NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT

1997

The cover photographs indicate GFP expression driven by
a-actin-GFP promoter in live transgenic zebrafish embryos.

Post Doctral Fellow
123
4

NIBB Research Fellow
JSPS Postdoctral Fellow
JSPS Reserch Associate
JST Fellow\

CONTENTS
INTRODUCTION............................................................................................................................................

1

ORGANIZATION OF THE INSTITUTE

3

GRADUATE PROGRAMS

4OFFICE OF DIRECTOR

5

Evolution of photoysnthesis and the mechanism of electron transfer ... .......... .......... ....... .. .. .. ............ ...

5

Mechanisms determining the outline shape of the adult lepidopteran wings

6

DEPARTMENT OF CELL BIOROGY7

DIVISION OF CELL MECHANISMS

8

I. Regulation at the level of protein transport to microbodies during
the microbody transition....................................................................................................................

8

II. Light dependent alternative splicing in higher plants

8III. A pumpkin 72-kDa membrane protein of precursor-accumulating vesicles
has characteristics of a vacuolar sorting receptor ......... .. .. ..... .. .. ...... ......... .. .... ............. ............ .... ....

9

IV. Expression and activation of the vacuolar processing enzyme
in Saccharomyces cerevisiae .... .. ..... .......... ............ .. ....................... .. .. ..... .... .. ....... ............ .. .. .... .........

11

V. An aspartic proteinase is involved in the maturation of storage
proteins in concert with the vacuolar processing enzyme ....... ........ ...... ............. ........ ........... ..........

11

VI. Role of molecular chaperones in organelle differentiation

11

DIVISION OF BIOENERGETICS

13

DIVISION OF CELL PROLIFERATION (ADJUNCT)16

I. Microtubule-binding protein which functions specifically during meiosis

16

II. Arabidopsis 14-3-3 isoform

16

III. Identification of fission yeast type II myosin heavy chain

17

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)18

DIVISION OF CELL FUSION (ADJUNCT)

20..............................................................................................

DEPARTMENT OF DEVELOPMENTAL BIOLOGYDIVISION OF REPRODUCTIVE BIOLOGY

2324

I. Endocrine regulation of oocyte growth and maturation

24

II. Endocrine regulation of spermatogenesis

24III. Endocrine regulation of gonadal sex differentiation

26

DIVISION OF CELL DIFFERENTIATION28

DIVISION OF MORPHOGENESIS

29

I. The role of follistatin in neural induction

29

II. Intracellular signaling of BMP through a novel MAPKKK, TAKl

29

III. Use of zebrafish as a model animal to study gene function ............................................................

30

IV. TGF-~ family in invertebrates

30

DIVISION OF DEVELOPMENTAL BIOLOGY

32

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

32

II. Mutants of Arabidopsis thaliana with altered patterns of the sugar-inducible
gene expression

32

III. Screening of mutants of Arabidopsis thaliana with defective
growth and development..................................................................................................................

32

DEPARTMENT OF REGULATION BIOLOGY35

DIVISION OF MOLECULAR NEUROBIOLOGY

36

I. Topographic map and synapse formation in the retinotectal system

36

II. Receptor-like protein tyrosine phosphatases and brain development

36

III. Gene targeting of Na-G, a voltage-gated sodium channel expressed in glial cells

38

DIVISION OF CELLULAR REGULATION

39

I. Improved performance of photosynthesis with polyunsaturated membrane lipids

39

II. Enhancement of stress tolerance in plants by genetic engineering:transformation with a gene that confers the biosynthesis of glycinebetaine

39

III. A new factor involved in the heat stability of the photosynthetic machinery

40

IV. Stress-dependent enhanced expression of cytochrome eM and suppression of
expression of cytochrome c6 and plastocyanin...............................................................................

41

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT)......................

42

I. Tissue-specific expression of plastid genes

42

II. Dynamic aspects of molecular organization of PS II ........................................................................

42

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

44................................................

44

II. Capricious, a novel LRR cell surface molecule expressed on subsets ofneurons and muscles ........... ... .. ....... ... .... ....... ... ............................................... ......... ......... .................

44

III. msh, a homeobox containing gene essential for neural and muscular development

45

I. Connectin can function as an attractive target recognition molecule

IV. M-spondin and G-spondin: a novel gene family of secreted molecules.....................

45

LABORATORY OF GENE EXPRESSION AND REGULATION47

DIVISION OF GENE EXPRESSION AND REGULATION I

48

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

48......... ......................................

48

III. Transposition of the maize AciDs transposable elements in transgenic rice plants
DIVISION OF GENE EXPRESSION AND REGULATION II

49
51

I. Analysis of E.coli recombinational hot spots, Hot

51

II. SOS inducibility by replication fork blocking at Ter site on plasmid
and its dependency on distance from ori to Ter sites .....................................................................

53

Ill. The E. coli genome project53.............. ... .................................................... .. ........... ............................

DIVISION OF SPECIATION MECHANISMS I

54

I. Evolution of cytokine receptor families in the immune and nervous systems

54

II. Gene expression and cerebellar long-term plasticity

54

DIVISION OF SPECIATION MECHANISMS II
I. Reproduction of animals

56.... .. ....... .. ..... ... .. ... ...... ... .. ... ........ ... .. .. ... .. .. .. ..... ........ .. ......... .. .... .. .. .... ..... .........

II. Morphological Evolution in plants

56
56

RESEARCH SUPPORT ...........................................................................................................

59

TECHNOLOGY DEPARTMENT.................................................................................................................

60

RESEARCH SUPPORT FACILITY.............................................................................................................

61........... ...................................................................... ..... ..................... .. ........... ........................

61

I. Facilities

II. Research activities..... .. .. .... .. .. .... .. ... ..... .. .... .. ... ...... ... ...... .. ... .. .. .. ... .. ....... .. .. .. .. ......... .. .. .. .. ............. .........

RADIOISOTOPE FACILITY (Managed by NIBB)...................................................................................

THE CENTER FOR ANALYTICAL INSTRUMENTS (Managed by NIBB)

62
64

68

INTRODUCTION
We present here the 1997 Annual Report describing
the research activities of the National Institute for
Basic Biology (NIBB) during the past year. The
NIBB is a government supported basic research institute established in 1977. It aims to stimulate andpromote the biological sciences, by conducting firstrate research as a center of excellence, and bycooperating with other universities and research organizations. The Institute concentrates on the study ofcellular functions, reproduction and development,
neuronal and environmental control and gene expression arid regulation in eukaryotic organisms at themolecular level.
In May of 1997, we celebrated the twentieth anniversary of the foundation of the NIBB together withthe opening of the newly built Okazaki ConferenceCenter on the campus of the Okazaki National Research Institutes. At the end of July, Professor
Yoshiaki Suzuki of the Division of Cell Differentiation left the NIBB . Thus, the first professors of all
Divisions, except those of the Laboratory of Gene
Expression and Regulation which was establishedmore recently have completely retired, and the NIBB
has entered into a new era. We are now making up
future plans for the NIBB including establishment of
new divisions such as molecular environmental biology, neurobiology and mechanism of biodiversity. At
the start of 1997, the activities and the future plan of
the NIBB were subjected to a peer review and highly
rated by authorities outside the institute.
The turnover of personnel was high during the past
year. At the beginning of the year, Associate Professor M. Mimuro moved to Yamaguchi University as aprofessor. In May, Professor Naoto Uneno of
Hokkaido University was appointed as Professor of
the Division of Morphogenesis, the position formerly
occupied by Professor Goro Eguchi . As Associate
Professor of the same Division, Dr. H. Shibuya was
appointed in July. At the end of March, Professor
Hitoshi Sakano (University of Tokyo) of the AdjunctDivision of Cell Fusion and Professor Kimiyuki Sato
(Okayama University) of the Adjunct Division of Biological Regulation completed their terms of office.
The former position was filled by Professor Issei
Mabuchi (University of Tokyo) in April. Drs. I.
Nishimura, N. Maeda, K. Mikami, K. Owaribe(Nagoya University), H. Abe (Chiba University) andY. Ozeki (Tokyo University of Agriculture and Technology) were respectively appointed as Associate
Professors in the Division of Cell Mechanism, the Division of Molecular Neurobiology, the Division of
Cellular Regulation, the Adjunct Division of Cellular
Communication, the Adjunct Division of Cell Fusion
and the Adjunct Division of Behavior and Neurobiology. On the other hand, Adjunct Associate Professors
T. Takai and H. Wada left the NIBB. In addition, we
recently appointed 5 research associates and 2 institute research fellows, while 3 research associates and2 institute research fellows moved to other institutions.
The NIBB plays important roles as a national andinternational center for biological research and is responsible for conducting research projects incooperation with research groups of various universities and institutes. As a part of such cooperative
activities, the NIBB carried out Special Programs
which are currently directed to "Adaptation and Resistance to Environment" and "Trans-differentiation of
Tissue Cell." Based on these programs, the NIBB
held the 38th Conference entitled "Plasticity in Differentiation and Morphogenesis" (organized by Professor
G. Eguchi and Dr. R. Kodama) and the 39th Conference "Dynamic Aspects of Seed Maturation .andGermination " (organized by Professor Mikio
Nishimura). In September, the Director-General of
the NIBB and the President of the Korea Basic Science Institute (KBSI) signed a "Memorandum ofUnderstanding between the NIBB and the KBSI" inorder to promote international cooperation between
the two institutions.
In addition, the Institute sponsors interdisciplinary
symposia and study meetings on current topics by inviting leading scientists national and international invarious related fields . In 1997, an International Symposium "Dynamic Aspects of Lysosomal/Vacuolar
System " (organized by Professor Yoshinori Ohsumi)
was held at the Okazaki Conference Center, sponsoredby the Ministry of Education, Science, Sports andCulture. The NIBB also provided a training course inbiological sciences for young investigators.
Our efforts in the future will continue to be directed

NATIONAL INSTITUTE

2

at supporting leading research in various fields of biological sciences. We welcome suggestions arid criticisms concerning the activities of the NIBB.

Hideo Mohri, D. Sc.
Director-General

3

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

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

research associates. A division forms, in principle, an
independent project team. Six of the divisions are for
adjunct professorship and are under professors who
hold joint appointments with other universities. The
adjunct division has resident research associates. The
arrangement aims to facilitate exchange in research
activities in Japan. The Technical Department manages the activities of research techniques and helps topromote research activities of each division and also
to maintain the research resources of the Institute. The
Department also undertakes the technical education ofits staff.

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

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

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

Department/Laboratory

Department of Cell Biology

Department of Developmental Biology

Department of Regulation Biology-1CampusThe Okazaki National Research Institutes covers an
area of 150,000 m2 with four principal buildings. The
NIBB's main research building has a floor space of
10,930 m2 • Two-thirds of the space was completed in1982 and the remaining third in June, 1983. The
buildings which house the research support facility
were also completed in June, 1983. A building forLaboratory of Gene Expression and Regulation (2,577m2) was newly built in December, 1996.

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
Laboratory of Gene Expression and Regulation
Speciation Mechanisms I
Speciation Mechanisms II

4

NATIONAL INSTITUTEGRADUATE PROGRAMSThe NIBB carries out two graduate programs.
1. Graduate University for Advanced Studies
The 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 whohave completed a master's course in a university or
are qualified at an equivalent or higher level are eligible to apply.
The Department consists of the following Divisions
and Fields:
DivisionsFieldsMolecular Cell
Biology

Biomolecular Systems
Cell Dynamics

Developmental
Gene Expression
and Regulation

Gene Expression
Morphogenesis
Transgenic Biology

Regulation
Biology

Biological Regulation
Biological Information

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

FOR BASIC BIOLOGY

5

OFFICE OF DIRECTOR
Director-General:
Hideo Mohri
Associate Professors: Shigeru ltoh
Ryuji Kodama
Katsunori Aizawa (on leave)
Research Associate:

Evolution of photoysnthesis and the mechanism ofelectron transfer
Shigeru Itoh
We study the evolution of molecular mechanism ofplant and bacterial photosynthesis. Anoxygenic photosynthesis of bacteria seems to have evolved in the
Precambrian Earth just after the evolution of life.
Oxygen-evolving photosynthesis was then establishedby cyanobacteria 3.5-2.7 billion years ago and increased atomospheric oxygen . Symbiosis of
cyanobacteria inside the larger cells produced the firstplant about 2 billion years ago.
What can we study to explore the past? We takethree approaches (1) Survey of electron transfer
mechanism in photosyntheic reaction center (RC) pigment-protein complexes. We replaced cofactors in thecomplex to see what happens. We studied light reactions by the ps-ns laser and the spin-echo ESR spectroscopy between 4 and 280 K in the cofactor-replaced RCs and found that the molecular architectureof plant and bacterial RCs are highly optimized indifferent directions. The result that was the first evidence of the energy-gap dependence of the ultra-fastelectron tunnnelling in plant, also suggeted us thedesign of prototype RC. (2) Site-directed mutagenesisof electron transfer proteins in attempts to constructprototype electron transfer protein. (3) Survey of everunknown photosynthesis as well as the comparative
studies of photosynthesis in purple, green sulfur,
hellio-, cyanobacteria and plant. A recent topic was
the discovery of a new bacterium, Acidiphilium
rubrum that undergoes photosynthesis with Zn-bacteriochlorophyll, instead of Mg-chlorophylls used in all
the ever-known plant and bacterial photosynthesis.
This finding was a supprise by itself, and led to thestudy of another, completely new oxygen-evolving
photosynthesis now.

Fig. I Space-filling (lower) and wire-frame (upper) models of Rhodopseudomonas capsulata ferredoxin (FdxN) . Pink-coloured
moiety in native (left) protein was altered (right) by site-directed mutagenesis. The mutation exposed the Fe-S cluster (yellow andred) and decreased its reducing power detectable by EPR.

6

Mechanisms determining the outline shape of theadult lepidopteran wings
Ryuji Kodama
Wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which isa hollow sac made of simple epithelium. When the
pupariation is completed, the wing, which was hidden
inside the body wall of the larvae, is exposed on thesurface of the pupa, which gradually turns into theadult wing. The outline shape of the adult wing isoften different from that of the pupal wing. This difference is brought about by the programmed cell
death of the marginal 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 internal area 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 al.,Roux's Arch. Dev. Biol. 204, 418-426, 1995).
The cells in the degeneration region are actively
engulfed by the macrophages in the cavity beneath thewing epithelium. Moreover, the macrophages seem tobe concentrated beneath the degeneration region by
the strong adhesion between basal surfaces of thedorsal 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 from the differentiation region , because the injected probes was found mostly concentrated in thedegeneration region when observed several minutes
later (Yoshida, A. (Biohistory Research Hall) andKodama, R., unpublished).
These studies were done using the small cabbage
butterfly, Pieris rapae, and the silk moth, Bombyx
mori. We have recently begun studies using anotherlepidopteren species, Orgyia recens approximans,
provided by Drs. Y. Arita and K. Yamada (Meijo
University). In this species, the wing is normallyformed until the beginning of the pupal period, but
becomes conspicuously degenerated only in the female adult (Fig. 2). In our preliminary study, it was
shown that the pupal wing is normally formed both inmale and female pupa, but after about two days, female pupal wing starts degeneration on its margin, as
if the degeneration region is continuously formed
deep into the center of the wing (Kodama, R. et al.,unpublished). It is thus suggested that the control
mechanism which demarcates the region to be degen-

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erated is defective in the female in this species. Further investigation using this species might give important insight into such mechanisms.

Fig. 2 The adult wing of the male (upper) and female(lower) moth, Orgyia recens approximans, are shown at thesame magnification. Only a part of the left forewing is shownfor the male wing. In the female, wings are degenerated tosmall protrusions (arrows) covered with scales similar to
those covering the body.

Publication List:
Dzuba, S. A., Hara,H., Kawamori, A., Iwaki, M., ltoh,
S. and Tsvetkov, Yu. D. (1997) Electron spin echoof spin-polarized radical pairs in the intact andquinone-reconstituted plant photosystem I reaction
centers. Chem. Phys. Leu. 264, 238-244.
Hara, H., Dzuba, S.A., Kawamori, A., Akabori, K.,
Torno, T. , Satoh, K., Iwaki, M. and ltoh, S. (1997)
The distance between P680 and QA in Photosystem
II determined by ESEEM spectrocscopy. Biochim.
Biophys. Acta 1322, 77-85 .
Ogawa, K. and Mohri, H. (1997) Establishment of adynein motor superfamily. In "Recent Advances inMarine Biotechnology. Vol. 1. Endocrinology andReproduction" (M. Fingerman, R. Nagabhushanamand M.-F. Thompson, eds.), pp. 249-281. Oxford &IBH Publishing Co., New Delhi, Calcutta.
Ohoka, H., Iwaki, M. and Itoh, S. (1997) Viscosity
dependence of the electron transfer rate from boundcytochrome c to P840 in the photosynthetic reaction
center of a green sulfur bacterium Chlorobium
tepidum. Biochemistry 36, 9267-9272.
Torno, T., Mimuro , M., Iwaki, M., Kobayashi, M. ,Itoh, S. and Satoh, K. (1997) Topology of pigmentsin the isolated photosystem II reaction center studied by selective extraction. Biochim. Biophys. Acta
1321, 21-30.

7

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Mikio Nishimura

The department consists of two regular divisions and three
adjunct divisions. An adjunct division was started in 1997. The
department conducts studies on molecular dynamics of the cellin higher plants and animals such as organelle differentiation,
autophagy, cell motility, cytokinesis and expression of cell cycle
genes.

8

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor:
Mikio Nishimura
Associate Professor: lkuko Hara-Nishimura
Associate Professor (adjunct): Masayoshi Maeshima
Research Associates: Makoto Hayashi
Tomoo Shimada
Post doctoral fellows: Akira Kato 1
Tetsu Kinoshita 2
Nagako Hiraiwa 3
Yasuko lshimaru 3
Kanae Shirahama 3
Graduate Students: Shoji Mano
Hiroshi Hayashi
Kenji Yamada
Daigo Takemoto (Nagoya University)
Yuki Tachibe (Hiroshima University)
Technical Staff:
Maki Kondo
Katsushi Yamaguchi
Visiting Scientists: Luigi De Bellis (Pis a Univ. Italy)
Claus Schnarrenberger (from Free
Univ. of Berlin, Germany)
Yasuko Koumoto
Miwa Kuroyanagi

Higher plant cells contain several distinct organelles
that play vital roles in cellular physiology. During
proliferation and differentiation of the cells, the organelles often undergo dynamic changes. The biogenesis of new organelles may occur, existing organelles
may undergo a transformation of function, while otherorganelles may degenerate. The dynamic transformationof organellar function (differentiation of organelles) is
responsible for flexibility of differentiation in higher
plant cells. 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 theshift from heterotrophic to autotrophic growth occur
in the greening that occurs during seed germination.
Accompanying these metabolic changes, many constitutive organelles are functionally transformed. For
example, etioplasts differentiate into chloroplasts andmitochondria acquire the ability to oxidize glycine.
Glyoxysomes which are microbodies engaged in the
degradation of reserve oil via ~-oxidation and theglyoxylate cycle, are transformed into leaf peroxisomes that function in several crucial steps ofphotorespiration. After the functional transition ofglyoxysomes to leaf peroxisomes during the greeningof pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs
during senescence. Microbody enzymes function after
their transport to microbodies. Since the enzyme compositions and functions of glyoxysomes and leaf
peroxisomes differ from each other, it was likely thatthe two types of microbodies possess different machineries for protein import.
Microbody proteins are synthesized in the cytosol

on free polysomes and are transported posttranslationally into microbodies. Two types oftargeting signals to microbodies have been reported.
One type of targeting signal is part of the mature protein. One such signal, the tripeptide Ser-Lys-Leu,
occurs at the C-terminal end has been identified as a 'targeting signal. Ser-Lys-Leu and related amino acidsequences commonly function in mammals, insects,
fungi, and plants. Glyoxysomal enzymes, such as
malate synthase and isocitrate lyase, and leaf peroxisomal enzymes, such as glycolate oxidase andhydroxypyruvate reductase, contain the targeting signal at their C-terminal ends.
To characterize the targeting signal, we have examined the ability of 24 carboxy-terminal amino acidsequences to facilitate the transport of a bacterial protein, ~-glucuronidase (GUS) into microbodies ingreen cotyledonary cells of transgenic Arabidopsis.
Immunocytochemical analysis of the transgenic plants
revealed that carboxy-terminal tripeptide sequences ofthe form [C/NS/P]-[K/R]-[1/L/M] function as amicrobody-targeting signal, although tripeptides withproline at the first amino acid position and isoleucine
at the carboxyl terminus show weak targeting efficiencies (Fig. 1). All known microbody enzymes that aresynthesized in a form similar in size to the mature
molecule, except catalase, contain one of these tripeptide sequences at their carboxyl terminus. These
carboxyl tripeptides function as a targeting signal tothe microbodies, not only to glyoxysomes but also toleaf peroxisomes. Therefore, it is unlikely thatglyoxysomes and leaf peroxisomes possess different
targeting machineries.

II. Light dependent alternative splicing inhigher plants.
We previously showed that two different forms ofhydroxypyruvate reductase which are localized in
microbodies and in the cytosol, are produced by alternative splicing. In 1997, we found that stromal(sAPX) and thylakoid-bound ascorbate peroxidases(tAPX) are also produced by alternative splicing.
eDNA for sAPX and tAPX were isolated and characterized. The eDNA for sAPX encodes a polypeptide
with 372 amino acids and shares complete sequence, identity with tAPX, except for the deletion of a putative membrane domain of tAPX. Southern blot
hybridization and analysis of intron structure indicated that the mRNAs for sAPX and tAPX, whose
suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot
analysis showed that the accumulation of sAPX andtAPX was differently regulated and that the alternative
splicing is regulated developmentally and by light.

