NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT
1998

The cover photograph shows the flower variegation of thecommon morning glory caused by excision of the transposable
element TiplOO from the CHS-D gene for flower pigmentation.
(See Division of Gene Expression and Regulation I)

Post Doctral Fellow
123
4

NIBB Research Fellow
JSPS Postdoctral Fellow
JSPS Reserch Associate
JST Fellow

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

1

ORGANIZATION OF THE INSTITUTE

2

GRADUATE PROGRAMS

3

OFFICE OF DIRECTOR

4

Evolution of photosynthesis... ...................................... .......... .................... ......... .. ....... .. ......... .................

4

Mechanisms determining the outline shape of the adult lepidopteran wings .......................................

5

Protein palmitoylation and cell development at embryogenesis

6

Analysis of meiosis

7.... .... .. ........................ ................................................................................................

DEPARTMENT OF CELL BIOROGY9DIVISION OF CELL MECHANISMS

10

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

10

II. Microbody defective mutant of Arabidopsis.

10

III. Transport of storage proteins to protein storage vacuoles is mediated by large PAC({lrecursor-accumulating) vesicles. ...... ..... ...... .................... ............ .. .. ........... ....... ........... ...................

11

IV. Vacuolar processing enzymes in protein-storage vacuoles andlytic vacuoles. .....................................................................................................................................

11

V. Role of molecular chaperones in organelle differentiation.

12

DIVISION OF BIOENERGETICS

14

DIVISION OF CELL PROLIFERATION (ADJUNCT)18

I. Developmental neuroanatomy
II. Functional neuroanatomy......... ....... .......... .................... ....... .... ......... ....... ...... ............. ............. .....

18....................................................................................................................

18

III. Contribution to the science community

18

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)20

DIVISION OF CELL FUSION (ADJUNCT)

22..............................................................................................

DEPARTMENT OF DEVELOPMENTAL BIOLOGYDIVISION OF REPRODUCTIVE BIOLOGY

2526

I. Endocrine regulation of oocyte growth and maturation

26

II. Endocrine regulation of spermatogenesis

26III. Endocrine regulation of gonadal sex differentiation and sex change

27

DIVISION OF CELL DIFFERENTIATION ...............................................................................................

30

I. Transcriptinal control of genes necessary for gonadal differentiation

30

II. Implication of Ad4BP/SF-1 in strctural organization of spleen.

30

DIVISION OF MORPHOGENESIS

32

I. The role of BMP in ectodermal pattern formation. ...........................................................................

32

II. The role of XIAP and Dlx5 in BMP signaling ........... ..... .. ........... .... .. .. .. .. .... .. .... .... .. ...... ..................

32

III. Eary pattern formation of the peripheral nervous system in Drosophila

33......................... ..............

IV. TGF-~ family in nematode33

DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)35

I. Molecular aQd genetic approaches for the analysis of the sugar-signalling during
growth and development of Arabidopsis thaliana .............................................................................

35

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

35

DEPARTMENT OF REGULATION BIOLOGY37

DIVISION OF MOLECULAR NEUROBIOLOGY

38

I. Molecular mechanism of retinotectal projection

38

II. Functional roles of protein tyrosine phosphatase I; and y in brain development

and brain function... ......... ..... .. ... ... .. .......... .... ... ... ...... ... .. ... .. .. .. ..... .. .. .. .. ....... ... ... .. ..... .. ... ........... ....... .. ..

Ill. Functional roles of subfamily 2 sodium channels

DIVISION OF CELLULAR REGULATION

40
40
43

I. Enhancement of the stress tolerance of plants by genetic engineering of
the synthesis of glycinebetaine .......... ................ .................... ....................... .... .. .......... .....................

43

II. Important roles of polyunsaturated membrane lipids in stress tolerance .... .. .. .. .. .... .........................

43

III. Molecular mechanisms of the protection of the photosynthetic machinery

against high-temperature stress ..........................................................................................................

44

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

45

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT)46

I. Cloning and characterization of blue-light photoreceptors in the fern
Adiantum capillus-veneris ...................................................................................................................

46

II. Chloroplast relocation.........................................................................................................................

46

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

49

I. Mechanism of Axonal Guidance ..........................................................................................................

49

II. Mechanism of Neuronal Migration

49....................................................................................................

LABORATORY OF GENE EXPRESSION AND REGULATION51

DIVISION OF GENE EXPRESSION AND REGULATION I

52

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

52...............................................

52

III. Characterization of the genes for anthocyanin pigmentation in morning glories.
DIVISION OF GENE EXPRESSION AND REGULATION II

52
54

I. Molecular mechanism of expansion/contraction of ribosomal RNA gene repeats in
S. cerevisiae. .........................................................................................................................................

54

II. The E. coli genome project56

DIVISION OF SPECIATION MECHANISMS I

57

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

57

II. Gene expression and cerebellar long-term plasticity

57

DIVISION OF SPECIATION MECHANISMS II

59

I. Molecular phylogeny of plants and lichen

59

II. Evolution of body plan in plants

59

RESEARCH SUPPORT63

TECHNOLOGY DEPARTMENTRESEARCH SUPPORT FACILITY
I. Facilities

64.............................................................................................................

65.. .... ......... ....... .. ... .... .. .. .... .. .. .......... ........ .......... ... ....... .......... ...... .. ..... .. ... ....... .. .. ....... .... .............

65

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

RADIOISOTOPE FACILITY (Managed by NIBB)

66
68

I. Research Supporting Activity

68

II. Academic activity

68.... ..... .... .. .. .. ........ .. ... .. .. ...... ........... ... ....... .. ......... ....... .. ..... ..... ......... ......... .. .. .............

THE CENTER FOR ANALYTICAL INSTRUMENTS (Managed by NIBB)

70

INTRODUCTION
We present here the 1998 Annual Report of the
National Institute for Basic Biology (NIBB). NIBB, agovernment-supported, basic research institute, wasestablished in 1977. It aims to stimulate and promote
the biological sciences by conducting first-rate research on site as well as by cooperating with otheruniversities and research organizations. The Instituteconcentrates on the study of cellular functions, reproduction and development, neuronal and environmental
control, gene expression and regulation, and molecular evolution of eukaryotic organisms.
In 1998, the Center for Transgenic Animals andPlants was established. Dr. E. Watanabe was appointed as an Associate Professor of the Center in
September. At the end of March, Professor Masayuki
Yamamoto (University of Tokyo) of the Adjunct Division of Cell Proliferation and Professor Masatoshi
Takeichi (Kyoto University) of the Adjunct Divisionof Behavior and Neurobiology successfully completed
their terms at NIBB. In turn, Professor Motoya
Katsuki of the University of Tokyo, Professor
Mitsumasa Wada of the Tokyo Metropolitan University and Professor Fujio Murakami of the Osaka University were respectively appointed as Adjunct
Professors in the Division of Cell Proliferation, theDivision of Cellular Regulation and the Division of
Behavior and Neurobiology in April. In September,
Professor Ken-ichirou Morohashi of the Division ofSpeciation Mechanisms II moved to the Division of
Cell Differentiation. Drs. Y. Ozeki and M. Nakafuku
were appointed as Associate Professors of the AdjunctDivision of Cell Proliferation and the Adjunct Division of Behavior and Neurobiology, respectively. The
positions were vacated when Adjunct Associate Professors M. Maejima and H. Kobayashi departed the
NIBB . We have also recently appointed 6 research
associates, 8 institute research fellows and 1 technician to replace 7 research associates, 9 institute research fellows and 2 technicians who moved to otherpositions (e.g. associate professors, senior scientists,
research associates, etc.) either within or outside of
NIBB. In addition numerous postdoctoral fellows areactively engaging in various research at NIBB. The
activities and future goals of NIBB, especially in thefield of plant science, were subjected to an external
peer review.
NIBB plays an important role as a national and international center for biological research and is responsible for conducting research projects incooperation with various research groups. As a part ofsuch cooperative activities, NIBB carried out a Special Program which is currently focused on "Adaptation and Resistance to the Environment." In support ofthis program, NIBB held the 40th NIBB Conference
entitled "Stress Responses. Sensing, Signal Transduction and Gene Expression" (Professor Norio Murata,
organizer). The 41st NIBB Conference was an International Symposium sponsored by the Ministry of

Education, Science, Sports and Culture and entitled"Frontier of the Genome Biology of Escherichia coli"(Professor Takashi Horiuchi, organizer). In August,the Director-Genelas of NIBB and the BiologicalResearch Center of the Hungarian Academy of Sciences signed a "Memorandum of Understanding between the NIBB and the Biological Research Centerof the Hungarian Academy of Sciences" to promoteinternational cooperation between the two institutions.
A similar agreement between NIBB and the Australian National University allowed the Australia-Japan
Binational Seminar entitled "Molecular Physiology of
Photosynthesis in Stress Environments" (Professors
C. Barry Osmond and N. Murata, organizers) to beheld in 1998. In addition, NIBB continues to sponsor
interdisciplinary symposia and study meetings on current topics by inviting leading scientists from aroundthe world to the institute. NIBB also provided a training course in biological sciences for young investigators. To continue our improvement welcome anysuggestions concerning the research activities of
NIB B.
Finally, I would like to congratulate Drs. S. Itoh, H.
Shibuya and M. Hayashi for being awarded the Plant
and Cell Physiology Award, the Young InvestigatorAward of the Japanese Biochemical Society and theBotanical Awards for Young Scientists, respectively.
Mr. Y. Tomoyasu received the Nagakura Award. Following Drs. T. Kinoshita and J. Yuasa, he is the third
graduate student from NIBB to win the Award.
Hideo Mohri, D.Sc.
Director-General

2

NATIONAL INSTITUTE

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology, NIBB, isa part 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 appointinents and on the Institute's
annual budget as well as its future prospect.

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--tresearch 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, Laboratory of Stress-Resistant
Plants and Center for Transgenic Animals and Plants.
In addition, seven facilities are operated jointly with
the NIPS; they consist of the Radioisotope Facility,
Electron Microscope Center, Center for Analytical Instruments, Machine Shop, Laboratory Glassware Facility, Animal Care Facility, and Low-Temperature
Facility.

CampusThe 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)

Department of Developmental Biology

Reproductive Biology- { Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)

Department of Regulation Biology

Molecular Neurobiology- { Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)

Gene Expression and Regulation I{
Laboratory of Gene Expression and Regulation
GSen~ Eti:preMssiohn a~d Reigulation II. pecta on ec amsms
Speciation Mechanisms II

3

FOR BASIC BIOLOGYGRADUATE 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.

4

NATIONAL INSTITUTE

OFFICE OF DIRECTOR
Director-General:
Hideo Mohri
Associate Professors: Shigeru Itoh
Ryuji Kodama
Kohji Ueno
Research Associate: Akinao Nose (-May 31, 1998)
Masua Go to (on leave)complex in collaboration with a Marine Biotechnology Institute Kamaishi . Although chlorophyll a thatabsorbs visible red light has been known to be indispensable for the oxygenic photosynthesis of plants
and cyanobacteria, this organism efficiently undergoes
oxygenic photosynthesis even with far-red light thathas lower quantum energy. We named the special pairchlorophyll-d of the newly identified photosystem I
reaction center P740 (Fig. 1). The organism can be a
missing link between the anoxygenic photosynthesisthat can use far-red light and the oxygenic photosynthesis that uses only visible-light. Molecular mechanisms and structures of the new photosynthetic
systems are now being studied.

Evolution of photosynthesis
Shigeru Itoh
We study the evolution of photosynthesis in molecular level. Oxygen-evolving photosynthesis of
cyanobacteria seems to be evolved from theanoxygenic bacterial photosynthesis at 2.7-3.5 billion
years ago just after the evolution of life. Symbiosis of
cyanobacteria inside the eukariotic cells, then, produced the first plant 2 billion years ago. We recently
focus our target of research on the evolutionary process from anoxygenic to oxygenic photosynthesis.

(2) Survey through optimization mechanism of photosynthesis.
We compared the structure-function relationshipsof the photosynthetic reaction center complexes ofplants, green sulfur bacteria, Heliobacteria and newly
found organisms. We replaced quinone cofactor molecules by artificial compounds inside the proteins andstudied their localization by the ps-ns absorption,
fluorescence and spin-echo ESR spectroscopy at 4280 K. Comparison of structure-function correlationin various photosynthetic systems also revealedunique cytochrome reactions in Green-sulfur bacteria.
Molecular architecture of plant and bacterial photosynthetic proteins are shown to be highly optimized
but in different directions . This gave us a hint for thedesign of ancestral photosynthetic apparatus .

(1) Hunting for missing links of evolution "New photosynthesis".
A newly discovered bacterium Acidiphilium rub rum
isolated from acidic mine drainage, was shown to use
Zn-bacteriochlorophyll in its anoxygenic photosynthesis by us. This was the first case of photosynthesis
based on pigments other than Mg-chlorophyll 's thatare ubiquitously used in the ever-known oxygenic andanoxygenic photosynthesis.
Acaryochloris marina, a newly discovered oxygenevolving cyanobacteria-like organism, was shown to
use a far-red absorbing new pigment chlorophyll-din
its photosystem I reaction center pigment protein

Photon energJ (e V)

A
~

CJ

=

..c 0.5

spinach .-·.

~

,.Q

PSI /

rJJ
,.Q

<
0

0
cA

,.Q

<
<l

flash
l Sms

D A740P700

Chloropllyll d
r:h/oroph vll a

P740-0.005700
600
Wavelength (nm)Rt
CHO
C II CI I2

800

Fig. I . Absorption spectrum (A) and light-induced difference spectrum of reaction center chlorophyll-d P740(B) of newly identified~hotosystem I (PSI) complex of Acaryochloris marina. Broken lines in (A) and (B) represent absorption and difference spectra (P700)

m spmach PS I reactiOn center containing chlorophyll-a, respectively. Molecular structures of chlorophylls are also shown.

5

FOR BASIC BIOLOGY

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 cellde<,1th 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 a!.,Raux's Arch. Dev. Bioi. 204, 418-426, 1995).
The cells in the degeneration region are actively
engulfed by the macrophages in the cavity beneath thewing epithelium. Moreover, the macrophages seem tobe concentrated beneath the degeneration region by
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).
Studies using another lepidopteran species, Orgyia
recens approximans, provided by Drs. Y. Arita and K.
Yamada (Meijo University) is underway. In this species, the wing is normally formed until the beginningof the pupal period, but becomes conspicuously degenerated only in the female adult. In our preliminary
study, it was shown that the pupal wing is normallyformed both in male and female pupa, but after about
two days, female pupal wing starts degeneration on its
margin, as if the degeneration region is continuously
formed deep into the center of the wing (Kodama, R.
et a!., unpublished). It is thus suggested that the control mechanism which demarcates the region to bedegenerated is defective in the female in this species.
Further investigation using this species might giveimportant insight into such mechanisms.
Another collaborative work with the laboratory of
Dr. K. Watanabe (Hiroshi.m a University) concerns
mostly on the development of trachea and tracheole
pattern in the swallow tail butterfly. According to theirobservations, the pattern formation of wing epithelium
is often dependent on tracheal and tracheole patterns.
Basic research on the development of tracheal patternformation is being done including SEM observation(Fig.2) and histological observations to provide thebasis of the morphogenetic analysis of wing epithelium as a whole.

Fig.2. The inside of the larval wing disc of a swallow tailbutterfly. The dorsal and the ventral epithelium of the discwere peeled apart with adhesive tape after critical-pointdrying. The upper panel shows a low magnification view,
whose lower right comer is shown in high magnification in
the lower panel. Thick and smooth-surfaced tubes in theupper panel are trachea. A bundle of much thinner tubes aretracheoles, whose details are shown in the lower panel.

6

NATIONAL INSTITUTE

Protein palmitoylation and cell development atembryogenesis
Kohji Ueno
Thus far I have studied the molecular mechanismsof abdominal leg development in the silkworm
Bombyx mori. From these studies, I have elucidated
that abdominal leg development is regulated by ahomeotic gene which specifies the identities of theabdominal segments. I have found that a high molecular weight protein, p260/270, is expressed in abdominal leg cells during early embryonic stages. The
purified p260/270 was found to transfer palmitate to
the cysteine residues of synthetic peptides in vitro.
Most small, GTP-binding, heterotrimeric G, and Gprotein-linked receptor proteins are known to bemodified with palmitate through thioester linkages.
Thus, these dynamic modifications may be importantin the regulation of signal transduction. Therefore, I
speculated that p260/270 may be involved in protein
palmitoylation and may function in abdominal leg
development (Ueno, K. and Suzuki, Y. J. Bioi. Chern.
272: 13519-13526, 1997).
To understand the molecular mechanisms of how
protein modification by palmitoylation regulates thedevelopment of cells and organs, I have attempted tofind a vertebrate homologue of Bombyx p260/270. In
order to accomplish this , I have searched an EST(Expressed Sequence Tag) data base for cDNAs encoding amino acid sequence residues corresponding top260 and p270. From these searches, I found thatseveral mouse embryonic eDNA clones were highly
homologous to the amino acid sequences of p260 and
p270. This suggested that a homologue of p260/270
was expressed in mouse embryos. One of these mousep260/270 cDNAs was therefore used for in situ hybridization analyses of mouse embryos. Transcripts
homologous to p260/270 were detected mainly in thecentral and peripheral nervous systems of mouse embryos from embryonic day 10 (ElO). These transcripts
were detected in the regions of the forebrain, midbrain, hindbrain and spinal cord of the central nervous
system as well as in the cranial ganglia and dorsal root
ganglia of the peripheral nervous system. The result ofin situ hybridization on one mouse embryo section isshown in Fig.3. From these results, I speculate that ahomologue of Bombyx p260/270 regulates development in the central and peripheral nervous system ofmouse embryos. Further study is necessary to identify
the specific cells which express these transcripts andunderstand the mechanisms of how this homologue
regulates cell development.
Akinao Nose
How individual nerve cells find and recognize theirtargets during development is one of the central issues
in modern biology. Our aim is to elucidate 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 fiber 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 identified two genes , connectin and capricious that playroles neuromuscular connectivity.
I. connectin

Fig.3. Localization of transcripts of a mouse homologueof p260/270 by in situ hybridization of a sagittal section ofmouse ElLS embryo. The transcripts are stained purple andnuclei are stained with methyl green. FB , forebrain ; MB,midbrain; CG cranial ganglia.

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

7

FOR BASIC BIOLOGY

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.
II. capricious
capricious (caps) is expressed in subsets of neurons
and muscles, including the RP5 motoneuron and its
target, muscle 12. The eDNA cloning and sequencing
revealed that caps encodes a novel transmembraneprotein that like connectin belongs to LRR family.
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 musculardevelopment. We found that in the loss-of-function
mutants of caps, the synaptic arborization pattern on
muscle 12, a caps-positive muscle, was abnormal. 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. Like in the lossof-function alleles, the muscle 12 terminal axon
branch often formed collaterals that turned back andinnervated muscle 13. These results suggest that Caps
is involved in neuromuscular target recognition and/or
stabilizaion of the synapses.

Analysis of meiosis
Masuo Goto
The major goal of our research 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 isolated and analyzed a number of cloned mouse genes which may be
relevant to spermatogenesis. In addition, efforts havebeen also paid to elucidate the regulatory mechanismsof meiosis in fission yeast in more detail. Some examples of the analysis are presented below.
I. Regulators of meiosis II in fission yeast.
We analyzed the mechanisms of fission yeast meiosis using a meiosis II deficient mutant mes 1. Even
though the mesJ+ gene is essential to progress tomeiosis II, its function has not yet been defined. To
better understand the downstream pathway of mesl
we decided to isolate a suppressor. We isolated our
suppressor, mesl suppressor, from a S. pombe genomic library. The mesl mutation was suppressed byoverexpression of the slpl gene. The slpl gene is relevant to spindle checkpoint in mitosis and radiation
sensitivity. This gene is homologous to S. cerevisiae
CDC20, but lacks the corresponding 35 amino acids
C-terminal. The authentic slpJ+ gene encodes 488

amino acids, and its carboxyl region has significant
homology to WD-repeat protein family. The C-terminal 35 amino acid portion includes a part of the seventh WD-repeat. In our experiments, full-length Slp1p
could not suppress the mes 1 defect. In addition, under
nitrogen starvation, the slpl mutant cells produce
mature asci containing one or two diploid spores. This
indicates that the slpJ+ gene is essential in both meiosis I and meiosis II.
We determined that the physical interaction between
Mes1p and Slp1p depends on the Slp1p C-terminal35
amino acids. This physical interaction was prevented
when an amino acid substitution occurred at aspartate457 or tryptophan-463 in the seventh WD-repeat. The
mutant Slp1p could suppress the mesl defect similarly
to the truncated 35 C-terminal amino acids form.
Therefore, the seventh WD-repeat of Slp1p is a key
region for mesl suppression and binding to Mes1p.
New insight into the crystal structure of G-protein
heterotrimer and the G ~ y dimer revealed that G ~ isshaped like a seven-bladed propeller (seven WD-repeats) with a central shaft tunnel connecting the two
faces. The seven WD-repeats of Slp1p could form asimilar structure. Mutations in the seventh WD-repeat
might destruct the propeller form since both the aspartate and the tryptophan residues are well conserved
between WD-repeat protein families. This result indicates that the Sip 1p may not be active in the sevenpropeller structure at the onset of meiosis II. In wildtype cells, the physical interaction between Meslp
and Slplp is a critical step in activating Slp1p andovercoming the meiosis II checkpoint. This study
implies that there are different cell cycle checkpointmechanisms between mitosis and meiosis even though
the same cell cycle regulators act at the appropriate
steps.
II. Molecular cloning and characterization of mouse
testis poly(A) binding protein II.
A eDNA clone from mouse testis eDNA library was
isolated by a transcomplementation method using a S.
pombe meiotic mutant (sme2) that arrests in the firstmeiotic division. The eDNA clone isolated has anopen reading frame encoding 302 amino acids and has
a strong similarity to mouse poly(A) binding protein
II (mPABII) and bovine poly(A) binding protein II(PABII). PABII is known to bind the growing poly(A)tail. Northern blot analysis of the eDNA clone identified by mPABII revealed a single transcript of 1.2kb,
detectable exclusively in adult testis. Immunohistochemical analysis using the polyclonal antibody
demonstrated that mPABII protein was expres~ed in
the nucleus at the specific stages from late pachytene
spermatocytes to round spermatids. Genetic mapping
showed the Pabp3 gene encoding mPABII locatednear at position 19.5 on mouse chromosome 14. Although mPABII function is not clearly linked tospermatogenesis, these results suggest that mPABII
might be involved in specific spermatogenetic celldifferentiation.

