NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT1999

The cover photograph shows the expression of a T-box genebrachyury in early Xenopus laevis embryos. The radial
expression is restricted to the equitorial region of embryo
which represents presumptive mesoderm.

Post Doctral Fellow
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NIBB Research Fellow
JSPS Postdoctral Fellow
JSPS Research Associate
JST Fellow

CONTENTS
INTRODUCTION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ORGANIZATION OF THE INSTITUTE · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·2

GRADUATE PROGRAMS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·3

OFFICE OF DIRECTOR · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·4Phylogeny of outer arm dynein in flagella and cilia · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·4

Molecular evolution of photosynthesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·5Mechanisms determining the outline shape of the adult lepidopteran wings · · · · · · · • · · • · · · · · · · • · ·6

Protein palmitoylation and developmental mechanism
at embryogenesis in invertebrate and vertebrate · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·7

DEPARTMENT OF CELL BIOROGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

DIVISION OF CELL MECHANISMS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·10

I. Regulation at the level of protein transport to microbodies during the
micro~ody transition. · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · • · · ·10

II. Microbody defective mutant of Arabidopsis. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·10Ill. Transport of storage proteins to protein storage vacuoles is mediated by large PAC(precursor-accumulating) vesicles. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · •12

IV. Vacuolar processing enzymes in protein-storage vacuoles and· · •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·lytic vacuoles.

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V. Role of molecular chaperones in organelle differentiation.

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DIVISION OF BIOENERGETICS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·14

I. Background • • · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·14

II. ApglO is a unique protein conjugating enzyme · • · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · ·14Ill. Apg16 cross1inks multiple Apg12-Apg5 conjugates · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · ·15

IV. Apg8 as a tracer of membrane flow in autophagosome formation · · · · · · · · · · · · · · · · · · · · · · · · · ·15

V. SKD 1 regulates morphology of endosomes and membrane traffic
through them in mammalian cells · · · · · · · • · · · • · · · · · · • · · · · · · · • • · · · · · · · • · · · · · • · · · · · · ·15

DMSION OF CELL PROLIFERATION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·17

I. Comprehensive identification and developmental tracing of brain cells· · · · · · · · · · · · · · · · · · · · · · ·17

II. Tools for comprehensive cell lineage analysis of the brain · · · · · · · · · · · · · • · · · · · · · · • · · · · · · · · ·17Ill. Analysis of genes for path fmding processes in the adult brain development · · · · · · · • · · · · • · · · ·18

IV. Contribution to the science community · · · · · · · · · • · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·18

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT) • · · • · · • · · • · · · · · · · · · · · • · • · · • · •20

I. Function of multiple axonemal dyneins · · • · · • • · · • · · · · · · · · · · · · · · • · · • · · • · · • · · · · · · · • · · · · ·20

II. Function of actin and an actin-related protein in Chlamydomonas • • • · • · · • · • • • · · • · • • • • · · • • • •20

DIVISION OF CELL FUSION (ADJUNCT) • · · · · • · · · · · · · · · • • · · • • • · • · · · · · · · · · · · · · · · • • · · · •22

DEPARTMENT OF DEVELOPMENTAL BIOLOGY

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I. Endocrine regulation of gonadal sex differentiation and sex change · · · · · · · · · · · · · · · o • • • • • • • • • •26

II. Endocrine regulation of spermatogenesis ·•26

III. Endocrine regulation of oocyte maturation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·27

DIVISION OF CELL DIFFERENTIATION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·29

I. Gene regulatory cascade in the steroidogenic tissue differentiation o • o • • • • • • • • • • • • • • • • • • • • • • •29

II. Sex-differentiation observed in adrenal cortex · · · · · · · · · · · · · · · · · · · · · • · · • · · · · · · · · · · · · • · · ·29

DIVISION OF MORPHOGENESIS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·31

DIVISION OF REPRODUCTIVE BIOLOGY · · · · ·

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I. Hierarchy of homeobox genes in pattening embryo • • · • · · · · · · · · · · · · · · • • · • ·

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• •o o o • o o o •II. Cross-talk of growth factor signaling in early development· • • · · • · · · · · · · · · · • · · ·

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III. Genetic dissection ofTGF-~ signaling in Drosophila model system · · · · · · · · • · · · · · ·

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IV. TGF-~ family in nematode·························

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DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·34

I. Molecular and genetic approaches for the analysis of the sugar-signalling during growthand development of Arabidopsis thaliana • o o • • • • • • • • • • • • • • o • • • • • • • • • o o • • • • • • • • • • o • • • • o

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II. Regulatory factors involved in the sugar-inducible expression of plant genes··················

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DEPARTMENT OF REGULATION BIOLOGY ·

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DIVISION OF MOLECULAR NEUROBIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·38

I. Molecular mechanism ofretinotectal map formation · o o • o o o • • • o • • • • • • • • • • • • • • • • • • • • • • • • • •38

II. Functional roles of protein tyrosine phosphatase~ ·•38

ill. Functional roles of subfamily 2 sodium channels · o o • • • • • • o o o • • • • • • o o o o • • • • • o o • • o o o • • • o • •39

DIVISION OF CELLULAR REGULATION · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·42

I. The perception and transduction oflow-temperature signals in Synechocystis · · o • • • • • • • • • • • • • • •42

II. Genetic modification of plants to enhance stress tolerance:
introduction of the capacity for the synthesis of glycinebetaine into Arabidopsis · · • · · • • • · ·•42

III. Mechanisms of inactivation of the photosynthetic machinery by salt stress in cyanobacteria······

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IV. Acclimation of the photosynthetic machinery to high temperature · · · · · ·44o o • o •• ••• • o •

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DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT) · · · · · · · · · · · · ·45

I. Cloning and characterization of blue-light photoreceptors · · · · · · · · · · · · · · · · • • · · · · • · · · · · • · · · ·45

II. Chloroplast relocation · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · • • · · · · · · · · · • · · · · ·45

DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)·······························

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I. Mechanism of axonal guidance·····················································

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II. Mechanism of neuronal migration · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · • • · · · · · · ·47

LABORATORY OF GENE EXPRESSION AND REGULATION · · · · · · · · · · · · · · · · · · · · ·49

DIVISION OF GENE EXPRESSION AND REGULATION I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·50

I. Identification and characterization of mutable alleles in the Japanese morning glory·············

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II. Identification of mutable alleles in the common morning glory · · · · · · · · · · · · · · · · · · · · · • · · · · · ·50III. Characterization of the genes for anthocyanin pigmentation in morning glories · · · · · · · · • · • • · · ·50

IV. An efficient transformation system and homologous recombination in rice· · · • · · · · · · · · · · · • · · ·51

DIVISION OF GENE EXPRESSION AND REGULATION II·······························

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I. Replication fork block protein (Fobl) acts as a rDNA specific recombinator · · · · · · · · · · · · · · · · · ·52

II. E. coli genomic structure and function · · · · · · · · · · · · · · · · · · · • · · · · · • · · · · · · · · · · · · · · · · · · · · •53

DIVISION OF SPECIATION MECHANISMS I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·54

I. Gene expression and cerebellar long-term plasticity · · · · · · · · · · · · · · • · · · · · · · · · · · · • · · · · · · · · ·54

II. Gene expression under audio-visual discrimination task · · · · · · · · · · • · · · • · · · · · · · · · · · · · · · · · ·54

III. Color-defective (dichromat) monkeys···············································

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IV. Brain Specific Repetitive (Bsr) RNA · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · • ·54

V. Genes expressed in specific areas of the neocortex · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · ·55

DIVISION OF SPECIATION MECHANISMS II · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·57

I. Evolution of reproductive organs in land plants·········································

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II. New hypothesis on the homology of Gnetum reproductive organ· · · • · · · · · · · · • • · · · · · · · · · · · · ·57

III. Evolution of gametophytic and sporophytic generations · · · · · · · · · · • • · · · · · · · · · · · · · · · · · · · · •58

IV. Establishment of tagged mutant library of the moss Physcomitrella patens···················

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CENTER FOR BIO-ENVIRONMENTAL SCIENCE·······························

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RESEARCH SUPPORT· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·67

CENTER FOR TRANSGENIC ANIMALS AND PLANTS· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·68

I. Research supporting activity · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • • · · · · · · · · · · · · ·68

II. Academic activity · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · ·68

TECHNOLOGY DEPARTMENT· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·69

RESEARCH SUPPORT FACILITY · · · · · · · · · • · · · · · · · · • · · • · · · · • · • · · · · · · · · · · · · · · · · · · • · · · ·70

I. Facilities· · • · · · • · · · · · • · · • · · · · · · · · • · · · · · • · · · · · · • · · · · · · · • · • · · · · · · · · · · · · · · · · · · · · · · ·70

II. Research activities · · · · · · · · · · · ; • · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · • · · · · • · • ·71

RADIOISOTOPE FACILITY (managed by NIBB) • · · • · • · · · · · · • • • • · • • · · · · · · · · · · · · · · · · · • · · ·73

I. Research supporting activity · • • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · • • · · · ·73

II. Ac~demic activity · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · • · · · · · ·73

THE CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB) · · · · · · · · · · · · · · · • · · · ·75

INTRODUCTION
The National Institute for Basic Biology (NIBB), a
government-supported, basic research institute, wasestablished in 1977. As a center of excellence, NIBB
aims to promote the biological sciences by conducting
first-rate research on site as well as by cooperating withother universities and research organizations. Researchers at NIBB investigate cell structure and function, reproduction and development, neuronal and environmental biology, gene expression and regulation, andmolecular evolution of eukaryotic organisms.
In 1999, the Center for Bio-environmental Science
was established to investigate pollution and other adverse effects of the activities of human beings on theearth, including the so-called "environmental hormones" (endocrine disruptors). The Center, managed
by NIBB, is operated jointly with the Institute for Molecular Science (lMS) and the National Institute forPhysiological Sciences (NIPS). The Center will belocated in the newly constructed center for biosciencesin 2000. The Director-General of the NIBB currently
acts as the head of the Center. In August, Professors
Teizo Kitagawa (lMS) and Minoru Kanehisa (Kyoto
University) were appointed Professor and Adjunct Professor of the Center, respectively. Drs. A. Hazama(NIPS) and H. Tsukaya (University of Tokyo) were
promoted to Associate Professor of the Center inAugust and October, respectively. Associate Professor
S. Saito (Nagoya University) was appointed Adjunct
Associate Professor in November. Professor Taisei
Iguchi (Yokohama Municipal University) is scheduled
to join this Center soon.
In addition to establishing the Center for Bioenvironmental Science, we have replaced 1 research
associate, 5 institute research fellows and 1 technician
with 7 research associates, 6 institute research fellows
and 1 technician. In the past several years, the total
number of personnel working at the NIBB, especially
graduate students and post doctoral fellows, has increased from about 200 to 300. We would also like tocongratulate Associate Professor lkuko Nishimura of
the Division of Cell Mechanism, Department of Cell
Biology, on her promotion to Professor at Kyoto University in November.
As a national and international center for biologicalresearch, NIBB is responsible for conducting research
projects in cooperation with various research groups.
As a part of such cooperative activities, NIBB held the42"d NIBB International Conference entitled "Tubulin·
30 Years Later" (Professors Y. Nagahama, R. Kamiya, I:
Mabuchi and H. Hotani, organizers) in March to celebrate the thirtieth anniversary of the naming of tubulin

by H. Mohri. In accordance with the "Memorandumof Understanding between the NIBB and the BiologicalResearch Center of the Hungarian Academy of Sciences", a Japanese-Hungarian Bi-national Symposium
entitled "Developmental and Environmental Control of
Cell Differentiation" (Professors Naoto Ueno andDenes Dudits, organizers) was held in Szeged, Hungaryin October with the support of the Japan Society for
Promotion of Science.
In addition, NIBB continues to sponsor interdisciplinary symposia and study meetings on current topics byinviting leading scientists from around the world to theInstitute. NIBB also provides a training course inbiological sciences for young investigators. To continue our improvement, the activities and future plan of
NIBB, especially in the field of animal science, were
subjected to an external peer review. We welcome anysuggestions concerning the research activities ofNIBB.
Finally, I would like to congratulate Professor Norio
Murata for being selected as a Foreign Member of the
Hungarian Academy of Sciences.
Hideo Mohri, D.Sc.
Director-General

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NATIONAL INSTITUTE

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology, NIBB, is apart of the Okazaki National Research Institutes(ONRI) located on a hill overlooking the old town ofOkazaki. The NIBB was established in 1977 and its
activities are supported by Monbu-sho (the Ministry of
Education, Science, Sports and Culture) of Japan. The
ONRI are composed of three independent organizations,
National Institute for Basic Biology (NIBB), National
Institute for Physiological Sciences (NIPS) and Institutefor Molecular Science (IMS).

Policy and Decision Making
The Director-General oversees the operation of theInstitute assisted by two advisory bodies, the Advisory
Council and Steering Council. The Advisory Council ismade up of distinguished scholars representing various
fields of science and culture, and advises the DirectorGeneral on the basic policy of the Institute. The
Steering Council is made up of professors of the Institute and an equal number of leading biologists in Japanoutside NIBB, and advises the Director-General on thescientific activities of the Institute. The Council advises on faculty appointments and on the Institute's
annual budget as well as its future prospect.

Research Support Facility
The research support facility of the NIBB consists of
the Large Spectrograph Laboratory, Tissue and Cell
Culture Laboratory, Computer Laboratory, Plant Culture Laboratory, Plant Cell Culture Laboratory, Experimantal Farm, Laboratory of Stress-Resistant Plants andCenter for Transgenic Animals and Plants. In addition,
seven facilities are operated jointly with the NIPS; they
consist of the Radioisotope Facility, Electron Microscope Center, Center for Analytical Instruments, Machine Shop, Laboratory Glassware Facility, Animal
Care Facility, and Low-Temperature Facility.

Administration
Administration of the Institute is undertaken by the
Administration Bureau of the Okazaki National Research Institutes under the direct auspices of the Ministry of Education, Science, Sports and Culture.

CampusThe Okazaki National Research Institutes covers an
area of 150,000m2 with four principal buildings. The
NIBB' s main research building has a floor space of
10,930m2 • Two-thirds of the space was completed in1982 and the remaining third in June, 1983. The
buildings which house the research support facility
were also completed in June, 1983. A building forLaboratory of Gene Expression and Regulation(2,577m2) was newly built in December, 1996.

Research
The Institute conducts its research programs throughthree departments and one laboratory organized into 17
divisions.
Each division has its own research project and isstaffed by a professor, an associate professor and two
research associates. A division forms, in principle, an

Department/Laboratry

Department of Cell Biology

Department of Developmental Biology

Department of Regulation Biology

Laboratory of Gene Expression and Regulation
Interim Center for Bio-environmental Science

independent project team. Six of the divisions are for
adjunct professorship and are under professors who
hold joint appointments with other universities. The
adjunct division has resident research associates. The
arrangement aims to facilitate exchange in research
activities in Japan. The Technical Department manages the activities of research techniques and helps topromote research activities of each division and also tomaintain the research resources of the Institute. The
Department also undertakes the technical education ofits staff.
In 1999 the Center for Bio-environmental Sciencehas attached to NIBB.

Divisions-iCell Mechanisms
Bioenergetics
Cell Proliferation (adjunct)
Cell Fusion (adjunct)
Cellular Communication (adjunct)

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Reproductive Biology
Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)
Molecular Neurobiology
Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)
Gene Expression and Regulation I
Gene Expression and Regulation II
Speciation Mechanisms I
Speciation Mechanisms II

3

FOR BASIC BIOLOGYGRADUATE PROGRAMSThe NIBB carries out two graduate programs.
1. Graduate University tor Advanced Studies
The NffiB constitutes the Department of Molecular
Biomechanics in the School of Life Science of the
Graduate University for Advanced Studies. The University provides a three year Ph. D. course. Those
who have completed a master's course in a university or
are qualified at an equivalent or higher level are eligible
to apply.
The Department consists of the following Divisions
and Fields:

DivisionsFieldsMolecular Cell
Biology

Biomolecular Systems
Cell Dynamics

Developmental
Gene Expression
and Regulation

Gene Expression
Morphogenesis
Transgenic Biology

Regulation
Biology

Biological Regulation
Biological Information

2. Graduate Student Training Program
Graduate students enorolled in other universities andinstitutions are trained to conduct research for fixed
periods of time under the supervision of NffiB professors.

4

NATIONAL INSTITUTE

OFFICE OF DIRECTOR
HideoMohri
Director-General:
Associate Professors: Shigeru Itoh
Ryuji Kodama
Kohji Ueno
Research Associate: Kaoru Ohno
Hiraki Kokubo (on leave)

Phylogeny of onter arm dynein in flagella andcilia
Hideo Mohri
Dynein is a motor protein essential for microtubuledependent cell motility. In axonemes of flagella andcilia, outer arm and inner arm dyneins attach to outerdoublet microtubules. The isolated outer arm dynein
molecule is a three-headed bouquet in protists (e.g.
Chlamydomonas and Tetrahymena), whereas the molecule is two-headed in multicellular animals (e.g.
Deuterostomia and Protostomia). The number ofheads corresponds to the number of heavy chain subunits. Alpha, [3 andy heavy chains compose the outerarm in protists and a and [3 heavy chains are present in
the outer arm of multicellular animals. We examined
the outer arm dynein isolated from sperm flagella of asea anemone, a species located just prior to the divergence of the two main branches in the animal kingdom
phylogenie tree. The outer arm was the two-headed
molecule typical of multicellular animals. Fig.l shows
schematic drawings of both animal (metazoan) andprotist (and some lower plant) flagella and cilia withspecial reference to the outer arms. Since molecularphylogenetic analyses of dynein heavy chains indicatethat animal a and [3 heavy chains are related to protist
y and [3 heavy chains, respectively, topographic relationship among these heavy chains in the cross-sections
are also indicated in the figure. Based on the phylogenetic tree of dynein genes, Chlamydomonas a and [3appear to be produced by gene duplication. Combinedwith other phylogenetic analyses, it is plausible that
there was (or is) the organism more ancient than Chlamydomonas and Tetrahymena, which has two-headed
outer arm dynein and consequently, the third heavy
chain acquired in protists may have been lost during theevolution of animals from their ancestral unicellularorganisms.

Fig.l. Schematic cross-sections of flagella or cilia in protists and lower plants (left) and in animals (right). Positions of each heavy chain subunit (head) are also shown.

FOR BASIC BIOLOGY

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Molecular evolution of photosynthesis.
Shigeru ltoh
We study the molecular evolution of photosynthesis
through physicochemical research of various types ofphotosynthetic organisms. Oxygen-evolving photosynthesis of cyanobacteria seems to be evolved from theanoxygenic bacterial photosynthesis at 2.7-3.5 billion
years ago just after the evolution of life. Symbiosis of
cyanobateria inside the eukariotic cells, then, produced the first plant 2 billion years ago. The process ofevolution from anoxygenic to oxygenic photosynthesis
has been our main target of research.
(l)Discovery of anomalous photosynthesis.
Acaryochloris marina, a newly discovered oxygenevolving cyanobacteria-like unicellular organism, wasfound to use a far-red absorbing new pigment chlorophyll d in its photosystem I reaction center pigmentprotein complex in collaboration with Marine Biotechnology Institute Kamaishi (Fig.2). Although chlorophylla that absorbs visible red light has been known to beindispensable for the oxygenic photosynthesis of all theplants and cyanobacteria, A. marina efficiently undergoes oxygenic photosynthesis even with far-red light oflower quantum energy compared to the red light absorbed by chlorophyll a. We named the special pairchlorophyll d of the newly identified photosystem I
reaction center P740. The organism can be a missing
link between the anoxygenic photosynthesis that uses
800-860 nm far-red light and the oxygenic photosynthesis that uses 650-700 nm red light.

Photosynthesis in a newly discovered bacterium
Acidiphilium rubrum isolated from acidic mine drainage, was also shown to be quite different from the photosynthesis in all the ever-known oxygenic and anoxygenic photosynthesis. A. rubrum uses Zn-containing
bacteriochlorophyll in its anoxygenic photosynthesis.
This was the first case of photosynthesis based on pigments other than chlorophylls that are Mg-containing
pigments. These new organisms show the wide variability of photosynthesis.
(2) Evolution of optimization mechanism of photosynthesis.
Structure-function relationships of the photosynthetic
light reaction in the newly found organisms as well as
plants and in anaerobic green sulfur bacteria Chlorobium and Heliobacteria, are studied by modem physicochemical techniques. We modified the reaction center
complexes of these organisms by replacing quinone(vitamin Kl) or chlorophyll cofactors by the artificial
compounds or by site-directed mutagenesis, and studied
the ultra-fast reaction kinetics by picosecond laser fluorometry, spectroscopy and spin-echo pulse ESR spectroscopy at 4 -280 K. Molecular architectures of plantand bacterial photosynthetic reaction center proteins areshown to be highly optimized in utilization of solar
energy, however, in somewhat different direction ineach case. The design of ancestral photosynthetic
apparatus that is now lost, is to be traced based on new
findings.

Photon energy (eV)
2.5

..=
II)

C!l
,Q

r...
0

"'

,Q

<
0
0
,;,
,Q

<t:

R,

<l

Chlorophyll dCHOr ·h/orophy/1 u

CII C'I Iz

Wavelength (nm)Fi 2Absorption spectrum (A) and light-induced difference absorption spectrum of reaction center chlorophyll P740(B) of newly identified

h~t~s stem 1 (PS I) reaction center complex of Acmyoch/oris marina that has far-red absorbing chlorophyll d. Broken hnes m (A) and (B)~epres:nt absorption and difference spectra (P700) in plant (spinach) PS I reaction center contammg ch lorophyll a, respectJvely. Molecular
structures of chlorophylls a and dare also shown.

6

Mechanisms determining the outline shape of theadult lepidopteran wings
Ryuji Kodama
Wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which is ahollow sac made of simple epithelium. When the
pupariation is completed, the wing, which was hidden
inside the body wall of the larvae, is exposed on thesurface of the pupa, which gradually turns into the adultwing. The outline shape of the adult wing is often
different from that of the pupal wing. This differenceis brought about by the programmed cell death of themarginal area of the pupal wing, while the internal area
develops as adult wing blade. The marginal dying
area is called the degeneration region and the internalarea is called the differentiation region, hereafter.
The cell deaths in the degeneration region proceeds
very rapidly and completes in a half to one day period
in Pieris rapae or several other species examined. It
was shown that the dying cells in the regeneration region have two characteristics common with the apoptotic cell death in mammalian cells. These are i) the
presence of apoptotic bodies, which are heavily condensed cells or their fragments engulfed by other cells
or macrophages, shown by transmission electron microscopy and ii) the presence of conspicuous accumulationof fragmented DNA evidenced by the TUNEL histological staining (Kodama, R. et al., Roux's Arch. Dev.
Biol. 204, 418-426, 1995).
The cells in the degeneration region are actively engulfed by the macrophages in the cavity beneath thewing epithelium. Moreover, the macrophages seem tobe concentrated beneath the degeneration region by thestrong adhesion between basal surfaces of the dorsal
and ventral epithelium in the differentiation region.
By injecting the india ink or ferritin solution to the body
cavity of the pupa, we have confirmed that this adhesion is tight enough to exclude the macrophages fromthe differentiation region, because the injected probes
was found mostly concentrated in the degeneration
region when observed several minutes later (Yoshida, A.
(Biohistory Research Hall) and Kodama, R., unpublished).
Studies using another lepidopteren species, Orgyia
recens approximans, provided by Drs. Y. Atita and K.
Yamada (Meijo University) is underway . In this species, the wing is normally formed until the beginning of
the pupal period, but becomes conspicuously degenerated only in the female adult. In our preliminary study,

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it was shown that the pupal wing is normally formed
both in male and female pupa, but after about two days,
female pupal wing starts degeneration on its margin, as
if the degeneration region is continuously formed deep
into the center of the wing (Kodama, R. et al., unpublished). It is thus suggested that the control mechanism which demarcates the region to be degenerated is
defective in the female in this species. Further investigation using this species might give important insight
into such mechanisms.
Another collaborative work with the laboratory of Dr.
K. Watanabe (Hiroshima University) concerns mostly
on the development of trachea and tracheole pattern inthe swallow tail butterflies. Trachea and trcheoles areboth important in delivering air into the wing and theirpattern coincide with that of the boundary of degeneration and differentiation zones at the distal end of thewing. According to the observations, the pattern formation of wing epithelium is often dependent on tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being done
through the scanning electron microscopy and thebright field light microscopy of the fixed or fresh specimens to describe the exact pathway and the time course of the formation of elaborate pattern of trachea andtracheoles and to establish the cytological and developmental relationship between the formation of tracheal
pattern and epithelial cell pattern, such as scale cellpattern. The Fig.3 depicts how the tracheoles protrude
from the primary trachea at the pre-pupa stage. Thesefine threads are arranged with even spaces and may
closely related with the scale cell pattern formation.

Fig.3 . The tracheoles (fme threads) and the primary trachea(thick tube in the center) at the late stage of the pre-pupa.

FOR BASIC BIOLOGY

Protein palmitoylation
anddevelopmental
mechanism at embryogenesis in invertebrate andvertebrate
Kohji Ueno
We have studied the molecular mechanisms of thedevelopment of cells and organs in the silkworm Bombyx mori and have elucidated that the abdominal leg
development was regulated by a homeotic gene which
specifies the identities of abdominal segments. We havefound that a high molecular weight protein (p260/270)
was expressed in abdominal leg cells during early embryonic stages. p260/270 was identified to be a protein
palmitoylase which transfers palmitate to cysteine residues of proteins. Almost of small GTP-binding, heterotrimeric G, and G-protein-linked receptor proteins are
known to be modified with palmitate through thioester
linkages. These dynamic modifications thought to beimportant in regulation of signal transduction. Therefore we speculated that p260/270 may be involved inregulation of signal transduction and may function in
abdominal leg development.
To better understand the molecular mechanism how
the modification of protein palmitoylation regulates thedevelopment of cells and organs, a search for a homolog of p260/270 in vertebrate was undertaken. Homology search of an ESTdb (Expressed Sequence Tags
data base) with the amino acid sequences of p260 and
p270 identified mouse embryonic eDNA clones whichwere highly homologous to the amino acid sequences ofp260 and p270. This result suggested that a homolog ofp260/270 was expressed in mouse embryos. In situ
hybridization of mouse embryos revealed that the transcripts are detected mainly in the central and peripheral
nervous system in mouse embryos from embryonic day
11.5. The transcripts were detected in the regions offorebrain, midbrain, hindbrain and spinal cord in thecentral nervous system and also detected in the cranial
ganglia and dorsal root ganglia in the peripheral nervous systems. Immunocytochemical analyses of cultured
mouse primary embryonic brain cells were performed

7
to identify which cells express mouse p260/270 homolog. This analysis revealed that mouse homolog was
expressed specifically in neural cells, but not in neuroepithelial and glial cells. Fig.4 showed a result of immunocytochemical analysis of primary cultured cells.
From these results, we speculate that a protein palmitoylase may function in the development of neural cells
in the central and peripheral nervous system during
mouse embryogenesis.
We found that protein palmitoylase were expressed
specifically in a few types of cells during early embryogenesis in invertebrate and vertebrate. Further study isnecessary to elucidate the common mechanism how themodification of protein palmitoylation regulates thedevelopment of the cells in invertebrate and vertebrate.

Fig.4. Immunocytochemical analysis of cultured primary cells.
Cells were double labeled with an antibody against mouse p260/270
homolog and anti-nestin antibody. Mouse homolog (green) was
detected in neurons whereas nestin (red) was detected in neuroepithelial cells. Bar represents 251J.m.

8Publication Lists:
Akiyama, M., Kobayashi, M., Kise, H., Takaichi, S.,
Watanabe, T., Shimada, K., Iwaki, M., Itoh, S.,
Ishida, N., Koizumi, M., Kano, H., Wakao, N. andHiraishi, A. (1999) Acidiphilium rubrum and Zincbacteriochlorophyll part I: Molecular structure of
the zinc-containing bacteriochlorophyll. In "Photosynthesis:Mechanism and Effects" (ed. Garab,G.), pp. 731-734. Kluwer.
Hu.Q., Ishikawa, T., Inoue, Y., Iwasaki, I., Miyashita,
H., Kurano, N., Miyachi, S., Iwaki, M. and Itoh, S.
(1999) Heterogeneity of chlorophyll d-binding
photosystem I reaction centers from the photosynthetic prokaryote Acaryochloris marina. In "Photosynthesis: Mechanism and Effects" (ed. Garab,G.), pp. 437-440. Kluwer.
Itoh, S., Iwaki,· M., Hu, Q., Miyashita, H. and Miyachi, S. (1999) A novel photosystem type reaction center from oxygenic prokaryote Acaryochloris
marina that contains chlorophyll d. In "The Photosynthetic prokaryotes" (eds. Peschek et al.), pp.
183-186. Kluwer.
Itoh, S., Iwaki, M., Hu, Q., Miyashita, H., Iwasaki, I.,
Kurano, N., Miyachi, S., Kobayashi, M., and Shimada, K. (1999) New types of reaction centers
using chlorophyll-d and Zn-bacteriochlorophyll a.
In "Photosynthesis: Mechanism and Effects" (ed.
Garab, G.), pp. 521-526. Kluwer
Iwaki, M., ltoh, S., Kamei, S., Matsubara, H. and Ohoka, H. (1999) Time-resolved spectroscopy ofchlorophyll-a like electron acceptor in the reaction
center complex of the green sulfur bacterium Chlorobium tepidum. Plant and Cell Physiol., 40, 10211028.
Kubo-Irie, M., Ohta, K., Meguro, T., Nakazawa, T.
and Mohri, H. (1999) Morphological evidence thatspermatogenic cells undergo apoptosis within testisof diapausing swallowtail pupa (Atropheneura alcinous). J. Exp. Zool.283, 194-201.

