NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT

2000

Development of isolation membrane into autophagosome
traced with GFP-Apg5 on the membrane. The top panels
show sequential images of autophagosome formation
visualized with GFP-Apg5 by time-laps microscopy. The
bottom panels show corresponding immunoelectron
microscopic images using anti-GFP antibody.

Post Doctral Fellow
1 NIBB Research Fellow
2 JSPS Postdoctral Fellow
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4

JSPS Research Associate
JSTFellow

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

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

OFFICE OF DIRECTOR · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·. · · · · · · · · · · · · · · · · · · · ·Mechanisms determining the outline shape of the adult lepidopteran wings · · · · · · · · · · · · · · · · · · · · · · · ·44Protein palrnitoylation and developmental mechanism at embryogenesis · · · · · · · · · · · · · · · · · · · · · · · · · ·5

DEPARTMENT OF CELL BIOROGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·7

DIVISION OF CELL MECHANISMS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Regulation at the level of protein transport to microbodies during the
microbody transition. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·8

II. Microbody defective mutant of Arabidopsis. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·8ill. Transport ofstorage proteins to protein storage vacuoles is mediated by large PAC(precursor-accumulating) vesicles. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

IV. Vacuolar processing enzymes in protein-storage vacuoles
and lytic vacuoles. · · · · · · · · · · · · · · · · :·. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

V. Role of molecular chaperones in organelle differentiation. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

DIVISION OF BIOENERGETICS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·11

I. Background · · · · · · · · · · · · · · · · · · · · ·-· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·. · · · · · · · · · · · · · · · · ·118

II. Apg1-Apg13 protein kinase complex mediates induction of autophagy in response to
starvation signal · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ill. Two Distinct Phosphatidylinositol 3-Kinase Complexes Function in Autophagy andCarboxypeptidase Y Sorting · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·12

IV. Discovery of novellipidation reaction mediated by ubiquitin-like system · · · · · · · · · · · · · · · · · · · · · ·12

V. Mammalian Apg proteins; localization and function · ·; · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·12

DIVISION OF CELL PROLIFERATION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

I. Comprehensive identification of cells in the aduilt brain· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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II. Mapping of neurotransmitters and receptors in the adult brain · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ill. Analysis of the cell lineage-dependent modular structures in the brain · · · · · · · · · · · · · · · · · · · · · · · · ·IV. Development of an improved red fluorescent protein (RFP) reporter system suitable11for double labelling and birth order analysis of neural fibres · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·V. Contribution to the science community · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·16
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DIVISION OF CELLULAR COMMUNICATION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·18

I. Function of Multiple Axonemal Dyneins · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Function of Actin and an Actin-related Protein in Chlamydomonas · · · · · · · · · • · · · · · · · · · · · · · · · · · ·18
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DIVISION OF CELL FUSION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·20

DEPARTMENT OF DEVELOPMENTAL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·23

DIVISION OF REPRODUCTIVE BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·24

I. Endocrine regulation of gonadal sex differentiation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·24

II. Endocrine regulation of spermatogenesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III. Endocrine regulation of oocyte maturation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·25
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DIVISION OF CELL DIFFERENTIATION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Gene regulatory cascade in the steroidogenic tissue differentiation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Sex-differentiation observed in adrenal cortex··············································

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III. Molecular mechanism for head formation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·29

DIVISION OF MORPHOGENESIS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Role of Xmsx -1 as a negative regulator of head formation· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Genetic dissection ofTGF-~/BMP signaling utilizing Drosophila model system · · · · · · · · · · · · · · · · · ·III. Brachyury downstream notochord differentiation in the asci dian embryo · · · · · · · · · · · · · · · · · · · · · · ·

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IV. TGF-~ family in nematode · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·31

DIVISION OF DEVELOPMENTAL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·33

DEPARTMENT OF REGULATION BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·35

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

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I. Molecular mechanism of the retinotectal projection · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Functional roles of protein tyrosine phosphatase ~ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III. Physiological roles ofN3.y2 ion channel · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·DIVISION OF CELLULAR REGULATION· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·40

I. A sensor for low-temperature signals · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Analysis of the regulation of gene expression using a DNA rnicroarray · · · · · · · · · · · · · · · · · · · · · · · · ·III. The importance of membrane lipids in protection of the photosynthetic

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machinery against salt stress · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·IV. Characterization of the Na+/H+ antiporters of Synechocystis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · ·V. The role of glycinebetaine in tolerance to freezing temperatures · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · ·I. Cloning and characterization of blue-light photoreceptors · · · · · · · · · · · · · · · · · · · · · · ' · · · · · · · · · · · · · ·4343

II. Chloroplast relocation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·43

DIVISION OF BEHAVIOR AND NEUROBIOLOGY··········································

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I. Mechanism of Axonal Guidance · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Mechanism of Neuronal Migration· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·45
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LABORATORY OF GENE EXPRESSION AND REGULATIONDIVISION OF GENE EXPRESSION ANDREGULATION I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Spontaneous mutants in the iapanese morning glory.·········································
II. A new procedure for isolation of a gene tagged by a transposable element belonging to
the Tpnl family in the Japanese morning glory. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III. Identification and characterization of the Purple gene encoding a vacuolar Na+/H+

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exchanger, InNHX1, for blue flower coloration in the Japanese morning glory. · · · · · · · · · · · · · · · · · ·48

DIVISION OF GENE EXPRESSION AND REGULATION II· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Identification of cis-essential sequences for amplification of the ribosomal RNA
gene repeats in yeast. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Enhanced homologous recombination caused by a non-transcribed spacer of theribosomal RNA gene in Arabidopsis. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III. E. coli genomic structure and function · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·51

DIVISION OF SPECIATION MECHANISMS I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Genes expressed in specific areas of the neocortex · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Gene expression under audio-visual discrimination task · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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DIVISION OF SPECIATION MECHANISMS II · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Evolution of reproductive organs in land plants · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Evolution of vegetative organs in land plants · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III. Biogeography of Coriaria · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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CENTER FOR INTEGRATIVE BIOSCIENCE
DEPARTMENT OF BIOENVIRONMENTAL RESEARCH I···································I Estrogen-induced irreversible changes· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II Effect of estrogen on amphibian and fishes. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·III Molecular Target Search · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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DEPARTMENT OF BIOENVIRONMENTAL RESEARCH II··································

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DEPARTMENT OF BIOENVIRONMENTAL RESEARCH III · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·63

RESEARCH SUPPORTCENTER FOR TRANSGENIC ANIMALS AND PLANTS· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I. Research supporting activity · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Academic activity · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

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TECHNOLOGY DEPARTMENT·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·67

RESEARCH SUPPORT FACILITY·························································

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I. Facilities · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·II. Research activities · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·68
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THE CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB) · · · · · · · · · · · · · · · · · · · · · ·71

CENTER FOR RADIOISOTOPE FACILITIES (CRF) ................................ · · · ... ·73

IN1RODUCTION
The National Institute for Basic Biology (NIBB), a
government-supported, basic research institute, wasestablished in 1977. As a center of excellence, NIBB
promotes the biological sciences by conducting firstrate research on site as well as in cooperation with otheruniversities and research organizations. Researchers
at NIBB investigate cell structure and function, reproduction and development, neuronal and environmental
biology, gene expression and regulation, and molecular
evolution of eukaryotic organisms.
In 2000, the Center for Integrative Bioscience(CIB) was established as a common center for interdisciplinary research among the NIBB and the otherinstitutes in Okazaki. CIB consists of the Departmentof Development, Differentiation and Regeneration, theDepartment of Strategic Methodology, and the Department of Bio-environmental Science (formerly a part of
NIBB). The Director-General of NIBB currently acts
as the head of CIB . Two professors in the Departmentof Development, Differentiation and Regeneration havebeen nominated and will join the Center soon. To
accommodate this new center together with the existing
three common centers, i.e. the Research Center for
Computational Science, the Center for Experimental
Animals, and the Center for Radioisotope Facilities, a
new campus space called the E-area was transferredfrom the Aichi University of Education to the Okazaki
National Research Institutes. Construction is scheduled to be completed by the end of March, 2002.
In March, Professor Taisei Iguchi was appointed
Professor of the Center for Bio-environmental Science.
In April, all the members of the Center for Bioenvironmental Science were reassigned to the Center
for Integrative Bioscience. In July, Associate Professor Mitsuyasu Hasebe was promoted to Professor of theDivision of Speciation Mechanisms II. Drs. H. Watanabe, M. Hayashi, Y. Sakai and T. Kiyosue wereappointed as Associate Professors of the Center for Bioenvironmental Science, the Division of Cell Mechanisms, the Adjunct Division of Cell Proliferation and
the Adjunct Division of Biological Regulation, respectively. Adjunct Associate Professors Y. Ozeki and K.
Owaribe departed NIBB .
We would like to congratulate Associate Professors Kaoru Itoh and Hiroshi Shibuya on their promotions to Professor at Nagoya University and Tokyo
Medical and Dental University, respectively. Three
research associates (equivalent to assistant professors)
were promoted to associate professors of other univerSities. In addition, we replaced 2 research associates,
8 institute research fellows and 3 technicians with 2
research associates, 11 institute research fellows and 1
technician. The total number of personnels working at
NIBB including graduate students and post doctoral
fellows has been kept at 300 for several years.
As an international center for biological research,
NIBB is responsible for conducting research projects incooperation with various research groups. As a part ofsuch cooperative activities, NIBB hosts International
Conferences. In March the 43ro NIBB International
Conference was sponsored by the Ministry of Education,
Science, Sports and Culture and entitled "Mechanisms
of Neural Network Formation" (Professors T. Yamamori, H. Fujisawa and F. Murakami, organizers). The
44m NIBB International Conference entitled "Evolution
and Development. Generality and Diversity of Development in Animals and Plants" (Professors M. Hasebe
and K. Agata and Dr. H. Tsukaya, organizers) was also
held in March. In addition, NIBB continues to sponsor interdisciplinary symposia and study meetings oncurrent topics by inviting leading scientists from aroundthe world to the Institute. NIBB also provides a
training course in biological sciences for young investigators. To assess our continuing improvement, theactivities and future plans of two professors who havespent 10 years at NIBB were subjected to peer review
by international scholars in related fields. We always
welcome any suggestions concerning . the research
activities ofNIBB.
Finally, I would like to congratulate Professor N.
Murata for being awarded the Terry Galliard Medalfrom the International Association of Plant Lipid Research. Mr. T. Kirisako received the Nagakura Award.
He is the fourth graduate student from NIBB to win theAward.

Hideo Mohri, D.Sc.
Director-General

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NATIONAL INSTITUTE

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology (NIBB), is a
one of the Okazaki National Research Institutes (ONRI)
located on a hill overlooking the old town of Okazaki.
NIBB was established in 1977 and its activities aresupported by Monbukagaku-sho (the Ministry of
Education, Science, Sports and Culture) of Japan.
ONRI is composed of three independent organizations,
NIBB, the National Institute for Physiological Sciences(NIPS) and the Institute for Molecular Science (IMS).
In 2000, the Center for Integrative Bioscience wasestablished as a common facility of the ONRI. The
Center for Bio-environmental Science, began by NIBB
in 1999, joined to this new Center.

research associates. A division forms, in principle, an
independent project team. Six of the divisions areadjunct and headed by professors who hold joint appointments with other universities. Adjunct divisions
have a resident research associate. This arrangement
facilitates exchange in research activities in Japan.
The Technical Department manages the activities ofresearch technicians and helps to promote research
activities of each division and to maintain the common
research resources of the Institute. The Department
also undertakes the technical education of staff.
Several members of the Center for Integrative Bioscience jointly work with the NIBB.

Policy and Decision Making

Research Support Facilities

The Director-General oversees the operation of theInstitute assisted by two advisory bodies, the Advisory
Council and Steering Council. The Advisory Council,
comprised of distinguished scholars representing
various fields of science and culture, advises the Director-General on basic policy. The Steering Council,
comprised of professors within the Institute and an
equal number of leading biologists outside NIBB,
advises the Director-General on the scientific activitiesof the Institute. The Council, also makes recommendations on faculty appointments, the Institute's annual
budget and future prospects.

The research support facilities of the NIBB consist of
the Large Spectrograph Laboratory, the Tissue and Cell
Culture Laboratory, the Computer Laboratory, the Plant
Culture Laboratory, the Plant Cell Culture Laboratory,
the Experimental Farm, the Laboratory of StressResistant Plants and the Center for Transgenic Animals
and Plants. In addition, five facilities are operated
jointly with NIPS; they consist of the Electron Microscope Center, the Center for Analytical Instruments, theMachine Shop, the Laboratory Glassware Facilities andthe Low-Temperature Facilities.
The Radioisotope
Facilities, the Computer Center and the Animal Care
Facilities became common facilities of ONRI.

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.

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

Department/Laboratory
Department of Cell Biology

Department of Developmental Biology

Department of Regulation Biology

Laboratory of Gene Expression and Regulation
Center for Integrative Bioscience (a common center of ONRI)

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 facilities
were also completed in 1983. A building for the
Laboratory of Gene Expression and Regulation(2,577m2) was built in 1996.

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

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FOR BASIC BIOLOGYGRADUATE PROGRAMSThe NIBB sponsors two graduate programs.
1. Graduate University for Advanced Studies
NIBB 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 or equivalent at
any university 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 eligible to conduct research for fixed
periods of time under the supervision of NIBB professors.

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NATIONAL INSTITUTE

OFFICE OF DIRECTOR
Director-General:
Associate Professors:
Research Associates:

Hideo Mohri
Ryuji Kodama
Kohji Ueno
Kaoru Ohno
Hiraki Kokubo (on leave)

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 grqdually 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 othercells or macrophages, shown by transmission electron
microscopy and ii) the presence of conspicuous accumulation of fragmented DNA evidenced by the TUNEL
histological staining (Kodama, R. et a!., Roux's Arch.
Dev. Bioi. 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 lepidopteran species, Orgyia
recens approximans, provided by Drs. Y. Arita and K.
Yamada (Meijo University) is underway . In thisspecies, 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, 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

Fig.!.

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

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 thecontrol mechanism which demarcates the region to bedegenerated is defective in the female in this species.
Further investigation using this species might giveimportant 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 patternformation 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 timecourse of the formation of elaborate pattern of trachea
and tracheoles and to establish the cytological anddevelopmental relationship between the formation oftracheal pattern and epithelial cell pattern, such as scale
cell pattern.
The figure depicts how the tracheoles protrude fromthe primary trachea at the pre-pupa stage. These finethreads are arranged with even spaces and may closely
related with the scale cell pattern formation (Fig. 1).

5

FOR BASIC BIOLOGY

Proteinpalmitoylation
andmechanism at embryogenesis

developmental

Kohji Ueno
We have studied the molecular mechanisms of thedevelopment of cells and organs in the silkworm Bombyx mori and have found that a high molecular weightprotein (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 ofsmall GTP-binding, heterotrimeric G, and G-proteinlinked receptor proteins are known to be modified withpalmitate through thioester linkages. These dynamic
modifications are thought to be important in regulationof signal transduction .
To understand the molecular mechanism how themodification 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
which were highly homologous to the amino acidsequences of p260 and p270. Since analysis of theseclones revealed that the eDNA contained a long open
reading frame encoding 2504 amino acids whichshowed 94% homology to rat fatty acid synthase (FAS),
it was concluded that a homologue of Bombyx p260/270,
i.e. FAS , is expressed during mouse embryogenesis.
In situ hybridization of mouse embryos revealed thatthe transcripts are detected mainly in the central andperipheral nervous system in mouse embryos fromembryonic day 11.5. Immunocytochemical analyses ofcultured mouse primary embryonic brain cells were
performed to identify which cells express mouse FAS .
This analysis revealed that mouse FAS was expressed
specifically in neur.al cells in which growth-associatedprotein (GAP)-43 was expressed. GAP-43 , by protein
palmitoylation, regulates G 0 signal transduction andneural axonal growth. In a cell-free system, purified
FAS from mouse embryos transferred palmitate to
GAP-43 through cysteine residues. Furthermore, cerulenin, an inhibitor of FAS, reduced axonal growth andin vivo palmitoylation of GAP-43 in cultured neurons.
Figure 2 shows the effect of cerulenin on neuron morphology.
From these results, mouse FAS was speculated to be
responsible for the palmitoylation of GAP-43 andsubsequent regulation of axonal growth in mouseembryos. Since we also found that p260/270 was
expressed in brain cells in insect embryos, a protein
palmitoylase was speculated to regulate the axonal
development during embryogenesis in invertebrate andvertebrate.

,

Publication List:
Kubo-lrie, M., Irie, M., Nakazawa, T. and Mohri, H.
(2000) Spermiogenesis in the stag beetle, Aegus
Lavicollis Waterhouse (Coleoptera, Lucanidae), withspecial reference to the centriole adjunct. Invert. Reprod.
Develop ..37, 223-231.
Ueno, K. (2000) Involvement of fatty acid synthase inaxonal development in mouse embryos. Genes. Cells 5,859-869

Fig.2. Effect of cerulenin on cultured primary neurons. Morphology was observed by phase-contrast microscopy after the addition of 400 J.!M
cerulenin. (A) ; 0 min. , (B) 120 min. Axonal outgrowths started to di sintegrate and disappeared by 120 min. Arrows indicate axonal outgrowths.
Bar represents 25 J.lm.

6

NATION:ALINSTII'UTE

7

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.

8

NATIONAL INSTITUTE

DNISION OF CELL MECHANISMS
Professor:
Associate Professor:
Research Associates:

Mikio Nishimura
Makoto Hayashi (April]-)Tomoo Shimada (-Feb. 28)
ShojiMano
Technical Staffs:
MakiKondo
Katsushi Yamaguchi (-Sept. 1)
Post doctoral fellows: Yasuko Hayashi-1shimaru
Kanae Shirahama
Hiroshi Hayashi
Kenji Yamada
Naoto Mitsuhashi (Oct. 1-)
Graduate Students:
Naoto Mitsuhashi (-Sept.30)
Kazumasa Nito
Etsuko Watanabe
Youichiro Fukao (April I-)
JSPS Technical Staffs: Miwa Kuroyanagi
Chizuru Ueda
Chihiro Nakamori
Rie Takei (April I-)
Akiko Komori (May 23-)
Visiting Scientists:
Yasuko Koumoto (-Feb. 28)

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 other organelles may degenerate. Because the dynamic transformation of organellar function (differentiation of organelles) is responsible for flexibility of differentiation
events in higher plant cells, the elucidation of regulatory
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 theability 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. Mter 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, alternative splicing,protein translocation and protein degradation.
To investigate the roles of microbody membraneproteins in the reversible conversion of glyoxysomes toleaf peroxisomes, we characterized several membrance
proteins of glyoxysomes. One of them was identified as
an ascorbate peroxidase (pAPX) that is localized on

glyoxysomal membranes. Its eDNA was isolated byimmunoscreening. The deduced amino acid sequence
encoded by the eDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX isimported by one or more PTS-independent pathways.
Subcellular fractionation of 3- and 5-d-old cotyledonsof pumpkin revealed that pAPX was localized not onlyin the glyoxysomal fraction, but also in the ER fraction.
A magnesium shift experiment showed that the densityof pAPX in the ER fraction did not increase in thepresence of Ml+ , indicating that pAPX is not localized
in the rough ER. Immunocytochemical analysis using atransgenic Arabidopsis which expressed pumpkin pAPX
showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure ingreen cotyledons. The overall results suggested thatpAPX is transported to glyoxysomal membranes via thisunknown membranous structure.

ll. 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 and characterized 2,4-dichlorophenoxybutyric acid (2,4-DB)-resistant mutants. It has
been demonstrated previously that 2,4-dichlorophenoxybutyric acid (2,4-DB) is metabolized to produce aherbicide, 2,4-D, by the action of peroxisomal fatty acid~-oxidation in higher plants. To isolate mutants thathave defects in peroxisomal fatty acid ~-oxidation, 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 beclassified 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 ~-oxidation during germination and subsequent
seedling growth. Ped2 gene was identified by positional
cloning .and complementation analysis. The amino acidsequence of the predicted protein product is similar tothat of human Pex 14p, which is a key component of theperoxisomal protein import machinery. Therefore, we
decided to call it AtPex14p. Analyses of the ped2
mutant revealed that At Pex 14p controls intracellulartransport of both peroxisome targeting signal (PTS) land PTS2-containing proteins into three different typesof microbodies, namely glyoxysomes, leaf peroxisomes
and unspecialized peroxisomes. Mutation in the PED2
gene results in reduction of enzymes in all of thesefunctionally differentiated microbodies. The reductionin these enzymes induces pleiotropic defects, such as
fatty acid degradation, photorespiration and the morphology of peroxisomes (Fig. 1). These data suggestthat the AT Pex14p has a common role in maintaining

9

FOR BASIC BIOLOGY

WT
/air

ped2

/air

ped2
/C02

Figure 1. Reduced activity of photorespiration in ped 2
mutant.
Effect of C02 on the growth of ped2 mutant. Wild-type
Arabidopsis (WT/air) and ped2 mutant (ped2/air) were grownfor 8 weeks in a normal atmosphere (36 Pa C02) underconstant illumination (IOO p. E/m 2/s) . The ped2 mutant wasalso grown for 8 weeks in an atmosphere containing 1000 Pa ·C02 (ped2/C0z) under constant illumination (50 p. E/m2/s).

physiological functions of each of these three kinds ofplant microbodies by determining peroxisomal protein
targeting.

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.
Despite of the accumulation of the fusion protein, thetransgenic Arabidopsis is still sensitive to phosphinothricin. Phosphinothricin acetyltransferase containedin the fusion protein is obviously compartmentalized in
the PAC vesicles that do not permit the detoxification ofthis herbicide. The.se 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.
In order to characterize the organelles in the vacuolar-sorting pathway, we constructed chimeric genes that
encode various GFP fusion proteins and expressed in
Tobacco BY -2 cells. The organelles in the vacuolarsorting pathway were able to be visualized in vitalconditions and were used for the characterization of thevacuolar-sorting pathway.

IV. 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 findings 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 }VPE promotersdirected the expression in senescent tissues, but not in10young intact tissues. The mRNA levels of both aVPEand "(VPE 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 "(VPE. Our
results suggest that vegetative VPE might regulate theactivation of some functional vacuolar proteins that are
known to respond to these treatments.
In order to clarify function of VPEs in vivo, theiranti-sence Arabidopsis were generated and the characterization is under experiments.

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.
Functional analyses of these chaperonin homologues indifferentiation of plastids are in progress.