8

Publication List:
Hara, H., Tang, J., Kawamori, A., lwaki, M. and Itoh,
S. (1998) Anomalous pulse-angle and phase dependence of Hahn's electron spin echo and multiplequantum echoes of the spin correlated radical pair
P700+Al- in Photosystem I. Applied Magnetic
Resonance 14, 367-379.
ltoh, S., lwaki, M., Wakao, N., Aoki, A. and Tazaki,
K. (1998) Accumulation of Fe, Cr and Ni metals
inside cells of acidophilic bacterium Acidiphilium
rubrum that produced Zn containing bacteriochlorophyll a. Plant Cell Physiol. 39, 740-744.
lwaki, M., ltoh, S., Hara, H., and Kawamori, A.
(1998) Spin-polarized radical pair in Photosystem I
reaction center that contains different quinones andfluorenones as the secondary electron acceptor. J.
Phys. Chern. 102, 10440-10445.
Kagami, 0., Gotoh, M., Makino, Y., Mohri, H.,
Kamiya, R. and Ogawa, K. (1998) A dynein lightchain of sea urchin sperm flagella is a homolog ofmouse Tctex 1, which is encoded by a gene of the
t complex sterility locus. Gene, 211, 383-386.
Kosaka, M., Kodama, R. and Eguchi, G. (1998) In
vitro culture system for iris-pigmented epithelial
cells for molecular analysis of transdifferentiation.
Exp. Cell Res. 245, 245-251.
Kubo-Irie, M., lrie M., Nakazawa, T. and Mohri, H.
(1998) Morphological changes in eupyrene andapyrene spermatozoa in the reproductive tract of themale butterfly Atrophaneura alcinous Klug. Invertebrate Reprod. Dev. 34, 259-268.
Nose, A., Isshiki, T. and Takeichi, M. (1998). Regional specification of muscle progenitors in Drosophila: the role of the msh homeobox gene.
Development 125, 215-223.
Ohoka, H., lwaki, M. and Itoh, S. (1998) Membranebound cytochrome cz couples quinol oxidoreductase
to the P840 reaction center complex in isolated
membranes of the green sulfur bacterium
Chlorobium tepidum. Biochemistry 37, 1229312300.
Qiang, H., Miyashita, H., Iwasaki, 1., Kurano, N.,
Miyachi, S., lwaki, M. and ltoh, S. (1998) A photosystem I reaction center driven by chlorophyll d inoxygenic photosynthesis. Proc. Nat/. Acad. Sci.
USA. 95, 13319-13323.
Shishido, E., Takeichi, M. and Nose, A. (1998).
Drosophila synapse formation: regulation by transmembrane protein with Leu-rich repeats, Capricious. Science 280, 2118-2121.
Yoshida, A., Arita, Y., Sakamaki, Y., Watanabe, K. andKodama, R. (1998) Transformation from the pupal
to adult wing in Oidaematophorus hirosakianus(Lepidoptera: Pterophoridae). Ann. Entomol. Soc.
Am. 91, 892-857.

NATIONAL INSTITUTE9

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Mikio Nishimura

The department consists of two regular divisions and three
adjunct divisions. One of the adjunct divisions started in 1998.
The department conducts studies on molecular dynamics of thecell in higher plants and animals such as organelle differentiation,
autophagy, cell motility, cytokinesis and neural development.

10

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

Mikio Nishimura
lkuko Hara-Nishimura
Makoto Hayashi
Tomoo Shimada
Post doctoral fellows: Tetsu Kinoshita
Akira Kato (- Aug. 30 )Nagako Hiraiwa (- March 30 )Yasuko lshimaru
Kanae Shirahama
Shoji Mano
Graduate Students;
Hiroshi Hayashi
Kenji Yamada
Naoto Mitsuhashi
Kazumasa Nito
Yuki Tachibe(from Hiroshima University)
Technical Staffs:
Maki Kondo
Katsushi Yamaguchi
JSPS Technical Staffs: Miwa Kuroyanagi
Kanako Toriyama (- Nov. 30 )Miki Kinoshita (April .1 - )Chizuru Ueda (April .1 - )Chihiro Nakamori (April .1 - )Visiting Scientists:
Roland R. Theimer(Bergische Univ. Germany )Yasuko Koumoto

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

I. Regulation at the level of protein transport tomicrobodies during the microbody transition.
Dramatic metabolic changes which underlie theshift from heterotrophic to autotrophic growth occur
in greening of seed germination. Accompanying thesemetabolic changes, many constitutive organelles arefunctionally transformed. For example, etioplasts differentiate into chloroplasts and mitochondria acquire
the ability to oxidize glycine. Glyoxysomes which aremicrobodies engaged in the degradation of reserve oil
via ~-oxidation and the glyoxylate cycle, are transformed into leaf peroxisomes that function in several
crucial steps of photorespiration. After the functionaltransition of glyoxysomes to_ leaf peroxisomes during
the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes toglyoxysomes occurs during senescence. Microbody
enzymes function after their transport to microbodies.
Since the enzyme compositions and functions ofglyoxysomes and leaf peroxisomes differ from eachother, it is likely that the 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 of targeting signals to microbodies have been reported. One
type of targeting signal is a part of the mature protein.
One such signal, the tripeptide Ser-Lys-Leu, occurs at
the C-terminal. has been identified as a targeting signal. Ser-Lys-Leu and related amino acid sequences
commonly function in mammals, insects, fungi, andplants.
To characterize the microbody targeting signal inplants, we have examined the ability of 24 carboxyterminal amino acid sequences to facilitate the transport of a bacterial protein, ~-glucuronidase (GUS)
into microbodies in green cotyledonary cells oftransgenic Arabidopsis. Immunocytochemical analysisof the transgenic plants revealed that carboxy-terminal
tripeptide sequences of the form [C/NS/P]-[K/R]-[11LIM] function as a microbody-targeting signal, while
tripeptides with proline at the first amino acid position
and isoleucine at the carboxyl terminus show weaktargeting efficiencies. In contrast, another small groupof microbody proteins are synthesized as precursors
with N-terminal cleavable presequences; these include
3-ketoacyl-CoA thiolase, glyoxysomal citrate synthase (gCS), glyoxysomal malate dehydrogenase(gMDH) and long-chain acyl-CoA oxidase. These Nterminal sequences also function as a targeting signal
to microbodies and are designated as PTS2. We generated a transgenic Arabidopsis plant that accumulated GUS-chimeric proteins with the N-terminal
presequences of the pumpkin gCS or pumpkin
gMDH. Immunocytochemical studies of thetransgenic plant showed that theN-terminal sequencesof gCS and gMDH function as targeting signals toplant microbodies. Site-directed mutagenesis studiesof the chimeric proteins indicated that several aminoacids in the consensus regions of the presequenceswere essential for targeting to microbodies or werenecessary for the processing of the presequence. The
fusion protein was transported into functionally different microbodies, such as glyoxysomes, leaf peroxisomes and unspecialized microbodies, and was subsequently processed. These observations indicated thatthe transport of gCS and gMDH is mediated by theiramino-terminal presequences and that the transportsystem is functional in all plant microbodies. Therefore, it is unlikely that glyoxysomes and leaf peroxisomes posses different targeting machineries.

II. Microbody defective mutant of Arabidopsis.
It has been suggested that the functional conversion
between glyoxysomes and leaf peroxisomes is controlled by gene expression, protein translocation, andprotein degradation. A genetic approach is an effective
strategy toward understanding the regulatory
mechanism(s) of peroxisomal function at the level ofFOR BASIC BIOLOGY

gene expression, protein translocation, and protein
degradation . We isolated and characterized 2,4dichlorophenoxybutyric acid (2,4-DB)-resistant mutants . It has been demonstrated previously that
2,4-dichlorophenoxybutyric acid (2,4-DB) is metabolized to produce a herbicide, 2,4-D, by the action ofperoxisomal fatty acid P-oxidation in higher plants. To
isolate mutants that have defects in peroxisomal fatty
acid b-oxidation, we screened mutant lines of
Arabidopsis seedlings for growth in the presence of
toxic levels of 2,4-DB. Twelve of the mutants survived; of these, four required sucrose for postgerminative growth (Figure I , A B). This result
suggests that these mutants have defects in peroxisomal fatty acid P-oxidation, because peroxisomal fatty
acid P-oxidation plays an important role in producing
sucrose from storage lipids during germination. Genetic analysis revealed that these mutants can be classified as carrying alleles at three independent loci,which we designated pedl, ped2, and ped3 , (where
ped stands for peroxisome defective). The pedl mutant lacks the thiolase protein, an enzyme involved infatty acid P-oxidation during germination and subsequent seedling growth, whereas the ped2 mutant has adefect in the intracellular transport of thiolase fromthe cytosol to glyoxysomes. Etiolated cotyledons ofboth pedl (Figure lD) and ped2 mutants haveglyoxysomes with abnormal morphology.

III. Transport of storage proteins to protein
storage vacuoles is mediated by large PAC(l!recursor-accumulating) vesicles.
Novel vesicles that accumulate large amounts ofproprotein precursors of storage proteins were purified
from maturing pumpkin seeds. These vesicles were designated urecursor-accumulating (PAC) vesicles and havediameters of 200 to 400 nm . We characterized them toanswer the question of how seed protein precursors areaccumulated in the vesicles to be delivered to protein
storage vacuoles. They contain an electron-dense core ofII

storage proteins surrounded by an electron-translucent
layer, and some vesicles also contained small vesiclelike structures. An immunocytochemical analysis revealed numerous electron-dense aggregates of storage
proteins within the endoplasmic reticulum (Figure 2). It
is likely that these aggregates develop into the electrondense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis
also showed that complex glycans are associated withthe peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These
results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates ofstorage proteins directly to protein storage vacuoles.

IV. Vacuolar processing enzymes in protein-storage
vacuoles and lytic vacuoles.
Vacuolar processing enzymes (VPEs), which are responsible for maturation of various vacuolar proteins
belong to a novel family of cysteine proteinases. Molecular characterization of castor bean VPE revealed
that the latent precursor of VPE (pro VPE) is converted
into an active VPE by self-catalytic proteolysis. Thus,
no other factor is necessary to produce active VPE,
and VPE itself is a key enzyme in determining thefinal conformation of the vacuolar proteins. The VPEmediated system is widely distributed in various plantorgans . The temporal and spatial expression of the
VPE system has been examined with three
Arabidopsis VPEs, aVPE, PVPE and yVPE. The
f3VPE gene is expressed in seeds, suggesting that
PVPE plays a role in maturation of seed proteins inprotein storage vacuoles. On the other hand, both theaVPE and yVPE genes are expressed in senescenttissues and their expression patterns are correlatedwith programmed cell death. The vegetative VPEs
might regulate the activation of senescence-associatedhydrolytic enzymes in the lytic vacuoles of cells preparing for death.

Figure 1. A microbody-defective mutant of Arabidopsis.
Wild-type Arabidopsis (A) can germinate and grow after supplying only water. In contrast, the pedl mutant (B), which has adefect in glyoxysomal fatty acid ~-oxidation, requires sucrose for post-germinative growth. Glyoxysomes in wild-type plant (C;arrowhead) are surrounded by a single membrane with approximately 1 j..lm in diameter, whereas glyoxysomes in the pedl mutant(D; arrowhead) become bigger, and contain tubular structures.

12

NATIONAL INSTITUTE

Figure 2. Electron microscopy of maturing pumpkin cotyledons showing numerous electron-dense aggregates within the ER.
(A) Electron-dense core aggregates (arrows) formed within the ER in cells at the middle stage of seed maturation. The electrontranslucent space between the aggregate and the ER membrane is loaded with ribosomes. PAC vesicles (PV) are also visible.
(B) Immunoelectron microscopy with 2S albumin-specific antibodies. The aggregates within the ER (arrows) are labeled withgold particles. PAC vesicles (PV) and a protein storage vacuole (V) are also visible.
CW, cell wall; Lb, lipid body. Bars in (A) and (B) = 500 nm.

V. Role of molecular chaperones in organelle
differentiation.
Molecular chaperones are cellular proteins thatfunction in the folding and assembly of certain otherpolypeptides into oligomeric structures 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 roles in the translocation of proteins into chloroplasts. In addition to mitochondrial chaperoninlO homologues, we isolated a eDNA for chloroplastic
chaperonin I 0 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 a 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 CpnlO homologue was abundant in leaves and was increased byheat treatment. To examine the oligomeric structure of
Cpn20, a histidine-tagged construct lacking the transit
peptide was expressed in E. coli and purified by affinity chromatography. Gel filtration and cross-linking
analyses showed that the expressed products form atetramer. The expressed products can substitute for
GroES to assist in the refolding of citrate synthase
under non-permissive conditions. Further analysis on
the subunit stoichiometry of the GroEL-Cpn20 complex revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.

Publication List:
Hara-Nishimura, I (1998) Asparaginyl endopeptidase.
In Handbook of Proteolytic Enzymes, edited by A.
J. Barrett, N. D. Rawlings and J. F. Woessner (Academic Press, London, UK, 1998) pp. 746-749.
Hara-Nishimura, 1., Shimada, T., Hatano, K.,
Takeuchi, Y. and Nishimura, M. (1998) Transport ofstorage proteins to protein-storage vacuoles is mediated by large precursor-accumulating vesicles.
Plant Cell 10, 825-836.
Hara-Nishimura, 1., Kinoshita, T., Hiraiwa, N . andNishimura, M . (1998) Vacuolar processing enzymes
in protein-storage vacuoles and lytic vacuoles . J.
Plant Physiol. 152, 668-674.
Hayashi , H., L. De Bellis, Yamaguchi, K. , Kato, A.,
Hayashi, M. and Nishimura, M. (1998) Molecularcharacterization of a glyoxysomal long-chain acylCoA oxidase that is synthesized as a precursor ofhigher molecular mass in pumpkin. J. Bioi. Chem.
273, 8301-8307.

FOR BASIC BIOLOGY

Hayashi, M., Toriyama, K., Kondo, M. andNishimura, M.(1998) 2,4-Dichlorophenoxybutyric
acid-resistant mutants of Arabidopsis have defects
on glyoxysomal ~-oxidation. Plant Cell 10, 183195.
Hayashi, M., Toriyama, K., Kondo, M., Kato, A.,
Mano, S., L. De Bellis., Hayashi-Ishimaru, Y.,
Yamaguchi, K., Hayashi,.H. and Nishimura, M.
(1998) Functional transformation of plant peroxisomes. Cell Biochem. Biophys. in press.
Kato, A., Hayashi, M., Kondo, M. and Nishimura,M.(1998) Transport of peroxisomal proteins that aresynthesized as large precursors in plants. Cell
Biochem. Biophys. in press.
Kato, A., Takeda-Yoshikawa, Y., Hayashi, M., Kondo,
M., Hara-Nishimura, I. and Nishimura, M. (1998)
Glyoxysomal malate dehydrogenase in pumpkin:cloning of a eDNA and functional analysis of its
presequence. Plant Cell Physiol. 39, 186-195.
Mano, S., Hayashi, M. and Nishimura, M. (1998) A
leaf-peroxisomal protein, hydroxypyruvate reductase, is produced by light-regulated alternative splicing. Cell Biochem. Biophys. in press.
Nishimura, M. (1998) Molecular chaperones and temperature stress. Stress Responses of Photosynthesis
Organisms: Molecular Mechanisms and Molecular
Regulations, Edited by K. Satoh and N. Murata,
Elsevier. pp. 83-92.
Nishimura, M., Hayashi, M., Toriyama, K., Kato, A.,
Mano, S., Yamaguchi, K., Kondo, M. and Hayashi,
H. (1998) Microbody defective mutants ofArabidopsis. J. Plant Res. 111, 329-332.

13

14

NATIONAL INSTITUTEDIVISION OF BIOENERGETICS
Professor:
Associate Professor:
Research As$ociate:

Yoshinori Ohsumi
Tamotsu Yoshimori
Takeshi Noda
Yoshiaki Kamada
Post Doctral Fellow: Noboru Mizushima 1
Takahiro Shintani 2 (from April)
Naotada Ishihara 1 (from July)
Graduate Students:
Takayoshi Kirisako(Graduate Univ. of Advanced Studies)
Yoshinobu lchimura(Graduate Univ. of Advanced Studies)Technical Staff:
Yukiko Kabeya
Visiting Fellow:
Fumi Yamagata(Teikyo Univ. of Sci. and Tech.)
Visiting Scientist:
Kazuaki Furukawa(from November)
Satsuki Okamoto

This division aims to elucidate the physiologicalrole and mechanism of intracellular protein degradation in a lytic compartinent. Up to recently, molecularbiologicaf studies on the topic of cellular regulation
have focused on protein synthesis, and (the control of)gene expression. However, it is clear that cellular activity is maintained by a delicate balance between
synthesis and breakdown of individual proteins. For
the last few years, protein degradation has become ahot field in cell biology, because it plays critical roles
in many aspects of cellular functions. Most of the selective protein degradation is believed to occur in thecytosol through the ubiquitin/proteasome system. In
contrast, major bulk protein degradation takes place inthe lysosome/vacuole. Non-specific protein turnover
has essential roles under nutrient stress or for cell differentiation of eukaryotic cells. Until now, the molecular details of protein degradation in the lytic
compartment remained to be uncovered due to thelack of a sensitive monitoring system, and the dynamic features of the lysosomal/vacuolar membrane
system.
Autophagy is defined as the process of bulk degradation of cytoplasmic proteins or organelles in thelytic compartment and seems to be a ubiquitous andbasic cellular activity in all eukaryotic cell types.
Autophagy has been described mostly in higher
eukaryotic cells by using electron microscopy. The
lysosome/vacuole is a single membrane bound compartment containing various hydrolytic enzymes. Segregation of lytic enzymes from the cytosol requires anobvious cell biological process how to deliver proteinsor organelles destined for degradation into the lumenof this compartment. Macroautophagy is the majorroute to the lysosome/vacuole under nutrient starvation conditions. This process involves the formation of
a double membrane structure called an autophagosome (AP). The AP fuses with a primary lysosome and matures to be an autophagolysosome, thenits contents are digested and recycled to be reused.
Autophagy has been studied extensively using various kinds of mammalian tissues and cultured cells. It
is highly regulated by various factors such as aminoacid or metabolites, hormones and growth factors, andsecond messengers. Because the modes of autophagy
are so diverse and the physiological role and regulation of autophagy depend upon the cell type andphysiological demands, it is difficult to present a general model for autophagy in higher eukaryotes. For
this reason, it has been helpful to develop a simple
system to elucidate the fundamental machinery of the
autophagic process.
Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, S. cerevisiae, induces
bulk protein turnover in the vacuoles under starvation
conditions. This whole process corresponds to that ofmacroautophagy in higher eukaryotic cells. Byelectron microscopic analyses we succeeded in
detecting double membrane structures in thecytoplasm enclosing a portion of cytosol. This
autophagosome immediately fuses with vacuoles,delivering single inner membrane structures,
autophagic body (AB) in the vacuole. When vacuolar
proteinase activities are blocked genetically or by
specific inhibitor ABs are accumulated in thevacuoles. They move around vigorously in thevacuoles by Brownian motion, and are easily
detectable by light microscope. Thus we can follow
the progression of autophagy as the accumulation of
ABs in real time. Biochemical and immunoelectron
microscopic analyses showed that starvation-induced
sequestration is non-selective, that is, cytosolic
enzymes and organelles are sequestered randomly tothe 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 amino acid depletion. Signal transduction of starvation for induction of
autophagy is a key question to be solved. Recently wefound rapamycin induces autophagy in the cellgrowing in a rich medium and that Tor, phosphatidylinositol kinase homologue plays essential role
for the regulation of autophagy.
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.
Genetical dissection of autophagic process
Yeast has made great contribution to solve 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 we started to isolate
mutants defective in the process of autophagy. Total
14 autophagy defective agp CAutQphagy) mutants

15

FOR BASIC BIOLOGY

APG 1 codes a novel Serffhr protein kinase essential
for the induction of autophagy. This provides the first
direct evidence for involvement of protein phosphorylation in the process of autophagy. Apg13p interacts
with Apglp and might function as a regulator of its
kinase activity. So far most APG genes are novel except APG6 and non-essential for vegetative growth.
The APG6 gene is allelic to VPS30 , which is required
for retrieval of the carboxypeptidase, CPY receptor to
the late Golgi from the endosome. APG 14 encodes anovel hydrophilic protein of 40.5kDa. Subcellular
fractionation experiments indicate that both Apg14p
and Apg6p are peripheral membrane proteins andform a stable complex. The apg14 mutant is normal
for CPY sorting. While overexpression of Apg14p
suppresses the autophagy defect of apg6-1, APG14
does not function as a multicopy suppressor of the
CPY sorting defect of this mutant. These results suggest that Apg6p has dual functions in autophagy andvacuolar protein sorting, and that Apg14p is specifically required for the autophagic process.
Using the assay system of autophagy we developed,
we are studying genetic interaction among APG
genes. Some APG gene products are starvation-inducible or change phosphorylation state by nutrient starvation. Analyses of intracellular localization of these
Apgps may provide us specific marker to elucidate themembrane dynamics during autophagy.

were isolated. They cannot induce protein degradation
upon shift from growth medium to the starvation
medium. They grow normally in a rich medium, but
start to die after 2 days in the starvation medium. Thissuggests that the autophagy is necessary for the maintenance of cell viability under starvation. Homozygous diploid of each apg mutant cannot undergo
sporulation, indicating that this cell differenciation
requires bulk protein degradation via autophagy.

APG genes are essential for Cvt pathway
One of vacuolar enzymes, aminopeptidase I, API issynthesized as a proform and sequestered to the vacuole directly from cytosol to the vacuole. All apg genes
are necessary for this process. Electron microscopic
analyses revealed that API first forms a complex in thecytosol and delivered to the vacuole by the distinct but
topologically similar mechanism to themacroautophagy. Though this Cvt pathway is biosynthetic and constitutive, and kinetically quite different
from the autophagy, but it must share most machinerywith the autophagy.

