1INTRODUCTION
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 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.
During the past year, we have built up
the Laboratory of Gene Expression andRegulation which was inaugurated in
1989 by newly appointing Prof. Takashi
Horiuchi (formerly Associate Professorof Kyushu University) who will continue
to work on chromosome duplication andreorganization in both prokaryotes andeukaryotes. The other Division of theLaboratory headed by Prof. Yoshiro
Shimura has been actively engaged instudies of genes involved in morphogenesis of the plant, Arabidopsis.
Two new professors were appointed
during the last year. Prof. Yukio Nishimura (former Associate Professor of
Kobe University) filled the position ofProfessor of Cell Mechanisms to promote molecular biological studies on formation of organelles in plant cells. Prof.
Yoshiki Hotta of Tokyo University wasappointed to be in charge of Division of
Cellular Communication (adjunct) tofurther molecular genetical studies ofDrosoplila development. On the other
hand, four associate professors of the Institute were promoted to professors inother universities during the period.

Active exchange of personnel with otheruniversities and institutions, I believe, isessential to keep the Institute active.
The NIBB is an inter-university research institute and plays many roles as anational center for the study of biology.
The Institute is responsible for conducting research projects in cooperation withresearch groups in different universities
and institutes. As a part of such cooperative activities, the NIBB carries out
Special Programs which are currently directed to "Biomembrane Research" arid
"Signal Transduction". Based on suchprograms, the NIBB organized the 24thand 25th Conferences in 1990, entitled"Membrane biogenesis and temperatureacclimation of plants" and "Inositol
phosphates and Ca signaling" respectively. The conferences were well attended by active scientists in the related
fields, from both inside Japan andabroad.
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 1990.

lkuo Takeuchi, Ph.D. & D.Sc.
Director General

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NATIONAL INSTITUTE

ORGANIZATION OF THE INSTITUTE
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.

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 (IPS) and Institute for Molecular Science (IMS).

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 15 divisions.

1

Divisions

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

Reproductive Biology
Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)Sensory Processing
Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)--[ Gene Expression and Regulation I
Gene Expression and Regulation II

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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 undertakes
the technical education of its staffs.
Research Support Facility

The research support facility of the
NIBB consists of the Large-scale Spectrograph Laboratory, Tissue and Cell

Culture Laboratory, Laboratory Computer Facility, Plant Culture Facility,
Plant Cell Culture Facility, and Experimental Farm. In addition, seven facilities are operated jointly with the National Institute of Physiological Sciences(IPS); they consist of the Radioisotope
Facility, Electron Microscope Center,
Center for Analytical Instruments, Machine Shop, Laboratory Glassware Facility, Animal Care Facility, and LowTemperature Facility.
CampusThe Okazaki National Research Institutes cover an area of 150,000 m2 withfour principal buildings. The NIBB's
main research building has a floor spaceof 10,930 m 2• 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

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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
Tasaka, M., Hasegawa,· M., Ozaki, T.,
Iwabuchi, M., and Takeuchi, I.
(1990). Isolation and characterization spore coat protein (SP96) gene of
Dictyostelium discoideum. Cell Differentiation and Development 31,
1-9.

Takemoto, K., Takeuchi, 1., and Tasaka,
M. (1990). cAMP regulation of theexpression of prespore specific genes,
SP96 and Dp87 in disaggregated slug
cells of Dictyoste/ium discoideum. Cell
Differentiation and Development 31,
89-96.

5

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Goro Eguchi

The department is composed of two research divisions and three adjunct research divisions. In this department members have been conducting researcheson the fundamentals of cell structures and functions inunicellular eukaryotic organisms, plants and animals at
molecular level utilizing modern technologies including
genetic engineering.

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NATIONAL INSTITUTEDMSION OF CELL MECHANISMS
Professor: Mikio Nishimura
Research Associate: Kazuo Ogawa
JSPS Post-doctoral fellow: Luigi De Bellis
Visiting Scientists: lkuko Hara-Nishimura
Kaori Inoue
Graduate Student: Ryuji Tsugeki

Eukaryotic cells contain several distinct organelles that play vital roles incellular physiology. During proliferationand differentiation of the cells, the organelles often undergo dynamic changes.
The biogenesis of new organelles may
occur, existing organelles may undergo atransformation of function, while otherorganelles may degenerate. Because thedynamic transformation of organellar
function (differentiation of organelles) is
responsible for differentiation events ineukaryotic cells, the following two research projects are currently being studied in this division.
1. Functional transformation of microbodies during greening of the seedlings.
In many seeds such as pumpkin andwatermelon, oil is stored as reserve material in the cotyledons. During thecourse of germination, this reserve oil isutilized as the primary energy source forthe growth of young seedlings. During
the period when cotyledons emerge fromsoil, the tissues become green andphotosynthetically active upon exposure
to light. Photosynthesis primarily supplies the energy needed for the growthof the seedlings after greening.
During the greening process, dramatic
metabolic changes which underlie theshift from heterotrophic to autotrophic
growth occur. Accompanying thesemetabolic changes, many constitutive organelles are functionally transformed.
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 ,8-oxidation and the glyoxylate cycle,
are transformed into leaf peroxisomesthat function in several crucial steps ofphotorespiration. Since cell division does
not occur in the cotyledons of these fatty
seedlings following germination, all ofthese developmental events take place ina fixed number of preexisting cells.
Therefore, greening cotyledon is an excellent model system for studying theregulatory mechanism which underlies
light-induced organelle differentiation.
Recently, we have found that the reversible transition of leaf peroxisomes toglyoxysomes takes place during the senescence of the cotyledons (De Bellisand Nishimura. Plant Cell Physiol. inpress).
As a step to understand the regulatory
mechanisms that operate during microbody transition in pumpkin cotyledons( Cucurbita sp., Amakuri Nankin), sevenmicrobody enzymes, including two
glyoxysomal enzymes, two leaf peroxisomal enzymes and three enzymes present
in both microbodies have been purified,and specific antibodies have been prepared for each enzyme. Immunocytochemical analyses demonstrated thatglyoxysomes are directly transformed toleaf peroxisomes. In 1990, we have isolated and characterized eDNA clones forthe glyoxyso~.al enzyme malate synthase, the leaf peroxisomal enzyme glycolate oxidase, and an enzyme present inboth microbodies, malate dehydrogenase, (Mori et al. Eur. J. Biochem. inpress; Takeda-Yoshikawa et al. J. Bioi.
Chern. in press). Using in vitro translocation experiments with these eDNA
clones, we have found that a specific degradation system for glyoxysomal
enzymes is induced in the microbodies of
cotyledons exposed to light. Studies of

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FOR BASIC BIOLOGY

Fig. 1.

In vitro fusion of isolated protein bodies.

the molecular aspects of this specific degradation system and of the signal transduction processes involved in light-induced microbody transition are in progress.
2. Reversible transformation between vacuoles and protein bodies during seedmaturation and germination.
Protein bodies are single membranebound organelles which accumulate seedstorage proteins. We have found that theprotein bodies are closely related to vacuoles which are the lytic compartmentof plant cells. Protein bodies are formedby budding from the vacuoles during
seed maturation, while vacuoles result
from the fusion of protein bodies during
seed germination. Therefore, protein
body is a specialized form of vacuoles indry seed. It must be emphasized that
dramatic transformation of protein
bodies from a storage to a lytic compart-

ment occurs during the maturation andgermination of seeds. We are studying
the regulatory mechanisms underlying
the reversible transformation between
protein bodies and vacuoles, and havefocused on the processes of budding andmembrane fusion. Protein bodies andvacuoles from various developmental
stages of maturing and germinating
pumpkin seeds have been isolated. Using
isolated protein bodies, we have recently
established an in vitro system to reconstitute the fusion process (Fig. 1). Experiments employing this in vitro system
are currently underway to elucidate themolecular mechanisms of membrane fusion during seed germination.
In parallel, the biogenesis of protein
bodies and the cellular components involved in the transport of seed storage
proteins are also being studied. Protein
bodies have been fractionated into three8
suborganellar components: membraneproteins, matrix proteins and crystalloid
protein. These three classes of proteins
are synthesized on rER as preproproteins. After co-translational processingof the signal peptide, proprotein precursors are transported via dense vesicles tovacuoles where specific processing
enzyme operates to form mature proteins. We have recently isolated dense
vesicles which are not contaminated withother cellular components and havecharacterized the protein composition.
The cellular components involved in thetransport of proteins to vacuoles, especially the specific processing enzyme, thedense vesicles, and the receptor for proprotein precursors, are currently being
characterized.

