ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT1991

No. 42Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1992

Annual Repori:of i:heNai:ional Insi:ii:ute of Genei:ics
No. 42, 1991

Published by

The National Institute of Genetics, Japan
1992

CONTENTS
General statement
Staff. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Council and Advisory committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Association for propagation of the knowledge of genetics
Projects of research for 1991. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Research activities in 1991.......... .. ..... . . . ... . ... . . . . . ... . . .
I.

1
3
6
8
9
14

Molecular Genetics
The promoter selectivity of Escherichia coli RNA polymerase: Twomodes of protein-protein communication between RNA polymerase and transcription factors. ISHIHAMA, A., FUJITA, N.,
IGARASHI, K., ZOU, C. and YAMAGISHI, M. . . . . . . . . . . . . . .
The promoter selectivity of Escherichia coli RNA polymerase: Fine
mapping of the contact site I for cAMP receptor protein(CRP). Zou, C., FUJITA, N., IGARASHI, K. and ISHIHAMA,A.
The promoter selectivity of Escherichia coli RNA polymerase:
Functional mapping of major sigma subunit dO. KUMAR, A.,
FUJITA, N., HAYWARD, R. S. and ISHIHAMA, A.
The promoter selectivity of Escherichia coli RNA polymerase:
Structure and function of stationary-phase RNA polymerase.
OZAKI, M., FUJITA, N. and ISHIHAMA, A.
Regulation of the Escherichia coli rmf gene encoding ribosome
modulation factor. YAMAGISHI, M., MATSUSHIMA, H., SAKAGAMI, M., WADA, A. and ISHIHAMA, A.
Structure and function of RNA polymerase II from Schizosaccharomyces pombe: Cloning and sequence determination of thesecond largest subunit gene. KAWAGISHI, M., YAMAGISHI,
M. and ISHIHAMA, A.
Structure and function of influenza virus RNA polymerase: Reconstitution of functional RNA polymerase from three subunits
expressed using a recombinant baculovirus system. KOBAYASHI, M., TUCHIYA, K., NAGATA, K. and ISHIHAMA, A. ..

14

16

17

18

19

20

21

II

Inhibition mechanism of influenza virus growth by lignin.
YASUDA, J., NAKAYAMA, M. and ISHIHAMA, A.
Inhibition mechanism of influenza virus growth by Mxl protein:
Self-assembly of Mx 1 and GTP-dependent conformationalchange.
NAKAYAMA, M., YAZAKI, K., KUSANO, A.,.
NAGATA, K., HANAI, N. and ISHIHAMA, A
Molecular architecture of rice stripe virus: Genome structure androle of RNA polymerase. ISHIHAMA, A., HAMAMATSU, c.,
NAKAMURA, I., BARBIER, P., TAKAHASHI, M. and TORIYAMA, S.
Intron 1 as a determinant of the cell cycle-dependent expression ofhuman thymidylate synthase. TAKAYANAGI, A., KANEDA,
S., AYUSAWA, D. and SENO, T.
Regulatory sequences in the human thymidylate synthase gene areclustered at the 5'-end of the first intron.
KANEDA, S.,
HORIE, N., TAKEISHI, K., TAKAYANAGI, A., AYUSAWA, D.
and SENO, T.
Complementation by a cloned human ubiquitin-activating enzyme
El of the S-phase-arrested mouse FM3A cell mutant with
KANEDA, S., SENO, T., AYUSAWA, D.,thermolabile E 1.
YASUDA, H. and MURAKAMI, Y. .
.
DNA degradation during thymidylate stress is coupled with DNA
synthesis in mammalian cells. YAMAO, E, NAGAI, Y,YoSHIDA, S., KANEDA, S. and SENO, T.
.
Structural analysis of wild type and mutant cDNAs for mouse
IMAI, N., KANEDA, S.,ubiquitin-activating enzyme E 1.
NAGAI, Y, SENO, T. and YAMAO, F.
Kinetic study on transcription by immobilized operons: Requirement for .8, r-phosphodiester bond of ATP in initiation byprokaryotic RNA polymerases. KUBORI, T., TERADA, H.
and SHIMAMOTO, N. .
.
Single-Molecule Dynamics of Transcription by Immobilized Operons: Sliding of RNA Polymerase on DNA. SHIMAMOTO, N.

21

22

23

24

2S

26

27

28

28

29
Morphological pathway of flagellar assembly in Salmonella thyphimurium. KUBORI, T., SHIMAMOTO, N., YAMAGUCHI, S.,
NAMBA, K. and AIZAWA, S. .
.

30

CONTENTS

iii

Structural study on E. coli single-stranded DNA binding protein.
KAMATA, K., TSUKIHARA, T. and SHIMAMOTO, N.
Negative supercoiling of DNA facilitates an interaction between
transcription factor lID and the fibroin gene promoter. MIZUTANI, M., OHTA, T., WATANABE, H., HANDA, H. and
HIROSE, S. . .
o,o'DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently. MIZUTANI, M.,
VRA, K. and HIROSE, S. .
Possible role of DNA topoisomerase II on transcription of thehomeobox gene Box-2.1 in F9 embryonal carcinoma cells.
VRA, K. and HIROSE, S.
Defining a sequence recognized with BmFTZ-Fl, a sequence specific DNA binding factor in the silkworm, Bombyx mori as
revealed by direct sequencing of bound oligonucleotides andgel mobility shift competition analysis.
VEDA, H. and
HIROSE, S.
o' o,Search for target genes of Drosophila homeotic genes. HAYASHI,
S., FUSE, N. and HIROSE, S.
Molecular cloning of escargot, a gene controlling segmentation oflarva and adult. HAYASHI, S., SHIRRAS, A. and HIROSE, S.
0

0

0

0

0

0

0

0

0

0

000000.

0

0

0

0

0"

0

•

0

0

0

0

0

0

0

0

0

00

••

•••

0

0

0

0

0

0

0

0

0

•

0

0

0

0

0

0

0

0000000000000000

0

0

0

•

0

••

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

••

0

0

0

0

0

0

0

0

0

0

0

0

•

0

•

0

0

0

0

0

0

•••

0

0

••••

0

•••

0

0

•••••

•

•

0

0

0

0

0

0

0

0

0

0

0

0

0

0

30

31

31

32

32
33

33
FTZ-Fl, a steroid hormone receptor-like protein implicated in theactivation of Jushi tarazuo LAVORGNA, G., VEDA, H., CLOS,
J. and Wu, Co.
o.
Accumulation of Wx protein and amylose in rice seeds increase in
response to cool temperatures. HIRANO, H.-Y. and SANO, Y.
0

0

0

0

0

0

0

0

0

0

0

0

0

0"

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

34

34
Some trans-acting loci (du) that regulate the expression of the wx +gene in rice. HIRANO, H.-Yo, SARUYAMA, H. and SANO, Y.
35

The promoter of the rice wx + gene is functional for tissue specificexpression in pollen in the transgenic petunia. HIRANO, H.o.
Y., KOMEDA, Y. and SANO, Y.
0000000000000000000.

II.

0

0

Microbial Genetics
Sequencing analysis of primer RNA for minus stand synthesis of

36

iv

bacteriophage fl. HIGASHITANI, N., HIGASHITANI, A. and
HORIUCHI, K.
.
.
A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein. HIGASHITANI, A. and
HORIUCHI, K.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
DnaK protein is involved in replication of Escherichia coli chromosome by protecting the initiation protein DnaA from heat
inactivation. YASUDA, S., HIGASHITANI, A. HORIUCHI, K.
and SAKAKIBARA, Y. . . .
In vitro protein phosphorylation in Escherichi coli cell cycle mutants. HIGASHITANI, A., YASUDA, S., NISHIMURA, Y. and
HORIUCHI, K.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Osmotic regulation of uptake of long-chain fatty acids in Escherichia coli: In vitro interaction of OmpR protein with a fatty acidreceptor gene fadL.
HIGASHITANI, A., HORIUCHI, K.,
HARA, H., AIBA, H., MIZUNO, T. and NISHIMURA, Y. ....
Correlation of a subset of the pLC plasmids to the physical map ofEscherichia coli K 12.
NISHIMURA, A., AKIYAMA, K.,
KOHARA, Y. and HORIUCHI, K.
III.

38

39

41

41

43

44

Mammalian Genetics

Genetic status of Mus musculus subspecies in Far East Russia.
MORIWAKI, K., SUZUKI, H., KAWASHIMA, T., TSUCHIYA, K.,
YONEKAWA, 1-1., YAKIMENKO, L. V., KORABLEV, V. P. andKRYUKOV, A. P. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 45
Male-specific suppressor of meiotic recombination in the MHC
region. SHIROISHI, T., SAGAI, T. and MORIWAKI, K.
46
Genetic organization of the recombinational hotspot within the
MHC: A tight linkage of the hotspot to the LMP-2, HAMI
and HAM2 genes. KOIDE, T., SHIROISHI, T. and MORIWAKI,
K
47
Gene dosage effect of recombinational hotspots on the rate ofrecombination frequency. YOSHINO, M., SAGAI, T., FISCHERLINDAHL, K., SHIROISHI, T. and MORIWAKI, K ..... , . . . .. 48
Mouse visible mutations of the intra-MHC recombinants. SHIROISHI, T., SAGAI, T., W AKANA, S. and MORIWAKI, K.
49
Establishment of embryonic stem cell lines from BI0.H-2 congenic

CONTENTS
strains. SAGAI, T., NAKATSUJI, N., SHIROISHI, T. and MORIWAKI, K.
Generation of a novel mutant mouse by transgenesis. GOTOH, H.,
SHIROISHI, T. and MORIWAKI, K.
Genetic analysis of susceptibility of urethane-induced pulmonary
adenomas in M.MOL-MSM (MSM) mice. MIYASHITA, N.
and MORIWAKI, K. .
.
Comparison of the DNA sequence in the D-Ioop region of mousemitochondrial DNA. II. Two distinct clusters in the fareast
population with musculus-type mtDNA. YONEKAWA, H.,
WATANABE, S., MIYASHITA, N. and MORIWAKI, K.

IV. Developmental and Somatic Cell Genetics
Cloning of Hydra interstitial stem cells developmentally restrictedto sperm production. NISHIMIYA-FuJISAWA, C. and SUGIYAMA, T.
A nerve factor( s) required for survival and proliferation of interstitial stem cells in Hyra. FUJISAWA, T.
.
Hereditary mosaic (mo) as a functional disorder in the differentiation process of meiotic oocytes in Bombyx. MURAKAMI, A.

v

49
51

52

52

55
56

56
A novel recessive multistar-spot mutant (m~) in Bombyx and its
genetic nature. MURAKAMI, A. and AKUZAWA, K.
A consideration of the hereditary mode of feeding behaviour on
artificial diets in Bombyx. MURAKAMI, A. .
.
Differential susceptibility of domesticated Bombyx silkworms to thetachina fly, Exorista sorbillans Wied. SHIMADA, J., OHSHIKI,
T. and MURAKAMI, A. .
.
Analysis of morphogenesis in postimplantation mouse embryos.
NAKATSUJI, N. and SHIRAYOSHI, Y.
.
Manipulation of embryogenesis using mammalian embryonic cells.
.
NAKATSUJI, N. and SHIRAYOSHI, Y.
Molecular analysis of cell differentiation in postimplantation mouseembryos. SHIRAYOSHI, Y. and NAKATSUJI, N. .
.
A eDNA project on the nematode Caenorhabditis elegans.
KOHARA, Y., MITSUKI, H. and NISHIGAKI, A.
Differential screening of founder cells in C. elegans embryo.

58
59

61
63
63
64
64

VI

TABARA, H. and KOHARA, Y. . . . . . . . . . . . . . . . . . . . . . . . . ..
Genetics of fluoride-resistance of the nematode Caenorhabditis elegans. KATSURA, I. and KAWAKAMI, M.
Caenorhabditis elegans larval lethal mutations that cause grossmorphological changes. KATSURA, I.

V.

66
67
68

Cytogenetics
Mutability of constitutive heterochromatin (C-bands) during eukaryotic chromosomal evolution and their cytological meaning. IMAI, H. T.
Nucleolar organizer regions (NORs) in the Australian ant Myrmecia croslandi (=M. (pilosula) n = 1). IMAI, H. T., HIRAI, H.,
SATTA, Y., SHIROISHI, T., YAMADA, M. and TAYLOR, R. W.

69

69
Cloning of rONA in the ant Myrmecia croslandi.
M., OGURA, K. and IMAI, H. T

YAMAMOTO,.

70

Abnormal differentiation and associated radiosensitivity of erythroid progenitor cells in the wasted mutant mouse. TEZUKA,
H., A YUSAWA, D. and SENO, T. .
.

72

VI.

VII.

Mutagenesis and Radiation Genetics

Population Genetics

Role of diversifying selection and gene conversion in evolution ofmajor histocompatibility complex loci. OHTA, T. .
.
Multigene famili 7s and the evolution of complexity. OHTA, T.
Overdispersed molecular clock at the major histocompatibility complex loci. TAKAHATA, N. .
.
.
A trend in population genetics theory. TAKAHATA, N
Statistical models of the overdispersed molecular clock. T AKAHATA, N.
Sampling errors in phylogeny. TAKAHATA, N. and TAJIMA, F.

73
73
74
75
75
76

Genealogy of neutral genes and spreading of selected mutations ina geographically structured population. T AKAHATA, N.
Trans-species polymorphism of HLA molecules, founder principle,

76

CONTENTS

vii

and human evolution. TAKAHATA, N.
Calibrating evolutionary rates at major histocompatibility complex
loco
SATTA, Y., TAKAHATA, N., SCHONBACH, C, GUTKNECHT, J. and KLEIN, J.
Persistence of repeated sequences that evolve by replication slip.
page. TACHIDA, H. and IIZUKA, M
Genetic variability and geographical structure in partially selfing
populations. MARUVAMA, K. and T ACHIDA, H. .
.
Determination of window size for analyzing DNA sequences.
TAJIMA, F.
Recent development of the neutral theory. KIMURA, M. .
.

77

VIII.

78

79
79
80
81

Evolutionary Genetics

Analysis of HLA data at the 11 th International Histocompatibility
Workshop. GOJOBORI, T., IMANISHI, T., INA, Y., SAITOU,.
N., TATENO, Y. and INOKO, H
Evolutionary origin of numerous kringles in human and simian
apolipoprotein (a). IKEO, K., TAKAHASHI, K. and GoJOBORI, T.
Mutation patterns of human immunodeficiency virus genes. MORIVAMA, E. N., INA, Y, IKEO, K., SHIMIZU, N. and GOJOBORI,

T

.

83

83

84

Two-color cytofluorometry and cellular properties of the urokinase
receptor associated with a human metastatic carcinomatous
cell line. TAKAHASHI, K., GOJOBORI, T. and TANIFUJI, M.
84
Evolutionary pathways of N2 neuraminidases of swine and human
influenza A viruses: Origin of the neuraminidase genes of two
reassortants (HIN2) isolated from pigs. NEROME, K., KANEGAE, Y, YOSHIOKA, Y, ITAMURA, S., ISHIDA, M., GoJOBORI, T. and OVA, A. .
.
Molecular evolution of hemagglutinin genes of HINI swine andhuman influenza A viruses. SUGITA, S., YOSHIOKA, Y., ITAMURA, S., KANEGAE, Y., OGUCHI, K., GOJOBORI, T.,.
NEROME, K. and OVA, A. .
Molecular cloning and characterization of a novel glycoprotein gp
34, that is specifically induced by the human T-cell leukemia

85

86

viii

virus type I transactivator p40tax. MIURA, S., OHTANI, K.,
NUMATA, N., NIKI, M., OHBO, K., INA, Y., GOJOBORI, T.,
TANAKA, Y., TOZAWA, H., NAKAMURA, M. and SUGAMURA,K ...
Reconstruction of molecular phylogeny of extent hominoids fromDNA sequence data. SAITOU, N.
Statistical methods for phylogenetic tree reconstruction. SAITOU,
0

••

0.0

••••

00

••

0000

••

0

••

00000

0

N.

0.0000000•••

000.00

••

•••

0

0

0

•••••

0

••••

00.00.0.000.00

••

0

0

••

0

00000.000.

0

00000

0

0

••

•••

0

0

00.

0

•

0

0

IX.

•••••

00

••

0

••••••

•

0

0

0

0

0

0

0

0

0

0

00.00.0.000.00.

•

0

0

•

0

••

0

0

0

•

0

•

0

0

0

0

0

••

0

0

0

•

0

0

0

••

000

•••

87
88

0"

Evolutionary rate of immunoglobulin alpha noncoding region isgreater in hominoids than in old world monkeys. KAWAMURA, S., TANABE, H., WATANABE, Y., KUROSAKI, K.,
SAITOU, N. and VEDA, S.
. ..
Integration of a SINE-like retroposon into a specific locus in rice
evolution. HIRANO, H.-Yo, MOCHIZUKI, K., VMEDA, M.,
OHTSUBO, H., OHTSUBO, Eo and SANO, y. .
Cytogenetic localization of zygotic hybrid rescue gene (Zhr) of
Drosophila melanogaster which rescues interspecific hybridsfrom embryonic lethality. SAWAMURA, K. and YAMAMOTO,
M.-T. .
0

87

0000

88

89

90

Human Genetics

Genetic polymorphisms in the polyadenylate tract of Alu repetitiveelements in the human genome. NAKASHIMA, H., SAKAI,
M., INABA, R., HASEGAWA, T. and IMAMURA, T.
Toward a transcriptional map of the human chromosome 21.
NAKASHIMA, H., SAKAi, M., INABA, R. and IMAMURA, T.
0

••••

0.0

91

92
Beta-thalassemia major resulting from compound heterozygosity
for the ,B-globin gene mutation: Further evidence for multiple
origin and migration of the thalassemia gene. NAKASHIMA,
H., CHIFU, Y. and IMAMURA, T.
Time of the deepest root for polymorphism in human mitochondrialDNA. HASEGAWA, M. and HORAI, S...
o. o'A new mtDNA mutation associated with mitochondrial myopathy,
encephalopathy, lactic acidosis and stroke-like episodes(MELAS). GOTO, Yo-I., NONAKA, I. and HORAI, S.
Heteroplasmy and polymorphism in the major noncoding region of
.0.

0

0

0

•

0

00

0

0

0

0

••

0.

0

•••••••••

0

0

•••

0

93
94

95

CONTENTS
mitochondrial DNA in Japanese monkeys: Association withtandemly repeated sequences. HAYASAKA, K., ISHIDA, T.
and HORAI, S. ..,".
A new type of mitochondrial DNA deletion in patients with enMIYABAYASHI, S., HANAMIZU, H.,
cephalomyopathy.
ENDO, H., T ADA, K. and HORAI, S. .
.
Phylogenetic affiliation of ancient and contemporary humans inferred from mitochondrial DNA. HORAI, S., KONDO, R.,
MURAYAMA, K., HAYASHI, S., KOIKE, H. and NAKAI, N.

ix

96

96

97
Extracellular matrix protein tenascin-like gene found in the human
MHC class III region. MATSUMOTO, K.-I., ISHIHARA, N.,
ANDO, A., INOKO, H. and IKEMURA, T. .
.
Giant G+C% mosaic structures in the HLA locus and a border
between the mosaic domains. MATSUMOTO, K.-I., ISHIHARA, N., ANDO, A. INOKO, H. and IKEMURA, T. .
.

X.

98

99

Applied Genetics
Responses of wild and cultivated rice populations to four different
races of pathogen Zanthomonas campestris. MORISHIMA, H.
and MIYABAYASHI, T. .
. 101
Constraints in using wild relatives in breeding: Lack of basic. 102
knowledge on crop genepools. SANO, Y.
Inheritance of low temperature resistance at the young seedling. 103
stage in rice. MORISHIMA, H. .
Genetic control of fertility reversions in cytoplasmic male sterilityof rice. SANO, Y., EIGUCHI, M. and HIRANO, H.-Y
. 104
Japonicas rice was born in East China. SATO, Y. I., TANG, X.
YANG, L. and TANG, L.
. 105
Developmental regulation of phenotypic plasticity in deepwater
rice. EIGUCHI, M., HIRANO, H.-Y. and SANO, Y. 105
An altitudinal cline of genetic variation found in Bhutan ricecultivars. SATO, T., SATO, Y.-I., YAMAGISHI, H., SHIMAMOTO, Y. and MORISHIMA, H. .
. 107
On the distribution patterns of selfing- and outcrossing-inclined. 108
genotypes in plant polulations. YONEZA W A, K. .
Analysis of chloroplast DNA from ancient rice seeds. NAKA-

x

MURA, I., CHEN, W.-B. and SATO, Y-1.
An accelerated internode overgrowth mutant in rice.
MURA, I.

XI.

, 108
NAKA109

Database

DNA database release 10 from DNA Data Bank of Japan.
UGAWA, Y.,YAMAZAKI, Y., HORIE, M., IWASE, M., SAITO,
M., SATO, Y, SUZUKI, S., HASEGAWA, Y., HATTORI, Y,
NOGUCHI, Y., SHIMOYAMA, M., HATADA, E., SUZUKI, R.,
UCHIDA, R., SHIDAHARA, Y, MATSUSHIMA, Y., WATANABE,
A., UEDA, Y., NAKAoKA, M., KAWAMOTO, T., KITAKAMI,
H., SAITOU, N., TATENO, Y and GOJOBORI, T
III
Building and searching biological information based on a relational
database system. KITAKAMI, H. and YAMAZAKI, Y ..... , III
Codon usage tabulated from GenBank genetic sequence Ddata.
W ADA, K., W ADA, Y, ISHIBASHI, F. GOJOBORI, T. and
IKEMURA, T. .
113
Publications
Abstracts of diary for 1991
Foreign visitors in 1991
Author index

, 114
125
128
131

GENERAL STATEMENT
Our institute was established 42 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofresearch on genetics, in particular population genetics, plant genetics andmolecular biology, by members of our institute have made it the center ofgenetic study in our country and a major institution with world-wide recognition. In 1984 the institute was reorganized into a National Inter-university
Research Institute to promote research activities through cooperation withother institutions. In addition, together with seven inter-university research
institutes, we formed the Graduate University for Advanced Studies, in 1988.
Our institute has admitted graduate students in the Department of Geneticsof the Graduate School of Life Science. We have now more than 20 such
students and the number is increasing. I consider it vital for our research
activities to have a steady flow of young scientists.
Recent rapid progress in the field of genetics with newly developed
approaches has greatly shifted the course of study in biology. I consider itnatural that researches in our institute are focused more on molecular studies
on various aspects of genetics. This is not to ignore the importance of more
traditional approaches on genetics. Our institute is uniquely suitable topursue cooperative works by scientists of various disciplines. Throughinteractions between these lines of researches, our institute will flourish.
We have been carrying out several research related services. The DNA
Data Bank of Japan (DDBJ) is one of the three banks in the world thatgather, annotate, store and distribute information on DNA sequences.
DDBJ will continue to be active as a regional center for such activities. Our
institute also stores and distributes various organisms with genetically characterized traits. Among them services on mice, rice and Escherichia coli are
particularly significant. These service activities will be advanced. However,as the responsible manager of these activities, I honestly feel pain in running
service operations in the conditions of understaffing, underpaying and underfunding. Nation-wide recognition of importance of these activities andappropriate actions to amend these conditions are essential.
I hope that with guidance from people in and outside this institute andfurther supports from governmental and private sources, I could lead the2institute into a more successful future.
In the past year, Associate Prof. S. Miyazawa, Dr. K. Nagata and Dr. H.
Hayashida were transferred to Gumma University, Tokyo Institute of Technology and Kyoto University, respectively. The Genetic Stock R.::;earchCenter gained Dr. N. Nakatsuji as professor and Dr. Y. Shirayoshi as
research member. Dr. A. Nishimura of the Center was promoted to associate
professor. Dr. I. Katsura joined the DNA Research Center as professor, from
where Prof. T. Ikemura and Dr. K. Matsumoto were transferred to the
Laboratory of Evolutionary Genetics. Dr. S. Horai (Laboratory of Human
Genetics) was promoted to the rank of associate professor.

STAFF
Director-General

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

OHTA, Tomoko, Ph. D., D. Sc.
Members1.

Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Laboratory of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
YAMAGISHI, Masahiro, D. Sc.
Laboratory of Mutagenesis
SENO, Takeshi, D. Sc., Professor
YAMAO, Fumiaki, D. Sc., Associate professor
TEZUKA, Hideo, D. Ag.
KANEDA, Sumiko, D. Pharm.
Laboratory of Nucleic Acid Chemistry (Guest member)
YASUDA, Hideyo, D. Pharm., Associate professor

2.

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

4

YASUDA, Seiichi, D. Sc., Associate professor
HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Laboratory of Cytoplasmic Genetics (Guest member)
OHTSUBO, Eiichi, D. Sc., Professor
YONEKAWA, Hiromichi, D. Sc., Associate professor

3.

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

4.

Department of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Head of the Department
Laboratory of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKAHATA, Naoyuki, D. Sc., Associate professor
TACHIDA, Hidenori, D. Sc.
TAJIMA, Fumio, Ph. D.
Laboratory of Evolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate professor
MATsuMoTo, Kenichi, D. Ag.
MORIYAMA, Etsuko, Ph. D.
Laboratory of Theoretical Genetics (Guest member)
KIMURA, Motoo, Ph. D., D. Sc., Professor
YASUNAGA, Teruo, Ph. D., Associate professor

STAFF

5.

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

6.

Research Facilities
Genetic Stock Research Center
MORISHIMA, Hiroko, D. Ag., Head of the Center
NAKATSUJI, Norio, D. Sc., Professor
MIYASHITA, Nobumoto, D. Med.
SHIRAYOSHI, Yasuaki, D. Sc.
WATANABE, Takao K., D. Sc., Associate professor
VEDA, Hitoshi, D. Ag.
NISHIMURA, Akiko, D. Ag., Associate professor
TATENO, Yoshio, Ph. D., D. Sc., Associate professor
DNA Research Center
SENO, Takeshi, D. Sc., Head of the Center
SHIMAMOTO, Nobuo, D. Sc., Associate professor
KATSURA, Isao, D. Sc., Professor
HIROSE, Susumu, D. Sc., Associate professor
HAYASHI, Shigeo, D. Sc.
GOJOBORI, Takashi, D. Sc., Professor
VGAWA, Yoshihiro, D. Sc.
KOHARA, Yuji, D. Sc., Associate professor
KITAKAMI, Hajime, D. Eng., Associate professor
YAMAZAKI, Yukiko, D. Sc.

5

6Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate professor, Head of the Center
Experimental Farm
SANa, Yoshio, D. Ag., Head of the Farm
NAKAMURA, Ikuo, D. Ag.
7.

Technical Section
TAMURA, Jinichi, Chief of the Section

8.

Department of Administration
EBIHARA, Akira, Head of the Department
KANDA, Tokio, Chief of the General Affairs Section
MABUCHI, Kenji, Chief of the Finance Section

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

ADVISORY COMMITTEE

7

SUGANO, Haruo; Director, Cancer Institute
SUGIMURA, Takashi; President, National Cancer Center
T AKANAMI, Mitsuru; Professor, Institute for Chemical Research, Kyoto
University
TAKEUCHI, Ikuo; Director-General, Institute for Basic Biology
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University
YURA, Takashi; Director, Institute for Virus Research, Kyoto University

ADVISORY COMMITTEE
ChairmanMORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
Vice-chairman
OSAWA, Shozo, Professor, Faculty of Science, Nagoya University
Members (Alphabetical order)
FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
ISHIWA, Sadao; Professor, Faculty of Science, Ochanomizu Women's
University
MIURA, Kin-ichiro; Professor, Faculty of Science, Gakushuin University
NAKAJIMA, Tetsuo; Professor, Faculty of Agriculture, Tamagawa University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
T AKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto University
TAKEUCHI, Takuji; Professor, Faculty of Science, Tohoku University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YOSHIKAWA, Hiroshi; Professor, Faculty of Medicine, Osaka University
HORIUCHI, Kensuke; Professor, Laboratory of Microbial Genetics
IMAMURA, Takashi; Professor, Laboratory of Human Genetics
ISHIHAMA, Akira; Professor, Laboratory of Molecular Genetics
MORISHIMA, Hiroko; Professor, Laboratory of Agricultural Genetics

8

OHTA, Tomoko; Professor, Laboratory of Population Genetics
SENO, Takeshi; Professor, Laboratory of Mutagenesis
SUGIYAMA, Tsutomu; Professor, Laboratory of Developmental GenetICS

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

PROJECTS OF RESEARCH FOR 1991
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular Genetics

Studies on regulatory mechanisms of gene transcription III prokaryotes(lSHIHAMA, FUJiTA and YAMAGISHI)
Studies on molecular architecture of transcription apparatuses in eukaryotes(ISHIHAMA and YAMAGISHI)
Studies on molecular mechanisms of transcription and replication of animaland plant viruses (lSHIHAMA, YAMAGISHI, FUJITA and NAKAMURA)
Laboratory of Mutagenesis

Molecular and genetic studies of cell-cycle associated genes (SENO, YAMAO
and KANEDA)
Genetic consequences of thymidylate stress imbalance in mammalian cells(SENO and YAMAO)
Radiation sensitivity in mammals (TEZUKA)
Laboratory of Nucleic Acid ChemisryCell-cycle regulation by cyclin and CDC2 kinase (YASUDA)
2.

DEPARTMENT OF CELL GENETICS
Laboratory of Cytogenetics

Genetic differentiation of mouse species (MORIWAKI, SHIROISHI and MIYASHITA*)
Structure and function of the mouse MHC high frequency genetic recombination (SHIROISHI and MORIWAKI)* Genetic stock research center.

10

Theoretical and experimental bases for karyotype evolution (lMAI)
Study of adrenal function in mouse development by transgene (GOTOH,SHIROISHI and MORIWAKI)
Laboratory of Microbial Genetics

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

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

DEPARTMENT OF ONTOGENETICS
Laboratory of Developmental Genetics

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

Genetic studies on the life history characters in Bombyx (MURAKAMI)
Genetic studies on the nerve system characters in Bombyx (MURAKAMI)
Studies on development and growth of insect (MINATO)
Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)
Genetic studies on maternal effect lethality in higher organisms (YAMADA)
Laboratory of Physiological Genetics

Nerve net formation in Hydra (KOIZUMI)PROJECTS OF RESEARCH FOR 1991
4.

11

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

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

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

Theoretical studies of population genetics and molecular evolution(KIMURA)
Computer analysis of genetic information (Y ASUNAGA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Genetic and physical mapping of human genome (IMAMURA and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA and NAKASHIMA)

12

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

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

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

RESEARCH FACILITIESGenetic Stock Research Center

Analysis of morphogenesis in postimplantation mouse embryos (NAKATSUJI
and SHIRAYOSHI)
Manipulation of embryogenesis using mammalian embryonic cells (NAKATSUJI and SHIRAYOSHI)
Molecular analysis of cell differentiation in postimplantation mouse embryos(SHIRAYOSHI and NAKATSUJI)
Genetic mechanisms for regulating tumor development in the laboratory andwild mice (MIYASHITA and MORIWAKI)
Evolutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA)
Genetic and Molecular studies of cell division mechanism in E. coli. (NIsHIMURA)
Theoretical studies in molecular phylogeny (TATENO)PROJECTS OF RESEARCH FOR 1991

13

DNA Research Center

Interaction between proteins and nucleic acids (SHIMAMOTO)
Genetics and molecular genetics of development and behavior of Caenorhabditis elegans. (KATSURA)
Computer analysis of DNA sequences (GOJOBORI and UGAWA)
Construction of DNA sequence database (GOJOBORI, Ugawa and TATENO)
Molecular genetics of insect development (UEDA and HIROSE)
Control of gene expression in eukaryotes (HIROSE, HAYASHI and UEDA)
Molecular Biology of C. elegans development (KOHARA)
Genome analysis of C. elegans (KOHARA)
Large-scale genetic information analysis project (KITAKAMI and Y AMAZAKI)Radioisotope Center

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

Molecular genetics of morphological mutants in rice (NAKAMURA)
DNA fingerprinting of plant species (NAKAMURA)RESEARCH ACTIVITIES IN 1991

I.

MOLECULAR GENETICS

The Promoter Selectivity of Escherichia coli RNA Polymerase:
Two Modes of Protein-Protein Communication between
RNA Polymerase and Transcription Factors
Akira ISHIHAMA, Nobuyuki FUJITA, Kazuhiko IGARASHI*, Chao ZotJ
and Masahiro Y AMAGISHI

The promoter selectivity control of RNA polymerase plays a key role inglobal control of gene transcription in prokaryotes. A number of accessorytranscription factors have been identified in E. coli, which interact with RNA
polymerase and modulate its promoter recognition properties. These factors
can be classified into two groups: one group factors form RNA polymerase
complexes in the absence of DNA; the other group factors bind to DNA andinteract with RNA polymerase only when both are aligned along the same
DNA strand (Ishihama, A. (1991) Molecules and Cells 1, 381-384; Ishihama,A. (1992) Mol. Mierobiol., in press). Regardless of apparent difference in theorder of molecular assembly, ternary complexes are formed between RNA
polymerase, transcription factor(s) and DNA signals prior to transcription
initiation.
In the course of the molecular anatomy of RNA polymerase subunits, wefound that the C-terminal proximal region of a is not necessary for both the
RNA polymerase assembly and the catalytic function of RNA synthesis, but
is needed for the molecular communication with some class I transcription
factors, which mostly bind to their target DNA sites located upstream of thepromoters (Igarashi, K. and Ishihama, A. (1991) Cell 65, 1015~1022;
Igarashi, K. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8958-8962). ClassII factors, however, do not require this contact site I on the a-subunit Cterminus, and instead interact with the still unidentified, putative site II. The
class II factors bind to the target DNA signals located near the promoter.

* Present address: Majorie B. Kolver Viral Oncology Lab., University of Chicago.

1.

Table 1.

MOLECULAR GENETICSClassification of transcription factors

Transcription factor/
Target gene(s) or promoter(s)CRP (cAMP receptor protein)/lac, gal, uxuAB, pBR-P4

OmpR/ompC, ompF

PhoBpstAda/alk, ada
OxyR/katGt, repressors/ AP L , APPR
MerR'mer
FadRifabA
FisjrrnB
GalR/gal
IHF (integration host factor)/ AP L
MetRjmet
RhaS, RhaRrha
TyrR/tyr, aro
N4 SSB/N4 late genes
NtrC, a 54 /glnA
NifA, a 54 !nifE. nijV

Table 2.
Subunit assembly
Cross-linking
Fluoresence polarization
Cys localization
Epitope mapping
X-ray crystallography
Electron microscopy
NMR
Module analysis

15

Collaborators(s)A. Hanamura/H. Aiba (Nagoya Univ.); S. Busby(Univ. Birmingham); R. Ebright (State Univ.
New Jersey); A. KolbjH. Buc (Pasteur Inst.);M. Thomas/R. E. Glass (Univ. Nottingham)
H. Aiba/T. Mizuno (Nagoya Univ.)
K. Makino/A. Nakata (Osaka Univ.)
K. Sakumi/M. Sekiguchi (Kyushu Univ.)
K. Tao (Univ. Tokyo)
G. N. Gussin/C. Olson (Univ. Iowa)
A. Z. Ansari/T. V. O'Halloran (Northwestern Univ.)
C. C. DiRusso (Univ. Tenessee)
R. L. Gourse/W. Ross (Univ. Wisconsin)
S. Adhya (NCI NIH)
A. Oppenheim/H. Giladi (Hebrew Univ.)
G. V. Stauffer (Univ. Iowa)
R. Schleif (Johns Hopkins Univ.)
A. J. Pittard (Univ. Melbourne)
L. B. Rothman-Denes (Univ. Chicago)
S. G. Kustu (UC-Berkeley)
W. Charlton/M. Buck (Univ. Sussex)

Structural studies of a subunit
R. S. Hayward (Univ. Edinburgh)
H. Buc/A. Kolb (Pasteur Inst.)
R. Ebright (State Univ. New Jersey)
O. Ozoline (Russian Acad. Sci.)
K. Sharif/J. S. Krakow (Hunter College),
R. R. Burgess (Univ. Wisconsin)
B. C. Wang (Univ. Pittsburgh)
A. Ikai (Tokyo Inst. Technology)
Y. Fujiyoshi (Prot. Engineer. Res. Inst.)
Y. Kyogoku (Osaka Univ.)
M. Go (Nagoya Univ.)