NATIONAL INSTITUTE

Matsunami, K., Kokubo, H., Ohno, K., Xu, P.-x.,
Ueno K. and Suzuki, Y. (1999) Embryonic silk
gland development in Bombyx: molecular cloning
and expression of the Bombyx trachealess gene.
Dev. Genes Evol. 209, 507-514.
Mohri, H. (1999) Tubulin 30 Years Later. Cell
Struct. Funct. 24, 265-267.
Mohri, H., Inaba, K., Kubo-Irie, M., Takai, H. and
Yano-Toyoshima, Y. (1999) Characterization ofouter arm dynein in sea anemone, Anthopleura midori. Cell Motif. Cytoskel. 44, 202-208.
Noguchi, T., Kamimura, Y., Inoue, Y. and Itoh, S.
(1999) Photoconversion of a water-soluble chlorophyll protein from Chenopodium Album: Resonance Raman and FTIR study of protein and pigment structures. Plant and Cell Physiol., 40, 305310.
Oh-oka, H. Iwaki, M., and Itoh, S. (1999) The tightcoupling of quinol oxidoreductase to the P840 reaction center complex in Chlorobium. In "Photosynthesis:Mechanism and Effects" (ed. Garab, G.),
pp. 1525-1528. Kluwer.
Shimada, K., Itoh, S., Iwaki, M., Nagashima, K. V. P.,
Matsuura, K. and Kobayashi, M., Wakao, N.
(1999) Reaction center complex based on Znbacteriochlorophyll from Acidiphi/ium rubrum. In"Photosynthesis: Mechanism and Effects" (ed. Garab G.), pp. 909-912. Kluwer.
Wakao, N., Hiraishi, A., Shimada, K., Kobayashi, M.,
Takaichi, S., Iwaki, M. & Itoh, S. (1999) Discovery, characteristics, and distribution of Zn-BChl inaerobic acidophilic bacteria including Acidiphilium
species and other related acidophilic bacteria. In" The Photosynthetic prokaryotes" (eds. Peschek
et al.), pp. 745-750. Kluwer.
Yoshii, T., Hara, H., Kawamori, A., Akabori K.,
Iwaki, M. & Itoh, S. (1999) ESEEM study of thelocation of spin-polarized chlorophyll-quinone
radical pair in membrane-oriented spinach photosystems I and II complexes. Appl. Magn. Reson.
16, 565-580.

9

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Mikio Nishimura

The department consists of two regular divisions and three adjunct divisiOns.

The department conducts studies on molecular dynamics of thecell in higher plants and animals such as organelle differentiation, autophagy, cell motility, cytokinesis and neural development.

10

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor:
Associate Professor:
Research Associates:

Mildo Nishimura
Ikuko Hara-Nishimura (-Sept. 30))
Makoto Hayashi(- March 31)
Tomoo Shimada
Shoji Mano (July 1-)
Technical Staffs:
MakiKondo
Katsushi Yamaguchi
Post doctoral fellows: Tetsu Kinoshita(- March 31)
Yasuko Hayashi-Ishimaru
Kanae Shirahama
Shoji Mano (-June 30)
Hiroshi Hayashi (April 1-)
Kenji Yamada (April1-)
Graduate Students:
Naoto Mitsuhashi
Kazumasa Nito
Etsuko Watanabe (April 1-)
Yuki Tachibe(from Hiroshima University)
Youichiro Fukao(from Fukui Pref Univ.)
JSPS Technical Staffs: Miwa Kuroyanagi
Mild Kinoshita (-Oct. 30)
Chizuru Ueda
Chihiro Nakamori
Michiyo Kato (April 1-)
Visiting Scientists:
Claudio Olivari(from Univ. ofMilano, Italy)
Yasuko Koumoto

Higher plant cells contain several distinct organelles
that play vital roles in cellular physiology. During
proliferation and differentiation of the cells, the organelles often undergo dynamic changes. The biogenesis of
new organelles may occur, existing organelles may
undergo a transformation of function, while otherorganelles may degenerate. Because the dynamictransformation of organellar function (differentiation oforganelles) is responsible for flexibility of differentiation events in higher plant cells, the elucidation ofregulatory mechanisms underlying organelle transformation are currently studied in this division.

I. Regulation at the level of protein transport tomicrobodies during the microbody transition.
Dramatic metabolic changes which underlie the shift
from heterotrophic to autotrophic growth occur ingreening of seed germination. Accompanying thesemetabolic changes, many constitutive organelles arefunctionally transformed. For example, etioplasts
differentiate into chloroplasts and mitochondria acquire
the ability to oxidize glycine. Glyoxysomes which aremicrobodies engaged in the degradation of reserve oil
via b-oxidation and the glyoxylate cycle, are transformed into leaf peroxisomes that function in several
crucial steps of photorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during
the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes
occurs during senescence. The functional transformation between glyoxysomes and leaf peroxisomes iscontrolled by gene expression, protein translocation andprotein degradation. In 1999, we showed that lightdependent alternative splicing is involved in the microbody transition.
Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that function in the glycolate pathwayof photorespiration. We have obtained two highly
similar cDNAs (HPRl and HPR2) for pumpkin HPR.
It has been revealed that two HPR mRNAs are produced by alternative splicing from a single type of premRNA. The HPRl protein, but not the HPR2 protein,
was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative
splicing control the subcellular localization of the two
HPR proteins. Analysis of transgenic Arabidopsis
expressing fusion proteins with green fluorescentprotein (GFP) revealed the different subcellular localizations of the two HPR proteins (Fig. 1). RT-PCR
analysis showed that the alternative splicing is regulated
by light, indicating that the microbody transition ispartly controlled in the level of splicing of the mRNAby the light dependent alternative splicing.

II. Microbody defective mutant of Arabidopsis.
It has been suggested that the functional conversion
between glyoxysomes and leaf peroxisomes is controlled by gene expression, protein translocation, andprotein degradation. A genetic approach is an effective
strategy toward understanding the regulatory mechanism(s) of peroxisomal function at the level of gene
expression, protein translocation, and protein degradation.
We
isolated
andcharacterized
2,4dichlorophenoxybutyric acid (2,4-DB)-resistant mutants.
It has been demonstrated previously that 2,4dichlorophenoxybutyric acid (2,4-DB) is metabolized to
produce a herbicide, 2,4-D, by the action of peroxisomal fatty acid b-oxidation in higher plants. To isolate
mutants that have defects in peroxisomal fatty acid Poxidation, we screened mutant lines of Arabidopsis
seedlings for growth in the presence of toxic levels of
2,4-DB. Genetic analysis revealed that these mutants
can be classified as carrying alleles at three independent
loci, which we designated pedl, ped2, and ped3, (where
ped stands for peroxisome defective). The pedl mutant
lacks the thiolase protein, an enzyme involved in fatty
acid b-oxidation during germination and subsequent
seedling growth, whereas the ped2 mutant has a defectin the intracellular transport of thiolase from the cytosol
to glyoxysomes. Further analysis on identification themutated genes of these ped mutants are now in progress.

FOR BASIC BIOLOGY

11

Figure I. Subcellular localizations of green fluorescent protein (GFP) fusions in root cells of transgenic Arabidopsis thaliana.
Root cells were observed in light field (Nomarski optics; E and F) and with blue light excitation (470-490 nm; A, B, C and D). (A, B) Threedimensional images of SGFP-H I which has the peroxisomal targeting signal (PTS I) at the C-tenninal end of GFP and SGFP-H2 which does
not have PTSI , respectively. (C, D) The same samples viewed with a confocal microscope. (E, F) The same samples viewed with a lightmicroscope. Peroxisomes were observed to be distributed diffusely throughout the cytosol of SGFP-H I (A, C), whereas the fluorescence of
GFP accumulated in the cytosol and the nucleus ofSGFP-H2 (B, D). Bar indicates 10 11m.

12

In addition of these genetic approaches, we started tocharacterize the enzymes of b-oxidation cycle. In
glyoxysomes, fatty acids are first activated to fatty acylCoA by fatty acyl-CoA synthetase. Fatty acyl-CoA is
the substrate for fatty acid b-oxidation, which consistsof four enzymic reactions. The first reaction is catalyzed
by acyl-CoA oxidase. The second and the third enzymatic reactions are catalyzed by a single enzyme thatpossesses enoyl-CoA hydratase and ~-hydroxyacyl­
CoA dehydrogenase activities. The fourth reaction iscatalyzed by 3-ketoacyl-CoA thiolase. We have characterized acyl-CoA oxidase that is active on long-chain
acyl-CoA and 3-ketoacyl-CoA thiolase in glyoxysomes.
In 1999, we reported an evidence that glyoxysomes
contain another acyl-CoA oxidase that can metabolize
short-chain acyl-CoA. These fmdings revealed that the
short-chain acyl-CoA oxidases function in fatty acid boxidation in glyoxysomes, and that by the cooperative
action of long- and short-chain acyl-CoA oxidases,
plant peroxisomes are capable to performing the complete b-oxidation of acyl-CoA, whereas mammal peroxisomes are not.

III. Transport of storage proteins to protein
storage vacuoles is mediated by large PAC (precursor-accumulating) vesicles.
Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified
from maturing pumpkin seeds. These vesicles were
designated precursor-accumulating (PAC) vesicles andhave diameters of 200 to 400 nm. We characterized
them to answer the question of how seed protein precursors are accumulated in the vesicles to be delivered toprotein storage vacuoles. They contain an electrondense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained
small vesicle-like structures. An immunocytochemical
analysis revealed numerous electron-dense aggregatesof storage proteins within the endoplasmic reticulum. It
is likely that these aggregates develop into the electrondense cores of the PAC vesicles and then leave theendoplasmic reticulum. Immunocytochemical analysis
also showed that complex glycans are associated withthe peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These
results suggest that the unique PAC vesicles might
mediate a transport pathway for insoluble aggregates ofstorage proteins directly to protein storage vacuoles.
In order to investigate the mechanism of the PAC
vesicle formation, we constructed chimeric genes that
encode fusion proteins consisting to both various
lengths of polypeptides derived from pumpkin 2S
albumin and a selectable marker enzyme, phosphinothricin acetyltransferase and expressed in Arabidopsis.
A fusion protein expressed by one of the chimeric genes
is accumulated as a proprotein-precursor form, andlocalized in novel vesicles of vegetative cells, that show
distinct features that well much to the PAC vesicles.

NATIONAL INSTITUTE

Despite of the accumulation of the fusion protein, thetransgenic Arabidopsis is still sensitive to phosphinothricin. Phosphinothricin acetyltransferase contained in
the fusion protein is obviously compartmentalized in
the PAC vesicles that do not permit the detoxification ofthis herbicide. These results indicate that PAC vesicle
can be induced in vegetative cells by ectopic expressionof the protein that is destined to be compartmentalized
into the PAC vesicles. Arabidopsis mutants that defectvesicular transport of the fusion protein are screened
and characterized by using the transgenic plants.

IY. Vacuolar processing enzymes in proteinstorage vacuoles and lytic vacuoles.
Vacuolar processing enzyme (VPE) has been shown
to be responsible for maturation of various seed proteinsin plant vacuoles. Arabidopsis has three VPE homologues; ~VPE is specific to seeds and aVPE and yVPE
are specific to vegetative organs. We expressed theyVPE in a pep4 strain of the yeast Saccharomyces
cerevisiae and found that yVPE has the ability to cleave
the peptide bond at the carbonyl side of asparagine
residues. An immunocytochemical analysis revealed thespecific localization of the yVPE in the lytic vacuoles of
Arabidopsis leaves. These fmdings indicate that yVPE
functions in the lytic vacuoles as the ~VPE does in theprotein-storage vacuoles. The ~VPE promoter was
found to direct the expression of the ~-glucuronidase
reporter gene in seeds of transgenic Arabidopsis plants.
On the other hand, both the aVPE and yVPE promotersdirected the expression in senescent tissues, but not inyoung intact tissues. The mRNA levels of both aVPE
and yVPE were increased in the primary leaves during
senescence in parallel with the increase of the mRNAlevel of a senescence-associated gene (SAG2). Treatment with wounding, ethylene and salicylic acid upregulated the expression of aVPE and yVPE. Our
results suggest that vegetative VPE might regulate theactivation of some functional vacuolar proteins that are
known to respond to these treatments.
To investigate a VPE system in protein-storage vacuoles, we isolated the PAC vesicles and characterized a
100-kDa component (PVlOO) of the vesicles.
Isolated eDNA for PVlOO encoded a 97,310-Da protein thatwas composed of a hydrophobic signal peptide and thefollowing three domains: an 11-kDa Cys-rich domain
with four CxxxC motifs (C, Cys), a 34-kDa Arg/Glurich domain composed of six homologous repeats, and a
50-kDa vicilin-like domain.
Mass spectrometry andpeptide sequencing of soluble proteins of the vacuoles
showed that two Cys-rich peptides, three Arg/Glu-rich
peptides and the vicilin-like protein were produced bycleaving Asn-Gln bonds of PVlOO and that all these
proteins had a pyroglutamate at their NH2 terminus.
VPE was responsible for cleaving Asn-Gln bonds of asingle precursor, PVl 00, to produce multiple seed
proteins. It is likely that the Asn-Gln stretches not
only provide cleavage sites for VPE but also produce
aminopeptidase-resistant proteins.
Cys-rich peptide

FOR BASIC BIOLOGY

function as a trypsin inhibitor and Arg/Glu-rich peptides
function as cytotoxic peptides. Our findings suggested
that PVIOO is converted into different functional proteins in the vacuoles of seed cells.

V. Role of molecular chaperones in organelle
differentiation.
Molecular chaperones are cellular proteins that function in the folding and assembly of certain other polypeptides into oligomeric structures but that are not,themselves, components of the final oligomeric structure. To clarify the roles of molecular chaperones onorganelle differentiation, we have purified and characterized chaperonin and Hsp70s and analyzed their rolesin the translocation of proteins into chloroplasts. In
addition to mitochondrial chaperoninlO homologues,
we isolated cDNAs for chloroplastic chaperonin 10
homologues from Arabidopsis thaliana. One of theeDNA insert was 958 bp long and encoded a polypeptide of 253 amino acids. The other eDNA insert was
603 bp and encoded a polypeptide of 139 amino acids.
The former was comprised of two distinct Gro-ES
domains whereas the latter had one Gro-ES domain.
Further analyses on the roles of these chaperonin
homologues in differentiation of plastids are underexperiments by using transgenic Arabidopsis which wasoverexpressed or was reduced these. chaperonin homologues

Publication List:
Ali, K., Nii, N., Yamaguchi, K. and Nishimura, M. (1999)
Levels of nonstructural carbohydrate in leaves and roots
and some characteristics of chloroplasts after applicationof different amounts of nitrogen fertilizer to peach seedlings. J. Jap. Soc. Hort. Sci. 68, 739-745.
De Bellis L., P. Giuntini, Hayashi, H., Hayashi, M. andNishimura, M. (1999) Purification and characterizationof pumpkin long-chain acyl-CoA oxidase. Physiol. Plant.
106, 170-176.
De Bellis, L., S. Gonzali, A. Alpi, Hayashi, H., Hayashi, M.
and Nishimura, M. (2000) Purification and characterization of a novel pumpkin short-chain acyl-CoA oxidase
which structure resembles acyl-CoA dehydrogenase.
Plant Physiol. in press.
Hayashi, H., L. De Bellis, Ciurli, A., Kondo, M., Hayashi,
M. and Nishimura, M.(1999) A novel oxidase that catalyzes oxidation of short-chain acyl CoA in plant peroxisomes. J. Bioi. Chern. 274, 12715-12721.
Hayashi, M., Toriyama, K., Kondo, M., Hara-Nishimura, I.
and Nishimura, M. (1999) Induction of precursoraccumulating vesicles by expression of chimeric genes
consisting of pumpkin 2S albumin and phosphinothricin
acetyltransferase. Plant Cell Physiol. 40, 263-272.
Hayashi, M., Toriyama, K., Kondo, M., Kato, K., Mano, S.,
L. De Bellis, Hayashi-Ishimaru, Y., Yamaguchi, K.,

13
Hayashi, H. and Nishimura, M. (2000) Functional transformation of plant peroxisomes. Cell Biochern. Biophys.
in press.
Hiraiwa, N., Nishimura, M. and Hara-Nishimura, I. (1999)
Vacuolar processing enzyme is activated selfcatalytically by sequential removal of the C-terminal
and N-terminal propeptides. FEES Lett. 447, 213-216.
Kato, A., Hayashi, M., Kondo, M. and Nishimura, M.
(2000) Transport of peroxisomal proteins that are synthesized as large precursors in plants. Cell Biochern.
Biophys. in press.
Kato, A., Hayashi, M. and Nishimura, M. (1999) Oligomeric proteins containing N-terminal targeting signals
are imported into peroxisomes in transgenic Arabidopsis.
Plant Cell Physiol. 40, 586-591.
Kinoshita, T., Yamada, K., Hiraiwa, N., Kondo, M.,
Nishimura, M. and Hara-Nishimura, I. (1999) Vacuolar
processing enzyme is up-regulated in the lytic vacuolesof vegetative tissues during senescence and under various stressed conditions. Plant J. 19, 43-53.
Koumoto, Y., Shimada, T., Kondo, M., Takao, T., Shimonishi, Y., Hara-Nishimura, I. and Nishimura, M.
(1999) Chloroplast Cpn20 forms a tetrameric structure in
Arabidopsis thaliana Plant J. 17, 467-4 77.
Marro, S., Hayashi, M. and Nishimura, M. (1999) Light
regulates alternative splicing of hydroxypyruvate reductase in pumpkin. Plant J. 17, 309-320.
Mano, S., Hayashi, M. and Nishimura, M.(2000) A leafperoxisomal protein, hydroxypyruvate reductase, is produced by light-regulated alternative splicing. Cell Biochern. Biophys. in press.
Minami, Y., Nishimura, 0., Hara-Nishimura, I., Nishimura,
M. and Matsubara, H. (2000) Tissue and intracellular
localization of indican and the purification and characterization of indican synthase from Indigo plants. Plant
Cell Physiol. in press.
Miyagishima, S., Itoh, R., Toda, K., Kuroiwa, H., Nishimura, M. and Kuroiwa, T. (1999) Microbody proliferation and segregation cycle in the single microbody-alga
Cyanidioschyzon merolae. Planta. 208, 326-336.
Takemoto, D., Hayashi, M., Doke, N., Nishimura, M. and
Kawakita, K. (1999) Molecular cloning of a defenseresponse-related cytochrome P450 gene from tobaccousing a fungal elicitor. Plant Cell Physiol. 40, 12321242.
Takemoto, D., Hayashi, M., Doke, N., Nishimura, M. and
Kawakita, K. (2000) Isolation of gene for EIR, an elicitor inducible receptor like protein, from tobacco by differential display. Plant Cell Physiol. in press.
Yamada, K., Shimada, T., Kondo, M., Nishimura, M. andHara-Nishimura, I. (1999) Multiple functional proteinsare produced by cleaving Asn-Gln bonds of a single
precursor protein by vacuolar processing enzyme. J. Bioi.
Chern. 274, 2563-2570.

14

NATIONAL JNSTITUTEDIVISION OF BIOENERGETICS
Professor:
Associate Professor:
Research Associates:

Yoshinori Ohsumi
Tamotsu Yoshimori
Yoshiaki Kamada
Takes hi Noda
Technical Staff:
Yukiko Kabeya
Post Doctoral Fellows: Noboru Mizushima l)
Takahiro Shintani
Naotada Ishihara
Akio Kihara 2!
Graduate Students:
Takayoshi Kirisako 3!
Yoshinobu /chimura 3J
Kuninori Suzufd 4J
Yoshinori Kobayashi4J
Atsuki Nara 4!
Hideki Hanaoka 4!
Visiting Fellow:
Hisashi Okada 5!
Fumi Yamagata 6!
Visiting Scientists:
Kazuaki Furukawa 7!
Satsuki Okamoto
IJ PRESTO, JST, Oct 1999-.
lJ Apr 1999-.
JJ Graduate University for Advanced Studies
4) Apr 1999-, Graduate University fo r Advanced Studies.
s; from Teikyo Univ. ofSci. and Tech.
6) from Teikyo Univ. of Sci. and Tech. -March 1999
7
) from Suntory Co. LTD, -July 1999.

This division aims to understand the autophagy in respects to its molecular mechanism and its physiologicalrole in multicellular organisms. Cells execute degradation processes of their constituents together with biosynthetic processes. These two processes are well coordinated to regulate the biological activities. In other
word, we must shed light on degradation process tofully understand the cell, because the study on the degradation has been retarded compared to the biosynthetic process. Autophagy is well conserved in eukaryotes and is a major route for bulk degradation of cytoplasmic constituents and organelles.

Apg5 , another Apg protein, via an isopeptide bond. This
conjugation reaction is distinct from but quite similar toubiquitination and the other related reaction such as
SUMO-I and Rub 1 system. All of these ubiquitinationlike (Ubl) reactions are catalyzed by a series of enzyme
system.
First, C-terminal glycine of Ubi protein is activatedby binding to active center of El -like enzyme via a
thioester bond. El-like enzymes are ATPase whichshare sequence similarity each other, and the ATPase
activity is prerequisite for the reaction. Collaboratingwith Dr. Kominami's laboratory(Juntendo Med. Univ.),
we have reported that Apg7 is an El-like enzyme involved in Apgl2 conjugation reaction.
Next, C-terminal glycine of Ubi protein is conjugatedto the active center of E2-like enzyme via thioester ·bond. We characterized ApglO, which is required for
conjugation reaction of Apg12 . ApglO binds to Apg12
depending on Apg7 function. We identified active center, Cys residue, of ApglO. C-terminal Gly of Apgl2
bind to this Cys via a thioester bond. The active Apg10
is required for Apg12-Apg5 conjugation, and necessary
for autophagy. Most notably, all previously known E2like enzymes show sequence similarity each other,
however ApglO does not show the similarity to any
known E2 enzymes. ApglO is a quite unique E2-likeenzyme. These findings prompted us the idea that Ubi
conjugation system is more prevailing than ever expected from sequence similarity.

I. Background
Upon nutrient starvation, autophagic process starts asbuilding up a membrane structure, an autophagosome,in the cytoplasm. The autophagosome sequesters cytosol and organelles nonselectively. Then it is delivered to
the vacuole/lysosome, and the cytoplasmic materials
inside are degraded by vacuolar/ lysosomal proteases.
We had discovered autophagy in a simple eukaryotic
model organism, Saccharomy ces cerevisiae and morphologically defined the whole process. We have already isolated a set of autophagy-deficient mutants(apg), and have cloned most of APG genes essential for
autophagy. We are now characterizing these geneproducts.

II. ApglO is a unique protein conjugating enzyme
Last year, we reported on novel protein conjugation
system required for autophagy. Apg12, one of Apg
proteins, is terminated with glycine at its C-terminus.
The C-terminal glycine is bound to a lysine residue of

Figure 1 Molecular apparatuses for autophagy in yeast. Upper
Panel: The diagram of Apg12 system. The numerals in the boxes
denotes the corresponding Apg proteins. The number of each
subunits in the final complex is still not determined. Arrowrelated to Apg16 does not necessarily represent the order of theevents, mther do show the concept of the organization. Lower
Panel: Immunofluorescence (Left) and Nomarsky Images(Right) of Apg8 localization. The vacuolar protease deficientmutants were starved and the tagged Apg8 is observed as the
FITC signal (the arrow in left panel). Autophagic body is indicated by the arrow in the right panel

FOR BASIC BIOLOGY

15

III. Apg16 crosslinks multiple Apg12-Apg5 conjugates
Despite these fmdings of novel Ubi system, themechanism how the system is involved in autophagicmechanism is still to be determined. However, an accessory part related to this system, which will aid us understanding the point, was discovered.
In a two-hybrid screen with Apgl2 as a bait, we
identified a novel protein, Apgl6, which is required for
autophagy. Thereafter, we noticed that the Apgl6 is
not bound to Apgl2 but Apgl2-Apg5 conjugate.
Moreover, Apgl6 is bound to Apg5 alone, but more
preferentially to Apgl2-Apg5 conjugate. The binding
occurs at theN-terminal region of Apgl6. On the other
hand, Apgl6 have a coiled coil domain at its C-terminus,
which generally functions for protein interaction. Actually Apgl6 forms an homooligomer via the coiled coildomain. In accordance with this scheme, two or more
independent Apgl2-Apg5 conjugates were shown to be
cross-linked via Apgl6. Hence, this hetero-oligomericlarge protein complex may play a role in autophagic
process.
IV. Apg8 as a tracer of membrane flow in autophagosome formation
Autophagosome is a double membrane structure. The
outer membrane fuses with the vacuolar membrane, andthe inner membrane structure is released into thevacuolar lumen. The released membrane structure,
autophagic body, is disintegrated depending on thevacuolar protease activity, and the cytosolic materials
inside are degraded. Therefore, when vacuolar proteaseactivity is blocked, autophagic bodies are accumulated
inside the vacuole.
Apg8, one of Apg proteins, is transcriptionally upregulated upon starvation via Tor-kinase dependent
signaling pathway, which controls induction of autophagy. Indirect immunofluorescence study showed thatduring a nutrient-rich growing phase, Apg8 is mostly
dispersed throughout the cytoplasm as tiny dot structures. Upon starvation, Apg8-positive structure appears
adjacent to the vacuole, which represents autophagosome or its intermediate. When vacuolar proteaseactivity is blocked, Apg8 was observed to be accumulated inside autophagic bodies. According to immuno
EM analysis in collaboration with Dr. M. Baba (Japan
Women's Univ.), Apg8 is mostly enriched on the membrane of forming intermediate of autophagosome. Uponcompletion of the formation, Apg8 is detached from themembrane and some are released to the cytosol and theothers are entrapped within the autophagosome. Thus,
Apg8 could be a useful tracer for membrane flow in
autophagosome formation. More detailed EM observation clarified that Apg8-positive electron lucent small
structures are gathering around forming autophagosome.
We postulate that these structures are one of the precursors of autophagosomal membrane. If this is the case,formation process of autophagosome is accompaniedwith multiple fusion of small precursor structures to the

Figure 2. TfR and the endocytosed dextran are accumulated in thecompartments where the mutant SK.Dl is localized. HeLa cells
transfected with the mutant SKD l fused with GFP were incubatedin the presence of l mg/ml TRITC-dextran at 37 oc for 8 hours.
The cells were then fixed , permeabilized, and subjected to immunofluorescence confocal microscopy for TfR. GFP-mutant SKDI
labeling (a), TfR staining (b), TRITC-dextran labeling (c), and amerged image (d) of a same field are shown. Arrows heads andarrows indicate the untransfected cells and the mutant SKD !positive compartments in the transfected cells, respectively. Bar,
20 J.lm.

intermediate structure. This contradicts to the generally
accepted scheme that autophagosome is formed byengulfing of the preexisting large membrane structure,such as ER. This hypothesis will be tested in our ongoing project.
V. SKDl regulates morphology of endosomes andmembrane traffic through them in mammaliancells
We also engage in the study of mammalian to pursue
cell biological and physiological issues of autophagy. In
the course of the study, we encountered an interesting
molecule, which regulates membrane dynamics in endosomal system.
The mouse SKDl is an AAA-type ATPase homologous to the yeast Vps4p/ Csclp implicated in bothautophagy and transport from endosomes to the vacuole.
To elucidate a possible role of SKDl in mammalianendocytosis, we generated a mutant SKD 1, harbouring amutation (E235Q) that is equivalent to the dominantnegative mutation (E233Q) in Vps4p/Csclp. Overexpression of the mutant SKDl in cultured mammaliancells caused defect in uptake of transferrin and lowdensity lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface
transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein
displayed a perinuclear punctate distribution in contrast
to a diffuse pattern of the wild type SKD 1. TfR, thelysosomal protein Iamp-l, endocytosed dextran andepidermal growth factor (EGF), but not markers for thesecretory pathway were accumulated in the mutant

16
SKDl-localized compartments. Degradation of EGF
was inhibited. Electron microscopy revealed that thecompartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and an endocytosed horseradish peroxidase.
The early endosome antigen EEAl was also redistributed to these aberrant membranes. Taken together, ourfindings suggest that SKD 1 regulates morphology ofendosomes and membrane traffic through them.