9

FOR BASIC BIOLOGY

A

B
Substitutions offirst amino acid

Substitutions ofsecond amino acid

Substitutions of
C-terminal amino acid

LRL
FRL
CRL
ARL
GRL
SRL
YRL
PRL
ERL
KRL

SIL
SGL
SSL
SHL
SKL
SRL

SRI
SRV
SRL
SRF
SRM
SRS
SRE
SRK

++
++
++
+

++
++

+
++

++

Fig. 1. Effects of Substitutions at the Carboxy-terminal Tripeptide on Subcellular Localization of Chimeric Proteins.
A) Subcellular localization of chimeric proteins in the cells of transgenic Arabidopsis was analyzed by immunoelectronmicroscopy. Label on each photograph represents the carboxy-terminal tripeptide sequence of the chimeric protein. Arrowheads
indicate microbodies. Bar = 1 fllll ·B) Summary of the targeting efficiencies of carboxy-terminal tripeptides. Amino acid sequences of carboxy-terminal tripeptides
are shown by single letter codes. Targeting efficiencies of proteins containing these tripeptides at the carboxy terminus were
presented by ++ (efficient), + (detectable) and - (inefficient).

III. A pumpkin 72-kDa membrane protein of precursor-accumulating vesicles has characteristics ofa vacuolar sorting receptor.
Seed proteins are synthesized on the rough endoplasmic reticulum and are then delivered to theprotein-storage vacuoles . Sorting and targeting themto the vacuoles requires the presence of a specific signal and a receptor. Previously we isolated transport

vesicles that mediate the transport of the precursor ofmajor storage proteins to protein-storage vacuoles indeveloping pumpkin cotyledons. We designated themruecursor-accumulating (PAC) vesicles, as shown in
Fig. 2A. The vesicles might contain a receptor protein
for the storage protein. We characterized two homologous proteins from PAC vesicles, a 72-kDa protein(PV72) and an 82-kDa protein (PV82). PV72 andPV82 showed an ability to bind to peptides derived

10

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from both an internal propeptide and a C-terminal
peptide of a proprotein precursor of 2S albumin, amajor storage protein (Fig. 2B). PV72 was predicted
to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs (Fig. 2A). These
results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage
vacuoles. Previous studies have identified a potential

receptor protein, BP-80, from the membranes ofclathrin-coated vesicles of developing cotyledons ofpea (Pisum sativum, Kirsch et al. 1994 ). Recently
molecular structures of BP-80 (Paris et al. 1997) andthe homolog from Arabidopsis (AtELP; Ahmed et al.
1997) were reported. They are membrane proteins thatare homologous to PV72.

A)
rER

pp2S
p2S

PAC Vesicles

at,oo -

p2S

motifsPro2S Albumin

p2S
2S

B)

Pro2S Albumin

28-N ,
28-I ,
28-C ,PAP ,YRTTITTVEVEENRQGRE
8RDVLQMRGIENPWRREG
KARNLP8MCGIRPQRCDFSSSFADSNPI RPVTDRAASTY

Column2S-I

EluentPAP

2S-C

Fig. 2. PV72 functions as a sorting receptor for intracellular transport of pro2S albumin.
A) A hypothetical scheme for the transport and processing of pro2S albumin. Pro2S albumin is synthesized on rER andtransported to protein storage vacuoles via precursor-accumulating (PAC) vesicles. PV72 and pro2S albumin co-exist in the PAC
vesicles. PV72 is predicted to be a type I integral membrane protein with three repeats of epidermal growth factor (EGF)-like
motifs.
B) PV72 binds the peptides derived from pro2S albumin. PV72 binds to an affinity column with 2S-I peptide as a ligand andis specifically eluted with an excess amount of each peptide of 2S-I, 2S-C or PAP, but not 2S-N peptide. PAP is a peptide derived
from a propeptide of barley aleurain, a thiol proteinase, that contained a vacuolar targeting signal. An NPIR (Asn-Pro-Ile-Arg)
has been shown to be a consensus signal sequence for vacuolar targeting that is found in both propeptides of barley aleurain andsweet potato sporamin.

11

FOR BASIC BIOLOGY

IV. Expression and activation of the vacuolar
processing enzyme in Saccharomyces cerevisiae.
Vacuolar processing enzymes (VPE) are cysteine
proteinases responsible for maturation of various
vacuolar proteins in plants. A larger precursor to VPE
synthesized on the rough endoplasmic reticulum isconverted to an active enzyme in the vacuoles. We
expressed a precursor to castor bean VPE in a pep4
strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE
proteins with 59 kDa and 46 kDa were detected in thevacuoles of the transformant. They were glycosylated
in the yeast cells, although VPE is not glycosylated inplant cells in spite of the presence of two N-linked
glycosylation sites. During the growth of thetransformant, the level of the 59-kDa VPE increasedslightly until a rapid decrease occurred after 9 h. By
contrast, the 46-kDa VPE appeared simultaneouslywith the disappearance of the 59-kDa VPE. Vacuolar
processing activity increased with the accumulation of
the 46-kDa VPE, but not an accumulation of the 59kDa VPE. The specific activity of the 46-kDa VPE
was similar to that of VPE in plant cells. The 46-kDa
VPE mediated the conversion of procarboxypeptidase
Y to the mature form instead of proteinase A. This
indicated that proteinase A which is responsible formaturation of yeast vacuolar proteins, can be replaced
functionally by plant VPE. These findings suggest thatan inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in theyeast cells and then processed to make an active VPE
by self-catalytic proteolysis within the vacuoles.

V. An aspartic proteinase is involved in thematuration of storage proteins in concert with thevacuolar processing enzyme.
A 48-kDa aspartic proteinase was purified fromprotein bodies of dry seeds of castor bean. Immunocytochemical and cell fractionation analyses of theendosperm of maturing castor bean seed showed thatthe aspartic proteinase was selectively localized in thematrix of the protein storage vacuoles, where a variety
of seed storage proteins were also present. To determine whether the aspartic proteinase is responsible formaturation of seed proteins, we prepared[ 35S]proproteins that were localized in the endoplasmic reticulum in pulse-labeled endosperm cells andused the authentic proproteins as substrate for in vitro
processing experiments. The purified aspartic proteinase was unable to convert any of three endosperm
proproteins into their mature forms, while the purified
vacuolar processing enzyme could convert all three
proproteins. We further examined the activity of aspartic proteinase on the cleavage of an internal
propeptide of Arabidopsis pro2S albumin, that was
post-translationally removed. The aspartic proteinasecleaved the propeptide at three sites at pH 3.0, but not
at pH 5.5. These results suggest that aspartic proteinase cannot directly convert pro2S albumin into themature form, but it may play a role in trimming the Cterminal propeptides from the subunits that are produced by the action of the vacuolar processing
enzyme. However, the activity of the aspartic proteinase in the protein storage vacuoles might be limited.

VI. Role of molecular chaperones in organelle
differentiation
Molecular chaperones are cellular proteins thatfunction in the folding and assembly into oligomeric
structures of certain other polypeptides but that are
not, themselves, components of the final oligomeric
structure. To clarify the roles of molecular chaperones
on organelle differentiation, we have purified andcharacterized chaperonin and Hsp70s and analyzed
their role in translocation of proteins into chloroplasts.
In addition to mitochondrial chaperonin10 homologues, we isolated a eDNA for chloroplastic
chaperonin 10 homologues from Arabidopsis
thaliana. The eDNA was 958 bp long and encoded apolypeptide of 253 amino acids. The deduced aminoacid sequence showed that the protein contained anNterminal chloroplast transit peptide and the mature region which was comprised of two distinct GroESdomains. The two halves of the Cpn20 show 42 %amino acid identity to each other. A Northern blot
analysis revealed that the mRNA for the Cpn10 homologue was abundant in leaves and was increased byheat treatment. A histidine-tagged construct lacking
the transit peptide was expressed in E. coli and thefunctional and structural characterization is inprogress.

Publication List:
Esaka, M., Yamada, N., Kitabayashi, M., Setoguchi,Y., Tsugeki, R., Kondo, M. and Nishimura, M.
(1997) eDNA cloning and differential gene expression of three catalases in pumpkin. Plant Mol. Bioi.
33: 141-155.
Fleurat-Lessard, P., Fragne, N., Maeshima, M.,
Ratajczak, R., Bonnemain, J. L. and Martinoia, E.
(1997) Increased expression of vacuolar aquaporin
and H+-ATPase related to motor cell function in Mimosa pudica L. Plant Physiol. 114: 827-834.
Hatano, K., Shimada, T., Hiraiwa, N., Nishimura, M.
and Hara-Nishimura, I. (1997) A rapid increase in thelevel of binding protein (BiP) is accompanied by synthesis and degradation of storage proteins in pumpkin
cotyledons. Plant Cell Physiol. 38: 344-351.
Hayashi, M., Aoki, M., Kondo, M. and Nishimura, M.
(1997) Changes in targeting efficiencies of proteinsto plant microbodies caused by amino acid substitution in the carboxy-terminal tripeptide. Plant Cell
Physiol. 38: 759-768.
Hiraiwa, N., Kondo, M., Nishimura, M. and HaraNishimura, I. (1997) An aspartic proteinase is involved in the maturation of storage proteins in concert with the vacuolar processing enzyme. Eur. J.
Biochem. 246: 133-141.

12

Hiraiwa, N., Nishimura, M. and Hara-Nishimura, I.
(1997) Expression and activation of the vacuolar
processing enzyme in Saccharomyces cerevisiae.
Plant J. 12: 819-829.
Inoue, K., Wada, Y., Nishimura, M. and HaraNishimura, I. (1997) Heterologous expression andsubcellular localization of pumpkin seed tonoplast
intrinsic proteins (TIP) in yeast cells. Plant Cell
Physiol. 38: 366-370.
Mano, S., Hayashi, M. Kondo, M. and Nishimura, M.
(1997) Hydroxypyruvate reductase with a carboxyterminal targeting signal to microbodies is expressed in Arabidopsis. Plant Cell Physiol. 38: 449455.
Mano, S., Yamaguchi, K., Hayashi, M. andNishimura, M. (1997) Stromal and thylakoid-boundascorbate peroxidases are produced by alternative
splicing in pumpkin. FEBS Lett. 413: 21-26.
Matsuoka, K., Higuchi, T., Maeshima, M. andNakamura, K. (1997) A vacuolar H+-ATPase in a
nonvacuolar organelle is required for the sorting ofsoluble vacuolar protein precursors in tobacco cells.
Plant Cell 9: 533-546.
Minami, Y., Takao, H., Kanafugi, T., Miura, K.,
Kondo, M., Hara-Nishimura, 1., Nishimura, M. andMatsubara, H. (1997) ~-glucosidase in the Indigo
plant: Intracellular localization and tissue specificexpression in leaves. Plant Cell Physiol. 38: 10691074.
Nozue, M., Yamada, K., Nakamura, T., Kubo, H.,
Kondo, M. and Nishimura, M. ( 1997) Expression ofa vacuolar protein (VP24) in anthocyanin-producing
cells of sweet potato in suspension culture. Plant
Physiol. 115: 1065-1072.
Odaira, M., Yoshida, S. and Maeshima, M. (1997)
Accumulation of a glycoprotein homologous toseed storage protein in mung bean hypocotyls in thelate stage of tissue elongation. Plant Cell Physiol.
38: 290-296.
Sato, M. H., Nakamura, M., Ohsumi, Y., Kouchi, H.,
Kondo, M., Hara-Nishimura, 1., Nishimura, M. and
Wada, Y. (1997) The AtVAM3 encodes a syntaxinrelated molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J. Bioi. Chern. 272:
24530-24535.
Shimada, T., Kuroyanagi, M., Kondo, M., Nishimura,
M. and Hara-Nishimura, I. (1997) A pumpkin 72kDa membrane protein of precursor accumulating
vesicles has characteristics of a vacuolar sorting receptor. Plant Cell Physiol. 38: 1414-1420.
Shiratake, K., Kanayama, Y., Maeshima, M. andYamaki, S. (1997) Changes in H+-pumps and atonoplast intrinsic protein of vacuolar membranes
during the development of pear fruit. Plant Cell
Physiol. 38: 1039-1045.
Takasu, A., Nakanishi, Y., Yamauchi, T. andMaeshima, M. (1997) Analysis of substrate bindingsite and carboxyl terminal region of vacuolar H+
translocating pyrophosphatase of mung bean withpeptide antibodies. J. Biochem. 122: 883-889.

NATIONAL INSTITUTE13

FOR BASIC BIOLOGY

to the lytic compartment (Fig.l ). Since cellular lytic
compartment, lysosome, was identified, biogenesis oflysosomal enzymes and their enzymatic characterization have been studied thoroughly. However, themechanisms of delivery of proteins to the lysosomes
are not known at a molecular level. Biochemical
analysis of lysosome/vacuole system is quite difficult
because of its complexity and dynamic feature. Autophagic process has been studied mostly by electron
microscopy, and molecular mech 1ism of autophagy
remains to be elucidated.

DIVISION OF BIOENERGETICS
Yoshinori Ohsumi
Tamotsu Yoshimori
Takeshi Noda
Yoshiaki Kamada
Post Doctral Fellow: Tomoko Funakoshi 1
Noboru Mizushima 2
Satoshi Kametaka (Univ. of Tokyo)
Graduate Students:
Takayoshi Kirisako (Univ. ofTokyo)
Yukiko Kabeya
Technical Staff:
Fumi Yamagata (Teikyo Univ. ofVisiting Fellow:
Sci. and Tech.)

Professor:
Associate Professor:
Research Associate:

Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, Saccharomyces
cerevisiae, induces bulk protein turnover in the vacuoles under starvation conditions. This whole process
corresponds to that of macroautophagy in higher eukaryotic cells. By electron microscopic analyses wesucceeded in detecting double membrane structures in
the cytoplasm enclosing a portion of cytosol. These
yeast autophagosomes immediately fuse with vacuoles, delivering single inner membrane structures, autophagic bodies in the vacuole. When vacuolar
proteinase activities are blocked genetically or by specific inhibitor such as PMSF autophagic bodies areaccumulated in the vacuoles. They moves around vigorously in the vacuoles by Brownian motion, and areeasily detectable by light microscope. Thus we canfollow the progression of autophagy as the accumula-This division aims to elucidate the mechanism andits regulation of intracellular protein degradation in alytic compartment. Recently it was realized that degradation process plays essential roles for various
physiological function. In the cytoplasm selective
protein degradation takes place by ubiquitin/
proteasome system. Short-lived or abnormal proteins
are selectively eliminated by this pathway. Whilevacuole/lysosome contributes to the bulk turnover ofcytosolic and organelles proteins.
Little is known about the mechanism of protein
degradation in contrast with that of biosynthesis.
Bulk protein degradation is induced by various nutrient starvation condition, which is obligatory to celldifferentiation and maintenance of cell viability. Autophagy is a major route for sequestration of proteins

(a)

(b)

Fig. l. (a) Freeze-Fracture Image of Yeast under Nitrogen Starvation.
When vacuolar proteinases are blocked, spherical membrane structures, autophagic bodies accumulate in the vacuole.
(b) Fusion of Autophagosome to the Vacuolar Membrane.
Double membrane structure, autophagosome (AP), encloses a portion of the cytosol, then fuses with the vacuole to deliver an
autophagic body (AB) in the vacuole. These membrane structures show very few intramembrane particles.

14

NATIONAL INSTITUTE

tion of autophagic bodies in real time. Biochemical
and immunoelectron microscopic analyses of vacuoles
containing autophagic bodies showed that starvationinduced sequestration is non-selective, that is, any
kind of cytosolic enzymes and organelles are sequestered randomly to the vacuoles to be degraded.
Autophagy in yeast is a kind of general stress response to adverse environmental condition and is induced by various starvation conditions such as nitrogen, carbon, sulfate, and even single amino aciddepletion. Signal transduction of starvation for induction of autophagy is a key question to be solved.
Recently we found rapamycin induces autophagy inthe cell growing in a rich medium and that Tor, phosphatidyl inositol kinase homologue plays essential
role for the regulation of autophagy (Fig.2).
Another crucial and controversial question is themechanism of formation of autophagosome. Still
nothing is clear about the origin of the membrane.
Freeze-fracture electron microscopy showed that
autophagosomal membrane has quite characteristicfeature and the density of intramembrane particles is
extremely low as compared with other intracellular
membrane.

Starvation

Genetical dissection of autophagic process
Yeast, S. cerevisiae, has made great contribution tosolve many fundamental problems in cell biology
because of tractability of genetic and molecular biological techniques . In order to dissect the complex
process of autophagy to its elementary steps westarted to isolate mutants in the process of autophagy,taking advantage of morphological selection underlight microscope. Total 14 autophagy defective mutants, agp, were isolated. They cannot induce protein
degradation upon shift from growth medium to the
starvation medium. They grow normally in a richmedium, but start to die after 2 days in the starvation
medium. This suggests that the autophagy is necessary for long time maintenance of cell viability. Homozygous diploid of each apg mutant is sporulationdefective as expected.
Analyses of APG genes
Now we are focusing on characterization of these
APG genes, essential for autophagy. So far we havefinished cloning and identification of twelve APG
genes. APGJ codes a novel Ser/Thr protein kinaseessential for the induction of autophagy. This pro-GrowingTor
inactive

Autophagosome

0

/

/Ap~

Cvt Vesicle®

Aminopeptidase I

r

i

l

Tor
active\Vacuole\\o~ oFig. 2 Schema of autophagy in yeast. When yeast cells face to starvation, isolation membrane starts to enclose a portion ofcytosol and form an autophagosome. Autophagosome fuses to the vacuolar membrane, delivering autophagic body in the vacuoles(Autophagic pathway). Under growing condition one of vacuolar enzymes, API is selectively sequestered to the vacuole (Cvt
pathway). Two distinct pathways share most molecular machinery.

FOR BASIC BIOLOGY

vides the first direct evidence for involvement of protein phosphorylation in the process of autophagy.
Apg13p interacts with Apg1p and might function as aregulator of its kinase activity. So far most APG
genes analyzed are novel and non-essential for vegetative growth. Using the assay system of autophagy wedeveloped, we are studying genetic interaction among
APG genes. Some APG gene products are inducible orchange phosphorylation state by nutrient starvation.
Analyses of intracellular localization of these Apgps
may provide us specific marker to elucidate the membrane dynamics during autophagy.
Diverse pathways of autophagy
Autophagy is ubiquitous and fundamental physiological response of every eukaryotic cell. We foundyeast cells induce macroautophagy under starvation,
which is a main route for bulk and non-selective protein degradation. While it is known that excessive
amount of organelles are also degraded by autophagic
process. In methylotrophic yeast, Pichia pastoris degrades peroxisomes by invagination of vacuolar membrane, microautophagic process. Further studies will
uncover more sophisticated pathways for the degradation in the lysosome/vacuoles.
Recently it was found that all APG genes are required for targeting of one of vacuolar enzyme, API,
from cytosol to the vacuole. Electron microscopic
analyses of API transport process revealed that API
first forms a complex in the cytosol and delivered to
the vacuole by the distinct but topologically similar
mechanism to the macroautophagy (Fig.2). However,
API transport is selective and constitutive process. It
is interesting that biosynthetic pathway shares themachinery with degradative process. Some selective
degradation may also be mediated by autophagy-related mechanism.
Perspective
Autophagy is essential for maintenance of cell viability during starvation. Degradation products may
provide essential nutrients necessary for minimal protein synthesis, or reduction of certain critical activities
in the cytosol may be essential for the maintenance ofviability. Molecules involved in the membrane dynamics are well conserved from yeast to higher eukaryotes. We realized some APG genes in yeast show
homologues in mammals or higher plants. Knowledge in yeast must give us key to uncover the mechanism of autophagy in higher eukaryotic organisms.
We are now developing systems for studying autophagy in mammalian cell culture.
Publication List:

Nakamura, N., Matsuura, A., Wada, Y., and Ohsumi,
Y. (1997) Acidification of vacuoles is required for
autophagic degradation in the yeast, Saccharomyces
cerevisiae. J. Biochem., 121, 338-344.
Wada, Y., Nakamura, N., Ohsumi, Y, and Hirata, A.
(1997) Vam3p, a new member of syntaxin related

15

protein, is required for vacuolar assembly in theyeast Saccharomyces cerevisiae. J. Cell Sci, 110,1299-1306.
Funakoshi, T., Matsuura, A., Noda, T., and Ohsumi, Y.
(1997) Analyses of APG13 gene involved in the
autophagy in yeast, Saccharomyces cerevisiae.
Gene, 192, 207-213.
Matsuura, A., Wada, Y., and Ohsumi, Y. (1997)
Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae.
Gene, 192, 245-250.
Scott, S. V., Baba, M., Ohsumi, Y., and Klionsky, D.
J. (1997) Aminopeptidase I is targeted to the vacuole by a nonclassical vescicular mechanism. J. Cell
Bioi. 138, 37-44.
Nakamura, N., Hirata, A., Ohsumi, Y., and Wada, Y.
(1997) Vam2/Vps41p and Vam6/Vps39p are components of a protein complex on the vacuolar membranes and involved in the vacuolar assembly in theyeast, Saccharomyces cerevisiae. J. Bioi. Chern.,
272,11644-11349.
Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., andOhsumi, Y. (1997) Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J. Cell Bioi. 139: 1687-1695.
Shirahama, K., Noda, T., and Ohsumi, Y. (1997)
Mutational analysis of Cscl!Vps2p-involvement ofendosome in regulation of the autophagy in yeast,
Cell Struc. Func. 22, 501-509.
Noda, T., and Ohsumi, Y. (1998) Tor, a phosphatidyl
inositol kinase homologue, controls autophagy inyeast. J. Bioi. Chern. 273, 3963-3966.

16

NATIONAL INSTITUTEDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor:
Research Associate:
Post-doctoral Fellow:
Visiting Fellow:

Masayuki Yamamoto
Masua Goto
Takashi Kuromori 1
Akira Yamashita
Satsuki Okamoto

The major goal of this Division is to elucidate regulatory mechanisms of meiosis. Meiosis is a crucial
step in gamete formation and is essential for sexual
reproduction . Meiotic steps are highly conserved
among eukaryotic species. We have been screening
for genes relevant to the regulation of meiosis in animals and plants, by isolating genes that either complement Schizosaccharomyces pombe meiotic mutants oraffect the meiotic behavior of this fission yeast. We
have isolated a number of animal and plant genes inthis manner, and analysis of the cloned genes is inprogress. In addition, efforts have been also paid to
elucidate the regulatory mechanisms of meiosis in fission yeast in more detail, so that the above strategy
may be reinforced. Some examples of the analysis arepresented below.

