NATIONAL INSTITUTE FOR BASIC BIOLOGY

E~ Small GTP-binmng Proteins.
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• 25 kDa. 0----....... (:;) 27 kDa
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ANNUAL REPORT1994

pG---. G

1

INTRODUCTION
The National Institute for Basic Biology (NIBB) is a government supported
basic research institute in the field of biology. It aims to stimulate and promotestudies of biology, by conducting firstrate research on its own, and by cooperating with other universities and researchorganizations. The Institute concentrates
on 1 the studies of cellular functions,
growth and development, homeostatic
control, and gene expression and regulation in eukaryotic organisms, to elucidate
the fundamental mechanisms underlying
various living phenomena at the molecular level.
That the Institute has been active inscience was evidenced by the fact that citation indices of papers published in
1981-1991 by the NIBB was highest
among Japanese universities and institutes and comparable to those of leading universities in the United States(Science, 258, 562). We are justly proudof this and would like to maintain a highlevel of research activity at the Institute.
A new professor joined the NIBB in
the past year. Prof. Tetsuo Yamamori(former Sub-Teamleader of the Riken)
filled the position of Professor of Speciation Mechanisms. His research is aimed
at an understanding of the molecularmechanisms for the evolution of information processing by the nervous system. During the past year, the Institutecontinued to be active in exchange of
personnel: we newly appointed 9 research associates from other universities,
while three research associates were promoted to associate professors at otheruniversities.
The NIBB is an inter-university research institute and plays many roles as anational and international center for the
study of biology. The Institute is responsible for conducting research projects incooperation with research groups in different universities and institutes. As apart of such cooperative activities, the
NIBB carries out Special Programs
which are currently directed to "Adaptation and Resistance to Environment"and "Transdifferentiation of Tissue

Cells". Based on such programs, the
NIBB held the 32nd and 33rd Conferences in 1994, entitled "Stress-induced
genes of plants with emphasis on theirfunctions" (organized by Prof. Murata)
and "Approaches to the cellular andmolecular mechanisms of regeneration"(organized by Prof. Eguchi), respectively.
In addition, the Institute sponsors
symposia and study meetings on current
topics at the interdisciplinary level by inviting leading scientists in various related
fields, both nationally and internationally. The NIBB also shares its researchresources to make them available to biologists both from Japan and around theworld. Through such activities, the NIBB
is and will continue to be a national andinternational center to promote basic research in the biological sciences.
This report describes an outline of recent research activities of the NIBB andcontains a list of papers published by themembers of the Institute in 1994.
lkuo Takeuchi, Ph.D. & D.Sc.
Director General

2

NATIONAL INSTITUTE

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

Institute. The Steering Council is madeup of professors of the Institute and an
equal number of leading biologists inJapan outside NIBB, and advises the Director-General on the scientific activitiesof the Institute. The Council advises on
faculty appointments and on the Institute's annual budget as well as its future prospect.
Administration
Administration of the Institute isundertaken by the Administration
Bureau of the Okazaki National Research Institutes under the direct auspices of the Ministry of Education,
Science and Culture.

Policy and Decision Making
The Director-General oversees theoperation of the Institute assisted by two
advisory bodies, the Advisory Council
and Steering Council. The Advisory
Council is made up of distinguishedscholars representing various fields ofscience and culture, and advises the Director-General on the basic policy of the

Department of Cell Biology

Department of Developmental Biology

Department of Regulation Biology

Laboratory of Gene Expression
and Regulation

Research
The Institute conducts its research
programs through three departments andone laboratory organized into 16 divisions.

i
{

{

-f

Divisions
Cell Mechanisms
Bioenergetics
Cell Proliferation (adjunct)
Cell Fusion (adjunct)
Cellular Communication (adjunct)
Reproductive Biology
Cell Differentiation·
Morphogenesis
Developmental Biology (adjunct)
Molecular Neurobiology
Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)
Gene Expression and Regulation I
Gene Expression and Regulation II
Speciation Mechanisms

3

FOR BASIC BIOLOGY

Each division has its own research project and is staffed by a professor, anassociate professor and two research associates. A division forms, in principle,
an independent project team. Six of thedivisions are for adjunct professorshipand 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 researchtechniques and helps to promote research activities of each division and also
to maintain the research resources of theInstitute. The Department also undertakes the technical education of its staffs.
Research Support Facility
The research support facility of the
NIBB consists of the Large Spectrograph
Laboratory, Tissue and Cell Culture

Laboratory, Computer Laboratory, Plant
Culture Laboratory, Plant Cell Culture
Laboratory, Experimental Farm, andLaboratory of Stress-Resistant Plants. In
addition, seven facilities are operated
jointly with the NIPS; they consist of theRadioisotope 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 cover an area of 150,000 m 2 withfour principal buildings. The NIBB's
main research building has a floor spaceof 10,930 m2• Two-thirds of the space
was completed in 1982 and the remaining third in June, 1983. The buildings
which house the research support facility
were also completed in June 1983.

GRADUATE PROGRAMSThe NIBB carries out two graduate
programs.
1. Graduate University for Advanced
Studies
The NIBB constitutes the Departmentof Molecular Biomechanics in the Schoolof Life Science of the Graduate University for Advanced Studies. The University provides Ph.D. course of three years
duration. Those who have completed
master's course in a university or are
qualified equivalent to or higher 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

4

NATIONAL INSTITUTE

2. Graduate Student Training Program
Graduate students enrolled in otheruniversities and institutions are trained

to conduct research for fixed periods oftime under the supervision of NIBB professors.

OFFICE OF DIRECTOR
Publication List:
Saran, S., Nakao, H., Tasaka, M., lida,
H., Tsuji, F. I., Nanjundiah, V. andTakeuchi, I. (1994) Intracellular freecalcium level and its response tocAMP stimulation in developing Dictyostelium cells transformed withjellyfish apoaequorin eDNA. FEBS
Letters 337, 43-47.
Araki, T., Nakao, H., Takeuchi, I. and
Maeda, Y. (1994) Cell-cycle-dependent sorting in the development ofDictyostelium cells. Develop. Bioi.
162, 221-228.
Traynor, D., Tasaka, M., Takeuchi, I.
and Williams, J. (1994) Aberrant pattern formation in myosin heavy chain
mutants of Dictyostelium. Development120, 591-601.

Morio, T., Takeuchi, I. and Tasaka, M.
(1994) Cooperation of positively andnegatively acting promoter elements
determines prespore-specific transcription of Dp87 gene in Dictyostelium.
Mechanisms of Development 45,
59-72.
Nakao, H., Yamamoto, A., Takeuchi, I.
and Tasaka, M. (1994) Dictyostelium
prespore-specific gene Dp87 encodesa sorus matrix protein. J. Cell Sci.
107, 397-403.
Takeuchi, I., Tasaka, M., Okamoto, K.
and Maeda, Y. (1994) Regulation ofcell differentiation and pattern formation in Dictyostelium development.
Int. J. Dev. Bioi. 38, 311-319.

5

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Yoshihiko Fujita

The Department is composed of two regular divisions and three adjunct divisions and conducts researches on the fundamentals of cell structures andfunctions in plants and animals at molecular level utilizing modem technologies including genetic engineermg.

6

NATIONAL INSTITUTEDMSION OF CELL MECHANISMS
Professor: Mikio Nishimura
Research Associates: Kazuo Ogawa
Makoto Hayashi
lkuko Hara-Nishimura
Graduate Students: Kaori Inoue
Akira Kato
Tomoo Shimada
Tetsu Kinoshita
Nagako Hiraiwa
Masahiro AokP!
Technical Staff: Maki Kondo
Katsushi Yamaguchi
Yasuko Koumoto
Visiting Scientists: Kyoko Hatanol!
Gerhard Bytofi( 1!from Shinshu University)

f 1from Kyoto University)[l!from Technischen Universitat Braunschweig,
Germany)

The cells of higher plants cells containseveral distinct organelles that play vitalroles in cellular physiology. During theproliferation and differentiation of thesecells, the organelles often undergo dynamic changes. The biogenesis of neworganelles may occur, existing organelles
may undergo a change in function,
which other organelles may degenerate.
The dynamic transformation of organellar functions (differentiation of organelles) is responsible for the flexibility ofdifferentiation events in higher plant
cells. Therefore, the efforts of this division are focussed on the elucidation ofregulatory mechanisms that underlie
such transformation.
I. Development of microbody membrane proteins during the microbody
transition.
Dramatic metabolic changes that
underlie the shift from heterotrophic toautotrophic growth occur during thegreening associated with the germinationof seeds. As these metabolic changes
take place, many constitutive organelles
are functionally transfomied. For
example, etioplasts differentiate into

chloroplasts and mitochondria acquire
the ability to oxidize glycine. Glyoxysomes, which are microbodies engaged
in the degradation of reserve oil via
j3-oxidation and the glyoxylate cycle, aretransformed into the leaf peroxisomesthat function at several crucial steps inphotorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during the greening of pumpkin
cotyledons, the reverse transition of leaf
peroxisomes to glyoxysomes occurs during senescence. As part of our attempts
to clarify the molecular mechanisms that
underlie the microbody transition, thatchanges with development in microbody
membrane proteins during the transformation of glyoxysomes to leaf peroxisomes were characterized. Two proteinsin glyoxysome membranes, with molecular masses of 31 kDa and 28 kDa respectively, were purified and polyclonal
antibodies were raised against the eachprotein. Analysis of these membraneproteins during development revealed
that the amounts of these proteins decreased during the microbody transition.
The larger one was retained in leaf peroxisomes, whereas the small one could
not be found in leaf peroxisomes after
completion of the microbody transition.
The results clearly showed that membrane proteins in glyoxysomes change
dramatically during the microbody transition, as do the enzymes in the matrix.
II. Characterization of the aconitasethat participates in the glyoxylate cycle.
Aconitase catalyzes the reversible interconversion of citrate, isocitrate andcis-aconitate. This enzyme is thought tofunction as a component of the glyoxylate cycle in glyoxysomes, as well as the
Krebs cycle in mitochondria. It was reported, however, that less than 0.5% ofFOR BASIC BIOLOGY

7

Fig. 1. Localization of aconitase and isocitrate lyase within a cell from a pumpkin cotyledon, as analyzed
by double immunofluorescence staining. Pumpkin seedlings were grown for 5 days in darkness. A thin sectionof a cotyledon was double-stained first with aconitase-specific antibodies which were visualized by use of
FITC-conjugated second antibody, and then with isocitrate lyase-specific antibodies that were visualized byuse of rhodamine-conjugated second antibody. (a) Immunofluorescent image due to FITC. (b) Immunofluorescent images in a and care superimposed. (c) Immunofluorescent image due to rhodamine of the same field
as in (a). Bar in panel c indicates lO!J.m. Magnification in a, band cis the same.

the total aconitase activity could berecovered in glyoxysomal fractions after
sucrose gradient centrifugation, withmost of the activity being found in

mitochondria and the cytosol. It remained to be confirmed whether mitochondria and glyoxysomes containdifferent and specific isoenzymes of8aconitase.
Three isoforms of aconitase (Acol,
Acoll, Acoiii) are found in etiolated
cotyledons of pumpkin, and we purified
two of them (Acol, Acoll). Antibodies
raised against Acol allowed us to isolatea eDNA for aconitase. The amino acidsequence deduced from the eDNA wasvery similar to those of mammalian ironresponsive element-binding proteins thatare also known as cytosolic aconitases.
Antibodies were raised against a fusion
protein that consisted of glutathione-Stransferase and the partial sequence (162amino acids) of the cloned aconitase. Not
only the antibodies against purified Acolbut also the antibodies against the fusion
protein cross-reacted with all three isoforms of aconitase. Further analysis bysubcellular fractionation, immunoblotting and immunofluorescene microscopy,
revealed that Acol and Acoiii werelocalized in the cytosol, while Acoll was
localized exclusively in mitochondria(Fig. 1). No aconitase was found inglyoxysomes. Acol was specificallyfound in etiolated pumpkin cotyledons
during the early stage of seedling growth,
while Acoll and Acoiii were present inall tissues of pumpkin plants. The pattern of expression of Acol was similar to
those of other enzymes of the glyoxylate
cycle. These data suggest that cytosolic
Acol functions as a component of theglyoxylate cycle despite the fact that allother enzymes of the glyoxylate cycle
appear to be localized in glyoxysomes.
ID. Mechanisms for vesicular transportof seed proteins.
i) Dense vesicles responsible for thetransport of precursors to seed proteins.
Most vacuolar proteins are synthesizedon the rough endoplasmic reticulum and

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are then delivered to vacuoles via
vesicle-mediated transport systems. Developing seeds, in which large amountsof seed proteins are synthesized andtransported to protein-storage vacuoles,
are particularly useful materials withwhich to explore the cellular machinery
involved in the sorting of vacuolar proteins in plant cells. Proprotein precursors
to seed proteins are delivered to thevacuoles via unique vesicles, known as
dense vesicles. Electron microscopic
studies of developing seeds of pumpkin( Cucurbita sp.) and castor bean (Ricinus
communis) have shown that the dense
vesicles have a highly electron-dense
core; they are about 300 nm in diameterand are different from Golgi vesicles.
Such dense vesicles were isolated fromdeveloping pumpkin cotyledons andwere shown to contain large amounts ofthe precursors to various seed proteins.
This result was supported by results ofimmunocytochemical analysis with specific antisera against two seed proteins,
namely, 2S albumin and 11S globulin.
The dense vesicles are directly targeted
to vacuoles and fuse to the vacuolar
membranes. Biochemical analysis of theisolated dense vesicles should provide
insight into the specific targeting and fusion of vesicles to vacuolar membranes.
ii) Isolation and characterization of thedense vesicles; association of small
GTP-binding proteins on the membrane of dense vesicles.
To date, a vesicle-mediated transportsystem through secretory pathway has
been investigated exclusively in yeast.
Distinct members of the ras superfamilyof small GTP-binding proteins facilitate
the targeting of vesicles with the appropriate membranes in a GTP-dependent
manner to regulate specific steps in the

9

FOR BASIC BIOLOGY~0
Dense Vesicle~
pG ----+- G

Fig. 2. Dense vesicles mediate the final step in the delivery of seed proteins to vacuoles in developing
pumpkin ( Cucurbita sp.) cotyledons. Two different, small GTP-binding proteins of 25 kDa and 27 kDa might
function in the targeting and/ or fusion of the vesicles to the vacuolar membranes or in the budding of thevesicles.

secretion pathway in yeast. However, themechanism of the vesicular transportthat is targeted to vacuoles is still obscure.
Isolation of these vesicles allows us toanalyze the components responsible forthe specific targeting of vesicles tovacuoles and for the fusion between
vesicles and vacuoles. To explore thevesicle-mediated transport system that istargeted to vacuoles in plant cells, we
isolated dense vesicles and examinedthem for the presence of guanine nucleotide-binding
proteins.
GTP-binding
proteins of 25 kDa and 27 kDa weredetected on the dense vesicles isolated
from developing pumpkin cotyledons.
The two different, small GTP-binding
proteins might function in the targeting
and/ or fusion of the vesicles to thevacuolar membranes or in the buddingof the vesicles (Fig. 2).

IV. A vacuolar processing enzyme responsible for conversion of proprotein
precursors into their mature forms.
Proprotein precursors of vacuolar
components are transported from theendoplasmic reticulum to the dense
vesicles and then they are targeted to thevacuoles. In the vacuoles, the proproteins are processed proteolytically totheir mature forms by a unique vacuolar
processing enzyme. A vacuolar processing enzyme responsible for the maturation of seed proteins was isolated fromcastor bean and soybean. The processing
enzyme is a novel cysteine proteinasewith a molecular mass of 37 kDa (castorbean) or 39 kDa (soybean). The enzyme
splits a peptide bond on the C-terminalside of an exposed asparagine residue ofa proprotein precursor to produce a mature seed protein such as llS globulin
and 2S albumin. The immunocytochemical localization of the enzyme in the vacuolar matrix of the maturing endosperm10of castor bean indicated that the maturation of the seed proteins occurs in thevacuoles. Molecular characterization
revealed that the enzyme is synthesized
as an inactive precursor with a larger
molecular mass. The results of immunoelectron microscopy suggested thatthe precursor is transported to vacuoles
via dense vesicles together with the proprotein forms of other seed proteins.
Upon arrival in the vacuole, the inactive
precursor is converted to the active
enzyme. This result suggests that processing of proprotein for the maturationof seed proteins involves a cascade ofreactions. The activity of the vacuolar
processing enzyme was found in various
plant tissues and several cDNAs corresponding to homologues of the enzyme
were isolated from different plants.
Thus, a similar processing enzyme appears to be widely distributed in planttissues and to play a crucial role in thematuration of a variety of proteins inplant vacuoles.
V. Role of molecular chaperones in thetranslocation of proteins into chloroplasts.
Molecular chaperones are cellular
proteins that function in the folding andassembly into oligomeric structures ofcertain other polypeptides but that are
not, themselves, components of the final
oligomeric structure. To clarify the rolesof chaperonin (Cpn) and Hsp 70 in thetranslocation of chloroplast proteins, we
examined the interaction of Cpn andHsp 70 with chloroplast proteins during
their import into chloroplasts. Ferredoxin NADP+ reductase (FNR), imported into pea chloroplasts in vitro, wasimmunoprecipitated not only with
antisera raised against Hsp 70, but alsowith against Cpn 60 in an ATP-depend-

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ent manner, indicating that newly imported FNR interacts physically with
Hsp 70 and Cpn 60 in chloroplasts.
Time-course and temperature-shift analysis of these interactions revealed thatimported FNR binds transiently to Hsp
70 and that the association of FNR with
Hsp 70 precedes that with Cpn 60.
These results suggest that Hsp 70 and
Cpn 60 in chloroplasts might sequentially assist in the maturation of newly
imported FNR in an ATP-dependent
matter.
Immunoblot analysis revealed that, in
germinating pumpkin seedlings, not only
chloroplasts but also etioplasts accumulate heat shock-induced Hsp 70 proteins.
Moreover, cDNAs for homologues of
Cpn60 and Cpn10 have been isolated
and characterized. Further investigations
on the function of Cpn10 in the transport of proteins into chloroplasts are in
progress.
Publication List:
De Bellis, L., M. Hayashi, I. HaraNishimura, A. Alpi and M. Nishimura(1994) Immunological analysis ofaconitase in pumpkin cotyledons: The
absence of aconitase in glyoxysomes.
Physiol. Plant. 90,757-762.
Kinoshita, T., I. Hara-Nishimura, H.
Shiraishi, K. Okada, Y. Shimura andM. Nishimura (1994) Nucleotide
sequence of a transmembrane protein(TMP-C) eDNA in Arabidopsis thaliana. Plant Physiol. 105, 1441-1442.
Maeshima, M., I. Hara-Nishimura, Y.
Takeuchi and M. Nishimura (1994)
Accumulation of vacuolar H+-pyrophosphatase and H+-ATPase during
reformation of the central vacuole ingerminating pumpkin seeds. Plant
Physiol.106, 61-69.

FOR BASIC BIOLOGY

Ozaki, K., A. Terakita, M. Ozaki, R.
Hara, T. Hara, I. Hara-Nishimura, H.
Mori and M. Nishimura (1994) Molecular characterization and functional
expression of squid retinal-binding
protein: A novel species of hydrophobic ligand-binding protein. J. Bioi.
Chem. 269, 3838-3845.
Shigemori, Y., J. Inagaki, H. Mori, M.
Nishimura, S. Takahashi and Y.
Yamamoto (1994) The presence ofthe precursor to the nucleus-encoded
30-kDa protein of photosystem II in
Euglena gracilis Z includes two hydrophobic domains. Plant Mol. Bioi. 24,
209-215.
Shimada, T., N. Hiraiwa, M. Nishimura
and I. Hara-Nishimura (1994) Vacuolar processing enzyme of soybean thatconverts proproteins to the corresponding mature forms. Plant Cell
Physiol. 35, 713-718.

11

Shimada, T., M. Nishimura and I. HaraNishimura (1994) Small GTP-binding
proteins are associated with thevesicles targeting to vacuoles indeveloping pumpkin cotyledons. Plant
Cell Physiol. 35, 995-1001.
Strzalka, K., R. Tsugeki and M.
Nishimura (1994) Heat shock inducessynthesis of plastid-associated hsp 70
in etiolated and greening pumpkin
cotyledons. Folia Histochem. Cytobiol. 32, 45-49.

