaster species subgroup.
Changes in effective population sizes have generally been considered age·nome"wide effect, but this is not always the case. Faster replacement" andsynonymous· substitution rates in D. melanogaster as compared with D.
simulanswere observed in many genes; no difference in synonymous substitu·
tion rates between the two species was observed in the four achaete·scutecomplex (AS"C) genes and ci gene, however. The AS·C and ci genes are locatedin regions ofseverely reduced recombination rates: AS-Con the tip of the X andci on the fourth chromosome. A lack of between·species difference in substitu·
tion rates at these loci is most likely due to the low efficacy of natural selec"tion, regardless of their effective population sizes. Thus, coupled with weak
selection against synonymous changes, fluctuation in effective population sizes
can contribute to the heterogeneous synonymous substitution rates among
genes to some degree. This study also provides evidence of remarkable local
changes in substitution patterns and in recombination rates on Drosophila
chromosomes. It is posited that these between"region variability and between"
lineage fluctuations of mutation pressures and recombination rates affected
evolutionary patterns of genes in the regions involved. In sum, the present
results suggest that changes in mutation, recombination, and effective POPU"
lation size all contribute to the significant locus"lineage interaction in thesynonymous substitution rates among the Drosophila lineages, but changes in70mutation pressures presumably have the greatest effects. I should add thatMs Yuriko Ishii has contributed significantly to this work. For details, seepublications 1.
(!)

Study of species difference as observed as interspecific hybrid anomaly
in Drosophila: genetic screens for genes involved in thebristle loss of hybrids

Toshiyuki T AKANO"SHIMIZU
Artificial selection for quantitative characters changes the phenotype far
beyond the range of variation in the original base population in mostcases. Environmental stresses (phenocopy) and mutant backgrounds also un"cover a surprising amount of hidden variation. Cryptic variation probably
exists because of stabilizing selection and genetic buffering ability, and cryp"tic variation and selection may provide an scenario for discontinuous evolu"
tionary changes in morphological characters. The clarification of the genetic
basis underlying the cryptic variation can shed a light on long"standing ques'tion of how morphology evolves while maintaining its homeostasis.
Interspecific"hybrid analysis is another powerful means to uncover hidden
within" and between"species variation in populations. One example is a bristle
loss phenotype of hybrids between Drosophila melanogasterand D. simulans,though both the pure species have exactly the same pattern of bristle forma"tion on the notum. There exists a large amount of genetic variability in thesimulans populations with respect to the number of missing bristles in hy"
brids, and the variation is largely attributable to simulansX chromosomes. I
have screened the simulans X chromosome for genetic factors affecting thebristle phenotype in melanogaster-simulans hybrids. Hybrid bristle'loss
anomaly is potentially caused by several distinct classes of genetic factors: (1)factors responsible for the within"simulansvariation in the number of miss'ing bristles, (2) fixed melanogaster-simulans differences involved in the hy'
brid bristle loss, and (3) factors that show dose'sensitive effects, especially ina hybrid background, without a between"species functional difference. Because
the pattern of notum bristles is fixed within each species and identical be'

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DEPARTMENT OF POPULATION GENETICS

71

tween species, these factors do not cause the anomaly in the original purespecies, but only the combination of the factors in hybrids does. Thus, we haveto make interspecific hybrids to screen for genetic factors responsible for thebristle loss phenotype. On the other hand, both male and female hybrids between the two species are completely sterile [but see Davis et al. (1996) for an
exception], allowing only F1 hybrid screening. I used two screening methods:(i) quantitative trait loci (QTL) mapping of X'chromosome factors affecting
the hybrid'bristle number by using a pair of simulans lines with high (H) andlow (L) missing bristle numbers and (ii) deficiency screening. The former is
one method for identifying class (1) factors; the second method is based on
phenotypic effects in melanogaster-deficiency'carrying hybrids. It has the po'tential to identify factors of the three classes.
The QTL mapping, together with duplication-rescue experiments, recovereda single major X'chromosome QTL (or block of QTL). Importantly, this region
accounted for most of the differences between Hand L lines in three otherindependent pairs. This suggests segregation ofH and L alleles (but not nee'essarily the same alleles) at the same locus in different populations. On theother hand, a deficiency screening uncovered several regions with factors that
potentially cause the hybrid bristle loss due to epistatic interactions with theother factors [class (2) or (3) factors].

Publications
1.
2.

TAKANO-SHIMlZU, T.: Local recombination and mutation effects on molecular evolution
in Drosophila. Genetics 153, 1285'1296, 1999.
YAMASHITA, S., TAKANO'SHIMIZU, T., KITAMURA, K., MIKAMI, T. and KISHIMA, Y.: Resistanceto gap repair of the transposon Tam3 in Antirrhinum majus: A role of the end regions.
Genetics 153, 1899-1908, 1999.

72

D-b. Division of Evolutionary Genetics(1) Fine replication-timing map of the entire human chromosome llq
at a genome sequence level: genome-wide approach to put landmarks
with biological significance along the human genome sequence bydefining transition zones for the replication-timing
during S phase and GC'/, level
Yoshihisa WATANABE, Asao FUJIYAMA 1,2, Masahira HATTORI 2, Yoshiyuki SAKAKI2
and Toshimichi lKEMURA (I Division of Human Genetics, 2Human Genome Research Group, RlKEN Genomic Sciences Center)
The human genome is composed oflarge-scale compartmentalized structures
including replication-timing zones and long-range G+C% (GC%) mosaic domains,that are thought to be related to chromosome bands. Using PCR-based quantification of newly-replicated DNA from cell-cycle fractionated human THP-1 cells,
replication timing during S phase for the entire human chromosome llq (ca. 80Mb) was determined at a genome sequence level, analyzing 212 sequence-taggedsites (STSs). This fine mapping could put approximately twenty five transition
zones of replication timing from the early to late S period, mainly in the regions
where ambiguous or inconsistent FISH mapping data have been reported. Global correlation of the replication timing with GC% level was also found, disclosing several characteristic exceptions. We proposed that genome-wide replication-timing map at a sequence level could provide fundamental knowledge ofthe human genome sequence inevitable for comprehensively understanding awide range of genome functions and evolutionary processes to have built up thepresent-day genome organization and therefore that the map construction is anindispensably important genome study that has now become possible.

(2)

Triplex-forming sticky DNAs In human Interphase nuclei; withspecial emphasis on trinucleotide repeat (GAA/TTC)nToshimichi lKEMURA, Mizuki OHNO l , Tatsuo FUKAGAWA (llnst. Genetic Information' Kyushu Univ,)

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DEPARTMENT OF POPULATION GENETICS

73

The (GAA/TTC)n repeat with the length equivalent to the expanded trio
nucleotide repeat in Friedreich's ataxia (FRDA) patients was reported to forma novel DNA structure (designated sticky DNA) through triplex formation. An
excellent correlation between the repeat length found in patients (n > 59) andthat required for formation of the novel DNA structure indicated that thestructure may be formed in living human nuclei and cause odd biological activo
ity resulting in FRDA. Many copies ofpolypurine/polypyrimidine (PuPy) tracts
such as (GAAlTTC)n are known to be present in the human genome and to
scatter among and within chromosomes. In PuPy"tract sequences, triplex for"mation occurs readily under physiological conditions, leaving single"stranded
DNAs available for hybridization with other unpaired DNAs and RNAs. Tri"plex"forming DNAs in the human interphase nucleus were analyzed by com"bining immunodetection by antitriplex antibodies and fluorescence in situ
"nondenaturing" hybridization employing PuPy"tract probes. The"nondenaturing" hybridization technique, which is used to detect RNA, candetect single"stranded DNAs in nondenatured nuclei. The probes such as (GAAI
TTC)n gave sequence"specific signals that overlapped with or were closely
associated with triplexes immunolocalized by known antitriplex antibodies.
Pretreatment of nuclei with antitriplex antibodies blocked the probe'signal
formation. The signal formation was resistant to pretreatment of nuclei with
RNases but sensitive to singe'strand"specific nucleases. These findings showed
triplex formation to occur in human interphase nuclei. For details, see Ref. 7.

(3)

Distribution of polypurlne/polypyrimldlne tract sequences In thehuman MHC region and their possible functions

Shigehiko KANAYA 1,2, Tatsuo FUKAGAWA, Asako AND0 3, Hidetoshi INOK0 3,
Yoshihiro KUD02 , Toshimichi IKEMURA(IDivision of Physiological Genetics, 2Department of Electrical and Informa"tion Engineering, Yamagata Univ., 3School of Medicine, Tokai Univ.>
To characterize distribution of polypurine/polypyrimidine (pur/pyr) tract se"quences in the human MHC region, we searched lOO'nt sequences with the
A+G% (AG%) higher than 85% for either DNA strands. In all human genome

74

sequences registered in DDBJ (ca. 32 Mb in totaD, we obtained 6247 A+Gtracts. There exists one tract per 51 kb and therefore roughly one tract per one
replicon size. 117 tracts (one tract per 33 kb) was found in the MHC region,that shows the level is significantly higher than levels of usual human genome
portions. One of the A+G tracts is found in DNA-replication switch region at
the junction ofMHC classes II and III. Other tracts were discussed in connection with polymorphism levels of several MHC genes. We also investigatedevidently long A+G tracts in the human genome. The longestA+G tract foundso far was the 2798-nt tract found in the 3' downstream ofrDNA. The biological significance of long A+G tracts was discussed in connection with triplex
formation, pausing of DNA replication and, enhancement of recombination.
For details, see Ref. 1.

(4)

Functional analyses of centromere proteins ofhigher vertebrate cells

Tatsuo FUKAGAWA, Atsushi OKAMURA, Toshimichi IKEMURA
The centromere is essential for the accurate segregation of eucaryotic chromosomes at cell division. CENP-C is an evolutionarily conserved centromeric
protein and has been predicted to play an important role in kinetochore assembly and function. We have used the chicken DT40 cell line to test the ideathat CENP-C is sufficient as well as necessary for the formation of a functional centromere. Firstly, we have used gene targeting in the DT40 cell line tocreate a cell line which expresses a fusion between CENP-C and a mousesteroid receptor and which behaves as a conditional loss offunction mutant of
CENP-C. We also have engineered the DT40 cell line that over-expressed CENPC using the tetracycline-repressible promoter system. This cell line over-expressed CENP-C in the absence of tetracycline, and expression was extinguished by the addition of tetracycline.
We have compared the effects of disrupting the localization of CENP-C withthose of inducibly over-expressing the protein. Removing CENP-C from thecentromere cause disassembly of the centromere protein complex and blocks
cells at the metaphase-anaphase junction. Over-expressed CENP-C is associ-D. DEPARTMENT OF POPULATION GENETICS

75

ated with an increase in errors of chromosome segregation and inhibits thecompletion of mitosis. However the excess CENP-C does not detectably dis"
rupt the native centromeres and does not associate with another conserved
centromere protein, ZW-10. The distribution ofthe excess CENP-C changes
during the cell cycle. In metaphase the excess CENP-C coats the chromosome
arms. At the metaphase anaphase transition the excess CENP-C clusters andduring interphase it is present in large bodies that form around pre"existing
centromeres which are also clustered. These results indicate that CENP-C isnecessary but not sufficient for the formation of a functional centromere andsuggest" that the structure of CENP-C may be regulated during the cell cycle.
For detail, Fukagawa et aI., EMBO J., 18, 4196-4209 (1999).
Recently, we attempt to make temperature"sensitive mutants for CENP-C
inDT40. When we put several point mutations into CENP-C, we can isolate
some temperature-sensitive mutants ofCENP-C. These cells grow well at 34'C,
but die at 43'C with chromosome missegragation. We also tried to isolate a
suppressor for these mutations using a retrovirus system. We are now characterizing suppressor genes.
In addition, we are trying to create knockout cell lines of several centromere
proteins, including ZWlO, CENP"H, Meteor and Mis6. From phenotype analy"
ses of these cell lines, we can understand how centromere works.

(5)

Development of mammalian artificial chromosomes

Tatsuo FUKAGAWA and Toshimichi IKEMURA
There are two alternative approaches to the construction of mammalianartificial chromosomes (MACs). In the first, "bottom-up", approach, homolo"
gous recombination in the yeast is used to assemble MACs from candidate
sequences that have been cloned in YACs. In the second, "top-down", approach,assembly of a chromosome from cloned constituents is avoided and instead, anatural mammalian chromosome is whittled down to a mini-chromosome bytelomere-directed chromosome breakage that is developed by Dr William Brown
in Oxford. Fukagawa has been developing a minichromosome vector in Brown
Lab. In this year, we created two counter selectable hprt-f- DT40 derived cell

76

lines. One of these cell lines contains a stably integrated gene encoding a
conditionally active cre recombinase and thus allows efficient manipulation oftargeted loci by site-specific recombination. These cell lines will cell lines will
enhance the utility of the hyper-recombinogenic DT40 cell line as a tool for
mammalian chromosome engineering. For detail, Fukagawa et al., Nucl. Acids
Res., 27, 1966-1969 (1999).
We are now modifying mini-chromosomes by double strand breaks inducedby the presence of a target site for a rare-cutting endonuclease, such as I -Sce- I.

(6)Molecular genetic analysis of remains of a Z,OOO-year-old
human population in China-and Its relevance for theorigin of the modern Japanese population

OOTA Hiroki!, SAITOU Naruya, UEDA Shintaroh 1 (IDepartment of Biological Sciences, Graduate School of Science, University of Tokyo)
We extracted DNA from the human remains excavated from the Yixi site
(approximately 2,000 years before the present) in the Shandong peninsula ofChina and, through PCR amplification, determined nucleotide sequences of
their mitochondrial D-loop regions. Nucleotide diversity ofthe ancient Yixi
people was similar to those of modern populations. Modern humans in Asiaand the circum-Pacific region are divided into six radiation groups, on thebasis of the phylogenetic network constructed by means of 414 mtDNA types
from 1,298 individuals. We compared the ancient Yixi people with the modern
Asian and the circum-Pacific populations, using two indices: frequency distribution of the radiation groups and genetic distances among populations. Bothrevealed that the closest genetic relatedness is between the ancient Yixi people
and the modern Taiwan Han Chinese. The Yixi people show closer genetic
affinity with Mongolians, mainland Japanese, and Koreans than with Ainu
and Ryukyu Japanese and less genetic resemblance with Jomon people andYayoi people, their predecessors and contemporaries, respectively, in ancientJapan. For details, see ref. 12.

D. DEPARTMENT OF POPULATION GENETICS

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77

Phylogenetic relationship of muscle tissues deduced fromsuperimposition of gene trees

OOTA Satoshi and SAITOU Naruya
Muscle tissues can be divided into six classes; smooth, fast skeletal, slow
skeletal and cardiac muscle tissues for vertebrates, and striated and smoothmuscle tissues for invertebrates. We reconstructed phylogenetic trees of six
protein genes that are expressed in muscle tissues and, using a newly devel'
oped program, inferred the phylogeny of muscle tissues by superimposition offive of those gene trees. The proteins used are troponin C, myosin essential
light chain, myosin regulatory light chain, myosin heavy chain, actin, and muscle
regulatory factor (MRF) families. Our results suggest that the emergence ofskeletal'cardiac muscle type tissues preceded the vertebrate/arthropod diver'gence (ca. 700 MYA), while vertebrate smooth muscle seemed to evolve inde'
pendent of other muscles. In addition, skeletal muscle is not monophyletic,
but cardiac and slow skeletal muscles make a cluster. Furthermore, arthropod
striated muscle, urochordate smooth muscle, and vertebrate muscles except
for smooth muscle share a common ancestor. On the other hand, arthropod
nonmuscle and vertebrate smooth muscle and nonmuscle share a common
ancestor. For details, see ref. 14.

(8)

Alphal,3-fucosyltransferase IX (Fuc-TIX) is very highly conserved
between human and mouse; molecular cloning,
characterization and tissue distribution ofhuman Fuc-TIX

KANEKO Mika, NISHIHARA Shoko 1, INOKO Hidetoshi2, SAITOU Naruya, NARIMATSU
HisashP(IDivision of Cell Biology, Institute of Life Science, Soka University,
2Department of Molecular Biology, Tokai University Medical Schoo))
The amino acid sequence of Fuc·TIX is very highly conserved between mouseand human. The number of non'synonymous nucleotide substitutions of the
Fuc·TIX gene between human and mouse was strikingly low, and almost equiva'
lent to that of the alpha'actin gene. This indicates that Fuc'TIX is under a78strong selective pressure of preservation during evolution. The human FucTIX (hFuc-TIX) showed a unique characteristics, i.e. hFuc-TIX was not activated by Mn2+ and Co2+, whereas hFuc-TIV and hFuc-TVI were activated by
the cations. The hFuc-TIX transcripts were abundantly expressed in brain
and stomach, and interestingly were detected in spleen and peripheral blood
leukocytes. For details, see ref. 15.

(9)Population Genetic Studies on Nine Aboriginal Ethnic Groups of
Taiwan. I. Red Cell Enzyme Systems

JIN Feng 1, SAITOU Naruya, ISHIDA Takafumi 2, SUN C.-S3, PAN I.-H4 and Satoshi

HORAl" (1 Laboratory for Human and Animal Genetic Studies, Institute of Genetics, Chinese Academy of Sciences, Beijing, China, 2Department of Biological Sciences, Graduate School of Science, University of Tokyo, 3Taidong Hospital, Taidong, Taiwan, 4Department of Bacteriology, College of Medicine, National Taiwan University, Taipei, Taiwan, "Department of Biosystems Science, Graduate University for Advanced Studies, Hayama)
Population genetic study of nine aboriginal ethnic groups of Taiwan (Ami,
Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiat, Tsou, and Yami) was carriedout. Twelve red cell enzymes (AcP, AK, CAl, CA2, EsD, GLO, GPT, GOT, LDH,
MDH, PGD and PGM1) were analyzed by isoelectric focusing method and starchgel electrophoretic method. Six loci (AcP, EsD, GLO, GPT, PGD, and PGM1)
were polymorphic. Three alleles (PGM1*6, GPT*6, and EsD*7) were in relatively higher allele frequencies in Taiwan aboriginal populations, and we foundhomozygotes for those alleles. Phylogenetic relationship based on genetic distances among those ethnic groups more or less fit to their geographical distribution, but not to linguistic classification. For details, see ref. 17.

(10)

Evolution of Rh Blood Group Genes Have Experienced Gene
Conversions and Positive Selection

KITANO Takashi and SAITOU Naruya

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DEPARTMENT OF POPULATION GENETICS

79

There are two tightly linked loci (D and CE) for the human Rh blood group.
Their gene products are membrane proteins having 12 transmembrane do'mains and form a complex with Rh50 glycoprotein on erythrocytes. We con'structed phylogenetic networks of human and nonhuman primate Rh genes,
and the network patterns suggested the occurrences of gene conversions. We
therefore used a modified site'by'site reconstruction method by using two
assumed gene trees and detected 9 or 11 converted regions. After eliminating
the effect of gene conversions, we estimated numbers of nonsynonymous andsynonymous substitutions for each branch of both trees. Whichever gene tree
we selected the branch connecting hominoids and Old World monkeys showedsignificantly higher nonsynonymous than synonymous substitutions, an indio
cation of positive selection. Many other branches also showed higher
nonsynonymous than synonymous substitutions; this suggests that the Rh
genes have experienced some kind of positive selection. For details, see ref.
19.

(11)

Phylogenetic Analysis of Pseudomonas syrlngae Pathovars
Suggests the Horizontal Gene Transfer of argK and the
Evolutionary Stability of hrp Gene Cluster

SAWADA Hiroyuki 1 and SAITOU Naruya (INational Institute of Agro'Environ'mental Sciences)
Pseudomonas syringae are differentiated into approximately 50 pathovars
with different plant pathogenicities and host specificities. To understand its
pathogenicity differentiation and the evolutionary mechanisms of pathogenic'
itY'related genes, phylogenetic analyses were conducted using 56 strains be'longing to 19 pathovars. gyrB and rpoD were adopted as the index genes todetermine the course of bacterial genome evolution, and hrpL and hrpS wereselected as the representatives of the pathogenicity'related genes located on
the genome (chromosome). Based on these data, NJ, MP, and ML phyloge'
netic trees were constructed and thus 3 trees for each gene and 12 gene trees intotal were obtained, all of which showed three distinct monophyletic groups:
Groups 1, 2 and 3. The observation that the same set ofOTUs constitute each80group in all four genes suggests that these genes had not experienced any
intergroup horizontal gene transfer within P. syringae but have been stable onand evolved along with the P. syringae genome. These four index genes werethen compared with another pathogenicity-related gene, argK (thephaseolotoxin-resistant ornithine carbamoyltransferase gene, which exists
within the argK·tox gene cluster). All 13 strains ofpv. phaseolicola and pv.
actinidiae used had been confirmed to produce phaseolotoxin and to have argK,
whose sequences were completely identical, without a single synonymous sub,stitution among the strains used. On the other hand, argK were not present on
the genomes of the other 43 strains used other than pv. actinidiae and pv.
phaseolicola. Thus, the productivity of phaseolotoxin and the possession of
the argK gene were shown at only two points on the phylogenetic tree: Group 1(pv. actinidiae) and Group 3 (pv. phaseolicola). At test between these twopathovars for the synonymous distances of argK and the tandemly combined
sequence of the four index genes showed a high significance, suggesting thatthe argK gene (or argK-tox gene cluster) experienced horizontal gene transfer
and expanded its distribution over two pathovars after the pathovars had
separated, thus showing a base substitution pattern extremely different fromthat of the noncluster region of the genome. For details, see ref. 20.

Publ icatians
1. KANAYA, S., FUKAGAWA, T., MDO, A, INoKo, H., KUDO, Y. and IKEMURA, T.: Distribution ofpolypurine/polypyrimidine tract sequences in the human MHC region and their possible functions. In: Major Histocompatibility Complex, Springer-Verlag BerlinlHeidelberg/New York/Tokyo, 131'145,2000.
2. KANAYA, S., YAMADA, Y., KUDO, Y. and IKEMURA, T.: Studies of codon usage and tRNA
genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene
expression level and species'specific diversity of codon usage based on multivariate
analysis. Gene, 238, 143-155, 1999.
3. NOGAMI, M., NOGAMI, 0., KAGOTANI, K., TAGUCHI, H., IKEMURA, T. and OKUMURA, K.:
Intranuclear arrangement of human chromosome 12 correlates to large'scale replication domains. Chromosoma, 108, 514-522, 2000.
4. SHIINA, T., TAMIYA, G., OKA,A, TAKISHIMA, N., YAMAGATA, T., KIKKAWA, E., IWATA, K., TOMlZAWA,
M., OKUAKI, N., KuwANo, Y., WATANABE, K., FuKUZUMI, Y., ITAKURA, S., SUGAWARA, C., ONO, A,
YAMAZAKI, M., TASHIRO, H., MOO, A, IKEMURA, T., SOEDA, E., KIURA, M., BAHRAM, S. andD.

DEPARTMENT OF POPULATION GENETICS

81

INOKO, H.: Molecular dynamics ofMHC genesis unraveled by sequence analysis ofthe 1.8
Mb HLA class I region. Proc Nat! Acid Sci USA, 96, 13282-13287, 1999.
5. FUKAGAWA, T., PENDON, C., MORRIS, J., BROWN, W.R.A.: CENP-C is necessary but not
sufficient to induce formation of a functional centromere. The EMBO Journal, 18,4196-4209, 1999.
6. SIMON, I., TENzEN, T. and CEDAR, H.: Asynchronous replication of imprinted genes isestablished in the gametes and maintained during development. Nature, 401, 929-932,
7.

8.

9.
10.

11.

12.

13.

14.
15.

16.

1999.
OHNO, M., TENzEN, T., WATANABE, Y., YAMAGATA, T., KANAYA, S. and IKEMURA, T.: Non-B
DNA structures spatially and sequence"specifically associated with individual centromeres in the human interphase nucleus. Chromosomes Today, 13, 57-69, 2000.
FUKAGAWA, T., HAYWARD, N., YANG, J., AzZALIN, C., GRIFFIN, D., STEWART, A.F., BROWN, B.:
The chicken HPRT gene: a counter selectable marker for the DT40 cell line. Nucleic
Acids Research, 27, 1966-1969, 1999.
NAKAMURA, Y., GOJOBORI, T. and IKEMURA, T.: Codon usage from the international DNA
sequence databases; its status 1999. Nucleic Acids Research, 27, 292, 1999.
ANDO, A., KIKUTI, Y.Y., ABE, K, SHIGENARI, A., KAWATA, H., IKEMURA, T., KIMURA, M. and
INOKO, H.: cDNA cloning, Northern hybridization and mapping analysis of a putative
GDS (guanine nucleotide dissociation stimulator of G proteins)-related protein gene at
the centromeric ends of the human and mouse MHC regions. Immunogenetics, 49, 354356, 1999.
WATANABE, Y., TENZEN T., NAGASAKA, Y. and IKEMURA, T.: Replication timing ofthe human Xinactivation center (XIC) region: correlation with chromosome bands. Gene, 252, 163-172,
2000.
OOTA, H., SAITOU, N., MATSUSHITA, T. and UEDA, S.: Molecular genetic analysis ofremains of a 2,OOO-year-old human population in China-and its relevance for theorigin of the modern Japanese population. American Journal of Human Genetics, 64,
250-258, 1999.
SATO, H., KOIDE, T., ISHIGURO, S.-I., TAMAI, M., SAITOU, N. and SHIROISHI, T.: The
organization of the type I keratin genes in the mouse Krt1 domain. Genomics, 56, 303309, 1999.
OOTA, S. and SAITOU, N.: Phylogenetic relationship of muscle tissues deduced fromsuperimposition of gene trees. Molecular Biology and Evolution, 16, 856-867, 1999.
KANEKO, M., KUDO, T., IWASAKI, H., IKEHARA, Y., NISHIHARA, S., NAKAGAWA, S., SASAKI, K,
SHIINA, T., INOKO, H., YOKOYAMA, Y., SAITOU, N. and NARIMATSU, H.: Molecular cloning,
characterization and chromosomal mapping of Human a 1,3-fucoslytransferase IX(hFuc-TIX); Fuc-TIX is very highly conserved between human and mouse. FEBS
Letter, 452, 237-242, 1999.
KOBAYASHI, K, IWASAKI, M., SUZUKI, Y., SUZUKI, H. and SAITOU, N.: An analysis ofpolymorphism for the ABO blood group genes in a Japanese population based onpolymerase chain reaction. Anthropological Science, 107, 109-121, 1999.