I. Microtubule-binding protein which functions
specifically during meiosis.
In fission yeast, an RNA species named meiRNA is
specifically required for the promotion of the firstmeiotic division. To dissect the function of this RNA
and its partner RNA-binding protein Mei2p, we
screened for high-copy-number suppressors of thearrest prior to the first meiotic division caused by lossof meiRNA. Analysis of one of the suppressors thus
isolated, named ssm4, suggested that it encodes acoiled-coil protein carrying a microtubule-binding
motif at its N-terminus. Expression of ssm4 was restricted to cells undergoing meiosis. Disruption of
ssm4 affected neither vegetative growth nor conjugation, but resulted in frequent generation of asci carrying less than four spores. Tagged Ssm4p localized atspindles during both the first and the second meiotic
division. The microtubule-binding motif was essential
for the association of Ssm4p with microtubules andfor its function during meiosis, but not for the suppression of loss of meiRNA. Ssm4p appeared to possess a potential to migrate to the nucleus. Thus,
Ssm4p is a microtubule-colocalizing protein that plays
a role specifically in meiosis. Ssm4p appears tomodify the structure or the function of nuclear microtubules in order to promote the meiotic nuclear division.

II. Arabidopsis 14-3-3 isoform.
14-3-3 proteins constitute a highly conserved family
among eukaryotes, and diverse biochemical activities
have been ascribed to them. We isolated a variety of
Arabidopsis 14-3-3 genes in a screening for cDNAs
that could block ectopic meiosis driven by the pat]mutation in fission yeast. In addition to suppressing
the pat] mutation, these cDNAs could cure the deformed morphology and raised UV sensitivity of fis sion yeast cells defective in rad24, which encodes a14-3-3 protein. This indicates that most Arabidopsis

Fig. 1. Cellular localization of S. pombe type II myosin heavy chain Myo2p fused to the jellyfish green fluorescent protein (GFP)
and that of S. pombe actin. Cells grown to the mid-log phase were fixed, immediately stained with Phalloidin-TRITC, and observed
under the microscope as rapidly as possible. Micrographs show GFP fluorescence of Myo2p (A) and phalloidin-TRITC-stained
actin (B) .

FOR BASIC BIOLOGY

14-3-3 proteins can execute the function of the fission
yeast 14-3-3 protein encoded by rad24, at least partially. We analyzed one of the Arabidopsis 14-3-3
clones in more detail, as it was not fully characterized
in literature. This 14-3-3 gene turned out to be expressed in all plant organs examined.

III. Identification of fission yeast type II myosin
heavy chain.
We cloned the myo2 gene of Schizosaccharomyces
pombe, which encodes a type II myosin heavy chain,by virtue of its ability to promote meiosis in certain
mutants. The myo2 gene encoded 1526 amino acids,
and deduced Myo2p showed homology to the head
domains and the coiled-coil tail of the conventional
type II myosin heavy chain, and carried putative binding sites for ATP and actin. Disruption of myo2 inhibited cell proliferation. myo2-defective cells showednormal punctate distribution of interphase actin, butthey produced irregular actin rings and septa, andwere impaired in cell separation. Overproduction of
Myo2p was also lethal, apparently blocking actin relocation. Nuclear division proceeded without actin
ring formation and cytokinesis in cells overexpressing
Myo2p, giving rise to multinucleated cells with dumbbell morphology. Analysis using tagged Myo2p revealed that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of
the ring and responsible for its contraction. Furthermore, genetic evidence suggested that the acto-myosin
system may interact with the Ras pathway, which
regulates mating and the maintenance of cell morphology inS. pombe.

Publication List:
Hirayama, T., Ishida, C., Kuromori, T., Obata, S.,
Shimoda, C., Yamamoto, M., Shinozaki, K., andOhto, C. (1997). Functional cloning of a eDNA
encoding Mei2-like protein from Arabidopsis
thaliana using a fission yeast pheromone receptor
deficient mutant. FEBS Lett. 413, 16-20.
Imai, Y., Davey, J., Kawagishi-Kobayashi, M., andYamamoto, M. (1997). Genes encoding farnesyl
cysteine carboxyl methyltransferase in
Schizosaccharomyces pombe and Xenopus laevis.
Mol. Cell. Bioi. 17, 1543-1551.
Kitayama, C., Sugimoto, A., and Yamamoto, M.
(1997). Type II myosin heavy chain encoded by themyo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J. Cell
Bioi. 137, 1309-1329.
Yamashita, A., Watanabe, Y., and Yamamoto, M.
( 1997). Microtubule-associated coiled-coil protein
Ssm4 is involved in the meiotic development in fission yeast. Genes Cells 2, 155-166.

17

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NATIONAL INSTITUTE

bule-based systems - should somehow cooperate in
the inner arm dynein, although the function of actin as
a subunit of dynein is totally unknown at present.
Recently we found that the mutant ida5, lacking fourout of the seven subspecies of inner-arm dyneins, has
a mutation in the actin-encoding gene. Intriguingly,
Chlamydomonas has only a single gene of conventional actin, and the mutant ida5 was found to express
no conventional actin at all. On close inspection, however, the cytoplasm ·and axoneme of this mutant were
found to contain a novel actin-like protein (NAP)
which has only a very low (64%) homology to conventional actin. Despite the lack of the conventional
actin, ida5 displays normal cell division and grows as
rapidly as wild type. However, the mating efficiencyof this mutant is much lower than in wild type, because it is deficient in the growth of the fertilization
tubule, an F-actin-containing structure that facilitatesmating. Thus conventional actin and NAP appear tooverlap in some, but not all , cellular functions .
The actin-null mutant ida5 should be of greatuse in elucidating the functional domains of actin. We
have recently succeeded in transforming this mutant
with a cloned actin gene, and found that inner arm
dynein, as well as the fertilization tubule in gametic
cells, became restored in the transformants. We are
currently trying to transforming the mutant with artificially modified actin gene, to see if polymerization
or interaction with myosin is important for the actinfunction in the dynein arms. We are also examining
expression and localization of actin and NAP in various stages of cell cycle. Studies on those lines will
enable us to determine whether actin or NAP is essential for cytokinesis, assembly and function of innerdynein arms, and other fundamental cellular phenomena in Chlamydomonas and other cells.

DIVISION OF CELLULAR COMMUNICATION(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Postdoctral Fellow:
Graduate student:

Ritsu Kamiya
Katsushi Owaribe
Takaka M inoura
Osamu Kagami 1
ltsushi Minoura (University of Tokyo)

The research in this laboratory, started in November, 1966, is aimed at an understanding of themolecular mechanisms that regulate the assembly andfunction of cytoskeletal proteins. Current research effort is centered on the function of actin and flagellar
dynein. The organism employed is Chlamydomonas, abiflagellate green alga particularly suited for genetic
and molecular biological studies.
A single flagellar axoneme contains at least
eleven kinds of dynein heavy chains in inner and outerarms . To understand the specific function of eachheavy chain, we have been isolating and characterizing mutants that lack different kinds of axonemal
dyneins. Their motility phenotypes have indicated thatdifferent dynein species significantly differ in func tion. For example, the outer arm heavy chains areimportant for flagellar beating at high frequency,
whereas the inner-arm heavy chains are important for
producing proper waveforms. Indirect evidence alsosuggests that the properties of force generation bydifferent heavy chains differ greatly. We are currently
carrying out micro-physiological experiments to assess the force production by each species of dynein.
The inner dynein arms are known to containactin as a subunit. Hence the two independent motility
systems of eukaryotes - the actin-based and microtu-

A

B

Fig. 1. 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 1 mM IBMX. Bar, 0.3 f.IID . Wild-type fertilization tubules have been shown to contain F-actin bundles.

FOR BASIC BIOLOGY

Publication list:

Hayashibe, K., Shingyoji, C. and Kamiya, R. (1997).
Induction of temporary beating in paralyzed flagellaof Chlamydomonas mutants by application of external force. Cell Motil. Cytoskel. 37:232-239.
Kato-Minoura, T., Hirono, M. and Kamiya, R. (1997).
Chlamydomonas inner-arm dynein mutant, ida5,
has a mutation in an actin-encoding gene. J. Cell
Bioi. 137: 649-656.
Takada, S. and Kamiya, R. (1997). Beat frequency
difference between the two flagella of Chlamydomonas depends on the attachment site of outerdynein arms on the outer-doublet microtubules. Cell
Motil. Cytoskel. 36, 68-75.
Wakabayashi, K., Yagi, T. and Kamiya, R. (1997).
Ca2+-dependent waveform conversion in the flagellar axoneme of Chlamydomonas mutants lacking
the centralpair/radial spoke system. Cell Motil.
Cytoskel. 38:22-;28.

19

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NATIONAL INSTITUTEDIVISION OF CELL FUSION(ADJUNCT)

Professor:
Issei Mabuchi
Associate Professor: Hiroshi Abe
Research Associate: Hirotaka Fujimoto

Cytokinesis in animal and some primitive eukaryotic cells is achieved by the progressive contraction of the cleavage furrow. The cleavage furrow contains a contractile apparatus, called the contractile
ring, which is composed of a bundle of actin filamentsthat lies in the furrow cortex beneath the plasma membrane. It has been established that the contractile ring
contracts as the result of interaction between actin
filaments and myosin. However, little is known aboutprocess of its formation, mechanism that controls its
formation, protein constituents, and its ultrastructure.
The goal of our research is to solve these problems
and thereby clarify the molecular mechanism of cytokinesis. For this purpose, we use three kinds ofcells, namely, sea urchin egg, Xenopus egg, and thefission yeast Schizosaccharomyces pombe.
The isolation of cleavage furrows from dividing
cells is one of the most important methods for elucidating the molecular mechanism underlying cytokinesis. We hand-isolated furrows from newt eggs andfound some unique proteins in the furrow preparation(Mabuchi, Tsukita, Tsukita, and Sawai, Proc. Nat!.
Acad. Sci. USA 85: 5966-5970, 1988). However, thismethod has the disadvantage that it is not easy toobtain a sufficient amount of the furrows for analysisof the protein constituents. We have also reported amass isolation method for cleavage furrows from eggsof the sand dollar, Clypeaster japonicus (Yonemura,
Mabuchi, and Tsukita, J. Cell Sci. 100: 73-84, 1991).
However, this method has not been applicable to theregular sea urchins.
We have recently developed an isolation method
for cleavage furrows by which we were able to isolate
furrows from various sea urchin eggs. The contractile
ring was included in the isolated cleavage furrows , asseen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and ~-glycero­
phosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation
was carried out without both of them, cleavage furrows without the mitotic apparatus were obtained(Fig. 1).
The development of a method of isolation ofcleavage furrows from regular sea urchin eggs enabled
us to compare protein constituents among furrows
from different sea urchin and sand dollar species. We
found that 32, 36, 51 kDa proteins were concentrated
in common in the cleavage furrows isolated fromeggs of the sand dollars , C. japonicus andScaphechinus mirabilis, and sea urchins ,Hemicentrotus pulcherrimus and Strongylocentrotus
nudus, on two-dimensional gel electrophoreses. We
are currently investigating the nature of these proteins.
Next we investigated the role of myosin in cytokinesis of S. pombe. Recently, it has been shown by
Kitayama et al. (J. Cell Bioi. 137: 1309-1319, 1997)
that Myo2, a type II myosin heavy chain plays a rolein the F-actin ring formation in this organism. We
found another myosin II called Myo3. Myo3 is thesame protein as Myp2 reported independently byBezanilla eta!. (Mol. Bioi. Cell, 8: 2693-2705, 1997).

Fig. 1 Cleavage furrows of Hemicentrotus pulcherrimus eggs isolated with the isolation medium that does not contain protein
phosphatase inhibitors. Fluorescence (A) and phase-contrast (B) micrographs of cleavage furrows stained with rhodamine-phalloidin
are presented. Bar, 50 f.Lm .

FOR BASIC BIOLOGY

Overexpression of Myo3 in the cell leads to formationof aberrant F-actin ring, F-actin cable, and septum.
We knocked out the myosin genes inS. pombe. Since
Myo3 is not essential, but Myo2 is essential for its
growth, we made a myo2myo3 null strain containing
pREP81-myo2 to control the expression of myo2.
After shut off the myo2, no F-actin ring formation
occurs during mitosis. Therefore, type-11 myosin isnecessary in the formation of the contractile ring in S.
pombe.
Publication List:

Fujimoto, H. and Mabuchi, I. (1997) Isolation ofcleavage furrows from eggs of regular sea urchinsand identification of furrow-specific proteins. J.
Biochem, 122, 518-524.

21

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 of developmental phenomena including:(1) differentiation and maturation of germ cells, (2) molecularbasis of body plans, and (3) gene regulation in cell differentiation and growth.

24

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

Yoshitaka Nagahama
Michiyasu Yoshikuni
Minoru Tanaka
Tohru Kobayashi
Postdoctoral Fellows:
Akihiko Yamaguchi 1
Yoshinao Katsu 2Mika Tokumoto 2
Takashi Todo 2
Zuxu Yao 2
Craig Morrey 2
JSPS Research Associates: Daisuke Kobayashi 3
Yuichi Ohba 3
Yasutoshi Yoshiura 3
NIBB Postdoctoral Fellows: Won-Kyo Lee
Jian-Qiao Jiang
Graduate Students: Jun Ding (Graduate University for
Advanced Studies)
Masatada Watanabe (Graduate
University for Advanced Studies)
Gui-Jin Guan (Graduate University
for Advanced Studies)
Monbusho Foreign Scientist: Allen Schuetz (Johns
Hopkins University)
Visiting Scientist:
Graham Young (University of Otago)

The pituitary-gonadal axis plays an important role
in regulating gametogenesis in vertebrates. In mostcases, however, gonadotropins act through the biosynthesis of gonadal steroid hormones which in turn
mediate various stages of gametogenesis. Studies using teleost fishes as experimental models provide new
information about the endocrine regulation of gametogenesis including oocyte growth, oocyte final maturation, spermatogenesis, and sperm maturation. Our research focuses on (1) the identification of steroidal
mediators involved in each process of gametogenesis,and (2) the mechanisms of synthesis and action of themediators. These studies collectively demonstrate theappropriateness of using teleost fishes as valid models
for examining hormonal influences on gametogenesis.
Such models could also have applications and validity
for vertebrates in general.

I.
Endocrine regulation of oocyte growth andmaturation
17a.,20~-dihydroxy-4-pregnen-3-one (17a.,20~-DP)

was identified as the maturation-inducing hormone ofseveral teleost fishes including salmonid fishes. Alongwith estradiol-17~, which was identified as the major
mediator of oocyte growth, we now have two known
biologically important mediators of oocyte growthand maturation in female fishes. Two cell type models
have been proposed for estradiol-17~ and 17a.,20~­
DP production, with the thecal layer providing precursor steroids to the granulosa layer. 17a.,20~-DP acts
via a receptor on the plasma membrane of oocytes. A
specific 17a.,20~-DP receptor has been identified andcharacterized from defolliculated oocytes of several
fish species. The concentrations of 17a.,20~-DP membrane receptors increase immediately prior to oocyte

maturation. The pertussis toxin-sensitive G-protein is
involved in the signal transduction pathway of
17a.,20~-DP. We have cloned two Gia. cDNAs from a
medaka oocyte eDNA library. Western blot analysis
showed that 17a.,20~-DP receptor concentrations andGia. content decreased concomitantly in membrane
preparations during oocyte maturation. We also foundthat significant amounts of 17a.,20~-DP receptors in
the immunoprecipitates, indicating that the 17a.,20PDP membrane receptors are directly coupled with Gi.
The early steps following 17a.,20~-DP action involve the formation of the major mediator of this steroid, maturation-promoting factor or metaphase-promoting factor (MPF), which consists of cdc2 kinase(34-kDa) and cyclin B (46- to 48-kDa). Goldfish immature oocytes contain only monomeric 35-kDa inactive cdc2 kinase and do not stockpile cyclin B, although immature oocytes contain mRNA for cyclin B.
17a.,20~-DP induces oocytes to synthesize cyclin B,
which in turn activates preexisting 35-kDa cdc2 kinase through its threonine 161 phosphorylation by athreonine kinase (M015), producing the 34-kDa active cdc2. We examined goldfish oocyte proteinsbound to cyclin B mRNA. Using oligo(dt)-cellulose
affinity chromatograpy and Southwestern and Northwestern analyses, we found a 54-kDa cyclin B
mRNA-binding protein (p54), which also bound to the
Y box DNA element. We cloned cDNAs encoding Y
box proteins, one of which is likely to encode p54 andis expressed specifically in germ cells. We suggestthat the 54-kDa Y box protein may have a role intranslational repression of cyclin B mRNA in immature oocytes, and 17a.,20~-DP stimulation releases
this repression, leading to the de nove synthesis of
cyclin B.
Immediately prior to the transition from metaphaseto anaphase, MPF is inactivated by degradation of
cyclin B. We investigated the role of proteasomes (anonlysosomal large protease) in cyclin degradation,using E. coli-produced goldfish cyclin B and purified
goldfish proteasomes (20S and 26S). The purified 26S
proteasome, but not 20S proteasome, cleaved bothmonomeric and cdc2-bound cyclin B at lysine 57(K57) restrictively in vitro, and produced a 42-kDa Nterminal truncated cyclin B, which was transiently
detected at the initial phase of the normal egg activation. The 42-kDa cyclin B, as well as full-length one,was degraded in Xenopus egg extracts, but a mutation
on K57 (K57R) inhibited both the digestion by 26S
proteasome and the degradation in Xenopus egg extracts. These findings strongly suggest the involvement of 26S proteasome in cyclin degradation throughthe first cleave on its N-terminus.

II.

Endocrine regulation of spermatogenesis

Under cultivation conditions, male Japanese eels
(Anguilla japonica) have immature testes containing
only type A and early type B spermatogonia together
with inactive Sertoli cells and Leydig cells. Using anorgan culture system for eel testes, we have shown

25

FOR BASIC BIOLOGY

that the hormonal regulation of spermatogenesis in eel
testes involves the gonadotropin stimulation of Leydig
cells to produce 11-ketotestosterone (11-KT), a potent
androgen in fish . In turn, 11-KT activates Sertoli cells
to stimulate premitotic spermatogonia to complete
spermatogenesis. Gonadotropin (human chorionic gonadotropin, hCG)-induced spermatogenesis was accompanied by marked, rapid increase in 11-KT production. Northern blot analysis revealed that both11~-hydroxylase and 11~-hydroxysteroid dehydrogenase mRNA transcripts were not present in testes ofeels prior to gonadotropin treatment, but were abundant in those after gonadotropin treatment for 1 day.
We used a subtractive hybridization method to clone
genes that are expressed differentially in eel testes in
the first 24 hr after gonadotropin treatment. One of the
up-regulated cDNAs was identified as coding theactivin ~B subunit. Activo ~B mRNA transcripts wereabsent in testes prior to gonadotropin treatment andwere abundant in Sertoli cells in testes of eels treatedwith gonadotropin for 1-6 days. 11-KT induced amarked production of activin B in cultured testes, indicating that eel activo ~B subunit production islargely for activin B formation . 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. Full-length cDNAs for eel activin type I
and II receptors were isolated and sequenced. The
type I receptor mRNA transcripts were present in testes prior to gonadotropin injection, with a marked
increase in spermatogonia and spermatocytes after
gonadotropin injection for 9 days. Taken together,
these results indicate that activin B produced by Ser-

D

I
Fig. 1. Immunocytochemical localization of 3~-hydroxysteroid dehydrogenase and cytochrome P450 aromatase in tilapia gonads
collected at 20 days (A, female; B, male), 23-25 days (C, female) , 30 days (D, female), 50 days (E, female), and 30 days (F, male)after hatching. A, B, D, E, and F, 3~-hydroxysteroid dehydrogenase. C, cytochrome P450 aromatase. g, gonium; Oc, ovarian cavity;
Ed, efferent duct. X 800.

26

toli cells under the influence of gonadotropin and 11KT plays an important role in the regulation of spermatogenesis in fish testes.

III. Endocrine regulation of gonadal sex
differentiation
The initial differentiation and development of steroid-producing cells during gonadal sexual differentiation in tilapia, Oreochromis niloticus, were examined immunohistochemically using the antibodies offour enzymes essential for steroidogenesis (P450scc,
3~-HSD, P450c17, and P450arom)(Fig. 1). There are
two patterns of immunoreactivity in gonads, with either positive reactions for all antibodies, or no reactions with any of the antibodies. Positive
immunoreactions for all four antibodies became evident for the first time in the gonads of some fish inmixed sex group examined several days before morphological sex differentiation (about 23 days after
hatching). However, positive reactions to the four antibodies increased in number during the process ofovarian differentiation. In contrast, positive
immunostaining for all four steroidogenic enzymes
could not be confirmed in the testis during sex differentiation. Weakly positive reactions to P450scc, 3~­
HSD and P450c17 antibodies appeared first in thetestis at 30 days and staining intensity increased
slowly during development of the testis. P450arom
immunoreactivity was never found. Based on the presence of P450arom, we presume that fish possessing
gonads with positive reactions are genetic females andfish with gonads that did not stain with the antibodies
are genetic males. These results strongly suggest that
endogenous estrogens act as the natural inducers ofovarian differentiation in tilapia. In contrast, the ability of steroid-producing cells to synthesize steroid
hormones in the testes appears at the time of testiculardifferentiation.
Sequential, protogynous hermaphroditism, i.e. female to male sex change in mature fish, exemplifies
the plasticity of teleost germ cell differentiation. To
begin investigating the importance of hormonal signals on germ cell differentiation during sex change,
we examined the expression of P450arom mRNA in
the Thalassoma duperrey. Prior to sex change, thegonad of T. duperrey contains primordial germ cells
and oogonia but not spertnatogonia. P450arom mRNAwas detected by Northern analysis and RT-PCR in theovary. In situ hybridization revealed that P450arommRNA is localized in the granulosa layer ofvitellogenic follicles. These results confirm a majorrole for arorriatase in ovarian physiology. Shortly after
the initiation of sex change; however, expression of
P450arom mRNA decreases to undetectable levels.
P450arom mRNA remains undetectable in the newlydifferentiated testis. These results demonstrate that theloss of aromatase is a permanent and critical step inthe sex change cascade. As such, they suggest that
aromatase/estradiol is important for oogonial differentiation, detrimental to spermatogonial differentiation

NATIONAL INSTITUTE

or a combination of both in T. duperrey.