A number of known transcription factors, listed in Table 1, are being tested
as to their requirement of the contact site I for transcription activation ofrespective target genes. In parallel, we started structural studies of thecontact site I on a subunit (Table 2). These studies are being carried out as16

an international collaboration including more than 30 laboratories from 10
different countries.
The Promoter Selectivity of Escherichia coli RNA Polymerase:
Fine Mapping of the Contact Site I for cAMP
Receptor Protein (CRP)
Chao Zou, Nobuyuki FUJITA, Kazuhiko IGARASHI* and Akira ISHIHAMA

The a subunit of E. coli RNA polymerase plays an essential role in RNA
polymerase assembly. The assembly domain is located within N-terminal 235
amino acid resides (intact a is composed of 326 amino acid residues) while
the C-terminal proximal region is needed for transcription activation byclass-I transcription factors, most of which bind at the respective cis-acting
DNA signals located upstream of promoter ~ 35 signal (this contact site isdesignated as "site I" for RNA polymerase-transcription factor interaction)(Igarashi, K. et al. (1991) J. Mol. BioI. 218, 1-6; Hayward, R.S. et al. (1991)
J. Mol. BioI. 221, 23-29; Igarashi, K. and Ishihama, A. (1991) Cell 65, 10151022; Igarashi, K. et al. (1991) Proc. Natl. A cad. Sci. USA 88, 8958-8962).
For detailed mapping of the contact site I for CRP (cAMP receptor
protein), we introduced random mutations in the contact site I region of asubunit gene (rpoA) using PCR mutagenesis method and, after transformation with this mutant library, screened for Lac· colonies on lactosetetrazolium agar plate. We have sequenced the contact site I region for 7
independent Lac - isolates, 4 of which rpoA 122, rpoA 124, rpoA 125, and rpoA
129, all carried single mutation at the same nucleotide position which
generates mutant a with single amino acid substitution, R265C for a 122, a 124,
and a 129, and R265H for am. Both rpoA 123 and rpoA 127 carried single
mutation, which led mutant a with N268D (a 123) and L270P (a 127) substitution. Interestingly, all these 6 mutations are clustered within a shortsegment, amino acid residue 265 to 270, suggesting that the contact site for
CRP is located in this small region. am had double mutations, one within
this CRP-contact site (R265H) and other (K297R) outside of this site,
suggesting that R265H is responsible for the mutant phenotype of thisparticular mutant.
The effects of these mutant a subunits on transcription were examined* Present address: Majorie B. Kolver Viral Oncology Lab., University of Chicago.

I.

MOLECULAR GENETICS

17

using reconstituted holoenzymes and lac and gal promoters. CRP-dependenttranscription in vitro from the lacPl promoter decreased markedly, while
CRP-dependent transcription from the galPl promoter was not affected atall. The results agreed well with the previous finding that the contact site I
on a is necessary for transcription activation of lacPl but not galPl(Igarashi, K. et al. (1991) Proc. Natl. A cad. Sci. USA 88, 8958-8962).
The Promoter Selectivity of Escherichia coli RNA Polymerase:
Functional Mapping of Major Sigma Subunit a 70
Ashok KUMAR*, Nobuyuki FUJITA, Richard S. HAYWARD** and Akira ISHIHAMA

The recognition of nucleotide sequences at promoters by RNA polymerase
is crucial to gene transcription and regulation. In E. coli, the major formof RNA polymerase (E0 70 ) interacts with most promoters by recognizing
two hexameric sequence elements, known as - 10 (TATAAT) and - 35
(TTGACA) signals, located around 10 and 35 bp, respectively, upstream oftranscription start site. It is considered that RNA polymerase (Edo) makes
contacts with the bases in these sequences, and the activity of promotersdecreases as the actual sequence deviates from the consensus. Among four
conserved amino acid sequences between multiple species of 0 subunits fromvarious prokaryotes, the region-2 and region-4 have been suggested to be
responsible for recognition of -10 and - 35 sequence, respectively.
Over the past few years, however, a number of E. coli promoters have been
identified, that do not obey these simple rules (Keilty, S. and Rosenberg, M.
(1981) J. BioI. Chern. 262, 6389-6395). In these cases, the RNA polymerase
Edo recognizes only the -10 sequence (and possibly a sequence upstream of-10 sequence). The basic question we asked was whether the region-4
located at the carboxy terminus of dO is dispensable for transcription
initiation at the "extended -10 promoters" with no significant - 35 sequence. We overexpressed the C-terminal truncated. dO (including region-4),
purified to homogenity and reconstituted various molecular species of mutant
holoenzyme containing the truncated dO subunits. In vitro transcription study
indicated that the region-4 is indeed not essential for transcription of the***

On leave of absence from the Institute of Cell and Molecular Biology, University of
Edinburgh (supported by the Royal Society and British Council).
Institute of Cell and Molecular Biology, University of Edinburgh.

18

extended ~ 10 promoter. We also found that TGN motif immediately
upstream of ~ 10 consensus sequence is important for the function of theextended ~ 10 promoter.
The Promoter Selectivity of Escherichia coli RNA Polymerase:
Structure and Function of Stationary-phase RNA Polymerase
Miwako OZAKI, Nobuyuki FUJITA and Akira ISHIHAMA

The promoter selectivity control of RNA polymerase plays a major role inglobal control of gene transcription in E. coli during stress response. As an
extension of this line study, we analyzed the growth phase-dependent modulation of RNA polymerase.
The overall level of gene transcription in stationary-phase cells decreased
to less than 10% the level of exponential growth phase. The synthesis andassembly of RNA polymerase are repressed almost completely (reviewed in
Ishihama, A. (1991) In: "Control of Cell Growth and Division", pp. 121140) and, in addition, the pre-existing RNA polymerase is converted intofunctionally different forms with altered promoter selectivity (Ozaki, M.
et al. (1991) Mol. Gen. Genet. 230, 17-23). The stationary-phase RNA
polymerase is unable to transcribe in vitro promoters associated with genes
expressed in exponentially growing cells, such as promoters of the genes for
rRNA, ribosomal proteins, tRNA and aminoacyl-tRNA synthetases. Only alimited number of promoters are transcribed, including promoters for thegenes encoding D-lactate dehydrogenase, glucose 6-phosphate dehydrogenase, NADH dehydrogenase, catalase hydroperoxidase, ribonuclease H,
RecA protein, NusA transcription termination factor, and heat-shock sigma
factor 0 32 (Ozaki, M. et a/. (1991) Nucleic Acids Res. 20,257-261).
All the stationary-phase form enzymes contained 0 7°, the major sigma
subunit, and three major core subunits, (3', (3 and a, but were very acidic in
net charge. The holoenzyme reconstitution experiments indicated that thefunctional difference was originated from the core enzyme portion. To oursurprise, the stationary-phase form enzymes were converted into the logphase type enzyme after treatment with nucleotides or pyrophosphate. Adenine nucleotides were most effective in this conversion. One candidate for thephosphorylated factor associated with the stationary-phase RNA polymerase
is poly- or oligophosphate. In collaboration with Drs. M. Akiyama and A.

I.

MOLECULAR GENETICS

19

Kornberg (Stanford Univ.), this possibility was indeed found to be the case.

Regulation of the Escherichia coli rmf Gene Encoding
Ribosome Modulation Factor
Masahiro YAMAGISHI, Hiroshi MATSlJSHIMA*, Masayuki SAKAGAMI**,
Akira WADA*** and Akira ISHIHAMA

Ribosome modulation factor (RMF) is a small protein of 55 amino acidresidues associated with lOOS ribosome dimers which appear in stationary
phase cells of Escherichia coli (Wada, A. et al. (1990) Proc. Natl. Acad. Sci.
USA 87, 2657~2661). The structural gene, rmf, has been cloned from
Kohara's phage library of the entire E. coli genome, sequenced and mapped
at 21.8 min of the E. coli chromosome. To examine the expression pattern of
rmf under various growth conditions, we carried out Northern blot hybridization and ,B-galactosidase assay for a rmf-lacZ fusion gene. In rapidly
growing cells in nutrient-rich media, the expression of rmf-lacZ was hardly
detected but during the growth transition from exponential to stationary
phase, both the level of rmf-lacZ RNA and the activity of ,B-galactosidase
rapidly increased in a parallel way. Thus, the rmf gene seems to betranscribed only in stationary phase.
Next, we examined the effect of growth rate on rmf expression. When cells
were grown at slow rates in poor media, rmf-lacZ was expressed even in
exponential growing phase. The level of ,B-galactosidase activity was inversely correlated to growth rate. The nucleotide sequence of the promoter
region deduced from the position of transcription start site deviates from theconsensus sequence recognized by all the known RNA polymerase, i.e.,
holoenzymes containing not only the major 0 70 subunit but also other minor
sigma subunits. In fact, rmf was not transcribed in vitro by purified RNA
polymerase containing 0 70 subunit. When rmf on the chromosome was disrupted, the resultant mutants lost viability during stationary phase, suggesting that rmf is one of the essential genes for survival in stationary phase. In
the rmf mutant, WOS ribosomes were not detected even in stationary phase.
Analysis of the function(s) of RMF and the activity of lOOS ribosomes
*
**
***

Present address: Health Science Research Institute, Yokohama.
Present address: Equine Research Institute, Japan Racing Association, Tokyo.
Department of Physics, Faculty of Science, Kyoto University, Kyoto.

20

dimers as well as search for cis-acting element(s) and trans-acting factor(s)required for the expression of rmf will be our future projects.
Structure and Function of RNA Polymerase II from Schizosaccharomyces
pombe: Cloning and Sequence Determination of the Second
Largest Subunit Gene
Makiko KAWAGISHI, Masahiro Y AMAGISHI and Akira ISHIHAMA

In spite of increasing knowledge of transcription factors, relatively little is
known on the structure and function of RNA polymerase in eukaryotes. We
then started cloning and DNA sequencing of the Schizosaccharomyces pombe
genes encoding the putative subunits of RNA polymerase II. Last year, wecloned the gene, rpbI, coding for the largest subunit and determined the
DNA sequence (Azuma, Y. et al. (1991) Nucleic Acids Res. 19,461-468).
This year, the gene, rpb2, encoding the second largest subunit of RNApolymerase II has been cloned from Schizosaccharomyces pombe using thecorresponding gene, RPB2, of Saccharomyces cerevisiae as a probe for crosshybridization. We have determined the complete nucleotide sequence of
rpb2, and parts of peR-amplified rpb2 eDNA. The predicted coding sequence, interrupted by a short intron, is able to code for a polypeptide of 1210
amino acid residues with a calculated molecular weight of 138 ki1odaltons.
Deduced amino acid sequence of rpb2 was nearly 70% identical with that ofS. cerevisiae RPB2. Detailed Southern analysis of genomic DNA digested
with various restriction enzymes showed that rpb2 is present as a single copyin the S. pombe genome. Northern analysis showed that the transcript of rpb2
is about 4 kb in length.
Recently it has been reported that a local sequence similarity exists between
the second largest subunit of RNA polymerase II and bacterial RNases(Shirai, T. and Go, M. (1991) Proc. Natl. Acad. Sci. USA 88,9056-9060).
We expressed the "RNase-like domain" of the second largest subunit of S.
pombe RNA polymerase II in E. coli. The product exhibited RNA binding
activity in a preliminary experiment.

I.

MOLECULAR GENETICS

21

Structure and Function of Influenza Virus RNA Polymerase: Reconstitutionof Functional RNA Polymerase from Three Subunits Expressed
Using a Recombinant Baculovirus System
Makoto KOBAYASHI, Kotara TUCHIYA*, Kyosuke NAGATA** and Akira ISHIHAMA

Influenza virus RNA polymerase catalyzes multiple step reactions in bothtranscription and replication of the genome RNA. The core enzyme iscomposed of each one of the three P proteins, PB I, PB2 and PA (Honda, A.
et al. (1990) J. Biochem.l07, 624-628). Genetic and biochemical studies
suggested that PBI is involved in polymerization of nucleotides to nascent
RNA chains while PB2 is involved in recognition of host cell capped mRNA.
The role of PA protein is not known yet, but temperature-sensitive mutationsin the PA gene affect only vRNA synthesis, but not mRNA synthesis.
For detailed analysis of the role of each P protein and the functional
domains on each P polypeptide, we expressed individual P proteins in
cultured insect cells after infection with recombinant baculovirses (Kobayashi, M. et al. (1992) Virus Res. 22,235-245). For this purpose, we inserted
the cDNA for each P gene into a baculovirus vector as to express under thecontrol of the polyhedrin promoter which is extremely active at the late stageof baculovirus infection. Using these recombinant viruses, we produced large
amounts of each P protein in insect cells. PBl and PB2 accumulated in cell
nuclei whereas PA stayed in cytoplasm. Both the PBl and PB2 proteins were
purified from aggregates in the respective nuclear extract, and the PA waspartially purified from the cytoplasm. RNA polymerase was reconstituted bymixing the three P proteins in a urea solution and then dialyzing against a
reconstitution buffer. The reconstituted enzyme was able to transcribe modelRNA templates in the presence of ApG as a primer. Minus-sense RNA wasa better template than plus-sense RNA.
Inhibition Mechanism of Influenza Virus Growth by Lignin
Jira YASUDA, Manabu NAKAYAMA and Akira ISHIHAMAPreviously we found that lignin, an abundant component in plant, inhibits***

Nippon Institute for Biological Science, Orne, Tokyo.
Present address: Faculty of Bioscience and Biotechnology, Tokyo Institute ofTechnology, Yokohama.

22

the growth of influenza virus and the actlVlty of virion-associated RNA
polymerase (Nagata, K. et al. (1990) Antiviral Res. 13, 11-22; Sakagami, M.
et al. (1990) Biochem. Biophys. Res. Commun. 172, 1267-1272). The
polymerized phenolic structure of lignified materials is responsible for thisanti-influenza virus activity (Harada, H. et al. (1991) Antiviral Res. 15, 4150). For detailed understanding of the virus growth inhibition, we attempted
to identify the target of lignin action by isolating lignin-resistant virus
mutants. After four passages of WSN strain (H 1N 1) in MDCK cells in thepresence of lignin, we isolated two mutants which were resistant to lignin up
to 200 flgfm/. The growth of wild-type parental virus. was completely
inhibited at this concentration.
After mixed infection of the resistant mutants with lignin-sensitive Aichi
strain (H3N2) in MDCK cells, lignin-resistant reassortants were isolated in
the presence of anti-HI antibodies. Although the growth of Aichi strain isretarded in MDCK cells, all the lignin-resistant reassortants were found tocarry RNA segment 7 from WSN strain. The results suggest that either Ml
or M2 encoded by WSN RNA segment 7 plays an essential role in growth of
influenza virus in MDCK cells, and that such fast growers are apparently
resistant to lignin.
Inhibition Mechanism of Influenza Virus Growth by Mx1 Protein:
Self-assembly of Mxl and GTP-dependent
Conformational Change
Manabu NAKAYAMA, Kazumori YAZAKI*, Akira KUSANO**, Kyosuke NAGATA***,
Nobuo HANAI** and Akira ISHIAHAMA

Murine Mxl protein, a host factor involved in resistance against influenza
virus intection, is an inferferon-induced nuclear protein. Last year, we
purified the Mxl protein from extracts of E. coli cells expressing Mxl eDNA,
and found that the Mxl protein is a GTP-binding protein with GTPase
activity (Nakayama, M. et al. (1991) J. Bioi. Chem. 266, 21404-21408;
Nakayama, M. et al. (1992) Virus Res. 22, 227-234). To confirm theconclusion, we purified the native Mxl protein from poly(l): poly(C)-treated
A2G mouse liver. The native Mxl exhibited GTPase activity, and both the
*
**
***

Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo.
Tokyo Research Laboratories, Kyowa Hakko, Machida, Tokyo.
Present address: Tokyo Institute of Technology, Midori-ku, Yokohama.

I.

MOLECULAR GENETICS

23

K m and Vmax are essentially identical with those of Mxl purified fromeDNA-expressing E. coli.
As judged from elution profile from gel filtration columns, the Mx 1 protein
was indicated to form self-assembled polymers. By negative staining
electron microscopy, both preparations of the Mxl protein were observed
as big polymers, forming "horseshoe"-like structure (Nakayama, M. et a/.,submitted for publication). By making a set of deletion derivatives, the motif
for self-assembly was mapped between amino acid residues 51-99. This motif
is highly conserved in not only Mx family proteins but also Mx-related
proteins.
Microscopic analysis of immunostained mouse liver revealed that the Mx 1
protein exists as giant complexes in nuclei. These observations altogether
suggest that the self-assembly of Mxl protein is essential for its biologicalactivities including antiviral function.
Molecular Architecture of Rice Stripe Virus:
Genome Structure and Role of RNA Polymerase
Akira ISHIHAMA, Chika HAMAMATSU, Ikuo NAKAMURA, Pascale BARBIER*,
Mami TAKAHASHI** and Shigemitsu TORIYAMA**

Rice stripe virus (RSV), the prototype of the tenuiviruses, is characterized
by its unique genome composition, consisting of four segment each of both
single-strand and double-strand RNA. Viral particles are composed of only
two viral proteins, 35 kDa coat protein (CP) and a minor polypeptide with anestimated size of 230 kDa, but contain RNA polymerase activity. Terminal/
sequences of both 5 - and 3' -proximal ends are conserved among four RNA
segments, and are complementary to each other (Takahashi, M. et a!. (1990)
J. Gen. Virol. 71, 2817-2821), indicating that RSV is a member of either
negative- or ambi-sense RNA viruses. Analysis of the complete sequence of
two small RNA segments indicated that both segment 3 and 4 are ambisence,containing an open reading frame at the 5' -terminal region of both vRNAand its complementary RNA (cRNA). This year, we sequenced RNA
segment 2 and the results suggest that it is also ambisence RNA (Takahashi,
M. et a/., submitted for publication).
***

Present address: Centre d'Etudes Nucleaire de Saclay, Gif-sur-Yvette.
National Institute of Agro-Environmental Sciences, Tsukuba.

24

As an attempt to reveal the molecular mechanism of virus multiplication,
we attempted to purify RNA polymerase and to analyze the mechanism of
RNA synthesis using purified RNA polymerase. The RNA polymerase wasdissociated from viral RNA (vRNA) by CsCI centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template of 50/
nucleotides in length, still carrying the 5 - and 3 -terminal conserved
sequences of all four RNA segments (Barbier, P. et al. (1992) J. Viral. 66,/
6171-6174). A 3 -terminal half molecule of the model template was also
active as a template. Hence, we proposed that the 3' -terminal conserved
sequence serves as a promoter for the RNA polymerase.
1

Intron 1 as a Determinant of the Cell Cycle-Dependent Expressionof Human Thymidylate Synthase
Atsushi TAKAYANAGI, Sumiko KANEDA, Dai AYASAWA and Takeshi SENO

Thymidylate synthase (TS) catalyzes the only pathway for de novo synthesis of dTMP and plays a key role in balancing the four nucleotide precursors
for DNA replication. Impairment of this enzyme causes various biologicaland genetic abnormalities. TS activity and mRNA content are associatedwith the DNA synthesis phase in the cell cycle: they increase sharply 20- to
40-fold at the G l-S border during serum-induced transition from the resting(GO) phase to the S phase in cultured mammalian cells.
We cloned biologically active human TS cDNA and the genomic DNA
clones covering the entire human TS gene of about 16 kb which is divided by
six introns. Determination of their structures enabled us to investigate theregion of the human TS gene responsible for S-phase-dependent expression
through the construction of various chimeric minigenes for TS and theexamination of their mode of expression during the cell cycle. For this, we
introduced each construct into a TS-negative mutant of rat 3Yl cells.
Through serum-restricted synchronization of the transformant cells, we
examined the expression of the introduced gene during the transition fromthe GO phase to the late S phase by measuring TS enzyme activity, mRNAlevel and transcription rate. We found that a minigene possessing the
genomic 5 -flanking region and intron 1 without other introns was sufficientfor S-phase specific expression at the normal extent and pattern at bothmRNA and enzymatic activity levels. A TS cDNA clone driven by an SV401

I.

MOLECULAR GENETICS

25

based expression vector showed constitutive expression. Insertion of intron 1
into the eDNA clone in the normal location converted the constitutive
expression to a S-phase dependent one, although the conversion was incomplete, that is, a considerable level of enzyme activity and mRNA were stilldetected in GO phase cells and also their levels in the S phase were lower thannormal. Replacement of the viral 5'-promoter region of the eDNA clone
with the genomic 5' -flanking sequence also gave rise to the same change. To
obtain the fully cell cycle-dependent expression equivalent to that of the bonafide TS gene, however, the coexistence of the two regions was required.
Results obtained by a nuclear run-on assay suggested that posttranscriptional
controls are also involved in this type of expression.
Regulatory Sequences in the Human Thymidylate Synthase Gene/
are Clustered at the 5 ·End of the First Intron
Sumiko KANEDA, Nobuyuki HORIE, Keiichi TAKEISHI, Atsushi TAKAYANAGI,
Dai A YUSAWA and Takeshi SENO

A human thymidylate synthase (TS) minigene containing 5 /- and 3'flanking sequences and only intron 1 showed a normal frequency of stabletransformation when transfected into TS-negative mutant cells, whereas
minigenes in which intron 1 was replaced by intron 2 or deleted in the above
construct showed a much lower frequency. Introduction of intron 1 into theabove intronless or intron 2-minigene restored transforming activities regardless of position and orientation. However, introduction of intron 1 into ts
cDNA on an SY40-based vector did not stimulate transformation frequency.
Deletion analysis revealed two positive and one negative regulatory sequencesin the 5'-end of intron 1, each of which seemed to bind specific proteins asshown by gel shift analysis. Intron 1 also stimulated expression of a TS
promoter-CAT gene construct but not that of an SY40 promoter-CAT geneconstruct. These results indicate that the multiple regulatory sequences
clustered in intron 1 stimulate TS gene expression in concert with thehomologous promoter.

26

Complementation by a Cloned Human Ubiquitin-Activating Enzyme
El of the S-Phase-Arrested Mouse FM3A Cell
Mutant with Thermolabile El
Sumiko KANEDA, Takeshi SENO, Dai A YUSAWA, Hideyo YASUDA
and Yasuko MURAKAMI

Duringe the genetic characterization of temperature-sensitive mutants ofmouse FM3A cells by the cell-cell hybridization test and DNA-mediatedgene transfer, we unexpectedly found that an unusually high proportion ofthese ts mutants, although showing different phenotypes, were of a single
complementation group to which the widely used FM3A cell mutant ts85
with a thermolabile ubiquitin-activating enzyme EI also belonged.
We identified one of the above mutants designated tsFS20, as a mutant
with thermolabile EI through biochemical analysis and transfection with afull-length eDNA encoding the human EI enzyme. Upon a shift-up of
temperature, asynchronously growing tsFS20 cells showed multiple points ofcell-cycle arrest as revealed with flow cytofluorometry. When the mutant
cells that had been synchronized at the G l-S phase were allowed to traverse
at a nonpermissive temperature, the cells accumulated in the mid-S phase.
The specificity of the accumulation was further evidenced by the absence ofG2-specific cdc2 kinase activity in the arrested cells. In contrast, ts85 cells
under the same conditions reached the G2-phase as reported previously andshowed an increase of cdc2 kinase activity. Thus, the EI mutation seems to
produce effects differentially, in S-phase progression and possibly in the G2phase, depending on the type of mutation occurring in the coding region. Itshould be mentioned in this context that the EI enzyme is a large protein of
1,058 amino acid residues and could be divided into multiple functional
domains including those for interaction with a family of the ubiquitin carrier
proteins E2. We also demonstrated genetically that EI is involved in the
degradation of abnormal protein in the cells: degradation of short-lived
abnormal proteins in tsFS20 cells decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level intransformant cells. The unusually high incidence of the ts E I mutation could
be explained by the fact that we mapped the El gene to human X chromosome (Xpl 1.3-pl 1.23) and by the recent report that the El is a determinantof heat tolerance in cells.

I.

MOLECULAR GENETICS

27

DNA Degradation during Thymidylate Stress is Coupledwith DNA Synthesis in Mammalian Cells
Fumiaki YAMAO, Yukiko NAGAI, Sei YOSHIDA, Sumiko KANEDA
and Takeshi SENO

Thymidylate stress is known to cause rapid cell death, so called thymineless death. In mammalian cells chromosomal DNA degradation occurs
during this stress, resulting in DNA fragments ranging from SO to 200 kb inlength. Previous studies suggest that the DNA cleavage is coupled with the
S phase of cell cycle. In order to investigate the molecular basis of the DNA
degradation, temperature sensitive growth mutants of the mouse mammary
carcinoma cell line FM3A were selected as conditional thymineless death
resistants at high temperature(39.SoC). Among the ts mutants were those
whose chromosomal DNA is not degradable under thymidylate stress even ata permissive temperature(33.S0C). Consequently, twenty-four mutants were
established and clarified into two complementing groups with cell fusion
analysis. The mutated gene in one group was identified as that for ubiquitin
activating enzyme El since mutants were not complemented with ts8S, anauthentic El mutant of FM3A. Simultaneously, another complementing
group was found to consist of mutants of the DNA polymerase alpha subunit.
A previous analysis of EI mutants of mouse cell lines suggested that ubiquitination is somehow involved in DNA synthesis and the cell cycle progression of the SjG2 phase. Thus, thymineless death resistance selected mutants
deficient in DNA synthesis in mammalian cells, which is in good contrast to
mutants of protein synthesis as predominantly selected by the same method in
E. coli. This indicates that the anomaly of DNA synthesis caused bymutational alteration of the replication machinery suppressed DNA degrada.tion under thymidylate stress. This is in accordance with previous observations that DNA degradation occurs during the S phase of the cell cycle. The
results also imply a substantial involvement of ubiquitin in DNA synthesis of
mammalian cells.

28

Structural Analysis of Wild Type and Mutant cDNAs for Mouse
Ubiquitin Activating Enzyme El.
Nobuyuki IMAI, Sumiko KANEDA, Yukiko NAGAI, Takeshi SENO
and Fumiaki Y AMAO

The ubiquitin system appears essential for cell growth because conditional
lethal mutants of enzymes involved in the conjugation pathway have been
isolated in mammalian somatic cell lines and yeast. In mouse cell lines, many
temperature sensitive growth mutants have been ascribed to a defect in the E
I enzyme in spite of multifarious phenotypes in terms of arresting points inthe cell cycle.
As a first step in clarifying the pleiotrophic effects of EI mutations on thecell cycle, we isolated functional mouse EI cDNA from the mouse mammary
carcinoma cell line FM3A. The cDNA was screened by hybridization with
a cDNA probe specific for human El. The complete sequence was found to
consist of 91 bp of 5'flanking sequence, a complete protein coding region of1058 amino acids(117.8kDa), and a 230bp 3'UTR with poly(A) at the end.
Amino acid sequences of mouse and· human El proteins were identical intheir length and shared 95% homology as a whole. The homology of themouse El amino acid sequence to that of yeast and wheat was 53% and 44%,
respectively. The cDNA fragment was re-cloned to pSG5, a mammalian
expression vector linked to the SV40 early promoter, and transfected totemperature sensitive E 1 mutants of FM3A. Although transfection efficiency
was rather variable among the mutant lines, most of the growth in the mutant
lines was rescued at restrictive temperature. Thus, the cDNA functions wellin the cell. Mutations in seven FM3A mutant cell lines were identified in thesequence, all of which were found to be localized in the C terminal half of the
El sequence.
Kinetic Study on Transcription by Immobilized Operons: Requirement
for ,r-phosphodiester Bond of ATP in Iinitiation
by Prokaryotic RNA Polymerases.aTomoko KUHORI, Hiroyuki TERADA* and Nobuo SHIMAMOTO

The requirements for the /3, r-phosphodiester bond of ATP in transcription*

Kyushu University.

I.

MOLECULAR GENETICS

29

InitiatIOn by prokaryotic RNA polymerases has long been questioned. A
method we devised for determining these requirements consists of immobilizing template DNA and separating the transcription complex from released
products.
A template DNA fragment was fixed to acrylamide beads at its ends withtight binding between avidin and biotin. The immobilized DNA was atemplate as good as free DNA for E. coli, bacteriophage T7, T3, and SP6
RNA polymerases. Transcription was started by adding a set of substrates,
and then interrupted by rapidly diluting substrates to 0.1 f1 M. Elongation was
resumed by adding the second set of substrates. Using either ATP or an ATP
analogues as one of four substrates, we determined that the 13, r-pyrophosphate bond of ATP increased the yield of long transcripts in the early stageof elongation, but did not affect it much in the late stage. Moreover, abortive
initiation was activated with such an ATP analogue. The requirement for
intact ATP in productive initiation was found in the all transcription system
examined. Thus ATP is likely to be used to keep the elongation complex inproductive form in the early period of initiation.
Single-Molecule Dynamics of Transcription by Immobilized Operons:
Sliding of RNA polymerase on DNA
Nobuo SHIMAMOTO

The first step of transcription is the search for and binding of RNApolymerase to a promoter, and this promoter search is the major determinant
in selective expression of genes. The enzyme could directly bind to apromoter on a long DNA, or alternatively, first bind to DNA in a nonspecificmanner and then form the promoter complex by one-dimensional diffusion
along the DNA chain. Unfortunately, kinetic evidence for the two conflicting models has been reported and further study should be based on more
confident evidence. The immobilized operon provides a critical method for
determining which case is true. Linear DNA immobilized on a slide glass was
prepared. Fluorescently labeled RNA polymerase was added to the linearly
fixed DNA, and the movement of a single molecule of RNA polymerase wasfollowed through microscopy and recorded on a video-tape. Onedimensional movement was detected and its contribution to promoter binding
is now being evaluated.

30

Morphological Pathway of Flagellar Assembly in Salmonella typhimurium
Tomoko KUBORI, Nobuo SHIMAMOTO, Shigeru YAMAGUCHI*, Keiichi NAMBA**and Shin-Ichi AIZAWA***The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar
mutants were purified by CsCI density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by twodimensional gel electrophoresis. The MS ring is formed in the absence of any
other flagellar components, including the switch complex and the putative
export apparatus. Four proteins previously identified as rod components,
FlgB, FlgC, FlgF, and FlgG, plus another protein, FliE, assemble cooperatively into a stable structure. Hook is formed in two distinct steps: formationof proximal part and elongation. Proximal part formation occurs, butelongation does not occur, in the absence of the LP ring. FlgD is necessary
for hook formation, but not for LP-ring formation. A revised pathway offlagellar assembly was proposed based on these and other results.
Structural Study on E. coli Single-Stranded DNA Binding Protein
Katsuhiko KAMATA****, Taketomi TSUKIHARA*****, and Nobuo SHIMAMOTO

E. coli Single-Stranded DNA Binding Protein (SSB) is a 19kda protein andessential to replication and recombination. In physiological conditions, itexists as a tetramer and binds to both single-stranded DNA and a group ofmRNAs which have a homologous RNA element called SSB box. Both of
the bindings are cooperative and the protein molecules form clusters on such
nucleic acid molecules. These bindings suggest a coupling between replication and translation by exchanging SSB between replication forks andmRNAs.
As the first step for understanding the mechanism of these characteristic
bindings, the protein was over-produced and purified to homogeneity. Crystals of the protein were formed with a salting out procedure and a larger
*
**
***
****
*****

Meiji University.
ERATO, Research and Development Corporation of Japan.
Teikyo University.
Tottori University.
Tokushima University.

I.

MOLECULAR GENETICS

31

crystal suitable for X-ray crystallography was obtained. A diffraction patternof 3.5 angstrom is now being analyzed. The protein see'11S to have two
structural domains although it is a small peptide.
Negative Supercoiling of DNA Facilitates an Interaction between
Transcription Factor lID and the Fibroin Gene Promoter
Mitsuko MIZUTANI, Tsutomu OHTA, Hajime WATANABE*, Hiroshi HANDA*
and Susumu HIROSE

Transcription of the fibroin gene can be reconstituted with partiallypurified components from HeLa cells. Transcription factors lIB, 110, andlIE and RNA polymerase II are required for accurate initiation of transcription. Linear and relaxed closed circular DNA show a similar level of template
activity.
However, transcription of closed circular DNA is stimulated when negative supercoils are introduced by the addition of DNA topoisomerase II anda supercoiling factor purified from the posterior silk gland of Bombyx mori.
Dissection of transcription into pre- and postinitiation steps by the use of
Sarkosyl reveals that DNA supercoiling promotes formation of a preinitiation complex. Furthermore, order of addition experiments suggest that DNA
supercoiling facilitates a functional binding of transcription factor 110 to thepromoter. For details, see Proc. N atl. Acad. Sci. USA 88, 718-722, 1991.
DNA Superhelicity Affects the Formation of Transcription Preinitiation
Complex on Eukaryotic Genes Differently
Mitsuko MIZUTANI, Kiyoe URA and Susumu HIROSE

In vitro transcription was reconstituted with HeLa cell transcription factors and RNA polymerase II, which were essentially free from DNA topoisomerase activities. DNA templates with defined negative superhelicaldensities were tested for transcription activity. Transcription of the Bombyx
mori fibroin gene increases and plateaus from templates of increasing superhelicity, and transcription from the adenovirus 2 major late promoter rises
and then falls, while transcription of the Drosophila hsp70 gene remains
unchanged. Dissection of transcription into pre- and postinitiation steps by*

Department of Bacteriology, Faculty of Medicine, The University of Tokyo, Tokyo.