NATIONAL INSTITUTE

Publication List
1. Mori, H. and Nishimura, M.
(1990). Purification of mRNA for
malate synthase and glycolate oxidase
by immunoadsorption of polysomes.
Plant
Physiol.
Biochem.,
28,505-508.
2. Hara-Nishimura, 1., Matsumoto, T.,
Mori, H., Nishimura, M., Hara, R.
and Hara, T. (1990). Cloning andnucleotide sequence of eDNA for
retinochrome, retinal photoisomerase
from the squid retina. FEES Letters,
271, 106-110.
3. Ogawa, K., Yokota, E., Hamada, Y.,
Wada, S., Okuno, M. and Nakajima,
Y. (1990). The outer arm dynein /3heavy chains of sea urchin sperm flagella and embryonic cilia are different.
Cell Motif. Cytoskel., 16, 58-67.

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
Professor: Yoshihiko Fujita
Associate Professor: Shigeru Itoh
Research Associates: Mamoru Mimuro
Kaori Ohki
NIBB Post-doctoral Fellow: Katsunori Aizawa
NIBB Visiting Scientists: Tiang-Qing Gu •Jonathan P. Zehr#
Fellows: Tohru lkeya
Yoko Nakamura
Technical Staffs: Akio Murakami
Masayo lwaki(*from Institute of Botany, Academia Sinica,
Beijing, China){'from Marine Sciences Research Center,
State University of New York, Stony Brook,
New York, U.S.A.)

Photosynthetic energy conversion has
been investigated at the cellular andmolecular levels. Our research is currently focused on (1) regulation of stoichiometry among thylakoid components in
response to the light regime for photosynthesis, (2) molecular mechanism of
thylakoid protein complexes functioning
in the primary process of photosyntheticenergy conversion, and (3) nitrogen-fumtion in cyanophyte directly coupled withphotosynthetic energy conversion.
(1) Regulation of thylakoid composition:
Previous study has indicated that assembly of photosystem I (PS I), the terminal
component of photosynthetic electrontransport system (ETS), is regulated in
response to the light regime for photosynthesis, so as to balance the two
photoreactions under each light regime(cf. Fig. 1). The signal for the regulation
was studied by examining the steadystate of photosynthetic electron transportin cells of the cyanophyte Synechocystis
PCC 6714 under various light regimes inrelation to the regulation. Flash-induced
oxidation-reduction of Cyt f and P700
under steady-state of photosynthesis indicated that the state of Q Of modified Q
cycle of Cyt b6 - f complex is, or is very
closely related to, the signal for the9

regulation. Study of intracellular localization of Cyt oxidase, the terminal of respiratory ETS in this organism, by usingchemical electron microscopy revealed
that Cyt oxidase is located only in thylakoids but not in cytoplasmic membranes
irrespective of growth conditions, indicating that Cyt oxidase is anotherterminal of thylakoid ETS. Assay of Cyt
oxidase in cells grown under various
conditions indicated that the level of Cyt
oxidase is also regulated, similar to thelevel of PS I. Thus, it is very probablethat levels of the two terminals of
thylkqid ETS are simultaneously regulated in response to the state of ETS.
(2) Molecular mechanism of electrontransfer in reaction center complexes:
Electron transfer mechanism in the PS I
reaction center complex was studied bymodification of intrinsic electron transfer
components, quinone and iron-sulfur
centers. After replacing intrinsic quinone
by artificial species or modifying theiron-sulfur proteins, the electron transfer
was analyzed by nanosecond-microsecond laser spectroscopy and by cryogenic
ESR spectroscopy. Over 100 species ofquinones and quinone analogs have beentested for replacement-experiments.
About 30 species were found to replace
fully the function of intrinsic phylloquinone in spinach and cyanophyte PS I reaction center, indicating that the reaction
center complex has a special quinone
binding site. Analysis of the relationshipbetween the redox potential and thefunction of quinone showed that free energy difference between the quinone andthe primary electron acceptor or the tertiary acceptor, iron-sulfur center, in PS I
complex is primarily important for thereaction. Quinone-reconstitution study'
was also done for PS II reaction center
complex.

10(3) Molecularmechanism of energy
transfer in light-harvesting protein complexes: The study in 1990 was focusedon the energy transfer from carotenoids
to CW a and structure of prosthetic
groups in bacterial reaction center. Studyof energy transfer in the fucoxanthin-CW
a/ c protein from brown algae indicatedthat the energy transfer from CW c to
Chi a and from fucoxanthin to CW a isachieved by two independent pathways
in respective cases. Structure of prosthetic groups in the reaction center
complex of a purple sulfur bacterium
Chromatium tepidum was deduced by linear dichroism and magnetic circulardichroism spectra. Results indicated that(1) the structure is essentially the sameas that in purple non-sulfur bacteria, and(2) magnetic coupling between different
energy levels of the special pair is polarization-plane dependent. An occurrenceof a "low-lying" antenna component was
found ubiquitous in photosynthetic bacteria having the reaction center of quinone-type. This evidence gives rise to aconcept of "antenna-reaction center interaction" in terms of energetics of energy transfer and charge separation processes.
(4) Nitrogen-fixation directly coupledwith oxygenic photosynthesis: The study
was focused on the regulation of nitrogenase activity in the non-heterocystous
cyanophyte Trichodesmium sp. NIBB
1067 in response to the nitrogen-sourcein the growth environments. Examination of nitrogenase activity and abundance of Fe- and Fe-Mo-proteins of
nitrogenase indicated that the regulationof nitrogenase activity occurs at least at
two different levels; one, at the level oftranscription of nif genes or translationof the transcripts, and the other, at the
post-translational level, probably modifi-

NATIONAL INSTITUTE

cation of Fe-protein. In the latter regulation, photosynthesis appears to be essential for maintaining the active form of
Fe-protein. Organization of nif genes( nifH, D and K) in Trichodesmium sp.
NIBB 1067 was also studied. Current results showed that (1) 3 genes, nifH, D
and K, form a cluster without excision
between nifD and K, and (2) the second
copy of nifH is not present. These features are different from those reported
for the heterocystous cyanophyte Anabaena sp. 7120.
Publication List
Adhikary, S. P., Murakami, A., Ohki, K.
and Fujita, Y. (1990) Photoregulation of respiratory activity in the
cyanophyte Synechocystis PCC 6714:
The possibility of the simultaneous regualtion of the amount of PS I complex and the activity of respiratory terminal oxidase in thylakoids. Plant Cell
Physiol. 31, 527-532.
Fujita, Y., Murakami, A. and Ohki, K.
(1990) Regulation of the stoichiometry of thylakoid components in thephotosynthetic system of cyanopytes:
Model experiments showing that control of the synthesis or supply of Chi acan change the stoichiometric relationship between the two photosystems.
Plant Cell Physiol. 31, 145-153.
Hoshina, S., Kunishima, N., Wada, K.
and ltoh, S. (1990) N-terminal sequence of 5 kDa polypeptide associated with photosystem I core complex
from spinach. In Current Research in
Photosynthesis, Baltscheffsky, M. ed.,vol. II, Kluwer Academic Publ. (Dordrecht), pp. 571-574.
Hoshina, S., Sakurai, R., Kunishima, N.,
Wada, K. and ltoh, S. (1990) Selective destruction of iron-sulfur centersby heat/ ethylene glycol treatment andFOR BASIC BIOLOGY