Publication List:
Original Articles
Tanida, 1., Mizushima, N., Kiyooka, M., Ohsumi, M.,
Ueno, T., Ohsumi, Y. and Kominami, E. (1999).
Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. Mol Bioi Cell. 10, 1367-1379.
Shintani, T., Mizushima, N., Ogawa, Y., Matsuura, A.,
Noda, T. and Ohsumi, Y. (1999). ApglOp, a novel protein-conjugating enzyme essential for autophagy in yeast.
EMBOJ. 18,5234-5241.
Mizushima, N., Noda, T. and Ohsumi, Y. (1999). Apg16p
is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. EMBO J. 18, 38883896.
Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T. and Ohsumi, Y.
(1999). Formation process of autophagosome is traced
with Apg8/Aut7p in yeast. J Cell Bioi. 147,435-446.
Tagawa, Y., Yamamoto, A., Yoshimori, T., Masaki, R.,
Omori, K., Himeno, M., Inoue, K. and Tashiro, Y.
(1999). A 60 kDa plasma membrane protein changes its
localization to autophagosome and autolysosome membranes during induction of autophagy in rat hepatoma
cell line, H-4-II-E cells. Cell Struct Funct. 24,59-70.
Yabe, I., Horiuchi, K., Nakahara, K., Hiyama, T., Yama-

NATIONAL INSTITUTEnaka, T., Wang, P. C., Toda, K., Hirata, A., Ohsumi, Y.,
Hirata, R., Anraku, Y. and Kusaka, I. (1999). Patchclamp studies on V-type ATPase of vacuolar membraneof haploid Saccharomyces cerevisiae. Preparation andutilization of a giant cell containing a giant vacuole. J
Bioi Chem. 274, 34903-34910.
Noda T., Kim J., Huang WP., Baba M., Tokunaga C.,
Ohsumi Y., and Klionsky D.J., (2000) Apg9p/Cvt7p is
an integral membrane protein required for transport
vesicle formation in the cvt and autophagy pathways. J
Cell Biol./148, 465-480
Yoshimori T., Yamagata F., Yamamoto A., Mizushima N.,
Kabeya Y., Nara A., Miwako I., Ohashi M., Ohsumi M.,
Ohsumi Y., (2000) The mouse SKDl, a homologue of
yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells. Mol Bioi Cell.
11, 747-763
George, M.D., Baba M., Scott, S.V., Mizushima N., Garrison B., Ohsumi Y., Klionsky D.J., (2000) Apg5p functions in the sequestration step in the cytoplasm to vacuole targeting and macroautophagy pathways. Mol Bioi
Cell. in press
Furukawa, K. Mizushima, N., Noda, T., Ohsumi, Y.,(2000) A protein conjugation system in yeast with homology to biosynthtic enzyme reaction ofprokaryotes. J
Bioi Chem. 275, 7462-7465· Review Articles
Klionsky, D. J. and Ohsumi, Y. (1999). Vacuolar import ofproteins and organelles from the cytoplasm. Annu Rev
Cell Dev Bioi. 15, l-32.
Ohsumi, Y. (1999). Molecular mechanism of autophagy inyeast, Saccharomyces cerevisiae. Philos Trans R. Soc.
Lond, B 354, 1577-1580; discussion 1580-1581.
Ohsumi, Y. (1999). Pictures in cell biology. Autophagosomes in yeast. Trends Cell Bioi. 9, 162.

FOR BASIC BIOLOGY17

DIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor:
Research Associate:
JST Research Staff:
JST Technical Staff:
Graduate Student:

Mataya Katsuki
Kei ItoTakes hi Awasaki (March-)
Kimiko Tanaka
Yusuke Uchiyama(Kyoto University, June-)
Nobuaki Tanaka(Graduate Univ. of Advanced
Studies, April-)
Hideo Otsuna(Nagoya University, February-)

The aim of this adjunct division, started in June 1998,
is to understand the basic rules by which elaborate neural circuits develop and function. With less than 1o5
neurones, and subject to powerful molecular and genetic techniques, the brain of the fruit fly Drosophilamelanogaster is a good model system for investigating
the whole of an easily-accessible nervous system thatshares certain of the architectural and functional features of the more complex vertebrate brains. Second
year of the five-year term, and with new research staff
and graduate students, we started a large-scale screening to fmd strains useful for this purpose.
I. Comprehensive identification and developmental tracing of brain cells
A comprehensive and detailed anatomical knowledgeof the brain is a prerequisite for 1) analysing the phenotypes of nervous system-related mutants, 2) identifying the cells that express the cloned genes, 3) understanding the way information is processed in the brain,
and 4) devising computer models that simulate brain
functions. In spite of the hundred years of efforts using
Golgi and other anatomical techniques, however, thecircuit structure of higher order associative regions of
the brain is still essentially unresolved. Moreover, traditional neuroanatomy tends to focus only on themature adult brain, leaving the developmental processes
largely uninvestigated. Since many nervous systemrelated mutants show structural defects, however, understanding the role of the responsible genes requiredetailed basic knowledge about when and how the brain
structure is formed. Thus, "developmental neuroanatomy" becomes all the more important in the age ofmolecular cloning.
The GAL4 enhancer-trap system, which is widelyused for mutagenesis and gene cloning of Drosophila, isalso a powerful tool for obtaining a vast array of transformant strains that label specific subsets of brain cells.
We screen such lines from a stock of 4500 GAL4
strains made by the "NP consortium" , a joint ventureof eight Japanese Drosophila laboratories organised byus. After initial pilot experiments, we employed a twostage screening process. In the first step, all the lines arecrossed with the flies carrying the UAS-GFP transgene,which fluoresces only in the cells where GAL4 expres-sion is active. The patterns of the GAL4-expressing
cells are recorded from freshly dissected, unfixed adultbrain tissue using a high-speed confocal microscope.
Photographs of between 20 and 100 optical sections aretaken. As of December 1999, ca. 50000 photographs
depicting 1612 of the total 4500 strains have been accumulated in a computer database. In the second step,
useful lines are selected from the database, and fixedand clealised brain specimens at various developmental
stages are subjected to confocal serial sectioning with aconventional confocal microscope and to threedimensional reconstruction with a UNIX workstation.
Among the 1612lines screened, 98% showed GAL4
expression in the brain, suggesting that most of thegenes in the genome are expressed in at least some
portion of the brain cells. Among them, less than 20 %showed expression in a small enough number of cells
for which identification is feasible. In many such strains,the expression pattern changes drastically during neurogenesis. Only a few percent of the whole strains label
identical neurons throughout larval, pupal and adultstages. These lines are found valuable to trace the development, path finding and synapse formation processes of the labelled cells.
Although the long-term aim of this project is to identify as many neurones and glial cells as possible to get
the comprehensive overview of the fly brain structure,at the initial stage a few brain regions are chosen for
intensive study. The first target is to identify projection
interneurons that connect lower-level sensory neuropileand higher-order associative regions. These fibres convey olfactory, gustatory, auditory and visual sensory
information. As for visual pathways, for example, fivenovel types of projection neurones have already been
identified by our screen.
The other target is to reveal the formation process of
one of the highest-order associative centres called the"central complex" . In this area, fibres from various
brain regions converge to form the sophisticated arraycircuit structure, providing a good model system forstudying the molecular mechanisms of path finding andcircuit formation. We have identified several strains thatlabel central complex neurons from rather early stages
and are tracing the developmental processes in moredetail (Fig. 1).

ll. Tools for comprehensive cell lineage analysis of
the brain
Combining the flippase-FRT recombination induction system and GAL4-UAS expression activation system, we have previously developed a novel technique,the "FRT-GAL4 system" , with which one can label asmall number of neural stem cells at any desired developmental stage and reveal the projection patterns of
their progeny at a later period. Though this system
proved useful for revealing the clonal architecture ofseveral brain regions, the performance of the transformant lines used for activating the flippase gene was not
optimal, showing a high level of spontaneous recombi-

18

nation. To solve this problem, we generated andscreened ca. hundred new lines and obtained several
that show no background jlippase expression and sharp
heat shock-induced activation. Using these lines, anattempt to make a comprehensive map of the lineagerelated circuit structures of the fly brain is underway.

III. Analysis of genes for path finding processes in
the adult brain development
Although many genes related to the neuronal pathfinding processes have been identified and analysed in
Drosophila, their functions have been studied primarily
in the simple motor neurons and the ventral nerve cordof embryos. Little has been investigated about theirfunctions when neurones compose highly complicatedcircuits of the adult brain.
The latest genetic mosaic system, the "MARCM(mosaic analysis with a repressible cell marker) system",can generate labelled clones with mutant genotype
whereas surrounding wild-type cells remain unlabelled.
This is a powerful technique for studying the functionof a gene in the path fmding processes of identified
fiber projection. Using this system, we analysed thefunction of the gene trio, which encodes a Db! family
protein. As a model system for analysing its possible
role in path fmding controle of developing axons, we
focused on the brain region called mushroom bodies(MB), whose clonal structure has been revealed in great
detail by us and other investigators.
An MB is made by four specific stem cells (neuroblasts) and thus consists of four clones. When one ofthem were labelled without introducing trio genotype,

NATIONAL INSTITUTE

the neurones of the clone extended their axons throughthe peduncle and arborised into the vertical and medial
lobes (Fig. 2A). In contrast, neurons of the trio mutant
clones appeared to navigate normally through the peduncle till the approximate region of the two lobes, butthere they either stalled or are misrouted around the endof the peduncle, forming only sparse arborisation into
the lobes (Fig. 2B). On the other hand, branching in thecalyx appeared unaffected. These suggest that trio plays
an essential role in projection and arborisation of axons
from the peduncle into lobes.

IV. Contribution to the science community
As a joint venture with German and US researchgroups, we maintain Flybrain, a web-based image daof the
Drosophila
nervoussystemtabase(http://flybrain.nibb.ac.jp). Over 2000 images has already been stored and served worldwide. Another database maintained here is Jjly, which is intended to help
the exchange of information among Japanese-speaking
Drosophila researchers (http://jfly.nibb.ac.jp). Archivesof research-related discussions, images and experimental protocols, as well as meetings and job announcements, are provided.

Publication List:
Awasaki, T., Saito, M., Sone, M., Suzuki, E., Sakai, R. , Ito,
K. , Hama, C. The Drosophilla Trio plays an essential
role in patterning of axons by regulating their directional
extention . Neuron . in press.

Fig. 1 Development of fibres innervating the central complex
A: In early pupae, only upper part of the fan-shaped body region of the central complex is formed. B: The adult fan -shaped body shows twolayers of tangential connections.

FOR BASIC BIOLOGY

19

Fig. 2 trio clones in the mushroom bodies (MB)A: The MB consists of neurons produced by four stem cells. One of the four wild-type clones is made to express CDB reporter gene and labelledwith anti-CD8 antibody (green). Fibres from all the four clones, together with some other tracts, are labelled with anti-Fasll (purple). B: In thetrio mutant clone (green), neurones extend axons in the peduncle but exhibit poor projection into the vertical and medial lobes. Anti-Fasii labelling (purple) shows that the other three clones form essentially normal MB structure.

20

NATIONAL INSTITUTEDIVISION OF CELLULAR COMMUNICATION(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Institute Research Fellow:
Postdoctoral Fellow:
Graduate student:

Ritsu Kamiya
Katsushi Owaribe
Takako Kato-Minoura
Osamu Kagami
Itsushi Minoura
Ken-ichi Wakabayashi(University of Tokyo)

The research in this laboratory is aimed at an understanding of the molecular mechanisms that regulate theassembly and function of cytoskeletal proteins. Specifically, we are currently studying the functional properties of axonemal dynein and actin in Chlamydomonas, anorganism ideally suited for genetic and molecular biological studies.

I. Function of Multiple Axonemal Dyneins
It is well established that the beating of cilia andflagella is based on sliding movements of outer-doublet
microtubules driven by motor proteins dyneins, but how
the sliding is converted into axonemal oscillatory
bending movement has not been made clear. Recently,
various lines of evidence have suggested that dynein is
crucially important also in the sliding-bending conversion mechanism. Thus our research effort is now focused on the properties of various dyneins.
Biochemical studies by us and other laboratories
have established that a single flagellar axoneme contains at least eleven kinds of dynein heavy chains ininner and outer arms. The question is how different
dynein heavy chains differ in function. To answer thisquestion, we have been isolating and characterizing
mutants that lack different kinds of axonemal dyneins.
During the last ten years, we have isolated as many as
15 genetically different mutants lacking various subsetsof dyneins. The isolation of these mutants greatly advanced our understanding of the function and organization of various dyneins within the axoneme, because
only three mutants had been known to lack dynein
heavy chains before we started mutant isolation.
The motility phenotypes of the isolated mutants haveindicated that different dynein species differ in function .
in a fundamental manner. For example, the outer-arm
heavy chains are important for flagellar beating at highfrequency, whereas the inner-arm heavy chains areimportant for producing proper waveforms. Indirect
evidence also suggests that the force generation properties differ greatly among different heavy chains. Interestingly, the axoneme can beat without some of theseheavy chains, but cannot beat if certain combinations ofheavy chains are lost. It appears that simultaneouspresence of dyneins with different properties is necessary for the axonemal beating. Thus, it should be important to understand the mechano-chemical property ofeach dynein. To this end, we are currently trying todirectly measure the force production in wild-type andmutant axonemes that lack various combinations ofdyneins; we have constructed an experimental device tomeasure minute axonemal force with fine glass needles.
Preliminary results indicate that the force produced in
the mutant axoneme lacking the outer arm or part of theinner arm is reduced to about 1/3 of that in the wildtype axoneme.
As a by-product of these experiments, we have recently succeeded in detecting elasticity between theouter-doublet microtubules. Our results confirmed that
there is an elastic component that connects adjacentouter-doublet microtubules, as has been postulated bytheoretical studies of cilia and flagella. Such an elastic
component has been considered crucial for axonemal
beating, since it is regarded as responsible for restricting
the amplitude of microtubule sliding and for generating
oscillatory movements.

II. Function of Actin and an Actin-related Protein
in Chlamydomonas
The inner dynein arms are known to contain actin as
a subunit. Hence the two independent motility systemsof eukaryotes - the actin-based and microtubule-based
motility systems - should somehow cooperate in the
inner arm dynein although the function of actin in
dynein arms is totally unknown at present. Recently wefound that the mutant ida5, lacking four out of theseven subspecies of inner-arm dyneins, has a mutationin the actin-encoding gene. Intriguingly, Chlamydomonas has been known to have only a single gene of conventional actin, and the mutant ida5 wa~ found to express no conventional actin at all. On close inspection,
the cytoplasm and axonemes of this mutant were foundto contain a novel actin-like protein (NAP) which displays exceptionally low homology (64%) to conventional actin. The mutant ida5 is deficient in the formation of the fertilization tubule and thus has a low mating
efficiency. However, it displays normal cell division andgrows as rapidly as wild type, possibly because NAP
can substitute for actin in important cellular functions.
Thus conventional actin and NAP may overlap in some,
but not all, cellular functions. It is interesting to notethat NAP is expressed in significant amount only in the
mutant lacking actin; i.e., the expression of NAP appears to depend on the presence of actin. We are currently investigating how such regulation takes place.
We have recently succeeded in transforming the mutant ida5 with cloned actin gene and found that innerdynein arms become restored upon transformation.
Transformation with NAP gene is underway. Studies
with artificially mutated actin gene will enable us todetermine what functions are carried out by actin and
NAP and, in particular, whether actin or NAP is really
essential for cytokinesis, assembly and function of innerdynein arms, or other fundamental processes in Chlamydomonas.

Publication List:
Minoura, I., Yagi, T., and Kamiya, R. (1999). Direct measurement of the inter-doublet elasticity in flagellar axo-

21

FOR BASIC BIOLOGY

nemes. Cell Struct. Funct. 24:27-33 .
Omoto, C. K. , Gibbons, I. R., Kamiya, R., Shingyoji, C.,
Takahashi, K., and Witman, G. B. (1999). Rotation of
the central pair microtubules in eukaryotic flagella. Molec. Bioi. CelllO:l-4.
Leivo, T., J. Lohi, A-L. Kariniemi, G. Molander, C. L.
Kiraly, M-L. Kotovirta, K. Owaribe, R. E. Burgeson,and Leivo, I. (1999) . Hemidesmosomal defect in derma-

titis herpetiformis; Decreased expression of BP230 andplectin/HD I in uninvolved skin. Histochem. J. 31: I 09116.
Okumura, M. , Uematsu, J., Hirako. Y., Nishizawa, Y.,
Shimizu, H. , Kido, N. , and Owaribe, K. (1999). Identification of the hemidesmosomal 500 kDa protein(HD1) as plectin. J. Biochem. 126:1144-1150.

8

Fig. l . Fertilization tubules in wild-type (A) and ida5 (B) mt+ gametes produced in response to a 1 hour exposure to l 0 rnM dibutyryl-cAMPand 1 rnM IBMX. Bar, 0.3 flill. Wild-type fertili zation tubules have been shown to contain F-actin bundles.

22

NATIONAL INSTITUTEDIVISION OF CELL FUSION (ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Insttitute Research Fellow:
Graduate Students:

Issei Mabuchi
Hiroshi Abe
Hirotaka Fujimoto
Izuru Yonemura
Tatsuhiko Noguchi(University of Tokyo)
Sawako yamashiro(University of Tokyo)
Ryutaro Yanase(University of Tokyo)

Cytokinesis in animal and some primitive eukaryotic
cells is achieved by the progressive contraction of thecleavage furrow. The cleavage furrow contains a contractile apparatus, called the contractile ring, which iscomposed of a bundle of actin filaments that lies in the
furrow cortex beneath the plasma membrane. It has
been established that the contractile ring contracts as theresult of interaction between actin filaments and myosin.
However, little is known about process of its formation,mechanism that controls its formation, protein constituents, and its ultrastructure. The goal of our research is tosolve these problems and thereby clarify the molecularmechanism of cytokinesis. For this purpose, we use
three kinds of cells, namely, sea urchin egg, Xenopus
egg, and the fission yeast Schizosaccharomyces pombe.
S. pombe is an excellent system to investigate thechanges in the actin cytoskeleton during cell cycle since
F-actin patches, F-actin cables and F-actin ring are only
visible structures in the cell (Fig. 1). The F-actin ring is
considered to correspond to the contractile ring in animal cells. It is formed during anaphase in this organism.
S. pombe cells have two IT-type myosin heavy chains
called Myo2 and Myo3/Myp2. Recently, we studied
how myosin accumulates at the division site. First, we
showed that Cdc4, an EF-hand protein, appears to be acommon myosin light chain associated with both Myo2
and Myo3. Loss of function of both Myo2 and Myo3
caused defect in the F-actin ring (contractile ring) assembly at the division site, like the phenotype of cdc4
null cells. It is suggested that Myo2, Myo3 and Cdc4
function in a cooperative manner in the formation of theF-actin ring during mitosis.
Next, we investigated dynamics of myosin-II duringmitosis in S. pombe cells. In early mitosis, Myo2 wasdetected primarily as dots widely located in the medialcortex. Myo2 fibers also became visible following theappearance of the dots. The Myo2 dots and fibers thenfuse with each other to form a medial cortical network.
Finally the network is packed into a thin contractile ring.
In mutant cells that cannot form the F-actin ring such as
cdc3, cdc8 and cdcl 2, Myo2 is able to accumulate as
the dots in the medial cortex, whereas no accumulationof the Myo2 dots was detected in cdc4 cells. Moreover,F-actin did not seem to be required for the accumulationof the Myo2 dots.
A truncated Myo2 which lacks putative Cdc4-bindingsites (Myo28.1Qs) was able to rescue myo2 null cells,
myo3 null cells, cdc4 mutant cells and cdc4 null cells.
The Myo28.1Qs could assemble into a normal-shapedring in these cells. Thus, its assembly at the division sitedoes not require function of either Cdc4 or Myo3.
On the other hand, we studied reorganization of actin-myosin cytoskeleton at the growing ends of thecleavage furrow of Xenopus eggs. At the the furrowformation, a cortical movement towards the division
plane occurs at the growing ends of the furrow. Immunofluorescence microscopy demonstrated that myosin II assembles at the growing end as spots probably asa result of the cortical movement. Actin filaments assemble a little later after the formation of the myosin
spots as small clusters which we call "F-actin patches",at the same positions as the myosin spots. The F-actin
patches seemed to be .formed and grow through new
actin polymerization rather than assembly of preexisting cortical F-actin. This was substantiated by
microinjection of rhodamine-G-actin near the growing
end: the microinjected G-actin was rapidly incorporated
in the F-actin patches. The F-actin patches then aligntandemly to form short F-actin bundles, and then the
short bundles form long F-actin bundles which compose
the contractile ring. The myosin spots are aligned on thelong F-actin bundles and fused each other to show fibrous appearance.
We also concentrate our study on function of actinregulatory proteins, including ADF/cofilin family proteins, during cytokinesis using Xenopus eggs and embryos. ADF/cofilin family proteins exist in all animals
and plants examined and have been shown to be essential. We found that ADF/cofilin family proteins are
essential for cytokinesis (Abe, Obinata, Minamide, andBamburg, J. Cell Bioi. 132: 871-875, 1996). Recent
studies revealed that ADF/cofilin accelerates turnoverof actin filaments both in vitro and in vivo. Most recently, we found a novel actin-regulatory protein which
induces disassembly of actin filaments cooperatively
with ADF/cofilin. eDNA analysis revealed that thisprotein is a Xenopus homologue of yeast actin interacting protein 1 (AIPl). Thus, we designated this protein
as Xenopus AIPl (XAIPl). Purified XAIP1 itself binds
to pure actin filaments to some extent, but it induces a
rapid, drastic disassembly of actin filaments associatedwith cofilin. Microinjection of this protein into Xenopus
embryos arrested development by the resulting actin
cytoskeletal disorder. XAIPl represents the first case ofa protein cooperatively disassembling actin filamentswith ADF/cofilin family proteins.

Publication List:
Suda, M., Fukui, M., Sogabe, Y., Sato, K., Morimatsu, A.,
Arai, R., Motegi, F., Miyakawa, T., Mabuchi, I., and Hirata, D. (1999). Overproduction of elongation factor Ia,
an essential translational component, causes aberrant cellmorphology by affecting the control of growth polarity
in fission yeast. Genes to Cells 4, 517-527.
Sayers, Lee G., Katayama, S., Nakano, K., Mellor, H.,
Mabuchi, I., Toda, T., and Parker, P. J. Rho-dependenceof Schizosaccharomyces pombe Pck2. Genes to Cells inpress.

23

FOR BASIC BIOLOGY

Kusano, K., Abe, H. and Obinata, T. (1999). Detection of asequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin. Mol. Cell.
Biochem. 190, 133-141.
Tsubakimoto, K., Matsumoto, K., Abe, H., Ishii, J., Amano,
M., Kaibuchi, K. and Endo, T. (1999). Small GTPase
RhoD suppresses cell migration and cytokinesis. Onco-gene 18, 2431-2440 .
Okada, K. , Obinata, T. and Abe, H. (1999). XAIP 1: a
Xenopus homologue of yeast actin interacting protein I(AlP I), which induces disassembly of actin filaments
cooperatively with ADF/cofilin family proteins. J. Cell
Sci. 112, 1553-1565.

Fig. I 3-D images of an interphase S.pombe cell. Each image is rotated by 12 degree from the neighbor. Green, F-actin. Blue, DNA. Red,
spindle pole body. Bar, 2 11m.

24

j

NATIONAL INSTITUTEl

25

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshitaka Nagahama

The Department is composed of three divisions and one adjunct division.
Department members conduct molecular analysis on various aspects ofdevelopmental phenomena including: (1) differentiation and maturation ofgerm cells, (2) gene regulation in cell differentiation and growth, and (3)molecular basis of body plans.

26

NATIONAL INSTITUTEDIVISION OF REPRODUCTIVE BIOLOGY
Professor:
Associate Professor:
Research Associate:

Yoshitaka Nagahama
Michiyasu Yoshikuni
Minoru Tanaka
Tohru Kobayashi
Institute Research Fellow:
Yoshinao Katsu
JSPS Postdoctoral Fellow: Mika Tokumoto
Daisuke Kobayashi
Masaru Matsuda
Craig E. Morrey
Catherine Dreanno
Balasubramarian
Senthilkumaran
JSPS Research Associate:
Akihiko Yamaguchi
Yuichi Oba
Toshitaka Ikeuchi
FumieSakai
Masatada Watanabe
Gui-Jin Guan
Ryo Horiuchi
Graduate Student:(Graduate University for
Advanced Studies)
Monbusho Foreign Scientist: Srinivas K. Saidapur(Karnatak University)
Visiting Scientist:
Shanbhag Bhagyashri(Karnatak University)
Ching-Fong Chang (National
Taiwan Ocean University)

The pituitary-gonadal axis plays an important role inregulating gametogenesis in vertebrates. Gonadotropins
typically act through the biosynthesis of gonadal steroid
hormones which in turn mediate various stages of gametogenesis. Their effects are particularly profound inteleost fishes which provide several excellent models
for investigating the basic hormonal mechanisms regulating gonadal sex differentiation and gametogenesis(spermatogenesis, sperm maturation, oocyte growth andoocyte maturation). Our research focuses on (I) the
identification of steroidal mediators involved in gonadal
sex differentiation and gametogenesis, and (2) the
mechanisms of synthesis and action of these mediators.

I. Endocrine regulation of gonadal sex differentiation and sex change
Sex determination and gonadal development varyconsiderably in fish. In addition to gonochorism, several types of hermaphroditism (protandry, protogyny andsynchronous hermaphroditism) are found in fish. Tilapia, Oreochromis niloticus, is an excellent example ofthe precise nature of steroidogenic actions during gonadal sex differentiation. In this fish, all genetic female(XX) or male (XY) broods can be obtained by artificial
fertilization of normal eggs (XX) and sex-reversed,
pseudo male sperm (XX}, or normal eggs (XX) andsuper male sperm (YY}, respectively. Fertilized eggs
hatch after 4 days at 26°C. On the day of hatching,primordial germ cells (PGCs}, which are morphologically distinguishable from somatic cells, are located in
the outer layer of the lateral plate mesoderm around thehind gut. At 3 days post-hatching, PGCs are located in

the gonadal anlagen after the formation of the coelomic
cavity in the lateral plate mesoderm rather than throughactive migration. Mitosis of germ cells begins 15-20
days post-hatching in genetic females, but can not be
confirmed until after sex differentiation in testes ofgenetic males.
During the course of morphological sex differentiation, the behavior of somatic cells in the gonad is of· often sex-specific. In these cases, the sex of the gonad iseasily distinguished. In tilapia, gonadal sex is morphologically distinct at 20-25 days post hatching.
Ovarian differentiation is initially marked by stromalelongations of the gonad for the formation of the ovarian cavity. Testicular differentiation is characterized by
the appearance of a narrow space in the stromal tissue
representing the formation of the efferent duct. Steroidproducing cells in ovaries, but not testes, at the undifferentiated and differentiating stages express all of thesteroidogenic enzymes required for estradiol-17~ biosynthesis from cholesterol. These results strongly suggest that endogenous estrogens act as the natural inducers of ovarian differentiation in tilapia. This hypothesis
is further supported by evidence of masculinization ofgenetic female tilapia by inhibition of estrogen synthesis using an inhibitor of cytochrome P450 aromatase(Fig. 1). In contrast, the ability of steroid-producing
cells to synthesize steroid hormones could not be confirmed in the testis during sex differntiation of testiculardifferentiation.
Sequential, protogynous hermaphroditism, i.e. female
to male sex change, provides an ideal comparativemodel for studies on the endocrine regulation of sexual
differentiation. Prior to sex change, the gonad of Thalassoma duperrey contains primordial germ cells andoogonia but not spermatogonia. To begin investigating
the importance of hormonal signals on germ cell differentiation during sex change, we examined the expression of P450arom and II ~-hydroxylase mRNAs in T.
duperrey. The ovarian form of P450arom is predominantly expressed in the ovary prior to the initiation ofsex change. Shortly thereafter, expression diminished toundetectable levels suggesting ovarian P450arornl
estrogen is critical for the maintenance of ovarian function and/or detrimental to testicular differentiation. In
contrast, P450 11~-hydroxylase is likely to be a major
factor regulating the gonadal changes. P450 II~­hydroxylase mRNA was relatively abundant in theovary; however, it was up-regulated concomitant withthe onset of testicular differentiation indicating theimportance of P450 11 ~-hydroxylase/11-ketotesto­
sterone (11-KT) to spermatogenesis and spermiation.
Consequently, the switch from an ovary to a testis corresponds exceptionally well to a switch in the steroidogenic pathway of the gonad thus providing further evidence of the critical nature of steroid hormones in teleost sexual differentiation.

ll. Endocrine regulation of spermatogenesis
Spermatogenesis is an extended process of differen-

27

FOR BASIC BIOLOGY

tiation and maturation of germ cells resulting in haploid
spermatozoa. The principal stimuli for vertebrate spermatogenesis are thought to be pituitary gonadotropins
and androgens. However, the mechanisms of action ofthese hormones remain unresolved. Using an organ
culture system for eel testes consisting of spermatogonia and inactive somatic cells, we have shown that thehormonal regulation of spermatogenesis in eel testes
involves the gonadotropin stimulation of Leydig cells to
produce 11-KT, a potent androgen in fish . In tum, 11KT activates Sertoli cells to stimulate production ofactivin B. Addition of recombinant eel activin B to theculture medium induced proliferation of spermatogonia,
producing late type B spermatogonia, within 15 days inthe same manner as did 11-KT. cDNAs encoding two
androgen receptors (eARa and eAR~) were cloned eel
testes. The amino acid sequences of these two ARs
share low homology. In transient transfection assays ofmammalian cells, both eAR proteins showed androgendependent activation of transcription with 11-KT being
the most effective activator. These results indicate thatthe cloned eAR cDNAs encode functional eel ARs,
whose native ligands are 11-KT. In situ hybridization
revealed that both eAR rnRNAs are present in testes
prior to HCG injection, only eARa transcripts increased
during HCG-induced spermatogenesis suggesting thateARa and eAR~ play different roles in spermatogeneSIS.