Publication List:
De Bellis, L., Gonzali, S., Alpi, A., Hayashi, H., Hayashi,
M. and Nishimura M. (2000) Purification and characterization of a novel pumpkin short-chain acyl-CoAoxidase which structure resembles acyl-CoA dehydrogenase. Plant Physiol. 123: 327-334
Hayashi-lshimaru, Y., Hayashi, M., Hayashi, H., HaraNishimura, I. and Nishimura, M. (2001) Glyoxysomespherosome interaction in etiolated cotyledons of Arabi-

NATIONAL INSTITUTE

dopsis Ped1 mutant that. has defect on a thiolase gene offatty acid ~-oxidation cycle. Protoplasma in press
Hayashi, M., Nito, K., Toriyama-Kato, K., Kondo, M.,
Yamaya, T. and Nishimura N. (2000) AtPex14 maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes. EMBO J. 19:
5701-5710
Hayashi, M., Toriyama, K., Kondo, M., Kato, A., Mano, S.,
De Bellis, L., Hayashi-lshimaru, Y., Yamaguchi, K.,
Hayashi, H. and Nishimura, M. (2000) Functional transformation of plant peroxisomes. Cell Biochem. Biophys.,
32:295-304
Kato, A., Hayashi, M., Kondo, M. and Nishimura, M.
(2000) Transport of peroxisomal proteins that are synthesized as large precursors in plants. Cell Biochem.
Biophys., 32: 269-275
Mano, S., Hayashi, M and Nishimura, M. (2000) A leafperoxisomal protein, hydroxypyruvate reductase, is produced by light-regulated alternative splicing. Cell Biochem. Biophys., 32: 147-154
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. 41: 218-225
Mitsuhashi, N., Shimada, T., Mano, S., Nishimura, M. andHara-Nishimura, I. (2000) Characterization of organellesin the vacuolar-sorting pathway by visualization with
GFP in tobacco BY-2 cells. Plant Cell Physiol. 41: 9931001
Nito, K., Yamaguchi, K., Kondo, M., Hayashi, M. andNishimura, M. (2001) Pumpkin peroxisomal ascorbate
peroxidase is localized on peroxisomal membranes andunknown membranous structures. Plant Cell Physiol.
42:20-27
Takemoto, D., Hayashi, M., Doke, N., Nishimura, M.
and Kawakita, K. (2000) Isolation of gene for EIR, anelicitor inducible receptor like protein, from tobacco bydifferential display. Plant Cell Physiol. 41: 458-464
Yamaguchi, K. and Nishimura, M. (2000) Reduction to
below threshold levels of glycolate oxidase activities intransgenic tobacco enhances photoinhibition during irradiation .. Plant Cell Physiol. 41: 1397-1406

11

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
Graduate University for Advanced Studies Professor:
Yoshinori Ohsumi
Tamotsu Yoshimori
Associate Professor:
Research Associates: Yoshiaki Kamada
Takeshi Noda
Technical Staff:
Yukiko Kabeya
Post Doctoral Fellows: Noboru Mizushima 1!
Takahiro Shintani
Naotada lshihara 2!
Akio Kihara 3!
Graduate Students:
Takayoshi Kirisako4!
Yoshinobu lchimura4!
Kuninori Suzukt><~!
Yoshinori Kobayasht><~!
Atsuki Nara4!
Hideki Hanaoka 4!
Akiko Kuma4·5!
Maho Hamasakt-4. 5!
Shisako Shojt><~. 6!
Visiting Scientists:
Satsuki Okamoto
I! PRESTO, JST, Oct 1999-.
! til March 2000
JJ til Dec 2000
4
! Graduate University for Advanced Studies.
5!from April 2000.
6! from Oct.2000
2

This division aims to understand the autophagy in respects to its molecular mechanism and its physiologicalrole in higher eukaryotes. 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 to fully understand the cell, because the study on the degradation
has been retarded compared to the bio-synthetic process.
Autophagy is well conserved in eukaryotes and is amajor route for bulk degradation of cytoplasmic constituents and organelles in a lytic compartment,
lysosome/vacuole.

duction of autophagy during growing condition, since
we observed that rapamycin, a Tor-specific inhibitor,
induced autophagy even under such a condition. Next
question is how inhibition of Tor function leads autophagy induction. Among 15 Apg proteins Apglp andApgl3p show close relations with Tor. Apglp is a protein kinase, and Apgl kinase activity is enhanced bynutrient starvation or rapamycin-treatment. This activation requires APGJ3. Apgl3p is highly phosphorylated
in a Tor-dependent manner. In starved or rapamycintreated cell, Apgl3p is immediately dephosphorylated.
And defect of autophagy by deletion of APGJ3 is rescued by overexpression of APG1. These suggest that
Apgl3p plays a key role in signal transduction from Tor
to Apglp. We found that only dephosphorylated form
(but not hyper-phosphorylated form) of Apgl3p associates to Apglp under starved condition. And we demonstrated that this association confers Apgl activation
which is required for the induction of autophagy. We
also identified a couple of Apgl-binding proteins
Apg17p and Cvt9p suggesting that Apgl makes a large
protein complex.

III. Two Distinct Phosphatidylinositol 3-Kinase
Complexes Function in Autophagy and Carboxypeptidase Y Sorting
Vps30p/Apg6p is required for both autophagy andsorting of carboxypeptidase Y (CPY). Although Vps30p
is known to interact with Apgl4p, its precise role hasremained unclear. We found that two proteins copurify
with Vps30p. By mass spectrometry they were identified to be Vps38p and Vps34p, a phosphatidylinositol(Ptdins) 3-kinase. Vps34p, Vps38p, Apgl4p, andVps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that
Vps30p functions as a subunit of a Vps34 Ptdins 3kinase complex. Phenotypic analyses indicated thatcomplex I<<

complex II

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, Saccharomyces cerevisiae and morphologically defined the whole process. We have isolated a set of autophagy-deficient mutants (apg), and havecloned most of the APG genes essential for autophagy.
We are now characterizing these gene products.

II. Apgl-Apg13 protein kinase CQJllPlex mediates
induction of autophagy in response to starvation
signalWe previously reported that Tor protein represses in-

Autophagy

CPY sorting

Fig. I PI3 kinase complex is required for autophagy.
PI3 kinase( vps34) and its activator (vps 15) form at least twocomplex. Complex I (Vps34, Vps15, Apg14, and Vps30) is
essential for autophagy, and Complex II (Vps34, Vps15,
Vps38, and Vps30) functions for vacuolar enzyme CPY
sorting.

12
Apg14p and Vps38p are required for autophagy and
CPY sorting, respectively, whereas Vps30p, Vps34p,
and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p
antibodies and pull-down experiments showed that two
distinct Vps34 Ptdlns 3-kinase complexes exist: one,
containing Vps15p, Vps30p, and Apg14p, functions in
autophagy and the other containing Vps15p, Vps30p,
and Vps38p functions in CPY sorting. The vps34 andvpsl5 mutants displayed additional phenotypes such as
defects in transport of proteinase A and proteinase B,
implying the existence of another Ptdlns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p
complexes associated with specific regulatory proteinsmight fulfil their membrane trafficking events at different sites (Fig. I).

VI. Discovery of novel lipidation reaction mediated by ubiquitin-like system
Apg8/Aut7, which plays an important role in theformation of autophagosome, tends to bind to membranes in spite of its. hydrophilic nature. We showedthat the mode of the association of Apg8 with membranes changes depending on a series of modificationsof the protein itself. First, the carboxy-terminal Arg of
newly synthesized Apg8 is removed by Apg4/ Aut2, anovel cysteine protease, and a Gly residue becomes the
carboxy-terminal residue of the protein that is now
designated Apg8FG. Subsequently, Apg8FG forms a
conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and
Apg7 , a ubiquitin El-like enzyme. Finally, the adduct
Apg8FG-X is reversed to soluble or loosely membranebound Apg8FG by cleavage by Apg4. The mode ofaction of Apg4, which cleaves both newly synthesized
Apg8 and Apg8FG-X, resembles that of deubiquitinating enzymes.
We then succeeded to identify molecule X and discovred a novel mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine (PE)through an amide bond between the C-terminal glycine
and the amino group of PE. This lipidation is mediatedby a ubiquitination-like system (Fig. 2). Apg8 is a ubiquitin-like protein that is activated by an El protein,Apg7, and is transferred subsequently to an E2 enzyme
Apg3/Autl. Apg7 activates two different ubiquitin-Iikeproteins, Apgl2 and Apg8, and assigns them to specificE2 enzymes, ApglO and Apg3, respectively. This reversible lipidation of Apg8 appears to be coupled to themembrane dynamics of autophagy and the Cvt pathway.

V. Mammalian Apg proteins; localization andfunction
Autophagy was fust described in mammalian ceiJs in
the 1960s and its morphology and regulation have been
extensively investigated. However, molecular mechanism underlying its process is poorly understood. Recent studies on yeast Apg proteins revealed that

NATIONAL INSTITUTE

Apg7

El

Apg4 .-.

Apg3 ApglO

.. E2

E2 .

Fig.2 Two Ubiquitin-like systems essential for autophagy
Apgl2 and Apg8 are activated by common El enzyme,
Apg7. Then they are assigned to different conjugating
enzymes, ApglO and Apg3 , respectively. Finally Apgl2
forms a conjugate with a single target molecule, Apg5 .
Apg8 conjugate with a membrane phospholipid, phosphatidylethanolamine (PE). Apg8-PE is reversed to Apg8 by
Apg4.

molecular machinery of autophagy seems highly conserved among eukaryotes. We are analyzing the mammalian orthologues of Apg proteins.
Apg8 has at least three mammalian homologues, oneof which, LC3, was identified to be involved in autophagy. Newly synthesized rat LC3, a 142 amino acidprotein, was immediately processed to remove the Cterminal 22 amino acids to generate the LC3-I form ,which was further converted to the LC3-II form . While
LC3-I was cytosolic, LC3-II was membrane bound andenriched in the autophagic vacuole fraction (Figure 3A
and B). LC3-II may correspond to yeast Apg8-PE (thePE-conjugated form of Apg8). Imrnunoelectron microscopy revealed LC3 was present both inside and outsideof autophagosomes in addition to cytoplasm. LC3 wasless detected on autolysosome, suggesting that someLC3 dissociated from the membrane after autophagosome formation (Figure 3B). Consistent with such
localization, the amount of LC3-II was correlated withthe extent of the autophagosome formation . Although
LC3 was originally identified as microtubule-associatedprotein 1 light chain 3, involvement of microtuble in
LC3 function and autophagy has remained to be clarified . LC3-II is the first mammalian protein identified
that specifically associates with the autophagosome
membranes.
The Apg 12-Apg5 protein conjugation system is another well conserved machinery. Most mammalian
Apg5 was present in the Apgl2-conjugated form in thecytoplasm. However, a small fraction of the Apgl2Apg5 conjugate localized to the isolation membranes
when autophagy proceeds (Figure 3A and B). Using
GFP-tagged Apg5 , we revealed that the cup-shaped
isolation membrane is developed from a small crescentshaped compartment. These small structures have neverbeen recognized unless labeled with Apg5, but they areFOR BASIC BIOLOGY

13

Fig.3.
A. Distribution of ApgS and LC3 in mouse embryonic stem cells.
YFP-LC3 (middle panel) localizes to spherical autophagosomes and semi-spherical isolation membranes, whereas CFP-ApgS (leftpanel) localizes to only isolation membranes. Bar, 2 J.!m.
B. Model of dynamic association of Apg 12-ApgS and LC3 with autophagic membranes.
Apgl2-Apg5 conjugate localizes to the crescent-shaped autophagosome precursors. While these structures elongate and maturate into
cup-shaped isolation membranes, cytosolic LC3-I is recruited ·to the membrane in the Apgl2-Apg5-dependent manner and accumulated
as the LC3-II form. ApgS biases its localization to the outer side of the membrane. ApgS plays an essential role in this membrane development. Immediately before or after the completion of autophagosome formation, ApgS detaches from the membrane. Some LC3
also dissociate from the autophagosomal membrane thereafter.

likely to be direct precursors of the autophagosome.
Apg5 localized on the isolation membrane throughout
its elongation process. Apg5 was preferentially distributed in the outer side of the membrane and detaches
from it immediately before or after autophagosome
formation is completed (Figure 3B). To examine therole of Apg5, we generated Apg5-deficient ES cells.
APG5 ·f· cells are viable but bulk protein degradation

was significantly reduced. Morphological analysis revealed that autophagosome formation was impaired in
APG5 .f. cells. We also showed that the covalent modification of Apg5 with Apg 12 was not required for its
membrane targeting but is essential for involvement of
Apg5 in elongation of the isolation membranes. Intriguingly, Apgl2-Apg5 was required for targeting of
LC3 to the isolation membranes and LC3-II generation.

14
Therefore, the Apg12-Apg5 conjugate plays essential
roles in isolation membrane development in cooperation with LC3. In addition, our studies provided
good molecular markers, LC3 and Apg12-Apg5, forautophagic membrane at all stages and isolation membranes, respectively, which so far have been defined
only by morphology.

Original papers :Yoshimori, T., Yamagata, F., Yamamoto, A., Mizushima, N., Kabeya, Y., Nara, A., Ishido, M., Ohashi,M., Ohsumi, M., and Ohsumi, Y. SKDl, an AAA
ATPase related to the yeast Vps4p, regulates morphology and transport function of early endosomes.
Mol. Biol. Cell., 11, 747-763 (2000)
George, M, D., Baba, M., Scott, S. V., Mizushima, N.,
Garrison, B., Ohsumi, Y. and Klionsky, D. J. Apg5p
functions in the sequestration step in the cytoplasm tovacuole targeting and macroautophagy pathways.
Mol. Biol. Cell, 11, 969-982 (2000)
Noda, T., Kim. J., Huang, W-P., Baba, M., Tokunaga,C., Ohsumi, Y., Klionsky, D. J. Apg9/Cvt7p is an
integral membrane protein required for transport
vesicle formation in the Cvt and autophagy pathways.
J. Cell Biol. 148,465-479 (2000)
Furukawa, K., Mizushima, N., Noda, T. and Ohsumi, Y.
A protein conjugation system in yeast with homology
to biosyntheic enzyme reaction of prokaryotes. J.
Bioi. Chem. 275, 11,747-763, (2000)·
Scott, S. V., Nice, ill, D. C., Nau, J. J., Weisman, L. S.,
Kamada, Y., Keizer-Gunnink, I., Funakoshi, T.,
Veenhuis, M., Ohsumi, Y., and Klionsky, D. J.
Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting. J. Biol. Chem. 275, 25840-25849(2000)
Kamada, Y., Funakoshi, T, Shintani, T, Nagano, T,
Ohsumi, M, Ohsumi, Y. Tor-mediated induction of
autophagy via an Apgl protein kinase complex. J.
Cell Biol. 150, 1507-1513 (2000)
Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mi-

NATIONAL INSTITUTE

zushima, N., Yoshimori, T., Ohsumi, M., Noda, T.,
Ohsumi, Y. Membrane association cycle of Apg8 isessential for authophagy and the Cvt pathway. J. Cell
Biol. 151, 263-275 (2000)
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A.,
Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y.,
and Yoshimori, T. LC3, a mammalian homologue of
yeast Apg8p is processed and localized in autophagosome membranes, EMBO. J. 19, 5720-5728,(2000)
Grote, E., Baba, M., Osumi, M. Ohsumi, Y., Novick,P. J. Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion. J. Cell Biol. 151, 453466 (2000).
Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi
Y, Ishihara N, Mizushima N, Tanida I, Kominami E,
Ohsumi M, Noda T, Ohsumi Y. A ubiquitin-likesystem mediates protein lipidation. Nature, 408, 488492 (2000)
Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y. Two
distinct Vps34 Ptdlns 3-kinase Complexes function
in autophagy and CPY sorting in Saccharomyces
cerevisiae. J. Cell Biol., 152, 519-530. (2001)
Kihara, A., Yamamoto, A., Ohsumi, Y., and Yoshimori
T., Beclin-Phsophatidylinositol 3-kinase complex
functions at the trans-Golgi Network. EMBO Report,
in press (2001)
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi,Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y.,
and Yoshimori, T. Dissection of autophagosome
formation using Apg5-deficient mouse embryonicstem cells. J. Cell Biol. in press (2001)
Komatsu,M., Tanida, 1., Ueno, T., Ohsumi, M., Ohsumi,
Y., and Kominami, E. The C-terminal region of an
Apg7p/Cvt2p is required for homodimerization andis essential for its E1-activity and E1-E2 complexformation. J. Biol. Chem. in press (2001)

Review:
Noda, T. Ohsumi, Y., Klionsky, D. J. The yeast vacuole: a paradigm for plant cell biologists? Annual
Plant Reviews, 5, 1-19 (2000)

15

FOR BASIC BIOLOGYDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor:
Research Associate:
JST Research Staff:
Postdoctoral Fellow:
Graduate Student:

Motoya Katsuki
Kei ItoTakeshi Awasaki (- Sep. 30, 2000
CREST, Oct. 1, 2000- PRESTO)
Ryuichi Okada
Yusuke Uchiyama(Kyoto University)
Nobuaki Tanaka(Graduate Univ. Advanced Studies)
Hideo Otsuna(Nagoya University)

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 105
neurones, and subject to powerful molecular andgenetic 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 functionalfeatures of the more complex vertebrate bmins. Third
year of the five-year term, we continued a large-scale
screening to find strains useful for this purpose.

I. Comprehensive identification of cells in theadult brain
A comprehensive and detailed anatomical knowledgeof the bmin is a prerequisite for 1) analysing thephenotypes of nervous system-related mutants, 2)
identifying the cells that express cloned genes, 3)
understanding the way information is processed in thebrain, and 4) devising computer models that simulate
brain functions. In spite of the hundred years of efforts
using Golgi and other anatomical techniques, however,the circuit structure of higher order associative regionsof the brain is still essentially unresolved. Moreover,traditional neuroanatomy tends to focus only on themature adult bmin, leaving the developmental processes
largely uninvestigated. Since many nervous systemrelated mutants show structural defects, however,understanding the role of the responsible genes requires
detailed basic knowledge about when and how the bminstructure 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,
is also a powerful tool for obtaining a vast array oftransformant strains that label specific subsets of bmincells. 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. Our screening consists of two stages. In the first step,
all the lines are crossed with the flies carrying the UASGFP transgene, which fluoresces only in the cells
where GAL4 expression is active. The patterns of the
GAL4-expressing cells are recorded from freshly
dissected, unfixed adult bmin tissue using a high-speed

confocal microscope. Photographs of between 20 and
100 optical sections are taken for each line. As of
December 2000, ca. 108,000 photographs depicting
3,400 of the total 4,500 strains have been accumulated
in a computer database. In the second step, useful lines
are selected from the database, and fixed and clealisedbmin specimens at various developmental stages are
subjected to confocal serial sectioning with aconventional confocal microscope and to threedimensional reconstruction with a UNIX workstation.
Although the long-term aim of this project is to
identify as many neurones and glial cells as possible toget the comprehensive overview of the fly bminstructure, at the initial stage a few brain regions arechosen for intensive study. The first target is to identify
projection intemeurons that connect lower-level sensory
neuropile and higher-order associative regions. These
fibres convey olfactory, gustatory, auditory and visualsensory information.
For visual pathways, we identified in total 30 types among which 22 were novel - of projection neurones(in total ca. 500 cells) that connect lower-level sensory
neuropile in the optic lobe to the higher order regions in
the central brain (Fig. 1). For olfactory pathways, we
identified 12 types of projection neurones (in total ca.
75 cells), that connect glomeruli in the first-order
sensory neuropile of olfaction (olfactory lobe, antenna!
lobe) to the second-order processing cites (mushroom
body and lateral hom). Further completion of suchconnection map would give us important insights about
how sensory information is conveyed and integrated
before association with signals of other sensory
modalities.

U. Mapping of neurotransmitters and receptors
in the adult brain
To understand the neural network of the bmin, it isalso important to reveal what kind of synapses is usedin each identified neurone. For this purpose,
information about the types of neurotransmitters andreceptors is indispensable. Previously, such information
is obtained by staining bmin tissue with various
antibodies. This approach, however, has the limitation
due to the availability of good andibodies. Antibodies
raised against transmitters or receptors of certain animal
species do not always label corresponding molecule inother species.
Taking the advantage of the completion of
Drosophila genome project, we thus employed a novel
approach. Using homology and other information, we
first search genes that code receptors or enzymes
associated to transmitter synthesis. Cells that express
those genes are labelled by using in-situ RNA
hybridisation. This technique can visualise only the cellbodies of the labelled cells. We then screen GAL4
enhancer-trap strains that label cells in thecorresponding area of the cortex, and perform double
labelling to certify the colocalisation of GAL4

16

NATIONAL INSTITUTE

Fig. 1 Neurones that connect optic lobe and the central brain Three-dimensional reconstruction of about 200 confocal sections.
Red-Cyan stereograph, posterior view of the whole-mount brain. To get 3-D image, use red filter for the left eye and green (or blue)
filter for the right eye. A: Neurones that connect the outer layer of medulla (me) to lateral horn (I ho). A subset of fibres project tocontralateral medulla. B: Columnar array of neurones that connect lobula (lo) and ventrolateral protocerebrum (v I pr). Note thatarborisation occurs only at defined layers in the outer region of the lobula neuropile. C: Another type of array neurons that connect
both sides of lobula via great commissure (g c). Arborisation occurs only in the inner region of lobula.

expression and in-situ label. This way, we can map the
position and morphology of cells that use certaintransmitters and receptors. First year of this project, weestablished a reliable and efficient protocol for in-situ
staining of the dissected whole brain, and successfully
visualised the expression pattern of three receptors andthree enzymes associated with transmitter synthesis.

III. Analysis of the cell lineage-dependent
modular structures in 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 a small number of neural stem cells at any
desired developmental stage and reveal the projection
patterns of their progeny at a later period.
The central brain of Drosophila melanogaster is
produced by an average of 85 stem cells (neuroblasts)
per hemisphere. We found that the majority of clones
keep their cell bodies in tightly packed clusters. In 30
out of the 32 clone types identified so far, the neurites
fasciculate to form a single bundle that runs from a cellbody cluster, and innervate a limited number ofneuropile regions in a stereotypic manner, forming
clearly defined units of neural circuits. These suggestthat in many cases the progeny of a single stem ceU
forms a lineage-dependent circuit structure unit, which
we named a "clonal unit."
The clustering of clonal cell bodies and thefasciculation of neurites are already apparent in thedeveloping larval brain. In larvae, glial cells form theborder of clonal ceU body clusters.

IV. Development of an improved red fluorescentprotein (RFP) reporter system suitable for double
labelling and birth order analysis of neural fibres
The green fluorescent protein (GFP) and its enhanced
mutants are widely used to monitor protein localisationand gene expression of various organisms. Recently,
red fluorescent protein, such as DsRed, was isolated
from corals. Its significantly red-shifted emission andabsorption maxima provide good complementation to
GFP. Its use, however, is still restricted by its rather low
quantum yield and parasitic green fluorescence peakthat cross-talks with GFP emission . To overcome thisproblem, we developed a new variant of DsRed (DsRed
S203Y), which is significantly brighter than the wildtype and free from green fluorescence peak, making it
an ideal reporter for double labelling with GFP.
DsRed requires significantly longer fluorescencematuration time than GFP. By co-expressing GFP and
DsRed under the control of the same promoter, timeafter the onset of gene expression can be monitored byits colour change from green (GFP only) to yellow(green by GFP + red by mature DsRed) over time. By
using a GAL4 enhancer-trap strain that drives
expression shortly after cells are born, it is possible tostudy birth order of the labelled cells. Though birthorder analysis has been possible by pulse-chaselabelling of DNA-replicating cells with 3H-thymidine
or bromodeoxyuridine (BrdU), the label was limited tocell bodies. Since GFP and DsRed spread even into finecellular-protrusions, the GFP/DsRed system makes it
possible to reveal birth order of fibre processes. We
applied this to the developing mushroom body neuronsof Drosophila brain and visualised, for the first time,
the formation order of fibres within a neural fiber
bundle (Fig. 2).
(This work is performed under collaboration with
Drs. Vladislav V. Verkhusha, Hiroki Oda and Shoichiro
Tsukita of ERATO Tsukita Cell Axis Project, Kyoto.)