Analyses of APG genes
Now we are focusing on characterization of APG
genes, essential for autophagy. So far we have finished cloning and identification of twelve APG genes.

Starvation

Growing
Tor

I'

·~

Tor

Cvt Vesicle
Aminopeptidase I
VacuoleFig. 1 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.

16

NATIONAL INSTITUTE

Novel protein conjugation system essential for autophagy
Recently we found that four APG genes are involved in a novel protein conjugation system essential
for autophagy and the Cvt pathway. Apgl2p is a
2lkDa hydrophilic protein. Western blot analysis of
HA-tagged Apgl2p shows an additional band at
70kDa in addition to a band with the expected molecular mass. Sequence analysis of APG5 shows thatit encodes a hydrophilic protein of 34kDa. A western
blot of Apg5p also shows the 70kDa band, suggestingcovalent binding between Apg5p and Apg12p. Elimination of a single Gly at the C-terminus of Apg12p
results in a complete loss of the higher molecularmass band. Similarly, substitution of Lys 149 of
Apg5p, among 19 Lys residues, with Arg also abolishes this band. These results strongly suggest that the
C-terminal Gly of Apg12p is conjugated to Lysl49 of
Apg5p via an isopeptide bond. Cells expressing thesemutant forms of Apg12p or Apg5p are defective in
autophagy -and also Cvt pathway, indicating that the
conjugate formation of Apg5p-Apg12p is essential forboth pathways.
Furthermore, among the apg mutants , apg7 andapglO, could not .form the Apg5p-Apg12p conjugate.
These two genes may encode enzymes required forthe conjugate formation . From sequence data, Apg7p
is predicted to be an E1 like activating enzyme for
Apgl2p. Recently, many ubiquitin-related molecules
have been identified, and shown to function in various
regulatory steps. However, Apg12p has no homology
with ubiquitin or ubiquitin-related molecules. Apg12p
is much larger than ubiquitin and seems to have asingle target, Apg5p. The Apg5p-Apg12p conjugation
reaction was reconstituted in vitro and shown to be
ATP-dependent. Similarly, mutations in the ATP-binding site of Apg7p block conjugate formation . The
function of this conjugate in the Apg and Cvt path-( Apg12p )G~GG

Approach to mammalian autophagy
Then, there is no reason for us hesitating to challenge mammalian autophagy again, by taking advantage of identified genes involved in yeast autophagy.
By searching databases, we have found several mammalian eDNA related to the yeast genes. Homologuesof Apgl2p and Apg5p are widely distributed fromyeast to higher organisms . We cloned recently thehuman homologues of Apg12p and Apg5p and demonstrated a similar conjugation reaction in mammaliancultured cells. This result strongly suggests that thenovel conjugation system is conserved in all eukaryotes. To elucidate roles of autophagy in development,
differentiation, physiology, pathology in a multi-cellular system, we are making Apg 12-deficient mice.
We also found that one of the Apgp homologues is
specifically associated to the autophagosome membrane in human cultured cells. Post-translational processing of the molecule regulates the association. Thisis the first identified protein that is localized in mammalian autophagosomes. Yeast Csc1p is an AAA-type
ATPase implicated in regulation of autophagy. We
showed evidences that mouse SKD1 protein homologous to Csc1p plays a pivotal role on membrane transport via early endosomes in human cultured cells.
This suggests involvement of endosomal system in the
autophagic process.
Knowledge in yeast must give us key to uncover themechanism of autophagy in higher eukaryotic organisms such as mammals. We are now developing anassay for monitoring autophagy in mammalian cultured cells to perform systematic evaluation of identified candidates for components in machinery of
mammalian autophagy.

( Apg1 2p )G( Apg12p )GI

I?

ATP AMP+PPi\. }ways is not known yet but that the conjugation may benecessary for autophagosome or Cvt vesicle formation.

... (ATP AM P+PPi

c

Apg7p

c

) ____. ( Apg10p)( Apg12p )G

I

... (

K

Apg5p

)

~GI

\___} ... (~1----. ( target protein )Fig. 2 The Apg12p conjugation system and ubiquitin system
Apgl2p is activated by Apg7p in an ATP-dependent manner. After formation of a thioester with Apg7p, and then possibly with
ApglOp, Apg l2p is fin ally conjugated to Apg5p via an isopeptide bond (upper).
Although Apgl2p has no homology to ubiquitin, the Apg12p conjugation system is quite similar to that of ubiquitin (lower) whichis involved in selective proteolysis.

FOR BASIC BIOLOGY

Publication List:
Kametaka, S., Okano, T., Ohsumi, M., and Ohsumi, Y.
Apg14p and Apg6Nps30p form a protein complex
essential for autophagy in the yeast, Saccharomyces
cerevisiae. J. Biol. Chern. 273, 22284-22291 (1998).
Mizushima, N., Noda, T, Yoshimori, T., Tanaka, Y.,
Ishii, T., Geoge, M. D., Klionsky, D. J. Ohsumi, M.,
Ohsumi, Y. A protein conjugation system essential
for autophagy, Nature 395, 395-398 (1998).
Mizushima, N., Sugita, H., Yoshimori, T., andOhsumi, Y. A New Protein Conjugation System in
Human. The counterpart of the yeast Apg12p conjugation sytem essential for autophagy. J. Biol. Chern.
in press.
Noda, T. and Ohsumi, Y. Tor, a phosphatidylinositol
kinase homologue, controls autophagy in yeast. J.
Biol. Chern. 273, 3963-3966 (1998).
Tagawa Y., Yamamoto A.,Yoshimori T., Masaki R.,
Omori K., Himeno M., Inoue K., Tashiro Y. A 60
kDa plasma membrane protein changes its localization to autophagosome and autolysosome membranes during induction of autophagy in rat
hepatoma cell line, H-4-11-E cells. Cell Struct.
Funct. in press.
Tanida, 1., Mizushima, N., Kiyooka, M., Ohsumi, M.,
Ueno, T., Ohsumi, Y., and Kominami, E. Apg7p/
Cvt2p: a novel protein activating enzyme essential
for autophagy. Mol. Biol. Cell, in press.
Yamamoto A., Tagawa Y., Yoshimori T., Moriyama Y.,
Masaki R., Tashiro Y. Bafilomycin Al preventsmaturation of autophagic vacuoles by inhibiting
fusion between autophagosomes and lysosomes in
rat hepatoma cell line, H-4-11-E cells. Cell Struct.
Funct. 23; 33-42 (1998).

17

18

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

Motoya Katsuki (April], 1998 -)Kei Ito (June 1, 1998 -)Kimiko Tanaka (October 1, 1998 -)Asami Kido
(Ochanomizu University; July], 1998 -)The aim of this new adjunct division, started in June
1998, is to understand the basic rules by which elaborate neural circuits develop and function. To addressthese questions, an effective approach is to investigate
the whole of an easily-accessible nervous system thatshares certain of the architectural and functional features of the more complex vertebrate brains. With lessthan 105 neurones, and subject to powerful molecularand genetic techniques, the brain of the fruit fly
Drosophila melanogaster is a good model system forsuch a study, particularly because it contains neuralarrangements, such as those in the olfactory and visual pathways, that are extraordinarily similar toequivalent parts of vertebrate brains.
I. Developmental neuroanatomy

A comprehensive and detailed anatomical knowledge of the brain is a prerequisite for 1) analysing thephenotypes of nervous system-related mutants, 2)
identifying the cells that express the cloned genes, 3)
understanding the way information is processed in thebrain, and 4) devising computer models that simulate
brain functions. In spite of the hundred years of efforts using Golgi and other anatomical techniques,
however, the circuit structure of higher order associative regions of the brain is still essentially unresolved.
The GAL4 enhancer-trap system, which is widelyused for mutagenesis and gene cloning of Drosophila, isalso a powerful tool for obtaining a vast array of molecular markers that label specific subsets of brain cells.
Forming a consortium with eight other Drosophila laboratories in Japan, we have established more than 4000
GAL4 strains. Different aspects of the morphology of
the GAL4-expressing cells can be visualised by crossingthese strains with the strains that carry various VASlinked reporter genes, such as nuclear-specific UASNLS-lacZ, axon-targeting VAS-tau, cytoplasmic
UAS-lacZ and UAS-GFP, and presynaptic site-specific
VAS-neuronal synaptobrevin-GFP.

The screening of the GAL4 strains is being performed by observing the expression pattern in theadult brain. Useful strains will be selected to analyse
the information pathway of different sensory modalities in the higher-order brain regions, as well as tostudy how the labelled neurones form their elaborate
fibre connections during neurogenesis.
II. Functional neuroanatomy

With the GAL4-UAS system, it is possible to alter
the normal cell functions by ectopically expressing

developmental switch genes specifically in the GAL4expressing cells. If this results in a change of certain
behaviour of the animal, it is likely that these alteredcells may play major roles in controlling thatbehaviour.
We use UAS-linked transformer (tra) gene, whichacts early in the sex determination cascade, to alter thesex of the GAL4-expressing cells. The courtshipbehaviour of Drosophila is very different between
male and female. Male flies actively try to attract females by presenting stereotyped steps of courtship,
whereas females stay passive. Does this male-specificbehaviour disappear, if particular brain cells arefeminised?
Previous studies by other investigators showed thatfeminisation of the mushroom body neurones may
make the male flies bisexual. Although this is an interesting phenomenon, the male-specific behaviour
did persist in these experiments. To fin~ the GAL4
strains that can really suppress male behaviour, we
performed a much larger behavioural screening. For
this purpose, we abandoned any assumption to preselect strains that drive tra gene in particular brain
structures. Instead, we crossed all the available homozygous viable GAL4 strains with UAS-tra. Amongthe 446 strains tested so far, we found only two strainsthat show near-total suppression of male-specificcourtship behaviour, regardless of the sex of the targets (Fig. 1).
The GAL4 expression patterns of these two strains,
however, were not very specific. Rather, GAL4 isexpressed in most of the brain cells. The results can be
explained by assuming the following: 1: The courtshipbehaviour is controlled by various biochemical typesof neurones, which may either be packed within asmall brain area, or scattered in many regions; and 2:
The male behaviour would manifest unless all the
relevant neurones are female. Since these neurones
may not share common enhancer activity, only GAL4
strains with near-ubiquitous expression pattern would
be able to feminise all of these cells simultaneously.
III. Contribution to the science community

As the number of identified neurones grows, it becomes important to develop a system with which science community can easily access the record ofcomplicated three-dimensional circuit structures. As ajoint venture with German and US research groups, we
maintain Flybrain, a web-based image database of the
Drosophila nervous system (http://flybrain.nibb.ac.jp).
Over 2000 images has already been stored and served
worldwide.
Another database maintained here is Jfly, which isintended to help the exchange of information among
Japanese-speaking Drosophila researchers (http://
jfly.nibb.ac.jp). Archives of research-related discussions, images and experimental protocols, as well as
meetings and job announcements, are provided.

19

FOR BASIC BIOLOGYAcs

B Np 218x tra

c cs

-¥g
-+d'

100I

~
SAP

Np218x tra

-¥~
-+d'

Mz490X tra

-¥1
-+d'

N=20

Cl
N=21

100I

N=20
N=20
100I

N=26
N=20

Fig. 1 Reduced male-specific activity caused by the ectopic expression of the transformer gene(A) Wild-type (CS) male flies actively court females when they are put together in small chambers. Within ten minutes, most(in this case four out of six) males reach the final step of the courtship: copulation. Other males are chasing and courting theirpartners. (B) Males of the GAL4 enhancer-trap strain Np218 crossed to UAS-tra, on the other hand, show no interests in females.
(C) Quantitative comparison of the courtship behaviour. The duration of the time when a male fly engaged in any kind of thecourtship-related behaviour (courtship index, CI), and the time when a male fly performed the courtship-specific wing vibration
behaviour (sex appeal parameter, SAP), were recorded during the first ten minutes after the subject (male) and the target (either
male or female) flies were put in the chamber. Wild type (CS) males spend long time courting females, but court males only
occasionally. The males of the Np218 and Mz 490 strains crossed to UAS -tra spend only a very short period for courting both maleand female.

Publication List:
Ito , K., Suzuki, K., Estes, P., Ramaswami, M.,
Yamamoto, D. and Strausfeld, N. J. (1998) The
organization of extrinsic neurons and their implications regarding the functional roles of the mushroom bodies in Drosophila melanogaster Meigen.
Learning and Memory 5, 52-77 .
Strausfeld, N. J., Hansen, L., Li , Y., Gomez, R. S. andIto, K. (1998) Evolution, discovery, and interpretations of Arthropod mushroom bodies. Learning andMemory 5, 11-37.

20

NATIONAL INSTITUTE

terns of eukaryotes - the actin-based and microtubulebased motility systems - should somehow cooperate in
the inner-arm dynein although the function of actin in
dynein arms is totally unknown at present. Several
years ago, 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 been known to have only asingle gene of conventional actin, and the mutant ida5
was found to express no conventional actin at all. On
close inspection, the cytoplasm and axonemes of thismutant were found to contain a novel actin-like protein (NAP) which displays exceptionally low homology (64% in amino acid identity) to conventional
actin. The mutant ida5 is deficient in the formation of
the fertilization tubule and thus has a low mating efficiency. However, it displays normal cell divisionand grows as rapidly as wild type, possibly because
NAP can substitute for actin in important cellularfunctions. Thus conventional actin and NAP may
overlap in some, but not all, cellular functions . It isinteresting to note that NAP is expressed in significant
amount only in the mutant lacking actin; i.e., the expression of NAP appears to depend on the presence ofactin. We are currently investigating how such regulation takes place.
We have recently succeeded in transforming the
mutant ida5 with cloned actin gene and found thatinner dynein arms become restored upon transformation. Transformation with the NAP-encoding gene isunder way. Studies with artificially mutated actin gene
will enable us to determine what functions are carriedout by actin and NAP and, in particular, whether actin
or NAP is really essential for cytokinesis, assemblyand function of inner dynein arms, or other fundamental processes in Chlamydomonas.

DIVISION OF CELLULAR COMMUNICATION(ADJUNCT)
Professor:
Ritsu Kamiya
Associate Professor: Katsushi Owaribe
Research Associate: Takaka Minoura
Institute Research Fellow: Osamu Kagami
Graduate student:
Its us hi Minoura (University of Tokyo)

The research in this laboratory, started in November, 1996, is aimed at an understanding of the molecular mechanisms that regulate the assembly andfunction of cytoskeletal proteins. Current research effort is centered on the function of axonemal dyneins,
microtubule-based motor proteins that produce force
for flagellar beating. The organism employed is
Chlamydomonas, a biflagellate green alga particularly
suited for genetic and molecular biological studies.
A single flagellar axoneme contains at least elevenkinds of dynein heavy chains in inner and outer arms.
To understand the specific function of each heavy
chain, we have been isolating and characterizing mutants that lack different kinds of axonemal dyneins .
Their motility phenotypes have indicated that different
dynein species differ in function in a fundamentalmanner. 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 force generation properties differ
greatly among different heavy chains. We are currently trying to directly measure the force production
by different dyneins using micro-physiological techniques.
The inner dynein arms are known to contain actin as
a subunit. Hence the two independent motility sys-A

8~Fig. 1. Fertilization tubules in wild-type(A) and ida5 (B) mt• gametes produced in response to a 1 hour expos~re to 10 mM
dibutyryl-cAMP and 1 mM IBMX. Bar, 0.3 IJffi. Wild-type fertilization tubules have been shown to contam F-actm bundles.

FOR BASIC BIOLOGY

Publication list:
Hayashi, M., Yagi, T., Yoshimura, K., and Kamiya, R.
(1998). Real-time observation of Ca 2+-induced
basal body reorientation in Chlamydomonas. Cell
Motil. Cytoskeleton 41, 49-56.
Kagami, 0., Gotoh, M., Makino, Y., Mohri, H.,
Kamiya, R., and Ogawa, K. (1998). A dynein lightchain of sea urchin sperm flagella is a homolog ofmouse Tctex 1, which is encoded by a gene of the
t complex sterility locus. Gene 211, 383-386.
Kato-Minoura, T., Uryu, S., Hirono, M., and Kamiya,
R. (1998). Highly divergent actin expressed in a
Chlamydomonas mutant lacking the conventional
actin gene. Biochem. Biophys. Res. Comm. 251, 7176.
Ohara, A., Kato-Minoura, T., R., Kamiya, R. andHirono, M. (1998). Recovery of flagellar inner-arm
dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.
Cell Struct. Funct., 23, 273-281.
Matsuda, A., Yoshimura, K., Sineshchekov, 0. A.,
Hirono, M., and Kamiya, R. (1998). Isolation andcharacterization of novel Chlamydomonas mutants
that display phototaxis but not photophobic response. Cell Motil. Cytoskeleton, 41, 353-362.
Okamoto, M., Ito, M., and Owaribe, K. (1998). Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells
from the adult newt, Cynops pyrrhogaster. Develop.
Growth and Differ. 40, 11-18.
Hirako, Y., Usukura, J., Uematsu, J., Hashimoto, T.,
Kitajima, Y., and Owaribe, K. (1998). Cleavage ofBP180, a 180 kDa bullous pemphigoid antigen,yields a 120 kDa collagenous extracellular polypeptide. J. Bioi. Chern. 273, 9711-9717.
Hirako, Y., and Owaribe, K. (1998). Hemidesmosomes and their unique transmembrane protein
BP180. Microscopy Research and Technique 43,
207-217.

21

22

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 thecleavage furrow. The cleavage furrow contains a contractile apparatus, called the contractile ring, which iscomposed of a bundle of actin filaments that lies in the
furrow cortex beneath the plasma membrane. It has
been established that the contractile ring contracts as theresult of interaction between actin filaments and myosin. However, little is known about process of its formation, mechanism that controls its formation, protein constituents, and its ultrastructure.The goal of our researchis to solve these problems and thereby clarify the molecular mechanism of cytokinesis. For this purpose, we
use three kinds of cells, namely, sea urchin egg, Xenopus egg, and the fission yeast Schizosaccharomyces
pombe.
S. pombe is an excellent system to investigate thechanges in the actin cytoskeleton during cell cycle
since F-actin patches, F-actin cables and F-actin ringare only visible structures in the cell (Fig. 1). The Factin ring is considered to correspond to the contractile ring in animal cells. It is formed during anaphasein this organism. In order to obtain basic features of
the actin cytoskeleton in S. pombe, we investigatedsubcellular localization and interactions of actin andtwo actin cytoskeleton-related proteins, Cdc8 tropomyosin and actin-related protein 3 (Arp3), using
specific antibodies and by gene disruption. Actin was
localized to medial microfilamentous ring in the region of the septum during cytokinesis and to corticalpatches by immunoelectron microscopy. F-actin
cables were detected throughout the cell cycle byfluorescent staining with Bodipy-phallacidin. The
cables were often linked to the patches and to themedial F-actin ring during its formation. Tropomyosin

was localized to the F-actin ring and the cables. It wasalso distributed in the cell as patches, although colocalization with F-actin was not frequent. In cdc8"
mutant cells, F-actin cables were not observed although the F-actin patches were detected and cell
polarity was maintained. These observations suggestthat the F-actin cables may be involved in the formation of the F-actin ring, and that tropomyosin plays animportant role in organizing both the F-actin ring andthe F-actin cable, but is not involved in the F-actin
patch formation or maintenance of cell polarity.
Binding of Arp3 to actin was revealed by immunoprecipitation as well as by DNasei column chromatography. Arp3 seemed to form a complex with several proteins in the cell extracts, as previousiy reported for other organisms. Arp3 was found to beconcentrated in the medial region of the cell fromearly anaphase to late cytokinesis, however, it was not
localized on the F-actin ring. Following arp3 genedisruption, F-actin patches were delocalized throughout the cell and cells did not undergo polarized
growth, suggesting that Arp3 influences the proper
localization of the actin patches in the cell and thereby
controls the polarized growth of the cell.
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 et al. (Mol. Bioi. Cell, 8: 2693-2705, 1997).
Overexpression of Myo3 in the cell leads to formationof aberrant F-actin ring, F-actin cable, and septum. We
knocked out the myosin genes in S. 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-II myosin is necessary
in the formation of the contractile ring in S. pombe.
We also concentrate our study on function of actinregulatory proteins, including ADF/cofilin family pro-

Fig. 1. 3-D images of an interphase S. pombe cell. Each image is rotated by 12 degree from the neighbor. Green, F-actin. Blue,
DNA. Red, spindle pole body. Bar, 2 ~J.m .

FOR BASIC BIOLOGY

teins, during cytokinesis using Xenopus eggs and embryos. We found that ADF/cofilin family proteins are
essential for cytokinesis (Abe, Obinata, Minamide, andBamburg, J. Cell Biol. 132: 871-875, 1996). Recent
studies revealed that ADF/cofilin accelerates turnoverof actin filaments both in vitro and in vivo. Most recently, we found a novel actin-regulatory protein
which induces disassembly of actin filaments cooperatively with ADF/cofilin. eDNA analysis revealed that
this protein is a Xenopus homologue of yeast actin
interacting protein 1 (AIP1). Thus, we designated thisprotein as Xenopus AIP1 (XAIP1). Purified XAIP1
itself binds to pure actin filaments to some extent, but
it induces a rapid, drastic disassembly of actin filaments associated with cofilin. Microinjection of thisprotein into Xenopus embryos arrested development by
the resulting actin cytoskeletal disorder. XAIP1 represents the first case of a protein cooperatively disassembling actin filaments with ADF/cofilin family proteins.
Publication List:

Hirata, D., Nakano, K., Fukui, M., Takenaka, H.,
Miyakawa, T. and Mabuchi, I. (1998). Genes thatcause aberrant cell morphology by overexpressionin fission yeast: a role of a small GTP-binding protein Rho2 in cell morphogenesis. J. Cell Sci. 111,
149-159.
Arai, R., Nakano, K. and Mabchi, I. (1998). Subcellular localization of actin, tropomyosin and actin-related protein 3 (Arp3) in Schizosaccharomyces
pombe. Eur. J. Cell Bioi. 76, 288-295.
Nishimura, Y., Nakano, K. and Mabuchi, I. (1998).
Localization of Rho GTPase in sea urchin eggs.
FEBS Lett. 441, 121-126.