In situ hybridization shows that activin type I and II
receptors are localized in spermatogonia. Activin B acts
via these receptors on spermatogonia to induce de novo
synthesis of Gl/S cyclins (cyclins A, D, and E) andCdks (cdc2, cdk2, and cdk4), leading to the initiation ofmitosis (spermatogonial proliferation). Despite theabove-mentioned progress in the . study of hormonal
regulation of spermatogenesis (mitosis), our attempts to
induce meiosis in vitro with recombinant eel activin B
have been unsuccessful. One possible explanation isthat another factor(s) , perhaps a meiosis-initiating substance produced in the testis in response to gonadotro-

pins or 11-KT, may be responsible for the mitosismeiosis transition. Interestingly, cyclin AI transcripts
were first detected in primary spermatocytes during
HCG-induced spermatogenesis in eel testes suggesting
an impOiiant role of cyclin AI in the progression tomeiosis of male germ cells. Further studies on the transcriptional regulation of cyclin A I expression may
therefore answer some of the basic questions related tothe initiation and progression towards meiosis during
spermatogenesis.

TIL Endocrine regulation of oocyte maturation
Meiotic maturation of fish oocytes is induced by theaction of maturation-inducing hormone (MIH). 17a,20~-dihydroxy-4-pregnen-3-one(17a,20~-DP)wasidentified as the MIHs of several fish species. The interaction of two ovarian follilce cell layers, the thecal
and granulosa cell layers, is required for the synthesis of
17a,20~ -DP. The thea) layer produces !?a-hydroxyprogesterone that is converted to 17a,20~-DP in
granulosa cells by the action of 20~-hydroxysteroid
dehydrogenase (20~-HSD). The preovulatory surge of
LH-like gonadotropin is responsible for the rapid expression of 20~-HSD mRNA transcripts in granulosa
cells during oocyte maturation. 17a,20~-DP induces
oocyte maturation by acting on a pertussis toxinsensitive G-protein-coupled membrane receptor. The
early steps of 17a,20~-DP action involve the formationof downstream mediator of this steroid, the maturationpromoting factor or metaphase-promoting factor (MPF)
consisting of cdc2 kinase and cyclin B. 17a,20~-DP
induces oocytes to synthesize cyclin B which activates apreexisting 35-kDa cdc2 kinase via phosphorylation of
its threonine 161 by a threonine kinase (M015), thus
producing the 34 kDa active cdc2. A 54-kDa Y box
protein and polyadenylation of cyclin B rnRNA are
thought to be involved in 17a,20~-DP-induced initiation of cyclin B rnRNA translation. Upon egg activation,
MPF is inactivated by degradation of cyclin B. It wasdemonstrated that the 26S proteasome initiates cyclin B

Fig. I. Masculinization of genetic female (XX) tilapia by inhibition of estrogen synthesis using an inhibitor of cytochrome P450 aromatase (P450arom), fadrozole (Fig. I ). Fadrozole was given to fry at 8 days after hatching for 22 days. A, Control (Female, ovary); B,
Fadrozole (Sex reversed male, testis). Gonads were stained with an antibody against P450arom (A) or an antibody against 3~­hydroxysteroid dehydrogenase (3~-HSD) (B).

28

NATIONAL INSTITUTE

degradation through the first cut of its NH2 terminus at
1ysme 57.
0

Publication List:
Chang, X.T., Kobayashi, T., Todo, T., Ikeuchi, T., Yoshiura, Y., Kajiura-Kobayashi, H., Morrey, C. and Nagahama, Y. (1999). Molecular cloning of estrogen receptors a and ~ in the ovary of a teleost fish, the tilapia(Oreochromis niloticus). Zoo/. Sci. 16, 653-658.
Guan, G., Tanaka, M., Todo, T., Young, G., Yoshikuni, M.
and Nagahama, Y. (1999). Cloning and expression of
two carbonyl reductase-like 20~-hydroxysteroid dehydrogenase cDNAs in ovarian follicles of rainbow trout(Oncorhynchus mykiss).
Biochem. Biophys. Res.
Comm. 255, 123-128.
Hondo, E., Kobayashi, T., Ishiguro, N., Kurohmaru, M.,
Kitamura, N., Yamada, J. and Nagahama, Y. (1999).
Prolactin induces protamine 2 mRNA expression in rat
testis. J. Reprod. Dev. 45,205-212.
Ikeuchi, T., Todo, T., Kobayashi, T. and Nagahama, Y.
(1999). eDNA cloning of a novel androgen receptor
subtype. J. Bioi. Chern. 274, 25205-25209.
Katsu, Y., Carnall, N., Nagahama, Y. and Standart, N.
(1999). Phosphorylation of p82 clam CPEB by MAP
kinase and cdc2 kinase. Dev. Bioi. 209, 186-199.
Katsu, Y., Yamashita, M. and Nagahama, Y. (1999).
Translational regulation of cyclin B mRNA during 17a,20~-dihydroxy-4-pregnen-3-one(maturation-inducing
hormone)-induced oocyte maturation in goldfish, Carassius auratus. Mol. Cell. Endocrinol. 158, 79-85.
Kitano, T., Takamura, K., Kobayashi, T., Nagahama, Y.
and Abe, S.-1. (1999). Suppression of P450 aromatase(P450arom) gene expression in sex-reversed males produced by rearing genetically female larvae at high temperature during a period of sex determination in Japanese flounder (Paralichthys olivaceus). J. Mol. Endocrinol. 23, 167-176.
Mita, M., Saneyoshi, M., Yoshikuni, M. and Nagahama, Y.
(1999). A methyl donor for 1-methyladenine biosynthesis in starfish ovarian follicle cells. Mol. Reprod.
Develop. 54, 63-68.
Mita, M., Yasumasu, I., Yoshikuni, M. and Nagahama, Y.
(1999).
1-Methyladenine production from ATP bystarfish ovarian follicles. Biochem. Biophys. Acta 1428,
13-20.
Oba, Y., Hirai, T., Yoshiura, Y., Yoshikuni, M., Kawauchi,

H. and Nagahama, Y. (1999). Cloning, functional
characterization and expression of a gonadotropin receptor eDNA in the ovary and testis of amago salmon(Oncorhynchus rhodurus). Biochem. Biophys. Res.
Comm. 263, 584-590.
Oba, Y., Hirai, T., Yoshiura, Y., Yoshikuni, M., Kawauchi,
H. and Nagahama, Y. (1999). The duality of fish gonadotropin receptors: cloning and functional characterization of a second gonadotropin receptor eDNA expressed in the ovary and testis of amago salmon (Oncorhynchus rhodurus). Biochem. Biophys. Res. Comm.
265, 366-371.
Osaki, A., Okida, N., Ishikawa, K., Tokumoto, M., Nagahama, Y., Lippert, T.H., Voelter, W. and Tokumoto, T.
(1999). Identification of ubiquitin in seminal plasma
from tilapia, Oreochromis niloticus. Biomed. Res. 20,
249-252.
Todo, T., Ikeuchi, T., Kobayashi, T. and Nagahama, Y.
(1999). Fish androgen receptor: eDNA cloning, steroid
activation of transcription in transfected mammaliancells, and tissue mRNA levels. Biochem. Biophys. Res.
Comm. 254, 378-383.
Tokumoto, M., Horiuchi, R., Nagahama, Y., Ishikawa, K.
and Tokumoto, T. (1999). Two proteins, a goldfish
208 proteasome subunit and the protein interacting with
268 proteasome, change in the meiotic cell cycle. Eur.
J. Biochem. 266, 1-8.
Tokumoto, M., Horiuchi, R., Nagahama, Y. and Tokumoto,T. (1999). Identification of the Xenopus 208 proteasome a4 subunit which is modified in the meiotic cell
cycle. Gene 239, 301-308.
Tokumoto, M., Nagahama, Y. and Tokumoto, T. (1999).
Molecular cloning of eDNA encoding a cyclin-sensitive
ubiquitin carrier protein (E2-C) from goldfish ( Carassius
auratus) and expression analysis of the cloned gene.
FEES Letters 458, 375-377.
Tokumoto, T., Tokumoto, M., Seto, K., Horiguchi, R.,
Nagahama, Y., Yamada, S., Ishikawa, K. and Lohka,
M.J. (1999). Disapperance of a novel protein component of the 268 proteasome during Xenopus oocyte
maturation. Exp. Cell Res. 247,313-319.
Watanabe, M., Tanaka, M., Kobayashi, D., Yoshiura, Y.,
Oba, Y. and Nagahama, Y. (1999). Medaka (Oryzias
latipes) FTZ-Fl potentially binds to promoter regions of
P-450 aromatase: eDNA cloning and functional characterization. Mol. Cell. Endocrinol. 149,221-228.

FOR BASIC BIOLOGY29

DIVISION OF CELL DIFFERENTIATION
Professor:
Research Associate:
Technical Staff:

Postdoctoral Fellow:
Graduate Student:

Visiting Fellow:

Ken-ichirou Morohashi
Akihiko Shimono
Satoru Ishihara
Sanae Oka
Hisae Tsuboi
Yuko Shinohara I)
Yoshie Takeda
Masatomo Kusaka
Tomoko Imura 2)
Akiko Iizuka JJ
Naohito Nakamura 4)
Ken Kawabe 5J
Tokuo Mukai 5J
Hirofumi Mizusaki
Ryouji Matsushima 6)
Akira Namba lJ
Yoshiyuki Kojima 8)

IJJSPS technical staff
2) JSPS secretary
3)NJBB Postdoctoral Fellow

JJSPS Postdoctoral Fellow
JGraduate School ofKyushu University
6JGraduate School ofNagoya University?)Graduate School ofTokyo University8) Nagoya City University
4

5

From an aspect that differentiation of cells is regulated at least in part by the functions of cell type- or tissuespecific transcription factors, mechanisms underlying
differentiation of the steroidogenic tissues such as the
gonads (testis and ovary) and adrenal cortex have been
under investigation in our division. Based on observations obtained in the past decade, we started in Okazaki new projects to understand mechanisms underlying
sex-differentiation of the gonads and mechanisms of
sex-differentiation of brain functions necessary forreproductive behaviour. To address these issues, wehave focussed our attention on the mechanisms how
steroidogenic-tissue specific transcription factors regulate their target genes which are essential for the tissue
differentiation and functions, and how the genes encoding the transcription factors are regulated during thetissue differentiation. In addition to the studies above,
Dr. Shimono joined to our division as a research associate from April 1999 and has started to investigate
mechanisms underlying head formation.
I. Gene regulatory cascade in the steroidogenic

tissue differentiation
When a differentiation process of a tissue is considered, it is reasonable to assume a gene regulatory cascade specific for each tissue in which certain genes
encoding transcription factors are involved as the components. In the cascades required for adrenal andgonadal differentiation, Ad4BP/SF-l is locates upstream of some tissue-specific genes, including thesteroidogenic CYP genes, and locates downstream ofother transcription factors regulating the Ad4BP/SF-lgene. Considering that the cascade flows from upstream to downstream during the tissue differentiation

and Ad4BP/SF-l is an essential transcription factor forthe tissue differentiation, identification of the components consisting the cascade as well as their genetical
relationship in the cascade are essential for fully understanding the mechanisms of the tissue differentiation.
From these points of view, the regulatory region of
the Ad4BP/SF-l gene was analysed by making transgenic mice or with cultured cells. However, our invivo investigation in a recent few years has not yet beensuccessful, probably because the regulatory region
locates far upstream or far downstream from the structural gene of Ad4BP/SF-l. On the contrary, our invitro study with cultured cells provided a novel mechanism regulating the Ad4BP/SF-l gene, which will be
expected to give us a new insight into metabolism of
biologically active lipophylic compounds.
Dax-1 is another transcription factor of our interest,
which is also implicated in the steroidogenic tissue
differentiation. Although our previous study revealed
that the factor acts as a suppressor of Ad4BP/SF-l,regulation of the suppressive effect has not yet clarified
at the molecular leveL We recently uncovered thefunction of the amino terminal half of Dax-1 containing
a unique repeated sequence instead of Zn-fmger DNA
binding domain, which enables us to understand how
Dax-1 suppresses the function of Ad4BP/SF-l and how
the activity ofDax-1 is regulated.
In addition to these transcription factors, factors suchas Sox-9, Wt-1, Emx-2, and GATA-4 are known to be
implicated in the gonad development. In order toisolate other factors interacting with the transcription
factors above, yeast two-hybrid screening was performed with a eDNA library constructed with an mRNA
prepared from mouse foetal gonads.
Extensive
screening resulted in isolation of interacting molecules
including coactivators and other type of transcription
factors, some of which were novel factors. Distributions and functions of these interacting molecules havebeen examined.

II. Sex-differentiation observed in adrenal cortex
Our previous study indicated that Ad4BP/SF-l is expressed in all three zones of the adrenal cortex while
Dax-1 is expressed in only outer zone, the zona glomerulosa, but not in inner zones, the zona fasciculata
and reticularis. However, this distribution revealed byimmunohistochemistry was quite distinct from thatobtained with in situ hybridization. To explain thediscrepancy between the two methods, close examination was carried out with a series of adrenal cortex ofboth sexes at several developing stages from foetal toadult. Although the distribution of Dax-1 was identical between the two sexes before puberty, distinct distribution was clearly observed after sexual maturation.
This sexually dimorphic expression disappeared bycastration and emerged again after testosterone replacement. Injection of testosterone into female micemake the expression profile altered into that of male.
Taken together, our in vivo studies suggested that an-

30

NATIONAL INSTITUTE

drogen and its receptor downregulate Dax-1 gene transcription, which is interestingly inconsistent with acommon understanding that androgen receptor activatestarget gene transcription in a ligand dependent manner.
The mechanism of suppression of the Dax-1 expression
by androgen receptor and its ligand is further investigating at a molecular level.
Investigations of head formation and brain sex differentiation are included as the targets of our study. These studies, same as the studies described above, havebeen performed based on examination of the functionsof transcription factors. Uncovering the molecularmechanisms will be elucidated by these efforts in thenear feature.

Publication List:
Morohashi, K., Tsuboi-Asai, H., Matsushita, S., Suda, M.,
Nakashima, M. , Sasano, H. , Hataba, Y., Li, C-L., Fukuta,J., Irie, J., Watanabe, T., Nagura, H., & Li, E. (1999)

Male

Fig.

Structural and Functional Abnormalities in the Spleen of
mFtz-Fl Gene Disrupted Mouse. Blood 93, 1586-1594.

Kawabe, K. , Shikayama, T., Tsuboi, H. , Oka, S., Oba, K.,
Yanase, T., Nawata, H., & Morohashi, K. (1999) Dax-1
as One of the Target Genes of Ad4BP/SF-l. Mol. Endocrinol. 13, 1267-1284.
Kabe, Y., Goto, M., Shima, D., Imai, T., Wada, T., Morohashi, K., Shirakawa, M., Hirose, H., & Handa, H.
(1999) The Role of Human MBF1 as a Transcriptional
Coactivator. J. Bioi. Chern. 274, 34196-34202.
Nagamine, C.M., Morohashi, K., Carlisle, C., & Chang,
D.K. (1999) Sex Reversal Caused by Mus musculus domesticus Y Chromosomes Linked to Variant Expressionof the Testis-Determining Gene Sry. Dev. Bioi. 216, 182194.
Morohashi, K. (1999) Gonadal and Extragonadal Functionsof Ad4BP/SF-1 -Developmental Aspects-. Trends in Endocrinol. Metab . 10, 169-173.

FemaleSex-dependent distribution ofDax-1 in mouse adrenal cortex.

Adult mouse adrenal glands from both sexes were immunostained with an antiserum for Dax-1.

31

FOR BASIC BIOLOGYDIVISION OF MORPHOGENESIS
Professor:
Associate Professor:
Research Associates:
Technical Stuff:

JSPS Postdoctoral Fellows:
Graduate Students:

Naoto Ueno
Hiroshi Shibuya
Makoto Nakamura
Makoto Mochii
Chiyo Takagi
Takamasa Yamamoto
AkikoHyodo
Miho Shimizu*
Shun-ichiro lemura
Kiyokazu Morita
Michiru Nishital)
Katsura Sugawara lJ
Satoru Yoshida 2J
Yasunori Tomoyasu 2J
Tomoko Kurata 2J
Bisei Nakano 2J
Masaki Takeuchi2J
Jyunko Hyodo 3J
Shin-ichi Nagai 3J
Yayoi Nishida 3J*until August, 1999
IJ Graduate School of Hokkaido University
2) Graduate University for Advanced Studies3) Graduate School ofNagoya University

The complex morphogenesis of organisms 1sachieved by consecutive cell-to-cell interactions ofgerm layers as well as tissues during development.
Recent studies suggest that several groups of molecules
called polypeptide growth factors (PGFs) are essential
component controlling such intercellular communications in a variety of organisms. These cell communications via PGFs are regulated by a number of processes
including secretion, activation, diffusion, reception by
specific receptors and intracellualr signaling of PGFs.
Our main interest is to know how pattern formation inmorphogenesis is regulated by PGFs during development. We address this problem using several model
animals, including frog, fly and worm with the view ofextracellular and intracllular signaling of PGFs. We
employ embryology, genetics, molecular and cellularbiology and biochemistry. In addition, we have recently
introduced a systematic and comprehensive way toscreen target genes of PGFs using arrayed cDNAs.

I. Hierarchy of homeobox genes in pattening embryo
Embryos are patterned along dorso-ventral (DV) axis
by the action of PGFs. Signaling triggered by PGFs
leads to the activation of their target genes. Several
homeobox genes are known to be induced in response
to PGFs in early Xenopus development. In particular,
Xmsx-1, an amphibian homologue of vertebrate Msx1, is well characterized as a target gene of bone morphogenetic protein (BMP). In order to clarify molecularbasis for ventalization by BMP and in vivo siginificanceof Msx-1, we examined whether Xmsx-1 activity isrequired in the endogenous ventralizing pathway, using
a dominant-negative form of Xmsx-1 (VP-Xmsx-1),
which is a fusion protein with the virus-derived VP16

activation domain. Interestingly, VP-Xmsx-1 induced asecondary body axis, complete with muscle and neuraltissues, when the fusion protein was overexpressed in
ventral blastomeres. The result suggests that Xmsx-1
activity is necessary for both mesoderm and ectoderm
to be ventralized. We also examined the epistatic relationship between Xmsx-1 and another ventralizing
homeobox protein Xvent-1 and showed that Xmsx-1 islikely to be acting upstream of Xvent-1. We proposethat Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation ofXvent-1.

II. Cross-talk of growth factor signaling in early
development
Signals generated by the members of the Wnt and
TGF-~ superfamilies mediate a diverse array of biologi-

cal responses. These secreted factors, via activation ofcell surface receptors, participate in the regulation ofcell fate specification and control cell proliferation
during development and tissue maintenance. In theearly amphibian embryo (Xenopus laevis), both Wnt
and TGF-~ superfamily signaling cascades are required
for establishment of a dorsal signaling center,
Spemann's organizer. In Xenopus, as in other systems,
both signaling pathways utilize extranuclear proteins
which, upon activation, translocate to the nucleus toparticipate in transcriptional complexes.
Wnt signaling is mediated by translocation of ~-catenin into the
nucleus together with members of the Lefl/Tcf class of
HMG box transcription factors to activate gene expression. Likewise, TGF-~ superfamily signaling is mediated by "pathway-specific" Smads which translocate
into the nucleus together with the "universal Smad"
Smad4 to activate gene expression. Recently, we havedemonstrated that Lefl/Tcf, a downstream componentof the Wnt signaling cascade can physically interact
with Smad4. In Xenopus, this interaction directly
affects transcriptional activity of the twin (Xtwn) geneduring Spemann's organizer formation. This is thefirst demonstration of a physical interaction between
TGF-~ and Wnt signaling components, and may represent a direct connection between the TGF-~ and Wnt
signaling pathways.

III. Genetic dissection of TGF-13 signaling in
Drosophila model system
We have reported that the morphogen gradients of
Dpp and Wg provide positional information to cells in
the notum region of the wing imaginal disc. dpp is induced at AlP boundary by a Hh dependent mechanism
in wing and leg discs, however, wg is induced in various ways. We revealed that initial notal wg induction isregulated by the function of two transcription factors,
Pannier and U-shaped. We also found that expression ofboth pnr and ush is regulated by Dpp signaling. We
propose that the Dpp gradient is utilized not only foranterior/posterior patterning but also contributes for
dorsal/ventral patterning through the induction of wg.

32

We also analyzed in vivo function of TAKl (dTAKl) in
Drosopihla. We generated transgenic flies which express vertebrate or Drosophila homolog of TAKl. Genetic and biochemical analyses indicated that the c-Junamino-terminal kinase (JNK) signaling pathway is
specifically activated by TAKl signaling. Expression ofa dominant negative form of dTAKl during embryonicdevelopment resulted invarious embryonic cuticle defects including dorsal open phenotypes. Our results
suggested that TAKl functions as a MAPKKK in the
JNK signaling pathway and participates in such diverseroles as control of cell shape and regulation of apoptosis
in Drosophila .

IV. TGF -~ family in nematode
Nematode C. elegans provides powerful genetic approaches to understand the role and mechanism ofTGF~ family signaling. We have identified a new member of
the TGF- ~ superfamily, CET-1 , from C. elegans. cet-1
null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants. Genetic
analysis suggested that CET-1 regulated body lengthand male ray pattern functioning as ligand in sma path-

NATIONAL INSTITUTE

way. cet-1 is mainly expressed in the ventral nerve cordand other neurons which are thought to secrete CET-1
ligand and induce specific gene expression in target
cells. To elucidate molecular mechanism regulating C.
elegans body length, we employed a differential hybridization of a eDNA array for identification of target
genes of the CET-1 signal.
C. elegans cDNAs representing 7,584 independent
genes were arrayed on a nylon membrane at high density, and hybridized with 33 P-labeled DNA probes synthesized from the rnRNAs of wild-type, cet-1, sma-2,
and lon-2 worms (Fig. 1, top). Signals for all the spots
representing hybridized DNA were quantified and compared between strains (Fig. 1, bottom). The screening
identified 22 and 2 clones, which were positively andnegatively regulated, respectively, by the CET-1 signal.
Northern hybridization confinned the expression profiles of most of the clones indicating good reliability ofthe differential hybridization using arrayed cDNAs. In
situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed thatat least 4 genes, including the gene for the type I receptor for CET-1 , sma-6, were transcriptionally regulated by the CET-1 signal.

Fig. 1. Screening for TGF-~-regulated genes in C. elegans. (Upper panel) Change of body length depending on cet-1 gene doses. Cet-1 overexpressing, wild-type, and cet-1 null mutant worms from top. (Bottom panel) Macroarray of C. elegans cDNAs. Hybridization signals were compared between wild-type and cet-1 null mutant worms. The arrowhead points a DNA spot down-regulated in the mutant.

FOR BASIC BIOLOGY

Publication List:
Adachi-Yamada, T., Nakamura, M., Irie, K., Tomoyasu, Y.,
Sano, Y., Mori, E., Goto, S., Ueno, N., Nishiada, Y.,
Matsumoto, K. (1999) p38 MAP kinase participates in
TGF-~ superfamily signal transduction in Drosophila
wing morphogenesis. Mol. Cell. Bio/.19, 2322-2329.
Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M.,
Nagai, S., Fujita, F., Shibuya, H., Ueno, N. and Akiyama,
T. (1999) Negative regulation of the Wingless signaling
by a Drosophila homologue of Axin. Science 283, 17391742.
Hamada, F., Murata, Y., Nishida, A., Fujita, F., Tomoyasu,
Y., Nakamura, M., Toyoshima, K., Tabata, T., Ueno,
N., Akiyama, T. (1999) Identification and characterization of E-APC, a novel Drosophila homologue of thetumour suppressor APC. Genes Cells 4, 465-474.
Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita,M., Fujisawa, J., Shibuya, H., Matsumoto, K. and Nishida, E. (1999) Involvement of the p38 MAP kinase
pathway in TGF-~-induced gene expression. J. Bioi.
Chern. 274,27161-27167.
Hirota, Y., Okabe, M., Imai, T., Kurusu, M. Yamamoto, A.,
Miyao, S., Nakamura, M., Sawamoto, K., Okano, H.
(1999) Musashi and Seven in absentia downregulate
Tramtrack through distinct mechanisms in Drosophila
eye development. Mech. Dev. 87(1-2) 93-101.
Iemura, S., Yamamoto, T. S., Chiyo, T. Kobayashi, H. andUeno, N. (1999) Isolation and characterization of bonemorphogenetic proteins from the early Xenopus embryo.
J. Bioi. Chern. 274,26843-26849.
Ishitani, T., Ninomiya-Tsuji, J., Nagai, S., Nishita, M.,
Meneghini, M., Baker, N., Waterman, M., Bowerman,
B., Clevers, H., Shibuya, H. and Matsumoto, K. (1999)
The TAKI-NLK MAP kinase-related pathway antagonizes ~-catenin-TCF signalling. Nature 399, 798-802.
Masuyama, N., Hanafusa, H., Kusakabe, M., Shibuya, H.
and Nishida, E. (1999) Identification of two Smad4s in
Xenopus: Their common and distinct properties. J.
Bioi. Chern. 274, 12163-12170.
Miyama, K., Yamada, G., Yamamoto, T., Takagi, C.,
Miyado, K., Sakai, M., Ueno, N. and Shibuya, H. (1999)
A BMP-inducible gene, Dlx-5 regulates osteoblast differentiation and mesoderm induction. Dev. Bioi.
208,123-133.
Mochii, M., Yoshida, S., Morita, K., Kohara, Y. and Ueno,
N. (1999) Identification of transforming growth factor~-regulated genes in Caenohabtidis elegans by differen-

33
tial hybridization of arrayed cDNAs. Proc. Nat!. Acad.
Sci. USA 96, 15020-15025.
Morita, K., Chow, K. and Ueno, N. (1999) Body lengthand male tail ray pattern formation of Caenohabditis
elegans are regulated by a mamber of TGF-~ family.
Development 126, 1337-1347.
Nagaso, H. Tada, M., Nikaido, M. and Ueno, N. (1999)
Dual specificity of activin type II receptor ActRIIb in
dorso-ventral patterning during zebrafish embryogeneisis. Dev. Growth Diff. 41, 119-133.
Nikaido, M., Masazurni, T. and Ueno, N. (1999) Restricted
expression of the receptor serine/threonine kinase
BMPR-IB in zebrafish. Mech. Dev. 82, 219-222.
Nikaido, M., Tada, M., Takeda, H., Kuroiwa, A. and Ueno,
N. (1999) 1n vivo analyisis using variants of zebrafish
BMPR-IA: range of action and involvement of BMP in
ectoderm patterning. Development 126, 181-190.
Nishita, M., Ueno, N. and Shibuya, H. (1999) Smad 8B
splice variant lacking the SSXS site that inhibits Smad8
mediated signalling. Genes Cells 4, 583-591.
Nishita, M., Hashimoto, M. K., Ogata, S., Laurent, M. N.,
Ueno, N. Shibuya, H. and Cho, K.W.Y. Interaction between Wnt and TGF-~ signalling pathways during formation ofSpemann's organizer. Nature (in press)
Shimasaki, S., Zachow, R. J., Li, D., Iemura, S., Ueno, N.,
Chang, R. J., Sampath, K., and Erickson, G. F. (1999)
Expression of a functional bone morphogenetic protein(BMP) system in the ovary. Proc. Nat!. Acad. Sci. USA
96,7282-7287.
Suzawa , M., Takeuchi, Y., Fukumoto, S., Kato, S., Ueno,
N., Miyazono, K., Matsumoto, T., Fujita, T. (1999) Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro. Endocrinology 140,2125-2133.
Takatsu, Y., Nakamura, M., Stapleton, M. Danos, M. C.,
Matsumoto, K. O'Connor, M. B., Shibuya, H. and Ueno,
N. TAKl participates in JNK signaling during Drosophila development. Mol. Cell. Bioi. (in press)
Yamaguchi, K., Nagai, S-I., Ninomiya-Tsuji, J. Noshita,M., Tarnai, K., Irie, K., Ueno, N., Nishida, E., Shibuya,
H. and Matsumoto, K. (1999) XIAP, a cellular memberof the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.
EMBOJ.18, 179-187.
Yamamoto, S.T., Takagi, C. and Ueno, N. Requirement of
Xmsx-1 in the BMP-triggered ventralization of Xenopus
embryos. Mech. Dev. (in press)

34

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL BIOLOGY

concerning the gene expression, in vitro flowering andchlorophyll content in leaves of in vitro-cultured plant.