32

the use of Sarkosyl reveals that formation of a preinitiation complex on the
fibroin gene or the adenovirus 2 major late promoter is slow on relaxed DNA
and accelerated by DNA superhelicity. On the contrary, the preinitiation
complex assembles rapidly on the hsp70 gene irrespective of DNA topology.
As is the case with the fibroin gene promoter, DNA superhelicity appears tofacilitate the interaction of transcription factor lID to the adenovirus 2 major
late promoter. For details, see Nucleic Acids Res. 19, 2907-2911, 1991.
Possible Role of DNA Topoisomerase II on Transcription of the Homeobox
Gene Hox-2.1 in F9 Embryonal Carcinoma Cells
Kiyoe VRA and Susumu HIROSE

The Hox-2.1 gene is one of the homeobox-containing genes located in the
Hox-2 cluster on mouse chromosome 11. In this study, we examinedtranscription of the Hox-2.1 gene during differentiation of F9 embryonalcarcinoma cells induced by treatment with retinoic acid. The level of Hox-2.1
mRNA increases rapidly after induction of differentiation and then falls.
Nuclear run-on experiments demonstrate that the rate of transcription for the
Hox-2.1 gene also increases upon differentiation. Treatment of F9 cells witha DNA topoisomerase II inhibitor etoposide (VP-16) during differentiation
blocks the accumulation of Hox-2.1 mRNA. Nuclear run-on analyses
revealed that etoposide inhibits transcription of the Hox-2.1 gene during F9cell differentiation. Measurements of the level of Hox-2.1 mRNA after
blocking transcription by actinomycin D show that etoposide does not affectthe stability of the mRNA. These observations indicate that DNA topoisomerase II is involved in the control of Hox-2.1 gene transcription. For
details, see Nucleic Acids Res. 19, 6087-6092, 1991.
Defining a Sequence Recognized with BmFTZ-Fl, a Sequence Specific DNA
Binding Factor in the Silkworm, Bombyx mori, as Revealed
by Direct Sequencing of Bound Oligonucleotides andGel Mobility Shift Competition Analysis
Hitoshi VEDA and Susumu HIROSE

BmFTZ-Fl is a Bombyx mori homologue of FTZ-Fl, a positive regulatorof the jushi tarazu gene of Drosophila melanogaster. In order to determine the

I.

MOLECULAR GENETICS

33

recognition sequence of this factor, we made three sets of oligonucleotide
mixture which contained 4 possible nucleotides at different positions within
the previously proposed 12-bp binding consensus sequence. Oligonucleotides
which bound to purified BmFTZ-Fl were separated by a gel mobility shift
procedure and a binding sequence was determined by direct sequencing
through the Maxam~Gilbert method. With this analysis, S positions showed
clear sequence preference, 4 positions showed partial sequence preference and3 positions showed no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition
analysis and the results were presented as a quantitative difference in binding
affinity. From these analyses, we concluded that the best binding sequence of
BmFTZ-Fl is S'-PyCAAGGPyCPu-3'. This method may be useful for thedetermination of a binding sequence in other sequence specific DNA binding
factors. For details, see Nucleic Acids Res. 19, 3689~3693, 1991.
Search for Target Genes of Drosophila Homeotic Genes
Shigeo HAYASHI, Naoyuki FUSE and Susumu HIROSE

Homeotic genes of Drosophila play a central role in the specification ofbody segments. Genes that are controlled by the homeotic genes, the target
genes, are not well understood. Weare using a genetic and molecularapproach to identify such genes. Formation of the anterior spiracle, which ispart of the tracheal system, is controlled by homeotic genes in the bithorax
complex. The study of genes required for the formation of the anterior
spiracle should lead to identification of the target genes. Using an "enhancer
trap" screen, we have identified lines which express j3-galactosidase specifically in the precursor of the anterior spiracle. Using those lines, genetic andmolecular characterization of genes expressed and required in the anterior
spiracle are in progress.
Molecular Cloning of escargot, a Gene Controlling Segmentationof Larva and Adult.
Shigeo HAYASHI, Allan SHIRRAS and Susumu HIROSE

Mutations in the escargot (formally called fleabag) locus cause a variety ofdefects in adult structures such as malformation of wing and leg and loss of34

abdominal cuticular structures. The escargot locus is more than 70 kb long
but contains a small transcription unit of about 2.7 kb which potentially
encodes a zinc finger protein highly homologous to the Drosophila snail
protein. Abdominal histblasts in escargot mutant larva become po!vploid,suggesting the normal function of escargot is maintainance of ploidy so that
histblasts can enter the mitotic cycle during metamorphosis.
FTZ-Fl, a Steroid Hormone Receptor-like Protein Implicatedin the Activation of fushi tarazu.
Giovanni LAVORGNA, Hitoshi VEDA, Joachim CLOS and Carl Wu

The Drosophila homeobox segmentation gene fushi tarazu. (ftz) is expressed in a seven-stripe pattern during early embryogenesis. This characteristic pattern is largely specified by the zebra element located immediately
upstream of the (ftz) transcriptional start site. The FTZ-Fl protein, one ofmultiple DNA binding factors that interact with the zebra element, is
implicated in the activation of ftz transcription, especially in stripes 1, 2, 3,
and 6. An FTZ-Fl complementary DNA has been cloned by recognition site
screening of a Drosophila expression library. The identity of the FTZ-FI
complementary DNA clone was confirmed by immunological cross-reaction
with antibodies to FTZ-Fl and by sequence analysis of peptides from purified
FTZ-Fl protein. The predicted amino acid sequence of FTZ-Fl revealed
that the protein is a member of the nuclear hormone receptor superfamily.
This finding raised the possibility that a hormonal ligand affects the expression of a homeobox segmentation gene in early embryonic development. For
details, see Science 252, 848-851, 1991.
Accumulation of Wx Protein and Amylose in Rice Seeds Increase
in Response to Cool Temperatures
Him-Yuki HIRANO and Yoshio SANOoWhen rice plants are exposed to cool temperatures (less than 20 e) during
seed development, the amylose content, relative to total starch, in theendosperm increase causing a lower quality of rice when cooked. We
examined this response to cool temperatures with respect to gene expression
at the wx locus, which controls amylose synthesis in the endosperm.

I.

MOLECULAR GENETICS

35

Rice plants were grown during seed maturation at two different temperatures, 28°C (normal) and 18°C (cool), after anthesis. The amounts of thegene product (Wx protein) of the wx locus were estimated by Western
blotting analysis. The results indicated that not only amylose content but also
the amount ofWx protein increased in seeds matured at 18°C compared withthose at 28°C. Then, plants were grown at 18°C for different lengths of time
during the period, in which Wx protein accumulated linearly, that is, for 1, 2,
4, 8, or 12 days from the 7th day after anthesis, and then grown at normal
temperatures until seed maturation. The longer plants were exposed to atemperature of 18°C, the higher the level of amylose and WX protein
accumulated in mature seeds. The extent of increase in the amylose content
was linearly correlated with that in the Wx protein level. These results clearly
indicates that the increase of amylose content at cool temperatures is brought
about by an elevated level of Wx protein, which catalyzes amylose synthesis.
The regulation responding to a slight difference of temperatures may bevery important for the growth and survival of plants. The study on the
response of the Wx protein accumulation responding to a cool temperature
may provide us one of the best clues for investigating this regulation.
Some Trans-acting Loci (du) that Regulate the Expressionof the wx Gene in Ricet

Hiro-Yuki HIRANO. Haruo SARUYAMA* and Yoshio SANO

The wx locus controls amylose synthesis in the endosperm and pollen of
rice. Amylose content, relative to total starch, greatly affects seed phenotype;seeds of the wild type look transparent but seeds of wx mutants, whichcontain no amylose, appear non-transparent when they are observed underpenetrating light. We found several mutants whose phenotypes were intermediate to the wild type and the null wx mutant by screening the mutants
induced with EMS.
These mutants were shown to have reduced levels of amylose content(3%-8%), compared with an original wild strain, Norin 8 (16%). Genetic
analysis indicated six mutants, designated dull (du), were independent of thewx locus, suggesting that the du loci are responsible for regulating wx gene
expression or modulating the enzymatic activity of the wx' gene product*

Hokkaido Green-Bio Institute, Sapporo.

36(Wx protein). Western blot analysis with anti-serum for Wx protein indicated that the amount of Wx protein was reduced in all mutants although
the extent of reduction is variable. This strongly supports the former
possibility, namely, the reduced levels of the amylose content are caused by
the lower amount of Wx protein in du mutants. Northern blot analysis
showed that this reduction of the Wx protein level was controlled at thetranscriptional level. The du loci may encode specific trans-acting factorsthat activate the transcription of the wx + gene.
We analyzed the total protein in mature seeds by two dimensional gel
electrophoresis and found that not only Wx protein but also a few additional
proteins decreased in du mutants. In addition, a few proteins were increasedin du mutants. These results suggest that the du loci may control thesynthesis of a few specific proteins including Wx protein through theirtranscription and may have both the function for activating and for repressing the genes that encode them. The function, which may be positive
regulation or negative, may depend on the genes affected. It is of greatinterest what kind of genes are controlled by the du loci and what mechanisms underlie this regulation.
The Promoter of the Rice wx" Gene is Functional for Tissue Specific
Expression in Pollen in the Transgenic Petunia
Hiro-Yuki HIRANO, Yoshibumi KOMEDA* and Yoshio SANO

Gene expression at the wx locus of rice is regulated in a tissue-specificmanner, namely, it is expressed only in the endosperm and pollen (Hirano
and Sano (1991) Plant Cell Physiol. 32,989-997). Of great interest to us is
the molecular mechanism of tissue-specific gene regulation in two distinctly
different and differentiated tissues. In order to examine whether this mechanism for tissue specificity is functional in a dicotyledonous plant, we introduced recombinant DNA consisting of the promoter region of the rice wx'
gene and the coding region for bacterial ,B-glucronidase (GUS) and assayed
the GUS activity for monitoring the wx + promoter function. The GUS
activities in the seed and leaf in all transgenic petunia were as low as that in
non-transformed plants. In the pollen, on the other hand, the GUS activities
were two to ten fold higher than those of a non-transformant. This result*

Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo.

1.

37

MOLECULAR GENETICS

indicates that the wx gene promoter in rice is functional in the pollen of the
dicotyledonous plant but not in the endosperm, reflecting the difference in thedevelopment of endosperm between dicotyledonous and monocotyledonous
plants. The molecular mechanism that switches on the wx gene in the pollen
and off in the leaf may be common to both monocotyledonous and dicotyledonous plants.
t

4

38

II.

MICROBIAL GENETICS

Sequencing Analysis of Primer RNA for Minus Stand
Synthesis of Bacteriophage f1
Nahoko HIGASHITANI, Atsushi HIGASHITANI and Kensuke HORIUCHI

The F-specific filamentous bacteriophage f1 possesses a circular, singlestranded genome. Upon infection, the viral single-stranded DNA (plusstrand) is converted to a double-stranded replicative form (RF) throughsynthesis of the complementary (minus) strand. Synthesis of the minus
strand is initiated at a unique site (Geider et a!. (1978) Proc. Nat!' A cad. Sci.
USA. 75, 645-649) on the single-stranded DNA by synthesis of an RNA
primer, which is catalyzed by the host RNA polymerase. The region required
for the function of the minus strand origin includes two inverted repeats,
which form two possible hairpin structures named Band C.
In order to determine which sequence(s) is essential for the origin function, we constructed a number of deletion and insertion mutants. The resultsof functional analyses of the mutant origins suggested that deletion of aregion in the 3' end of hairpin C, which contains the previously reported
initiation point of the primer RNA (nucleotide 5756 of f1 DNA) (Geider etal., 1978), did not seriously affect the origin function (Higashitani et a!.
(1990) Annual Rrep. 41, 31-32). R411 is such a mutant which carries adeletion ranging from nucleotide 5755 to 5766. Thus, the R411 deletionshould include nucleotides that correspond to the first 2 nucleotides of theprimer RNA. Yet, R411 showed normal plaque morphology, and grewnormally.
These results prompted us to re-examine the start site of the primer RNA
synthesis. We determined the sequence of the primer RNA by synthesizing
it in the presence of chain-terminating ribonucleotide triphosphate analogues(3' -deoxy NTP's). The results indicated that the primer RNA of R411 had
a sequence identical to the wild-type primer: pppAGGGCGAUGGCCCACUACGU. The size of the primer RNA was 20 nucleotides and the sequence
was complementary to the f1 DNA sequence from nucleotide 5736 to 5717.
This meant that the primer synthesis started at a site 20 bases downstream
from the site previously reported. Therefore, R411 does not lack the normal

II.

MICROBIAL GENETICS

39

initiation point of the primer RNA.
We also sequenced the primer RNA of an N-specific filamentous phage
IKe. The DNA sequence of IKe has a high degree of homology to fl,particularly in hairpins Band C. This suggests that the initiation ofcomplementary strand synthesis in IKe is accomplished by the same RNA
polymerase-dependent mechanism (Peeters, B. et al. (1985) J. Mol. BioI. 181,27-39). However, this homology ends at position 5751 of fl, and as aconsequence the sequences around the previously reported initiation site aredifferent between fl and IKe. Our results showed that the sequence of the
IKe primer was pppAAGGCGAUGGCCCACUACGU, complementary to
the region from the nucleotide 6441 to 6422 of IKe DNA. A high degree ofhomology between the fl and IKe primer RNAs is obvious.
In summary, our results indicate that the start site of the RNA primer islocated 20 bases downstream from the site previously reported by Geider etal. (1978).
A Single Amino Acid Substitution Reduces the Superhelicity
Requirement of a Replication Initiator Protein
Atsushi HIGASHITANI and Kensuke HORIUCHI

The origin of rolling circle replication in filamentous coliphage consists ofa core origin that is absolutely required and an adjacent replication enhancer
sequence that increases in vivo replication 30 to lOO-fold. The core origin
binds the initiator protein (gpII) which either nicks or relaxes the negatively
superhelical replicative form DNA (RFI). Nicking at the origin, but notrelaxation, leads to initiation of DNA replication. Our results indicate thatthe ratio of nicking to relaxation (nicking-closing) in vitro depends on the
superhelical density of the substrate (Fig. 1). We studied the effect of singleamino acid substitutions in gpII, which allow wild-type levels of replication in
the absence of the enhancer, on origin nicking and binding. These mutations
map to three positions within the N-terminal portion (codon 40,41, and 73)
of gene II (410 codons). The mutation M40I (Met40--Ile; previously calledmp-l) has been shown to increase the co-operativity with which the protein
binds the origin to form a functional complex for the nicking reaction(Greenstein. D & Horiuchi. K. (1990) J. Mol. Bioi. 211,91-101).
We further characterized the enhancer-independent mutants, and the40

100 . - - - - - - - - - - - - - - - - - - ,Nicked (G73A gpll)

80

~
<C

60

z

0

0
C

:::>

40

0

E

<C

20
Relaxed (G73A gpll)

o

1.0

2.0

3.0

4.0

5.0

6.0

Negative superhelical density x1 ()'2

Fig. I.

nicking actlVlty of an enhancer-independent G73A (Gly73-->-Ala) mutant
gpII. The G73A mutation yielded more nicking and less relaxation of RFI,
compared to the wild-type protein. The mutant gpII also showed a reduced
requirement for superhelicity of the substrate in the nicking reaction (Fig.1).
We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both the increase in the negative superhelicity of thesubstrate and the action of the replication enhancer may antagonize therelaxation activity.

II.

MICROBIAL GENETICS

41

DnaK Protein Is Involved in Replication of Escherichia coli
Chromosome by Protecting the Initiation Protein
DnaA from Heat Inactivation
Seiichi YASUDA, Atsushi HIGASHITANI, Kensuke HORIUCHI
and Yoshimasa SAKAKIBARA*The DnaK protein is one of the major heat shock proteins of E. coli andparticipates in various cellular functions including phage and chromosomal
DNA replication. Although the role of the DnaK protein in A phage DNA
replication is well established, it is not clear how it participates in chromosomal DNA replication. Since a mutant of the dnaK gene defective in
replication initiation has been isolated, it is probable that the DnaK protein
is involved in the initiation of DNA replication. On the other hand, there isan increasing number of evidences which shows that the DnaK protein,
acting as a 'chaperone', can protect and reactivate various proteins from heat
inactivation. From these facts we postulated that the DnaK protein may be
involved in chromosomal DNA replication by protecting the initiation protein DnaA from heat. We examined this possibility by in vivo and in vitro
experiments, and the results obtained are in support of this hypothesis.
In vitro Protein Phosphorylation in Escherichia coli
Cell Cycle Mutants
Atsushi HIGASHITANI, Seiichi YASUDA, Yukinobu NISHIMURA**
and Kensuke HORIUCHI

Protein phosphorylation and dephosphorylation play important roles in theregulation of physiological functions of various proteins in both eukaryotes
and prokaryotes. Bacterial phosphorylation systems can be classified into
three different types, the classical protein kinase systems, the two-component
systems in adaptive responses, and the sugar phosphotransfer systems. In
several eukaryotes, cdc2 kinase has been identified as a key enzyme commonly
involved in the regulation of the cell cycle. In prokaryotes, however, cdc2like kinase has not been defined and kinase systems for cell cycle regulation
have not been identified. In order to search for cell cycle-dependent protein***

National Institute of Health, Tokyo.
Faculty of Science, Toho University, Chiba.

42

phosphorylation systems in E. coli, we carried out SDS-PAGE analyses ofproteins that are phosphorylated in vitro in crude cell extracts. The extracts
were prepared from logarithmic phase and stationary phase cells, and from
cells carrying a temperature-sensitive (ts) mutation in various cell cyclerelated genes (dnaA, dnaB, dnaC, JtsI,ftsZ, unidentified Jts mutations, andunidentified par mutations).
In the wild type cell extracts, we detected about 20 phosphorylated proteinsafter incubation with r_ J2 p ATP in vitro. Two of them, 18 kd and 10 kd
phosphorylated proteins, were identified as phosphotransferase enzyme II(IIIg1c) and histidine phosphotransferase (HPr), respectively, by sequencing
the amino-terminal amino acids followed by computer analysis. 100 kd and
40 kd phosphorylated proteins were specifically detected only in extracts fromthe stationary phase. Phosphorylation of three proteins, 29 kd, 12 kd, and 10
kd proteins, also increased in stationary cell extracts as compared withlogarithmic phase extracts. The 29 kd phosphorylated protein was identified
as the a-subunit of succinyl-CoA synthetase from its amino-terminal sequence.
Extracts from a temperature-sensitive partition mutant par530 specificallylacked a 62 kd signal of protein phosphorylation, while its temperatureresistant revertant regained the 62 kd spot. In crude extracts of dnaA mutant
cells grown at a nonpermissive temperature (42°C), phosphorylation of 58
kd, 34 kd, and 17 kd proteins decreased. The three proteins did not show anychanges in the degree of phosphorylation in extracts of the wild type cells orother mutants such as dnaB, ftsI, and JtsZ cultured at 42°C. The phosphorylation of the 34 kd protein increased upon addition of purified DnaK
protein to the in vitro reaction with wild-type cell extracts. The DnaK protein
is a phosphorylated protein by itself. We are in the process of identifying thephosphorylated proteins described above and their physiological roles.

II.

43

MICROBIAL GENETICS

Osmotic Regulation of Uptake of Long-Chain Fatty Acids in Escherichia
coli.: In vitro Interaction of OmpR Protein with a Fatty
Acid Receptor Gene fadL
Atsushi HIGASHITANI, Kensuke HORIUCHI, Hiroshi HARA, Hirofumi AIBA*,
Takeshi MIZUNO* and Yukinobu NISHIMURA**

ThermosensitivefabB mutant of E. coli can grow at 42°C when supplemented with oleate. We found that this particular growth was drasticallydepressed in a medium of high osmolarity. The growth response wasapparently dependent on the function of OmpR, which is known to osmotically regulate the biosynthesis of major outer membrane proteins, OmpF andOmpe. The fadL gene of E. coli codes for an outer membrane protein
involved in the uptake oflong-chain fatty acids. We investigated whether the
uptake of long-chain fatty acids is regulated by environmental osmolarity,and if the fadL gene is controlled by the OmpR protein.
The uptake of long-chain fatty acids in E. coli decreased when the cells
HpaI I- 16;=-5,,----.,..,,-------,-----,=c--=-cc:-~GGAAAGTGCTGCTCCAGTTGTTAATTCTGCAAAATCGGATAA~TGACCGAAATCACACTTAAAAATGATCT-136-8;::-6_

AAAACAA~ATTCACCCGAATCCATGAGTGCGCCACCTCCAAATTTTGCCCAGCTG~TCGCGTTTCTTAGAT_ _ _ _ _ _ _ _ _--.:4-.::,6

AvaIl

-35

- 16

+1

CATATTTGAAAAAAGATAGAAACAT~CTTGCAACATTCCAGCTGGTCCGACCTATACTCTCGCC~CTGGTCT+4;=-5_GATTTCTAAGATGTACCTCAGACCCTACACTTCGC~TCCTGTTACAGCACGTAACATAGTTTGTATAAAAA
+96
+166
+127

T~A~A~A~TC~A~T=T~G~GGTTATGG[CATGAGCCAGAAAACCCTGTTTACAAAPTCTGCTCTCGCAGTCGCAGTGGCA
Met----Hpall

CTTATCTCCACCCAGGCCTGGTCGGCAGGCTTTCAGTTAAACGAATTTTCTTCCTCTGGCCTGGG~-~_0-{80==-~'~il+4C5==+:J90 +100 +127~

fadL- Transcription

Met----

c::=J : OmpR binding site
Fig. 2.

***

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

44

were cultured under high osmotic conditions. Gel retardation assays andDNase I protection experiments showed that phosphorylated OmpR specifically bound to four DNA regions, two upstream and two downstream fromthe fadL-transcriptional start site (Fig. 2). These results suggest that theexpression of thefadL gene may be both positively and negatively controlled
by OmpR-EnvZ proteins in the absence of the FadR repressor.
Correlation of a Subset of the pLC Plasmids to the Physical
Map of Escherichia coli K12
Akiko NISHIMURA, Kiyotaka AKIYAMA, Yuji KOHARA and Kensuke HORIUCHI

We determined map positions of the Escheichia coli Kl2 portions of asubset of the hybrid E. coli-ColE 1 plasmids constructed by Clarke andCarbon. The probe DNA of pLC plasmids was labeled with digoxigeninedUTP, hybridized to the 476 phage clones of the E. coli ordered clones bankminiset, which was adsorbed on a strip of nylon membrane filters, anddetected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed
color reaction. The total number of Clarke-Carbon plasmids we analyzed
was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed
with those reported previously, and the remaining 41 plasmids gave map
positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it waspresented, as well as a table which correlates the pLC plasmids with theclones of the E. coli ordered clone bank miniset on the basis of the hubridization data. We estimate that approximately one-half of the entire genome of
E. coli was covered by the pLC plasmids used in this study. For details, see
Microbial. Rev.56, 137-151, 1992.

45

III.

MAMMALIAN GENETICS

Genetic Status of Mus musculus Subspecies in Far East Russia
Kazuo MORIWAKI, Hitoshi SUZUKI*, Tsuyoshi KAWASHIMA, Kimiyuki TSUCHIYA**,
Hiromichi YONEKAWA***, Ludmila V. YAKIMENKO****,
Vladimir P. KORABLEV**** and Alexei P. KRYUKov****

Our previous survey demonstrated that the Chang Jiang River is a good
geographical barrier between the two subspecies of Mus musculus, castaneus
and musculus. The former inhabits areas south of the river and the latterareas north of it. In the north western areas such as Xinjian Uygur province,
however,the mtDNA RELPs of wild mice are often of the castaneus type.
This finding suggests the possibility that the castanceus subspecies had widely
inhabited east Asia including the northern areas in the past. To accumulate
more data supporting this notion, the present study attempted to find out
whether the wild mice inhabiting Far East Russia are related to the castaneus
subspecies or not. As summarized in Table I, the wild mice collected fromthe Primorye region including Rudnaya and Vladivostok, revealed a definite
relationship to the castaneus subspecies in mtDNA, rDNA and HbbDNA,
though none of them was a complete Mus musculus castaneus. The occurrence of castaneus genomes in the wild mice collected from Primorye and also
those from northern localities in China likely supports the above idea. But
reservation is that the Primorye mice could have been transferred from Japanor southern China in the relatively recent past. The molecular comparison of
the mtDNA D-Ioop between wild mice from Primorye and those from Japanis now underway.
In Novii, Teli and Krementi which adjoin northern China, a new allele ofHbb, wi, which was first found in northwestern China (Kawashima et al.,1991, Jpn. J. Genet 66, 491-500), is frequently observed. This may suggestthat a similar type of mouse as those in northwestern China in habits theseregions of Russia. In those regions, both the mtDNA and rDNA are wholely
*
**
***
****

Jikei Medical College, Tokyo.
Miyazaki Medical College, Miyazaki.
Tokyo Metropolitan Institute of Medical Science, Tokyo.
Institute of Biology and Pedoloty, Vladivostock, Russia.

46
RFLPs of mtDNA, rONA and HbbDNA in house mice collectedfrom Far East Russia

Table 1.

mtDNA

rONA

cas dom musmus mus/cas-dom

Place ofcollection

No. ofsampleNorth caucasus
Novii
Teli
Krementi
BirakanRudnaya
Gorny
Vladivostok

I
3

3

5

5

3
4
2
4
7

3
4

3

Total

29

5

1

I
3

5
3
4

2
4
6

2
2

2
4

2

22

19

10

HbbDNA
did

1
I
2
1
4
2
4
6
21

dip d/wl

222

7

the musculus type. Nevertheless their HBB frequently shows a d-type which
could have originated from the castaneus subspecies. This seems to again
support the above hypothesis.

Male-Specific Suppressor of Meiotic Recombinationin the MHC Region
Toshihiko SIROISHI, Tomoko SAGAI and Kazuo MORIWAKIThe wm7 MHC haplotype enhance meiotic recombination at the specificsites, the so called "hotspot" in the proximal region of the MHC. A uniquecharacteristic of this enhancement is sex-specificity in that high rate ofrecombination frequency is observed only in female meiosis but not in malemeiosis. Recombinants generated from the cross involved by the wm7
haplotype retain the ability to enhance the recombination at the hotspot in thesame region when their chromosomal segments proximal to the hotspot arederived from the wm7 haplotype. On the other hand, recombinants to carry
the wm7-derived chromosomal segment in the distal region to the hotspot lostthe ability to enhance the recombination. Taking these facts into account, weconcluded that enhancer activity of the wm7 haplotype resides in the region
proximal to the hotspot, while suppressor activity is located in the distal
region (see EMBO J. 10, 681-686, 1991). In the present study, we attempted
to examine whether the recombination suppressor of the'wm7 haplotype acts

III.

MAMMALIAN GENETICS

47

only in the cis-position relative to the enhancer. In order to address thisquestion, we estimaed the recombination frequency of the cross between B10.
A(R209) and BIO.A(R201) strain. The former mouse retained the activity
to enhance recombination but lost the suppressor. The latter mouse haspreserved the wm7-derived chromosome in the region distal to the hotspot, inwhich the suppressor is expected to be present. The result from this mating
experiment clerarly indicated that the rate of recombination frequency at the
hotspot in female meiosis is as high as the cross between the wm7 and inbred
strain. On the contrary, there was no recombination in male meiosis. Thus,
it appeared that male-specific suppressor of recombination act both in cis andtrans position relative to the recombination enhancer.
Genetic Organization of the Recombinational Hotsopt within the MHC:
A Tight Linkage of the Hotspot to the LMP-2,
HAMI and HAM2 Genes
Tsuyoshi KOIDE*, Toshihiko SHIROISHI and Kazuo MORIWAKI

It is known that homologous recombination during meiosis occurs preferentially at the hotspot between Pb and Ob loci within the MHC region ofmouse. Two sequence elements characteristic to the hotspot, MT-consensus
and repeat of TCTG tetramer, have been identified at the hotspot. There isa cluster of genes encoding for the MHC class II molecules in this region as
well as Pb and Ob. Recent reports have shown that there are several
functional genes, such as LMP-2, HAMI and HAM2 genes, in the same
region. Our preliminary southern hybridization analysis of a cosmid DNA
including the hotspot indicated that the LMP-2 gene and a part of HAM I
gene are located to the same cosmid clone. Sequencing analysis of this clone
demonstrated that the TCTG repeat is adjacent to the LMP-2 gene. It is
located to 1.9 kb downstream from polyadenylation signal of LMP-2 gene.
Other groups have reported that the HAM I gene is located to the upstreamof the LMP-2 gene in head to head manner. Furthermore HAM2 gene,which is homologous to the HAMI gene, is located to the just downstream of
the HAMI gene in the same orientation. The LMP-2 gene is considered to be
one of the subunits of proteasome having multiple proteolytic activities in_._--------~*

Present Address; Institute of Anymal Physiology & Genetics Research, Cambridge,
U.K.

48

cells. The HAM I and HAM2 genes are thought to be required for fortranslocating peptide fragments of endogenous antigens into the endoplasmic
reticulum for association with MHC class I molecules. Because the hotspot
is located between the MHC class I gene and genes mentioned above,
recombination at the hotspot produce new assortments of the class I molecules and proteins involving antigen digestion, transportation and presentation to the T-cell receptor by the MHC class I molecules.
Gene Dosage Effect of Recombination Hotspots on the Rrate
of Recombination Frequency
Masayasu YOSHINO, Tomoko SAGAI, Kirsten FISCHER-LINDAHL*,
Toshihiko SHIROISHI and Kazuo MORIWAKI

Recocmbinational breakpoints in the MHC class II region are clustered at
restricted sites, the so-called recombinational hot spots. At least four
different hotspots have been identified in this region. The location of eachhotspot differs, depending on the MHC haplotypes in the genetic crosses. For
example, recombination occurs preferentially at a hotspot located between
the Ab3 and the Ab2 genes when the wm7 and the cas3 MHC haplotypes,
both of which are derived from Asian wild mice, are used in the genetic cross,while recombination takes place at another hotspot located between the H2K and the Ab3 genes when the cas4 haplotype is used in the genetic cross.
In these mating experiments, a high rate of recombination frequency at therespective hotspots was observed if one of the parental strains carry the above
haplotypes. Thus, we concluded that a high rate of recombination is a
genetically dominant trait. In the present study, we attempted to test thegene dosage of the hotspots on the rate of recombination frequency. For thispurpose, two genetically different crosses were made. First, the recombination frequency in (wm7 X cas3) F I mice were examined to test the effect of adouble dosage of the hotspots at the same position. Second, (wm7 X cas4)F
I were examined for recombination frequency. Our preliminary results
indicated that neither of the two crosses yielded augmented recombination
frequencies, suggesting that a double dosage of the hotspots does not show anadditive effect on the rate of recombination frequency. Further studies arebeing continued.

* Howard Hughes Medical Institute, University of Texas, USA.

III.

MAMMALIAN GENETICS

49

Mouse Visible Mutations of the Intra-MHC Recombinants
Toshihiko SHIROISHI, Tomoko SAGAI, Shigeharu WAKANA* and Kazuo MORIWAKI

Intra-MHC recombinats between wild-derived wm7 and standard laboratory chromosomes have exhibited a high incidence of visible mutation. Since
neither of the parental strains, BI0. MOL-SGR nor laboratory strains, haveyielded any mutation, high mutation rate is characteristic to the intra-MHC
recombinants. Preliminary linkage analysis indicated that six independent
mutant genes are not linked to the MHC in the chromosome 17. Thus itappeared that the mutations occurred on the genetic background of C57BL/
toJ strain, because the mouse strain used were Bto. H-2 congenic strains. In
the present study, we carried out the gene mapping to locate the dominantmutant genes, Ram3, Ram4 and Ram5. The mouse stocks harboring thesemutant genes were crossed with DBA/2J strain and resultant Fl mice weresuccessively backcrossed to DBA/2J strain. After the phenotype of the
backcross progeny was typed, genetic linkage of respective mutant phenotypes with a series of genetic markers were analyzed based on a length
polymorphism of microsatellite sequences which distributes in mouse genome
with high frequency. As a result, Ram3 and Ram5 were mapped tochromosomes 11 and 2, respectively. Ram3 is tightly linked to Mpo andGfap. Gene order and distance between each markers is Mpo-7.4cM-Ram34.2cM-Gfap. Ram5 is linked to micarossatellite marker, Mit30. The result of
mapping experiment can provide crucial information for guiding molecularcloning of the genes in order to understand the molecular mechanism of ahigh incidence of mutation in intra-MHC recombinants.
Establishment of Embryonic Stem Cell Lines from BI0.H-2
Congenic Strains
Tomoko SAGAI, Norio NAKATSUJI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Embryonic stem (ES) cell lines, established from preimplantation mouseembryos, can colonize both the somatic and germ-cell line ages of chimaeric
mice following injection into host blastocysts. These ES cells are accessible
for experimental genetic manipulation by homologous recombination in vitro
and the modified ES cells can be used to introduce any mutation into the*Central Institute for Experimental Animals, Kawasaki.

50

germ lines of chimaeric mice.
Such ES cells should provide a useful system for the various studies on
BlO.H-2 congenic strains. In particular, the study of the molecular mechanism of meiotic recombination in the H-2 region definitely requires thissystem. Thus far, ES cell lines have been produced from limited mousestrains, mainly 129/Sv, and not from BlO.H-2 congenic strains. Therefore we
tried to establish new ES cell lines from several different BlO.H-2 congenic
strains. As the result, we obtained total 46 ES cell lines from 5 BlO.H-2
congenic strains (14; C57BL/1O, 1; BlO.MOL-SGR, 19; BlO.A(R209), 7;
BlO(R233), 5; BlO.CAS-TCH). These cells showed fine growth and retained
the undifferenciated figure on feeder cells.
For the chimaera formation, the cell lines are desirable to have normal andTable 1.

Karyotype analysis of ES cell line

Exp.

Cell line

Passage

2n%a)

4n%b)

Karyotype

I
2
2
2
2
3
4
5
5
5
5
5
6
6
6
6
7
8
9
10
11
12
13

ES.BIO-2
ES.BIO-11
ES.BIO-12
ES.BIO-13
ES.BIO-14
ES.SGR-I
ES.R209-1
ES.R209-2
ES.R209-4
ES.R209-9
ES.R209-15
ES.R209-16
ES.R209-17
ES.R209-18
ES.R209-19
ES.R209-20
ES.R233-1
ES.R233-2
ES.R233-6
ES.R233-7
ES.TCH-I
ES.TCH-2
ES.TCH-5

5
7
6
6
7
8
5
5
6
6
7
4
5
5
6
4
7
5
5
6
6
8
5

92

94
93
90
94
95
94
93
93
91
90
91
96
88
95
94
98
91
96
94
89
94
94

6
6
7
IO
6
5
6
7
7
9
IO
9
4
12
5
6
2
9
4
6
II
6
6

40XY (5)')
40XY (5)40XY (6)40XY (5)40XY (4)40XY (I)40XY (3)4IXY-Ts6d ) (11)40XY (4)40XY (7)40?X? (I)40XY (2)40XY (3)40XY (2)40XY (2)40XY (3)40XY (2)40XY (3)40XY (5)40XY (2)40XY (5), 40?XY (1)40XY (6)40XY (11)

a),b)
oj

d)

2n and 4n indicate approximate diploidy and tetraploidy.
Figure in parentheses represents the number of observations.
Ts represents trisomy.

III.

MAMMALIAN GENETICS

51

male karyotypes. So the karyotypes of 23 cell lines were examined byG-banding method using urea solution. The result showed that 21 of them
have normal and male karyotypes and there is no XX cell line. (Table 1)
Generation of a Novel Mutant Mouse by Transgenesis
Hidea GOTOH, Tashihika SHIROISHI and Kazua MORIWAKIThe genes for the steroid 21-hydroxylase (21-0Hase) and for the fourthcomplement component (C4) are located in the H-2 class III region. Boththe genes for 21-0Hase and for C4 are duplicated in tandem array. Of thetwo 21-0Hase genes, Cyp21 and Cyp-ps, the Cyp21 gene is functionally active
while the other is inactive. The gene family for C4 consists of the C4 and Sip
genes. Immunogenetic studies have reported that the C4 gene is haemolytically active, and that the Sip gene is inactive.
The C57BLjlOSnSlc_H_2aW18jH_2aW18 mouse is lethal in the early postnatal
period due to a lack of 21-0Hase. We previously introduced DNA fragmentsof the active form of 21-0Hase into the H_2aw18jH_2aw18 homozygous mice,and rescued the lethal mutant mice. The chromosome of the H_2 Gw18
haplotype has been reported to have a deletion of about 80 kilobases encompassing the Cyp21 gene. Further genetic analysis of the rescued mousehas been performed using specific DNA sequences for the C4 gene and the Sip
gene. The genome of the transgenic mouse has a DNA fragment specific for
the Sip gene but lacks one specific for the C4 gene. Detection of transcriptsof the C4 gene also indicated a deletion of the C4 gene in the transgenic H2aw18jH_2aw18 mouse genome.
In humans, several HLA haplotypes containing a null allele for the C4 gene
have been reported. In most cases, homozygocity for the HLA haplotype isrelated to autoimmune disease, and the correlation between C4 deficiency andautoimmune disease has been discussed. We produced a novel mutant mousewith a deletion for the C4 gene through transgenesis.. The mouse will provide
a novel means to investigate the unknown function of the C4 protein in
relation to autoimmune disease in humans.