Publication List:
1. Original Papers
Chang, X.T., Kobayashi, T., Kajiura, H., Nakamura,
M. and Nagahama, Y. (1997). Isolation and characterization of the eDNA encoding the tilapia( Oreochromis niloticus) cytochrome P450
aromatase (P450arom): changes in P450arom
mRNA, protein and enzyme activity in ovarian follicles during oogenesis. J. Mol. Endocrinol. 18, 5766.
Ge, W., Peter, R.E. and Nagahama, Y. (1997). Activin
and its receptors in the goldfish. Fish Physiol.
Biochem. 17, 143-153.
Ge, W., Miura, T., Kobayashi, H., Peter, R.E. andNagahama, Y. (1997). Cloning of eDNA for goldfish activin ~B subunit, and the expression of its
mRNA in gonadal and non-gonadal tissues. J. Mol.
Endocrinol. 19, 37-45.
Ge, W., Tanaka, M., Yoshikuni, M., Eto, Y. andNagahama, Y. (1997). Cloning and characterizationof goldfish activin type liB receptor. J. Mol.
Endocrinol. 19, 47-57.
Haraguchi, S., Naito, K., Azuma, S., Sato, E.,
Nagahama, Y., Yamashita, M. and Nagahama, Y.
(1996). Effects of phosphate on in vitro 2-cell blockof AKR/N mouse embryos based on changes in
cdc2 kinase activity and phosphorylation states.
Bioi. Reprod. 55, 598-603.
Iwao ,Y., Yasumitsu, K., Narihira, M., Jiang, J.-Q. andNagahama, Y. (1997). Changes in microtubule
structures during the first cell cycle of physiologically polyspermic newt eggs. Mol. Reprod. Develop.
47, 210-221.
Katsu, Y., Yamashita, M. and Nagahama, Y. (1997).
Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA binding protein. Eur.
J. Biochem. 249, 854-861.
King, P.D., Zhao, Y., Sangoram, A.M., Wilsbacher,
L.D., Tanaka, M., Antoch, M.P., Steeves, T.D.L.,
Vitaterna, M.H., Kornhauser, J.M., Lowrey, P.L.,
Turek, F.W. and Takahashi, J.S. (1997). Positional
cloning of the mouse circadian Clock gene. Cell 89,
641-653.
Matsuyama, M., Morita, S., Hamaji, N., Kashiwagi,
M. and Nagahama, Y. (1997). Diurnal spermatogenesis and spawning in the secondary male of a
protogynous wrasse, Pseudolabrus japonicus(Teleostie, labridae). Zool Sci. 14, 1001-1008.
Mita. M., Yasumasu, I., Nagahama, Y. and Saneyoshi,
M. (1996). Change in the levels of adenine-related
compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance.
Dev. Growth Differ. 38, 413-418.
Oba, Y., Yoshikuni, M., Tanaka, M., Mita, M. andNagahama, Y. (1997). Inhibitory guanine-nucleotidebinding-regulatory protein a subunits in medaka(Oryzias latipes) oocytes: eDNA cloning and de-FOR BASIC BIOLOGY

creased expression of proteins during oocyte maturation. Eur. J. Biochem. 249, 846-853.
Tokumoto, M., Horiguchi, R., Yamashita, M.,
Nagahama, Y. and Tokumoto, T. (1997). Involvement of 26S proteasome in oocyte maturation ofgoldfish Carassius auratus. Zoot. Sci. 14, 347-351.
Tokumoto, T., Yamashita, M., Tokumoto, M., Katsu,
Y., Horiguchi, R., Kajiura, H. and Nagahama, Y.
(1997). Initiation of cyclin B degradation by the
26S proteasome upon egg activation. J. Cell Biol.
22, 1313-1322.

2. Reveiws and Proceedings
Chang, X.T., Nakamura, M., Kobayashi, T. andNagahama, Y. (1997). Immunocytochemical localization of steroidogenic enzymes in gonads during
sex differentiation in tilapia. J. Reprod. Develop. 43,
Suppl. 73-74.
Iwao, Y., Yasumitsu, K., Narihira, M., Jiang, J.Q. andNagahama, Y. (1997). Changes in microtubule
structures in physiologically polyspermic newt
eggs. J. Reprod. Develop. 43, Suppl. 157-158.
Mita, M., Yoshikuni, M. and Nagahama, Y. (1997). 1Methyladenine biosynthesis in ovarian follicle cellsof the starfish Asterina pectinifera. In Advances in
Comparative Endocrinology (Kawashima, S. andKikuyama, S., eds.), pp. 317-320, Monduzzi
Editore, Bologna, Italy
Mita, M., Yoshikuni, M. and Nagahama, Y. (1997).
Methylation process in 1-methyladenine production
by starfish ovarian follicle cells. J. Reprod. Develop.
43, Suppl. 99-100.
Nagahama, Y. (1997). 17a,20f3-Dihydroxy-4-pregnen3-one, a maturation-inducing hormone in fish oocytes: Mechanisms of synthesis and action. Steroids
62, 190-196.
Nagahama, Y., Miura, T., Kobayashi, T. and Ding, J.
(1997). The role of activin in spermatogenesis infish. In Inhibin, Activin and Follistatin (Aono, T.,
Sugino, H. and Vale, W.W., eds.), pp. 196-203,
Springer-Verlag, New York.
Yao, Z., Chang, X.T., Hirai, T., Todo, T., Yoshikuni,
M., Kobayashi, T. and Nagahama, Y. (1997). eDNA
cloning and protein expression of the gonadotropin
receptor from tilapia, Oreochromis niloticus. In Advances in Comparative Endocrinology (Kawashima,
S. and Kikuyama, S., eds.), pp. 317-320, Monduzzi
Editore, Bologna, Italy.
Young, G., Todo, 1;'., Kobayashi, T., Guan, G. andNagahama, Y. (1997). Steroidogenesis by the salmonid ovarian follicle: the two-cell type model revisited. In Advances in Comparative Endocrinology(Kawashima, S. and Kikuyama, S., eds.), pp. 14431449, Monduzzi Editore, Bologna, Italy.

27

28

NATIONAL INSTITUTEDIVISION OF CELL DIFFERENTIATION
Professor:
Yoshiaki Suzuki (until July)
Associate Professor: Kohji Ueno
Research Associates: Kaoru Ohno
Hiraki Kokubo (on leave)

The E complex is a homeotic gene complex which
specifies the identities of abdominal segments in thesilkworm, Bombyx mori. We have reported that the E
complex is similar to the Drosophila bithorax complex. The E complex comprises three homeobox
genes: Bombyx Ultrabithorax (Bm Ubx), Bombyx abdominal-A (Bm abd-A), and Bombyx Abdominal-B(Bm Abd-B). Since some types of mutations in theEcomplex cause abnormal development of abdominal
legs, theE complex regulates development of abdominal legs. Analyses of two mutants, the Eca;Eca (additional crescent) and ENfEN (new additional crescent)mutants, which lack abdominal legs demonstrated thatthe Bm abd-A is deleted within the mutant chromosomes. Therefore, Bm abd-A may be an essential genefor development of abdominal legs.
The homeodomain proteins encoded by homeotic
genes are proposed to act as transcriptional regulators
on target genes. To determine the target genes of the
Bm abd-A gene product, we biochemically analyzed
the proteins that are expressed specifically in abdominal legs and found that a high molecular weight protein (p260/270) was expressed specifically in abdominal leg cells during early embryonic stages and disappeared by a late embryonic stage. p260/270 consistsof two polypeptides with molecular weights of
260,000 and 270,000 daltons. We have established apurification procedure and have raised an antibody
against p260/270. Immunoblot analysis of the Eca;Eca
and ENfEN mutants demonstrated that the two mutants
lacked p260/270.
eDNA cloning and sequencing demonstrated thatp260 and p270 have structures similar to rat fatty acidsynthase, which synthesizes palmitate. Most of enzymatic domains for palmitate synthesis were well conserved in the amino acid sequences of p260 and p270.
Many viral, cytoskeletal and cell surface receptor proteins have been reported to be modified with palmitateat their cysteine residues. Some small GTP-binding,heterotrimeric G and G-protein-linked receptor proteins are known to be modified with palmitate via
thioester linkages. Such modifications may be important in regulation of signal transduction. Purified
p260/270 can transfer palmitate to cysteine residues ofsynthetic peptides in vitro. We propose that p260/270
may be involved in protein palmitoylation and may
function in abdominal leg development.

Publication List:

Kokubo, H., Ueno, K., Amanai, K. and. Suzuki, Y.
(1997) Involvement of the Bombyx Scr gene in development of the silk gland. Develop. Biol. 186, 4657
Ueno, K. and Suzuki, Y. (1997) p260/270 expressed inembryonic abdominal leg cells of Bombyx mori cantransfer palmitate to peptides J. Biol. Chern. 272,
13519-13526

FOR BASIC BIOLOGYDIVISION OF MORPHOGENESIS
Professor:
Naoto Ueno
Associate Professor: Hiroshi Shibuya
Research Associates: Makoto Nakamura
Makoto Mochii
Graduate Students: Hideyuki Nagaso (Hokkaido
University)
Masataka Nikaido (Hokkaido
University)
Yoshihiro Takatsu (Hokkaido University)
Michiru Nishita (Hokkaido University)
Katsura Sugawara (The Graduate
University for Advanced Studies)
Satoru Yoshida (The Graduate
University for Advanced Studies)
Shinichi Ohki (Hokkaido University)
Yoshinori Tomoyasu (Hokkaido
University)
Visiting Scientists: Shinichi Higashijima (PRESTO, STA)
Kiyokazu Morita (Hokkaido
University)
Fumihiko Hamada (Osaka University)
Technical Staff:
Chiyo Takagi
Takamasa Yamamoto
Shunichiro Iemura
Yuko Takahashi

Our laboratory joined the NIBB in May of 1997
from Hokkaido University. We have been studying
about the roles of growth factors in early embryogenesis.
Early developmental processes are regulated by anumber of endocrine and paracrine factors that mediate cell-to-cell interactions. Particularly, polypeptide
growth factors are believed to be essential for the
regulation of cell proliferation and differentiation inmorphogenesis during embryogenesis. One of the intriguing features of polypeptide growth factors is that
their functions are well conserved in animal species.
This allows us to study developmental mechanisms
governed by growth factors using multiple animalmodels such as mouse, Xenopus, Drosophila, and C.
elegans. We focus on a group of growth factors, theTGF-~ superfamily and investigate molecular mechanisms for their actions in early embryogenesis. Our
study involves biochemistry, embryology, molecularbiology and genetics ..

I. The role of follistatin in neural induction
It had been shown that bone morphogenetic protein
BMP is a key regulator of mesoderm patterning by
inducing ventral mesoderm in early Xenopus embryo.
BMP also has an essential role in neural tissue formation. Presumptive ectoderm of Xenopus embryo gives
rise to epidermis unless it is exposed to endogenous
factors(s) from dorsal mesoderm or exogenous stimuli
such as neural inducers. Therefore, it was believedthat epidermis is a ground (default) state and neuraltissue is induced by the neural inducers. However, it
has been demonstrated by us and other groups that
BMP specifies epidermis and inhibits neural fate. Thisanti-neural activity of BMP was later found to be

29

conserved in animal species, from fly to mammals.
More recently, it turned out that neural inducers
chordin and noggin are BMP-binding proteins which
inhibit BMP to interact with its receptor. These observations led to a model that not epidermal but neuralfate is the default state. Follistatin, an activin-binding
protein that inhibits activin activity had also beenshown to act as a neural inducer. Because activin itself
has no inhibitory action to neural induction, we questioned about the mechanism by which follistatin induces neural fate. Does follistatin mediate neuralinducing signal through its own receptor yet unknown? Or, does it inhibit the neural inhibitor BMP as
do chordin and noggin?
To address this problem, we hypothesized that binding specificity of follistain may be broader and bind
BMP as well as activin. In fact, follistatin was foundto inhibit ventralization caused by BMPs (Figure la,b). After a series of analyses employing an instrument
called surface plasmon resonance (SPR) sensor, wehave successfully demonstrated that follistatin interacts with three subtypes of BMP, namely homodimers
of BMP-2, BMP-4, and BMP-7 and a heterodimeric
BMP, BMP-4/7. Interestingly, we have also shownthat follistatin bind to a complex of BMP and BMP
receptor, thereby inhibiting receptor activation (Figurelc). It was previously shown that there are two
isoforms of follistatin generated by alternative splicing, one having an extended C-terminal. During thecourse of this study, we have found that the C-terminally extended form has much weaker inhibitory activity against BMP. We are currently undertaking a
study to understand the structure-function relationship.

II. Intracellular signaling of BMP through a novel
MAPKKK, TAKl
TGF-~ superfamily members including BMP are
known to elicit signals through simulation of a complex of serine/threonine kinase receptors consists oftype I and type II receptors. Recent studies of thissignaling pathway have identified two types of novel
mediating molecules, the Smads and TGF-~ activatedkinase 1 (TAKl). Smads are cytoplasmic proteins thatbind type I receptors but translocate to nucleus upon
stimulation of ligands. They have been shown to
mimic the effect of BMP, activin and TGF-~. In collaboration with groups of Dr. Matsumoto (Nagoya
University) and Dr. Nishida (Kyoto University), we
have identified TAKl and TAB 1 as a MAPKKK and
its activator, respectively, which might be involved in
the up-regulation of TGF-~ family-induced gene expression.
To understand biological significance, we isolated
Xenopus counterpart of TAKl and TABl and examined their role in the dorsoventral patterning of early
Xenopus embryo (Shibuya, H. et al. (1998) EMBO 1.,
17, 1019-1028). Ectopic expression ofTAKl in early
embryo induced cell death. Interestingly, however,concomitant overexpression of bcl-2 with the acti-

30

NATIONAL INSTITUTE

cllchordln

follistatin)(
Figure 1. Inhibition of BMP-induced ventralization of Xenopus embryo by follistatin . (a), ventralized embryo caused by BMP7 ectopic expression. (b), embryo rescued to normal by follistain coexpression with BMP-7. (c), proposed mechanism by which
follistatin inhibits receptor activation by BMP. In contrast to chordin that competes with the BMP receptor for BMP-binding,follistatin forms a ternary complex with BMP and its receptor.

vated TAKl or both TAK1 and TAB I in dorsal blastoni.eres not only rescued the cells but also caused theventralization of the embryo. In addition, a kinasenegative form ofTAKl (TAK1KN) which is known to
inhibit endogenous signaling could partially rescued
phenotypes generated by the expression of a constitutively active BMP-2/4 type I receptor. Moreover,
TAK I KN could block the expression of ventral markers normally induced by Smad 1 or 5 that mediates
BMP signal. Taken together, we proposed that TAKland TAB 1 function in the BMP signal transduction
pathway in Xenopus embryo in a cooperative manner.
Further study is currently undertaken to identify
downstream targets of BMP signals.
III. Use of zebrafish as a model animal to study
gene function
In addition to Xenopus, zebrafish is a useful model
animal because of several experimental advantages
over other vertebrates. Function of BMP in early
embryogenesis is also being studied using zebrafish.
Taking advantage of transparency of zebrafish embryos , we have been able to show that not relay but
direct diffusion is the mechanism by which BMP influences surrounding cells. Moreover, a visiting scientist Dr. Higashijima (PRESTO, STA) has succeeded inestablishing transgenic technology for zebrafish ·(Higashijima, S. et al. (1997) Dev. Bioi. 192, 289299). He used muscle actin promoter fused to green

fluorescent protein (GFP) gene to show that the geneis transmitted to germ line and maintained in offspringof at least five generations. This technology will allow us to study how organs and tissues are formed,
enabling us to observe promotor specific expression inlive embryos and adult animals.

IV. TGF-~ family in invertebrates
Nematode C. elegans and Drosophila provide powerful genetical approaches to understand the role of
TGF-~ family ligands and their signaling mechanism.
We identified a BMP/nodai-Iike ligand in C. elegans
and designated as cet-1 (.C.. glegans IGF-~). To understand the function of cet-1 , we isolated a null mutant
for cet-1 gene and found that the loss-of-function
mutant worms have shortened body length, suggestingthat cet-1 encodes a ligand that regulates body lengthof C. elegans. The molecular basis of the regulation ofbody length is our current interest. We plan to identify
genes that are up- or down-regulated in the mutant by
a newly developed method Differential Panel Display(DPD), using arrayed eDNA of C. elegans.
In Drosophila, decapentapledgic (dpp) encodes a
homologue of BMP. We have been studying the involvement of dpp in the formation of mechanosensory
sensory organ. Ectopic activation of Dpp signal by aconstitutively activated form of a dpp receptor tkv
Dpp receptor tkv increased the number ofmachanosensory organ. It was found that Dpp signal

FOR BASIC BIOLOGY

31

Figure 2. Interaction of dpp and wingless in the formation of the mechnosensory organ of Drosophila (a) increased mechanosensory
bristles in the nortal region by ubiquitous expression of. a constitutively active Dpp receptor Tkv. (b), induction of SOP cells(shown in red) nearby wingless expression domain (shown in green).

suppresses expression of another growth factor wingless. As a result, dpp induces sensory organ precursor(SOP) cells near by wingless expressing region. Position of SOP cells seems to be determined by the cooperation of at least two secreted growth factors , dpp
and wingless.

Publications List:
Akiyama, S., Katagiri, T., Namiki, M., Yamaji, N.,
Yamamoto, N., Miyama, K., Shibuya, H., Ueno,
N., Wozney, J. M. , Suda, T. (1997) Constitutively
active BMP type I receptors transduce BMP-2 signals without the ligand in C2C12 myoblasts. Exp.
Cell. Res. 15, 362-369.
Higashijima, S. , Okamoto, H., Ueno, N. , Hotta, Y.
and Eguchi, G. (1997) High-frequency generationof transgenic zebrafish which reliably express GFP
In whole muscles or the whole body by using promoters of zebrafish origin. Dev. Bioi. 192, 289-299
Miya, T., Morita, K., Suzuki, A., Ueno, N. and Satoh,N. (1997) Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its rolein the inhibition of neural fate specification. Development 124, 5149-5159.
Mochii , M., Ono, T., Matsubara, Y. and Eguchi, G.
(1998) Spontaneous transdifferentiation of quailpigmented epithelial cell is accompanied by a mutation in the Mitf gene. Dev. Biol. 193, 47-62.
Namiki, M. , Akiyama, S., Katagiri, T., Suzuki, A.,
Ueno, N., Yamaji , N., Rosen , V. , Wozney, J. M.,
Suda, T. (1997) A kinase domain-truncated type I
receptor blocks bonemorphogenetic protein-2-induced signal transduction in C2C12 myoblasts. J.
Biol. Chern. 272, 22046-22052.
Natsume, T., Tomita, S., lemura, S., Kintou. N.,
Yamaguchi, A. and Ueno, N. (1997) Interaction
between soluble type I receptor for bone morphoge-

netic protein and bone morphogenetic protein-4. J.
Bioi. Chern. 272, 11535-11540.
Nikaido, M., Tada, M., Saji, T. and Ueno, N. (1997)
Conservation of BMP signaling in zebrafish mesoderm patterning. Mech. Dev. 61, 75-88.
Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y. ,Yamaguchi, K., Irie, K., Matsumoto, K., Nishida, E.
and Ueno, N. Role of TAKl and TAB1 in BMP
signaling in early Xenopus development. (1998)
EMBO J. 17, 1019-1028.
Suzuki, A., Kaneko, E., Maeda, J. , Ueno, N. (1997)
Mesoderm induction by BMP-4 and -7 heterodimer.
Biochem. Biophys. Res. Commun. 232, 153-156.
Suzuki, A., Kaneko, E., Ueno, N., HemmatiBrivanlou, A. (1997) Regulation of epidermal induction by BMP2 and BMP7 signaling. Dev. Bioi.
189, 112-122.
Suzuki, A. , Ueno, N. , and Hemmati -Brivanlou, A.
(1997) Xenopus msxl mediates epidermal induction
and neural inhibition by BMP4. Development 124,
3037-3044.
Tonegawa, A., Funayama, N., Ueno, N., Takahashi, Y.
(1997) Mesodermal subdivision along themediolateral axis in chicken controlled by different
concentrations of BMP-4. Development 124, 19751984.

32

DIVISION OF DEVELOPMENTAL BIOLOGY
Professor: Kenzo Nakamura (Nagoya University)
Associate Professor: Tsukaho Hattori (Mie University)
Research Associate: Masa-aki Ohto
Graduate Students: Yasuhiro Koide (Nagoya University)
Yukiko Iwata (Nagoya University)

Availability of sugars and the inter-organ transportand distribution of sugars are essential in the growthand development of the plant body. Expression of avariety of plant genes is regulated, either positively or
negatively depending on the gene, by the level ofsugars at the sugar-importing sink sites as well as at
the sugar-exporting source sites of the plant body.
Thus sugars are not only important as sources forcellular energy and the synthesis of macromolecules
but also as a signal controlling the growth and development of plants by changing the pattern of gene
expression. Our research attention is focused toeludicate the mechanisms involved in the regulation ofgene expression in response to sugars, especially theactivation of gene expression by increased-levels ofsugars, and the role of such regulation in the organ
development in plants. In addition, mechanisms involved in the accumulation of storage proteins in plantvacuoles are also studied.