(ADJUNCT)
Professor:

Kenzo Nakamura(Nagoya University)
Associate Professor: Tsukaho Hattori (Mie University)
Assistant Professor: Masa-aki Ohto
Etsuko Aoki
Technician Staff
Yasuko Furukawa
Postdoctoral Fellow: Kiyoshi Onai
Graduate Students: Yasuhiro Koide (Nagoya University)
Yukiko Iwata (Nagoya University)
Shingo Hayashi (Nagoya University)

Availability of sugars and the inter-organ transportand distribution of sugars are essential in the growthand development of the plant body. Expression of avariety of plant genes is regulated, either positively or
negatively depending on the gene, by the level of sugars
at the sugar-importing sink sites as well as at the sugarexporting source sites of the plant body. Thus sugars are
not only important as sources for cellular energy and thesynthesis of macromolecules but also as a signal
controlling the growth and development of plants bychanging the pattern of gene expression. Our researchattention is focused to elucidate the mechanisms
involved in the regulation of gene expression in
response to sugars, especially the activation of gene
expression by increased-levels of sugars, and the role ofsuch regulation in the organ development in plants. In
addition, mechanisms involved in the accumulation ofstorage proteins in plant vacuoles are also studied.

I. Molecular and genetic approaches for the
analysis of the sugar-signalling during growth anddevelopment of Arabidopsis thaliana.
During the growth of plants, new organs develop as
carbohydrate sink, and many vegetative organs shows
sink to source transition after their maturation. Many
aspects of the organ development in higher plants arethought to be affected, to some degree, by the levels ofsugars. Sugars seem to have influence on themeristematic transition in long day plants fromvegetative to reproductive growth. To obtain insights
into the role of sugar-regulated gene expression in thegrowth and organ development in plants, we arescreening for mutants of Arabidopsis with defects oranomalies both in the sugar-regulated gene expression
and in the developmental processes such as leaf
development and the determination of the flowering
time. To aid this purpose, we have established morethan 7,000 independent lines of Arabidopsis plants
transformed with T-DNA containing multiple copies ofthe enhancer sequence. We have identified more than 20of mutant lines with defects in the development ofleaves or anormalies in the flowering time which also
show the altered patterns of the expression of sugarinducible genes, such as ~-amylase gene (Atf3-Amy)(Fig. 1). The mutants were named as uns (unusual
sugar response) after their abnonnal responses to sugars

II. Regulatory factors involved in the sugarinducible expression of plant genes
Expression of genes coding for sporamin and ~­amylase, two major proteins of the storage roots ofsweet potato, is inducible by high levels of sugars invarious vegetative tissues. The GUS reporter genes
under the control of the promoters of these genes arealso inducible by sugars in leaves of transgenic tobacco
plants, and these fusion genes are expressed in tubers oftransgenic potato plants. Although the induction of
expression of these fusion genes requires the activity ofhexokinase, phosphorylation of hexose by hexokinase is
not sufficient to cause the induction. The induction
requires ca2+-signalling and the activity of protein
kinase.
Eight different cDNAs for the isoforms of calciumdependent protein kinase (CDPK) were isolated fromleaves of tobacco, and transcripts of two of them were
found to be increased upon treatment of leaves withvarious metabolizable sugars. Antibodies against afragment of one of these isofonns cross-reacted strongly
with the 57 kDa-protein in the soluble fraction from theyoung leaves. The level of this 57 kDa-proteiu
decreased significantly as leaf matures, while the levelof this 57-kDa protein in mature leaves increased
significantly after the treatment of leaves with sugars.
The sugar-induction of the 57-kDa protein occured
preceding the induction of expression of the ~­amylase:GUS reporter gene. In addition, a 54 kDaprotein with autophosphorylation activity in the plasma
membrane of mature leaves also increased significantly
upon treatment of leaves with sugars. This protein was
purified to about 1,000-fold compared to the crudeNT

WID Sbc

Qc

Rc

Fig.! Phenotypes of wild type and homozygous mutant line
(u ns l) grown for 3 weeks. The chlorophyll content of the mutant
plants (Fig. lA, right) is much lower than that of wild type plants(Fig. I A, left). Mutant plants also showed the reduced levels of
the sugar-inducible increase of ~-a mylase (Fig.IB, left) andanthocyanin (Fig.IB, right) when leaf explants were treated withhigh levels (5 %) of sugars.

FOR BASIC BIOLOGY

extract. It phosphorylated histone IllS in a ca2+dependent manner and cross-reacted with an antibody
against CDPK of Arabidopsis thaliana. These results
suggest the possible involvement of CDPKs in thesugar-inducible gene expression and the development ofleaves.

Selected Publication:
Iwata, Y., Kuriyama, M., Nakakita, M., Kojima, H., Ohto,

35
M. and Nakamura, K., (1998) Charaqterization of a
calcium depedent protein kinase of tobacco leaves that isasociated with the plasma membrane and is inducible bysucrose. Plant Cell Physiol. 39(11): 1176-1183.
Koide, Y., Matsuoka, K., Ohto, M. and Nakamura, K.,(1999) TheN-terminal propeptide and the C terminusof the precursor to 20-kilo-dalton potato tuber protein
can function as different types of vaculor sorting signals.
Plant Cell Physiol. 40(11): 1152-1159.

NATIONALINSTITUTE37

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman:

Masaharu Noda

The Department is composed of two regular divisions and two adjunctdivisions.

The study of this department is focused on molecular mecha-

nisms for the development of central nervous systems in vertebrates, andalso on molecular mechanisms for the response of plants toward external
environments, such as light, temperature and salinity.

38

NATIONAL INSTITUTEDIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Associate Professor:
Research Associates:

Masaharu Noda
Nobuaki Maeda
Takafumi Shintani
Junichi Yuasa
Technical Staff:
Shigemi Takami
Hiroko Iyoda (- Jyn. 31, 2000)
Post Doctoral Fellows:Akihiro Fujikawa
Hiroyuki Kawachi4
Hiraki Sakuta4
Mohamad Zubair4
Minoru Ujita4
Graduate Students:
Chika Saegusa
AkiraKato
Ryoko Suzuki
Masahide Fukada
Takeshi Hiyama
Hiroshi Tamura
Takeshi Ohkawara (Oct. I, 1999-)
Visiting Scientist:
Ikuko Watakabe (-Sept. 31, 1999)
JST Technical Staff:
Masae Mizoguchi
Megumi Goto
Minako Ishida

We have been studying the molecular and cellularmechanisms underlying the development and functioning of the vertebrate central nervous system. We are
currently searching for and analyzing the functions ofmolecules involved in various cellular events in brain
morphogenesis and brain function, such as generationof neuroblasts, migration to form the laminar structure
and various nuclei, elongation and path-finding of neural processes, the formation and refmement of specificconnections between neurons, and also synaptic plasticity. We have been using various techniques including molecular biology (e.g. eDNA cloning, site-directedmutagenesis Yeast two-hybrid system), biochemistry·(protein, carbohydrate), immunological methods(monoclonal-antibody production), neuroanatomy, celland organotypic culture (immortalized cell-line production), and embryo manipulation (classical embryology,
gene transfer with viral vectors, and gene targeting).

I. Molecular mechanism of retinotectal map formation
Topographic maps are a fundamental feature of neuralnetworks in the nervous system. Understanding the molecular mechanisms by which topographically ordered
neuronal connections are established during development
has long been a major challenge in developmental neurobiology. The retinotectal projection of lower vertebrates
including birds has been used as a readily accessible model
system. In this projection, the temporal (posterior) retina
is connected to the rostral (anterior) part of the contralateral optic tectum, the nasal (anterior) retina to the caudal(posterior) tectum, and likewise the dorsal and ventral
retina to the ventral and dorsal tectum, respectively. Thus,
images received by the retina are precisely projected onto
the tectum in a reversed manner. In 1963, Sperry proposed that topographic mapping could be guided by complementary positional labels in gradients across pre- andpostsynaptic fields. Although this concept is widely
accepted today, and Eph families of receptor tyrosine kinases and their ligands were recently identified as candidates for such positional labels, the molecular mechanismof retinotectal map formation remains to be elucidated.
Since 1993, we have been devoting our efforts tosearching for topographic molecules which show
asymmetrical distribution in the embryonic chick retina.
In the first-round screening, using a eDNA subtractive
hybridization technique, we identified two winged-helix
transcriptional regulators, CBF-1 and CBF-2, expressedin the nasal and temporal retina, respectively. Furthermore, our misexpression experiments using a retroviral vector suggested that these two transcription factors determine the regional specificity of the retinal
ganglion cells, namely, the directed axonal projections
to the appropriate tecta! targets along the anteroposterior axis. We examined whether CBF-1 and CBF-2
control the expression ofEphA3, a promising candidate
for a topographic guidance label in the retina, using in
ovo electropolation. We obtained results suggestingthat these two transcription factors affect the topographic expression of EphA3 indirectly.
In order to further search for topographic moleculesin the embryonic retina, we performed a large-scale
screening using a new eDNA display system called
Restriction Landmark eDNA Scanning {RLCS). A
number of molecules displaying various asymmetricalexpression patterns along the nasotemporal axis or
dorsoventral axis in the retina have been identified.
These included already known topographic molecules
such as EphA3, CBF-2, etc, expressed along the nasatemporal axis, and ephrin-B2, EphB3, etc, expressedalong the dorsoventral axis. We have identified all of
the eDNA clones isolated by this screening and examined their expression patterns during development.
Furthermore, with respect to the topographic molecules
which might have important roles in formation of theretinotectal map and/or the neural network in the retina,
we conducted over- and misexpression experiments
using viral vectors and in ovo electropolation to elucidate the molecular function. In 1999, with the assistance of a computer image-processing software (Fig. 1),
we found additional-30 and -80 candidate eDNA spots
for topographic molecules along the nasotemporal anddorsoventral axes, respectively. Currently, projects to
identify them and analyze the expression pattern are
underway.
We expect that our studies will lead to elucidation of
the molecular mechanism underlying formation of theregional specificity in the retina, and ultimately to uncovering the basic principles for establishing complicated but extremely precise neural networks.

II. Functional roles of protein tyrosine phosphatase~

Protein tyrosine phosphorylation plays crucial roles
in various aspects of brain development. The level oftyrosine phosphorylation is determined by the balance

FOR BASIC BIOLOGY

39

.·
B

Fig. 1 Genes expressed in a region-specific fashion in thechick retina(A) RLCS profiles for the dorsal and ventral retina. The dorsal profile (black) was overlaid with the ventral profile (green)using an image-processor. Dorsal specific clones are detectable as rare black spots among abundant green spots.
(B) Region-specific gene expression in the retina. Wholemount in situ hybridization showed that these genes were
specifically expressed in the nasal, temporal, dorsal or ventral
region of the retina, respectivel y.

between the activities of protein tyrosine kinases andprotein tyrosine phosphatases. Many types of receptor-type protein tyrosine phosphatases (RPTP) havebeen cloned and characterized. In 1994, we found that
PTPs/RPTPP , a nervous system-specific RPTP, is expressed as a chondroitin sulfate proteoglycan in thebrain. An RNA splice variant corresponding to theextracellular region of PTPs is secreted as a major proteoglycan in the brain known as 6B4 proteoglycan/
phosphacan. The extracellular region of PTPs consistsof a carbonic anhydrase-like domain, a fibronectin-type
III-like domain and a serine-glycine-rich region, whichis considered to be the chondroitin sulfate attachment
region.
In an attempt to reveal the signal transduction
mechanism of PTPs, we tried to identify the ligand
molecules of this receptor. To date, we have foundthat PTPs binds pleiotrophin!HB-GAM and midkine,
closely related heparin-binding growth factors whichshare many biological activities.
The chondroitin
sulfate portion of PTPs is essential for the high affmity
binding (Kd = - 0.25 nM) to these growth factors, andremoval of chondroitin sulfate chains results in amarked decrease of binding affinity (Kd = - 13 nM).
We further revealed that chondroitin sulfate interacts
with Arg78 in Cluster I, one of the two heparin-bindingsites in the C-terminal half domain of midkine. This is
the first demonstration that chondroitin sulfate plays animportant regulatory role in growth factor signaling.
In the embryonic rat brain, pleiotrophin and midkine
are localized along the radial glial fibers , a scaffold forneuronal migration. On the other hand, PTPs is expressed in the migrating neurons, suggesting that theligand-receptor relationship between these molecules
plays a role in migration of neurons during brain development. Thus, we examined the roles of pleiotrophinmidkine-PTPs interaction in neuronal migration using
the glass fiber assay and Boyden chamber cell migration assay. Pleiotrophin and midkine on the substratum stimulated migration of neurons in these assays.
Polyclonal antibodies against the extracellular domainof PTPs, 6B4 proteoglycan (a secreted extracellular
fom1 of PTPs) and sodium vanadate (a protein tyrosine
phosphatase inhibitor) added to the culture medium
strongly suppressed this migration. Experiments using
various midkine mutants with various affmities for
PTPs indicated that the strength of binding affinities
and the neuronal migration-inducing activities arehighly correlated. These results suggested that PTPs
is involved in migration as a neuronal receptor forpleiotrophin and midkine distributed along radial glial
fibers.
Next, in order to reveal the intracellular signalingmechanism of PTPs, we performed yeast two-hybrid
screening using the intracellular region of PTPs as bait.
By screening a rat brain eDNA library, we found that
PTPs interacts with PSD-95/SAP90 family molecules,
SAP102, PSD-95/SAP90 and SAP97/hDlg.
These
proteins are composed of three PDZ domains, a SH3
domain and a guanylate kinase-like domain, and areconcentrated in the central synapses mediating proteinprotein interactions to form large synaptic macromolecular complexes. Using the yeast two-hybrid
binding assay, we found that the C-terminal sequence of
PTPs binds to PSD-95/SAP90 proteins through thesecond PDZ domain (Fig. 2). Immunohistochemical
analysis revealed that PTPs and PSD-95/SAP90 are
similarly distributed in the dendrites of pyramidal neurons of the hippocampus and neocortex (Fig. 2). These results suggest that PTPs is also involved in theregulation of synaptic function.
To study the physiological function of PTPs in vivo,
we generated PTPs-deficient mice in which the PTPI;
gene was replaced with the LacZ gene in 1997. By
investigating the expression of LacZ in heterozygous
mutant mice, we demonstrated that neurons as well as
astrocytes express PTPs in the central nervous system.
We are cun·ently studying the phenotype of PTPsdeficient mice biochemically, anatomically, physiologically and ethologically, and have already found abnormalities in behaviour, circadian rhythm, LTP in the
hippocampus, etc.

Ill. Functional roles of subfamily 2 sodium channels
Voltage-gated sodium channels (NaChs) are responsible for generating action potentials in excitable cells
and play many important physiological roles. Cloning

NATIONAL INSTITUTE

40A
CAH FNIII

TM D1

D2

PTPI;-A2316

1. pGBT9-D1 D2C

1748 ~ 2316

2. pGBT9-01

174BC)-1 994

3. pGBT9-D2C

2003-c=J-- 2316

4 . pGBT9-C

2292 - 23 16

5 . pG BT9-C6 42292-2312

6. pGBT9·C-S2314A

2292-2316(SLV- ALV)

B

c
2
3
4
5

To date, subfamily 2 channels have not been expressedin a functionally active form using in vitro expression
systems, and therefore the functional properties of these
NaChs are not yet clear. To clarify the cells expressing
subfamily 2 channels and their physiological functions
in vivo, we planned to generate knock-out mice deficient in the channel gene.
We successfully generated mutant mice in which themouse subfamily 2 channel gene (mNaG) was replacedwith the LacZ or neo gene by gene targeting. From theanalysis of LacZ knock-in mice, we demonstrated thatmNaG gene expression is restricted to the dorsal root
ganglion and lung during the embryonic stage (see Fig.
lA in the part of Center for Transgenic Animals andPlants). During the postnatal period, in addition tothese tissues, Schwann cells in the sensory afferent
nerve fibers (ibid. Fig. lB, C) and a subset of neurons in
the central nervous system were positive for mNaG
gene expression.
We are currently examining thephenotypes of mNaG-null mutant mice to gain insight
into the physiological functions of this channel.

6

Publication List:

Fig. 2
Interaction of PTP/;; with PSD-95/SAP90
Schematic representations of PTPI;; and yeast two-hybrid constructs are shown in (A). Various bait constructs of PTPI;;
were tested for interaction with PSD-95/SAP90 by inductionof reporter genes, HIS3 (B) and LacZ (C). Numbers in (B)and (C) correspond to the construct numbers in (A). These
results and the other experiments indicated that the C-terminal
portion of PTPI;; interacts with the second PDZ domain of
PSD-95/SAP90. Immunohistochemical staining with theantibodies against PTPI;; (D) and PSD-95/SAP90 (E) indicatedthat both proteins were present in the dendrites of hippocampal pyramidal cells. Scale bars: 40 j.lm.

of NaChs revealed marked conservation in primarystructure that underlies their functional similarity.
Thus, all NaChs cloned had been grouped into a single
gene family. However, recently, novel NaChs, humanNav2.1 , mouse Nav2.3/mNaG and rat SCLll/NaG,
were cloned from inexcitable cells such as glial cells.
These molecules closely resemble each other but are
divergent from the previously cloned sodium channels
even in the regions involved in activation, inactivationand ion selectivity. Thus, these molecules have beengrouped into a new subfamily of NaChs (subfamily 2).

Takahashi, M., Yamagata, M. and Noda, M. (1999)
Specific expression of ezrin, a cytoskeletal-membrane
linker protein, in a subset of chick retinotectal and sensory projections. Eur. J. Neurosci. 11, 545-558.
Yamakawa, T., Kurosawa, N., Kadomatsu, K ., Matsui, T.,ltoh, K., Maeda, N., Noda, M., and Muramatsu, T.
(1999) Levels of expression of pleiotrophin and protein
tyrosine phosphatase 1;, are decreased in human colorectal cancers. Cancer Letter, 135, 91-96.
Maeda, N., Ichihara-Tanaka, K. , Kimura, T., Kadomatsu,
K., Muramatsu, T. and Noda, M. (1999) A receptor-likeprotein tyrosine phosphatase PTP1;,/RPTPI3 binds a heparin-binding growth factor midkine: Involvement of arginine 78 of midkine in the high affinity binding to PTP1;,.
J. Biol. Chern. , 274, 12474-12479.
Revest, J.-M., Faivre-Sarrailh, C. , Maeda, N ., Noda, M. ,Schachner, M. and Rougon, G. (1999) The interaction
between F3 immunoglobulin domains and protein tyrosine phosphatase 1;,113 triggers bidirectional signalling
between neurons and glial cells. Eur. J. Neurosci. 11,1134-1147.
Kawachi, H., Tamura, H., Watakabe, 1., Shintani, T.,
Maeda, N. and Noda, M. (1999) Protein tyrosine phosphatase 1;,1RPTPI3 interacts with PSD-95/SAP90 family.
Mol. Brain Res., 72, 47-54.
Yamagata, M., Mai, A. , Pollerberg, G.E. and Noda, M.
(1999) Regulatory interrelations among topographic
molecules CBF I, CBF2 and EphA3 in the developing
chck retina. Dev. Growth Differ. 41, 575-587.
Meng, K., Rodorigues-Peiia, A., Dimitrov, T., Chen, W.,
Yamin, M., Noda, M. and Deuel, T.F. (2000) Pleiotrophin signals increased tyrosine phoshorylation of 13catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase1311;,. Proc. Nat!. Acad.Sci. USA 97, 2603-2608.

FOR BASIC BIOLOGY

Shintani, T., Maeda, N. and Noda, M. (2000) Receptorlike protein tyrosine phosphatase "{ (RPTPy), but not
PTPQRPTPp, inhibits NGF-induced neurite outgrowthin PC12D cells. Dev. Neurosci., in press.
Nishiwaki, T., Maeda, N., and Noda, M. (1999) Characterization and developmental regulation ofproteoglycantype protein tyrosine phosphatase siRPTPP isoforms. In
Neural Development (Uyemura, K., Kawamura, K., andYazaki, T. eds.) pp.291-297. Springer-Verlag Tokyo.

I

I.

41

42

NATIONAL INSTITUTEDIVISION OF CELLULAR REGULATION
Norio Murata
KojiMikami
Atsushi Sakamoto
Yoshitaka Nishiyama!wane Suzuki
Hideko Iinuma
Technical Staff:
Mikio Kinoshita
Institute Research Fellow:
Monbusho Foreign Scientists: Suleyman I. Allakhverdiev1!
Dmitry A. Los2!
Vladimir Shuvalovl)
Richard A. Dilley!
JSPS Visiting Scientists:
Vyacheslav V. Klimovl)
Silvia Franceschelfi4!
EU Fellow:
Masami lnaba
HFSP Fellow:
Tony H.H. Chen 5!
Visiting Scientists:
Gabor Galiba 6!
Adam Gilmore 7!
Cai-Xia Hou 8!
Alexander Ivanov!
Larissa Kisseleva 2!
Sachio Miyairi 10!
Sayamrat Panpoom 11!
George C. Papageorgiou 12!
Hiroshi Yamamoto
Graduate Students:
Yuji Tanaka
Ryoma Suzuki
Yu Kanesaki

Professor:
Associate Professor:
Research Associates:

JJ from

the Institute of Basic Biological Problems, Pushchino, Russia~from the Institute of Plant Physiology, Moscow, Russia
1 from Purdue University, Lafayette, IN, USA
4! from the International Institute of Genetics and Biophysics, Naples, Italys; from Oregon State University,, Corvallis OR USA
6)''from the Agricultural Research Institute of the Hungarian Academy of Sciences, Martonwisar, Hungary
7! from the Australian National University, Canberra, Australia
8; from Xinjiang University, Urumqi, China
9! from the University of Western Ontario, London, ON,
Canadaw; from the National Institute of Bioscience and HumanTechnology, Tsukuba, Japann; from the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand
12! from the National Center for Scientific Research "Demokritos ", Athens, Greece

The research efforts of this division are aimed at developing a full understanding of the molecular mechanisms by which plants are able to acclimate to and tolerate stresses that arise from changes in environmental
conditions, with particular emphasis on temperaturestress and salt stress. In 1999, using higher plants andcyanobacteria as our experimental materials, we madesignificant progress in the following areas.

However, mechanisms for the perception and transduction of low-temperature signals remain to be characterized. In the cyanobacterium Synechocystis sp. PCC
6803, expression of the genes for fatty acid desaturases
is enhanced by low temperature. Moreover, the decrease
in membrane fluidity induced by low temperature appears to be a primary signal for induction of the expression of the genes for these desaturases.
In various bacteria, yeast and plants, physical andchemical stimuli are perceived by a group of proteinsthat includes histidine kinases. We attempted to disrupt
putative genes for histidine kinases in Synechocystis sp.
PCC 6803 and monitored the subsequent response of
the promoter of the gene for the ro3 fatty acid desaturase,
desB, to low temperature. Among 41 mutant lines with
disrupted genes for histidine kinases, we identified two
mutants, in which, respectively, the hikl9 gene and thehik33 gene had been inactivated and in which no induction of the reporter gene for luciferase occurred upon
exposure of cells to low temperature. This result indicated that mutation of the two genes for histidine kinases abolished the inducibility by low temperature of
the activity of the desB promoter. Mutation of these twogenes also reduced the extent of induction at low temperature of other low temperature-inducible genes, suchas the desD gene for .6.6 desaturase and the crh gene for
RNA helicase.
We also introduced an antibiotic-resistance gene cassette randomly into the chromosomes of Synechocystis
and screened mutants for altered expression of the desBgene. In one of the mutants, 2C, in which the desB promoter was insensitive to low temperature, we found thatthe gene for a response regulator, Rerl, had been inactivated. The extent of the low temperature-dependent
increase in the level of the desB transcript in LirerI cells
was reduced to half of that in wild-type cells. By contrast, the inducibility by low temperature of the desDand crh genes was unaffected by the mutation. Thus, it
is possible that Rerl might regulate the expression of
the desB gene specifically and might not affect the expression of the other genes examined.
Figure 1 shows a hypothetical scheme for the transduction of low-temperature signals. In this scheme,
Hik33 spans the plasma membrane twice and forms a
dimer, whose structure. is influenced by the physicalcharacteristics of the lipids in the plasma membrane.
Such characteristics include fluidity, which is controlled
by temperature. When the temperature is decreased, thehistidine residue in the histidine kinase domain is phosphorylated. The phosphate group is then transferred to
Hik19 and eventually to Rerl which regulates the expression of the desB gene.

I. The perception and transduction of lowtemperature signals in Synechocystis

II. Genetic modification of plants to enhancestress tolerance: introduction of the capacity forthe synthesis of glycine betaine into Arabidopsis

Low temperature is an important environmental factor that affects the growth of all living organisms. Manyorganisms are known to acclimate to low temperaturesby expressing various low temperature-inducible genes.

Metabolic acclimation via the accumulation of compatible solutes is considered to be a fundamental strategy for the protection and survival of plants when they

43

FOR BASIC BIOLOGY- - - - - - Plasma- - - - - - membrane@

IHik19l~~[I]

IRerll./'V"\..liiiiii

desB gene

more, enhancement of such tolerance was not confined
to a specific stage of development but was apparent
throughout developmental stages that included the imbibition and germination of seeds and the early and laterstages of vegetative growth.
In transformed Arabidopsis, enhanced tolerance tostress was also evident at the cellular level. Photosystem II in the chloroplasts of transformed plants was lesssusceptible than that of wild-type plants to photoinhibition by high-intensity light. We demonstrated that theenhanced tolerance of the photosynthetic machinery tohigh-intensity light resulted from the accelerated recovery of the photosystem II complex from photoinduced
inactivation. The extent of such accelerated recovery
was reduced when the synthesis of proteins was blockedby lincomycin, suggesting that betaine, accumulated invivo, might promote protein synthesis de novo, which isconsidered to be crucial for the recovery of cells fromstress-induced damage.

III. Mechanisms of inactivation of the photosynthetic machinery by salt stress in cyanobacteria~~

desD genecrh gene

Figure I . A hypothetical scheme showing the pathway for transduction of low-temperature signals in Synechocystis. The histidine
kinase domains of Hik33, Hikl9, the receiver domains of Hikl9
and Rerl , and the histidine phosphate-transfer domain of Hik 19
are shown in turquoise, pink and blue, respectively. The plasma
membame is shown in yellow. The histidine and aspartate residues that might be involved in the phospho-relay reaction are
indicated by H and D in circles, respectively. Adapted froin I.
Suzuki, D.A. Los, Y. Kanesaki, K. Mikami and N. Murata, EMBO
J, 19, 1327-1334 (2000).

are exposed to extreme environments. In response tostress, such as high levels of salt, cold and drought,
certain plants accumulate glycinebetaine (hereafter
called betaine), a quaternary amine that protects cellularcomponents, such as complex proteins and membranes,from the harmful effects of high levels of salt or extreme temperatures in vitro.
In order to evaluate the role of betaine in stress tolerance in vivo, we transformed Arabidopsis thaliana,
which does not normally accumulate betaine, with thecodA gene for choline oxidase (which catalyzes theconversion of choline to betaine) from the soil bacterium Arthrobacter globiformis . In transformed Arabidopsis, the accumulation of betaine, which was the result of
the expression of functional choline oxidase in chloroplasts, significantly enhanced the tolerance of the plants
to a wide variety of environmental stresses, such as highlevels of salt, and low and high temperatures. Further-

High-salt stress is an environmental factor of major
importance that limits the growth and productivity ofplants. We studied the mechanism of the salt-induced
inactivation of the photosynthetic machinery in the
cyanobacterium Synechococcus sp. PCC 7942. Incubation of cells in medium prepared with 0.5 M NaCl resulted first in the rapid and reversible inactivation ofphotosystems I and II, which was followed by the slow
and irreversible inactivation of both photosystems. The
rapid inactivation resembled the effects of 1.0 M sorbitol. The slow inactivation was prevented by a blocker of
Na+ channels. The presence of blockers ofNa+ channels
and water channels together protected both photosystems I and II against the short-term and the long-term
effects of 0.5 M NaCI. Thus, it seems likely that NaCl
has both osmotic and ionic effects. Our results suggest
the following mechanism for the salt-induced inactivation of photosynthesis. The osmotic effect of NaCl
decreases the amount of water in the cytosol, rapidly
increasing the intracellular concentrations of salts. The
ionic effect of NaCl is caused by an influx of Na+ ionsthrough K+/Na+ channels that also increases concentrations of salts in the cytosol, which results in the irreversible inactivation ofphotosystems I and II.
We also investigated the role of polyunsaturated lipids in cell membranes in the tolerance of the photosynthetic machinery to high-salt stress by comparing thedesA-IdesD- mutant of Synechocystis sp. PCC 6803,
which contained monounsaturated fatty acids, with thewild-type strain, which contained the full complementof polyunsaturated fatty acids. The oxygen-evolving
activity of des£/desD- cells was more sensitive tohigh-salt stress than was that of wild-type cells. Moreover, the activity of the Na+/H+ anti port in desA-/desDcells was suppressed to a greater extent than that of the
antiport in wild-type cells under high-salt stress. Theseobservations suggest that polyunsaturated fatty acids in44
membrane lipids might stimulate the activity and/or thesynthesis of the Na+/H+ antiport system to protect thephotosynthetic machinery against salt-induced inactivation.