52

Genetic Analysis of Susceptibility to Urethane-induced Pulmonary
Adenomas in M.MOL-MSM (MSM) Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIIn mice, It IS evident that multiple genes control susceptibility to thedevelopment of pulmonary adenomas, because Fl and F2 progeny gave
responses intermediate to those of the parental laboratory strains. In crosses
between strain A and C57BL mice, resistance to adenoma development in the
C57BL strain show marked behavior as a single recessive gene. To analyze
the mode of inheritance of adenoma susceptibility, we crossed one of the
adenoma-resistant, wild-derived M.MOL-MSM (MSM) strain with the adenoma susceptible A/Wy strain. All of these mice were sacrificed in the fifthmonth after a single subcutaneous injection of 1.5 mg urethane/g bodyweight. After the lungs were fixed in ethanol/formaldehyde (9: 1), thenumber of adenoma foci were counted.
MSM and A/Wy mice have tumor multiplicities of O.16±O.06 (range: 0-2)
and 27.07±O.96 (10-43), respectively. The Fl generation mice were intermediate to the parental strains in their susceptibility. All of the Fl mice hadtumors (range: 1-9) and a tumor multiplicity of 3.91 ±0.40. A/Wy-backcross mice showed responses intermediate to those of the Fl and A/Wy strain
with a range of 4-43. F2 mice showed all gradations in tumor multilicity
between the parental strains with a range of 0-27. There is no indication ofresistant: susceptible = 3 : 1 distribution as would be expected with a dominant single major factor for resistance in MSM mice. However there is apossibility a major genetic factor expressing no dominance, because 11 out of
56 F2 progeny had no tumors and 14 had more than 9 tumors. Multiplicitiesof which overlap those of the resistant MSM and susceptible A/Wy mice,
respectively.
Comparison of the DNA Sequence in the D-loop Region of Mouse
Mitochondrial DNA. II. Two Distinct Clusters in the
Fareast Population with musculus-type mtDNA
Hiromichi YONEKAWA, Sumiyo WATANABE, Nobumoto MIYASHITA
and Kazuo MORIWAKI.

Based on mtDNA haplotype analysis with restriction enzymes, we previously proposed the hypothesis that Japanese wild mouse which had been

III.

MAMMALIAN GENETICS

53

recognized as an independent subspecies "Mus musculus molossinus", is ahybrid from crosses between ancestral colonies, possibly very small, of M. m.
musculus and M. m. castaneus (Yonekawa et al. (1988) Mol. BioI. Evol. 5,63). To prove this hypothesis, it is necessary to analyze more precisely the
population structure of Japanese "molossinus" mouse and to examine theevolutionary relationship of mouse populations between Japan and its neighboring countries. Due to very low degree of restriction polymorphism in theJapanese population, it is difficult to analyze them through restriction enzymes. Thus, we introduced a new technique, DNA sequencing analysis
combined with PCR technique, to this subject. The D-loop region of
mtDNA was chosen for this analysis, because we found that there was astretch of 200 nucleotides with a very high degree of nucleotide substitutions(approximately 10%) in this region when we compared the entire sequenceof mtDNA between M m. domesticus and M. m. musculus.
Through this analysis we previously showed the following lines of evidence: 1) substituted nucleotides in the mtDNA D-loop region, like restriction site variation, can be used as a diagnostic marker for subspecies identification when we construct phylogenic trees using the unweighted pairgroupe method with arithmetic mean (UPGMA) or the neighbor-joining(NJ) method. 2) at least four different mtDNA types exist in the Southern
population of Japanese wild mice (Yonekawa et al. (1990) Ann. Rep. Natl.
Inst. Genetics, lpn. 41, 46-47), although no restriction polymorphisms havebeen detected in the population (Yonekawa et al. (1988) Mol. BioI. Evol. 5,
63).
The next question is how the populations in Japan are evolutionally related
to those in its neighboring countries. To address this question, we sequenced
35 mtDNA samples, in total, of M. m. musculus collected from China, Korea
and Russia including the samples which were collected from Japan and had
been sequenced. Using the sequence divergence values among the samples,
we constructed a phylogenic tree by UPGMA, and found two distinct
clusters, cluster I and cluster II, in the musculus populations. Although thedegree of sequence divergence values between the two clusters was small,they showed a distinct geographical distribution; e.g. all Japanese mice belong
to cluster I, in which all Russian mice are included, whereas all Chinese andKorean mice belong to cluster II. Based on this result, we conclude thatJapanese populations with musculus-type mtDNA are evolutionally closer to
Russian populations than Chinese-Korean ones and the ancestral colony

54(ies) of Japanese mice possibly came from Russia.
We also sequenced 18 mtDNA samples of M. m. castaneus collected fromSouth East Asia including 5 samples from Japanese mice with castaneus-type
mtDNA. We have found so far that M. m. castaneus did not show any
distinct clusters in the phylogenic tree. However, it still remains a question
whether or not this is true in populations of M. m. castaneus, because M. m.
castaneus also widely distributes in the Southern part of China with very large
populations. To clarify this, the sequence analysis of these Chinese castaneus
mice is now in progress.

55

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Cloning of Hydra Interstitial Stem Cells Developmentally
Restricted to Sperm Production
Chiemi NISHIMIYA-FuJiSAWA and Tsutomu SUGIYAMAInterstitial cells in hydra are undifferentiated stem cells which divide
rapidly. At the same time they give rise by differentiation to somatic (nerve,
nematocyte and gland) cells in asexual animals, and also to gametic cells insexually differentiated animals. It is not clear at present whether all membersof the interstitial stem cell population can differentiate into both somatic andgametic cells (Bosch and David, 1987), or whether a separate germline
subpopulation exists for gametic cell differentiation (Littlefield, 1985).
Strain chim-Cl is a chimeric strain of Hydra magnipapil/ata which contains wild-type epithelial cells and temperature-sensitive interstitial cell lineage (see Marcum, Fujisawa and Sugiyama, 1989). At 18°C, it grows andmultiplies by budding normally. If cultured at non-permissive temperature of
25 °C, it is expected to lose all interstitial stem cells and their differentiation
products (nerve cells and nematocytes), and turn into "epithelial" hydra
unable to move or feed.
When Chim-Cl was actually cultured at 25°C for several days, and thenreturned to 18°C, many polyps turned into epithelial hydra as expected. The
same treatment, however, also produced an unexpected type of polyps,
termed "pseudo-epithelial hydra". These polyps, like epithelial hydra, lacked
nerve cells and nematocytes in the tissue, and could not move or feed. In
contrast to epithelial hydra, however, they contained interstitial cells in thetissue. These interstitial cells, however, were apparently unable to differentiate into nerve cells or nematocytes. Similar pseudo-epithelial hydra was also
produced from another strain, nem-l, by reducing interstitial cell numbers byhydroxyurea treatment.
Clones of pseudo-epithelial hydra were maintained by force-feeding over
130 days for chim-Cl and over 400 days for nem-I. In both cases, interstitialcells proliferated throughout the entire period without producing any nerve
cells or nematocytes. These interstitial cells, however, differentiated into
sperms. Thus, the interstitial cells present in pseudo-epithelial hydra were56

able to differentiate into gametic but not into somatic cells.
These observations suggest that the interstitial stem cell population in H.
magnipapillata includes a subpopulation whose differentiation is restricted togametic cell production. This subpopulation was cloned in this study.
A Nerve Factor(s) Required for Survival and Proliferationof Interstitial Stem Cells in Hydra
Toshitaka FUJISAWA

Interstitial cells (I-cells) in hydra are undifferentiated multipotent stem
cells which proliferate to maintain their own population as well as differentiate nerve cells and nematocytes during an asexual growth phase. When asmall number of I-cells alone are introduced into epithelial tissue which lackall the cell types in the I-cell lineage, the majority of the I-cells cannot survive.
In contrast, when I-cells are introduced into epithelial tissue together withnerve cells, they proliferate exponentially. These results indicate that somenerve factor(s) is required for maintaining the I-cell population.
In the present study, such a factor was searched for in hydra extracts byexamining whether or not they can support the survival of I-cells introduced
into epithelial tissue. It was found that methanol-extracts effectively supported the survival of I-cells. The extracts were further fractionated according to molecular weights. The fraction with a molecular weight of around
1,000 daltons was active. This activity was destroyed by proteolytic enzymes.
These results prompted us to test known hydra neuropeptides with a similarmolecular weight range; head activator (Schaller and Bodenmuller, 1981)
and hydra RF-amide (Grimmerikhuijen et al., 1992). Both neuropeptides
used here were synthetic substances supplied by Schaller and Grimmerikhuijen, respectively. The head activator but not hydra RF-amide was foundto be active. These results indicate that at least the head activator is an active
component of the nerve factor(s) which supports I-cell growth.
Hereditary Mosaic (mo) as a Functional Disorder in the Differentiation
Process of Meiotic Oocytes in Bombyx
Akio MURAKAMI

In one batch culture of F 1 hybrid slikworms from a cross between an moIV.

DEVELOPMENT AND SOMATIC CELL GENETICS

57

mutant strain female and a wild-type male, 136 sex-mosaic or gynandromorph moths with both male and female sex organs appeared in addition to
37 females and 28 males among a total of 201 adult moths. The occurrenceof gynanders is strong evidence of fertilization, an ovum with a sperm in one
part of a mosaic individual and a polar-body with another sperm in the otherpart, must be involved in abnormal development. The latter case is a resultof abnormal fertilization between the sperm and polar-body suggests that thepolar-body in this rno strain has the ability to attract sperm.
In the course of normal meiotic divisions, a mature oocyte results in two
types of cells: an ovum and three polar-bodies. The ovum will unite with asperm in general, with the polar-body being uninvolved. Accordingly, thisbiological event principally depends on the ability of the ovum to receive asperm, but the other meiotic products (or polar-bodies) do not retain thisability. Thus, it can be said that the function of the polar-body in the rno
mutant strain is different from that in the wild-type. The morphology of thepolar-body in insects is identical with that of ova, while the polar-body andova in mammals are generally different in shape. We can assume that theovum maintains the function of producing a sperm attractant, while othermeiotic products acquire an inhibitory function. Thus, the rno gene provokes
a functional disorder in the polar-body which usually rejects sperm.
Meiosis is the special process of the reductive division of chromosome
number and it also provides for an interchange of segments of the chromosome. Normal meiosis in females seems to have in addition a process offunctional differentiation of gametocytes into two types of cells in which one
cell or ovum accepts a sperm and the other (or one of three polar-bodies)rejects sperm. In other words, the meiotic process is defined as the ovum
maintaining the ability to produce a sperm attracting substance and to accept
the sperm, while the polar-bodies suspend the functions which results in therejection of sperm. If this is the case, it is likely that the action of the rno gene
involves the hindrance of the normal differentation of oocytes into polarbodies. In short, the rno mutant gene is an impediment factor on the suppression sperm attractant production in polar bodies.
In the rno mutant, there often appear polyploids, mostly tetraploid females.
This biological phenomenon is an additional type of abnormal development
probably due to fusions between the doublly fertilized zygotes mentioned.
Spontaneous parthenogenesis in Bornbyx is a common biological event andthe resultant parthenotes developed are mostly triploidal females, suggesting

58

that this abnormal development is a result of nuclear fusions between thesecondary oocytes (probably due to an arrest in meiotic division II) and oneof the first polar-bodies depending on affinity (or a flexible discrimination)
among the meiotic products. Accordingly, the mechanism responsible for theappearance of polyploids and parthenotes is not the same as that in the double
fertilization mentioned.
A Novel Recessive Multistar-spot Mutant (ms A ) in Bombyx
and Its Genetic Nature
Akio MURAKAMI and Kiyomi AKUZAWA*

A standard Bombyx + P has a pair of larval "star-spot" markings on the 8thsegment (numbering is assigned by counting all through the thoracic andabdominal segments in this report), besides a pair of "crescent" markings on
the 5th segment. In this insect, a new multistar-spot mutant with a high levelof embryonic lethality, fixed as a triplicated star-sport type on segments 6, 7,
8, was found twenty years ago in a commercial Japanese race hybrid line,
Miyama and Shinshu (Akuzawa, K., 1972). Since then the multistar has
been preserved and several multistar types, 7-8, 8-9, 7-8-9, 6-7-8-9-10,
appeared in addition to the original 6-7-8 type during the course of numerous breeding generations. In preservation, this mutant has shown a tendency
to select larvae having a greater number of segments marked with star-spots.
In spite of continued selections, the multistar types have not remained pure,with a fair number of various multistar types in every generation. In
addition, some exhibited an unpaired star-spot on one or more segments incertain cases.
To learn the dominant-recessive relationship for this mutant, a test crossbetween the standard (+p) type females and 6-7-8-9-10 type multistar
males was made and the resultant offspring were subjected to genetic analyses.
The results of breeding experiments showed that no multistar type larvae
appeared in the F] generation, but various multistar types corresponding to alittle over 20% of the total observed larvae were detected in the F 2 generation. Lethality in embryos obtained from sibmatings of the hexaplicated* Gumma-ken Sericult. Expt. Stn., Maebashi (the present address: AgricultureaJ division.
the Gumma Pref. Government, Maebashi).

IV.

DEVELOPMENT AND SOMATIC CELL GENETICS

59

types was about 50% and that of F, embryos between the standard typefemales and hexaplicated type males was about 35 %. These findings andother observations clearly indicate that the gene responsible for the newmultistar mutant is recessive and that the chromosome on which the gene islocated is accompanied by high lethality, indicating that the mutant gene, m~(Akuzawa's multistar), is not the same as those of coventional multistar
mutants, ms (Tanaka, 1963), msn (Chikushi, 1953) and Ems (Tsujita, 1953).
The offspring obtained from sibmatings of the hexaplicated type were
cultured and subjected an analysis of the spectrum of star-spot marking
patterns. The multistar type on the 6-7-8-9 segments was the most common(62.5%) followed by the 6-7-8-9-10 type (26.7%). The frequencies of the
7-8-9 and 7-8-9-10 types were about 8 and 2.5%, respectively. The 8-9 type
was found in only two out of the total 562 larvae observed (0.4%). Theseobservations suggest that the expressivity of star-spot markings on the 10th
segment is the most unstable followed by those on the 6th segment. It isnatural that the star-spot on the 8th segment is the most stable as the normal(standard) trait. Such a feature in the m~ mutant appears to be almost thesame as ms rather than msn which only affects the 6, 7 and 8th segments.
However, the m~ mutant seems to be associated with a certain chromosomal
aberration similar to the mutant Ems.
From the genetic analysis of the m~ mutant and other related mutants inBombyx, it became clear that the phenotypic expression of the ms gene islimited to five segments, with the 8th segment at the center and including thetwo infront of it (6-7) and the two behind it (9-10). The star-spot
expressivity of the m~ gene on the 8th segment is the most stable andfollowed by those of the 7 and 9th segments. The 6 and 10th segments, which
are more remote from the 8th segment, are unstable. This variance may
depend on the action probability of the ms gene on each segment and/or theaccessibility of each segment to the gene action, inferring that such differences are essentially connected with the time sequence for segment formation
or affinity between segments.

A Consideration of the Hereditary Mode of Feeding Behaviour
on Artificial Diets in Bombyx
Akio MURAKAMI

The domesticated mulberry silkworm (B. mori L.) has widely been viewed

60

as a monophagous insect which feeds only on mulberry leaves. In 1972,
Yokoyama detected a polyphagous line named as Sawa J from among
Japanese stocks through persistent selection efforts. This polyphagous stockshows a wide spectrum in feeding on various kinds of plant leaves and evensome fruits other than mulberry leaves. In addition, the stock feeds well oneither a semi-artificial diet containing a high percentage of powdered mulberry leaves or a specifc artificial diet (LT-l) without the mulberry leaves.
Cultured larvae with the artificaial diet can be be raised to the adult stage tosome extent. It is worth-while to note that the feeding ability of Bombyx
larvae on artificial diet is a strong racial difference (e. g., Kanda et al., 1988):
the Japanese race has a strong ability as a rule, while the Chinese race is poor.
The ability of the European race is differs stock to stock. This racialdifference is a strong indication of the participation of some genetic factor( s)responsoble for the feeding behaviour.
According to the reports of Fujimori et al. (1982) and Kanda et al. (1988),
the larvae of Sawa J stock fed well on semi-artificial (ca. 100%) and artificial(ca. 89%) diets, whereas, Kansen, one of the Chinese races, showed a low
feeding ability (ca. 16%) on the semi-artificial diet and another Chinese
stock, Chinese No.2, did not feed on the atrificial diet (LT-l). These stock
differences among the Chinese stocks in feeding response is due to the qualityof diets used for the experiments in relation to the presence of powderedmulberrry leaves. It is also likely that the racial difference between theJapanese and Chinese races in the feeding ability depends on a differential
response to the smell as to the taste of the diet. Fujimori et al. (1982)
indicated that the high feeding ability of Sawa J on a semi-artificial diet was
dominant over the low ability of Kansen, while the feeding ability on theartificial diet (LT-1) appeared a recessive in relation to the poor ability ofChinese No.2 (Kanda et al., 1988). This contradiction may have been due
to the constituency of the diets used for the tests.
A number of factors, olfaction, gustation, and other senses are involved in
feeding behavior. It is natural to assume that the physical properties of thetwo types of diets used for the experiments did not markedly differ except fora few factors, especially in the presence or absence of powdered mulberryleaves. Thus it is not unreasonable to suppose that olfaction and gustation
are the main factors influencing feeding behaviour.
Newly hatched F 1 larvae obtained from a mating between Sawa J and
Kansen stocks fed well on the semi-artificial diet and about 68% of the BF 1

IV.

DEVELOPMENT AND SOMATIC CELL GENETICS

61

larvae [(S X K) X K] fed on the diet (Fujimori et al., 1982), while the F 1 andBF[ larvae [(S X C) X C] from a cross of Sawa J and Chinese No.2 did not
feed on the artificial diet (LT-l) (Kanda et al., 1988). From these results, it
can be assumed that the Sawa J stock larvae acceptance of the artificial dietis due to dull senses of olfaction and gustation and that the genetic factor
responsible for the dull sense of smell is recessive (0) and for taste it isdominant (G ), whereas the Chinese stocks are particularly fastidious about
the artificial diet on account of keen senses of smell and taste and the former
trait is under the control of a dominant factor (0+), but the latter trait isrecessive (g' ).
Differential Susceptibility of Domesticated Bombyx silkworms
to the Tachina Fly, Exorista sorbillans Wied
J. SHIMADA*, T. OHSHIKI* and A. MURAKAMI

The domesticated mulberry silkworm, Bombyx mari L., has been developed
from its ancestral form through selection and other ways on the basis ofhuman economic needs since before the time of recorded history, while thenon-domesticated mulberry silkworm, B. mandarina M., seems to have been
free of any domestication. F[ hybrid obtained from a cross between B. moriand B. mandarina are fertile so that these can be regarded as subspecies. This
infers that their common ancestral form had a number of genetic traits whichmight more closely resemble those of B. mandarina than these of B. marL In
the wild, B. mandarina frequently suffers from infestations of the tachina fly,E. sarbillans. The fly is occasionally parasitic on B. mari too. Tachina flies
infect Bambyx silkworms in the daytime, but not in darkness (Prasad, N. R.,
1990), suggesting that the fly's visual recognition plays an important role inhost selection, among other factors. Thus, the fly may selectively and/or
inherently infect B. mandarina or some mutant stocks of B. mari through its
ability to recognize preferential larval-body marking patterns (texture) as
well as colour. If so, by putting its instinct to use, the tachina can detect thelarval-body marking pattern and colour of B. mari which may closely
resemble the ancestnal form of silkworms.
Recently, two papers reported independently that the fly has a preference
for the larval-body marking patterns in B. mari (Ohsiki et al., 1989; Prasad,* Faculty of Agric., Tokyo Univ. of Agric. and Techno\., Tokyo.

621990), but there seemed to be no consistent tendency for a preference among
the mutant stocks used in their experiments.
The present experiment was carried out to clarify the relationship between
susceptibility to the tachina fly and Bombyx larval-body marking patterns.
Eight different mutant stocks in larval-body marking patterns and colour
were used. The p3 (or p t ) stock was used as the standard type. It has a pairof black crescent markings and a pair of black star spots on a whitebackground p (was plain with no crescent or star spot on a white background). pM (moricaud),ps (striped),pB (black), U (ursa-like body marking
pattern), pSa (this trait, sable, to some extent resembles the larval-bodymarking of B. mandarina) and Ze (or zebra) were also used in the present
study. The host preference of the fly on B. mori larvae at the 5th instar wasmeasured on five larvae for each mutant stock on mulberry treetops in the
Tsukui Experimental Farm of Tokyo Univ. of Agric. & Techno!. A similar
procedure was repeated at least three times for each stock.
The result of the experiments showed that the number of eggs laid on thelarval-body surface of the mutant stock by the tachnia fly was greater on thep3 stock larvae than those of the p3 stock or the mutant stocks pM, pS, pB and
U. The body colour in those mutant stocks (pM,pS,pB and U) is prominently
black in comparison with the p stock. The Ze mutant was susceptible to thefly to the same degree as the p3 stock. E. sorbillans showed a tendency tomore frequently to lay eggs on the mutant stock with the sable marking (pSa)
than on the the normal pJ. This finding suggested that the tachina fly has an
instinctive preference for silkworn larvae with a larval-body marking patternsimilar to B. mandarina. Although the marking pattern of the pSa resembles
that of B. mandarina, the colour is to some extent different.
In addition to the experiment above, we also examined whether the fly issusceptible to the tone of the larval marking colour in B. marL There is apositive correlation between colour on larval-body marking in Bombyx andthe concentration of juvenile hormones during the moulting period (Kiguchi,1972). On the allatectomized pSa mutant larvae immediately before the 4th
moulting, the body marking pattern darkens in the next instar as comparedwith the sham operated ones, while the larvae aplied with Methoprene (JHA,
20/lg per capita) just before the 4th molt changed into brownish. The
allatechtomized pSa mutant larvae hardly suffered from infection of tachina
fly in contrast to the sham operated ones. The JHA applied larvae also did
not show any significant change in infection as compared with the non-IV.

DEVELOPMENT AND SOMATIC CELL GENETICS

63

treated group. These findings indicated that the tachina fly has a preference
for silkworms having a similar colour of the larval-body marking pattern as
B. mandarina, but does not have a preference on larvae having an extremely
deviated tone of colour from the ordinary trait of the pM mutant, which is like
B. mandarina.
Analysis of Morphogenesis in Postimplantation Mouse Embryos
Norio NAKATSUJI and Yasuaki SHIRAYOSHI

The postimplantation period of mammalian embryogenesis includes many
important events such as formation of the mesoderm cell layer, central
nervous system and primordial germ cells. For experimental analysis of thisperiod, we utilized various methods including whole embryo culture, manipulation of cultured embryos, and isolation and culture of embryonic tissues
and cells (Nakatsuji, N. and Hashimoto, K. (1991) In "Gastrulation: Movement, Patterns and Molecules," 43-56). In collaboration with anotherlaboratory, we studied migration patterns of neuroblasts isolated from thecentral nervous system (Nagata, I. and Nakatsuji, N. (1991) Development
112, 581-590). We are setting up a new laboratory of mammalian development, in which we are planning to analyze morphogenetic events during thepostimplantation period of normal and mutant strain mice, by using whole
embryo culture and micromanipulation techniques. Particular attention will
be paid to the formation of mesoderm cells at the primitive streak and theirmigration, the appearance and migration of the primordial germ cells, anddifferentiation of the central nervous system.
Manipulation of Embryogenesis Using Mammalian Embryonic Cells
Norio NAKATSUJI and Yasuaki SHIRAYOSHI

The isolation and manipulation of embryonic cells in culture and reincorporation of such manipulated cells into embryos would enable many experimental strategies in basic research on embryogenesis and also in more appliedresearch in the medical and agricultural sciences.
We used embryonic stem cells established from mouse blastocysts foranalysis of embryogenesis. We isolated a cell line which expresses 13galactosidase, and made chimaeras using these labeled cells. For example, we64analyzed chimaeric brains to study the migration of neuroblasts during theformation of the cerebral cortex (Nakatsuji, N. et al. (1991) Devel. GrowthDiffer. 33, 571-578).
We also studied primordial germ cells which may be used for manipulationof the mouse germ line. As the first step, we isolated germ cells and somatic
cells from fetal ovaries (12.5-14.5 days post coitum), cultured them asdispersed cells, and made reaggregates. Such reaggregates (reconstituted
fetal ovaries) were transplanted into ovary capsules of adult female mice.
Then, they gave birth to offspring from transplanted germ cells (oogonia or
oocytes) (Hashimoto, K. et al. (1992) Devel. Growth Differ. 34, 233-238).
In the new laboratory, we continue to use embryonic stem cells and germ
cells for analysis of embryogenesis through manipulation of these cells andtheir reincorporation into embryos or mice. New attention will be paid toprimordial germ cells during early stages in postimplantation embryos andcells in the neural plate and neural tube.
Molecular Analysis of Cell Differentiation in Postimplantation
Mouse Embryos
Yasuaki SHIRAYOSHI and Norio NAKATSUJI

Determination of cell fate and cell differentiation are crucial events in early
embryogenesis. In our new laboratory, we have started experiments toapproach such problems from their molecular aspects. One example would
be to identify genes involved in determination and differentiation of thecentral nervous system during the earliest stages in postimplantation stage
mouse embryos. As a first step, we have been constructing a eDNA libraryfrom 7.5-8.5 day mouse embryos, in which the early events of the central
nervous system development are initiated, including neural plate induction
and neural tube formation.
A cDNA Project on the Nematode Caenorhabditis elegans
Yuji KOHARA, Hironobu MiTSUKI and Akiko NISHIGAKI

C. elegans is a small, free-living soil nematode, whose biology has beenstudied extensively. The accumulated information includes an extensive
genetic map which now contains about 1,000 genes, the entire somatic cell

IV.

DEVELOPMENT AND SOMATIC CELL GENETICS

65

lineage from fertilized egg to adult consisting of 959 cells, the complete
neural network at the ultrastructural level, and, more recently, the physicalmap of the 100 Mb (megabase-pairs) genome. A project to sequence theentire genome is now under way at two laboratories in the U.K. and the U.S ..
In order to have easy access to all the genes of this organism and to
ultimately understand the network of gene expression in its development, wehave started to classify and characterize all cDNA species, whose number is
now believed to be around 15,000. To this end, the following systematic
strategies were taken.
(1) First of all, abundant cDNA species were identified by picking up theclones which gave strong signals in the hybridization of a worm cDNA
library with total cDNA as a probe. The 4 most abundant species, which
each occupied 1-2% of the library, turned out to be in the vit (vitellogenin)
gene family. Dozens of other abundant cDNA species were also identified.
(2) We made a cDNA library (AZAPII vector) from the mRNA of amixed-stage population which included all developmental stages of thisorganism. To get as full length cDNA as possible, cDNA longer than 1.5 kb
were selected on agarose gel before cloning. On a first attempt, we picked up
8,000 independent clones and stored them separately in the 96-well format.
They were gridded onto the lawn of indicator bacteria, blotted onto nylon
membranes and probed with the abundant clones to discover and remove theabundant ones. As a result, a subset of some 5,000 less abundant or rare
cDNA clones was established and we have started to analyze it using thefollowing procedures.
(a) Restriction mapping: A procedure to efficiently make restriction
maps of cDNA inserts was developed, applying PCR with one labeled primer
and partial digestion with 4 base cutter enzymes. The maps are used as
fingerprints for the classification of clones. This was used because we wishedto avoid sorting methods which depend solely on DNA-DNA hybridization,
considering the potential importance of the gene family and alternative
splicing.
(b) Pattern of expression: We amplified virtually all cDNA species froma series of single embryos of various developmental stages and dot-blotted
them onto strips of nylon membrane. The stage of expression of a given
cDNA was able to estimate simply by probing the strip with the cDNA clone.
(c) Gene mapping: The position of cDNA clones on the genome were
determined by hybridization to the "YAC polytene filter", on which the66ordered 956 YAC clones that covered virtually the whole genome were
blotted.
(d) DNA sequencing: An efficient protocol for sequencing a cDNA
insert directly from a single plaque was established.
(3) After completion of the analysis of the current set of 8,000 clones, weplan to carry out a subtraction between the current set and the larger numberof clones, 100,000 or more, from the same library to find out rarer clones
which were not presented in the current set and then to continue the above
analyses.
Differential Screening of Founder Cells in C. elegans Embryo
Hiroaki TABARA* and Yuji KOHARA

A fertilized egg of C. elegans divides unequally to give rise to a larger
somatic founder cell named AB and a smaller germ-line daughter PI. The P
I cell further divides 3 times in a stem-cell fashion to produce three othersomatic founder cells, EMS, C and D, and the germ-line precursor cell, P4.
The founder cells continue to divide to finally give rise to 959 somatic cells at
the adult stage. The somatic cell lineage is invariant and is known in its
entirety. Accumulated evidence has shown that the fate of the founder cells
can be determined both by internally segregating factors and by cell-cellinteractions. The fate-determining factors may be some mRNA, protein or
small molecules, but the nature of the factors are largely unknown.
Aiming to identify the fate-determining factors, we have started, as a firststep, to search mRNA that segregate unequally among founder cells. For
this purpose, we established a procedure to isolate individual founder cells
from 2- and 4-cell stage embryos using a micromanipulater. cDNA was madeand amplified from individual founder cells. The amplified cDNA, which
represents most of the mRNA species in individual founder cells, werelabeled and used for screening a cDNA library that had been made from a
populatiton of early embryos; most were before gastrulation. In preliminary
experiments using cDNA probes from AB and PI cells, we got manydifferentially positive signals. Analyses of these clones are in progress.

*

Department of biology, Tsukuba University, Ibaraki.

IV.

DEVELOPMENT AND SOMATIC CELL GENETICS

67

Genetics of Fluoride-resistance of the Nematode Caenorhabditis elegans*
Isao KATSURA and Minoru KAWAKAMI

Sodium fluoride (NaF) is toxic to almost all organisms. Biochemical
reactions in vitro suggest that it may interfere with interesting regulatory
mechanisms, because it depletes Ca I I , inhibits phosphatases, and activates G
proteins. We are trying to discover the real target of NaF in vivo and to
elucidate related regulatory mechanisms through the isolation and characterization of C. elegans mutants resistant to NaF.
We have isolated 13 recessive fluoride-resistant mutants that map in 5 newgenes, fir-J X, fir-2 V, fir-3 IV, fir-4 X and fir-5 (linkage group not yet
determined). They are grouped into two categories, class 1 and class 2. Class
1 mutants (jir-J,fir-3, andfir-4) are resistant to lOmM NaF, but they growtwice as slowly as wild type worms and have a small brood size even in theabsence of NaF. In contrast, class 2 mutants (jir-2 and fir-5) are notcompletely resistant to lOmM NaF. They are almost normal in growth rate
and brood size in the absence of NaF. Double- and triple-mutants in thefir
genes are resistant to lOmM NaF and almost normal in growth rate andbrood size if they contain both class 1 and class 2 mutations, whereas they
have the same phenotype as the single mutants if they contain mutations of
only one class. Namely, class 1 mutations are epistatic to class 2 mutations
concerning the degree of fluoride-resistance but hypostatic concerning
growth rate and brood size. We interpret this relationship by assuming ahypothetical metabolic or transport pathway that confers fluoride sensitivity
to C. elegans. Class 1 mutations give rise to fluoride-resistance by blocking
the pathway. At the same time they cause accumulation of a toxic metabolite
or an intermediate state of transport that delays growth and decreases broodsize. Class 2 gene products act before the intermediate in the pathway, andhence mutations in those genes restore the growth rate and brood size of class
1 mutants by preventing the accumulation of the hypothetical toxic intermediate. The weak fluoride-resistance of class 2 mutants can be explained by thepresence of a parallel bypass to the intermediate.
We cloned a 3.7 kb DNA fragment containing one of the fir-J mutationsites by the transposon-tagging method and are determining the nucleotide* Continuation of the work performed at Department of Biology, College of Arts andSciences, University of Tokyo in collaboration with Dr. Kazunori Kondo and Mr.
Tosikazu Amana.

68

sequence.
Caenorhabditis elegans Larval Lethal Mutations That Cause
Gross Morphological Changes*
Isao KATSURA

We isolated 24 mutants which die as larvae with abnormal shapes, to study
the essential developmental genes and their functions at the late embryonicand post-embryonic stages. They were classified according to shape into (1)2-fold short body mutants (3 alleles), (2) mutants with irregular outer shape(2 alleles), (3) a mutant that dies during the first molt (l allele, lowpenetrance), (4) mutants that are bloated around the pharynx (2 alleles), (5)4-fold mutants (3 alleles), (6) coiled mutants (3 alleles), and (7) mutants inwhich the outer surface of the intestine gradually detaches from the innersurface of the body wall (10 alleles). These mutations were mapped by crossand complementation with known mutants. They were maintained asbalanced stocks using known visible mutations that map near the lethal
mutations.
Genetic mapping studies showed that most of the mutations classified
under (5) and (6) above are in known muscle-related (unc-I5, mup-I) andneural (unc-I7, unc-104) genes, respectively. Most of the mutants of class(l) to (3) seem to be defective in hypodermis or cuticle, judging from thephenotype. Some of the class (7) mutants map in genes that are thought to
act in signal transduction systems, such as let-23 (tyrosine kinase), let-34I,
and elr-I. We are now concentrating our study on mutants of this phenotype.

* Continuation of the work performed at Department of Biology, College of Arts andSciences, University of Tokyo in collaboration with Dr. Kazunori Kondo and Miss
Yuko Kawase.

69

V.