23

25

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshitaka Nagahama

The Department is composed of three divisions and one adjunct division. Department members conduct molecular analysis on various aspects 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.

26

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

Yoshitaka Nagahama
Michiyasu Yoshikuni
Minoru Tanaka
Tohru Kobayashi
Institute Research Fellow: Akihiko Yamaguchi
JSPS Postdoctoral Fellows: Mika Tokumoto
Yoshinao Katsu
Daisuke Kobayashi
Craig Morrey
Catherine Dreanno
Balasubramarian
Senthilkumaran
JSPS Research Associates: Yuichi Ohba
Yasutoshi Yoshiura
Masaru Matsuda
Toshitaka Ikeuchi
Graduate Students: Masatada Watanabe (Graduate
University for Advanced Studies)
Gui-Jin Guan (Graduate University
for Advanced Studies)
Ryo Horiuchi (Graduate University
for Advanced Studies)
Monbusho Foreign Scientists: Allen Schuetz (Johns
Hopkins University)
Srinivas K. Saidapur(Kamatak University)
Visiting Scientist:
Graham Young (University of Otago)

Gametogenesis of teleost fishes, as in other oviparous vertebrates, is controlled primarily by pituitary
gonadotropins. In most cases, gonadotropins act via
biosynthesis of gonadal steroidal hormones which, inturn, mediate various stages of gametogenesis. Using
fish as a primary study model, our research focuses on(1) the identification of steroidal mediators involvedin gonadal sex differentiation and gametogenesis, and(2) the mechanisms of synthesis and action of these
mediators.

I. Endocrine regulation of oocyte growth andmaturation
Two follicular steroidal mediators, estradiol-17~(oocyte growth) and 17a.,20~-dihydroxy-4-pregnen-3one (17a.,20~-DP; oocyte maturation), were identified
in several teleost fishes. Two-cell type models inwhich the thecal layer provides precursor steroids to
the granulosa layer, have been proposed for estradiol17~ and 17a.,20~-DP production. There is a distinct
shift in expression of steroidogenic enzyme genes
from cytochrome P450 aromatase (P450arom) to 20~­hydroxysteroid dehydrogenase (20~-HSD) in granulosa cells immediately prior to oocyte maturation. The
presence of orphan nuclear binding sites in the
medaka P450arom promoter and the similar expression patterns of FTZ-Fl and P450arom transcripts inovarian follicles suggest that FTZ-Fl is involved in
the transcriptional regulation of medaka P450arom
during oocyte growth. The preovulatory surge of LHlike gonadotropin is responsible for the rapid expression of 20~-HSD mRNA transcripts in granulosa cells
during oocyte maturation.

17a.,20~-DP induces oocyte maturation by acting
on a pertussis toxin-sensitive G-protein-coupled membrane receptor. The early steps of 17a.,20~-DP action
involve the formation of downstream mediator of thissteroid, the maturation-promoting factor or
metaphase-promoting factor (MPF) consisting of cdc2
kinase and cyclin B. 17a.,20~-DP induces oocytes tosynthesize cyclin B which activates a preexisting 35kDa cdc2 kinase via phosphorylation of its threonine
161 by a threonine kinase (M015), thus producing the
34 kDa active cdc2. A 54-kDa Y box protein andpolyadenylation of cyclin B mRNA are thought to be
involed in 17a.,20~-DP-induced initiation of cyclin B
mRNA translation.
Upon egg activation, MPF is inactivated by degradation of cyclin B. The availability of biologically-active goldfish cyclin B produced in E. coli
and purified goldfish proteasomes allowed the roleof the proteasome in the regulation of cyclin degradation to be examined for the first time. It wasdemonstrated that the 26S proteasome initiates
cyclin B degradation through the first cut of its
NH 2 terminus at lysine 57.

II. Endocrine regulation of spermatogenesis
The importance of gonadotropins and androgens
for spermatogenesis is generally accepted in vertebrates; however, the mechanism of action of these
hormones remains unresolved. Using an organ culture system for eel testes consisting of spermatogonia and inactive somatic cells, we haveshown that the hormonal regulation of spermatogenesis in eel testes involves the gonadotropin
stimulation of Leydig cells to produce 11ketotestosterone (11-KT), a potent androgen infish. In turn, 11-KT activates Sertoli cells to stimulate premitotic spermatogonia to complete
spermatogenesis (Fig. 1). A subtractive hybridization method was used to clone genes that are expressed differentially in eel testes in the first 24 hr
after gonadotropin treatment. Six down-regulated
and two up-regulated eDNA clones were isolated.
Two eDNA clones suppressed by gonadotropin
stimulation exhibited ZP (zona pellucida spermbinding protein)2- and ZP3-like structures. Transcripts of ZP2- and ZP3 are detected in immature
testes and disappear immediately after HCG injection, suggesting that ZP2 and ZP3 may play important roles in the prevention of spermatogenesis in
the eel. One of the up-regulated cDNAs was identified as coding the activin ~B subunit. Activn ~B
mRNA transcripts were absent in testes prior togonadotropin treatment, but were abundant in Sertoli cells in testes of eels treated with gonadotropin
for 1-6 days. 11-KT induced a marked productionof activin B in cultured testes, indicating that eel
activin ~B subunit production is largely for activin
B formation. An androgen receptor localized in
Sertoli cells. Addition of recombinant eel activin B
to the culture medium induced proliferation of

27

FOR BASIC BIOLOGY

to the culture medium induced proliferation of
spermatogonia, producing late type B spermatogonia, within 15 days in the same manner as did 11KT. Activin B also acts on spermatogonia to
induce de novo synsthesis of G liS cyclins (cyclins
A, D , and E) and Cdks (cdc2, cdk2, and cdk4),
leading to the initiation of mitosis (spermatogonial
proliferation) (Fig. 1).

III. Endocrine regulation of gonadal sex
differentiation and sex change
The initial differentiation and development of steroid-producing cells during gonadal sexual differentiation were examined by in situ hybridization andimmunohistochemistry in tilapia, Oreochromis
niloticus. Transcripts of P450arom became evident for
the first time in the gonads of genetic females two
weeks before morphological sex differentiation (Fig.
2) . These gonads were also positive to antibodies

Gonadotropin

Leydig cell

::;:

~

11-Ketotestosterone~------------------~~-----------------~~--------------------+------------------~~

Sertoli cell~
Activin B
~
T

~
Q1

Clik/CyC!in~

Sp(ermatogonium

~

ai

~
_/
substanc~Mitosi~(!)

Meiosis-initiating

11\4

-.. •

y

M eiosis (Spermatocyte)

Fig. 1 Hormonal regulation of spermatogenesis initiation in the Japanese eel.

28
against P450scc, 3~-HSD , P450c17, and P450arom.
Thus, steroid-producing cells in ovaries, but not testes,at the undifferentiated and differentiating stages express all of the steroidogenic enzymes required for
estradiol-17~ biosynthesis from cholesterol. These results strongly suggest that endogenous estrogens act
as the natural inducers of ovarian differentiation intilapia. In contrast, weakly positive reactions toP450scc, 3~-HSD and P450c17 antibodies appeared
first in the testis at 30 days ; however, staining intensity increased slowly during development of the testis.
Therefore, the ability of steroid-producing cells tosynthesize steroid hormones in the testes only appears
at the time of testicular differentiation.
Sequential, protogynous hermaphroditism, i.e.
female to male sex change, provides an ideal comparative model for studies on sexual differentiation.
Fully-functional females are induced to develop functional testes by non-invasive social conditions, thus
allowing simultaneous investigation into the normal
endocrine requirements of ovarian maintenance andtesticular differentiation . As steroid hormones are considered major mediators of sexual differentiation andgonadal function, molecular studies of key enzymes inthe steroidogenic pathways were completed. Two different P450arom mRNAs as well as P450 11~-hy­droxylase mRNA have been cloned and characterizedthroughout sex change in Thalassoma duperrey, aprotogynous hermaphrodite. The ovarian form ofP450arom is predominantly expressed in the ovary
prior to the initiation of sex change. Shortly thereafter,
expression diminished to undetectable levels suggesting that ovarian P450arom/estrogen is critical for the
maintenance of ovarian function and/or detrimental totesticular differentiation. Although intriguingly com-

NATIONAL INSTITUTE

plex and potentially relevant, the neural form ofP450arom, named for its abundance in the brain, does
not appear to be directly related to the gonadal restructuring seen during sex change . Nevertheless, its
function and significance are being actively pursued.
In contrast, P450 11 ~-hydroxy lase is likely to be amajor factor regulating the gonadal changes. P450
11 ~-hydroxylase mRNA was relatively abundant in
the ovary; however, it was up-regulated concomitantwith the onset of testicular differentiation indicating
the importance of P450 11~ -hydroxylase/11-KT tospermatogenesis and spermiation. Consequently, theswitch from an ovary to a testis corresponds exceptionally well to a switch in the steroidogenic pathwayof the gonad thus providing further evidence of thecritical nature of steroid hormones in teleost sexual
differentiation.
Publication List:
Horiguchi, R., Tokumoto, M., Yoshiura, Y. , Aida, K. ,Nagahama, Y. and Tokumoto, T. (1998). Molecularcloning of eDNA encoding a 20S proteasome a2
subunit from goldfish (Carassius auratus) and its
expression analysis. Zool. Sci., 15, 773-777.
Jiang, J.Q., Young, G. , Kobayashi , T. and Nagahama,Y. (1998). Eel (Anguilla japonica) testis 11~-hy­droxylase gene is expressed in interrenal tissue andits product lacks aldosterone synthesizing activity.
Mol. Cell. Endocrinol., 146, 207-211.
Kobayashi, T. , Kajiura-Kobayashi, H. and Nagahama,Y. (1998). A novel stage-specific antigen is expressed only in early stages of spermatogenesis inJapanese eel, Anguillajaponica testis. Mol. Reprod.
Develop., 51 , 355-361

Fig. 2 Expression of cytochrome P450 aromatase mRNA in the gonad of tilapia two weeks before morphological sex differentiation.
Note strong signals in steroid-producing cells (arrows).

FOR BASIC BIOLOGY

Kobayashi, T., Nakamura, M., Kajiura-Kobayashi, H.,
Young, G. and Nagahama, Y. (1998). Immunolocalization of steroidogenic enzymes (P450scc,
P450c17, P450arom, and 3j3-HSD) in immature andmature testes of rainbow trout (Oncorhynchus
mykiss). Cell Tissue Res., 292, 573-577.
Mita, M., Yasumasu, 1., Saneyoshi, M., Yoshikuni, M.
and Nagahama, Y. (1998). Production of the oocyte
maturation-inducing substance of starfish by heat
treatment of S-adenosylmethionine. Zool. Sci., 15,
117-122.
Mita,. M., Yoshikuni, M. and Nagahama, Y. (1998).
Ecto-ATP diphosphohydrolase (apyrase) in ovarianfollicle cells of starfish Asterina pectinifera. Comp.
Biochem. Physiol. Part B, 119, 577-583.
Miura, T., Kudo, N., Miura, C., Yamauchi, K. andNagahama, Y. (1998). Two testicular eDNA clones
suppressed by gonadotropin stimulation exhibit
ZP2- and ZP3-like structures in Japanese eel. Mol.
Reprod. Develop., 51, 235-242.
Morrey, C.E., Nakamura, M., Kobayashi, T., Grau,
E.G. and Nagahama, Y. (1998). P450scc-like immunocytochemistry throughout gonadal restructuringin the protogynous hermaphrodite Thalassoma
duperrey. Int. J. Develop. Bioi., 42, 811-816.
Nakamura, M, Kobayashi, T., Chang, X.T. andNagahama, Y. (1998). Gonadal sex differentiation inteleost fish. J. Exp. Zool., 362-372.
Parhar, I.S., Nagahama, Y., Grau, E.G. and Ross, R.M.
(1998). Immunocytochemical and ultrastructural
identification of pituitary cell types in the
protogynous Thalassoma dupperrey during adultsexual ontogeny. Zool. Sci., 15, 263-276.
Parhar, I.S., Soga, T., Ishikawa, Y., Nagahama, Y. and
Sakuma, Y. (1998). Neurons synthesizing gonadotropin-releasing hormone mRNA subtypes havemultiple developmental origins in the medaka. J.
Comp. Neurol., 401, 217-226.
Todo, T., Ikeuchi, T., Kobayashi, T. and Nagahama, Y.
(1998). Fish androgen receptor: eDNA cloning, steroid activation of transcription in transfected mammalian cells, and tissue mRNA levels. Biochem.
Biophys. Res. Comm., 254, 378-383.

29

30

NATIONAL INSTITUTEDIVISION OF CELL DIFFERENTIATION
Professor:
Ken-ichirou Morohashi
Research Associate: Satoru Ishihara
Technical Staff:
Sanae Oka
Post Doctral Fellow: Naoe Kotomura
Graduate Students: Ken Kawabe (Kyushu University)
Tokuo Mukai (Kyushu University)
Hirofumi Mizusaki (The Graduate
University for Advanced Studies)
Tatsuji Shikayama (Kyushu
University)
Hayato Yokoi (Nagoya University)
Akira Nanba (University of Tokyo)
Ryoji Matsushima (Nagoya
University)
Visiting Fellow:
Yoshiyuki Kojima (Nagoya City
University)
Tetsushi Dodo (Eisai Co. Ltd)
Assistant:Hisae Tsuboi

Steroid hormones, which act as endocrine messengers, are mainly synthesized in steroidogenic tissues
such as the gonads and adrenal cortex. Because of thepivotal function of sex steroids and corticosteroids
secreted from these tissues, several groups have investigated the regulation of hormone production. Specifically, in the past decade, extensive research has been
directed into the area of transcriptional regulation ofgenes implicated in steroidogenesis. As one of thesestudies, we identified Ad4BP/SF-l, which belongs toa nllclear hormone receptor family, as a major steroidogenic tissue-specific transcription factor. Further
studies have suggested that this versatile transcription
factor is central to the regulation of a reproductive
behavior thorugh functioning in the pituitary
gonadotroph and ventromedial hypothalamic nucleus
in addition to the steroidogenic tissues. Based onthese observation, our attention has been directed tounderstanding mechanisms underlying differentiationof the reproductive system and establishment of reproductive behavior of animals.

I. Transcriptinal control of genes necessary forgonadal differentiation.
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-l 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,a particular cell population immunoreactive for
Ad4BP/SF-l (AGP, adreno-gonadal primordium) was
identified to be present from the dorsal aorta to coelomic epithelia of rodent fetauses and succesively they
were shown to give rise to two distinct primordia, thegonads and adrenal cortex. In the case of the gonadal
primodium, it finally gives rise to the testis or ovary
according to the sex chromosomal composition. Theseobservations gave us many interesting issues as follows. What is the signal for the onset of the Ad4BP/
SF-1 gene transcription in the particular cells comprising AGP? What is the mechanism underlying theseparation of the AGP into the adrenal and gonadal
primordia? How is the mechanism employed for sex
differentiation of the gonads? Why dose such sex
dependent differentiation not occur in the adrenal cortex? To address these issues, nuclear transcription
factors (WT-1, SRY, SOX-9, DAX-1, Emx-2, and
GATA-4, as well as Ad4BP/SF-1), all of which are
critically implicated in gonadal and adrenocortical differentiation, havebeen underinvestigation from the· molecular and morphological aspects.
As one of these efforts, we investigsted Dax-1 gene
regulation and identified a novel Ad4 site, which isrecognized by Ad4BP/SF-l, by transient transfectionand electrophoretic mobility shift assays. In addition,
immunohistochemical analyses with antibodies specific for Dax-1 indicated the presence of immunoreactive cells in the steroidogenic tissues, pituitary glandand hypothalamus. Although the distributions of Dax1 and Ad4BP/SF-l were highly similar, they were notcompletely identical. Namely a certain population ofcells immunoreactive for Ad4BP/SF-l is negative forDax-1 expression. Such inconsistent distributions between the two transcription factors were observed in
all tisseus. To confirm the positive regulation of theDax-1 gene by Ad4BP/SF-l, the Dax-1 expression
was investigated with the mFtz-F1 gene disrupted
mice. The Dax-1 expression was significantly impaired in the mFtz-F1 gene disrupted mice (Fig. 1),
confirming our understanding that Ad4BP/SF-l controls the transcription of the Dax-1 gene.

II. Implication of Ad4BP/SF-1 in strcturalorganization of spleen.
During characterization of the mFtz-F1 gene disrupted mice, we were aware of the abnormal spleen of
the mFtz-F1 gene disrupted mice in addition to thetissues above (Fig. 2). The spleen has normally two
main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, while
the second involves clearance of abnormal, damaged,
and aged erythrocytes. Immuno-histochemical examination of the mammalian spleens confirmed the expression of Ad4BP/SF-l in endothelial cells of thesplenic venous sinuses and pulp vein. In the mFtz-F1
gene disrupted mice, several structural abnormalities
were detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes.
Examination of the spleen of KO fetuses showed failure of development of certain tubular structuresduring embryogenesis. These structures are normallyassembled by Ad4BP/SF-l immunoreactive cells, andmost likely form the vascular system during laterstages of development. Other structural abnormalities

31

FOR BASIC BIOLOGY

Fig. I, Immunohistochemical detection of Dax-1 in the pituitary. Sections of the pituitary glands from the newborn were preparedfrom wild-type (A and C) and mFtz-F1 gene disrupted (B and D) mice. They were stained with anti-Ad4BP/SF-l antisera (A andB) or anti-Dax-1 antibodies (C and D) as described. Bars; 50 Jlm.

Fig. 2, Comparison of macroscopic features of spleens ofwild-type (Wild) and mFtz-F1 gene disrupted (KO) newborn
mice. Arrowheads show red spots characteristic of the KO
spleen.

mRNA encoding cholesterol side chain cleavage P450
in the spleen, resembling the findings in steroidogenic
tissues such as the gonads and adrenal cortex. The
mRNA transcript was not involved in splenic structural defects as it was detected in the spleens of bothnormal and the mFtz-Fl gene disrupted mice, indicating that the regulatory mechanism of the P450 gene in
the spleen is different from that in steroidogenic tissues. Our results indicate a lack of the mFtz-Fl genein mice is associated with structural and functional
abnormalities of the splenic vascular system (In press,
Blood, 1999).

Publication List:in the spleen of the mFtz-Fl gene disrupted mice included defects in the tissue distribution of type IV
collagen, laminin, c-Kit, and vimentin. These morphological defects in the vascular system were associatedwith a decrease in the proportion of hematopoietic
cells although differentiation of these cells was notaffected significantly. A high number of abnormal red
blood cells containing Howell-Jolly bodies were notedin the mFtz-Fl gene disrupted mice, indicating impaired ability for clearance due to the affected splenic
vascular system. We also detected the presence of an

Kawano, K. , Miura, I., Morohashi, K., Takase, M. ,and Nakamura, M. (1998) Molecular cloning andexpression of the SF-l/Ad4BP gene in the frog,
Rana rugosa. Gene 222, 169-176.
Nomura, M. , Kawabe, K., Matsushita, S., Oka, S.,
Hatano , 0., Harada, N., and Nawata, H., andMorohashi, K. ( 1998) Adrenalcortical and gonadal
expression of the mammalian Ftz-F 1 gene encoding
Ad4BP/SF-l is independent of pituitary control. J.
Biochem. 124, 217-224.

32

NATIONAL INSTITUTEDIVISION OF MORPHOGENESIS
Professor:
Associate Professor:
Research Associates:

Naoto Ueno
Hiroshi Shibuya
Makoto Nakamura
Makoto Mochii
Ken W. Y. Chon
JSPS Visiting Scientist:
Shinichi Higashijima 2!
Visiting Scientists:
Fumihiko Hamada3!
Takeshi Suzuki4!
Kathy NeaZSJ
JSPS Postdoctoral Fellow: Miho Shimizu
Graduate Students:
Hideyuki Nagaso 6!
Masataka Nikaido 6!
Yasuhiro Takatsu 6!
Michiru Nishita 6i
Katsura Sugawara
Satoru Yoshida
Yasunori Tomoyasu
Shinichi NagaV!
Yayoi Nishida 7!
Chiyo TakagiTechnical Stuff:
Takamasa Yamamoto
Shunichiro Iemura
Kiyokazu Morita
Yuko Takahashin from University of California, Irvine

21 PRESTO, JST
3!

Osaka University

41 Medical School, Nagoya University
5! MRC,Mill Hill

61 Graduate School of Hokkaido University
7! Graduate School of Nagoya University

Early embryogenesis is regulated by a number of
endocrine and paracrine factors that mediate cell-tocell communications. Particularly, polypeptide growth
factors are believed to be essential for the regulationof cell proliferation, differentiation and apoptosis inmorphogenic events during early development. The
most striking feature of growth factor is that theirfunctions are extremely well conserved among animal
species, which allows us to investigate developmental
mechanisms governed by growth factors using multiple model animals, such as mouse, zebrafish,
Xenopus, Drosophila, C. elegans etc. To understandmolecular basis of morphogenesis, we focus on agroup of growth factors Transforming growth factor-~(TGF-~) superfamily and investigate molecularmechanisms of their actions in early embryogenesis.
Our goal is to unveil species-specific and conserved
mechanism of early development.

I. The role of BMP in ectodermal patternformation.
Bone morphogenetic proteins belonging to the
TGF-~ superfamily have critical roles in the determi-

nation of cell fate during early development. It is
widely accepted that ventrally expressed BMPs are
essential f<;>r epidermal differentiation. Overexpression
BMP in dorsal blastomeres of Xenopus laevis resultsin the loss of neural tissues including head and eyes.

To investigate whether BMP can determine epidermal
differentiation by acting directly on ectoderm or acting through the conversion of mesodermal cell fate,
we performed bead transplantation experiment using
zebrafish as a model. BMP-soaked beads were transplanted into presumptive neural region of early gastrula (shield stage) embryo and marker gene expression was examined. The result indicated that BMP
induces epidermal fate and inhibits neural fate, acting
directly on ectoderm (Figure 1). We also found that
action range of BMP in zebrafish ectoderm is rather
restricted, denying the possibility of relay mechanismof signal propagation.
Neural induction takes place because BMP activity
is inhibited by organizer factors emanated from
Spemann organizer. Such organizer factors include
Chordin, Noggin and Follistatin. Chordin and Noggin
have been shown to bind BMPs directly and thus inhibit their epidermal inducing activity. We have beenable to show that Follistatin can also bind BMPs directly albeit with a low affinity and high dissociation
constant.