12

NATIONAL INSTITUTEDMSION OF BIOENERGETICS
Professor: Yoshihiko Fujita
Associate Professor: Sigeru Itoh
Research Associates: Mamoru Mimuro
Katsunori Aizawa
JSPS-Post-doctral Fellow: Tohru Ikeya
NIBB Visiting Scientist: Sung-Jun Kim 1!
Technical Staff: Akio Murakami(l)from Kyngpook National University)

Primary processes of photosynthesis
have been studied. A special attention
has been paid for the regulation of PSI/
PSII stoichiometry in thylakoid electrontransport system (ETS) in response tophotosynthetic environments. Biophysical studies of the excitation energy transfer in light-harvesting system and theelectron transfer in photosynthetic reaction center complexes have also been
carried out.
I. Regulation of PSIIPSII stoichiometryin oxygenic photo-synthesis.
In our previous study, we proposed ahypothesis that (1) stoichiometry between the two photosystems is regulated
in response to light regime for photosynthesis so as to maintain the efficiency oflight-energy conversion in photosynthesis, and that (2) the stoichiometry is
adjusted by regulation of PSI abundance,
mainly regulation of PSI formation;
redox state of electron component(s),probably cytochrome b6 -! complex, ismonitored, and PSI formation is regulated by the signal from monitoring system (cf. Fig. 1). As one of trials forevaluating this hypothesis, the identification of the step to be regulated in PSI
formation was carried out. Assay ofmRNA activity for synthesis of PsaA/B
polypeptides by the in vitro translation
showed that the mRNA abundance remains constant irrespective of the PSI/
PSII regulation while rates of PsaA/B
polypeptides sysnthesis varies by a factorof 2. Sensitivity of PsaA/B synthesis tothe inhibitors for peptide elongation was
different in differently regulated synthesis; the synthesis was less sensitive
when the synthetic rate remained at low
level under the light mainly exciting PSI,
but the sensitivity became high when thesynthesis was accelerated under the lightmainly for PSII excitation. Results indicated that the apoprotein synthesis isregulated at the step of translation, andfurther suggest that the translation issuppressed under PSI light and is released from the suppression under PSII
light. Since apoprotein synthesis depends
on Chl a supply, we also analyzed Chl abiosynthesis. The step before Chl a synthetase was not regulated. However, amarked accumulation of Pchlide upon
suppression of PSI synthesis suggested a
possibility for regulation of Chl a supply
to each photosystem assembly. We suspect that the step(s) for formation of ribosome-mRNA complex or installationof Chl a into peptides is the target(s) for
the regulation.
In the cyanophyte Synechocystis PCC
6714, NaCl- and C0 2-stresses for autotrophic growth were found to induce
variation of PSI/PSII stoichiometry
besides light regime. Again, PSI abundance varied in these cases. Further, theactivity of cytochrome c oxidase, anotherterminal of ETS in thyalkoids (Ohki and
Fujita (1995) Protoplasma in press),varied simultaneously in the same direction to PSI variation in all cases. Since
these stresses affect the steady state ofthylakoid ETS in a similar manner to thelight regime, we suspect that the mechanism is common among regulations of
PSI/PSII stoichiometry in response to
three different environmental stresses,
and further that cytochrome c oxidase
activity is also regulated under the same13

FOR BASIC BIOLOGY
Light

D

Photosynthetic_:::~,~i·~:y

D

Light

Photosynthetic

efficiency
H,O _ _ _ _ _ NAOP'

0 .4

H:O -

NAOP•

Fig. 1. Schematic presentation of regulation of PSIIPSII stoichiometry and electron transport state.
Shaded area in each circle indicates level of reduced state of each component. Reduced PSII and oxidized PSI
are photochemically inactive, respectively, and large occurrence of such states causes low efficiency of photosynthesis. PQ, plastoquinone; Cyt b6 -f, cytochrome b6 -f complex; PC, plastocyanin.

mechanism.
II. Energy transfer in light-harvesting
pigment system.
Energy transfer processes in photosynthetic pigment systems have been studied
using steady-state and time-resolved
spectroscopy.
Carotenoid-containing
pigment systems in algae and chlorosomes of photosynthetic green bacteria
were studied. A new method for spectral
analysis was also introduced to biologicalstudy.
Dynamics of excited state of ~­carotene in n-hexane were analyzed in
subpico second time-range. Decay kinetics of the S2 state were single-exponential with a lifetime of 195 fs ± 10 fs, anda dynamic stokes shift was not observedin a 50-fs time range. Spectral analysis
was made for peridinine-Chl a-protein
complex newly isolated from the dinoflagellate Alexandorium cohorticula.
Magnetic circular dichroism indicatedthat magnetic mixing of excited states ofperidinine, S1 and S2, did not occur, andChi a did not interact with other pigments. In the light-harvesting pigmentprotein complex II of the siphonous
green alga Bryposis maxima, siphoneinand siphonaxanthin were found to transfer the energy directly to Chi a. The fea-

ture is the same as the energy transferfrom fucoxanthin to Chi a in the fucoxanthin-containing pigment protein complex of brown algae, suggesting a generalproperty of the energy transfer in carotenoid-Chi pigment-protein complex.
Based on the in vitro self assembly of
Bchl c and the in vivo time-resolved
fluorescence polarization spectrum in theps time-range, a model was proposed formolecular structure of Bchl c in chlorosomes.
A new method for spectral analysis,
principal multi-component spectral estimation method, was found to valid inapplication to biological system. The
method is advantageous in that the analysis does not require the assumption onnumber of components and spectral
shape.

m. Electron transfer in reaction center
complexes.
The study has been focussed on theelectron transfer in photosystem I complex. Phylloquinone in PSI complex
functions in the electron transfer from
Chi a to iron-sulfur centers. After replacing phylloquinone with various artificial quinones, rates of the electrontransfer from Chi a to respective quinones were determined in a pstime-

14
range. The rates were found to be afunction of free energy difference aspredicted by the quantum mechanicalMarcus theory. Protein environments
regulate the quinone energy levels. The
matching between the energy difference
and the reorganization of reaction coordinates is a key for the optimal rate.
Photodamage of PSI in vivo was alsostudied.
Reaction center complexes in the anaerobic green sulfur bacteria were alsostudied. The highly purified complex
from Chlorobium limico/a consisted ofonly one kind of polypeptide and forms
a "homo-dimer complex". The feature
may be characteristic to primitive photosynthetic system in these organisms.
Publication List:
Abe, S., Murakami, A., Ohki, K.,
Aruga, Y. and Fujita, Y. (1994)
Changes in stoichiometry among PSI,
PSII and Cyt b6 -! complexes in
response to chromatic light for cell
growth observed with the red alga
Prophyra yezoensis. Plant Cell
Physiol. 35:901-906.
Fujita, Y., Murakami, A., Aizawa, K.
and Ohki, K. (1994) Short-term andlong-term adaptation of the photosynthetic apparatus: Homeostatic properties of thylakoids. In The Molecular
Biology of Cyanobactria (D.A.Bryanted.) Kluwer Academic Publishers,
Dordrecht, pp.677-692.
Gu, T.-Q., Iwama, Y., Murakami, A.,
Adhikary, S.P. and Fujita, Y. (1994)
Changes in the cytochrome c oxidase
activity in response to light regime forphotosynthesis observed with the
cyanophyte Synechocystis PCC 6714.
Plant Cell Physiol. 35:1135-1140.

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Ikeya, T., Ohki, K., Takahashi, M. andFujita, Y. (1994) Photosynthetic characteristics of marine Synechococcus
spp. with special reference to lightenvironments near the bottom of theeuphotic zone of the open ocean.
Marine Bioi. 118: 215-221.
Iwaki, M. and Itoh, S. (1994) Reactionof reconstituted acceptor quinone anddynamic equilibration of electrontransfer in the photosystem I reaction
center. Plant Cell Physiol. 35:
983-993.
Kandori, H., Sasabe, H. and Mimuro,M. (1994) Direct determination of alifetime of the S2 state of ~-carotene
by femtosecond time-resolved fluorescence spectroscopy. J. Am. Chem.
Soc. 116: 2671-2672.
Kumazaki, 1., Iwaki, M., Ikegami, 1.,
Kandori, H., Yoshihara, K. and Itoh,
S. (1994) Rates of primary electrontransfer reactions in the photosystem I
reaction center reconstituted withdifferent quinones as the secondary
acceptor. J. Chem. Phys. 98:
11220-11225.
Mimuro, M., Hirota, M., Nishimura, Y.,
Moriyama, T., Yamazaki, 1., Shimada,K. and Matsuura, K. (1994) Molecular organization of bacteriochlorophylls in chlorosomes of the green
photosynthetic bacterium Ch/oroflexus
aurantiacus: Studies of fluorescence
depolarization accompanied with theenergy transfer processes. Photosyn.
Res. 41: 181-191.
Mimuro, M., Nozawa, T., Tarnai, N.,
Nishimura, Y. and Yamazaki, I.
(1994) Presence and significance ofminor antenna components in theenergy transfer sequence of a green
photosynthetic bacterium Chloroflexus
340:
aurantiacus.
FEBS
Lett.
167-172.

FOR BASIC BIOLOGY

Nakayama, K. and Mimuro, M. (1994)
Chlorophyll forms and light energy
transfer in light-harvesting chlorophyll
alb protein complexes from the siphonous green alga Bryopsis ·maxima.
Biochim.
Biophys. Acta 1184:
103-110.
Nakayama, K., Mimuro, M., Nishimura,Y., Yamazaki, I. and Okada, M.
(1994) Kinetic analysis of energy
transfer processes in LHCII isolated
from a siphonous green alga Bryopsis
maxima with use of a picosecond
fluorescence spectroscopy. Biochim.
Biophys. Acta 1188: 117-124.
Ogata, T., Kodama, M., Nomura, S.,
Kobayashi, M., Nozawa, T., Katoh, T.
and Mimuro, M. (1994) A novel
peridinine-chlorophyll a protein (PCP)
isolated from the dinoflagellate
Alexandorium cohorticula: A higher
pigment content and plural spectral
forms of peridinine and chi a. FEBS
Lett. 356: 367-371.

15
Uehara, K., Mimuro, M., Ozaki, Y. and
Olson, J.M. (1994) The formation andcharacterization of the in vitro
polymeric aggregates of bacteriochlorophyll c homologs from Chlorobium
limicola in aqueous solution in thepresence of monogalactosyl diglyceride. Photosyn Res. 41: 235-243.
Yamazaki, T., Nishimura, Y., Yamazaki,
1., Hirano, M., Matsuura, K.,
Shimada, K. and Mimuro, M. (1994)
Energy migration in a allophycocyanin
B trimer with linker: Analysis byprincipal multicomponent spectral
separation method. FEBS Lett. 353:
43-47.

16

NATIONAL INSTITUTEDIVISION OF CELL PROLIFERATION(ADJUNCr)
Professor: Masayuki Yamamoto
Research Associates: Hidetoshi Iida
Yoshiyuki ImaiNIBB Post-doctoral Fellows:
Masuo Goto
Makiko Kawagishi-Kobayashi
Graduate Student: Tomoko Ono (from The...
. .
University of Tokyo)
Vzsltmg Sczentzst: Makiko S. Okumura

This Division aims to explore theregulation of meiosis in higher organisms. Meiosis is a crucial step in gamete
formation and is essential for sexual
reproduction. Meiotic steps are highly
conserved among eukaryotic species.
The major strategy taken by us to isolate
genes that may be relevant to the regulation of meiosis in animals or plants
depends upon trans-complementation
between heterologous organisms. In the
Schizosaccharomyces
fissionyeast
pombe, which is a unicellular eukaryotic
microorganism, genes involved in controlof meiosis have been well characterized
and many of them are cloned. Mutants
defective in these genes, isolated either~y classical genetics or by gene disruptiO~ and chromosome manipulation, areavailable. We have thus set out to isolate
homologs of these S. pombe genes fromanimals and a plant, by using either
similarity in nucleotide sequences orfunctional complementation of the mutants. To facilitate this strategy, we also
paid efforts to elucidation of the regulatory mechanisms of meiosis in the fission
yeast. In addition to the above, a projectto characterize the effect of calcium ion
on cell proliferation of the budding yeast
has been performed.

I. Animal and plant genes that transcomplement meiotic defects in thefission yeast
Using eDNA libraries prepared frommouse testis, Xenopus oocyte andArabidopsis thaliana, we screened forgenes that can rescue loss of function of
the S. pombe sme2 gene, which encodes
an RNA product essential for the promotion of meiosis I (see section II).
Several genes have been isolated frommouse and Arabidopsis in this screening.
Some of the mouse genes thus obtained
apparently encode RNA binding proteins, and one of the Arabidopsis genes
encodes a putative single strand DNAbinding protein. We also characterized
three Arabidopsis eDNA clones that
could rescue the meiosis-deficientphenotype of the S. pombe pdel mutant
which lacks cAMP phosphodiesterase:
The first clone apparently encodes protein phosphatase 2C (PP2C). The second
encodes a homolog of human Drl. Drl
is a protein that interacts with TATAbinding protein (TBP) and blocks its
ability to activate transcription. The third
encodes Arabidopsis TBP itself. Furthermore, we obtained mouse and Xenopus
eDNA clones that can suppress a defectin meiosis II in the fission yeast. Further
characterization of these isolated genes isin progress and their relevance to theregulation of meiosis in the originalorganisms is to be examined.
II. Identification of an RNA-protein
complex as a critical regulator ofmeiosis in the fission yeast
S. pombe mei2 has been shown to be
essential for the initiation of premeiotic
DNA synthesis. We demonstrated that atemperature-sensitive mei2 mutant performs premeiotic DNA synthesis but
does not undergo meiotic divisions'

17

FOR BASIC BIOLOGY

G1

premeiotics~meiosis I
Figure 1. A model for the regulation of meiosis by Mei2-RNA complexes in the fission yeast
The function of Mei2 is required both prior to premeiotic DNA synthesis and prior to meiosis I. Mei2
complexes with meiRNA to promote meiosis I. This RNA is essential for the promotion, but it is not requiredfor premeiotic DNA synthesis. Mei2 is likely to couple with another RNA partner to fulfill its function prior
to premeiotic DNA synthesis, because the RNA-binding ability of Mei2 is indispensable to perform thisfunction. (Adopted from ref. Watanabe and Yamamoto, 1994)

suggesting that Mei2 is required also formeiosis I. We screened for high copy
suppressors of this mei2 ts defect, andisolated a gene as an efficient suppressor.
Surprisingly, this gene, named sme2,
does not encode a protein product. It
encodes a novel, polyadenylated RNA
species of 0.5-kb in length (meiRNA),
which specifically binds to Mei2 both invivo and in vitro. Cells in which thesme2 gene is deleted perform premeiotic
DNA synthesis but cannot undergo
meiosis I. Therefore the meiRNA-Mei2
complex is apparently required for
promotion of meiosis I but not for premeiotic DNA synthesis. However, mutations introduced in the RNA-binding
motifs in Mei2 blocked premeiotic DNA
synthesis. This suggests that Mei2 islikely to couple with another RNA
species to promote premeiotic DNA
synthesis (Figure 1).

ill. Analysis of budding yeast genes
involved in Ca2+ signaling
The midi and mid2 mutants were isolated as those defective in Ca2+ signaling,on the basis that Ca2+ is essential for
maintaining viability in Saccharomyces
cerevisiae cells which are exposed to themating pheromone released from themating partner. The MIDI and MID2
genes were cloned and their nucleotide
sequences determined. The deduced
MIDI gene product is a protein of 548
amino acid residues, which is localized in
the plasma membrane. This protein isessential for Ca2+ influx and mating but
not for vegetative cell growth. The
MID2 gene product is a putative integral
membrane protein of 376 amino acids
residues. It carries a potential transmembrane domain and a conserved Ca 2+binding domain in its C-terminal half.
This protein is not essential for vegetative cell growth but is required to keep
viability of the cell in the presence of the

18mating pheromone. Moreover, expression of MID2 was found to be stimulated
three fold by the mating pheromone
signaling.
Publication List:
Davey, J., Davis, K., Imai, Y.,
Yamamoto, M. and Matthews, G.
(1994) Isolation and characterizationof krp, a dibasic endopeptidase required for cell viability in the fission
yeast Schizosaccharomyces pombe.
EMBO J. 13, 5910-5921.
Hughes, D. A., Yabana, N. andYamamoto, M. (1994) Transcriptional
regulation of a Ras nucleotideexchange factor gene by extracellularsignals in fission yeast. J. Cell Sci. 107,3635-3642.
Iida, H., Nakamura, H., Ono, T.,
Okumura, M. S. and Anraku, Y.
(1994) MIDI, a novel Saccharomyces
cerevisiae gene encoding a plasma
membrane protein, is required forCa2+ influx and mating. Mol Cell
Bioi. 14, 8259-8271.
Imai, Y. and Yamamoto, M. (1994)
The fission yeast mating pheromone
P-factor: Its molecular structure, genestructure, and ability to induce gene
expression and G 1 arrest in the mating
partner. Genes Dev. 8, 328-338.
Kuromori, T. and Yamamoto, M.
(1994) Cloning of cDNAs from
Arabidopsis thaliana that encode
putative protein phosphatase 2C and ahuman Drl-like protein by transformation of a fission yeast mutant. Nucl.
Acids Res. 22, 5296-5301.
Maeda, T., Watanabe, Y., Kunitomo,
H. and Yamamoto, M. (1994)
Cloning of the pkal gene encoding thecatalytic subunit of the cAMPdependent protein kinase in Schizosaccharomyces pombe. J. Bioi. Chern.

NATIONAL INSTITUTE

269, 9632-9637.
Mineyuki, Y., Iida, H. and Anraku, Y.
(1994) Loss of microtubules in theinterphase cells of onion root tips fromthe cell cortex and their appearance inthe cytoplasm after treatment withcycloheximide. Plant Physiol. 104,281-284.
Nakajima-Shimada, J., Iida, H., Anraku,
Y. and Aoki, T. (1994) Determinationof intracellular calcium concentrationof individual host cells infected with
Trypanosoma cruzi using microfluorimetry in conjunction with digital
image processing. Jpn. J. Parasitol. 43,
136-139.
Nocero, M., Isshiki, T., Yamamoto, M.
and Hoffman, C. S. (1994) Glucose
repression of fbpl transcription in
Schizosaccharomyces pombe is partially regulated by adenylate cyclase
activation by a G protein a subunit
encoded by gpa2 (git8). Genetics 138,
39-45.
Ono, T., Suzuki, T., Anraku, Y. andIida, H. (1994) The MID2 geneencodes a putative integral membraneprotein with a Ca2+-binding domain
and shows mating-pheromone stimulated expression in Saccharomyces
cerevisiae. Gene 151, 203-208.
Saran, S., Nakao, H., Tasaka, M., lida,
H., Tsuji, F. 1., Nanjundiah, V. andTakeuchi, I. (1994) Intracellular freecalcium level and its r~sponse tocAMP stimulation in developing
Dictyostelium cells transformed withjellyfish apoaequorin eDNA. FEES
Lett. 337, 43-47.
Watanabe, Y. and Yamamoto, M.
(1994) S. pombe mei2+ encodes anRNA-binding protein essential forpremeiotic DNA synthesis and meiosis
I, which cooperates with a novel RNA
species meiRNA. Cel/78, 487-498.

FOR BASIC BIOLOGYDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Botta
Associate Professor: Hitoshi Okamoto
Research Associates: Mika Tokumoto
Shin-ichi Higashijima
Institute Research Fellow: Akira Chiba
Nobuyoshi Shimoda
Graduate Students: Keita KoizumP
Yoko Yasuda2(l!from Tokyo University)
f'from Aichi University of Education)

Brain can be seen as an integrated circuit where neurons of various identities
are interconnected in a highly orderedmanner by their axons. We have beeninterested in how individual neurons
aquire their own identities and how theiraxons find their own pathways and finally recognize their proper targets.
Using two different animals, zebrafish(Danio rerio) and fly (Drosophila me/anogaster), both of which are suitable forgenetic analysis and gene manipulation,
we are trying to address these questions
both at the molecular and cellular levels.
I. Differential expression of islet-1 homologs during specification of primary
motoneuron
Isiet-1 (Isl-1) is a LIM domain/homeodomain-type transcnptton regulator
originally identified as an insulin geneenhancer binding protein. Isl-1 is considered to be involved in the differentiation of the neuronal cells. We previously
cloned a is/-1 homolog from zebrafish
eDNA library, named it zebrafishis/-1.
Recently, we have isolated two novel is/1 homologs from zebrafish eDNA library, named them zebrafishis/-2 and 3.
We examined the mRNA expression
pattern of each homolog using in situ hybridization to whole-mount embryos. All
three homologs are expressed in RohonBeard neurons. However the expression

19
in primary motoneurons diverged. Zebrafishis/-1 mRNA is expressed in the
rostral primary motoneuron (RoP). is/-2
mRNA is expressed in the caudal primary motoneuron (CaP) and its variant
sibling (VaP). is/-3 mRNA is expressedin the ventral region of the myotome but
not in the primary motoneurolis. The
ventral myotome is the region that theaxon of the CaP extend into. is/-3
mRNA is also expressed throughout thedeveloping eye and tectal region of the
midbrain, the target for the retial axons.
These results raise possibilities that theis/-1 homologs may be involved in thespecification and/ or target recognitionby the primary motoneurons.

n. Cloning and expression of AN34/FSpondin family in zebrafish
The floor plate is a cell group implicated in the control of neural cell pattern
and axonal growth in the developing vertebrate CNS. By subtractive hybridization technique, Klar et al. identified
F-Spondin, a novel secreted protein expressed at high levels in the floor plate.
The C-terminal half of the protein contains six TSRs (Thrombospondin Type 1
repeats), while the N-terminal half exhibited no homology to other proteins.
Although F-Spondin was shown to promote neural cell adhesion and neurite
extension in vitro, its functions in vivo
remain largely unknown.
Nose performed an enhancer trap
screen to search for genes expressed by asubset of muscle cells in Drosophila.
One of such genes, AN34 was cloned
and found to encode a secreted protein
sharing homology in two parts (H1 andH2) with the N-terminal half of F-Spondin (Nose, personal communication).
We expected the homologous reigions
to be novel domains important for20neuronal development, and thus, searched for AN34/F-Spondin family fromzebrafish (Danio rerio). We identified
two novel genes (zfANl and zfAN2) inaddition to zf-F-Spondin. zfANl andzfAN2 consist of Hl, H2 and one TSR.
The overall structure are highly similar
to AN34 and more related to AN34
than F-Spondin, suggesing that zfANl,
zfAN2 and AN34 constitute a novelsubfamily. zfANl is expressed weakly inthe floor plate and a small subset ofneuronal cells at 24-30 hr. zfAN2 expression is first observed broadly aroundthe axial mesoderm (notochord) at 1012 hr. Then, the expression becomes restricted to the floor plate and hypochordat 14 hr and continues at high levels up
to 30 hr. F-Spondin is expressed in thefloor plate as seen in rat. These findings
suggest that the AN34/F-Spondin family play important roles in the midline
development in the vertebrate.

m. Search for new cell surface recognition molecules expressed in a subset of
CNS axons
During neuronal development, growthcones are known to have ability to recognize and extend along specific axonal
surfaces. Several lines of evidence suggest that cell surface glycoproteins play
important roles in this process. Immunocytochemical studies have demonstrated
that the monoclonal antibody HNK-1,
originaly raised to a human lymphoblastoma, recognizes a subset of CNS andPNS axons in many vertebrates including
zebrafish by binding to a carbohydrate
determinant in several glycoproteins.
Thus, we started to characterize glycoproteins recognized by HNK-1, hoping
to isolate new cell adhesion molecules(CAMs) expressed in a subset of CNS
and PNS axons during neuronal development.