CYTOGENETICS

Mutability of Constitutive Heterochromatin (C-bands) During Eukaryotic
Chromosomal Evolution and Their Cytological Meaning
Hirotami T. IMAIA quantitative analysis of the alterations of constitutive heterochromatinin eukaryotic chromosomal evolution was attempted using the accumulated
C-banding data available for mammals, amphibians, fish, ants, grasshoppers,
and plants. It was found that these eukaryotes could be classified into two
types by their C-banding patterns: Type I including mammals, fish, and ants,
and Type II including amphibians, grasshoppers, and plants. C-bands wererather scarce in Type I eukaryote chromosomes and were found around the
pericentromeric region when found in Type II eukaryote chromosomes. The
Type I and II C-banding patterns can best be interpreted by assuming that in
the former group of eukaryotes the saltatory increase in constitutive heterochromatin, occurs preferentially at the pericentromeric regions of telocentricchromosomes induced by centric fission, with C-bands being eliminatedalmost completely through centric fusion and/or pericentric inversion. On
the other hand, C-bands appear in Type II eukaryotes both interstitially andin the telomeric regions of chromosomes, and there may be no effective
mechanism for eliminating these bands once they are integrated. For details
see Jpn. J. Genet. 66, 635-661, 1991.
Nucleolar Organizer Regions (NORs) in the Australian Ant
Myrmecia croslandi (= M. (pilosula) n = 1)
Hirotami T. IMAI, Hirohisa HIRAI*, Yoko SATTA, Toshihiko SHIROISHI,
Masa-aki YAMADA and Robert W. TAYLOR**

Localization of NORs on metaphase chromosomes of the Australian ant
Myrmecia croslandi (previously discussed as M. (Pilosula) n = 1) were examined using the rDNA (Drosophila): DNA in situ hybridization (FISH
method). It was revealed that in individuals with 2K = IM(1 +2) + ISM! + 1M 2***

Department of Parasitic Diseases, Kumamoto University, Medical School, Kumamoto.
Division of Entomology, CSIRO, Australia.

70

NOR[

NOR[

SM 1
Fig. 1.

(2n=3) NORs are located at the proximal region of the short arm heterochromatin block in 1M(I . 2), and at the chromosomal gap in the heterochromatic short arm of M 2 (Fig. 1). These findings were supported cytologicallyby silver staining, i.e., nucleoli appeared exactly at the expected NOR sites.
There was no silver staining at any of the metaphase NORs in this ant, butinstead the kinetochores stained faintly.
Cloning of rDNA of the Ant Myrmecia croslandi
Masa-Toshi YAMAMOTO*, Keiji OGlJRA* and Hirotami T. IMAI

We tried to isolate DNA fragments of Myrmecia croslandi homologous torDNA of Drosophila melanogaster. Southern hybridization analysis of M.
croslandi revealed a strongly hybridized 6.0 kb band, possibly a doublet, anda weakly hybridized 7.0 kb fragment when probed with a mixture of internal
coding region of 18S and 28S of D. melanogaster at a lower stringency.
Genomic DNA of M. croslandi extracted from a sample HI89-030 with 2n =2 by Y. Satta (1989) was digested with £CoRl, and the DNA fragments were

* Division of Biology, Miyazaki Medical College, Miyazaki.

V.

CYTOGENETICS

71

ligated into a phage vector A ZAP II. Eight positive clones were isolated
through plaque hybridization using the same probe as that for the Southernblot analysis. Two of them were isolated and purified as recombinant
plasmids named pMc' rl and pMc' r2. Restriction maps of the inserted
fragments have been determined. They however both lack a 18S coding
region but carry that of the 28S and the spacer region. It may be necessary
to clone the entire unit of rDNA repeats in the future but the two clones withthe 28S region will be valuable molecular markers for analyses of karyotype
evolution in ants.

72

VI.

MUTAGENESIS AND RADIATION GENETICS

Abnormal Differentiation and Associated Radiosensitivity of Erythroid
Progenitor Cells in the Wasted Mutant Mouse
Hideo TEZUKA. Dai A YUSAWA and Takeshi SEND

Erythropoietic cell differentiation and its relationship to radiation induced
cell death was studied in a mouse mutant, "wasted". Previous reports notedthat bone marrow cells of the wasted mouse became hypersensitive to ionizing
radiation in an age dependent fashion, and the radiosensitivity increased
four-fold over that in a control mouse with erythroid colony forming units(CFUE) but not with other progenitor cells. Here we report that the increasein radiosensitivity is closely associated with abnormal erythroid differentiation in the wasted mouse. Stem cells (day8 spleen colony forming units, anancestor of CFUE) increased in number, followed by decreasing numbers of
the stem cells and CFUE, and lastly CFUE responsiveness to erythropoietin(Epo) decreased. In temporal accordance with the change of Epo responsiveness, the radiosensitivity of the CFUE increased. Experimental results
showed that radiosensitivity in the wasted and control marrow CFUE isdependent on the Epo concentration in culture, and the radiosensitivity of thewasted CFUE is probably due to a decrease in Epo responsiveness in the cells
themselves. The wasted mutation is closely related to the development ofhematopoietic stem cells and erythropoietic progenitor cells. This mutant
mouse may be a good model animal for studying the mechanism of hematopoiesis and of radiation induced cell death in mice.

73

VII.

POPULATION GENETICS

Role of Diversifying Selection and Gene Conversion in Evolutionof Major Histocompatibility Complex Loci
Tomoko OHTA

Genes at the major histocompatibility complex (MHC) in mammals are
known to have exceptionally high polymorphism and linkage disequilibrium.
In addition, these genes form highly complicated gene families that have
evolved through gene conversion and unequal crossing-over. It has beenshown recently that amino acid substitution at the antigen recognition site
(ARS) is more rapid than synonymous substitution, suggesting some kind of
positive natural selection working at the ARS. It is highly desirable to know
the interactive effect of gene conversion and natural selection on the evolution and variation of MHC gene families. A population genetic model isconstructed that incorporates both selection and gene conversion. Diversifying selection is assumed in which sequence diversity is enhanced not only
between alleles at the same locus but also between duplicated genes. Expressed and nonexpressed loci are assumed as in the class I gene family of MHC,
with gene conversion occurring among all loci. Extensive simulation studies
reveal that very weak selection at individual amino acid sites in combination
with gene conversion can explain the unusual pattern of evolution andpolymorphisms. Here both gene conversion and natural selection contributeto enhancing polymorphism. For details, see Proc. Natl. Acad. Sci. USA 88,
6716~6720.

Multigene Families and the Evolution of Complexity
Tomoko OHTA

Higher organisms are complex, and their developmental processes arecontrolled by the sequential expression of genes that often form multigene
families. Facts are surveyed on how functional diversity of genes is related to
duplication of genes or segments of genes, by emphasizing that diversity isoften enhanced by alternate splicing and proteolytic cleavage involving

74

duplicated genes or gene segments. Analyses of a population genetics modelfor the origin of gene families suggest that positive Darwinian selection isneeded for acquiring gene families with desirable functions. Based on these
considerations, examples that show acceleration of amino acid substitution
relative to synonymous change during evolutionary processes are surveyed.
Some of such examples strongly suggest that positive selection has worked.
In other cases it is difficult to judge whether or not acceleration is caused bypositive Darwinian selection. As a general pattern, acceleration of aminoacid substitution is often found to be related to gene duplication. It isthought that complexity and diversity of gene function have been advantageous in the long evolutionary course of higher organisms. For details, see J.
Mol. Evol. 33, 34-41.
Overdispersed Molecular Clock at the Major Histocompatibility
Complex Loci
Naoyuki TAKAHATA

The extent of amino acid differences of major histocompatibility complex
molecules within species is unusually high, consistent with the finding thatsome pairs of alleles have persisted for more than ten million years and theview that the polymorphism has been maintained by natural selecution. The
disparity between synonymous and nonsynonymous substitutions in theantigen recognition site, however, suggests that some nonsynonymous sites
have undergone a number of substitutions whereas others have little or none.
To describe statistically such an overdispersed underlying process, commonlyused Poisson processes are inadequate. An alternative process leads to thesurprising conclusion that each nonsynonymous site has accumulated as
many as 2.6 substitutions, on the average, in the two lineages leading tohumans and mice. The standard deviation is also very large (6.6) and thedispersion index (the ratio of the variance to the mean) is at least 17. The
substitution process thus inferred qualitatively agrees with the disposition (aboomerang pattern) of substitutions between HLA-A2 and Aw68 alleles, andquantitatively agrees well with that expected where the evolution of major
histocompatibility complex molecules has long been driven mostly by balancing selection. (See Proc. R. Soc. Lond. B 243, 13-18, 1991 for details.)

VII.

POPULATION GENETICS

75

A Trend in Population Genetics Theory
Naoyuki TAKAHATA

Recent studies in theoretical population genetics focus on retrospective
properties of stochastic processes occurring in finite populations in conjunction with DNA sequence data from which the ancestry of homologous genes
is inferred. Under neutrality (selective equivalence), two stochastic theories(the coalescent and the lines-of-descent) have been developed and extensively
applied to various population genetics problems. There can, however, be athird category of such stochastic processes which may be called allelicgenealogy that describes the ancestral relationships among different alleliclines. Here, I summarize several results of this stochastic process subjected tofairly strong selection, although neutral allelic genealogy is also considered.
(See New Aspects of the Genetics of Molecular Evolution, eds. M. Kimura
and N. Takahata, pp. 27-47. Japan Sci. Soc. Press, Tokyo/Springer-Verlag,
Berlin, 1991 for details.)
Statistical Models of the Overdispersed Molecular ClockNaoyuki TAKAHATA

The most commonly used statistical model to describe the rate constancy
of molecular evolution (molecular clock) is a simple Poisson process in which
the variance of the number of amino acid or nucleotide substitutions in aparticular gene should be equal to the mean and henceforth the dispersion
index, the ratio of the variance to the mean, should be equal to one. Recent
sequence data, however, have shown that the substitutional process in molecular evolution is often considerably overdispersed and have called into
question the generality of using a simple Poisson process. Several efforts havebeen made to develop more realistic models of molecular evolution. In thispaper, I will show that the spatial (site-specific) variation in the rate ofmolecular evolution is an improbable cause of the overdispersion and thenreview various statistical models which take the temporal variation into
account. Although these models do not immediately specify what themechanisms of molecular evolution might be, they do make qualitatively
different predictitions and give some insight into the inference. One way to
distinguish them is suggested. In addition, effects of selected substitutions

76

that presumably occur after a major change in a molecule are quasiquantitatively examined. It is most likely that the overdispersion of molecular clock is due either to a major molecular reconfiguration (fluctuating
neutral space) led by a series of subliminal neutral changes or to selected
substitutions fine-tuning a molecule after a major molecular change. Although the latter possibility, of course, violates the simplest neutrality assumption, it would not impair the neutral theory as a whole. (See Theor. Pop.
Bioi. 39,329-344, 1991 for details.)
Sampling Errors in Phylogeny
Naoyuki TAKAHATA and Fumio TAJIMA

The sampling variance of nucleotide diversity or branch length in aphylogenetic tree constructed by any distance method provides a criterion tojudge whether a deduction or an inference made from data is statisticallysignificant. However, computation of the sampling variance is usually
tedious, particularly when the number of operational taxonomic units(OTUs) or DNA sequences is large, and must rely on computers. Recently,
Nei and Jin (1989) have developed a computer algorithm, but it can beapplied only to a simple substitution model. In this paper, we derive simple
formulas for the minimum and maximum values of the sampling variance,
which are independent of underlying substitution models. Application ofthese formulas demonstrates satisfactorily accurate estimates of the sampling
variances and therefore their practical use. (See Mol. Bioi. Evol. 8(4), 494502, 1991 for details.)
Genealogy of Neutral Genes and Spreading of Selected Mutations
in a Geographically Structured Population
Naoyuki TAKAHATA

In a geographically structured population, the interplay among gene migration, genetic drift and natural selection raises intriguing evolutionary problems, but the rigorous mathematical treatment is often very difficult. Therefore several approximate formulas were developed concerning the coalescence process of neutral genes and the fixation process of selected mutations in
an island model, and their accuracy was examined by computer simulation.

VII.

POPULATION GENETICS

77

When migration is limited, the coalescence (or divergence) time for sampled
neutral genes can be described by the convolution of exponential functions, as
in a panmictic population, but it is determined mainly by migration rate andthe number of demes from which the sample is taken. This time can be much
longer than that in a panmictic population with the same number of breeding
individuals. For a selected mutation, the spreading over the entire population
was formulated as a birth and death process, in which the fixation probability
within a deme plays a key role. With limited amounts of migration, evenadvantageous mutations take a large number of generations to spread.
Furthermore, it is likely that these mutations which are temporarily fixed insome demes may be swamped out again by non-mutant immigrants fromother demes unless selection is strong enough. These results are potentially
useful for testing quantitatively various hypotheses that have been proposed
for the origin of modern human populations. (See Genetics 129, 585-595,
1991 for details.)

Trans-species Polymorphism of HLA Molecules, Founder Principle,and Human Evolution
Naoyuki TAKAHATA

There are three main hypotheses for the origin of Homo sapiens, called thecandelabra, multiregional and Noah's Ark. Their essential difference is in therole and extent of gene migration which might have occurred during the
Middle and Upper Pleistocene. The candelabra assumes no migration andthus parallel evolution of H. sapiens in several regional localities while the
Noah's Ark assumes the complete replacement of Homo erectus populationsin the Old World, around 200,000 years ago, by anatomically modern
humans from Africa. The multiregional hypothesis allows continuous but
presumably infrequent gene migration throughout the Pleistocene. It had
been difficult to distinguish these hypotheses based solely upon morphologicalcharacters, because of limited amounts of fossil records and little knowledge
about the evolutionary mechanisms. This situation in the study of human
evolution has recently been changed dramatically by the introduction of
DNA technology. It seems now possible to examine more deeply the historyof organisms and the population dynamics including speciation processes. It
is argued that although the data of mitochondrial (mt) DNA sequences

78

sampled from human populations contradict the candelabra hypothesis, they
can be consistent with the other alternatives and do not provide further
information about what might have happened during the Middle and Upper
Pleistocene. In contrast, HLA polymorphisms exhibit trans-species modes of
evolution and therefore have a great potential in examining the human
evolution in much longer time scales. The trans-species polymorphisms
clearly contradict the extreme form of the Noah's Ark as well as the founderprinciple as being important in speciation. Combined with DNA sequence
data of HLA, population genetics theory predicts that the long-term effective
size of the human population is of the order of 105 • This figure is about ten
times larger than that inferred from the allele frequency data of otherproteins and enzymes, suggesting either that the population had been structured, not to a small extent, until the Late Pleistocene, or that the number ofbreeding individuals per se had been large. (See NATO ASI Series, Vol. H59
Molecular Evolution of the Major Histocompatibility Complex, Edited by J.
Klein and D. Klein, Springer-Verlag Berlin Heidelberg, 1991 for details.)
Calibrating Evolutionary Rates at Major Histocompatibility
Complex Loci
Yoko SATTA, Naoyuki TAKAHATA, Christian SCHONBACH,
Jutta GUTKNECHT, and Jan KLEIN

Unlike alleles at many other loci, major histocompatibility complex (Mhc)
locus alleles often differ by nucleotide substitutions at more than one site,
often as many as 88 sites. The substitutions accumulate gradually during
evolution by the same process that leads to the divergence of genes in two
biological species. The difference between the inter-and intraspecific variation is that in the former, substitutions become fixed in the population (reacha frequency of 1.0), whereas in the latter, they reach polymorphic frequencies( ~ 0.01, < 1.0). Since accumulation of interspecific differences is believed bymany geneticists to proceed with a clock-like regularity within certain taxonomic groups, there is no a priori reason why the same should not be true forthe accumulation of polymorphic differences. Here we demonstrate thevalidity of this assumption by comparing alleles at the Mhc-DRBI and
Mhc-DQBI loci of different primate species. We then estimate the evolutionary rates at the DRBI and DQBI loci; the overall rates of these loci are 0.97

VII.

POPULATION GENETICS

79±O.17 and 1.2±O.39 (site/billion years), respectively. However, the rate of
the sites (both synonymous and nonsynonymous) encoding the peptide(antigen)-binding region (PBR) is 4 to 7 times higher than in the rest of thegene. As previously suggested, the enhanced nonsynonymous rate at the
PBR is most likely due to balancing selection, but the PBR as a whole may be
a hot spot of nucleotide substitutions. (See NATO ASI Series, Vol. H59
Molecular Evolution of the Major Histocompatibility Complex, Edited by J.
Klein and D. Klein, Springer-Verlag Berlin Heidelberg, 1991 for details.)
Persistence of Repeated Sequences That Evolve by Replication Slippage
Hidenori T ACHIDA and Masaru IIZUKA*

The evolution of short repeated sequences by replication slippage under theassumption of selective neutrality is modeled using a linear birth and deathprocess. The equilibrium distribution, the distribution of the life expectancy
of a repeated sequence when the process starts from two repeats, the age
distribution of repeats, the probability of obtaining two genes with i and jcopies which diverged t generations ago and the conditional variance of copy
number given the repeat number is more than one are computed. The
distributions of life expectancy and age are shown to have long tails. Also thestatistic which estimates the conditional variance is shown to have a largecoefficient of variation. Using these theoretical results, we develop an
approximate test of our model and analyze persistent repeated sequences
found in the primate ;3-g1obin gene region and Oenothera chloroplast DNA
which are polymorphic within species. We found one sequence in Onenotherachloroplast DNA which does not fit to our neutral model. For details, see
Genetics 131: 471-478.
Genetic Variability and Geographical Structure in Partially
Selfing Populations
Kishiko MARUYAMA** and Hidenori TACH IDA

In order to clarify the effect of selfing on genetic variation in geographical***

General Education Course, Chikushi Jogakuen Junior College, Fukuoka.
Department of Statistical Science, The Graduate University for Advanced Studies,Tokyo.

80

ly structured populations, Wright's island model is generalized to include theeffects of extinction, mutation, selfing, and migration of pollen and seed. The
equilibrium probabilities of identity by descent for two genes are obtained. It
is found that selfing causes a decrease of the variation within colonies and anincrease of the variation between colonies. Our model partly explains the"heterozygosity paradox" but other factors such as selection are necessary to
fully account for the paradox. For details, see Jpn. J. Genet. 67: 39-51.
Determination of Window Size for Analyzing DNA Sequences
Fumio T AJIMA

DNA sequences are generally not random sequences; the G + C content
may not be the same for all regions of a sequence, and there may beconservative and variable regions in it. Such nonrandomness can be tested byusing several statistical methods. On the other hand, to show such nonrandomness visually, data are often plotted as moving averages for a certain
length of window slid along sequence. For example, if the window size is 5,
the first point is the average over nucleotide sites 1-5, the second is that of 26, the third is that of 3-7, and so on. In this case, however, the window size
is arbitrary and no method is known for determining it. Here a simple
algorithm is presented for determining the window size and for finding anonrandom region of sequence. This method can be applied to various
studies in a wide field.
Consider a DNA sequence with N nucleotides. If the window size is Landif the window is moved one by one, then we have N - L+ 1 average points. If
these points are independent, we can test whether or not each point deviatesfrom the average over these points in a usual way such as binomial test.
When L = 1, all points are independent, so that we can use an ordinary test.
In this case, if C is the confidence level (= 1- significance level) for the entire
sequence, then C I /N is the confidence level for each point. To simplify thealgorithm, I suggest C I/N to be used as the confidence level even for L> 1,
although this causes the test to be conservative when the window size is large.
The two-tailed test for nonrandomness can be conducted by using thebinomial, normal, or Poisson distribution, depending on the data and theaccuracy we seek.
To determine the window size, we first compute for each L how many

VII.

POPULATION GENETICS81

average points significantly deviate from the average for the entire sequence
by using the above algorithm. Then we choose the window size that has thelargest number of significantly deviated points. In the case where there are
two or more window sizes that have the same largest number, we choose thesmallest window size among them. This procedure causes the smaller
window size to be chosen, noting also that the expected number of significantly deviated points for the entire sequence is approximately given by (N - L)
(I - eN) if the sequence is made by a random distribution of nucleotides.
Since a large window size may overlook small regions which deviate fromrandomness, this tendency may be preferable. When the sequence underconsideration is long, it may be better to divide the sequence into a certain
number of units with equal size to simplify the computations. In this case N
and L become the total number of units in the sequence and the window sizein terms of the number of units, respectively.
The present method for determining the window size and for finding
nonrandom regions in the sequence is quite simple, and the computations
required are straightforward, so that we can easily apply this method tovarious cases not only in biology but also in many fields of science. The
computer program is available on request. The statistical properties of thismethod, however, remain to be solved. One way to study them is to use thebootstrap resampling method. In any case there might be room for improvement and more extensive studies on this subject must be done. For details,
see J. Mol. Evol. 33, 470-473.
Recent Development of the Neutral Theory
Motoo KIMURAIn sharp contrast to the Darwinian theory of evolution by natural selection, the neutral theory claims that the overwhelming majority of evolutionary changes at the molecular level are caused by random fixation (due torandom sampling drift in finite populations) of selectively neutral (i.e.,
selectively equivalent) mutants under continued inputs of mutations. The
theory also asserts that most of the genetic variability within species at themolecular level (such as protein and DNA polymorphism) are selectively
neutral or very nearly neutral and that they are maintained in the species by
the balance between mutational input and random extinction. The neutral

82

theory is based on simple assumptions, enabling us to develop mathematicaltheories based on population genetics to treat molecular evolution andvariation in quantitative terms. The theory can readily be tested against
actual observations. The neutral theory was proposed nearly a quarter
century ago. Since then, strong opposition against the theory has been
expressed by the neo-Darwinian establishment. However, supporting evidence for the theory has gradually accumulated as time went on. Particularly, with outpouring of DNA sequence data starting some fifteen years ago,
many pieces of favorable evidence for the theory have been obtained. They
include such observations as high evolutionary rates of pseudoglobin genes ofthe mouse, aA-crystalIin genes of the blind mole rat and genes of influenza A
virus. In addition, it was shown that the evolution of the exceptional,
non-universal coding system in Mycoplasma capricolum, as discovered by S.
Osawa and his group, can readily be explained by the neutral theory.
Recently, the famous physicist, F. Dyson proposed a new theory on the originof life, in which stochastic processes involving neutral evolution plays acrucial role. Also, I recently proposed what I called the "four-stage scenario"
theory of macroevolution in which neutral evolution plays an essential role.
Based on these theoretical considerations as well as favorable observations
for the neutral theory, I claim that neutral evolutionary changes havepredominated over Darwinian evolutionary changes, at least in number,throughout the whole history of life on Earth. For details, see Kimura, M.
(1991) Jpn. J. Genet. 66,367-386.

83

VIII.

EVOLUTIONARY GENETICS

Analysis of HLA Data at the 11th International
Histocompatibility Workshop
Takashi GOJOBORI, Tadashi IMANISHI, Yasuo INA, Naruya SAITOlJ,
Yoshio T ATENO and Hidetoshi INOKOWe tried to compile all the data that were sent to the Central Data Analysis
Committee by the participants of the 11 th International Histocompatibility
Workshop. Although we could successfully list most of the raw data of HLA
typing at the serological and DNA levels in the data book, we could notinclude all score data of serological typing and other information. This ismainly because we had to deal with an extremely large amount of data, toolarge to be in one volume of reasonable size, and because of a shortage of time
for processing the data. Thus, the data book was regarded as a tentative
version of the data for the Workshop. It was also possible that the data fromsome laboratories were missing from the book. Despite the incompletenessmentioned, we still believe that the information in the book was very helpful
for discussion by all the participants at the Workshop.
To provide the participants with more comprehensive data, we compiled
them in the form of CD-ROM. The CD-ROM was distributed to the allparticipants together with this data book. It was noted, however, that thedata compiled in the CD-ROM were also tentative, possibly including someerrors. For details, see the Data Book of the 11th International Histocompatibility Workshop, Volumes 1 and 2.
Evolutionary Origin of Numerous Kringles in Humanand Simian Apolipoprotein (a)Kazuho IKEO, Kei TAKAHASHI, and Takashi GOJOBORI

Human apolipoprotein (a) has a great size heterogeneity and consists of 38
kringle domains in the amino terminal and a serine protease domain in the
carboxyl terminal. All but one kringle of apolipoprotein (a) are homologous
to the fourth kringle of plasminogen. However, the 38th kringle resembles
the fifth kringle of a plasminogen and it seems to have been deleted in simian

84

species. The phylogenetic trees suggested that an ancestral apolipoprotein(a) may have started with a duplicate of a plasminogen type protein. It also
implies that deletion of the three kringles in the amino terminus followed, andthat one of the remaining two kringles was duplicated in both human andsimian species and the other was processed by a deletion in simian species,
after species separation. Thus, the number of kringles in other mammals not
yet studied may vary considerably from species to species. For details, see
FEBS Lett. 287, 146-148.
Mutation Patterns of Human Immunodeficientcy Virus Genes
Etsuko N. MORIYAMA, Yasuo INA, Kazuho IKEo, Nobuaki SHIMIZU
and Takashi GOJOBORI

Human immunodeficiency viruses (HIVs) show extensive genetic variation. This feature is the fundamental cause of pathogenicity of HIVs andthwarts efforts to develop effective vaccines. To understand the mutation
mechanism of these viruses, we analyzed nucleotide sequences of env and gag
genes of the viruses by use of molecular evolutionary methods and estimatedthe direction and frequency of nucleotide substitutions. Results obtained
showed that the frequency of changes between A and G was extremely highand the mutation pattern of HIVs was distinct from those of nuclear genes of
their host cells. This distinction may be caused by the characteristics of thereverse transcription of HIVs. The mutation pattern obtained would be
helpful in constructing effective antiviral drugs. For details, see J. Mol. Eval.
32, 360-363.
Two-Color Cytofluorometry and Cellular Properties of the Urokinase
Receptor Associated with a Human Metastatic
Carcinomatous Cell LineKei TAKAHASHI, Takashi GOJOBORI and Masaki T ANIFUJI

Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-l25 and used as a probe for binding to the human metastatic
carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand
bound preferentially to cells that had been exposed to acidic pH. The binding
was competitive and decreased after mild tryptic digestion. The bound ligand

VIII.

EVOLUTIONARY GENETICS

85

could be removed by restoration of the cells to a low pH. Therefore, the cells
had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed bycounterstaining with propidium iodide. Results suggested that there weredifferent cell populations that had different numbers of receptors andamounts of DNA. We cloned cells and found that single clones had
homogeneous levels of receptors with different dissociation constants (from10 13 10 11 mol/mg protein) for different clones. Cells of one clone, C5,
which had high levels of receptor production, moved characteristically on a
glass substratum coated with gold particles and reacted with wheat germ
agglutinin, but not with concanavalin A. The receptors were found together
with adhesion proteins at the sites where the cells adhered to the substrate.
These results and the data obtained by zymography of the cellular proteinssuggested that the urokinase-type plasminogen activators were bound to thereceptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cells across the substrate. For
details, see Exp. Cell Res. 192, 405-413.
Evolutionary Pathways of N2 Neuraminidases of Swine and Human
Influenza A Viruses: Origin of the Neuraminidase Genesof Two Reassortants (HIN2) Isolated from Pigs.
Kuniaki NEROME, Yumi KANEGAE, Yasuyuki YOSHIOKA, Shigeyuki ITAMURA,
Masatoshi ISHIDA, Takashi GOJOBORI and Akira OVA

The complete nucleotide sequences of the neuraminidase genes of two
reassortants (HIN2) and two H3N2 influenza A viruses isolated from pigs,
were determined. The phylogenetic relationships between these genes and thepreviously reported N2 NA genes were investigated. On the basis of pairwise
comparisons of nucleotide sequences, the NA genes of two reassortants,
A/sw/Kanagawa/2/78 and A/sw/Ehime/l/80, were most closely related tothose of human influenza A virus strains isolated in 1972 and the earliest
available swine H3N2 influenza A viruses, respectively. Phylogenetic trees
showed that the NA genes can be separated into three groups, (i) swine
strains, (ii) the earliest human strain, and (iii) recent human strains. The
evolutionary tree for the 11 amino acid sequences suggested that the NAs of
A/sw/HK/4/76 and A/sw/Kanagawa/2/78 belong to the lineage of recent

86human viruses. In contrast, the NA genes of the A/sw/HK/3/76 and HIN2
reassortant A/sw/Ehime/l/80 viruses were found to be of a swine lineage. The
swine virus NA genes were further characterized by the cocirculation of two
distinct lineages. Although the rates of synonymous (silent) substitutions forthe swine and human viruses were nearly equal (0.00946 to 0.00884 per site
per year), the rate of nonsynonymous (amino acid changing) substitutions
for swine virus NA genes was about 60% of that for the human virus. For
details, see J. Gen. Viral. 72, 693-698.
Molecular Evolution of Hemagglutinin Genes of HtNt Swineand Human Influenza A Viruses
Shigeo SUGITA, Yasuaki YOSHIOKA, Shigeyuki ITAMURA, Yumi KANEGAE,
Keiko OGUCHI, Takashi GOJOBORI, Kuniaki NEROME
and Akira OyA

The hemagglutinin (HA) genes of influenza type A (HINl) viruses
isolated from swine were cloned into plasmid vectors and their nucleotide
sequences were determined. A phylogenetic tree for the HA genes of swine
and human influenza viruses was constructed by the neighbor-joining
method. It showed that the divergence between swine and human HA genes
might have occurred around 1905. The estimated rates of synonymous(silent) substitutions for swine and human influenza viruses were almost thesame. For both viruses, the rate of synonymous substitution was muchhigher than that of nonsynonymous (amino acid changing) substitution. It isthe case even for only the antigenic sites of the HA. This feature is consistent
with the neutral theory of molecular evolution. The rate of nonsynonymous
substitution for human influenza viruses was three times the rate for swine
influenza viruses. In particular, nonsynonymous substitutions at antigenic
sites occurred less frequently in swine than in humans. The difference in therate of nonsynonymous substitution between swine and human influenza
viruses can be explained by the different degrees of functional constraint
operating on the amino acid sequence of the HA in both hosts. For details,
see J. Mol. Eval. 32, 16-23.

VIII.

EVOLUTIONARY GENETICS

87

Molecular Cloning and Characterization of a Novel Glycoprotein, gp34,
that is Specifically Induced by the Human T·Cell leukemia
Virus Type I Transactivator p40 tax
Shigeo MIURA, Kiyoshi OHTANI, Noboru NUMATA, Masaru NIKI, Kazuyuji OHBO,
Yasuo INA, Takashi GOJOBORI, Yuetsu TANAKA, Hideki TOZAWA,
Masataka NAKAMURA and Kazuo SUGAMURA

We cloned and sequenced a cDNA encoding gp34, a novel glycoprotein
expressed in cells bearing human T-cell leukemia virus type I (HTLV-I).
HTLV-I has a trans-acting transcriptional activator, p40 ta X, that is thought tobe implicated in leukemogenesis through the activation of cellular enhancers.
With a subline (JPX-9) of the human T-cell line Jurkat, in which p40 tax is
inducible, gp34 was shown to be of cellular origin and to be transcriptionallyactivated by p40'ax. It was also demonstrated that two species of mRNA are
generated from one copy of the gp34 gene and that these mRNAs encode theidentical gp34 product and differ in the 3' untranslated region. Analysis of
the deduced amino acid sequence of gp34 showed that it lacks typical signal
peptides; however, it has a hydrophobic stretch for membrane anchoring andfour possible N-linked glycosylation sites at the carboxy-terminal portion,
indicating that it belongs to the family of membrane proteins whose carboxyterminal portion protrudes out of the cell. The gp34 gene displayed relatively
delayed induction compared with other genes activated by p40'ax. Added tothe observation of the dependence of gp34 expression on HTLV-I p40'ax,
unlike other p40,ax-dependent genes, such as those for the interleukin-2
receptor a chain and c-fos which are expressed or induced under physiological conditions, these results imply that the mechanism involved in the
induction of gp34 expression by p40'ax is distinct from and more intricate thanthose for previously characterized genes. For details, see Mol. Cell. Boil. 11,
1313-1325.
Reconstruction of Molecular Phylogeny of Extant Hominoids
from DNA Sequence Data
Naruya SAITOU

Evolutionary distance matrices of the extant hominoids are computed fromDNA sequence data, and hominoid DNA phylogenies are reconstructed byapplying the neighbor-joining method to these distance matrices. The chim-

88

panzee is clustered with the human in most of the phylogenetic trees thus
obtained. The proportion of the distance between human and chimpanzee tothat between human/chimpanzee and orangutan is estimated. Both mitochondrial DNA and nuclear DNA show a similar value (0.44), which is close tovalues derived from DNA-DNA hybridization data. For details, seeAmer. J.
Phys. Anthropol. 84, 75-85, 1991.
Statistical Methods for Phylogenetic Tree Reconstruction
Naruya SAITOU

Reconstruction of the phylogeny of organisms is one of the most important
problems in evolutionary study. A phylogeny is usually illustrated by atree-like figure. Thus we call it "phylogenetic tree" or simply "tree."Previously phylogenetic trees were reconstructed mostly by using morphological data. With the advent of the study of molecular evolution, however, it is
now customary to construct phylogenetic trees from molecular data, especially from nucleotide sequences. We are therefore concerned primarily with
nucleotide sequence data in this review. Theoretical aspects of phylogenetic
trees are first discussed, and distance matrix methods and character-statemethods are explained in the followings. Lastly, the efficiency of different
methods is discussed. For details, see Statistical Methods for Biological andMedical Sciences (c. R. Rao and R. Chakraborty eds.), pp. 317-346, Elsevier
Science Publishers RV., Amsterdam, 1991.
Evolutionary Rate of Immunoglobulin Alpha Noncoding Region Is
Greater in Hominoids than in Old World Monkeys
Shohji KAWAMURA*. Hideyuki TANABE*. Yuji WATANABE*. Kunihiko KUROSAKI*,
Naruya SAITOU and Shintaroh UEDA*

Recent studies on the molecular evolution of primates show that theevolutionary rate among hominoids is considerably slower than that among
nonhominoid primates. However, this observation at the nucleotide sequence level is restricted to the ,B-globin family region. In this study, wesequenced orthologous immunoglobulin alpha (Ca) genes of chimpanzee,
gorilla, orangutan, and crab-eating macaque (an Old WorId monkey) and*

Department of Antoropology. Faculty of Science. The University of Tokyo.

VIII.

EVOLUTIONARY GENETICS

89

compared them with that of the human by using noncoding regions foranalysis. Since significant differences in rates among hominoids were notfound by using the relative rate test, we evaluated the ratio (R) of theevolutionary distance between Old World monkey and human to the distance
between orangutan and human. The R value (1.12) for the Ca gene wasmuch smaller than the expected value (1.38-2.33), showing that the nucleotide substitution rate (=mutation rate per year under selective neutrality) of
the Ca gene is greater in the human lineage than in the Old World monkey
lineage. We also did a similar analysis for the rl-, 12-, cjJTj-, and o-globin genes
and found a considerable heterogeneity (1.12-2.37) among the R values,
including that for the Ca gene. This indicates that the hominoid slowdownof the evolutionary rate is not a universal phenomenon in primate evolution.
For details, see Mol. BioI. Evol., 8, 743-752, 1991.
Integration of a SINE-like Retroposon into a Specific Locus
in Rice Evolution
Hiro-Yuki HIRANO, Kayoko MOCHIZUKI*, Masaaki UMEDA*,
Hisako OHTSUBO*, Eiichi OHTSUBO*'**and Yoshio SANO

p-SINEI, which is found in the wx locus of rice (Oryza sativa), belongs toa class of retroposon transcribed by RNA polymerase III such as the Alu or
Bl family in mammals (Umeda, M., Ohtsubo, R., Ohtsubo, E. (1991) Jpn. J.
Genet. 66, 569-586). Retroposons transcribed from the master member are
believed to be integrated into another position via a eDNA intermediate.
This event of retroposition and amplification is quite rare and has been
occurred in the evolutionary time scale. The time of retroposition in theevolutionary process can be inferred by analyzing the distribution of a
retroposon at a specific locus among related species at present, because
retroposons appear not to be deleted. Rice is a very suitable organism for thisanalysis, because many strains of various species have been collected from allover the world and stocked at the Genetic Stock Research Center in our
institute, and their relationships have been well described.
Two members of p-SINEI were located at the untranslated 5' -region(p-SINEI-rl) and at the 9th intron (p-SINEI-r2) of the wx + gene of rice.
***

Institute of Applied microbiology, The University of Tokyo, Tokyo.
Laboratory of Cytoplasmic Genetics.