V. Contribution to the science community
As a joint venture with German and US researchgroups, we maintain Flybrain, a web-based image
database of the
Drosophila nervous
system(http ://flybrain.nibb.ac.jp). Over 2000 images has

FOR BASIC BIOLOGY

17

Fig. 2 Birth order analysis of neural fibres in the mushruum body Late third-instar larval brain of the GAIA enhancer-trap strain
20Jy expressing UAS-GFP S65T and UAS-DsRed S203Y. A-C: Reconstruction of horizontal optical sections. D-F: Vertical section of

the axon bundle of pedunculus. GFP labels a majority of cell bodies in the larval mushroom body (A). Arborisation and axons are alsolabelled. Accordingly, most fibres are labelled in the section of the axon bundle (D). DsRed, on the other hand, labells only cell bodies
that are far from the stem cells, which are older than those near the stem cells (B). In the section, Only fibres near the periphery of thebundle are labelled (E). Comparison of GFP and DsRed labelling (C, F) reveals that older fibres run in the periphery. Thus, it is likelythat newly elongating fibres run into the core, rather than along the outer surface, of the fibre bundle.

already been stored and served worldwide. Anotherdatabase maintained here is Jfly, which is intended tohelp the exchange of information among Japanesespeaking Drosophila researchers (http://jfly.nibb.ac.jp).
Archives of research-related discussions, images,
movies and experimental protocols, as well as meetingsand job announcements, are provided.

Publication List:
Awasaki, T ., Saito, M ., Sone, M ., Suzuki, E., Sakai, R., Ito,

K., Hama, C. (2000) The Drosophilla Trio plays anessential role in patterning of axons by regulating theirdirectional extention . Neuron.26: 119-131
Awasaki, T. and Kimura, K. (2001) Multiple function of
poxn gene in larval PNS development and in ad ult
appendage formation of Drosophila, Dev. Genes. Evol.
211: 20-29

18

NATIONAL INSTITUTE

DNISION OF CELLULAR COMMUNICATION(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Postdoctoral Fellows:
Graduate student:

Ritsu Kamiya
Katsushi Owaribe (-Aug.l5)
Takako Kato-Minoura
Yoshiaki Hirako
ltsushi Minoura
Ken-ichi Wakabayashi(University ofTokyo)

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 forqe with a fine glass needle.
Preliminary results indicate that the force produced in
the mutant axoneme lacking the outer arm or part of theinner arm is reduced to about 113 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 the sevensubspecies of inner-arm dyneins, has a mutation in theactin-encoding gene. Intriguingly, Chlamydomonas has
been known to have only a single gene of conventional
actin, and the mutant ida5 was found to express noconventional actin at all. On close inspection, the cytoplasm and axonemes of this mutant were found to 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 inChlamydomonas.

Publication List:
Inoue, M., Tarnai, K., Shimizu, H., Owaribe, K., Nakama,

19

FOR BASIC BIOLOGY

T., Hashimoto, T., and McGrath, J. A. (2000)

A homozygous missense mutation in the cytoplasmic tail of ~4
integrin, G931D, that disrupts hemidesmosome assembly and underlies non-Herlitz junctional epidermolysis
bullosa without pyloric atresia? J. Invest. Dermatol.
114, 1061-1064.
Isogai, N., Karniya, R., and Yoshimura, K. (2000) Dominance between the two flagella during phototaxis in
Chlamydomonas: dependence on the light/dark cycle
caused by bodily rotation. Zoo/ Sci. 17, 1261-1266.
Karniya, R. (2000) Analysis of cell vibration for assessing axonemal motility in Chlamydomonas. Methods 22,

383-387.
Okamoto-Nakazato, A., Takahashi, K., Kido, N.,
Owaribe, K., and Katou, K. (2000) Molecular cloningof yieldins regulating the yield threshold of cowpea cell
walls: eDNA cloning and characterization of recombinant yield in. Plant, Cell Environment 23, 155-164.
Yagi, T. and Kamiya, R. (2000) Vigorous beating ofChlamydomonas axonemes lacking central pair/radial
spoke structures in the presence of salts and organic
compounds. Cell Motil. Cytoskeleton 46, 190-199.

.·

8

Fig. I . Fertilization tubules in wild-type (A) and ida5 (B) mt+ gametes produced in response to a 1 hour exposure to 10 mM dibutyryl-cAMP and I mM IBMX. Bar, 0.3 11m. Wild-type fertilization tubules have beenshown to contain F-actin bundles.

20

NATIONAL INSTITUTEDIVISION OF CELL FuSION (ADJUNCT)
Professor:
Associate Professor:
Research Associate:
lnsttitute Research Fellow:
Graduate Students:

Issei Mabuchi
Hiroshi Abe
Hirotaka Fujirrwto
Izuru Yonemura
Naeko Shinozaki(University ofTokyo)

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. 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-11 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 cdcl2, 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 (Myo2MQs) was able to rescue myo2 null cells,
myo3 null cells, cdc4 mutant cells and cdc4 null cells.
The Myo2MQs 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 11 assembles at the growing end as spots probably
as a 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 XAIPl itself binds
to pure actin filaments to some extent, but it induces a
rapid, drastic disassembly. of actin fil'aments 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., andHirata, D. (1999). Overproduction of elongation factor
1a, an essential translational component, causes aberrant
cell morphology by affecting the control of growth polarity in fission yeast. Genes to Cells 4, 517-527.
Sayers, L. G., Katayama, S., Nakano, K., Mellor, H., Mabuchi, I., Toda, T., and Parker, P. J. (2000). Rhodependence of Schizosaccharomyces pombe Pck2. Genes to Cells 5, I 7-27.
Motegi, F., Nakano, K., and Mabuchi, I. (2000). Molecularmechanism of myosin-11 assembly at the division site in21

FOR BASIC BIOLOGY

Schizosaccharomyces pombe. J. Cell Sci. 113, 18131825.
Le Goff, X., Motegi, F., Salimova, E., Mabuchi , 1., andSimanis, V. (2000). The S. pombe riel gene encodes aputative myosin regulatory light chain that binds the type
Il myosins myo3p and myo2p. J. Cell Sci. 113, 41574163.
Calonge, T. M., Nakano, K., Arellano, M., Arai , R., Katayama, S., Toda, T., Mabuchi , I. , and Perez, P. (2000).
Schizosaccharomyces pombe Rho2 GTPase regulates

Fig. I

cell wall a-g1ucan biosynthesis, through the protein
kinase Pck2. Mol. Bioi. Cell11, 4393-4401.
Bernstein, B. W., Painter, W. B., Chen, H., Minamide, L.
S., Abe, H. and Bamburg, J. R. (2000) . Intracellular pH
Modulation of ADF/Cofilin Proteins. Cell Motil. Cytoskel. 47: 319-336.
Takahashi, T., Koshimizu, U., Abe, H., Obinata, T. andNakamura, T. (2000) . Functional Involvement of
Xenopus LIM-Kinases in Progression of Oocyte Maturation. Dev. Bioi. in press.

First cleavage of a Xenopus egg.

22

NATIONAL INSTITUTE

23

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.

24

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

Yoshitaka Nagahama
Michiyasu Yoshikuni
Tohru Kobayashi
Hiroko Kobayashi
Naomi Sumikawa
Institute Research Fellows: Craig E. Morrey
Yasuji Shibata
JSPS Postdoctoral Fellows: Masaru Matsuda
Catherine Dreanno
BalasubramanianSenthilkumaran
JSPS Research A ssociates: Akihiko Yamaguchi
Yuichi Oba
Toshitaka lkeuchi
Fumie Sakai
Gui-Jin Guan
Ryo Horiguchi (Graduate
Graduate Students:
University for Advanced
Studies)
Yasuhisa Kobayashi
Hai-Guang Fan
Cheni Chery Sudhakumari
Visiting Scientists:
Chika Matsuda

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 ofgametogenesis. Their effects are particularly profoundin teleost 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 (1) the
identification of steroidal mediators involved in gonadal
sex differentiation and gametogenesis , and (2) the
mechanisms of synthesis and action of these mediators.

family of putative RNA .helicase and is present in thegerm line of several animal species. Vas (a Drosophila
vasa homologue) gene expression pattern in germ cells
during oogenesis and spermatogenesis has bee examined using all genetic females and males of tilapia. In
the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in
auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, foilwed by a decrease from vitellogenic to postvitellogenic
oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes.
No vas RNA expression is evident in either diplotene
primary spermatocytes, secondary spermatocytes,
spermatids or spermatozoa. The observed differences in
vas RNA expression suggest a differential function ofvas in the regulation of meiotic progression of female
and male germ cells. We have also generated medaka(Oryzias latipes) transgenic lines with green fluorescentprotein (GFP) fluorescence controlled by the regulatory
regions of the olvas gene in the germ cells (Fig. 1). The
intensity of GFP t1uorescence increases dramatically in
PGCs located in the ventrolateral region of the posterior
intestine around stage 25 (the onset of brood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescencesuggest that 1) the increase in zygotic olvas expression
occurs after PGC specification and 2) PGCs can maintain their cell characteristics ectopically after stages 2025. The GFP expression persists throughout the laterstages in the mature ovary and testis.
In til apia, mitosis of germ cells begins around I 0days post-hatching in genetic females, but can not be

I. Endocrine regulation of gonadal sex differentia-tion
Sex determination and gonadal development varyconsiderably in fish. In addition to gonochorism, several
types of hermaphroditism (protandry, protogyny andsynchronous hermaphroditism) are found in fish. Niletilapia, Oreochromis niloticus, is an excellent exampleof the precise nature of steroidogenic actions during
gonadal sex differentiation. In this fish, all genetic female (XX) or male (XY) broods can be obtained byartificial fertilization of normal eggs (XX) and sexreversed, pseudo male sperm (XX), or normal eggs
(XX) and super male sperm (YY) , respectively. Fertilized eggs hatch after 4 days at 26 oC. On the day ofhatching, primordial germ cells (PGCs), which aremorphologically distinguishable from somatic cells, arelocated in the outer layer of the lateral plate mesoderm
around the hind gut. At 3 days post-hatching, PGCs arelocated in the gonadal anlagen after the formation of the
coelomic cavity in the lateral plate mesoderm rather
than through active migration.
The gene vasa encodes a DEAD (Asp-Giu-Aia-Asp)

Fig. 1 GFP fluorescent germ cells in the ovary of female
medaka, Oryzias latipes. The ovary contains numerous oocytes which can be seen as a green fluorescence.

25

FOR BASIC BIOLOGY

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 often sex-specific. In these cases, the sex of the
gonad is easily distinguishable. In tilapia, gonadal sex ismorphologically distinct at 20-25 days post-hatching.
Ovarian differentiation is initially marked by storomalelongation of the gonad for the formation of the ovariancavity. Testicular differentiation is characterized by theappearance of a narrow space in the storomal 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. In
contrast, the ability of steroid-producing cells to synthesize steroid hormones in the testes only appears at thetime of testicular differentiation. Transcripts of estrogen
receptors (ER) a and ~ first appear in both female andmale gonads of fry 10-15 days post-hatching. ERa andER~ exhibit different expression patterns suggesting
differential roles of ERa and ER~ in estrogen action on
gonadal sex differentiation. We have isolated two DM(Doublesex/Mab-3 DNA-binding mitif)-domain cDNAs
from tilapia testis and ovary, named DMRTJ and DMO,
respectively. DMRTJ is expressed only in Sertoli cells
and DMO is detected only in oocytes by in situ hybridization. The correlation between expression of DMRTJ
and testicular differentiation of both normal XY-maleand sex reversed XX-males suggest that DMRTJ is acandidate testis determining gene in tilapia. In contrast,
abundant DMO expression in pre- and early vitellogenic
oocytes in XX- and sex reversed XY-females indicatesa relationship between DMO and oocyte growth.

II. Endocrine regulation of spermatogenesis
Spermatogenesis is an extended process of differentiation 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-ketotestosterone (11-KT), a potent androgenin fish. In tum, 11-KT activates Sertoli cells to stimulate
the production of activin B. Addition of recombinant eel
activin B to the culture medium induced proliferation of
spermatogonia, producing late type B spermatogonia,
within 15 days in the same manner as did 11-KT.
cDNAs encoding two androgen receptors (ARa andAR~) have been cloned, for the first time in any verte-

brates, from eel and tilapia testes. In situ hybridization
reveals that although both AR mRNAs are present in eel
testes prior to HCG injection, only ARa transcripts
increase during HCG-induced spermatogenesis suggesting that ARa and AR~ play different roles in spermatogenesis. Activin B binds to activin type I and II
receptors on spermatogonia to stimulate de novo synthesis of Gl/S cyclins and CDKs leading to the initiation of mitosis. Interestingly, cyclin A1 transcripts are
first detected in primary spermatocytes during HCGinduced spermatogenesiss in eel testes suggesting animportant role for cyclin AI in the progression to meiosis of male germ cells. Overexpression of GFP-labeled
cyclins A and E in type A spermatogonia induces spermatogonial proliferation, followed by meiosis
ill. 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) has
been identified as the MIHs of several fish species. Theinteraction of two ovarian follilce cell layers, the thecal
and granulosa cell layers, is required for the synthesis of
17a,20~-DP.
The theal layer produces 17ahydroxyprogesterone 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. Two types of 20~-HSD
cDNAs are expressed in ovarian follicles of rainbow
trout (Oncorhynchus mykiss). Recombinant proteinsproduced by expression in E. coli in vitro show that one(type A) has 20~-HSD activity but that the other (typeB) does not. Among the three distinct residues between
the protein products encoded by the two cDNAs, two
residues (positions 15 and 27) are located in the Nterrninal Rossmann fold, the coenzyme binding site. We
have then generated mutants by site-directed mutagenesis at the following positions: MutA/115T, MutB/Tl5I,
and MutB/Q27K. Enzyme activity of wild-type A isabolished by substitution oflle-15 by Thr (MutA/115T).
Conversely, enzyme activity is acquired by the replacement of Thr-15 with Ile in type B (MutB/Tl5I).
MutB/T151 mutant shows properties similar to the wildtype A in every aspect tested. Mutation MutB/Q27K has
only partial enzyme activity, indicating that Ile-15 plays
an important role in enzyme binding of cofactor
NADPH.
17a,20~-DP induces oocyte maturation by acting ona pertussis toxin-sensitive G-protein-coupled membrane
receptor. The early steps of 17a,20~-DP action involvethe formation of downstream mediator of this steroid,
the maturation-promoting factor or metaphasepromoting factor (MPF) consisting of cdc2 kinase andcyclin B. 17a,20~-DP induces oocytes to synthesize
cyclin B which activates a preexisting 35-kDa cdc2
kinase via phosphorylation of its threonine 161 by athreonine kinase (M015), thus producing the 34 kDa26
active cdc2. The function of the MOS/MAPK pathway
during 17a,20~-DP-induced oocyte matration has been
investigated using goldfish oocytes. Mos is absent inimmature oocytes. It appears before the onset of germinal vesicle breakdown (GVBD), increases to a maximum in mature oocytes arrested at Mil and disappears
after fertilization. MAPK is activated after Mos synthesis but before MPF activation, and its activity reaches
maximum at Mil. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage
Xenopus and goldfish embryos induces metaphase arrest, suggesting that goldfish Mos has a cytostatic factor(CSF) activity. Injection of constitutively active
Xenopus c-mos mRNA into immature goldfish oocytes
induced MAPK activation, but neither MPF activation
nor GVBD occurs. Conversely, the injection of goldfish
c-mos antisense RNA inhibits both Mos synthesis and
MAPK activation in the 17a,20~-DP-treated oocytes,
but these oocytes undergo GVBD. These results indicate that the Mos/MAPK pathway is not essential for
initiating goldfish oocyte maturation despite its generalfunction as a CSF.

Publication List:
Guan, G., Todo, T., Tanaka, M., Young, G. and Nagahama, Y.
(2000). Isoleucine-IS of rainbow
trout carbonyl reductase-like 20~-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH)
binding. Proc. Natl. Acad. Sci. USA 97, 3079-3083.
Guan, G., Kobayashi, T. and Nagahama, Y. (2000).
Sexually dimorphic expression of DM (Doublesex/Mab-3)-domain genes during gonadal differentiation in tilapia. Biochem. Biophys. Res. Comm.
272, 662-666.
Kajiura-Kobayashi, H., Yoshida, N., Sagata, N., M.
Yamashita and Nagahama, Y. (2000).
The
Mos/MAPK pathway is involved in metaphase II arresst as a cytostatic factor but is neither necessary nor
sufficient for initiating oocyte maturation in goldfish.
Dev. Genes Evol. 210, 416-425.
Kitano, T., Takamune, K., Nagahama, Y. and Abe, S.
(2000).
Aromataseinhibitor
and
17amethyltestosterone causes sex-reversal from geneticalfemales to phenotypic males and suppression of P450
aromatase gene expression in Japanese flounder(Paralichthys olivaceus). Mol. Reprod. Develop. 56,
1-5.
Kobayashi, T., Kajiura-Kobayashi, H. and Nagahama,Y. (2000). The vasa homologue RNA in the germline of a teleost fish, tilapia, Oreochromis niloticus:
its differential expression during oogenesis andspermatogenesis. Mech. Develop. 99, 139-142.
Liu, SJ., Govoroun, M., D'Cotta, H., Ricordel, MJ.,
Lareyre, J.J., McMeel, O.M., Smith, T., Nagahama,Y. and Guiguen, Y. (2000). Expression of cytochrome P450~ (11~-hydroxylase) genes during gonadal sex differentiation and spermatogenesis ofrainbow trout, Oncorhynchus mykiss. J. Steroid

NATIONAL INSTITUTE

Biochem Mol. Bioi., in press.
Matsuyama, M., Sasaki, A., Nakagawa, K., Kobayashi,T., Nagahama, Y. and Chuda, H. (2000). Maturationinducing hormone of the tiger puffer, Takifugu rubripes (Tetraodontidae, Teleostei): biosynthesis ofsteroids by the ovaries and the relative effectivenessof steroid metabolites for germinal vesicle breakdown in vitro. Zool. Sci., in press.
Nakahata, S., Mita, K., Katsu, Y., Nagahama, Y. andYamashita, M. (2000). Immunological detection
and characterization of poly(A) polymerase, poly(A)binding protein and cytoplasmic polyadenylation
element-binding protein in goldfish and Xenopus oocytes. Zool. Sci., in press.
Nakayama, Y., Yamamoto, T., Oba, Y., Nagahama, Y.
and Abe, S.-1.(2000). Molecular cloning, functional
characterization, and gene expression of a folliclestimulating hormone receptor in the testis of newt
Cynops pyrrhogaster. Biochem. Biophys. Res. Comm.
275, 121-128.
Oba, Y., Hirai, T., Yoshiura, Y., Kobayashi, T. andNagahama, Y. (2000). Cloning, functional characterization, and expression of thyrotropin receptors in
the thyroid of amago salmon (Oncorhynchus rhodurus). Biochem. Biophys. Res. Comm. 216, 258-263.
Okida, N., Tokumoto, M., Tokumoto, T., Nagahama, Y.,
Ohe, Y., Miyamoto, K. and Ishikawa, K. (2000).
Cloning of eDNA encoding thimet oligopeptidasefrom Xenopus oocytes and regulation of the rnRNAduring oogenesis. Zool. Sci. 17, 431-436.
Shibata, Y., Iwamatsu, T., Oba, Y., Kobayashi, D.,
Tanaka, M., Nagahama, Y., Suzuki, N. and Yoshikuni, M. (2000). Identification and eDNA cloningof alveolin, an extracellular metalloproteinase responsible for chorion hardening of medaka (Oryzias
latipes) eggs upon fertilization. J. Bioi. Chem. 215,
8349-8354.
Shinomiya, A., Tanaka, M., Kobayashi, T., Nagahama,Y. and Hamaguchi, S. (2000). The vasa-like gene,olvas, identifies migration path of primordial germ
cells during embryonic body formation stage in the
medaka, Oryzias latipes. Develop. Growth Differ. 42,
317-326.
Tanaka, M., Kinoshita, M. and Nagahama, Y. (2000).
Establishment of medaka (Oryzias latipes) transgeniclines with the expression of green fluorescent protein
fluorescence exclusively in germ cells: A usefulmodel to monitor germ cells in a live vertebrate.
Proc. Natl. Acad. Sci. USA, in press.
Todo, T., Ikeuchi, T., Kobayashi, T., Kajiura-Kobayashi,
H., Suzuki, K., Yoshikuni, M. and Nagahama, Y.
(2000).
Characterization of a nuclear 17a,20~­
dihydroxy-4-pregnen-3-one (a spermiation-inducing
steroid in fish) receptor eDNA from the testis of ateleost, Japanese eel (Anguilla japonica). FEBS
Letters 465, 12-17.
Tokumoto, M., Horiuchi, R., Nagahama, Y., Ishikawa,
K. and Tokumoto, T. (2000). Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic

27

FOR BASIC BIOLOGY

cell cycle. Eur. J. Biochem. 267, 97-103.
Tokumoto, M., Nagahama, Y. and Tokumoto, T. (2000).
Molecular cloning of eDNA encoding a ubiquitinactivating enzyme (El) from goldfish (Carassius auratus) and expression analysis of the cloned gene.
Bioch. Biophys. Acta 1492, 259-263.
Tokumoto, M., Yamaguchi, A., Nagahama, Y. and Tokumoto, T. (2000). Identification of the goldfish
20S proteasome ~6 subunit bound to nuclear matrix.
FEBS Letters 472, 62-66.
Yamaguchi, A., Yamashita, M., Yoshikuni, M. andNagahama, Y. (2000). Identification and molecularcloning of germinal vesicle B3 in goldfish (Carassius
auratus) oocytes. Eur. J. Biochem, in press.

Oba, Y., Hirai, T., Yoshiura, Y. and Nagahama, Y.
Fish pituitary glycoprotein hormone receptors: cloning and characterization of two different gonadotropin receptors from gonads and two thyrotropin-likereceptors from thyroid of amago salmon (Oncorhynchus rhodurus). In Reproductive Physiology of Fish
(Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E. and Stefansson, S.O. eds.), pp. 164-166, Bergen (2000).
Nakamura, M., Kobayashi, T., Yoshiura, Y. and Nagahama, Y. Role of endogenous steroid hormoneson gonadal sex differentiation in fish. In Reproductive Physiology of Fish (Norberg, B., Kjesbu, O.S.,
Taranger, G.L., Andersson, E. and Stefansson, S.O.
eds.), pp. 247-249, Bergen (2000).

Proceedings:
Morrey, C.E. and Nagahama, Y. 11~-hydroxylase andandrogen receptor mRNA expression in the ovary,testes and brain of the protogynous hermaphrodite
Thalassoma duperrey. In Reproductive Physiologyof Fish (Norberg, B., Kjesbu, O.S., Taranger, G.L.,
Andersson, E. and Stefansson, S.O. eds.), pp. 157159, Bergen (2000).

Review:
Nagahama, Y.
Gonadal steroid hormones: major
regulators of gonadal sex differentiation and gametogenesis in fish. In Reproductive Physiology of Fish
(Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E. and Stefansson, S.O. eds.), pp. 211-222, Bergen (2000).

28

NATIONAL INSTITUTEDIVISION OF CELL DIFFERENTIATION
Professor:
Research Associate:
Technical Staff:

Secretary:
Postdoctoral Fellow:

Graduate Student:

Visiting Scientist:

Ken-ichirou Morohashi
Akihiko Shimono
Satoru Ishihara
Sanae Oka
ChiyoNoda
Yuko Shinohara I)
Masatomo Kusaka
MioSugiura
Azusa Ishikawa
Hiroko Kusaka
Tomoko Imura 21
Akiko Iizuka 31
Zubair Mohamad 41
Noriyuki Sugiyama 41
Naohito Nakamura 41
Ken Kawabe 41
Tokuo Mukai 51
Hirofumi Mizusaki
Taiga Suzuki 61
Yuichi Shima 51
Makoto Matsuyama 71
Takafumi Gotoh 81
Tsutomu Daito 91

nJSPS technical staff
1JSPS secretary
3iNIBB Postdoctoral Fellow
4iJSPS Postdoctoral Fellow
51 Graduate School of Kyushu University
61 Graduate School of Kyoto University
71Graduate School ofNagoya University
8iAssociate Professor of Kyushu University
9iBIACORE Co. Ltd.
2

Cell and tissue differentiation proceeds systematically based on a number of gene expressions that commence successively along with the passage of time.
As the consequence, a fertilised egg develops into avariety of tissues and organs comprising specialised
cells in terms of their structures and functions. Accordingly, it is no doubt that investigation of themechanisms underlying the cell and tissue-specific gene
expression at a molecular base is essential for obtaining
a proper understanding of the process of tissue differentiation. In our division of Cell Differentiation, twodistinct but closely correlated studies at their central
concepts have proceeded. One of them is the study forcomprehensive understanding of sex differentiation at
the levels from the gonad function to reproductive behavior, and the other is the study focussing on head
formation through characterization of the function ofhead organizer.