I. Regulatory factors involved in the sugarinducible expression of plant genes
Expression of genes coding for sporamin and Pamylase, two major proteins of the storage roots ofsweet potato, is inducible by high levels of sugars invarious vegetative tissues. The GUS reporter genes
under the control of the promoters of these genes arealso inducible by sugars in leaves of transgenic tobacco plants, and these fusion genes are expressed in
tubers of transgenic potato plants. Although the induction of expression of these fusion genes requires theactivity of hexokinase, phosphorylation of hexose by
hexokinase is not sufficient to cause the induction.
The induction requires Ca2+-signalling and the activityof protein kinase. Eight different cDNAs for theisoforms of calcium-dependent protein kinase(CDPK) were isolated from leaves of tobacco, andtranscripts of two of them were found to be increased
upon treatment of leaves with various metabolizablesugars. Antibodies against a fragment of one of these
isoforms cross-reacted strongly with the 57 kDa-protein in the soluble fraction from the young leaves. The
level of this 57 kDa-protein decreased significantly asleaf matures, while the level of this 57-kDa protein in
mature leaves increased significantly after the treatment of leaves with sugars. The sugar-induction of the
57-kDa protein occured preceding the induction of
expression of the P-amylase:GUS reporter gene. In
addition, a 54 kDa-protein with autophosphorylation
acitivity in the plasma membrane of mature leaves .
also increased significantly upon treatment of leaves
with sugars. This protein was purified to about 1,000-

NATIONAL INSTITUTE

fold compared to the crude extract. It phosphorylated
histone IllS in a Ca2+-dependent manner and crossreacted with an antibody against CDPK of
Arabidopsis thaliana. These results suggest the possible involvement of CDPKs in the sugar-inducible
gene expression and the development of leaves.

II. Mutants of Arabidopsis thaliana with alteredpatterns of the sugar-inducible gene expression
Expression of the P-amylase gene of Arabidopsis
thaliana (At{3-Amy) in vegetative tissues occurs in response to high levels of sugars. A recessive mutation,lbal, caused significantly reduced-level of expressionof At{3-Amy under high levels of sugars, while a recessive mutation, hbal, caused increased levels of expression in response to lower levels of sugars. It issuggested that HBAJ might function to maintain lowlevel expression of At{3-Amy until the level of sugars
reaches some high levels and that the expression ofAt{3-Amy is regulated by a combination of both positive and negative regulation depending on the level ofsugars.
A sequence between -172 and -61 of At{3-Amy was
not only essential to drive the sugar-inducible expression of the GUS reporter gene but also able to confer
sugar-inducibility to the core promoter of the constitutive gene. Linker scanning analysis identified two
cis-regulatory elements between -172 and -62 of At{3Amy, namely the negative element A and the positiveelement B. The element A contained a sequencewhich is highly homologous to the sequence motifthat is conserved between sporamin and P-amylase
genes of sweet potato and required for their sugarinducible expression. The gel-mobility shift assay of
the nuclear extracts identified activities binding tothese regions.
Neither lbal nor hbal mutation affected the sugarregulated gene expression in general suggesting thatsugar-regulated expression of a variety of plant genes
is mediated by multiple mechanisms. Nevertheless,lbal and hbal mutant plants showed pleiotropic effects on the sugar-inducible accumulation of anthocyanin. Furthermore, these mutant plants showed several characteristic growth properties. For example,
lbal mutant plants showed defective leaf development
under the carbohydrate-limiting growth conditions
and altered patterns of root development. The flowering time of the Arabidopsis plants under different
carbohydrate availability was altered in the hbal mutant plants. These results suggest that products of
LBAJ and HBAJ genes might affect the organ development either directly or indirectly.

III. Screening of mutants of Arabidopsis thalianawith defective growth and development.
During the growth of plants, new organs develop as
carbohydrate sink, and many vegetative organs shows
sink to source transition after their maturation. Many
aspects of the organ development in higher plants are

33

FOR BASIC BIOLOGY

thought to be affected, to some degree, by the levelsof sugars. Sugars seem to have influence on the meristematic transition in long day plants from vegetative
to reproductive growth. To obtain insights into the roleof sugar-regulated gene expression in the growth andorgan development in plants, we are screening formutants of Arabidopsis thaliana with defects oranomalies both in the sugar-regulated gene expression
and in the developmental processes such as leaf development and the determination of the flowering time.
To aid this purpose, we have established more than
4,000 independent lines of Arabidopsis plants transformed with T-DNA containing multiple copies of theenhancer sequence. We have identified several plantlines with defects in the development of leaves oranormalies in the flowering time which also show thealtered patterns of the sugar-regulated gene expression(Fig. 1).

Selected Publications:
Mita, S., Murano, N.,Akaike, M. and Nakamura, K.
(1997) Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for ~­amylase and of the accumulatio_n of anthocyanin
that are inducible by sugars. Plant J. 11: 841-851.
Matsuoka, K., Higuchi, T. , Maeshima, M. andNakamura, K. (1997) A vacuolar-type H+-ATPasein non -vacuolar organelle is required for the sortingof soluble vacuolar protein precursors in tobacco
cells. Plant Cell 9: 533-546.
Mita, S., Hirano, H. and Nakamura, K. (1997) Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana; a recessive mutation causing enhanced expression of a gene for bamylase. Plant Physiol. 114: 575-582.
Koide, Y., Hirano, H. , Matsuoka, K. and Nakamura,
K. (1997) The N-terminalpropeptide of the precursor to sporamin acts as a vacuolar targeting signal
even at the C-terminus of the mature part in tobacco
cell s. Plant Physiol. 114: 863-870.

(A)

(B)

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Fig 1 Phenotypes of the wild type plants (WT) and homozygous mutant line (#154) that were grown for 3 weeks. The

chlor~phyll content of the mutant plants is much lower than that of the wild type plants (Fig.lA). Mutant plants also showed ~ereduced levels of the sugar-inducible increase of ~-amyl ase (left) and of anthocyanin (right) when leaf explants were treated Withhigh levels (5 %) of sucrose (Fig.lB).

34

NA'ITONALINS.TITU'TE

35

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Norio Murata

The Department is composed of two regular divisions andtwo adjunct divisions. The study of this department isfocused on the molecular mechanisms for the development ofcentral nervous systems in mice, chickens and Drosophila, andalso on the molecular mechanisms for the response of plants
toward external environments, such as light, temperature andsalinity.

36

NATIONAL INSTITUTE

Our efforts have been devoted to studying
molecular and cellular mechanisms underlying thedevelopment of the vertebrate central nervous system.
We are screening for molecules and structures that
regulate various cellular events in brain morphogenesis such as generation of neuroblasts, their migration
to form the laminar structure and various nuclei, elongation and path-finding of neural processes, and theformation and refinement of specific connections between neurons. Our research has been conductedusing various techniques including molecular biology(e.g. eDNA cloning, site-directed mutagenesis), biochemistry (protein, carbohydrate), immunologicalmethods (monoclonal-antibody production), neuroanatomy, cell and organotypic culture (immortalized
cell production), and embryo manipulation (classicalembryology, gene transfer with viral vectors, and genetargeting).

retina, respectively, and our misexpression experiments using a retrovirus vector suggested that thesetranscription factors direct the retinal ganglion cellaxons to choose the appropriate tectal targets along
the antero-posterior axis.
To examine topographic molecules which show
asymmetrical distributions in the embryonic retina, wehave undertaken a large-scale screen using a new
eDNA display system called Restriction Landmark
eDNA Scanning (RLCS) (Fig. 1). A number of molecules displaying asymmetrical expression along thenaso-temporal axis or dorso-ventral axis in the retina
have been identified. This approach seems to bepromising because topographic molecules which are
already known such as CBF-1, CBF-2, EphA3, aldehyde dehydrogenase etc. were included among those
identified. Sequence analyses of the eDNA clones
and examination of their expression patterns during
development are currently underway. We expect that
our studies will lead to systematic identification of aseries of topographic molecules in the retina, and to
elucidation of the molecular mechanisms underlying
formation of the topographic retinotectal projection.
After reaching their appropriate sites along therostro-caudal and dorso-ventral axes of the tectum,
retinal axons begin to seek their appropriate termination sites among 15 distinct laminae within the tectum. The molecular and cellular bases of such discrete target choice are poorly understood, although
our previous experiments suggested that molecularcomplementarity between retinal axons and tectal targets underlies lamina-specific synapse formation. We
have generated a series of monoclonal antibodies thatrecognize one of these retinal termination laminae
or a subset of retinal ganglion cells. We hope thatcharacterization of these antigens and their cellular
localizations in this relatively accessible system
will provide insight into the complicated and stillunresolved aspects of neural specificity.

I. Topographic map and synapse formation in theretinotectal system

II. Receptor-like protein tyrosine phosphatases andbrain development

Topographic maps are a fundamental feature ofbrain organization. In the avian visual system, thetemporal (posterior) retina is connected to the rostral(anterior) optic tectum, nasal (anterior) retina to thecaudal (posterior) tectum, and likewise dorsal andventral retina are connected to the ventral and dorsal
tectum, respectively. About half a century ago, Sperry
proposed that topographic mapping could be guided
by complementary positional labels in gradients
across pre- and postsynaptic fields. Although thisconcept is widely accepted today, the developmental
mechanism has not been fully characterized at themolecular level. In 1996, using a subtractive hybridization technique, we discovered several distinct transcripts which are topographically expressed in thedeveloping chick retina. Among these molecules, twowinged-helix transcriptional regulators termed CBF-1
and CBF-2 were expressed in the nasal and temporal

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. Recently, many typesof receptor-like protein tyrosine phosphatases(RPTPs) have been cloned and characterized. In
1994, we found that PTP~/RPTP~, a nervous systemspecific RPTP, is expressed as a chondroitin sulfate
proteoglycan in the brain. An RNA splice variant
corresponding to the extracellular region of PTP~ issecreted as a major proteoglycan in the brain known
as 6B4 proteoglycan!phosphacan. The extracellularregion of PTP~ consists of a carbonic anhydrase-likedomain, a fibronectin type III-like domain and aserine-glycine-rich region, which is considered to be
the chondroitin sulfate attachment region. This
unique structure of PTP~ has attracted a great deal of

DIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Masaharu Noda
Associate Professor: Nobuaki Maeda (Oct. 15, 1997 -)Research Associates: Masahito Yamagata
Eiji Watanabe
Post doctral Fellow: Junichi Yuasa 1
Takafumi Shintani 4
Masakazu Takahashi 4
Hiroyuki Kawachi 4
Hiraki Sakuta 4 (Oct. 1, 1997 -)Graduate Students: Taeko Nishiwaki
Chika Saegusa
Akira Kato
Ryoko Suzuki (Oct. 1, 1997 -)Visiting Scientists:
Angela Mai (Apr. 1 - Oct. 31,
1997)
Ikuko Watakabe
Akiko Kawai
Technical Staff:
Shigemi Ohsugi
JST Technical Staff: Masae Mizoguchi (May 1, 1997 -)Megumi Goto (Jun. 11, 1997 -)37

FOR BASIC BIOLOGY

Temporal

Nasal

..

.-

•

/

•

"'... .

--.._....,.:_. ~

•

•

•

Fig.! RLCS profiles for the chick nasal and temporal retina
Topographic molecules were detected as spots specifically found on one side. As an example, see the spot arrowed in the right
below panel, the enlargement of a part of the above picture.

attention but little is known about the functional rolesof this molecule.
In 1996, we found that PTP~ binds pleiotrophin/heparin-binding growth-associated molecule (HBGAM) . In the embryonic rat brain, pleiotrophin is
localized along the radial glial fibers, a scaffold forneuronal migration. On the other hand, immunohistochemical studies of normal rat brain indicated that
PTP~ is expressed in the migrating neurons, suggesting that the ligand-receptor relationship between
PTP~ and pleiotrophin plays a role in migration ofneurons during brain development. Thus, we examined the roles of pleiotrophin-PTP~ interaction in neuronal migration using the Boyden chamber cell migration assay. Pleiotrophin coated on the membranes
stimulated migration of cortical neurons. Polyclonal
antibodies against the extracellular domain of PTP~,
6B4 proteoglycan, a secreted extracellular form of
PTP~, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium
strongly suppressed the pleiotrophin-induced neuronalmigration. Furthermore, chondroitin sulfate C but not
chondroitin sulfate A inhibited pleiotrophin-induced
neuronal migration, in accordance with our previous
findings that chondroitin sulfate constitutes part of thepleiotrophin-binding site of PTP~, and PTP~-

pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. These results
suggest that PTP~ is involved in neuronal migration asa neuronal receptor of pleiotrophin distributed alongradial glial fibers.
Furthermore, to examine the functions of PTP~ invivo, we generated PTP~-deficient mice in which the
PTP~ gene was replaced by the LacZ gene by genetargeting. Firstly, we examined the cell types expressing PTP~ by investigating the expression of LacZ inheterozygous PTP~-deficient mice. Throughout development from the early stage of embryogenesis,
LacZ staining was restricted to the nervous system.
At embryonic day 12.5 (El2.5), LacZ staining wasobserved in the forebrain, midbrain, hindbrain andspinal cord (Fig. 2). In the adult heterozygous mice,
LacZ expression was abundant in the olfactory bulb,
cerebral cortex, hippocampus, thalamus and cerebellum. In the hippocampus, strong reporter gene expression was observed in the pyramidal neurons andin the granule cell layer of the dentate gyrus. We are
currently examining the phenotype of homozygous
PTP~-deficient mice.

38

NATIONAL INSTITUTE

Fig.2 LacZ expression in El2.5 heterozygous PTPs-deficient mice.

III. Gene targeting of Na-G, a voltage-gatedsodium channel expressed in glial cells
Glial cells have been considered to be inexcitable.
Despite the lack of electrical excitability, they express
voltage-gated sodium channels with properties similar
to the sodium channels in excitable cells. The cellularfunction of these voltage-gated sodium channels is notclear. An in vitro study raised the possibility that glial
sodium channels serve as a pathway for sodium ion
entry to fuel Na-K ATPase, which requires three sodium ions for each pair of potassium ions transported:a drop in the intracellular concentration of sodium
ions through this channel may hamper ATPase activity. To test this possibility and clarify the roles of the
glial sodium channel in vivo, we are currently attempting to generate a knock-out mouse deficient in
the glial sodium channel, Na-G . We have successfully
produced chimeric mice which contain the targeted
embryonic stem-cells.
By applying gene targeting technology to othernovel genes expressed in the brain, we hope to shedlight on the molecular mechanisms underlying thedevelopment and function of the brain.

Publication List:
Hamanaka, H., Maeda, N. and Noda, M. (1997)
Spatially and temporally regulated modification of
the receptor-like protein tyrosine phosphatase ~IP
isoforms with keratan sulfate in the developing
chick brain. Eur. J. Neurosci. 9, 2297-2308 .
Matsui , F. , Nishizuka, M ., Yasuda, Y. , Aono , S. ,Watanabe, E. and Oohira,A. (1998) Occurrence ofanN-terminal proteolytic fragment of neurocan, nota C-terminal half, in a perineuronal net in the adultrat cerebrum. Brain Res. , 790, 45-51 (1998).
Nishiwaki, T., Maeda, N. and Noda, M. (1998)
Characterization and developmental regulation ofproteoglycan-type protein tyrosine phosphatase ~/RPTPP isoforms. J. Biochem., 123, 458-467.
Noda, M. , Yamagata, M. , Yuasa, J. and Takahashi, M.
(1997) Topographic and laminar connection in thechick retinotectal system. In Molecular basis ofaxon growth and nerve pattern formation (H .
Fujisawa, ed.) pp. 197-214. Japan Scientific Societies Press, Tokyo.
Shintani, T., Maeda, N. , Nishiwaki, T. and Noda, M.
(1997) Characterization of rat receptor-like protein
tyrosine phosphatase y isoforms. Biochem. Biophys.
Res. Comm. 230, 419-425 .

FOR BASIC BIOLOGYDIVISION OF CELLULAR REGULATION
Professor:
Associate Professor:
Research Associates:

Norio Murata
Koji Mikami
Atsushi Sakamoto
Yoshitaka Nishiyama!wane Suzuki
Monbusho Foreign Scientists: Zolttin Gombosn
Rajinder S. Dhindsa2!
Institute Research Fellow:
Akira Katoh
JSPS Visiting Scientists:
Govindjee31
Vyacheslav V. Klimov4!
P. Pardha SaradhP1
Peddisetty Sharmila 5!
JSPS Postdoctoral Fellows: Alia51
Michael P. Malakhov 6!
Ana Maria Otero CasaF1
EC Fellow:
Visiting Scientists:
Prasanna MohantyB1
George C. Papageorgiou9!
Tony H.H. Chen 101
Sisinthy Shivaji111
Tom Wydrzynski 121
Balazs SzalontainDmitry A. Los61
Rajni Govindjee3!
Suleyman I. Allakhverdiev4!
Julian Eaton-Rye 131
Kostas Stamatakis 91
Liberato Marzullo 141
Nikolai Moseiko 151
Otto Zsiros 11
Oxana Malakhova 16!
Visiting Fellow:
Yasushi Tasaka
Sayamrat Panpoom
Graduate Students:
Masami lnaba
Hiroshi Yamamoto
Yuji Tanaka
Fumiyasu Yamaguchi
Ryoma Suzuki
Sho-ichi Higashi
Technical Staff:
Hideko Nonaka
from Biological Research Center, Szeged, Hungary
from McGill University, Montreal, Canada
31 from University of Illinois, Urbana, USA
41 from Institute of Soil Science and Photosynthesis,
Pushchino, Russia
51 from Jamia Millia Islamia University, New Delhi, India
6 ! from Plant Physiology Institute, Moscow, Russia
71 from Universidad de Santiago, Santiago deCompostela, Spain
81 from Jawaharlal Nehru University, New Delhi, India
91 from National Centre for Scientific Research"Demokritos", Athens, Greece
101 from Oregon State University, Corvallis, USA
111 from Centre for Cellular and Molecular Biology,
Hyderabad, India
121 from Australian National University, Canberra,
Australia
131 from Otago University, Dunedin, New Zealand
141 from International Institute of Genetics and Biophysics,
Naples, Italy
151 from Institute of Molecular Genetics, Moscow, Russia
161 from Institute of Common Genetics, Moscow, Russia
11

21

The research effort of this division is aimed at developing a full understanding of the molecular mecha-

39

nisms by which plants are able to acclimate to andtolerate stresses that arise from changes in environmental conditions, with particular emphasis on temperature and salt stresses. In 1997, significant progress
was made in research on the following topics in studies with cyanobacteria and higher plants as experimental materials.

I. Improved performance of photosynthesis withpolyunsaturated membrane lipids
We previously inactivated genes for fatty-aciddesaturases by targeted mutagenesis in the
cyanobacterium Synechocystis sp. PCC 6803 and we
produced several mutant strains that contained abnormal numbers of unsaturated bonds in the fatty acids oftheir membrane lipids. By comparing these strains, wedemonstrated that polyunsaturated membrane lipids
are important in the ability of the photosynthetic machinery to tolerate low temperatures. In order to extend this finding by an alternative approach, we introduced the desA gene for the ~12 fatty-acid desaturaseof Synechocystis sp. PCC 6803 into cells of
Synechococcus sp. PCC 7942 that normally containmonounsaturated fatty acids but no polyunsaturated
fatty acids. This transformation resulted in replacement of half of the monounsaturated fatty acids in themembrane lipids by diunsaturated fatty acids. Comparison of the transformed cells with the wild-type
cells revealed that the increase in the number ofdouble bonds in the membrane lipids enhanced theability of the cells to resist photoinhibition at low temperatures by accelerating the recovery of the photosystem II complex from the photoinhibitory damage.
These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic
machinery from damage caused by strong light at low
temperatures.
To characterize biochemically the M2 desaturase of
Synechocystis sp. PCC 6803, we overexpressed the
enzyme in Escherichia coli. The overexpressed enzyme
was active; it was associated with cell membranes andrepresented about 10% of the total cellular protein. The
activity of desaturase was very stable in the presenceof 2 M sorbitol between pH 7 and pH 8. Purificationof the desaturase is in progress.

II. Enhancement of stress tolerance in plants bygenetic engineering: transformation with a genethat confers the biosynthesis of glycinebetaine
Glycinebetaine (hereafter abbreviated as betaine) is
a compatible solute that is found in a number of
halotolerant species of plants and bacteria. It has been
implicated in the protection of cellular functions
against salt and other types of environmental stress.
To examine the effect of betaine in vivo on the protection of the photosynthetic machinery against salt
stress, we transformed Arabidopsis thaliana, which
does not normally accumulate betaine, with the codA
gene for choline oxidase (which catalyzes the conver-

40

NATIONAL INSTITUTE

sion of choline to betaine) from Arthrobacter
globiformis. Transgenic Arabidopsis plants had theability to synthesize betaine and to tolerate salt stress.
Furthermore, transformation with the codA gene enhanced the protection against low-temperature
photoinhibition and accelerated the recovery fromphoto-induced damage. Thus, accumulation of betaine
both enhanced salt tolerance and contributed to resistance to photoinhibition at low temperature. Betaine
was also accumulated in the seeds of transformed
plants and, as a result, the seeds were more tolerant
than seeds of wild-type plants to low temperaturesduring imbibition and germination. Furthermore,transformation with the codA gene accelerated thegrowth of young seedlings at low temperatures.
We also introduced the codA gene into rice plants
by Agrobacterium-mediated transformation, aiming toenhance the tolerance of this important crop to salt
stress. Transformed rice plants accumulated betaine
and, consequently, exhibited enhanced ability to tolerate salt stress. Figure 1 shows wild-type rice plants
and transformed plants after growth under salt-stressconditions. During the stress treatment with 0.15 M

NaCl , growth of both wild-type and transformed
plants was inhibited and obvious damage, such as
wilting, bleaching of chlorophyll and necrosis, wasvisible. After removal of the salt stress, the growthrate of transformed plants returned to normal but thatof wild-type plants did not recover. The oxygenevolving machinery of the transformed plants wasmore tolerant to salt stress than that of the wild-type
plants. Our results in Arabidopsis and rice demonstrate the potential usefulness of the codA gene in theengineering of stress tolerance in a wide variety ofagronomically important crops.