NATIONAL INSTITUTE

involved in the acclimation of the photosynthetic machinery to high temperature.

Publication List:
IV. Acclimation of the photosynthetic machinery
to high temperature
High-temperature stress causes the irreversible inactivation of the photosynthetic machinery. However,when photosynthetic organisms have acclimated tomoderately high temperatures, their photosynthetic
machinery exhibits enhanced thermal stability. We havebeen studying the molecular mechanisms that underlie
the acclimation of the photosynthetic machinery to hightemperature in a cyanobacterium and in Chlamydomonas.
In the cyanobacterium Synechococcus sp. PCC 7002,
cytochrome c550 and PsbU, the extrinsic proteins of thephotosystem II complex, stabilize the oxygen-evolving
machinery at high temperatures. To clarify the role of
PsbU in vivo, we inactivated the psbU gene in Synechococcus sp. PCC 7002 by targeted mutagenesis. Not only
the thermal stability of the oxygen-evolving machinery
did not increase in the mutated cells at a moderately
high temperature, but these cells were also ·unable todevelop cellular thermotolerance upon acclimation tosuch temperatures. These results suggest that PsbU
might play an important role in enhancing the thermal
stability of the oxygen-evolving machinery at hightemperatures and, moreover, that the stabilization of themachinery might be crucial for the acquisition of cellular thermotolerance.
In Chlamydomonas reinhardtii, enhancement of thethermal stability of the oxygen-evolving machinery wasprevented by cycloheximide and lincomycin. The specificity and effects of these drugs suggest that the synthesis of proteins encoded by both the nuclear genome andthe chloroplast genome is required for this enhancement.
No synthesis of homo logs of three heat-shock proteins,
namely, Hsp60, Hsp70 and Hsp22, was induced at themoderately high temperatures that induce the enhanced
thermal stability of the oxygen-evolving machinery.
Thus, it appears likely that heat-shock proteins are not(1) Original articles
Alia, Kondo, Y., Sakamoto, A., Nonaka, H., Hayashi, H.,
Saradhi, P.P., Chen, T.H.H. and Murata, N. (1999) Enhanced tolerance to light stress of transgenic Arabidopsis
plants that express the codA gene for a bacterial choline
oxidase. Plant Mol. Bioi., 40, 279-288.
Allakhverdiev, S.l., Nishiyama, Y., Suzuki, 1., Tasaka, Y.
and Murata, N. (1999) Genetic engineering of the unsaturation of fatty acids in membrane lipids alters thetolerance of Synechocystis to salt stress. Proc. Nat!.
Acad. Sci. U.S.A., 96, 5862-5867.
Malakhov, M.P., Malakhova, O.A. and Murata, N. (1999)
Balanced regulation of expression of the gene for cytochrome eM and that of genes for plastocyanin and cytochrome c6 in Synechocystis. FEES Lett., 444,281-284.
Nishiyama, Y., Los, D.A. and Murata, N. (1999) PsbU, aprotein-associated with photosystem II, is required forthe acquisition of cellular thermotolerance in Synechococcus species PCC 7002. Plant Physiol., 120, 301-308.
Papageorgiou, G.C., Govindjee, Govindjee, R., Mimuro,
M., Stamatakis, K., Alygizaki-Zorba, A. and Murata, N.
(1999) Light-induced and osmotically-induced changes
in chlorophyll a fluorescence in two Synechocystis sp.
PCC 6803 strains that differ in membrane lipid unsaturation. Photosynth. Res., 59, 125-136.
Yamamoto, H., Miyake, C., Dietz, K.-J., Tomizawa, K.-1.,
Murata, N. and Yokota, A. (1999) Thioredoxin peroxidase in the cyanobacterium Synechocystis sp. PCC 6803.
FEES Lett., 447, 269-273.

(2) Review article
Los, D.A. and Murata, N (1999) Responses to cold shockin cyanobacteria. J. Mol. Microbial. Biotechnol., 1, 221230.

45

FOR BASIC BIOLOGYDIVISION OF BIOLOGICAL REGULATION AND
PHOTOBIOLOGY (ADJUNCT)
Professor (Adjunct):
Research Associate:
Postdoctoral Fellow:

Masamitsu Wada
Tomohiro Kiyosue
Takatoshi Kagawa(- Sep. 30, 1999)
BRAIN Postdoctoral Fellow: Kazuhiro Kikuchi
Graduate Students:
Takata Imaizumi(Tokyo Metropolitan
University)
Kazusato Oikawa(Tokyo Metropolitan
University)
PRESTO Fellow:
Takatoshi Kagawa(Oct. 1, 1999 -)Visiting Scientists:
John M Christie(Carnegie Institutionof Washington)
Steen Christensen (Universityof California, Santa Cruz)
Tilman Lamparter(Free University, Berlin)
Winslow R. Briggs(Carnegie Institutionof Washington)

Plants use light as an environmental factor which
controls their development as well as their other physiological phenomena. Phytochrome and blue light receptors, such as cryptochrome and phototropin (NPHl),are main photoreceptors for plant photomorphogenesis.
The goal of our research is to clarify the signal transduction pathways of photomorphogenesis. One of ourmajor subjects is chloroplast photo-relocation movement which is thought to be one of the simplest phenomena in this field. We use the fern Adiantum gametophytes as a model plant for our cell biological approach not only because the gametophytes are very
sensitive to light, but also because the organization of
the cells is very simple. We also use Arabidopsis formutant screening to clarify the genes regulating chloroplast photo-relocation. We have begun to use Physcomitrella patens as a model system for which gene targeting is available.

I. Cloning and characterization of blue-light photoreceptors
We have described many blue-light induced photomorphological responses in gametophytes of the fern
Adiantum capillus-veneris. As the first step in understanding the molecular mechanisms of these various
blue-light responses .in Adiantum, we are working on
the cloning and sequencing of the genes of blue light
receptors and on the gene expression and intracellular
distributions of the gene products.
1- 1

Cryptochromes

We identified two different genes, designated Adiantum cryptochrome4 and 5 (CRY4 and 5), from agenomic DNA library, and found stage specific andlight dependent gene expression of the five CRY genes(two newly isolated and three previously isolated genes)in Adiantum. The expression of CRY4 and CRY5 isregulated by light and is under phytochrome regulation.
The intracellular distribution of reporter GUS-CRY
fusion proteins indicates that GUS-CRY3 and GUSCRY4 localize in gametophyte nuclei. The nuclear
localization of GUS-CRY3 is regulated in a lightdependent manner (lmaizumi et al, Plant Cell12: 81-96,
2000).
Cryptochrome genes of Physcomitrella patens havealso been identified.
1- 2

Phototropin

Phototropin is another blue light photoreceptor isolated recently in higher plants, and is a flavin binding
protein with light sensitive protein kinase activity. A
eDNA of Adiantum phototropin has been sequenced.
The complete eDNA clone is 3492 bp in length andencodes a protein of 1092 amino acids. Southern blot
analysis showed that Adiantum phototropin is likely to
be a single copy gene. RT-PCR analysis revealed that itwas expressed in various developmental stages (imbibed spores, protonemata, dark grown young leaves,and light grown young leaves). The chromophore attached to LOV (Light, Oxygen, or Voltage) domains
was flavin mononucleotide (FMN).
We also cloned and sequenced two cDNAs and respective genomic DNAs ofphototropin from Physcomitrella patens.
1- 3 Adiantum Phytochrome 3
Adiantum phytochrome3 (PHY3) is a unique kimeric
protein with a phytochrome structure in the N-terminal
half and a phototropin structure in the C-terminal half.
From the analytical study of fusion protein expressed in
E. coli, LOV domains of PHY3 also bind with a FMN,
suggesting that phy3 absorbs blue light as well as red/
far-red light.

II. Chloroplast relocation2- 1

Arabidopsis

Chloroplasts accumulate at the cell surface under
weak light and escape from strong light to optimize
photosynthesis. The mechanism of chloroplast relocation, however, is not known. We studied light induced-chloroplast relocation in leaves of Arabidopsis
mutants such as cryl, cry2, crylcry2 and nphl andfound that the chloroplast relocation movement was
normal in these mutants, meaning that another (i.e. anew) blue light receptor for chloroplast relocation must
exist. To find the photoreceptor for blue light-induced
chloroplast relocation, we screened several mutantsfrom T-DNA tagging lines as well as EMS lines of
Arabidopsis. The identification of the mutated genesof Arabidopsis mutants is now in progress.

46

2-2

NATIONAL INSTITUTE

Publication List:

AdiantumA heavy-ion-beam induced deletion mutant of Adiantum which does not show phototropic response andchloroplast photorelocation movement under red light
was revealed to lack the PHY3 gene by genomic PCRand Southern blot analysis. It is suggested that phy3 is
the photoreceptor mediating red light-induced tropic
response and chloroplast photorelocation movement.
However, these phenomena induced by blue light arenormal, meaning that phy3 might or might not absorb
blue light. In .the former case, Adiantum phototropin
may also work on blue light-induced response as phy3
does.

Christie, J.M., M. Salomon, K. Nozue, M. Wada and W.R.
Briggs (1999) LOY (light, oxygen, or voltage) domains of the blue-light photoreceptor
phototropin (nph1): Binding sites for the chromophore
flavin mononucleotide.
Proc. Nat!. Acad. Sci. USA
96: 8779-8783.
Imaizumi, T., T. Kiyosue, T. Kanegae and M. Wada
(1999)
Cloning of the eDNA encoding the blue-light
photoreceptor (cryptochrome) from the moss Physcomitrella patens (Accession No. AB027528) (PGR99-11 0)
Plant Physiol. 120: 1205.

GUSCRY1GUSCRY2GUSCRY3GUSCRY4GUSCRY5

GUS

FIG. Representative Images of the Intracellular Distribution of GUS-CRY Fusion Proteins in Germinating Cells of Adiantum.
Intracellular distribution of GUS-CRY fusion proteins under different light conditions. GUS-CRY I (A and B), GUS-CRY2 (C and D), GUSCRY3 (E and F), GUS-CRY4 (G and H), GUS-CRYS (I and J), GUS (K and L). Two-celled protonemata expressing various GUS-CRY fusion
proteins were incubated under blue light (A, C, E, G, I, and K) or in the dark (B, D, F, H, J, and L) for 16 hr and stained under the same light
conditions. The cells showing GUS activity were photographed using Nomarski optics (left panels) and fluorescence micrographs show theposition of the nuclei in the same cells stained with 4',6-diamidino-2-phenylindole (right panels). Note that under fluorescence, chlorophyll
autofluoresces red and spore coats appear bluish white. The bar shown in L represents 20 11m for all panels.

47

FOR BASIC BIOLOGYDIVISION OF BEHAVIOR AND NEUROBIOLOGY(ADJUNCT)
Professor:
Fujio Murakami
Associate Professor: Masato Nakafuku
Research Associate: Atsushi Tamada 1
Postdoctoral Fellows: Tatsuro Kumada 4
Hiraki Taniguchi 4
Yumiko Hatanaka
Graduate Student:
Hiroshi Ono(from Osaka University)

The vertebrate nervous system contains a large
number of neurons that are connected to each other byspecific axonal projections. We are interested in how
the complex but precise neuronal network, which isindispensable for functioning of the nervous system, isconstructed during development. The goal of ourresearch in this division is to elucidate the cellular andmolecular mechanisms underlying the neuronal network
formation in vertebrates, with current focuses on themechanisms of axon guidance and neuronal migration.
I. Mechanism of Axonal Guidance

During development of the nervous system, growing
axons are guided through specific pathways to correct
targets. Our research interest focuses on the cellularand molecular mechanisms of axon guidance in thevertebrate nervous system.
We are particularlyinterested in how guidance mechanisms operating indifferent phases of axonal growth are assembled togenerate the complex but precise axonal wiring patterns
in the brain. To address these issues, we are first
trying to understand axonal wiring patterns during
development with reference to the structural organization of the brain. Using whole-mounted preparationsof embryonic rat brains, we will reveal whole axonal
trajectories by labeling with neuronal tracers, such as
Dil, or by immunohistochemical stammg with
antibodies against molecular markers expressed in a
subset of neurons. We will next explore the axonal
guidance mechanisms generating these wiring patterns.
1) WhatFollowing questions will be addressed.
structures along the pathway have key roles in axonal
guidance? 2) What guidance cues exist in thesestructures? Are these attractive cues or repulsive cues?
Are these short-range cues or long-range cues? 3)
What molecules are responsible for these cues? 4)
How do multiple guidance mechanisms work in concert
to generate specific wiring patterns? To answer thesequestions, we use neuroanatomical techniques, in vitro
culture techniques including dissociated cell, explant
and whole-mount cultures, and biochemical andmolecular biological techniques.

II. Mechanism of Neuronal Migration
A variety of neurons migrate from their birthplace tothe position where they fmally settle.
Neuronal
migration in the vertebrate central nervous systemoccurs both along the radial axis and along thetangential axis of the neural tube. Another interest of
our research focuses on the cellular and molecularmechanisms underlying the radial and tangential
neuronal migration. In many regions of the central
nervous system, neurons migrate radially from theventricular zone where they are born toward the pial
surface. We are investigating the mechanisms ofradial migration, using neurons in the neocortex as amodel system. In addition to the radial migration,some neurons in the brain, such as interneurons in theforebrain, move tangentially for a long .distance. We
are also investigating how tangentially migratingneurons are guided for a long distance through specificroutes to the fmal positions, using neurons that migratefrom the rhombic lip at the dorsal rim of the hindbrain,such as cerebellar granule cells and precerebellar
neurons.
To address these issues, we developed in vitro culture
systems that reconstruct migratory events occurring in
We labeled migrating neurons with green
vivo.
fluorescent protein (GFP) by transplantation of small
piece of explant taken from a transgenic rat expressing
GFP or by introducing GFP eDNA into limited regionsof the brain by electroporation. Slices of the brain or
flat-mounted brain preparation are then cultured on
permeable membrane filters. These culture systems
enable us to analyze migration pattern of neurons in realtime (Fig. 1). Using these in vitro culture systems, weare currently investigating the cellular and molecularmechanisms of neuronal migration.

Figure 1. Migrating neocortical neurons visualized with green
fluorescent protein (GFP) in vitro.
(Left) Slice culture ofrat neocortex that was introduced with GFPeDNA into the ventricular zone by electroporation. A broken
line shows the outer margin of the slice. GFP-positive neurons
were moving radially from the ventricular zone (bottom) toward
the pial surface (top).
(Right) High power view of labeled migrating neurons.
Leading processes of neurons extended toward the pial surface.

48

49

FOR BASIC BIOLOGYLABORATORY OF GENE EXPRESSION AND REGULATION

Head: Takashi Horiuchi

The laboratory consists of four regular divisions and conducts research
into regulatory mechanisms of gene expression in microorganisms, plants
and animals.

so

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND

REGULATION I

Professor:
Shigeru !ida
Associate Professor (adjunct): Yoshihiro Ozeki
Research Associate:
Rie Terada
Yoshiki Habu
Yoshishige Inagaki
Atsushi Hoshino
Sachiko Tanaka-Fukada
Technical Staff:
Kazuhiko Furukawa
Yasuyo Johzuka-Hisatomi 2
Postdoctoral Fellow:
Hong-Qing Li2
Kazuo Tsugane 1
Jeong-Doo Choi2
Graduate Student:
Naoko Ishikawa
Takashi Kagami
Toshio Yamaguchi
Yasumasa Morita
Visiting Fellows:
Laurel Caitlin Coberly)
Mary L. Durbin 2J
1ljrom Duke University
2J_{rom University of California, Riverside

The main interest of the group is in understanding thebiology of the dynamic genome, namely, genome organization and reorganization and its impact on gene
expression and regulation. Although there are many
elements affecting organization and reorganization of
the genomes, we are currently focused on mobile
genetic elements in general and plant transposable controlling elements in particular.

I. Identification and characterization of mutablealleles in the Japanese morning glory
The Japanese morning glory is a traditional horticultural plant in Japan, and extensive physiological andgenetical studies on the plant have been conducted. A
number of mutants related to the color and shape of its
flowers and leaves have been isolated since the 17th
century, and more than 200 genetic loci including about
20 mutable loci have been documented. We have identified that the mutable flecked allele bearing white flowers with colored flecks is the DFR-B gene having the
En/Spm-related transposable element Tpnl inserted into
its second intron. The non-autonomous Tpnl element
carries a part of the genomic sequence for an HMG-box.
Thus Tpnl can be regarded as a specialized transducing
transposon.
It is known that the frequency and the timing of the
flecking phenotype tend to be heritable by their progeny,
although conversion of these phenotypes is sometimes
observed. From the variegated flower lines, white variants bearing white flowers free from variegation
throughout their plant lives occasionally appeared.
Since some of the selfed progeny of the white variants
produced a few flecked flowers together with whiteflowers (others bore only white flowers), the plants
were called as variants rather than mutants. We haveexamined the structure of the DFR-B region in the whitevariants and found that Tpnl excision rarely occurs in

the variants. DNA methylation in the subterminal repetitive regions of Tpnl in the DFR-B gene of the whitevariant appeared to be similar to that of a heavily variegated line, suggesting that the DNA methylation within
Tpnl is probably not the primary cause of deficiency inexcision and that the Tpnl-related autonomous elementin the white variant is likely to become inactive. The
results also suggest that the frequency and the timing of
the flecking phenotype of the variegated flowers in themutable flecked line are mainly dependent on the activity of the Tpnl-related autonomous element.
The plants with the recessive mutable speckled allele,in the presence of the dominant Speckled-activator,
produce colorless flowers with fine and round coloredspots distributed over the corolla, while plants carrying
the speckled allele without active Speckled-activator
bear pale yellow flowers. We have found that the
speckled allele is the CHI gene containing a TpnJrelated non-autonomous element of 6.5 kb, termed Tpn2.
Interestingly, Tpn2 is also a specialized transducing
transposon containing a part of the genomic sequence
encoding ~-galactosidase. The results indicate that thedominant Speckled-activator is the TpnJ-related autonomous element acting on not only Tpn2 on the mutable speckled allele but also Tpnl on the mutable flecked allele.

II. Identification of mutable alleles in the common
morning glory
The mutable flaked line of the common morning glory also bears white flowers with colored flakes andsectors. We showed that the mutable flaked allele iscaused by insertion of a 3.9 kb transposable element,
TipJOO, into the CHS-D gene intron and that TipJOO
belongs to the AciDs family. It has been postulated thatthe timing and the frequency of the variegation aredetermined by the active state of another genetic element modulator. As an initial step to elucidate the genetic system to determine the timing and the frequencyof the flower variegation, we examined whether TiplOO
is able to transpose in transgenic tobacco plants andfound that TiplOO can be excised from the introduced
vector carrying TipJOO. The results strongly indicatethat TiplOO is an autonomous element.

III. Characterization of the genes for anthocyanin
pigmentation in morning glories.
The CHS genes encoding chalcone synthase for flavonoid biosynthesis comprise a multigene family in thecommon and Japanese morning glories. Among these
Ipomoea CHS genes, the CHS-D gene is the most abundantly expressed in the pigmented young flower buds
and is primarily responsible for flower pigmentation.
The majority of the remaining CHS transcripts in theflower buds are produced from the CHS-E gene.
Moreover, the CHS-D and CHS-E genes are expressed
predominantly in flower limbs and tubes, respectively.
The recombinant CHS-D and CHS-E proteins obtainedby expressing the CHS-D and CHS-E genes in Escher-

51

FOR BASIC BIOLOGY

ichia coli with various expression systems showed CHS
activity to produce naringenin chalcone. These results
are consistent with the notion that the CHS-D and CHSE genes encode the chalcone synthases for anthocyanin
biosynthesis in flowers (see also II).
We have characterized the genomic DNA segmentsof the CHS-D and CHS-E genes in the Japanese andcommon morning glories. Both genes have two exons
with identical intron positions and carry several copies
of two mobile element-like sequences with short terminal inverted repeats, MELSJ and MELS6 of around 200- 300 bp. Small tandem repeats were also found in these
CHS gene regions. Gene duplication and subsequent
divergence are regarded to play important roles in evolution of multiple genes. Comparison of the genomic
sequences suggests that gene duplication and major
divergence in these CHS genes occurred prior to thespeciation of the Japanese and common morning glories.
Subsequent DNA rearrangements are likely to havetaken place after the speciation, and the MELS elements
appear to play a role in generating such DNA rearrangements.

IV. An efficient transformation system and homologous recombination in rice.
In higher plants, efficient and reliable gene targeting
procedures for "reverse genetics" remains to be established. As an initial step to develop such procedures, weare trying to develop an efficient rice transformation
system and searching for factors to enhance homologous recombination in rice. We employed an Agrobacterium-mediated transformation system using vigorously dividing calli derived from mature seed scutellum.
Under the optimum condition, approximately 1200
independent transformed calli were obtainable from 150
matured seeds.

Publication List:
Hasebe A. and Iida, S. The novel insertion sequences
IS1417, IS1418 and IS1419 from Burkholderia glumae
and their strain distribution. Plasmid in press.
lida, S., Hoshino, A., Johzuka-Hisatomi, Y., Habu Y. andInagaki, Y. (1999) Floricultural traits and transposable
elements in the Japanese and common morning glories.
Annal. New York A cad. Sci. 870, 265-274.
Iida S. and Hoshino, A. Spontaneous mutagenesis andtransposable elements in the Japanese morning glory.
Gamma Field Symposia in press.
Inagaki, Y., Johzuka-Hisatomi, Y. , Mori, T. , Takahashi, S.,
Hayakawa, Y., Peyachoknagul, S., Ozeki Y. and lida, S.

•• • •

Tpn1~=========~~

•

~ ·

-

DFR·B

•

Tpn1

~

•

~ transposase

-

•

DFR·B

Tpn2

t

autonomous element
•

•

•

e • ~

~~

.~ ~
CHI

Fig. I. Mutable alleles and transposable elements in the Japanese and common morning glories. In the Japanese morning glory,
activities of a putative autonomous element act on the nonautonomous Tpnl and Tpn2 elements and determine the frequency and the timing of flower variegation. In the common
morning glory, TiplOO is an autonomous element.

(1999) Genomic organization of the genes encoding dihydroflavonol4-reductase for flower pigmentation in theJapanese and common morning glories. Gene 226, 181188.
Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu Y. andIida, S. (1999) Characterization of the chalcone synthasegenes expressed in flowers of the common and Japanesemorning glories. Genes Genet. Syst. 74, 141-147.
Shiokawa, K., Inagaki, Y., Morita, H., Hsu, T.-J., lida S.
and Noguchi, H. The functional expression of the CHSD and CHS-E genes of the common morning glory(Ipomoea purpurea) in Escherichia coli and characterization of their gene products. Plant Biotechnology inpress.
Takahashi, S., Inagaki, Y., Satoh, H., Hoshino A. and lida,S. (1999) Capturing of a genomic HMG domain sequence by an En/Spm related transposable element
Tpnl in the Japanese morning glory. Mol. Gen. Genet.
261 , 447-451.

52

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION II
Professor:
Research Associate:

Technical Staff:
Graduate Student:

Takashi Horiuchi
Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama*Katsuki Johzuka
Kohji Hayashi
Yasushi Takeuchi
Hiroko Urawa*to Department of Biological Chemistry, University ofCalifornia-Irvine, Irvine, California (Feb. I. 1999-)

Homologous recombination, which may occur in allorganisms, apparently involves genetic exchange between two parent-derived chromatids in addition to therepair of DNA damage induced by physical and chemical reagents. As deduced from our analyses of recombinational hotspots of E. coli and S. cerevisiae, in particular the activity related to DNA replication fork
blocking events, the physiological function of homologous recombination (especially in normally growing
cells) is better understood. In 1999, work on the two
following subjects has advanced our knowledge of both
the dynamics and function of genome.

I. Replication fork block protein (Fobl) acts as arDNA specific recombinator.
We previously found that some recombinational hotspots in E. coli are regions where progress of a DNA
replication fork is severely impeded. Detailed analysisof this phenomenon revealed that when the fork isblocked, recombinational enzymes construct a new
replication fork after breakdown of the arrested fork byintroducing a double-stranded break at either of thesister chromosomes. Consequently, this recombination
reaction for renewal of the replication fork is a primarycause resulting in recombinational hotspots.
Next, we showed that this phenomenon occurred not
only in prokaryotes but also in eucaryotes. In mosteucaryotes, the DNA replication fork blocking site,
called RFB (replication fork harrier), is located in each
unit of rRNA multi-genes (rDNA).
It has been reported that in budding yeast (S. cerevisiae) when a nontranscriptional region (NTS) of the rDNA, in which
RFB site is located, is inserted into a non-rDNA regionof chromosomes, recombination of a region close to theinsertion site is enhanced. This enhancement is called
HOTJ activity and the DNA fragment required for activation is named HOTJ DNA. We isolated a number ofmutants, which are defective in HOTJ activity, andfound that some of them had a concomitant defect infork blocking activity at the RFB site. Both of these
defective phenotypes were restored to wild type byintroducing a single gene, named FOBJ, thereby suggesting that fork blockage acts as a trigger for recombination in yeast, as well. In addition to this fmding, thedetection of RFB activity within the rDNA cluster invarious higher eucaryotic cells indicates that the fork
renewal cycle occurring after the blockage is moregeneral than expected.
A copy nmnber of rDNA repeats is unstable. Until
recently, though an increase and a decrease in a nmnberof rDNA copies have been observed in various eucaryotes, the underlying mechanism remains unknown.
Because fork arrest at the RFB site was a trigger ofrecombination, as mentioned above, we suspected that afork-blocking event was involved in changing the copy
nmnber. Upon examination it turned out that in afobl
mutant neither an increase nor a decrease in the rDNA
copy nmnber took place; thereby suggesting that thefork blocking event is required for changing the copy
nmnber, probably through recombination process.
Since fork blocking system has only been understood toprevent replication from "colliding" with rDNA transcription so far, this study sheds light a novel physiological role for fork blocking system. The newly
discovered role may be quite a common in most of
eucaryotes.
This year we conducted a molecular examination as
to whether the fork blocking step is actually essential
for homologous recombination within the rDNA repeated region.
In this case, recombination means intra- orinter-sister chromosomal exchange. Because the intrasister chromosomal recombination between two repeatsof the rDNA produces a covalently closed circular (ccc)
molecule, its recombination activity can be assayedeasily by measuring the quantity of ccc molecules produced.
In the case of inter-chromosomal recombination, since the structure of the rDNA cluster remains
unchanged whether inter-recombination occurs or not,the recombination product is hard to detect. However,
because rDNA has a repeated structure, if any selective
maker (URA3 marker in this experiment) is inserted
anywhere in the rDNA region, unequal inter-sister
chromosomal recombination can be assayed by detecting duplicated molecules of the inserted marker.
Using these two assay methods, we investigated whether FOBI or other recombination genes were involved inrecombination of the rDNA region and the following
results were obtained: (1) the frequency of intra- andinter-recombination in both fobl and rad52 mutants
decreased to one tenth of those in the wild type strain(Figure A), (2) while the RAD52 gene was a generalrecombinator, the FOBI gene was a rDNA regionspecific recombinator, (3) a major type of recombination was of the inter-sister chromosomal type, while
intra-recombination was minor, (4) at least an interrecombination occurred uniformly throughout therDNA region, (5) enhancement of rDNA region-specificrecombination by the sir2 (silencer gene 2) mutation
was found to be FOBJ-dependent.
Guarente's group at MIT has reported that the life
span of budding yeast is determined by the nmnber of
ccc molecule of rDNA accumulated in mother cells.
ccc rDNA molecules, which is excised from the rDNA
cluster on the chromosome, are not segregated equally
into daughter and mother cells but are accmnulated only

53

FOR BASIC BIOLOGY

in the mother cells. Thus, mother cells "die" after
about 20 generations. If the group's speculation iscorrect, thefobl mutant is expected to have a longer life
span than the wild type strain. Actually, we found thatthe fob] mutant could live 60 % longer than the wild
type strain (Figure B).
Independently, Guarente's
group also reported a similar result. These findings
raised the possibility that the ccc rDNA molecule mightalso be involved in determining the life span of multicellular organisms.
In order to investigate this possibility, isolation and analysis of their FOBJ-like genedefective mutants should prove to be a successful approach.

source, (2) bio-informatics, (3) database, and (4) systematic functional analysis. At present, we are determining the sequences of a remaining region (about 370kb) of the genome of E. coli W311 0 strain and willcomplete it until this summer. The resource group hasconstructed a large number of gene disruptants using arandom transposon insertion method, in which the insertion sites will be determined by sequencing in part inthis laboratory. Our immediate goal is to establish asystematic method for screening the phenotypes ofabout 4000 different disruptants, each of which has
disrupted mutation in each of about 4000 nonessential
genes.

II. E. coli genomic structure and function

Publication List:

A project named "Structure and function of the E.
coli genome", supported by a grant termed CREST
from JST for a duration of five years, began in January
1999. The team is composed of twelve independent
research groups that are divided into four main categories of research. Namely, these divisions are: (1) re-

Taki, K., Horiuchi, T
The SOS response is induced by replication fork blockage
at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites. Mol.
Gen. Genet. (1999) 262, 302-309.