II. The role of XIAP and Dlx5 in BMP signaling
TAKl, a member of the MAP kinase kinase kinase
family, and its activator, TABl, participate in the bonemorphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early
Xenopus embryos. However, the events leading fromreceptor activation to TAKl activation remain to be
identified. A yeast interaction screen was used tosearch for proteins that function in the pathway linking the receptors and TABl-TAKl. The human Xchromosome-linked inhibitor of apoptosis protein(XIAP) was isolated as a TAB 1-binding protein.
XIAP associated not only with TAB 1 but also with the
BMP receptors in mammalian cells. Injection of XIAPmRNA into dorsal blastomeres enhanced theventralization of Xenopus embryos in a TABl-TAKldependent manner. Furthermore, a truncated form of
XIAP lacking the TABl-binding domain partially
blocked the expression of ventral mesodermal marker
genes induced by a constitutively-active BMP type I
receptor. Taken together, we identified that XIAP participates in the BMP signaling pathway as a positive
regulator linking the BMP receptors and TABl-TAKl.
Recently, we isolated a murine homeobox-containing gene, distal-less 5 (mDlx5), as a BMP-inducible
gene in osteoblastic MC3T3-El cells. Stabletransfectants of MC3T3-El that overexpress mDlx5
mRNA showed increase in various osteogenic markers; a 4-fold increase in alkaline phosphatase activity,
a 6-fold increase in osteocalcin production and appearance in mineralization of extracellular matrix.
Furthermore, mDlx5 was induced orthotopically inmouse embryos treated with BMP-4 and in fractured
bone of adult mice. Consistent with these observations, we also found that injection of mDlx5 mRNA in
dorsal blastomeres enhanced the ventralization of
Xenopus embryos. Our results indicated that mDlx5 is

33

FOR BASIC BIOLOGY

Figure l Effect of BMP in ectodermal patterning. (A) BMP-soaked bead in neural region suppressed a neural marker otx-2
expression. (B) Control bead soaked in BSA does not affect otx-2 expression. (C) BMP-bead transplantation into shiled stageembryos does not perturb expression of mesodermal marker genes goosecoid (light brown) and no tail (dark brown). (D) BMPbead ectopically induces an epidermal marker gata-3 .

a target gene of the BMP signaling pathway and acts
as an important regulator of both osteogenesis anddorso-ventral patterning of embryonic axis.

III. Eary pattern formation of the peripheral
nervous system in Drosophila
Large mechanosensory bristles (macrochaetes) develop in fixed numbers at constant positions on theadult notum of Drosophila melanogaster. This patternformation of macrochaetes has provided an ideal
model system for studying two-dimensional patterning. Precise positioning of macrochaetes depends onthe complex expression pattern of proneural genes in
the wing disc. Prepattern genes are thought to regulatethese expression of proneural genes. A proneural cluster of dorsocentral bristles forms adjacent to the dorsalside of wingless (wg)-expressing cells in the notum
region of the wing imaginal disc. It has been shownthat wg activity is required for these structures toform . However, the restriction of this pro neural cluster
to the dorsal posterior side of the wg expression
domain in the anterior compartment of the wing
imaginal disc has suggested that W g signaling itself isinsufficient to establish the dorsocentral proneuralcluster. Some factor(s) from posterior side must participate in this action in cooperation with W g signaling. We have examined the role of Dpp (Drosophila

BMP2/4 homolog) signaling in dorsocentral bristle
formation by either ectopically activating or conditionally reducing Dpp signaling. Ubiquitous activationof Dpp signaling in the notum region of the wing
imaginal disc induced additional dorsocentral
proneural cluster all along the dorsal side of the wg
expression domain, and altered wg expression. Conditional loss-of-function of Dpp signaling during discdevelopment resulted in the inhibition of dorsocentral
proneural cluster formation and expansion of the wg
expression domain. These results indicate that Dpp
signaling has two indispensable roles in dorsocentral
bristle formation, induction of the dorsocentral
proneural cluster in cooperation with Wg signaling
and restriction of the wg expression domain in thenotum region of the wing imaginal disc.

IV. TGF-~ family in nematode
Nematode C. elegans provides powerful genetical
approaches to understand the role of TGF-~ family
ligands and their signaling mechanism. We have identified a new member of the TGF-~ superfamily, CET1, from C. elegans, which is expressed in the ventral
nerve cord and other neurons. cet-1 null mutants haveshortened bodies and male tail abnormal phenotype
resembling sma mutants. Overexpression experiments
demonstrated that cet-1 function requires wild type34

sma genes, suggesting that CET-1 functions as ligand
in sma pathway. Interestingly, CET-1 appears to affectbody length in a dose dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpressionof CET-1 in wild type worms elongated body length
close to ion mutants. Epistasis analysis concerning
male sensory ray pattern revealed that mab-21 lies
downstream and is negatively regulated by the cet-11
sma pathway. Our results show that cet-1 controls
diverse biological processes during C. elegans development probably through different target genes.
Recently, almost entire genome sequence of C.
elegans was published. The sequence information,
therefore, allows us comprehensive approach in gene
expression analysis. To identify the target genes ofcet-1/sma pathway and understand the molecular basisof the regulation of body length, we performed a differential hybridization between wild-type and cet-11
sma mutants, using a high-density filter on which over
7,000 different cDNAs were arrayed. We are now
analyzing functions of candidate genes whose expressions are up- or down-regulated in cet-1/sma mutants.

Publication List:
Iemura, S., Yamamoto, T. S., Takagi, C., Uchiyama,
H., Natsume, T., Shimasaki, S., Sugino, H. andUeno, N. (1998). Direct binding of follistatin to acomplex of bone morphogenetic protein and its receptor inhibits ventral and epidermal cell fates inearly Xenopus embryo. Proc. Natl. Acad. Sci. USA
95, 9337-9342.
Kurozumi, K. Nishita, M., Yamaguchi, K., Fujita, T.,
Ueno, N. and Shibuya, H. (1998) BRAM1, a BMP
receptor-associated molecule involved in BMP signalling. Genes Cells 3, 257-264.
Mizuno, N. Mochii, M. Takagi, C., Takahashi, C. T.,
Eguchi, G. and Okada, T.S. (1998) A critical role
for the optic vesicle in lens development; a reinvestigation of free lens formation in Cynops
pyrrhogaster. Differentiation 63, 247-52.
Mochii, M., Mazaki, Y., Mizuno, N., Hayashi H. andEguchi, G. (1998) Role of Mitf in differentiationand transdifferentiation of chicken pigmented epithelial cell. Dev. Biol. 193, 47-62.
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. Bioi. 196, 145-159
Nikaido, M., Tada, M., Takeda, H., Kuroiwa, A. andUeno, N. (1998) In vivo analysis using variants ofzebrafish BMPR-IA: range of action and involvement of BMP in ectoderm patterning. Development
126, 181-190.
Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y.,
Yamaguchi, K., Irie, K., Matsumoto, K., Nishida, E.
and Ueno, N. (1998). Role of TAK1 and TAB1 in
BMP signaling in early Xenopus development.
EMBO J. 17, 1019-1028.

NATIONAL INSTITUTE

Tomoyasu, Y. Nakamura, M. and Ueno, N. (1998)
Role of Dpp signaling in prepattern formation of thedorsocentral mechanosensory organ in Drosophilamelanogaster. Development 125, 4215-4224.
Wakamatsu, Y., Mochii, M., Vogel, K. S. and Weston
J. A. (1998) Avian neural crest-derived neurogenic
precursors undergo apoptosis on the lateral migration pathway. Development 125, 4205-4213.
Yamaguchi, K., Nagai, S., Ninomiya-Tsuji, J., Nishita,M., Tarnai, K., Irie, K., Ueno, N., Nishida, E.,
Shibuya, H. and Matsumoto, K. (1999). XIAP, acellular member of the inhibitor of apoptosis protein
family, links the receptors to TAB 1-TAK 1 in the
BMP signaling pathway. EMBO J. 18, 179-187.

35

FOR BASIC BIOLOGYDIVISION OF DEVELOPMENTAL BIOLOGY(ADJUNCT)
Professor:

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

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. Molecular and genetic approaches for the

analysis of the sugar-signalling during growth anddevelopment of Arabidopsis thaliana.
During the growth of plants, new organs develop as
carbohydrate sink, and many vegetative organs shows
sink to source transition during their maturation.
Many aspects of the organ development in higher
plants are thought to be affected, to some degree, by
the levels of sugars. Sugars seem to have influence on
the meristematic transition in long day plants fromvegetative to reproductive growth. To obtain insights
into the role of sugar-regulated gene expression in thegrowth and organ development in plants, we arescreening for mutants of Arabidopsis with defects oranomalies both in the sugar-regulated gene expression
and in the developmental processes such as leaf development and the determination of the flowering time.
To aid this purpose, we have established more than
7,000 independent lines of Arabidopsis plants transformed with T-DNA containing multiple copies of theenhancer sequence. We have identified more than 20of mutant lines with defects in the development ofleaves or anormalies in the flowering time, which also
show the altered patterns of the expression of sugarinducible genes, such as 13-amylase gene (At/3-Amy)(Fig. 1). The mutants were named as uns (unusual

sugar response) after their abnormal sugar responses
with the gene expression.

II. Regulatory factors involved in the sugarinducible expression of plant genes
Expression of genes coding for sporamin and 13amylase, 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 the isoforms of calciumdependent protein kinase (CDPK) were isolated fromleaves of tobacco, and transcripts of two of them were
found to be increased upon treatment of leaves withvarious metabolizable sugars. Antibodies against afragment of one of these isoforms cross-reacted
strongly with the 57 kDa-protein in the soluble fraction from the young leaves. The level of this 57 kDaprotein decreased significantly as leaf matures, while
the level of this 57-kDa protein in mature leaves increased significantly after the treatment of leaves withsugars. The sugar-induction of the 57-kDa protein
occured preceding the induction of expression of the
13-amylase:GUS reporter gene. In addition, a 54 kDaprotein with autophosphorylation acitivity in theplasma membrane of mature leaves also increased significantly upon treatment of leaves with sugars. This
protein was purified to about 1,000-fold compared tothe crude extract. It phosphorylated histone IllS in aCa2+-dependent manner and cross-reacted 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.

Selected Publications:
Iwata, Y., Kuriyama, M., Nakakita, M., Kojima, H.,
Ohto, M. and Nakamura, K., (1998) Characterization of a calcium depedent protein kinase of tobacco
leaves that is asociated with the plasma membrane
and is inducible by sucrose. Plant Cell Physiol.
39(11): 1176-1183.

36

NATIONAL INSTITUTE

A

B

3

~-·c
-~ ·~
- ... 2

o wr

i

8

eli

6

• wr

12] UJ!s1CTS)Q>

IZJ UJIS 1CTSJ

~

~~

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NT

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Fig.l Phenotypes of wild type and homozygous mutant plants (unsl) grown for 3 weeks. The chlorophyll content of the mutant
plants (Fig.lA, right) is much lower than that of wild type plants (Fig.lA, left). Mutant plants also showed the reduced levels of
the sugar-inducible increase of ~-amylas e (Fig.lB, left) and anthocyanin (Fig.lB, right) when leaf explants were treated with highlevels (5%) of sugars.

37

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.

38

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

Masaharu Noda
Nobuaki Maeda*Masahito Yamagata(- Sep. 30,
1998)**Eiji Watanabe(- Aug. 31, 1998)
Takafumi Shintani (Nov. 1,
1998 -)
Junichi Yuasa (Nov. 1, 1998 -)Post doctral Fellows:
Akihiro Fujikawa 1 (Aug. 16,
1998 -)
Masakazu Takahashi 4
Hiroyuki Kawachi 4
Hiraki Sakuta 4
Mohamad Z!ibair 4
Shuhei Kan 4 (Jun. 1, 1998 -)Graduate Students:
Chika Saegusa
Akira Kato
Ryoko Suzuki
Visiting Scientists: ***Elisabeth G. Pollerberg (Mar. 22,
1998- Apr. 19, 1998)
Ikuko Watakabe
Hiroshi Tamura
Technical Staff:
Akiko Oda (- Dec. 31, 1998)
Shigemi Takami
JST Technical Staff:
Masae Mizoguchi
Megumi Goto
Minako Ishida (Jan. 1, 1999 -)*to Washington University School of Medicine (Oct. 1,
1998 -)** to NIBB Center for Transgenic Animals and Plants(Sept. 1, 1998 -)***from Institute for Zoology, University of Heidelberg

We have been studying the molecular and cellularmechanisms underlying the development and functioning of the vertebrate central nervous system. We
are currently searching for and analyzing the functions of molecules involved in various cellular eventsin brain morphogenesis and brain function, such as
generation of neuroblasts, their migration to form thelaminar structure and various nuclei, elongation andpath-finding of neural processes, the formation andrefinement of specific connections between neurons,
and also synaptic plasticity. We have been using various techniques including molecular biology (e.g.
eDNA cloning, site-directed mutagenesis), biochemistry (protein purification, carbohydrate analysis),
immunological methods (monoclonal-antibody production), neuroanatomy, cell and organotypic culture(immortalized cell-line production), and embryo manipulation (classical embryology, gene transfer withviral vectors, and gene targeting).
I. Molecular mechanism of retinotectal projection
A. Topographic projection
Topographic maps are a fundamental feature ofneural networks in the nervous system. Understanding
the molecular mechanisms by which topographicallyordered neuronal connections are established during
development has long been a major challenge in de-

NATIONAL INSTITUTE

velopmental neurobiology. The retinotectal projectionof lower vertebrates including birds has been used as
an readily accessible model system. In this projection,
the temporal (posterior) retina is connected to the rostral (anterior) part of the contralateral optic tectum,
nasal (anterior) retina to the caudal (posterior) tectum,
and likewise dorsal and ventral retina are connected to
the ventral and dorsal tectum, respectively. Thus, images received by the retina are precisely projected
onto the tectum in a reversed manner. In 1963, Sperry
proposed that topographic mapping could be guided
by complementary position labels in gradients acrosspre-and postsynaptic fields. Although this concept iswidely accepted today, and Eph receptor tyrosine kinases and their ligands were recently identified as
candidates for such positional labels, the molecularmechanism of retinotectal map formation remains tobe elucidated.
In 1993, we began to screen for topographic molecules which show asymmetrical distribution in theembryonic chicken retina. In the first-round screening,
using a eDNA subtractive hybridization technique, we
discovered several distinct transcripts which were topographically expressed along the nasotemporal (anteroposterior) axis in the retina. Among these, twowinged-helix transcriptional regulators termed CBF-1
and CBF-2 were expressed in the nasal and temporal
retina, respectively, and our misexpression experiments using a retroviral vector suggested that these
two transcriptional factors direct the retinal ganglion
cell axons to project to the appropriate tectal targets
along the anteroposterior axis.
In 1997, to further search for topographic moleculesin the embryonic retina, we performed a large-scale
screen using a new eDNA display system called Restriction Landmark eDNA Scanning (RLCS) (Fig.
1A). A number of molecules displaying various asymmetrical expression patterns along the nasotemporalaxis or dorsoventral axis in the retina have been identified. These included already known topographic
molecules such as EphA3, CBF-2, etc. expressedalong the nasotemporal axis, and ephrin-B2, EphB3,
etc. expressed along the dorsoventral axis.
In 1998, we continued our efforts to identify all of
the topographic eDNA clones and examine their expression patterns during development (Fig. 1B). Furthermore, with respect to the topographic molecules
which might have important roles in formation of theretinotectal map and/or the neural network in theretina, we have started over- and misexpression experiments using viral vectors and in ovo
electroporation.
B. Layer-specific projection
After reaching their appropriate target zones along
the rostrocaudal and dorsoventral axes of the tectum,
retinal axons begin to seek their appropriate termination sites among 15 distinct laminae within the tectum, of which only three or four receive retinal projections. The molecular and cellular bases of such

39

FOR BASIC BIOLOGY

A

Ventral

Dorsal-..•--------- -,.. - --•.:..-~.»-~ ~ .

...,..:..:_-_
a::cL

.
--.

•

.. . .

.· ·_. .. ·._ :-. ·. ;. ~.

... -~ ----

• -=--=- ~

.a

.

, _

-~

--.. .

-. •• ·.

_: ~:---=--­- ·-

~-

•

•

•

B

Fig. 1 Genes expressed in a region-specific fashion in the chick retina(A) RLCS profiles for the dorsal and ventral retina. The lower panels are enlargements of corresponding regions of the top
pictures. Spots indicated by arrows in the left panel are those of dorsal-specific expression . (B) Region-specific gene expression
in the retina. Whole-mount in situ hybridization showed that these topographic genes were specifically expressed in the nasal ,temporal, dorsal, or ventral region of the retina, respectively.

discrete target choice are poorly understood.
In 1994, we screened for monoclonal antibodies
that recognize one of these retinal termination laminae. Among these, we found three clones TBS, TB2
and TB4, which labeled laminae B, D and F, respectively. eDNA cloning and immunochemical analysis
revealed that the TB4 antigen molecule was ezrin, acytoskeletal-membrane linker molecule belonging to
the ezrin-radixin-moesin family. Ezrin was selectively
expressed in a subset of retinal ganglion cells thatproject to the lamina F. Similar subset-selective expression and resultant lamina-selective distribution of
ezrin were also observed in the lamina-specific central
projections from the dorsal root ganglia. The staining
pattern for TB4 in the dorsal root ganglia and spinal
cord indicated that high expression of ezrin was restricted to cutaneous sensory neurons, but not muscle
sensory neurons . Since ezrin modulates cell morphology and cell adhesion profiles by linking specificmembrane proteins with the cytoskeleton, it was sug-40

gested that ezrin may be involved in the formation
and/or maintenance of lamina-specific connections forneuronal subsets in the visual and somatosensory systems.
We expect that our studies will lead to elucidationof the molecular mechanism underlying formation of
the retinotectal projection, and ultimately to uncover
the basic principles for establishing complicated but
extremely precise neural networks in the nervous system.
II. Functional roles of protein tyrosine phosphatase~ and y in brain development and brain function
Protein tyrosine phosphorylation plays crucial roles
in various aspects of brain development. The level oftyrosine phosphorylation is determined by the balance
between the activities of protein tyrosine kinases andprotein tyrosine phosphatases. Many types of receptor-like protein tyrosine phosphatases (RPTP) havebeen cloned and characterized. In 1994, we found that
PTP~P~. a nervous system-specific RPTP, is expressed as a chondroitin sulfate proteoglycan in thebrain. An RNA splice variant corresponding to theextracellular region of PTP~ is secreted as a major
proteoglycan in the brain known as 6B4 proteoglycan/
phosphacan. The extracellular region of PTP~ consistsof a carbonic anhydrase (CAH)-like domain, afibronectin type III (FN-III)-like domain and a serineglycine-rich region, which is considered to be thechondroitin sulfate attachment region. PTP~ has another family member, RPTPy, which also contains
CAH-like and FN-III-like domains. We found that
RPTPy has four splice variants including an extracellular secreted form. However, they are not synthesized
as proteoglycans and are expressed in various tissues
including the brain, kidney, lung and heart.
In 1996, we found that PTP~ binds pleiotrophin/heparin-binding growth-associated molecule (HBGAM) with high affinity (I\d=0.25 nM). The chondroitin sulfate portion of PTP~ is essential to achieve
high affinity binding between PTP~ and pleiotrophin,and removal of chondroitin sulfate chains results in adecrease in the binding affinity (Kd=l3 nM). This is
the first demonstration that chondroitin sulfate plays
an important regulatory role in growth factor signaling.
In the embryonic rat brain, pleiotrophin is localizedalong the radial glial fibers, a scaffold for neuronalmigration. On the other hand, PTP~ is expressed in
the migrating neurons, suggesting that the ligand-receptor relationship between PTP~ and pleiotrophin
plays a role in migration of neurons during brain development. Thus, we examined the roles ofpleiotrophin-PTP~ interaction in neuronal migration
using the glass fiber assay and Boyden chamber cell
migration assay. Pleiotrophin on the substratum
stimulated migration of cortical neurons in both assays. Polyclonal antibodies against the extracellulardomain of PTP~. 6B4 proteoglycan (a secreted extracellular form of PTP~) and sodium vanadate (aNATIONAL INSTITUTE

protein tyrosine phosphatase inhibitor) added to theculture medium strongly suppressed pleiotrophin-induced neuronal migration. These results suggested
that PTP~ is involved in neuronal migration as a neuronal receptor for pleiotrophin distributed along radial
glial fibers.
Furthermore, to study the physiological functions of
PTP~ in vivo, we generated PTP~-deficient mice inwhich the PTP~ gene was replaced by the LacZ geneby homologous recombination in mouse embryonicstem (ES) cells. First, we examined the cell types
expressing PTP~ by investigating the expression of
LacZ in heterozygous PTP~-deficient mice. Throughout development from the early stage of embryogenesis, LacZ staining was restricted to the nervous
system. On embryonic day 12.5 (E12.5), LacZ staining was observed in the forebrain, midbrain, hindbrain
and spinal cord (Fig. 2A). Examination of the cerebral
cortex at higher magnification indicated that subsetsof neurons including pyramidal neurons expressed
LacZ (Fig. 2B). At the early postnatal stages, subsetsof neurons and astrocytes in the brain including pyramidal cells in the hippocampus expressed LacZ (Fig.
2C). Both neurons and astrocytes were positive for
LacZ in primary culture of cells from the fetal cerebral cortex. From these results, we concluded thatmany neurons as well as astrocytes express PTP~. We
are currently studying the phenotype of homozygous
mutant mice using biochemical, anatomical, physiological and ethological techniques, and various abnormalities have been found.
III. Functional roles of subfamily 2 sodium
channels
Voltage-gated sodium channels (NaChs) are responsible for the depolarizing phase of action potentials in
excitable cells and are essential for many physiological functions. Cloning of NaChs revealed marked conservation in the primary structures that underlies theirfunctional similarity. Thus, all NaChs cloned had beengrouped into a single gene family. However, recently,
novel NaChs, human Nav2.1, mouse Nav2.3 and rat
SCLll/Na-G, were cloned from inexcitable cells suchas glial cells. These molecules closely resemble eachother but are divergent from the previously cloned
sodium channels including the regions involved in
activation, inactivation and ion selectivity. Thus, thesemolecules have been grouped into a new subfamily of
sodium channels (subfamily 2). To date, subfamily 2channels have not been expressed in a functionallyactive form using in vitro expression systems, andtherefore the functional properties of these NaChs are
not yet clear. To clarify the cells expressing subfamily
2 sodium channels and their physiological functions
in vivo, we planned to generate knock-out mice deficient in channel genes.
We successfully produced mutant mice in which themNav2.3 gene was replaced with the LacZ or neo
gene by gene targeting. Using these mice, we foundthat mNav2.3 gene expression was restricted to theFOR BASIC BIOLOGY

41

Fig. 2 LacZ expression in PTPs+'- mice (A-C), and in mNav2.J+'- mice (D-F)(A) El2.5 whole embryo, (B) P7 cerebral cortex section, and (C) section of adult hippocampus. (D) El5 whole embryo, (E) adultsympathetic nerve and ganglia, and (F) section of adult tongue. In (B), the section was counterstained with cresyl violet, and thearrows indicate the pyramidal cells. In (C), p and g indicate the pyramidal cell layer and granule cell layer, respectively. In (F),the section was counterstained with cresyl violet

42

NATIONAL INSTITUTE

dorsal root ganglion (DRG) and lung during the embryonic stage (Fig. 2D). During the postnatal period,in addition to these tissues, Schwann cells in the sensory afferent nerve fibers (Fig. 2E, F) and a subset ofneurons in the central nervous system were positive
for mNav2.3 expression. We are currently examining
the phenotypes of homozygous mNav2.3-deficient
mice to gain insight into the physiological functionsof this channel.