isolation of photosystem I core complex. Biochim. Biophys. Acta 1015,
61-68.
Ikegami, I., Itoh, S. and Katoh, S.
(1990) Properties of P700-enriched
particles isolated from the thermophilic cyanobacterium Synechococcus
elongatus. In Current Research in
Photosynthesis, Baltscheffsky, M. ed.,vol. II, Kluwer Academic Publ. (Dordrecht), pp. 643-646.
Isogai, Y., ltoh, S. and Nishimura, M.
(1990) Location of D+ and distribution of surface charge in photosystem
II. Biochim. Biophys. Acta 1017,
204-208.
Isogai, Y., Itoh, S. Nishimura, M.
(1990) Structural organization andcharge distribution on the donor sideof photosystem II. In Current Research in Photosynthesis,
Baltscheffsky, M. ed., vol. I, Kluwer
Academic Publ. (Dordrecht), pp.
813-816.
Itoh, S. and Iwaki, M. (1990) Competitive bindings of herbicide and quinone
to photosystem I phylloquinone (A-1)
binding site. In Current Research in
Photosynthesis, Baltscheffsky, M. ed.,vol. II, Kluwer Academic Publ. (Dordrecht), pp. 651-654.
Iwaki, M. and ltoh, S. (1990) Functionof substituted quinones as the acceptor
A-1 (phylloquinone) in photosystem I
reaction center. In Current Researchin Photosynthesis, Baltscheffsky, M.
ed., vol. II, Kluwer Academic Publ.
(Dordrecht), pp. 647-650.
Miki, K., Ezoe, T., Masui, A., Yoshisaka, T., Mimuro, M., FujiwaraArasaki,T.
andKasai,
N.
(1990) Crystallization and preliminary X -ray diffraction studies of
C-phycocyanin from a red alga Porphyra tenura. J. Biochem. (Tokyo).

11108, 646-649.
Mimuro, M., Katoh, T. and Kawai, H.
(1990) Spatial arrangement of pigments and their interaction in the fucoxanthin-chl ale protein assembly(FCPA) isolated from the brown alga
Dictyota dichotoma: Analysis bymeans of polarized spectroscopy. Biochim. Biophys. Acta 1015, 450-456.
Mimuro, M., Tarnai, N., Ishimaru, T.
and Yamazaki, I. (1990) Characteristic fluorescence components in thepigment system of the marine dinoflagellate Protogonyaulax tamarensis
and energy flow arpong them: Studies
by means of steady-state and time-resolved fluorescence spectroscopy. Biochim. Biophys. Acta 1016, 281-287.
Mimuro, M. (1990) Excitation energy
flow in the photosynthetic pigment
systems: Structure and energy transfer
mechanism. Bot. Mag. Tokyo 103,
233-253.
Mimuro, M., Shimada, K., Tarnai, N.
and Yamazaki, I. (1990) Excitationenergy flow in purple photosynthetic
bacteria: Analysis by the time-resolved
fluorescence spectroscopy. In Molecular Biology of Membrane-Bound
Complex in Phototrophic Bacteria,
Drews, G. and Dawes, E. A. eds., Plenum Press (New York), pp. 365-374.
Mimuro, M., Tarnai, N., Ishimaru, T.
and Yamazaki, I. (1990) Excitation
enegy flow in the marine dinoflagellate
Protogonyaulax tamarensis. In Current Research in Photosynthesis, Baltscheffsky, M. ed., val. II, Kluwer
Academic Publ. (Dordrecht), pp.
309-312.
Nozawa, T., Mimuro, M., Kobayashi, M.
and Tanaka, H. (1990) Magnetic circular dichroism and linear dichroism
for elucidation of electronic transitions
and their orientations in a reaction

12
center isolated from a tahermophilic
purple sulfur photosynthetic bacterium
Chromatium tepidum. Chern. Lett.
2125-2128.
Rueter, J. G., Ohki, K. and Fujita, Y.
(1990) The effect of iron nutritionon photosynthesis and nitrogen fixation in cultures of Trichodesmium(cyanophyceae). J. Phycol. 26, 3035.
Shimada, K., Yamazaki, 1., Tarnai, N.
and Mimuro, M. (1990) Excitationenergy flow in a photosynthetic bacterium lacking B850: Fast energy transfer from B806 to B870 in Erythrobacter sp. strain OCh 114. Biochim.
Biophys. Acta 1016, 266-271.

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Shimada, K., Yamazaki, 1., Tarnai, N.
and Mimuro, M. (1990) Excitationenergy flow in the aerobic photosynthetic bacterium, Erythrobacter sp.
OCh 114: A fast energy transfer from
B806 to B870. In Current Research in
Photosynthesis, Baltscheffsky, M. ed.,vol. II, Kluwer Academic Publ. (Dordrecht), pp. 193-196.
Sonoike, K., Hatanaka, H., Katoh, S.
and ltoh, S. (1990) Heat-stability ofiron-sulfur centers and P-700 in
photosystem I reaction center complexes isolated from the thermophilic
cyanobacterium Synechococcus elongatus. Plant Cell PhysioL 31, 865-870.

FOR BASIC BIOLOGYDMSION OF CELL PROLIFERATION(ADJUCNT)
Professor: Yasuhiro Anraku
Associate Professor: Souichi Nakamura
Research Associate: Hidetoshi /ida
NIBB Post-doctoral Fellow: Hiro Nakamura
JSPS Post-doctoral Fellows:
Junko Nakajima-Shimada
Shuichi Sakaguchi
Tohru Yashihisa

Ca2+ is a growth-regulating substancein the cell cycle of mammalian cells. For
example, when the availability of Ca2+
becomes limiting in the presence of a
sufficient amount of growth factors,
mouse fibroblastic cells are arrested in
the G 1 phase of the cell cycle. Most of
the growth factors elicits transient increase in the cytosolic free Ca2+ concentration ([Ca2+]J There is also a rise in[Ca2+]i during mitosis. In recent years,
several Ca2+ channels have been identified and methods for the monitoring of[Ca2+]i have been developed, but molecular mechanisms of how Ca2+ is mobilized and how the mobilized Ca2+ acts
to regulate the cell cycle are still obscure.
In this Division recent research has focused on the role of Ca2+ in the cell
cycle and mating process of the yeast
Saccharomyces cerevisiae and on the establishment of measuring systems for[Ca2+]i in yeast cells, with the hope thatcombination of cell biological andgenetic approaches will uncover themolecular mechanism of Ca2+ signaling.
Cell Cycle Control by Ca2+
Unlike in mammalian cells, the role ofCa2+ during the cell cycle in the yeast S.
cerevisiae has been unknown for many
years. The main difficulty in investigating
its role by direct growth studies was thatyeast cells can grow indefinitely inCa2+-deficient medium. In this project,
we performed systematic cell cycle ana-

13

lysis using media containing various concentrations of Ca2+, a Ca2+-ionophore(A23187) and a Ca2+-chelator (EGTA),
and established experimental conditions
for controlling yeast cell proliferation bylowering the intracellular Ca2+ content.
Simultanenous addition of 10 ,uM
A23187 and 10 roM EGTA to cells
growing in a Ca2+-deficient medium at
zzoc caused rapid decrease in intracellular Ca2+ content and resulted in transient
G 1 arrest followed by block mostly at the
G 2/M phase, as revealed by flow cytometry. Recovery from G 1 arrest was notdue to coordinated initiation of DNA
synthesis and bud emergence: unbudded
cells with S or G 2 /M DNA were observed. Examination of terminal phenotype suggested that Ca2+ was required at
all the stages of the cell cycle except for
the initiation of DNA synthesis. The intracellular cAMP level decreased within
10 min after addition of A23187 and
EGTA. No significant transient G 1 arrest was observed in cells incubated with
8-Br-cAMP, or RAS2val1 9 and bcyl mutants, which produce a high level ofcAMP and have constitutively activatedcAMP-dependent protein kinase, respectively. These results indicate that
Ca2+ is essential for cell cycle progression in yeast and suggest that G 1 events
may be mediated by cross-talk between
Ca2+ and cAMP pathways.
The Role of Ca2+ in the Mating Process
In S. cerevisiae, the mating process of
haploid cells is controlled by the mating
pheromones, a- and a-factors, that aresynthesized and secreted by a and amating type cells, respectively. The mating pheromones induce several responses
in cells of opposite mating type. The
mating pheromone response pathway of