NATIONAL INSTITUTE

Our strategy is as follows: Manybrains of adult zebrafish are homogenized, and from this extract, molecules
which binds to HNK-1 are intensely enriched by using HNK-1 affinity chromatography. The affinty-purified fractions
are used to immunize mice to get a seriesof monoclonal antibodies (MAbs). The
MAbs, thus obtained, are screened withimmunohistochemistry to zebrafish tissues. Hopefully, those MAbs which recognize peptides and not carbohydrates
are used to isolate and characterize newmolecules.
Immunogen which represents several
protein families on Western Blotting was
prepared. Mice were immunized with it,
and hybrydoma cells were generated bycell fusion. A few MAbs each of which
recognizes different protein species havebeen obtained. One of them could recognize a subset of CNS axons in theadult brain. Efforts are currently underway to obtain more MAbs.
IV. Toll: Tel-like element of Oryzias
latipes
The zebrafish and the medaka havethe potential to become important organisms for molecular genetic analysis ofearly vertebrate development. This is because both fishes offer the possibility of
combining genetics with excellent embryology. The goal of this research is to
use transposon to perform mutant
screens in these fishes in order to identify, and subsequently clone genes important in early development, especially inneurogenesis.
A transposon, Tel, which moves via
DNA intermediate has been a useful toolin C. elegans genetics. Recently homologs of Tel were found in some fishes,raising the possibility that insertionalmutagenesis would also be possible invertebrate.

FOR BASIC BIOLOGY

We discovered a Tel-like element in
the genome of medaka. Southern hybridization analysis showed that the element, Toll, exists as multi copy in medaka genome but did not exist in zebrafish genome. Although the element
seems to encode a protein similar to the
Tel family of transposes, the putativetransposase is probably not functional
due to mutations as other putative transposases encoded by Tel-like elements infishes.
V. Fasciclin m as a synaptic recognition molecule in Drosophila
The larval neuromuscular system of
the Drosophila consists of uniquely
identified cells, and is a powerful model
system for studying selective synapse
formation. During synaptogenesis, thecell adhesion molecule Fasciclin III appears in both motoneuron RP3 and its
targets, muscle 6 and 7. We have tested
whether fasciclin III is necessary and/ or
sufficient for RP3 targe selection using
intracellular dye injection.
First, we have found that in the existing fasciclin III null mutant RP3 reliably
formed synapse with its normal targets.
Therefore, fasciclin III is either irrelevant for the process, or playing a positiverole but its absence can be compensatedfor another redundant mechanism ('X').
We now have demonstrated that thelatter is the case. We generated transgenic flies which misexpresses fasciclin
III extopically on all skeltal muscles during neuromuscular synaptogenesis. This
was accompanied by creating a constructwhich placed the fasciclin III gene under
the control of the myosin heavy chain
promoter, and introducing this construct
into the fly genome by P-element mediated genomic transformation. In theseflies, RP3 often innervated non-target

21
muscle cells while other identified motoneurons innervated targets normally.
Our results provide the single identified-cell level evidence that a cell adhesion molecule functions as a specific synaptic target recognition molecule invivo.
VI. no optic lobes (no[), a gene that isessential for larval neurogenesis in
Drosophila
We have identified a larval/pupal lethal line by P-element insertion mutagenesis. Although embryonic neurogenesis of the homozygous mutant is fairly
normal, BrdU incorporation analysis revealed they lack proliferating larval neuroblasts and some non-neuroblasts cells(neurons?) start DNA synthesis at latelarval stage. On the other hand, imaginaldiscs differentiate normally, so that rareescapers become normal looking pupae
with a small central nervous system. The
most characteristic feature is that they
have no optic lobes. In such an individual, retinular axons lose their targets andare directly guided into the tiny brain.
We conclude that there is an interesting
gene named as no optic lobes (no/) near
the P-insertion, which is essential for
proliferation and differentiation of larval
neuroblasts.
We cloned genomic regions aroundthe P-element to find a transcription unit
whose expression is significantly reducedin the homozygotes. The putative no/gene encodes a novel protein with a hydrophobic signal sequence. The deduced
amino acid sequence has a mucin-likeregion, but its significance remains to beanalyzed.

22

NATIONAL INSTITUTE

The expression pattern of F-Spondin mRNA in the spinal cord.
F-Spondin mRNA is expressed in the floor plate cells.

Publication List:
Inoue, A., Hatta, K., Hotta, Y. andOkamoto, H. (1994) Developmental
regulation of Islet-1 mRNA expression during neuronal differentiation inembryonic zebrafish. Devel. Dynam.
199, 1-11.
Goto, S., Tanimura, T. and Hotta, Y.
(1995) Emhancer trap detection of

positional information corresponding
to the polar coordinate system in theimainal disc of Drosophila melanogaster. Raux's Archiv Develop. Bioi. (Inthe Press).
Chiba, A., Snow, P., Keshishian, H. andHotta, Y. (1995) Fasciclin III as a synaptic target recognition molecule mDrosophila. Nature 374, 166-168.

FOR BASIC BIOLOGYDMSION OF CELL FuSION(ADJUNCT)
Professor: Hitoshi Sakano
Research Associate: Masahiro Ishiura
Fumikiyo Nagawa
Kanae Muraiso
Akio Tsuboi
Institute Research Fellow: Hiroaki Kasai
Graduate Student: Setsuyuki Aoki
(from Kyoto University)
Nika Yamazaki(from Tokyo Institute of Technology)

Our research interest is focused on theregulatory mechanisms of multigene
families both in the immune and in thecentral nervous systems. In the immune
system, somatic DNA recombinationand gene conversion play importantroles in the expression of antigen receptor genes. Gene rearrangement, known
as V-(D)-J joining, not only generates avast diversity in the receptor genes, butalso activates a particular member of thegene family by bringing an enhancer element and promotor element into close
proximity.
For the olfactory system, hundreds ofodorant receptor genes have been reported, although it is yet to be studied
how this multigene family is regulated
for expression. It is known that eachmember of the gene family is expressedin one of the four topographically distinct zones, where olfactory neurons expressing one particular kind of receptor
are randomly distributed~ Furthermore,
it is assumed that a limited number ofthe receptor genes (possibly one) is activated in each olfactory neuron.
We have been studying how each neuron activates a limited number of the receptor genes keeping the rest of thegenes silent. One obvious regulatory
mechanism is at the level of transcription. In the mammalian immune system,
DNA rearrangement is also utilized to23
activate one particular member of the receptor genes, and to exclude one of thetwo alleles (allelic exclusion). It is of interest to study whether such a mechanism is involved in the regulation of theolfactory system.
In order to study selective expressionof the odorant receptor genes, we havebeen characterizing the genomic structure of the murine odorant receptor
genes using genomic clones from the Pl
phage library. Many Pl clones were
found to contain multiple receptor
genes, indicating that these genes arelinked in tandem on the chromosome.
We have extensively analyzed a Pl clonethat contains two highly related (92%homologous) receptor genes, No.28 and
No.lO. It was found that these genes
were expressed in the same zone in theolfactory epithelium, yet each gene wasexpressed in a different olfactory neuron. We are trying to understand themolecular mechanisms for the differential expression of the two highly related
receptor genes.
Some odorant receptor genes are
known to be expressed also in the testes,suggesting that they may be involved inmaturation and/ or chemotaxis of sperm
cells. In order to elucidate the expressionof the odorant receptor genes in the testis, we are currently characterizing amurine olfactory receptor gene whichshares a significant homology with the
HGMP07J gene isolated from a humantesticular eDNA library.
For the study of mutually exclusive expression of the odorant receptor genes,
we have generated transgenic mice
which are forced to express one particular member of the receptors in every
single olfactory neuron. The transgene
contains the mouse receptor No.28 gene
under the control of OMP promoter.

24
Since the OMP gene is expressed specifically in the olfactory epithelium, thetransgenic receptor gene is expected tobe activated in all mature neurons in thetransgenic mouse. These studies will give
us a new insight into the molecularmechanisms not only of the receptor
gene expression, but also of neuronal
projection in the olfactory bulb. Whenthe olfactory neurons are regenerated,
they send axons to the specific region
called glomeruli in the olfactory bulb. It
is amazing that axons are able to find aright target glomerulus out of two thousand similar structures. We are hopingthat our transgenic approach will
become a useful clue to study the target
specificity and selectivity in the synapses
formation.
Publication List:
Akamatsu, Y., Tsurushita, N., Nagawa,F., Matsuoka, M., Okazaki, K., Imai,
M. and Sakano, H. (1994) Essential
residues in V(D)J recombination signals. J. lmmunol., 153, 4520-4529.
Heike, T., Nishikomori, R., Kawai, M.,
Tsuboi, A., Arai, N. and Mikawa, H.
(1994) Molecular basis for developmental changes of GM-CSF gene inducibility in embryonal carcinoma
cells. Bioche. Biophys. Res. Comm.
198, 473-479.
Heiki, T., Nishikomori, R., Kawai, M.,
Tsuboi, A., Arai, N. and Mikawa, H.
(1994) Developmental changes of
GM-CSF gene inducibility in embryonal carcinoma cells. Mol. Immun.
31, 1269-1275.
Kondo, T. and Ishiura, M. (1994) Circadian Rhythms of Cyanobacteria:

NATIONAL INSTITUTE

Monitoring the Biological Clocks of
Individual Colonies by Bioluminescence. J. Bacterial. 176, 1881-1885.
Kondo, T., Tsinoremas, N.F., Golden,
S.S., Johnson, C.H., Kutsuna, S. andIshiura M. (1994) Circadian Clock
Mutants of Cyanobacteria. Science
266, 1233-1236.
Matsuda, 1., Masuda, E.M., Tsuboi, A.,
Behnam, S., Arai, N. and Arai, K.
(1994) Characterization of NF(P), thenuclear factor that interacts withthe regulatory P sequence (5'CGAAAATTTCC-3') of the humaninterleukin-4 gene: relationship to NFKB and NF-AT. Bioche. Biophys. Res.
Comm. 199, 439-446.
Tsuboi, A., Matsuda, E.S., Naito, Y.,
Tokumitsu, H., Arai, K. and Arai, N.
(1994) Calcineurin potentiates activation of the granulocyte-macrophagecolony-stimulating factor gene in T
cells: involvement of the conserved
lymphokine element 0. Mol. Bioi. Cell5, 119-128.
Tsuboi, A., Muramatsu, M., Tsutsumi,A., Arai, K. and Arai, N. (1994) Calcineurin activates transcription fromthe GM-CSF promoter in synergy
with either protein kinase C or NFKB! AP-1 in T cells. Bioche. Biophys.
Res. Comm.199, 1064-1072.
Watanabe, M., Muramatsu, M., Tsuboi,
A. and Arai, K. (1994) Differential
response of NF-KB1 p105 and NFKB2 p100 to HTLV-1 encoded Tax.
FEBS Letters 342, 115-118.
Yoshimatsu, T. and Nagawa, F. (1994)
Effect of artificially inserted introns on
gene expression in Sacchrom yces cerevisiae. DNA Cell Bioi. 13, 51-58.

25

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshitaka Nagahama

The Department is composed of three regular divisions and one adjunct division and conducts molecularanalysis on various aspects of developmental phenomena; differentiation and maturation of the germ
cells, molecular basis of the body plan, and gene regulation in the differentiated tissues, changes of thedifferentiated states, and different phases of the cell
cycle.

26

NATIONAL INSTITUTELABORATORY OF
REPRODUCTIVE BIOLOGY
Professor: Yoshitaka Nagahama
Research Associates: Michiyasu Yoshikuni
Minoru Tanaka
Tohru Kobayashi
JSPS Post-doctoral Fellows: Takeshi Miura
Chiemi Miura
Graduate Students: Yoshinao Katsu
Shinji Onoe
Daisuke Kobayashi
Xiao- Tian Chang
Yuichi Ooba
Institute Research Fellows: Akihiko Yamaguchi
Toshinobu Tokumoto
Visiting Scientists: Mishiya Matsuyama 11
Jian-quiao Jiantf!
Wei Gel!
Technical Staffs: Hiroko Kobayashi
Sachiko Fukada
1!from Kyushu University
2!from Wuhan University
3ifrom University of Alberta

The division of reproductive biology
conducts research on the endocrine
regulation of differentiation, growth andmaturation of germ cells in multicellularanimals, using fish as a primary study
model.
I. Endocrine regulation of oocyte differentiation, growth and maturation
Our research effort in previous years
concentrated on the identification andcharacterization of the molecules (gonadotropin hormones and gonadal steroid
hormones) that stimulate and control
germ cell growth and maturation. It wasin 1985 that we identified, for the firsttime in any vertebrate, 17 a,20~-dihy­
droxy-4-pregnen-3-one (17 a,20~- DP)
as the maturation-inducing hormone ofamago salmon (Oncorhynchus rhodurus). Along with estradiol-17~, which
was identified as the major mediator of
oocyte growth, we now have two known
biologically important mediators ofoocyte growth and maturation in female
salmonid fishes. It is established that the

granulosa cells are the site of productionof these two mediators, but their production by the ovarian follicle depends on
the provision of precursor steroids by thethecal cell (two-cell type model). A dramatic switch in the steroidogenic pathway from estradiol-17~ to 17a,20~-DP
occurs in ovarian follicle cells immediately prior to oocyte maturation. This
switch is a prerequisite step for thegrowing oocyte to enter the maturation
phase, and requires a complex and integrated network of gene regulation involving cell-specificity, hormonal regulation, and developmental patterning.
We have isolated and characterized
the eDNA encoding several ovarian steroidogenic enzymes of rainbow trout(Oncorhynchus mykiss) and medaka( Oryzias latipes) which are responsible
for estradiol-17~ and 17a,20~-DP biosynthesis: cholesterol side-chain cleavage
cytochrome P450 (P450scc), 3~-hy­
dro:xysteroid dehydrogenase (3~-HSD),
17a-hydroxylase/ C17 ,20-lyase cytochrome P450 (P450c17), P450 aromatase(P450arom) and 20~-hydroxysteroid
dehydrogenase (20~-HSD). We also isolated the structural gene encoding
P450arom, for the first time from a nonmammalian vertebrate, the medaka. The
medaka P450arom gene consisted ofnine exons, but spans only 2.6 kb, being
much smaller than the human P450arom
gene (at least 70 kb), as the result of extremely small introns. Genomic Southem blots revealed the presence of asingle medaka gene. Promoter analyses
indicated two major transcription initiation sites 60 and 61 bp upstream from aputative initiation codon. The promoter
region of medaka P450arom gene contain Ad4BP sites and estrogen responsive element half-sites.
The eDNA clones obtained have been

27

FOR BASIC BIOLOGY

used for Northern and whole mount insitu hybridization to investigate themolecular basis of differential production of estradiol-17~ and 17a,20~-DP
during oocyte growth and maturation in
rainbow trout and medaka. In both
species, P450scc and P450c17 (also 3~­
HSD in rainbow trout) mRNA transcripts were increased in follicles towards
the end of oocyte growth phase and during oocyte maturation. Furthermore, incubations of isolated thecal layers withgonadotropin resulted in the elevation of
P450scc mRNA. The effect of gonadotropin becomes more dramatic when theexpression of P450scc mRNA is examined in granulosa cells; P450scc mRNA
is not detected in the absence of gonadotropin, but markedly expressed in thepresence of gonadotropin. The increases
in the amount of P450scc, 3~-HSD and
P450c17 transcripts provide an explanation for the dramatic increase in

17a,20~-DP production in follicles during naturally- and gonadotropin-induced
oocyte maturation. In contrast, levels ofmRNA for P450arom were high duringoocyte growth, but rapidly decreased
during oocyte maturation. This decreasein P450arom mRNA levels appears to
be correlated with the decreased abilityof maturing follicles to produce estradiol-17~ (Fig. 1).
17a,20~-DP acts via a receptor on theplasma membrane of oocytes. We haveidentified and characterized a specific
17a,20~-DP receptor from defolliculated oocytes of several fish species.
Scatchard analysis reveled two different
receptors: a high affinity with a Kd of 18
nM and a Bmax of 0.2 pmoles/ mg protein, and a low affinity receptor with a
Kd of 0.5 f-LM and a Bmax of 1 pmole/
mg protein. 17a,20~-DP receptor concentrations increase during oocyte maturation. The interaction between

Fig. 1 Whole mount in situ hybridization showing P450 aromatase mRNA in medaka ovarian follicles.

28

NATIONAL INSTITUTE

17a,20~-Dihydroxy-4-pregnen-3-one~

135kDaI i

cdc2 kinase
mRNA~

Cyclin B!I

3SkDa I cdc2 kinase\tkDj
Cyclin B synthesis
1~~Qic,·
Translation

Serine kinase(cdc2 kinase)yc m 8.-----<-P Threonine kinase(p4QM015)'IIIII(

Active MPF
Fig. 2 Molecular mechanisms of 17a,20~-dihydroxy-4-pregnen-3-one induced MPF activation in goldfish
oocytes.

17a,20j3-DP receptors and G-proteins
was examined. Pertussis toxin (PT) catalyzed the ADP ribosylation of a 40 kDa
protein in crude membranes from rainbow trout oocytes. The 40 kDa protein
was recognized by an antibody against a
subunit of inhibitory G-protein. Treating
the membrane fraction with 17a,20j3DP decreased the PT-catalyzed ADP ribosylation of the 40 kDa protein. The
specific binding of 17a,20j3-DP was
decreased by PT. We conclude that the
PT-sensitive Gi is involved in the signaltransduction pathway of 17a,20j3-DP infish oocytes.
The early steps following 17a,20j3-DPaction involve the formation of the
major mediator of this steroid, maturation-promoting factor or metaphase-promoting factor (MPF). MPF activity
17a,20j3-DP-induced
cycles duringoocyte maturation with the highest activity occurring at the first and second

meiotic metaphase. Studies from our laboratory and others have shown that
MPF activity is not species-specific andcan be detected in both meiotic andmitotic cells of various organisms, fromyeast to mammals.
Fish MPF, like that of amphibians,
consists of two components, catalytic
cdc2 kinase (34 kDa) and regulatory
cyclin B (46- to 48 kDa). Immature
goldfish oocytes contain only monomeric
35 kDa cdc2 and do not stockpile cyclin
B, although immature oocytes containmRNA for cyclin B. In maturingoocytes, activation of cdc2 is associatedwith its phophorylation of threonine 161(Thr161) after binding to cyclin B,
producing 34 kDa cdc2. Using mutant
cdc2, we showed that Thr161 phosphorylation is required for both the downward shift of cdc2 (35- to 34 kDa) andthe kinase activation. Since thyrosine 15
of cdc2 is not phosphorylated after

FOR BASIC BIOLOGY

binding to cyclin B, it does not requiredephosphorylation. This situation is obviously different from that in immature
Xenopus oocytes, in which the cdc2cyclin B complex preexists with cdc2
phosphorylated on both Tyr15 andThr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25
phosphatase for MPF activation. Recently we have isolated a eDNA clone
encoding a goldfish homolog of p40Mots,
the catalytic subunit of a protein kinase
which activates cdc2 kinase throughphosphorylation of Thr161, from a goldfish oocyte eDNA library. Northern andWestern blot analyses revealed that both
p40M015 mRNA and protein are alreadypresent in goldfish immature oocytes anddo not exhibit any changes during hormonally induced maturation.
Immediately prior to the transition
from metaphase to anaphase, M-phasepromoting factor (MPF) is inactivated bydegradation of cyclin B. We investigated
the role of proteasomes (a nonlysosomal
large protease) in cyclin degradation,using Eschelicia coli-produced goldfish
cyclin B and purified goldfish proteasomes (20S and 26S). The purified 26S
proteasome, but not 20S proteasome,cleaved both monomeric and cdc2bound cyclin B at lysine 57 (K57) restrictively in vitro, and produced a 42
kDa N-terminal truncated cyclin B,
which was transiently detected at the initial phase of the normal egg activation.
The 42 kDa cyclin B, as well as fulllength one, was degraded in Xenopus
egg extracts, but a mutation on K57(K57R, the 26S proteasome-catalyzed
digestion site K57 was substituted witharginine) inhibited both the digestion by
26S proteasome and the degradation in
Xenopus egg extracts. These findings
strongly suggest the involvement of 26S

29
proteasome in cyclin degradation
through the first cleave on its N-terminus.