III. A new factor involved in the heat stability ofthe photosynthetic machinery
The evolution of oxygen is one of the reactions ofphotosynthesis that is extremely susceptible to hightemperature. The molecular mechanism underlying
the stabilization of the photosynthetic machinery
against heat-induced inactivation has been studied in
the cyanobacterium Synechococcus sp. PCC 7002. We
demonstrated previously that cytochrome c 550, located

ChiCOD CytCOD

WT

Fig. l. Wild-type rice plants (WT) and rice plants (ChiCOD and CytCOD) transformed with the codA gene from Arthrobacter
globiformis after growth under salt stress. Three-week-old plants that had been grown under normal conditions were subjected tosalt stress by exposure to 0.15 M NaCI for one week. They were then allowed to grow under normal conditions for three weeks.

41

FOR BASIC BIOLOGY

in the lumenal side of thylakoid membranes, is involved in the stability of the oxygen-evolving machinery at high temperatures. We then initiated a searchfor other factors that might enhance heat stability and
we identified a protein of 13 kDa as an importantfactor. The gene for the 13-kDa protein was cloned
from Synechococcus, and the deduced amino-acid sequence revealed that this protein was homologous to
PsbU, an extrinsic protein of the photosystem II complex. Targeted mutagenesis of the psbU gene in
Synechococcus sp. PCC 7002 resulted in cells inwhich oxygen-evolving activity was particularly susceptible to high temperature. These results indicatethat PsbU plays an important role in stabilizing theoxygen-evolving machinery at high temperatures.

IV. Stress-dependent enhanced expression ofcytochrome eM and suppression of expression ofcytochrome c 6 and plastocyanin
Cytochrome eM is a c-type cytochrome with a molecular mass of 8 kDa. We identified and cloned thecytM gene for cytochrome eM from Synechocystis sp.
PCC 6803. In 1997, we cloned homologs of the cytM
gene from other cyanophytes and a prochlorophyte,
providing evidence that the cytM gene is probably
distributed universally in oxygenic photosynthetic
prokaryotes. Northern-blotting analysis revealed thatthe cytM gene of Synechocystis sp. PCC 6803 wasbarely expressed under normal growth conditions butthat expression was induced when cells were exposed
to low temperature and/or high-intensity-light. By
contrast, expression of the pet] gene for cytochromec6 and the petE gene for plastocyanin, which are electron carriers that transport electrons from the cytochrome b/f complex to the photosystein I complex,
was suppressed at low temperatures or under highintensity-light. These observations suggest that regulation of the expression of the cytM gene was the mirror
image of regulation of the pet] and petE genes under
the stress conditions examined.

Publication List:(1) Original articles
Deshnium, P., Gombos, Z., Nishiyama, Y. and Murata,
N. (1997) The action in vivo of glycine betaine inenhancement of tolerance of Synechococcus sp.
strain PCC 7942 to low temperature. J. Bacteriol.
179, 339-344.
Gombos, Z., Kanervo, E., Tsvetkova, N., Sakamoto, T.,
Aro, E.-M. and Murata, N. (1997) Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane
glycerolipids. Plant Physiol., 115, 551-559.
Hayashi, H., Alia, Mustardy, L., Deshnium, P., Ida, M.
and Murata, N. (1997) Transformation of
Arabidopsis thaliana with the codA gene for choline
oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J., 12,
133-142.

Hideg, E. and Murata, N. (1997) The irreversible
photoinhibition of the photosystem II complex inleaves of Vi cia faba under strong light. Plant Sci.,
130, 151-158.
Kanervo, E., Tasaka, Y., Murata, N. and Aro, E.-M.
(1997) Membrane lipid unsaturation modulates processing of photosystem II reaction-center protein
D1 at low temperature. Plant Physiol., 114, 841849.
Los, D.A., Ray, M.K. and Murata, N. (1997) Differences in the control of the temperature-dependent
expression of four genes for desaturases in
Synechocystis sp. PCC 6803. Mol. Microbiol., 25,1167-1175.
Nishiyama, Y., Los, D.A., Hayashi, H. and Murata, N.
(1997) Thermal protection of the oxygen-evolving
machinery by PsbU, an extrinsic protein of photosystem II, in Synechococcus species PCC 7002.
Plant Physiol., 115, 1473-1480.
Sakamoto, T., Higashi, S., Wada, H., Murata, N. andBryant, D.A. (1997) Low temperature-induced
desaturation of fatty acids and expression ofdesaturase genes in the cyanobacterium
Synechococcus sp. PCC 7002. FEMS Microbiol.
Lett., 152, 313-320.
Sato, N., Maruyama, K., Nishiyama, Y. and Murata,
N. (1997) Identification of a cold-regulated RNAbinding protein from the marine cyanobacterium
Synechococcus sp. PCC 7002. J. Plant Res., 110,405-410.

(2) Review articles
Hayashi, H. and Murata, N. (1997) Genetically engineered enhancement of salt tolerance in higher
plants. In Stress Responses of Photosynthetic Organisms (K. Sato and N. Murata, eds.) pp. 133-148.
Elsevier Science, Amsterdam.
Murata, N. and Los, D.A. (1997) Membrane fluidity
and temperature perception. Plant Physiol., 115,
875-879.
Murata, N. and Nishiyama, Y. (1997)" Molecularmechanisms of the low-temperature tolerance of thephotosynthetic machinery. In Stress Responses of
Photosynthetic Organisms (K. Sato and N. Murata,
eds.) pp. 93-112. Elsevier Science, Amsterdam.
Murata, N. and Tasaka, Y. (1997) Glycerol-3-phosphate acyltransferase in plants. Biochim. Biophys.
Acta, 1348, 10-16.

42

NATIONAL INSTITUTEDIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADJUNCT)
Associate Professor (adjunct): Hirokazu Kobayashi
Research Associate:
Noritoshi Inagaki

The conversion of light energy into chemical energy
used for C02 assimilation is the process of photosynthesis, one of the most important biological events.
The photosynthesis utilizing water as an electron donor occurs in cyanobacteria and in chloroplasts in all
eukaryotic algae and plants. The architecture of machinery for photosynthesis is directed by both nuclear
and plastid genes in plants. The extremely efficientenergy conversion is ensured by the highly orderedorganization of molecules in photochemical reaction
centers. The projects in this division are aiming toelucidate the mechanisms underlying the biogenesisof the photosynthetic machinery and the dynamic organization of molecules in the photosystem II (PS II)reaction center.
I. Tissue-specific expression of plastid genes(1) Mechanisms of suppressed levels of transcripts
for plastid photosynthesis genes in nongreen tissues
such as roots and calli were analyzed employing thehigher plant Arabidopsis thaliana which is suited forfurther genetical dissection. A region encoding promoters of rbcL for the large subunit of ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco) andatpB!E operon for ~ and E subunits of coupling factor
one was cloned and sequenced. Transcripts both for
rbcL and psbA (gene for D 1 protein in PS II reaction
center) were undetectable and 16S rRNA was detected
at a low level in roots of A. thaliana. The run-ontranscription experiment revealed that expression of
rbcL and psbA was regulated at transcription or upper
steps in gene expression. The copy number of plastid
DNA in roots was one-fifth of that in green leaves on
the basis of total cellular DNA. Digestion of DNA
with methyl-sensitive and -insensitive isoschizomericendonucleases and subsequent polymerase chain reaction (PCR), as well as in vitro run-off transcription,
resulted in no evidence of the regulation by DNA
modification. In spite of predominant suppression of
expression of rbcL and psbA at transcription in roots
and calli, 16S rRNA levels there decreased in theresult of low RNA stability.
(2) Genes for s-like factors of bacterial-type RNA
polymerase have not been characterized from any
multicellular eukaryotes, although they likely play a
crucial role in the expression of plastid photosynthesis
genes. We have cloned three distinct cDNAs designated SIG 1, SIG2, and SIG3 for polypeptides possessing amino acid sequences for domains conserved in
cr70 factors of bacterial RNA polymerases from A.
thaliana. Each gene is present as one copy per haploid
genome without any additional sequences hybridizedin the genome. Transient expression assays using
green fluorescent protein (GFP) demonstrated that

N-terminal regions of the SIG2 and SIG3 open reading frames could function as transit peptides for import into chloroplasts. Transcripts for all three SIG
genes were detected in leaves but not in roots, andinduced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of
the analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, expressed levelsof the genes may influence transcription by regulating
RNA polymerase activity in a green tissue-specificmanner.
II. Dynamic aspects of molecular organization of
PS II
We have focused on elucidation of molecularmechanism of the C-terminal processing of the D1
protein known to be an integral subunit of the PS II
reaction center. D1 protein is synthesized as a precursor form furnished with a C-terminal extension. The
specific excision of the C-terminus is well characterized to be an essential event for construction of thephotosynthetic oxygen-evolving machinery. Therefore, the protease, CtpA, involved in this process wascharacterized in biochemical and molecular biological
techniques. However, the catalytic mechanism of theprotease has not yet been demonstrated. We haveconstructed a series of systematic mutants of the
cyanobacterium Synechocystis sp. PCC 6803, in
which one of the putative catalytic residues of theprotease was substituted to alanine by conventionalsite-directed mutagenesis. Biological and biochemical analyses of these mutants suggest that essential
residues for the protease reaction are Ser313 andLys338 in the Synechocystis CtpA sequence. It is
speculated that the residues constitute Ser-Lys dyad in
the tertiary structure and the Ser residue of the dyadhydrolyze a peptide bond with a similar manner of ~­lactamase, a Ser-Lys dyad type hydrolase for cleavageof the ~-lactam ring of antibiotics.
Publication List:
Isono, K., Niwa, Y., Satoh, K. and Kobayashi, H.
( 1997) Evidence for transcriptional regulation ofplastid photosynthesis genes in Arabidopsis
thaliana roots. Plant Physiol., 114, 623-630.
Isono, K., Shimizu, M., Yoshimoto, K., Niwa, Y.,
Satoh, K., Yokota, A. and Kobayashi, H. (1997)
Leaf-specifically expressed genes for polypeptides
destined for chloroplasts with domains of cr70 factorsof bacterial RNA polymerases in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA, 94, 1494814953.

FOR BASIC BIOLOGY

43

Fig. 1. Localization of GFPs fused toN-terminal regions of S/G2 and SIG3. GFP fusion constructs with theN-terminal regionsof SIG2 ORF (SIG2-GFP, panels C and G) and S/G3 ORF (SIG3-GFP, panels D and H), and the transit peptide of small subunitof Rubisco (RBCS-GFP, panels B and F), as well as GFP alone (GFP, panels A and E), were introduced into tobacco leaves byparticle bombardment. Guard cells were observed using MRC-1024 Confocal Imaging System (X 480) with excitation at 488 nm
and emission at 520 nm (panels A-D), as well as excitation at 647 nm and emission at 666 nm (panels E-H). The same objects
are shown in each pair of upper and lower panels.

44

NATIONAL INSTITUTEDIVISION OF BEHAVIOR AND
NEUROBIOLOGY (ADJUNCT)
Professor:
Research Associate:
Postdoctoral Fellow:
Graduate Students:

Masatoshi Takeichi
Akinao Nose
Kazuaki Tatei
Emiko Shishido 1
Takaka /sshiki 2
Hiraki Taniguchi
Takeshi Umemiya (from Kyoto
University)

How individual nerve cells find and recognize theirtargets during development is one of the central issues
in modern biology. The aim of our division is toelucidate the molecular mechanism of axon guidance
and target recognition by using the simple and highly
accessible neuromuscular system of Drosophila.
The musculature of Drosophila embryos consists of
30 identifiable muscle fibers per hemisegment. Eachmuscle fib~r is innervated by a few motoneurons in ahighly stereotypic manner. The high degree of precision and previous cellular manipulations of neuromuscular connectivity suggest the presence of recognition
molecules on the surface of specific muscle fibers
which guide the growth cones of motoneurons. We
have previously isolated several enhancer trap linesthat express the reporter gene ~-galactosidase (~-gal)in small subsets of muscle fibers prior to innervation.
By molecularly characterizing these lines, we are trying to identify genes that play roles in the specification of the muscles and neuromuscular connectivity.
Previous studies showed that two of the lines are insertions in the connectin and Toll genes, that encode
cell recognition molecules which belong to the leucine-rich repeat (LRR) family. We have been studying the function of these genes, and also characterizing other lines by molecular and genetic analysis.

II. Capricious, a novel LRR cell surface molecule
expressed on subsets of neurons and muscles.
We have been conducting molecular and genetic
analysis of other muscle enhancer trap lines. One ofthem, P750 expresses ~-gal in subsets of neurons andmuscles, including the RP5 motoneuron and its target,
muscle 12. The eDNA cloning and sequencing revealed that the corresponding gene, termed capricious(caps), encodes a novel transmembrane protein thatbelongs to LRR family. It is interesting that three ofthe five muscle enhancer trap lines that we have thus
far characterized contain LRRs. Within the LRR family, Caps protein was found to be most related to the
product of the Drosophila tartan gene that have been
implicated in neural and muscular development. We
found that in the loss-of-function mutants of caps, thesynaptic arborization pattern on muscle 12, a capspositive muscle, was abnormal (Fig.1A, B). The
nerve terminal failed to stabilize on muscle 12, andinstead extended to and arborized on the adjacentmuscle, muscle 13. Ectopic expression of caps in all
muscles by GAL4-UAS system also resulted in aberrant synapse formation on muscle 12 (Fig. 1C). Likein the loss-of-function alleles, the muscle 12 terminal
axon branch often formed collaterals that turned backand innervated muscle 13. These results suggest that
Caps is involved in neuromuscular target recognition
and/or stabilizaion of the synapses.

I. Connectin can function as an attractive target
recognition molecule.
Connectin is expressed on a subset of muscle fibers(primarily lateral muscles) and on the axons, growthcones of the motoneurons which innervate thesemuscles (primarily SNa motoneurons) and on several
associated glial cells. When coupled with its ability tomediate homophilic cell adhesion in vitro, these results led to the suggestion that Connectin functions as
an attractive signal for SNa pathfinding and targeting .
To study the role of Connectin in vivo, we
ectopically expressed Connectin on all muscles byusing MHC (myosin heavy chain) promoter (MHCconnectin) in the P-element ·mediated transformants .
In MHC-connectin, SNa nerves were observed to send
extra axon branches that form ectopic nerve endings
on muscles 12, muscles they would never innervate inwild type. Furthermore, the ectopic innervation on
muscle 12 was dependent on the Connectin expression
on SNa. These results showed that Connectin functions as an attractive and homophilic guidance molecule for SNa in vivo .

Fig. 1 Neuromuscular defects associated with loss-offunction and ectopic expression of caps. Body wall fillet ofwild -type (A), caps los s-of-function (B) and caps
misexpressing (C) 3rd instar larvae, stained with MAb 22CIO
to visualize motor nerve projection and synaptic endings.
Ectopic synapses formed by muscle 12 motoneurons areindicated by arrows.

FOR BASIC BIOLOGY

III. msh, a homeobox containing gene essential forneural and muscular development.
Another line rH96 was found to be a P-element
insertion in the muscle segment homeobox (msh)gene, that was previously cloned as a homeobox containing gene. By generating and analyzing both lossof-function and gain-of-function (ectopic expression)
mutants, we showed that msh is essential for neuraland muscular development. During CNS development, msh is specifically expressed in the dorsal neuroectoderm and subsequently in many neuroblasts and ·their progenies derived from this region. We foundthat the loss of msh results in the failure of the properdifferentiation of many neural and glial progenies
derived from the dorsal neuroectoderm. Conversely,
ectopic expression of msh in the entire neuroectoderm
severely disrupts the formation of midline structureand differentiation of neuroblasts located in the ventral neuroectoderm. These results suggest that msh
plays crucial roles in the dorso-ventral (DV) specification of the CNS. The vertebrate homologues of msh,
Msxs are also know to be expressed in the dorsal
portion of the spinal cord. Our work on msh raises a
possibility that this family of genes may play a conserved role during DV patterning of the CNS.
IY. M-spondin and G-spondin: a novel gene familyof secreted molecules
By molecularly characterizing another enhancer
trap line, AN34 ·which is also expressed in a subset ofmuscles and neurons, we identified a novel secreted
protein, termed M-spondin, that is highly homologous
to rat F-spondin. F-spondin is a secreted molecule
expressed at high levels in the floor plate and has beenshown to promote neural cell adhesion and neurite
extension in vitro. We found three regions that arehighly conserved between M-spondin and F-spondin.
One of them is a known repeating motif called
thrombospondin type I repeats (TSRs). The other two
domains (termed FSl and FS2) are novel conserved
sequences that we identified. By using PCR, wecloned two more genes that share similar overall
structure with M-spondin and F-spondin in that they
possessed FSl, FS2 and one to six TSRs. The identification of these genes thus defines a novel genefamily of secreted protein with potential roles in celladhesion. One of the newly cloned genes, termed Gspondin, is expressed in a subset of glia that sit along
the longitudinal axon tracts in the CNS. The specificexpression pattern of G-spondin suggests that it may
play a role in the guidance of specific axons.

Publication List:
Oda, H., Uemura, T. and Takeichi, M. (1997) Phenotypic analysis of null mutants for DE-cadherin andArmadillo in Drosophila ovaries reveals distinct aspects of their functions in cell adhesion andcytoskeletal organization. Genes to Cells 2, 29-40.
Inoue, T., Chisaka, 0., Matsunami, H. and Takeichi,

45

M. (1997) Cadherin-6 expression transiently delineates specific rhombomeres, other neural tube subdivisions and neural crest subpopulations in mouseembryos. Develop. Bioi. 183, 183-194.
Fushimi, D., Arndt, K., Takeichi, M and Redies, C.
( 1997) Cloning and expression analysis of
cadherin-10 in the CNS of the chicken embryos.
Develop. Dynamics 209, 1-17.
Nose, A., Umeda, T. and Takeichi, M. (1997) Neuromuscular target recognition by a homophile interaction of Connectin cell adhesion molecules in Drosophila. Development 124, 1433-1441.
Radice, G.L., Rayburn, H., Matsunami, H., Knudsen,
K.A., Takeichi, M. and Hynes, R.O. (1997) Developmental defects in mouse embryos lacking Ncadherin. Develop. Bioi., 181, 64-78.
Umemiya, T., Takeichi, M. and Nose, A. (1997) Mspondin, a novel ECM protein highly homologous
to vertebrate F-spondin, is localized at the muscle
attachment sites in the Drosophila embryos. Develop. Bioi. 186, 165-176.
Nakagawa, S. and Takeichi, M. (1997) N-cadherin is
crucial for heart formation in the chick embryo.
Develop. Growth Differ. 39, 451-455.
Iwai, Y., Usui, T., Hirano, S., Steward, R., Takeichi,
M. and Uemura. T. (1997) Axon patterning requires
DN-cadherin, a novel neuronal adhesion receptor, in
the Drosophila embryonic CNS. Neuron 19, 77-89.
Isshiki, T., Takeichi, M. and Nose, A. (1997) The roleof the msh homeobox gene during neurogenesis:
implication for the dorsoventral specification of theneuroectoderm. Development 124, 3099-3109.
Suzuki, S.C., Inoue, T., Kimura, Y., Tanaka, T. andTakeichi, M. (1997) Neuronal circuits are subdivided by differential expression of type-11 classic
cadherins in postnatal mouse brains. Mol. Cell.
Neuroscience 9, 433-447.
Higashijima, S., Nose, A., Eguchi, G., Hotta, Y. andOkamoto, H. (1997) Mindin/F-spondin family:
novel ECM proteins expressed in the zebrafish
embryonic axis. Develop. Bioi., 192, 211-227.

4(iNATIONAL INSTITUTE47

FOR BASIC BIOLOGYLABORATORY OF GENE EXPRESSION AND REGULATION

Head: Takashi Horiuchi

The laboratory consists of four regular divisions andconducts research into regulatory mechanisms of gene
expression in higher plants and animals.

48

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION I

Professor:
Shigeru !ida
Associate Professor (adjunct): Yoshihiro Ozeki
Research Associate:
Rie Terada
Yoshiki Habu
Yoshishige Inagaki
Postdoctoral Fellow:
Yasuyo Hisatomi 2
Atsushi Hoshino
Graduate Student:
Sachiko Tanaka-Fukada
Technical Staff:
Tomoko Mori
Yoshiko Inoue
Ken-ichi Shiokawa
Visiting Fellows:
Yasumasa Morita
Fumiyoshi Myoga
Makiyo Yamagishi

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

I. Identification and characterization of mutablealleles in the Japanese morning glory
The Japanese morning glory (Pharbitis nil or Ipomoea nil) is a traditional horticultural plant in Japan,
and extensive physiological and genetical studies on
the plant have been conducted. Among several mutable loci that condition variegated flower phenotypes,
two mutable alleles, flecked and speckled have beenchosen for molecular elucidation of the variegatedphenotypes.
The flecked mutant bears white flowers with colored
flecks and sectors (Fig. lA). The flecking was regarded as recurrent somatic mutation from therecessive white to the dominant pigmented allele, accompanied by changes in genotypes from thehomozygous recessive to the heterozygous condition.
We have molecularly characterized the flecked mutation in one of the anthocyanin genes, A-3, and foundthat the mutable a-Jfl•cked (jlecked) allele carries thetransposable element, Tpni, in the anthocyanin biosynthesis gene for dihydroflavonol4-reductase (DFR).
Among the three copies of the DFR gene in the genome of the Japanese morning glory, Tpni resides
within the second intron of the DFR-B gene. The
flower variegation is due to somatic reversion of the
DFR-B gene by Tpni excision. The 6.4 kb Tpnl element belongs to the En/Spm family and is likely to bea non-autonomous element deficient to produce active
transposases.
It is known that the frequency and the timing of the
flecking are generally heritable by their progeny but
sometimes conversion of these phenotypes is also
observed. Thus the flecking caused by the excision of

Tpnl from the DFR-B gene may be determined either
by trans-acting activities of its related autonomous
element or by heritable modifications of Tpnl itself.
We are currently focusing on these lines of investigations.
Fig. lB shows another flower variegation caused by
the mutable speckled allele. Our genetic studies indicate that another element termed speckled-activator
acting in trans on the speckled allele is necessary toconfer the speckled variegation phenotype. Our current hypothesis is that the recessive speckled allele
carries a non-autonomous transposable element andthe dominant speckled-activator must be a related autonomous element. The plants carrying the speckled
allele without active speckled-activator bear pale yellow flowers. Based on the chemical analysis of thepale yellow flowers, it was proposed that the mutationin the gene for flavanone 3-hydroxylase (F3H) is alikely candidate for the speckled allele. We have tested
this hypothesis and shown that the speckled allele isneither F3H gene nor a regulatory gene acting on the
F3H gene expression. Currently, we are characterizing
the CHI gene which contains an insertion in the mutable speckled lines.