14the yeast has been shown to involve
mechanisms similar to those found in
mammalian cells, such as the pheromone-receptor interaction and the function of the pheromone receptor-coupledguanine nucleotide-binding regulatory(G) protein. However, nothing is known
about the role of extracellular and intracellular Ca2+ in this pathway except that
Ca2+ influx in a cells is stimulated by afactor. Thus, the study in this project was
designed to investigate whether Ca2+ influx induced by a-factor correlates withchanges in [Ca2+L that may have a regulatory role for signal transduction of themating pheromone and to elucidate therole of changes in [Ca2+];· To measure[Ca2+]; in individual yeast cells, we employed fura-2 as a Ca2+-specific probe,in conjunction with digital image processing. The results showed that [Ca2+]irose to about 500 nM from a basal levelof about 100 nM, depending upon an influx of Ca2+ which is induced by a-factor. When this influx and consequent riseare prevented by incubating a cells witha-factor in a Ca2+-deficient medium, thecells died specifically after they hadchanged into shmoos, cells morphologically changed by the action of a-factor.
The duration of the Ca2+ requirement
for maintaining viability is short and therequirement is specific to this limited
stage. Mating between a and a cells wasthereby impaired due to cell death at or
before the stage of conjugation. These
results clearly indicate that induced Ca2+
influx followed by a rise in [Ca2+]; is essential for maintaining viability of yeast
cells in the mating process. Taking advantage of the finding that yeast cells die
when exposed to a-factor in the absenceof a rise in [Ca2+];, we are isolating mutants defective in Ca2+ signaling.

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Monitoring of Intracellular Ca2+ Usingan Apoaequorin eDNA Expression
SystemThe use of fura-2 in conjunction withdigital image processing enabled us toquantify [Ca2+]; in a single yeast cell, as
described above. However, the fura-2
method has some drawbacks: 1) fura-2 issequestered into an organellar compartment, the vacuole, and 2) fura-2 requires
excitation at 340 and 380 nm. These two
problems make it difficult to monitorchanges in [Ca2+]; for a long period. To
circumvent these problems, we havechosen a second Ca2+-specific probe,
aequorin. It is a Ca2+-binding proteinthat emits light upon reacting with Ca2+and consists of three components:
apoaequorin (apoprotein), molecularoxygen and coelenterazine (chromophore). The eDNA for apoaequorin of
the jellyfish Aequorea victoria has beencloned and sequenced. Apoaequorin iscomposed of 189 amino acid residues
with three EF-hand structures. Althoughaequorin has been used as a probe, theuse of this protein has been limited by
the difficulty of introducing sufficientamount of it into small cells, including
yeast. To overcome this difficulty, we
have designed the present study to express the apoaequorin eDNA in yeast
cells under the control of the GALl or
the GAP promoters (see Figure) andtested whether apoaequorin can be accumulated in high enough concentration in
the cells to detect a Ca2+ signal. The results showed that the cells accumulated
sufficient amounts of recombinantapoaequorin and that the protein was
active and not toxic to the cells (Nakajima-Shimada et a!. (1991) Biochem.
Biophys. Res. Commun., in press). For
an in vivo method for monitoring ofchanges in [Ca2+];, introduction of

FOR BASI C BIOLOGY

15

A cartoon indicating expression of the apoaequorin eDNA from the jellyfish A equorea victoria under thecontrol of the GAP promoter in yeast cells and regeneration of aequorin from expressed apoaequorin and achromophore, coelenterazine. The binding of Ca2+ to aequorin generates a transient luminescence (A.=470nm). Intensity of the luminescence correlates with the concentration of intracellular Ca2+. On the plasmid, theopen box represents the GAP promoter, the closed portion the apoaequorin eDNA, and the line the vector
DNA.

coelenterazine into intact yeast cells followed by regeneration of aequorin is an
essential step. We found that aequorin
can be effectively regenerated by simply
incubating yeast cells with coelenterazine. Coelenterazine-incorporated cells
responded to extracellular stimuli. A
mating pheromone, a-factor, generated
extracellular Ca2+-dependent luminescence specifically in a mating type cells,with maximal intensity occurring 45-50
min after addition of a-factor. Glucose
added to glucose-starved G 0 / G 1 cells
stimulated an extracellular Ca2+-dependent luminescence with maximal intensity
occurring 2 min after the addition. These
results demonstrate the usefulness of theaequorin system in monitoring [Ca2+];
response to extracellular stimuli in yeast
cells. Using this system, we are investigating mechanisms of Ca 2+ mobilizationin the initiation of the cell cycle triggered
by glucose and in the initiation of mating
triggered by a -factor.
Publication List
Iida, H., Sakaguchi, S., Yagawa, Y., andAnraku, Y. (1990) Cell cycle control
by Ca2+ in Saccharomyces cerevisiae.
J. Bioi. Chern. 265, 21216-21222.
!ida, H., Yagawa, Y., and Anraku, Y.
(1990) Essential role for induced
Ca2+ influx followed by [Ca2+]i rise inmaintaining viability of yeast cells latein the mating pheromone response
pathway. J.
Biol.
Chern. 265,
13391- 13399.
Umemoto, N., Yoshihisa, T., Hirata, R. ,and Anraku, Y. (1990) Roles of the
VMA3 gene product, subunit c of thevacuolar membrane H+-ATPase on
vacuolar acidification and protein

16transport: A study with vma3-disrupted mutants of Saccharomyces
cerevzszae. J. Bioi. Chern. 265,
18447-18453.
Yashihisa, T., and Anraku, Y.

NATIONAL INSTITUTE(1990) A novel pathway of import ofa-mannosidase, a marker enzyme of
vacuolar membrane, in Saccharomyces
cerevisiae. J. Bioi. Chern. 265,
22418-22425.

FOR BASIC BIOLOGYDIVISION OF CELL FuSION(ADJUNCT)
Professor: Yoshio Okada
Research Associate: Masahiro /shiura
Graduate Student: Koide Tsuyoshi(from School of Medicine, Osaka University)