82
17. JIN, F., SAITOU, N, ISHlIJA, T., SUN, C.-S., PAN, 1.-H., OMOTO, K and HORAI, S.: Population genetic studies on nine aboriginal ethnic groups of Taiwan. 1. Red blood cell
enzyme systems. Anthropological Science, 108, 229-246, 1999.
18. KANEKo, M., KUDO, T., IWASAKI, H., SHIINA, T., INOKO, H., KOZAKI, T., SAITOU, N. andNARIMATSU, H.: Assignment of the human alpha 1,3-fucosyltransferase IX (FDT9) geneto chromosome band 6q16 by in situ hybridization. Cytogenetics and Cell Genetics, 86,
329-330, 1999.
19. KITANO, T. and SAITOU, N.: Evolution of the Rh blood group genes has experienced geneconversions and positive selection. Journal of Molecular Evolution, 49, 615-626, 1999.
20. SAWADA, H., SUZUKI, F., MATSUDA, 1. and SAITOU. N.: Phylogenetic analysis ofPseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster. Journal of Molecular Evolution, 49, 627-644,
1999.
21. KITANO, T., OOTA, S. and SAITOU, N.: Molecular evolutionary analyses of the Rh bloodgroup genes and Rh50 genes. Zoological Studies, 38, 379-386, 1999.
22. JIN, F., SAITOU, N. and OMOTO, K: Population genetic studies on the Chinese. In
Omoto K (edJ, "Interdisciplinary Perspectives on the Origin of Japanese", International Research Center for Japanese Studies, Kyoto, Japan, pp.15-24, 1999.
23. OOTA, H., SAITOU, N., KUROSAKI, K, POOKAJORN, S., ISHIDA, T, MATSUSHITA, T. and DEDA,
S.: Ancient DNA: a New Strategy for Studying Population History. In Omoto K (edJ,"Interdisciplinary Perspectives on the Origin of Japanese", International ResearchCenter for Japanese Studies, Kyoto, Japan, pp.25-41, 1999.
24. KITAKAMI, H., MORI, Y, OOTA, S. and SAITOU, N.: Satisfying the Zero-Crossover Constraints for Reconciliation across Heterogeneous Trees Gn Japanese). Database, 40, 114, 1999.
25. YUAN, Y-D., JIN F. and SAITOU, N.: The study of the distribution of Chinese surnames
and the diversity of genetic population structure in the Song Dynasty Gn Chinese).
Acta Genetica Sinica, 26, 187-197, 1999.

D-c. Division of Theoretical Genetics(1)

Effect of gene conversion on polymorphic patterns atmajor histocompatibility complex lociTomoko OHTA
The pattern of polymorphisms at MHC loci was studies by computer simu'
lations and by DNA sequence analyses, with special reference to synonymous

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DEPARTMENT OF POPULATION GENETICS

83

and non"synonymous divergence among alleles. A model of overdominanceplus short"term selection was simulated for a gene family with allelic and non"allelic gene conversion. It was found that the ratio ofnon"synonymous"to"syn"onymous divergence among alleles decreases as the non"allelic conversion be"comes more frequent. On the other hand, the ratio of synonymous divergenceat the antigen recognition site to that at non"coding region increases by non"allelic conversion. By comparing such a pattern with the observed values ofmammalian genes, it is suggested that non"allelic conversion is less frequentin human genes than in genes of other mammals. A quantitative analysis on
the patchwork pattern was attempted by using identity excess among nucle"
otide sites of a gene. Comparison of simulation results with DNA data analy'
sis shows that the rate of allelic conversion is high. For details, see Immuno"
logical Reviews 167, 319"325.

Publ ication
1.

Effect of gene conversion on polymorphic patterns at major histocompatibility complex
loci. Immunological Reviews 167, 319"325, 1999.

Oral presentations
Near neutrality and the molecular clock. Symposium, "Molecular Clock," at the Ameri"can Society for the Study of Evolution meeting, June, Madison, US.
2. Near neutrality in the interactive systems. Symposium, "Neutralism and Selectionism:
the end of a debate?" at the European Society of Evolutionary Biology meeting, August,Barcelona, Spain, and at the seminar of the Uppsala University, September, Uppsala,Sweden.
3. Usefulness of identity coefficient for analysing gene diversity. At the Malecot Sympo"
sium, August, Paris, France, and at the seminar ofthe Lund University, September,
Lund, Sweden.
1.

84

E.

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

Cloning and structurai anaiysis of themouse 7F4/F5 imprinted domain

Hiroyuki SASAKI, Reiko KATO l , Chikako SUDA, Hisao SHIROHZU, Wahyu
PURBOWASIT0 2, Takaaki YOKOMINE 2, Shin-ichi MIZUN0 2, Tsunehiro MUKAI 3,
Masahira HATTORI I and Yoshiyuki SAKAKI 1 (I Genome Science Center Riken,
2Inst. of Genetic Information, Kyushu Univ., 3Dept. of Biochemistry, Saga Medical College)
Genomic imprinting, an epigenetic gene marking phenomenon, causes parental-origin-specific mono allelic expression of a subset of mammaliangenes. Imprinted genes tend to form clusters in specific regions ofthe genome,
which may be related to the mechanism of imprinting or the reason for theevolution of imprinting. As a step to understand the structural and functional
characteristics ofthe imprinted genome domains, we are studying an imprinted
domain in mouse chromosome band 7F41F5. This region is syntenic to human
llp15.5, which contains genetic loci responsible for Beckwith-Wiedemann syndrome and childhood and adult tumors. To date, at least eight imprinted
genes have been mapped to this mouse region. Therefore, YAC, BAC, andcosmid contigs covering the 1-Mb region were constructed and a physical mapof the region was produced (Ref. 1). Large-scale sequencing of selected BAC
clones is now ongoing. The obtained sequence information will be used to
identify new imprinted genes and their regulatory elements.

(Z)

Regulation of imprinting of the mouse [IIZ/HII sub-domain

Hiroyuki SASAKI, Ko ISHIHARA, Reiko KAT0 1, Hiroyasu FuRUUMI, Mizuki OHN0 2,
Naoya HATAN0 2 and Tohru IWAKI 3 (I Genome Science Center Riken, 2In8t. of

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DEPARTMENT OF INTEGRATED GENETICS

85

Genetic Information, Kyushu Univ., 3Dept. of Neuropathology, NeurologicalInst., Faculty of Medicine, Kyushu Univ,}
At the centromeric end of the above imprinted domain, there are two recipro'
cally imprinted genes, Igf2and H19: Igf2is paternally expressed and H19ma'
ternally expressed. It was previously shown that the allele'specific expressionof these genes occurs through a competition for a set of endoderm'specific en'hancers located downstream of H19. To look for enhancers responsible for the
imprinted expression in other tissues, we sequenced both the mouse and humanH19regions over 40 kb. A comparison of the sequences from the two species
revealed a total of ten evolutionarily conserved non'coding segments, two of
which precisely coincided with the known endoderm enhancers. We then tested
the function of the remaining eight segments in transgenic mice and found thatat least five of them possessed tissue'specific enhancer activity. We also identified a conserved 39'bp element that appeared repeatedly within the upstream
differentially methylated region (DMR) and formed complexes with specificnuclear factors. Binding of one of the factors was inhibited when the target
sequence contained methylated CpGs. These complexes may contribute to thepresumed boundary function of the unmethylated DMR, which is proposed toinsulate maternal Igf2from the enhancers.

(3)

Cloning of a family of novel mammalian methyltransferases

Hiroyuki SASAKI, Takahito CHIJIWA, Naomi TSUJIMOTO, Shin'ichi MIZUNO 1 andShoji TAJIMA2 (IInst. of Genetic Information, Kyushu Univ. 2Inst. of Protein
Biochemistry, Osaka Univ,)
In the imprinting process, DNA methylation works as an important mark,ing mechanism to distinguish the parental alleles of imprinted genes. We
performed a TBLASTN search of the dbEST database using the catalytic do'main of the known mammalian DNA(cytosine'5) methyltransferase Dnmti,
and identified cDNAs that potentially codes for novel types ofmethyltransferase. We cloned full'length cDNAs for the mouse proteins andconfirmed their methyltransferase activity using an in vitro assay. It will be86interesting to investigate the roles of these novel methyltransferases in ge"
nomic imprinting.

(4)

Is the chicken genome imprinted?

Hiroyuki SASAKI, Takaaki YOKOMINE 1, Yoichi MATSUDA2 and Masaoki TSUDUIU 3(1Inst. of Genetic Information, Kyushu Univ., 2Chromosome Research Unit,
Hokkaido Univ., 3Faculty ofAnimal Production, Hiroshima Univ.)
Although it is generally thought that genomic imprinting has evolved only inmammals, this has not been tested in other vertebrate species. We have asked
whether the homologs of the mammalian imprinted genes are imprinted inchicken. Using DNA plymorphisms identified between various breeds andstrains, it was shown that both IGF2 and MPR are expressed biallelically inchicken embryos. We are currently looking at other genes.

(5) Are the polycomb group genes involved in genomic imprinting?
Hiroyuki SASAKI, Naomi TSUJIMOTO and Haruhiko KOSEKI 1 (IChiba Univ. Gradu"
ate School)
Polycomb group proteins (PcGs) are the components of chromatin and in"volved in gene silencing and insulator functions in Drosophila. Previous ex"
periments using transgenic flies suggested that mammalian PcGs may be
involved in the allele"specific repression of imprinted genes. We have exam"
ined the allele"specific expression of Igf2and H19in mice disrupted with two
PcG genes Mel-18 and Bmi"l. In both single homozygotes ([Mel-18-t] and[Bmi-l-t)) and double homozygotes ([Mel-18-j -, Bmi-l-t)), the proper im"
printed expression patterns ofboth Igf2and H19were maintained, suggestingthat imprinting was not influenced by these PcG genes.

E. DEPARTMENT OF INTEGRATED GENETICS

(6)

87

X-Inactivation In the mouse embryo deficient for
DNA methyltransferase t (Dnmtt)

Takashi SADO, Martin H. FENNER 1, Seon-Seng TAN 2, Patric P. L. TAM 3, Toshi
SHIODA 1, En LI4 (ICancer Center., MGH, USA, 2Melbourne Univ., Aus., 3CMRI,
Aus., 4CVRC, MGH, USA)It has been suggested that DNA methylation plays a crucial role in genomic
imprinting and X-inactivation. Using DNA methyltransferase 1 (Dnmtl) deficient mouse embryos carrying X-linked lacZtransgenes, we studied effects ofgenomic demethylation on X-inactivation. Based on the expression pattern oflacZ, the imprinted X-inactivation in the visceral endoderm, a derivative of theextraembryonic lineage, was found unaffected in Dnmtl mutant embryos at
the time other imprinted genes showed aberrant expression. Random X-inactivation in the embryonic lineage of Dnmtl mutant embryos, however, wasunstable as a result of hypomethylation, causing reactivation of, at least, one
lacZtransgene that had initially been repressed. Our results suggest thatmaintenance of imprinted X-inactivation in the extraembryonic lineages cantolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X-inactivation in the embryonic lineage.

(7)

Analysis of X-Inactivation center (Xlc) In mouse

Takashi SADO, Asuka MIURA, Hiroyuki SASAKI, En Lr 1 (ICVRC, MGH, USA)It has been shown that the region called X-inactivation center (Xic) on an X
chromosome is essential for X-inactivation to occur in cis. Available evidence
suggests that the Xist gene, which is mapped to Xic and exclusively expressedfrom the inactive X chromosome, is one of components ofXic. In our attempt toisolate a gene(s) involved in X-inactivation other than Xistin Xic region, we foundantisense transcripts to Xist, which cover whole the transcription unit ofXist andapparently do not encode for a protein. To elucidate the function of this novel RNA
in X-inactivation, we disrupted the antisense transcripts in ES cells by targetedmutagenesis. Production ofthe knockout mouse is currently in progress.

88

(8)

Buman Genome Resources and Their Application to the Completionof Buman Chromosome !1 Sequencing

Asao FUJIYAMA, Akiko ENDO and Ayuko MOTOYAMA 1 (lGenome Science Center
Riken)The goal of human genome analysis is not only sequencing entire genome nor
cataloging protein coding regions, but to understand functions retained in thehuman genome and chromosomes. Since most of human chromosomes can be
purified by means of dual-laser cell sorting system, such isolated chromo·
somes are good resources for the studies to understand biological functions
retained in individual chromosome. Using purified chromosomes, we haveconstructed human mono-chromosomal cosmid libraries (except for CM#9 12) and #21 fosmid library, and BAC library. Unlike other libraries, our cosmidand fosmid libraries employed random fragmentation/cloning protocol thatenabled us to efficiently construct highly randomized libraries. Using theseresources, sequencing of human chromosome 21 long arm has beenaccomplished. A total of 33,546,361 bp of DNA, distributed over four contigs,
were sequenced with very high accuracy. The largest of these contigs is
28,515,322 bp long. Only three small clone gaps remain, which together com·prise approximately 100 kb. Thus, we achieved a coverage of 99.7% of21q. In
addition, 281,116 bp from the short arm were also sequenced. The chromo·
some 21·specific libraries were particularly useful for mapping the centro·
meric and telomeric regions which contain repeats, as well as sequences show·
ing homology with other human chromosomes.

(9)

Whole Genome Analysis of Signal-Transduction Pathways In
Fission Yeast

Yong·Sik BONG, Inaho DANJ0 1, Masahiro INOUYE and Asao FUJIYAMA (IRadiation Effect Research Foundation, Hiroshima, Japan)

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DEPARTMENT OF INTEGRATED GENETICS

89

In fission yeast, Schizosaccharomyces pombe, deficiency of rasl gene causes
abnormal cell shape and abolishes mating ability. However, the signaling
pathway in the cell and its target genes are largely unknown because ofthelack of appropriate analysis system. To overcome this problem, we catego'
rized genes based on their expression levels in the presence or absence of therasl gene product under different growth conditions. We utilized arrays ofclones covering entire genome of the fission yeast. Here, we demonstrate thedetection oflow molecular weight heat shock protein gene, hsp16, and showthat it is regulated by a ras 'mediated signaling pathway, not by the heatshock response in fission yeast.

Pub II ca t ions
1. KATO, R, SHIROHZU, H., YOKOMINE, T., MIZUNO, S., MUKAI, T. and SASAKI, H.: Sequence'
ready l'Mb YAC, BAC and cosmid contigs covering the distal imprinted region ofmouse chromosome 7. DNA Res. 6, 401'405,1999.
2. DANJOH, 1. and FUJIYAMA, A.: Ras'mediated signaling pathway regulates the expres'sion of a low molecular weight heat shock protein in fission yeast. Gene, 236, 347'532,
1999.

E-b. Division of Brain Function(1)

Mechanisms of Collateral Branching and Target Invasion of
Mitral Cell Axons during Development

Tatsumi HIRATA
During development, mitral cell axons, the major efferents of the olfactorybulb, exhibit a protracted "waiting period" in the lateral olfactory tract (LOT)
before giving off collateral branches and innervating the target olfactory cortex. To investigate the target invasion mechanism, a series of heterochronicand heterotopic co'cultures of olfactory bulbs with various olfactory cortical
strips were conducted. These experiments indicated that development of collateral branches is triggered by environmental cues but not by intrinsic mecha'
nisms in mitral cells. The collateral'inducing cues are apparently different

90from the cues directing outgrowth of primary mitral cell axons. CO'culture
experiments also indicated that the target olfactory cortex undergoes a devel'
opmental change to become accessible to mitral cell fibers. Primary mitralcell axons, however, still preferred the LOT position over such accessible piri'
form cortex when encountered both the locations. These results suggest thatmitral cell projection comprises multiple steps which are controlled by various
environmental cues.

Publ icaticn
1.

HIRATA, T. and FUJISAWA, H.: Environmental control of collateral branching and target
invasion of mitral cell axons during development. J. Neurobiol. 38, 93-104, 1999.

E-c. Division of Appl ied Genetics(1)

Characterization of /3 -amylase and its deficiency invarious rice cultlvars

Junji YAMAGUCHI!, Shin'Ichi ITOH 1 , Toshikazu SAITOH 1, Akira IKEDA 1, ToruTASHIRO 1 and Yasuo NAGAT0 2 (IBioScience Center, Nagoya University, 2 Gradu'
ate School ofAgricultural and Life Sciences, University of Tokyo){3 'amylase deficiency in various cultivars of rice was examined. Although{3'amylase is a starch'hydrolyzing enzyme, the {3 'amylase gene is expressed
differently from a 'amylase gene, i.e. (1) {3 'amylase is synthesized only inaleurone cells, (2) the enzyme production in the embryo'less half'seeds is notunder hormonal control. We identified several cultivars that are deficient for{3 'amylase activity. We present new evidence that the synthesis is blocked at
the level of mRNA synthesis. For details, see Ref. 1.

E. DEPARTMENT OF INTEGRATED GENETICS

(2)

91

Differential in situ expression of three ABA-responsive
genes of rice, Rab16A, REG2 and OSBZ8,
during seed development

Kazumaru MIYOSHI 1, Eijirou NAKATA l, Yasuo NAGATO l and Tsukaho HATTORI 2(lGraduate School ofAgricultural and Life Sciences, University of Tokyo, 2Cen'
ter for Molecular Biology and Genetics Mie University)
The spatial and temporal expression pattern of three rice abscisic acid (ABA).
responsive genes, Rab16A, REG2 and OSBZ8, which are expressed during
seed maturation, were studied by in situ hybridization. Tissue'specificityofthese three genes in embryo was much different from each other. The onset of
Rab16A and REG2 expression was observed after organogenesis had started.
By contrast, the expression ofOSBZ8 was already observed in the whole embryo at the globular stage when organogenesis had not been initiated. Whilethe expression of OSBZ8 at the earlier stages was uniform throughout theembryo, a clear tissue specificity was established at the later stage. Interestingly,
an exactly opposite relationship in the spatial pattern of expression was ob'
served between OSBZ8 and REG2. For details, see Ref. 2.

(3)

Initiation of shoot apical meristem in rice: characterization offour SHOOTLESS genes

Namiko SATOHl, Soon'Kwang HONG2, Asuka NISHlMURA2, Makoto MATSUOKA2,
Hidemi KITAN03and Yasuo NAGAT0 1 (IGraduate School ofAgricultural and Life
Sciences, University of Tokyo, 2BioScience Center, Nagoya University, 3Fac'
ulty ofAgriculture, Nagoya University)
We characterized nine recessive mutations derived from four independent
loci (SHLI-SHL4) causing the deletion of the SAM. Concomitant with theloss of SAM, two embryo'specific organs, coleoptile and epiblast, were lost,
but the scutellum was formed normally. Therefore, the differentiation ofradicle
and scutellum is regulated independently of SAM, but that of coleoptile andepiblast may depend on SAM. An in situ hybridization experiment using a ricehomeobox gene, OSHl, as a probe revealed that shU and shl2 modified the92
expression domain of OSHI, but normal expression of OSHI was observed insh13 and sh14 embryos. Accordingly, SHLI and SHL2 function in the upstreamof OSHl, and SHL3 and SHL4 in the downstream or independently of
OSHl. These shl mutants are useful for elucidating the genetic program driving SAM initiation. For details, see Ref. 3.

(4) Population Genetic Studies on Nine Aboriginal Ethnic Groups of
Taiwan. I. Red Cell Enzyme Systems
Feng JIN l, Naruya SAITOU2, Takafumi ISHIDA3, Cheih-Shan SUN4, I-Hung PAN5,
Keiichi OMOT06 and Satoshi HoraF (IChinese Academy of Sciences, Beijing,
China, 2National Institute of Genetics, Mishima, 3University of Tokyo, Tokyo,
4Taidong Hospital,Taiwan, 5National Taiwan University, Taiwan, 6International
Research Center for Japanese Studies, Kyoto, 7The Graduate University for
Advanced Studies, Hayama)
Population genetic study of nine aboriginal ethnic groups of Taiwan (Ami,
Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiat, Tsou, and Yami) was carriedout. Twelve red cell enzymes (AcP, AK, CAl, CA2, EsD, GLO, GPT, GOT, LDH,
MDH, PGD, and PGMl) were analyzed by isoelectric focusing method andstarch gel electrophoretic method. Six loci (AcP, EsD, GLO, GPT, PGD, andPGMl) were polymorphic. Three alleles (PGMl*6, GPT*6, and EsD*7) werein relatively higher allele frequencies in Taiwan aboriginal populations, andwe found homozygotes for those alleles. Phylogenetic relationship based on
genetic distances among those ethnic groups more or less fit to their geo·
graphical distribution, but not to linguistic classification. For the details, see
Ref. 7.

(5)

The presence of ancient human T-cell lymphotropic virus type I
provirus DNA in an Andean mummy

Hong-Chuan LII, Toshinobu FUJIYOSHI 1, Hong LouI, Shinji YASHIKI l, Shunro
SONODAl, Luis CARTIER2, Lautaro NUNEZ 3, Ivan MUNOZ4, Satoshi HORAI5 andKazuo TAJIMA6 (IKagoshima University, Kagoshima, 2Chile University,

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DEPARTMENT OF INTEGRATED GENETICS

93

Santiago, Chile,3North Catholic University, San Pedro de Atacama, Chile,
4Tarapaca University, Arica, Chile, 5The Graduate University for Advanced
Studies, Hayama, 6Aichi Cancer Center Research Institute, Nagoya)
The worldwide geographic and ethnic clustering of patients with diseases
related to human T-celllymphotropic virus type I (HTLV- 1) may be explainedby the natural history of HTLV-I infection. The genetic characteristics of indigenous people in the Andes are similar to those of the Japanese, and HTLVI is generally detected in both group. To clarify the common origin ofHTLV-I in
Asia and the Andes, we analyzed HTLV-I provirus DNA from Andean mummies about 1,500 years old. Two of 104 mummy bone marrow specimens yieldeda band of human {3 -globin gene DNA 110 base pairs in length, and one of thesetwo produced bands of HTLV-I-PX (open reading frame encoding p401 , p271) and
HTLV-I-LTR (long terminal repeat) gene DNA 159 base pairs and 157 basepairs in length, respectively. The nucleotide sequences of ancient HTLV-I·PX
and HTLV-I-LTR clones isolated from mummy bone marrow were similar to
those in contemporary Andeans and Japanese, although there was micro-heterogeneity in the sequences of some mummy DNA clones. This result provides
evidence that HTLV-I was carried with ancient Mongoloids to the Andes before
the Colonial era. Analysis of ancient HTLV'I sequences could be a useful tool
for studying the history of human retroviral infection as well as human prehistoric migration. For the details, see Ref. 8.

Publications
1. YAMAGUCHI, J., ITOH, S."I., SAITO, T.-K, IKEDA, A., TASHIRO, T. and NAGATa, Y. (1999)
Characterization of 13 -amylase and its deficiency in various rice cultivars. Theor.
Appl. Genet. 98: 32-38.
2. MIYOSHI, K, NAKATA, E., NAGATa, Y. and HATTORI, T. (1999) Differential in situ expres"sion of three ABA-responsive genes of rice, Rabl6A, REG2 and OSBZ8, during seeddevelopment. Plant Cell Physiol. 40: 443-447.
3. SATOH, N., HONG, S.·K, NISHIMURA, A., MATSUOKA, M., KITANO, H. and NAGATO, Y. (1999)
Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes.
Development 126: 3629'3636.
4. KINAE, T., MATSUOKA, M. and NAGATO,Y. (1999) apical displacement 1 affects the shoot94
position and the number of radicles in rice embryo. Rice Genet Newslett. 16: 27-30.
ITOH, J.-I., KITANO, H. and NAGATO, Y. (1999) Leaf development in shoot organization2
mutant of rice. Rice Genet Newslett. 16: 32-34.
6. SUNOHARA, H., Dozu, S., KITANO, H. and NAGATO, Y. (1999) Interaction between panicle
development and internode elongation in rice. Rice Genet Newslett. 16: 30-32.
7. JIN, F., SAITOU, N., ISHIDA, T., SUN, C-S., PAN, I-H., OMOTO, K and HORAI, S.: Population
genetic studies on nine aboriginal ethnic groups of Taiwan. I. Red cell enzyme systems.
Anthropological Science 107(3): 229'245, 1999.
8. LI, H·C., FUJIYOSHI, T., Lou, H., YASHIKI, S., SONODA, S., CARTIER, L., NUNEZ, L., MUNOZ,
I., HORAI, S. and TAJIMA, K: The presence of ancient human T-celllymphotrophic virus
type I provirus DNA in an Andean mummy. Nature Medicine 5(12): 1428'1432, 1999.
5.