II. Endocrine regulation of male germ
cell development and maturation
We have identified two steroidal
mediators of male germ cell development in salmonid fishes (11-ketotestosteroneforspermatogenesis
and17a,20~-DP for sperm maturation). A
steroidogenic switch, from 11-ketotestosterone to 17a,20~-DP, occurs in salmonid testes around the onset of final
maturation. In vitro incubation studies
using different testicular preparations
have revealed that the site of 17 a,20~­
DP production is in the sperm, but its
production depends on the provision of
precursor steroids by somatic cells. The
site of 11-ketotestosterone production isin the testicular somatic cells.
In the cultivated male Japanese eel(Auguilla japonica), spermatogonia are
the only germ cells present in the testis.
A serum-free, chemically defined organ
culture system developed for eel testes
was used to investigate the effect of various steroid hormones on the inductionof spermatogenesis in vitro. We obtained
evidence that 11-ketotestosterone can
induce the entire process of spermatogenesis in vitro from premitotic spermatogonia to spermatozoa within 21days.
We have used subtractive hybridization to identify genes that are expressed
differentially in eel testes in the first 24
hr after HCG treatment in vivo, which
ultimately induces spermatogenesis. One
up-regulated eDNA was isolated fromsubtractive eDNA libraries derived frommRNA extracted from control testes andtestes one day after a single injection of
HCG. From its deduced amino acid30
seqence, this clone was identified ascoding for the activin ~B subunit. Using
Northern blot analysis and in situ hybridization techniques, we examined sequential changes in transcripts of testicular activin ~B during HCG-induced
spermatogenesis. No transcripts for activin ~B were found in testes prior to
HCG injection. In contrast, 3.3 kb
mRNA transcripts were prominent intestes one day after the injection. The·transcript concentration began to decrease three days after the injection,
followed by a further sharp decrease bynine days. The HCG-dependent activin~B mRNA expression in the testes was
confirmed by in situ hybridization using
a digoxigenin-labelled RNA probe: thesignal was restricted to Sertoli cells intestes treated with HCG for one to threedays. A marked stimulation of activin B
production, but not either activin A or
activin AB, was observed in testes after
HCG and 11-ketotestosterone treatment. Addition of recombinant humanactivin B induced spermatogonial proliferation in vitro. Taken together, thesefindings suggest the following sequenceof the hormonal induction of spermatogenesis in the eel. Gonadotropin
stimulates the Leydig cells to produce
11-ketotestosterone, which, in tum, activates the Sertoli cells to produce activin
B. Activin B then acts on spermatogonia
to induce mitosis leading to the formation of spermatocytes.
In salmonid fishes, spermatozoa takenfrom the testes are immotile, but acquire
motility during their passage through thesperm duct. Using male masu salmon(Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17a.,20~-DP is responsible forthe acquisition of sperm motility;
17a.,20~-DP acts to increase sperm duct

NATIONAL INSTITUTE

pH, which in tum increases the cAMP
content of sperm, allowing the acquisition of motility.
Publication List:

Fukada, S., Sakai, N., Adachi, S. andNagahama, Y. (1994) Steroidogenesis
in the ovarian follicle of medaka ( Oryzias latipes, a daily spawner) duringoocyte maturation. Develop. GrowthDiffer. 36, 81-88.
Furukawa, K., Inagaki, H., Naruge, T.,
Tabata, S., Tomida, T., Yamaguchi,A., Yoshikuni, M., Nagahama, Y. andHotta, Y. (1994) eDNA cloning andfunctional characterization of ameiosis-specific protein (NMS 1) withapparent nuclear association. Chromosome Res. 2, 99-113.
Iwamatsu, T., Nakashima, S., Onitake,K., Matsuhisa, A. and Nagahama, Y.
(1994) Regional differences in granulosa cells of preovulatory medaka follicles. Zoo/. Sci. 11, 77-82.
Matsuyama, M., Fukada, T., Ikeura, S.,
Nagahama, Y. and Matsuura, S.
(1994) Spawning characteristics andsteroid hormone profiles in the wild
female Japanese sardine Sardinops
melanostictus. Fish. Sci. 60, 703-706.
Mita, M., Yoshikuni, M. and Nagahama,Y. (1994) G-proteins and adenylate
cyclase in ovarian granulosa cells of
amago salmon (Oncorhynchus rhodurus). Mol. Cell. Endocrinol. 105,
83-88.
Miura, T., Kobayashi, T. and Nagahama,Y. (1994) Hormonal regulation of
spermatogenesis in the Japanese eel(Anguilla japonica). Perspecitive in
Comparative Endocrinology, pp.
631-635.
Nagahama, Y. (1994) Molecular biologyof oocyte maturation in fish. Perspective in Comparative Endocrinology,

FOR BASIC BIOLOGY

193-198.
Nagahama, Y. (1994) The onset of spermatogenesis in fish. In Germline Development, Ciba Foundation Symposium No. 182, 255-270.
Nagahama, Y. (1994) Endocrine regulation of gametogenesis in fish. Int. J.
Develop. Bioi. 38, 217-229.
Nagahama, Y., Yamashita, M., Tokumoto, T. and Katsu, Y. (1994) Regulation of oocyte maturation in fish.
Current Topics in Developmental Biology, 30.
Nagahama, Y., Yoshikuni, M., Yamashita, M. and Nagahama, Y. (1994)
Regulation of oocyte maturation infish. In Molecular Endocrinology ofFish, Fish Physiology XIII (N .M. Sherwood and C.L. Hew, eds.), Academic
Press, New York, pp.393-439.
Nakamura, M., Mariko, T. and Nagahama, Y. (1994) Ultrastructure and invitro steroidogenesis of the gonads inthe protandrous anemonefish Amphiprion frenatus. Japan. J. Ichthyol. 41,
47-56.
Sakai, N., Tanaka, M., Takahashi,M., Fukada, S., Mason, J.l. and31
Nagahama, Y. (1994) Ovarian 3~­hydro:xysteroid dehydrogenase/~:::,. 5-4isomerase of rainbow trout: its eDNA
cloning and properties of the enzyme
expressed in a mammalian cell. FEBS
Lett. 350, 309-313.
Yamashita, M., Kajiura, H., Tanaka, T.,
Onoe, S. and Nagahama, Y. (1994)
Molecular mechanisms of the activation of maturation-promoting factor
during goldfish oocyte maturation.
Develop. Bioi. 166 (in press).
Yoshikuni, M. and Nagahama, Y.
(1994) Involvement of an inhibitory
G-protein in the signal transduction
pathway of maturation-inducing hormone (17 a,20~-dihydroxy-4-pregnen3-one) action in rainbow trout (Oncorhynchus rhodurus) oocytes. Develop. Bioi. 166, 615-622.
Yoshikuni, M., Matsushita, H., Shibata,N. and Nagahama, Y. (1994) Purification and characterization of 17a,20~­
dihydroxy-4-pregnen-3-one-binding
protein from plasma of rainbow trout,
Oncorhynchus mykiss. Gen. Comp.
Endocrinol. 96, 189-196.

32

NATIONAL INSTITUTEDIVISION OF CELL
DIFFERENTIATION
Professor: Yoshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Shigeharu Takiya
Toshiharu Suzuki
Kaoru Ohno
Hiraki Kokubo
JSPS Postdoctoral Fellow: Vticlav Mach
Visiting Scientists: Pin-Xian Xu
XinXu
Graduate Students:
Hiraki Kokubo (Graduate University for
Advanced Studies)
X in Xu (Graduate University for Advanced
Studies)
Yoshinori Ueno (Graduate University for
Advanced Studies)
Katsuyoshi Matsunami (Graduate University
for Advanced Studies)Technical Staffs: Miyuki Ohkubo
Chikako Inoue

Members of the Division have been
involved in two well associated projects.
One initiated in 1968 is to understandhow a special tissue like the silk gland of
Bombyx mori is differentiated along thedevelopmental programs and results intranscribing a specific set of genes likethe silk fibroin,fibroin L-chain' P25>.
sericin-1, and sericin-2 genes. The otherinitiated at the time when the Division
was established in 1978 is concernedwith how the body plan of the silkworm
is controlled and how the developmental
regulatory genes regulate a set of target
genes in specifying the identities of various regions of the embryos.
I. Genes and factors that control thesilk gland development and the silk
genes transcription
We have been trying to understandthe networks of gene regulation hierarchy that function in the processes ofsilk gland development and differentiation. As a bottom-up type approach forthis project, analyses on the molecularmechanisms that control the differential
transcription of the fibroin and sericin-1

genes in the silk gland should shed lighton a part of the networks. In complementing this approach, a top-down typeapproach should also help understanding
the networks; analyses of regulation
hierarchy of the homeobox genes andother regulatory genes, and identificationof their target genes expressed in thelabial segment, where the silk gland isoriginated.
Among many factors proposed to bindand control the fibroin and sericin-1
genes, the POU-M1 which accommodates a POU-domain identical to Drosophila Cf1-a was cloned previously andcharacterized. The POU-M1 binds to theSC site of the sericin-1 gene and is assumed to enhance the transcription. This
protein also binds to the PB element of
the POU-Ml gene and suppresses thetranscription. The expression of the
POU-Ml gene has been analyzed in
Bombyx embryos by in situ hybridizationand immunohistochemistry. The gene
was expressed for the first time at stage
18-19 in a restricted site of the labial
segment; the primordial cells of prothoracic gland. This location matches withthe site where the Bombyx Scr expression disappears which was detected in theentire labial segment in the preceding
stage. Later the POU-Ml expression isobserved along with developing silk
gland and is confined to the anterior andmiddle portions of the silk gland by lateembryonic stages. These observations
suggest that the POU-Ml gene may
have multiple functions.
Silk gland specific transcription factor
SGF-1 interacts with the SA site of thesericin-1 gene and FA and FB sites ofthe fibroin gene. Two related SGF-1
polypeptides were purified of observed
molecular weights 40 kDa and 41 kDa(V. Mach et al. (1995). J. Bioi. Chern.,

33

FOR BASIC BIOLOGYin press). These proteins interact specifically with the SA site in electrophoretic
mobility shift assay and DMS methylation interference assay. The SGF-1 40
kDa protein was partially sequenced andfound to be a new member of the fork
head/HNF-3 family. This information
facilitated cloning of several full length
cDNAs coding for the SGF-1 40 kDa
and possibly also for the SGF-1 41 kDa
proteins (V. Mach et al. (1995). J. Bioi.
Chem., in press). Sequencing of the fulllength cDNAs revealed three domains
conserved between Drosophila Forkhead protein and mammalian HNF-3
factors (Fig. 1). SGF-1 mRNA is expressed consitently with the presumedrole of its protein product in the regulation of sericin-1 gene and SGF-1 protein
contains putative transactivation domains. We conclude that regulation of
the sericin-1 gene transcription via SA
site is exerted by the SGF-1 40 and 41

kDa proteins.
The expression patterns of SGF-1
mRNA and its protein were analyzed inembryos. The transcripts and protein
were detected firstly in the most anterior
and posterior domains at the time when
the germ anlage is formed, and at laterstages in the entire region of the foregut,in the most anterior and posterior
regions of the elongated midgut, and in
the entire region of the hindgut (Fig.
2A). At the time when the embryo
retraction is finished (stage 20) the transcripts and protein are also detected in
the invaginated silk gland (Fig. 2A).
By the time when the blastokinesis is finished (stage 25) the transcripts andprotein are restricted to the middle andposterior regions of the silk gland (Fig.
2B). These results suggest that the Bombyx fork head/SGF-1 might play important roles in the gut and silk glanddevelopment.

I
SGF-1

KTYRRSYTHAKPPYSYISLI'IMAIQNNPSRMLTLSEIYQFIMDLFPF'YR,QNQQRW

FKH
HNF-~

T .•..•...•.............•.••• T •••....••.....•..•...•....

••••••••••••••••••••••.•• QS.NK ••••••.•• W•••.•••••••••••************************* ********* ***************SGF-1

QNSIRHSLSFNIX:FVKVPRTPDKPGKGSFWI'LHPDSGNMFENFLRRQKRFKDE

FKH•......•........ I •.........•.....•.........• Y ..•••.....

HNF-313.•....•.•.•••. L ...• A •••.•••••••••••..••••.•• Y •••.•••• C.

************** * ** ************************ ******** *

II

III

SGF-1

LKQEPSGYAPAQHPFSITRL

SGF-1

FKH

.••••.•• T.SS ••.•• N .•FKH

S .... LG. --- .. P .GITS

HNF-3a.
HNF-313
HNF-3'Y.. GD.H-.SF-N ..... NN.
.. P.HH- •• F-N ..... NN.
•• LDAP- .NF-N ..•.. NN.

HNF-3a.
HNF-3~A .. • GV-. ---SRPVLNI'SS ••• GV-.---SRPIMNSS

HNF-3'Y

V .... L-. ---SRSLLNAS

**

*

*****

*

NYYQSPLYHHHHAHA-QPPL****

Fig. 1 Comparison of SGF-1 with other members of Fork head/HNF-3 family.
The three domains conserved between Drosophila Fork head and mammalian HNF-3 factors are present
also in SGF-1. The domain boundaries in SGF-1 are Lys103/Glu212 (1), Leu286/Leu303 (II), and Asp331/
Leu349 (III). Domain I is the DNA binding domain, and domains II and III participate in transactivation by
HNF-3 beta.

34

NATIONAL INSTITUTE

Fig. 2 Expression of Bombyx Fork head/ SGF-1 protein.
A. An embryo at the embryo retraction stage (stage 20). The triangles from left to right indicate positive
signals at the foregut, the anterior most of the midgut, the posterior most of the midgut, and the hindgut,
respectively. The arrowheads indicate the signals at the invaginated silk glands.
B. A silk gland dissected out from an embryo of stage 25. The arrowhead indicates the border between theanterior (negative) and the middle (positive) silk gland. The positive signal continues all the way to the end of
the posterior silk gland.

Cloning and expression pattern analysis of the Bombyx Scr have been described previously. Regulation of the
Bombyx Scr gene itself in the labial segment and search for the target genes of
the Bombyx Scr have been intiated.

II. Other genes of developmental
interest
Following the previous discovery thatthe Bombyx cad transcripts and protein
form concentration gradients at different
timing of development than in Droso-

FOR BASIC BIOLOGY

phi/a, our attention has been forwarded
to Bombyx pair-rule genes. We havecloned the Bombyx even-skipped andBombyx hunchback cDNAs. After
examining the expression patterns ofthese genes, we propose a possible
molecular explanation for Bombyx
segmentation mechanism. At the top ofinterests, our data suggest that Bombyx
even-skipped might serve a doublesegment defining role and determine on
the odd-numbered Bombyx engrailed
stripes.
In continuation of the abdominal
segments identification, we have concentrated in the study of morphogenesis ofembryonic abdominal legs. We haveanalyzed proteins in the wild type embryos by SDS-PAGE, and found that
two high molecular weight proteins of
270 and 260 kDa (p270/260) are expressed specifically in the abdominal
legs. These proteins are not detectable in
the Eca I Eca embryo which lacks Bombyx abd-A gene as reported previously,suggesting a control under the Bombyx
abd-A. We have purified the p270/260
to greater than 90% homogeniety byammonium sulfate fractionation, hydroxyapatite column chromatography
and gel filtration column chromatography. Since p270 and p260 were
coeluted through these procedures, we
assume that p270 and p260 participate
in the formation of a stable complex.

35
Immunohistochemical analysis demonstrated that p270/260 was detected in a
few cells in each abdominal leg anlage ofthe first to the eighth abdominal segments at an early developmental stage.
The number of positive cells increased
and they were integrated into abdominal
legs of the third to the sixth abdominal
segments in later stages. From these results we speculate that p270/260 may
have an important role in the development of abdominal legs.
Publication List:
Amanai, K., Suzuki, Y. and Ohtaki, T.
(1994) Involvement of a maternallytranscribed lectin gene in the early
development of Bombyx mori. Roux'sArch. Dev. Bioi. 203, 397-401.
Suzuki, Y. (1994) Genes that are involved in Bombyx body plan and silk
gene regulation. Int. J. Dev. Bioi. 38,
231-235.
Xu, P.-X., Fukuta, M., Takiya, S.,
Matsuno, K., Xu, X. and Suzuki, Y.
(1994) Promoter of the POU-Mll
SGF-3 gene involved in the expression of Bombyx silk genes. J. Bioi.
Chem. 269, 2733-2742.
Xu, X., Xu, P.-X. and Suzuki, Y. (1994)
A maternal homeobox gene, Bombyx
caudal, forms both mRNA and protein concentration gradients spanning
anteroposterior axis during gastrulation. Develop.120, 277-285.

36

NATIONAL INSTITUTEDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Associate Professor: Ryuji Kodama
Research Associates: Makoto Mochii
Mitsuko Kosaka
Visiting Scientists: Takamasa S. Yamamoto
Kaichiro Sawada
Akiolio
Toshiyuki Nagamoto
Graduate Students:
Nobuhiko Mizuno (Graduate University for
Advanced Studies)
Yuuichi Mazaki (Graduate University for
Advanced Studies)
Harutoshi Hayashi (from School of Agriculture,
University of Tokyo)
Jatupol Kositsawat (from School of Medicine,
University of Tokyo)
Technical Staffs: Chiyo Takagi
Hisae UraiIn the newt and some other limited
animal species, the lens and neural retina
can be completely regenerated throughtransdifferentiation of pigmented epithelial cells (PECs). Such a phenomenon,transdifferentiation, as observed in regeneration of ocular tissues seems to bea highly powerful model for studying
stability and instability in differentiationof tissue cells. From this view point, lenstransdifferentiation of PECs of vertebrate has been studied in vivo and invitro systems, and our in vitro studies
have revealed that dormant potential totransdifferentiate into lens cells is widelyconserved throughout vertebrate species,
and that the cell type-specific genes arecompletely inactivated in the multipotent(at least bipotent) dedifferentiated cells
originated from pigmented epithelial
cells.
Our studies have been conducted toclarify the molecular mechanism controlling the lens transdifferentiation invertebrate PECs and also to search thereason why the pigmented epithelia ofspecies other than the newt and so forth
never regenerate the lens in the in situ
eyes. Based on findings accumulated up

to the last year, we have conductedanalysis of the lens transdifferentiationof PECs in vivo and in vitro and thefollowing results have been established.
I. Multipotency conserved in thehuman PECs
It has been proved that retinal PECs
isolated from the eye of 80-year-old
human can transdifferentiate into lens
cells to construct lentoids when dissociated and cultured under the culture
condition established in our laboratory(cf. Itoh and Eguchi, Dev. Biol. 115:
353-362, 1986). Human PECs canreadily transdifferentiate and form
lentoids, which express major humancrystallin genes and ultrastructures characteristic to lens, through the dedifferentiated state in the similar manner as observed in chicken retinal PECs. We haveisolated clones from the progeny ofdedifferentiated human retinal PECs toestablish cell lines. Reaggregates of cellsof one of established cell lines were
found to express lens specificities when
maintained under the culture condition
permissive for lens cell differentiation(cf. Itoh and Eguchi, 1986), althoughthese dedifferentiated cells could not
develop to lentoid in the monolayer culture. However, when maintained withthe ordinary medium, Eagle's minimum
essential medium supplemented with
10% of fetal bovine seruqt, for longtime, they express neuronal cellmorphology (Fig. 1). Neuronal specificities of these cells with neuronal morphology could be detected by indirect
immunofluorescent staining using antibodies against 70, 150 and 200 kd subunits of neurofilament protein. This
result is the first evidence that humanretinal PECs can express neuronalphenotype by themselves and is strongly

FOR BASIC BIOLOGY

37

Fig. 1. Human retinal PECs showing a neuron-like morphology. Cells were isolated from the eye of an
80-year-old donor and their growth was enhanced by the EdFPH medium. Cells were then cultured in thestandard medium for about 30 days.