II. Identification of the mutable alleles in thecommon morning glory
The mutable afiaked line of the common morning
glory (Pharbitis purpurea or Ipomoea purpurea) alsobears white flowers with colored flakes and sectors(Fig. lC). The mutable aflaked allele is known to exhibit
incomplete dominance. Interestingly, not only intensely colored flakes but also white spots and sectors
were often observed in lightly colored flowers of themorning glory with the heterozygous state AJafi•ked(Fig. lD). To identify the mutable afi•ked allele, wehave characterized DNA rearrangements found at the
DFR and ANS (Anthocyanidin synthase) gene regionsin the anthocyanin biosynthesis and identified several
new mobile genetic elements. However, subsequent
genetic studies revealed that these elements have notassociated with the mutable afi•ked allele.
Based on amplified restriction fragment length
polymorphism (AFLP) technology, we have also developed a new protocol termed AMF (AFLP-basedmRNA fingerprinting) for the fingerprinting of mRNAto detect differentially expressed genes. Using both
AMF and the differential display (DD) .method, wewere able to identified new CHS (chalcone synthase)
genes, CHS-D and CHS-E, in the anthocyanin biosynthesis. Both CHS-D and CHS-E are expressed in theflower buds of both the common and the Japanesemorning glory, and CHS-D is more abundantly expressed than CHS-E. In the mutable afi"ked line of thecommon morning glory, the CHS-D gene carries anabout 4 kb insertion and its mRNA accumulation is
drastically reduced. Further characterization of these
newly identified CHS genes is in progress.

49

FOR BASIC BIOLOGY

Fig. ! Flower variegation.
(A) a - 3fiecke<~; (B) speckled; (C) afl"k"' [aflaked/afl"k'd]; (D ) afiaked [Nafl"k'd]III. Transposition of the maize AciDs transposable
elements in transgenic rice plants.
To understand the behavior of the transposable elements, we are characterizing transposition in heterologous system using transgenic plants. A transgenic
rice carrying a non-autoJ;Iomous Ds element wascrossed with another transgenic rice containing theactive Ac transposase (T. Izawa, et al., (1997) Plant
Mol. Bioi. 35, 219-229), we are currently investigating
somatic transpositio'n of Ds and its target selectivity in
the F1 plants.

Publication List:
Abe, Y., Hoshino, A. and Iida, S. (1997) Appearanceof flower variegation in the mutable speckled line of
the Japanese morning glory is controlled by two
genetic elements. Genes Genet. Syst. 72, 57-62.
Fukada-Tanaka, S., Hoshino, A. , Hisatomi, Y. , Habu,

Y. , Hasebe, M. and Iida, S. (1997) Identification of
new chalcone synthase genes for flower pigmentation in the Japanese and common morning glories.
Plant Cell Physiol. 38, 754-758.
Habu, Y. , Fukada-Tanaka, S., Hisatomi, Y. and Iida, S.
(1997) Amplified restriction fragment length polymorphi sm-based mRNA fingerprinting using asingle restriction enzyme that recognizes a 4-bp
sequence. Biochem. Biophys. Res. Commun. 234,516-521.
Hisatomi, Y. , Hanada, K. and Iida, S. (1997) The
retrotransposon RTipl is integrated into a novel typeof mini satellite MiniSipl in the genome of the common morning glory, and carries another new type ofmini satellite MiniSip2. The01: Appl. Genet. 95,
1049-1056.
Hisatomi, Y., Yoneda, Y. , Kasahara, K. , Inagaki, Y. andIida, S. (1997) DNA rearrangements at the region of
the dihydroflavonol 4-reductase gene for flower

50

pigmentation and incomplete dominance in morning
glory carrying the mutable flaked mutation. Theor.
Appl. Genet. 95, 509-515.
Hoshino, A., Abe, Y., Saito, N., Inagaki, Y. and Iida, S.
(1997) The gene encoding flavanone 3-hydroxylase
is expressed normally in the pale yellow flowers of
the Japanese morning glory carrying the speckled
mutation which produce neither flavonol nor anthocyanin but accumulate chalcone, aurone and flavanone. Plant Cell Physiol. 38, 970-974.

NATIONAL INSTITUTE

51

FOR BASIC BIOLOGYDIVISION OF GENE EXPRESSION
AND REGULATION II
Professor:
Research Associates:

Post Doctoral Fellow:
Graduate Students:
Technical Staff:

Takashi Horiuchi
Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama
Katsuki Johzuka
Katufumi Ohsumi 1
Keiko Taki
Hiroko Urawa
Kohji Hayashi
Yasushi Takeuchi

in japan. In January 1997, we reached the region at
69 minutes, sequence data from where to 13 min region being registered in databank. Thus, we expressed the entire E. coli genome in a single continuous nucleotide sequence.
I. Analysis of E. coli recombinational hot spots,
Hot.

Elucidation of dynamic molecular mechanism of
replication, recombination and repair processes ofgenome in prokaryotes and eucaryotes requires extensive research. We focus on interactive processes between replication and recombination. In 1997, thefollowing three interactive subjects were given detailed attention. (1) We analyzed molecular mechanisms of a recombinational hotspot in E. coli, named
HotH and we found that collision between replication
and rRNA gene transcription is responsible for activation of HotH. (2) We developed a new system, inwhich the SOS response was induced by replication
fork blocking at the Ter site located on pUC derivativeplasmids. (3) I (T. H) became head of Japan E. coligenome project in April 1995 and we began to define
nucleotide sequences from 13 minutes on the E. coli
circular linkage map, in the clockwise direction, together with eleven independent groups of investigators

In E. coli RNase H defective (rnh ·) mutants, we
identified specific DNA fragments, termed Hot DNA,
when DNA in the ccc form is integrated into the E.
coli genome by homologous recombination to form a
directly repeated structure; a strikingly enhanced
excisional recombination between the repeats occurs.
We obtained 8 groups (HotA-H) of such Hot DNA, 7of which (HotA-G) were clustered in a narrow region,
known as replication terminus region (about 280 kb)
located on the circular E. coli genome. Analysis of
the HotA, B and C revealed that blocking of the replication fork at the Ter (replication terminus) site is
responsible for these Hot activities. Further analysis
led to design of a putative model , in which the ds(double stranded)-break occurs at the fork arrested at
the DNA replication fork blocking (Ter) site. Throughthis site the RecBCD recombinational enzyme complex enters the ds-DNA molecule and enhances recombination between directly repeated Hot DNA
when the enzyme complex meets an appropriatelyoriented recombinational hotspot sequence, calledChi.

number
25

20
15

ter

•

or1
508

10
5
0

5

10

15
20

500

25

30

Figure 1. Orientation-dependent distribution of Chi sequence on E. coli genome
Blue and purple bars indicate the number of the Chi site in clock- and counter-clock-wise orientations in a each 100 kb intervalof the genome, respectively. The total number of each oriented Chi site is almost the same. However, number of one oriented
Chi are apparently dominant over that of the other in one of two half ori-ter sections and vise versa. Black arrow indicates directionof replication fork initiated from replication origin (ori) and an oriented fork is efficiently repaired by recombination using reverseoriented Chi sequence when it is broken down.

52

NATIONAL INSTITUTE

rrnE
rrnB/ J r
rrnA /

0/100

Terl

rrnc /
84

E. coli genome........

rrnG

TerG

TerF

Figure 2. Arrangement of rrn operons and Ter sites on the E. coli genome.
Reds arrows indicate locations and transcriptional direction of rrn operons. ~ means DNA replication terminus (Ter) site which
can block replication fork approaching from the right.

The Chi sequence had been considered to distributeand orient randomly over the E. coli genome. Dr.
Fred Blattner's group in the U. S. A. noted an orientation-dependent symmetrical distribution of the Chi
sequence related to replication origin (oriC), at leastin a relative narrow region. Our E. coli genome
analysis, as will be described below, revealed that thisfeature extends all over the genome, as shown in Figure 1. This finding is consistent with our model
mentioned above; that is if the replication fork isimpeded by one cause or another, the ds-break occurred cannot be repaired by recombination until theChi sequence in an orientation opposite that of replication fork is present at an appropriate site.
To determine how other termination event-independent Hots can be activated, we selected HotH because
it locates outside of replication terminus region. HotH
is a 11.2 kb EcoRI DNA fragment and locates at about
91 minutes on the E. coli genetic map. We found thatit contains the 3' end region of a rRNA operon, rrnD,
and also a Chi sequence at the downstream region of
the rrnD. HotH activity was abolished, when a mutation was introduced into the Chi sequence to destroy
its activity, which means that the Chi is probably required for HotH activity. To investigate events near
the HotH site, we asked whether progress in the DNA
replication fork is blocked or retarded in or around of
HotH region, and for this we used 2 dimensional (2D)
agarose gel electrophoresis. We detected the replica-tion fork passing through this region, in the counterclockwise direction specifically in the rnh- mutant andnot in wild type strain. Movement of the counterclockwise directed fork was retarded in a latter part of
the rrnD operon, the transcriptional orientation ofwhich is opposite to that of the fork, thereby suggesting that collision between replication fork and rRNA
gene transcription may be responsible for retardation .
Inactivation of the promoter of the rrnD operon reduced simultaneously retardation of fork movementand HotH activity. Thus , we concluded that HotH
activation is caused by collision between DNA replication fork and reverse-oriented rrnD transcription,
through the same mechanism related to fork blocking
at the Ter site, as described above. This conclusionsuggests that the collision is deleterious to the cells,
because continuous recombinational repair has to takeplace to maintain integrity of the replication fork. In
Figure 2, because Ter sites are arranged as if they
prevent replication fork from colliding with reverseoriented transcription of rrn operons; circular E. coligenome can be divided into two parts, one is rrn
operon side and other is Ter side and they never overlap, which strongly suggests that prevention of collision between fork and transcription may be the physiological role of the Ter site.

53

FOR BASIC BIOLOGY

II. SOS inducibility by replication fork blocking at
Ter site on plasmid and its dependency on distancefrom ori to Ter sites.
We developed a new system, in which the SOS response was induced by replication fork blocking at the
Ter site located on pUC derivative plasmids. In thesystem, production of Ter binding protein, known as
Tus protein, can be controlled by placing the structural gene, tus, under the araC promoter and SOS
induction is fully controlled by omitting or adding
arabinose. The extent of SOS response was controlled
by measuring the activity of ~-galactosidase, the expression of which is under control of the sfiA gene, atypical SOS responsive one, the product of which inhibits cell division. SOS inducibility dependent on
recA+ and le.xA+ genes. Using this system, we foundthat plasmids at a long distance from plasmid ori to
Ter were SOS positive but ones with a short distancewere SOS negative. Analyses revealed that up to 2.5
kb, the SOS response is hardly induced, but in a more
extended range to at least 4.8 kb, inducibility showed
good linear correlation to the distance. Replicationintermediate molecules of the SQS+ and sos- plasmids were purified through CsCl density gradient centrifugation and tertiary structures were analyzed using
2D gel electrophoresis and electron-microscopy. In
particular we examined the region of single stranded(ss) DNA found at theY-junction at the ori or Ter site,
because ssDNA functions as a cofactor for in vitro
activation of RecA protein; none of differences between them found. While the cause of ori-Ter length
dependent SOS induction needs more study, one possibility is that a ds-break occurs at the arrested
fork and degradation product from the ds-end maytrigger SOS induction. In such case, a longer DNA tobe degraded may more efficiently induce the SOS
response. Another possibility is that some specifictertiary structure of the 8 type DNA molecule may be
responsible for SOS induction, and to form the structure, a longer ori-Ter DNA may be needed.

III. The E. coli genome project
E. coli genome project in Japan was begun in 1989,
the objective being to analyze, in an independently
living organism, nucleotide sequences from 0 min ina clockwise direction. In April1995, DNA sequencesof the 0-12.7 min region in the genetic map were
determined, published or registered. At that time, I (T.
H.) was appointed leader of the Japan E. coli genome
project and a group of researchers was re-organized toanalyzed DNA sequences of the region from 12.7 min
to 70 min, using Kohara lambda clones and the shotgun sequence technique.
In January 1997, we determined about a 2.2 mega
nucleotide sequence of a region corresponding to12.7-69.0 min (including DNA replication terminus
region). Combining these data with data (69.0-100min) of Dr. Fred Blattner and colleagues in the USA,the entire E. coli genome was expressed in a single,

continuous nucleotide sequence, in which about 3400
putative open reading frames were identified. At thistime, Blattner's group independently determined theentire E. coli DNA sequence. Figures 1 and 2 show
arrangements of Chi sequences and Ter sites on wholegenome, respectively; these are some results of the
genomic sequence analysis. Our sequence data, registered in the DNA Data Bank in Japan (DDBJ) arealso available through Web sites (http://
mil.geges.nig.ac.jp/ecoli/, http://www.ddbj.nig.ac.jp/,
http://bsw3.aist-nara.ac.jp/).

Publication List:
Yamamoto, Y., Aiba, H., et al. (1997) Construction ofa contiguous 874-kb sequence of the Escherichia
coli-K12 genome corresponding to 50.0-68.8 minon the linkage map and analysis of its sequence
features. DNA research 4, 91-113.

54

DIVISION OF SPECIATION MECHANISMS I
Professor:
Tetsuo Yamamori
Research Associates: Satoshi Koike
Yuriko Komine
Akiya Watakabe
Institute Research Fellow: Takashi Kitsukawa
Postdoctoral Fellow: Rejan Vigot 2
Visiting scientist:
Kimiko Yamamori
Yoshinori Shirai
Graduate student:
Shiro Tochitani
Katsusuke Hata
Akishi Onishi (Kyoto University)
Shuzo Sakata (Kyoto University)
Nobuaki Tanaka (Kyoto University)
Hiroshi Fujita (Kyoto University)
Hideko Utsumi
Technical Staffs:
Miki Ida

Our research goal is to understand mechanisms
underlying evolution of the nervous system. In order
to approach this question, we are currently focusing
on two systems.

I. Evolution of cytokine receptor families in the
immune and nervous systems
It has been recognized that cytokines, defined asinter-cellular mediators in the immune system, have avariety of roles in the nervous system as well. One
such a factor, LIF (leukemia inhibitory factor) known
also as CDF (Cholinergic Differentiation Factor), is apleiotropic factor which shows a remarkable repertoire of activities from embryonic stem cells to neurons (Yamamori, T., 1996). Recent study have revealed that CDFILIF and its receptors belong to theIL-6 family and the receptor family.
Based on Bazan's model which predicted thecytokine receptor family as a member of immunoglobulin super gene family (1990) and the model of theinteraction among the members of the IL-6 family(ligand) and the IL-6 receptor family (Taga andKishimoto, 1992; Stahl and Yancopoulos,
1993), we proposed that the evolution of the IL-6/class IB receptor family may have occurred in at least
two major steps (Yamamori and Sarai, 1994). Firstly,binding subunits of an IL-6 receptor and for a CDF/
LIF receptor evolved and secondly, a third binding
subunits of a CNTF receptor evolved. Our model
predicts that the binding subunits generally determine
the specificity of the receptors and it is possible thatnovel members of the cytokine family and their receptors exist in the nervous system. In order to prove thishypothesis, we are currently working to identify new
members of the family which are specifically expressed in the nervous system.

II. Gene expression and cerebellar long-term
plasticity
In order to know roles of the genes involved inlong-term memory, we choose the cerebellum as amodel system. In the cerebellum the conjunctive

NATIONAL INSTITUTE

stimuli of parallel fibers and a climbing fiber to a
Purkinje cell induce prolonged reduction of a synaptic
efficacy between the paralleled fiber and the Purkinje
cell (LTD; long-term depression, Ito et al., 1982).
Previously, we examined the expression of 10 immediate early genes (lEGs) including all the known
Fos and Jun family in cerebellar slices under the pharmacological condition that causes long-term desensitization of the Purkinje cell to AMPA (a glutamate
analogue). Among the lEGs examined, Fos and JunB were predominantly induced under the conjunctive
condition (Nakazawa et al., 1993).
Recently, we have examined Jun-B expression invivo under a conjunctive protocol of AMPA, a pharmacological substitute for parallel fiber stimulation,and climbing fiber stimulation via electric Inferior
Olive stimulation. June-B are predominantly induced
around the local area where the AMPA and climbing
fiber stimulation were conjunct (Yamamori et al.,1995). These results suggest that the coincidence
mechanism may exist at gene expression level andlead to a cerebellar long-term plasticity (Fig. 1).
Toward further confirming this hypothesis, we are
currently identifying several molecules which are induced in Purkinje cells 3 hours after the conjunction
in collaboration with Dr. Ryoji Yano's group (Brain
Research Institute, RIKEN). One of these genes iscurently further being characterized. We are also examining the expression of the genes under cerebellar
or hippocampal learning paradigm.

Publication List
Fujiwara, T., Yamamori, T., Yamaguchi, K., andAkagawa, K. (1997) Interaction of HPC-1/syntaxin
1A with the cytoskeletal protein, tubulin. Biochem.
Biophy. Res. Comm. 231, 352-355.
Matsuzawa, M., Muramatsu, T., Yamamori, T., Knoll,
W. and Yano, R.(in press) Novel neuronal activities
of midkine were examined on embryonic cerebellar
neurons using a difined culture system. Cellul. andMolec. Neurobiol.

FOR BASIC BIOLOGY

55

Parallel fiber

Climbing Fiberlong-term syna
plasticity

Expression oflate genes

AP-1 complex(c-Fos/Jun-B)

Purkinje Cell
Fig. 1

56

NATIONAL INSTITUTEDIVISION OF SPECIATION MECHANISMS II
Professor:
Ken-ichirou Morohashi
Associate Professor: Mitsuyasu Hasebe
Research Associate: Satoru Ishihara
Sanae Oka
Technical Staff:
Post Doctral Fellow: Naoe Kotomura 4
Graduate Students: Satomi Shindo
Kumi Aso (Univ. of Tokyo)
Ken Kawabe (Kyushu Univ.)
Tokuo Mukai (Kyushu Univ.)
Mikihiko Kato (Nagoya Univ.)
Hirofumi Mizusaki (Kumamoto Univ.)
Tomoaki Nishiyama (Univ. of Tokyo)
Keiko Sakakibara ( Univ. of Tokyo)
Ryosuke Sana (Chiba Univ.)
Tatsuji Shikayama (Kyushu Univ.)
Kazuyoshi Someya ( Univ. of Tokyo)
Hayato Yokoi (Nagoya Univ.)
Visiting Fellow:
Rumiko Kofuji
Yoshiyuki Kojima
Assistant:Hisae Tsuboi

This division was established recently with two research groups. These two groups managed by theprofessor, Ken-ichirou Morohashi (1), and the associate professor, Mitsuyasu Hasebe (II), have adopted
animals and plants, respectively, as the materials forthe research. Our attention has been and will be directed to understanding mechanisms underlying differentiation of the reproductive system of animals andevolution of the morphology in plants. These efforts
with a variety of the materials are expected to give us
deeper insights into speciation mechanisms of organism.

I.

Reproduction of animals.

Because of the fundamental and pivotal function of
the gonads, extensive efforts have been made to characterize the differentiation processes including sexdependent differentiation. By focusing on thesteroidogenesis as one of the gonad specific functions,
we identified a steroidogenic tissue specific transcription factor designated Ad4BP/SF-l. Recent studies
with an antiserum to Ad4BP/SF-1 and a gene disrupted mice clearly demonstrated that the transcription factor is essential for differentiation of thesteroidogenic tissues, the gonads and the adrenal cortex. Showing a good correlation with the observation
above, a particular cell population identified as
Ad4BP/SF-1 immunoreactive cells (AGP, adrenogenital primordium) was revealed to give rise to these
distinct tissues. These observations gave us many
interesting issues as follows. What is the signal forthe onset of the Ad4BPISF-1 gene transcription in theparticular cells, AGP? What is the mechanism underlying the separation of the AGP into the adrenal andgonadal primordia? How is the mechanism employed
for sex differentiation of the gonads? Why dose suchsex dependent differentiation not occur in the adrenal
cortex? To address these issues, functions and regulation of nuclear transcription factors, WTl, SRY,

SOX9, and DAXl as well as Ad4BP/SF-l, all ofwhich are critically implicated in gonadal and adrenocortical differentiation, have been investigated fromthe molecular and morphological aspects.

II.

Morphological Evolution in plants.

Morphological diversity among taxa is the mostprominent characteristics in the biodiversity. Ourgroup aims to explore genetical and molecularchanges which caused renovative morphologicalchanges during the course of land plants evolution.
We selected Arabidopsis (angiosperm), Gnetum (gymnosperm), Ginkgo (gymnosperm), Ceratopteris (pteridophyte), Physcomitrella (bryophyte), and Chlamydomonas (green algae) as model plants to compare thegenes related to morphogenesis. We are focusing on
the following three topics; (1) Evolution of floral
homeotic genes (MADS gene family and LEAFY
gene homologue which is a regulator of the MADS
genes), (2) Comparison of homeobox gene functions
between sporophyte and gametophyte generations toreveal the evolution of the both generations, and (3)establishment of mutant library of Physcomitrella patens to get new morphological mutants which have notbeen obtained from the study of the higher plants
having only reduced gametophytic generations. Some
MADS, LEAFY and homeobox genes have beencloned from the gymnosperms, Ceratopteris,
Physcomitrella and the analyses of their functions arein progress. Comparisons of the gene functions andswapping experiments of genes among the organisms
will make possible to infer what kinds of changes ofdevelopmental genes caused morphological diversityof oragnisms.