90

We examined the distribution of p-SINEl in seven related species carrying
the AA genome in the Oryza genus by polymerase chain reaction (peR), andfound that a member of p-SINEl (p-SINEl-r2) was present in only two
closely related species, O. sativa and 0. rufipogon, but not in the other species.
This result indicates that p-SINEl-r2 was integrated into the wx locus after
0. sativa and O. rufipogon diverged from the other species with the AA
genome. In contrast to p-SINEl-r2, another member (p-SINEl-rl) at theuntranslated 5'-region was present in all species, indicating that p-SINEl-rl
has been integrated into that position before the divergence of all rice species
with the AA genome. These results, therefore, demonstrate that each pSINEl member retroposed to a specific position at a different period in rice
evolution.
Cytogenetic Localization of Zygotic hybrid rescue Gene (Zhr) of
Drosophila melanogaster which Rescues InterspecificHybrids from Embryonic Lethality
Kyoichi SAWAMURA and Masa-Toshi YAMAMOTO

Hybrid females from a cross between D. melanogaster males and females of
its sibling species, D. simulans, D. mauritiana or D. sechellia die as embryos.
Their lethality is supposed to be caused by incompatibility between the X
chromosome of D. melanogaster and the maternal cytoplasm. Zygotic hybrid
rescue (Zhr) prevents this embryonic lethality and has been genetically
mapped at a proximal region of the X chromosome of D. melanogaster, andcytologically it has ,?een suggested that it is localized in the centromericheterochromatin. Thus, we carried out a high resolution cytological mapping
using deficiencies and duplications in the X heterochromatin. A deletion of
the Zhr + gene from a hybrid genome exhibits the Zhr phenotype. On thecontrary, the addition of the gene to the hybrid genome causes embryoniclethality regardless of sex. In order to minimize variation in tests on theviability of hybrid females, interspecific crosses were performed using D.
sechellia females and D. melanogaster males since escapers rarely emerged
from them. The Zhr locus has been narrowed down to the region covered byDp(l;f)1l62 but uncovered by Dp(l;f)l205, a chromosome carrying a duplication of heterochromatin slightly distal to the In(l)sc 8 heterochromatic
breakpoint.

91

IX.

HUMAN GENETICS

Genetic Polymorphisms in the Polyadenylate Tract of Alu
Repetitive Elements in the Human Genome
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA, Tomoko HASEGAWA
and Takashi IMAMURA

As a step toward constructing the complete genetic linkage map of chromosome 18, we made a gene library from a mouse hybrid cell line with an
extra human chromosome 18, using the pWE15 cosmid cloning vector. We
have attempted to place these clones at distinct bands of chromosome 18
using in situ hybridization techniques and high sensitivity microscopic fluorescent imaging apparatus. Genetic polymorphisms in regions that are identified with these clones were analysed with some of the clone located either on
the short or on the long arm of chromosome 18. To identify DNA
polymorphism that are abundant in the human genome and are detectable bypolymerase chain reaction amplification of genomic DNA, we tested the
hypothesis that the polydeoxyadenylate tract of the Alu family of repetitiveelements is polymorphic among human chromosomes and found that ten
examined thus far were polymorphic. This novel class of polymorphisms is
indeed representing one of the most useful and informative group of DNA
markers in the human genome. Mapping of the human genome is greatly
facilitated by the enormous normal variability of the DNA sequences between two randomly chosen homologous chromosomes. This DNA polymorphism provides the basis for the large number of markers currently being
used to construct linkage maps and search for the location of unknown genesthat causes hereditary disorders.
In this study we explored the possibilities that the tracts of polyadenylate
residues within the 3' ends of Alu repetitive elements in the human genome
contain a variable number of adenine residues and a short reiterated units on
the different chromosomes. The Alu sequences examined were isolated fromcosmid clones mapped on Chromosome 18p 13 region, and those in the HPRT
gene region on the Xq28. The locus-specific oligonucleotide primers weresynthesized according to the information obtained by sequencing the subclones or by searching for that in the DDBJ and EMBL DNA database. In

92order to detect non-radioactively the different alleles, we utilized the methodof asymetric polymerase chain reaction followed by SSCP detection onpolyacrylamide gel electrophoresis. For the amplification of specific single
stranded DNA fragments it was impoartant to use the repetitive and nonrepetitive oligonucleotides at a concentration of I : 100. Because Alu repeats
comprise about 1% of the human DNA, a large number of Alu variable poly(A) polymorphism may exist in the human genome, which should serve as amajor marker system in almost any DNA fragment that contains Alu
repetitive elements.
Toward a Transcriptional Map of the Human Chromosome 21
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA and Takashi IMAMURA

The present study is an approach for identifying genes within the humangenome. While considerable progress has been made in genetic and physicalmapping of the human genome, approaches are now needed to identify theprotein-coding sequences contained therein. The ultimate goal would be an
integrated genetic, physical and transcriptional map of the genome, combining information in the position of a gene, its sequence and its pattern ofexpression. Three basic techniques have been used to identify protein-coding
sequences from genomic DNA: particular restriction enzymes such as Not!
can be used to search for regions rich in CpG residues (HTF islands), which
are frequently associated with the 5' ends of constitutively expreessed genes;
genomic DNA of different species such as mouse can be probed with agenomic clone (zoo blots) to detect highly conserved sequences, which are
likely to be protein-coding; and northern blots and cDNA libraries can bedirectly probed with a genomic clone. We chose for this study the cosmid
clones of genome DNA that have been mapped by the method of fluorescentin situ hybridization on the long arm of chromosome 21 (2lq22). Among 8of these clones examined, 5 were apparently expressed in the mouse brain,
liver, and kidney cells. Interestingly, these clones positive for zoo blot
testings had at least one Not! site within the insert. Although the results
await further confirmation, these clones may possibly encode genes on thehuman chromosome 21.
While these strategies are feasible for small number of genomic clones, newapproaches are necessary for rapid detection of transcribed sequences in

IX.

HUMAN GENETICS

93

longer streches of DNA, whole chromosomes or entire genome. Other
approaches to generate region-specific cDNA libraries used hybridization of
cDNA inserts to genomic DNA from specific chromosomes, and subsequentcloning of annealed cDNAs. In 'one application of this scheme, the humanbrain cDNA library was hybridized to biotinylated DNA fragments from thehuman-mouse hybrid cell line (NA08854) containing chromosome 21 as the
only human chromosome. Hybrid molecules were then immobilized on themagnet-coated beads, non-specific hybrids were eliminated, and selected
cDNA inserts were amplified by PCR before cloning. Using this strategy,
substantial enrichment of a low abundance cDNA encoded by a gene on ahuman chromosome 21 was performed. Because of the problem of carryover of non-specific cDNAs, the cDNA library has to be subtracted for
cDNA clones encoded by the human genomic material. We will continue thisline of research, aiming that the resulting cDNA library will consist of
expressed sequences from one chromosome. Each clone may then be mappedon the restricted part of the chromosome by in situ hybridization.
Beta-Thalassemia Major Resulting from Compound Heterozygosity
for the ,8-globin Gene Mutation: Further Evidence for Multiple
Origin and Migration of the Thalassemia Gene
Hitoshi NAKASHIMA, Yutaka CHIFU and Takashi IMAMURA

Thalassemias are a heterogeneous group of inherited disorders of hemoglobin synthesis, all being characterized by the absence or reduced output of one
or more of the globin chains of hemoglobin. This leads to an imbalanced
globin chain synthesis. Thealassemias are the most common single genedisorder in the world population. Genetic studies at the DNA level disclosed
more than 40 different molecular varieties of thalassemia. Thus, it is common
for individuals to receive genes for more than one type of thalassemia. We
have encountered a patient with severe hypochromia and hemolysis resulting
from heterozygosity for two types of .B-thalassemia. One mutation was a
C-to-T transition at IYS-2 nucleotide position 654 on the backgroud of
Mediterranean RFLPs haplotype IX. Another mutation was a G-to-Atransition at IYS-2 nucleotide position 1, associated with a novel haplotype
XI. The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, inter-94

allelic gene conversion, and multiple origins of the same mutation.
The IYS-2 654 mutation is commonly found in the Chinese population,
associated with either haplotype I or II, which predicts the ,B-globin geneframework (Frl). We have previously reported the same mutation in a
Japanese family, which was associated with framework (Frl) on the background of haplotype I. Although the presence of IYS-2 654 mutant in two
different haplotypes I and IX is consistent with recurrence of the mutation,interchromosomal recombination between and ,B-globin genes is more likely
in view of the nonrandom assortment of the 5' and 3' collection of polymorphic restriction sites in the ,B-globin gene cluster. Interestingly, all three
haplotypes I, II and IX found so far associated with the IYS-654 mutation aregrouped into the same ,B-globin gene framework (Frl). Thus, the presence ofthe same mutation in a restricted region of the world (China and Japan)
might be explained by gene migration in populations, and subsequent interallelic gene conversion or recombination 5' to the ,B-globin gene changing
haplotype linkage. The IYS-2 I mutation has been found in several Mediterranean families associated with haplotypes I (Frl), III (Frla) and Y (Fr2),
but until now there had been no report in Asiatic population. The occurrenceof identical mutation on different ethnicity, and the association of themutation with a variety of frameworks strongly suggest multiple, independent origins of this mutation.
Time of the Deepest Root for Polymorphism in Human
Mitochondrial DNA
Masami HASEGAWA and Satoshi HORAI

A molecular clock analysis was carried out on the nucleotide sequences ofparts of the major noncoding region of mitochondrial DNA (mtDNA) fromthe major geographic populations of humans. Dates of branchings in the
mtDNA tree among humans were estimated with an improved maximum
likelihood method. Two species of chimpanzees were used as an outgroup,and the mtDNA clock was calibrated by assuming that the chimpanzee/human split occurres 4 million years ago, following our earlier works. A modelof homogeneous evolution among sites dose not fit well with the data even
within hypervariable segments, and hence an additional parameter that
represents a proportion of variable sites was introduced. Taking account of

IX.

HUMAN GENETICS

95

this heterogeneity among sites, the data for the deepest root of the mtDNA
tree among humans was estimated to be 280,OOO±50,OOO years old (± 1 SE),although there remains uncertainty about the constancy of the evolutionaryrate among lineges. The evolutionary rate of the most rapidly evolving sites
in mtDNA was estimates to be more than 100 times greater than that of a
nuclear pseudogene. For details, see J. Mol. Eva!. 32, 37-42, 1991.
A New mtDNA Mutation Associated with Mitochondrial Myopathy,
Encephalopathy, Lactic Acidosis and Stroke-like
Episodes (MELAS)
Yu-ichi GOTO, Ikuya NON AKA and Satoshi HORAIMELAS (mitochondrial myopathy, encephalopathy, lactic acidosis andstroke-like episodes) is a clinical entity of mitochondrial diseases. Recently,
a transition mutation in the mitochondrial tRNA Leu(UUR) gene was found in 26
of 31 MELAS patients and by a comparative analysis on the affected andnormal cultured muscle cell lines from a patient. We identified another 9
MELAS patients and found that 32 of the 40 patients (80%) had thismutation, but the remaining 8 patients did not. We, therefore, tried to detect
other molecular abnormalities specific to MELAS. Sequence analysis was
performed in all mitochondrial tRNA regions as described previously, and we
found T-to-C transition mutation at nucleotide position 3271 in three unrelated patients.
To develop a simple test to detect this mutation, we synthesized a modified
PCR primer, with a G-to-T mismatch at bp3275 and a T-to-C at bp3276. If
there was a transition mutation at bp3271, the PCR products (170bp) withthis mismatch reverse primer and the forward primer, could be cleaved into
two fragments (140 and 30 bp) by endonuclease Aft II because a newrecogmition site emerged. This test revealed that three patients had mutation
DNA in heteroplasmic fashion. However, 32 MELAS patients with the mostcommon mutation at 3243, 6 MERRF (myoclonus epilepsy associated withragged-red fibers) with the mutation at 8344, 32 and 6 CPEO (chronic
progressive external ophathalmoplegia) with and without large scale deletions, respectively, and 50 normal controls from different racial groups did
not have this nutataion.
The nucleotide in the anticodon stem are not invariant in contrast to the96most common mutation in the dihydrouridine loop during evolution. However, the preserved nucleotides forming the anticodon stem are complementary to each other, e.g., nucleotides T at 3271 and A at 3261 in normal humanindividuals. As the tRNA with the most common mutataion at 3243 in
MELAS patients is deprived of the hydrogen bond to T-residue in theamino-acid acceptor arm, the new transition should have a similar adverse
effect on the structure and the function of tRNA Leu(UUR). Accordingly,
this mutation is thought to be an additional candidate for genetic defects
in MELAS. For details, see Biochimica et Biophysica Acta 1097, 238-240,
1991.
Heteroplasmy and Polymorphism in the Major Noncoding Regionof Mitochondrial DNA in Japanese Monkeys: Association
with Tandemly Repeated Sequences
Kenji HAYASAKA, Takafumi ISHIDA and Satoshi HORAI

We have sequenced the major noncoding region of mitochondrial DNA(mtDNA) offour Japanese monkeys and have found length polymorphism inthe sequenced region. The length polymorphism resulted from two tandem
duplications of 160-bp sequences which contained the conserved sequence
blocks 2 and 3 and the light-strand transcription-promoter region. We also
found polymorphisms in this mtDNA region among 100 Japanese monkeys
from 12 localities, 90 of which were analyzed using DNA amplified throughthe polymerase chain reaction. In two localities, we found individuals withheteroplasmic mtDNAs which had different numbers of the 160-bp repeats
mentioned above. The 100 samples were classified into six types in term oflength and presence/absence of the recognition site of two restriction enzymes
in the major noncoding region. For details, see Mol. BioI. Evl. 8(4),399-415,
1991.
A New Type of Mitochondrial DNA Deletion in Patientswith Encephalomyopathy
Shigeaki MIYABAYASHI, Hiroshi HANAMIZU, Hitoshi ENDO,
Keiya TADA and Satoshi HORAI

We performed Southern blot analysis of muscle mitochondrial DNAs fromIX.

HUMAN GENETICS

97

47 patients who were clinically diagnosed as having mitochondrial disorders,and found deletions in five patients. Four patients with mitochondrial DNA
deletion had muscle weakness, myoclonic seizure, sensorineuronal hearing
loss, brain atrophy, delay of neurological development or neurological deterioration, but neither external ophthalmoplegia nor ragged-red fibers inbiopsied muscle tissues. The sites of deletions in mtDNAs from KSS wereclarified at hotspot regions found by Schon et al. or between the nucleotide
positions from 5,770 to 16,020. The size of deletion ranged from 1.3 to 7.6
kb. The deletions were flanked mainly by perfect direct repeats, located at
the edges of the deletion. The deleted mtDNAs from our patients were larger
in size, and surprisingly included the origin of light-strand replication in threepatients. The D-Ioop region was preserved as well as that in the previously
described patients with mtDNA deletion. Three patients with the deletion
including the origin of light-strand replication were characterized as having
an early onset of symptoms before the age of one year and a subsequently
severe clinical course. The pathogenesis of the mitochondrial DNA, including the origin oflight-strand replication, was unclear. However, these deleted
mitochondrial DNA may be made during the process of the replication of
normal mitochondrial based on abnormalities within the nuclear DNA or onderangements of the "cross-talk" between the nuclear and the mitochondrial
genomes, because they are thought to be unable to replicate in vivo. For
details, see J. Inher. Metab. Dis. 14, 805-812, 1991.
Phylogenetic Affiliation of Ancient and Contemporary Humans
Inferred from Mitochondrial DNA
Satoshi HORAI, Rumi KONDO, Kumiko MURAYAMA, Seiji HAYASHI,
Hiroko KOIKE and Nobuyuki NAKAI

Nucleotide sequence analysis of the major noncoding region of humanmitochondrial DNA (mtDNA) from three major races was extended withdata from 27 contemporary Mongoloids (20 from Southeast Asia, 7 from
America) and 11 ancient Japanese bones (5 from Jomon Age; 3,000-6,000
years RP., 6 from the early modern Ainu; 200-300 years RP.). In bothcases, the sequence was determined directly from the peR products. Basedon a comparison of the 482-bp sequences from a total of 128 contemporary
humans, the nucleotide diversity is estimated to be 1.46%, which is three

98

times higher than the corresponding value estimated from restriction-enzyme
analysis of the whole mtDNA genome. The phylogenetic tree revealed that
all lineages are classified into at least five clusters designated as CI to C5. C
I consists exclusively of Africans, and the majority of Asians and Europeans
formed C2, C3, CS and C4, respectively. Phylogenetic analysis also indicatedthat part of the Asians, including the Japanese, subsequently diverged fromthe majority of Africans, and that overall Asians can therefore be separated
into two distinct groups. Native Americans, on the other hand, appeared
only in C3 and CS, suggesting that the size of the founder population was not
so large during the peopling of America. Nucleotide sequences derived fromancient bones in a highly polymorphic region were also compared with thoseof contemporary humans. The nucleotide diversity among the 139 sequencesin the region was estimated to be 2.26%. A group of ancient Japanese,
including both Jomon peoples and the Ainu, showed a close phylogenetic
affiliation with one group of contemporary Japanese and Southeast Asians.
Moreover, all of the ancient Japanese were clearly placed in a larger cluster,
indicating their genealogical difference from another group of contemporary
Japanese. This observation supports the immigration theory, which postulates that immigrants from the Asian continent made a considerable contribution to the formation of modern Japanese. Finally, the presence of an
Asian-specific deletion was examined through PCR analysis of native American samples. The deletion was found in one mummy sample. This, together
with the present sequence analysis and published information, clearly showsthat the Circum-Pacifican populations (Asians, Oceanians and native Americans) can be separated into two distinct mitochondrial lineages. For details,
see Phil. Trans. R. Soc. Lond. B 333, 409-417, 1991.
Extracellular Matrix Protein Tenascin-Like Gene Foundin the Human MHC Class III Region
Ken-ichi MATSUMOTO, Nanayo ISHIHARA, Asako ANDO*, Hidetoshi INoKo*and Toshimichi IKEMURA

We previously reported a cluster of fibronectin type III repeats located ca.
20-S0 kb centromeric of CYP21 B in the class III region which showed
sequence similarity with human tenascin. Because of the high degree of*Dept. of Transplantation II, School of Medicine, Tokai University, Isehara.

IX.

HUMAN GENETICS

99

similarity and general parallelism of their organizations, the existence of atenascin-like gene was proposed. Tenascin is a multi-domain and multifunctional extracellular matrix glycoprotein and is made up of heptad, EGF,
and fibronectin type III repeats, and a fibrinogen domain, from the N- to
C-terminus. If the newly found gene in MHC is actually a tenascin-like gene,
heptad and EGF repeats may exist upstream of the clustered type III repeats.
Both heptad and EGF repeats, showing evident similarity with those oftenascin, were found in the 2.6 kb sequence ca. 60 kb centromeric of CYP21
B. There were 4 heptad and 18.5 EGF repeats. This work thus presented
evidence that a tenascin-like gene exists in the human MHC class III locus.
We recently found RNA transcripts from this gene and designated the geneas "tenascin-MHC". The existence of an extracellular matrix protein gene in
the MHC locus is of interest because not yet identified disease-susceptible
genes for such A diseases as narcolepsy are expected to be present within ornear the MHC class II region. For details, see Genomics 12, 485~491 (1992)
and also Immunogenetics (1992, in press).
Giant G+C% Mosaic Structures in the HLA Locus and a Border
between the Mosaic Domains
Ken-ichi MATSUMOTO, Nanayo ISHIHARA, Asakao ANDO*, Hidetoshi INOKO*
and Toshimichi IKEMURA

We previously showed that the genomes of higher vertebrates are composed of G+C% mosaic structures and that the mosaic structure is a factor
producing G + C% diversity at the codon third position. The correlation ofthis mosaic structure with chromosome band structures and with the'isochore' proposed by Bernardi was also established. An example of bordersof the giant G + C% mosaic structures of the human genome was identified
fairly precisely between class II and III of the MHC locus: The contiguousclasses I and III belong to the evidently GC-rich domain which appears tocorrespond to T band sequences, and the G + C% levels of class II weresignificantly lower than those of classes I and III. Thus a border between the
G + C % mosaic domains was assigned at the junction area between classes II
and III. About 350kb of this junction area has not yet been sequenced, andwe are now attempting to clone the entire junction in order to clarify the*Dept. of Transplantation II, School of Medicine, Tokai University, Isehara.

100

characteristics of the border region of the G + C% mosaic structures (e.g.,
how sharp the boundary is and how precisely it can be defined). At present,
the border was localized within lOOkb. For details, see HLA1991 (ed. T.
Sasazuki) Oxford Univ. Press, in press.

101

x.

APPLIED GENETICS

Responses of Wild and Cultivated Rice Populations to Four Different
Races of Pathogen Zanthomonas campestris
Hiroko MORISHIMA and Toshie MIYABAYASHI

Comparative studies of host-pathogen interaction in natural and agricultural ecosystems can provide perspectives on the evolutionary dynamics ofplant-pathogen systems. We are studying the genetic variation of wild andcultivated rice populations in response to infection by Xanthomonas eampestris pv. oryzae (Xeo). Xeo causes serious disease in rice plants (bacterial leaf
blight), and different pathogenic races are known.
We have already reported that wild and landrace populations of rice are
more or less polymorphic in response to Xeo. In 1990-1991, a number of
populations collected in Thailand and Bangladesh were tested for resistanceto four different Xeo races which are the major pathogen races prevailing inJapan. These plant materials were collected in delta areas in Thailand and
Bangladesh which are deeply flooded in rainy season. Deepwater condition
should favor epidemics this disease.
It was found that perennial populations of Thai wild rice adapted to
deepwater condition are generally resistant to all four races, while annualtype populations adapted to drier condition in the same area contained
individuals susceptible to different races. In the Bangladesh materials, ten
different infection types in response to the four races were found. Cultivars
grown in rainy season (aman) tended to show higher resistance than thosegrown in dry season (bora). Weedy wild rice populations associated withaman cultivars, which are most probably hybrid derivatives of natural crosses
between wild and cultivated rices, were found to be highly polymorphic inresponses to four Xeo races. The host-pathogen interactions we studied areinteractions between Japanese pathogens and exotic plants which have neverencountered each other. Yet, the above results indicate that the genetic
variation dealt with in this study has been subjected to certain kinds ofnatural selection. Further study is planned using pathogen races prevailing in
the area from which host plants were collected.
To determine the genetic basis for this disease resistance, resistant and102

susceptible plants sampled from an annual wild population were crossed, andthe F 2 population was investigated. It was confirmed that at least one
dominant major gene responsible for resistance segregated. In addition,
parent-offspring regression was detected in a cultivar population whichshowed continuous variation in resistance. This suggests that polygenes
controlling quantitative resistance exist.
Constraints in Using Wild Relatives in Breeding: Lack of Basic
Knowledge on Crop Genepools
Yoshio SANG

The utilization of alien germplasm became an urgent problem after recent
developments in technology. Abnormalities in hybrids have resulted fromdisharmonious interactions of genes expressed in various developmental
stages. Genetic comparisons of internal barriers suggest that different species
carry genes leading to genetic discontinuities. A profound barrier candevelop from an accumulation of genes with relatively small effects. Genecomplexes often show complicated features of incompatibilities depending
upon the accumulated genes. Despite the complexity of developmental
constraints, adequate selection makes it possible to transfer a simply inheritedgene from distantly related taxa.
Plant breeders use local elite lines rather than wild relatives since it is noteasy to select favorable gene combinations through hybridization. The
nature of the primary genepool in crops predicts the importance of gene flow
between subdivided populations. Isolating barriers act as genetic sieves
which protect balanced gene sets for the developmental patterns preserved in
species. Nevertheless gene flow through the genetic sieve seems to play asignificant role in crop evolution. Law genetic materials supplied from alien
species could be utilized for the acquisition of new characteristics with othergenetic modifications, even though it may appear slightly deleterious. Local
cultivars have probably received some alien genes accompanying genetic
assimilation under natural conditions. If so it may be easy to utilize suchgenes in crop improvement. When we consider that genetic erosion isaccelerating all over the world, there is no reason why we should wait toutilize alien genes until the primary genepool absorbs them by means of
naturally occurring hybridization and selection. This study will appear in

X.

APPLIED GENETICS

103

International Crop Science I (1992).

Inheritance of Low Temperature Resistance at the Young
Seedling Stage in Rice
Hiroko Morishima

The Japonica and Indica types are the two major varietal groups of Asian
cultivated rice which can be distinguished by several diagnostic character andby genes. The former type is known to be less damaged by low temperature
treatment followed by exposure to high temperature at the young seedlingstage, than the latter type is. Strains of wild rice (Oryza rujipogon), except
for some Chinese strains, are generally more susceptible to the abovementioned treatment than cultivars.
An upland cultivar collected in Yunnan, China, was found to be highly
polymorphic in various Indica-Japonica diagnostic character including theabove mentioned low temperature resistance, within the population. To
study the inheritance of these key characters, five lines derived from different
individuals of the original population sample were intercrossed, and six F 2
populations were studied regarding low temperature resistance. Five to tenprogeny seedlings (1.0-1.5 cm length) derived from each F 2 plant were kept
at 0-1 Cc for 4 days and at 30°C for the following 3 days. The degree of
injury was scored individually, dividing into 4 classes (O:resistant-I-2-3:
susceptible), and the average value was used as the index for resistance of thecorresponding F 2 plant. Each F 2 population tended to show a bimodal
segregation. Four susceptible (S) X resistant (R) crosses segregated into
resistant and susceptible plants giving lOR: 54S or IR: 3S ratios, an S X S
cross giving a IR: ISS ratio, and a TXT cross giving a 7R: 9S ratio. These
results suggest that recessive genes with a complementary nature are responsible for this resistance. If we assume that when at least two out of three locicarry recessive alleles in a homozygous state the carrier exhibits resistance,
Then the segregation patterns observed in the six F 2 populations can beconsistently interpreted.
To confirm the genetic basis of this resistance, further analysis is under way
using F 3 lines. Various types of low temperature resistance so far reported inrice seedlings are all under the control of a single dominant gene. The
resistance dealt with in this report seems to be controlled by a different genic

104

system.
Genetic Control of Fertility Reversions in Cytoplasmic
Male Sterility of Rice
Yoshio SANO, Mitsugu EIGUCHI, and Hiro-Yuki HIRANO

The present study was carried out to examine the genetic mechanism
responsible for reversions to a fertile phenotype which were detected incytoplasmic male-sterile plants of rice. The cms-bo cytoplasm of Chinsurahboro II gave rise to male-sterility in plants without a gametophytic restorer
gene (Rfj). Taichung 65 (T65A) is known to be a maintainer which carries
no restorer. However, Taichung 65 preserved in our laboratory (T65B)
showed partial fertility (about 8% seed set) when crossed with male-sterile
plants. Unexpectedly, the seed fertility gradually increased with repeatedselfings and almost fully fertile plants were obtained in the F 6 generation.
Cytoplasmic substitution lines revealed that reversions to a fertile phenotype
resulted from mutational events at the nuclear level. Genetic experiments
indicated that the partial fertility observed in the F 1 hybrid was controlled bya dominant gene, Ifrj, which was carried by T65B. The results obtainedsuggested that Ifrj itself is associated with the instability of fertility restoration in the presence of cms-bo cytoplasm, since partially fertile plants carrying
Ifrj always showed a tendency toward gradual increases in fertility in later
generations. Backcrossings are under way to introduce Ifrj into male-sterile
plants. Although the backcross generation is still BC2 at present, theresultant plants with Ifrj have shown a gradual increase in selfings.
Pollen gametophytic selection is apparently more effective than sporophytic selection in leading to fertile revertants. An advantage of gametophytic
selection may be minimization of segregational cost. Such an intensified
selection could be carried out automatically in the case of self-fertilizing
plants. New mutations could be effectively selected for and maintained in thepresence of cms-bo cytoplasm only through intensified gametophytic selection, otherwise they quickly become extinct. This might give one of thepossible explanations for diversified genotypes preserved in plant populations
in relation to complex features in fertility relationships. Whatever themechanism for instability is, the present results confirmed that intensified
gametic selection combined with instability leads to a rapid change in thex.

APPLIED GENETICS

105

genetic content of rice. For details, see Genetical Research 60 (No.3), 1992.
Japonica Rice was Born in East China
Y. I. SATO, x. TANG*, L. YANG** and L. TANG***

The middle and lower basins of Yangtze river are argued to be the place oforigin for cultivated rice. We examined ancient rice spikelets excavated at
Homedu site in Zhejiang province (as old as 7,000 yrs.) to examine whether
wild rice existed at this site. It was found under a scanning electric
microscope (SEM) that five out of eighty-five spikelets examined, hadcharacteristics of wild rice, although previous observations had not indicatedthis. Some other seeds showed combined characteristics of wild and cultivated rices, suggesting the frequent occurrence of natural hybridization between
wild and cultivated rice or the existence of an intermediate type.
Archaeological records of the excavations in China show that initial ricecultivation began in the middle and lower basins of Yangtze river seven tonine thousand years ago. Records from tropical Asia suggests that it waspracticed also in multiple areas of the tropics. Rice cultivation appears tohave begun at different sites in Asia independently.
Data taken from the genetic viewpoint also support a hypothesis of adiphyletic origin of Asian rice. Restriction fragment length polymorphisms(RFLPs) of chloroplast DNAs suggest that indica andjaponica evolved fromdifferent strains of on ancestral species, O. rufipogon. and that strains of O.
rufipogon having japonica-like chloroplast DNAs are largely inhabitants ofChina (Dally and Second 1990). Combining our results and previous work,
we propose a new hypothesis thatjaponica was born in the middle and lower
basins of Yangtze river seven to nine thousand years ago.
Developmental Regulation of Phenotypic Plasticity in Deepwater Rice
Mitsugu EIGUCHI, H.-Y. HIRANO and Yoshio SANa

Plants utilize environmental signals to alter the timing of gene expression
so as to respond to changing environments. A drastic change in morphology* China National Rice Research Institute, Hagzhou, China.
** Zhejiang Museum, Hangzhou, China.
*** Jiangsu Academy of Agriculture Sciences, Nanjiang, China.

106

and physiology due to changing environments is known as phenotypic
plasticity which is particularly important in plants because of their sessile life
style. Although phenotypic plasticity has been assumed to playa significant
role in evolution, more information is needed about the genetic control ofplasticity as well as plastic changes which occur during ontogeny.
Submergence in water induces drastic changes in the morphology ofdeepwater or floating rice, including a rapid internodal elongation. A floating
habit is widely observed in Oryza rufipogon which is the wild progenitor of
Asian cultivated rice, O. sativa. The wild progenitor shows an annualperennial continuum and the perennial type generally grows in deep water,while the annual type grows in temporal swamps which are parched in the dry
season. Experiments were carried out using a perennial wild rice accession
with deepwater tolerance and a non-deepwater rice cultivar. Selection for
deepwater tolerance was performed under 1m-deepwater conditions andbackcrosses were made up to the BC s generation to transfer a gene(s) for
deepwater tolerance into the intolerant cultivar. The results indicated that arecessive gene (dw]) was responsible for deepwater tolerance and plants withdw] induced internodal elongation in response to submergence rather thanenhancement of elongation. Plants without dw] showed no internodal
elongation and soon died.
A most striking difference between submerged and air-grown T65dw] was
the number of elongated internodes. When submerged before 10 weeks ofage, 8 to 9 internodes elongated. After 10 weeks of age the number of
elongated internodes became almost the same (5 to 6) as that of air-grownplants. Panicle primordia covered with bract hairs were first recognized onshoot apices at 11 weeks of age in both air-grown T65 and T65dw], suggestingthat floral initiation occurred between 10 and 11 weeks of age. After floral
initiation, elongation of developing internodes was similarly enhanced bysubmergence, irrespective of the presence or absence of dw]. This indicatedthat the dw] gene changes the timing of internodal elongation depending on
environmental conditions in the vegetative phase. A mutation changing therelative timing of developmental events is known as a heterochronic gene.
Plants are metameric or modular in their growth and the plant body is
indeterminant in its growth, giving an opportunity for a high level of plastic
response to environments. The elongation patterns of T65dw] in response toflooding markedly changed around the time of floral initiation. This suggeststhat plastic response by dw] in rice is restricted to the vegetative phase or toX.

APPLIED GENETICS107

the stage prior to the determination of panicle formation in the shoot apicalmeristem. This study will appear in J. Heredity, 1993.
An Altitudinal Cline of Genetic Variation Found in Bhutan Rice Cultivars
Tadashi SATO*, Yo-Ichiro SATO, Hiroshi YAMAGISHI**, Yoshiya SHIMAMOTO***and Hiroko MORISHIMA

Rice plants are cultivated in diverse environments ranging from low lying
delta to mountainous highland. One-hundred and fifty eight rice strains
collected in three major valleys in Bhutan (12QO-2600m alt.) were investigated regarding various key characters and genes which have been used
for distinguishing the two subspecies Asian rice (indica and japonica).
Samples contained cultivated as well as weedy (naturally propagated incultivated fields) types of rice.
The following were pointed out.
1. Samples from high altitudes (2200 m < ), which were collected in Paroand Thimpu valleys, tended to have japonica characters, such as KCIO)
resistant, and negative phenol reaction (Ph), while samples from lower
altitudes (l600m », collected in Punakha valley, tended to have indica
characters.
2. Isozyme analysis clearly separated all the samples into japonica andindica. It was confirmed from this analysis that samples from high altitudes
were mostly japonica and those from low altitudes were mostly indica.
3. Apiculus hair length, which is another indica-japonica key character,
did not show altitudinal variation.
4. In KelO) resistance, the frequency of the ph gene and length of thebasic vegetative phase, variations in cultivar populations and in coexisting
weedy populations were positively correlated among collection sites, but nosuch trend was found with apiculus hair length and grain shape.

*
**
***

Institute of Genetic Ecology, Tohoku University, Sendai.
Kyoto Sangyo University, Kyoto.
Hokkaido University, Sapporo.

108

On the Distribution Patterns of Selfing- and Outcrossing-Inclined
Genotypes in Plant Populations
Katsuei YONEZAWA

Development of spatial patterns in plant populations of selfing-inclined(highly fertile with selfing) and outcrossing-inclined (showing no or low
fertility with selfing) genotypes was theoretically investigated based on a one
dimensional population model where three genotypes, i.e., selfing-inclined(aa), outcrossing-inclined (AA), and intermediate (Aa) genotypes were
distributed with decreasing population density from central toward peripheral regions. Mating among plants was assumed to be random with a limited
dispersion distance for pollen and seeds. The equilibrium distribution of the
genotypes was calculated based on an initial state of the three genotypes being
randomly distributed in the population. Four major types of distribution
patterns were derived according to combinations of the parameters constituting the population model; in two of the four, selfing- or outcrossing-inclined
genotypes occupied the entire population (gene a or A became fixed), and in
the other two, selfing- and outcrossing-inclined genotypes coexisted in a
mixture, or separately with selfing-inclined genotypes predominating in peripheral regions. It was found that the distribution pattern was determinedprimarily by the dispersion distance of pollen rather than that of seeds.
Population size also regulated the spatial pattern. Populations with a large
size tended to be more selfing-inclined.
Analysis of Chloroplast DNA from Ancient Rice Seeds
Ikuo NAKAMURA, Wen-Bing CHEN and Yo-Ichiro SATO

We have reported that DNA could be extracted from ancient rice seeds
preserved under anaerobic conditions and the extracted DNA could be usefulas a template for PCR amplification (Ann. Rep. 41). We could specify thesubspecies of the ancient rice into indica or japonica by the amplification of aparticular chloroplast DNA fragment from the ancient rice seed.
Restriction fragment length polymorphisms of chloroplast DNA between
indica andjaponica were reported by Ishii et al. (1988) and Dally and Second(1990). We focused on the Pst-12 fragment of japonica chloroplast DNA,because the RFLP data suggested the presence of a small deletion inside thisX.