suggesting that the human retinal PECs,
like the newt PECs, conserve multipotency for differentiation, althoughneuronal transdifferentiation of humanretinal PECs by transfection with H- ras
proto-oncogene has been reported byDutt, Scott et al (DNA and Cell Biology
12(8): 667-673, 1993). The human
PECs grow well and endure genetic
manipulations, such as gene transfection,
thus are supposed to be a promisingmaterial to study transdifferentiation.
We are now extending our studies oftransdifferentiation using this cell line
established from human retinal PECs.
II. Establishment of in vitro model system of lens transdifferentiation nsing
chicken iris PECs
As the main material to study the process of transdifferentiation in our laboratory, we have been utilized retinal PECs
isolated from approximately 10 day-old

chick embryos, because we can obtainlarge number of PECs through an easy
manipulation, owing to the large size ofthe eye of the chick embryo. Recently,
however, we have come to recognizethat cell culture of the retinal PECs has
gradually become difficult. Cells are susceptible to the change of culture conditions, such as the product lot of the fetal
calf serum or other components of theculture medium, and often stop growing,embedded in irregularly accumulated extracellular matrices. Besides trials tomodify culture conditions to enable
more efficient culture of the retinal
PECs, other sources of PECs were alsosought. The iris PECs of the chick has
emerged as a promising material as
briefly described below.
Because it is the iris PECs that giverise to lens regeneration in the newt, theiris PECs of the chicken was tested fortheir ability to transdifferentiate in vitro.
The iris was isolated from newly hatched

38

NATIONAL INSTITUTE

Fig. 2. Lentoids formed in the culture of the iris PECs of newly hatched chicks. Cells passaged in the
EdFPH medium for several times were cultured with ascorbic acid for about 30 days.

chicks, and the pigmented epithelium
was cleanly separated from the stroma
using dispase. The iris PECs slowly growand stably maintain the phenotype in astandard culture medium. When the
EdFPH medium, which is effective in inducing dedifferentiation of retinal PECs
(cf. ltoh and Eguchi, 1986), was applied,
the iris PECs grow rapidly and dedifferentiate. By further addition of ascorbic acid, such dedifferentiated cells accumulate and form large number of lentoids (Fig. 2). The growth and transdifferentiation of the iris PECs are highly
reproducible. Preliminary studies haveshown that all the genes we have analyzed in the retinal PECs are expressedin the culture of the iris PECs.
III. Detailed analysis of the gap junctions in the W olffian lens regeneration
On the basis of the results accumulated through the studies of lens transdifferentiation of the retinal PECs of thechick embryo, detailed studies of themechanisms regulating the lens regener-

ation in the newt have become possible.
Our recent observation in the chick
PECs has shown that the dePECs aredevoid of both the structure and conductance of the gap junction. There wasalso a preliminary study that showed atransient absence of the electric coupling
between dorsal iris PECs prior to thelens regeneration. We have started astudy on the gap junction between theiris PECs during lens regeneration, andhave obtained preliminary results shown
below.
A significant decrease in the numberof gap junctions was detected in the dorsal iris pigmented epithelium, but not inthe ventral, on approximately 10 days
after lens removal. This result confirms
the preliminary result in the electric
coupling, and is in accordance with theresult in the chick retinal PECs. However, we also found that, just after thelens removal, there is a period during
which gap junctions are increased allover the iris. This increase was accompanied with the increase in the transcript of

FOR BASIC BIOLOGY

the connexin gene, which codes for amajor component of the gap junction (R.
Kodama, unpublished).
Similar study in the partial hepatectomy showed that, shortly after the hepatectomy, there is a quick decrease ingap junction number, which is then followed by the raise in DNA synthesis andmitoses. Our result in the lens regeneration suggests that the regeneration process is biphasic, consisting of a period of
emergency repair in the whole iris and a
period of regeneration only in the dorsal
iris. Through such renewed studies,
which are also focused on growth factors
and tissue proteases, a clearer image ofthe lens regeneration process will be obtained.
Publication List:
Kodama, R. and Eguchi, G. (1994) The
loss of gap junctional cell-to-cell communication is coupled with dedifferentiation of retinal pigmented epithelial
cells in the course of transdifferentiation into the lens. Int. J. Dev. Bioi. 38,
357-364.
Kodama, R. and Eguchi, G. (1994)
Gene regulation and differentiation invertebrate ocular tissues. Curr. Opin.
Genetics Dev. 4, 703-308.

39
Iio, A., Mochii, M., Agata, K., Kodama,
R. and Eguchi, G. (1994) Expressionof the retinal pigmented epithelial cellspecific pP344 gene during development of the chicken eye and identification of its product. Develop. GrowthDiffer. 36, 155-164.
Ono, T., Murakami, T., Mochii, M.,
Agata, K., Kino, K., Otsuka, K., Ohta,
M., Mizutani, M., Yoshida, M. andEguchi, G. (1994) A complete culture
system for avian transgenesis, supporting quail embryos from the single-cellstage to hatching. Dev. Bioi. 161,
126-130.
Ono, T., Muto, S., Mizutani, M., Agata,K., Mochii, M., Kino, K., Otsuka, K.,
Ohta, M., Yoshida, M. and Eguchi, G.
(1994) Production of quail chimera bytransfer of early blastodermal cells andits use for transgenesis. Jap. Poultry
Sci. 31, 119-129.
Orii, H., Hyuga, M., Mochii, M., Kosaka, J., Eguchi, G. and Watanabe, K.
(1994) Predominant melanogenesis
and lentoidogenesis in vitro frommultipotent pineal cells by dimethyl
sulfoxide and hexamethylene bisacetamide. Int. J. Dev. Bioi. 38, 397-404.

40

NATIONAL INSTITUTEDMSION OF DEVELOPMENTAL
BIOLOGY (ADJUNCT)
Professor: Masaki lwabuchi
Associate Professor: Masao Tasaka
Research Associates: Norihiro Ohtsubo
KojiMikami
Visiting Scientist: Michael Filosa1J
Graduate Students: Takuya Itoll
Ken-ichiro Taoka 2J( 11from University of Toronto)(llfrom Kyoto University)

Genes encoding four types of core histones (H2A, H2B, H3, H4) and a linker
histone (Hl) are coordinately expressed
during S phase of the cell cycle. To
understand how this coordinate expression is regulated, we have been continuously studying the transcriptional regulation of wheat histone genes.
Within the promoter region of an H3gene (TH012), three types of positive
cis-acting elements, the hexamer(ACGTCA),theoctamer(CGCGGATC) and the nonamer(CATCCAACG) have been found. The
hexamer and the octamer constitute asequence unit designated as type I element. This combined sequence is often
found within the promoter region ofplant histone H2B, H3 and H4 genes.
We have also found several sequencespecific DNA-binding proteins interacting with the type I element; HBP-laand HBP-lb specific for the hexamer'
and OBRFs for the octamer. Analyses ofcloned cDNAs for HBP-la and HBP-lb
revealed that they have the characteristicfeature of bZIP (basic region/leucine
zipper) type transcription factors.
To analyze the temporal patterns ofplant histone gene expression during thecell cycle, we have established the cell
cycle synchronization system using suspension culture of rice Oc cells and theinhibitor of DNA synthesis, aphidicolin.
Sl analyses of mRNAs transcribed fromchimeric genes composed of the H3 promoter and protein coding region of ~­glucuronidase gene revealed that thetype I element is necessary and sufficientfor the S phase-specific activation of theH3 gene expression.
I. Auxin-inducible expression of thewheat histone H3 geneTo define the functions of the type I
element in vivo, we introduced the H3/
GUS chimeric gene (-185WTH3/GUS)
into Arabidopsis plants via Agrobacterium Ti plasmid, then analyzed their expressions in roots of regenerated plants.
Histochemical
staining
experimentshowed that the WTH3/GUS gene isexpressed at root apical meristem andvascular bundle (Fig. lA) whereas expression in the latter region was not observed when we examined using regenerated roots of transgenic rice plants. Expression in vascular bundle may occur inthe dividing cells that constitute secondary vascular bundle specific for dicots
and gymnosperms. We concluded thatthe H3 gene expression in roots is detected only in dividing cells whose cell
cycle is actively progressing.
To determine whether the type I element is also involved in the meristemspecific gene expression, transgenic
plants harboring mutant genes, HexM,
OctM, and HexM/ OctM were tested.
Base-substitutions introduced into thehexamer or the octamer abolished theexpression in roots (data not shown),suggesting that the type I element is necessary for the H3 gene expression in
dividing cells.
Since auxin acts as a mitogen, progression of the cell cycle and expression ofcell cycle-dependent genes such as histone genes would be expected to be
regulated by auxin. To address thisFOR BASIC BIOLOGY

possibility, we examined the effect ofexogenously provided auxin on the meristem-specific expression of the H3 /GUS genes. As shown in Fig. lB, expression of the WTH3/GUS gene wasenhanced in both root apical meristem
and vascular bundle. This result indicatesthat the action of auxin is involved in thesignal transduction pathway from thepassage of G/S boundary to the H3gene expression. Detailed analyses of theH3 promoter sequence that functions inthis induction pathway are currently
underway.
II. Functional analysis of HBP-lb(c38)
When the HBP-lb(c38) expression
plasmid was co-transfected into tobacco
mesophyll protoplasts with reporter plasrnids containing the binding site of HBPlb(c38), expression of the reporter gene
was repressed. However, when the hexamer (a binding site of HBP-lb) was
mutated, HBP-lb(c38) acted as an activator. This result represents a dual function of HBP-lb(c38) through directbinding to DNA and protein-protein interactions with the octamer-binding protein(s) or some other transcription factors. In addition, we investigated thefunctional difference between HBPlb(c38) and its tobacco homologue,
TGA-la, in this expression system.

41
Swapping experiments aiming at thefunctional regions of HBP-lb(c38) and
TGA-la showed that the activation ofgene expression through the hexamer
motif depends on the acidic region of
TGA-la.
To elucidate the functional roles of
HBP-lb(c38) in plant development, wemade transgenic Arabidopsis harboring
the plasmids that overexpress HBPlb(c38) or its antisense RNA and observed morphological effects of transgenic plants. Transformants overexpressing HBP-lb(c38) showed significant
morphological changes such as bent
stems, leafless plants, and abnormal production of flowers. This result suggeststhat HBP-lb(c38) is involved in theregulation of cell fate determination,flower development and/ or othergrowth control mechanisms.

m. s phase-specific expression of thewheat histone Hl gene
The genomic clones, TH 315 andTH325, for wheat Hl genes, have not
the type I element within their promoter
region. Instead, they have another typeof characteristic sequence unit composedof the octamer and the CAAT-box. Thissequence, designated as the type III element, is found not only within the promoter region of wheat histone Hl genes

42
but also within those of Arabidopsis(AtHl-1) and tomato H1 genes andwheat H2B genes (TH123 and TH153).
Conservation of the order and the spacing (14 bp) between the two components(the octamer and CAAT) represents thepossibility that the type III element is involved in the S phase-specific regulationof H1 and H2B gene expression.
Transient expression analyses of 5'deletion and base-substitution mutantsof H1 promoter/GUS chimeric genes
revealed that the type III element acts as
a positive cis-acting element of the H1(TH315) gene.
To determine whether the type III element is involved in the regulation of S
phase-specific gene expression, and tounderstand how the regulation of core
histone gene expression during the Sphase is different from that of H1 genes,
we analyzed the expression of the H1/
GUS chimeric genes in transformed ricecells. S1 analyses of the Hl/GUS
mRNA from synchronized culture oftransformed rice cells showed that thepeak of Hl!GUS mRNA accumulation
appeared slightly later (by an hour) thanthat of H3/GUS mRNA. In addtion,
when the type III element of this promoter was deleted by truncation from its
5' end, the S phase-specific change ofmRNA level was no longer observed.
These results suggest the difference inthe regulatory mechanism between theH1 gene and H3 gene, and also repre-

NATIONAL INSTITUTE

sent the contribution of the type III element to the S phase-specific regulationof the H1 gene expression.

Publication List:
Mikami, K., Sakamoto, A. and Iwabuchi, M. (1994) The HBP-1 family of
wheat basic/leucine zipper proteinsinteracts with overlapping cis-acting
hexamer motifs of plant histone genes.
J. Bioi. Chem. 269, 9974-9985.
Morio, T., Takeuchi, I. and Tasaka, M.
(1994) Cooperation of positively andnegatively acting promoter elements
determines prespore-specific transcription of Dp87 gene in Dictyostelium.
Mech. Dev. 45,59-72.
Nakao, H., Yamamoto, A., Takeuchi, I.
and Tasaka, M. (1994) Dictyostelium
prespore-specific gene Dp87 encodesa sorus mattix protein. J. Cell Sci.
107, 397-403.
Ohtsubo, N. and Iwabuchi, M. (1994)
The conserved 3'-flanking sequence,
AATGGAAATG, of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA,
Nuc/. Acids Res. 22, 1052-1058.
Saran, S., Nakao, H., Tasaka, M., Iida,
H., Tsuji, F.I., Nanjundiah, V. and Takeuchi, I. (1994) Intracellular free calcium level and its response to cAMP
stimulation in developing Dictyostelium cells transformed with jellyfish
apoaequorin eDNA. FEBS Lett. 337,
43-47.

43

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Norio Murata

The Department is composed of two regular divisions and two adjunct divisions. The study of this department is focused on the molecular mechanisms forthe response of organisms including tissues and cells
toward external and internal environments, such as
light, temperature, hormones, signal-transducing substances.

44

NATIONAL INSTITUTEDIVISION OF MOLECULARNEUROBIOLOGY
Professor: Masaharu Noda
Research Associates: Nobuaki Maeda
Shinji Hirano
Masahito Yamagata
Graduate Students: Hiraki Hamanaka
Junichi Yuasa
Takafumi Shintani
Taeko Nishiwaki
Visiting Fellow: Haruyuki Matsunaga •Technical Staff' Tomoko Mori
Shigemi Ohsugi
Masae Mizoguchi(*from Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd.)Our efforts have been devoted to
studying molecular and cellular mechanisms which underlie the development of
the vertebrate central nervous system.
We are seeking for the molecules andstructures that regulate various cellular
events requisite for the brain morphogenesis, such as generation of neuroblasts, their migration to form the laminar structure and various nuclei, theelongation and path-finding of neuralprocesses, and the formation and refinement of specific connections between
neurons. The research has been conducted using various techniques, including molecular biology (e.g. eDNA cloning, site-directed mutagenesis), biochemistry (protein, carbohydrate), monoclonal
antibodies, neuroanatomy, cell and organotypic culture, and embryo manipulation (classical embryology, gene transfer
with viral vectors, and gene targeting).
I. Topographical and laminar connection in the chick retinotectal system
Neural connection in the vertebrate
brain is selective in two ways, which werefer to as topographical and laminar.
Topographical specificity determines theorderly maps of connectivity, in whichpresynaptic neurons arrayed along

spatial axes project to corresponding arrays of target cells in the target area. By
contrast, laminar specificity determines
the local circuitry, in which presynaptic
neurons form synapses on the particular
cells/ portions of the particular target
layer/cell's surface. However, we still
have no knowledge about the crucial factors that determine the development ofthese two classes of neural connection.
We have chosen to study a retinotectal
system in chickens which have many experimental advantages.
Most intensively studied in this system
has been the retinotopic map, where retinal ganglion cells from various parts of
the retina project topographically onto
the tectal surface. For example, retinal
axons from the temporal (posterior) retina connect to the anterior part of thetectum. Many works, since the formulation of 'chemoaffinity theory' by Roger
Sperry, have supported the involvementof gradient molecules in this topographically organized connection. In positing
this hypothesis, we performed subtractive screening to isolate the cDNAs thatis graded along the anterio-posterior axis
in the retina. Recently, we have foundseveral known and novel molecules,
which are selectively expressed in thenasal or temporal half region of theretina. Starting with these molecules, wehope to understand the molecular mechanism by which retinotectal topographical connection is established.
Less well studied, but comparatively
striking, is a laminar selectivity of retinotectal connection in the orthogonal direction. Retinal axons enter the tectum
through the most superficial of its 15 different laminae. Once axons reachdefined loci on the tectal surface, theybranch inwards and make connections in
only 3-4 of the 15 laminae. Moreover,FOR BASIC "BIOLOGY

each axon confines most of its synapses
to one of these 'retinoreceptive' laminae,
andneurochemically-distinguishable
subsets of the ganglion cells connect todistinct laminae to establish a functional
network (Yamagata et al. submitted).
The laminar selectivity in this system
may require an axon to recognize particular target cells (cellular specificity)and particular portions of the target
cell's surface (subcellular specificity). To
identify the cellular and molecular mechanism underlying these synaptic specificities, we are taking two interrelated
approaches. First, we are trying to establish culture systems to analyze the recognition cues between presynaptic axons
and their target cells in vitro. The second
approach is to use molecular techniques,particularly by developing useful probes.
We are now preparing new monoclonal
antibodies that mark the subsets of laminae or cells, eventually to identify themolecules that guide the lamina-specificconnection. To understand the functionof these molecules, we will employ a variety of gene transfer techniques, including retroviral and adenoviral vectors.
II. Proteoglycan and brain development

Proteoglycans are a family of proteinsbearing sulfated glycosaminoglycans,which bind many extracellular matrix
components and growth factors throughtheir core protein and glycosaminoglycan
portions. We have been interested in thefunctional roles of the brain-specific proteoglycans in the development of thenervous tissue because they are themajor extracellular matrix components inthe tissue.
Previously, we identified a phosphatebuffered saline-soluble chondroitin sulfate proteoglycan with a 300-kDa coreprotein (6B4 proteoglycan) using a45

monoclonal antibody (MAb6B4 ). Immunohistochemical analysis of the adult rat
hindbrain showed that 6B4 proteoglycan
is expressed in fairly restricted areas suchas pontine nuclei and lateral reticular
nucleus. Developmental studies of the
rat hindbrain indicated that the expression of 6B4 proteoglycan is spatiotemporally correlated with the circuit formation of the mossy fiber system. In theembryonal rat cerebral cortex, in contrast, 6B4 proteoglycan is expressed on
the radial glial fiber, a scaffold for neuronal migration.
Recently, we cloned cDNAs encoding
this proteoglycan from a A.gt11 rat wholebrain eDNA library. Nucleotide sequence analysis of the isolated eDNA
clones revealed that 6B4 proteoglycan ishighly homologous to human receptorlike protein tyrosine phosphatase(PTPase), PTPl; (also known as RPTP[3).
In parallel, Maurel et al. reported aeDNA encoding a chondroitin sulfate
proteoglycan termed phosphacan, fromrat brain, and it turned out that 6B4 proteoglycan is identical to phosphacan.
The eDNA analysis including our own
revealed that 6B4 proteoglycan/ phosphacan is an alternatively spliced extracellular variant of PTPl;. We then attempted to identify proteoglycan-type
PTPases in the rat brain, where many
types of proteoglycans and PTPases are
known to be present.
Membrane-bound proteoglycan fractions were obtained from the postnuclear
membrane preparation of the 8-day-old
rat brain by DEAE ion exchange chromatography and CsCl density gradientcentrifugation. The isolated proteoglycan
fraction contained a high PTPase activity
with typical PTPase characteristics. Protein renaturation experiments from SDS
gels demonstrated that chondroitin

46

NATIONAl: INSTITUTE

Fig. 1 A) Lamina-specific projection of retinal axons in the chick optic tectum. The anterogradely-labeledretinal terminals (brown) make synapses in some restricted laminae. B) Recombinant adenovirus-mediatedgene transfer into the chick optic tectum. Adenovirus carrying lacZ (E. coli ~-galactosidase) was injected into
the mesencephalic ventricle. After one week, many cells in various laminae express the introduced gene
(blue).

sulfate proteoglycans with a 380- and
170-kDa core proteins carried the
PTPase activity. The proteoglycan with
380-kDa core protein was identified as
PTP~ with the specific antibodies. The
PTPase with a 170-kDa core protein did
not cross-react with the antibodies
against PTP~, suggesting that these two
are not closely related structurally. Thesefindings show that early postnatal rat
brain indeed contain multiple proteoglycan-type PTPases.
Proteoglycans may play crucial roles incell adhesion, motility, growth and differentiation through the process of binding to various extracellular matrix molecules and growth factors. On the other
hand, the level of protein tyrosine phosphorylation is regulated by cellular interactions with these molecules through themodulation of protein tyrosine kinase
and PTPase activities. Our findings thatsome membrane-bound PTPases areproteoglycans would explain the functional overlap between the two classes ofmolecules.

Publication List:
Maeda, N., Hamanaka, H., Shintani, T.,
Nishiwaki, T. and Noda, M. (1994)
Multiple receptor-like protein tyrosine
phosphatases in the form of chondroitin sulfate proteoglycan. FEBS
Lett. 354, 67-70.
Maeda, N., Hamanaka, H., Oohira, A.
and Noda, M. (1995) Purification,
characterization and developmental
expression of a brain-specific chondroitin sulfate proteoglycan, 6B4 proteoglycan/ phosphacan. Neuroscience.
in press.
Oohira, A., Matsui, F., Watanabe, E.,
Kushima, Y. and Maeda, N. (1994)
Developmentally regulated expressionof a brain specific species of chondroitin sulfate proteoglycan, neurocan,
identified with a monoclonal antibody
1G 2 in the rat cerebrum. N euroscience. 60, 145-157.