I. Gene regulatory cascade in the steroidogenic
tissue differentiation
When a differentiation process of a tissue is considered, it is reasonable to assume a tissue-specific gene
regulatory cascade in which certain genes encoding
transcription factors are involved as the critical components. In the cascades required for adrenal and gonadal differentiation, Ad4BP/SF-1 is locates upstreamof tissue-specific genes, including the steroidogenic

CYP genes, and locates downstream of other transcription factors regulating the Ad4BP/SF-1 gene. Considering that the cascade flows from upstream to downstream through out the tissue differentiation and
Ad4BP/SF-1 is an essential transcription factor in the
gonadal cascade, identification of the components consisting the cascade as well as their genetical relationshipis essential for fully understanding the mechanisms ofthe tissue differentiation.
Based on the aspect above, the regulatory region of
the Ad4BPISF-1 gene has been analysed in vivo bymaking transgenic mice. However, our in vivo study
in a recent few years has not yet been successful,probably because the regulatory region locates far upstream or far downstream from the structural gene of
Ad4BP/SF-1. On the contrary, our in vitro study with
cultured cells provided a novel mechanism regulating
Ad4BP/SF-1 gene, in which an activating signal fromone of growth factors, Wnt, is implicated. Although
the fine mechanism has been under investigation, thestudy from this aspect will give us a novel insight into
gonad differentiation.
Dax-1 is another transcription factor of our interest,
which is also implicated in the steroidogenic tissue
differentiation. Our previous study revealed that thefactor acts as a suppressor of Ad4BP/SF-l. However,regulation of the suppressive effect has remained to beclarified at the molecular level. We recently uncovered the function of the amino terminal half of Dax-1
containing a unique repeated sequence. When Dax-1
functions as the suppressor, a certain amino acid sequence in the repeats is essential for interaction withtarget nuclear receptors, and thereby the transcription
mediated by the nuclear receptors are largely inhibited.
Although it remains unclear how the inhibition activity
is regulated in a variety of physiological conditions, thisinteraction gave us a cue to address it.
In addition to these transcription factors, Sox-9, Wt-1,
Ernx-2, and GATA-4 are known to be implicated in the
gonad development. In order to isolate novel factors
interacting with all these transcription factors above,
yeast two-hybrid screening has been performed using aeDNA library constructed with an mRNA prepared frommouse fetal gonads. Extensive screening resulted inisolation of molecules including coactivators and othertype of transcription factors. Some of them are novel
factors and have interesting structures making us toanticipate novel regulations of the transcription. Distributions and functions of these interacting molecules
have been examined.

11. Sex-differentiation observed in adrenal cortex
Our previous study indicated that Ad4BP/SF-1 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 andreticularis. However, this distribution revealed byimmunohistochemistry was quite distinct from thatobtained with in situ hybridization. To explain the

29

FOR BASIC BIOLOGY

target 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.

III. Molecular mechanism for head formation

Expression of Factor X, which was isolated by two-hybrid
screening as a molecule interacting with Ad4BP/SF-l.
Factor X is expressed in the developing gonads (E 12.5 testis(upper) and ovary (lower)).

It has been clarified that the anterior visceral endoderm (AVE) and mesendoderm (AME) plays criticalroles for head formation by the induction of specificgene expressions in anterior neuroectoderm. Lim I, a
LIM homeodomain-containing transcription factor, isexpressed at the AVE and primitive streak-derived cells
during gastrulation. Since a striking function of Lim!
was revealed by making gene disrupted mice whichdisplayed headless phenotype, molecular events after
commencing the Liml expression have been investigated through identifying a set of downstream genes of thetranscription factor.
For identification , differential
screen was performed using subtraction between eDNA
pools generated from a small number of cells corresponding to the AVE of wild and KO individuals. A
number of candidate genes showing a loss of expression
in the KO and a close correlation with Liml in terms of
their expression profiles have been isolated so far.
The functions of these genes are investigating extensively by gene disruption studies.

Publication List:
discrepancy between the two methods, close examination was carried out with a series of adrenal cortex of ·both sexes at several developing stages from fetal 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 androgen and its receptor downregulate Dax-1 gene transcription, which is interestingly inconsistent with acommon understanding that androgen receptor activates

Identification of Novel First Exons in Ad4BPISF-1(NR5Al) Gene, and Their Tissue- and SpeciesSpecific Usage. (2000) Rina Kimura, Hironori Yoshii,
Masatoshi Nomura, Naoe Kotomura, Tokuo Mukai,
Satoru Ishihara, Koichi Ohba, Toshihiko Yanase,Osamu Gotoh, Hajime Nawata, & Ken-ichirou
Morohashi Biochem. Biophys. Res. Commun. 278,
63-71
Comparative localization of Dax-1 and Ad4BP/SF-lduring development of the hypothalamic-pituitarygonadal axis implies their closely related and distinct
functions . (2001) Yayoi Ikeda, Yospie Takeda, Tatsuji Shikayama, Tokuo Mukai, Setsuji Hisano, &Ken-ichirou Morohashi Develop. Dynam. in press

30

NATIONAL INSTITUTE

DNISION OF MORPHOGENESIS
Professor:
Associate Professor
Research Associates:
Technical Stuff:

Visiting Sicentist:
Postdoctoral Fellows:

Graduate Students:

Naoto Ueno
Hiroshi Shibuya*
Makoto Nakamura
Hiraki Takahashi
Chiyo Takagi
Takamasa Yamamoto
AkikoHyodo
Michiko Ichikawa
Yukiko Sugihara
Chie Terasaka
Nobuhiko Mizuno
Shun-ichiro lemura
Kiyokazu Morita
Atsushi Kitayama
Jun Nakabayashi
Katsura Sugaward!
Satoru Yoshida 2!
Yasunori Tomoyasu 2!
Yasuo Mitan;J!
Tomoko Kurata 2!
Bisei Nakano2!
Masaki Takeuchi2!
Jyunko Hyodo 4!

*until August 3/, 2000
!jrom Osaka University
2! Graduate University for Advanced Studies
3! Graduate School of Kyoto University
4! Graduate School of Nagoya University
1

The complex morphogenesis of organisms isachieved by consecutive cell-to-cell interactions ofgerm layers as well as tissues during development.
Recent studies suggest that polypeptide growth factors(PGFs) are essential component controlling such intercellular communications in a variety of organisms.
These cell communications via PGFs are regulated by anumber of processes including secretion, activation,diffusion, reception by specific receptors and intracellualr signaling. In addtion to secretory factors, transcription factors which act cell-autonomously havecritical roles in the determination of cell fates. Our maininterest is to know how pattern formation in morphogenesis is regulated by PGFs and transcription factors
during development. We address this problem using
several model animals, including frog, fly, acidians andnematode with the view of extracellular and intracellular signaling, employing embryology, genetics,
molecular and cellular biology and biochemistry. In
addition, we are currently introducing micro/macroarray
technology to understand precise genetic program controlling early development.

I. Role of Xmsx-1 as a negative regulator of head
formation
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 induced in response to PGFs inearly Xenopus development. In particular, Xmsx-1, an
amphibian homologue of vertebrate Msx-1, is wellcharacterized as a target gene of bone morphogenetic
protein (BMP). In order to clarify molecular basis forventalization by BMP and in vivo significance ofMsx-1,
we examined whether Xmsx-1 activity is required in theendogenous ventralizing pathway, using a dominantnegative form of Xmsx-1 (VP-Xmsx-1). The VP-Xmsx1 induced a secondary body axis, complete with muscle
and neural tissues, when the fusion protein was overexpressed in ventral blastomeres. Interestingly, a morepotent dominat negative Xmsx-1 induced ectopic headwith eyes and cement gland. These results suggest that
Xmsx-1 activity is necessary for both mesoderm andectoderm to be ventralized and that head formation may
represent a default state of BMP/Msxl activity. We are
currently investigating the molecular machanism ofhead repression in ventral side of embryo.

II. Genetic dissection of TGF-WBMP signaling
utilizing Drosophila model system
Fruit fly (Drosophila melanogaster) is one of theideal model organisms to elucidate certain intracellularsignal transduction pathways by genetic methods. A
Drosophila BMP homolog, Decapentaplegic (Dpp)plays crucial roles in a number of developmental events
including patterning of the adult wing. We have established two transgenic lines that express activated
Dpp/BMP receptors. These mutants exhibit stable Dppgain-of-function phenotypes in the wing. One of thetransgenic lines, tkv-CA::7IB, expressed constitutively
activated Tkv (Dpp type-I receptor) shows ectopic wingvein formation in the entire wing blade (Fig. lA). Another line, ALK2::69B, expresses activated vertebrate
BMP type-I receptor and shows abnormal wing outgrowth toward anterior and posterior direction (Fig. IB).
These phenotypes are sensitive to the gene dosage of
the Dpp signal component such as Mad (Fig. I C).
Combinatorial screening has been done utilizing a Deficiency-kit (minimum set of the deficiency mutant linesthat covers about 70 % of the Drosophila genome) andP-element insertion lethal stocks from public stock
centers. We isolated 19 dominant suppressers to either
or both tkv-CA::71B and ALK2::69B. We named thesemutants Suppresser of constitutively activated Dpp
signaling (Scad). Alleles of punt, Mad, shn and dCrebA
were found in the isolated Scad mutants. It has beenreported that the function of these genes are involved in
the Dpp signaling. We focused to further analyze twonovel loci, Scad67 and Scad78 at a molecular level.
Candidate genes for Scad67 and Scad78 were isolated.
Scad67 encodes a novel putative nuclear protein of aputative Zn-finger motif with a weak homology to the
PIAS family transcriptional regulators. Scad78 encodesa homolog of vertebrate putative transcription co-factor
1RAP240. Mosaic analyses of Scad67 and Scad78 mutants suggest that these putative transcriptional regulators are involved not only in the Dpp signal transduction but also in other signal transduction cascades.
We have also been interested in the function of a
MAPKKK class protein kinase, TGF-13 activated31

FOR BASIC BIOLOGY

kinasel (TAKI) . TAK1 was originally isolated as adownstream signal component of TGF-~ and BMP
receptors. TAK1 function was extensively studied incultured cells, however, the in vivo function of TAKl
has not yet been fully understood. To dissect in vivo
function of TAKI , we generated transgenic flies which
express an activated form and a dominant negative formof TAK1 s (Drosophila and mouse TAK1s) in the fly
visual system. Ectopic activation of TAKl signaling led
to ectopic apoptosis and resulted in small eye phenotype.
Genetic and biochemical analyses indicated that the
JNK signaling pathway is specifically activated byTAKl . Loss-of-function analysis using dominant negative TAK1s suggested that TAKl participates in cell
movement, cell shape control and apoptosis in Drosophila. Recently candidates of dTAKJ mutants wereisolated. Phenotypic analysis of these mutants is ongoing.

A) tkv1-ca::71BGa/41 +III. Brachyury downstream notochord differentiation in the ascidian embryo
Ascidians, urochordates, are one of the three chordategroups, and the ascidian tadpole is thought to represent
the most simplified and primitive chordate body plan.
It contains a notochord, which is a defining characteristic of chordate embryo composed of only 40 cells. To
understand the morphogenesi ~ in this simple system, we
have focused on a gene, Brachyury, which is known toplay an important role in the notochord development.
In ascidian, Brachyury is expressed exclusively in the
notochord and the misexpression of the Brachyury gene
(Ci-Bra) of Ciona intestinalis is sufficient to transform
endoderm into notochord. This gene encodes a sequence-specific activator that contains a T -box DNAbinding domain, and in vertebrates, it is initially expressed throughout the presumptive mesoderm andgradually restricted to the developing notochord andtailbud. The phenotype of the Brachyury mutants inmice and zebrafish revealed that thi s gene is essential
for notochord differentiation. Our goal is to elucidate
the down stream pathway of this important gene inascidian in order to set the stage for understanding not
only the formation and function of the notochord but
how this important structure has evolved. We conducted
the subtractive hybridization screens to identify potential Brachyury target genes that are induced upon CiBra overexpression. Out of 501 independent eDNA
clones that were induced cDNAs, 38 were specifically
expressed in notochord cells (Fig. 2). We characterized
20 of them by determining the complete nucleotide
sequences and in situ hybridization analyses whichshow the spatial and temporal expression patterns of thecDNAs. These potential Ci-Bra downstream genes
appear to encode a broad spectrum of divergent proteinsassociated with notochord formation and function.

IV. TGF-~ family in nematode~B) ALK2-ca::69BGa/4 I+

C) ALK2-ca::69BGa/4 I Mad12
Figure 1. The wing phenotypes genetic interaction utilized
for the screening. Expression of activated Dpp/BMP type-I
receptors are resulted in the stable wing phenotypes (A, B).
These phenotype is sensitive to the dosage of Dpp signal
comonent Mad (C).

We have previously shown that CET-1/DBL-1, amember of TGF-~ superfamily regulates body length in
C. elegans. To understand molecular mechanism ofbody length regulation by the secreted factor, we aimed
to identify target genes regulated by CET-1/DBL-1
signaling using a eDNA-based array analysis followed
by functional analysis with double stranded RNAi . We
identified yk298h6 to be one of genes suppressed in
CET-1/DBL-1 overexpressing worm with a shortened
body length. Disruption of yk298h6 resulted in long
worm, suggesting that it encodes a negative regulator ofbody length.
yk298h was then mapped to, and shown to be identical with, lon-1 , a known gene that affects body length.
LON-1 encodes a protein with a motif
sequence thatis conserved from plants to human. Expression studies
confirm that LON-I is repressed CET-1/DBL-1 suggesting that LON-1 is a novel downstream componentof the C. elegans TGF-~.

32

NATIONAL INSTITUTE

Ci-cdc45 (406g)

Ci-flhrn (408h)

Ci-Notol (507h)

Ci-NotQII (210d)

1 Ci-trop (llOg)

903h

Ci-pellino (212g)

Ci-Noto2 (006c)

309b

Figure 2. Expression of Ci-Bra and its downstream notochord genes in C. intestinalis tai lbud embryos revealed by whole-mount insitu hybridization. Scale bar, I 00 11m.

Publication List
Haraguchi, R. , Suzuki, K., Murakami, R. , Sakai, M.,
Kamikawa, M., Kengaku, M ., Sekine, K. , Kawano,
H., Kato, S., Ueno, N. and Yamada, G. (2000)
Molecular analysis of external genitalia formation:
the role of fibroblast growth factor (Fgf) genes during
genital tubercle formation . Development 127, 24712479.
Hotta, K., Takahashi, H. , Asakura, T ., Saitoh, B., Takatori, N., Satou , Y. and Satoh, N . (2000) Characterization of Brachyury-downstream notochord genes inciona intestinalis embryo. Dev. Bioi. 224, 69-80.
Kurata, T ., Nakabayashi, J., Yamamoto, T. S., Mochii ,M . and Ueno, N . (2000) Visualization of endogenous
BMP signaling during Xenopus development Differentiation 67, 33-40.
Masuda Y, Sasaki A, Shibuya H, Ueno N, Ikeda K,
Watanabe K. (2000) Dlxin-1, a novel protein thatbinds Dlx5 and regul ates its transcriptional function .
J Bioi Chem. 276, 5331-5338.
Nishita, M. , Hashimoto, M . K., Ogata, S., Laurent,
M . N,. Ueno, N., Shibuya, H. and Cho, K. W.
(2000) Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's or-

ganizer. Nature 403, 781-785.
Takatsu, Y., Nakamura, M ., Stapleton, M ., Danos,
M. C., Matsumoto, K. , O'Connor , M.B ., Shibuya,
H. , Ueno, N. (2000) TAK1 participates in c-Jun Nterminal kinase signaling during Drosophila development. Mol. Cell. Bioi. 20, 3015-3026,
Tomoyasu, Y., Ueno, N. and Nakamura, M . (2000)
The decapentaplegic morphogen gradient regulates
the notal wingless expression through induction ofpannier and u-shaped in Drosophila. Mech. Dev. 96,
37-49.
Wada T, Kagawa T, Ivanova A, Zalc B, Shirasaki R,
Murakami F, Iemura S, Ueno N, Ikenaka K. (2000)
Dorsal spinal cord inhibits oligodendrocyte development. Dev Bioi. 227, 42-55.
Yagi, Y. Tomita, S. , Nakamura, M . and Suzuki, T .
Over-expression of human amyloid precursor protein
in Drosophila. Molecular Cell Biology ResearchCommunications, (in press)
Yamamoto, S. T., Takagi , C. and Ueno, N. Requirementof Xmsx-1 in the BMP-triggered ventralization of
Xenopus embryos. (2000) Mech. Dev. 91, 131-141.

33

FOR BASIC BIOLOGYDIVISION OF DEVELOPMENTAL BIOLOGY(ADJUNCT)
Professor:

Kenzo Nakamura(Nagoya University)
Associate Professor: Tsukaho Hattori (Mie University)
Assistant Professor: Masa-aki Ohto
Technician Staff
Etsuko Aoki
Yasuko Furukawa
Postdoctoral Fellow: Akiko Ohta

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 andthe synthesis of macromolecules but also as a signal
controlling the pattern of gene expression in various
parts of the plant body. Our research attention is focused to elucidate the mechanisms involved in the regulation of gene expression in response to sugars and therole of such regulation in the growth and developmentof plants.
During the growth of plants, new organs develop as
carbohydrate sink, and many vegetative organs show
sink-to-source transition after their maturation. Sugar
affects many aspects of these organ developments inplants. To obtain insights into the role of sugarregulated gene expression in the growth and development of plants, we took genetic approach using Arabidopsis thaliana. To aid this purpose, we establishedmore than 7,000 independent lines of Arabidopsis plants
transformed with T-DNA containing multiple copies ofthe enhancer sequence. Among these lines, we screened
for mutants that not only showed defects in thedevelopment of leaves or anomalies in the flowering
time but also showed the altered patterns of the sugarinducible expression of a gene for ~-amylase (At/3-Amy).
More than 20 such mutants, designated as uns (unusual
sugar response), were isolated. Similar to lbal andhbal mutants of Arabidopsis isolated previously [Mita,
S., Murano, M., Akaike, M. and Nakamura, K., Plant J.

11: 841-851 (1997); Mita, S., Hirano, H. and Nakamura,
K. Plant Physiol. 114: 575-582 (1997)], the sugarinducible expression of At/3-Amy in uns mutants waseither significantly reduced or enhanced compared tothat in the wild-type plants depending on the mutantline.
In several of uns mutants, we could identify mutatedgenes responsible for the mutant phenotypes. One ofthem, UNS6, codes for a protein that shows amino acidsequence similarity to a gene of Saccharomyces cerevisiae, which is essential in yeast and has been suggested
to play a role in the assembly of the transcription
preinitiation complex. The uns6 mutant plants showenhanced sugar-inducible expression of At/3-Amy andseveral other genes and defects in leaf development
However, despite UNS6 is a single-copy gene in
Arabidopsis, mutant plants do not show severe
developmental defects other than leaf development.
Furthermore, the sugar-responsible expression of several other genes is not affected in the mutant plants. Thus,
UNS6 may have some unique function other than ageneral role in transciption initiation in Arabidopsis.

Selected Publication:
Iwata, Y., Kuriyama, M., Nakakita, l\1., Kojima, H., Ohto,
M. and Nakamura, K., (1998) Characterization 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) The N-terminal propeptide and the C terminus of
the precursor to 20-kilo-dalton potato tuber protein canfunction as different types of vaculor sorting signals.
Plant Cell Physiol. 40(11): 1152-1159.
Nakamura, K., Morikarni, A., Ohto, M., Tomiya, T.,
Hayashi, S., Saijo, T., Yamada, M. and Furukawa, Y.
(2000) Mutants of Arabidopsis thaliana with alteredresponse to sugars. In "Proceedings of the 2000 JapanKorea Joint Symposium of Plant Science; Plant Responses to Environments", The Symposium on Plant
Biotechnology 14: 91-94.

34

NATIONAL INSTITUTE

35

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.

36

NATIONAL INSTITUTE

DNISION OF MOLECULAR NEUROBIOLOGY
Professor:
Associate Professor:
Research Associates:

Masaharu Noda
Nobuaki Maeda
Takafumi Shintani
Junichi Yuasa
Technical Staff:
Yasushi Takeuchi (May 8, 2000 -)Shigemi Takami (-Jun. 23, 2000)
Kaoru Yamada (Oct. I, 2000-)
Post Doctoral Fellows: Hiraki Sakuta4
Akihiro Fujikawa4
Hiroyuki Kawachz4(-Aug. 31, 2000)
Mohamad Zubair4(-Mar. 31, 2000)
Minoru Ujita4 (-Mar. 31, 2000)
Graduate Students:
AkiraKato
Ryoko Suzuki
Masahide Fukada
Takeshi Hiyama
Hiroshi Tamura
Takeshi Ohkawara
Hi roo Takahashi (Apr. 1, 2000 -)Chika Saegusa (-Sept. 30, 2000)
Takuji Yoshitomi.
(Apr. 1 -Oct. 31, 2000)
Visiting Scientist:lkuko Watakabe (Sept. 18, 2000-)
JST Technical Staff:
Masae Mizoguchi
MegumiGoto
Minako Ishida(- Dec. 31, 2000)

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 refinement of specificconnections between neurons, and also synaptic plasticity. We have been using various techniques including
molecular biology, biochemistry, immunological methods, neuroanatomy, cell and organotypic culture, embryo manipulation (gene transfer and gene targeting),
and behavioral analysis.
I. Molecular mechanism of the retinotectal projection
Topographic maps are a ~damental feature of neural networks in the nervous system. Understanding themolecular mechanisms by which topographically ordered neuronal connections are established during development has long been a major challenge in developmental neurobiology. The retinotectal projection oflower vertebrates including birds has been used as areadily accessible model system. In this projection,
the temporal (posterior) retina is connected to the rostral(anterior) part of the contralateral optic tectum, thenasal (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 thetectum in a reversed manner. In 1963, Sperry proposed that topographic mapping could be guided bycomplementary positional labels in gradients across preand postsynaptic fields.
Although this concept iswidely accepted today, and Eph families of receptor
tyrosine kinases and their ligands were recently identified as candidates for such positional labels, themolecular mechanism of 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 showed that these two transcription factors
determine the regional specificity of the retinal ganglion
cells, namely, the directed axonal projections to theappropriate tecta! targets along the anteroposterior axis.
To further search for topographic molecules in the embryonic retina, we next performed a large-scale screening using a new eDNA display system called Restriction
Landmark eDNA Scanning (RLCS). With the assistance of a computer image-processing software, we
successfully identified 33 molecules along the nasotemporal axis and 20 molecules along the dorsoventral axis,
with various asymmetrical expression patterns in thedeveloping retina. We have elucidated the primarystructures of all these eDNA clones and examined theirexpression patterns during development. These included many novel molecules together with the known
molecules: transcription factors (CBF-2, COUP-TFII,etc.), receptor proteins (EphA3, EphB3, etc.), secretory
factors, intracellular proteins, and so on.
Among them, we recently identified a novel retinoic
acid (RA)-generating enzyme, RALDH-3, which is
specifically expressed in the ventral region of the retina,
together with a dorsal-specific enzyme RALDH-1. In
chick and mouse embryos, the expression of Raldh-3 isobserved first in the surface ectoderm overlying theventral portion of the prospective eye region earlier thanthat of Raldh-1 in the dorsal retina, and then the Raldh3 expression shifts to the ventral retina (Fig. 1). Furthermore, we found that Raldh-3 is a downstream target
of Pax6 which is known to be the master gene for theeye development in many species. It is well known
that RA is essential for the eye development. These
results suggest that RALDH-3 and RALDH-1 are thekey enzymes for the dorsoventral patterning at the earlystage of the retinal development.
Currently, with respect to the identified topographic
molecules, we are conducting over- and misexpression
experiments using viral vectors and in ovo electroporation to elucidate their molecular functions. We expectthat our studies will lead to elucidation of the molecularmechanism underlying the retinal patterning and topographic retinotectal projection, and ultimately to uncovering the basic principles for establishing complicatedFOR BASIC BIOLOGY
A

Fig.! Expression patterns of Raldh-3 (A) and Raldh-1 (B)during chick development
All embryos were hybridized with DIG-labeled antisense
riboprobes. Developmental stages of embryos are stage I 0(A, a), stage II (B, a), stage 12 (A and B, b), stage 14 (A andB, c, d), stage 16 (A and B, e), stage 20 (A and B, f, g), E6 (Aand B, h), and E8 (A and B, i), respectively. The expressionof Raldh-3 is observed first in the surface ectoderm overlying
the ventral portion of the prospective eye region earlier thanthat of Raldh-1 in the dorsal retina. Coronal sections areoriented in dorsal up and ventral down. so, somites; ot, otic
vesicle. Scale bars: 100 Jlm (A and B, d, g, h); 200 Jlm (Aand B, i).

but extremely precise neural networks.