Publication List:
Shintani, T., Watanabe, E., Maeda, N., and Noda, M.
(1998) Neurons as well as astrocytes express
proteoglycan-type protein tyrosine phosphatase
RRTPj3: Analysis of mice in which the PTP r;RRTP
13 gene was replaced with the lacZ gene. Neurosci.
Lett. 241, 135-138.
Yamagata, M. and Noda, M. (1998) The winged-helix
transcription factor CWH-3 is expressed in developing neural crest cells. Neurosci. Lett. 249, 1-4.
Katoh-Semba, R., Matsuda, M., Watanabe, E., Maeda,
N., and Oohira, A. (1998) Two types of brain chondroitin sulfate proteoglycan: their distribution andpossible functions in the rat embryo. Neurosci. Res.
31, 273-282.
Maeda, N. and Noda, M. (1998) Involvement of receptor-like protein tyrosine phosphatase ~IRPTPI3
and its ligand p1eiotrophin/HB-GAM in neuronalmigration. J. Cell Bioi., 142, 203-216.
Matsui, F., Nishizuka, M., Yasuda, Y., Aono, S.,
Watanabe, E. and Oohira, A. (1998) Occurrence ofan N-terrninal proteolytic fragment of neurocan, nota C-terminal half, in a perineuronal net in the adultrat cerebrum. Brain Res., 790, 45-51.
Nishiwaki, T., Maeda, N. and Noda, M. (1998) Characterization and developmental regulation ofproteoglycan-type protein tyrosine phosphatase ~/RPTPI3 isoforms. J. Biochem., 123, 458-467.
Yasuda, Y., Tokita, Y., Aono, S., Matsui, F., Ono, T.,
Sonta, S., Watanabe, E., Nakanishi, Y., and Oohira,A. ( 1998) Cloning and chromosomal mapping of
the human gene of neuroglycan C (NGC), a neuronal transmembrane chondroitin sulfate
proteoglycan with an EGF module. Neurosci. Res.,32, 313-322.
Takahashi, M., Yamagata, M.,and Noda, M. (1999)
Specific expression of ezrin, a cytoskeletal-membrane linker protein, in a subset of chick retinotectal
and sensory projections. Eur. J. Neurosci. 11, 545558.
Maeda, N., Ichihara-Tanaka, K., Kimura, T.,
Kadomatsu, K., Muramatsu, T. and Noda, M.
(1999) A receptor-like protein tyrosine phosphatase
PTPr;RPTPj3 binds a heparin-binding growth factor
midkine: Involvement of arginine 78 of midkine in
the high affinity binding to PTP~. J. Bioi. Chern., inpress.

r;

Yamakawa, T., Kurosawa, N., Kadomatsu, K., Matsui,T., Itoh, K., Maeda, N., Noda, M., and Muramatsu,
T. (1999) Levels of expression of pleiotrophin andprotein tyrosine phosphatase ~ are decreased in human colorectal cancers. Cancer Letter, in press.
Nishiwaki, T., Maeda, N., and Noda, M. (1999) Characterization and developmental regulation ofproteog1ycan-type protein tyrosine phosphatase
RPTPI3 isoforms. In Neural Development(Uyemura, K., Kawamura, K., and Yazaki, T. eds.)
pp.291-297. Springer-Verlag Tokyo.

r;FOR BASIC BIOLOGYDIVISION OF CELLULAR REGULATION
Norio Murata
Koji Mikami
Atsushi Sakamoto
Yoshitaka Nishiyama
Iwane Suzuki
Monbusho Foreign Scientist: Suleyman I. Allakhverdievl!
Institute Research Fellows: Akira Katoh
Mikio Kinoshita
Sayamrat Panpoom
Vyacheslav V. Klimovl!
JSPS Visiting Scientist:
JSPS Postdoctoral Fellow: Eira Kanervo 2!
Silvia Franceschelli3!
EU Fellow:
Adam Gilmore4!
Visiting Scientists:
Cai-Xia Hou 5!
Larissa Kisseleva 6!
Dmitry A. Los6!
Zoltdn Magyar7!
Bruno Maresca 3!
Liberato Marzullo 3!
Balazs Szalontaf!
Roumiana Todorova 8!
Tom Wydrzynski4!
Masami Inaba
Graduate Students:
Hiroshi Yamamoto
Yuji Tanaka
Ryoma Suzuki
Yu Kanesaki
Sho-ichi Higashi
Technical Staff:
Hideko Nonaka

Professor:
Associate Professor:
Research Associates:

1! from the Institute of Basic Biological Problems,

Pushchino, Russia
2! from the University of Turku,

Turku, Finlandfrom the International Institute of Genetics andBiophysics, Naples, Italy
4! from the Australian National University, Canberra,
Australia
5! from Xinjiang University, Urumqi, China
6! from the Institute of Plant Physiology, Moscow, Russia
7! from the Biological Research Center, Szeged, Hungary
B! from the Institute of Biophysics, Sofia, Bulgaria

31

The research efforts of this division are aimed atdeveloping a full understanding of the molecularmechanisms by which plants are able to acclimate toand tolerate stresses that arise from changes in environmental conditions, with particular emphasis on
temperature stress and salt stress. In 1998, we madesignificant progress in the following areas, using
higher plants and cyanobacteria as our experimental
materials.
I. Enhancement of the stress tolerance of plants bygenetic engineering of the synthesis ofglycinebetaine

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 high concentrations of salt and other types ofenvironmental stress. To examine the effects of betaine in vivo on the protection against high-salt stress,

43

we transformed Arabidopsis thaliana, which does notnormally accumulate betaine, with the codA gene forcholine oxidase (which catalyzes the conversion ofcholine to betaine) from Arthrobacter globiformis.
Transformed Arabidopsis plants were able to synthesize betaine and to tolerate high-salt stress. Moreover,transformation with the codA gene enhanced theprotection of the photosynthetic machinery against
low-temperature photoinhibition and accelerated therecovery of the machinery from photo-induced damage. Betaine also accumulated in the seeds of thetransformed plants. Such seeds were more tolerant
than seeds of wild-type plants to low temperaturesduring imbibition and germination. The growth ofyoung seedlings of transformed plants was also accelerated at low temperatures. Furthermore, transformation with the codA gene significantly enhanced thetolerance of seeds to high temperatures during both
imbibition and germination (Fig. 1), and young seedlings also exhibited enhanced tolerance to high temperatures. Thus, accumulation of betaine increased thetolerance of Arabidopsis to various stresses, namely,
high-salt stress, light stress, low-temperature stressand high-temperature stress.
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, as a consequence, exhibited tolerance to high-salt
stress. During treatment with 0.15 M NaCl (high-salt
stress), 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, transformed
plants began to grow again at the normal rate after asignificantly shorter time than the wild-type plants.
The photosynthetic machinery of the transformed
plants was more tolerant to salt stress and low-temperature stress than was that of the wild-type plants.
Our results in Arabidopsis and rice demonstrate thepotential usefulness of the codA gene in the genetic
engineering of stress tolerance in a wide variety ofagronomically important crops.
II. Important roles of polyunsaturated membrane
lipids in stress tolerance
In studies conducted prior to 1998, we succeeded ininactivating genes for fatty-acid desaturases bytargeted mutagenesis in the cyanobacterium
Synechocystis sp. PCC 6803 and we produced several
mutant strains in which the fatty acids of the membrane lipids contained abnormal numbers of unsaturated bonds. By comparing various properties of thesestrains, we demonstrated 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 Al2
fatty-acid desaturase of Synechocystis sp. PCC 6803
into the wild-type strain Synechococcus sp. PCC

44

NATIONAL INSTITUTE

Wild typeTransformed

Fig. 1. Effects of high temperature during imbibition on the subsequent germination of seeds from wild-type and transformed
plants of Arabidopsis thaliana . Dry seeds were allowed to imbibe water at 22, 40, 50 and 55°C for 60 min. Then seeds were
inoculated on plates of MS medium. After incubation at 4°C for 2 days, the seeds were incubated at 22°C. Three days later, after
germination had occurred, plates were photographed. Each plate was 9 em in diameter.

7942, which normally contains monounsaturated fatty
acids and no polyunsaturated fatty acids. We designated the transformed strain de sA+. The transformation resulted in replacement of half of themonounsaturated fatty acids in the membrane lipids
by diunsaturated fatty acids. Comparison of de sA+
cells with wild-type cells revealed that the increased
number of double bonds in the membrane lipids enhanced the ability of cells to resist photoinhibition at
low temperatures by accelerating the recovery of thephotosystem II complex from photoinhibitory damage. Our findings indicate that polyunsaturated membrane lipids are important in protecting the photosynthetic machinery from damage caused by strong light
at low temperatures.
The Dl protein at the catalytic center of the photosystem II complex of Synechococcus sp. PCC 7942
exists as two isoforms, designated D1 :1 and Dl:2, andtransfer of cells to low temperatures results in thereplacement of D 1: 1, the prevailing form, by D 1:2.
The extent of such replacement in desA+ cells at low
temperatures was greater than that in wild-type cells.
It seems likely that polyunsaturated membrane lipids
facilitate the exchange of isoforms of the D 1 protein
and, in this way, they maintain the photochemical
activity of the photosystem II complex at low temperatures.
We investigated the role of polyunsaturated membrane lipids in the tolerance of the photosynthetic
machinery to high-salt stress by comparing the desk

/desD· mutant of Synechocystis sp. PCC 6803, whichcontained monounsaturated fatty acids, with the wildtype strain, which contained a full complement ofpolyunsaturated fatty acids. The oxygen-evolving activity of desk/desD· cells was more sensitive to highsalt stress than was that of wild-type cells. Moreover,
the activity of the Na+fH+ antiport in desk/desD· cells
was suppressed to a greater extent than that of the
antiport in wild-type cells under high-salt stress.
These observations suggest that polyunsaturated
membrane lipids might stimulate the activity and/or
the synthesis of the Na+fH+ antiport system and protect the photosynthetic machinery against salt-induced
inactivation.

III. Molecular mechanisms of the protection of thephotosynthetic machinery against hightemperature stress
The oxygen-evolving machinery of the photosystem
II complex is extremely susceptible to inhibition athigh temperatures. We have been studying the molecular mechanisms that underlie the stabilization of
the photosynthetic machinery against the heat-induced
inactivation in the cyanobacterium Synechococcus sp.
PCC 7002. Biochemical investigations of thylakoid
membranes allowed us to identify two proteins, cytochrome c550 and PsbU, as factors that stabilize theoxygen-evolving machinery at high temperatures. To
elucidate the role of PsbU in vivo, we inactivated thepsbU gene in Synechococcus sp. PCC 7002 by tar-

45

FOR BASIC BIOLOGY

geted mutagenesis. Mutated cells were not only unable to increase the thermal stability of their oxygenevolving machinery but they were also unable todevelop cellular thermotolerance upon acclimation tohigh temperatures. These results suggest that PsbU
might play an important role in enhancing the thermal
stability of the oxygen-evolving machinery at hightemperatures and, moreover, that the stabilization of
the machinery might be crucial for the acquisition ofcellular thermotolerance.

IV. Stress-dependent enhanced expression ofcytochrome eM and suppression of the expressionof cytochrome c6 and plastocyanin
Cytochrome eM is a c-type cytochrome with a molecular mass of 8 kDa. We previously identified
and cloned the cytM gene for cytochrome eM from
Synechocystis sp. PCC 6803. We cloned homologs ofthe cytM gene from other cyanophytes and aprochlorophyte, providing evidence that suggests thatthe cytM gene might be distributed universally in oxygenic photosynthetic prokaryotes. Northern blotting
analysis revealed that the cytM gene of Synechocystis
sp. PCC 6803 is barely expressed under normal
growth conditions. However, expression of the gene
was induced when cells were exposed to low temperature and/or high-intensity light. By contrast, the expression of the petl gene for cytochrome c6 andexpression of the petE gene for plastocyanin, which
are electron carriers that transport electrons from thecytochrome bif complex to the photosystem I complex, were suppressed at low temperatures and also
under high-intensity light. These observations suggestthat regulation of the expression of the cytM genemight be the mirror image of regulation of the pet]and petE genes under the stress conditions examined.

Publication List:

Kanervo, E, Murata, N. and Aro, E.-M. (1998) Massive breakdown of the photosystem II polypeptides
in a mutant of the cyanobacterium Synechocystis sp.
PCC 6803. Photosynth. Res., 57, 81-91.
Panpoom, S., Los, D.A. and Murata, N. (1998) Biochemical characterization of a ill2 acyl-lipid
desaturase after overexpression of the enzyme in
Escherichia coli. Biochim. Biophys. Acta, 1390,
323-332.
Papageorgiou, G.C., Alygizaki-Zorba, A., Ladas, N.
and Murata, N. (1998) A method to probe the cytoplasmic osmolality and osmotic water and solute
fluxes across the cell membrane of cyanobacteriawith chlorophyll a fluorescence: Experiments with
Synechococcus sp. PCC 7942. Physiol. Plant., 103,
215-224.
Sakamoto, A., Alia and Murata, N. (1998) Metabolicengineering of rice leading to biosynthesis ofglycinebetaine and tolerance to salt and cold. Plant
Mol. Biol., 38, 1011-1019.
Sakamoto, T., Shen, G., Higashi, S., Murata, N. andBryant, D.A. (1998) Alteration of low-temperaturesusceptibility of the cyanobacterium Synechococcus
sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation. Arch. Microbial., 169, 2028.
Sippola, K., Kanervo, E., Murata, N. and Aro, E.-M.
(1998) A genetically engineered increase in fatty
acid unsaturation in Synechococcus sp. PCC 7942
allows exchange of D 1 protein forms and sustenance of photosystem II activity at low temperature.
Eur. J. Biochem., 251, 641-648.
Yokoi, S., Higashi, S., Kishitani, S., Murata, N. andToriyama, K. (1998) Introduction of the eDNA for
Arabidopsis glycerol-3-phosphate acyltransferase(GPAT) confers unsaturation of fatty acids andchilling tolerance of photosynthesis on rice. Mol.
Breeding, 4, 269-275.

(1) Original articles(2) Review articles

Alia, Hayashi, H., Chen, T.H.H. and Murata, N.
(1998) Transformation with a gene for choline oxidase enhances the cold tolerance of Arabidopsis
during germination and early growth. Plant Cell
Environ., 21, 232-239.
Alia, Hayashi, H., Sakamoto, A. and Murata, N.
( 1998) Enhancement of the tolerance of Arabidopsis
to high temperatures by genetic engineering of thesynthesis of glycinebetaine. Plant 1., 16, 155-161.
Fukuchi-Mizutani, M., Tasaka, Y., Tanaka, T.,
Ashikari, T., Kusumi, T. and Murata, N. (1998)
Characterization of il9 acyl-lipid desaturase homologues from Arabidopsis thaliana. Plant Cell
Physiol., 39, 247-253.
Hayashi, H., Alia, Sakamoto, A., Nonaka, H., Chen,
T.H.H. and Murata, N. ( 1998) Enhanced germination under high-salt conditions of seeds oftransgenic Arabidopsis with a bacterial gene (codA)
for choline oxidase. J. Plant Res., 111, 357-362.

Gombos, Z. and Murata, N. (1998) Genetic engineering of the unsaturation of membrane glycerolipid:
effects on the ability of the photosynthetic machinery to tolerate temperature stress. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A.
Siegenthaler and N. Murata, eds.) pp. 249-262.
Kluwer Academic Publishers, Dordrecht.
Los, D.A. and Murata, N. (1998) Structure and expression of fatty acid desaturases. Biochim.
Biophys. Acta, 1394, 3-15.
Murata, N. and Siegenthaler, P.-A. (1998) Lipids inphotosynthesis: an overview. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A.
Siegenthaler and N. Murata, eds.) pp. 1-20. Kluwer
Academic Publishers, Dordrecht.
Wada, H. and Murata, N. (1998) Membrane lipids incyanobacteria. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A. Siegenthaler andN. Murata, eds.) pp. 65-81. Kluwer Academic Publishers, Dordrecht.

46

NATIONAL INSTITUTEDIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADJUNCT)
Professor (Adjunct): Masamitsu Wada
Research Associate: Tomohiro Kiyosue
Postdoctoral Fellow: Takatoshi Kagawa
Graduate Students: Takata Imaizumi(Tokyo Metropolitan University)
Kazusato Oikawa(Tokyo Metropolitan University)

Plants use light as an environmental factor which
controls their development as well as their otherphysiological phenomena. Phytochrome and bluelight receptors, such as cryptochrome and phototropin(NPHI ), are main photoreceptors for plant photomorphogenesis. The goal of our research is to clarify thesignal transduction pathways of photomorphogenesis.
We are focusing our study on chloroplast
photorelocation movement which is thought to be oneof the simplest phenomena in this field. We use thefern Adiantum gametophytes as a model plant not
only because the gametophytes are very sensitive tolight, but also because the organization of the cells isvery simple and easy to manipulate. We also use
Arabidopsis for mutant screening to clarify the genes
regulating chloroplast photorelocation.

regulated expression patterns. CRY4 was down regulated by light throughout the life cycle of Adiantum.
The eDNA expression level was reduced by approximately 100 times when dark imbibed spores were
irradiated with red light, which caused spore germination. CRY5 expression was induced rapidly after light
exposure and kept increasing for 12 hours. The expression level was reduced for the next 12 hours andafter that it did not vary much.
The major change in CRY4 and CRY5 expression
level occurred before spore germination, so transcriptsof these two genes might act somewhat in the processof light dependent germination of Adiantum.
1-2 Phototropin
NPHI (phototropin) is another blue-light photoreceptor recently isolated in higher plants, which is aflavin binding protein with a light sensitive protein
kinase activity. By RT-PCR with primers designed
from highly conserved sequence motifs in NPHl
photoreceptors, we cloned a eDNA fragment from
Adiantum. The RT-PCR experiment suggested that theaccumulation of mRNA for the Adiantum NPH I isdown regulated by light. The structural and biochemical characterization of Adiantum NPH1 is in progress.

II. Chloroplast relocation
I. Cloning and characterization of blue-light
photoreceptors in the fern Adiantum capillusveneris

I -1 Cryptochromes
We have described many blue-light-induced
photomorphological responses in gametophytes of thefern Adiantum capillus-veneris. As the first step inunderstanding the molecular mechanisms of thesevarious blue-light responses in Adiantum, we isolated
blue-light photoreceptor-related genes as homologuesof Arabidopsis cryptochromel.
We cloned five different genes, which are designated as Adiantum cryptochromel, 2, 3, 4, and 5(CRYJ, 2, 3, 4, and 5), from a genomic DNA library.
The N-termini of the five genes showed remarkablehomology to Arabidopsis cryptochrome 1, as well as
to other cryptochromes which have already been identified. However, these genes have every distinct Ctermini from each other.
To analyze the correlation between blue-light-induced responses and cryptochromes, we investigatedthe expression pattern of these genes in various developmental stages and also in different light conditions
using RNA blot analysis and competitive PCR analysis.
The results showed CRYJ, CRY2 and CRY3 to have
similar expression patterns. Transcripts of these genes
were elevated after spores imbibed water in the dark,
but after that the level of these transcripts did not vary
much. The character of this expression pattern is very
similar to those of previously characterized plant
cryptochromes. CRY4 and CRY5 showed unique light-

2- I Arabidopsis

Chloroplasts accumulate at cell surface under weak
light and escape from strong light to optimize photosynthesis. Which photoreceptors are active in chloroplast relocation, however, is not known.
We are studying light-induced chloroplast relocation in Arabidopsis leaves. In a light-adapted mesophyll cell, chloroplasts spread over the cell surface,but in a dark-adapted cell they escape from the cellsurface. When a cell is partly irradiated with blue lightat a strength of 30Wm·2 , chloroplasts move out fromthe irradiated spot. In comparison, chloroplasts accumulate in a spot irradiated with blue light at a strengthof 3Wm·2 • Simultaneous irradiation with red light increased the sensitivity to the blue light, although the
red light irradiation alone did not induce chloroplast
relocation.
Photoreceptor mutants, such as cry], cry2, crylcry2
double mutant and nphl, were demonstrated to benormal for blue-light-induced chloroplast relocation
for both weak and strong blue light, meaning thet
CRY!, CRY2 and NPHI are not the photoreceptors
for chloroplast relocation. To find the photoreceptor
for blue-light-induced chloroplast relocation, we arescreening mutants from T-DNA tagging lines. Identification of the mutated genes of Arabidopsis mutants
is now in progress.
2-2 Adiantum
Mutants of chloroplast relocation movement werescreened and analyzed is Adiantum gametophytes.