FOR BASIC BIOLOGYDIVISION OF CELLULARREGULATION
Professor: Norio Mura~a.
.
Associate Professor: H1denon Hayashi
Research Associates: Takao Kondo
lkuo Nishida
Dmitry A. Los
Atsushi Sakamoto
Monbusho Foreign Scientist: Laszlo Mustardy1
Visiting Scientists: Yasushi Tasaka
Byoung Yong Moon 21
Michael P. Malakhov11
Zoltan Gombos"!
Carl H. Johnson 51
Graduate Students: Katsuzo Noguchi
Patcharaporn Deshnium
Sayamrat Panpoom
Technical staffs: Sho-/chi Higashi
Mikilda(1) from Biological Research Center, Szeged,
Hungary)
fl from Inje University, Pusan, Korea)(31 from Plant Physiology Institute, Moscow,
Russia)(41 from Biological Research Center, Szeged,
Hungary)(51 from Vanderbilt University, TN, USA)The research effort of this division is
directed toward understanding the tolerance and acclimation of plants to temperature extremes, with particular emphasis on the molecular mechanisms bywhich plants acclimate to or toleratetemperature conditions. In 1994, several
significant achievements were made ~the following areas with cyanobactenaand higher plants as experimental materials.
I. Molecular cloning and characterization of cyanobacterial desaturases.
Higher plants and most cyanobacteria
have high levels of membrane lipids containing polyunsaturated fatty acids,
which are important in their response toambient temperature. We have isolated
the desA, desB, and desC genes of Synechocystis sp. PCC 6803 that encode theA12, oo3, and A9 desaturases, respectively, of the acyl-lipid type. We overexpressed the desA and desC genes in47
Escherichia coli using the bacteriophage
T7 RNA polymerase system. The desaturases, thus overexpressed in E. coli,
were active in vitro when reduced ferredoxin was supplemented as an electron
donor. The mode of fatty acid desaturation in the transformed E. coli cells demonstrates that the A9 and A12 desaturases are specific to the sn- 1 position of
the glycerol moiety and the C18 fatty
acids, and are nonspecific with regard to
the polar head group of the lipid.

II. Regulation of the expression of thedesaturase genes.
Living organisms are exposed tochanges in ambient temperatures. However, they can maintain levels of molecular motion, or "fluidity," of membrane
lipids by regulating the level of their fatty
acid unsaturation. For example, cyanobacterial cells respond to a temperature
decrease by introducing double bonds
into the fatty acids of membrane lipids,
thus compensating for the temperatureinduced decrease in the molecular motion of membrane lipids. Desaturases are
responsible for the introduction of these
specific double bonds. We have demonstrated that the low temperature-induced
desaturation of the fatty acids of themembrane lipids is regulated at the levelof the expression of the desaturase
genes.
We have shown that the levels of thetranscripts of the desA, desB, and desD
(for A6 desaturase) genes increase about
10-fold during a downward shift of temperature from 34"C to 22"C. However,the level of the transcript of the desC
gene remains constant after the temperature shift, suggesting that the expressionof the gene is constitutive. Determination of the 5'-termini of the desaturases
indicated that the sites of initiation of

48

NATIONAL INSTITUTE

WT

desA ·tdesD·

Figure 1. Changes in composition of lipid molecular species by step-wise depletion of desaturases in Synechocystis sp. PCC 6803. WT, wild-type strain; desD-, a mutant of ~6 desaturase in which the desD gene wasdisrupted by insertion of a chloramphenicol resistance gene cartridge; desA-I des!J, double mutant of ~6and ~12 desaturases in which the desA gene in the des!J mutant was mutated by insertion of a kanamycin
resistance gene cartridge.

transcnpt10n are significantly similar
among all four desaturase genes. Identification of the regulatory elements of thedesaturase genes and cloning of theirtransacting factors are under way.
III. Importance of . membrane-lipid onsaturation in tolerance to low-temperature photoinhibition.
To understand the role of unsaturationof membrane lipids, we disrupted thedesA and desD genes in Synechocystis
sp. PCC 6803 by inserting antibiotic resistance gene cartridges. This mutation
greatly modified the extent of unsaturation of the fatty acids of the membrane
lipids (Figure 1). In the wild-type strain,each of the saturated, monounsaturated,diunsaturated, and triunsaturated lipid
molecules amounted to 15% of the total
membrane lipids; in the desv- mutant,
the triunsaturated lipid molecule was notpresent; in the desA-I desv- mutant,
80% of the total membrane lipids weremonounsaturated lipid molecules andonly 15% were fully saturated molecules. These decreases in the unsaturation of membrane lipids greatly reduced

the tolerance of the cyanobacterium to
low temperature in the presence of light.
These results together with other observations suggest that unsaturated fatty
acids play an important role in protection of the photosynthetic machinery
meant to protect against low-temperature photoinhibition. Because the extent
of photoinhibition in intact cells reflects
a balance between the light-induced inactivation of the photosystem II protein
complex and the recovery of the complex from the inactivation, we separated
the two processes using protein synthesis
inhibitors. The results demonstrated thatthe unsaturation of the membrane lipids
has no effect on the light-induced inactivation process, but accelerates the recovery of the photosystem II complex
from the photoinactivated state. Essentially, the same results were obtained
from studies of a higher plant, namely,
tobacco, in which the level of unsaturation of the thylakoid membrane lipids
was genetically modulated by overexpression of glycerol-3-phosphate acyltransferase from squash.

FOR BASIC BIOLOGY

IV. Heat stability of photosynthesis.
We also focused on the response ofplants to high-temperature stress. Because photosynthesis is one of the physiological processes that is most susceptible to heat stress in plants, stabilizationof the photosynthetic machinery against
heat helps plants to tolerate higher temperature.
We have studied an effect for the heatstability of photosynthesis in the cyanobacterium, Synechococcus sp. PCC
7002. When the thylakoid membranes
isolated from the cyanobacterial cells
were treated with a low concentration of
Triton X-100, the heat stability ofoxygen evolution was decreased by 4"C.
From the extracts obtained with Triton
X-100, we purified a protein that re-established the heat stability of oxygen
evolution. The protein was identified as
cytochrome c-550 which has a low redox
potential and a molecular mass of 16
kDa. These results indicate that cytochrome c-550 is involved in the mechanismof heat stability of oxygen evolution and,
therefore, in the heat stability of photosynthesis. We isolated the gene encoding
this cytochrome of Synechococcus sp.
PCC 7002. Disruption of this gene in
Synechococcus sp. PCC 7002 is in progress in this laboratory. We also studied
the heat tolerance of photosynthesis incultured soybean cells. The heat stabilityof oxygen evolution in these cells wasenhanced by 3"C upon an increase of
growth temperature from 25"C to 35"C.
V. Two genes homologous to groEL in
cyanobacteria.
GroEL is a highly conserved heatshock protein in prokaryotes and a homologue of the eukaryotic heat shockprotein with a molecular mass of about
60 kDa (HSP60). These heat shock49
proteins act as molecular chaperones
that assist proper folding and assemblyof other proteins. We discovered two
kinds of groEL homologue in Synechococcus sp. PCC 7002 and designatedthem as groEL-a. and groEL-B. The .
groEL-a. gene forms an operon together
with the upstream component of thegroES gene, as is the case with thegroEL of E. coli. The groEL-B gene is
not accompanied by the groES gene, but
groEL-B retains the carboxyl terminal
repeat of Gly-Gly-Met as in the E. coli
GroEL.
We mutated the groEL-B gene in
Synechococcus sp. PCC 7002 by inserting an antibiotic resistance gene cartridge and examined the heat shock response of the mutant cells. The heatshock response was greatly reduced in
the groEL-B mutant cells. This wasevaluated from examining the viability athigh temperatures after heat shock treatment, for example, viability at 48"C after
exposure to 45"C. These results demonstrate that not only the groEL-a. but also
the groEL-B gene products contribute to
the heat shock response in the cyanobacterium. However, the thermal toleranceof photosynthetic oxygen evolution was
not modified by the groEL-B mutation,
indicating that GroEL-B does not contribute to the thermal tolerance ofphotosynthesis.
VI. Genetic modification of salinity
tolerance of a cyanobacterium.
Under saline conditions, some plants
produce compatible solutes to avoid
deleterious effects caused by salt intolerance. Glycine betaine, a quaternary ammonium compound, is one of such solute
found in halotolerant plants and bacteria. We discovered that this compound
protects the photosystem II protein

50

NATIONAL INSTITUTE

Figure 2. Growth under saline conditions in control cells (PAM) and transformed cells (PAM COD) in
which the codA gene (for choline oxidase) was introduced in the chromosome of Synechococcus sp. PCC
7942 and was overexpressed under control of the Conll promoter. Cells were grown at 30•c for 6 days in a
medium containing 0.4 M NaCI.

complex from inactivation of oxygen
evolution in saline conditions. To
examine the effect of glycine betaine on
photosynthesis in vivo, we transformed
the salinity-sensitive cyanobacterium,
Synechococcus sp. PCC 7942, with thecodA gene for choline oxidase of Arthrobacter globiformis, which can oxidize
choline into glycine betaine. The transformed cells accumulated glycine betaine

to a concentration of about 85 mM andgrew in the medium containing 0.4 M
NaCl (lethal conditions for the wild-type
cells) (Figure 2). The salinity tolerance ofthe oxygen-evolving activity was enhanced by the transformation. These observations demonstrate the in vivo actionof glycine betaine in protecting the
cyanobacterial cells from salinity-induced stresses.

FOR BASIC BIOLOGY

Publication List:(1) Original papers
Gombos, Z., Wada, H. and Murata, N.
(1994) The recovery of photosynthesis
from low-temperature photoinhibitionis accelerated by the unsaturation of
membrane lipids: a mechanism ofchilling tolerance. Proc. Nat/. Acad.
Sci. U.S.A. 91, 8787-8791.
Gombos, Z., Wada, H., Hideg, E. and
Murata, N. (1994) The unsaturationof membrane lipids stabilizes photosynthesis against heat stress. Plant
Physiol. 104, 563-567.
Kondo, T. and lshiura, M. (1994) Circadian rhythms of cyanobacteria: Monitoring the biological clock of individual colonies by bioluminescence. J.
Bacteriol. 176, 1881-1885.
Kondo, T., Tsinoremas, N.F., Golden,
S.S., Johnson, C.H., Kutsuna, S. andIshiura, M. (1994) Circadian clock
mutants of cyanobacteria. Science
266, 1233-1236.
Los, D.A. and Murata, N. (1994) Lowtemperature induced accumulation of
the desaturase gene transcript in Synechocystis PCC 6803 results from both
acceleration of transcription and increase in mRNA stability. Russian J.
Plant Physiol. 41, 147-151.
Malakhov, M.P., Wada, H., Los, D.A.,
Semenenko, V.E. and Murata, N.
(1994) A new type cytochrome c from
Synechocystis PCC 6803. J. Plant
Physiol. 144, 259-264.
Nishiyama, Y., Hayashi, H., Watanabe,
T. and Murata, N. (1994) Photosynthetic oxygen evolution is stabilized bycytochrome c-550 against heat inactivation in Synechococcus sp. PCC
7002. Plant Physiol. 105, 13131319.
Sakamoto, T., Los, D.A., Higashi, S.,
Wada, H., Nishida, 1., Ohmori, M.

51
and Murata, N. (1994) Cloning of ro3
desaturase from cyanobacteria and its
use in altering the degree of membrane-lipid unsaturation. Plant Mol.
Bioi. 26, 249-264.
Sakamoto, T., Wada, H., Nishida, I.,
Ohm?~' ~· and Murata, N. (1994)
IdentificatiOn of conserved domains in
the L\12 desaturase of cyanobacteria.
Plant Mol. Bioi. 24, 643-650.
Sakamoto, T., Wada, H., Nishida, I.,
Ohmori, M. and Murata, N. (1994)
L\9 acyl-lipid desaturase of cyanobacteria: Molecular cloning and substratespecificities in terms of fatty acids, snpositions, and polar head groups. J.
Bioi. Chern. 269, 25576-25580.
Wada, H., Gombos, Z. and Murata, N.
(1994) Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress. Proc. Nat/. Acad. Sci.
U.S.A. 91, 4273-4277.
(2) Reviews
Murata, N. (1994) Genetic and temperature-induced
modulationofcyanobacterial membrane lipids. In
Temperature Adaptation of Biological
Membranes (A.R. Cossins, ed.), Portland Press, London, pp. 155-162.
Murata, N. (1994) Genetic engineeringof phosphatidylglycerol and chilling
sensitivity in higher plants. In Temperature Adaptation of Biological
Membranes (A.R. Cossins, ed.), Portland Press, London, pp. 163-167.
(3) Proceeding
Hayashi, H., Nishida, 1., Ishizaki-Nishizawa, 0., Nishiyama, Y. and Murata,
N. (1994) Genetically engineered
modification of plant chilling sensitivity ~d characterization of cyanobactenal heat shock proteins. In

52

Biochemical and Cellular Mechanismsof Stress Tolerance in Plants (J.H.
Cherry, ed.), Springer-Verlag, Berlin,
pp. 543-555.

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDMSION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY(ADJUNCT)
Professor (adjunct): Kimiyuki Satoh
Associate Professor (adjunct):
Hirokazu Kobayashi
Research Associates: Satoru Tokutomi
Noritoshi lnagaki
Graduate Students: K yoichi Isono •Yoshihiro Narusaka*
Tatsuya Tomo•
Akira Matsumoto •(*from Okayama University)

From an energetic point of view, theconversion of light energy into chemicalenergy in photosynthesis is the most important biological process on earth. The
highly efficient energy conversion in thisprocess is ensured by the highly orderedorganization of molecules in the photochemical reaction center, in a physical,
chemical and biological sense. The project in this division is aiming to elucidate
the organization of photosystem II reaction center of oxygenic photosynthesis
which has a unique property to generate
a strong oxidant for utilizing the watermolecule as electron donor. In the first
approach, molecular organization of thephotosystem II reaction center, which
has been identified in our study, will beanalyzed by several methods which include crystallographic analysis, chemical
modification & cross-linking analysis andoptical & EPR spectroscopies. Structurefunctional analysis will also be conducted
for the reaction center using random andsite-directed mutagenesis for transformable algae, Synechocystis PCC 6803
and Chlamydomonas reinhardtii. The
principal target of these analyses will be
the structure and molecular environmentof P-680, the primary donor, which
determine the redox potential of thisunique photosystem.
In the second approach, the effort

53
will be focused on the elucidation ofmolecular mechanism of light-regulated
metabolic turnover of a subunit of thephotosystem II reaction center, Dl protein. Some of unique steps are involvedin this process; i. e., photo-damage ofthe function of photochemical reaction
center, specific degradation of the impaired protein subunit, light-regulated
gene expression at the translational level,
post-translational cleavage of the C-terminal extension and the incorporation of
cofactors and subunits into multi-component pigment-protein complexes.
I. Structural organization of photosystem IT reaction center
The structure and molecular interactions of the primary donor in the photosystem II reaction center have been
investigated by detecting light-induced
FT- IR difference spectra upon the formation of its triplet state. From the band
positions of the keto and carbomethoxy
C=O stretches, the hydrogen-bonding
properties of the two chlorophylls of
P-680 were found to be asymmetrical; in
one chlorophyll (Pl) both the keto andcarbomethoxy C=O groups form hydrogen bonds, while in the other chlorophyll (P2) the keto C=O is nothydrogen-bonded whereas the carbomethoxy C=O probably is hydrogenbonded. Considering the orientation of
P-680 analyzed by EPR and the structure of bacterial reaction center determined by X-ray crystallography, together
with the sequence homology between the
Dl and D2 subunits of photosystem II
and the L and M subunits of purple bacteria, a model of the structure of P-680
and its interactions with apoproteins has
been proposed (Noguchi, T., Inoue, Y.
and Satoh, K., 1993, Biochemistry, 32,
7186-7195). Site-directed modification

54
using Chlamydomonas reinhardtii of theamino acid side chains on D 1 protein
presumably involved in the hydrogenbonding interaction (Ser-191 and Thr192) has been succeeded and the analysis of these mutants is now in progressin order to prove this hypothesis.
Chemical cross-linking analysis has
also been conducted for the isolated
photosystem II reaction center to analyze
the gross structure; i. e., the nearest
neighbors of the D1 and D2 subunits
and amino acid residues in cross-contact
in the reaction center complex.
D. Dynamic aspects of the organizationof photosystem ll reaction center(1) The D1 subunit of photosystem II reaction center has a C-terminal extension
consisting of 8-16 amino acids. Posttranslational removal of this part of theprotein is essential for the manifestationof oxygen-evolving function in photosystem II. The enzyme involved in theprocessing has been identified and thepartial amino acid sequences have been
determined for spinach. Based on these
data, a gene coding for the enzyme has
been identified and sequenced (paper in
preparation).
(2) The recognition signal on substrate
was analyzed for the C-terminal processing protease using substituted synthetic
oligopeptides corresponding to the
C-terminal sequence of D1 precursor
protein. The efficiency of these synthetic
oligopeptides, both as substrate and as
inhibitor for the C-terminal cleavage,
were examined. The result suggests thatthe presence of a hydrophobic aminoacid side chain at the -2 position, in addition to the secondary structure aroundthe cleavage site, is crucial in the recognition (Taguchi, F., Yamamoto, Y. and
Satoh, K., J. Biol. Chem., in press).

NATIONAL INSTITUTE(3) The synthesis of D1 precursor protein is regulated by light at the stage oftranslation. The mechanism of this light
regulation was analyzed using isolated
pea chloroplasts. The isolated chloroplasts was shown to accumulate translation intermediate(s) of D1 protein in thepresence of externally added A TP. The
result suggested that the translation of
D 1 protein at the specific stage of polypeptide elongation requires factor(s)caused by illumination, in addition toenergy supply.
(4) Photo-tolerant mutants of an unicellular cyanobacterium Synechocystis PCC
6803 were obtained by in vitro randommutagenesis of psbAII (gene for D1 protein) by PCR under a condition for reduced fidelity of amplification, in orderto analyze the damage-repair cycle ofD 1 protein in photoinhibition of photosystem II reaction center. The mechanistic analysis of photo-tolerance caused bythese amino acid displacements on D 1
protein is now in progress. Figure showsa photo-tolerant mutant (lower), together with the control strain (upper),grown at the photon flux density ofSO,uE (left) and those after exposure to
320,uE (right).
(5) Synthesis of some photosystem proteins are regulated by light signals received by phytochrome. Molecularmechanism of the photoreception ofphytochrome were studied by circulardichroism and resonance Raman spectroscopies. The former shows that thephototransformation of pea phytochrome from Pr to Pfr induces unfolding ofa-helix in the 6-kDa N-terminal domain
which is requisite for its function. The
latter proposes that a proton associatedto the chromophore in Pr migrates to theapoprotein during a phototransformation
process(s) to the bleached intermediate,FOR BASIC BIOLOGY

which presumably triggers conformational changes in the molecule.
Publication List:
Deforce, L., Tokutomi, S. and Song,
P.-S. (1994) Phototransformation ofpea phytochrome A induces an increase in a-helical folding of the apoprotein: Comparison with a monocot
phytochrome A and CD analysis bydifferent methods. Biochemistry 33,
4918-4922.
Mizutani, Y., Tokutomi, S. and Kitagawa, T. (1994) Resonance Raman
spectra of the intermediates in phototransformation of large phytochrome:
Deprotonation of the chromophore in55

the bleached intermediate. Biochemistry 33, 153-158.
Nozawa, T., Kobayashi, M., Wang,
Z.-Y., Itoh, S., lwaki, M., Mimuro,
M. and Satoh, K. (1994) Magnetic circular dichroism investigation on
chromophores in reaction centers ofphotosystem I and II of green plant
photosynthesis. Spectrochimica A eta,
in press.
Torno, T . and Satoh, K. (1994) Nearest
neighbor analysis of D 1 and D2 subunits in the photosystem II reaction
center using bifunctional cross-linker,
hexamethylene diisocyanate. FEES
Lett. 351, 27-30.