95

F.

GENETIC STRAINS RESEARCH CENTER
F-a. Mammal ian Genetics Laboratory(1)

Phenotype characterization of male sterHlty of a consomic strain
86.MSM-ChrX and fine mapping of the causative gene(s)in the X chromosome

Ayako OKA, Nobuo TAKAGI!, Akihiko MITA, Yoichi MIZUSINA, Noriko SAKURAI
and Toshihiko SHIROISHI (IRes. Cent. for Mol. Genet. And Grad. Schl. OfEnv.
Earth Sci. Hokkaido Univ.)
Hybrid sterility is the mechanism that safeguards the integrity of species
by preventing a free flow of genetic information between related species. The
infertility of inter-specific hybrids usually affects the heterogametic sex, whichin the case of mammals is the male. In the mouse, male infertility has beenfound in hybrids between Mus spretus and laboratory mouse strains (Musmusculus). Understanding hybrid sterility might give us an insight into not
only how a new species evolves, which is one of the fundamental questions
about the evolution, but also how terminal differentiation of germline cells is
operated.
Since several years ago, we have set out to construct a series of consomic
strains that substitute each chromosomes of Japanese wild mouse'derived
MSM strain (Mus musuculus molossinusJ for the chromosomes of the stan"
dard laboratory strain C57BLl6J (Mus musculus domesticus). The consomic
strains would be of great use to study all kind of multigenic traits. During theprocess ofproduction ofX'chromosome consomic strain, in which the X chromo'
some ofC57BU6J (B6) was replaced by the MSM'derived one, we noticed thatmales of the strain have lowered fertility. It seemed attributable to the dis'turbance of coordination between the X chromosome of MSM (X,MSM) andthe autosome background gene(s) from B6 strain, which has separately evolved
over a long period, approximately one million years.

96

Previously the severe reproductive disadvantage and morphological anomaly
in spermatozoa ofB6 strain that carries the X chromosome ofMSM strain has
been reported (Takagi et aI., 1994). The decline in the fertility was emphasized as backcross generations proceeded. Unlike X'MsMYmales, X-BSY males
in the same backcross generation were fully fertile. Thus, the X chromosome
from MSM is responsible for the reduced fertility ofthe consomic males. We
inferred that certain autosomal or Y chromosomal gene(s) of B6 could notcoordinate with a gene(s) on X chromosome ofMSM strain.
In this study, we first examined whether the X'chromosome consomic strainshows male sterility that was previously reported. We observed that mostepididymal spermatozoa from B6 and X-BSY males had a well-balanced, hooklike head. The distal part of the hook tended to be slightly short and lesscurved in X- MSMY males of N2 (BC1) generation. Malformation of the distal
part ofthe sperm head was accelerated in X-MSMY males ofN3 (BC2) and N4(BC3). At the N3 or N4 generation, certain sprmatozoa from X-MSMY males
did not have the hook-like heads, instead almost the entire distal part werelost, and in extreme cases they had small and round heads. In order to evalu'
ate the fertility of the consomic males, in vitro fertilization (IVF) and IVF
after partial zona pellucida dissection (PZD) were performed with capacitatedsperms ofX-MSMY and X-BSY males, and the number of progeny was counted innatural mating with B6 females for 4-8 months. Significant low fertility of
IVF was observed in X- MSM Y males (5.9%; N=6), though the X'BSY males
showed high frequency (79 %; N=6). In the IVF-PZD, fertility of the X-MSMY
males was recovered slightly, but it remained to be still lowered (32.9 %;N=4). In addition, significantly small testis were observed in the X'MSMY males,
which were histologically normal (76.4 ± 13.5mg; p<O.05). In the natural mating, the number of live birth and offspring ofthe X-MSMY males were smaller
than those of the X- B6Y males. The defect of the sperms of the consomic males
seemed to be much severer in the IVF experiment than that in the naturalmating. In the TYH medium for IVF, most of the capacitated spermatozoa of
the X- MSMY males was no longer active after 2 hours, whereas sermatozoa fromthe X· B6Y males kept on swimming for several hours. An environment of thefemale reproductive tract is different from that of in vitro, which is possibly
much milder.

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We intended to map gene(s) responsible for this morphological anomalies.
QTL<quantitative trait loci) analysis with 120 recombinants on the X Chro'mosome, whose autosomes were mostly replaced by ones from B6, demonstrated a tight linkage of X chromosomal loci and the anomaly in spermatozoa. The most intense linkage with DXMit166 and the weaker linkage with aninterval between DXMit97 and DXMit217were shown. It is of interest to notethat the locus that controls the testis weight was linked with an interval
between DXMit217-DXMitl60, which was possibly separated from the locithat control sperm morphology.

(Z)

Phenotype characterization and fine mapping of apreaxial polydactylous mouse mutantion,
X-linked polydactyly (XpJ)

Yukari YADA 1, Hiroshi MASUYA 2, Shigeru MAKINO and Toshihiko SHIROISHI(IOchanomizu Univ., 2Mouse Funct. Genomics. Res. Cent., RIKEN GSC)
The anteroposterior axis patterning on vertebral limb morphogenesis iscontrolled by the formation of the zone of polarizing activity (ZPA), which islocated at the posterior margin oflimb buds. It is established that the Sonic
hedgehog (Sh~ gene that is expressed at the posterior margin oflimb bud andencodes a secreted signaling molecule mediates the ZPA activity. However, it ispoorly understood how the polarized expression of Shh in developing limb
buds is regulated.
X'linked polydactyly (XpJ) is a spontaneous mouse mutation that exhibits
preaxial polydactyly only on the hindlimb and was mapped to X chromosome.
in situ hybridization with the Xpl embryos revealed ectopic expression of Shh,Fgf4, Ore and Hoxdll genes at the anterior margin of the hindlimb bud. Suchectopic ZPA activity may results in the preaxial polydactyly of Xplmutants.
To isolate the Xpl gene, we have carried out a linkage analysis using 1,262
backcross progeny generated from cross between B6-Xpl/+ and Japanese wild
mouse-derived MSM strain. As a result, XpJ gene was mapped to a 0.48cM region
between microsatellite markers, DXMgc39 and DXMit5. In the human syntenic
region, chromosome Xp22, a congenital face and limb deformity, Oral-facial-digi'

98

tal syndrome type1 (OFD1: OMIM311200), has been mapped. Therefore, Xplis
likely to be a mouse model for OFDI. Now we are constracting YAC and BAC
contigs covering the causative gene for Xpl(3)

Developmental genetics of a mouse mutant,mesenchymal dysplasia (mes)

Shigeru MAKINO, Yukari YADA, Hiroshi MASUYA 1, Junko ISHIJlMA, Masaru
TAMURA, Toshihiko SHIROISHI (IMouse Funct. Genomics Res. Group, RIKEN
GSC)
mesenchymal dysplasia (mes) is a recessive mouse mutant that exhibits
abnormality of mesenchyme-derived tissues, including preaxial polydactyly,
shortened face, bifurcate sternum and shortened kinky tail. To determine thechromosomal location of mes, we carried out a linkage analysis. As a result,mes was mapped to a region between D13Mit318 and D13Mit187, where
patched (pte) has been mapped. To examine a possibility that ptc is a candi'date for mes, we investigated the nucleotide sequence of ptc in the mes
mutation. In this study, we identified a 32 bp-deletion in the C-terminal cyto'
plasmic domain of pte. Ptc is a transmembrane receptor protein for Shh thatis expressed in the organizing centers in developing mouse embryo, such as
notochord, floor plate, ZPA of limb buds and lung, and it mediates a key signal'
ing for cell growth and differentiation in developing embryos. Ptc functionallyantagonizes the Shh activity. Subsequently, we performed allelism test ofmes, using a ptc knockout mutant (ptc-) (Goodrich et aI., 1996). As a consequence, the compound heterozygotes (pte-I+,mes- I+) exhibited severe polydac'tyly, though neither pte-I+ nor mes-I+ mice exhibited such visible phenotype. This
result showed that mesis hypomorphic allele for pte, and that the cytoplasmic
domain plays an indispensable role in antagonistic activity of Ptc for Shhsignaling.
pte-1m.. mice died soon after birth because ofinability ofbreathing. Histologicalanalysis demonstrated that lung mesenchyme overgrew after about 17.5
dpc. With the observation of increased body weight in ptc-Im.., the results sug'gested that Ptc has a function of negative regulation in mesenchymal cell growth.

F. GENETIC STRAINS RESEARCH CENTER

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In addition, whole mount in situ hybridization at 11.5 dpc showed that shh andfgf4 were ectopically expressed at the anterior margin of the limb buds. This
result suggested that ectopic expression of shh was induced by subtle activationof Shh signaling evoked by weakened Ptc activity.

(4)

Positional cloning of the mouse skeletal mutation,
Tail-short (Ts)Kunihiko SHIMIZU l , Tsuyoshi KOIDE, Akihiko MITA, Kikue UCHIDA, Junko
ISHIJIMA, Shigeharu WAKANA2, TsutomuHIRASAWA3,YoshiakiKIKKAwA4, Hiromich
YONEKAWA4, Hiroki SASAKI5 and Toshihiko SHIROISHI (IDept. of Pediatric Dent.,
Nihon Univ. Sch. of Dent. at Matudo, 2Central Institute for Experimental Animals, 3Shionogi lnst. For Med. Sci., 4Dept. oflab. Exp. Animal Sci., The Tokyo
Metropol. lnst. for Med. Sci., 5Genet. Div., Natl Cancer Res. lnst.)
A mouse mutation Tail·short (Ts) exhibits shortened kinky tail and numerous skeletal abnormalities including homeotic anteroposterior patterning problem along the axial skeleton. Ts gene was mapped to the teromeric region of
the chromosome 11. To elucidate the function of the 18 gene in mouse embryogenesis, we intended to clone the gene by means of positional cloning. First,
we employed a fine mapping of this gene based on a large scale inter-subspecific backcross between the mutant stock TsJlLe- Ts/+ and Japanese wild mousederived MSM strains. Ts gene was mapped to a O.16cM region between two
microsatellite markers, DllMit128 and DllMit256. We screened mouse YAC
and BAC libraries with microsatellite markers tightly linked to the Ts locus
and have obtained YAC and BAC clones. Further chromosome walking with
the isolated clones allowed us to construct a complete BAC contig covering the
18 causative gene, and the critical region of the Ts was narrowed down to aninterval between two new STSs, DllRin56 and DllNigl7. This contig consists of 3 BAC clones, spanning a 250kb DNA fragment.
We have isolated several cDNA clones as Ts candidate genes from the critical region by directed cDNA selection using the corresponding BAC and mouseembryonic cDNA library. Among these candidate genes, expression level of
RpJ38gene, which encodes a subunit ofthe large component of ribosomal pro-100

tein complex, was reduced in 18 mutant mouse. Analysis ofthe genomic struc"ture of Rp138 in Tsmutant revealed that it has an 18kb long deletion contain"
ing Rp138. Other two mutations, Rabo torcido (Rbt) and Tail"short shionogi(Tss), were reported to have phenotype similar to Ts and to be mapped to thesame region in the chromosome 11. We found that Rbt and Tss have a frame
shift mutation and an insertional mutation in Rp138, respectively. In order toconfirm that Rp138 is the causative gene for Ts, we tried to rescue the lethalityof Ts mutant by transgenesis with the wild"type gene of Rp138. Over expres"sion both of the BAC containing Rp138 and the Rp138 cDNA appeared to reo
store the viability of the Ts mutant in a certain genetic background and com"pletely rescued the morphological phenotype. Thus, it appeared that the Ie"
thality and the morphological anomalies of Ts mutants, including their
homeotic transformation, are caused by the Rp138 mutation.

(5)

Positional cloning of the preaxial polydactyly mutation,
hemimelic extra toes (Hx)

Tomoko SAGA!, Hiroshi MAsUYA 1, Kunihiko SHIMIZU2 and Toshihiko SHIROISHI(lMouse Funct. Genomics Res. Group, RIKEN GSC, 2Nihon Univ. Sch. of Dent.
at Matsudo)
The zone of polarizing activity (ZPA) located at the posterior limb margin
controls patterning of the vertebrate limb along the anterior-posterior axis. Oneof the hedgehog family member, Shh has been shown to be able to mediate thefunction of the ZPA. Several preaxial polydactyly mutations of mouse, exhibit
ectopic expression of shh at the anterior limb margin. One of the mutations,hemimelic extra"toes (HJd have been mapped to the region very closely linked
to shh locus. In the human syntenic region ofChr7q36, a complex poly-syndac"
tyly mutation has been mapped. The data of our physical mapping using YAC
and BAC clones localized Hx to a telomelic region approximately 1Mb awayfrom the shh locus. By genetic mapping based on several intra'subspecies
crosses and use of newly isolated STS markers, we were able to narrow down
the Hxcritical region to a single BAC clone. To identify the transcripts in theapproximately 140kb of the BAC, we carried out cDNA selection with mouse

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embryonic cDNA library. We obtained many cDNA clones. Recently, anothergroup has identified eleven transcripts in the human syntenic region 7q36.
From a search of sequence homology, we found the two mouse homologs of thetranscripts in compiling several cDNA fragments. Because one homolog was
located in the Hxcritical region, we anticipated that it should be a good candi'date of Hx. We cloned the full'length cDNAofthis transcript, and compared
the coding sequence of Hx mutant with the wild type sequence. We found nodifference in the two sequences. It is likely that there is limb specific isoforms
through alternative splicing of the same gene, and there are unknown genes inthe Hxcritical region. Now, we are employing genomic sequencing ofthe whole
BAC clone.

(6)

Gasdermin (Csdm) localizing to mouse chromosome 11 is
predominantly expressed in upper gastrointestinal tract
but significantly suppressed inhuman gastric cancer cells

Norihisa SAEKI 1, Yoshitaka KUWAHARA 2, Hiroki SASAKI2, Aya AOKI 3, Yoichi
MIZUSHINA, Haruhiko KOSEKI 4 and Toshihiko SHIROISHI (IDepart. of Biochem.,
Faculty of Med., Univ. of the Ryukyu, 2Genet. Div., Nat!. Cancer Cent. Res.
Inst., 3Dept. of Neurosurgery, Sch. of Med., Juntendo Univ. 4Div. of Develop'
mental Biology, School of Medicine, Chiba University)
Amplification of proto' oncogenes associated with their over-expression is
one of the critical carcinogenic events identified in human cancer cells. In manycases of human gastric cancer, a proto'oncogene ERBB'2is co' amplified with
CABl genes physically linked to ERBB'2, and both genes are over'expressed. The amplified region containing ERBB'2 and CABl was named
17q12 amplicon from its chromosomal location. The syntenic region corre'
sponding to the 17q12 amplicon is well conserved in mouse. We isolated andcharacterized a novel mouse gene that locates telomeric to the mouse syntenic
region. Northern blot analysis using the mouse cDNA and a cloned partial
cDNA of human homologue disclosed unique expression pattern of thegenes. They are expressed predominantly in the gastrointestinal (GI) tract

102

and in the skin at a lower level. Moreover, in the GI tract, the expression ishighly restricted to the esophagus and stomach. Thus, we named the mousegene Gasdermin (Gsdm). Interestingly, in spite of its expression in normal
stomach, no transcript was detected by Northern blot analysis in human gas·
tric cancer cells. These data suggest that the loss of the expression of thehuman homologue is required for the carcinogenesis of gastric tissue and thatthe gene has an activity adverse to malignant transformation of cells. The
function of Gsdm is unknown. To investigate the function of this gene thatposses unique feature, we are preparing a knock"out mouse mutant with dis·
rupted Gsdm gene.
(7)

Behavioral study of inbred strains established from wild mice. III.
Active avoidance test

Tsuyoshi KOIDE, Kazuo MORIWAKI 1 and Toshihiko SHIROISHI (1 Grad. Univ. Adv.
Stud.)
Many aspects of mouse behavior have been studied using only a relatively
small sample of laboratory strains. An inherent problem in analyzing mouse
behavior is that genetic diversity is limited among currently available
strains. In this respect, the use of strains that are derived from a variety ofwild mice should provide a means to identify novel behavioral phenotypes. We
have been investigating several behavioral phenotypes using females of anum·
ber of mouse strains, BFM/2, NJL, BLG2, HMI, CAST/Ei, KJR, SWN and
MSM, which were derived from wild mice of different subspecies. A fancy mice
derived strain JFl as well as two standard laboratory strains, C57BLl6 andDBA/I, have been also used in our study.
In the previous report, we have shown that the two strains, CASTlEi and
BLG2, are poor learners, while five strains, BFM/2, KJR, SWN, MSM and C57BU
6 are good learners in the passive avoidance test. In order to investigate theability for learning and memory in different ways, we conducted an active avoidance test. The test (fear-motivated learning test) was performed in a shuttle
avoidance apparatus (O'hara & Co. Ltd., Tokyo, Japan) made of polyvinyl chlo·ride (300 x 90 x 150 mm). The conditioned stimulus (CS) consisted of a signal

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light (30 V, 0.1 A, 3 W) placed on the lid and sound (600 Hz, 60 dB) for 5 seconds,
preceding the unconditioned stimulus (US; continuous electric stimulation, 30V
constant; 0.05 rnA average, through the grid floor) for 3 seconds. For the US,
mild electric stimulation was carefully chosen to minimize the occurrence of
freezing. Both CS and US were terminated when the animal ran to the otherside of the apparatus. The mice were tested individually. Each animal received
60 trials per day for 4 consecutive days, a total of 240 trials with an inter-trial
interval of 22 sec. The JF1 strain was not included in this analysis due to ahearing defect associated with a coat color mutation. Significant differenceswere noted in the learning curves between the strains. The two poor performers,
NJL and MSM strains, exhibited rearing or jumping (R-type response), while
the other strains showed an increase in horizontal locomotion (L-type response)
following the electric shock in the active avoidance test. Therefore, it is possiblethat the poor learning ability shown by the NJL and MSM strains in this test
would be improved if the test is modified such that the R-type response effectively terminates the CS signal and electric shock. The wild mouse derived
BLG2 strain also performed relatively poorly in this test, but did not show an R·
type response. Instead, increased L-type activity was observed. The data obtained from two tests for passive and active avoidance behavior suggesting that
BLG2 strain is impaired for the ability oflearning and memory, contrasting to
two other strains, KJR and SWN, which are good performers in both tests.

Pub I i ca t ions
1. SATO, H., KOlDE, T., SAGAI, T., ISHIGURO, S.I., TAMAI, M., SAITO, N., SHIROISHI, T.: The
Genomic organization oftype I keratin genes in mice. Genomics, 56: 303-309, 1999.
2. KOMINE, Y., TANAKA, N.K, YANO, R., TAKAI, S., YUASA, S., SHIROISHl, T., TSUCHIYA, K and
YAMAMORI, T.: A novel type of non-coding RNA expressed in the rat brain. Mol. Brain
Res. 66: 1-13, 1999.
3. MORIWAKI, K, MIYASHITA, N., YAMAGUCHI, Y. and SHIROISHI, T.: Multiple genes governing
biological functions in the genetic backgrounds of laboratory mice and Asian wild mice.
Prog. Exp. Tumor Res. Basel, Karger, 35: 1-12, 1999.
4. SHUM, A.S.w., POON, L.L.M., TANG, W.W.T., KOIDE, T., CHAN,B.W.H., LEUNG, Y·C.G.,SHIROISHI, T. and COPP, A.J.: Retinoic acid induces down-regulation ofWnt-3a, apoptosis
and diversion oftail bud cells to a neural fate in the mouse embryo. Mech. Dev. 84: 1730, 1999.

104
5. SHIMIZU, K, TANI, M.m WATANABE, H., NAGAMACHI, Y., NIINAKA, Y, SHlROlSHI, T., OHWADA,
S., RAZ, A. and YOKOTA, J.: The autocrine motility factor receptor gene encodes a novel
type of seven transmembrane protein. FEBS letter 456: 295-300, 1999.
6. UEDA, Y., MIYASHITA, N., IMAI, K, YAMAGUCHI, Y, TAKAMURA, K, NOTOHARA, M., SHIROISHI,
T., KAWASHIMA, T., NING, L., WANG, C., Wu, X. and MORIWAKI, K: Nucleotide sequencesof the mouse globin beta cDNAs in a wild derived new haplotype Hbb w1 • Mammal.
Genome 10: 879-882, 1999.
7. HATTORI, M., YAMATO, E., ITOH, N., Fu.JISAWA, T., YOSHINO, M., FUKUDA, M., MATSUMOTO,
E., TOYONAGA, T., NAKAGAWA, 1., PETRUZZELLI, M., MCMURRAY, A., WEINER, H., SAGAI, T.,
MORIWAKI, K, SHIROISHI, T., MARON, R. and LUND, T.: Cutting edge: homologous recombination of the MHC class I K region defines new MHC-linked diabetogenic susceptibility gene(s) in nonobese diabetic mice. J. Immunol. 163: 1721-1724, 1999.

F-b. Plant Genetics Laboratory(1)

Analysis of genetic mechanisms controlling embryogenesis andregeneration of rice (Oryza sativa)(I)-a. Regulation of expression of KNl-type homeobox genes in rice
Yukihiro ITO, Yasuo NIWA 1, Nori KURATA (IShizuoka Pref. Univ,)
To elucidate genetic programmes that control embryogenesis and regeneration of rice, we conducted cloning, structural and functional analyses of genes
which encode transcription factors and protein kinases. We previously identified eight KNl-type homeobox genes. Previous studies of our and others' indicated that KNl-type homeobox genes are specifically expressed in a shootmeristem and function in formation or maintenance ofthe shoot meristem. To
understand a shoot meristem specific expression of aSHl, a KNl-type
homeobox gene of rice, GUS or GFP reporter genes driven by the aSHl pro'
moter were introduced into rice. In the transgenic rice GUS or GFP activities
were detected in the leaves in addition to the shoot meristem. Thus, the aSHl
promoter region alone does not confer the endogeneous expression of aSHl
and other regions are necessary for the precise expression. To gain insights
into the function of aSHl, the aSHl cDNA driven by the CaMV 35S promoter
in the antisense orientation was introduced into rice. Transgenic rice showed
an abnormal leaf phenotype similar to those in which aSHl was

F. GENETIC STRAINS RESEARCH CENTER

105

overexpressed. RT·PCR analysis indicated that the OSHl sense mRNA wasexpressed in such leaves. The expression of OSHl in the leaves does not de"pend on the 35S promoter and deletion of the 5' half of the cDNA lost such
effects. These results suggest that exons of OSHl contain certain activity on
the OSHl expression in the leaves. Our current hypothesis is that the OSHl
promoter region is active both in a shoot meristem and in leaves, and thecoding region has a repressor activity in leaves. By these two activities theexpression of OSHl is restricted in the shoot meristem and the introduction of
the extra copy of the coding sequence somehow interfares its repressor activo
ity.
(1)·b. Isolation of a mutant for a KNHike homeobox gene in rice
Yukihiro ITO, Hirohiko HIROCHIKA 1, Nori KURATA (lNational Institute ofAgrobiological Resources, MAFF)
The most effective method to isolate a mutant of a gene with known se'quence in rice is to screen insertional lines of Tosl7retrotransposon by PCR
with primers specific for 1bsl7 and for the target gene. We identified a line inwhich 1bsl7was inserted in a 3' untranslated region of HOS59, a KNl-typeclass 2 homeobox gene. Characterization of this line will be carried out.
(1)·c. Generation and screenig ofretrotransposon insertion lines in rice
Nori KURATA, Kazumaru MIYOSHI, Mitsugu EIGUCHI, Hidemi KITAN0 1, Makoto
MATSUOKA l, Yasuo NAGAT0 2, Yasumoto IWASAKI 3 (lNagoya Univ. ,2Univ. Tokyo,
3Fukui Prefectural Univ.)
To obtain a large number of mutations affecting the steps of embryogenesis,
we also used transposon-induced mutagenesis system of rice using
retrotransposon Tosl7. To isolate valuable genes taking roles for early em'
bryogenesis, we screened for segregation of embryo defect mutation with each
20 M2 seeds of about 2800 Tosl71ines. We obtained about 100 candidatelines defective in early embryogenesis. Twenty individuals from each 100
embryo defect lines were grown. DNAs of all individuals were extracted andSouthern hybridization using 1bsl7 probe was conducted. However, no link

106

between 1bs17insertion and embryo defectiveness was seen in about 30lines
screened so far. Further investigation for screening insertional mutations has
been continuing.
(I)-d. Isolation and structural analysis of OSLEGl genes offunctional importance in embryogenesis
Kazumaru MIYOSHI, Nori KURATA

LEGl (LEAFY GOTYLEDON1) gene is thought to be a central regulator ofseed development in Arabidopsis thaliana. LEGl encodes HAP3 subunit protein, a component of transcriptional factor, CBF (CCAAT-binding factor), which
is ubiquitously present in many eukaryotes. Most striking feature of thisgene is that the overexpression of LEGl protein causes ectopic embryogenesis
on leaves of transgenic plant. This is the first case of ectopic induction of plant
embryogenesis by gene manipulation. It must be significant to analyse thesignaling pathway before and after the LEGl homologous gene expression
during embryogenesis of rice. We have isolated several rice cDNA clones showing high similarity to the LEGl gene from a 3DAP seed cDNAlibrary. Sequencing
analysis revealed that at least three different LEGl homologues (OSLEGla,
OSLEGlb, LEG1-EST) are expressed during early seed development inrice. The temporal and spatial expression pattern analysed by in situ hybridization revealed that OSLEGla transcripts are detectable in developing globular embryo as early as at 3 days after pollination, in which no organ differentiation was observed. OSLEGla expression was also detected in the regenerating calli at I day after transplantation, whereas the morhological changes ofregenerated cells were observed at 4-5 days after transplantation. The early
onset ofthe gene expression in both zygotic embryogenesis and somatic regeneration process might suggest that OSLECla plays important role in earlyrice embryogenesis like as Arabidopsis LEGl gene. Overexpression experiment and search for OSLEGl knock-out plants would unravel the real functionof these genes.
(I)-e. Isolation and structural analysis of genes for receptor-like kinases in
rice

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GENETIC STRAINS RESEARCH CENTER

107

Kazuhiko TAKAYA, Yukihiro ITO, Nori KURATA
In plant development, positional information and communication withneighbouring cells are assumed to be important. Receptor-like protein ki'
nases are suitable candidates to transduce such signals and mutations ofsome ofthese genes are reported to cause developmental defects. We cloned acDNA for a receptor'like kinase gene from a 3DAP (3 days after pollination)embryo cDNA library using a carrot SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene as a probe. Southern blot of rice genomic DNA
probed with the cloned cDNA showed several bands and three independent
genomic sequences that hybridize with the cDNA were obtained from a ricegenomic DNA library. These results suggested that several SERKlike genes
exsist in a rice genome. We analyse functions of these genes with a reverse
genetic technique.
(!)