II. Functional roles of protein tyrosine phosphatase t;,
Protein tyrosine phosphorylation plays crucial roles
in various aspects of brain development. The level oftyrosine phosphorylation is determined by the balance
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/RPTP~. a nervous system-rich RPTP, is expressed as a chondroitin sulfate proteoglycan in the brain.
An RNA splice variant corresponding to the extracellular region of PTP~ is secreted as a major proteoglycan
in the brain known as 6B4 proteoglycan/phosphacan.
The extracellular region of PTP~ consists of a carbonic
anhydrase-like domain, a fibronectin-type Ill-like domain and a serine-glycine-rich region, which is considered to be the chondroitin sulfate attachment region .
PTP~ is expressed from the early developmental stage
to the adulthood. This suggests that this molecule
plays variegated roles in the brain development andbrain functions.
In an attempt to reveal the signal transduction

37
mechani sm of PTP~, we first tried to identify the ligand
molecules of this receptor. We found that PTP~ binds
pleiotrophin!HB-GAM · and midkine, closely related
heparin-binding growth factors which share many biological activities. The chondroitin sulfate portion of
PTP~ is essential for the high affinity binding (Kd =-0.25 nM) to these growth factors, and removal ofchondroitin sulfate chains results in a marked decreaseof binding affinity (Kd = -13 nM). We further revealed that chondroitin sulfate interacts with Arg 78 in
Cluster I, one of the two heparin-binding sites in the Cterrninal half domain of midkine. This is the first
demonstration that chondroitin sulfate plays an important regulatory role in growth factor signaling.
Next, we examined the roles of pleiotrophin/midkinePTP~ 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 domain of PTPI;;, 6B4proteoglycan (a secreted extracellular form of PTPI;;)and sodium vanadate (a protein tyrosine phosphataseinhibitor) added to the culture medium strongly suppressed this migration . Experiments using various
midkine mutants with various affinities for PTPI;; indicated that the strength of binding affinities and the neuronal migration-inducing activities are highly correlated.
These results suggest that PTPI;; is involved in migration
as a neuronal receptor for pleiotrophin and midkine.
In order to reveal the intracellular signaling mechanism of PTPI;;, we performed yeast two-hybrid screening using the intracellular region of PTPI;; as bait. We
found that PTP~ interacts with PSD-95/SAP90 family
members, SAP102, PSD-95/SAP90 and SAP97 /hDig,
which are concentrated in the central synapses mediating protein-protein interactions to form large synaptic
macromolecular complexes. Here, the C-terminus of
PTPI;; binds to PSD-95/SAP90 proteins through thesecond PDZ domain. This suggests that PTPI;; is involved in the regulation of synaptic function. However, PSD-95/SAP90 family members are not likely tobe the substrate for PTPI;; because this family members
are not tyrosine-phosphorylated.
To identify the substrate molecules of PTPI;;, we haverecently developed the yeast substrate-trapping system.
This system is based on the yeast two-hybrid systemwith two essential modifications: conditional expressionof v-src to tyrosine-phosphorylate the prey proteins andscreening using a substrate-trap mutant of PTPI;; as bait
(Fig. 2). Using this system, we identified GITl/Cat-1
as a PTP~ substrate. PTPI;; and GITl/Cat-1 were colocalized in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B 103 neuroblastoma cells (Fig. 2). Moreover,pleiotrophin increased tyrosine phosphorylation of
GITl/Cat-1 concomitantly with its recruitment to thestimulated PTPI;; in B103 cells. It is known that Cat-1
regulates Pak, a serine threonine kinase which serves asa target for the small GTP-binding proteins , Cdc42 andRae, and is implicated in a wide range of cellular events

38

NATIONAL INSTITUTE

A

Sr9.

gous mutant mice, we demonstrated that neurons aswell as astrocytes express PTPs in the central nervous
system. We are currently studying the phenotype of
PTP/;;-deficient mice biochemically, anatomically,physiologically and ethologically, and have alreadyfound abnormalities in behavior, learning and memory,etc.

GAL4AD. ' ~Prey_c::::::5Prote1n.
PTP~ICR-D1902A/

LexA _...;.~-'

8

Promoter

Reporter Gene

Zincfinger

ProlinerichAnkyrinrepeat

GRK
Binding

GIT1/Cat-1

770

251

555

c

Fig. 2 The yeast substrate-trapping system and GITl/Cat-1(A) In the presence of 1 mM methionine when v-src is not
induced, only the standard two-hybrid bindings occur. In theabsence of methionine, prey proteins (substrates for PTP~)
could be phosphorylated by the induced v-src, and trapped by
the bait consisting of the whole intracellular domain with an
Asp1902Ala mutation (PTP~ ICR-D1902A). This complex
formation leads to activation of transcription of the reporter
genes, HIS3 and LacZ. We performed screening in two steps
and selected the colonies which showed an increase in blue
·color development upon induction of v-src.
(B) GITl/Cat-1 contains a zinc-finger region, three ankyrinrepeat regions, a potential SH3-binding site and a GRK binding site. The region encoded by the clone isolated by the
yeast substrate-trapping system is shown with a solid line
underneath.
(C) The mossy fibers extending from pontine explants weredoubly imrnunostained with anti-GITl/Cat-1 (a) and anti-6B4
PG (b) antibodies. The combined image is shown in (c).
The colocalization of both proteins was evident at the peripheral regions (arrows) and filopodia! processes (arrowhead)of the growth cones. 8103 cells were doubly imrnunostainedwith anti-GITl/Cat-1 (d) and anti-6B4 PG (e) antibodies.
The combined image is shown in (f). GITI/Cat-1 and PTP~
were colocalized in the processes (arrowhead) and ruffling
membranes (arrows) of the cells.
Scale bars: 5 J.Lm (a-c),
10 J.Lm (d-f).

including the cell adhesion and cell morphologicalchange.
Pleiotrophin, PTPs and GITl/Cat-1 might
regulate the neuronal migration and neurite extension
by controlling the Pak signaling pathway.
To further study the physiological function of PTPs
in vivo, we generated PTPI;;-deficient mice in which the
PTPs gene was replaced with the LacZ gene in 1997.
By investigating the expression of LacZ in heterozy-

ill. Physiological roles of Nav2 ion channel
Voltage-gated sodium channels (NaChs) are responsible for generating action potentials in excitable cells
and play many important physiological roles. Cloningof NaChs revealed marked conservation in the primarystructures that underlies their functional similarity.
Thus, all cloned NaChs had been grouped into a single
gene family. However, recently, novel NaChs, humanNa.2.1, mouse Na.2.3 and rat NaG/SCLll , were cloned
from inexcitable cells such as glial cells.
These
molecules closely resemble each other but are divergent
from the previously cloned sodium channels including
the regions involved in activation , inactivation and ion
selectivity. Thus, these molecules should be grouped
into a new subfamily of NaChs (Na.2). To date, Na.2
channels have not been expressed in a functionallyactive form using in vitro expression systems, andtherefore the functional properties of these NaChs are
not yet clear.
To clarify the physiological function and the cells
expressing Na.2 channels in vivo , we generated knockout mice in which Na.2 channel gene was replaced with
the LacZ or neo gene by gene targeting. In this study,
we found that mouse Na.2.3 and rat NaG/SCLll genes
are species counterparts. Analysis of the targeted miceallowed us to identify Na.2-producing cells by examining the lacZ expression. Besides in the lung, heart,
dorsal root ganglia and Schwann cells in the peripheral
nervous system, Na.,2 was expressed in neurons andependymal cells in restricted areas of the central nervous system, particularly in the circumventricuJar organs
that are involved in body-fluid homeostasis (see Fig. lA
in the part of Center for Transgenic Animals and Plants).
Under water-depleted conditions, the mutant mice
showed markedly elevated c-fos expression in neurons
in the subfornical organ and organum vasculosum
laminae terrninalis compared with wild-type animals.
This suggests that these neurons are in a hyperactive
state in the Na.,2-null mice . Moreover, the null mutants showed abnormal intakes of hypertonic saline
under both water- and salt-depleted conditions (Fig. lB,
ibid.). These findings suggest that the Na.2 channel
plays an important role in the central sensing of thebody-fluid sodium level, and in regulation of salt intake
behavior.
We are currently examining the electrophysiological property of the cells derived from Na.2null mutant mice and wild-type mice to gain insight into
the channel property of Na.2 .

FOR BASIC BIOLOGY

Publication List:
Suzuki, R., Shintani, T., Sakuta, H., Kato, A., Ohkawara, T.,
Osumi, N. and Noda, M. (2000) Identification of
RALDH-3, a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina. Mech. Dev. 98,
37-50.
Fukada, M., Watakabe, 1., Yuasa-Kawada, J., Kawachi, H.,
Kuroiwa, A., Matsuda, Y. and Noda, M. (2000) Molecular characterization of CRMP5, a novel member of thecollapsin response mediator protein family. J. Bioi.
Chern. 275, 37957-37965.
Watanabe, E., Fujikawa, A., Matsunaga, H., Yasoshima, Y.,
Sako, N., Yamamoto, T., Saegusa, C. and Noda, M.
(2000) Na,2/NaG channel is involved in control of saltintake behavior in the CNS. J. Neurosci. 20,
39
7751.
Meng, K., Rodriguez-Pefia, A., Dimitrov, T., Chen, W.,
Yamin, M., Noda, M. and Deuel, T. F. (2000) Pleiotrophin signals increased tyrosine phosphorylation of ~-39
catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase
W~- Proc. Natl. Acad. Sci. USA 91, 2603-2608.
Shintani, T., Maeda, N. and Noda, M. (2001) Receptorlike protein tyrosine phosphatase y (RPTPy), but not
PTP1;/RPTP~. inhibits NGF-induced neurite outgrowthin PC12D cells. Dev. Neurosci., 23, 55-69.
Goldin, A. L., Barchi, R. L., Caldwell, J. H., Hofmann, F.,
Howe, J. R., Hunter, J. C., Kallen, R. G., Mandel, G.,
Meisler, M. H., Netter, Y. B., Noda, M., Tarnkun, M. M.,
Waxman, S. G., Wood, J. N. and Catterall, W. A. (2000)
Nomenclature of voltage-gated sodium channels. Neuron,
28,365-368
Qi, M., Ikematsu, S., Maeda, N., lchihara-Tanaka, K.,
Sakuma, S., Noda, M., Muramatsu, T. and Kadomatsu,
K. (2001) Haptotactic migration by midkine: Invovement of protein-tyrosine phosphatase ~. mitogen-activated protein kinase and phosphatidylinositol 3-kinase.
J.Biol. Chern., in press

40
DNISION OF CELLULAR REGULATION

Professor:
Associate Professor:
Research Associates:

Norio Murata
KojiMikami
Atsushi Sakamoto
Yoshitaka Nishiyama/wane Suzuki
Technical Staff:
Hideko linuma
Katsushi Yamaguchi
Monbusho Foreign Scientist:
Dmitry A. Lo/!
JSPS Fellows:
Suleyman I. Allakhverdievl!
S. Shivaji 3!
Prasanna Mohanty 4!
HFSP Fellow:
Masami Inaba
Tony H. H. Chen 5!
Visiting Scientists:
Patcharapom Deshnium 6!
Richard A. Dilley 7)
Martin Fulda 8!
Gabor Galiba 9!
Martin Hagemann IOJ
Lisa Heins II!
Cai-Xia Hou I 2J
Larissa Kisseleva nAlexander A. Lyukevich IJ
Jogadhere S. S. Prakash 3!
Kostas Stamatakis 13!
Balazs Szalontai 14!
Graduate Students:
Yuji Tanaka
Ryoma Suzuki
YuKanesaki
Ali Ferjani
IJ from the Institute of Plant Physiology, Moscow,

Russiafrom the Institute of Basic Biological Problems,
Pushchino, Russia
3! from the Centre for Cellular and Molecular Biology,
Hyderabad, India
4J from the Regional Plant Resource Centre,
Bhubaneswar, India
5! from Oregon State University, Corvallis, OR, USA
6J from the National Center for Genetic Engineeringand Biotechnology, Bangkok, Thailand
7! from Purdue University, Lafayette, IN, USA
8J from the University of Hamburg, Hamburg, Germany
9J from the Agricultural Research Institute, Martonvasar, Hungary
IOJ from the University of Rostock, Rostock, Germany
IIJ from the University of Kiel, Kiel, Germany
I 2Jfrom Xinjiang University, Urumqi, China
I3J from the National Center for Scientific Research"Demokritos", Athens, Greece
I 4Jfrom the Biological Research Center, Szeged,
Hungary
2J

The research efforts of this division are devoted todeveloping a full understanding of the molecularmechanisms that allow plants to acclimate to andtolerate various kinds of stress that arise from changes
in environmental conditions, with particular emphasison extreme temperatures and high salinity. In 2000,
significant progress was made in the following areas as·a result of studies of higher plants and cyanobacteria.

I. A sensor for low-temperature signals
Low temperature is an important environmental
factor that affects the growth and behavior of all living

NATIONAL INSTITUTE

organisms. Many organisms are able to acclimate to lowtemperatures by regulating the expression of various
genes. However, mechanisms for the perception andtransduction of low-temperature signals remain to be
characterized. In the cyanobacterium Synechocystis sp.
PCC 6803 (hereafter Synechocystis), expression of thegenes for fatty acid desaturases is enhanced by low
temperature. Moreover, the decrease in membrane
fluidity that occurs upon a downward shift intemperature appears to be a primary signal for inductionof 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
individually all the putative genes for histidine kinase in
Synechocystis and then we examined the subsequent
response to low temperature of the promoter of the desBgene, which encodes the ro3 fatty acid desaturase, bymonitoring the activity of a reporter gene for luciferase.
Among 41 mutant lines with disrupted genes forhistidine kinase, we identified two mutants, in which,respectively, the hik19 gene and the hik33 gene had
been inactivated. Mutation of these two genes also
reduced the extent of induction at low temperature ofother genes whose expression is induced by low
temperature, such as the desD gene for the L\6desaturase and the crh gene for RNA helicase. Hik33
has two membrane-spanning domains at its amino
terminus and it is likely that this enzyme is localized on
the cell membrane. By contrast, it is likely that Hik19 isa soluble protein. Therefore, Hik33 might be a primarysensor and Hik19 might be a signal transducer. This is,
to our knowledge, the first discovery of a cold-sensing
protein in any type of cell.

II. Analysis of the regulation of gene expression
using a DNA microarray
The Synechocystis DNA microarray, CyanoCHIPTM,
covers 97% of all the open reading frames (ORFs)
found in the genome of this cyanobacterium. Analysisof temperature-dependent gene expression with thisDNA microarray revealed that genes whose expression
is enhanced by low temperature encode proteins that are,for the most part, subunits of the transcriptional andtransiational machinery, such as the a subunit of the
RNA polymerase, a cr factor, and some protein ·subunitsof the ribosome. These proteins might be essential forreversal of suppression of protein-synthesizing activity
by low temperature. By contrast, most cold-repressible
genes were found to encode components of thephotosynthetic machinery, such as subunits ofphotosystem I and of phycobilisomes. These changes ingene expression might represent the acclimative
responses of photosynthetic organisms that allow
maintenance of photosynthetic activity at a certain level
regardless of temperature. To our surprise, we foundthat the expression of a number of genes for proteins of
unknown function was also enhanced or repressed by
low temperature.

41

FOR BASIC BIOLOGY

Low temperature

Hi k 3 3

- .

D

D

Plasmamembra/n

Unidentified
cold sensor

~? /
•

\

Na•/H+ antiport systems . The enhanced tolerance wasmediated, at least in part, by the increased tolerance ofthe photosynthetic machinery to salt-induced damage
and by an increase in the cell' s ability to repair thephotosynthetic and Na•/H• antiport systems . These
results serve to emphasize the physiological importanceof the repair of the photosynthetic machinery and theNa•/H+ antiport system in the protection of
Synechococcus cells from salt-induced damage .

Signal~ tm"'d"'"~--

Genes(Group II)

Acclimation to low temperature

Figure 1. A hypothetical scheme for transduction of lowtemperature signal.

Our examination of the cold-dependent regulation ofgene expression with the DNA microarray also revealed
that the cold-regulated genes could be divided into threegroups by reference to the effect of inactivation by
mutation of Hik33. In the first group, regulation of gene
expression by low temperature was totally abolishedupon inactivation of Hik33 ; in the second group, theextent of such regulation was reduced by half; and, inthe third group, such regulation was totally unaffected.
These observations indicate that Hik33 might regulate
the expression of certain cold-regulated genes, while
expression of those genes that were unaffected byinactivation of Hik33 might be regulated by anothersystem for transduction of the low-temperature signal(Fig. l ; Suzuki, Kanesaki , Mikami, Kanehisa, and
Murata Mol. Microbial. in press).

III. The importance of membrane lipids inprotection of the photosynthetic machinery
against salt stressHigh salinity is one of the main environmental
factors that severely limit the growth and the
productivity of plants and microorganisms. We showed
previously that membrane lipids are intimately involvedin the protection of both plants and microorganisms
against low-temperature stress . To provide further
insight into the importance of membrane lipids in stress
tolerance, we examined the tolerance to salt stress of thephotosynthetic machinery of Synechococcus sp. PCC
7942 in which we had genetically enhanced theunsaturation of fatty acids in the membrane lipids. Our
results revealed that the unsaturation of fatty acids
enhanced the salt tolerance of cells at least in terms of
the activities of photosystem I, photosystem II and theIV. Characterization of the Na•;u• antiporters of
Synechocystis
Control of membrane permeability to Na• ions and tocounteracting K+ ions is one of the most important
aspects of the acclimation of cells to high-salt
environments. Na+/H+ antiporters are membrane-boundproteins that play an important role in maintenance ofthe balance of intracellular concentrations of Na• andK+ ions in plant, fungal and bacterial cells. We used amutant of Escherichia coli that is deficient in Na+/H+
antiport activity as a host to examine functional
complementation with genes for putative Na•/H•
antiporters from the genome of Synechocystis. We
tested five genes and found that at least two genes
encoded Na•/H+ antiporters with different respective
affinities for Na• and Lt ions . To our knowledge, this is
the first · functional characterization of Na•/H•
antiporters from a cyanobacterium. The coexistence ofhigh-affinity and low-affinity Na•/H• antiporters in
Synechocystis is consistent with the ability of thisorganism to acclimate to a wide range of extracellularconcentrations of Na• ions (lnaba, Sakamoto, and
Murata J. Bacterial. in press).

V. The role of glycinebetaine in tolerance tofreezing temperatures
Gl ycinebetaine (GB) is a zwitterionic quaternaryamine that is found in a large variety of microorganisms,
plants and animals. It belongs to a group of compounds
known as compatible solutes, which act very efficiently
to stabilize the structure and function of cellular
macromolecules such as protein complexes andmembranes . In a previous study, we cloned the codA
gene for choline oxidase from Arthrobacter globiformis.
Choline oxidase catalyzes the conversion of choline to
GB. We introduced the gene for this enzyme into
Arabidopsis thaliana, which does not synthesize GB .
The resultant transformed plants not only accumulated
high levels of GB but also exhibited significantly
elevated tolerance to a broad spectrum of environmental
stresses, such as high salt, low and high temperatures,and strong light.
In 2000, we found that transformation of A. thalianawith the codA gene also enhanced the freezing toleranceof transformed plants. The accumulation of GB inchloroplasts dramatically improved the survival of
mature plants, stabilized membranes, and efficiently
protected the photosynthetic machinery at freezing
temperatures (Fig. 2).
The observation that the42

NATIONAL INSTITUTE

List of publication:(1) Original articles

lOcm
Figure 2. Enhanced freezing tolerance of Arabidopsis plantsthat have been transformed with the codA gene for choline
oxidase.
Wild-type (Wt) and transformed (Tf) plants were grown at
22'C for 33 days. They were then incubated at 20' C or -S' C
for 2 h and subsequently incubated at 22' C for seven days to
examine survival. The transformed plants accumulated
glycinebetain (GB) at 0.90 J.lmol g·' fresh weight, whereas
wild-type plants contained no GB . Adapted from Sakamoto etal.,

Plant 1., 22, 449-453 (2000).

transformed plants exhibited tolerance to various kindsof abiotic stress suggests that, in addition to its
stabilizing effect on proteins and membranes, GB might,
under stress conditions, contribute to the maintenanceof cellular functions of fundamental importance, such astranscription and translation . We are now focusing ourattention on the molecular mechanisms by which GB
enhances the tolerance to various kinds of stress , withparticular emphasis on possible protection of thetranscriptional and translational apparatus from stressinduced damage.

Allakhverdiev, S.I., Sakamoto, A. , Nishiyama, Y., Inaba,
M ., and Murata, N. (2000) Ionic and osmotic effectsof NaCl-induced inactivation of photosystems I and
II in Synechococcus sp. Plant Physiol., 123, 10471056.
Allakhverdiev, S.I., Sakamoto, A., Nishiyama, Y., and
Murata, N. (2000) Inactivation of photosystems I and
II in response to osmotic stress in Synechococcus.
Contribution of water channels. Plant Physiol., 122,
1201-1208.
Gao, M., Sakamoto, A., Miura, K. , Murata, N., Sugiura,
A. , and Tao, R. (2000) Transformation of Japanesepersimmon (Diospyros kaki Thunb.) with a bacterial
gene for choline oxidase. Mol. Breed., 6, 501-510.
Pardha Saradhi, P., Suzuki, 1., Katoh, A., Sakamoto, A.,
Sharmila, P., Shi, D.-J., and Murata, N. (2000)
Protection against the photo-induced inactivation of
the photosystem II complex by abscisic acid. Plant
Cell Environ., 23,711-718.
Sakamoto, A., Valverde, R. , Alia, Chen, T.H.H., and
Murata, N. (2000) Transformation of Arabidopsis
with the codA gene for choline oxidase enhances
freezing tolerance of plants. Plant J., 22, 449-453.
Suzuki, 1., Los, D.A., Kanesaki, Y., Mikami, K., and
Murata, N. (2000) The pathway for perception andtransductionof
low-temperature
signalsin
Synechocystis . EMBO J., 19, 1327-1334.
Szalontai, B., Nishiyama, Y., Gombos, Z., and Murata,
N. (2000) Membrane dynamics as seen by Fouriertransform infrared spectroscopy in a cyanobacterium,
Synechocystis. PCC 6803. The effects of lipid
unsaturation and the protein to lipid ratio. Biochim.
Biophys. Acta, 1509, 409-419.
Tanaka, Y., Nishiyama, Y., and Murata, N. (2000)
Acclimation of the photosynthetic machinery to hightemperature in Chlamydomonas reinhardtii requires
synthesis de novo of proteins encoded by the nuclear
and chloroplast genomes. Plant Physiol., 124, 441449.