47

FOR BASIC BIOLOGY

Mutants which were deficient either in high fluence
response or in low fluence response were obtained
independently, meaning that the signal transduction
pathways of both responses are different.

Publication:
Nozue, K., Kanegae, T., lmaizumi, T., Fukada, S.,
Okamoto, H., Yeh, K.C., Lagarias, J.C. and Wada,
M. (1998) A phytochrome from the fern Adiantum
with features of the putative photoreceptor NPH 1.
Proc. Nat!. Acad. Sci. USA, 95, 15826-15830.

Figure l. Serial photographs of chloroplast relocation in a dark-adapted (for 2 days) prothallial cell induced by one minillumination with a red microbeam (10 J.lm in diameter) of 30 Wm· 2 Chloroplast moved toward the beam after the beam was
switched off over 70 min and then dispersed. Bar, 20 J.lm.

48

NATIONAL INSTITUTE

Figure 2. Serial photographs of chloroplast relocation in a dark-adapted prothallial cell induced by continuous blue microbeam
irradiation (10 Wm·2, 27 Jlm in diameter). The illumination spot is shown only time 0 but the light was given from time 0 to 90
min. The numbers shown in the photographs are the time after onset of blue light irradiation. Chloroplasts move toward the beam,
but they can not get into the beam irradiated area until the microbeam was switched off. Bar, 20 Jlm.

49

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

The vertebrate nervous system contains a large
number of neurons that are connected to each other byspecific axonal projections. We are interested in how
the complex but precise neuronal network, which isindispensable for functioning of the nervous system,
is constructed during development. The goal of ourresearch in this division is to elucidate the cellular andmolecular mechanisms underlying the neuronal network formation in vertebrates, with current focuses on
the mechanisms of axon guidance and neuronal migration. Our laboratory was just started in April 1998
by two of the members and others joined thereafter.
Plans of research but not results of experiments will
be described below.

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

II. Mechanism of Neuronal Migration
A variety of neurons migrate from their birthplace
to the position where they finally settle. We are alsointerested in the mechanisms underlying the neuronalmigration in vertebrate central nervous system, inparticular, how neurons are guided through specificroutes to final positions. To address these issues, weare planning to develop in vitro culture systems thatreconstruct migratory events occurring in vivo.

50

NATIONAL INSTITUTE

51

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.

52

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION I
Professor:
Shigeru /ida
Associate Professor (adjunct): Yoshihiro Ozeki
Research Associate:
Rie Terada
Yoshiki Habu
Yoshishige lnagaki
Atsushi Hoshino
Postdoctoral Fellow:
Yasuyo Johzuka-Hisatomi 2
Hong-Qing Li 2
Graduate Student:
Naoko Ishikawa
Takashi Kagami
Toshio Yamaguchi
Technical Staff:
Sachiko Tanaka-Fukada
Tomoko Mori
Yoshiko Inoue
Visiting Fellows:
Yasumasa Morita
Fumiyoshi Myoga

The main interest of the group is in understanding
the biology of the dynamic genome, namely, genome
organization and reorganization and its impact on
gene expression and regulation. Although there aremany elements affecting organization and reorganization of the genomes, we are currently focused on
mobile genetic elements in general and plant transposable controlling elements in particular.
I. Identification and characterization of mutablealleles in the Japanese morning glory
The Japanese morning glory (Pharbitis nil or Ipomoea nil) is a traditional horticultural plant in Japan,
and extensive physiological and genetical studies on
the plant have been conducted. A number of mutants
related to the color and shape of its flowers and leaves
have been isolated since the 17th century, and morethan 200 genetic loci including about 20 mutable locihave been assigned to one of the 10 linkage groups.
We have identified that the mutable flecked allele
bearing white flowers with colored flecks is the DFRB gene having the En/Spm-related transposable element Tpnl inserted into its second intron. The nonautonomous Tpnl element carries a genomic DNA
segment containing at least four exon sequences encoding part of a HMG-box sequence. Spliced hybridtranscripts containing the DFR-B exon(s) and the
HMG exons from Tpnl were detected in the flower
buds of the flecked line, and they were polyadenylated
within Tpnl. Thus Tpnl can be regarded as a specialized transducing transposon carrying a part of the
genomic sequence for a HMG-box (Takahashi et al.
(1999) Mol. Gen. Genet. in press).
The plants with the recessive mutable speckled allele, in the presence of the dominant Speckled-activator, produce colorless flowers with fine and roundcolored spots distributed over the corolla, while plants
carrying the speckled allele without active Speckledactivator bear pale yellow flowers. Currently, we are
analyzing the CHI gene containing another En/Spmrelated element Tpn2 in the mutable speckled lines.

We are also characterizing not only other mutablealleles such as purple-mutable but also stable alleles
displaying white flowers.
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. 1). The mutable afiaked 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 A!afiaked. We
showed that the mutable afiaked allele is caused by insertion of a new transposable element, TiplOO, into the
CHS-D gene intron. TipJOO is 3.9 kb long and belongs
to the AciDs family. Although the timing and frequency of the flower variegation vary in different lines(Fig. lA and B), they carry the identical mutable allele (Fig. IE).
It has been postulated that the timing and the frequency of the variegation are determined by the active
state of another genetic element modulator. It was
further postulated that some lines bearing white flowers must be a modulator inactive line whereas otherwhite-flower lines must be deficient at the A locus,(Fig. IC and D), because in the presence of active
modulator the latter can produce variegated flowers
w~ile the former still display white flowers. Indeed,
the structure of the CHS-D gene in one of the formerlines we analyzed was found to be indistinguishable
from that in the mutable afiaked lines (Fig. IE) whereasa white-flower line deficient at the A locus carries
double insertions of TiplOO (Fig. IF). Presumably,
excision of one of the two copies of TipJOO from the
CHS-D gene in the latter line appears to be insufficient to restore the CHS-D function and probably bothelements are rarely excised in the same tissue.
III. Characterization of the genes for anthocyanin
pigmentation in morning glories.
As an initial step to characterize the genes for anthocyanin pigmentation in morning glories, we sequenced segments of about 17 kb genomic DNA containing three DFR genes in the Japanese and common
morning glories. The three DFR genes in both plants
are arranged in tandem array, and all of them comprise six exons with identical intron positions. Their
DFR-B genes, which carry longer introns than the
DFR-A and DFR-C genes, are responsible for pigmentation in flowers, stems and coloring leaves. Indeed,
the DFR-B gene of the common morning glory isexpressed extensively in the young buds of pigmented
flowers, considerably in stems, and moderately in sepals and leaves, whereas the DFR-A and DFR-C genes
were expressed scarcely but significantly in the young
flower buds and stems. Several novel mobile elementlike sequences of around 200 bp were found in the

53

FOR BASIC BIOLOGY

B

A

c

0

F

Tip tOO

CHS-0

ootdflFig. 1 The mutable lines of the common morning glory: heavily (A) and lightly (B) variegated lines; white-flower lines (C andD) ; structures of the CHS-D genes in the lines A, B and C (E) and the line D (F).

genomic DFR regions.
A phylogenetic tree indicated that each DFR genein the Japanese morning glory is most closely related
to the corresponding DFR gene in the common morning glory and that the DFR-B gene is the most diversified gene among the three DFR genes. At least twogene duplication processes must be involved in generating three tandem copies from a single copy, andsignificant sequence divergence in both exons andintrons of the duplicated DFR genes might occur during evolution of the genomes of these morning glories. Since homologies between the corresponding
DFR genes extend beyond the exon sequences to introns and intergenic sequences, the results can beinterpreted to mean that duplications and major divergence occurred prior to speciation of the Japanese andcommon morning glories (lnagaki et a!. ( 1999) Genein press).
Publication List:

Habu , Y. , Hisatomi Y. and Iida, S. (1998) Molecularcharacterization of the mutable flaked allele forflower variegation in the common morning glory.
Plant J. 16, 371 -376.
Habu Y. and lida, S. (1998) AFLP (flmplified restriction fragment length ,120lymorphism)-based mRNA
fingerprinting. Plant Biotechnology 15, 249-251 .
Hasebe, A., Tsushima S. and Iida, S. (1998) Isolationand characterization of IS1416 from Pseudomonas
glumae, a new member of the IS3 family. Plasmid
39, 196-204.

Iida, S., Hiestand-Nauer, R., Sandmeier, H ., Lehnherr
H . and Arber, W. (1998) Accessory genes in thedarA operon of bacteriophage P1 affect anti-restriction function, generalized transduction, head morphogenesis and host cell lysis. Virology 251 , 49-58.
Nakai , K., Inagaki, Y., Nagata, H. , Miyazaki C. andlida, S. (1998) Molecular characterization of thegene for dihydroflavonol 4-reductase of Japonica
rice varieties. Plant Biotechnology 15, 221-225.
Saito, N. , Tatsuzawa, F. , Kasahara, K. , Iida S. and
Honda, T. (1998) Acylated cyanidin 3-sophorosides
in the brownish-red flowers of Ipomoea purpurea.
Phytochemistry 49, 875-880.

54

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION II
Professor:
Takashi Horiuchi
Research Associates: Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama
Katsuki Johzuka
Postdoctoral Fellow: Katsufumi Ohsumi
Keiko Taki
Graduate Student:
Hiroko Urawa
Technical Staff:
Kohji Hayashi
Yasushi Takeuchi

Homologous recombination which may occur in allorganisms apparently involves exchange between twoparent-derived chromatids plus the repair of DNA
damage induced by physical and chemical reagents.
As deduced from our analyses of recombination
hotspots of E. coli and S. cerevisiae, in particular theactivity related to DNA replication fork blocking
events, the physiological function of homologous recombination, especially in normally growing cells is
better understood. In 1998, work on the two followinginterrelated subjects have advanced our knowledge ofrelated factors.
I. Molecular mechanism of expansion/contractionof ribosomal RNA gene repeats inS. cerevisiae.
HOTI is a mitotic recombinational hotspot in theyeast S. cerevisiae and was first identified by Keil andRoeder (1984). HOTl stimulates both intra- and interchromosomal recombination nearby regions when inserted at a non-rDNA location. HOT1 was originallycloned on a 4.6 kb Bglii-A fragment which locates inrRNA repeated genes (about 150 copies) on chromosome XII. A single rRNA unit consists of two
transcribed 35S and 5S rRNA genes and two nontranscribed regions, NTS 1 and NTS2, the former isbetween 3'-ends of 35S and 5S rRNA genes, and thelatter is 5' ends of these two genes. The HOT1 DNA
fragment contains the NTS 1, 5SRNA and NTS2 region but it was later found to be composed of two
non-contiguous cis-elements, E and I, located in
NTS1 and NTS2, respectively. Because E and I positionally and functionally overlapped with the enhancer and initiator of the 35S rRNA transcription,
respectively, Roeder's group suggested that transcription by RNA polymerase I, initiated at the 35S rRNA
promoter site may stimulate recombination of thedownstream region, thereby revealing Hot1 activity.
The NTS 1 has a site at which the replication fork isblocked and we termed this site SOG, but later called
RFB (replication fork barrier). By assaying activity
for various DNA fragments derived from the NTS I
and cloned on plasmids, we determined the minimal
region, about 100 bp long, located near or within theenhancer region of the 35S rRNA transcription andessential for blocking replication fork advancing in a
direction opposite that for transcription. The RFB se-quence has no homology to any other known sequence and has no characteristic structure such 2-foldsymmetry, repeated structure, etc.; hence, transfactor(s) may have a role in blocking the fork. Interestingly, this region is included in one of two ciselements required for a recombinational hotspot,
Hot1, activity.
To investigate functional relationships between thefork blocking activity in RFB and the hotspot activity
in HOT1, we first isolated mutants defective in Hotlactivity and examined whether these mutations would
also affect RFB activity. Using a colony color sectoring assay method, we isolated 23 Hot defective mutants from approximately 40,000 mutagenized colonies. Among these Hor- mutants, one proved to be arad52 mutant; the other 4 mutants lose fork blocking
activity. Genetic analysis of these mutants revealed
that all four were recessive for the RFB phenotype
and defined one complementation group. This mutation was designated fob] (fork b.locking function) and
one of the fob] mutants,fobl-4, was further analyzed.
First, from yeast eDNA bank, we cloned FOBJ geneby selecting a DNA fragment, which had suppressive
activity for Hot deficiency of the mutant. The minimal
FOBJ plasmid was shown to complement both theHor- and RFB- phenotype of the fobl-4 mutant, suggesting that both phenotypes are caused by a mutationin the FOBJ gene. DNA sequencing of the FOBJ
gene revealed that the putative Fobl protein consistsof 566 amino acids and has a molecular mass of
65,000 daltons. This gene has no homology with any
DNA sequence registered in Genbank. Sequencing of
the fobl-4 mutant gene revealed two mutationalchanges in the open reading frame, one is non-sense(amber) and other is a miss-sense mutation. The
amber mutation may account for the two defective
phenotypes of the fobl-4 mutant and why it is nonleakyness.
Because there is an extraordinary functional similarity between factors required for the hotspot activity inE. coli and S. cerevisiae, in both organisms ds-break
and repair would occur when the DNA replication
fork is arrested at fork blocking sites and probably
was spontaneously blocked.
Next we investigated relationship between FOBJ
gene and expansion/contraction of rRNA gene
(rDNA) repeats in collaboration with the laboratory of
Dr. Masayasu Nomura in Univ. of California (Irvine).
As described above, S. cerevisiae carries approximately 150 copies of rRNA gene in tandem repeats . .It
was found that the absence of an essential subunit of
RNA polymerase I (Poll) in rpa135 deletion mutants
triggers a gradual decrease in rDNA repeat number toabout one-half the normal level. Reintroduction of themissing RPA135 gene induced a gradual increase inrepeat number back to the normal level. Gene FOBJ
was shown to be essential for both the decrease andincrease of rDNA repeats (Fig.1). Thus, DNA replication fork blockage appears to stimulate recombinationand play an essential role in rDNA expansion/contrac-

FOR BASIC BIOLOGY

55

(A)
POL1
FOB1

(B)

- + - +
- - + + W.T.
2 3 4 5 6 7

FOB1 -POL1

8 9 10 11

M 1

1

+ - +
- + + W.T.
2
6 7
3 4 5

8 9 10 11

2.7 -2.351.8 1.661.371.05(Mb)

ETBr stain

Autoradiography (rONA)

Figure 1. Increase of the size of chromosome by Fob l and Po !I proteins. (by Pulse-field gel electrophoresis)

Fork Block Dependent Recombination model

(A)

(8)

(C)

ori-1 358-1

•

ori-2
I [

C

358-2

RFB

RFB

:=

• (-.,

RFB

t: •
1[ •

l

·~

•

358-3
E: ori-3
e
I [

I

l

l [

~~

l [

~~

(C')

[

.r

[ r. . .xq=
358-2

(D)

•
•

358-1

•E•
l [

•

!J:l.'"E IQl [358-31•El [Figure 2. Fork block-dependent recombination model for rDNA expansion/contraction.

56

tion and sequence homogenization, and possibly, in
the instability of repeated sequences in general (ex.
expansion of triplet repeats). RNA polymerase I, on
the other hand, appears to control repeat numbers,
perhaps by stabilizing rONA with the normal repeat
numbers as a stable nucleolar structure.
We propose a model, shown in Fig.2, we call thefork block dependent recombination model to show
how the blocked replication fork changes the copy
number of rONA.
II. The E. coli genome project
E. coli genome project in Japan was started in 1989,
the objective being to analyze, in an independently
living organism, nucleotide sequences from 0 min ina clockwise direction. lli April 1995, the DNA sequences of the 0-12.7 min region in the genetic map
were determined and they were published or registered. At that time I (T. H.) was appointed leader of
the Japan E. coli genome project and a new group of
researchers was reorganized to analyze DNA sequences of the region from 12.7 min to 70 min of
W3110 strain, using Kohara lambda clones and a newprotocol, including a shot-gun sequence technique.
In January 1997, we had completed a sequence
analysis of E. coli genome by combining our sequence data of 2.2 mega base of a region corresponding to 12.7-69.0 min (including DNA replication terminus region) with those (69.0-100 min) previously
registered in GenBank by other groups. The entire E.
coli genome was expressed.in a single, continuous
nucleotide sequence (4,636,552 bp), in which about
4.300 putative open reading frames were identified. At
this same time, Blattner's group in Wisconsin Univ.
had determined independently the sequence of entire
genome of another closely related strain, MG1655.
Now we are sequencing the entire genome ofW3110(it will be completed within this year) and their genomic structural changes between W311 0 and
MG1655 strains, which occurred during a very shortperiod after being separated from a common ancestor,
are being compared. It may reveal about mechanismsof micro-evolution in bacteria.

Publication list;
Kobayashi, T., Rein, T., & Depamphilis, ML. (1998)
Identification of primary initiation sites for DNA
replication in the hamster dihydrofolate reductase
gene initiation zone. Mol Cell Bioi 18, 3266-77
Kobayashi, T., Heck, J.D., Nomura, M., and Horiuchi,
T. (1998) Expansion and contraction of ribosomal
DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein
and the role of RNA polymerase I. Genes & Development 12, 3821-3830.

NATIONAL INSTITUTE

57

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS I
Professor:
Tetsuo Yamamori
Research Associates: Yuriko Komine
Akiya Watakabe
Takashi Kitsukawa
Postdoctoral Fellow: Rejan Vigot (JSPS)
Visiting scientist:
Kimiko Yamamori
Yoshinori Shirai
Graduate student:
Shiro Tochitani
Katsusuke Hata
Yusuke Komatsu
Akishi Onishi (Kyoto University)
Shuzo Sakata (kyoto University)
Nobuaki Tanaka (Kyoto University)
Hiroshi Fujita (Kyoto University)
Ryohei Tomioka (Kyoto University)
Hideko Utsumi
Technical Staffs:

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 haverevealed 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 CD FILIP receptor evolved and secondly, a third binding subunits ofa CNTF receptor evolved. Our model predicts that thebinding subunits generally determine the specificity of
the receptors and it is possible that novel members of
the cytokine family and their receptors exist in the
nervous system. In order to prove this hypothesis, weare currently working to examine the expression pattern of the family 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
stimuli of parallel fibers and a climbing fiber to a

Purkinje cell induce prolonged reduction of a synaptic
efficacy between the paralleled fiber to the Purkinje
cell (LTD; long-term depression, Ito et al., 1982).
Previously, we examined the expression of 10 immediate early genes (lEGs) including all the known
Fos and Jun family in cerebellar slices under the pharmacological condition that cause long-term desensitization of the Purkinje cell to AMPA (a glutamate analogue). Among the lEGs examined, Fos and Jun-B
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
Science Institute, RIKEN). One of these genes iscurrently further being characterized. We are also examining the expression of the genes under different
types of, cognitive and procedural, learning tasks.
During our attempt to isolate LTD-related genes, we
accidentally found repetitive genes which were specifically expressed in the rat brain (Bsr: b.rain ~pecificrepetitive gene). To our surprise, the genes are only
found in the rattus but no other species so far examined including murine species. Although we do notknow the function of this new type of gene at the
moment, it may play some important role in the rat
brain and we hope further characterization of the genereveals it.

Publication List
Komine, Y., Tanaka, N., Yano, R., Takai, S., Yuasa, S.,
Shiraishi, T., Tsuchiya, K., and Yamamori, T. (inpress) A novel type of non-coding RNA expressedin the rat brain. Mol. Brain Res.

58

NATIONAL INSTITUTE

Fig. I Expression of Bsr in the rat hippocampus. In situ hybridization with an antisense probe of Bsr.

59

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS II
Associate Professor: Mitsuyasu Hasebe
Post Doctral Fellow: Rumiko Kofujil
Graduate Students: Satomi Shindo
Yuji Hiwatashi
Satoko Kobayashi-Arakawa
Ryosuke Sana (Chiba Univ.)
Tomoaki Nishiyama ( Univ. of Tokyo)
Kumi Aso (Univ. of Tokyo)
Fernando-Rivadavia Lopes (Univ. ofTokyo)
Kazuyoshi Someya ( Univ. of Tokyo)
Keiko Sakakibara ( Univ. of Tokyo)
Saiko Himi (Kanazawa Univ.)
Youichi Tanabe ( Chiba Univ.)
Yukiko Tanikawa
Technical Staff:
Masae Umeda
Chigusa Ono

All living organisms evolved from a common ancestor more than 35 billion years ago, and accumulated
mutations on their genomes caused the present
biodiversity. The traces of evolutionary processes areremained in the genomes of extant organisms and we
can infer (1) the phylogenetic relationships of organisms and (2) the genetic changes having caused thephenotypic evolution by comparing the genomes ofdifferent organisms. The inferred phylogenetic relationships give important insights on problems in various fields of evolutionary biology and our group is
now focusing on biogeography, evolution of morphological traits and systematics in wide range of taxa.
On the phenotypic evolution, we are especially interested in the morphological evolution and aim to explore genetic changes led the evolution of plant bodyplan. We selected Arabidopsis (angiosperm), Gnetum(gymnosperm), Ginkgo (gymnosperm), Ceratopteris(pteridophyte), and Physcomitrella (bryophyte) as
models to compare the genetic cascades regulating
morphogenesis especially in the reproductive organs
and shoot apical meristem of land plants.
I. Molecular phylogeny of plants and lichen(a) Phylogeny of maple trees and their disjunct distribution between North America and eastern Asia.
The maple tree genus, Acer is the largest genus in
broad-leaved deciduous trees and contains about 200
species. The delimitation of the genus is clear but the
intrageneric classification was controversial becauseof homoplasies in morphological characters. The phylogenetic relationship in Acer was inferred based on
chloroplast DNA restriction site polymorphisms with17 restriction endonucleases and previously proposed
intrageneric classifications were evaluated. Based on
the estimated evolutionary rate of chlotoplast DNA,
divergence of eastern Asian and North American species in two different sections were estimated to havetaken place in late Miocene. In consideration withprevious data, multiple migrations and disjunctions
are likely to have formed the eastern Asian and North
American similar but disjunct distribution.