Positional cloning of a heterochronic gene, Plat, regulating theplastochron and the duration of vegetative phase in rice

Byoung-Ohg AHN, Kazumaru MIYOSHI, Jun-Ichi ITOH', Yasuo NAGATa' and Nori
KURATA(lGraduate School ofAgricultural and Life Science, Univ. Tokyo)
Heterochronic mutations affecting the timing of developmental events may
be of major significance in ontogeny and evolution. In plants, several
heterochronic mutations that affect step wise development of vegetative tis'sue and therefore alter shoot architecture have been identified. The recessive
mutations at the rice PIastochron 1 (PIal) locus cause the short plastochronand ectopic expression of vegetative programs in the reproductive phase. To
understand the molecular aspects of PIal during plant development, we havestarted to isolate the PIal gene by map·basedcloning.
To generate mapping population, the indica variety Kasalath was crossedby the japonica variety Fukei 71 carrying pIal-l mutation. Since the pIal-lhomozygous plants are sterile, the heterozygous plants were used in this cross'ing. Among 579 F 2 segregants, pIal-l homozygous plants, which have narrower leaves and a shorter plastochron than wild type siblings, were selected.

108

Preliminary studies for rough mapping using 30 Pial homozygotes and 24
RFLP markers located on all 24 chromosomes arms indicated that the Pial
locus was relatively close to both marker R2174 and R1629 on the short armof chromosome 10. And, By using the 117 pooled sampling method which eachleaf of five individuals is one pool to save cost and laborious, it showed that
Pial locus is in between the marker C961 and Rl738A on chromosome 10.
To construct a high'resolution genetic map of Pial locus, we isolated ge'
nomic DNA of 279 plants occurred recombinant on two marker above andlinkage analysis was conducted. These results indicated that Pial is closely
linked between the marker C96l(0.9cM) and R1738A(3.0cM) on centromere
region of chromosome 10.
(3)Study for the Chromosome and Nucleus Organization in Rice(3)·a. Structural analyses for the centromeric region of rice chromosome 5
and construction of rice artificial chromosomes
Ken'Ichi NONOMURA, Nori KURATA
One of profitable features of plant cells for biotechnology is to induce thereproductive adults easier than animal cells. Fusing this feature with mo'lecular biotechnology such as construction of stable plant artificial chromo'
somes will introduce us to tremendous progression of chromosome technology.
In this study, we analyzed the structure of the centromeric region of rice chro'mosome 5, ultimate goal of the analysis is to construct the cereal artificial
chromosome.
We selected Y6514 (220kb), one of rice yeast artificial chromosome (YAC)
clones derived from the centromere region ofrice chromosome 5 (Saji et a11995).
This clone includes two kinds of centromeric repeats RCS2 and RCE1, which
was 160bp'short tandem repeats and 1.9·kb dispersed repeats localizing on
all centromeres, respectively. The contiguous clones including Y6514 wasmapped just in the centromere region by the analysis of secondary trisomics(Harushima et a1. 1998). Furthermore, the crossing over events within theregion was severely reduced. These facts indicate that the contig region would
be a functional center for the chromosome 5 centromere.

F.

GENETIC STRAINS RESEARCH CENTER109

The contig was composed of at least four YAC clones including Y6514 andoccupied about 860kb length in the minimum path. It was also revealed that
RCS2 repeats formed two tandem arryed clusters within 100 kb length and at
least nine copies of RCE 1 were scattered within 600 kb in this path. Many
copies similar to long terminal repeat (LTR) ofRIRE3, which was Ty3/gypsy
type retrotransposon of rice, were found out to be distributed in thiscontig. Recent research revealed that a part ofRCE1 repeat unit was identical with LTR sequence ofRIRE4, another retroposon of rice.
Structural and functional importance of the detected centromere repetitive
sequences is waiting to be resolved in the analysis of centromere organization
through the processes of construction, intruduciton and maintenance of riceartificial chromosomes in rice. Retrofitting the vector sequences of the centromeric YAC clones to those with rice reporter genes and transferring into ricecells are now in progress.
(3)-b. Analysis of chromosome dynamics in the nucleus by using GFP-tags
NoriKURATA
Focusing to the chromosome movement in the nucleus of critical stages ofdevelopment would show some rationales for controlling nuclear organization.
To analyse such chromosome movements, I constracted and introduced a GFPLac repressor fusion gene (GFP- LR) into 17 rice plants and a Lac operator
repeats (LOR) into another 6 rices. After self-pollination of the transgenic
heterozygotes, GFP-LR and LOR homozygous lines were crossed each others
to generate GFP-tagged chromosomes. IF this system will work well in therice cells, we could analyse GFP-tagged chromosome movements under thecircumstances of specific differentiation stages and under mutants phenotypes, aneuploids and genomic hybrids conditions. Then we could expect toextract principals necessary for establishing nucleus organization and chromosome dynamics.

110

(4)

Analysis of Genetic Factors causing Segregation
Distortion

Yoshiaki HARUSHlMA, Nori KURATA
Genetic mechanisms for isolation of "species" are called as reproductive
barriers and these include hybrid incompatibility, hybrid inviability, hybrid
sterility, hybrid breakdown, etc. These reproductive barriers cause segrega"tion distortion of linked marker genotypes in the population derived fromhybrid of inter- or intra" species cross. In the last year, we developped a new
method to conduct a genome"wide survey of reproductive barriers by a multi"
response non"liner regression analysis. This method attempts to explain seg"regation distortions of a linkage map by adopting mathematical models andto estimate the number, position and strength ofthe reproductive barriers oneach chromosome. We succeed to explain the segregation distortions on all
chromosomes in F 2 population derived from a cross between a japonica culti"var, Nipponbare, and an indica cultivar, Kasalath by 34 reproductive barriers(in preparation for publication).
In this year, we have been constructing linkage maps using reciprocal back"cross populations between Nipponbare and F I of Nipponbare and Kasalath toconfirm whether the detected each reproductive barrier affects male gameto"
phyte, female gametophyte or zygote. The most pronounced segregation dis"tortion was observed at C582 on chromosome 3 in the F 2 population and wehave aimed to isolate the causal reproductive barrier by positional
cloning. However, segregation distortion of C582 was not observed either back"cross populations. This situation of the C582 segregation in the F 2 and thebackcross populations can be explained by the interaction between the malegametophyte reproductive barrier and genotype of mother plant. This expla"
nation was confirmed by the progeny test of the back crossed populations. The
C582 segregation in self pollinated progenies from the back crossed plants
those were heterozygote at C582 were segregated into not"distorted and dis"torted depending on the genotype of the interactive mother locus. The interac"
tive locus in mother plant was located on the long arm of chromosome 6.
To isolate the above male gametophyte gene on chromosome 3, we have

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GENETIC STRAINS RESEARCH CENTER

111

selected 61 plants with recombination that occurs heterozygote to homozygote
in 1.9 cM regions near the gene, from 473 backcross plants and 1000 F 2 plants
and then obtained their self pollinated progenies for detailed mapping the
gene.

(5)

Generation of enhancer trap lines of rice

Yukihiro ITO, Mitsugu EIGUCHI, Non KURATA
To isolate valuable mutants defective in various steps of rice development
and to clone the corresponding genes, we are generating enhancer trap lines ofrice. We employ an enhancer trap system used in Arabidopsis with somemodifications. This system is based on the Agrobacterium'mediated transformation using AciDs transposable elements of maize. The enhancer trap
construct contains, in the T-DNAregion, the Dselement that harbours a GUS
coding region with a P35S minimal promoter and a hygromycin resistancegene. We also use a P35S·Ac transposes (AcTPase) gene together with abialaphos resistance gene to supply transposase which is essential and sufficient for transposition of the Ds. We generated more than two hundreds
transgenic rice with Ds (Ds line) and six with P35S-Ac (AcTPase line). The
four Ds lines were clossed with each of the six AcTPase lines and somatic
excision of the Ds in leaves of the F 1 plants were examined. The excision of theDs was detected in most of the combinations but in one Ds line the excision
was not detected in any combinations with the AcTPase lines and one AcTPaseline could not excise the Ds in any combinations with the Ds lines. This result
indicates that this enhancer trap system is functional in rice. Screening ofgerminal transposants is now in progress.

Pub I i ca t ions
1.
2.

NONOMURA, K.I. and KURATA, N.: Organization of 1.9-kb repeat unit RCEI in thecentromeric region of rice chromosomes. Mol. Gen. Genet., !61, 1-10, 1999.
MIYOSHI, K., NAKATA, E., NAGATO, Y. and HATTORI, T.: Differential in situ expression of
three ABA-responsive genes ofrice, Rab16A, REG2and OSBZ8, during seed develop'
ment. Plant Cell Physio!., 40, 443'447, 1999.

112

3. ITo, Y., EIGUCHI, M. and KURATA, N.: Expression of novel homeobox genes in early
embryogenesis in rice. Biochim. Biophys. Acta, 1444, 445-450, 1999.
4. AsHIKAWA, I., KURATA, N., SAJI, S., UMEHARA, Y. and SASAKI, T.: Application ofrestriction
fingerprinting with a rice microsatellite sequence to assembling rice YAC clones. Genome, 4l!, 330-337, 1999.
5. SENTOKU, N., SATO, Y., KUHATA, N. ITO, Y.. KITANO, H. and MATSUOKA, M.: Regional
expression of the rice KNl-type homeobox gene family during embryo, shoot and flower
development. The Plant Cell, 11, 1651-1664, 1999.
6. MIYOSHI, K and KURATA, N.: Cloning of two cDNAs encoding the HAP3 subunit proteinfrom developing rice seeds. Rice Genetics Newsletter, 16, 103-104, 1999.
7. AHN, B. 0., MIYOSHI, K., IToH, J-I., NAGATo, Y. and KUHATA, N.: Mapping of a rice
heterochronic gene, PIal, regulating the plastochron and the duration of vegetative
phase. Rice Genetics Newsletter, 16, 36-37, 1999.

F-c. Microbial Genetics Laboratory(1)

The kdsA mutations affect the FtsZ-ring formation in Escherichia coliK12

Kana NISHIMORI', Hideyuki TAKAGI, Masaaki WACHI 1 , Hiroshi FUJISHIMA,
Takeshi KAWABATA, Ken NISHIKAWA, Akiko NISHIMURA (ITokyo Institute of Technology)
The outer membrane of Gram negative bacteria, such as Escherichia coh is
integral in controlling the interaction of the bacteria with the external environment, serving as a barrier, controlling the absorption of ions and nutrients,
and transmitting signals to the inside of cell. Instabilities of the outer membrane adversely affects multiple cellular responses. Proper cell division requires both the construction of division machinery, physically separating internal cellular components, and the division of cell membrane structures. Bothprocesses are closely related; instabilities in membranes may affect both theconstruction of division machinery and the expression of gene(s) essential forcell division. The mechanism, however, controlling the relationship between
cell division and the nature of the cell membrane remains unknown. No one
has, as yet, addressed the relationship between the nature of the outer membrane and cell division. We have created seven unique fts/kdsA mutants allowing analysis of the discrimination mechanism governing membrane con-F.

GENETIC STRAINS RESEARCH CENTER

113

struction and cell division.
The outer membrane of Gram negative bacteria is comprised principally of
lipopolysaccharide (LPS) and phospholipids. LPS, a major component of theouter membrane, comprises approximately 30% of the outer membrane bygross weight. LPS, comprised of hydrophorbic Lipid A, the hydrophilic coreoligosaccharide chain and 3'deoxy'D'manno'octulosonic acid (KDO), connect·
ing Lipid A to the oligosaccharide chain, is important in determining outermembrane barrier function.
KDO is synthesized by the condensation of D'arabinose'5'phosphate andphosphoenol pyruvate, followed dephosphorylation. The first step of the reac'tion is catalyzed by KDO 8'phosphate synthetase, encoded by the kdsA gene inboth Salmonella typhimurium and E. coli. E. coli kdsA, however, was first
identified as a gene complementing the kdsA mutation of Salmonella, althoughno similar mutants have yet been identified in E. coli. Mutations in kdsA of
Salmonella typhimurium causes the accumulation of Lipid A in the periplasm,
also resulting in the disappearance ofLPS from the outer membrane. LPS isan essential membrane component; only conditional lethal mutants in KDO
biosynthesis can be isolated in. The biosynthesis ofLPS is growth phase regu'
lated at the transcriptional level in E. colI: no evidences, however, has uncov'
ered a relationship between LPS biosynthesis and cell division. In this study,
we isolated temperature sensitive mutants of kdsA, to determine that memo
brane instability resulting from the defect in KDO biosynthesis affected the
FtsZ-ring formation.
Seven temperature sensitive mutants, fts/kdsA, contained mutations mapped
at approximately 27 min in the E. coli genetic map. These mutants stopped cell
division at 41°C, producing filamentous cells lacking FtsZ-rings. The mutations were co-transduced with trp into the wild type strain, MG 1655, at an
efficiency of 13%. All seven mutants contained missence mutations in kdsA, tobe complemented only by the plasmid'born, wild type kdsA. The mutations
affected the production of KDO; the defect was reversed by the plasmid-born,wild type kdsA. Western blotting analysis, however, demonstrated that theamount of FtsZ protein was not affected by the mutation. Therefore, we concluded that the instability of the outer membrane affects the FtsZ-ringformation. The FtsZ-ring, however, is constructed onto the inner membrane,

114

maintaining a position at the leading edge of invaginating septum; the protein(s)governing this cell division at the outer membrane have not been found as yet.
As E. coli can divide without outer membrane, it is not likely the structure ofouter membrane would affect the construction of FtsZ-rings. The mechanism
whereby the instability of outer membrane results in the loss of FtsZ-ring formation, therefore, remains to be established.
While the mutants also displayed sensitivity to hydrophobic materials, including Novobiosine, Eosine Y and SDS, at 36°C, Methylene blue, however, restored the colony forming ability ofthe mutants, indicating that the instabilityof the outer membrane creates the altered cellular responces to theirenvironment. The expression of a subset of genes may overcome the sensitivity
to the hydrophobic materials. The kdsA mutation may also affect the transcription of a cell division gene(s), required for FtsZ-ring formation. Membrane instability is expected to affect the transcription of genes, including those involvedin cell division. Because decreases in lipopolysaccharide bare phospholipids tothe external environment, and phospholipids are important in the transfer ofsignals from the extracellular environment to the transcription machinary.

Publication
None

F-d.
(1)

Invert~brate

Genetics Laboratory

Proximal to distal cell communication In the Drosophila leg
provides a basis for an Intercalary mechanism of
11mb patterning

Satoshi GOTO and Shigeo HAYASHI
Proximodistal patterning in the Drosophila leg is elaborated from the circular arrangement of the proximal domain expressing escargot and homothorax,
and the distal domain expressing Distal-less that are allocated during
embryogenesis. The distal domain differentiates multiply segmented distal
appendages by activating additional genes such as dachshund. Secreted sig-

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GENETIC STRAINS RESEARCH CENTER

115

naling molecules Wingless and Decapentaplegic, expressed along the ante'rior-posterior compartment boundary, are required for activation of Distal'less and dachshund and repression of homothoraxin the distal domain. However,whether Wingless and Decapentaplegic are sufficient for the circular patternof gene expression is not known. Here we show that a proximal gene escargot
and its activator homothorax regulate proximodistal patterning in the distal
domain. Clones of cells expressing escargot or homothorax placed in the dis'tal domain induce intercalary expression of dachshund in surrounding cells
and reorient planar cell polarity of those cells. Escargot and homothorax-ex'pressing cells also sort out from other in the distal domain. We suggest thatinductive cell communication between the proximodistal domains, which ismaintained in part by a cell,sorting mechanism, is the cellular basis for anintercalary mechanism of the proximodistal axis patterning of the limb. For
detail, see ref 2.
(2)

Interplay of Notch and FGF signaling restricts cell fate and
MAPK activation in the DrosopblJa trachea

Tomoatsu IKEYA and Shigeo HAYASHIThe patterned branching in the Drosophila tracheal system is triggered by
the FGF·like ligand Branchless that activates a receptor tyrosine kinase
Breathless and the MAP kinase pathway. A single fusion cell at the tip of eachfusion branch expresses the zinc'finger gene escargot, leads branch migration
in a stereotypical pattern and contacts with another fusion cell to mediate
fusion of the branches. A high level of MAP kinase activation is also limited tothe tip of the branches. Restriction of such cell specialization events to the tip
is essential for tracheal tubulogenesis. Here we show that Notch signaling
plays crucial roles in the singling out process of the fusion cell. We found that
Notch is activated in tracheal cells by Branchless signaling through stimula'tion of Delta expression at the tip of tracheal branches and that activatedNotch represses the fate of the fusion cell. In addition, Notch is required torestrict activation of MAP kinase to the tip of the branches, in part throughthe negative regulation of Branchless expression. Notch'mediated lateral in'116

hibition in sending and receiving cells is thus essential to restrict the induc"tive influence of Branchless on the tracheal tubulogenesis. For detail, see ref3.

(3)

Repression of the wing vein development In Drosophila by the
nuclear matrix protein Plexus

Hitoshi MATAKATSU, Ryosuke TADOKORO, Sumiko GAM0 1 and Shigeo HAYASHI(IOsaka Prefecture University. )The wing of Drosophila is separated into several sectors by the wing veins.
Vein primordia are specified by the positional information provided by hedge"
hog and decapentaplegicin the wing imaginal disc and express the key regula"tory gene rhomboid. One model of this process is that boundaries of gene
expression regulated by hedgehogor decapentaplegicprovide reference points
where rhomboid transcription is activated. We present an analysis of the geneplexus, whose loss of function causes an excess vein phenotype. Molecularcloning revealed that plexus encodes a novel 1990"amino acid protein withcysteine"rich motifs. Plexus protein was ubiquitously expressed and wastightly associated with the nuclear matrix. In plexus mutant wing imaginaldiscs, an anteroposterior positional coordinate was established normally as
revealed by the wild"type pattern of spalt major and knirpsexpression. However,
the expression of several vein"specific and intervein"specific genes was
misregulated, as if they had neglected the positional coordinate. These reo
suIts suggest that Plexus is an essential component of a global repressor ofvein differentiation. Although Plexus protein was expressed in vein primordia of the wing disc, it does not appear to interfere with vein differentiation in
the normal position. A genetic epistasis test between px and knirps suggeststhat plexus acts downstream of knirps. We propose that the vein differentia"
tion takes place by inactivation of the plexus"mediated repression by
prepattern genes such as knirps. Plexus may regulate transcription of vein"
and intervein"specific genes by tethering transcriptional regulators to specificlocations in the nucleus. For detail, see ref 4.

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GENETIC STRAINS RESEARCH CENTER

117

Snail-type zinc finger proteins prevent neurogenesis in
Sea/old and transgenic animals of Drosophl/aNaoyuki FUSE, Hitoshi MATAKATSU, Misako TANIGUCHI and Shigeo HAYASHI

Scutoidis a classical dominant gain'of'function mutation of Drosophila,
causing a loss of bristles and roughening of the compound eye. Previous ge'
netic and molecular analyses have shown that Scutoidis associated with achromosomal transposition resulting in a fusion of no'oceli and snail genes. How
this gene fusion event leads to the defects in neurogenesis was not known until
now. Here have found that snail is ectopically expressed in the eye'antennal
and wing imaginal discs in Scutoidlarvae, and that this expression is reducedin Scutoidrevertants. We have also shown that the expressivity of Scutoidis
enhanced by zeste mutations. snail and escargot encode evolutionarily con'served zinc'finger proteins involved in the development of mesoderm and limbs.
Snail and Escargot proteins share a common target DNA sequence with thebasic helix'loop'helix (bHLH) type proneural gene products. When expressedin the developing external sense organ precursors of the thorax and the eye,these proteins cause a loss of mechanosensory bristles in the thorax and perturbed the development of the compound eye. Such phenotypes resemble those
associated with Scutoid Furthermore, the effect of ectopic Escargot on bristle
development is antagonized by coexpression of the bHLH gene asense. Thus,
our results suggest that the Scutoid phenotype is due to an ectopic snail ex'pression under the control of no'oceli enhancer, antagonizing neurogenesis
through its inhibitory interaction with bHLH proteins. For detail, see ref 5.

(5)Enhancer trap screen for genes involved in pattern formation

Satoshi GOTO, Misako TANIGUCHI, Yukiko SADO and Shigeo HAYASHI
To identify novel genes and gene functions in the pattern formation of theimaginal disc and the trachea, we are conducting an enhancer trap screen
using the Ga14'UAS system. About 4500 lines were established in collabora'

118

tion with groups in Japan and were examined for the activity of enhancers
flanking the inserts in embryos, larvae and adults. In order to identify genes
responsible for those expression, we are mapping insertion sites of eachtransposons onto the genomic sequence of Drosophila.

Pub I i ca t ions
HAYASHI, S. and YAMAGUCHI, M.: Kinase'independent activity of Cdc2/Cyclin A prevents
S phase in the Drosophila cell cycle. Genes to Cells, 4, 111-122, 1999.
2. GOTO, S.and HAYASHI, S.: Proximal to distal cell communication in the Drosophila leg
provides a basis for an intercalary mechanism of limb patterning. Development, 126,
3407-3413, 1999.
3. IKEYA, T. and HAYASHI, S.: Interplay of Notch and FGF signaling restricts cell fate and
MARK activation in the Drosophila trachea. Development, 126, 4455-4463, 1999.
4. MATAKATSU, H., TADOKORO, R., GAMO, S. and HAYASHI, S.: Repression of the wing veindevelopment in Drosophila by the nuclear matrix protein Plexus. Development, 126,
5207-5216, 1999.
5. FUSE, N., MATAKATSU, H., TANIGUCHI, M. and HAYASHI, S.: Snail-type zinc finger proteinsprevent neurogenesis in Scutoid and transgenic animals of Drosophila. Development
Genes and Evolution, 209, 573-580, 1999.
1.

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G.

CENTER FOR GENETIC RESOURCE INFORMATION
G-a. Genetic Informatics Laboratory(1)

ORYZABASE--INTEGRATED RICE SCIENCE DATABASE--

Takahiro YAMAKAWA, Kazu MITSUI, Nori KURATA, Atsushi YOSHIMURA 1, Yasuo
NAGAT0 2 and Yukiko YAMAZAKI(I Kyushu University, 2 University of Tokyo)
The Oryzabase is a comprehensive rice science database established in thecollaboration with rice researcher's committee in Japan. The database is originally aimed to gather as much knowledge as possible ranging from classicalrice genetics to recent genomics and from fundamental information to hot
topics.
The current Oryzabase consists of five parts, which are (1) genetic resource
stock information, (2) gene dictionary, (3) chromosome maps, (4) mutant images, and (5) fundamental knowledge of rice science.
The database includes more than 10000 collections of germplasms collected
or developed by classical breeding and/or new molecular biological method asa result oflong history of rice breeding in Japan. The Oryzabase map represents the integration of seven different maps from classical linkage map to
the latest YAC physical map provided by Rice Genome Project. We've completed the internal cross"linking of related information such as common mark"ers on different maps, genes and the respective mutant images, strains andtheir marker genes, and so on. Using Web-based good interface, the rice genedictionary has been constantly updated by the specialists coupled with theactivity of Rice Gene Cooperative.
Oryzabase uses an object"oriented database management software with
Java2D for application.
We are planning to do more extensive cross"referencing of Oryzabase to themajor DNA sequence database, literature database and other rice databasesuch as Ricegenes in order to provide the wealth of information to rice reo

120

searchers.
Oryzabase is available from http://www.shigen.nig.ac.jp/rice/oryzabase/(2)

PEC:Profillng of Escherichia coli Chromosome

Yukiko YAMAZAKI, Toru IKEGAMI', Takehiro YAMAKAWA, Kazu MITSUI, Takeshi
KAWABATA, Ken NISHIKAWA, Tadahiro MORI 2, Akiko NISHIMURA and Junichi KATO
(1 University of Tokyo, 2Nara Institute of Science and Technology)
Profiling of Escherichia coli chromosome (PEC) is a database that compiles
all necessary information for functional analysis ofE. coli genes. The Japa'
nese E. coli genetic resource committee has extensively contributed to thedevelopment of the database, which provides a comprehensive interface forexperimental researchers. Available from the database are (i) deletion mu'
tant information obtained from experimental research, (ii) annotated gene
information including gene classification, Gii) genetic stock information, Gv)
similarity search results for each genes/orfs, (v) structural information pro'
posed by bioinformatics researchers and (vi) a tool for graphical display ofdifferent gene classes in different colors. PEC is available at http://
www.shigen.nig.ac.jp/ecolilpecl
Since the whole genome sequence ofE.coli was published in 1997, enormous
efforts have been made to analyze the functions of the genes/orfs. Further
innovative research is still needed to complete those tasks. Many different
E.coli databases are now available through the Internet, but most databases
have been constructed by computer scientists with the contents derived fromsequence analysis. Experimental biologists, however, are not always satisfied
with such databases. On the other hand, computational scientists face prob'
lems on how to annotate their analytical results generated and/or calculated
from sequence data.
To resolve the dilemma, we've developed the PEC aiming to provide experi'mental scientists a chance to meet and communicate with computer scientists
and vice versa through the Internet. The database includes original deletionmutant data and genetic stock available through the Internet. Most genes/
orfs in the database are classified into three groups, essential, non-essential,

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121

and unknown according to either experimental results or data published injournal articles. The basic information of genes/orfs are acquired from resources available to the public and annotated by researchers before their incorporation into the database. BLASTIPSI-BLAST homology search and motif search using PROSITE and Pfam database have also been performed for all
genes/orfs periodically and the results incorporated into PEC. The databasealso provides analytical data such as nucleotide composition proposed by computer scientists with a comprehensive interface. To create an overview, we areplanning to develop off-line tool by which the user can create an original image
file by applying his/her own data set.