56

NATIONAL INSTITUTEDMSION OF BEHAVIOR AND
NEUROBIOLOGY(ADJUNCT)
Professor: Masatoshi Takeichi
Research Associate: Akinao Nose
Kazuaki Tatei
Institute Research Fellow: Tomoko Tominaga
Graduate Student: Tatsuo Umeda
Visiting Scientist: Takeshi Umemiya (from Kyoto
University)

How individual nerve cells find andrecognize their targets during development is one of the central issues in modem biology. The aim of our division is toelucidate the molecular mechanism ofaxon guidance and target recognition byusing the simple and highly accessible
neuromuscular system of Drosophila.
The musculature of Drosophila embryos consists of 30 identifiable muscle
fibers per hemisegment. Each muscle
fiber is innervated by a few motoneurons
in a highly stereotypic manner. The highdegree of precision and previous cellular
manipulations of neuromuscular connectivity suggest the presence of recognition
molecules on the surface of specificmuscle fibers which guide the growthcones of motoneurons.
By using an enhancer trap method,
several genes have previously been
identified that are expressed in small
subsets of muscle fibers prior to innervation, and are thus good candidates forsuch recognition molecules. Two ofthem, connectin and Toll, were shown to
encode cell recognition molecules which
belong to the leucine-rich repeat (LRR)
family. In paticular, Connectin is expressed not only on a subset of muscle
fibers (primarily lateral muscles) but also
on the axons and growth cones of thevery motoneurons which innervate thesemuscles (primarily SNa motoneurons,
see Figure 1) and on several associatedglial cells. When coupled with its ability

to mediate homophilic cell adhesion invitro, these results led to the suggestionthat Connectin functions as an attractive
signal for SNa pathfinding and targeting.
We are currently studying the functionof Connectin by molecular genetic methods and also trying to clone novel
genes implicated in the neuromuscularconnectivity.
I. Molecular genetic analysis of thefunction of Connectin
To study the role of Connectin in vivo,
we first ectopically expressed Connectinon muscle fibers that normally do not
express the molecule by using Tollenhancer. Toll is expressed on ventral
muscles 6, 7, 14-17 and 28. These
muscle fibers do not normally express
Connectin, and are innervated by SNb
motoneurons. We used a 7 kb Toll upstream sequence sufficient for the muscle
expression to express Connectin on thesemuscle fibers in P-element mediatedtransgenic flies (Figure 1).
The analysis of the transformants( Toll-connectin) showed that the development of SNb is abnormal. The SNb
growth cones change their morphology
and their trajectory as they encounterectopic Connectin-positive ventral muscles, displaying "bypass", "detour" and"stall" phenotypes. Moreover, SNb
synapse formation is prevented by Connectin expression on ventral muscles.
These results revealed a repulsive function for Connectin during motoneuron
growth cone guidance and synapse formation.
In Toll-connectin, we did not observe
any abnormality in the SNa motoneurons_ To further study the possible roleof Connectin as an attractive guidance
molecule for SNa, we then ectopically
expressed Connectin on all muscles by

57

FOR BASIC BIOLOGY

using MHC (myosin heavy chain) promoter (MHC-connectin, see Figure 1).

In MHC-connectin, SNa nerves were observed to send an extra axon branch that

MHCconnectin

Tollconnect inwild type15

15

Fig. 1. Ectopic expression of Connectin.
Schematic diagram showing wild type and ectopic Connectin expression on embryonic muscles and motor
nerves. In wild type, Connectin is expressed on a subset of motoneurons (SNa and SNc, shown in red) andmuscles (primarily lateral muscles, shown in yellow). In Toll-connectin, Connectin is ectopically expressed ona subset of ventral muscles (also shown in yellow). In MHC-connectin, Connectin is eCtopically expressed on
all muscles.

Fig. 2. Connectin functions as an attractive guidance molecule.
In wild type, muscle 12 is innervated by SNb motoneurons (short arrow). Connectin-positive SNa moteneurons innervate more distal muscles. Ectopic Connectin expression on muscle 12 in MHC-connectinchanged SNa trajectories: SNa sends an extra axon branch that forms an ectopic nerve ending on muscle 12(large arrow).

58
forms an ectopic nerve endings on
muscle 12, a muscle they would neverinnervate in wild type (Figure 2). Thisphenoype was highly penetrant and wasobserved in over 60% of the segments
examined. Furthermore, the ectopic innervation on muscle 12 was dependent
on the Connectin expression on SNa.
These results showed that Connectinfunctions as an attractive homophilic
guidance molecule for SNa in vivo.
Taken together, the results suggestthat Connectin plays bifunctional roles
during motoneuronal pathfinding andtargetting. One is an attractive andhomophilic function, and another is repulsive and perhaps heterophilic function. Single recognition molecule having
bifunctional roles may be an effective
and economical way to establish precise
patterns of neural connection.
II. Cloning of novel genes implicated in
the neuromnscnlar connectivity in Drosophila
We are conducting molecular andgenetic analysis of two other enhancer
trap lines that are expressed in specificsubsets of muscles and/ or motoneurons.
One of the line, AN34 expresses the reporter gene (,8-gal) in a single muscle
fiber18 per hemisegment. The remarkable specificity in its expression pattern(one out of 30 muscle fibers) makes it agood candidate for the muscle target recognition molecule. The eDNA cloning
and sequencing revealed that AN34 encodes a putative secreted protein withextensive amino acid similarity to rat
F-spondin. F-spondin is a secreted molecule expressed at high levels in the floor
plate and has been shown to promoteneural cell adhesion and neurite extension in vitro. The~e results strongly
suggest that AN34 is involed in moto-

NATIONAL INSTITUTE

neuronal gidance and/ or targeting.
Another line, rQ224 expresses ,8-gal
in a small subset of neurons including an
identified motoneuron RP3. The eDNA
cloning and sequence analysis showedthat it encodes a type II membrane protein homologous to vertebrate dopamine
beta-hydroxylase, an enzyme involved in
catecholamine synthesis. rQ224 thus
may be involved in specific synaptic
functions.
We are currently trying to isolate theloss-of-function mutants of these twolines as well as the transgenic flies that
ectopically express these molecules (asdescribed for Connectin) to study theirroles in the neuromuscular development
and function.
Publication List:
Takaishi, K., Sasaki, T., Kato, M.,
Yamochi, W., Kuroda, S., Nakamura,T., Takeichi, M. and Takai, Y. (1994)
Involvement of Rho p21 small GTPbinding protein and its regulator in the
HGF-induced cell motility. Oncogene
9, 273-279.
Shibamoto, S., Hayakawa, M., Takeuchi,
K., Hori, T., Oku, N., Miyazawa, K.,
Kitamura, N., Takeichi, M. and Ito, F.
(1994) Tyrosine phosphorylation ofb-catenin and plakoglobin enhancedby hepatocyte growth factor andepidermal growth factor in humancarcinoma cells. Cell Adhesion &Commun. 1, 295-305.
Uchida, N., Shimamura, K., Miyatani,
S., Copeland, N.G., Gilbert, D.J.,
Jenkins, N.A. and Takeichi, M.
(1994) Mouse aN-catenin, two isoforms, specific expression in the nervous sytem and chromosomal localization of the gene. Develop. Bioi. 163,
75-85.

FOR BASIC BIOLOGY

Shiomi, K., Takeichi, M., Nishida, Y.,
Nishi, Y. and Uemura, T. (1994) Alternative cell fate choice induced by
low-level expression of a regulator ofprotein phosphatase 2A in the Drosophila peripheral nervous system.
Development 120, 1591-1599.
Uchiyama, N., Hasegawa, M., Yamashina, T., Yamashita, J., Shimamura,
K. and Takeichi, M. (1994) Immunoelectron microscopic localization ofE-cadherin in dorsal root ganglia,
dorsal root and dorsal hom of postnatal mice. J. Neurocytol. 23, 460-468.
Shimamura, K., Hirano, S., McMahon,
A.P. and M. Takeichi. (1994) Wnt1-dependent regulation of local
E-cadherin and aN -catenin expression
in the embryonic mouse brain. Development 120, 2225-2234.
Watabe, M., Nagafuchi, A., Tsukita, S.
and Takeichi, M. (1994) Induction ofpolarized cell-cell association and retardation of growth by activation of
the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J.

59
Cell Bioi. 127, 247-256.
Tong, K.l., Yau, P., Overduin, M.,
Bagby, S., Porumb, T., Takeichi, M.
and Ikura, M. (1994) Purification andspectroscopic characterization of arecombinant amino-terminal polypeptide fragment of mouse epithelial
cadherin. FEBS letters 352, 318-322.
Nose, A., Takeichi, M. and Goodman,
C.S. (1994) Ectopic expression ofconnection reveals a repulsive function
during growth cone guidance and
Neuron 13,
synapse formation.
525-539.
Oda, H., Uemura, T., Harada, Y., Iwai,
Y. and Takeichi, M. (1994) A Drosophila homolog of cadherin associated with Armadillo and essential forembryonic cell-cell adhesion. Develop.
Bioi. 165, 716-726.
Hatta, M. and Takeichi, M. (1994)
Complex cell type-specific transcriptional regulation by the promoter andan intron of the mouse P-cadherin
gene. Develop. Growth & Differ. 36,
509-519.

61

FOR BASIC BIOLOGYLABOLATORY OF GENE EXPRESSION
AND REGULATION

Head: Goro Eguchi

The Laboratory consists of three regular divisions
and conducts research into regulatory mechanisms ofgene expression in higher plants and animals.

62

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION
AND REGULATION I
Associate Professor: Kiyotaka Okada
Research Associate: Sumie lshiguro
Graduate Students: Azusa Yano
Yoichi Ono
Tashiro /tal)
Takuji Wada
Tokitaka Oyama11
Sinichiro Sawa21
Technical Staff: Hideko Nonaka
AkikoKawai(I) from Kyoto University)~~from Nagoya University)

The principal interest of this laboratory is molecular genetic studies on theregulatory systems of organ development
and on growth control by several environmental stimuli in higher plants. For
these studies, we have mainly used asmall crusifer, Arabidopsis thaliana. This
plant is called "botanical Drosophild',
because it has some remarkable features,such as a small genome size (1 X 108
base pairs per haploid), short life-cycle(5-6 weeks), small size (20-30 em inheight), and ease of propagation. Thesefeatures make the plant ideally suited forgenetic and molecular biological studies.
In addition, more than 360 loci andmore than 650 RFLP and RAPD markers are mapped on 5 chromosomes. Experimental techniques such as transformation, regeneration of transgenic plants
and gene tagging have been improved.
Using this plant, we have isolated andcharacterized many mutants defective inflower development and morphogenesis
or in root formation and responses toward physical stimuli such as gravity,
light or touching.
I. Development and morphogenesis offlowers
The process of flower development
can be divided into four major steps:
phase transition from vegetative toreproductive growth, formation of inflorescence meristem, formation and identity determination of floral organs, andgrowth and maturation of floral organs.
Several different types of signalingmechanism, between or within cells,
must have important roles in each stepof flower development, because eachstep requires cell division, cell growthand cell differentiation in a concerted
fashion. In order to unveil the signalingmechanism and its genetic regulatory
systems, we are carrying out genetic, biochemical, anatomical and physiologicalanalyses on the processes of flower development using A. thaliana.
The filamentous flower (fil) mutant
shows several structural defects inflowers and inflorescences. After flower
initiation, fil mutant elongates an inflorescence axis normally, and forms about
10 flowers that show some structural abnormalities (phase 1). Then formation offloral buds is stopped at the inflorescence meristem, and instead, more than
10 filaments and more than 10 sepal-likestructures are formed (phase 2). After
forming the phase 2 cluster, a cluster offlowers is formed again (phase 3). One
possible explanation of the phase shift isthat the amount of the factor(s) or theamount of receptor molecules of the factor(s) changes to high-low-high as plants
grow. In order to examine the genetic relationships of FIL gene with otherknown flower genes, we have constructed a series of double mutants. The
results indicate that FIL gene is required
for formation and maintenance of floral
meristem in combination with APETALA 1, LEAFY and CAUL/FLOWER.
The AGAMOUS (AG) gene of A.
thaliana is a homeotic gene involved in
the development of stamens and carpels.

FOR BASIC BIOLOGYThis gene encodes a putative DNAbinding protein sharing a homologous
region with the DNA-binding domains,
MADS boxes, of yeast MCM1 andmammalian SRF. We confirmed that theAG protein is localized in the nuclei ofthe cell in stamen- and carpel-primodia.
In order to identify AG target genes, we
isolated the DNA fragments bound by
AG protein in native chromatin by immunopurification, and then identified
genes within or near the isolated fragments. Characterization of these genes isin progress.
D. Development and stimulus-response
reactions in root

The root of A. thaliana has many advantages of simplicity, transparency andstrict organization of cells to analyze thedevelopment of roots. One of the thickroot mutant, RH32, has 10 cortical cells
in the root, whereas wild type has 8 cortical cells. The epidermal cells of RH32
mutant in different tissues, such as root,
hypocotyl, stem and sepal, are rounderthan those of wild type. RFLP mapping
has shown that RH32 gene is located on
the lower portion of chromosome 1.
A T-DNA-tagged mutant K293 has a
few root hairs of the primary root. Map
position of K293 gene is on the lower
portion of chromosome 2. We are now
in progress of cloning of this gene.
Normal root hairs of A. thaliana areshort and formed perpendicular to theroot surface, when roots grow on agar
plates stood vertical ("agar" form). Thelong and tilted root hairs are formed
when roots grow in the air ("air" form).
We showed that the "agar" form hairs
are formed by the cells that have passed
through the elongation zone, whereas
the "air" form hairs are generated within
the elongation zone. Mutants defective

63

in these morphological alteration of root
hairs are isolated and classified into
three categories: namely, mutants generating root hairs of either the "agar" or
the "air" form in both condition (class I);
mutants defective in the "agar" form
hair formation but normal in the "air"form hair formation (class II); and mutants defective in both hair formation(class III).
Roots alter their growth direction
when their relative orientation against
gravity is changed (gravitropic response),
when they are illuminated from aside(phototropic response), or when they encounter obstacles (obstacle-escaping response). These responses were analyzed
using agar plates. Young seedlings grownon vertical agar plates have roots which
grow straight downward on the agar surface. When the plates are put aside,roots bend 90° and grow to the altereddirection of gravity. If the plates arecovered with black cloth and illuminatedfrom a side, roots grow to the opposite
direction of the incoming light. On thesurface of agar plates which are set at anangle of 45o to the vertical, the roots exhibit a wavy growth pattern that iscaused by periodic reversion of rotationof the root tip. Using these systems, mutants which show abnormal graviresponse, photoresponse, or obstacle-escaping responses were isolated. Several
mutants with abnormal obstacle-escaping
response also show abnormal gravitropism and/ or phototropism. These results
indicate that root gravitropic, phototropic and obstacle-escaping responses
share at least in part a common genetic
regulatory mechanism. Physiologicalcharacterization and molecular cloningof these mutants are in progress.
A mutant hy5 of A. thaliana, which
was isolated as a long hypocotyl mutant,

64

NATIONAL INSTITUTE

Filamentous flower mutant forms clusters of deformed flowers and of filaments with a sepal-like structure
at its top (left). In leafy mutant, flowers are homeotically transformed to shoots (middle). A double mutant
carrying mutations in both FILAMENTOUS FLOWER and LEAFY genes fails to generate either flowers or
shoots, and instead continues to form filaments and sepal-like structures (right).

shows a variety of abnormalities in development and stimulus-response ofroots. HYS gene therefore plays a key
function in signaling pathways of root
morphogenesis as well as photomorphogenesis of hypocotyl. We have isolated a hy5 mutant allele from T-DNA
inserted lines and cloned the putative
HYS gene. This gene encodes an open
reading frame of 168 amino acidresidues which include a bZIP motif at
the C-terminus. Therefore, HYS gene
may function as a transcriptional regulator in the signaling pathways.
III. Technical improvement for molecnIar cloning
In order to isolate the genes responsible for the mutants, some technical improvements for gene tagging systems in
A. thaliana were attempted. For such experiments, it is absolutely necessary todevelop a good, efficient system of transformation mediated by Agrobacterium.
We carried out so called in planta transformation, in which adult A. thaliana
plants are directly infected with Agrobacterium and gene transfer occurs during floral development. In this system, it
is expected that problems caused by somaclonal variation are avoided. About
1,100 lines of transformants were generated. Several interesting mutants havebeen identified and characterized.
Publication List:
Okamoto, H., Yano, A., Shiraishi, H.,
Okada, K. and Shimura, Y. (1994)
Genetic complementation of a floral
homeotic mutation, apetala 3, with an
Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum
majus. Plant Mol. Bioi. 26, 465-472.

FOR BASIC BIOLOGY

Ito, T., Shiraishi, H., Okada, K. andShimura, Y. (1994) Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different
organs. Plant Mol. Bioi.
26,529-533.
Okada, K. and Shimura, Y. (1994)
Genetic analyses of signaling in flower

65
development using Arabidopsis. Plant
Mol. Bioi. 26, 1357-1377.
Okada, K. and Shimura, Y. (1994)
Modulation of root growth by physicalstimuli. In Arabidopsis (E.M. Meyerowitz and C. Somerville, eds. ). Cold
Spring Harbor Lab. Press, pp.665684.

66

NATIONAL INSTITUTE

DNISION OF GENE EXPRESSION
AND REGULATION II
Professor: Takashi Horiuchi
Research Associate: Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodilma
Institute Research Fellow: Katsuki Johzuka
Graduate Student: Katsufumi Ohsumi
Keiko Taki
Technical Staff: Yasushi Takeuchi

Homologous
recombination may
occur in all organisms. While related
functions apparently involve exchange
between two parent-derived chromatids
and repair of DNA damage incurred byphysical and chemical reagents, manyquestions remain unanswered. As deduced from our analyses of recombinational hotspots of E. coli & S. cerevisiae,in particular the activity related to DNA
replication fork blocking events, thephysiological function of homologous
recombination, especially in normallygrowing cells is better understood.
I. Analysis of E. coli recombinational
hot spots, Hot.
We identified hotspots of recombination (termed Hot) on the E. coli chromosome. These sites are specifically activated under rnh- (RNase H-defective)
conditions (Nishitani et al., MGG 240,
307, 1993). Analysis of these Hot led todesign of a putative model, in which theds(double stranded)-break occurs at thefork arrested at the DNA replication
fork blocking (called Ter) site, throughwhich the RecBCD recombinational. enzyme enters the ds-DNA moleculeand enhances recombination between directly repeated Hot DNA, when the
enzyme meets an appropriately oriented
Chi sequence.
If the ds-break is induced by replication fork arrest, physiological effects on
the cell would be evident. To elucidate

effects of fork arrest at Ter sites, we constructed an E. coli strain, termed ablocked strain, in which oligonucleotides
with the TerA sequence were inserted
into the lacZ gene in an orientation so as
to block the fork of clockwise (normal
direction) replication. In this strain, replication forks were expected to be
blocked at both the TerL in the lacZ andat TerA, D and E sites. While theblocked strain grew somewhat more
slowly than a control strain, it had abnormal phenotypes similar of E. coli
dam- mutants, i.e., hyper-Rec phenotype, recA +- and recB+ ( c+)-dependent
growth, and constitutive SOS expression.
We propose that the following sequential
events may occur in both strains (seeFigure 1). A ds-break occurs at theblocked replication fork in the blockedstrain and at the ongoing fork in thedam- mutant, through which RecBCD
enzyme enters and degrades the dsDNA molecule, and the degradation
product serves as the signal molecule for
SOS induction. When RecBCD enzyme
meets an appropriately oriented .Chi sequence, its DNase activity is converted
to recombinase enzyme, which is able toform a new replication fork, recombinationally with RecA and SSB proteins.
This model (i) explains the puzzling
phenotype of recA- and recB- (c-) mutants and SOS-inducing phenotype of
po/A, lig and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence,and (iii) suggests a possible key role forhomologous recombination with regard
to cell survival following the arrest of
DNA replication (Horiuchi and Fujimura; J. Bacteriol. in press).

67

FOR BASIC BIOLOGY

degradation

SOS signal?

to Chi by
RecBCD

RecBCD enzyme

Chi

1)

!)

Chi

I

(4)

.

(3)
(5)

D loop formation(RecA)~~ ds-break

(

(1)

(6)

(7)

~

•

new fork formation(RuvABC)

•

(8)

: \

~

DNA fork blocking·····)lo.J

mo.ement~

Fig. 1. Model of rescue from blocked DNA replication in E. coli, as applicable to a spontaneously stalled
replication fork. Essentially the same sequential reactions would probably occur in S. cerevisiae.