Publication List:
S. Leers-Sucheta, K. Morohashi, J. I. Mason, & M. H.
Meiner. ( 1997) Synergistic Activation of the Human
Type II 3~-Hydroxysteroid Dehydrogenase/D5D4
Isomerase Promoter by the Transcription Factor
Steroidogenic Factor-11Adrenal 4-Binding Protein
and Phorbol Ester. J. Bioi. Chern. 272, 7960-7967.
J. Lund, M. Bakke, G. Mellgren, K. Morohashi, & S.0. Doskeland. (1997) Tmascriptional regulation of
the bovine CYP17 gene by cAMP. Steroid 62, 4345.
J. I. Mason, D. Keeney, I. M. Bird, W. E. Rainey, K.
Morohashi, S. Leers-Sucheta, & M. H. Meiner.
(1997) The regulation of 3~-hydroxysteroid dehydrogenase expression. Steroid 62, 164-168.
K. Morohashi. (1997) The ontogenesis of the steroidogenic tissues. Genes Cells 2, 95-106.
Hasebe, M. (1997) Molecular phylogeny of Ginkgo
biloba. In T. Hori, ed., Ginkgo biloba (SpringerVerlag), pp173-181.
Hasebe, M. and Banks, J.A. (1997) Evolution of
MADS gene family in plants. In K. Iwatsuki and
P.H. Raven eds., Evolution and Diversification inLand Plants (Springer-Verlag), pp 179-197.

FOR BASIC BIOLOGY

Fukada-Tanaka, S., Hoshino, A., Hisatomi, Y., Habu,
Y., Hasebe, M. and Iida, S. (1997) Identification of
new chalcone synthase genes forflowerpigmentation in the Japanese and common
morning glories. Plant Cell Physiol. 38: 754-758.

57

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NATIONAL INSTITUTE

'

L

59

FOR BASIC BIOLOGYRESEARCH SUPPORT60

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT

Head: Hiroyuki Hattori
Common Facility Group
Chief: Kazuhiko Furukawa

Research Support Group

Reseach Support Facilities
Mamoru Kubota (Subunit Chief)
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
Tomoki Miwa
Makiko Itoh (Technical Assistant)Keiko Suzuki (Technical Assistant)
Misayo Masuda (Technical Assistant)

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

Radioisotope Facility
Yousuke Kato (Subunit Chief)
Yoshimi Matsuda
Takayo Itoh (Technical Assistant)Center for Analytical Instruments
Akio Murakami (Unit Chief)
Sonoko Ohsawa
Yumiko Makino
Takeshi Mizutani
Hatsumi Moribe (Technical Assistant)
Glassware Washing Facility
Masayo Iwaki(Kazuhiko Furukawa)(Toshiki Ohkawa)

The Technology Department is a supporting organization for researchers and research organization
within the NIBB. The Department develops and promotes the institute's research activities and at thesame time, maintains the research functions of theinstitute.
The department is organized into two groups:
one, the Common Facility Group,which supports andmaintains the institute's common research facilities
and the other, the Research Support Group, whichassists the research activities as described in individual reports.
Technical staffs participate, through the department, in mutual enligtement and education increase
their capability in technical area. Each technical staff
is proceeded to the fixed division usually and they
support the various research with their special biological and biophysical techniques.

Developmental Biology Group
Hiroko Kobayashi(Unit Chief)
Chiyo Takagi
Naomi Sumikawa
Regulation Biology Group
Shoichi Higashi(Subunit Chief)
Hideko Nonaka
Akiko Kawai
Shigemi Ohsugi
Gene Expression and Regulation Group
Sachiko Tanaka
Tomoko Mori
Koji Hayashi
Miki Ida
Yasushi Takeuchi
Hideko Utsumi
Sanae Oka
Yoshiko Inoue

The Department hosts an annual meeting fortechnical engineers who work in various fields of biology at universities and research insititutes throughout Japan. At this meeting, the participants present
their own activities and discuss technicalproblems.The Proceedings are published soon after
the meeting.

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Head of Facility:
Takashi Horiuchi
Associate Professor: Masakatsu Watanabe
Research Associates: Yoshio Hamada, (Tissue and Cell
Culture)Atsushi Ogiwara (Computer)
Post Doctral Fellow: Mineo lseki 1 (Large Spectrograph)
Masayo lwaki (-August 31, 1997)
Technical Staffs:
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
Tomoki Miwa
Makiko Ito (April 14, 1997-)
Misayo Masuda (September 1, 1997-)

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 biological processes.The spectrograph runs on a 30kW
Xenon arc lamp and has a compound grating composed of 36 smaller individual gratings. It projects aspectrum of a wavelength range from 250nm (ultraviolet) to l,OOOnm (infrared) onto its focal curve oflOrn in length. The fluence rate (intensity) of themonochromatic 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,
491-498).
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
specifically require ultra-high fluence rates as well as
ultra-high spectral-, time-and spatial resolutions.lt iscomposed of a high-power Ar-ion laser (Coherent,
Innova 20) (336.6-528.7 nm, 20W output), two CW
dye lasers (Coherent, CR599-0l) (420-930nm, 250IOOOmW output), A/0 modulators (up to 40MHz) tochop the laser beam, a beam expander, and a tracking
microbeam irradiator (up to 200mm s· 1 in tracking
speed, down to 2mm in beam diameter) with an infrared phase-contrast observation system.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely
rooms which satisfy the P2/P3 physical containment
level. This facility is routinely used for DNA recombination experiments.
3. Computer Laboratory
To meet various computational needs and to provide
means of electronic communication, many kind ofcomputers are equipped: UNIX servers and engineering workstations (Sun Ultra Enterprise server, SPARC
stations, IRIS machines, NEWS machines, etc.), and61

some personal computers (Macintosh's and Windows
machines). All of these machines, as well as almost
every PC in each laboratory, are connected each otherwith local area networks, which are also linked to thehigh performance multimedia backbone network ofOkazaki National Research Institutes.
The Computer Laboratory provides various computational services to the institute members: file servers
for Macintosh and NetWare users, print servers for PC
and UNIX users, computational servers that provides
sequence analyses, database retrievals, homology
search services, and so on. Providing network communication services is also an important task of thislaboratory. We maintain the World-Wide Web serverthat contains the NIBB home pages (URL is 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
for plant culture. In some of these facilities androoms, experiments can be carried out at the PI 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, threegreen houses (each 6m2) at the Pl physical containment level, a small farm, two greenhouses (45 and 88m2) with automatic sprinklers, two open aquariums(30 and SOt) and several smaller tanks. The laboratory
also includes a building with office, storage and work
space.
6. Plant Cell Laboratory
Autotrophic and heterotrophic culture devices andare equipped for experimental cultures of plant andmicrobial cells. A facility for preparation of plant cell
cultures including an aseptic room with cleanbenches,is also provided.

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

62
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 single-celled, flagellate algae are studied action spectroscopically(Watanabe 1995, In CRC Handbook of Organic Photochemistry and Photobiology) by measuring computerized-videomiceographs of the motile behavior of thecells at the cellular and subcellular levels (Erata et al.
1995, Protoplasma). Photo-receptive and signal transduction mechanisms of algal gametogenesis are alsostudied by action spectroscopy.
(2) Developmental Biology: Notch is an integral cellsurface membrane protein that is known to play a keyrole in developmental cell-cell interactions in Drosophila, particularly in lateral specification of neuralversus epidermal cell fates, a process described thus
far only in invertebrates. It is thought to act by a direct
signaling pathway rather than through one of the classical signal transduction cascades. The mammalian
genome is known to contain three Notch homologues
but their developmental significance is not clear. To
investigate their role in mammalian development, wehave sequenced the murine Notch 2 eDNA, determined the primary sequence of its protein, and haveinvestigated its genomic organization. We are now
attempting to produce a mutant in which the ankyrin
repeat region of Notch 2 is replaced by lacZ. Analysisof the mutant phenotype will provide us with insights
about the significance of the repeat in Notch 2 signaltransduction in relation to its developmental importance.
(3) Computational Biology: The genome sequencing
project of an important Gram-positive bacterium Bacillus subtilis had been completed in 1997. This
project was carried out under the international collaboration, and seven research teams participated in
the sequencing from Japan. We took part in thisproject for assembly and analysis of the sequence dataof Japan. The genome contains about 4,200 ORFs,
and about a half of them seemed to constituteparalogous clusters. According to the comprehensive
sequence analysis, some protein families like transporters, regulators, and sensors showed a tendency tothe paralog. A database system called BSORF has
been constructed in collaboration with Nara Institutefor Science and Technology and the Human Genome
Center of the University of Tokyo (http://
bacillus.genome.ad.jp). Since the Bacillus genome
project has been shifted to the functional analysis, weare also trying to integrate the functional information
into the database.

NATIONAL INSTITUTE

2. Cooperative Research Program for the Okazaki
Large Spectrograph
The NIBB Cooperative Research Program for the
Use of 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 regulatory and 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

Furuhashi, K., Tada, Y., Okamoto, K., Sugai, M.,
Kubota, M. and Watanabe, M. (1997). Phytochromeparticipation in induction of haustoria in Cuscuta
japonica, a holoparasitic flowering plant. Plant Cell
Physiol. 38, 935-940
Kunst, F., Ogasawara, N., Moszer, 1., ..... , Ogiwara, A.,..... ,Yoshikawa, H., and Danchin, A. (1997) The
complete genome sequence of the Gram-positivebacterium Bacillus subtilis. Nature 390, 249 - 256.
Nishizaki, Y., Kubota, M., Yamamiya, K. and
Watanabe, M. (1997). Action spectrum of lightpluse-induced membrane depolarization in pulvinar
motor cells of Phaseolus. Plant Cell Physiol. 38,
526-529.
Ogiwara,A., Ogasawara,N., Watanabe,M., and Takagi,
T. (1997) Comprehensive Sequence Analysis of B.
subtilis Genome Using the BSORF Database. Proc.
Genome Informatics 1997, 320- 321.
II. Cooperative Research Program for the Okazaki
Large Spectrograph
Furuhashi, K., Tada, Y., Okamoto, K., Sugai, M.,
Kubota, M. and Watanabe, M. (1997). Phytochromeparticipation in induction of haustoria in Cuscuta
japonica, a holoparasitic flowering plant. Plant Cell
Physiol. 38, 935-940
Hamazato, F., Shinomura, T., Hanzawa, H., Chory, J.
and Furuya, M. (1997). Fluence and wavelength
requirements for Arabidopsis Cab gene induction
by different phytochromes. Plant Physiol. 115,
1533-1540.
Nishizaki, Y., Kubota, M., Yamamiya, K. and
Watanabe, M. (1997). Action spectrum of lightpluse-induced membrane depolarizatioh in pulvinar
motor cells of Phaseolus. Plant Cell Physiol. 38,
526-529.
Quintern, L. E., Furusawa, Y., Fukutsu, K., andHoltschmidt, H. (1997). Characterization and application of UV detector spore films: the sensitivity
curve of a new detector system provides good similarity to the action spectrum for UV-induced
erythema in human skin. J. Photochem. Photobiol.
B: Biol. 37, 158-166.

FOR BASIC BIOLOGY

Saitow, F. and Nakaoka, Y. (1997). The photodynamic
action of methylene blue on the ion channels of
Paramecium causes cell damage. Photochem.
Photobiol., 65, 902-907.
Takeda, J., Ozeki, Y. and Yoshida, K. (1997). Action
spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot
suspension cells. Photochem. Photobiol. 66, 464470.
Tazawa, E., Fujiwara, A., Kamata, Y., Konishi, K.,
Ohta, H., Shimma, H. and Yasumasu, I. (1997).
Does light-induced relief of cytochrome c oxidasefrom CO-induced inhibition result in photo-reactivation of CO-inhivited respiration in sperm of sea
urchin, oyster and fish? Zoot. Sci. 14, 629-636.

63

64

NATIONAL INSTITUTE

RADIOISOTOPE FACILITY(Managed by NIBB)Head:Masaharu Noda
Associate Professor: Kazuo Ogawa
Technical Staff:
Kazuhiko Furukawa (Radiation
Protection Supervisor),
Yosuke Kato (Radiation Protection
Supervisor),
Yoshimi Matsuda (Radiation
Protection Supervisor)
Supporting Staff:
Takayo Ito, Risako Shirai

This Facility consists of a central experimental station which is placed in the Common Building I andthree smaller substations which are placed in National
Institute for Basic Biology (NIBB) Building, Laboratories of Gene Expression and Regulation (LGER)
Building which opened in May 1, 1997, and National
Institute for Physiological Sciences (NIPS) Building.
These spaces are controlled areas and only the persons
who are permitted by the Radiation Protection Supervisor can enter. The members of the Radioisotope

Figure 1A

Facility have a variety of businesses such as monitoring and recording the going in and out the controlledareas of the persons, purchasing the radioisotopes forthe users from the Japan Radioisotope Association(JRA), and passing the radioisotope wastes to theJRA. They also serve as lecturers of the training
course required for the registration of beginners andfor annual registrations of users. At every opportunity,
the Radiation Protection Supervisors are providingthem appropriate guidance for radioisotope handlingand the law of interest.
The total number of users in and out the controlledareas from April to December, 1997 was 8,922. As
shown in Figure 1A, the number of users in the LGER
substation was comparable to that in the central station. The total radioisotopes purchased in this period
for each controlled area and nuclide are shown in
Figure 1. Although a variety of nuclides can be usedin the central station, only 125I, 35 S, 32 P, 14C, 33p , and 3H
were purchased within this period. From April , 1997,
we have been allowed to use 33 P in all the controlledareas .

NIPS
13%

Center
40%

LGER
35%

NIBS
12%

Figure 1 B

3H
14C
32P
33 p
355

kBq

FOR BASIC BIOLOGY

The teaching staff of the Radioisotope Facility is also
engaged in their own research. They are interested inclarifying the structure and function of dynein motor
protein. Dyneins are a group of microtubule-activatedATPases that serve to convert chemical energy intomechanical energy. They have been divided into two65large subgroups, namely, the axonemal and cytoplasmic
dyneins. Figure 2 shows the localization of two dyneinsin the outer arms (Ogawa et al., 1977) and the mitotic
apparatus (Mohri et al., 1976) that have been visualized
by the same antibodies directed against the motor domain of axonemal dynein (fragment A).

Fig. 2The native dyneins are very large. They range inmolecular mass up to 1 to 2 mega da1tons and they arecomplex proteins as shown in Figure 2. Each dynein
contains two or three heavy chains (HCs) with ATPase
activity, which range in molecular mass up to 500
kDa. The motor activity of dynein is associated withthese chains. Some functional differences have beenreported between HCs of outer arm dynein. Sea urchin outer arm dynein is a heterodimer of HCs (alphaand beta) and at least the beta-HC is able to induce
gliding of microtubules in vitro. The alpha-HC might
amplify the function of beta-HC and it has been reported to have no motile activity. After the first cloning of beta-HC from sea urchin ciliary axonemes(Gibbons et al., 1991; Ogawa, 1991), the sequences of
HCs of axonemal and cytoplasmic dyneins from avariety of organisms were determined in their entirety.
Without exception, all the HCs cloned to date containfour P-loop (ATP-binding) sequences in the midregionof the molecule. Thus, they can be classified as a fourP-loop family.
The outer arm dyneins contain two or three proteinsthat range in molecular mass from 70 to 120 kDa andcopurify with HCs . ICs of sea urchin outer-arm
dynein are abbreviated as ICI, IC2, and IC3. Those ofChlamydomonas are called IC78 and IC69, and ICs ofcytoplasmic dynein are called IC74. Chlamydomonas
IC78 and IC69 were cloned by Wilkerson et al. (1995)
and Mitchell and Kang (1991), respectively. Thesequences of sea urchin IC2 and IC3 were determinedby Ogawa et al. (1995). Finally, the sequence of IC1
was determined this year by Ogawa et al. (1996).
Thus, all the ICs found in the axonemal and cytoplasmic dyneins of the model organisms used for studies
of dynein function have been completely sequenced.
Comparison of amino acid sequences of IC2 and IC3
with those of IC78 and IC69 and with that of IC74
showed that, although all five ICs are homologous,
IC2 is much more closely related to IC78, and IC3 ismuch more closely related to IC69, than either sea
urchin chain or either Chlamydomonas chain is related to each other. Regions of similarity between all
five ICs are limited to the carboxy-terminal halves ofthe molecules. Similarity are due primarily to conservation of the WD repeats in all of these chains. The
WD repeats are involved in protein-protein interactions in a large family of regulatory molecules (Neeret al. , 1994). A parsimony tree for these chains(Ogawa et al. , 1995) shows that, although thecarboxy-terminal halves of all of these chains contain
WD repeats, the chains can be divided into three dis-

66

NATIONAL INSTITUTE

Fig . 3. Substructures of outer arm dyneins from sea urchin sperm flagella and Chlamydomonas flagella .

tinct subclasses (IC3 plus IC69, IC2 plus IC78, andIC78).
By contrast, sea urchin ICl is not a member of the
WD family. ICl has a unique primary structure, theN-terminal part is homologous to the sequence ofthioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic

acid residues . Thus, ICl is a novel dynein intermediate chain distinct from IC2 and IC3 and may be amultifunctional protein . Then, a question arises as
whether Chlamydomonas outer arm dynein contains
ICl. The answer is "no". Because it consists of just
two intermediate.chains. Alternative answer is "yes".
Because the sequences of two light chains (LC 16 andLC14) from Chlamydomonas outer arm dynein showsthat they are members of novel family of thioredoxin

FOR BASIC BIOLOGY(Patel-King et al. 1996). The thioredoxin-related partof IC1 is more closely related to those of two redoxactive Chlamydomonas light chains than thioredoxin.
Antibodies were prepared against the N-terminal
and middle domains of ICl expressed as His-tagged
proteins in bacteria. These antibodies cross-reactedwith some dynein polypeptides (potential homologuesof ICl) from distantly related species. They propose
here that the three intermediate chains are the basic
core units of sperm outer arm dynein because of theirubiquitous existence.
They are now isolating eDNA clones that encode
LCs of sea urchin outer arm dynein as part of an effort
to understand why dynein is so very large and complex. The outer arm dynein of sea urchin sperm flagella contains six light chains with molecular massesof 23.2, 20.8, 12.3, 11.5, 10.4, and 9.3 k:Da, respectively. They have cloned the eDNA for the 23.2 k:Da
(LCI) and the 12.3 kDa (LC3) polypeptides andfound that they are highly homologous to mouse
Tctex2 and Tctex1, respectively. These mouse proteins are encoded by the t complex region that is involved in transmission ratio distortion (TRD), malesterility and the development of the germ cells. Theirfinding raises the possibility that axonemal dyneins
are involved in this phenomenon. TRD may be causedby the dysfunction of multiple axonemal dyneins.
Publication List:

Kazuo Ogawa, and Hideo Mohri (1997) Establishment of a dynein motor superfamily. In Recent Advances in Marine Biotechnology. Vol I. Endocrinology and Reproduction (Edited by M. Fingerman, R.
Nagabhushanam, and M.-F. Thompson), pp 249281. Oxford & IBH Publishing Co. New Delhi.

67

68

THE CENTER FOR ANALYTICAL INSTRUMENTS(Managed by NIBB)Head of Facility: Tetsuo Yamamori
Technical Staffs: Akio Murakami
Sonoko Ohsawa
Yumiko Makino
Takeshi Mizutani
Hatsumi Moribe (Apr. 21, 1997 -)The Center, equipped with various types of instruments, serves for general use and supports biochemical structural analyses of biomolecules of proteins,
nucleic acids and lipids. Amino acid sequence analysis, amino acid analysis, and chemical syntheses of
peptides and DNA/RNA are carried out to support
researchers in NIBB and NIPS . In April 1997, theCenter started new services; site-specific modificationof synthetic peptides (biotinylation and fluorophorelabeling) and mass spectrometric analysis of peptides
and proteins. Newly installed instruments in 1997 are
Biomolecular Interaction Analysis System, Flow Cytometer, FT-IR Spectrophotometer and Microplate
Luminometer. Some of instruments of the Center are
opened to researchers outside the Institute.
Representative instruments are listed below.

(Pharmacia DELFIA Research)
Microplate Readers (Corona MTP-120, MTP-100F)
Spectropolarimeter (JASCO J-40S)
Ff-IR Spectrophotometer (Horiba Ff-730)
Laser Raman Spectrophotometer (JASCO R-800)
Biomolecular Interaction Analysis Systems(Pharmacia BIAcore 2000, Affinity Sensors IAsys)
Bio Imaging Analyzer (Fujifilm BAS2000)
Electrophoresis Imaging Systems (PDI Discovery Series, BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Microscope Photometer (Carl Zeiss MPM 03-FL)

Protein Sequencers (ABI 492, ABI 473A)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A, ABI 432A)
Plasmid Isolation System (Kurabo PI-100)
DNA Sequencers (ABI 373S, ABI 310)
DNA/RNA Synthesizers (ABI 394, ABI 392)
Thermal Cyclers (Perkin Elmer PJ-1000, Takara TP300)
Integrated Thermal Cycler (ABI CATALYST Turbo800)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs(Shimadzu LC-10AD, Shimadzu LC-6AD)
Integrated Micropurification System (Pharmacia
SMART)
Flow Cytometer (Coulter EPICS XL)NMR Spectrometer (Bruker AMX-360wb)
EPR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS1200A)
Spectrofluorometers (Hitachi 850, Simadzu RF-5000)
Spectrophotometers (Hitachi 330, Hitachi 557, Varian
Cary 5G, Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB
96P)Time-resolved Fluorescence Microplate Reader

Figure 1. Amino acid sequence analysis of proteins by anautomated protein sequencer.

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