Publ icatianNone

G-b. Genome Biology Laboratory(1)

Analysis of gene expression patterns In Caenorhabd/tls eleKans

Yuji KOHARA, Tadasu SHIN'I, Ikuko SUGIURA, Tokie OBA, Masumi OBARA, Takami
SUZUKI, Sayuri KITAYAMA, Tomoko MOTOHASHI, Keiko HIRaNO, Tamami NAGAOKA,
Masahiro ITO, Yohei MINAKUCHI, Jean and Daniell THIERRY-MIEG1(lCNRS,Montpellier, France)
The nematode G.elegans is a good model system to study functional genomics
with respect to animal development, nervous system and behavior at the levelof single cells. Although C.elegans has the basic structure of animals, it has
only about 1,000 somatic cells. This simplicity has led to the description of theentire cell from embryo to adult, which has allowed us to study gene functionsin individual cells. The genome consists of six chromosomes whose total size
is about 100 Mbp and total number of genes is estimated to be about 19,000.
Almost the entiret of the genome had been sequenced by the consortium of theSanger Centre and Washington University by the end of 1998.
We think the main targets in the post genomics era are as follows:(l) to
integrate the information on the expression and the function of all the genes of

122

the genome in the context of development, (2) to extract the rules that govern
the molecular mechanisms of development that are carried out by a finite
number of genes, (3) ultimately to reconstruct the developmental process inthe computer.
We have identified about 10,000 cDNA species (more than a half of all ofthe
genes) of C.elegans through our EST project. About 4,000 cDNA species mostly
from chromosome 3 (autosome) and X (sex chromosome) have been analyzed
by use of whole mount in situ hybridization for mRNA distribution throughout
the life ofthe worm. (Generation time is about 3 days). The mRNA patterns
were classified into several categories: maternal expression, zygotic expres"sion, expression in a specific cell lineage but at different times, expression in
specific cell(s) and so on. Based on the information, we are performing cluster"ing analyses on subsets of the genes to elucidate the network of gene regula"tion. As a trial, focusing on the early embyrogenesis, we selected a subset of
100 genes on the criteria that the mRNA was expressed maternally and disap"
peared quickly before gastrulation, which seemed to play important roles inthis period. For these genes, we are analyzing (i) the phenotypes caused by thesystematic RNAi (dsRNA mediated interference) experiments, (ii) the protein
distribution through the systematic raising of antibodies, and (iii) interacting
genes by the yeast two hybrid technology. To integrate and analyze the information, first we have established a WWW"based database NEXTDB (http://
watson.genes.nig.ac.jp:SOSO/db/index.htmD, and recently we have been con"structing a computer graphics based 4 dimensional (3D + time course) data"base that covers the early embryogenesis.

(Z)

Studies on translational regulation of maternal mRNA

Ken"ichi OGURA, Yuji KOHARA
Accumulation of maternal messenger RNAs in oocytes is widely observed inmany species, and the temporal and spatial control of their translation inearly embryos is crucial for development. Although extensive studies ofvari"
ous maternal mRNAs, including glp-l, have shown that the 3' UTR(untranslated region) is important for the translational control, the molecularG. CENTER FOR GENETIC RESOURCE INFORMATION

123

mechanism is largely unknown. Previously we found that a Celegans maternal gene, pos-l, which has two CCCH zinc finger motifs, plays an importantrole in the translational control of the maternal polarity gene aprl. We further characterized the function of the protein POS-I and of its interacting
proteins that we identified by the yeast two hybrid method. Here we report on
PIP-I, a novel RNP type RNA binding protein that interacts with POS-I. We
show that PIP-I is a positive regulator and POS-I is a negative regulator of
the translation of the maternal glp-l mRNA.
(3)

Application of cDNA array of C.eJegans

Makoto MOCHII 1, Satoru YOSHIDA I, Kiyokazu MaRITAl, Hiroko UESUGI, Yuji
KOHARA, Naoto UENo1(INational Institute of Basic Biology)
Members of the transforming growth factor-b family play critical roles inbody patterning, in both vertebrates and invertebrates. One transforming
growth factor- 13 -related gene dbl-l, has been shown to regulate body lengthand male ray patterning in Caenorhabditis elegans. We screened arrayed
cDNAs to identify down-stream target genes for the DBL-I signaling by using
differential hybridization. C.elegans cDNAs representing 7,584 independent
genes were arraydd on a nylon membrane at high density and hybridized with
32P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-l,
sma -2 and lon-2worms. Signals for all the spots representing hybridized DNA
were quantified and compared among strains. The screening identified 22 and2 clones, which were positively and negatively regulated, respectively, by the
DBL-I signals. Northern hybridization confirmed the expression profiles ofmost of the clones, indicating good reliability ofthe differential hybridizaiton
using arayed cDNAs. In situ hybridization analysis revealed the spatial andtemporal expression patterns of each clone and showed that at least fourgenes, including the gene for the type I receptor for DNB-I, sma-6were transcriptionally regulated by the DBL-I signal.

124

(4)

Functional analysis of the C. eJeffBns T-box gene tbx-fl

Yoshiki ANDACHIMembers of the T-box family that share a DNA binding motif play critical roles
in developmental phenomenon in metazoans. Some of the members have beenshown to encode transcription factors. The complete sequence of the C. elegans
genome indicates 20 T-box genes in this organism, and nearly half of them havebeen identified by the C. elegans EST project. I have been studying one of the C.
elegansT-box genes, the cDNA group CELK02736 or tbx-9, which is the first T-box
gene found by the EST project and seems to be the most highly expressed T-box
gene as deduced from the number of isolated cDNA clones corresponding to eachgene. I previously showed that tbx-gencodes a transcription activator, that tbx-9
is expressed in a few cells in embryogenesis, and that the tbx-9deletion mutant
generated by gene disruption shows aberration of morphogenesis predominantlyin the posterior body, including abnormality ofbody"wall muscle.
To further elucidate the expression pattern of tbx-9, I performed in situ hy"
bridization double-staining analysis using the pos-] and hlh-] genes asmarkers. The onset of the tbx-gexpression is at the a"cell stage in the E cell, theancestor cell of gut, and the expression lasts until daughters of the E cell at the26"cell stage. At this stage, the Ca and Cp cells start to express tbx-9, which
produce body"wall muscle and hypodermis. On the other hand, at about the
200"cell stage four MS descendant cells express both tbx-9and hlh-l. hJh-]is ahomolog of vertebrate MyoDand is expressed in the cells whose clonal descendants give rise only to striated muscle cells. This indicates that tbx-9is also
expressed in precursors of MS"lineage body"wall muscle cells. Among thesetissues whose precursor cells express tbx-9, body"wall muscle that shows abnormality in the tbx-9 mutant was examined with respect to formation byimmunostaining with an antibody against body"wall muscle myosin. In wildtype
embryos, the muscle cells move and form four rows along the length ofthe ani"
mal by the 1.5"fold stage. In tbx-9mutant embryos, some of the muscle cells
that differ form worm to worm turned out to be positioned apart from the rows
at this stage, suggesting that tbx-9is involved in the proper arrangement of thebody"wall muscle cells.

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CENTER FOR GENETIC RESOURCE INFORMATION

125

Pub I i ca t ionsTabara, H., Hill, RJ., Mello, C., Priess, J. and Kohara, Y.: Pas'] encodes a cytoplas'
mic zinc'finger protein essential for germline specification in C.elegans. Development
126, l'll (1999).
2. Fields, S., Kohara, Y. and Lockhart, D.J.: Functional Genomics. Proc. Nat!. Acad. Sci.
USA 96, 8825-8826 (1999).
3. Motii, M., Yoshida, S., Morita, K, Kohara, Y. and Ueno, N.: Identification of TGF-b'
regulated genes in Caenorhabditis elegans by differential hybridization of arrayed
cDNAs. Proc. Nat!. Acad. Sci. USA, 96, 15020-15025 (1999).
1.

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H.

STRUCTURAL BIOLOGY CENTER

H-a. Biological Macromolecules Laboratory(1)

Single Molecule Imaging of Biological Functions

Makio TOKUNAGA
Single molecule techniques have been recently developed, and importanceof visualization, manipulation, as well as force measurement of single bio"
logical molecules are being realized. These new techniques were born mainly
at in vitro researches of motor proteins, and we are trying to spread out thisnew technique from in vitro to in vivo and from motor proteins to other bio"
logical molecules. Moreover, by using green fluorescent protein (GFP), realtime
imaging of biological molecules has been achieved in live cells quite recently.
Conjunction of these new techniques, on which much attention is now fo"
cused, will provide new powerful and universal tools for researches of moo
lecular mechanisms of biological functions in the post-genome era. With whichmolecule does a molecule interact? When? Where? And How? In this project,
we will answer these questions by directly visualizing not only molecules them"selves but also their interactions.
Imaging of single fluorescent molecules has been achieved in a relatively
simple manner using objective"type total internal reflection fluorescence microscopy (TIRFM). It is found that single molecule imaging in vivo is realizedusing the objective"type TIRFM. Furthermore, I'm developping new micros"
copy to achieve followings: 1) Quantitative imaging of molecular numbers orconcentrations in vivo. The key points are minimized background light, highsensitivity and wide dynamic range. 2) Imaging in vivo of interacting mol"ecules and activated molecules.

H.

(!)

STRUCTURAL BIOLOGY CENTER

127

Single molecule imaging of nuclear transport

Naoko IMAMOT0 1 and Makio TOKUNAGA (IDepartment of Anatomy III, Osaka
University Medical School)
Using objective-type total internal reflection fluorescence microscopy(TIRFM), it has been found that singe molecules can be visualized in vivo. Thismethod allowed visualization of single molecules up to the depth of about 10/.1. m from the glass·water interface. We are now trying to visualize molecules
involved in nuclear transport of macromolecules. Recent years have seen considerable progress in researches of macromolecule import into the nucleus
and export from the nucleus. NLS proteins are transported into the nucleus
by importin alpha/beta heterodimer. Importin alpha binds the NLS, while
importin beta mediates translocation through the nuclear pore complex. Aftertranslocation, RanGTP, whose predicted concentration is high in the nucleus
and low in the cytoplasm, binds importin beta and displaces importin alpha.
Importin alpha must then be returned to the cytoplasm, leaving the NLS
protein behind.
NLS proteins bound to the nuclear pore have been well visualized using
our single molecule technique. This method is useful to elucidate molecularmechanisms of nuclear transport.

(3) Single molecule measurement of intermolecular and intramolecular interactions using subpiconewton intermolecular force microscopy
Michio HIROSHIMA and Makio TOKUNAGA
For a detailed understanding of the interactions between biological macro·molecules and of intramolecular interactions, it is required to obtain infor·
mation about the properties of these forces at the molecular level. We havedeveloped intermolecular force microscopy (IMF) by refining atomic force microscopy (AFM) to improve the sensitivity of force detection and control ofprobe position. Force resolution of subpiconewton has been achieved, whichis over lOO-fold more sensitive than that of conventional AFM. IMF has also

128

overcome the disadvantage of AFM that the cantilever changes its position
when forces are exerted, because IMF system keeps the cantilever position
unchanged. Thus, we can measure intermolecular or intramolecular forces
exerted on a single molecule as a function of the distance with subpiconewton
resolution.
We have refined the IMF system to improve the following points: 1) expansion of force ranges up to 100 - 200 pN, 2) development of simultaneous measurement with single molecule fluorescence imaging, and 3) development of a
new horizontal system in which cantilever moves horizontally and horizontal
positions are controled by the feedback system.
(4)Single molecule measurement of protein folding using intermolecularforce microscopy

Isao SAKANE, Kunihiro KUWAJIMA 1 and Makio TOKUNAGA (IDepartment ofPhysics, University of Tokyo)
The understanding of the mechanism of protein folding has remarkably
progressed during the past decade. Many globular proteins have been foundto accumulate intermediate states during refolding from the unfolded state to
the native state. Although the molten globule state has been proposed as amajor intermediate of protein folding, it has proven difficult to obtain thermodynamic data characterizing this state. Certain globular proteins showcomplex multiphasic refolding kinetics that are not directly related to theaccumulation of the folding intermediates; the complex folding kinetics ofthese proteins have often been interpreted in terms of the heterogeneity of
the unfolded state. Thus, a novel technique is required to study the folding
process. Our single molecule technique using intermolecular force microscopy could characterize dynamic structures of intermediate states during folding.
Staphylococcal Nuculease (SNase) is a small globular protein, and is one ofthe proteins most generally used for folding researches. Its folding kinetics iswell characterized. and also its atomic structure is available. Therefore, SNaseis thought to be suitable for single molecule measurement. We have preparedH. STRUCTURAL BIOLOGY CENTER

129

a mutant protein ofSNase, which lacks the C-terminal helix and has cysteinyl
residues at the both N- and C-terminal end.

Pub I i ca t i on
1.

KITAMURA, K., TOKUNAGA, M., IWANE, A. H. and YANAGIDA, T.:A single myosin head moves

along an actin filam en t with regu Jar steps of 5.3 nanOO1 etres. Nature, 397, 129-134,
1999.

H-b. Molecular Biomechanism Laboratory(1) The arrest of E. colJ RNA polymerase at promoters, and the effectof the Gre factors

Motoki SUSA 1, Tomoko KUBORl l, Hiroki NAGAI l, Tamas GAAL 2, V. Georgi
MISKELISHIVILI 3 and Nobuo SHIMAMOT0 1 (lStructural Biology Center, National
Institute of Genetics, 2Microbiology, Wisconsin-Madison, 3Institute fms Genetik
und Mikrobiologie)
The initiation frequency oftranscription is conventionally attributed to thestability of holoenzyme-promoter binary complex, the rate/degree of strand
opening, and the rate of promoter clearance. We previously showed that a
portion of an otherwise homogeneous holoenzyme preparation following thebinding to the APR promoter is converted into a complex incapable of synthe'
sizing full-length RNAs but capable of synthesizing abortive transcripts. This
non-productive complex, designated moribund complex, is converted into deadend complex which has lost any activity for elongation, constituting a deadend branched pathway of transcription initiation (Nuc. Acids Res. 24, 1380'
1381 (1996): J. Mol. BioI. 256, 449-457 (1996)).
We kinetically and biochemically isolated the nonproductive complexes atseveral promoters. But no such complexes are found at T7Al and rrnBPl
promoters, indicating the existence oftwo classes of promoter. Binary com'
plexes with APR -type promoters are resistant to heparin while T7Al type isnot (SEN et aI., J. BioI. Chern. 1998).

130

DNA and protein footprinting analyses showed that the dead-end complex isbacktracked from the promoter, lacks the strand opening and has more exposed
conserved region 3 of a 70 (Sen et aI. J. BioI. Chem. in press). This inactivation
was mitigated by a a 70 factor with S506F mutation in region 3 (SEN et aI., J.
BioI. Chem. 1998) or the Gre factors (GreA and GreB). These factors introduce
reversibility between productive and non-productive pathways so that moribund complex can be converted into productive complex at a high concentrationof the initiating nucleotide. This result shows that two branching pathways
diverge before binding ofthe initiating nucleotide and RNA synthesis.
The Gre factors that have been assigned to be elongation factors, therefore,
also work in initiation. GreB has stronger activity as an elongation factor
than GreA, but GreA turns out to be stronger than GreB as an initiation
factor in vitro. We have constructed single and double disruptants of greA
and greB, and the disruption of GreA shows stronger phenotype. This indicates that the Gre factors are initiation factors also in vivo. An analysis of 2dimensional protein gels and an analysis using gene array indicate 300 candidates for the target of Gre action.

(2)

Role of CI) subunit of E. coli RNA polymerase

Dipankar CHATTERJI 2 , Kakoli MUKHERJEE 2 , Hiroki NAGAI!, and Nobuo
SHIMAMOTO' ('Structural Biology Center, National Institute of Genetics, 2Centre of Cellular and Molecular Biology)
The function of w subunit of E. coli RNA polymerase has not been identified for decades, and a disruption of its gene rpoZ does not have any clear
phenotype. We have purified RNA polymerase core enzyme from a rpoZ
disruptant and found the enzyme is tightly bound to GroEL shaperon. The
core enzyme can bind to a 70 and active. It is inactivated, however, with a lossof binding activity to a 70 if the associated GroEL is removed with a dyeaffinity column or denaturation by urea. Reconstitution of activity is greatly
stimulated by the presence of w . These results show w is structurally required for maturation of core enzyme. The lack of any phenotype of rpoZ
mutant may be due to the functional supression by groEL.

H. STRUCTURAL BIOLOGY CENTER

131(3) Inactivation of (J70 by oligomerization and identification of therole of its spacer region
Hiroki NAGAI l , Taciana KAUSCIUKOVIC 2, Richard S. HAYWARD 2 Yumiko SAT0 1
and Nobuo SHIMAMOT0 1 (INat. Inst. of Genet./Grad. School ofAdv. Stud., 2lnst.
Of Cell and Molec. BioI., Edinburgh Univ., Scotland)
We have found that 0 70 , the major 0 factors ofE. coli, forms aggregate invivo and in vitro at a high temperature within physiological condition. The
oligomeric 0 70 has little transcriptional activity and the oligomerization invitro showed a sharp temperature dependence. We have constructed a strain
with a disrupted rpoD ( 0 70 ), and plasmid born 0 70 supports its growth. We
have observed a positive correlation between the intracellular concentrationof 0 70 and upper limit of growing temperature. This raises a possibility that0 70 is a molecular thermometer.
The major 0 factors of proteobacteria mostly have a big spacer region be'
tween the conserved regions 1 and 2 which is not conserved in eubacteria. In
E. coli 0 70 this region has extensive acidic patches which may be concernedwith the property of 0 70 to readily interact with nonspecific and specificsurfaces. An rpoD 'disrupted strain was constructed to test the viability ofstrains expressing plasmid-borne mutated rpoDor another sigma factor. E.
coli rpoS and M tubecuJosis sigA failed to complement the disruption, and weare now testing B. subtiJis sigA and others. The mutant 0 70 lacking the spacer
region of aa130-374 ( toSR) complemented the disruption at 30 and 25°C,
proving that the region is not essential for growth at low temperature. At all
tested temperature this protein predominantly exists as aggregate which arein equilibrium with a small fraction of monomer. Therefore, the role of thespacer region is the maintenance of active monomeric form.

(4) Single-Molecule Dynamics of Transcription: Sliding ofproteins along DNA
Takashi KINEBUCHI 1 , Kumiko SOGAWA 1 , Hiroyuki KABATA 1,2, Nobuo
SHIMAMOT0 1, Osamu KUROSAWA 2,3, Hironori ARAMAKI4, Masao WASHIZU 2(1 Structural Biology Center, National Institute of Genetics, 2Department of132

Mechanical Engineering, Kyoto University, 3Advance Co., 4Department ofMo"
lecular and Life Science, Daiichi College of Pharmaceutical Science)
We have showed the existence of a sliding motion ofprotein along DNA throughdirect visualization of single molecules of E. coli RNA polymerase (Science 262,
1561"1563 (1993». To check the generality of sliding, we applied similar single
molecule dynamics to a bacterial repressor protein, P. putida CamR, which wasobserved to slide along DNA. In the absence of its inducer, d"camphor, CamR
molecules were trapped at three positions on a A. DNA, one was its cognate
operator cloned in A. DNA, and the other two are likely to be homologous to theoperator. All trapping occurred at specific sites, and only sliding complexes
were observed at non"specific sites. This observation indicates that the non"
specific complex is the sliding complex itself.
The most distinct difference between the movements of RNA polymerase and
CamR was the pathway of dissociation from their specific sites. RNA polymerase
slides out of its specific site into nonspecific sites and then dissociate from non"
specific sites into bulk. This twO"step dissociation was not observed in the caseof CamR. CamR seemed to dissociate directly into bulk and its sliding upon
dissociation from the specific site was not long enough to be detected. CamR
also slides extensively upon association to the specific site, and thus long non"
specific DNA segment flanking the specific site accelerate association but notdissociation, making its affinity for the specific site on longer DNA
stronger. Thus long DNA can harvest CamR like an antenna.
There is a long"standing contradiction on E. coliTrpR that its specificity is
too small to compete binding to its operator against the predominant nonspe"
cific sites with the copy number present in the cell. We challenged to solve
this contradiction by introducing the concept of antenna effect by sliding. We
found that the affinity of TrpR for trpO strongly depends on the length of
DNA and is enhanced more than 10,000 "fold. A control experiment showedthat this enhancement is not due to the stabilization by an additional interaction with a long DNA. Therefore, antenna effect by sliding is really present
in vivo. This effect open up several new ways of gene regulation and further
proof of antenna effect will be obtained.

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133

Publ i cat ions
1.

2.

SHIMAMOTO, N.: One dimensional diffusion of proteins along DNA: its biological andchemical significance revealed by single-molecule measurements. J. BioI. Chern. 274,
15293-15296, 1999.
MUKHERJEE, K, NAGAI, H., SHIMAMOTO, N. & CHATTERJI, D.: GroEL is involved in activation of Escherichia coli RNA polymerase devoid of omega subunit in vivo. Eur. J.
Biochem. 266, 228-235, 1999.

H-c. Multicellular Organization Laboratory(1)

Fluoride-resistant Mutants of the Nematode Caenorhabdit/s eJegans

Akane OISHI, Takeshi ISHIHARA and Isao KATSURA
Fluoride-resistant (flr) mutations of C.elegans are recessive and grouped
into two categories: class 1 mutations, which map in fJrl,fJr3 and fJr4, andclass 2 mutations, which map in flr2 and flr5 (Katsura, 1. et aI.: Genetics
136, 145-154, 1994). Class 1 fJrmutations show many phenotypes: slow growth,short defecation cycle periods, frequent skip of the expulsion step of defecation' and synthetic abnormality in dauer formation (See (2) below), besides
strong resistance to fluoride ion. The flrl gene encodes an ion channel belonging to the DEGIENaC (G. elegansdegenerins and mammalian amiloridesensitive epithelial sodium channels) superfamily, while fJr4 and fJr3 code
for a novel Ser/Thr protein kinase and a kinase-like molecule, respectively,
both having a hydrophobic domain on the carboxyl-terminal side. A functional
flrl::GFP fusion gene is expressed only in the intestinal cells from the comma
stage of embryos to the adult stage, while flr4=:GFP is expressed in the intes'
tinal cells from the 1.5·fold stage, in the isthmus of the pharynx from the 3'
fold stage and in a pair of head neurons called ADA from L1larvae to adults.
Moreover, the expression of flr3::1acZis detected mainly in the intestine. We
therefore think that class 1 fJrgenes constitute a regulatory system that actsin the intestine and that controls many food'related functions. Class 2 flr
mutations, which confer weak resistance to fluoride ion, suppress the slow
growth and dauer formation abnormality but not the defecation abnormality

134

and strong fluoride-resistance of class 1 fJrmutations. Hence, it seems thatthe regulatory pathway bifurcates after class 1 genes, and that class 2 genes
control only one of the two branches. Of the class 2 genes, fJr -2 encodes asecreted protein belonging to the gremlinlDAN/cerberous family, most of which
are TGF- {3 antagonists.
We obtained the following results in 1999.
(j) Measuring the probability of climbing the petri dish of plate cultures, wefound that class 1 fJrmutants have lower tendency to stay on bacterial lawn.
This phenotype is suppressed by class 2 mutations. We are now investigating the relationship between this phenotype and the phenotype of dauer formation abnormality, which also suggests abnormality in chemoreception or
its regulation.
(ii) The four mutant alleles of fJr4have a mutation at a splice-acceptor site(ut3), a missense mutation in the C-terminal hydrophobic domain (ut7), andmissense mutations in the protein kinase domain (n2259, sa201 ),respectively_ While the former two mutants grow slowly, the latter two mutants grow at a normal rate, which is exceptional for class 1 fJrmutants. Of
the heterozygotes of these alleles, ut7/n2259and ut7/sa201 unexpectedly grewat a reduced rate, while ut3/n2259 and ut3/sa201 grew at a normal rate, as
expected. It is known that the heterozygotes n2259,4 and sa201,4 grow at a
normal rate, i. e., n2259 and sa201 are not haplo-insufficient. Rather, it seems
that ut7 acts as a dominant-negative allele to n2259 and sa201. The result
suggests that the FLR-4 protein may act as an oligomer or that the FLR-4(ut7)mutant protein may deprive FLR-4(n2259) and FLR-4(sa201 ) of the substrate.
(iii) We carried out temperature-shift experiments concerning the phenotype of defecation cycle periods, using the temperature-sensitive allele n2259.
The results showed that the active FLR-4 is required at the time of the defecation cycle assay. This suggests that fJr4 plays a role in a cell function that
controls defecation cycle periods, rather than in the generation or differentia"
tion of cells that are required for the normal defecation cycle period.
(iv) FLR-2 has homology to TGF- {3 antagonists. We therefore introduced
each of the four TGF- (3 genes in G.elegans(daf-7, dbl-1/cet-1, unc"129, F39G3.8)
into the fJr4(ut7) mutant as an extra chromosomal array. The transformants

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grew as slowly as the flr4 mutant. The results were the same when a heatshock promoter was used for driving the expression of the TGF· {3 genes. Thus,
there is so far no evidence showing that FLR'2 acts as a TGF' {3 antagonist.
(v) Afunctional flr2::GFP fusion gene was expressed in a small number ofneurons in the pharynx, head and tail. We are identifying these neurons.
(!)