APPLIED GENETICS

109/
fragment of indica chloroplast DNA. Two oligonucleotides (5 -AGTCC/ACTCAGCCATCTCTC-3' and 5 -GGCCATCATTTTCTTCTTTAG-3')
were synthesized and used as primers for PCR reactions. A 1.0 kbp fragment
was amplified from japonica cultivars and a 0.9 kbp fragment was found inindica cultivars. The detail restriction analysis of amplified fragment showedthat a small deletion of about l00bp was present in indica chloroplast DNA,corresponding to the ORF 100 region of japonica chloroplast DNA. We
considered this small deletion a convenient DNA marker for distinguishing
the subspecies of ancient rice or rice cultivars, because rice chloroplast DNA
is transmitted by maternal inheritance and no recombination events between
maternal and paternal chloroplasts is known.
DNA was extracted from a total of 11 ancient rice seeds, 90-1300 years
old, which were excavated and found from four different archaeological sites
in Japan and about 1 ng of DNA was used as a template for PCR reactions.
Amplification of chloroplast DNA fragments was carried out in a thermal
programer for 42 cycles under these conditions: 1 min. denaturation at 92°C;
1min. annealing at 37 °C; and 2 min. polymerization at 72 °C. Aliquots offour III were analyzed by agarose gel electrophoresis. The results showedthat a 1.0 kb fragment was detected in all ancient rice seeds, revealing that all
11 ancient rice seeds had japonica type chloroplast.
Many ancient rice seeds and plant remains have been excavated fromarchaeological sites in east and south Asia. In the Tai-hu area of China, there
are many excavations where rice seeds 5,000-7,000 years old have beenfound. If the genotypes of such ancient rice seeds could be identified by the
DNA analysis described as above, it would greatly contribute to the investigation of domestication of wild rice and varietal diversification of ricecultivars.

An Accelerated Internode Overgrowth Mutant in Rice
Ikuo NAKAMURA

A rice mutant of potential biochemical interest, showing greatly accelerated internode overgrowth characteristics, was isolated in the cultivar
Koshi-hikari, following treatment with r-irradiation.
This mutant named Awa-odori was isolated as follows; forty-thousand M 3
seeds which were descendants derived from seeds irradiated with 25 kR or 30

110

kR r-ray were kindly provided by Professor H. Nakai of Shizuoka University.
These seeds were directly and densely sown into a paddy field and grown tomaturity. A total of 21,190 panicles, with a single mature panicle derived
from each stock, were collected and then ten seeds were randomly pickedfrom every panicle. The resulting ca 200,(X)() M 4 seeds were germinated inplastic cases and screened for stature of seedling growth. Only one plantshowed extreme extension growth of the internode and leaf at the seedlingstage. This mutant plant did not form an panicle or panicle-like structure,but it remained alive for a period as long as normal plants. Since the mutant
could not produce seeds, two heterozygous plants were identified in the same
panicle from which the overgrowth mutant was derived. A progeny test ofheterozygosity yielded a segregation ratio of 3 normal: 1 mutant, indicatinga monofactorial recessive inheritance of the mutant phenotype.
The mutant phenotype is characterized by very rapid extension growth ofinternodes and by extremely long and narrow leaf blades both in the seedlingand adult plant stages. The mature plant is tall, slender, extremely lax, andunable to support itself. At the four leaf stage the mutant plants are up to 2.5
times as tall as these with a normal phenotype. The phenotype of Awa-odori
mutants resembles that of the slender mutant in barley (Foster 1977).
Awa-odori mutant plants like barley slender do not respond to exogenous
gibberellin, whereas non-mutant sibs respond in the usual manner with rapid
stem and leaf sheath extension growth occurring. Mesocotyle and first andsecond internodes elongate in the slender mutant, but these organs do notshow such elongation in the Awa-odori mutant. In the case of Awa-odori,
the higher internodes over second node show rapid extreme extension
growth.

III

XI.

DATABASE

DNA Database Release 10 from DNA Data Bank of Japan
Yoshihiro UGAWA, Yukiko YAMAZAKI, Molono HORIE, Masako IWASE, Mari SAITO,
Yumiko SATO, Shigeko SUZUKI, Yuko HASEGAWA, Yoshie HATTORI, Yuriko NOGUCHI,
Mary SHIMOYAMA, Eriko HATADA, Rikiko SUZUKI, Reiko UCHIDA, Yoko SHIDAHARA,
Yoko MATSUSHIMA, Akino WATANABE, Yoko UEDA, Machiko NAKAOKA,
Talsuko KAWAMOTO, Hajime KITAKAMI, Naruya SAITOU,
Yoshio TATENO and Takashi GOJOBORIThe DNA database Release 10 includes 59,317 entries which correspond to
77,805,556 bases. Unlike past releases, this release contains the newest dataprepared by DDBJ, GenBank and the EMBL Data Library as of January,
1992. Thanks to international collaboration between the three data banks,
the International Nucleotide Sequence Database was organized, resulting ina unified database published in this release.
All the entries designated by the accession numbers with a "D" have beencollected and processed by DDBJ, and the rest have been prepared by
GenBank and the EMBL Data Library. It should particularly noted that due
to unresolved technical problems, at present some entries in the threedatabases overlap. We will resolve the problem by working together with theother data banks.
Because the release contains unified data, all the entries have been annotated with common feature keys. The feature keys used in the previous
release were updated beforehand. The present release was prepared byprocessing the data on the relational data base management system (Sybase).
It does not include amino acid sequence data, because the genetic code system
is known to be no longer uniform among the species and organella, and theInternational Nucleotide Sequence Database is not yet prepared for this.
Building and Searching Biological Information based on a Relational
Database System
Hajime KITAKAMI and Yukiko YAMAZAKI

The DNA database has been managed in a flat-file system at the DDBJ

112

since 1985. The flat-file system is inadequate for building and searching the
DNA database which is receiving an explosive increase entries. We carriedout a transformation from the flat-file system to the relational databasesystem with GenBank staff. The schema of the relational database wasdesigned as follows:(1) Decomposing the DNA data into both structuralized and non-structuralized data.
(2) Partitioning large tables into small tables without redundancies.
(3) Making a flexible relationship among tables to represent complex
data.
This schema provided the capability for building and searching the DNA
database with less memory on the relational database system. However, theschema was implemented as a complex network structure with about 60
tables. It is difficult to use the SQL search language of the relational databasesystem with this schema.
We defined and simplified the schema for easy use of the commands using
the view function of the relational database system on the existing schema.
The simplified schema implemented in the view function was defined as
LOCUS, DEFINITION, ACCESSION, KEYWORDS, SOURCE, REFERENCE, FEATURE, ORIGIN, and SEQUENCE tables which are virtualtables without storing real data. It represents aspects of the traditional
DDBJ/EMBL/GenBank data format which are familiar to biologists using
the flat-file system.
Users can easily join these virtual tables using attribute storing accession
numbers. Since we developed the simplified schema, users are able to use the
SQL search command easily and get quick response in DNA data searches.

XI.

DATABASE

113

Codon Usage Tabulated from GenBank Genetic Sequence Data
Ken-nosuke WADA*, Yoshiko WADA**, Fumie ISHIBASHI, Takashi GOJOBORI
and Toshimichi IKEMURA

Codon usages in 22361 genes were analyzed using nucleotide sequence dataobtained from the GenBank Genetic Sequence Data Bank (Release 69.0,
Sept., 1991). The database is called the CUTG Databas~, and is distributedon EMBL CD-ROM (December 1991; CODON by Wada et al.) as amember of the NAR Sequence Supplement Databases. The CUTG codon
database is also available with on-line access to DDBJ. For details, see Nucl.
Acids Res. 20, Supplement, 2111-2118 (1992).

* Chykyo Univ., School of Computer and Congnitive Science, Nagoya.
**

Niigata University, School of Medicine, Niigata.

114

PUBLICATIONSABO, T, INAMOTO, S. and OHTSUBO, E.: Specific DNA binding of the TraM protein in the oriT
region of plasmid RlOO. J. Bacteriol. 173,6347-6354, 1991.
AMEMURA-MAEKAWA, J. and OHTSUBO, E.: Functional analysis of the two domains in theterminal inverted repeat sequence required for transposition of TnJ. Gene 103, 11-16,
1991.
AZlIMA, Y., YAMAGISHI, M., UESHIMA, R. and ISHIHAMA, A.: Cloning and sequence
determination of the Schizosaccharomyces pombe rpbl gene encoding the largest subunit of
RNA polymerase II. Nucl. Acids Res. 19(3),461-468,1991.
BARBIER, P. and ISHlHAMA, A.: Relatedness of annual and perennial strains of Oryza rufipogoninvestigated with nuclear DNA sequences. In "Rice Genetics II" (lRRI, ed.), pp. 731-733,
Manila, 1991.
BARBIER, P., MORISHlMA, H. and ISHlHAMA, A.: Phylogenetic relationships of annual andperennial wild rice: probing by direct DNA sequencing. Theor. Appl. Genet. 81(5), 693702, 1991.
BRENNER, M. and TOMlzAwA, J.: Quantitation of ColEI-encoded replication elements. Proc.
Natl. Acad. Sci. USA. 88, 405-409, 1991.
BROWN, J. L., SONODA, S., UEDA, H., SCOTT, M. P. and Wu, c.: Repression of the Drosophila
fushi tarazu (ftz) segmentation gene. EMBO J. to, 665-674, 1991.
CHIFU, Y., NAKASHIMA, H., HARA, H., YOKOTA, E. and IMAMURA, T: ,B-thalassemia major
resulting from a compound heterozygosity for the ,B-globin gene mutation: further evidence
for multiple origin and migration of the thalassemia gene. Human Genetics 89,343-346,
1992.
DAVID, C. N., FUJISAWA, T and BOSCH, T. C. G.: Interstitial stem cell proliferation in Hydra:
Evidence for strain-specific regulatory signals. Dev. BioI. 148, 501-507, 1991.
EGUCHI, Y., IToH, T and TOMIZAWA, J.: Antisense RNA. Annu. Rev. Biochem. 60, 631-652,
1991.
EGUCHI, Y and TOMIZAWA, J.: Complexes formed by complementary RNA stem-loops: Their
formations, structures, and interaction with ColEI Rom protein. J. Mol. BioI. 220, 831-842,
1991.
FUJIOKA, M., HIRATA, T and SHIMAMOTO, N.: Requirement for the ,B,y-pyrophosphate bondof A TP in a stage between transcription initiation and elongation by Escherichia coli RNA
polymerase. Biochemistry 30, 1801-1807, 1991.
FUJISAWA, T: Homeostatic recovery of interstitial cell populations in Hydra. Dev. BioI. 150,
185-192, 1992.
FUJIYAMA, A., TSUNASAWA, S., TAMANOI, F. and SAKIYAMA, F.: S-farnesylation and Methyl
Esterification of C-terminal Domain of Yeast RAS 2 Protein Prior to Fatty Acid Acylation.
J. BioI. Chem. 266, 17926-17931,1991.
GOJOBORI, T, IMANISHI, T., INA, Y., SAITOU, N., TATENO, Y, KIMURA, A., KAMIKAWAJI,
N., WAKISAKA, A., AKAZA, T., MIYOSHI, H., MURA, T., SATO, K., TAKATA, H. andPUBLICATIONSliSINOKO, H. (1991): HLA-IIth, CD-ROM (HMC-717) of 11th International Histocompatibility Workshop and Conference, Yokohama.
GOJOBORI, T. and INOKO, H. (1991): The data book of the II th International Histocompatibility Workshop. (ed.) Volume I, pp. I-480. Central Data Analysis Committee, Yokohama.
GOJOBORI, T. and INOKO, H. (1991): The data book of the 11th International Histocompatibility Workshop (ed.) Volume II, pp. 481-827. Central Data Analysis Committee, Yokohama.
GOJOBORI, T., MORIYAMA, E. N., IKEO, K., INA, Y., IMANISHI, T., TATENO, Y., KAWAI, M.,
KISHINO, A., NAITOU, K., SHIOBARA, M. and MATSUBARA, Y. (1991): Alignment ofhomologous amino acid sequences, SODHO Ver. 1.0. CD-ROM (HMC-709), Fujitsu
Limited, Tokyo, Japan.
GOTO, Y., NONAKA, I. and HORAI, S.: A new mtDNA mutation associated with mitochondrial
myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Biochimica
et Biophysica Acta 1097, 238-240, 1991.
HARA, H., YAMAMOTO, Y., HIGASHITANI, A., SUZUKI, H. and NISHIMURA, Y.: Cloning,
mapping, and characterization of the Escherichia coli prc gene, which is involved in
C-terminal processing of penicillin-binding protein 3. J. Bacteriol. 173, 4799-4813, 1991.
HARADA, H., SAKAGAMI, H., NAGATA, K., OH-HARA, T., KAWAZOE, Y., ISHIHAMA, A.,
HATA, N., MlsAwA, Y., TERADA, H. and KONNO, K.: Possible involvement of lignin
structure in anti-influenza virus activity. Antiviral Res. 15(1), 41-50, 1991.
HARIHARA, S., IMANISHI, T., SAITOU, N., OMOTO, K., VARAVUDHI, P. and TAKENAKA, 0.:Phylogenetic analysis of Macaca fascicularis in Thailand, using data of mitochondrial DNA
variation. In "Primatology Today" (Ehara, A. et al., eds.), pp. 611-612, Elsevier Science
Publishers B. V., Amsterdam, 1991.
HASEGAWA, M. and HORAI, S.: Time of the deepest root for polymorphisms in humanmitochondrial DNA. J. Mol. Evol. 32, 37-42, 1991.
HASEGAWA, M., KISHINO H. and SAITOU, N.: On the maximum likelihood method in
molecular phylogenetics. J. Mol. Evol. 32, 443--445, 1991.
HASHIMOTO, K., NOGUCHI, M. and NAKATSUJI, N.: Mouse offspring derived from fetal ovaries
or reaggregates which were cultured and transplanted into adult females. Develop. GrowthDiffer. 34, 233-238, 1992.
HAYASAKA, K., ISHIDA, T. and HORAI, S.: Heteroplasmy and polymorphisms in the major
noncoding region of mitochondrial DNA in Japanese monkeys: Association with tandemly
repeated sequences. Mol. BioI. Evol. 8(4), 399-415, 1991.
HAYASHI, J.-I., YONEKAWA, H., WATANABE, S., NONAKA, I., YOSHIDA MOMOI, M.,
KAGAWA, Y., OHTA, S.: Somatic Cell Genetical Approaches to Mitochondrial Diseases.
Mitochondrial Encephalomyopathies, In "The Series Progress in Neuropathology". Vol. 7(Raven Press, New York), 93-102, 1991.
HAYWARD, R. S., IGARASHI, K. and ISHIHAMA, A.: Functional specialization within the asubunit of Escherichia coli RNA polymerase. J. Mol. BioI. 221(1), 23-29, 1991.
HIGASHITANI, A., GREENSTEIN, D. and HORIUCHI, K.: A single amino acid substitution
reduces the superhelicity requirement of a replication initiator protein. N ucl. Acids Res. 20,
in press.
HIGASHITANI, N., HIGASHITANI, A., ROTH, A. and HORIUCHI, K.: SOS induction in Escherl'-

116chia coli by infection with mutant flamentous phage that are defective in initiation of thecomplementary strand DNA synthesis. J. Bacteriol. 174, 1612-1618, 1992.
HIRANO, H.-Y: Flower color patterns and transposable element. Protein Nucl. Acid. Enzym.
36, 657-659, 1991 (in Japanese).
HIRANO, H.-Y.: Genes responsible for starch synthesis. Plant Cell Tech. 3, 588-594, 1991 (inJapanese).
HIRANO, H.-Y. and SANO, Y: The wx locus of rice (Oryza sativa )-from genetics to molecularbiology- Plant Cell Tech. 3, 35-42, 1991 (in Japanese).
HIRANO, H.-Y. and SANO, Y.: Molecular characterization of the waxy locus of rice (Oryza
sativa). Plant Cell Physiol. 32, 989-997, 1991.
HIROSE, S.: DNA supercoiling facilitates initiation of transcription from certain eukaryotic
promoters. In "Gene Expression", p. 289, Mochida Memorial Foundation, Tokyo, 1991.
HIROSE, S. and SUZUKI, Y.: In vitro transcription of eukaryotic genes is affected differently by
the degree of DNA supercoiling. In "Transcription Regulation Mechanism in Eukaryotic
Genes" (MlJRAMATSU, M., ed.), pp. 43-47, Nakanishi, Tokyo, 1991.
HORAI, S.: A genetic trail of human mitochondrial DNA. In "New Era of Bioenergetics"(MUKOHATA, Y., eds.), pp. 273-299, Academic Press, Tokyo, 1991.
HORAI, S.: Mitochondrial DNA and human evolution. In "Mitochondrial encephalomyopathies"(SATO, T. and DIMAURO, S., eds.), pp. 9-20, Raven Press, New York, 1991.
HORAI, S., KONDO, R., MURAYAMA, K., HAYASHI, S., KOIKE, H. and NAKAI, N.: Phylogenetic affiliation of ancient and contemporary humans inferred from mitochondrial DNA.
Phil. Trans. R. Soc. Lond. B333, 409-417, 1991.
IGARASHI, K., FUJITA, N. and ISHIHAMA, A.: Identification of a subunit assembly domain in
the alpha subunit of Escherichia coli RNA polymerase. J. Mol. BioI. 218(1), 1-6, 1991.
IGARASHI, K., HANAMURA, A., MAKINO, K., AlBA, H., AlBA, H., MIZUNO, T., NAKATA, A.
and ISHIHAMA, A.: Bipartite functional organization of the a subunit of Escherichia coli
RNA polymerase: Two modes of transcription activation by positive factors. Proc. Natl.
Acad. Sci. USA. 88(20), 8958-8962, 1991.
IGARASHI, K. and ISHIHAMA, A.: Bipartite functional map of the E. coli RNA polymerase a
subunit: Involvement of the C-terminal region in transcription activation by cAMP-CRP.
Cell 65, 1015-1022, 1991.
IKEMlJRA, T.: Correlation between codon usage and tRNA content in Microorganisms. In"Transfer RNA in Protein Synthesis" (HATFIELD, D., ed.), pp. 87-111, CRC Press,
Florida, USA.
IKEMURA, T, MATSUMOTO, K., ISHIHARA, N., AN DO, A. and INOKO, H.: Giant G+C%
mosaic structures in HLA locus and a border between the mosaic domains. In "HLA 1991"(SASAZUKI et al., eds.), Oxford Univ. Press, in press.
IKEMlJRA, T and WADA, K.: Evident diversity of codon usage patterns of human genes withrespect to chromosome banding patterns and chromosome numbers; relation between
nucleotide sequence data and cytogenetic data. Nucl. Acids Res. 19(16), 4333-4339, 1991.
IKEO, K., TAKAHASHI, K. and GOJOBORI, T: Evolutionary origin of numerous kringles inhuman and simian apolipoprotein (a). FEBS Lett. 287, 146-148, 1991.
IMAI, H. T: Mutability of constitutive heterochromatin (C-bands) during eukaryotic chromosomal evolution and their cytological meaning. Jpn. J. Genet. 66, 635-661, 1991.

PVBLICATIONS117

INAGUMA, Y., MIYASHITA, N., MORIWAKI, K., WANG, C.-H., JIN, M-L., HE, X-Q. and IKEDA,
H.: Acquisition of two endogenous ecotropic murine leukemia viruses in distinct Asian wild
mouse populations. J. Virol. 65(4), 1796-1802, 1991.
INAMOTO, S., YOSHIOKA, Y. and OHTSUBO, E.: Site- and strand-specific nicking in vitro at oriTby the TraY-TraI endonuclease of plasmid Rloo. J. BioI. Chern. 266, 10086-10092, 1991.
ISHIHAMA, A.: Global control of gene expression in bacteria. In "Control of Cell Growth andDivision" (lSHIHAMA, A. and YOSHIKAWA, H., eds.), pp. 121-140, Jpn. Sci. Press, Tokyo!
Springer-Verlag, Berlin, 1991.
ISHIHAMA, A.: Genes for cell growth and division in Escherichia coli. In "Control of Cell Growthand Division" (ISHIHAMA, A. and YOSHIKAWA, H., eds.), pp. 199-203, Jpn. Sci. Soc. Press,
Tokyo/Springer-Verlag, Berlin, 1991.
ISHIHAMA, A.: Protein-protein communications within the transcription apparatus. Molecules
and Cells 1, 381-384, 1991.
ISHIHAMA, A., FUJITA, N., IGARASHI, K., VESHIMA, R., NAKAYAMA, M. and YAMAZAKI, Y.:
Molecular mechanisms of transcription regulation: Promoter selectivity of RNA polymerase. In "Recent Advances in Biochemistry" (BYUN, S. Y., LEE, S. Y. and YANG, C. H.,eds.), pp. 179-185, Biochem. Soc. Rep. Korea, Seoul, 1991.
ISHIHAMA, A., IGARASHI, K., OZAKI, M., HAYWARD, R. S. and YAMAGISHI, M.: The
promoter selectivity of Escherichia coli RNA polymerase. In "Gene Expression" (MATSUBARA, K. et al., eds.), pp. 17-34, Mochida Memorial Found., Tokyo, 1991.
ISHIHAMA, A. and YOSHIKAWA, H. (eds.): Control of Cell Growth and Division. Jpn. Sci. Soc.
Press, Tokyo/Springer-Verlag, Berlin, 1991.
ISHIKAWA, R., MORISHIMA, H., KINOSHITA, T, HARADA, T, NIIZEKI, M. and SAITO, K.:
Linkage analysis of nine isozyme genes on the conventional linkage map in rice. Jpn. J.
Breed. 41, 265-272, 1991.
IWATA, A., VEDA, S., ISHIHAMA, A. and HIRAI, K.: Sequence determination of cDNA clonesof transcripts from the tumor-associated region of Marek's disease virus genome. Virology
187, 805-808, 1992.
KAJITANI, M. and ISHIHAMA, A.: Identification and sequence determination of the host factor
gene for bacteriophage QIJo Nucl. Acids Res. 19(5), 1063-1066, 1991.
KATAYAMA, S., HIRANO, H.-Y., TSUKAYA, H., NAITO, S. and KOMEDA, Y.: Analysis ofintergenic spacer regions in the nuclear rDNA of Pharbitis nil. Genome 35, 92-97, 1992.
KATOH, Y. and SANO, Y.: Effects of 3-amino-I,4-dimethyl-5H-pyrido (4,3-b) indole (Trp-P-I)
and 3-amino-I-methyl-5H-pyrido (4,3-b)-indole (Trp-P-2) on somatic mutation in soybean
YIIYII plants. Agric. BioI. Chern. 55, 1669-1670, 1990.
KATSU RA, I.: Development and genes (special issue for the advances in genetics of nematodes).
The Heredity (Iden) 45(11), 28-33,1991 (in Japanese).
KATSURA, I. and KONDO, K.: Morphogenetic genes in the nematode C. elegans. Cell
Technology (Saibou Kougaku) 10(9),667-672, 1991 (in Japanese).
KAWAMURA, S., TANABE, H., WATANABE, Y., KUROSAKI, K., SAITOU, N. and VEDA, S.:
Evolutionary rate of immunoglobulin alpha noncoding region is greater in hominoids thanin Old World monkeys. Mol. BioI. Evol. 8, 743-752, 1991.
KAWASHIMA, T, MIYASHITA, N., WANG, c., HE, X., JIN, M., Wu, Z. and MORIWAKI, K.: A
new haplotype of the j3-globin gene complex, Hbb WI, in Chinese wild mouse. Jpn. J. Genet.

118
66, 491-500, 1991.
KIMURA, M.: Neutral evolution. In "Evolution of Life" (OSAWA, S. and HONJO, T., eds.), pp.
67-78, Springer-Verlag, Tokyo, 1991.
KIMURA, M.: Some recent data supporting the neutral theory. In "New Aspects of The Geneticsof Molecular Evolution" (KIMURA, M. and TAKAHATA, N., eds.), pp. 3-14, Japan Sci. Soc.
Press, Tokyo/Springer-Verlag, Berlin, 1991.
KIMURA, M. and TAKAHATA, N. (eds.): New Aspects of The Genetics of Molecular Evolution.
Japan Scientific Soc. Press, Tokyo/Springer-Verlag, Berlin, 1991.
KIMURA, M.: Recent development of the neutral theory viewed from the Wright ian tradition oftheoretical population genetics. Proc. Natl. Acad. Sci. USA. 88, 5969-5973, 1991.
KIMURA, M.: The neutral theory of molecular evolution: A review of recent evidence. Jpn. J.
Genet. 66, 367-386, 1991.
KOBAYASHI, M., TSUCHIYA, K., NAGATA, K. and ISHIHAMA, A.: Reconstitution of influenza
virus RNA polymerase from three subunits expressed using recombinant baculovirus
system. Virus Res. 22, 235-245, 1992.
KOGA, A., KUSAKABE, S., TAJlMA, F., TAKANO, T., HARADA, K. and MUKAI, T.: A methodfor detecting effect of beneficial mutations in natural populations of Drosophila melanogaster. Genetical Research 58, 145-156, 1991.
KOHARA, Y.: The Genome Map of Escherichia coli. K-12. In "Control of Cell Growth andDivision" (lSHIHAMA, A. and YOSHIKAWA, H., eds.), pp. 185-197, Springer-Verlag, Tokyo,
1991.
KOIZUMI, 0., MIZUMOTO, H., SUGIYAMA, T. and BODE, H. R.: Nerve net formation in theprimitive nervous system of hydra~An overview. Neuroscience Research, Supplement 13,
165-170, 1990.
KOSEKI, H., ASANO, H., IN ABA, T., MIYASHITA, N., MORIWAKI, K., FISCHER LINDAHL, K.,
MIZUTANI, Y., IMAI, K. and TANIGUCHI, M.: Dominant expression of a distinctive V14 14
T-cel1 antigen receptor a chain in mice. Proc. Natl. Acad. Sci. USA. 88, 7518-7522, 1991.
KUBORI, T., SHIMAMOTO, N., YAMAGUCHI, S., NAMBA, K., and AIZAWA, S-I.: Morphologicalpathway of flagel1ar assembly in Salmonel1a typhimurium. J. Mol. BioI. 224, in press.
KUBOTA, M., YAMAZAKI, Y. and ISHIHAMA, A.: Random screening of promoters from
Escherichia coli and classification based on the promoter strength. Jpn. J. Genet. 66(4),399-409, 1991.
KURIHARA, Y., NAITO, T., OBAYASHI, K., HIRASAWA, M., KURIHARA, Y. and MORIWAKI,
K.: Caries susceptibility in inbred mouse strains and inheritance patterns in Fl andbackcross (N2) progeny from strains with high and low caries susceptibility. Caries
Research 25,341-346,1991.
MATSUMOTO, K., ISHIHARA, N., AN DO, A., INOKO, H. and IKEMURA, T.: Clustered occurrence of fibronectin type-III repeats in the human major histocompatibility complex class
III region: a tenascin-like gene in the MHC locus. In "HLA 1991" (SASAZUKI et al., eds.),
Oxford Univ. Press, in press.
MISHINA, Y., AYUSAWA, D., SENO, T. and KOYAMA, H.: Thymidylate stress induces homologous recombination activity in mammalian cel1s. Mutation Res. 24,215-220, 1991.
MIURA, S., OHTANI, K., MURATA, N., NIKI, M., OHBO, K., INA, Y., GOJOBORI, T., TANAKA,
Y., TozAwA, H., NAKAMURA, M. and SUGAMURA, K.: Molecular cloning and character-PUBLICAnONS

119

ization of a novel glycoprotein, gp34 that is specifically induced by the human T-cellleukemia virus type I transactivator p40tax. Mol. Cell BioI. 11, 1313-1325, 1991.
MIYABAYASHI, S., HANAMIZU, H., ENDO, H., TADA, K. and HORAI, S.: Mitochondrial DNA
deletion in patients with myopathy. In "Mitochondrial encephlaomyopathies" (SATO, T. and
DIMAURO, S., eds.), pp. 153-160, Raven Press, New York, 1991.
MORISHIMA, H.: Association between Pox-l variation and seed productivity potential in wild
rice. In "Rice Genetics II" (IRRI, ed.), pp. 1-9, Manila, 1991.
MORIYAMA, E. N. and GOJOBORI, T.: Molecular evolution of human and simian immunodeficiency viruses. In "New Aspects of the Genetics of Molecular Evolution" (KIMURA, M. and
TAKAHATA, N., eds.), pp. 291-301,Japan Scientific Societies Press, Tokyo/Springer-Verlag,
Berlin, 1991.
MORIYAMA, E. N. and GOJOBORI, T.: Rates of synonymous substitution and base compositionof nuclear genes in Drosophila. Genetics 130, 855-864, 1992.
MORIYAMA, E. N., INA, Y., IKEO, K., SHIMIZU, N. and GOJOBORI, T.: Mutation pattern of
human immunodeficiency virus genes. J. Mol. Evol. 32, 360-363, 1991.
NAGATA, I. and NAKATSUJI, N.: Rodent CNS neuroblasts exhibit both perpendicular andparallel contact guidance on the aligned parallel neurite bundle. Development, 112, 581590, 1991.
NAKAMURA, I. and SATO, Y.-I.: Amplification of DNA fragments isolated from single seed ofancient rice (AD 800) by polymerase chain reaction. Chinease J. Rice Sci. 5, 175-179,
1991.
NAKAMURA, K., MIYASHITA, S., OZAKI, M., IWAYA, H., NAKAZAWA, S., aKA, J., KAMADA,
N., TANAKA, K., KOBAYASHI, N. and MIZUTANI, S.: Molecular studies of chronic
myelogenous leukemia using polymerase chain reaction. Cancer 68, 2426-2430, 1991.
NAKASHIMA, H., CHIFU, Y., and IMAMURA, T.: Beta-Thalassemia major resulting fromcompound heterozygosity for the ,,-globin gene mutation: further evidence for multiple
origin and migration of the thalassemia gene. Amer. J. Human Genet. 49, 199, 1991.
NAKATSUJI, N. and HASHIMOTO, K.: Culture of embryonic cells for analysis of amphibian andmammalian early embryogenesis. In "Gastrulation: Movement, Patterns and Molecules"(KELLER, R., CLARK, W. and GRIFFIN, F., eds.), pp. 43-56, Plenum Press, New York,
1991.
NAKATSUJI, N., KADOKAWA, Y. and SUEMORI, H.: Radial columnar patches in the chimeric
cerebral cortex visualized by use of mouse embryonic stem cells expressing ,,-galactosidase.
Develop. Growth Differ. 33, 571-578, 1991.
NAKAYAMA, M., FUJITA, N., aSAWA, S. and ISHIHAMA, A.: Identification and characterization of RNA polymerase sigma factor from Micrococcus luteus. J. BioI. Chern. 266(5),2911-2916, 1991.
NAKAYAMA, M., NAGATA, K. and ISHIHAMA, A.: Enzymatic properties of mouse Mxl
protein-associated GTPase. Virus Res. 22, 227-234, 1992.
NAKAYAMA, M., NAGATA, K., KATO, A. and ISHIHAMA, A.: Interferon-inducible mouse Mx
I protein that confers resistance to influenza virus is GTPase. J. BioI. Chern. 266, 2140421408, 1991.
NATSUUME-SAKAI, S., OKADA, M., SEYA, T., NONAKA, M., NONAKA, M., HARADA, Y. and
MORIWAKI, K.: The mouse factor H allotypes with multiple amino acid replicement, H.I

120
and H.2 show indistinguishable co-factor activity for factor I. European. J. Immunogenet.
18,399-403, 1991.
NEROME, K., KANEGAE, Y., YOSHIOKA, Y., ITAMURA, S., ISHIDA, M., GOJOBORI, T. andOYA, A.: Evolutionary pathways of N2 neuraminidases of swine and human influenza A
viruses: Origin of the neuraminidase genes of two reassortants (HIN2) isolated from pigs.
J. Gen. Virol. 72, 693-698, 1991.
NINOMIYA-TsUJI, J., NOMOTO, S., YASUDA, H., REED, S. I. and MATSUMOTO, K.: Cloning ofa human cDNA encoding a cdc2-related kinase by complementation of a budding yeast cdc
28 mutation. Proc. Natl. Acad. Sci. USA. 88, 9008-9010, 1991.
NISHIMURA, A., AKIYAMA, K., KOHARA, Y. and HORIUCHI, K.: Correlation ofa subset of thepLC plasmids to the physical map of Escherichia coli K-12. Microbiol. Rev. 56, 137-151,
1992.
NISHIMURA, A., AKIYAMA, K., KOHARA, Y., TAKEDA, Y., NISHIMURA, Y., HIGASHITANI, A.,
YASUDA, S., HORIUCHI, K., and HIROTA, Y.: In "Control of Cell Growth and Division"(lSHIHAMA, A. and YOSHIKAWA, H., eds.), pp. 205-223, Springer-Verlag/Tokyo, 1991.
NISHITANI, H., OHTSUBO, M., YAMASHITA, K., IIDA, H., PINES, J., YASUDA, H., HUNTER, T.
and NISHIMOTO, T.: Loss of RCCI, a nuclear DNA-binding protein, uncouples thecomplementation of DNA replication from the activation of cdc2 protein kinase andmitosis. EMBO J. 10, 1555-1564, 1991.
NODA, A., COURTRIGHT, J. B., DENOR, P. F., WEBB, G., KOHARA, Y. and ISHIHAMA, A.:
Rapid identification of specific genes in E. coli by hybridization to membranes containing
the ordered set of phage clones. Bio Techniques 10(4), 474-476,1991.
NOMURA, N., MASAI, H., INUZUKA, M., MIYAZAKI, C., OHTSUBO, E., ITOH, T., SASAMOTO,
S., MATSUI, M., ISHIZAKI, R. and ARAI, K.: Identification of eleven single-strand initiation
sequences (ssi) for priming of DNA replication in the F, R6K, RlOO and CoIE2 plasmids.
Gene 108, 15-22, 1991.
NUNOMURA, K., MAEDA, Y. and OHTSUBO, E.: The interaction of platinum complexes with
DNA studied by differential scanning calorimetry. J. Gen. Appl. Microbiol. 37, 207-214,
1991.
OHTA, T.: Evolution of the multigene family: A case of dynamically evolving genes at major
histocompatibility complex. In "Evolution of Life" (OSAWA, S. and HONJO, T., eds.), pp.
145-149, SpringerNeriag/Tokyo, 1991.
OHTA, T.: Multigene families and their implications for evolutionary theory. In "New Aspectsof The Genetics of Molecular Evolution" (KIMURA, M. and TAKAHATA, N., eds.), pp. 1525, Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin, 1991.
OHTA, T.: Multigene families and the evolution of complexity. J. Mol. Evol. 33, 34-41,1991.
OHTA, T.: Role of diversifying selection and gene conversion in evolution of major histocompatibility complex loci. Proc. Natl. Acad. Sci. USA. 88, 6716-6720, 1991.
OHTSUBO, H., UMEDA, M. and OHTSUBO, E.: Organization of DNA sequences highly repeatedin tandem in rice genomes. Jpn. J. Genet. 66, 241-254, 1991.
OKADA, K., OHTA, T. and HIROSE, S.: Negative supercoiling of DNA by eukaryotic DNA
topoisomerase II and dextran sulfate. J. Biochem. 109, 365-369, 1991.
OMOTO, K., HIRAI, M., SAITOU, N. and HARIHARA, S.: Extensive polymorphism of complement C6 among crab-eating monkeys. In "Primatology Today" (EHARA, A. et al., eds.), pp.