Molecular and cellular biology of
mammalian cells, aided by cell engineering and recombinant DNA technology,
are the research projects of this division.
In relation to the action mechanism for
diphtheria toxin in mammalian cells, thestructure and function of the elongation
factor 2 (EF2) involved in the protein
synthesizing system of eucaryotes are
currently being extensively studied at themolecular level using cloned EF2 genes.
Cosmid cloning has been established byconstructing simplified cosmid vectors
and selecting suitable Escherichia coli
host cells. Elucidation of the mechanisms
for recombination and DNA repair in
mammalian cells is also in progress.
1. Cosmid Cloning(1) Recombinant cosmid DNA suffersfrom high-frequency deletions in the
DNA when propagated in Escherichia
coli hosts. This deletion occurs even in
recA-deficient E. coli hosts. As reported
previously, a recB recC sbcB reel quadruple mutation in the host prevents such
deletion (Ishiura et al., 1989, J. Bacterial. 171: 1068-1074). To clarify thesequence motif(s) which is involved incosmid-deletion events, we propagated
two test cosmids, cMB 15 carrying themouse insert No. 15 and cHEF2-15 carrying the human EF2 gene (Ishiura et al.,
1989, J. Bacterial.), in the recA E. coli
HB101, isolated the deletion derivativesof the test cosmids, and analyzed the nucleotide sequence surrounding eachdeletion junction. We found that there
are T -stretch (or A -stretch) and/ or17
a specific 8-bp sequence around thedeletion junctions.
(2) Multi-copy EF2-related genes
which exist in the mouse genome wereanalyzed by cosmid cloning. We constructed a cosmid library of mouse L cell
genomic DNA with pDC105 vector(Ishiura et a!., 1989, Gene 85:429-435), isolated the EF2-related
genes by colony hybridization using theEF2 gene as a probe, and analyzed thenucleotide sequences.
(3) Recombinational hotspots specificto female meiosis in the murine major
histocompatibility complex were analyzed by cosmid cloning. We constructedcosmid libraries of the genomic DNAs
from recombinant mice with pDC104
vector (Ishiura et al., 1989, Anal.
Biochem. 176: 117-127), isolated cosmid clones covering the major histocompatibility complex region, and analyzed
the nucleotide sequences sorrounding
recombination junctions.
(4) The Neurospora prd-1 gene which
control the period length of circadian conidiation rhythm was isolated by cosmid
cloning and sib selection (Nakahsima, H.
[Division of Biological Chronology] andIshiura, M., in preparation). For genetransfer experiments in Neurospora
crassa, we constructed pDC107 vector
by inserting the Neurospora benomyl-resistance gene as a dominant selective
marker into the pDC1 vector (Ishiura etal., 1989, Anal. Biochem.). We constructed a cosmid library of the genomic
DNA from wild-type Neurospora with
pDC107, and isolated about 5,000 single
clones and stored them separately. Cosmid DNAs were prepared from a set ofclones stored and transferred into prd-1
mutant cells (sib selection). Single colonies of the benomyl-resistant transformants carrying the transferred cosmid

18
DNAs were isolated by plating the cells
on benomyl-agar plates, and the period
length of conidiation rhythm of thetransformants was analyzed. By thisprocedure, we succeeded in isolating
single cosmid clone which complemented the prd-1 mutation.
2. Genetic Analysis of Fabry's disease
We analyzed the case of Fabry's
disease of a Japanese family at the molecular level by cloning eDNA which encodes human a-galactosidase A andfound that this case of the disease wasresulted from a single amino-acid substitution of Ser for Pro-40.
3. Others(1) Human
enzymesynthesizing
blood-group A substance was purified tohomogeneity from human plasma, andits enzymatic properties were characterized.
(2) Starfish cyclin eDNA was cloned
from eggs and sequenced. Expression of
cyclin during meiosis was also analyzed.
Publication List
1. Ishiura, M., Hazumi, N., Shinagawa,
H., Nakata, A., Uchida, T., andOkada, Y. (1990) RecA-independent high-frequency deletions in recombinant cosmid DNA propagated in

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Escherichia coli. J. Gen. Microbiol.
136:67-79.
2. Koide, T., Ishiura, M., Iwai, K., Inoue,
M., Kaneda, Y., Okada, Y., andUchida, T. (1990) A case of Fabry's
disease in a patient with null a-galactosidase A caused by a single aminoacid substitution of Pro-40 by Ser.
FEBS lett. 259:353-356.
3. Koide, T., Ishiura, M., Hazumi, N.,
Shiroishi, T., Okada, Y., and Uchida,T. (1990) Amplification of a long sequence that includes a processed pseudogene for elongation factor 2 in themouse. Genomics 6:80-88.
4. Shiroishi, T., Hanazawa, N., Sagai, T.,
Ishiura, M., Gojobori, T., Steinmetz,
M., and Moriwaki, K. (1990) Recombinational hotspot specific to female meiosis in the murine major histocompatibility
complex.
Immunogenet. 31:79-88.
5. Tachibana, K., Ishiura, M., Uchida,T., and Kishimoto, T. (1990) Starfish
egg mRNA responsible for meiosis reinitiation encodes cyclin. Dev. Bioi.
140:241-252.
6. Takeya, A., Hosomi, 0., and Ishiura,
M. (1990) Complete purification andcharacterization of a-3-N-acetylgalactosaminyl-transferase encoded by thehuman blood group A gene. J.
Biochem. 107:369-368.

FOR BASIC BIOLOGYDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Hotta

The division just started its new projects from September 1990, and is currently engaging in the study of cellularcommunications in the development anddifferentiation processes of multicellularorganisms. The project is aiming at thegenetic dissection and molecular analysisof sensory receptors, nervous systems
and muscles. Many Drosophila mutations have been isolated and analyzed
which harbor defects in the morphology
and functions of these structures.
Among them are many visual transduction mutants isolated by their abnor-

19
mal visual behavior. Their molecular defects have been identified in the phosphatidyl-inositol metabolisms in the eye.
It is, therefore, suggested that the visualsignal transduction system is closely related to the phospholipid turnover, most
likely that of in ositol 1,4,5-trisphosphate (InsP3).
Other genes of interest are the embryonic lethals which are normal in embryonic polarities and segmentations but
are causing defects in the early differentiation of mesoderm, neuroblasts, glia
and muscle precursor cells. They are
currently investigated by using enhancer
trap technique, single embryo primaryculture method and various other cell biological and molecular technologies.

NATIONAL INSTITUTE~---~--~--~~~·~-·._~..____

21

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshiaki Suzuki

The Department is composed of 3 regular divisions
and 1 adjunct division and conducts molecular analysis
on various aspects of developmental phenomena.

22

NATIONAL INSTITUTEDIVISION OF REPRODUCTIVE
BIOLOGY
Professor: Yoshitaka Nagahama
Research Associates: Michiyasu Yoshikuni
Masakane Yamashita
Minoru Tanaka
JSPS Post-doctoral Fellows: Thomas F. Hourigan
Noriyoshi Sakai
Teresa Telecky
Ian G. Gleadall
Johannes Kamen
Graduate Students: Akihiko Yamaguchi
Mika Takahashi
Visiting Scientists: Jennifer L. Specker
Naoki Shibata
Tashiro Hirai
Takeshi Miura(from Hokkaido University)
Shyuji Ohno
(from Hoshi University)
Chiemi Inoue(from Okayama University)

The germ cell is the most importantcell in the body of any species, since ittransmits genetic information from one
generation to the next. The male gameteis specialized to deliver its nuclear package to the egg, but the egg contains not
only a haploid nucleus but also everything else that constitutes the zygote.
Thus, the origin, differentiation, growthand maturation of these essential cells
are of fundamental interest and importance to developmental biology. The
sequence of events leading to the production of fertile gametes takes place
while germ cells reside within the gonads, and occurs in close association
with gonadal somatic cells, by which
various endocrine products (particularly
steroid hormones) are secreted. The division of reproductive biology conducts
research on the endocrine regulation ofdifferentiation, growth and maturation ofgerm cells in multicellular animals, using
fish as a primary study model.
1) 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,20P-dihydroxy-4-pregnen-3-one (17 a,20P-DP)
as the maturation-inducing hormone ofamago salmon (Oncorhynchus rhodurus). Along with estradiol-17 p (E2),
which was identified as the major mediator of oocyte growth, we now have two
known biologically important mediatorsof oocyte growth and maturation in female salmonid fishes. It is establishedthat the granulosa cells are the site ofproduction of these two mediators, but
their production by the ovarian follicle
depends on the provision of precursor
steroids by the thecal cell (two-cell typemodel). A dramatic switch in the steroidogenic pathway from E 2 to 17 a,20 pDP 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. To
investigate the molecular basis for thisswitch, our current efforts center on thecloning and sequencing of the genes encoding five steroidogenic enzymes (cholesterol side-chain cleavage enzyme,
17 a-hydroxylase, c17,20-lyase, aromataseand 20p-hydroxysteroid dehydrogenase)responsible for E 2 and 17a,20P-DP
biosynthesis. We have isolated and sequenced the eDNA encoding the rainbow trout aromatase. Northern blot analyses of rainbow trout ovarian follicles atdifferent stages of development haveshown that the levels of mRNA specificFOR BASIC BIOLOGY