Analysis of Synthetic Dauer-eonstitutive Mutations

Kouji MIYAHARA, Kiyotaka OHKURA, Takeshi ISHIHARA and Isao KATSURA
If newly hatched larvae of C. elegans are in a crowded state and given alimited food supply, they sense the environmental signal of pheromone andfood with head sensory organs called amphids and deviate from the normallife cycle, ending in non'feeding larvae called dauer larvae. Since the ll.ssay ofdauer formation is much less time'consuming than behavioral assays such as
chemotaxis assays, we are analyzing the head neural circuit by detecting dauer
formation as the output. We found that mutations in more than 50 known
genes show synthetic dauer'constitutive (Sdf'c) phenotypes, i.e.,they develop
to dauer larvae in a certain mutant background, regardless of the environ'mental conditions. The synthetic nature of the phenotype, we think, is basedon the structure ofthe neural circuit, in which neural signals are transmitted
through parallel routes consisting of different types of neurons so that two
mutations may be required to block the signals. We are studying the combi'
nations of mutations for the Sdf·c phenotype and the pattern of suppressionof the Sdf·c phenotype by various suppressor mutations. In this way we hope
to determine the functional neural network for dauer regulatory signals andthe function of relevant genes in the network. Furthermore, we isolated andmapped 44 new mutations that show the Sdf·c phenotype in combination withthe unc-31(e169) mutation. Many of them are expected to cause defects in ASI
neurons, since the unc-31 (e169) mutant shows dauer-constitutive phenotype,if ASI neurons are killed. Eight of the mutations mapped in 4 known genes(tax-2, osm-6, che'l1, and aex-3), but most of the remaining 36 mutations,which map in at least 13 genes, seem to be alleles of novel genes. Of thesemutants, that in sdl'1 gene (1 allele) avoids benzaldehyde, isoamyl alcohol,

136butanone. NaCl, and lysine, which are attractants of wild-type animals. It
behaved as thermophilic in thermotaxis. Mutants in sdf-13 gene (2 alleles)
showed defects in adaptation of chemotaxis to benzaldehyde, isoamyl alcoholand butanone, although they showed normal chemotaxis to these attractants.
This gene encodes a homolog of mammalian Tbx2 and Drosophila Omb, atranscription factor containing the T-box domain.
We obtained the following results in 1999.
(i) The two sdf-13alleles (ut180, utl92) had amino acid substitutions for thesame lysine residue in the T-box domain: glutamic acid in one allele and arginine in the other. For expression studies, GFP cDNA was inserted at the beginning and the end ofthe sdf-13 coding region, respectively, and sdf-13 mutant animals were transformed with the DNA constructs which also contained
a 1.5-kb 5' upstream sequence. While the former transformant showed mutant phenotypes, the latter transformant showed wild type phenotypes. BothGFP fusion genes were expressed in M2 and 15 neurons in the pharynx. We
are now making antibodies against SDF-13 to confirm the expression pattern
and to test if SDF-13 acts in pharyngeal neurons or amphid sensory neurons.
(ii) Of the genes identified by the new synthetic dauer-constitutive mutations, sdf-9has the largest number (five) of alleles. Mutants in sdf-9produce
many dauer larvae in the presence of a chromosomaIJy integrated array of adaf-7.:GFP transcriptional fusion gene, where daf-7encodes a TGF- 13 protein that represses dauer formation. Interestingly, these dauer larvae recover
spontaneously and grow to adults.

(3)

Analysis of Mutants That Show Abnormality In the Selection
between Two Behaviors

Takeshi ISHIHARA, Yuichi IIN0 1 and Isao KATSURA (I Molecular Genetics Laboratory, University of Tokyo)
The nematode G.elegans shows avoidance of copper ion and chemotaxis toodorants by perceiving them with different sensory neurons in the head. We
devised a behavioral assay method for the interaction between the two responses to learn a possible role of interneurons. Wild-type animals change

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their preference between the responses, depending on the relative concentration of copper ion and odorants. This suggests that the two sensory signals
interact with each other in a neural circuit consisting of about 10 neurons, on
the basis of the neural circuitry of C. elegans and the identity of the sensory
neurons that act in these behaviors.
While these experiments were performed with well-fed animals, animals
starved for 5 hours showed a stronger preference for odorants, because they
responded more weakly to copper ion. This starvation effect was not detectedin the presence of serotonin, which is considered to induce a well'fed state.
The behavioral change upon starvation seems appropriate, because starved
worms can look for food over a wider area.
To elucidate the mechanism of the complex behaviors mentioned above, we
are isolating and analyzing mutants that show abnormality in thesebehaviors. The mutant ut236had a tendency to choose avoidance of copper ion
rather than chemotaxis to odorants, although it showed no abnormality in eachbehavior in case only one of the stimuli was given. The result shows that ut236
is abnormal in the interaction between the two behaviors. Furthermore, ut236
seems to be abnormal in the conditioning (associative learning) by the paired
presentation of starvation and NaC!. Thus, it probably has abnormality in many
aspects of sensory signal processing. We identified the gene for ut236by positional cloning. It encoded a novel secretory protein having an LDL receptor
ligand-binding domain. A functional GFP fusion gene was expressed in a small
number of sensory neurons and interneurons. We are investigating the expres'sion of ut236by antibodies against its recombinant protein.
The mutant ut235 lacked the effect of starvation in the above assay, but wasessentially normal in the increase of locomotion speed by starvation and invarious responses to food. Namely, this mutant has defects only in part of the
behavioral responses to starvation. The double-mutant ut235; ut236 showed apreference to avoidance of copper ion, regardless of starvation. We are now
trying to identify the gene responsible for the ut235 mutation by positional
cloning.

138

Publ icatians
1.

FUJIWARA, M., ISHIHARA. T. and KATSURA, I.: A novel WD40 protein, CHE-2, acts cellautonomously in the formation of C.eleganssensory cilia. Development 1%6, 4839-4848,
1999.

2.

WINNIER, A.R., MEIR, J.Y.-J., Ross, J.M., TAVERNARAKIS. N., DRISCOLL. M., ISHIHARA, T.,
KATSURA. I., and MILLER, III D.M.: UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans. Genes Dev. 13,
2774-2786, 1999.

3.

HASHIMOTO, H., NISHINO, A., SHINTANI, N., HAGIHARA, N., COPELAND, N,G" JENKINS, N,A.,
YAMAMOTO, K, MATSUDA, T., ISHIHARA, T., NAGATA, S. and BABA. A.: Genomic organizationand chromosomal location of the mouse vasoactive intestinal polypeptide 1 (VPACI)
receptor. Genomics, 58, 90-93, 1999.

4.

OKUDA, T., HAGA, T., KANAI, Y., ENDOU, H., ISHIHARA, T. and KATSURA. I.: Identificationand characterization of the high-affinity choline transporter. Nature Neurosci., 3, 120125,2000.

H-d. Biomolecular Structure Laboratory(1) Crystallographic Study of F1·ATPase:Structurai Analysis of Sopramolecole
Yasuo SHIRAKIHARA and Aya SHlRATORIFI-ATPase, with a subunit composition of 0: 3/3 3 Y a E, is a catalytic sectorof the membrane bound ATP synthase. The ATP synthase plays a central rolein energy conversion in mitochondria, chloroplasts and bacteria, generating
ATP from ADP and inorganic phosphate using energy derived from a transmembrane electro'chemical potential. We previously solved the structure of
the 0: 3/3 3sub'assembly of FI-ATPase from a thermophilic bacterium Ba.cillus PS3. We have been extending the structural study to a nucleotide'boundform of the 0: 3/3 3 sub-assembly and to 0: 3/3 3 Y and 0: 3/3 3 Y E sub-assemblies.
We previously attempted to look at the structure of the nucleotide'boundform of the 0:3/33 sub-assembly by analyzing x'ray diffraction data from thecrystals, originally grown in absence of nucleotide but later soaked in nucleotide'containing media. We could trap a state where the over-all structure is

H. STRUCTURAL BIOLOGY CENTER

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very similar to that ofthe nucleotide"free state, but where phosphate binding
to the {3 "subunit gets weaker than in the nucleotide-free state. In order tolook at other states distinctly different from the above, we have tried to crys·tallize the sub-assembly in presence of non"hydrolyzable nucleotide analog
AMPPNP, and got crystals. We plan to examine a structure the crystals will
present.
In recent years, we have made considerable efforts to get good crystals of thea 3 {3 3 Y sub"assembly, as the sub"assembly exhibits kinetic properties very
similar to those ofF1, and a number of interesting mutant sub"assemblies havebeen engineered. Both preparation method and crystallization conditions havebeen examined extensively in this year as well. It has now been establishedthat treatments to the crude extract such as heat treatment and ammonium
sulfate fractionation are very effective. A different combination of chromato"graphic columns, like gel filtration (1st) and weak hydrophobic (2nd) columns
gave performance slightly better than the usual combination of hydrophobic
and ion exchange columns. Inclusion of sodium sulfate, which is thought tostabilize the sub"assembly, in the chromatographic solution had a good effecton crystallization, but inclusion of ATP, assumed to playa similar role of SO"
dium sulfate, was not effective. A single most important point in improving
crystallization found this year was inclusion ofnon"ionic detergent like LAO
and Decyl-trio"maltoside in the mother liquor. When they were included, thecrystals grown in those conditions were much larger, at least in two directions,and better-shaped, that is, like plates or columns rather than petals whichwere usually seen in the previous crystallization experiments. It took sometime to be able to record diffraction pattern from these crystals, because, as
found later, the crystals were sensitive to changes of environment and seemed
to have very weak diffraction capability (to 20 Aresolution). It is being planned
to do diffraction experiment in SPRING8 with very brilliant x"ray source onthese crystals, and, for that purpose, search for the cooling conditions for thesecrystals is in progress.
Eighteen mutant a 3 {3 3 y sub· assemblies were subjected to crystallization
study this year. The mutations were in the {3 ·subunit and may be classified
into 3 groups: 1) a hinge region of the subunit was changed to interfere withthe hinge bending motion of the subunit, 2) the DELSEED region which inter-140

act with the y -subunit was modified, and 3) cross link between two {3 "subunits could be under control. Mutants belonging groupl gave somewhat larger
crystals than the wild type.
The a 3 {3 3 y E sub"assembly preparation got available due to overproduction system of this multi"subunit assembly established by Dr S. Suzuki at
Tokyo Institute of technology. The preparation crystallized fortunately at theconditions for the a 3 {3 3 Y sub-assembly. Further purification with a weakhydrophobic column produced better preparation which could be crystallized
into much larger bi-pyramids with sizes of about O.3mm. The hydrophobic
column removed contaminating a3{33 sub-assembly which could have interfered with crystallization of the a 3 {3 3 Y E sub"assembly. Further efforts arebeing made to tune up the crystallization conditions.
These structure studies were done in collaboration with Masasuke Yoshida,
Toyoki Amano, Eiro Muneyuki, Tomoko Masaike, Kiyotaka Hara, Shunji
Suzuki and Satoshi Tsunoda at Research Laboratory of Resource Utilization,
Tokyo Institute of technology.
(2)

Crystallization of ATPsynthase

Kazuyasu SHINDO and Yasuo SHIRAKIHARA
ATPsynthase may be described as a combination of soluble Fl and a membrane"embedded Fo (with subunit composition of ab2c8-l2), and is therefore amembrane protein, a challenging target for crystallization. We started to ex"
amine purification method for the ATPsynthase extracted with Dodecylmaltosidefrom the PS3 membrane. Also we started to screen crystallization conditions
for an assembly comprising Fl and a soluble portion of the b subunit (b '), of
which preparation were provided by Dr S. Suzuki at Tokyo Institute oftechnol"ogy.
The structure study were done in collaboration with Yoshida, Shunji Suzuki
and Dirk Bald at Research Laboratory of Resource Utilization, Tokyo Institute of technology.

H. STRUCTURAL BIOLOGY CENTER

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141

Crystalllzation of cytoplasmic domain of CD40 receptor In B-cell

Yasuo SHlRAKlHARA
CD40 signalings play crucial roles in B-cell function. Signals through CD40
rescue B cells from apoptosis induced by cross-linking ofthe surface immunoglobulin complex and also induce B cells to differentiate. TRAF5 and TRAF6,
signal transducers, interact with a cytoplasmic tail (about 70 amino acid residue) of CD40. We tried to crystallize the cytoplasmic tail to understand themechanism of signal transduction using that part. The cytoplasmic portion(aa 216'277)was expressed in E.coli as GST fusion protein, and was purified
with Glutathione-Sepharose. It was anticipated that the tendency of GST
part to crystallize could be utilized to crystallize the fusion protein, as in thecase ofMrellfragment-GST fusion protein reported last year. The crystallization condition search was extensive, however, ended up in finding only
dendlytes formation conditions (ammonium sulfate at pH5).
The crystallization study were done in collaboration with Jun-ichiro Inoue,
Asuka Naitoh at the Institute of medical science, The university of Tokyo.
(4)

Crystallization of D-amlnoacylase from Aleallgenes

Yasuo SHlRAKIHARA
D-aminoacylase catalyzes hydrolysis of N'acylderivatives of various neu'
tral D-amino acids to D-amino acids and fatty acids. The enzyme has not
only industrial relevance with potential to produce large amount ofD'aminoacids, but also possibly unique active site structure that distinguishes L-aminoacids that are abundant in nature. The enzyme from Alcaligenes xylosoxydanssuhsp. xylosoxydans A -6was purified from cell extract of overproducing Eeali
with DEAE ion exchange and butyl Toyopearl columns. The crystallization
condition search was done with grid screen and crystal screen from Hampton
Research. Small crystals grew from MPD or isopropanol solutions, however,they were not reproducibly obtained. Purity of preparations was found to bevariable, and therefore efforts have been made to establish a solid prepara'142

tion procedure.
The crystallization study was done in collaboration with Mituaki Moriguchi,
Akiko Sato, at Oita University, and Mamoru Wakayama, at Ritsumeikan
University.

(5)

Crystallization of Na+-translocating ATPase

Yasuo SHlRAKlHARA
Na+·translocating ATPase from Enterococcus hirae is classified as a Vacuo
olar-type ATPase (V·type), and is expected to have a structure similar to Fl'
ATPase (F·type ATPase) from amino acid sequence comparison. Na+·translo·
eating ATPase was highly purified and was subjected to crystallization
experiment. Plate shaped crystals with largest dimension of O.2mm was pro'
duced from polyethylene glycol solution, and was found to diffract to 6 A
resolution. Further diffraction studies conducted this year allowed us to record
weak diffraction spots at 3.6 A resolution. Preparation methods are still be'
ing improved to get better crystals. This work has been done in collaboration
with Toshiyuki Meguro and Ichiro Yamato, Science University of Tokyo.

Publ ication
1.

SHIRAKIHARA, Y.:

ATP synthase. Protein, Nucleic acid and Enzyme. 44, 538'545, 1999.

143

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I-a. Laboratory for DNA Data Analysis(1)

Reconstruction of the Ancestral Genome and Evolutionary Study ofGenome Structures by Comparison of Complete Genome Sequences

Takeshi ITOH and Takashi GOJOBORI
A number of complete genome sequences have been determined in various
species such as microbes. These sequence data gave us a unique opportunity
to investigate the evolution of the whole genome structures. By comparison oforthologous gene orders, we elucidated that gene orders had been frequently
shuffled during long-term evolution, and that operon structures, which arethought to be functionally important, had been evolutionarily unstable.
In this study, in order to effectively detect orthologous gene pairs between
distantly related species, we improved our method for ortholog-finding in thefollowing two points: (1) we employed log-normalized Smith-Waterman scoresfor evaluation of sequence similarity significance, (2) gene fusion events wereconsidered. By comparing gene orders of orthologs defined by this method, wereconstructed the ancestral genome structure between eubacteria andarchaebacteria, and estimated the degrees of genome stability. As a result,
we found that the degrees of genome instability were positively correlatedwith the occurrence frequencies ofinsertion sequences (ISs). This implies thatISs strongly affect genome stability. Moreover, it was revealed that frequent
rearrangements of genomes diminished strand"specific nucleotide biases such
as GC-skew.
In a comparison of gene fusion between eukaryotes and prokaryotes, it wasstrongly suggested that eukaryotes possess more fused genes than prokaryotes, and almost all of fused genes in eukaryotes appeared to be derived fromprokaryotic operons. These observations indicate that the prokaryotic genome
structure is ancestral, and eukaryotes have lost the structure but partly re-144tained them as forms of fused genes. Drastic changes of the genome structureand gene regulation may have happened in the early evolution of the eukaryotic ancestor.

(Z)

Different evolutionary histories In two subgenomlc regions of the
major histocompatiblllty complex

Silvana GAUDIERI, Jerzy K. KULSKI l, Roger L. DAWKINS l and Takashi GoJYOBORI(lCenter for Molecular Immunology and Instrumentation, University of Western Australia)
Two subgenomic regions within the major histocompatibility complex, thealpha and beta blocks, contain members of the multicopy gene families HLAclass I, human endogenous retroviral sequence (HERV-16; previously known
as P5 and PERB3), hemochromatosis candidate genes (RCG) (II, IV, VIII, IX),
3.8-1, and MIC (PERBll). In this study we show that the two blocks consist ofimperfect duplicated segments, which contain linked members of the different
gene families. The duplication and truncation sites of the segments are associated with retroelements. The retroelement sites appear to generate theimperfect duplications, insertions/deletions, and rearrangements, most likely
via homologous recombination. Although the two blocks share several characteristics, they differ in the number and orientation of the duplicatedsegments. On the 62.1 haplotype, the alpha block consists of at least 10 duplicated segments that predominantly contain pseudogenes and gene fragmentsof the HLA class I and MIC (PERBI0 gene families. In contrast, the beta
block has two major duplications containing the genes HLA-B and HLA-C,
and MICA (PERBl1.1) and MICB (PERBl1.2). Given the common origin between the blocks, we reconstructed the duplication history of the segments tounderstand the processes involved in producing the different organization in
the two blocks. We then found that the beta block contains four distinct duplications from two separate events, whereas the alpha block is characterized bymultisegment duplications. We discussed these results in relation to the genetic content of the two blocks. See Gaudieri et al. (Genome Res 9:541-549,
1999) for details.

I.

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145

Identification of a ribonuclease H gene In both Mycoplasma
genltaIJum and Mycoplasma pneumonJae by a new
method for exhaustive Identification of ORFs In thecomplete genome sequences

Matthew I. BELLGARD and Takashi GOJOBORI
Exhaustive identification of open reading frames in complete genome sequences is a difficult task. It is possible that important genes are missed. In
our efforts to reanalyze the intergenic regions of Mycoplasma genitalium andMycoplasma pneumoniae, we have newly identified a number of new open
reading frames (ORFs) in both M genitalium and M pneumoniae. The mostsignificant identification was that of a ribonuclease H enzyme in both species
which until now has not been identified or assumed absent and interpreted assuch. We discusses the biological importance of RNase H and its evolutionary
implication. We also stressed the usefulness of our method for identifying
new ORFs by reanalyzing intergenic regions of existing ORFs in complete
genome sequences. See Bellgard and Gojobori (FEBS Letters 445:6-8, 1999)
for details.

(4)

Significant differences between the G+C content of synonymouscodons In orthologous genes and the genomic G+C content

Matthew I. BELLGARD and Takashi GOJOBORIThe relationship between the overall G+C content of the genome (GC) and
the GC content at the third codon positions (GC3) of genes, which we refer to asa GC3"plot, was examined using 15 currently available complete genome
sequences. A remarkably linear relationship was found between these twoquantities, confirming previous observations of a strong positive correlationin the GC3-plot. In order to conduct a more detailed analysis ofthe GC3-plot,
we examined the GC3 content by separating orthologous codons into three
categories: synonymously different codons (namely identical amino acids, W,
different amino acids (DA), and identical codons (IC), for a pairwise compari"146

son of two closely related species. When we took pairwise species comparisons between Mycoplasma genitalium (Mg) and Mycoplasma pneumoniae (Mp)and between Mycobacterium tuberculosis (Mt) and Mycobacterium leprae (Ml)as examples, we found that for Mp and MI, the GC3 for IA deviated the mostfrom the linear expectation in the GC3-plot, whereas for Mg and Mt the deviation was minimal. These findings suggest that the major changes of GC content took place in Mp and MI, but not in Mg and Mt. This analysis alsoenables us to predict the future direction of the evolutionary changes of the
genomic GC content. See Bellgard and Gojobori (Gene 238:33-37, 1999; Trends
in Genetics 15:254-256, 1999) for details.

(5)

MITEs, interspersed nuclear elements, in the plant genomes andtheir significance in the piant evolution

Katsuhiko MINETA, Kazuho IKEO, Yasunari OGlHARA l and Takashi GOJOBORI(IYokohama City University)
The chloroplast is the organelle where photosynthesis takes place, and thechloroplast genome is highly suitable for molecular evolutionary analyses ofplants. Focusing on the chloroplast genome in higher plants, we looked forevidence of genomic rearrangements in the evolutionary history of thisorganelle. We compared the complete chloroplast genomes of five species. We
found one large inversion evident between the genomes of monocotyledons anddicotyledons. We also found that the physical ordering of orthologous genes
was strongly conserved. To investigate possible mechanisms of genome rearrangement in the chloroplast genome, we searched for the transposable
elements. Consequently, we found a putative MITE sequence, that seems tohave been inserted following the emergence of monocotyledons. This discoveryof MITE sequences in the chloroplast genome of monocotyledons is the first of
its find. However, transposable elements in organella genomes are extremely
uncommon, and may not playa role in rearrangement of these genomes.
Alternatively, a mechanism may exist whereby transposable elements are
rapidly eliminated from the chloroplast genome after rearrangements havetaken place.

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CENTER FOR INFORMATION BIOLOGY147

Pub I i ca t ions
1.
2.

3.

4,
5.

6.

7.

8.

9.

10,

11.
12.

GOJOBORI, T: Evolution, in The Encyclopedia of Molecular Biology, T,E. Creighton,
Editor. John Wiley & Sons: NY. (1999).
HATIA, Y, OHASHI, J., IMANISHI, T., KAMIYAMA, H., IHA, M., SIMABUKURO, T., OGAWA, A.,TANAKA, H., AKAZA, T, GOJOBORI, T., JUJI, T. and TOKUNAGA, K: HLAgenes and haplotypes
in Ryukyuan suggest a recent gene flow to the Okinawa islands. Human Biology. 71(3):353-365 (1999).
GAUDIERI, S., KULSKI, J.K, DAWKINS, RL. and GOJOBORI, T.: Extensive nucleotide vari'ability in a 370kb sequence within the central region of the major histocompatibility
complex. Gene Special Issue. 238(1): 157-161 (1999).
SUZUKI, Y. and GOJOBORI, T.: A method for detecting positive selection at single aminoacid sites. Mol. Bol. Evol. (16): 1315-1328 (1999).
REN, F" TANAKA, H., OKAYAMA, T. and GOJOBORI, T.: Reconstructing molecular phylogenetic tree with multifurcation by using minimum complexity principle. DNA and Molecular Computing. :1825-1828 (1999).
TANAKA, H., REN, F., OKAYAMA, T. and GOJOBORI, T.: Topology selection in unrooted
molecular phylogenetic tree by minimum model-based complexity method. in PacificSymposium on Biocomputing'99. Hawaii, USA. (1999).
SATO, S., TOYODA, R, IKEO, K, GOJOBORI, T., KATSUYAMA, Y, SAIGA, H., NUMAKUNAI, T.,
YAJIMA, I. and YAMAMOTO, H.: Structure and developmental expression of the ascidian
TRP Gene: Insights into the evolution of pigment cell-specific gene expression. Developmental Dynamics. 215: 225-237 (1999).
BABA, T., SHIINA, T., ANDo, A., IMANISHI, T., MATSUNO, N., SAKURAI, E., NAGAo, T., TANAKA,
K., GOJOBORI, T. and INOKO, H.: Isolation and characterization of a pig major histocompatibility complez (SLA) class II DNA cDNA clone. Immunogenetics. 49: 915-917(1999).
TAZAKI, A., GAUDIERI, S., IKEO, K, GOJOBORI, T., WATANABE, K and AGATA, K: Neuralnetwork in planarian revealed by an antibody against planarian synaptotagmin homologue. Biochemi. Biophys. Res. Commun. 260(2): 426-432 (1999).
GAUDIERI, S., KULSKI, J., DAWKINS, Rand GOJOBORI, T.: Different evolutionary histories
in two subgenomic regions of the major histocompatibility complex. Genome Research.
9(6): 541-549 (1999).
BELLGARD, M. and GOJOBORI, T.: Inferring the direction of evolutionary changes of genomic base composition. Trends in Genetics. 15(7): 254-256 (1999).