D. Analysis of recombinational hot
spot in S. cerevisiae.
HOT1 is a mitotic recombinational
hotspot in the yeast S. cerevisiae and was
first identified by Keil and Roeder.
HOT1 stimulates both intra- and interchromosomal recombination, and for a
precise analysis enhancement of excisional recombination between directly
repeated DNAs at its nearby site was investigated. HOT1 was originally cloned
on a 4.6 kb Bglll fragment which locatesin rRNA repeated genes (about 140
copies) on chromosome XII. A single
rRNA unit consists of two transcribed
35S and 5S rRNA genes and two nontranscribed regions, NTS1 and NTS2,
the former is between 3'-ends of 35S and
5S rRNA genes, and the latter is 5' endsof these two genes. The HOT1 DNA
fragment contains the NTS1, 5SRNA
gene 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 functionallyoverlapped 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 is blocked and we termed
this site SOG. By assaying Sog activity
for various DNA fragments derived fromthe NTS 1 and cloned on plasmids, we
determined the minimal region, about
100 bp long, located near the enhancer
region of the 35S rRNA transcription
and essential for blocking replication
fork advancing in a direction oppositethat for transcription. The SOG sequence has no homology to any otherknown sequence and has no characteris~tic structure such 2-fold symmetry,

68
repeated structure, etc.; hence, trans-factor(s) may have a role in blocking thefork. Interestingly, this region is included
in one of two cis-elements required for a
recombinational hotspot, Hot1, activity.
To investigate functional relationships
between the fork blocking activity in
SOG and the hotspot activity in HOT1,
we first isolated mutants defective in
Hot1 activity and examined whether
these mutations would also affect Sog
activity. Using a colony color sectoring
assay method, we isolated 23 Hot defective mutants from approximately 40,000
mutagenized colonies. Among these
HoC mutants, one proved to be a rad52
mutant; the other 4 mutants lose fork
blocking activity. Genetic analysis ofthese mutants revealed that all four wererecessive for the Sog phenotype anddefined one complementation group.
This mutation was designated fobl (fork
Qlocking function) and one of the foblmutants, fob 1-4, was further analyzed.
First, from yeast eDNA bank, we cloned
FOB gene by selecting a DNA fragment
which had suppressive activity for Hot
deficiency of the mutant. The minimal
FOG plasmid was shown to complement
both the Hoc and Sog- phenotype ofthe fob 1-4 mutant, suggesting that both

NATIONAL INSTITUTE

phenotypes are caused by a mutation in
the FOBJ gene. DNA sequencing of the
FOBJ gene revealed that the putative
Fob1 protein consists of 566 amino acids
and has a molecular mass of 65,000 daltons. This gene has no homology withany DNA sequence registered in Genbank. Sequencing of the fobl-4 mutant
gene revealed two mutational changes in
the open reading frame, one is non-sense(amber) and other is a miss-sense mutation. The amber mutation may accountfor the two defective phenotypes of thefobl-4 mutant and why it is non-leakyness.
Because there is an extraordinary
functional similarity between factors required for the hotspot activity in E. coliand S. cerevisiae, in both organisms dsbreak and repair would occur when the
DNA replication fork is arrested at fork
blocking sites and probably was spontaneously blocked (Figure 1).
Publication List:
Horiuchi; T., Fujimura, Y., Nishitani, T.,
Kobayashi, T. and Hidaka, M. (1994)
The DNA replication fork blocked at
the Ter site may be entrance for the
RecBCD enzyme into duplex DNA. J.
Bacteriol. 176, 4656-4663.

FOR BASIC BIOLOGYDIVISION OF SPECIATIONMECHANISMS
Professor: Tetsuo Yamamori

Our research goal is to understandmechanisms underlying evolution of thenervous system. To approach this question, we are currently focusing on two
different subsystems which may respectively represent some molecular and cellular aspects of the system.
I. Evolution of cytokine receptor
families in the immune and nervous
systems
Recently, it has been recognized thatcytokines, defined as inter-cellular mediators in the immune system, have a variety of roles in the nervous system aswell. 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. Recent
study have revealed that CDF/LIF and
its receptors belong to the IL-6 family
and the receptor family.
Bazen, first pointed out that the cytokine receptor family may be evolved
from an ancestral module of immunoglobulin (1990). Based on Bazen's modeland the model of the interaction 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. Firstly, binding subunits ofan IL-6 receptor and for a CDF/LIF receptor evolved and secondly, a thirdbinding subunits of a CNTF receptor
evolved. Our evolutional consideration
predicts that the binding subunits gene-69
rally determine the specificity of the receptors and it is possible that novel
members of the cytokine family andtheir receptors exist in the nervous system.
D. Gene expression and cerebellar
long-term plasticity
In order to know roles of the genes involved in long-term memory, we choose
the cerebellum as a model system. It has
been demonstrated by Ito and his colleagues (1982) that in the cerebellum theconjunctive stimuli of a parallel fiber anda 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).
Previously, we examined the expression of 10 immediate early genes (lEGs)
including all the known Fos and Jun
family in cerebellar slices under thepharmacological 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 in vivo under a conjunctive
protocol of AMPA, a pharmacologicalsubstitute for parallel fiber stimulation,and climbing fiber stimulation via electric Inferior Olive stimulation. Jun-B are
predominantly induced around the local
area where the AMPA and climbing
fiber stimulation were conjuncted(Yamamori eta/., 1995; Neuroreport, inpress). These results suggest that thecoincidence mechanism may exist at
gene expression level and lead to a cerebellar long-term plasticity.

70

NATIONAL INSTITUTE~

1

classiAIL-ZR,IL-3R,ILSR,GM-CSFR~Cytokine

original gp 1 30 (Receptors-like molecule
G-CSFR
hematopoietic+classlla primordial, cell surface
lg domain

gene duplication

hematopoietic/l•
Receptors
forll-6, IL-11(gp 130+gp 130
SO/IL- 11 R? )+gp·

original module of IGSF~
Receptors for~ronal

CDF/LIF, ONC-M(gp 1 30+LIFR )+Receptors
forCNTF
Receptors
forONC-M?

(gp130+LIFR+CNTFR)(gp130+LIFR+ONC-M?)
Fig. 1. Evolution of the IL-6 receptor family in the immune and nervous system (a Model). Modified fromthe figure 4 of Yamamori and Sarai (1994) including recent results from Hilton et al. (EMBO J., 13,4765-4775, 1994.)

71

FOR BASIC BIOLOGY

Pararell Fiber(AMPA)

Fig. 2. Gene Expression and Long-term Memory (a Model)

Publication List:

Yamamori, T. and Sarai, A. (1994)
Evolution of the IL-6/ class IB cytokine receptor family in the immune
and nervous systems. J. Physiology(Paris). 88, 165-171.

72

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head: Hiroyuki Hattori
Common Facility Group
Chief: Kazuhiko Furukawa
Research Support Facilities
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
TomokiMiwa
Kaoru Sawada
Kimiko Yamamiya
Radioisotope Facility
Y ousuke Kato
Yosimi Matsuda
Center for Analytical Instruments
Yukiko Kabeya
Yumiko Makino
Sonoko Ohsawa
Takeshi Mizutani
Glassware Washing Facility(Kazuhiko Furukawa)(Toshiki Ohkawa)

The Technology Department is a supporting organization for researchers andresearch organization within the NIBB.
The Department develops and promotes
the institute's research activities and at
the same time, maintains the researchfunctions of the institute.
The Department is organized into twogroups: one, the Common FacilityGroup, which supports and maintains
the institute's common research facilities
and the other, the Research Support
Group, which assists the research activities as described in individual reports.
Technical staffs participate, throughthe department, in mutual enlightenment

Research Support Group
Cell Biology Group
Akio Murakami (Unit Chief)
Masayo Iwaki
MakiKondo
Katsushi Yamaguchi
Developmental Biology Group
Hiroko Kobayashi (Unit Chief)
Miyuki Ohkubo
Sachiko Fukada
Chikako Inoue
Chiyo Takagi
Hisae UraiRegulation Biology Group
Shoichi Higashi
MikiidaTomokoMori
Shigemi Ohsugi
Gene Expression and Regulation Group
Hideko Nonaka
Koji Hayashi
AkikoKawai
Yasushi Takeuchi

and education increase their capability intechnical area. Each technical staff isproceeded to the fixed division usuallyand they support the various researchwith their special biological and biophysical techniques.
The Department hosts an annual
meeting for technical engineers who
work in various fields of biology atuniversities and research institutes
throughout Japan. At this meeting, theparticipants present their own activities
and discuss technical problems. The Proceeding are published soon after the
meeting.

73

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Head of Facility: Yoshiaki Suzuki
Associate Professor: Masakatsu Watanabe
Research Associates:
Yoshio Hamada (Tissue and Cell Culture)
Kenta Nakai (Computer)
Technical Staff: Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
TomokiMiwa
Kimiko Yamamiya

The Facility provides large- and medium-scale equipments and facilities forbiophysical, molecular biological, andcomputational analyses as well as for
growing and maintaining biological specimens. The facility is shared among the
research members, and has sevenlaboratories, among which the Large
Spectrograph Laboratory and the Laboratory of Stress-Resistant Plants are
dedicated to cooperative use under the
NIBB Cooperative Research Programs.
I. Facilities
1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, the Okazaki Large Spectrograph (OLS), which is the largest spectrograph in the world, dedicated toaction spectroscopical studies of various
light-controlled biological processes. The
spectrograph runs on a 30 kW-Xenon
arc lamp and has a compound gratingsurface composed of 36 smaller individual gratings. It projects a spectrum of awavelength range from 250 nm (ultraviolet) to 1,000 nm (infrared) onto its
focal curve of 10 m in length. The
fluence rate (intensity) of the monochromatic light at each wavelength is morethan twice as much as that of the corresponding monochromatic component of
tropial sunlight at noon (Watanabe etal., 1982, Photochem. Photobiol., 36,

491-498).
A tunable two-wavelength CW laser
irradiation system is also available as acomplementary light source to OLS tobe used in irradiation experiments which
specifically require ultra-high fluence
rates as well as ultra-high spectral-, timeand spatial-resolutions. It is composed ofa high-power Ar-ion laser (Coherent,
Innova 20) (336.6-528.7 nm, 20 W output), two CW dye lasers (Coherent, CR599-01) (420-930 run, 250-1000 mW
output), A/0 modulators (up to 40
MHz) to chop the laser beam, a beam
expander, and a tracking microbeam
irradiator (up to 200 11m s- 1 in tracking
speed, down to 2 11m in beam diameter)with an infrared phase-contrast observation system.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell
culture are provided. This laboratory isequipped with safety rooms which satisfy
the P2/P3 physical containment level.
This facility is routinely used for DNA
recombination experiments.
3. Computer Laboratory
To meet various computational needes
in this Institute, various computers areequipped: VAX/VMS machines, UNIX
workstations (SPARCstation 10 andothers), and personal computers of various vendors. Through the Ethernet or
the CDDI, all of them are linked to the
backbone FDDI of this institute, which isfurther linked to the hyper-multimedia
network of Okazaki National ResearchInstitutes (the ORION network). Eachlaboratory has several computers connected to the network and full resourcesof the Internet are accessible from there.
In addition, NetWare server machines

74
work as file servers and printer servers.
Latest databases and various useful softwares are also maintained. Informationof this institute, including this document
is open to the world through the World
Wide Web (WWW; URL ishttp:/ I
www.nibb.ac.jp).
4. Plant Culture Laboratory
There are a large number of cultureboxes, cubicles, and a limited number ofrooms with environmental control forplant culture.
5. Experimental FarmThis laboratory consists of two 20 m2
glass-houses with precision temperatureand humidity control, a limited farm,
two greenhouses (45 m2, 88 m2) with
automatic sprinklers and window control
two open aquariums (30 t, 50 t) and several smaller tanks. The laboratory also
includes a building with office, storage
and work-space.
6. Plant Cell Culture Laboratory
Autotrophic and heterotrophic culture
devices and equipped for experimental
cultures of plant and microbial cells.
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 (30 km from the National Institutefor Basic Biology). The laboratory provides a variety of growth chambers thatprecisely control the conditions of plant
growth and facilities for molecular biological, biochemical, and physiologicalevaluations of transgenic plants. The laboratory is also a base of domestic andinternational collaborations devoted tothe topic of Stress-Resistant Transgenic
Plants.

NATIONAL INSTITUTE

D. 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 andsignal transduction mechanisms ofphototaxis of single-celled, flagellate
algae are studied action spectroscopically(Watanabe (1995), in CRC Handbookof Organic Photochemistry and Photobiology, in press) by measuring computerized-videomiceographs of the motile
behavior of the cells at the cellular andsubcellular levels (Erata et al. (1995)
Protoplasma, in press). Photoreceptive
and signal tranduction mechanisms ofalgal gametogenesis are also studied byaction spectroscopy.
(2) Developmental Biology: Myosin is aubiquitous protein in eukaryotes. It is
presumed that this molecule plays a keyrole in cell motility as well as other functions. The role of myosis in non-muscle
cells might be better understood if wildtype cell could be compared with mutant
cells with altered myosin molecules,
though such mutants have not been isolated. The faculty intends to examine thefunction of the myosin heavy chain invertebrate non-muscle cells by interrupting its synthesis with the introduction ofantisense RNA molecules.
(3) Computational Biology:. Efforts todevelop new methodology for sequence
analysis have been continued. One of the
projects is to develop an expert system
to predict various protein localizationsites from amino acid sequence data.
This work is done in collaboration with
Drs. S. Miyano (Kyushu Univ.), T.
Shimizu (Hirosaki Univ.) and A. Bairoch(Geneva Univ.). Some results have been
Published in Shimizu and ZK. Nakai

FOR BASIC BIOLOGY(Proc. Genome Informatics Workshop
1994, 148-149) and in K. Nakai, A.
Shinohara, and S. Miyano (Proc. GIW
1994, 170-171). Another is to constructa prediction system of mature mRNA
sequence from their precursors. It is incollaboration with Drs. H. Sakamoto(Kobe Univ.), Y. Akiyama (Kyoto
Univ.) and S. Stamm (Cold Spring
Harbor Lab.). The activities of computerassistance for other experimental researchers can be exemplified in M.
Tomita, J. Kobayashi, AA. Mori, K.
Hagiwara, K. Nakai, Y. Suzuki, and S.
Miyajima, Protein Eng., 7, in press.
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 conductedby visiting scientists including foreignscientists as well as those in the Institute.
Action spectroscopical studies forvarious regulatory and damaging actionsof light on living organisms, biologicalmolecules, and organic molecules havebeen conducted (Watanabe (1995), in
CRC Handbook of Organic Photochemistry and Photobiology, in press).
Publication List:
I. Faculty
Nakai, K. and Sakamoto, H. (1994)
Construction of a novel database containing aberrant splicing mutations ofGene, 141,mammalian genes,
171-177.
Nakai, K., Tokimori, T., Ogiwara, A.,
Uchiyama, 1., and Niiyama, T. (1994)
Gnome - an Internet-based sequence
analysis tool, Comp. Appl. Biosci., 10,547-550.

75
II. Cooperative Research Program for
the Okazaki Large Spectrograph
Andrady, A.L., Amin, M.B., Hamid,
S.H., Hu, X. and Torikai, A. (1994)
Effects of increased solar ultraviolet
radiation on materials. In Environmental Effects of Ozone Depletion:
1994 Assessment (J.C. van der Leun,
X. Tang and M. Tevini, eds.), United
Nations Environment Programme(UNEP), Nairobi. pp. 101-110.
Hashimoto, T. (1994) Requirements ofblue, UV-A, and UV-B light for normal growth of higher plants, as assessed by action spectra for growth
and related phenomena. In Proceedings of International Lighting in Controlled
Environments
Workshop(T.W. Tibbitts, ed.), NASA Ames
Research Center, Moffett Field, CA.,
USA, pp. 143-157.
Hashimoto, T. (1994) UV-B effects onplant: The present knowledge andprospect for future studies. In Proceedings of the Tsukuba Ozone Workshop - Grobal Environment Tsukuba'94, (K. Takemoto and S. Nishioka,eds.), Center for Grobal Environmental Research, Japan, pp. 134-145.
Nikaido, 0., Ohta, M. and Matsunaga,T. (1994) Detection of the Dewar
photoproducts in solar light-exposed
DNA by a damage specific monoclonal antibody and the repair of thephotoproducts in human cells. In The
13th UOEH International Symposium& The 2nd Pan Pacific Cooperative
Symposium on Inpact of Increased
UV-Exposure on Human Health andEcosystem (Y. Kodama and Si Duk
Lee, eds.), University of Occupational
and Environmental Health, pp. 190193.
Sasaki, M., Takeshita, S., Sugiura, M.
and Sakata, T. (1994) An increase in

76
the global solar ultraviolet-B irradiance at 35oN in Japan since 1990.
J. Geomag. Geoelectr. 46, 827-834.
Todo, T., Ryo, H., Takemori, H., Toh,
H., Nomura, T. and Kondo, S. (1994)
High-level expression of the photorepair gene in Drosophila ovary and its
evolutionary implications. Mutation
Research, DNA Repair 315, 213228.

NATIONAL INSTITUTE

77

FOR BASIC BIOLOGY

facility is being used for molecular analyses of organisms. A laboratory facility
for recombinant DNA research is installed in the center. The sub center in
the NIBB is also equipped with a recombinant DNA research laboratory. The
members of the Radioisotope Facility
maintain and control the center and subcenter, and provide users appropriate
guidance for radioisotope handling.

RADIOISOTOPE FACILITY(managed by NIBB)Head of Facility: Takashi Horiuchi
Techinical Staffs: Kazuhiko Furukawa (Radiation
Protection Supervisor)
YosukeKato
Yoshimi Matsuda (Radiation
Protection Supervisor)

The facility is composed of a main
center and two subcenters, one in the
NIBB and the other in the NIPS. The

Appendix: Maximum Permissible Quantities and Nuclides
Maximum Permissible Quantity
Place

NuclideYear

3 Months

Week

Day

liquidliquidliquidliquidliquid150 MBq
2GBq
2GBq
500MBq

150 MBq
2GBq
2GBq
500 MBq

150 MBq
1400MBq
1400MBq
350 MBq

15GBq

7.215 GBq

555 MBq

50MBq
200 MBq
200 MBq
50MBq
185 MBq2sMg
32p

liquidliquid500 MBq
30GBq

500MBq
30GBq

100 MBq
3500MBq

35S
42K

30GBq
500MBq
20GBq
5GBq

30GBq
500 MBq

3H
14C

liquidliquidliquidliquid. 20 GBq
5GBq

3500 MBq
500MBq
3500MBq
3500MBq

500MBq
500MBq
100MBq
500 MBq
500 MBq32p

liquid

7.4 GBq

7.4 GBq1295 MBq

185 MBq

35S

liquid3H
14C

liquidliquid

7.4 GBq
7.4 GBq
740MBq

7.4 GBq
6.73 GBq
740MBq1295 MBq
518 MBq
518 MBq

185 MBq
74MBq
74MBq

45Ca
1251
32p

liquidliquidliquid

3.7 GBq
1.85 GBq
3.7 GBq

3.36 GBq
1.68 GBq

259 MBq
129.5 MBq

37MBq
18.5 MBq
111 MBqliquid

3.7 GBq

3.7 GBq
3.7 GBq

777MBq

35S

518 MBq

74MBq

3H
14C

liquidliquid

7.4 GBq
740MBq

6.73 GBq
740 MBq

518 MBq
518 MBq

74MBq
74MBq

22Na
36Q
45Ca
89Sr
1251
Main center

NIBB
subcenter

NIP
subcenter

State

50MBq

78

NATIONAL INSTITUTECENTER OF FACILITY FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head of Facility: Mikio Nishimura
Technical Staffs: Yukiko Kabeya
Yumiko Makino
Sonoko Ohsawa
Takeshi Mizutani

This center provides analytical instruments for biological and biophysicalstudies. These instruments consists of thefollowing four groups and are supported
by experienced technical staff. Eachgroup is equipped with instruments forgeneral use as listed below.
Instruments for Protein and Gene
Analysis
Amino Acid Analyzer
HITACHI 835
Automatic Plasmid Isolation System
KURABO PI-100~
DNA Sequencer
ABI 370A, 373A-36
DNA/RNA Synthesizer
ABI 381A, 392
Nucleic Acid Extractor
ABI 340A
Peptide Synthesizer
ABI 430A, 431A
Portein Sequencer
ABI 470A, 473A
Instruments for Chemical Analysis
Capillary Electrophoresis System
ABI 270A
Gas Chromatograph
SHIMADZU GC-7APfF,
GC-14APFSC
Glycoprotein Analysis System
TAKARA Glyco-Tag
High Performance Liquid
Chromatograph
SHIMADZU LC-lOAD, 6AD
Micro Preparative Electrophoresis
System
ABI 230A
Preparative Ultracentrifuge
BECKMAN L8-80
Micro Preparative System
Pharmacia SMART System

Instruments for Physical andSpectroscopic Analysis
Atomic Absorption
Spectrophotometer
PERKIN-ELMER 603
Dual Wavelength Spectrophotometer
HITACHI 557
EPR Spectrometer
BRUKER ER 200D
GC/MS Spectrometer
HITACHI M-80,
JEOL DX-300
Inductively Coupled Plasma
Spectrometer
SEIKO SPS 1200A
Infrared Spectrophotometer
JASCO A-302
Laser Raman Spectrophotometer
JASCO R-800
Light Scattering Photometer
CHROMATIX KMX-6DCNMR Spectrometer
BRUKER AMX 360wb
Spectrofluorometer
HITACHI 850
SHIMADZU RF-5000
Spectrophotometer
HITACHI 330
Spectropolarimeter
JASCO J-40S
Instruments for Microscopic andImage Analysis
Bio Imaging Analyzer
FUJIFILM BAS 2000
Confocal Laser Scanning Microscope
BIO-RAD MRC-500
Imaging Analyzer
KONTRON IBAS-1 & II
Electrophoresis Imaging System
PDI The Discovery Series
Microdensitometer
JOYCE LOEBL 3CS
Microscope
CARL ZEISS Axiophot,
ICM405s
Microscope Photometer
CARL ZEISS MPM 03-FL

Rou ter\o . :l -H~

National Institute for Basic Biology
Okazaki 444, Japan' ..