for aromatase were abundant in vitellogenic follicles, but undetectable inpostvitellogenic follicles, follicles duringoocyte maturation, and postovulatory
follicles.
Our microinjection experiments haverevealed that 17 a,20/3-DP acts at theoocyte surface: 17 a,20/3-DP binding was
found in oocyte cortices. The early steps
following 17 a,20f3-DP action involve theformation of the major mediator of thissteroid, maturation-promoting factor or
metaphase-promoting factor (MPF).
MPF activity cycles during 17 a,20/3-DPinduced oocyte maturation; the highest
activity occurs at the first and second
meiotic metaphase. Studies from our laboratories have shown that MPF activity
is not species-specific and can be detected in both meiotic and mitotic cellsof various organisms, from yeast to
mammals. We recently purified MPF
from unfertilized eggs of carp ( Cyprinus
carpio) using four chromatographic steps(Q-Sepharose FF, p13-Sepharose, Mono
S, Superose 12). MPF activity at eachpurification step was assessed by injecting samples into immature cycloheximide-treated Xenopus oocytes. On a
Superose 12 gel filtration column, MPF
and H1 histone kinase eluted as a single
peak with an apparent molecular mass of
100 kd. SDS-PAGE analysis of the active fractions after Superose 12 showedthat they contained 4 proteins of 33, 34,
46, and 48 kd. A monoclonal antibody
against the PSTAIR sequence of cdc2
kinase immunoblotted the 33 and 34 kd
components of purified carp MPF, indicating that cdc2 kinase homologs arecomponents of carp MPF. The 46 and48 kd proteins are endogenous substrates of the kinase. We have also isolated cyclin A and B genes from aeDNA library constructed from mature

23
goldfish oocytes, and their products wereexpressed in E. coli. The deduced aminoacid sequences for cyclin A and B of
goldfish have a homology of 35%. The
46 and 48 kd proteins were recognized
by a monoclonal antibody against E.
coli-produced goldfish cyclin B, but notby an anti-cyclin A antibody. These results indicate that fish MPF cosists of
cdc2 kinase and cyclin B.
Our PSTAIR monoclonal antibody
also cross-reacted with cell homogenates
in several species, including mouse,
Japanese quail, Xenopus, goldfish, medaka, starfish, and a higher plant, lily
(Fig. 1). The cdc2 kinase homolog had
several forms which differed in their mobility on SDS-PAGE. In most species
examined, the form with the fastest mobility appeared or became dominant
when cells entered metaphase. These results suggest that MPF generality discussed earlier is caused by the presenceof cdc2 homolog proteins.
2) Endocrine regulation of male germ
cell development and maturation
Using salmonid fishes, we have recently identified two steroidal mediatorsof male germ cell development ( 11-ketotestosterone for spermatogenesis and
17 a,20/3-DP for sperm maturation). A
steroidogenic switch, from 11-ketotestosterone to 17a,20/3-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 PDP 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 cell elements.
Recent studies have revealed a role of
11-ketotestosterone in spermatogenesis

24

NATIONAL INSTITUTE

94~

67~

43~

36.5 ....

.....

-

~

30 ..

20:.
~

en

"'C
0

C!J

c
0

E

en

:l

0.
0

ro
(/)

c

0

X

<1>

0)

~
(/)

0.
L...

en

L...

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0
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ro

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Fig. 1. Immunoblotting with a monoclonal antibody against anti-cdc2 kinase (PSTAIR).
Left to right: unfertilized eggs of goldfish, amago salmon, Xenopus and starfish, carp testis, mouse myeloma
cell and lily microsporocyte.

and of 17 a,20,8-DP in sperm motility. In
the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. We have developed aserum-free, chemically defined organ
culture system for eel testes, and used
this to investigate the effect of various
steroid hormones on the induction of
spermatogenesis in vitro. It was foundthat 11-ketotestosterone could induce allstages of spermatogenesis (spermatogonial proliferation, meiotic division andspermiogenesis) in vitro within 21 days
(Fig. 2). This is the first animal system inwhich the entire process of spermatogenesis has been induced by hormonal manipulation in vitro (Miura et al. (1991)
Proc. Nat!. Acad. Sci. USA, in press).
Addition of 11-ketotestosterone to theculture medium also caused a marked
cytological activation of Sertoli cells, but
not Leydig cells, suggesting that the action of 11-ketotestosterone on spermatogenesis is mediated through the actionof Sertoli cells. The organ culture systemdeveloped for eel testes should aid us indissecting out the complex of factors involved in spermatogenesis and the roles

FOR BASIC BIOLOGY

25

Fig. 2. Light micrographs of toluidine blue-stained 1 _urn sections of testes before culture (A) and cultured
in media containing 10 ng/ ml 11-ketotestosterone for 36 days (B).

Leydig cells and Sertoli cells play in thisprocess.
Sperm maturation is the developmentof spermatozoa from non-functional
gametes to mature spermatozoa fully
capable of vigorous motility and fertilization. Although the importance of hormones, such as androgens, for this process is generally accepted in vertebrates,
the specific role played by specific hormones has not yet been clarified. In salmonid fishes, spermatozoa taken fromthe testes are immotile, but acquire motility during their passage through thesperm duct. Using male masu salmon,
we found that gonadotropin-induced testicular production of 17 a,20f3-DP is responsible for the acquisiti<m of sperm
motility. However, neither 11-ketotestosterone nor testosterone were effective.
We also obtained evidence that
17 a,20 /3-D P acts to increase sperm ductpH, which in turn increases the cAMP
content of sperm, allowing the acquisition of motility. This appears to be thefirst example in which the sequence ofendocrine mediators and related mechanisms resulting in the acquisition of

sperm motility have fully been established. Thus, 17a,20f3-DP, which was
identified as the steroidal mediator responsible for the acquisition of sperm
motility, has now been proven to be thecommon endocrine mediator of oocyte
and sperm maturation in salmonid fishes.
Our recently-initiated studies include
work on the molecular mechanisms of(1) gonadal differentiation, (2) initiationof meiosis, and (3) vitellogenin uptake
into the oocyte from the vascular system.
Publication List
Adachi, S., and Nagahama, Y.
(1990) Involvement of pH in the enhancement of 20/3-hydroxysteroid dehydrogenase activity in isolated granulosa cell layers from ovarian follicles of
salmonid fishes. Biomed. Res. 11,
327-335.
Adachi, S., Kajiura, H., Kagawa, H., andNagahama, Y. (1990) Identificationof aromatizable androgens secreted byisolated thecal cell layers from vitellogenic ovarian follicles of a teleost,amago salmon (Oncorhynchus rhodurus). Biomed. Res. 11, 359-363.