GOJOBORI, T.: Part VI. Summary, in Molecular Starategies in Biological Evolution, L.H.
Caporale, Editor. Annals ofthe New Thrk Academy ofSciences: New York. p. 339-340(1999).
13. BELLGARD, M., ITOH, T., WATANABE, H., IMANISHI, T. and GOJOBORI, T.: Dynamic Evolu-

148
tion of Genomes and the Concept of Genome Space, in Molecular Starategies in Biological Evolution, L.H. Caporale, Editor. Annals of the New York Academy ofSciences:
New York. p.293-300 (1999).
14. GOJOBORI, T.: Part VI. Introduction, in Molecular Starategies in Biological Evolution,
L.H. Caporale, Editor. Annals ofthe New York Academy ofSciences: New York. p.292(1999).
15.

ITOH, T., OKAYAMA, T., HASHIMOTO, H., TAKEDA, J.-I., DAVIS, R.W., MORI, H. and GOJOBORI,
T.: A low rate of nucleotide changes in Escherichia coliK-12 estimated from a comparison of the genome sequences between two different substrains. FEBS Letters. 450: 7276 (1999).

16.

NAKAMURA, Y., GOJOBORI, T. and IKEMURA, T.: Codon usage tabulated from the international DNA sequence databases: its status 1999. Nucl. Acids Res. 27(1): 292 (1999).
SHIINA, T., OKA, A, lMANISHI, T., HANZAWA, K., GOJOBORI, T., WATANABE, S. and INOKO, H.:
Multiple class I loci expressed by the quail MHC. Immunogenetics. 49(5): 456-460(1999).
TAKEZAKI, N. and GOJOBORI, T.: Correct and incorrect vertebrate phylogenies obtainedby the entire mitochondrial DNA sequences. Mol. Bol. Evol. 16(5): 590-601 (1999).
SUGAWARA, H., MIYAZAKI, S., GOJOBORI, T. and TATENO, Y.: DNA Data Bank of Japan
dealing with large'scale data submission. Nucl. Acids. Res. 27(1): 25-28 (1999).
BELLGARD, M. and GOJOBORI, T.: Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive
identification of ORFs in the complete genome sequences. FEBS Letters. 445(1): 6-8(1999).
ITOH, T., TAKEMOTO, K., MORI, H. and GOJOBORI, T.: Evolutionary instability of operon
structures disclosed by sequence comparisons of complete microbial genomes. Mol.
Biol. Evol. 16(3): 332-346 (1999).
SUZUKI, Y., KATAYAMA, K., FUKUSHI, S., KAGEYAMA, T., OYA, A, OKAMURA, H., TANAKA, Y.,MIZOKAMI, M. and GoJOBORI, T.: Slow evolutionary rate ofGB virus ClHepatitis G virus.
J. Mol. Evol. 48(4): 383-389.(1999).
OPHIR, R., ITOH, T., GRAUR, D. and GOJOBORI, T.: A simple method for estimating theintensity of purifying selection in protein-coding genes. Mol. Biol. Evol. 16(1): 49-53(1999).
SHIINA, T., SHIMIZU, C., OKA, A, TERAOKA, Y., IMANISHI, T., GOJOBORI, T., HANZAWA, K.,
WATANABE, S. and INOKO, H.: Gene organization of the quail major histocompatibility
complex (MhcCoja) class I gene region. Immunogenetics. 49(5): 384-394 (1999).
MIZOKAMI, M., lMANISHI, T_, IKEO,K., SUZUKI, Y., ORITO, E., KUMADA, T., UEDA, R., IINO, S.
and NAKANO, T.: Mutation patterns for two flaviviruses: hepatitis C virus and GB
virus Clhepatitis G virus. FEBS Letters 450: 294-298 (1999).
GEIiRING, W.J., IKEO, K.: Pax 6: mastering eye morphogenesis and eye evolution.
Trends Genet. 1999 Sep:15(9): 371-7 (1999).

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I-b. Laboratory for Gene-Product Informatics(1)

Cooperative approach for the protein fold recognition

Motonori OTA, Takeshi KAWABATA, Akira R. KINJO and Ken NISHIKAWA
We, four independent predictors, organized a team and tackled blind protein
structure predictions using fold recognition methods. We tried to assign thehomologous or analogous folds in the protein structure database for a numberof target sequences that showed no apparent sequence homology to the pro'teins of known folds. After primary analyses by conventional softwares, thesesequences were threaded through the structural library using three different
programs developed by ourselves, which employed different compatibility
functions. Collecting the results of our individual analyses, and the available
biological knowledge about the target, we held meetings and discussed allplausible structures for the target. For 25 target sequences, we submitted 56
models including NONE: This was the first time the fold was determined. At
the time of the meeting (CASP3), 19 protein structures (21 domains) catego'
rized as the threading targets were available. We succeeded in predicting
eight out of 18 targets (20 domains) that we submitted; however, alignment
accuracies were not satisfactory for some of the models. We often obtainedcorrect answers even if some of us missed the right prediction; therefore it
would appear that our threaders compensated each other. When all the information is managed effectively, the prediction gains more accuracy. See Ref. 2
for the details.

(2)

Strong hydrophobic nature of cysteine residues in proteins

Nozomi NAGANO l , Motonori OTA, Ken NISHIKAWA (IBiochemistry and Molecu'
lar Biology Department, University College London)
The differences between disulfide'bonding cystine (Cys_SS) and free cys'
teine (Cys_SH) residues were examined by analyzing the statistical distribu'
tion of both types of residue in proteins of known structure. Surprisingly, Cys_

150

SH residues display strong hydrophobicity than Cys_SS residues. A detailed
survey of atoms which come into contact with the sulfhydryl group (sulfuratom) of Cys_SH revealed that contacting atoms are essentially the same innumber and variety as those of the methyl group of isoleucine, but are quite
different to those of the hydroxyl group of serine. Moreover, the relationships
among amino acids were also determined using the 3D-profile table of known
protein structures. Cys_SH was located in the hydrophobic cluster, along withresidues such as Met, Trp and Tyr, and was clearly separated from Ser and Thrin the polar cluster. These results imply that free cysteines behave as stronglyhydrophobic, and not hydrophilic, residues in proteins. See Ref. 3 for the details.

(3)

Design and synthesis of a globin foldYasuhiro ISOGAI 1, Motonori OTA, Tetsuro FUJISAWA2, Hiroyuki IZUN0 3, Masahiro
MUKAI 1, Hiro NAKAMURA 1, Tetsutaro IIZUKA 1 and Ken NISHIKAWA (IThe Institute of Physical and Chemical Research (RIKEN), 2RIKEN Harima Institute,
3Gakushuin University)
We propose a simple method to find an amino acid sequence that is foldable
into a globular protein with a desired structure based on a knowledge-based
3D-1D compatibility function. An asymmetric a -helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues waschosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The
heme-binding site was designed by fixing His64 and His93 at the distal andproximal positions, respectively, and by penalizing residues that protrude into
the space with a repulsive function. The apparent bumps among side chainsin the computer model of the converged, self-consistent sequence were removed
by replacing some of the bumping residues with smaller ones according to thefinal 3D profile. The finally obtained sequence shares 26% of sequence with
the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia
coli. Analyses using size-exclusion chromatography, circular dichroism spec-

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troscopy, and solution X-ray scattering showed that DG 1 folds into a monomeric, compact, highly helical, and globular form with an overall molecularshape similar to the target structure in an aqueous solution. Furthermore, itbinds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A,
slightly larger than that of natural apoMb, and decreased to 19.5A upon heme
binding based on X-ray scattering analysis. However, the heme-bound DG1
did not stably bind molecular oxygen as natural globins do, possibly due tohigh conformational diversity of side-chain structures observed in the NMR
and denaturation experiments. These results give insight into the relation'
ship between the sequence selection and the structural uniqueness of naturalproteins to achieve biological functions. See Ref. 4 for the details.

(4)

Experimental verification of the . stability profile of mutant
protein' (SPMP) data using mutant human lysozymes

Kazufumi TAKANO l , Motonori OTA, Kyoko OGASAHARA l , Yuriko YAMAGATA2,Ken NISHIKAWA and Katsuhide YUTANI l (IInstitute for Protein Research, Osaka
University, 2Graduate School of Pharmaceutical Science, Osaka University)
The stability profile of mutant protein (SPMP) (Ota, M., Kanaya, S. andNishikawa, K., 1995, JMol.Biol., 248, 733'738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudoenergy potential developed for evaluating structure-sequence compatibility inthe structure prediction method, the 3D'lD compatibility evaluation. Nine
mutant human lysozymes expected to significantly increase in stability from
SPMP were constructed, in order to experimentally verify the reliability of
SPMP. The thermodynamic parameters for denaturation and crystal struc'tures of these mutant proteins were determined. One mutant protein wasstabilized as expected, compared with the wild-type protein. However, theothers were not stabilized even though the structural changes were subtle,
indicating that SPMP overestimates the increase in stability or underesti'mates negative effects due to substitution. The stability changes in the othermutant human lysozymes previously reported were also analyzed by

152

SPMP. The correlation of the stability changes between the experiment andprediction depended on the types of substitution: there were some correIa"
tions for proline mutants and cavity'creating mutants, but no correlation formutants related to side'chain hydrogen bonds. The present results may indio
cate some additional factors that should be considered in the calculation of
SPMP, suggesting that SPMP can be refined further. See Ref. 5 for the details.

Pub I i ca t ions
1.

NISHIKAWA, K: "Protein structure prediction", in The Encyclopedia ofMolecular Biology(Ed., T.E. Creighton), John Wiley & Sons, Inc, 1999.

2.

OTA, M., KAWABATA, T., KINJO, A.R. and NISHIKAWA, K: "Cooperative approach for theprotein fold recognition", Proteins: Str. Func. Genet., Suppl. 3, 126-132, 1999.

3.

NAGANO, N., OTA, M. and NISHIKAWA, K: "Strong hydrophobic nature of cysteine resi-

4.

dues in proteins", FEBS Lett., 458, 69-71, 1999.
ISOGAI, Y., OTA, M., FUJISAWA, T., IzuNo, R., MUKAI, M., NAKAMURA, R., IIZUKA, T. andNISHIKAWA, K: "Design and synthesis ofa globin fold", Biochemistry, 38,7431-7443,

5.

1999.
TAKANO, K, OTA, M., OGASAHARA, K, YAMAGATA, Y., NISHIKAWA, K and YUTANI, K:"Experimental verification of table for 'Stability Profile of Mutant Protein' (SPMP)
using mutant human lysozymes", Protein Eng., tZ, 663-672, 1999.

6.

TATENO, Y., MIYAZAKI, S., OTA, M., SUGAWARA, R. and GOJOBORI, T.: "DNA data bank ofJapan (DDBJ) in collaboration with mass sequencing teams", Nucleic Acids Res. Z8,
24-26, 2000.

I-c. Laboratory of Gene Function(1)

DNA Data Bank of Japan dealing with large-seale data submission

Hideaki SUGAWARA, Satoru MIYAZAKi, Takashi GOJOBORI and Yoshio TATENOThe DNA Data Bank of Japan (DDBJ) has developed a software system formass submission to cope with a recent expansion of EST and genome datasubmissions. The system is composed of four parts, the WWW data submis'
sion, large'scale submission, submission management and storing. Using thissystem one can submit data on a large number of sequences or a very longI.

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sequence while checking the consistency between the annotation and sequence
without much effort. DDBJ has received large-scale data of Homo sapiens,
Arabidopsis and Pyrococcus from Japanese researchers who made full use of
the new submission system. See Ref. 1 for details.
(!)

A phosphate-induced gene which promotes Pen/cHI/am mediatedbioconversion of cis-propenylphosphonic acid to Fosfomycin.
Applied and Environmental Microbiology

M.WATANABE 1, N. SUMIDA 1, S. MURAKAMI!, H.ANzAII, C.J. THOMPSON2 , Y. TATENO

and T. MURAKAMI l (IMeiji Seika Kaisha, Ltd., 2University of Basel)

Penicillium decumbensis able to epoxidize cis-propenylphosphonic acid (cPA)to produce the antibiotic fosfomycin [FOM; also referred to as phosphonomycinand (-)-cis-l,2-epoxypropylphosphonic acid], a bioconversion of considerable
commercial significance. We sought to improve the efficiency of the process byoverexpression of the genes involved. A conventional approach of isolating thepresumed epoxidase and its corresponding gene was not possible since cPA
epoxidation could not be achieved with protein extracts. As an alternative
approach, proteins induced by cPA were detected by two"dimensional gel
electrophoresis. The observation that a 31-kDa protein (EpoA) was both cPA
induced and overaccumulated in a strain which more efficiently converted cPA
suggested that it might take part in the bioconversion. EpoA was purified, itsamino acid sequence was partially determined, and the corresponding gene
was isolated from cosmid and cDNA libraries with oligonucleotide probes. The
DNA sequence for this gene (epoA) contained two introns and an open reading
frame encoding a peptide of 277 amino acids having some similarity tooxygenases. When the gene was subcloned into P. decumbens, a fourfold in"crease in epoxidation activity was achieved. epoA-disruption mutants whichwere obtained by homologous recombination could not convert cPA to FOM. To
investigate the regulation of the epoA promoter, the bialaphos resistance gene(bar, encoding phosphinothricin acetyltransferase) was used to replace the
epoA-coding region. In P. decumbens, expression ofthe bar reporter gene was
induced by cPA, FOM, and phosphorous acid but not by phosphoric acid. See
Ref. 2 for details.

154

(3)

Genomic organization around the centromeric end of the
HLA class I region: Large-scale sequence analysis

Masa-aki YAMAZAKI l,2, Yoshio TATENO and Hidetoshi INOK0 2 (lFujiya Co, Ltd.,
2Tokai University)
We previously sequenced two regions around the centromeric end of HLAclass I and the boundary between class I and class III. We analyzed the tworegions of about 385 kb and confirmed, giving a new line of evidence, that thefollowing two pairs of the genomic segments were duplicated in evolution: (i) a
43-kb genomic segment including the HLA-B gene showing the highest polymorphism among the classical HLA class I loci (class la) and a 40-kb segment
including the HLA-C locus showing the lowest polymorphism and (ii) a 52-kb
segment including the MIC (MHC class I chain related gene) B and a 35-kb
segment including MICA. We also found that repetitive elements such as SINEs,
LINEs, and LTRs occupy as much as 47% of nucleotides in this 385-kb
region. This unusually high content of repetitive elements indicates that repeat-mediated rearrangements have frequently occurred in the evolutionary
history of the HLA class la region. Analysis of LINE compositions within thetwo pairs of duplicated segments revealed that (i) LINEs in these regions had
been dispersed prior to both the duplication of the HLA-Band -C loci and the
duplication of the MICB and MICA loci, and (ii) the divergence of the HLA-B
and -C loci occurred prior to the duplication of the MICA and MICB loci. To
find novel genes responsible for HLA class I -associated or other diseases, we
performed computer analysis applying GenScan and GRAIL to GenBank's
dbEST. As a result, at least five as yet uncharacterized genes were newly
mapped on the HLA class I centromeric region studied. These novel genes
should be analyzed further to determine their relationships to diseases associated with this region. See Ref. 3 for details.

(4)

Domain dislocation: a change of core structure In perlplasmlc
binding proteins In their evolutionary history

Kaoru FUKAMI-KoBAYASHI, Yoshio TATENO and Ken NISHIKAWA

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Periplasmic binding proteins (PBPs) serve as receptors for various watersoluble ligands in ATP"binding cassette (ABC) transport systems, and form
one of the largest protein families in eubacterial and archaebacterial
genomes. They are considered to be derived from a common ancestor, judging
from their similarities of three"dimensional structure, their mechanism ofligand binding and the operon structure of their genes. Nevertheless, there
are two types of topological arrangements ofthe central beta"sheets in theircore structures. It follows that there must have been differentiation in thecore structure, which we call "domain dislocation", in the course of evolution of
the PBP family. To find a clue as to when the domain dislocation occurred, weconstructed phylogenetic trees for PBPs based on their amino acid sequences
and three"dimensional structures, respectively. The trees show that the pro"teins of each type clearly cluster together, strongly indicating that the change
in the core structure occurred only once in the evolution of PBPs. We also
constructed a phylogenetic tree for the ABC proteins that are encoded by thesame operon of their partner PBP, and obtained the same result. Based on thephylogenetic relationship and comparison ofthe topological arrangements of
PBPs, we obtained a reasonable genealogical chart of structural changes in
the PBP family. The present analysis shows that the unidirectional change ofprotein evolution is clearly deduced at the level of protein three"dimensional
structure rather than the level of amino acid sequence. See Ref. 4 for details.

(5) Evolutionary pattern of influenza B viruses based on
the HA and NS genes during 1940 to 1999: origin of the NS
genes after 1977
C. Luo l , T. MORISHITA2, K SATOU2, Y. TATENO, K NAKAJIMA!, K andE. NOBUSAWA1(INagoya City University Medical School, 2Aichi Prefectural Institute of Pub"
lie Health)Phylogenetic analysis was carried out for genes encoding hemagglutinin (HA)
(24 new and 25 previously reported sequences) and nonstructural proteins(NS) (22 new and 14 previously reported sequences) of influenza B virus iso"lates obtained from 1940 to 1999. Two antigenically and genetically distant156

HA lineages are presently known to exist. Divergence into these two lineages
was estimated to have occurred around 1969. Phylogenetic analysis of NS
genes revealed that their phylogenetic relationships were not linked to the two
HA lineages but suggested that reassortment of viral genes between the viruses of two HA lineages had occurred. In addition two distinct NS lineages
which were not linked the two HA lineages were observed. Viruses isolated
after 1997 formed their own lineage in combination with BlHouston/84 whileother virus isolates obtained from 1973 to 1995 comprised the other NS lin'
eage. See Ref. 5 for details.

Publications
1.

SUGAHAWA, H., MIYAZAKI, S., GOJOBORI, T. and TATENO, Y.: DNA Data Bank of Japan
dealing with large'scale data submission. Nucleic Acids Res. 27: 25'28, 1999.
2. WATANABE, M., SUMIDA, N., MURAKAMI, S., ANZAI, H., THOMPSON, C.J., TATENO Y. and
MURAKAMI, T.: A phosphate' induced gene which promotes Penicillium mediated bio'
conversion of cis'propenylphosphonic acid to Fosfomycin. Applied and Environmental
Microbiology. 65: 1036'1044, 1999.
3. YAMAZAKI, M., TATENO, Y. and INOKO, H.: Genomic organization around the centromericend of the HLA class I region: Large'scale sequence analysis. J. Mol. Evol. 48: 317'
327, 1999.
4. FUKAMI'KoBAYASHI, K TATENO, Y. and NISHIKAWA, K: Domain dislocation: a change ofcore structure in periplasmic binding proteins in their evolutionary history. J. Mol.
BioI. 286: 279'290, 1999.
5. Luo, C., MORISHITA, T., SATOU, K., TATENO, Y., NAKAJIMA, K. and NOBUSAWA,
E.: Evolutionary pattern ofinfluenza B viruses based on the HA and NS genes during
1940 to 1999: origin of the NS genes after 1977. Arch. Virol. 144: 1881'1891, 1999.

I-d. Laboratory for Molecular Classification(1)

Data processing system for DDBJ

Hideaki SUGAWARA, Satoru MIYAZAKI
We enhanced the core system of the data processing in DDBJ to capture,review, accumulate and disseminate massive data produced by genome projects

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and biodiversity study in collaboration with three laboratories in the Center
for Information Biology.
(2)

WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)

Hideaki SUGAWARA, Satoru MIYAZAKI and Yumi FUJISAWA
We construct the Web site ofWDCM for the World Federation for Culture
Collections (WFCC) and the Microbial Resources Centers Network(MIRCEN). We provide our original databases of CCINFO and STRAIN andalso a search engine named ARMI!. The site was highly evaludated by thejournal HELIX and the American Society for Microbiology and the number ofaccesses increased by 50% this year.
(3)

Workbench for the microbial systematics

Hideaki SUGAWARA, Satoru MIYAZAKI and Hiroyuki OGAWA
Biodiversity is one of key subjects in biology in the 21st century. It is also an
important issue on socioeconomic aspects such as sustainable use and equitable benefit sharing. However, we have only a limited knowledge on organisms on the planet. In the case of microorganisms, for example, it is said that
we know less than 10% of all the strains that are alive in the environment andin other organisms. Therefore, taxonomy of known strains and identificationof unknown strains is the infrastructure of the study and socioeconomic judgement about biodiversity. Without the systematics, we can not make a rightdecision on any biodiversity issues. We aimed at the development of an information system that would improve the systematics by the integration of analy·
sis based on phenotypic characteristics (physiology, biochemistry, genetics andetc) and phylogenetic analysis based on sequence data. The final product provides such functions as: the integration of data sources distributed on theInternet; the integration of numerical taxonomy and phylogenetic analysis;
the integration of probabilistic identification and the identification by a decision tree.

158

We actually sequenced 16sRNA of some fungi and did identification andclassification in the course of the system development to put the system topractical use. We could also find the diversity ofinsertion ofintrons at genus
level and species level.
For the novel function of the workbench, we define and study properties of
the new measure based on Shannon entropy to evaluate the significance of thepartial region on multiple aligned sequences for the reconstruction ofphylogentictree.

Pub I i ca t ions
1. SUGAWARA, H., MIYAZAKI, S., GOJOBORI, T. and TATENo, Y.: DNA Data Bank of Japan
dealing with large-scale data submission. Nuc Acids Res, 1, 24-26, 1999.
2. SUGAWARA, H.: Culture Collections and Data Activities. Protein, Nucleic Acid and Enzyme. 44, 175-180, 1999.
3. SUGAWARA, H: Proposal of Information Biology. Iden 53, 39-43, 1999.
4. MIYAZAKI, S. and SUGAWARA, H.: Reconstruction oflarge-scale phylogenetic trees: problems and solutions, Amino Acids Vo1.l7, No.1, pp119-120.

Oral presentations
1. SUGAWARA, H. and MIYAZAKI, S.: An information system for data integration andpolyphasic analysis of microbes. The Proceedings of the 99th General Meeting ofthe
American Society for Microbiology, Chicago, 1999/5/30-6/3
2. MIYAZAKI, S. and SUGAWARA, H.: From linking to integration of biological databases,
The International Joint Workshop for Studies on BIODIVERSITY (Species 2000,
CODATA and Global Environment Tsukuba), Tsukuba, 1999/7/14-16
3. SUGAWARA, H.: Links between microbial resources and gene sequences, The proceedings oflXth International Congress of Bacteriology & Applied Microbiology,Sydney, 1999/
8/16-20
4. SUGAWARA, H.: Bio-resources are strategic information resources, the proceedings ofBio
Resource Network Symposium, pp2-IQ, Tokyo, 1999/12/17
5. MIYAZAKIi, S. and SUGAWARA, H.: Development of a prototype system for sharing andanalysing data of microbial cultures, the proceedings ofBio Resource Network Symposium, pp36-45, Tokyo, 1999/12/17

159

J.
(1)

EXPERIMENTAL FARM

Development, propagation and distribution of thegenetic stocks of rice

Ken-Ichi NONOMURA, Mitsugu EIGUCHI, Toshie MIYABAYASHI and Nori KURATA
We have conducted development, reproduction, reevaluation and distribution of genetic stocks of wild and cultivated strains of rice. About last two
years, we have also been introducing new projects to propagate geneticallyengineered and transposon-tagged rice strains for utilizing them as genetic
materials for functional analyses of a large number of rice genes. Resources
we are generating are enhancer trap lines of AciDs system and insertional
mutant lines tagged by the endugenous transposon Tos17. Another trial toanalyze the composition of the centromeric heterochromatin in rice chromosomes is also progressed. These projects are cooperatively conducted with themembers of the plant genetics laboratory. For detail information, see the reports of plant genetics laboratory.

Publ ications
1.
2.

3.
4.

5.

6.