PUBLICATIONS

121

603-604, Elsevier Science Publishers B. V., Amsterdam, 1991.
OZAKI, M., WADA, A., FUJITA, N. and ISHIHAMA, A.: Growth phase-dependent conversion of
RNA polymerase in Escherichia coli. Mol. Gen. Genet. 230, 17-24, 1991.
OZAKI, M., FUJITA, N., WADA, A. and ISHIHAMA, A.: Promoter selectivity of the stationaryphase forms of Escherichia coli RNA polymerase and conversion in vitro of the SI form
enzyme into a log-phase enzyme-like form. Nucleic Acids Res. 20, 257-261, 1992.
QADOTA, H., ISHII, I., FUJIYAMA, A., OHYA, Y. and ANRAKU, Y.: RHO gene products,
putative small GTP-binding proteins, are important for activation of the CAL I/CDC 43
gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae. Yeast, inpress.
RUSSELL, D. W. and HORIUCHI, K.: The mutH gene regulates the replication and methylationof the pMBI origin. J. Bacteriol. 173,3209-3214, 1991.
SADAIE, Y., TAKAMATSU, H., NAKAMURA, K. and YAMANE, K.: Sequencing reveals similarityof the wild-type div + gene of Bacillus subtilis to the Escherichia coli secA gene. Gene 98,
101-105, 1991.
SAEKI, T., AZUMA, Y., SHIBANO, T., KOMANO, T. and UEDA, K.: Human p-glycoprotein(hMDRI) expression in yeast. Agr. BioI. Chern. 55(7), 1859-1865, 1991.
SAITOU, N.: Phylogeny of extant hominoids reconstructed from molecular data. In "Primatology Today" (EHARA, A. et al., eds.), pp. 627-630. Elsevier Science Publishers B. V.,
Amsterdam, 1991.
SAITOU, N.: Statistical methods for phylogenetic tree reconstruction. In "Handbook of
Statistics, Volume 8: Statistical Methods for Biological and Medical Sciences" (RAO, C. R.
and CHAKRABORTY, R., eds.), pp. 317-346, Elsevier Science Publishers B. V., Amsterdam,
1991.
SAITOU, N.: Reconstruction of rnolecular phylogeny of extant hominoids from DNA sequence
data. American Journal of Physical Anthropology 84,75-85, 1991.
SANO, Y., HIRANO, H.-Y. and NISHIMURA, M.: Evolutionary significances of differential
regulation at the wx locus of rice. In "Rice Genetics II" (IRRI, ed.), Manila, pp. II-20,
1991.
SANO, Y., SANO, R. and HIRANO, H.-Y.: Evolution of the intergenic spacer region in cultivatedand wild rice species. In "Rice Genetics II" (IRRI, ed.), Manila, pp. 653-654,1991.
SATO, T., NAKAMURA, S., HIRAWAKE, H., SEKI, K., ISHIGAKI, Y., HORAI, S. and OZAWA, T.:In situ hybridization and immuno-electron microscopic study of mitochondrial DNA
mutations. In "Mitochondrial encephalomyopathies" (SATO, T. and DIMAURO, S., ed.), pp.
195-204, Raven Press, New York, 1991.
SATO, Y. I.: How was rice differentiated into indica and japonica? In "Rice Genetics II" (lRRI,ed.), Manila, pp. 45-53, 1991.
SATO, Y.-1. and YAMAGATA, H.: Genetic analysis of culm length by its regression on days toheading in rice. SABRAO J. 22, 17-24, 1991.
SATTA, Y., TAKAHATA, N., SCHONBACH, C., GUTKNECHT, J. and KLEIN, J.: Calibrating
evolutionary rates at major histocompatibility complex polymorphism. In "Molecular
Evolution of the Major Histocompatibility Complex" (KLEIN, J. and KLEIN, D., eds.), pp.
51-62, Springer-Verlag, Heidelberg, 1991.
SEKINE, Y. and OHTSUBO, E.: Translational frameshifting in IS elements and other genetic

122
systems. In "New Aspects of the Genetics of Molecular Evolution". (KIMURA, M. and
TAKAHATA, N., eds.), pp. 243-261, Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin,
1991.
SEONG, B. L., KOBAYASHI, M., NAGATA, K., BROWNLEE, G. G. and ISHIHAMA, A.: Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.
J. Biochem. 111,496-499, 1992.
SHIMAMOTO, N., TERADA, H. and FUJIOKA, M.: An early period in elongation by prokaryotic
RNA porymerases is specified by the requirement for 13, r-pyrophosphate bond of ATP. J.
Cell Biochem. sup 15G, 245, 1991.
SHIROISHI, T., SAGAI, T., HANZAWA, N., GOTOH, H. and MORIWAKI, K.: Genetic control of
sex-dependent recombination in the major histocompatibility complex of the mouse.
EMBO J. 10,681-686, 1991.
SUGITA, S., YOSHIOKA, Y., ITAMURA, S., KANEGAE, Y., OGUCHI, K., GOJOBORI, T., NEROME,
K. and OYA, A.: 1991 Molecular evolution of hemagglutinin genes of HINI swine andhuman influenza A viruses. J. Mol. Evol. 32, 16-23, 1991.
STY RNA, J., IMAI, H. T. and MORIWAKI, K.: An increased level of sperm abnormalities in micewith a partial deletion of the Y chromosome. Genet. Res. Camb. 57, 195-199, 1991.
STY RNA, J., KLAG, J. imd MORIWAKI, K.: Influence of partial deletion of the Y chromosome
on mouse sperm phenotype. J. Reprod. Fer!. 92, 187-195, 1991.
TABU CHI, H. and HIROSE, S.: DNA supercoiling facilitates formation of the transcription
initiation complex on the fibroin gene promoter. In "Transcription Regulation Mechanism
in Eukaryotic Genes" (MURAMATSU, M., ed.), pp. 48-53, Nakanishi, Tokyo, 1991.
T ACHIDA, H.: A study on a nearly neutral mutation model in finite populations. Genetics 128,183-192,1991.
T ACHIDA, H. and IIZUKA, M.: Fixation probability in spatially changing environments.
Genetical Research 58, 243-251, 1991.
TAJIMA, F.: Determination of window size for analyzing DNA sequences. J. Mol. Evol. 33,
470-473, 1991.
TAKAHASHI, K., GOJOBORI, T. and TANIFUJI, M.: Two-color cytofluorometry and cellular
properties of the urokinase receptor associated with a human metastatic carcinomatous cellline. Exp. Cell Res. lin, 405-413, 1991.
TAKAHATA, N.: A trend in population genetics theory. In "New Aspects of the Genetics ofMolecular Evolution" (KIMURA, M. and TAKAHATA, N., eds.), pp. 27-47, Japan Sci. Soc.
Press, Tokyo/Springer-Verlag, 1991.
TAKAHATA, N.: Trans-species polymorphism of HLA molecules, founder principle and human
evolution. In "Molecular Evolution of the Major Histocompatibility Complex" (KLEIN, J.
and KLEIN, D., eds.), pp. 29-49, Springer-Verlag, Heidelberg, 1991.
TAKAHATA, N.: Book Review for Evolutio at the molecular Level. edited by R. K. Selander, A.
G. Clark and T. S. Wittam, Sinauer Associates Inc, Mol. BioI. Evol. 8, 901-903, 1991.
TAKAHATA, N.: Overdispersed molecular clock at the major histocompatibility complex loci.
Phil. Trans. R. Soc. Lond. B 243, 13-18, 1991.
TAKAHATA, N.: Statistical models for the overdispersed molecular clock. Theor. Pop. BioI. 39,
329-344, 1991.
TAKAHATA, N.: Genealogy of neutral genes and spreading of selected mutations in a geograp-

PUBLICATIONS

123

hically structured population. Genetics 129, 585-595, 1991.
TAKAHATA, N. and TAJlMA, F.: Sampling errors in phylogeny. Mol. BioI. Evol. 8,494-502,
1991.
TANAKA, S., SUZUKI, T., SAKAIZUMI, M., HARADA, Y, MATSUSHIMA, Y., MIYASHITA, N.,
FUKUMORI, Y., INAI, S., MORIWAKI, K. and YONEKAWA, H.: Gene responsible for
deficient activity of the f3 subunit of C8, the eighth component of complement, is located onmouse chromosome 4. Immunogenetics, 33, 18-23, 1991.
TENZEN, T. and OHTSUBO, E.: Preferential transposition of an IS630-associated composite
transposon to TA in the 5'-CTAG-3' sequence. J. Bacteriol. 173, 6207-6212, 1991.
TERADA, H. and SHIMAMOTO, N.: Dynamic analysis of transcription by immobilized operon.
Seibutubuturi 31,335-338, 1990 (in Japanese).
TERADA, K., OKUHARA, E., KAWARADA, Y. and HIROSE, S.: Demonstration of extrinsic
DNA from immune complexes in plasma of a patient with systemic lupus erythematosus.
Biochem. Biophys. Res. Commun. 174, 323-330, 1991.
TEZUKA, H.: Studies on radiation sensitivity in mouse mutant, "wasted". Ph. D. Thesis at
University of Tokyo, 1991.
TSUCHIYA, K., MATSUURA, Y., KAWAI, A., ISHIHAMA, A. and UEDA, S.: Characterization ofrabies virus glycoprotein expressed by recombinant baculovirus. J. Gen. Virol. 25, 1-13,
1992.
TSUJI, H., YASUSHI, M., AJlRO, K., YASUDA, H., HANAOKA, F., HAYASHI, A., UTSUMI, S.,
OHBA, Y. and HORI, T.: A temperature-sensitive CHO-Kl cell mutant (tsTM13) defective
in chromosome decondensation and spindle decon struction in M phase. Exp. Cell Res. inpress.
TSUKIYAMA, T., VEDA, H., HIROSE, S. and NIWA, 0.: Embryonal LTR-binding protein is amurine homologue of FTZ-Fl, a member of the steriod receptor superfamily. Mol. Cell
BioI. in press.
VEDA, H. and HIROSE, S.: Defining the sequence recognized with BmFTZ-FI, a sequencespecific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis. Nucl. Acids Res.
19,3689-3693, 1991.
VMEDA, M. and OHTSUBO, E.: Four types of lSI with difference in nucleotide sequences residein the Escherichia coli K-12 chromosome. Gene 98, 1-5, 1991.
VMEDA, M., OHTSUBO, H. and OHTSUBO, E.: Diversification of the rice Waxy gene by insertionof mobile DNA elements into introns. Jpn. J. Genetics 66,569-586, 1991.
VRA, K. and HIROSE, S.: Possible role of DNA topoisomerase II on transcription of thehomeobox gene Hox-2.1 in F9 embryonal carcinoma cells. Nucl. Acids Res. 19, 6087-6092,
1991.
WADA, K., WADA, Y., DOl, H., ISHIBASHI, F., GOJOBORI, T. and IKEMUTA, T.: Codon usage
tabulated from the GenBank genetic sequence data. Nucl. Acids Res. 19, Supplement,
1981-1986, 1991.
WADA, M. Y and IMAI, H. T.: On the Robertsonian polymorphism found in the Japanese
raccoon dog (Nyctereutes procyonoides viverrinus). Jpn. J. Genet. 66, I-II, 1991.
WINKING, H., WEITH, A., BOLDYREFF, B., MORIWAKI, K., FREDGA, K. and TRAUT, W.:
Polymorphic HSRs in chromosome I of the two semispecies Mus musculus musculus and124
M. m. domestic us have a common origin in an ances tral population. Chromosoma 100,147~151, 1991.
YAMAGUCHI, M., HAYASHI, Y, HIROSE, F., MATSUOKA, S., MORIUCHI, T., SHIROISHI, T.,
MORIWAKI, K. and MATSUKAGE, A.: Molecular cloning and structural analysis of mousegene and pseudogenes for proliferating cell nuclear antigen. Nuclei Acids Res. 19, 24032410, 1991.
YAMANAKA, K., OGASAWARA, N., YOSHIKAWA, H., ISHIHAMA, A. and NAGATA, K.: In vivo
analysis of the promoter structure of the influenza virus RNA genome using a transfection
system with an engineered RNA. Proc. Natl. Acad. Sci. USA. 88(12), 5369-5373, 1991.
YAMAO, F., ADACHI, Y, MUTO, A., IKEMURA, T. and OSAWA, S.: Levels of tRNAs inbacterial cells as affected by amino acid usage in proteins. Nuc1. Acids Res. 19, 6119-6122,
1991.
YAMAZAKI, Y., TRUONG, V. L. and LOWENSTEIN, J. M.: 5'-nucleotidase I from rabbit heart.
Biochemistry 30, 1503-1508, 1991.
YASUDA, H., KAMIJO, M., HONDA, R., NAKAMURA, M. and OHBA, Y: A point mutation in
C-termina1 region of cdc2 kinase causes a G2-phase arrest in a mouse temperature-sensitive
FM3A cell mutant. Cell Struct. Func. 16, 105-112, 1991.
YASUDA, J., SHORTRIDGE, K. F., SHIMIZU, Y. and KIDA, H.: Molecular evidence for a role ofdomestic ducks in the introduction of avian H3 influenza viruses to pigs in southern China,
where the A/Hong Kong/68 (H3N2) strain emerged. J. Gen. Virol 72(8), 2007-2010, 1991.
YONEDA, M., TSUJI, S., TANNO, Y., HORAI, S., OZAWA, T. and MIYATAKE, T.: Molecular
genetic analysis of myoclonus epilepsy associated with ragged-red fibers. In "Mitochondrial
encephalomyopathies", (SATO, T. and DIMAURO, S., eds), pp. 169-180, Raven Press, New
York, 1991.

125

ABSTRACTS OF DIARY FOR 1991

Biological Symposium
Paxl and undulated: a genetic approach to study the337th-Jan. 28
axial skeleton development of the mouse (Rudolf Balling)
338th-Feb. 1
Models for genomic rearrangements (David Sankoff)
Promoter analysis of influenza RNA transcription and339th-Feb. 27
replication (Baik L. Seong)
Transposons, DNA sequencing and reverse genetics in
340th-Mar. 19
E. coli (Douglas E. Berg)
341st-Mar. 19
Population Genetics of HLA (Phil Hedrick)342nd-Mar. 22 Initiation of transdifferentiation in jelly fish (Volker
Schmid)343rd-Apr. 5
American human genome project-genome information(David Benton)
344th-May 9Morphogenesis in hydra: Morphogenetic and mitogenic
activity of head activator (Engerbert Hobmayer)
345th-May 17
X-chromosome inactivation (Mary F. Lyon)
346th-Sept. 6
Development of a simple nervous system (Gunther S.
Stent)
347th-Sept. 27
Regulation of bacterial chromosome replication: The
initiator titration model (Flemming G. Hansen)
348th-Sept. 27
Growth phase regulated genes in E. coli: Genes induced
by energy limitation (Tove Atlung)349th-Dct. 11
msDNA, retron and retronphage (Masayori Inouye)
350th-Dct. 31
Electronic data publishing at Los Alamos (Michael
Cinkosky)
35 1st-Nov. 8
Cancer genetics and the human genome (Walter
Bodmer)352nd-Nov. 21 DNA supercoiling in vivo and its biological ramification(James C. Wang)
353rd-Nov. 22 The origin and evolution of MHC genes (Masatoshi
Nei)
354th-Dec. 5
Statistical considerations for mapping complex dis-

126355th-Dec. 13

orders (Partha P. Majumder)
Yeast RNA polymerase (Masayasu Nomura)Mishima Geneticists' ClubTransposition and its control mechanism of P elements
376th-Jan. 17
in Drosophila melanogaster (Eiji Nitasaka)
Mariner: a transposon found in Drosophila species (Aki377th-Jan. 28
hiko Koga)
378th-Feb. 4
Transcriptional activator of plant genes (Ken-ichi Yamazaki)
379th-Feb. 5
Transcription regulation of Moloney Leukemia Virus
during differentiation of mouse embryonal carcinoma
cells (Toshio Tsukiyama)
Analysis of embryogenesis using cell culture and gene380th-Feb. 15transfer of early embryonic cells (Norio Nakatsuji)
381st-Mar. 26
Coupled pattern generators in lateral inhibition models(Masayasu Mimura)
382nd-Mar. 29 Life cycle of HIV-1 and the mechanisms of posttranslational regulation of processing of the virus proteins (Tominori Kimura)
383rd-June 20
Transcriptional regulation through TATA box binding
protein, TFIID (Masami Horikoshi)
384th-June 27
Morphogenesis and segment polarity genes of Drosophila (Yoshiro Nakano)385th-July 17
Control of yeast cell proliferation via a RAS-cAMP
system (Suguru Morishita)
Regulation of the mouse P-cadherin gene transcription
386th-Ju1y 18(Masayuki Hatta)
387th-Aug. 6
Regulation of mouse fetomodulin gene expression (Yasuaki Shirayoshi)
388th-Aug. 7
Comparison of the structure of organella genomes(Haruo Ozeki)
Cloning of the mouse Ly-5 (CD45) gene and cell type389th-Aug. 14
specific expression of its isoforms (Yumiko Saga)
390th-Aug. 14
Genomic G+C-content and genetic code variation ineubacteria (Yoshiki Andachi)
391st-Sept. 12
1. Structure and function of molluscs neuropeptides

ABSTRACTS OF DIARY FOR 1991

CARP and MIP
X. laevis AS protein and the growth of optic nerve
fibers (Tatsumi Hirata)
ATP hydrolysis by a3[33 Subcomplex of ATP synthase(thermophilic Bacterium) (Yuji Ito)
Developmental neurobiology in Caenorhabditis e1egans-a new signal transduction pathway (Ichiro Maruyama)
Biomolecular Machines-Loose coupling story (Fumio
Osawa)
2.

392nd-Dct. 3
393rd-Nov. 1

394th-Nov. 28

127

128

FOREIGN VISITORS IN 1991

Oct. 4, 1990Aug. 3, 1991
Jan. 9, 1991Jan. 28
Feb. 1Feb. 18-Mar. 10
Mar. I-May 31
Mar. 10-31
Mar. 18-20
Mar. 19
Mar. 22Mar. 28Mar. 29
Apr. 2
Apr. 2
Apr. 2
Apr. 2

Apr. 5
Apr. 17
Apr. 17

Jotun J. Hein, University of Montreal, Canada
Christopher J. Basten, North Carolina State University,
U.S.A.
Rudolf Balling, Max-Planck Inst. of Biophys. Chern.,GermanyDavid Sankoff, University of Montreal, Canada
Baik L. Seong, Oxford University, U.K.
Ashok Kumor, University of Edinburgh, Scotland,
U.K.
Woojin Jeong, Korean Advanced Institute for Scienceand Technology, Korea
Philip W. Hedrick, Pennsylvania State University,
U.S.A.
Douglas E. Berg, Washington University, U.S.A.
Volker Schmid, University of Basel, Switzerland
Engelbert Hobmayer, University of Munchen, Germany
Tominori Kimura, MRC Laboratory of Molecular Biology, U.K.
Xu Xin-Lai, Biologivsl Development Center, China
Xing Rui-Chang, Inspective Institute of Medicines &Chemical Reagents & Biological Products, China
Xu Zhen-Lan, Jing Feng Institute of Medicine & Laboratory Animal, China
Feng Zhao-Liang, International Cooperative Department of National Science & Technology Commission,
China
David Benton, NC.H.G.R., National Institutes of
Health, U.S.A.
Ning Lei, Lanzhou Institute of Biological Product, Ministry of Public Health, China
Song Kai Zhong, Gan Su Sheng Science and Technology Committee, China

FOREIGN VISITORS IN 1991

Apr. 17
Apr. 17
Apr. 17
Apr. 19
Apr. 27-May 25
May 17
Jun. 15-Sept. 14
Aug. 23-26
Aug. 26-28
Sept. 1Sept. 6-7
Sept. 27
Sept. 27
Oct. 11
Oct. 13-Nov. 16
Oct. 31
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 14
Nov. 17-19

129

Sun Yi Fang, Lanzhou Institute of Medicine, China
Wang Zhong Yu, Gan Su Sheng Institute for New
Medicine and Pharmacy, China
Zhao He, Lanzhou Institute of Biological Product, Ministry of Public Health, China
Larry Weber, National Science Foundation, U.S.A.
William B. Provine, Cornell University, U.S.A.
Mary F. Lyon, MRC Radiobiology Unit, U.K.
Karim Sharif, The City University of New York, U.S.A.
G. Second, International Rice Research Institute, Philippines
Wang Bin, Institute of Genetics, Academia Sinica,
China
Aseem Ansari, Northwestern University, U.S.A.
Gunther S. Stent, University of California, U.S.A.
Flemming G. Hansen, The Technical University of Denmark, Denmark
Tove Atlung, The Technical University of Denmark,Denmark
Masayori Inouye, University of Medicine and Dentistryof New Jersey, U.S.A.
James F. Crow, University of Wisconsin, U.S.A.
Michael Cinkosky, Los Alamos National Laboratories,
U.S.A.
Wesley Brown, University of Michigan, U.S.A.
Thomas Whittam, Pennsylvania State University,
U.S.A.
Bruce Walsh, University of Arizona, U.S.A.
Austin Hughes, Pennsylvania State University, U.S.A.
Charles Aquadro, Cornell University, U.S.A.
Chung-I. Wu, University of Rochester, U.S.A.
Colm OhUigin, Max-Planck Institut fUr Biologie Abteilung Immungenetik, Germany
Richard Hudson, University of California, U.S.A.
Andrew Clark, Pennsylvania State University, U.S.A.
Brain Golding, York University, Canada
Walter Bodmer, Imperial Cancer Research Laborato-

130

Nov. 17-19
Nov. 19-Dec. 6
Nov. 21-22
Dec. 5
Dec. 12~13
Dec. 26

ries, U.K.
Colm OhUigin, Max-Planck Institut flir Biologie Abteilung Immungenetik, Germany
Masatoshi Nei, Pennsylvania State University, U.S.A.
James C. Wang, Harvard University, U.S.A.
Partha P. Majumder, Indian Statistical Institute, India
Masayasu Nomura, University of California at Irvine,
U.S.A.
Kiyoshi Mizuuchi, NIDDK, National Institutes of
Health, U.S.A.

131

AUTHOR INDEXAlBA, H . • . . . . . . . . . . . . . . . . . . . . • . . . . . .43

30
AKIYAMA, K.
• . . . . . . • . . . . . . • . . . . . . .44
AKUZAWA, K.
. • . . . . . . • . . . . . • . . . . . . • 58
ANDO, A. • . . . . . . • . . . . . . . . • . . . . . • . 98, 99
AYUSAWA, D
24,25,26,72
BARBIER, P.
.
23
CHEN, W.-B.
108
CHIFU, Y.
.
93
Cws, J
34
EIGUCHI, M.
. . . . . . . . . . . . . . . . . • 104, 105
ENDO, H.
. . . . . . . . . . . . . . . . . . • • . . . . . . 96
FISCHER-LINDAHL, K. • . . . . . . . . . . . . . • .48
FUSISA WA, T.
56
FUJITA, N. ..
14, 16, 17, 18
FUSE, N33
GOJOBORI, T. ..83, 84, 85, 86, 87, 111, 113
GOTO, Y.-I.
95
GOTOH, H . • . . . . . . • . . . . . . • . • . . . . • . • . . 51
GUTKNECHT, J. .
78
HAMAMATSU, C.
23
HANAI, N
22
HANAMIZU, H. . • . . . . . • . . . . . . . . . . . . . . 96
HANDA, H . . . . . . . . . . . . • . . . . . • . . . . . . . • 31
HARA, H.
. . • • . . . . . . • . . . . . . . . • . . . . . .43
HASEGA WA, M. . . . • . . . . . . . . . . . . . . . • • . 94
HASEGAWA, T
91
HASEGAWA, Y.
..
111
AIZA WA, S.

.

HATADA, E . . . . . . . • . . . . . . . . . • . . . . . . • 111
HATTORI, Y.

..

HA YASAKA, K.

111• . . . . . • . . . . . . . . • • . . . . . 96

97
33

HAYASHI, S.

.
.
HA YW ARD, R. S.

.

HIGASHITANI, A.

. . . . . . • . . . 38,39,41,43

HIGASHITANI, N

38

HA YASH I, S.

HIRAI, H.

• . . . . . . • . . . . . . • . • • . . . . . . • . 69

HIRANO, H. Y.
HIROSE, S

17.

34, 35, 36, 89, 104, 105
31,32,33

HORAI, S.

94, 95, 96, 97
111
HORIE, N
25
HORIUCHI, K . . . . • • . . . . . 38,39,41,43,44
IGARASHI, K. .
14, 16IIZUKA, M. . . . . . . • • . . . . . . . . . . . . . . . . . . 79
IKEMURA, T.
.
98, 99, 113
IKEo, K. .
83, 84
IMAI, H. T.
69, 70
IMAI, N28
IMAMURA, T.
.
91,92,93
IMANISHI, T. .
83
INA, Y.
83, 84, 87
INABA, R
91, 92INOKO, H. .
83, 98, 99
ISHIBASHI, F.
'"
113
ISHIDA, M. . . . . . . . . . . • . . . . • . . • . . . . . . . 85
ISHIDA, T. .
96
ISHIHAMA, A . • . . . 14, 16, 17, 18, 19,20,21,
22, 23
ISHIHARA, N. .
98, 99ITAMURA S
85,86
IWASE,M,. . . . . . . . . . . . . . • . • . . . . . . . 111
KAMATA, K . . • • . . . . . . . . . . . . . . . . . . . . . 30
KANEDA, S. .
24, 25, 26, 27, 28
KANEGAE, Y.
85, 86
KATSURA, I.
67, 68KAW AGISHI, M.
. . . . . . . . . . . . . . . . . . . • 20
KAWAKAMI, M.
. . . . . . • • • . . . . . . • . • . . 67
KAWAMOTO, T.
.
111
KAWAMURA, S
88
KAWASHIMA, T.
'".45
KIMURA, M . • • • . . . . . . . . . . . . . . . . . . • . . 81
KIT AKAMI, H. . . . . . • . . • . . . . . . . . . . . . . 111
KLEIN, J.
.
78
KOBAYASHI, M.
• • . . . . . . . • . . . . . . . . . . 21
KOHARA, Y.
.44, 64, 66
KOIDE, T.
.
47
KOIKE, H . . . . • . . . . . . . . . . . . . . . . . . . . . . 97
HORIE, M.

.

.

132
KOMEDA, Y.

.

36
97
KORABLEV, V. P. .
.45
KRYUKOV, A. P.
.
.45
KUBORI, T.
28, 30KUMAR, A.
.
17
KUROSAKI, K.
. . . . . • • . . . . . . • . . • . . . . . 88
KUSASO, A.
.
22
LAVORGNA, G
34
MARUYAMA, K.
. ....•....•.••....•• 79
MATSUMOTO, K. I.
98, 99
MATSUSHIMA, H. .
19
MATSUSHIMA, Y.
III
MITSUKI, H. .
64
MIURA, S . . . . . . • • . . . . . . • . . . . . . • • . . . . 87
MIYABA YASHI, S. . • . . . . . • . . . . . . . . • . . . 96
MIYABAYASHI, T.
.
101
MIYASHITA, N . . . . . • • . . . . . . . • . . . . . . . • 52
MIZUNO, T.
.
43
MIZUTANI, M.
. • . . . . . . • . . . . . . . • . . . . • 31
MOCHIZUKI, K. • . . . . . . . . . . . . . . . . . . . • . 89
MORISHIMA, H. .
101, 103, 107
MORIWAKI, K.
. .45,46,47,48,49, 51, 52
MORIYAMA, E. N.
. ••.....•.•....•.. 84
MURAKAMI, A
56,58,59,61
MURAKAMI, Y
26
MURAYAMA, K.
. .•.....•.•......•.. 97
NAGAI, Y.
.,
27,28
NAGATA, K.
. . . . . . . . . . . . . . • . • . . . 21,22
NAKAI, N . . . . . . . • • • . . . . • • . . . . . . . • . . . 97
NAKAMURA, I.
23, 108, 109
NAKAMURA, M.
. •...•..... , ....•... 87
NAKAOKA, M . . . . . • . . . . . • . • . . . . . • . . . 111
NAKASHIMA, H.
.
91,92,93
NAKATSUJI, N .•.....••...•••. .49, 63, 64
NAKAYAMA, M . . . . . . . • . . . . • . . • • . . 21,22
NAMBA, K.
. ....••.•.....•.....•.•. 30
NEROME, K.
. .•.•......•.....•.. 85, 86
NIKI, M . . . • . . . . . . • • . . . • . • . . . . . • . • . . . 87
NISHIGAKI, A.
.
64
NISHIMIYA-FuJiSAWA, C.
.
55
NISHIMURA, A
44
NISHIMURA, Y.
.41, 43
KONDO, R

NOGUCHI, Y.

.

III

NONAKA, I.

95

NUMATA, N . . . . . . • . • . . . • . • . . . . • . . . . . 87
. .......•.....••.....••. 86

OGUCHI, K.

OGURA, K . . . . . • • . • . . . . . • . . . . • . . . . . . . 70
OHBO, K.

. . . . . . . . • . • . . . . . • . . . . . . • . . 87

OHSHIKI, T.

61
31
OHTA, T.
73
OHTANI, K.
. . • . . . . . • . . . . . . • . . . . . . • . 87
OHTSUBO, E. .
89
OHTSUBO, H. .
89
OVA, A.
.
85,86
OZAKI, M . . . . . . • . . . . . • . . . . . . • • . . . . • • 18
SAGAI, T. .
.46, 48, 49
OHTA, T.

.
.

SAITO, M. . . • • . . . . • . . . . . . . . . . . . • • . . . III
SAITOU, N.

• •.....•...... 83, 87, 88,

SAKAI, M.

III. . . . . • . . . . . . . . . . . . . . . . 19

SAKAGAMI, M.

. . . . . . . . . . . • . . . . . • . . . . 91,92

SAKAKIBARA, Y.

.
41.... 34, 35, 36, 89, 102, 104, 105
SARUYAMA, H. .
35
SATO. T.
.
107
SATO, Y.
111
SATO, Y.-I.
105, 107, 108
SATTA, Y.
.
69, 78SAWAMURA, K . . • . . . . . . . . . . . . . . . . . . . . 90
SCHON BACH, C. .
78
SENO, T.
24,25,26,27,28,72
SANO, Y.

SHIDAHARA, Y.
SHIMADA, J.

.

III

.

61

SHIMAMOTO, N.

. ....•......•. 28,29,30

SHIMOYAMA, M.

. ..•......•.....••.. III

SHIMAMOTO, Y.

.

107. ••.....•......•.......• 84

SHIMIZU, N.

SHIRAYOSHI, Y.

.

63,64
.46,47,48,49, 51, 69
SHIRRAS, A.
.
33
SUGAMURA, K. . . . • . . . . . . . . • . . . . . • • . . 87
SUGITA, S. . . . . . . . . . • . . . . . . . • . . . . . . . . 86
SUGIYAMA, T.
.
55
SUZUKI, H. .
.45
SHIROISHI, T

SUZUKI, R.

.

SUZUKI, S.

. ..•.....•.•.....•.....• III

III

T ABARA, H.

.

66

AUTHOR INDEX
T ACHIDA, H.

.

79
96
TAJIMA. F. .
76, 80
TAKAHASHI, K. .
83, 84
TAKAHASHI, M. .
23
TAKAHATA, N.
.
74. 75, 76, 77. 78
TAKAYANAGI, A.
.
24.25
T AKEISHI, K. .
25
TANABE, H.
.
88
TANAKA, Y.
. . . . . . . . . . . . • . . . . . . • • . . • 87
TANG. L.
105
TANG, X
105
T ANI FUJI. M
84
TATENO. Y.
. • . . . . . . • . . . . • . . . . . • 83,111
TAYLOR, R. W . . . . . . . . . . . . . . . . . • . . . . . 69
TERADA. H.
.
28

TADA, K.

.

TEZUKA. H.
TORIY AMA, S.

.

72

23
87
TSUCHIYA, K.
.
45
TSUKIHARA, T. . . . . . . . • • . . . . . . . • • . . . . 30
TUCHIY A, K.
21
UCHIDA, R. .
111
UEDA, H. .
32, 34
UEDA, S
88
UEDA, Y . . • . . . . • • . . . . . . . . . . . . • • . . . . 111
UGAWA. Y • • . . . . . . . . . • . . . . . • . • • . . . . 111
UMEDA, M.
.
89
URA, K.
.
31.32
TOZAWA. H.

133

WADA, A

19
113
WADA, Y . . . . . . . . • • . . . . . . • . . . . . . . . • . 113
W AKANA. S.
.
49
WATANABE, A
111
WATANABE, H
31
WATANABE. S
52
WATANABE, Y . . • • . . . . . . • . . . . . . . . . . . . 88
Wu, C.
.
34
YAKIMENKO, L. V.
. . . . . . . . • . . . . . . . . .45
YAMADA, M. .
69
Y AMAGISHI, H.
.
107
YAMAGISHI. M.
.
14, 19,20
YAMAGUCHI, S
30
YAMAMOTO, M. T . . • . . . . . . . . . . . . • . 70. 90
YAMAO, F
27,28
YANG, L.
105
YAMAZAKI, Y . . . . . . . . . . . . . . . . • . . . . . . 111
YASUDA, H.
.
26
YASUDA. J
21
YASUDA, S.
.
.41
Y AZAKI, K.
22
YONEKAWA, H.
.
.45, 52
YONEZAW A, K.
.
108
YOSHIDA, S.
.
27
YOSHINO, M.48
YOSHIOKA, Y . . • . . . . . . . . . . . . . . . • • . 85.86
Zou, C.
.
14, 16
WADA, K.

.

m

IEft5l~~i1f~m~m
42 ~3jlRJ(; 4 ~ 10.R 26 B I'IJ,Ij!U
3jlRJ(; 4 ~ 10 .R 30 B :3£fi:3£filt

'i'

?R

~

1Ej'[~~$iIf~FJfP3

lI~lt

li~tm~· tlJ~)OJj

lEj'[jI~$iIf~FJfP3m

I'IJ,Ij!Ult '1Iit5.&
310X:M! If mg j';1Ij EB .~ ~ 3-8-8
I'IJ,Ij!UFJf :~ lE I~t>t mx i=P l1JiH±
JRJii::M!lfmg i\1Ii EB .~~ 3-8-8:3£ fi FJf
31. i:I ffi ~ iiJf ~ PJT

m

T411

m.Jli1JJli'<=~m~EB 1111

'it illi ~* (0559) (75) 077 1

to Hakonef
1:25,000