26

Matsuyama, M., Adachi, S., Nagahama,Y., Maruyama, K., and Matsuura, S.
(1990) Diurnal rhythm of serum steroid hormone levels in the Japanesewhiting, Sillago japonica, a dailyFish Physiol.
spawning teleost.
Biochem. 8, 329-338.
Matsuyama, M., Adachi, S., Nagahama,Y., Kitajima, C., and Matsuura, S.
(1990) Annual reproductive cycle ofthe captive female Japanese sardine
Sardinops melanostictus: relationshipto ovarian development and serum levels of gonadal steroid hormones.
Marine Bioi. (in press).
Matsuyama, M., Adachi, S., Nagahama,Y., Kitajima, C., and Matsuura, S.
(1990) Testicular development andserum levels of gonadal steroids inJapanese sardine during the annual reproductive cycle in captivity. Japan J.
Ichthyol. (in press).
Nagahama, Y. (1990) Endocrine con-

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trol of oocyte maturation in teleosts.
Prog. Comp. Endocrinol. 1990,
385-392.
Nagahama, Y., Takemura, A., Takano,K., Adachi, S., and Kusakari, M.
(1990) Serum steroid hormone levels
in relation to the reproductive cycle of
Sebastes taxzanowskii and S. schlegeli.
Env. Bioi. 30, 31-38.
Shirai, H., Hosoya, N., Sawada, T., Nagahama, Y., and Mohri, H. (1990)
Dynamics of mitotic apparatus formation and tubulin content during oocyte
maturation in starfish. Develop.
Growth Differ. 32, 521-529.
Yamashita, M., Onozato, H., Nakanishi,T., and Nagahama, Y. (1990) Breakdown of the sperm nuclear envelope isa prerequisite for male pronucleus
formation: direct evidence from thegynogenetic crucian carp Carassius
auratus langsdorfii. Dev. Bioi. 137,
155-160.

FOR BASIC BIOLOGYDIVISION OF CELL
DIFFERENTIATION
Professor: Yoshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Shigeharu Takiya
Toshiharu Suzuki
JSPS Postdoctoral Fellows:
Chi-chung Hui
Kenji Matsuno
Visiting Scientist: Ya-Huan Lou
Graduate Students:
Toshifumi Nagata (from Kyushu University)
Kazuto Amanai (from Kanazawa University)
Masakazu Fukuta(Graduate University For Advanced Studies)
Kaoru Ohno
(Graduate University For Advanced Studies)
Pin-Xian Xu (from Tottori University)
Kensuke Suzuki (from Nagoya City University)

Members of the Division have been
involved in two well associated projects.
One, which was initiated in 1968, is tounderstand how a special tissue like thesilk gland of Bombyx mori is differentiated and results in transcribing a specific set of genes, like the silk fibroin,fibroin L chain, P 25 , sericin-1, andsericin-2 genes. In 1990, some of theregulatory factors for these genes wereidentified as homeobox- or POU domain-containing proteins. The other initiated at the time when the Division wasestablished in 1978 is concerned withhow the body plan of the silkworm iscontrolled and how the homeotic genes
regulate a set of target (structural) genes
in specifying segment identities. In 1990,
the E complex genetically known as acluster of homeotic genes on the sixth
chromosome was identified as the Bombyx equivalent of the bithorax complex
accommodating the Bombyx Ultrabithorax, abdominal-A, and Abdominal-B
homeobox genes. The Nc locus which islocated about 1.4 eM apart from the E
complex was also identified to accommodate the Bombyx Antennapedia
homeobox gene.

27
Several cis-acting elements and transacting factors of the fibroin and sericin-1
genes that are transcribed in the posterior silk gland and middle silk gland, respectively, have been identified by thedevelopment and use of the cell-freetranscription systems from various tissues of different developmental stages
coupled with techniques of footprinting
and gel shift assays. Those factors are
FF1, FF2, SGF-1, -2, -3, -4, -5, and
FBF-A1; some of them function specifically on the respective genes, and someact on both genes. Tissue distribution ofthese factors was found also complex; anactive form of FF1 which constructs inthe presence of FF2 a gel shift complex
with the fibroin gene enhancer element I(-238/ -73) is present specifically in theposterior silk gland but an inactive form
is present ubiquitously, SGF-2 is present
specifically in the posterior silk gland,
and SGF-3 is present most abundantly inthe middle silk gland, in lesser amount in
the posterior silk gland, and not detectable in cultured cells derived from embryos.
From the posterior silk gland extracts
FF1 has been purified to homogeneity
which, in association with a partiallypurified FF2, specifically binds to -205/-185 of the fibroin gene (T. Suzuki et al.
(1991a) J. Bioi. Chern., in press). An
antibody raised against the purified FF1
inhibited the transcription enhancement
governed by the enhancer I (T. Suzuki etal. (1991b) J. Bioi. Chern., in press) that
accommodates 6 · copies of homeodomain
proteinbinding sequences,
TCAATTAAAT or its variants. The
FF1 binding region accommodates 2
copies of these. An introduction of twobase substitution mutation on all 6 repeats destroyed the transcription enhancement function. Competition assays

28
using oligonucleotides of the wild typeand mutant types on the gel shift complexes suggested that SGF-2, -3, and -4
are borneo- or POU-domain containing
proteins.
Besides the enhancer I and the proximal upstream element, at least two otherparts serve for the preferential transcription of the fibroin gene in the posteriorsilk gland extract; one is the core-promoter and the other is the intronic
modulator (the enhancer II). The enhancer II is composed of AT-rich sequences and multiple octamer-like sequences. By footprint experiments thesesequences were protected differentiallyin extracts prepared from different tissues and cells, suggesting its interactions
with cell type specific factors. The corepromoter was indispensable for the preferential transcription and functioned synergistically with the enhancer elements.
eDNA clones that contain homeobox
sequences homologous to Drosophila
Antennapedia, engrailed, and invected
were obtained from the middle silk glandof the fifth larval instar. By using a PCR
technique, a eDNA clone containing a
POU-domain sequence, POU-M1, homologous to Drosophila Cfl-a or rat
POU-III Brn-2 was obtained (see Fig.
1). The POU-M1 protein synthesized invitro from the eDNA binds to the SC
element of the sericin-1 gene, the key
element controlling the sericin-1 genetranscription, as does SGF-3. Footprint
and gel shift assays using various elements and their mutant oligonucleotides
indicated that the POU-M1 protein is
indistinguishable from SGF-3. Messenger RNA for POU-M1 is present at ahigh concentration in the fourth moulting stage and becomes lower in the fifth
larval instar in the middle silk gland. In
the posterior silk gland the mRNA was

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not detectable in the early stage of thefifth instar but a trace amount was detected in the fourth moulting stage.
eDNA clones covering the entire open
reading frame for Bombyx engrailed, invected, and Antennapedia were obtained
and sequenced. Developmental expression patterns of the respective mRNAs
were analyzed by Northern blotting. The
Bombyx Antennapedia mRNA was 4.8
kb long in the middle silk gland RNA
from the wild type larvae, while an additional mRNA 1.8 kb long was detectedin the middle silk gland RNA from the
Nc!+ heterozygote larvae. Cloning andsequencing of the 1.8 kb mRNA revealed that it lacks the YPWM, borneobox, and carboxyl end regions. A Southern blot analysis on genomic DNAs indicated that the missing sequence on themRNA is deleted on the Nc DNA. Fromthese results we conclude that the Bombyx Antennapedia gene is mapped to the
Nc locus which is located about 1.4 eM
apart from the E complex on the 6thchromosome. As described previously by
N. Itikawa in 1944, the Nc!Nc homozygote is embryonic lethal showing defects
in the thoracic segments (see Fig. 2). The
silk glands in the lethal embryos were
found shrunk and compacted in the affected thoracic regions, while those fromwild type embryos were extended into
abdominal regions. At this moment we
can not determine whether the effect on
the silk glands was caused directly by the
Nc mutation or simply by the physicalabnormality of the thorax segments.
Candidate clones for SGF-2 are being
screened from the posterior silk gland
eDNA library by the DNA-protein binding screening method. PCR and sequencing analyses revealed that borneobox sequences homologous to Drosophila Deformed, proboscipedia, labial,

29

FOR BASIC BIOLOGY
1: CCGTAGCGCACGCACGCGCATGTCGCTCGCTG'J'CGAATCCOGTGATGTGAGGTGOCCCCCr,): GCCGCTACCGCAGCGCACCGGGACCCCCGCGCAGTTTTCGCGMAGM'l'MGCCAGTTGA
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Fig. 1 The DNA sequences of eDNA encoding the POU-Ml protein The amino-acid sequences werededuced from the DNA sequences. Entire open reading frame sequence accommodates repeats of histidinerich sequences followed by proline-rich sequences in the N-terminal half, POU specific domain and POU
homeodomain in the middle, and histidine- and proline-rich sequences in the C-terminus.