(b) Phylogeny of Coriaria and the biogeography.
Coriaria is distributed in four separate areas in theworld, which is the most conspicuous disjunct distribution in flowering plants. The phylogenetic relationships of 12 Coriaria species collected from the representative disjunct areas were inferred by comparing
the combined data set of rbcL (a large subunit of ribulose 1,5-bisphosphate carboxylase I oxygenase) andmatK (maturase K) genes. The estimated divergencetime between the Eurasian species and other species
distributed in the Southern Hemisphere was estimatedas 63 and 59 million years ago using rbcL and matK
molecular clocks, respectively. These results do notsupport the previously proposed hypotheses to explain
the disjunct distribution based on the continental drift,
but suggest that the distribution pattern was formed byseveral geographical migrations and separations in the
Cenozoic.
(c) Phylogeny of lady ferns. The lady fern group,
Physematieae is one of five tribes in the
Dryopteridaceae and contains about 700 species distributed mainly in temperate forests. The classificationof the group is controversial and nucleotide sequencesof the chloroplast gene rbcL from 42 species of thefern tribe Physematieae (Dryopteridaceae) were analyzed to provide insights into the inter and intra generic relationships and the generic circumscriptions of
the group. Phylogenetic relationships of several enigmatic genera were revealed.
(d) Other phylogenetic studies
The Hydrostachiacear and the Podostemaceae haveunusual morphology in angiosperms and their
phylogentic relationships are controversial. During thecourse of field expedition to Madagascar (August to
September, 1998), more than 10 species of Hydrostachis and three species of Podostemaceae were collected and the phylogenetic analyses are in progress.
Molecular phylogeny of Myelochroa (lichen),Diplazium (ferns), major groups in bryophytes, andthe Droseraceae (carnivorous plants) are also inprogress.
II Evolution of body plan in plants(a) Evolution of reproductive organs. A flower isthe most complex reproductive organ in land plants
and composed of sepals, petals, stamens, and gynoecium (s). Female haploid reproductive cells are covered with a sporangium (nucellus) and two integuments, and further enclosed in a gynoecium. Male
haploid reproductive cells (pollens) are covered with asporangium (pollen sack). On the other hand, gymnosperms and ferns have simpler reproductive organs
than angiosperms and lack sepals and petals. Female
sporangia (nucellus) of gymnosperms are covered
with only one integument. Sporangia of ferns have no
integuments and are naked on the abaxial side of aleaf.
The development of floral organs is mainly regulated by the members of the MADS gene family
whose members are transcription factors containing

60

the conserved MADS and K domains. MADS genesof angiosperms are divided into more than 10 groups
based on the gene tree.
What kind of changes of the MADS genes caused
the evolution of the complex reproductive organs in
the flowering plant lineage? To address the function of
the MADS genes in a plant with primitive reproductive structures lacking specialized floral organs,
MADS genes were newly sought from the fern
Ceratopteris richardii. A MADS gene tree, incorporating the seed plant and fern MADS genes, revealed
that the known fern MADS genes form only three
gene groups. The number is considerably less than thenumber of MADS gene groups in seed plants. The
expression patterns of five genes, representing all
three fern MADS gene groups, during sporophyte andgametophyte development were examined. In flowering plants, some MADS genes are expressed in specific floral organ primordia as homeotic selector
genes, while other MADS genes are expressed in both
reproductive and vegetative organs. Like the latter
type of flowering plant MADS genes, most of the fern
MADS genes are expressed in both reproductive andvegetative organs. The ubiquitously-expressed MADS
genes may be more primitive than the reproductive
organ-specific MADS genes. If so, it is likely thatsome of the MADS genes were co-opted as homeotic
selector genes of specialized reproductive organs andtheir expression restricted to specific floral organ primordia, events that occurred after the divergence offerns and angiosperms. To verify the hypothesis,
analyses of MADS genes in gymnosperms (Gnetum
and Ginkgo) with more complexed reproductive organs than ferns, but simpler ones than angiosperms,
are in progress. The restriction of MADS gene expression may have been caused by the evolution of othergenes that regulate the MADS genes. LEAFY gene is
one of the regulators of Arabidopsis floral homeotic

NATIONAL INSTITUTE

MADS genes. The LEAFY gene homolog of Gnetum
has been cloned and the characterization is inprogress. The roles of MADS genes in non-flowering
plants are mysterious and characterization of MADS
gene functions in the moss Physcomitrella patens will
give insights for the point.
(b) Establishment of tagged mutant library of themoss Physcomitrella patens. Mosses have the different body plan from flowering plants. Leafy shoots ofmosses are similar to the ones of angiosperms, butdevelop in the gametophytic generation instead of the
sporophytic generation as angiosperms. Organs ofmosses are much simpler than flowering plants. For
example, the leaves are composed of one layer ofcells . Therefore, the body plan of mosses may beregulated by different genes from angiosperms. In
addition to analyses of the homologs of angiosperm
genes governing morphogenesis , it is necessary to
screen specific genes in the moss . We establishedenhancer and gene trap lines and tagged mutant libraries of Physcomitrella patens to clone genes related toleafy shoot development. P. patens is known by its
high rate of homologous recombination and suitable
for molecular biological analyses using the gene targeting. Our libraries should be also useful for otherpurposes.
Publication List:
Hasebe, M., Ando, T. and Iwatsuki, K. (1998)
Intrageneric relationships of maple trees based on
the chloroplast DNA restriction fragment length
polymorphisms. J. Plant Res. 111: 441-451.
Hasebe, M., Wen, C.-K., Kato, M. and Banks, J.A.
(1998) Characterization of MADS homeotic genes
in the fern Ceratopteris richardii. Proc. Natl. Acad.
Sci. USA 95: 6222-6227.
Wolf, P.G., Pryer, K.M., Smith, A.R. and Hasebe, M.

Fig. 1. Phylogeny of Acer. (A) A. albopurpurascens, (B) A. laevigatum, (C) A. pseudosieboldianum. (A) and (B) have beenclassified in the same group because of their peculiar simple leaves in maple trees. Phylogenetic tree based on RFLPs of chloroplast
genome revealed that species with simple leaves (A and B) are polyphyletic and (B) is more closely related to the species withpalmate leaves (C) .

FOR BASIC BIOLOGY

61

Fig. 2. Patterns of CMADS 1 RNA expression in sporophyte tissues as detected by in situ hybridization. MADS genes of
Ceratopteris richardii are expressed in both reproductive (C) and vegetative (D, E, and F) tissues. (A) C. richardii sporophyte. A
whole plant with reproductive (r) and vegetative leaves. (B) Naked sporangia (sp) on the abaxial surface of a reproductive leaf.
(C) Longitudinal section of a sporophyll showing sporangia (sp). (D) Longitudinal section of the growing tip of a root (ro). (E)Transverse section of a petiole showing vascular bundles (v). (F) A longitudinal (I) and a transverse (t) section of two young
vegetative leaves.

(1998) Phylogenetic studies of extant Pteridophytes.
In D . Soltis et al. eds, Molecular Systematics ofPlants (2nd), Chapmann and Hall, New York. Pp.

541-556

62

NATIONAL INSTITUTE

63

FOR BASIC BIOLOGYRESEARCH SUPPORT64

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT

Head: Hiroyuki Hattori
Common Facility Group
Chief: Kazuhiko Furukawa
Reseach Support Facilities
Mamoru Kubota (Subunit Chief)
Shoichi Higashi (Subunit Chief)
Tomoki Miwa (Subunit Chief)
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
Makiko ltoh (Technical Assistant)Keiko Suzuki (Technical Assistant)
Misayo Masuda (Technical Assistant)Radioisotope Facility
Yousuke Kato (Subunit Chief)
Yoshimi Matsuda (Subunit Chief)
Naoki Morooka
Takayo Itoh (Technical Assistant)Center for Analytical Instruments
Sonoko Ohsawa
Yumiko Makino
Tomoko Mori
Takeshi Mizutani
Hatsumi Moribe (Technical Assistant)

Research Support Group
Cell Biology Group
Maki Kondo (Subunit Chief)
Yukiko Kabeya
Katsushi Yamaguchi
Developmental Biology Group
Hiroko Kobayashi (Unit Chief)
Chiyo Takagi
Sanae Oka
Naomi Sumikawa
Regulation Biology Group
Hideko Nonaka
Akiko Oda
Shigemi Takami
Gene Expression and Regulation Group
Sachiko Tanaka
Koji Hayashi
Yasushi Takeuchi
Hideko Utsumi

Glassware Washing Facility
Masayo lwaki(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 theircapability in technical area. Each technical staff isproceeded to the fixed division usually and they support the various research with their special biologicaland biophysical techniques.
The Department hosts an annual meeting for technical engineers who work in various fields of biology
at universities and research insititutes throughout
Japan. At this meeting, the participants present theirown activities and discuss technical problems. The
Proceedings are published soon after the meeting.

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Yoshinori Ohsumi (April], 1998-)
Head of Facility:
Associate Professor: Masakatsu Watanabe
Research Associates: Yoshio Hamada, (Tissue and Cell
Culture)Atsushi Ogiwara (Computer)
Post Doctral Fellow: Mineo lseki 1 (Large Spectrograph)(-October 31, 1998)
Mamoru Kubota (Large
Technical Staffs:
Spectrograph)
Chieko Nanba (Plant Culture,
Farm, Plant Cell)
Toshiki Ohkawa (Computer)
Kaoru Sawada (Tissue and Cell
Culture)Tomoki Miwa (Computer)
Sho-ichi Higashi (May 1, 1998-)(Large Spectrograph)
Makiko Ito (Large Spectrograph)
Misayo Masuda (Computer)
Keiko Suzuki (Plant Culture, Farm,
Plant Cell)65

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.), andsome 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 ).

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 com. posed of 36 smaller individual gratings. It projects aspectrum of a wavelength range from 250nm (ultraviolet) to l,OOOnm (infrared) onto its focal curve of
10m 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), NO modulators (up to 40MHz) tochop the laser beam, a beam expander, and a tracking
rnicrobeam 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.

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 Pl 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 inter-

66
national collaborations devoted to the topic of stressresistant transgenic plants.

II. Research activities
1. Faculty
The faculty of the Research Support Facility conducts its own research as well as scientific and administrative public services.
(1) Photobiology: photoreceptive and signal transduction mechanisms of phototaxis of 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 (Matsunaga
et al., 1998). Photo-receptive and signal transduction
mechanisms of algal gametogenesis are also studied
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: Today, many kinds ofgenome structures of various organisms have been
revealed. Using these genomic sequence data, we performed a comprehensive sequence analysis especiallyfor Bacillus subtilis. Microbial genomics has entereda new era of functional analysis, and bioinformatics is
also shifting to support the functional analysis. We
have developed a knowledge base system for thetransporter system of B. subtilis. The system not only
represents functional information but also helps todiscriminate transporter genes in newly determined
genomic sequences.
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.

NATIONAL 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
Fujibuchi, W., Goto, S., Migimatsu, H., Uchiyama, 1.,
Ogiwara, A., Akiyama, Y., and Kanehisa, M. (1998).
DBGET/LinkDB: an Integrated Database Retrieval
System. Pacific Symposium on Biocomputing '98,
683-694.
Atsushi Ogiwara (1998). Systematic Classification of
B. subtilis Genes and Construction of a Knowledge
Base to Represent Functional Information. Proc.
Genome Informatics 1998, 310-311.
Furukawa, T., Watanabe, M. and Shihira-Ishikawa, I.
(1998). Green- and Blue-Light-mediated chloroplastmigration in the centric diatom, Pleurosira laevis.
Protoplasma. 203, 214-220.
Matsunaga, S., Hori, T., Takahashi, T., Kubota, M.,
Watanabe, M., Okamoto, K., Masuda, K. and Sugai,
M. (1998). Discovery of signaling effect of UV-B/C
light in the extended blue-UV-A-type action spectra
for step-down and step-up photophobic responses in
the unicellular flagellate alga Euglena gracilis.
Protoplasma 201, 45-52.
Mimuro, M., Tarnai, N., Murakami, A., Watanabe, M.,
Erata, M., Watanabe, M.M., Tokutomi, M. andYamazaki, I. (1998) Multiple pathways of the excitation energy flow in the phtosynthetic pigment system of a cryptophyta, Cryptomonas sp. (CR-1).
Phycological Res., 46, 155-164.
II. Cooperative Research Program for the Okazaki
Large Spectrograph
Furukawa, T., Watanabe, M. and Shihira-Ishikawa, I.
(1998). Green- and Blue-Light-mediated chloroplastmigration in the centric diatom, Pleurosira laevis.
Protoplasma. 203, 214-220.
Furusawa, Y., Quintern, L.E., Holtschmidt, H.,
Koepke, P. and Saito, M. (1998). Determination oferythema-effective solar radiation in Japan and Germany with a spore monolayer film optimized for thedetection of UVB and UVA -results of a field campaign. Appl. Microbial Biotechnol. 50, 597-603.
Matsunaga, S., Hori, T., Takahashi, T., Kubota, M.,
Watanabe, M., Okamoto, K., Masuda, K. and Sugai,
M. (1998). Discovery of signaling effect of UV-B/C
light in the extended blue-UV-A-type action spectra
for step-down and step-up photophobic responses in
the unicellular flagellate alga Euglena gracilis.
Protoplasma 201, 45-52.
Munakata, N., Ono, M. and Watanabe, S. (1998).
Monitoring of solar-UV exposure among schoolchildren in five japanese cities using spore dosimeter and UV-coloring Labels. Jpn. J. Cancer Res.
89, 235-245.

FOR BASIC BIOLOGY

Shinomura, T., Hanzawa, H., Schafer, E. and Furuya,
M. (1998). Mode of phytochrome B action in thephotoregulation of seed germination in Arabidopsis
thaliana. Plant J. 13, 583-590.
Takeuchi, Y., Murakami, M., Nakajima, N., Kondo, N.
and Nikaido, 0. (1998). The photorepair andphotoisomerization of DNA lesions in etiolated
cucumber cotyledons after irradiation by UV-B depends on wavelength. Plant Cell Physiol. 39, 745750.
Torikai, A. and Hasegawa, H. (1998). Wavelength effect on the accelerated photodegradation ofpolymethylmethacrylate. Polym. Degradn. Stab. 61,
361-364.
Torikai, A., Kobatake, T. and Okisaki, F. (1998).
Photodegradation of polystyrene containing flame
retardants. J. Appl. Polym. Sci. 67, 1293-1300.
Torikai, A. ( 1998) Wavelength sneistivity in thephotodegradation of polymethylmehtacrylate: Accelerated degradation and gel formation. In "Science and Technology of Polymers and Advanced
Materials". (Prasad, P. N. et al. ed.), Plenum Press,
New York, pp. 581-586.

67

68

NATIONAL INSTITUTE

RADIOISOTOPE FACILITY(Managed by NIBB)Head (Professor, concurrent post): Shigeru !ida
Associate Professor: Kazuo Ogawa
Technical Staff:
Kazuhiko Furukawa (Radiation
Protection Supervisor),
Yosuke Kato (Radiation Protection
Supervisor),
Yoshimi Matuda (Radiation
Protection Supervisor),
Naoki Morooka
Supporting Staff:
Takayo Ito, Risako Shirai

I. Research Supporting Activity
Technical and Supporting Staffs of this facility areserving the purchase of radioisotopes from JRA (Japan Radioisotope Association) and the transfer of radioisotope wastes to JRA. The physical maintenanceof the controlled areas where radioisotopes are usedby the registered users of NIBB (National Institute for
Basic Biology) and NIPS (National Institute forPhysiological Science) for research is also one of ourbusiness.
This facility consists of four controlled areas: Center, NIBB-sub, LGER (Laboratory of Gene Expression and Tegulation)-sub, and NIPS-sub. Users goingin and out the controlled areas counted by the monitoring system are 8,482 in 1998. This count is comparable to that (8,922) in 1997 . The items in eachcontrolled area is presented in Figure 1.

II. Academic activity
Academic activity by Associate Professor is focusedon the analysis of the structure and function of adynein motor protein. Dyneins are a group of microtubule-activated ATPases that serve to convert chemical energy into mechanical energy and divided into
axonemal and cytoplasmic dyneins. Figure 2 shows

the localization of two isoforms of dynein in the outerarms of sperm axonemes (Ogawa et al., 1977) and themitotic apparatus of cleaving egg (Mohri et al., 1976)
visualized by anti-axonemal dynein (Fragement A)
antibodies.
The native dyneins are very large and range inmolecular mass up to 1 to 2 mega Da. They are complex proteins containing heavy, intermediate, and lightchains defined by the molecular mass. Our prensent
project is the molecular cloning of polypeptides contained in outer arm dynein of sea urchin sperm
flagella to understand the mechanism how dynein interacts with microtubules, resulting in producing theforce.
Outer arm dynein consists of two heavy chains with
ATPase activity. The motor activity is closely related
to this polypeptide. The first successful molecularcloning of this huge polypeptide (520 kDa) was performed in our laboratory in 1991 . Since then eDNA
clones for axonemal and cytoplasmic dyneins havebeen isolated in a variety of organisms. The sequencesof heavy chains, without exception, contain four Ploop motives referred to as ATP-binding sites in themidregion of the molecules. Figure 3A and B draw
the structure of heavy chain deduced from the aminoacid sequence (Ogawa, 1992). Taking the recent
works by Koonce et al. (1998) and Vallee et al. ( 1998)
into consideration, this model might be seen as depicted in Figure 3C. In particular, Vallee et al. (1998)
have described the importance of a hairpin structure
formed between M and C domains which binds tomicrotubules and presented a novel mechanism fordynein force production different from that of myosin
and kinesin.
Outer arm dynein contains three intermediate chains(IC I, IC2, and IC3) that range in molecular mass from70 to 120 kDa. IC2 and IC3 were cloned by Ogawa etal. (1995) and contain the WD repeats in the carboxyterminal halves of the molecules. By contrast, IC1 is

NIPS-subCenterNIBB-sub
LGER-sub
NIPS-subTotal

Actual counts
2,628
843
3,739
1,273
8,483

Figure 1. Percentage of users going in and out the cntrolled areas during April to December, 1998.

69

FOR BASIC BIOLOGY

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

A

N

M

c

B

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

not a member of the WD family. IC 1 has a uniquesequence such that theN-terminal part is homologous
to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to thesequence of NDP kinase, and the C-terminal part contains a high proportion of negatively charged glutamic
acid residues (Ogawa et al. , 1996). Thus, IC1 is a
novel dynein intermediate chain distinct from IC2 and· IC3 and may be a multifunctional protein.
Six light chains with molecular masses of 23 .2,
20.8, 12.3, 11.5, 10.4, and 9.3 kDa are associatingwith outer arm dynein . We have already isolated
eDNA clone of five LCs. LC1 (23 .2 kDa) and LC3(12.3 kDa) are highly homologous to mouse Tctex2
and Tctexl, respectively. These mouse proteins are
encoded by the t complex region that is involved intransmission ratio distortion (TRD), male sterility andthe development of germ cells. Our finding raises thepossibility that axonemal dynein proteins are involvedin this phenomenon. TRD may be caused by the dysfunction of multiple axonemal dynein proteins.

Publication List:
0. Kagami, M. Goto , Y. Makino, H. Mohri , R .
Kamiya, and K. Ogawa. A dynein light chain of sea
urchin sperm flagella is a homolog of mouse
Tctex 1, which is encoded by a gene of the t complex sterility locus. Gene 211 (1998) 383-386.

70

NATIONAL INSTITUTE

THE CENTER FOR ANALYTICAL INSTRUMENTS(Managed by NIBB)Head of Facility:
Technical Staffs:

Tetsuo Yamamori
Akio Murakami (-Ap1: 30, 1998)
Sonoko Ohsawa
Tomoko Mori (Jun.1, 1998 -)Yumiko Makino
Takeshi Mizutani
Technical Assistant: Hatsumi Moribe

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. On June 1 in 1998, theCenter merged with the facility of Molecular Biological Analysis in the Laboratory of Gene Expression
and Regulation. Consequently, instruments transferredto the Center are Particle Delivery System, DNA Sequencers, DNA Synthesizer, Plasmid Isolation Systems, etc. Newly installed instruments are Fluorescence Bio Imaging Analyzer and DNA Sequencer(ABI 310). Some of instruments of the Center are
opened to researchers outside the Institute.

Representative instruments are listed below.
Protein Sequencers (ABI 494, ABI 473A)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A, ABI 432A)
Plasmid Isolation Systems (Kurabo PI-100 I)
DNA Sequencers (ABI 377, 373S, ABI 310)
DNA/RNA Synthesizers (ABI 394, ABI 392)
Thermal Cyclers (Perkin Elmer PJ-9600, Takara TP300)
Integrated Thermal Cyclers (ABI CATALYST Turbo
800)
Particle Delivery System (Bio-Rad BiolisticPDS1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs (Shimadzu
LC-10AD, 6AD) ·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, Shimadzu RF5000)
Spectrophotometers (Hitachi 330, Hitachi 557, Varian
Cary 5G, Perkin Elmer Lambda-Bio)
Microplate Lurninometer (Berthold MicroLumat LB
96P)

Figure 1. Amino acid sequence analysis of protein byprotein sequencer.

Time-resolved Fluorescence Microplate Reader(Pharmacia DELFIA Research)
Microplate Readers (Corona MTP-120, MTP-100F)
Spectropolarimeter (JASCO J-40S)
FT-IR Spectrophotometer (Horiba FT-730)

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t 0 0

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I "

•

"

;. •

"

~ I , t• :. "• 0I

0 o • •,~ 0

I o o t •o ' .: • pLaser Raman Spectrophotometer (JASCO R-800)
Biomolecular Interaction Analysis Systems(Pharmacia BIAcore 2000, Affinity Sensors IAsys)
Bio Imaging Analyzers (Fujifilm BAS2000)
Fluorescence Bio Imaging Analyzer (Takara FMBIOU)Electrophoresis Imaging Systems (PDI Discovery Series, BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Microscope Photometer (Carl Zeiss MPM 03-FL)
Figure 2. DNA sequencing electropherogram.

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