B

A

C\.1

g. I-1-Js

Mortality Curve

lt)

..Q

-2

""Q

~

-e- fob7- - - Wild Type

100

(%)

tri
mer
dim_
mer

Q)

75

..a
ro

>

-1-'

c

50

Q)

u
!.....

Q)0..

monomer

25

0

0

10

20

30

40

Gen~rations

Figure:fobl is a long life mutant.
(A) Production of covalently closed circular (ccc) rDNA molecules (mono-, di-, and trimmer) of wild type,fobl and other mutants. Guarente's
group have been argued that amount of accumulated ccc rDNA molecules in a mother cell determines her life span. (B) Mortality curves ofwild type andfobl mutants. Life span was determined by scoring the number of daughter cells produced by each mother cell until she lost her
abil ity to bud. Average life spans were as follows: wild type (black square), 17.9 generations andfobl (red circle), 28.2 generations.

54

NATIONAL INSTITUTEDIVISION OF SPECIATION MECHANISMS I
Professor:
Research Associates:

Tetsuo Yamamori
Yuriko Komine
Akiya Watakabe
Takas hi Kitsukawa
Technical Staffs:
Hideko Utsumi
Kaoru Sawada
NIBB Postdoctoral Fellow: Rejan Vigot
Visiting scientist:
Yoshinori Shirai
Graduate student:
Shiro Tochitani
Katsusuke Hata
Yusuke Komatsu
Shuzo Sakata(kyoto University)
Ryohei Tomioka(Kyoto University)

Our research goal is to understand mechanisms underlying evolution of the nervous system. In order toapproach this question, we are studying in two major
subjects. 1) One approach is to understand informational processing in the brain underlying learning behaviors.
2) The second approach is to study the genes that are
expressed in the specific areas of the monkey neocortex.
Here, we report our findings of this year (1999).

L Gene expression and cerebellar long-term plasticity
In order to know roles of the genes involved in longterm memory, we choose the cerebellum as a model
system. In the cerebellum the conjunctive stimuli ofparallel fibers and a climbing fiber to a Purkinje cell
induce prolonged reduction of a synaptic efficacy between the paralleled fiber to the Purkinje cell (LTD;long-term depression, Ito et al., 1982). Previously, we
examined the expression of 10 immediate early genes(lEGs) including all the known Fos and Jun family in
cerebellar slices under the pharmacological condition
that cause long-term desensitization of the Purkinje cellto AMPA (a glutamate analogue). Among the lEGs
examined, Fos and Jun-B were predominantly induced
under the conjunctive condition (Nakazawa et al.,1993).
We further examined Jun-B expression in vivo undera conjunctive protocol of AMPA, a pharmacologicalsubstitute for parallel fiber stimulation, and climbing
fiber stimulation via electric Inferior Olive stimulation.
June-B are predominantly induced around the local area
where the AMPA and climbing fiber stimulation wereconjunct. These results suggest that the coincidence
mechanism may exist at gene expression level and leadto a cerebellar long-term plasticity. However, it has notbeen still clear how these newly synthesized gene products are involved in cerebellar LTD. We are currently
investigating the mechanisms.

II. Gene expression under audio-visual discrimination task
We are studying the gene expression of c-Fos underaudio-visual discrimination tasks in collaboration with
Dr. Yoshio Sakurai (Kyoto University). We found thatthe visual and audio tasks enhanced the specific expression of c-Fos in the visual and audio cortex, respectively. Among the early visual and auditory pathways
examined, c-Fos was specifically induced in the cortexes but not in the earlier pathways, suggesting the neuralmodulation of the neocortex depending on the types of
the tasks (manuscript in preparation).

III. Color-defective (dichromat) monkeys
Several percent of humans, most of whom are Xchromosome linked, have been reported to be color
deficient. However, despite the color pigment genes ofmacaque monkeys are very similar to human in the
nucleotide sequence, no color-deficient monkeys havebeen found. We have examined 3153 macaque monkeys
using an assay to identify a deletion of exon 5 of thelong wave (red) pigment and middle wave (green) pigment genes by PCR amplification with a common
primers in both genes and discriminating them by thefollowing Mbo I restriction enzyme digestion, andfound three monkeys that lacked the exon five of thelong wave pigment genes. Further genetic analysis
revealed that the three monkeys possessed a hybrid oflong (exon 1 to 4) and middle wave (exon 5 and 6)
pigment genes (Fig. 1). Analysis of photobleachingdifference absorption spectra showed that the absorbency maxima (A.max) of reconstituted L, M and hybrid
pigments are 564, 532 and 538 run, respectively. The
A.max for the hybrid was red-shifted by 6 nm from thatof the M pigment, indicating that these monkeys arealmost protanopic (insensitive to red). The ratio of dichromats in macaque monkeys (approximately
0.1%=3/3153) is significantly lower than that of 1% inhumans. These results suggest that there may be somemechanisms which do not exist in humans to reduce theoccurrence of color-deficiency in macaque monkeys.

IV. Brain Specific Repetitive (Bsr) RNA
During our attempt to isolate LTD-related genes incultured cerebellar Purkinje cells, we accidentally foundrepetitive genes which were specifically expressed in
the rat brain (Bsr: brain .specific repetitive gene).
Among the cells of the rat brain Bsr RNA tends to express in the relatively large cells and in phylogeneticallyold structures, such as the pareo- and archicortex,
amygdala, thalamus and hypothalamus (see Fig. 2., forexample). To our surprise, the genes are only found in
the rattus but no other species so far examined including murine species. Although we do not know the function of this new type of gene at the moment, it may playsome important role in the rat brain and we hope further
characterization of the gene reveals it.

55

FOR BASIC BIOLOGY

kenaka, 0., Hanazawa, A., Komatau, H., Mikami, A.,
Figure 1

aNo. 1114(Normal)
E
I

MW
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I I

I

EI

189bp

No. 2727(Dichromatic)

LW/ MN

E

E
I

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I

4kb
189bp
b

Macague
1

2

3

Human
4

5

6

7

<ll

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00 ;::: ,.._
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189- MW137 ] LW

52

Fig. I Physical maps of the Land the M genes. a, Top, the 77-kilobase (kb) region of a wild-type macaque, showing exons of the L (red) and M(green) genes. Enlarged exon-5 regions are shown underneath. The PCR primers and products for exon 5 of the Land M genes are shown in red(137 and 52 base pairs (bp), cleaved by Mboi digestion) and green boxes (189 bp, uncleaved), respectively. An Mbo I site specific for exon 5 of
the L gene is conserved among Old World monkeys. Bottom, the 36-kb region of a dhichromatic candidate. The enlarged PCR-amplified fragment is shown underneath. B, Gel elctrophoresis analysis. The bands of 189, 137 and 52 bp shown in a are indicated as M (green) or L (red).
Lane I, macaque wild type; lanes 2-4, dichromats, showing only the exon 5 fragment of theM gene; lane 5, human wild type; lanes 6 and 7,
deuteranopic (M cone absent) and protanopic (L cone absent) humans, respectively. [Published in Onishi et al., Nature 402, 139-140, 1999]

V. Genes expressed in specific areas of the neocortex
The neocortex is most evolved in mammals, particularly in primates, and thought to play the major role inhigher functions of the brain. The neocortex in the
mammal can be divided into distinct functional andanatomical areas. It has been a matter of debate what
extent the areas of the neocortex are genetically andenvironmentally determined. To access this question,
we started to isolate the genes that are expressed in
specific areas of the brain. Using differential display
methods, we found at least two genes that indicated thearea specific expression. One, designated OCC-1 isexpressed in the occipital cortex in the brain and theother gene is expressed in the motor area. We are currently examining the detailed expression pattern of thegenes. We are also further isolating area specific genes
with RLCS (Restriction Landmark eDNA Scanning).

Publication List:
Liang, F., Hatanaka, Y. , Saito, H., Yamamori, T. andHashikawa, T. Differential expression of gammaaminobutyric acid type B receptor-1a and 1b mRNAvariants in GABA and non-GABAergic neurons of the
rat brain. (2000) J. Camp. Neural. 416, 475 -495
Onishi, A., Koike, S. , Ida, M., lmai, H., Shichida, Y., Ta-

;··,

. .·
~

•'•.

Fig. 2 Expression of Bsr in the rat hippocampus. In situ hybridization with an antisense probe of Bsr. Inset: higher magnification.
[published in Komine et al., Mol. Brain Res., 66, 1-13, 1999]

Goto, S., Suryobroto, B., Kitahara, K. and Yamamori, T.
(1999) Dichromatism in macaque monkeys. Nature 422,140-141.
Tochitani, S., Watakabe, A., Liang, F. and Yamamori, T.
(1999) A molecular marker for a neuronal population
dominantly distributed in the macaque occipital neocortex. 29th Society for Neuroscience (North America) Abstract 2190.
Komine, Y., Tanaka, N. , Yano, R ., Takai, R., Yuasa, S. ,

56
Shiroishi, T., Tsuchiya, K. and Yamamori, T. (1999) A
novel type of non-coding RNA expressed in the rat brain.
Mol. Brain Res., 66, 1-13.
Matsuzawa, M., Muramatsu, T., Yamamori, T., Knoll, W.
and Yano, R. (1999) Novel neuronal activities ofmidkine were examined on embryonic cerebellar neurons using a defined culture system. Cell. Mol. Neurobiol., 19,
209-220.
Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y.,
Sanbo, M., Yagi, T., Fujisawa, H. (1999) A requirement
for neuropilin-1 in embryonic vessel formation. Development 126,4895-902

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS II

Associate Professor:
Assistant Professor:
Technical Staff:

Mitsuyasu Hasebe
Tomomichi Fujita
Yukiko Tanikawa
Masae Umeda
Yoshimi Bitoh
Makiko Kondo
Post Doctoral Fellow: Rumiko Kofoji 1
Graduate Students:
Satomi Shindo
Yuji Hiwatashi
Satoko Kobayashi-Arakawa
Keiko Sakakibara
Ryosuke Sano (Chiba Univ.)
Tomoaki Nishiyama(Univ. ofTokyo)
Takaka Tanahashi (Univ. ofTokyo)

All living organisms evolved from a common ancestor more than 35 billion years ago, and accumulated
mutations on their genomes caused the present biodiversity. The traces of evolutionary processes are remained in the genomes of extant organisms, and we caninfer (1) the phylogenetic relationships of organismsand (2) the genetic changes having caused the phenotypic evolution by comparing the genomes of different
organisms. The inferred phylogenetic relationships
give important insights on problems in various fields ofevolutionary biology, and our group is now focusing on
biogeography, evolution of morphological traits, andsystematics in wide range of taxa. On the phenotypicevolution, we are especially interested in the morphological evolution and aim to explore genetic
changes led the evolution of plant body plan. We
selected Arabidopsis (angiosperm), Gnetum (gymnosperm), Ginkgo (gymnosperm), Ceratopteris (pteridophyte), and Physcomitrella (bryophyte) as models tocompare the genetic cascades regulating morphogenesis
especially in the reproductive organs and shoot apicalmeristem of land plants.

I. Evolution of reproductive organs in land plants
A flower is the most complex reproductive organ inland plants and composed of sepals, petals, stamens,
and gynoecium. Female haploid reproductive cells arecovered with a sporangium (nucellus) and two integuments, and further enclosed in a gynoecium. Male
haploid reproductive cells (pollens) are covered with asporangium (pollen sack). On the other hand, gymnosperms and ferns have simpler reproductive organs thanangiosperms and lack sepals and petals. Female sporangia (nucellus) of gymnosperms are covered with
only one integument. Sporangia of ferns have no integuments and are naked on the abaxial side of a leaf.
The development of floral organs is mainly regulatedby A-, B-, C-function genes, which are members of the
MADS-box gene family. These genes are transcription
factors containing the conserved MADS and K domains.
MADS-box genes of angiosperms are divided into morethan 10 groups based on the gene tree. The LEAFY

57

gene is the positive regulator of the MADS-box genes
in flower primordia.
What kind of changes of the MADS-box genes
caused the evolution of the complex reproductive organs in the flowering plant lineage ? Comparisons of
MADS-box and LFY genes in vascular plants suggestthat the following sequential changes occurred in theevolution of reproductive organs (Hasebe and Ito 1999,
Hasebe 1999). (I) Plant-type MADS-box genes withboth MADS and K domains were established. (2) The
number of MADS-box genes increased, and the threeancestral MADS-box genes that later generate A-, B-,
C-functions genes were likely originated before thedivergence of ferns and seed plants. (3) Specifically
expressed MADS-box genes in reproductive organs
evolved from generally expressed ones in the seed plantlineage. (4) The ancestral gene of the AG group of
MADS-box genes acquired the C-function before thedivergence of extant gymnosperms and angiosperms.
(5) The gene duplication that formed the AP3 and PI
groups in MADS-box genes occurred before the diversification of extant .gymnosperms and angiosperms. (6)The ancestral gene of angiosperm A-function gene waslost in extant gymnosperm lineage. (7) LFY gene
becomes positively regulate MADS-box genes before
extant gymnosperms and angiosperm diverged. (8)Spatial and temporal patterns of A-, B-, C-function gene
expression were established in the angiosperm lineage.
Homeobox genes play indispensable roles for development in metazoa, instead of MADS-box genes. Thisdifference is likely caused by the fact that metazoa andland plants established multicellular organs independently after their last common ancestor, which waspresumably a unicellular organism or a multicellularorganism without multicellular organs. Of note, inboth land plants and metazoa, an increase in the numberof specific transcription factors (MADS-box genes inland plants and homeobox genes in metazoa) and thesubsequent diversification of their expression patterns
and regulation of downstream genes are the principal
mechanisms for the evolution of body plans.

II. New hypothesis on the homology of Gnetum
reproductive organ
Gnetales, one of the extant gymnosperm orders has
traditionally been recognized to be most closely related
to flowering plants, because the reproductive organ of
Gnetales has some morphological characters similar toflowering plants. Most recent molecular phylogenetic
studies do not support the sister relationship of the
Gnetales and flowering plants, but instead support aclose relationship between Gnetales and other extant
gymnosperms. The MADS-box genes are transcription factors, some of which are involved in reproductive
organ qevelopment in flowering plants. To resolve thediscrepancy in phylogenetic inferences, and to provide
insights into the evolution of reproductive organs inseed plants, four MADS-box genes (GpMADSl - 4)
were cloned from Gnetum parvifolium (Fig. lA, B).

58
GpMADS2 is likely to be a pseudogene and the otherthree genes were characterized. A MADS-box genetree based on partial amino acid sequences showed that
GpMADS3 is included in the AGL6 group, but the othertwo genes do not cluster with any previously reported
MADS-box gene. The three GpMADS genes wereexpressed during the early stage of ovule developmentin the differentiating nucellus and three envelopes (Fig.
1C-F). A comparison of MADS-box gene expression
among conifers, Gnetum, and flowering plants suggeststhat the comparable reproductive organs in Gnetum andflowering plants evolved in parallel, and is likely tosupport the homology between the ovule-ovuliferous
scale complex of conifers and the Gnetum ovules including the three envelopes (Fig. 1G).

III. Evolution of gametophytic and sporophytic
generations
Plants have the haploid gametophytic and diploidsporophytic generations, both of which are usuallymulticellular and morphologically diversified eachother. Origin and evolution of the two generations
have been discussed and some hypotheses are proposed,
although the molecular basis of the both generations
have not been studied. For example, it is not known
whether different sets of genes contribute to the morphological differences of the two generations or similar
genes are differently used. Ferns are good materials to
study the evolution of gametophytic and sporophytic
generations, because individuals of the both generations
grow independently and it is much easier to analyze
than other plats in which one of the two generations isenrolled in the tissue of another generation.
We have cloned 11 homeodomain - leucine zipper(HD-Zip) genes of the fern Ceratopteris richardii (Crhb
genes) encoding a transcription factor and are characterized by the presence of a homeodomain closely
linked to a leucine zipper motif. Most of Crhb genes
belonging to the HD-ZIP I or II subfamilies are almost
ubiquitously expressed in both sporophytes and gametophytes, while the amounts of expression are different
among the tissues. The similarly ubiquitous expression patterns among different sporophytic tissues havebeen reported in some angiosperm HD-Zip genes, although the patterns of expression in gametophytic tissues in angiosperms are not known. Our study clearly
showed that most of Ceratopteris HD-Zip genes are
ubiquitously expressed even in gametophytes fromwhich morphologically different tissues and organs of
sporophytes develop. It may indicate that Crhb genes
have unknown functions common in the both generations. On the contrary, Crhbl and Crhb3 genes are
expressed only in the gametophytes and it may suggest
the existence of gametophyte specific regulatory systems which cause morphological diversity between the

NATIONAL INSTITUTE

two generations. Further studies on the regulatory
elements of the Crhb genes and on more detailed spatial
patterns of expression will give useful information toreveal the evolutionary aspects of differences between
sporophytic and gametophytic generations.

IV. Establishment of tagged mutant library of themoss Physcomitrella patens.
Mosses have the different body plan from flowering
plants. Leafy shoots of mosses are similar to the onesof angiosperms, but develop in the gametophytic generation instead of the sporophytic generation as angiosperms. Organs of mosses are much simpler thanflowering plants. For example, the leaves are composed of one layer of cells. Therefore, the body planof mosses may be regulated by different genes fromangiosperms. In addition to analyses of the homo logs
of angiosperm genes governing morphogenesis, it isnecessary to screen specific genes in the moss. We
established enhancer and gene trap lines and tagged
mutant libraries of Physcomitrella patens to clone genes
related to leafy shoot development. P. patens is known
by its high rate of homologous recombination andsuitable for molecular biological analyses using thegene targeting. Our libraries should be also useful forother purposes.

Publication List:
Hasebe, M. (1999) Evolution of reproductive organs inland plants. J. Plant Res. 112: 463-474.
Hasebe, M. and Ito, M. (1999) Evolution of reproductive
organs in vascular plants. In The Biology of Biodiversity(M. Kato ed.), pp.243-255. Springer-Verlag, Tokyo.
Shindo, S., Ito, M., Ueda, K., Kato, M. and Hasebe, M.
(1999) Characterization of MADS genes in the gymnosperm Gnetum parvifolium and its implication on theevolution of reproductive organs in seed plants. Evolution and Development 1 : 180-190.
Aso, K., Kato, M., Banks, J.A. and Hasebe, M. (1999)
Characterization of homeodomain-leucine zipper genes
in the fern, Ceratopteris richardii and the evolution of
the homeodomain-leucine zipper gene family in vascularplants. Mol. Bioi. Evol. 16: 544-552
Salzman, R., Fujita, T., Zhu-Salzman, K., Hasegawa, M.
and Bressan, R.A. (1999) An improved RNJ\ isolation
method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol. Bioi.
Repter 17: 11-17.
Nishiyama, T. and Kato, M. (1999) Molecular phylogenetic analysis among bryophytes and tracheophytes
based on combined data of plastid coded genes and the
18S rRNA gene. Mol. Bioi. Evol. 16: 1027-1036.

FOR BASIC BIOLOGY59/

Fig. 1 New hypothesis on the homology between Gnetum and conifer reproductive organs. (A) Gnetum parvifolium. Female strobili andvegetative branches with leaves. (B) A whorl of nearly mature ovules subtended by annular collars. (C-F) In situ localization of GpMADS3mRNA in developing ovules of Gnetum parvifolium. (G) Scenario for the evolution of ovule-bearing structures in Gnetum and conifers from a
Cordaitales-like prototype. b, bract; bs, bracteole scale; ca, cone axis; co, collar; i, inner integument; ie, inner envelope; me, middle envelope; n,nucellus; o, ovule; op, ovule primordium; oe, outer envelope; os, ovuliferous scale; ss, sterile scale.

60

NATIONAL.INS.'I'ITIJ'fE61

FOR BASIC BIOLOGYCENTER FOR BIO-ENVIRONMENTAL SCIENCEInterim Head: Hideo Mohri

The center is currently managed by NIBB and is scheduled to be included in a new center for biosciences which will be jointly managed by NIBB
and two other institutes in Okazaki, IMS and NIPS.

The following pro-jects will be the main focus ofthe Center: 1) Endocrine disrupters. 2) Other chemicals affecting the environment. 3) Biomolecular sensors of environmental factors. 4) Database ofbio-environmental science.

62
Professor : Teizo Kitagawa

I. Biomolecular sciense
Elucidation of a structure-function relationship of metalloproteins is a current subject of this group. The primary
technique used for this project is the stationary and timeresolved resonance Raman spectroscopy monitored bynear IR to UV lasers. The main themes that we want to
explore are (1) mechanism of oxygen activation by enzymes, (2) mechanism of active proton translocationand its coupling with electron transfer, (3) couplingmechanism of proton- and electron transfers by quinones
in photosynthetic reaction center, (4) higher order protein
structures and their dynamics, and (5) reactions of biological NO.
In category ( 1), we have examined a variety of terminal
oxidases, cytochrome P450s, and peroxidases, and also
treated their enzymatic reaction intermediates by using
the mixed flow transient Raman apparatus and the Raman/absorption simultaneous measurement device. For(2) the third generation UV resonance Raman (UVRR)
spectrometer was constructed and we are going to use it to
the peroxy and ferryl intermediates of cytochrme c oxidase. In (3) we succeeded in observing RR spectra ofquinones A and B in bacterial photosynthetic reaction
centers for the first time last year, but we focused ourattention on tyrosine radical this year. For (4) we developed a novel technique for UV resonance Raman measurements based on the combination of the first/second
order dispersions of gratings and applied it successfully to
235-nm excited RR spectra of seveml proteins including
mutant hemoglobins and myoglobins. Nowadays we cancarry out time-resolved UVRR experiments with nanosecond resolution to discuss protein dynamics. We havesucceeded in isolating the spectrum of ~145-Tyr, ~35-Tyrand a.140-Tyr of Hb A separately and their changes uponquaternary structure transition. For (5) we purified soluble guanylate cyclase from bovine lung and observed its
RR spectra. To understand the implication, we examined Raman spectra of NO adducts of various mutant
Mbs.

D. Fast dynamics of molecules in a solution phase
Picosecond time-resolved resonance Raman (ps-TR3)
spectroscopy is a promising technique to investigate ultrafast structural changes of molecules. However, thistechnique has not been used as widely as nanosecond TR3
spectroscopy, mainly due to the lack of light source which
has suitable repetition rates of pulses and wavelength
tunability. In order to obtain qualified TR3 spectra, first
we need two independently tunable light sources forpump and probe pulses. Second, the repetition rate should
be higher than kilohertz to keep a moderate average laser
power without allowing the photon density of probe pulse
to be too high. We succeeded in developing light sources
for ps-TR3 spectroscopy having wide tunability and kHz
repetition, and applied them to study fast dynamics ofphoto-excited molecules. For carbonmonoxy myoglobin(MbCO), vibrational relaxation with the time constant of

NATIONAL INSTITUTE

1.9 ps was observed for CO-photodissociated heme. For
Ni-octaethylporphyrin in benzene, appreciable differencesin the rise times of population at vibrationally excitedlevels among various modes were observed in the antiStokes spectra for the first time. For the same moleculein piperidine, coordination of two solvent molecules wasobserved in the transient (d,d) excited state. The ps-TR3
experiments were also applied to Zn-porphyrin dimers,
for which some evidence for the 1t-1t interaction in the sl
state was obtained. The UV ns-TR3 experiments on
MbCO demonstrated the presence of a transient open
form of the ligand pathway.

Publication List:
Itoh, S., Bandoh, H., Nagatomo, S., Kitagawa, T. and Fuku-·
zumi, S. (1999) Aliphatic hydroxylation by a bis(Jloxo)dinickel(III) complex. J. Am. Chern. Soc. 121, 89458946.
Itoh, S., Taki, M., Kumei, H., Takayama, S., Nagatomo, S.,
Kitagawa, T., Sakurada, N., Arakawa, R. and Fukuzurni, S.
(1999) Model complexes of the active form of galactose
oxidase. Physicochemical properties and reactivity ofCu(II)- and Zn(li)-phenoxyl radical complexes of thenovel organic cofactor. Angew. Chem.Intl. Ed. 38, 27752776.
Iwase, T., Varotsis, C., Shinzawa-Itoh, K., Yoshikawa, S.
and Kitagawa, T. (1999) Infrared evidence for Cu8 ligationof photodissociated CO of cytochrome c oxidase at ambient temperatures and accompanied deprotonation of a carboxyl side chain of protein. J. Am. Chern. Soc. 121, 14151416.
Kaminaka, S., Imamura, Y., Shingu, M., Kitagawa, T. andToyoda, T. (1999) Studies of bovine enterovirus structure
by ultraviolet resonance Raman spectroscopy. J. Virol. Met.
77, 117-123.
Kohzuma, T., Inoue, T., Yoshizaki, F., Sasakawa, Y., Onodera, K., Nagatomo, S., Kitagawa, T., Uzawa, S., Isobe, Y.,
Sugimura, Y., Gotowda, M. and Kai, Y. (1999) The structure and unusual pH-dependence of plastocyanin from the~fern Dryopteris crassirhizoma: The protonation of an active-site histidine is hindered by 1t-1t interactions. J. Bioi.
Chern. 274,11817-11823.
Mizutani, Y., Uesugi, Y. and Kitagawa, T. (1999) Intramolecular vibrational energy redistribution and intermolecular energy transfer in the (d,d) excited state of nickel octaethylporphyrin. J. Chern. Phys. 111, 8950-8962.
Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S. and Kitagawa, T. (1999) Quaternary structuresensitive tyrosine residues in human hemoglobin: UV
resonance Raman studies of mutants at a.l40, ~35, and~ 145 tyrosine. Biochemistry 38, 1243-1251.
Nagatomo, S., Nagai, M., Tsuneshige, A., Yonetani, T. andKitagawa, T. (1999) UV resonance Raman studies of a.nitrosyl hemoglobin derivatives: Relation between thea.l-~2 subunit interface interactions and the Fe-histidine
bonding of a. heme. Biochemistry in press,
Nakashima, S., Taniguchi, S., Okada, T., Osuka, A., Mizutani, Y. and Kitagawa, T. (1999) Evidence for 1t-1t interac-

FOR BASIC BIOLOGY

tions in the S 1 state of zinc porphyrin dimers revealed bypicosecond time-resolved resonance Raman spectroscopy.
J. Phys. Chern. A 103, 9184-9189.
Suga, M., Oku, J., Masuda, H., Takaki, M., Mukai, M. andKitagawa, T. (1999) Mechanism of the anionic cyclopolymerization of bis(dimethylvinylsilyl)methane. Macromolecules 32, 1362-1366.
Suzuki, N., Higuchi, T., Urano, Y., Kikuchi, K., Uekusa, H.,
Ohashi, Y., Uchida, T., Kitagawa, T. and Nagano, T.
(1999) Novel iron porphyrin-alkanethiolate complex withintramolecular NH... S hydrogen bond: Synthesis, spectroscopy, and reactivity. J. Am. Chern. Soc. 121, 11571-11572.
Tomita, T., Hirota, S., Ogura, T., Olson, J. S. and Kitagawa,T. (1999) Resonance Raman investigation of Fe-N-0
structure of nitrosylheme in myoglobin and its mutants. J.
Phys. Chem.B 103, 7044-7054.
Wada, A., Ogo, S., Watanabe, Y., Mukai, M., Kitagawa, T.,
Jitsukawa, K., Masuda, H. and Einaga, H. (1999) Synthesis
and characterization of novel alkylperoxo mononuclear
iron(III) complexes with a tripodal pyridylamine ligand: Amodel for peroxo intermediates in reactions catalyzed by
non-heme iron enzymes. Inorg. Chern. 38, 3592-3593

63

64
Professor (Adjunct):Minoru Kanehisa (Kyoto University)

Though intermediary metabolism common to most organisms has been deeply investigated so far, variety of
species specific pathways in secondary metabolism,
which may work only in specific environmental conditions, are still unclear. The aim of this laboratory is todevelop a database system for environmental biology,
which integrates knowledge about organic compounds,chemical reactions between these compounds in vivo,
enzymes (genes) involved in these reactions, and species
whose genomes contain these genes. Through this database combining with data from transcriptome or proteome analyses in various environmental conditions, we
intend to elucidate the principle of interactions between
organisms and environmental chemical compounds topredict or design novel interactions.

Publication list:
Goto, S., Nishikoka, T., and Kanehisa, M. (1999) LIGAND
database for enzymes, compounds, and reactions. Nucleic
Acids Res. 27, 377-379.
Wackett, L.P., Ellis, L.B.M., Speedie, S.M., Hershberger,
C.D., Knackmuss, H.-J., Sporrnann, A.M., Walsh, C.T.,
Forney, L.J., Punch, W.F., Kazic, T., Kanehisa, M., andBerndt, D.J. (1999) Predicting microbial biodegradation
pathways. ASM News 65, 87-93.

NATIONAL INSTITUTE

Associate Professor : Akihiro Hazama
Research Associate : Kensuke Nakahira
Research Associate : Takuya Takahashi

This division has just started in August, 1999. The cells
which constitute our bodies accomadate their environment
by sensing the conditions surrounding them. The cells use
the special membrane proteins, such as receptors, channels, and transporters, for sensing not only many substances but also heat, osmolarity, and mechanical stimuli.
The aim of this division is to understand the sensing
mechanims of such membrane proteins sense and how thecells accomodate their environment after sensing theoutside conditions.