(2) Review articles
Los, D.A., and Murata, N. (2000) Regulation of
enzymatic activity and gene expression by membrane
fluidity. Science's STKE, http ://www .stke.org/cgi/
content/fuli/OC_sigtrans ;2000/62/pe 1.
Sakamoto, A., and Murata, N. (2000) Genetic
engineering of glycinebetaine synthesis in plants:
current status and implications for enhancement ofstress tolerance. J. Exp. Bot., 51, 81-88.

43

FOR BASIC BIOLOGYDIVISION OF BIOLOGICAL REGULATION AND

tonema. Gametophore induction and its growth anddevelopment were also regulated.

PHOTOBIOLOGY (ADJUNCT)
I- 2
Professor (Adjunct):
Masamitsu Wada
Associate Professor(Adjunct):
Tomohiro Kiyosue
Kikuchi Kazuhiro
Research Associate:
Masahiro Kasahara
Postdoctoral Fellow:(Jul. 1, 2000-)
Takatoshi Kagawa
PRESTO Fellow:
BRAIN Postdoctoral Fellow: Kazuki Terauchi(Aug.l, 2000-)
Takata lmaizumi
Graduate Students:(Tokyo Metropolitan
University)
Kazusato Oikawa(Tokyo Metropolitan
University)
Noriyuki Suetsugu(Tokyo Metropolitan
University)
Tilman Lamparter
Visiting Scientist:(Free University, Berlin)

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 the main photoreceptors for plant photomorphogenesis. The goal of our research is to clarify the signaltransduction pathways of photomorphogenesis. One ofour major subjects is chloroplast photo-relocation
movement which is thought to be one of the simplest
phenomena in this field. We use the fern Adiantum
capillus-veneris and the moss Physcomitrella patens asmodel plants for our cell biological approach not only
because the gametophytes are very sensitive to light, butalso because the organization of the cells is very si~ple.
We also use Arabidopsis mutants to clarify the genes
regulating chloroplast photo-relocation movement.

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 blue-light
responses, we are cloning and sequencing the genes ofblue light receptors, and are studying intracellular distributions of the gene products and their function in
Adiantum and Physcomitrella.

1- 1 Cryptochromes
We identified two cryptochrome genes from Physcomitrellapatens, designated CRYla and CRYlb genes,
and made single and double disrupants of these genes
using gene targeting by means of homologous recombination. Using these disruptants, trwas revealed that blue
light signals via cryptochromes inhibited the transitionof cell types of the moss protonema from chloronema tocaulonema, but induced side-branching of the pro-

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 NPLJ, a homologue of phototropin
has been sequenced. The complete eDNA clone is 3492
bp in length and encodes a protein of 1092 amino acids.

II. Chloroplast relocation
2- 1 Arabidopsis
Chloroplasts accumulate at the cell surface under
weak light and escape from the cell surface to the anticlinal wall under strong light to optimize photosynthesis.
The mechanism of chloroplast relocation, however, is
not known. We screened several mutants from T-DNA
tagging lines as well as EMS lines of Arabidopsis. Geneanalysis of several mutants defective in chloroplast
relocation movement showed that npll is the blue light
receptor for the avoidance response under strong light.
Both nphl and npll are the blue light receptors for thechloroplast accumulation response under weak light.
Gene analysis of mutants defective in accumulation
response are also under way.
2- 2Adiantum

Adiantum phytochrome3 (PHY3) is a unique kim ericprotein with a phytochrome structure in the N-terminal
half and a phototropin structure in the C-terrninal half.
Heavy-ion-beam- or EMS-induced mutants of Adiantum
which do not show phototropic response and chloroplast
photorelocation movement under red light were revealed to lack the PHY3 gene or to have a mutation in
the PHY3 gene. It was confirmed by gene silencing thatphy3 is the photoreceptor mediating red light-induced
tropic response and chloroplast photorelocation movement. However, since these phenomena induced by blue
light are normal in these mutants, it is still unknown
whether phy3 might or might not absorb blue light as aphotoreceptor.

Publication List:
Imaizumi, T., T. Kanegae and M. Wada (2000) Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum
capillus-veneris. Plant Cell12: 81-96.
Kagawa, T. and M. Wada (2000) Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by
microbeam irradiation. Plant Cell Physiol. 41: 84-93.
Kiyosue, T. and M. Wada (2000) LKPl (LOV kelch protein 1): a factor involved in the regulation of flowering
time in Arabidopsis. Plant Journal23: 807-815.

44

NATIONAL INSTITUTE

WT
A

cry1a-1B

cry1b-1ccry1 acry1 b-1
D

White

E

Blue

Red

Figure. Protonemal Colonies of the Cryptochrome Disruptants under Different Light Conditions.
The protonemal colonies (ca. 1 mm in diameter) of wild type, cry1a, cry1b, and cry1acry1b strains were inoculated on the agar plates. These
plates were incubated under continuous white, blue, or red light for 10 days. Representative colony images of one of each cryptochrome disruptant were shown. The colonies are the following strains: (A , E, and I) the wild type (WT); (B , F, and J) cry1a-l; (C, G, and K) crylb-1 ; and (D,H, and L) crylacry1b-1. The light conditions are given: (A-D) white light; (E-H) blue light; and (I-J) red light.
The blue-light grown protonemata are shown in M-0. These are (M) wild type ; (N) cry1a-1; and (0) cry1acry1b-l . The protonema cell walls
were stained with a drop of 0.1% (v/v) Calcofluor White to help distinguish each cell. The appearance of protonemata of e~yl b strains was similar to that of cryl a strain protonemata.
The bar in L represents 2 mm for the panels A-L, and the bars in M-0 represents 200 IJ.m.

45

FOR BASIC BIOLOGYDIVISION OF BEHAVIOR AND NEUROBIOLOGY(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Postdoctoral Fellows:

Graduate Student:

Fujio Murakami
Masato Nakafuku
Atsushi Tamada
Tatsuro Kumada 1
Hiroki Taniguchi 4
Yumiko Hatanaka 4
Tadashi Kizaki 4
Hiroshi Ono(from Osaka University)
Hiroyuki Konishi(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 staining with
antibodies against molecular markers expressed in a
subset of neurons. We will next explore the axonal
guidance mechanisms generating these wiring patterns.
Following questions will be addressed.
1) Whatstructures 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 finally 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 final 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 invivo.
We labeled migrating neurons with green
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.

Publication List:
Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M.,
Heizmann, C.W. and Murakami, F. (2000) Afferentsof cranial sensory ganglia pathfind to their target
independent of the site of entry into the hindbrain. J.
Com. Neurol. 417,491-500.
Nakamura, S., Ito, Y., Shirasaki, R. and Murakami, F.
(2000) Local directional cues control growth polarityof dopaminergic axons along the rostrocaudal axis. J.
Neurosci. 20, 4112-4119.
Wada, T., Kagawa, T., Ivanova, A., Zalc, B., Shirasaki,R., Murakami, F., lemura, S., Ueno, N. and Ikenaka,
K. (2000) Dorsal spinal cord inhibits oligodendrocyte
development. Dev. Biol. 227, 42-55
Song, W.-J., Baba, Y., Otsuka, T. and Murakami , F.
(2000) Characterization of Ca2+ channels in rat
subthalamic nucleus neurons. J. Neurophysiol. 84:
2630-2637.
Yamamoto, N., Inui, K., Matsuyama, Y., Harada, A.,
Hanamura, K., Murakami, F., Ruthazer, E.S.,
Rutishauser, U. and Seki, T. (2000) Inhibitory
mechanism by polysialic acid for lamina-specific

46

NATIONAL INSTITUTE

branch formation of thalamocortical axons J.
Neurosci. 20: 9145-9151.
Tashiro, Y., Miyahara, M., Shirasaki, R., Okabe, M.,
Heizmann CW., and Murakami F. Local

Nonpermissiveand Oriented Permissive Cues Guide
Vestibular Axons to the Cerebellum. Development(in press)

Figure 1. Migrating neocortical neurons visualized with green fluorescent protein (GFP) in vitro.
(Left) Slice culture of rat neocortex that was introduced with GFP eDNA 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.

I47

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.

48

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

Postdoctoral Fellow:

Graduate Student:

Technical Staff:
Visiting Fellows:

Shigeru !ida
Rie Terada
Yoshishige Inagaki
Atsushi HoshinoKazuo Tsuganel
Hong-Qing Li2
Jeong-Doo Choi2
Naoko Ishikawa
Takashi Kagami
Toshio Yamaguchi
Makoto Ohnishi
Sachiko Tanaka-Fukada
Kazuhiko Furukawa
Yasumasa Morita
Laurel Caitlin Cobert/)

1YJ,.om Duke University

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 genome, we are currently focused on mobile genetic
elements in general and plant transposable controlling
elements in particular.

I. Spontaneous mutants in the Japanese morning
glory.
The Japanese morning glory (Ipomoea nil or Pharbitis nil), displaying blue flowers, is believed to be originated from southeast Asia and has an extensive historyof genetic and physiological studies. The plant had been
introduced into Japan from China in about 8th century
as a medicinal herb, seeds of which were utilized as alaxative, and has become a traditional horticultural plantin Japan since around 17th century. A number of its
spontaneous mutants related to the colors and shapes ofthe flowers and leaves have been isolated, and about
10% of these mutants carry mutable alleles conferring
variegated phenotypes. Several lines of evidence indicate that an En!Spm-related transposable element Tpn1
and its relatives, which we termed Tpn1-farnily elements, are major sources of these spontaneous mutations. Indeed, we have succeeded to identify two ofthese mutable alleles for flower pigmentation, flecked
and speckled, which are caused by integration of Tpn1related elements, Tpn1 and Tpn2, respectively. Both
Tpn1 and Tpn2 are non-autonomous elements and theirtransposition is mediated by a Tpn1-related autonomous
element Among selfed progeny of a mutable flecked
line, a white variant displaying white flowers occasionally appeared. Some of the selfed progeny of the whitevariant bore only white flowers whereas others produced a few flecked flowers together with white flowers.
In these white variant derivatives, the excision of Tpn1
occurred rarely. We are speculating that appearance of
the white variant is probably due to epigenetic inactiva-tion of the autonomous element In accordance with thisnotion, we also found that the apparent stable r-1 allele
conferring white flowers is caused by insertion of a
non-autonomous Tpn1-family element, Tpn3, into the
CHS-D gene encoding a chalcone synthase for anthocyanin biosynthesis.

II. A new procedure for isolation of a gene tagged
by a transposable element belonging to the Tpnl
family in the Japanese morning glory.
Transposable elements are regarded as a powerful
mutagen and as an effective tool to isolate genes taggedby transposon insertions. The Japanese morning glory
contains around 500-1000 copies of an En/Spm-related
element Tpn1 and its relatives, which act as major
spontaneous mutagens. We have previously developed
an amplified restriction fragment length polymorphism(AFLP)-based mRNA fingerprinting (AMF) procedure
which is based on the systematic comparison of differently expressed transcripts in the same tissue in different lines, and succeeded in applying AMF for the identification of a new mutable allele caused by integrationof a transposable element into an anthocyanin biosynthesis gene. Since transposon mutagenesis has become apowerful tool for the isolation of genes of interest, wehave attempted to develop a new protocol for identifying tagged genes by insertion of Tpnl-related elementsin the Japanese morning glory. Our transposon tagging
method, named simplified transposon display (STD),
was based on our AMF procedure and is simple andrequires neither biotinylated oligonucleotides nor
streptavidin-capturing which are essential in othertransposon display methods published recently.

m. Identification and characterization of the
Purple gene encoding a vacuolar Na+JH+ ex-changer, InNHXl, for blue flower coloration in
the Japanese morning glory.
We have applied STD for identification of a mutableallele, purple-mutable (pr-m), which confers purple
flowers with blue sectors (Fig. 1). The flower variegation is regarded to be due to recurrent somatic mutationfrom the recessive purple to the blue revertant allele,
Purple-revertant (Pr-r) and we assumed that the pr-m
allele is caused by insertion of a Tpnl-family element.
To characterize the Purple (Pr) gene, we chose pairs ofsiblings carrying either the pr-m or Pr-r allele homozygously. No alterations were detected in the anthocyanin
pigment compositions between the pr-m and Pr-r lines.
The pr-m mutant showed partial increase in the vacuolar pH during flower opening and its reddish-purple
buds change into purple open flowers. The vacuolar pH
in the purple open flowers of the mutant was significantly lower than that in the blue open flowers, indicating that the pr mutant fails to increase the vacuolarpH.
We have succeeded in identifying the pr-m mutationthat is caused by integration of an En/Spm-relatedtransposable element, Tpn4, into the Pr gene encoding a

49

FOR BASIC BIOLOGY

eating that the Pr gene product bears the NHXl activity(Fig. 1). The NHXl proteins are shown to be importantfor salt tolerance and intracellular protein trafficking inyeast and plants. We have thus added a new biologicalrole for blue flower coloration in the Japanese morning
glory by the vacuolar alkalization. The vacuolar pH has
been regarded to play an important role in the blue
flower coloration, and InNHXl is the first major component characterized for increasing vacuolar pH responsible for blue flower coloration.

vacuolar Na+/H+ exchanger, InNHXl (Fig. 1). The Pr
gene comprises 15 exons, and Tpn4 is integrated into
the first untranslated exon. The genome of I. nil carries
one copy of the Pr (or lnNHXl) gene and its pseudogene. The Pr gene is most abundantly expressed ataround 12 h before flower opening in the petals, whichmust correlate with the increase in the vacuolar pH for
the blue flower coloration. The isolated Pr gene is able
to show functional complementation to a deletion mutation in the NHXJ gene encoding a vacuolar Na+/H+
exchanger in yeast (Saccharomyses cerevisiae), indi-

A

B
Tpn4

NHX1

c

-4 hr

-3 hr

-2 hr

1kb

-1 hr

0 hr

Figure I. Blue flower coloration and the vacuolar pH in the Japanese morning glory.
A, Flower variegation and the Pr gene product, InNHXJ, for blue flower coloration. B, The genomic structure of the mutable pr-m
allele. C, Blue flower coloration during flower opening in the Pr-r plant. Petals in the upper line are the artificially opened petals of
the buds in the lower line.

50

Publication List:
Fukada-Tanaka, S., Inagaki, Y., Yamaguchi T. and Iida,
S. Simplified transposon display (STD): a new procedure for isolation of a gene tagged by a transposable element belonging to the Tpnl family in the Japanese morning glory. Plant Biotech. (in press).
Fukada-Tanaka, S., Inagaki, Y., Yamaguchi, T. Saito N.
and Iida, S. (2000) Colour-enhancing protein in blue
petals. Nature 407, 581.
Hasebe A. and Iida, S. (2000) The novel insertion sequences IS1417, IS1418 and IS1419 from Burkholderia glumae and their strain distribution. Plasmid
44,44-53.
Hoshino, A., Johzuka-Hisatomi Y. and Iida, S. Geneduplication and mobile genetic elements in themorning glories. Gene (in press).
Ignacimuthu, S., Arockiasamy, S. and Terada, R (2000).
Genetic transformation of rice: Current status andfuture prospects. Curr. Sci. 79, 186-195.
Iida S., Hoshino, A. and Choi, J.-D. (2000) Floricultural
traits and transposable elements in morning glories.
Kor. J. Hart. Sci. & Tech. 18, 845-848.
Iida S. and Hoshino, A. Spontaneous mutagenesis andtransposable elements in the Japanese morning glory.

NATIONAL INSTITUTE

Gamma Field Symposia (in press).
Inagaki, Y., Myouga, F., Kawabata, H., Yamai S. and
Watanabe H. (2000) Genomic differences in Streptococcus pyogenes serotype M3 between recent isolates
associated with toxic shock-like syndrome and pastclinical isolates. J. Infect. Dis. 181, 975-983.
Kojima, T., Habu, Y., Iida S. and Ogihara, Y. (2000)
Direct isolation of differentially expressed genes
from a specific chromosome region of common
wheat: Application of the amplified fragment length
polymorphism-based mRNA fingerprinting (AMF)
method in combination with a deletion line of wheat.
Mol. Gen. Genet. 263,635-641.
Shiokawa, K., Inagaki, Y., Morita, H., Hsu, T.-J., Iida S.
and Noguchi, H. (2000) Thefunctional expression of
the CHS-D and CHS-E genes of the common morning glory (Ipomoea purpurea) in Escherichia coli andcharacterization of their gene products. Plant Biotech.
17, 203-210.
Terada, R., Nakajima, M., Isshiki, M., Okagaki, R.J.,
Wessler, S.R. and Shimamoto, K. (2000) Antisense
Waxy genes with highly active promoters effectively
suppress Waxy gene expression in transgenic rice.
Plant Cell Physiol. 41, 881-888.

51

FOR BASIC BIOLOGYDIVISION 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
Naomi Serizawa* to Department of Biological Chemistry, University ofCalifornia-Irvine, Irvine, California (Feb. 1. 1999-)** to Division of Molecular Neurobiology in NIBB (May.
1 2000-)*** She obtained her doctorate Sept. 2000.
Homologous recombination, which may occur in allorganisms, involves genetic exchange between twoparent-derived homologous chromosomes in meiosis inaddition to the repair of DNA damage induced byphysical and chemical agents. As a result of 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 ofhomologous recombination (especially in normallygrowing cells) is better understood. In 2000, work on
the following three subjects has advanced our knowledge of the dynamics and function of the genome.

I. Identification of cis-essential sequences for amplification of the ribosomal RNA gene repeats inyeast.
We previously found that some recombinational hotspots in E. coli present in regions where progress of a
DNA replication fork is severely impeded. Detailed
analysis of this phenomenon revealed that when thefork is blocked, recombinational enzymes construct a
new replication fork after breakdown of the arrested
fork by introducing a double-stranded break at either ofthe sister chromatids. Consequently, this recombination
reaction for renewal of the replication fork is a primarysource of recombinational hotspots. Next, we showedthat this phenomenon occurred not only in prokaryotes
but also in eucaryotes. In most eucaryotes, the DNA
replication fork blocking site, called RFB (replication
fork harrier), is located in each unit of ribosomal multigenes (rDNA). It has been reported that in budding
yeast (S. cerevisiae) when a non-transcriptional region(NTS) of the rDNA, in which the RFB site is located, isinserted into a non-rDNA region of chromosomes, recombination of a region close to the insertion site isenhanced. This enhancement is called HOTl activity
and the DNA fragment required for activation is named
HOT/ DNA. We isolated a number of mutants, which
are defective in HOTl activity, and found that some ofthem had a concomitant defect in fork blocking activity
at the RFB site. Both of these defective phenotypes

were restored to wild type by introducing a single gene,
named FOBJ, thereby suggesting that fork blockage
acts as a trigger for recombination in yeast, as well as inbacteria. In addition to this finding, the detection of
RFB activity within the rDNA cluster in various higher
eucaryotic cells suggests that the fork renewal cycle
occurring after the blockage is more general than expected.
The copy number of rDNA repeats is unstable.
Although variation in the number of rDNA copies has
been observed in various eucaryotes, the underlying
mechanism remains unknown. Because fork arrest at
the RFB site was a trigger for recombination, as mentioned above, we suspected that a fork-blocking event
was involved in changing the copy number of rDNA
repeats. Neither an increase nor a decrease in rDNA
repeats occurred in fobl mutants, suggesting that thefork blocking event is required for changing the copy
number, probably through a recombination process.
As the fork blocking system has only been understoodto prevent replication from "colliding" with rDNA transcription so faf, this study indicates that the fork block
system may have an entirely new function. If this is
the case, RFB deleted rDNAs should not amplify.
This year, we tested this prediction and also attempted to identify cis-sequences, required for amplificationof the copy number of rDNA. However, this kind ofanalysis was not feasible for a repeated gene with highcopy number. Therefore, we constructed a strain with
a rDNA copy number reduced to two copies of theoriginal genomic locus by using a multi-copy plasmid,carrying a hygromycin B resistant copy of rDNA.
Then, each sub-region in a single NTS 1 region among
the two rDNAs on the genome were replaced with a
URA3 marker gene in order to determine which sequences would be required for rDNA amplification,
especially around the RFB site. Mutational analysis
using this system showed that not only the RFB site but
also the adjacent -440 bp region in NTS 1 (together
called the EXP region) are required for the FORIdependent repeat expansion. This -400 bp DNA element is not required for the fork arrest activity or theHOT1 activity and therefore defines a function unique
to rDNA repeat expansion (and presumably contraction)
separate from HOTJ and RFB activity. Our model,
which indicates a mechanism by which the copy number of rDNA increases from two to 150, is shown in theaccompanying Figure.

II. Enhanced homologous recombination caused
by a non-transcribed spacer of the ribosomal
RNA gene in Arabidopsis.
In yeast, a single rDNA unit structure consists of tworRNA genes, 35S pre-rRNA and 5S rRNA genes, andtwo non-transcribed spacer (NTS) regions, NTS 1 and
NTS2.
NTSl .is flanked by the 3'-ends of the tworRNA genes and NTS2 is flanked by the 5'-ends of thetwo rRNA genes. A region containing NTS 1-SSrRNA
gene-NTS2, named HOT1 DNA, has activity, that

52

NATIONAL INSTITUTE

Eucaryotic rRNA genes (rONA: __. ) have a typical repeated structure.
The mechanism of a change in roN! copy numbers has remained unknown.

t

There are fork block barriers (RFB:(l in the rONA repeats. Fob1 protein(~) is
requ1red for the fork block event and also for the changing copy numbers. ~+A model, in which the two copies of rON As increase to the normal level of rONA copies(about 150 copies) is shown below.
No. XII chromosome@>,. ' 2(N) copies of the rONAs

,;/

~.:

Treplication is blocke a\~~ . \A.
'. Ill!:
~

•

\/'-

~

•

fV"'

Ta ds-break at either sister choromachids•

one rONA increase I one cycle reaction,,~~~~~~~~~~~

copiesa normal(150
level
copies)of the rONA

Figure. An increase mechanism for rDNA copy number. The figure shows that one reaction cycle increases the rDNA by one
copy. Therefore, 150 reaction cycles can increase the copy number of the rDNA from two to 150.

stimulates recombination at a region close to a nonrDNA locus where the HOTJ DNA is inserted. The
stimulated recombination activity is called HOTJ activity. Further analysis revealed that only two small noncontiguous regions, called E and I, are required for
HOTJ activity. E and I are located in NTSl and NTS2,
respectively and correspond to enhancer and initiator of
RNA polymerase I dependent 35S rRNA transcription.
The rDNA repeated structure is essentially the same invarious eucaryotes as in yeast, except in other eucaryotes 5S rRNA genes are located at a different locus fromthe 35S rRNA gene cluster.
In addition, forkblocking activity has been detected in the NTS regionsof the rDNA array in all eucaryotes tested so far. Here,
we investigated whether the non-transcribed spacer

region in the rDNA cluster of higher eucaryotes has
hotspot activity similar to that observed in yeast. We
chose Arabidopsis as an experimental model, because areliable recombination assay system had been developed and recombination event, if it occurs anywherewhole body of the plant, is easily visible. To analyze
the effect of the NTS on mitotic homologous recombination in the plant, we constructed transgenic lines of
Arabidopsis containing the NTS region and a recombination substrate, in which two 3'- and 5'- deleted uidA
genes, with partially overlapping sequences are separatHistochemical GUS-staining
ed by a Hyg' gene.
monitored reconstitution of functional uidA genes byhomologous recombination. We found that recombination occurred more frequently in all organs tested in F

53

FOR BASIC BIOLOGY(Fork Block) lines transgenic for NTS than in C (Control) lines without NTS. The average number of GUS+
spots on leaves in F lines was more than nine-foldhigher than in C lines. Furthermore, by genomic
Southern blotting analysis, post-recombinational
molecules were detected in a transgenic line, F43,
which had an extremely high number of Gus+ blue
spots.
These results strongly suggests that NTSdependent enhancement of homologous recombination
could be a common feature in higher plant as well as
yeast.