NONOMURA, K.I. and KURATA, N.: Organization of 1.9-kb repeat unit RCE1 in thecentromeric region of rice chromosomes. Mol. Gen. Genet., 261, 1-10, 1999.
MIYOSHI, K., NAKATA, E., NAGATO, Y. and HATTORI, T.: Differential in situ expression of
three ABA-responsive genes ofrice, Rab16A, REG2 and OSBZ8, during seed development. Plant Cell Physiol., 40, 443-447, 1999.
ITO, Y., EIGUCHI, M. and KURATA, N.: Expression of novel homeobox genes in early
embryogenesis in rice. Biochim. Biophys. Acta, 1444, 445-450, 1999.
AsHiKAWA, I., KURATA, N., SA-II, S., UMEHARA, Y. and SASAKI, T.: Application ofrestriction
fingerprinting with a rice microsatellite sequence to assembling rice YAC clones. Genome, 42, 330-337, 1999.
SENTOKU, N., SATO, Y., KURATA, N. ITO, Y., KITANO, H. and MATSUOKA, M.: Regional
expression of the rice KNl-type homeobox gene family during embryo, shoot and flower
development. The Plant Cell, 11, 1651-1664, 1999.
MIYOSHI, K and KURATA, N.: Cloning oftwo cDNAs encoding the HAP3 subunit protein

160
from developing rice seeds. Rice Genetics Newsletter, 16, 103'104, 1999.
7. AHN, B.O., MIYOSHI, K., ITOH, J.I., NAGATO, Y. and KURATA, N.: Mapping of a rice
heterochronic gene, PIsl, regulating the plastochron and the duration of vegetative
phase. Rice Genetics Newsletter, 16, 36'37, 1999.

161

ABSTRACTS OF DIARY FOR 1999
Biological SymposiumJan. 6
Circadian clock gene regulation in silkworm differs from Drosophila (Ivo Sauman)
How does the extra-nuclear transportations of protein and RNA
occur? <Mutsuhito Ohno)
Jan. 8
Histone acetylase complex and transcriptional control (Reiko
Ohba)
Jan. 11
The Roles of PKI in the formation of a bilateral axis and meso·dermal somite differentiation of chick embryo (Minoru
Kawakami)Jan. 18
Metabolic type glutamic acid receptor can be also activated byextracellular Ca 2+ ! (Yoshihiro Kubo)
Feb. 1
Intracellular Traffic Control (Shahid S. Siddiqui)
Feb. 8Polymerization control of actin filaments in the process of forming myofibrils: investigations on actin-binding protein using C.
elegans as a model <Ichiro Onomasa)
Feb. 9Finding gene regulatory signals in vertebrates using the com"pact genome of Fugu (Sydney Brenner)
Feb. 15
Analysis on the interaction between protein and DNA using
atomic force microscope: visualization of nucleosome (Kunio
Takeyasu)Feb. 22
Effect of Demethylation on Imprinted X-inactivation (Takashi
Sado)
Feb. 23
Effect oflow concentration of guanidine hydrochloride upon thetranscriptional activity ofT7 RNA polymerase (Dipak Dasgupta)
Molecular Anatomy of rrnE PI: Transcription activation and regulation without factors besides RNA polymerase and promoter(Tamas Gaal)
Transcriptional activation mediated by the carboxy·terminal
domain of RNA polymerase ex subunit: Multipoint monitoring

162

Feb. 24
Mar. 1Mar. 5Mar. 15

Mar. 24

Mar. 31
Apr. 5

Apr. 20

May 10
May 19with a fluorescent probe (Olga N. Ozoline)
Biomathematical approaches on developmental phenomena (YohIwasa)
Chromosome Pairing and Synapsis in Yeast (Beth Rockmill)Olfactory Conditioning in Drosophila (Obaid Siddiqi)
Site- Directed Mutagenesis of Active Site of Yeast Replicative
DNA Polymerases 0 and E t'iouri Pavlov)
Patterns of Single Nucleotide Polymorphisms in Human Genes<Aravinda Chakravarti)
New Ca indicator Chameleon using GFP energy transfer (Atsushi
Miyawaki)
Molecular mechanism of protein transfer between nucleus andcytoplasm (Naoko Imamoto)Mechanism of regulating JAKISTAT pathway by CIS/JAB:
cytokine signal transmission (Akihiko Yoshimura)
Ploughing a lonely furrow: the curious case ofthe E.coliproUPI
promoter (J. Gowrishankar)
Physiological roles of aggregation of sigma 70 in E.coli(Hiroki
Nagai)
GroEL is involved in maturation of E.coliRNA polymerase devoid of omega subunit (Dipankar Chatterji)Gene Express: intelligent system on gene expression regulation(Nikolay A. Kolchanov)
Cytoplasmic signaling in neuronal growth cone guidance (Muming Poo)
Protein Machines Directing Myosin Assembly (Henry F. Epstein)
Applications of XML in the replacement and searching for of
information on about XML genome sequence of XML (Hideo
Matsuda)Activity control ofRas family GTP binding protein Rapl: Whatis the meaning of diverse control mechanisms? (Michiyuki
Matsuda)Generation time of HIV-l (Yun-Xin Fu)
The Molecular Mechanisms ofthe Organelle Transport in Cells:

ABSTRACTS OF DIARY FOR 1999

May 21
May 28June 4

June 7
June 9June 10
June 16
June 24
June 28
July 1
July 12

July 13
July 14
July 27
July 28

July 29

163

Identification and Characterization of New Motor Proteins, RIFs
{Nobutaka Hirokawa}
Neuronal mechanism of molecular smell discrimination (Kensaku
Mori)
Assembly and disassembly of protein complexes in homologous
recombination (Akira Shinohara)
A new type of eukaryotic protein phosphatase required for dephosphorylation of the CTD and transcription by RNAPolymeraseII in Saccharomyces cerevisiae {Jack Greenblatt}
Structure ofRecA protein-DNA complex {Katsumi Morimatsu}
Control mechanism of asymmetrical division in yeast {Kenji lrie}
Binding of "Sonic hedgehog" to "Patched" and patterning activity of neural tube {Naoyuki Fuse}.
Survival signaling pathways in neuronal cells (Yukiko Goto)
Development of HIV vector and gene therapy (Hiroyuki Miyoshi)
Towards Mining Gene Expression Database (Shinichi Morisita)
Regulation of cytoskeleton and adhesion by intracellular signal(Kozo Kaibuchi).
p53 family genes: how to explore the biological functions (Yoji
19awa)
Transcriptional control mechanism in eucaryotic cell: with particular emphasis on TFIID, histone acetylase (Yoshihiro
Nakatani)
Design of animal skin and Turing pattern (means and the limitsof mathematical analyseis in biological science) {Shigeru Kondo}
Conserved Mechanism of Induced Mutagenesis: Newly Emerging Mutagneic DNA polymerases (Gomori Haruo)
Genetic Analysis of TGF- 13 Signaling - the long and short ofit{Richard W. Padgett}Multifunctional membrane molecule Neuropirin-l that acts on
the formations of neuronal circuits and blood vessels (Takahiko
Kawasaki)
Asymmetric Cell Divisions and the Generation of Neuronal Diversity (William Chia)

164

Aug. 2
Aug. 6
Aug. 9Aug. 19
Aug. 26

Aug. 30
Sep. 1
Sep. 2
Sep. 13
Oct. 4Oct. 5Oct. 15
Oct. 20

Nov. 1

Artificial life and development of an evolutionary computer(Katsunori Shimohara)
Downstream factor "ebi" ofEGF receptor regulates "Notch" sig'
nals (Reo Tsuda)
Studies on Learning and memory, and ActivitY'dependent Devel'
opment of the Visual System with Genetically Engineered Mice:
Multilevel Analysis of Hippocampus'dependent Memory(Susumu Tonegawa)
Making a difference: the asymmetric division of stem cells in the
germline (Haifan Lin)
Control mechanism of neuronal system with JMK, p38MAP ki·nase cascade employing nematode C. elegans as model systems(Kunihiro Matsumoto)
Structure and function of human killer cell immunoglobulin reo
ceptors (Katsumi Maenaka)
Regulation of cytoskeleton with phosphorylation of myosin II(Shinichiro Kojima)
Midline signaling in Zebrafish development (Atsushi Kawakami)
Directional cues that guide axons in the zebrafish brain (John Y.
Kuwada)How Cdc20 regulates cell cycle progression in Saccharomyces
cerevisiae (Masaki Shirayama)
Control mechanisms in of microtubular dynamics with Xenopus
MAP 4 and identification and analysis of on factor that accumu'
lates on centromere (Nobuyuki Shiina)
Establishment of large' scale in' situ hybridization system andsearch for genes being expressed in small intestine and orchis ofmouse in a site- or cell'specific manner through the use of such asystem (Tohru Komiya)Brain mechanisms of behavior (Masakazu Konishi)
Intracerebral mechanism of attention (Tadashi Isa)
The extent to which language acquisition is modularized (Nobuo
Masataka)
TGF'beta signaling in vertebrate development: Insights from

ABSTRACTS OF DIARY FOR 1999

Nov. 8
Nov. 12
Nov. 15

Nov. 16
Nov. 18

Nov. 22
Nov. 24

Nov. 29
Dec. 13

Dec. 14165

zebrafish (Alexander Schier)
Genetic analysis offorebrain development in zebrafish (Stephen
Wilson)Computer simulation of cell: follow' up report and future pros'
pects for ofE'CELL project (Masaru Tomita)
Steps Common to Genetic Recombination in Eukaryotes, Prokary'otes, Archaea, and Phage (Stephen Kowalczykowski)
The Multiple Genomic Origins of the Mitochondrial Proteome(C.G.Kurland)
DNA DAMAGE CHECKPOINTS AND DNA REPAIR IN BUD·DING YEAST(Giovanna Lucchini)
Discovery of new transcriptional control stage (Tadashi Wada)
DNA replication', transcription', and translation'coupled biases
from intra'strand Parity Rule2 (A=T and G=C) do not explaininter-specific variations of DNA G+C content(Noboru Sueoka)
Transcription factories and the path of transcripts to the cyto'
plasm (Peter R. Cook)
Strategies for analyzing large DNA clones: A Transposon-mediated insertion technique for analyzing the murine Hoxb Cluster(Timothy M. Jinks)
Intracerebellar model and evolution of communication (Mitsuo
Kawahito)
REGULATING CHROMOSOMAL DNA REPLICATION IN EUKARYOTIC CELLS(John F.X. Diffley)
Role of de novomethyltransferases Dnmt3a and Dnmt3b in development and disease (En Li)
Structural and Kinetic Mechanisms ofT7 RNA Polymerase Regu'
lation (Rui J. Sousa)
Biochemical and Physiological/Genomic Studies of Nitrogen Signalling in Enteric Bacteria (Sydney Kustu)
Integration of Regulatory Signals at Bacterial Promoters (as revealed by the study of CRP- and FNR-dependent promoters)(Steve Busby)
New gene that controls bilaterally axis (Yuji Yokouchi)

166

Dec. 15
Dec. 16
Dec. 17

Mechanism of preserving chromatin structure associated with
DNA replication (Yasukazu Shibahara)
Genetic analysis of on RNAi and inhibition of transposon trans"
fer in nematode (Hiroaki Tahara).
Study on gene"transfer recombinant molecular mechanism(Kiyoshi Mizuuchi)

167

FOREIGN VISITORS IN 1999
Jan. 6'7
Jan. 8Jan. 11-12
Jan. 21-Mar. 25
Feb. 8-9
Feb. 9-23
Feb. 18-Mar. 24
Mar. 5-6
Mar. 6'23
Mar. 6'23
Mar. 14-15
Mar. 15-30

Ivo Sauman, Institute of Entomology, Czech Academy of
Sciences, Czech Republic
Reiko Ohba, University of Virginia, Health Science Cen'ter, U.S.A.
Minoru Kawakami, Harvard Medical School, U.S.A.
Thomas Daniel Andrews, Australian National University,
Australia
Sydney Brenner, Molecular Sciences Research InstituteInc., U.S.A.
Lim Sun-Hee Univ. of Dong-a Department of Biology, Korea
Richard Hayward, University of Edinburgh, U.K.
Obaid Siddiqi, Tata Institute of Fundamental Research,
India
Jean Thierry-Mieg, CNRS Laboratoire de Physique
Mathematique, University MontpellierII, France
Danielle Thierry-Mieg, CNRS Laboratoire de Physique
Mathematique, University MontpellierII, France
Youri Pavlov, Sainkt-Petersburg State University, Russia
Nikolay A. Kolchanov, Russian Academy of Science, RusSla

Mar. 24
Mar. 30-31
Apr. 5-6
June 10'11
July 27-28
July 29·Aug. 4
Aug. 2'13
Aug. 6-7

Sandro J. de Souza, Ludwig Institute for Cancer Research,
Brazil
MU'ming Poo, University of California San Diego, U.S.A.
Henry F. Epstein, Baylor College of Medicine, U.S.A.
Naoyuki Fuse, Johns Hopkins University, U.S.A.
Richard W. Padgett, Waksman Institute Rutgers University, U.S.A.
William Chia, National University of Singapore,
Singapore
Giorgio Bernardi, Stazione Zoologica Anton Dohrn, Italy
Leo Tsuda, University of California Los Angeles, U.S.A.

168

Aug. 9-10
Aug. 19-20
Sep. 12-13
Oct. 3-4
Oct. 14-15
Nov. 13-20
Nov. 14-16
Nov. 21-22
Nov. 24-25
Dec. 4-14
Dec. 13-14
Dec. 13-14
Dec. 15
Dec. 15-18
Dec. 15-19

Susumu Tonegawa, Massachusetts Institute of Technology, U.S.A.
Haifan Lin, Duke University Medical School, US.A.
John Y. Kuwada, University of Michigan, U.S.A.
Masaki Shirayama, Institute of Molecular Pathology,
Austria
Masakazu Konishi, California Institute of Technology,
US.A.
Giovanna Lucchini, Universita delgi Studi di Milano, Italy
Charles G. Kurland, Evolutionary Biology Centre, University of Uppsala, SwedenPeter R. Cook, The Sir William Dunn School of Pathology
The University of Oxford, U.K.
Timothy M. Jinks, National Institute for Medical Research, UK.
John F.X. Diffley, ICRF Clare Hall Laboratories, UK.
Chun-Fang Wu, University oflowa, U.S.A.
En Li, Massachusetts General Hospital, UB.A.
Keiichi Shibahara, Cold Spring Harbor Laboratry, U.S.A.
Hiroaki Tabara, University of Massachusetts, US.A.
Kiyoshi Mizuuchi, NIH, US.AASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS
President
MORIWAKI, Kazuo; Vice-President, The Graduate University for Advanced
StudiesManaging Director
GOJOBORI, Takashi; Professor, National Institute of Genetics
SHIROISHI, Toshihiko; Professor, National Institute of Genetics
ManagerFOREIGN VISITORS IN 1999

169

HOTTA, Yoshiki; 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
ISHlHAMA, Akira; Professor, National Institute of Genetics
SHlGETO, Gakuji; Managing Director, Inoue Foundation for Science
NAKATSUJI Norio; Professor, National Institute of Genetics

Trustee
TAZIMA, Yataro; Director Emeritus, Institute of Silkworm Genetics andBreeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Director and Professor, Institute of Medical Mycology, Teikyo
University
MATSUNAGA, Ei; Professor Emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, Shokabo Publishing Co.
TAKAGAKI, Yoshio; Full-time Adviser, Chugai Pharmaceutical Co.
KURODA, Yukiaki; Professor Emeritus, National Institute of Genetics
TATENO, Yoshio; Professor, National Institute of Genetics
Auditor
IMAMURA, Takashi; Professor Emeritus, National Institute of Genetics
IKEMURA, Toshimichi; Professor National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
Adviser
MORIWAKI, Daigoro; Professor Emeritus, Tokyo Metropolitan University

170

AUTHOR INDEX
AHN, B.O

AKAsAKA. K
ANDACHI. Y
ANDO.AANZAI. H
AOKI. A.

ARAKI. H
ARAMAKI. H
BELLGARD. M.1.
BoNG. Y.-SCARTIER. L
CHATTERJI. D
CIDJIWA, T
COLLAND. F
DANJO. I.
DASGUPTA. D
DAWKINS. RL
EIGUCHI. M
ENDO.A
ENDO.A
ENDO. S
FENNER. M.H
FRITZ. A
FuJISAWA. T
FuJISAWA. T
FuJISAWA. Y
FuJISHIMA. H
FuJITA. N
FuJIYAMA. A
FuJIYOSHI. T
FuKAGAWA. T
FuKAMI-KOBAYASHI. K.
FURUUMI. H
FuSE. N
GAAL. T

107
63
124
73
153
101
44.45.46
131
145
88
92
130
85
17
88
16
144
105.111.159
27
88
11
87
66
53.54
150
157
112
10.11.13.14.17
72.88
92
72.73.74.75
154
84
117
129

GAMO. SGAUDIERI. S,
GoJOBORI. T
GoTO. M
GoTO. SHANDA, H
HARAFUJI. N
HARUSHIMA. Y
HATANO. N
HATTA, M
HATTORI. M
HATTORI. T
HAYASID. S
HAYWARD. RS
HIRAMOTO. M
HIRASAWA, T
HIRATA. T
HIROCIDKA, H
HIROMI. Y
HIRONO. K
HIROSE. S
HIROSHIMA, M
HONDA, A.
HONG. S.-K
HORAI. SHOSOYA. T
HOTTA. Y.
HWANG. J.-S
lINO. YlIZUKA, T
IKEDA. A.
lKEGAMI. T
lKEGAMI. T
lKEMURA. T
lKEO. K

116
144
143.144.145.146.152
60
114.117
61
53
110
84
53.55
72.84
91
114.115.116.117
131
57
99
89
105
50.51.52.53
121
60.61.63
127
26.27.28.30
91
78.92
58
56,57.58
28
136
150
90
61
120
68.72.73.74.75
146

AUTHOR INDEXIKEYA, 1.

115
60
IMAMOTO, N
127
INOKO, H
73,77,154
INOUYE, M
88
ISHIDA, 1.
78,92
ISHIGURO, A
23
ISHIHAMA, A
10,11,13,14,16,17,18,19,20,
22,23,25,26,27,28,30
ISHIHARA, K.
84
ISHIHARA, T
133,135,136
ISHIJIMA, J
98, 99ISOGAI, Y.
150
ITO, M
121
ITO, Y.
104,105,107,111
ITOH, J.I.
107
ITOH, S.'I.
90
ITOH, T
143
IWAKI, T
84
IWANAMI, M
50
IWASAKI, Y
105
IWATA, A
18,20
IZUNO, H
150
JIANG, Y.'J
66
JIN, F
78,92
JISHAGE, M
14,16
KABATA, H
131
KABE, Y.
60,61
KAMIMURA, Y.
45,46
KANAYA, S
68,73
KANEKO, M
77
KATAYAMA, A
10,14
KATO, J
120
KATO, R.
84
KATSURA, I.
133,135,136
KAUSCIUKOVlC, T
131
KAWABATA, T
112,120,149
KIKKAWA, Y
99
KIMURA, M
19,20,22
KINEBUCHI, T
131
KINJO, AR.
149
IMAI, 1.

KISHI, T

171

36
91,105KITANO, T
78
KITAYAMA, S
121
KOBAYAKAWA, Y.
53
KOBAYASHI, M
60
KOHARA, Y
121,122,123
KOIDE, T
99,102
KorZUMI, O
53
KOSE, H53
KOSEKI, H
86,101KUBORI, T
129
KUDO, Y73
KULSKI, J.K
144
KURATA, N
104,105,106,107,108,109,
110,111,119,159
KUROSAWA, O
131
KUWAHARA, Y
101
KUWAJIMA, K.
128
LI, E
87
Lr, H·C
92
Lou, H
92
LUO, C
155
MAEDA, H
14
MAKINO, S
97,98
MASUMOTO, H
44
MASUYA, H
97,98,100
MATAKATSU, H
116,117
MATSUDA, S
41
MATSUDA, Y
86
MATSUNO, M
53
MATSUOKA, M
91,105
MEARES, C.F
17
MIBUKA, F
63
MINAKUCHI, Y.
121
MINETA, K
146
MISHIMA, M
61
MISKELISHIVlLI, VG
129
MITA, A
95,99
MITOBE, J
23
MITSUI, K
119,120KITANO, H172
63

NISHIMURA, A

23,25

NISHIMURA, A.

91

MIURA, A.

87

NISHIMURA, A.

112,120MIYABAYASHI, T

159

NIWA, N52

MIYAHARA, K.

135

NIWA, Y104

MITSUNAGA, K.
MITSUZAWA, H

63

MIYAZAKI, S

152,156,157

NOBUSAWA, E

155

MIYOSHI, K.

91,105,106,107

NOBUSAWA, K.

155

MIZUMOTO, K.

26

MIZUNO, S

84,85

MIZUNO, T

64

MIZUSHINA, Y.

95,101

NOG!, T.
NOMURA, T
NONOMURA, K.I

23
10,14
108,159

NUNEZ, L

92

OBA, T

121

MOCHII, M

123

MOCHIZUKI, K

54

OBARA, M

121

MORI, T.

120

OGASAHARA, K

151

MORISHITA, T

155

OGAWA, H

157

MORITA, K

123

OGAWA, T

38,41

MORIWAKI, K.

102

OG!HARA, Y

146

MOROHASHI, K

61

OGURA, K.

122

MOTOHASHI, T.

121

OHKURA, K

135

MOTOYAMA, A.

88

OHNO, M

72,84

MUKAI, M

150

OHTA, T

38,41

MUKAI, T

84

OHTA, T82

MUKHERJEE, K.

130

OISHI, A133

MUNOZ, I.

92

OKA, A.

MURAKAMI, S

153

OKABE, M

MURAKAMI, T

153

OKAMOTO, T

46

OKAMURA, A.

MURAMATSU, S

30
74

NAGANO, N

149

NAGAOKA, T

121

NAGATO, Y.

90,91,105,107,119

NAKADE, K

28

OTA, M

92
76
77
38
149,150,151

155

OZAKI, J

61

NAKAMURA, H

150

OZOLINE, O.N

NAKATA, E

91

PAN, I.-H

NARIMATSU, H

77

PARK, J.H

34

NIKI, H

48

PURBOWASITO, W.

84

NISHIHARA, S

77

SADO, T.

87

NISHIKAWA, K.

112,120,149,150,151,154

SADO, Y

117

54

SAEKI, N

101

112

SAGA, Y66

NAGAI, H

129,130,131

NAKAJIMA, K

NISHIMIYA-FUJISAWA, C
NISHIMORI, K

OMOTO, K

95
50,51,52

OOTA, HOOTA, SOSHIUMI, H

13
78,92

AVTHORINDEXSAGAI, T.
SAITOH, T
SAITOU, N
SAKAKI, Y.
SAKANE, I.
SAKURAI, H
SAKURAI, N
SASAKI, H
SASAKI, H
SASAMURA, T.
SATO, Y.
SATOH, N
SATOU, K
SAWADA, A
SAWADA, H
SEINO, H
SHIMA, D

100
90
76,77,78,79,92

TAJIMA, K

72,84
128
20
95
84,85,86,87
99,10156

SHIRAKAWA, M

SHIRATORI, A
SHIROHZU, HSHIROISHI, T.
SOGAWA, K
SONODA, S
SUDA, CSUGAWARA, H
SUGIURA, I.

TAKANO, K.
TAKANO-SHIMIZU, T
TAKATA, K.
TAKAYA, K.
TAKAYAMA, Y

107
46

98
87

TANIGUCHI, M

117

140
121
65
87

SHIRAKIHARA, Y.

95
53
151
69,70
63

TAKAHASHI, T

TAN, S.-S

99,100SHIODA, T

112

TAKAGI, N

TAMURA, M

SHINDO, K
SHINYA, M

TAKAGI, H

25,34,3560

SHIMIZU, K
SHIN·I, T.

92
85

TAKEDA, H

SHIMIZU, HSHIMAMOTO, N

TAJIMA, S

131
91
155
66
79

63
129,130,131
53,55

SHIMADA, H

173

61
138,140,141,142
138
84
95,97,98,99, 100, 101, 102
131
92
84
152,156,157
121

TAKEMARU, K.
TAKIZAWA, K.
TALUKDER, AA
TAM, P.P.L.

TASHIRO, T
TATENO, Y.

64,65,66
61
56,57
18
87

90
152,153,154,155

TATSUDA, D

38

THIERRY-MIEG, D

121
121

THIERRY-MIEG, J
THOMPSON, C.J
TOKUNAGA, M
TOMIZAWA, J
TSUDUKI, M
TSUJIMOTO, N
TSUTSUI, K.
VCHIDA, K
VEDA, H
VEDA, S

153
126,127,128
38
86
85,86
63
99
61,63
18,20

VEDA, S76

VENO, N

123
123

UESUGI, H

153
78,92
129

VMESONO, Y

56

WACHI, M

112

WADA, ASUZUKI, H

22

WADA, T

37
61

SUZUKI, T.

121

WAKANA, S

99

TADOKORO, R.

116

WASHIZU, M

131

SUMIDA, N
SUN, C.-S
SUSA, M

174
WATANABE, K.
WATANABE, M

41
153

WATANABE, Y.

72

WIGNESHWERARAJ, S.R.

17

WLASOFF, W.A
YADA, Y.

19
97,98

YAMADA, K

28

YAMAGATA, Y

151

YAMAGUCHI, J

90

YAMAHA, E

64

Y AMAKAWA, T
YAMAO, F

119,120

YAMAZAKI, M

25,34,35,36
154

YAMAZAKI, Y

119,120YASHIKI, S
YOKOMINE, T

92

84,86

YONEKAWA, H

99

YOSHIDA, S

123

YOSHIMURA, A

119

YUM, S

53
151

YUTANI, K.

Annual Report of the National Institute of Genetics. No.50

Issued by Director'General : Yoshiki Hotta, D.Med.
National Institute of Genetics
Yata 1,111 Mishima, Shizuoka'Ken, 411-8540 Japan
Tel. 0559'81'6718
Fax. 0559'81'6715
Edited by Hiroyuki Sasaki & Nori Kurata
Published by

National Institute of Genetics

Printed by Hobundo Insatsu Co., Ltd.

Ito Hakone

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