Publications:
Yasui M, Hazama A, Kwon TH, Nielsen S, Guggino WB,
Agre P. Rapid gating and anion permeability of an intracellular aquaporin. Nature. 402: 184-187, 1999
Hazama A, Shimizu T, Ando-Akatsuka Y, Hayashi S, Tanaka S, Maeno E, Okada Y. Swelling-induced, CFTRindependent ATP release from a human epithelial cell line:
lack of correlation with volume-sensitive Cl- channels. J
Gen Physiol. 114:525-533.1999
Baba H, Akita H, Ishibashi T, Inoue Y, Nakahira K, Ikenaka
K. Completion of myelin compaction, but not the attachment of oligodendroglia! processes triggers K+ channel
clustering. J Neurosci Res. 58:752-764. 1999
Shibata R, Wakazono Y, Nakahira K, Trimmer JS, Ikenaka K.
Expression ofKv3.1 and Kv4.2 genes in developing cerebellar granule cells. Dev Neurosci. 21:87-93. 1999

FOR BASIC BIOLOGY

Associate Professor:
Assistant Professor:

65

Hirokazu Tsukaya, Ph.D.
Gyung- Tae Kim, Ph.D.

Diversity of plant form is mostly attributable tovariation of leaf and morphology of floral organs, which
are modified, leaves. The leaf is the fundamental unit of
the shoot system, which is composed with leaf and stem.
So the leaf is the key organ for a full understanding ofplant morphogenesis. However, the genetic control ofdevelopment of these shapes had remained unclear.
Recently, studies of leaf morphogenesis has been in aturning point, after our successful application of thetechniques of developmental genetics and molecular
genetics to it, using model plants such as Arabidopsis
thaliana (L.) Heynh. Our purpose is to understand
Plants from view point of molecular genetic control ofleaf morphogenesis.
Focusing on mechanisms that govern polarized
growth of leaves in a model plant, Arabidopsis thaliana ,we found that the two genes act independently to eachother on the processes of polar growth of leaves: the ANgene regulates width of leaves and the ROT3 gene
regulates length of leaves. The AN gene controls thewidth of leaf blades and the ROT3 gene controls length.
The AN gene seems to control orientation of cortical
microtubules in leaf cells. Cloning of the AN gene revealed that the gene is a member of gene family foundfrom animal kingdom (Tsukaya et al., in prep). The
ROT3 gene was cloned by us in 1998. Transgenic experiments proved that the ROT3 gene regulates leaflength without affect on leaf-width (Kim et al., 1999).
We are trying to identify molecular function of theabove genes which are essential for leaf morphogenesis.
While ROT3 regulates the length of both leaf blades
and petioles, ACL2 appears to regulate petiole length
exclusively. Genes for perception of environmental
stimuli such as light and/or phytohormone perception

also affect the petiole length relative to the length of theleaf blade. Genetic studies suggested that petioles andleaf blades share some regulatory pathways but petioles
also have their own developmental programs that are
independent of those of leaf blades (Tsukaya and Kim,
submitted).
Apart from polar elongation, we identified the following genes involved in leaf expansion process. The
ASJ and AS2 genes are needed for proportional growthof the leaf. Molecular and anatomical analysis of theas2 mutant is now underway, in collaboration with Prof.
Machida, Nagoya University.
On the other hand, we are trying to identify molecular mechanisms which distinguish developmental pathway of leaves from that of shoots. For such purposes,
we introduced tropical plants having queer developmental program for leaf morphogenesis, namely, Chisocheton, Guarea and Monophy llaea, as materials formolecular studies.
In addition, we are interested in roles of such genes
for environmental adaptation. Leaf index, relative
length of leaf to width, is one of the most diverse factor
of leaf shape. For instance, rheophytes are characterizedby narrow leaves, which represent an adaptation to theirhabitats. Are AN and ROT3 genes are involved inregulation of adaptive change of leaf index in natural
condition? Are these genes the responsible for evolutionof rheophytes? So called "Evo/Devo" study of leaf
morphogenesis is also one of our research project in
NIBB .

Publication List:
Tsukaya, H., Shoda, K. , Kim, G.-T. and Uchimiya, H.
(2000) Heteroblasty in Arabidopsis thaliana (L.) Heynh.
Planta 210 : 536-542.

Figure: Single gene, the ROT3, controls leafform (modified from Kim et al. , 1999)

66'

l:fATIONPiL.lNSTITU.TE

67

FOR BASIC BIOLOGYRESEARCH SUPPORT68

NATIONAL INSTITUTECENTER FOR TRANSGENIC ANIMALS AND PLANTS
Head: Masaharu Noda
Associate Professor:
Supporting Staff:

Eiji Watanabe
Mie Yasuda (Sept. 1, 1999-)

I. Research supporting activity
NIBB Center for Transgenic Animals and Plants, established in April, 1998, aims to support researches in
NIBB using transgenic and gene-targeting techniques.
We are now planning on the construction of new facilities.
The expected activities of the Center at present are as
follows:!.Provision of information, materials and techniques toresearchers.
2.Equipment of various instruments to analyze mutant
animals and plants.
3.Development of novel techniques related to transgenic

and gene targeting technology.

II. Academic activity
We are studying the functional role of subfamily 2channels in collaboration with Division of Molecular
Neurobiology. Subfamily 2 channels are a group of
voltage-gated sodium channels (NaChs) that generateaction potentials in electrically excitable cells such asneurons and muscle cells. Comparing with the other
NaChs, this channel species has unique amino acidsequences in the regions, which are known to be involved in ion selectivity and voltage-dependent activation and inactivation, suggesting that subfamily 2 channels must have specific functional properties. To clarify the functional role in vivo , the subfamily 2 channeldeficient mice were generated by gene targeting. We
are now examining the physiological phenotypes.

Fig. I LacZ expression in mNaG+/- mice (A-C). .
.
.
The expression pattern of mouse subfamily 2 channel gene (mNaG) was revealed by LacZ gene expressiOn m mNaG+/- mtce. The blue signals
represent the site expressing LacZ gene. (A) E 15 whole embryo, (B) tissue section of adult tongue, and (C) adult sympatbettc nerve trunk m thethoracic region. In (B), the tissue section was counterstained with cresyl violet.

69

FOR BASIC BIOLOGYTECHNOLOGY DEPARTMENT

Head:Hiroyuki Hattori

Common Facility Group

Research Support Group

Chief: Kazuhiko Furukawa

Chief: Hiroko Kobayashi

Reseach Support Facilities
Shoichi Higashi (Unit Chief)
Tomoki Miwa(Subunit Chief)
Chieko Nanba(Subunit Chief)
Hiroyo Nishide
Makiko ltoh(Technical Assistant)Keiko Suzuki(Technical Assistant)
Misayo Masuda (Technical Assistant)
Hideyuki Gotoh(Thecnical Assistant)
Yasuyo Kamiya(Thecnical Assistant)

Cell Biology Group
Maki Kondo(Subunit Chief)
Yukiko Kabeya
Katsushi Yamaguchi

Radioisotope Facility
Yousuke Kato(Subunit Chief)
Yoshimi Matsuda(Subunit Chief)
Naoki Morooka
Takayo Itoh(Technical Assistant)Center for Analytical Instruments
Mamoru Kubota (Unit Chief)
Sonoko Ohsawa(Subunit Chief)
TomokoMori
Yumiko Makino
Takeshi Mizutani
Hatsumi Moribe(Technical Assistant)

Developmental Biology Group
Chiyo Takagi
Sanae Oka
Naomi Sumikawa
Regulation Biology Group
Hideko linuma
Akiko Oda
Shigemi Takami
Gene Expression and Regulation Group
Sachiko Tanaka(Subunit Chief)
Koji Hayashi
Yasushi Takeuchi
Kaoru Sawada
Hideko Utsumi
Makiko Kondo(Technical Assistant)

Glassware Washing Facility
Masayo lwaki(Kazuhiko Furukawa)

The Technology Department is a supporting organization for researchers and research organization within
the NffiB. The Department develops and promotes theinstitute's research activities and at the same time,
maintains the research functions of the institute.
The department is organized into two groups: one,
the Common Facility Group,which supports and maintains the institute's common research facilities and theother, the Research Support Group, which assists theresearch activities as described in individual reports.
Technical staffs participate, through the department,

in mutual enligtement and education increase theircapability in technical area. Each technical staff is proceeded to the fixed division usually and they support thevarious research with their special biological and biophysical techniques.
The Department hosts an annual meeting for technical engineers who work in various fields of biology atuniversities and research insititutes throughout Japan.
At this meeting, the participants present their own activities and discuss technical problems. The Proceedings
are published soon after the meeting.

70

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY
Head ofFacility:
Associate Professor:
Research Associates:

Technical Staffs:

Yoshinori Ohsumi
Masakatsu Watanabe
Yoshio Hamada(Tissue and Cell Culture)
Ikuo Uchiyama(Computer) (Marc~ 1, 1999-)
Sho-ichi Higashi(Large Spectrograph)
Mamoru Kubota (Large Spectrograph) (-March 31, 1999)
Tomoki Miwa (Computer)
Chieko Nanba(Plant Culture, Farm, Plant Cell)
Hiroyo Nishide(Computer) (June 1, 1999-)
Toshiki Ohkawa(Computer) (-May 31, 1999)
Kaoru Sawada (Tissue and Cell
Culture) (-June 30, 1999)
Hideyuki Go to (Large Spectrograph)(May 17, 1999-)
Makiko Ito (Large Spectrograph)
Yasuyo Kamiya (Tissue and Cell
Culture) (May 17, 1999-)
Misayo Masuda (Computer)
Keiko Suzuki(Plant Culture, Farm, Plant Cell)

I. Facilities
1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, the Okazaki Large Spectrograph (OLS), which is the largest
spectrograph in the world, dedicated to action spectroscopical studies of various light-controlled biologicalprocesses.The spectrograph runs on a 30kW Xenon arc
lamp and has a compound grating composed of 36 smaller
individual gratings. It projects a spectrum of a wavelength
range from 250nm (ultraviolet) to l,OOOnm (infrared) onto
its focal curve of 10m in length. The fluence rate (intensity) Of the monochromatic light at each wavelength ismore than twice as much as that of the corresponding
monochromatic component of tropical sunlight at noon
(Watanabe et al., 1982, Photochem. Photobiol., 36, 491498).
A tunable two-wavelength CW laser irradiation system
is also available as a complementary light source to OLS
to be used in irradiation experiments which specificallyrequire ultra-high fluence rates as well as ultra-high spectral-, time-and spatial resolutions.It is composed of ahigh-power Ar-ion laser (Coherent, Innova 20) (336.6528.7 nm, 20W output), two CW dye lasers (Coherent,
CR599-0l) (420-930nm, 250-lOOOmW output), A/0
modulators (up to 40MHz) to chop the laser beam, a beam
expander, and a tracking microbeam irradiator (up to 200
Jlm s·' in tracking speed, down to 2 Jlm in beam diameter)with an infrared phase-contrast observation system.

2. Tissue and Cell Culture Laboratory
Notch is an integral cell surface memberane proteinthat is known to play a key role in developmental cell-cellinteractions in Drosophila, particulaly in lateral specification of neural versus epidermal cell fates, a process described thus far only in invertebrates. It is thought to act
by a direct signaling pathway rather than through one of
the classical signal transduction cascades. The mammalian genome is known to contain four Notch homologues but their developmental significance is not clear.
To investigate their role in mammalian development, wehave sequenced the murine Notch2 eDNA, determined theprimary sequence of its protein, and have produced
Notch2 mutant mice by gene trageting procedures. The
mutant mice die prior to embryonic day 11.5. Thus,
Notch2 palys an essential role in postimplantation development in mice. Chimeric analysis revealed that embryonic lethality of the mutant mice is due to defect in placenta development and Notch2 function is involved information of the roof plate of diencephalon and mesencephalon in development later than the lethal stage.
3. Computer Laboratory
Computer laboratory maintains several computers toprovide computation resources and means of electronic
communication in this Institute. This year, new computer systems were introduced to meet requirement of large
scale biological data analysis, and now the system mainly
consists of three servers and two terminal workstations:biological information analysis server (SGI Origin 2000),
database server (Sun Enterprise 450), file server (SunEnterprise 3000), data visualization terminal and molecular simulation terminal (both are SGI Octanes). Somepersonal computers (Macintoshes and Windows PCs) andcolor/monochrome printers are also equipped. Various
biological databases and data retrievaVanalysis programs
are available on this system.
Computer laboratory also provides network communication services in the Institute. Most of PCs in eachlaboratory as well as all of the above service machines areconnected each other with local area network (LAN),
which is linked to the high performance multimedia backbone network of Okazaki National Research Institute(ORION). Many local services including sequence analysis service, file sharing service and printer service areprovided for the Institute members through this LAN.
We also maintain a public World Wide Web server that
contains the NIBB home pages (http://www.nibb.ac.jp).
4. Plant Culture Laboratory
There are a large number of culture boxes, and a limited number of rooms with environmental control for plant
culture. In some of these facilities and rooms, experiments
can be carried out at the Pl physical containment level
under extraordinary environments such as strong lightintensity, low or high temperatures.

71

FOR BASIC BIOLOGY

5. Experimental FarmThis laboratory consists of two 20 m2 glass-houses with
precise temperature and humidity control, three green
houses (each 6m2) at the PI physical containment level, asmall farm, two greenhouses (45 and 88 m2) with automatic sprinklers, two open aquariums (30 and 50 t) andseveral smaller tanks. The laboratory also includes abuilding with office, storage and work space.
6. Plant Cell Laboratory
Autotrophic and heterotrophic culture devices and areequipped for experimental cultures of plant and microbial
cells. A facility for preparation of plant cell cultures including an aseptic room with cleanbenches, is also provided.

7. Laboratory of Stress-Resistant Plants
This laboratory was founded to study transgenic plants
with respect to tolerance toward various enviromental
stresses. It is located in the Agricultural Experimental
Station of Nagoya University (30 km from National Institute for Basic Biology). The laboratory provides a variety of growth chambers that precisely control the conditions of plant growth and facilities for molecular biological, and physiological evaluations of transgenic plants.
The laboratory is also a base of domestic and international collaborations devoted to the topic of stressresistant transgenic plants.

cused on the identification of orthologous genes between
multiple genomes, which is a crucial step for comparative
genomics. Since considerable number of genes consistof multiple domains, we are now developing a hierarchical clustering algorithm that can automatically split fusion
genes into orthologous domains using all-against-all homology search results. In parallel, we also make detailed comparison of closely related microbial genomes toinvestigate the genomic polymorphisms or evolutionarychanges in collaboration with Dr. I. Kobayashi's group(Univ. Tokyo). By comparing genomes of two Helicobacter pyroli strains, we could fmd iilteresting insertion/deletion patterns that frequently include restrictionmodification genes.

2. Cooperative Research Program for the Okazaki
Large Spectrograph
The NIBB Cooperative Research Program for the Useof the OLS supports about 30 projects every year conducted by visiting scientists including foreign scientists aswell as those in the Institute.
Action spectroscopical studies for various regulatoryand damaging actions of light on living organisms, biological molecules, and organic molecules have been
conducted (Watanabe, 1995, In CRC Handbook of Organic Photochemistry and Photobiology).

Publication List:
II. Research activities
1. Faculty
The faculty of the Research Support Facility conducts
its own research as well as scientific and administrative
public services.
(1) Photobiology: photoreceptive and signal transduction mechanisms of phototaxis of unicellular algae arestudied action spectroscopically (Watanabe 1995, In CRC
Handbook of Organic Photochemistry and Photobiology)
by measuring computerized-videomiceographs of the
motile behavior of the cells at the cellular and subcellularlevels (Horiguchi et a!., 1999; Matsunaga et a!., 1999;
Choi eta!., 1999). Photo-receptive and signal transduction
mechanisms of algal gene expression were also studied byaction spectroscopy (Leblanc eta!., 1999).
(2) Computational Biology: Explosively growing biological data produced from various genome projects
should contain many clues to understanding complex anddiverse biological systems. Comparative genomics is a
useful approach to find s~ch clues. We are constructing
a database system for comparative analysis of many of
microbial genomes ever sequenced and developing newcomputational techniques for large-scale genome sequence comparison. Currently, our research aim is fo-

I. Faculty
Choi, J-S., Chung-Y-H, Moon, Y-J., Kim, C., Watanabe, M.,
Song, P-s., Joe, C-0, Bogorad, L., and Park, Y. M. (1999)
Photomovement of the gliding cyanobacterium Synechocystis sp. PCC 6803. Photochem. Photobiol. 70, 95-102.
Hamada,Y., Kadokawa,Y., Okabe, M., Ikawa, M., Coleman,
J.R. & Tsujimoto, Y(1999). Mutation in ankyrin repeats ofthe mouse Notch2 gene induces early embryonic lethality.
Development 126, 3415-3424.
Horiguchi, T., Kawai, H., Kubota, M., Takahasi, T. and
Watanabe, M.(l999). Phototactic responses of four marine
dinoflagellates with different types of eyespot and chloroplast. Phycol. Res. 47, 101-107.
Kobayashi, I., Nobusato, A., Kobayashi-Takahashi, N.,
Uchiyama, I. (1999) Shaping the genome - restrictionmodification systems as mobile genetic elements. Curr.
Opin. Genet. Dev. 9, 649-656.
Leblanc, C., Falciatore, A., Watanabe, M. and Bowler, C.
(1999). Semi-quantitative RT-PCR analysis ofphotoregulated gene expression in marine diatoms. Plant Mol. Bioi
40 (6), 1031-1044.
Matsunaga, S., Takahashi. T., Watanabe, M., Sugai, M. andHori, T. (1999). Control by ammonium ion of change fromstep-up to step-down photophobically responding cells in
the flagellate alga Euglena gracilis. Plant Cell Physiol.
40(2) , 213-221.
Shimizu, K., Chiba, S., Kumano, K., Hosoya,N., Takahashi,

72T., Kanda,Y., Hamada,Y., Yazaki, Y. & Hirai, H.(1999).
Mouse Jagged1 physically interacts with Notch2 and otherNotch receptors. J. Bioi Chern. 274, 32961-32969.
Tanaka,M., Kadokawa, Y., Hamada,Y. & Marunouchi,
T.(1999). Notch2 expression negatively correlates withglial differentiation in the postnatal mouse brain. J. Neurobiol. 41, 524-539.

II. Cooperative Research Program for the Okazaki
Large Spectrograph
Andrady, A L. and Torikai, A (1999). Photoyellowing of
mechanical pulps III. Intensity effects and dose-response
relationships. Polym. Degradn. Stab., 66, 317-322.
Andrady, A L., Hamid, S. H., Hu, X. and Torikai, A (1999).
Effects of icreased solar ultraviolet radiation on materials.
J. Photochem. Photobial. B:Biol. 46, 96-103.
Choi, J-S., Chung-Y-H, Moon, Y-J., Kim, C., Watanabe, M.,
Song, P-s., Joe, C-0, Bogorad, L., and Park, Y. M. (1999)
Photomovement of the gliding cyanobacterium Synechocystis sp. PCC 6803. Photochem. Photobiol. 70,95-102.
Fujiwara, A, Tazawa, E., Kamata, Y. and Yasumasu, I.
(1999). Photo-actvation of respiration in degenerated
sperm of echiuroid, oyster and sea urchin. Zoo!. Sci. 16,
237-246.
Hashimoto, T. and Shichijo, C. (1999). Amplification ofphytochrome-induced photomorphogenesis by red light
pre-irradiation, and a cryptic red-light signal production.

NATIONAL INSTITUTE

Trend in Photochem. and photobiol. vol. 6, 15-27.
Horiguchi, T., Kawai, H., Kubota, M., Takahashi, T. and
Watanabe, M.(1999). Phototactic responses of four marine
dinoflagellates with different types of eyespot and chloroplast. Phycol. Res. 47, 101-107.
Ishigaki, Y., Takayama, A,Yamashita, S. and Nikaido, 0.
( 1999). Development and characterization of a DNA solar
dosimeter. J. Photochem. Photobiol. B: Bioi. 50, 184188.
Leblanc, C., Falciatore, A, Watanabe, M. and Bowler, C.
(1999). Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms. Plant Mol. Bioi
40 (6), 1031-1044.
Matsunaga, S., Takahashi. T., Watanabe, M., Sugai, M. andHori, T. (1999). Control by ammonium ion of change fromstep-up to step-down photophobically responding cells in
the flagellate alga Euglena gracilis. Plant Cell Physiol.
40(2) '213-221.
Shichijo, C., Onda, S., Kawano, R., Nishimura, Y. and Hashimoto, T. (1999). Phytochrome elicits the cryptic redlight signal which results in amplification of anthocyanin
biosynthesis in sorghum. Planta. 208, 80-87.
Torikai, A and Hasegawa, H. (1999). Accelerated.photodegradation of poly(vinyl chloride). Polym. Degradn. Stab.
63,441-445.
Torikai, A. and Shibata, H. (1999). Effect of Ultraviolet
radiation on photodegradation of collagen. J. Appl. Polym.
Sci. 73, 1259-1265.

73

FOR BASIC BIOLOGYRADIOISOTOPE FACILITY (managed by NIBB)Head (Projess01; concurrent post): Shigeru !ida
Associate Professor:
Kazuo Ogawa
Technical Staffs:
Yosuke Kato
(Radiation Protection
Supervisor),
Yoshimi Matsuda(Radiation Protection
Supervis01) ,Naoki Morooka
Supporting Staff:
Takayo Ito
Risako Shirai
Yumiko !ida

I. Research supporting activity
Technical and Supporting Staffs of this facility areserving the purchase of radioisotopes from JRA (Japan
Radioisotope Association) and the transfer of radioisotope wastes to JRA. The physical maintenance of thecontrolled areas where radioisotopes are used by theregistered users of NIBB (National Institute for Basic
Biology) and NIPS (National Institute for Physiological
Science) for research is also one of our business.
This facility consists of four controlled areas : Center,
NIBB-sub, LGER (Laboratory of Gene Expression andRegulation)-sub, and NIPS-sub. Users going in and out
the controlled areas counted by the monitoring systemwere 7,912 in 1999. This count was comparable to that
(8,483) in 1998. The items in each controlled area arepresented in Figure 1.

II. Academic activity
Academic activity by Associate Professor is focusedon the analysis of the structure and function of a dynein
motor protein. Dyneins are a group of microtubuleactivated ATPases that serve to convert chemical energy
into mechanical energy and divided into axonemal andcytoplasmic dyneins . Figure 2 shows the localization of
two isoforms of dyne in in the outer mms of sperm axonemes (Ogawa eta!., 1977) and the mitotic apparatus ofcleaving egg (Mohri et a!., 1976) visualized by antiaxonemal dynein (Fragment A) antibodies.
The native dyneins are very large and range in molecular mass up to 1 to 2 mega Da. They are complex
proteins containing heavy, intermediate, and lightchains defined by the molecular mass. Our present
project is the molecular cloning of polypeptides contained in outer arm dynein of sea urchin sperm flagellato understand the mechanism how dynein interacts withmicrotubules, resulting in producing the force .
Outer arm dynein consists of two heavy chains with
ATPase activity. The motor activity is closely related tothis polypeptide. The first successful molecular cloningof this huge polypeptide (520 kDa) was perfonned in
our laboratory in 1991. Since then eDNA clones foraxonemal and cytoplasmic dyneins have been isolated
in a variety of organisms. The sequences of heavy
chains, without exception, contain four P-loop motives
referred to as ATP-binding sites in the midregion of themolecules. Figure 3A and B draw the structure of heavy
chain deduced from the amino acid sequence (Ogawa,
1992). Taking the recent works by Koonce et a!. (1998)
and Vallee et al. (1998) into consideration, this model
might be seen as depicted in Figure 3C. In particular,
Vallee eta!. (1998) have described the importance of a
hairpin structure formed between M and C domains
which binds to microtubules and presented a novel
mechanism for dynein force production different fromthat of myosin and kinesin.
Outer arm dynein contains three intennediate chains(IC1 , IC2 , and IC3) that range in molecular mass from70 to 120 kDa. IC2 and IC3 were cloned by Ogawa eta!.
(1995) and contain the WD repeats in the carboxyterminal halves of the molecules. By contrast, IC1 is nota member of the WD family. IC1 has a unique sequencesuch that the N-terrninal part is homologous to the sequence of thioredoxin, the middle part consists of three
repetitive sequences homologous to the sequence of
NDP kinase, and the C-terrninal part contains a highproportion of negatively charged glutamic acid residues(Ogawa et a!., 1996). Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be
a multifunctional protein.
Six light chains with molecular masses of 23.2, 20.8 ,
12.3, 11.5, 10.4, and 9.3 kDa are associating with outeram1 dynein. We have already isolated eDNA clone offive LCs. LC1 (23.2 kDa) and LC3 (12.3 kDa) arehighly homologous to mouse Tctex2 and Tctex1, respectively. These mouse proteins are encoded by the tcomplex region that is involved in transmission ratio
distortion (TRD), male sterility and the development ofgerm cells. Our finding raises the possibility that axonemal dynein proteins are involved in this phenomenon.
TRD may be caused by the dysfunction of multiple
axonemal dynein proteins.

NIPS-sub

NIBB-subActual countCenter

3,336

NIBB-sub871

LGER-sub2,885

NIPS-sub
total

820
7,912

Fi gure I. Percentage of users going in and out the controlledareas during April to December, 1999.

74

NATIONAL INSTITUTE

Figure 2. Localization of two dynein isotypes on outer arm of sperm axonemes and mitotic apparatus of cleaving egg.

Publication List:
S. Watabe, Y. Makino, K. Ogawa, T. Hiroi, Y. Yamamoto,and S. -Y. Takahashi. Mitochondrial thioredoxin reductase In bovine adrenal cortex: Its purification, properties,
nucleotide/amino acid sequences, and Identification ofselenocysteine. Eur. J. Biochem. 204, 74-84 (1999) .
K. Inaba, 0. Kagami, and K. Ogawa. Tctex2-related outerarm dynein light chain Is phosphorylated at activation of
sperm motility. Biochem. Biophys. Res. Commun. 256,
177-183 (1999).

A

P4

N

M

a2

c

B

c
Figure 3. Structure of dynein heavy chain. A; Analysis of aminoacid sequence of heavy chain reveals that it consists of three major
domains referred to as N, M, and C from theN-terminus. B; M andC domains make larger domain (motor domain) by intramolecularassociation. C, According to Koonce et al. ( 1998) showthat recombinant motor domain would be spherical. Vallee et al. (1998) propose that a2 region corresponds to the B-link which is the stalkprojected from the globular head structure of dynein, by demonstrating that the recombinant a2 actually binds to microtubules.

75

FOR BASIC BIOLOGYTHE CENTER FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head ofFacility:
Technical Staffs:

Technical Assistant:

Tetsuo Yamamori
Mamoru Kubota
Sonoko Ohsawa
TomokoMori
Yumiko Makino
Takeshi Mizutani (- Feb. 15, 2000)
Hatsumi Moribe

The Center serves for amino acid sequence analysis, and chemical syntheses of peptides and nucleotids
to support researchers in NffiB and NIPS . Newly installed instruments in 1999 are Biomek 2000 Laboratory Automation System and Automatic Nucleic Acid
Isolation System. Instruments of the Center can be usedby researchers outside the Institute upon proposal.

Figure I. Procise 494 Protein Sequencer.

Figure 2. Biomek 2000 Laboratory Automation System.

Representative instruments are listed below.
Protein Sequencers (ABI Procise 494, ABI 473A)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A, ABI 432A)
Plasmid Isolation Systems (Kurabo PI-1 00 L:)
Automatic Nucleic Acid Isolation System (Kurabo NA2000)
DNA Sequencers (ABI 377,373S, ABI 310)
DNA/RNA Synthesizers (ABI 394, ABI 392)
Thennal Cyclers (Perkin Elmer PJ-9600, Takara TP300)
Integrated Thermal Cyclers (ABI CATALYST Turbo
800)
Particle Delivery System (Bio-Rad BiolisticPDS1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs(Shimadzu LC-10AD, 6AD, Waters 600E)
Integrated Micropurification System (Phannacia
SMART)
Flow Cytometer (Coulter EPICS XL)
Biomolecular Interaction Analysis Systems(Pharmacia BIACORE 2000, Affinity Sensors IAsys)
Laboratory Automation System(Beckman Coulter Biomek 2000)
NMR Spectrometer (Bruker AMX-360wb)
EPR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS1200A)
Spectrofluorometers (Hitachi 850, Simadzu RF-5000)
Spectrophotometers (Hitachi 330, Hitachi 557, Varian
Cary 5G, Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB
96P)Time-resolved Fluorescence Microplate Reader(Pharmacia DELFIA Research)
Microplate Readers (Corona MTP-120, MTP-lOOF)
Spectropolarimeter (JASCO J-40S)
FT-IR Spectrophotometer (Horiba FT-730)
Laser Raman Spectrophotometer (JASCO R-800)
Bio Imaging Analyzers (Fujifilm BAS2000)
Fluorescence Bio Imaging Analyzer (Takara FMBIO II)Electrophoresis Imaging Systems (PDI Discovery Series,
BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Microscope Photometer (Carl Zeiss MPM 03-FL)

76

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