III. E. coli genomic structure and function
Analysis of whole genomic sequence of E. coli had
been completed in 1997. While a strain Japan teamsequenced is W3110, the other US team did is MG1655.
These strains were separated from a common ancestor
during or right after World War II. In order to understand the micro-evolution of these strains, we are sequencing a previously undetermined region of theW3110 genome and will finished this spring. Our goal
is to compare this genome with the MG1655 genomes
at the nucleotide level.
In order to identify a minimal set of genes required
for the duplication of a single cell and to elucidate thefunction of those genes, we proposed a new project anda established a new collaboration team, headed by Mori
(NAIST). Since last year our project has been supported by CREST from JST and is being carried out by
the following four groups: (1) resources, (2) infor-.
matics, (3) database, and (4) functional analysis. At
present, the resources group (1) has cloned all ORF
candidates, constructed E. coli micro-arrays using thecloned ORFs and started to analyze gene expression
under various conditions, obtaining results that will be
published. Other plans, such as the construction of aset of mutant strains, in which each gene is disrupted,
are in progress. These strains will be distributed toworkers world wide, stimulating functional analysis of
the E. coli genome, and eventually contributing to defining an integrated functional network of genes in E.
coli.

Publication list:
Mori, H., Isono, K., Horiuchi, T., and Miki, T. (2000)
Functional genomics of Escherichia coli in Japan.
Res. Microbiol. 151, 121-128.
Kobayashi,. T., Nomura, M., and Horiuchi, T. (2001)
Identification of DNA cis-elements essential for expansion of ribosomal DNA repeats in Saccharomyces
cerevisiae. Mol. Cell. Biol. 21, 136-147.

54

NATIONAL INSTITUTE

DNISION OF SPECIATION MECHANISMS I
Professor:
Research Associates:

Tetsuo Yamamori
Yuriko Komine
Akiya Watakabe
Takashi 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 the genes thatare expressed in the specific areas of the primate neocortex. Using differential display method, we haveobtained genes that showed marked differences within
the primate neocortex.
Our second approach is to understand informational
processing in the brain underlying learning behaviors
with gene expression techniques. Here, we report ourfindings in the year of 2000.

I. 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. It is known to be divided
into distinct functional and anatomical areas and has· been a matter of debate what extent the areas of the
neocortex are genetically and environmentally determined. It is also puzzling why, during the evolution of
mammals, the neocortex was most markedly expanded
while the number of the genes in the mammal was littlechanged. To access these questions, we studied gene
expression within different areas of the neocortex .
1) In collaboration with Professor Hiroyuki Nawa
(Nigata university), we used DNA macroarray technique to examine gene expression in the areas of humanprefrontal, motor and visual cortexes. We found almost
all the genes among 1088 genes examined showed only
less than a factor of two in the difference of their expressions. Only one gene showed more than three folddifference and another one was between two and threefold difference within the three areas. These results
suggest that the genes that are expressed among thedifferent areas of the human neocortex are very similar.
However, the question remained whether there are any
genes that show marked difference within areas of
neocortex.
2) In order to answer this question, we employed
differential display methods and found at least two
genes that indicated the area specific expression.
i) One, designated occl is specifically expressed in

the occipital cortex, particularly in Vl area, in the
primate brain. Furthermore, the expression of occl
turned out to be activity dependent, because, in the
monocularly deprive-monkeys with being injected
TTX into one of eyes, the expression of occl is markedly decreased in the ocular dominance columns of the
primary visual cortex (Vl).
ii) The other gene that showed marked difference
within the neocortex, is gdf7, a member of BMP!fGF-P
family , which is specifically expressed in the motor
cortex of the African green monkey. We are currently
examining the detailed expression pattern of the bothgenes.
3) We have also further isolated several area specificgenes with RLCS (Restriction Landmark eDNA Scanning).

II. Gene expression under audio-visual discrimination task
We are studying gene expression of c-Fos underaudio-visual discrimination tasks in collaboration withprofessor Yoshio Sakurai (Kyoto University). We foundthat the visual and audio tasks enhanced the specificexpression 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 neural modulation of the neocortex depending on
the types of the tasks. We are currently identifying theneuronal cell types in the cortical area that induced cFos depending on the relevant task.

Publication list:
Original Articles
Watakabe, A., Sugai, T., Nakaya, N., Wakabayashi, K. ,Takahashi, H ., Yamamori, T. and Nawa, H. Similarity and variation in gene expression among humanFig. 1 Expression pattern of occl in the visual cortex.
In situ hybridization pattern of occl in the primate visualcortex. Occl is markedly expressed in the layer IVc~ andmoderately in the layers of II, III and IVa in area VI. The
boundary between VI and V2 is shown by an arrow.

55

FOR BASIC BIOLOGY

cereberal cortical subregions: an mRNA expression
profiling study using DNA macroarrays. Malec.
Brain Res. (in press)
Tochitani, S., Liang, F., Watakbe, A., Hashikawa, T.
and Yamamori, T. occl is preferentially expressed in
the primary visual cortex in an activity-dependent
manner: a pattern of gene expression related to thecytoarchitectonic area in adult macaque neocortex.
European J. Neuroscience (in press).
Watakabe, A., Fujita, H., Hayashi, M., and Yamamori,
T. GDF7, a BMPffGF beta family member, is enriched in the primary motor area of monkey neocortex. J. Neurochem. (in press).
Karachot, L., Shirai, Y., Vigot, R., Yamamori, T. andIto, M . Rapidly turned over protein maintains metabotropic synaptic transmission. NeuroReport 11,2903-2906,2000
Serizawa S, Ishii T, Nakatani H, Tsuboi A, Nagawa F,
Asano M, Sudo K, Sakagami J, Sakano H, Ijiri T,
Matsuda Y, Suzuki M, Yamamori T, Iwakura Y. and
Sakano S. Mutually exclusive expression of odorant
receptor transgenes. Nature Neurosci. 3, 687-693,
2000
Liang, F., Hatanaka, Y., Saito, H., Yamamori, T. andHashikawa, T. Differential expression of gamma-

aminobutyric acid type B receptor-la and lb mRNAvariants in GABA and non-GABAergic neurons of
the rat brain. J. Comp. Neural. 416, 475-495, 2000

Abstracts
Watakabe, A., Komatsu Y., Fujita, H., Hayashi, M.,
Yamamori, T. GDF7, a BMPffGF~ family member,is enriched in the primary motor area of monkey neocortex. In the abstracts of the 30th Society for Neuroscience ofNorthAmerica. pp609, 2000
Tochitani, S., Liang, F., Watakabe, A., Hashikaw, T.,
Yamamori, T. The unique neuronal organization of
the primary visual cortex revealed by occl mRNA
activity-dependent expresssion. In the abstracts of the
30th Society for Neuroscience of North America.
pp1614,2000
Sakata, S., Kitsukawa, T., Sakurai, Y., Yamamori, T.
Attention modulates fos expression in rat auditory
cortex. In the abstracts of the 30th Society for Neuroscience of North America. pp2230, 2000
Vigot, R., Batini, C., Kado, R.T., Yamamori, T. Long
lasting LTD recorded in the unanesthetized rat cerebellum. In the abstracts of the 30'h Society for Neuroscience of North America. pp 1394, 2000

56

NATIONAL INSTITUTE

DNISION OF SPECIATION MECHANISMS II
Professor:
Mitsuyasu Hasebe
Assistant Professor:
Tomomichi Fujita
Post Doctoral Fellow: Rumiko Kojujjl(-Mar. 31)
Tomoaki Nishiyamcl (Apr. 1-)
Misako Mishima 1 (Dec. 1-)
Technical Staff:
Naomi Sumikawa (Oct. 1-)
Yukiko Tanikawa
Masae Umeda
Yoshimi Bitoh
Makiko Kondo (-Sept.30)
Mayumi Naruse (Sept. 30-)
Kazuko Kabeya (July 1-)
Graduate Students:
Satomi Shindo(-Mar. 31)
Yuji Hiwatashi
Satoko Kobayashi-Arakawa
Keiko Sakakibara
Ryosuke Sano (Chiba Univ.)
Tomoaki Nishiyama(Univ. ofTokyo)(-Mar. 31)
Takako Tanahashi(Univ. ofTokyo) (-Mar. 31)
Hisako Sakaguchi(Shinshu Univ.) (Apr. 1-)

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 arefound in the genomes of extant organisms. By comparing the genomes of different organisms, we can infer(1) the phylogenetic relationships of extant organismsand (2) the genetic changes having caused the evolutionof morphology and development. The inferred phylogenetic relationships give important insights on problems in various fields of evolutionary biology, and ourgroup focuses on biogeography, evolution of morphological traits, and systematics in wide range of taxa.
On the evolution of morphology and development, we
aim to explore genetic changes led the evolution ofplant body plan. We selected Arabidopsis (angiosperm), Gnetum (gymnosperm), Ginkgo (gymnosperm),
Ceratopteris (pteridophyte), Physcomitrella (bryophyte),
and some green algae as models to compare the genefunctions involved in development of the reproductive
organs and shoot apical meristem 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 simp!ef 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 more than 10 groups based on the gene tree.
The LEAFY gene is the positive regulator of the MADSbox 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 the(1) Plant-type
evolution of reproductive organs.
MADS-box genes with both MADS and K domains
were established. (2) The number of MADS-box
genes increased, and the three ancestral MADS-box
genes that later generate A-, B-, C-functions genes werelikely originated before the divergence of ferns and seedplants. (3) Specifically expressed MADS-box genes in
reproductive organs evolved from generally expressed
ones in the seed plant lineage. (4) The ancestral geneof the AG group of MADS-box genes acquired the Cfunction before the divergence of extant gymnosperms
and angiosperms.
(5) The gene duplication thatformed the AP3 and PI groups in MADS-box genes
occurred before the diversification of extant gymnosperms and angiosperms. (6) The ancestral gene ofangiosperm A-function gene was lost in extant gymno(7) LFY gene becomes positively
sperm lineage.
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. Evolution of vegetative organs in land plants
The ancestor of land plants was primarily haploid.
The only diploid cell was the zygote, which immediately underwent meiosis. It is believed that early during land plant evolution, zygotic meiosis was delayed
and a multicellular diploid sporophytic generation became interpolated into the life cycle. In the early stages of land plant evolution, sporophytes are epiphytic togametophytes, as observed in extant bryophytes.
During the course of evolution, both generations started
to grow independently at the stage of pteridophytes.
Finally gametophytes became much reduced and epi-

57

FOR BASIC BIOLOGY

phytic to sporophytes in seed plant lineage. Molecularmechanisms of development in a diploid generation
have been well studied in some model angiosperms, but
we have scarce information on those in a gametophyte
generation. For example, mosses have leaf- and stemlike organs in their haploid generation, but it is completely unknown whether similar genes involved inangiosperm leaf and stem development are used in thegametophytic generation of mosses or not. To understand the evolution of body plans in diploid and haploid
generation at the molecular level, we focus on the comparison of molecular mechanisms governing shoot development between Arabidopsis and the moss Physcomitrella patens. P. patens is known by its high rateof homologous recombination and suitable for analyze
gene functions using the gene targeting. The moss

homologs of SHOOTMERISTEMLESS, ZWILLE, andHD-Zip genes, which are involved in Arabidopsis shootdevelopment, have been cloned and their characterization IS m progress. We also established enhancer andgene trap lines and tagged mutant libraries of P. patens
to clone genes involved in the leafy shoot development(Nishiyama et al. 2000).

III. Biogeography of Coriaria
Coriaria is, which has been mentioned to be the mostconspicuous disjunct distribution in flowering plants
distributed in four separate areas in the world. The
phylogenetic relationships of 12 Coriaria species collected from the representative disjunct areas were inferred by comparing 2416 base pairs of the combined
data set of rbcL (a large subunit of ribulose 1,5-

Figure 1. Example of mutant strains and gene-trap lines of Physcomitrel/a patens. A, transformant morphologically indistinguishable from the wild type (19980725084-3). B (TNI) and C(TN2), morphological mutants obtained by shuttle mutagenesis. D-H, histochemical staining of
gene-trap lines. D, YH330, mucilage hairs are stained. E, YH78, young leaves are stained. F,
YH229, a young bud is stained. G and H, YH206, the chloronema (G) and caulonema (H) arestained. Bar in B = 0.5 mm for A and B. Bars in C and D = 0.1 mm. Bar in E = 0.2 mm. Bar in F
= 20 Jlm. Bar in H = 50 Jlm for G and H.

58bisphosphate carboxylase I oxygenase) and matK
(maturase K) genes (Yokoyama et a!. 2000). The
phylogenetic tree shows that the Chile - Papua New
Guinea - New Zealand - Pacific islands species and theCentral America -northern South America species forma sister group, and the Eurasian clade is more basal tothem. The divergence time between the Eurasiangroup and the other species was estimated as 63 or 59
million years ago using rbcL and matK molecular
clocks, respectively. These results do not support thepreviously proposed hypotheses to explain the disjunctdistribution based on the continental drift, but suggestthat the distribution pattern was formed by several geographical migrations and separations in the Cenozoic.

IV. Molecular phylogeny of athyrioid ferns
Nucleotide sequences of the chloroplast gene rbcL
from 42 species of the fern tribe Physematieae (Dryopteridaceae) were analyzed to gain insights into the interand intrageneric relationships and the generic circumscriptions in the tribe. The phylogenetic relationships
were inferred using the neighbor joining and maximum
parsimony methods, and both methods produced largely
congruent trees (Sano et a!. 2000). These trees reveal
that: 1) Athyrium, Comopteris, Pseudocystopteris, andAnisocampium form a clade and Athyrium is polyphyletic; 2) Deparia sensu lato is monophyletic andDictyodroma formosana is included in the Deparia
clade; 3) Diplaziopsis forms a clade with Homalosorus,
which is isolated from the other genera of the Physematieae; 4) Monomelangium is included in the monophyletic Diplazium clade; 5) Rhachidosorus is not
closely related to either Athyrium or Diplazium.
It has been suggested that Diplazium tomitaroanum
Masam. is a hybrid arising from Diplazium subsinuatum(Wall. ex Hook. et Grev.) Tagawa and Deparia petersenii (Kunze) M. Kato. Di. subsinuatum's basic
chromosome number differs from that of Diplazium but
is consistent with that of Deparia, suggesting that Di.
subsinuatum is closely related to Deparia but not to
Diplazium.
Traditional taxonomy based on morphological characteristics sometimes encounters difficulty in inferring phylogenetic relationships when
dealing with taxa having few diagnostic morphologicalcharacteristics, such as Di. subsinuatum with its simple
leaves. We obtained the rbcL nucleotide sequences

NATIONAL INSTITUTE

from Di. subsinuatum and Di. tomitaroanum in order toinfer their phylogenetic relationships to other Deparia
and Diplazium species (Sano et a!. 2000). We also
examined the morphological characteristics of both thescales and leaf axes, which are regarded as diagnostic
characteristics for Deparia but have not been describedin detail for Di. subsinuatum. According to the rbcL
nucleotide sequences, Di. subsinuatum and Di. tomitaroanum are included in the Deparia clade, not in the
Diplazium clade. Furthermore, the articulate multicellular hairs on leaves, the shape of the rachis groove,the basic chromosome number and the spore morphology are all more similar to Deparia rather than to
Diplazium, indicating that Di. subsinuatum should beclassified as Deparia. We therefore propose a new
taxonomic treatment of the two Diplazium species.

Publication List:
Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M.
and Hasebe, M. (2000) Tagged mutagenesis andgene-trap in the moss, Physcomitrella patens by
shuttle mutagenesis. DNA Res. 7: 1-9.
Yokoyama, J., Suzuki, M., Iwatsuki, K. and Hasebe, M.
(2000) Molecular phylogeny of Coriaria, with special emphasis on the disjunct distribution. Molec.
Phyl. Evol. 14:11-19.
Sano, R., Takamiya, M., Ito, M., Kurita, S. and Hasebe,M. (2000) Phylogeny of lady fern group, tribe Physematieae (Dryopteridaceae) based on chloroplastrbcL gene sequences. MoZee. Phyl. Evol. 15: 403413.
Sano, R., Ito, M., Kurita, S. and Hasebe, M. (2000)
Deparia formosana (Rosenst.) as the new name for
Diplazium formosanum. Acta Phototaxa Geobot. 51:
17-20.
Sano, R., Takamiya, M., Kurita, S., Ito, M. and Hasebe,M. 2000. Diplazium subsinuatum and Di. tomitaroanum should be moved to Deparia according tomolecular, morphological, and cytological characters.
J. Plant Res. 113: 157-163.
Yoshimoto, Y., Higeta, D., Ito, Y., Yoshida, H., Hasebe,
M. and Ozeki, Y. (2000) Isolation and characterization of a eDNA for Phenylalanine ammonia-lyase(PAL) from Dianthus caryophyllus (carnation).
Plant Biotechnology 17: 325-329.

59

FOR BASIC BIOLOGYCENTER FOR INTEGRATIVE BIOSCIENCEInterim Head: Hideo Mohri

The center is jointly managed by NIBB and two other institutes in Okazaki, IMS and NIPS.

The following projects will be the main focus of

the center: 1) Development, Differentiation and Regeneration. 2) Strategic
Methodology.

3) Bio-environmental Science.

60

NATIONAL INSTITUTE

DEPARTMENT OF BIOENVIRONMENTALRESEARCH I
Professor:
Taisen Iguchi
Associate Professor:
Hajime Watanabe
Research Associate:
Yoshinao Katsu
Technical Staffs:
Takeshi Mizutani
Institute Research Fellow: Hiraki Okumura
Post doctoral fellow:
David Buchanan
Graduate Students:
Hiroshi Urushitani 11
Satomi Khono 11
n Graduate School of Yokohama City University

Synthetic chemicals found in the environment havethe capacity to disrupt endocrine system development
and function in both wildlife and humans. This has
drawn public concern since many of these chemicals
may bind to estrogen receptors and evoke estrogenic
effects.
Early evidence that estrogenic chemicals
could pose a threat to human health during development
came from studies of diethylstilbestrol (DES), which
was used to prevent premature birth and spontaneousabortion. Laboratory experiments have demonstrated
that exposure of animals to sex hormones during perinatal life can cause permanent and irreversible alterations of the endocrine and reproductive systeins as wellas the immune system, nervous system, bone, muscle,
and liver in both sexes. Although many of thesechemicals may bind to estrogen receptors and evoke
estrogenic effects in wildlife and humans, the effects ofestrogen are not well understood even now. Thus,
understanding the effects of sex hormones at themolecular level, especially during development, is very
important to resolve these problems.

I Estrogen-induced irreversible changes
Perinatal sex-hormone exposure has been found to
induce lesions in reproductive tracts in female mice.
The possible relevance of the mouse findings to theNormal Mouse

FIG. 1development of cancer in humans has been emphasized.
In the early seventies, a close correlation between
occurrence of vaginal clear cell adenocarcinoma inyoung women and early intrauterine exposure to diethylstilbestrol (DES) was demonstrated. Many chemicals released into the environment have the potential todisrupt endocrine function in wildlife and humans.
Some of these chemicals induce estrogenic activity bybinding to the estrogen receptor (ER). The neonatal
mouse model has been utilized especially to demonstrate the long-term effects of early sex hormone exposure on the female reproductive tract.
Neonataltreatment of female mice with estrogens induces various
abnormalities of the reproductive tract: ovaryindependent cervicovaginal keratinization, adenosis,
uterine hypoplasia, epithelial metaplasia, oviductal
tumors, polyovular follicles and polyfollicular ovaries.
Female reproductive tracts in mice exposed prenatallyto estrogen show altered expression of Hoxa genes andWnt genes and the analysis of knockout mice lacking
Hoxa-10 or Wnt7a show uterine hypoplasia. The
growth response of neonatally DES-exposed reproductive organs to estrogen is reduced, as are ER levels and
EGF receptor levels, in addition to other hormone
receptor levels.
Estrogenic compounds such as bisphenol A (BPA)
and nonylphenol as well as dioxins and PCBs were
found in the human umbilical cord. BPA can easily
cross the placenta and enter the fetus in Japanese monkey and mice. Bisphenol-A (BPA) can be found in fetal
brain, testis and uterus when given to pregnant mice.
Neonatal exposure to a high BPA dose induced ovaryindependent vaginal changes, polyovular follicles andinfertility lacking corpora lutera. Thus, the developing
mammal is sensitive to exposure to estrogenic agents asevident by the induction of long-term changes in female
reproductive organs.
In order to clarify the molecular mechanisms of theseeffects, we are studying changes in gene expression
patterns induced by perinatal exposure to chemicals or
estrogen using differential display and DNA microarrytechiniques . We have found genes possibly related tothe ovary-independent changes by differential display.
We also have clustered groups of genes that are respon-

FIG. 2 Fluorescence image of an array

61

FOR BASIC BIOLOGYIII Molecular Target Search
Abnormalities caused by endocrine disrupting
chemicals are reported but the molecular mechanisms of
the effects are not well studied. Although estrogen
receptor is one of the strongest candidates possibly
responsible for the endocrine disrupting function ofmany chemicals, it alone cannot explain the variety ofphenomena induced by endocrine disrupting chemicals.
Thus, we are also looking for new target molecules that
may be responsible for endocrine disruption.
In
parallel, we also are studying the ligand-binding
mechanisms of nuclear receptors to hormones and otherchemicals using Surface Plasmon Resonance technology.

.. . .
.
'·i . •.. ... J..

~

<:1)

••

ioow 1000 r •

~

I

:

~ ~- ...
.
100

•

•

10L_~_.___.c.....;..._._ _ , _ _
10

100

1000

10000

10
100000Control
FIG.3 Scatter plot of average expression levels in control
and chemical-treated uterus

sive to estrogenic stimuli in uterus by using the DNA
microarray system.
We need to understand themolecular background of the critical period during
development, the low dose effect of estrogenic chemicals and the molecular metabolism of hormones andhormone-like agents in animals including humans.

II Effect of estrogen on amphibian and fishes.
During embryogenesis, exogenous estrogen exposure
induces abnormal sex differentiation and the abnormalbone formation in African clawed frog, Xenopus laevis
and the cyprinodont fish, mummichog (Fundulus
heteroclitus) . In these animals estrogen receptor ispresent in the embryo. To analyze the function of
estrogen, we have isolated eDNA clones of estrogen
receptor a and ~- The estrogen-responsive genes must
play important roles. So, we try to isolate the estrogenresponsive genes to understand the molecular physiology of estrogen action. Vitellogenin has been wellcharacterized in avian, amphibian, and fish as a precursor of egg yolk. As the vitellogenin gene is responsive
to estrogen, we can examine the effect of endocrine
disruptors in the environment using a vitellogeninspecific and sensitive enzyme-linked immunosorbent
assay (ELISA). Japanese tree frog (hyla japonica) takes
water through ventral skin. We found that sex steroids
and endocrine disruptors interfere with water absorption
through ventral skin in frogs. Further, using the amphibian and fish as model animals we aim to analyze
the effects of numerous chemicals released into theenvironment on endocrine system function in wildlife.

Publication List:
Hashimoto, S., Bessho, H., Hara, A., Nakamura, M.,
Iguchi, T. and Fujita, K.: Elevated serum vitellogenin
levels and gonadal abnormalities in wild flounder(Pleurunectes yokohamae) from Tokyo Bay, Japan.
Marine Environ. Res., 49: 37-53, 2000.
Iguchi, T.: Book review: Hormonal Chaos. Nature Med.,
6: 246-247, 2000.
Yamamura, Y., K. Sayama, Y. Takeda, A Matsuzawa,T. Iguchi and Y. Ohta: Further study of methallothionein expression in transplantable mouse mammary tumors. Anticancer Res., 20: 379-384, 2000.
Iguchi, T .: Developmental effects of estrogenic agentson mice, fish and frogs. Trabajos del Instituto Cajal.
Torno LXXVII: 52-53, 2000.
Iguchi, T.: Embryonic and neonatal exposure to endocrine-altering contaminants: effects on mammalian
female reproduction. In: Environmental Endocrine
Disrupters. Eds. L. Guillette,Jr. and D.A Crain,
Taylor & Francis, New York, pp. 234-268, 2000.
Iguchi, T. and T . Sato: Endocrine disruption and developmental abnormalities of female reproduction. Am.
Zoologist, 40:402-411,2000.
Shimizu N, Sugimoto K, Tang J, Nishi T, Sato I,
Hiramoto M, Aizawa S, Hatakeyama M, Ohba R,
Hatori H, Yoshikawa T, Suzuki F, Oomori A, Tanaka
H, Kawaguchi H, Watanabe H, Handa H: Highperformance affinity beads for identifying drug receptors. Nature Biotechnol. 18:877-81,2000
Ishizu KI, Watanabe H, Han SI, Kanesashi SN, Hoque
M, Yajima H, Kataoka K, Handa H. Roles of disulfide linkage and calcium ion-mediated interactions inassembly and disassembly of virus-like particles
composed of simian virus 40 VP1 capsid protein. J
Viral. 75:61-72.2001
Iguchi, T., H. Watanabe and Y. Katsu: Developmental
effects of estrogenic agents on mice, fish and frogs: amini review. Horm. Behav. (in press).

62

NATIONAL INSTITUTE

DEPARTMENT OF BIOENVIRONMENTALRESEARCH II
Associate Professor:
Assistant Professor:
Technical Staff:
Post Doctoral Fellow:
Graduate Student:
Secretary

Hirokazu Tsukaya, Ph.D.
Gyung-Tae Kim, Ph.D.
Makiko Kondo
Kiuhyon Cho, Ph.D.
Takeshi Furuhashi (Nagoya Univ.)
Kazuko Kabeya

Diversity of plant form is mostly attributable to variation of leaf and 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 of plant morphogenesis.
However, the genetic control of development of theseshapes had remained unclear. Recently, studies of leaf
morphogenesis has been in a turning point, after our successful application of the techniques of developmental
and molecular genetics to it, using model plants, Arabidopsis thaliana (L.) Heynh. Our purpose is to understand
Plants from view point of molecular genetic control of leaf morphogenesis .
Focusing on mechanisms that govern polarized growthof leaves in a model plant, Arabidopsis thaliana, wefound that the two genes act independently to each otheron the processes of polar growth of leaves: the AN gene
regulates width of -leaves and the ROT3 gene regulates
length of leaves. The AN gene controls the width of leaf
blades and the ROT3 gene controls length. The AN geneseems to control orientation of cortical rnicrotubules inleaf cells. Cloning of the AN gene revealed that the geneis a member of gene family found from animal kingdom(Tsukaya et a!., in prep). The ROT3 gene was cloned by
us in 1998. Transgenic experiments proved that the ROT3
gene regulates leaf-length without affect on leaf-width(Kim et al., 1999). We are trying to identify molecularfunction of the above genes which are essential for leaf
morphogenesis.

While ROT3 regulates the length of both leaf blades
and petioles, A CL2 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 the leaf
blade. Genetic studies suggested that petioles and leaf
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 growth of theleaf. Molecular and anatomical analysis of the as2 mutant
is now underway, in collaboration with Prof. Machida,
Nagoya University (Endang eta!., submitted).
On the other hand, we are trying to identify molecularmechanisms which distinguish developmental pathway ofleaves from that of shoots. For such purposes, we introduced tropical plants having queer developmental program for leaf morphogenesis, namely, Chisocheton,
Guarea and Monophyllaea , as materials for molecularstudies.
In addition, we are interested in roles of such genes forenvironmental adaptation, from view point of biodiversity.
Leaf index, relative length of leaf to width, is one of themost diverse factor of leaf shape. For instance, rheophytes
are characterized by narrow leaves, which represent anadaptation to their habitats. Are AN and ROT3 genes are
involved in regulation of adaptive change of leaf index innatural condition? Are these genes the responsible for
evolution of rheophytes? So called "Evo/Devo" study ofleaf morphogenesis is also one of our research project in
NIBB.

Publication List:
Tsukaya, H., Shoda, K., Kim, G.-T. and Uchirniya, H. (2000)
Heteroblasty in Arabidopsis thaliana (L.) Heynh. Planta
210 : 536-542.
Tsukaya, H. (2000). The role of meristematic activities in theformation of leaf blades. J. Plant Res. 113 : 119-126.
Ito, T. , Kim, G.-T. and Shinozaki, K. (2000). Disruption of
an Arabidopsis cytoplasmic ribosomal protein S 13homologous gene by transposon-mediated mutagenesis
causes aberrant growth and development. Plant J. 22: 257264.
Tsukaya, H. (2000). Orchidaceae. In: Ohba, H. and Ikeda,
H. eds. "The Flora of Hinku Valleys, East Nepal" The
University Museum, Univ. Tokyo, Nature and Culture,
No. 6, pp. 257-266.
Tsukaya, H. (2000) Utricularia furcellata Oliver (Lentibulariaceae), a new record for Nepal. Newslet.Himalayan Bot.
27: 6-7.
Ohba, H. and Tsukaya, H. (2000) A taxonomic note on Habenaria albomarginata King & Pant!. (Orchidaceae). J.
Jap(Jpn) . Bot. 75:311-313.
Tsukaya, H. and Ohba, H. (2000) Malaxis monophyllos (L.)
Sw. (Orchidaceae), a new record from Nepal with description of a new variety. J. Jap(Jpn). Bot. 75 : 314-316.

63

FOR BASIC BIOLOGYDEPARTMENT OF BIOENVIRONMENTALRESEARCH Ill
Professor (Adjunct):Minoru Kanehisa (Kyoto University)

Publication list:

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 to predictor design novel interactions.

Goto, S., Nishioka, T., and Kanehisa, M. (2000). LIGAND:
chemical database for enzyme reactions. Nucleic Acids
Res. 28, 380-382.
Kanehisa, M. (2000). Pathway databases and higher orderfunction. Adv. Protein Chem 54, 381-408.
Kihara, D. and Kanehisa, M. (2000). Tandem clusters ofmembrane proteins in complete genome sequences. Genome Res. 10, 731-743.
Ogata, H., Fujibuchi, W., Goto, S., and Kanehisa, M. (2000).
A heuristic graph comparison algorithm and its application
to detect functionally related enzyme clusters. Nucleic
Acids Res. 28, 4021-4028.
Fujibuchi, W., Ogata, H., Matsuda, H., and Kanehisa, M.
(2000). Automatic detection of conserved gene clusters in
multiple genomes by graph comparison and P-quasi
grouping. Nucleic Acids Res. 28, 4029-4036.

64

NATIONAL INSTITUTE65

FOR BASIC BIOLOGYRESEARCH SUPPORT66

NATIONAL INSTITUTECENTER FOR TRANSGENIC ANIMALS AND PLANTS

Head: Masaharu Noda
Associate Professor:
Supporting Staff:

Eiji Watanabe
Mie Yasuda
Ayako Tozaki (June 1, 2000-)

I. Research supporting activity
NIBB Center for Transgenic Animals and Plants toestablished in April 1998 to support researches usingtransgenic and gene targeting techniques in NIBB . We
are now planning on the construction of the centerbuilding.
The expected activities of the Center are as follows:

1Provision 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 Na,2 ionchannel in collaboration with Division of Molecular
Neurobiology . Na,2 belongs to a group of voltage-gated sodium channels (NaChs) that serve to generateaction potentials in electrically excitable cells such asneuronal and muscle cells. Comparing with the other
NaChs, Na,2 has unique amino acid sequences in theregions, which are known to be involved in ion selectivity and voltage-dependent activation and inactivation,suggesting that it must have specific functional properties. To clarify the functional role of Na,2 in vivo, theNa,2-deficient mice were generated by gene targetingand the physiological phenotypes have been examined.
It was suggested that the Na,2 channel plays an important role in the central sensing of body-fluid sodium
level and regulation of salt intake behavior. Details of
this study are described in the part of Division of
Molecular Neurobiology .

Publication List
Watanabe, E., Fujikawa, A., Matsunaga, H., Yasosruma, Y., Sako, N., Yamamoto, T., Saegusa, C.,
Noda, M. (2000) Na,2/NaG channel is involved in control of salt intake behavior in the CNS. J. Neurosci. 20,
7743-7751.

A

Fig. 1 Na,2 channel is expressed in specialized neurons and ependymal cells in the adult CNS (A), and the null mutant shows an abnormal ingestion of hypertonic sal ine (B).
The expression pattern of mouse Na,2 channel gene was revealed by lacZ gene expression in Na,2+/- mice. The blue signals represent the cells
expressing lacZ gene. Fig. I A shows the specific expression of Na,2 in the subfomical organ. The null mutants showed excessive ingestionof hypertonic saline under both thirst and acute salt appetite conditions. Fig. I B shows the null mutant ingesting hypertonic saline under thethirst condition.

67

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)
Chieko Nanba(Subunit Chief)
Hiroyo Nishide
Makiko Itoh(Technical Assistant)Keiko Suzuki(Technical Assistant)
Yasuyo Kamiya(Technical Assistant)
Yumi Hashimoto (Technical Assistant)
Ayako Tosaki(Technical Assistant)
Nobuko Hattori (Technical Assistant)
Yukiko Tanigawa(Technical Assistant)

Cell Biology Group
Maki Kondo( Unit Chief)
Yukiko Kabeya

Radioisotope Facility
Yoshimi Matsuda( Unit Chief)
Yousuke Kato(Subunit Chief)
Naoki Morooka
Takayo Itoh(Technical Assistant)

Developmental Biology Group
Chiyo Takagi
Sanae Oka
ChiyoNoda
Regulation Biology Group
Hideko Iinuma
Katsushi Yamaguchi
Yasushi Takeuchi
Shigemi Takami
Kaoru Yamada

Center for Analytical Instruments
Sonoko Ohsawa(Unit Chief)
Tomoko Mori(Subunit Chief)
Yumiko Makino
Hatsumi Moribe(Technical Assistant)Gene Expression and Regulation Group
Tomoki Miwa(Unit Chief)
Sachiko Tanaka( Subunit Chief)
Kaoru Sawada( Subunit Chief)
Koji Hayashi
Hideko Utsumi
Naomi Sumikawa

Glassware Washing Facility(Tomoko Mori)(Kazuhiko Furukawa)

Integrated Bioscience Group
Takeshi Mizutani
Makiko Kondo(TechnicalAssistant)

The Technology Department is a supporting organization for researchers and research organization within
the NIBB. 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 institutes throughout Japan.
At this meeting, the participants present their own activities and discuss technical problems.The Proceedings
are published soon after the meeting.

68

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY
Head of Facility:
Associate Professor:
Research Associates:

Technical Staff:

Norio Murata
Masakatsu Watanabe
Yoshio Hamada(Tissue and Cell Culture)lkuo Uchiyama(Computer)
Sho-ichi Higashi(Large Spectrograph)
Tomoki Miwa (Computer)
Chieko Nanba(Plant Culture, Farm, Plant Cell)
Hiroyo Nishide(Computer)
Nobuko Hattori(Large Spectrograph)
Makikolto(Large Spectrograph)
Yasuyo Kamiya (Tissue and Cell
Culture)Misayo Masuda (Computer)
Keiko Suzuki(Plant Culture, Farm, Plant Cell)

I. Facilities
1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, theOkazaki 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 1,OOOnm (infrared) onto
its focal curve of 1Om in length. The fluence rate
(intensity) of the monochromatic light at each wavelength
is more 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-highspectral-, 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,
CR599-01) (420-930nm, 250-1000mW output), A/0
modulators (up to 40MHz) to chop the laser beam, a beam
expander, and a tracking microbeam irradiator (up to 200
J.lm s· 1 in tracking speed, down to 2 J.lm in beam diameter)with an infrared phase-contrast observation system.

facility is routinely used for DNA recombination experiments.

3. Computer Laboratory
Computer laboratory maintains several computers toprovide computation resources and means of electronic
communication in this Institute. Currently, the main
system consists of three servers and two terminal workstations: biological information analysis server (SGI
Origin 2000), database server (Sun Enterprise 450), fileserver (Sun Enterprise 3000), data visualization terminal
and molecular simulation terminal (both are SGI Octanes).
Some personal computers and color/monochrome printers
are also equipped. On this system, we provide various
biological databases and data retrieval/analysis programs,
and support large-scale data analysis and database
construction for the Institute members.
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
are provided through this LAN. We also maintain apublic 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 alimited number of rooms with environmental control forplant culture. In some of these facilities and rooms,
experiments can be carried out at the P1 physical containment level under extraordinary environments such as
strong light intensity, low or high temperatures.

5. Experimental FarmThis laboratory consists of two 20 m2 glass-houses with
precise temperature and humidity control, three green
houses (each 6m2) at the P1 physical containment level, asmall farm, two greenhouses (45 and 88 m2) with
automatic sprinklers, two open aquariums (30 and 50 t)
and several 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 clean benches, is also
provided.

2. Tissue and Cell Culture Laboratory

7. Laboratory of Stress-Resistant Plants

Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely rooms
which satisfy the P2/P3 physical containment level. This

This laboratory was founded to study transgenic plants
with respect to tolerance toward various enviromental
stresses. It is located in the Agricultural Experimental

69

FOR BASIC BIOLOGY

Station of Nagoya University (30 km from National
Institute for Basic Biology). The laboratory provides avariety of growth chambers that precisely control theconditions of plant growth and facilities for molecularbiological, 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.

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. Photo-receptive and signal transduction mechanisms of algal gene expression were also studied byaction spectroscopy.
(2) Developmental Biology: Replacement of theankyrin repeats of mouse Notch2 gene with E.coli bgactosidase gene induces early embryonic lethality aroundE10.5. The lethality was suggested due to defects inextraembryonic tissues, because the mutant embryo grew
and differentiated further in vitro. Histological examination and in situ hybridization analysis with trophoblast
subtype-specific probes revealed that the development ofgiant and spongiotrophoblast cell layers are normal in the
mutant placenta, while vasculogenesis in the labyrinth
layer apperaed compromised at E9.5. Since the lethality
was circumvented by production of chimeric mice with tetraploidy wild type embryos, weconcluded that the embryonic lethality is due to defect ingrowth and/or differentiation of labyrinthine trophoblast
cells. The mutant embryo, however, could not be rescuedin the tetraploid chimeras beyond El2.5 because of
insurfficient developmentof umbilical cord, indicating another role of Notch2
signaling in the mouse development. Chimeric analysis
with diploid wild type, however, revealed contribution ofmutant cells to these affected tissues by El3.5. Thus,
Notch2 are not cell autonomously required for the earlycell fate determination of labyrinthine trophoblast cells
and allantoic mesodermal cells, but plays an indispensablerole in the further formation of functional labyrinth layer
andumbilical cord.
(3) Computational Biology: Comparative genomics is a
useful approach to find clues to understanding complex

and diverse biological systems from rapidly growing
genome database.
We have constructed a databasesystem for comparative analysis of many of microbial
genomes ever sequenced and are developing new computational techniques for large-scale genome sequencecomparison. Especially, we are developing a method fororthologous grouping among multiple genomes, which isa crucial step for comparative genomics. Since
considerable number of genes consist of multiple domains,
we have developed a hierarchical clustering algorithm
that can automatically split fusion genes into orthologous
domains.
In parallel, we are developing a tool to incorporate
various sequence features such as G+C contents, codon
usage bias and locations of repetitive elements into the
genome comparison. By this approach, we 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 and two Pyrococcus species, we
could find interesting insertion/deletion patterns thatfrequently include restriction-modification 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 as well 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:
I. Faculty
Chinen A., Uchiyama, I., Kobayashi, I. (2000). Comparison
between Pyrococcus horikoshii and Pyrococcus abyssi
genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms. Gene 259,
109-121.
Kumano, K ., Saito, T., Kurokawa, M.m Kanda, Y., Hamada,Y. and Hirai, H. (2000) Binding of Delta!, Jagged!, andJagged2 to Notch2 rapidly induces cleavage, nuclear
translocation, and hyperphosphorylation of Notch2. Mol.
Cell. Bioi. 20, 6913-6922.
Nobusato, A., Uchiyama, 1., Kobayashi, I. (2000). Diversity
homologues
inof restriction-modification gene
Helicobacter pylori. Gene 259, 89-98
Nobusato, A., Uchiyama, 1., Ohashi, S., Kobayashi, I. (2000).
Insertion with long target duplication: a mechanism forgene mobility suggested from comparison of two related

70bacterial genomes. Gene 259, 99-108.
Uchiyama, I. (2000). Hierarchical clustemg procedure forgrouping orthologous domains in multiple genomes. Currents in Computational Molecular Biology, 146-147.
Uchiyama, 1., Miwa, T., Nishide, H., Suzuki, 1., Ornata, T.,
Ikeuchi, M., Murata, N., Kanehisa, M. (2000). Data
submission system for cyanobacterial DNA chip consortium, Genome Informatics 11,235-236.
Uchiyama, I., Higuchi, T., Kobayashi, I. (2000). CGAT:
Comparative genome analysis tool for closely related
microbial genomes. Genome Informatics 11, 341-342.
Takinami, S., Mochizuki, M., Hayatsu, H., Nikaido, 0.,
Kubota, M., Watanabe, M., Bieda, K. and Negishi, T.
(2000) Somatic cell mutation and photoproduct formation
in Drosophila induced by monochromatic UV light insunlight, Environmental Toxicology, 15, in press

II. Cooperative Research Program for the Okazaki
Large SpectrographHada, M., Iida,Y.,and Takeuchi,Y.(2000). Action spectraof DNA photolyases for photorepair of cyclobutane pyrimidine dimers in sorghum and cucumber. Plant Cell
Physiol. 45: 644-648 .
Kawai, H. and Kreimer, G. (2000). Sensory mechanisms:
Phototaxes and light perception in algae. In ''The Flagellates: Unity, Diversity and Evolution." (Edited by J.

NATIONAL INSTITUTE

Green and B.S.C. Leadbeater), pp. 124-146. Taylor &Francis Publishing.
Shinomura, T., Uchida, K., and Fruuya, M. (2000).
Elementary process of photoperception by phytochorome
A for high-irradiance response of hypocotyl elongation in
Arabidopsis. Plant Physiol. 122, 147-156.
Tada, Y., Wakasugi, T., Nishikawa, A., Furuhashi K. and
Yamada K. (2000) Developmental regulation of a genecoding for a low-molecular-weight heat shock protein
during haustorium formation in the seedlings of aholoparasitic plant, Cuscuta japonica. Plant Cell Phisiol.
41, 1373-1380.
Takeuchi, Y., lida, Y., Nakajima, N. and Nikaido, 0. (2000).
Formation of DNA lesions in cucumber cotyledons
exposed to solar UV radiation. Environ. Sci. 13, 351-355
Takinami, S., Mochizuki, M., Hayatsu, H., Nikaido, 0.,
Kubota, M., Watanabe, M., Hieda, K. and Negishi, T.
(2000) Somatic cell mutation and photoproduct formation
in Drosophila induced by monochromatic UV light insunlight, Environmental Toxicology, 15, in press
Torikai, A., (2000), Wavelength sensitivity of photodegradation of polymers. In "Handbook of Polymer
Degradation Second Ed.," (Edited by S. Halim Hamid),
pp.573-603, Marcel Dekker, Inc. New York.

71

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

Technical Assistant:

Tetsuo Yamamori
Sonoko Ohsawa
TomokoMori
Yumiko Makino
Hatsumi Moribe

The Center serves for amino acid sequence analysis, and chemical syntheses of peptides and nucleotids
to support researchers in NIBB and NIPS. Instrumentsof the Center can be used by researchers outside theInstitute 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 2: )Automatic Nucleic Acid Isolation System (Kurabo NA2000)
DNA Sequencers (ABI 377, 373S, ABI 31 0)
DNA/RNA Synthesizers (ABI 394, ABI 392)
Thermal 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 (Pharmacia
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 SPS 1200A)
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)Electrophoresis Imaging Systems (BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Microscope Photometer (Carl Zeiss MPM 03-FL)

72

Center for Radioisotope Facilities (CRF)
Head (Professor, concurrent post): Shigeru !ida
Associate Professor:
Kazuo Ogawa
Technical Staffs:
Yoshimi Matsuda(Radiation Protection
Supervisor)
Yosuke Kato
(Radiation Protection
Supervisor)
Naoki Morooka(Radiation Protection
Supervisor)
Supporting Staff:
Takayo Ito
Yumi !ida

I. Research supporting activity
In this year, the Radioisotope Faci lity managed by
NIBB (National Institute for Basic Biology) was reorganized to the CRF which is included in one of theCommon Research Centers belonged to ONRI (Okazaki
National Research Institutes).
Technical and supporting staffs of the CRF are serving 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 is also
one of our business.
The CRF consists of four controlled areas: Center,
NIBB-sub, LGER (Laboratory of Gene Expression andRegulation)-sub, and NIPS (National Institute forPhysiological Science)-sub. Users going in and out thecontrolled areas counted by the monitoring system are
6,273 in 2000. This count is comparable to that (7,912)
in 1999. The items in each controlled area is presented
in Figure 1.

II. Academic activity
Academic activity by teaching staff is focused on theanalysis of the structure and function of a dynein motor
protein. Dyneins are a group of microtubule-activatedATPases that serve to convert chemical energy intomechanical energy and divided into axonemal and cytoplasmic dyneins. Figure 2 shows the localization of two
isoforms of dynein in the outer arms of sperm axonemes(Ogawa et al. , 1977) and the mitotic apparatus ofcleaving egg (Mohri et al., 1976) visualized by antiaxonemal dynein (Fragement A) antibodies.
The native dyneins are very large and range inmolecular mass up to 1 to 2 mega Da. They are complex proteins containing heavy, intermediate, and lightchains defined by the molecular mass. Our prensent
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 performed in

NATIONAL INSTITUTE

our laboratory in 1991. Since then cDN A 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 et al. (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 intermediate chains(IC1, IC2, and IC3) that range in molecular mass from70 to 120 kDa. IC2 and IC3 were cloned by Ogawa et al.
(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-terminal part is homologous to the sequence of thioredoxin, t!le middle part consists of three
repetitive sequences homologous to the sequence of
NDP kinase, and the C-terminal 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 outerarm dynein. We have already isolated eDNA clone offive LCs. LC1 (23.2 kDa) and LC3 (12.3 kDa) arehighly homologous to mouse Tctex2 and Tctex I, 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.

Actual countCenter

1,917

NIBB-sub756

LGER-sub

2,780

NIPS-sub
total

820
6,273

Figure l. Percentage of users going in and out the controlled areas
during April to December, 2000.

73

FOR BASIC BIOLOGY

Mitotic

Sperm
Axoneme

Egg

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

A

Pl P2 P3 P4

N

M

a2

c

B

c
Figure 3. Structure of dynein heavy chain. A; Analysis of aminoacid sequence of heavy chain reveal s that it consists of three major
domains referred to as N, M, and C from the N-terrninus. 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.

74

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