caudal, and others are expressed in theposterior silk gland. They are homologous genes mostly expressed at aroundthe head region of Drosophila. Since theBombyx silk gland is derived from thelabial segment, it is important to identify

the roles of these genes and others mentioned above in the silk gland differentiation and/ or the silk gene transcription.
The E complex in B. mori is a homeotic gene comple,x specifying the

30

NATION AL INSTITUTE

Fig. 2 Morphological appearances of the wild type embryo and the Nc/ Nc homozygote embryo

Fig. 3 Morphological appearances of the wild type embryo and the £ N/ £ N homozygote embryo

FOR BASIC BIOLOGY

identities of the larval abdominal segments. We presumed that theE complex
might be similar in structure and function to the Drosophila bithorax complex,
which specifies from the mesothoracic
segment to the eighth abdominal segment in Drosophila melanogaster. To
test the assumption, we isolated and sequenced three homeobox genes from
Bombyx. The deduced amino-acid sequences of homeodomain are almost
identical to Drosophila Ultrabithorax,
abdominal-A, and Abdominal-B in thebithorax complex. Interesting mutants
EN and Eca were described by N. ltikawa in 1943 and an ENI EN homozygote embryo is shown in Fig. 3. Analyses
on EN and Eca chromosomes indicatethat the Bombyx Ultrabithorax and abdominal-A genes are deleted in the embryos homozygous for EN, and the Bombyx abdominal-A gene is deleted in theembryos homozygous for Eca. Fromthese results we conclude that the E
complex consists of the Bombyx Ultrabithorax, abdominal-A, and possibly Abdominal-B genes, which may play similar roles to their analogues in the Drosophila bithorax complex.
Continuation of these projects would
reveal us how the head, thoracic, and abdominal segments are designed, and specifically how the silk gland differentiatesfrom the labial segment and results intranscribing a specific set of genes.
Publication List
Hui, C.-c. and Suzuki, Y. (1990)
Homeodomain protein binding sites in
the promoter region of a group of silk
protein genes. Develop. Growth &Differ., 32, 263-273.
Hui, C.-c., Matsuno, K., and Suzuki, Y.
(1990) Fibroin gene promoter contains a cluster of homeodomain bind-31
ing sites that interact with three silk
gland factors. J. Mol. Bioi. 213,
651-670.
Hui, C.-c., Suzuki, Y., Kikuchi, Y., andMizuno, S. (1990) Homeodomainbinding sites in the 5' flanking regionof the Bombyx mori silk fibroin lightchain gene. J. Mol. Bioi. 213,395-398.
Iwami, M., Adachi, T., Kondo, H., Kawakami, A., Suzuki, Y., Nagasawa,
H., Suzuki, A., and lshizaki, H.
(1990) A novel family C of thegenes that encode bombyxin, an insulin-related brain secretory peptide of
the silkmoth Bombyx mori: Isolationand characterization of gene C-1. Insect Biochem. 20, 295-303.
Matsuno, K., Takiya, S., Hui, C.-c., Suzuki, T., Fukuta, M., Ueno, K., andSuzuki, Y. (1990) Transcriptional
stimulation via SC site of Bombyx
sericin-1 gene through an interaction
with a DNA binding protein SGF-3.
Nucl. Acids Res. 18, 1853-1858.
Suzuki, Y., Takiya, S., Suzuki, T., Hui,C.-c., Matsuno, K., Fukuta, M., Nagata, T., and Ueno, K. (1990) Developmental regulation of silk gene expression. In Molecular Insect Science,
Hagedorn, H. H., Hildebrand, J. G.,
Kidwell, M. G., and Law, J. H., eds.,
Plenum Publ. Corp. (New York), pp.
83-89.
Suzuki, Y., Obara, T., Takiya, S., Hui,C.-c., Matsuno, K., Suzuki, T., Suzuki,
E., Ohkubo, M., and Tamura, T.
(1990) Differential transcription of
the fibroin and sericin-1 genes in cellfree extracts. Develop. Growth & Differ. 32, 179-187.
Takiya, S., Hui, C.-c., and Suzuki, Y.
(1990) A contribution of the corepromoter and its surrounding regions
to the preferential transcription of the32
fibroin gene in posterior silk gland extracts. EMBO J. 9, 489-496.
Tamura, T., Kanda, T., Takiya, S.,
Okano, K., and Maekawa, H.
(1990) Transient
expressionofchimeric CAT genes injected into
early embryos of the domesticatedsilkworm Bombyx mori. Jpn. J. Genet.
65, 401-410.

NATIONAL INSTITUTE

Yamaguchi, M., Nishida, Y., Moriuchi,T., Hirose, F., Hui, C.-c., Suzuki, Y.,
and Matsukage, A. (1990) Drosophila proliferating cell nuclear antigen(cyclin) gene: Structure, expression
during development, and specificbinding of homeodomain proteins to
its 5'-flanking region. Mol. Cell. Bioi.
10, 872-879.

FOR BASIC BIOLOGYDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Research Associate: Ryuji Kodama
Kiyokazu Agata
Makoto Mochii
Visiting Scientist: Takamasa S. Yamamoto
Kaichiro Sawada
Graduate Student: fun Kosaka
Masashi Hyuga
Akio Iio
Keiko lshikawan
Yutaka Imokawa 2!
(nTsurumi University)( 2!Nagoya University)

The metaplastic changes of some ocular tissues during regeneration of lostparts of the urodele eye are perhaps theclearest examples of differentiation froma once-specialized tissue ·into another.
Actually the lens and neural retina in the
newts can be completely regenerated
through transdifferentiation of pigmented epithelia. Hence evidence
derived from analysis of these phenomena might make a valuable contribution to a much deeper understanding of
the mechanisms both of cell differentiation and probably of cellular transformation during carcinogenesis in vertebratetissues as well. From such a view point
members have engaged in cellular andmolecular analyses of transdifferentiation
utilizing the lens transdifferentiation of
the vertebrate pigmented epithelial cells
in vivo and in vitro as a model system, inorder to understand the regulatory
mechanisms of stabilization in cell differentiation of animal tissue cells, whichmust be one of fundamental requisites
for stable maintenance of multicellularorganization of tissues and organs. The
following results and development havebeen achieved by members of this division in 1990.
(1) Factors controlling the lens transdifferentiation of pigmented epithelial cells
in vitro

33
Since ltoh and Eguchi introduced aculture method by which the lens transdifferentiation of pigmented epithelial
cells of chicken embryos is greatly enhanced, we have utilized this method tostudy biochemical and molecular biological aspects of the transdifferentiation
phenomenon. This culture method uses
phenylthiourea and testicular hyaluronidase as important components of theculture medium. When different productlots of commercially available preparations of testicular hyaluronidase werecompared, we noticed that they show
large variation in their ability to enhance
the transdifferentiation, though they
have similar activity to degrade hyaluronate. We tried to show whether thehyaluronidase activity itself is importantin enhancing the transdifferentiation orwhether there are other factors which arereally effective.
We purified crude preparation of
testicular hyaluronidase by column chromatography and obtained a pure specimen of hyaluronidase, however it
showed no activity to enhance the transdifferentiation. According to recent reports that the basic fibroblast growth
factor (bFGF) is important in controlling
cellular differentiation, we examined
whether there is a bFGF-like component
in the crude hyaluronidase. We could
purify a 18kd protein whose amino acidsequence at N-terminal is the same asthat of bFGF. Further, we could showthat bFGF purified from bovine brain isalso effective in enhancing the transdifferentiation.
Since bFGF alone can not enhance thetransdifferentiation and phenylthiourea
is needed as well, there must be a special
condition of cells where bFGF can exert
its effect. As described in the next section, a possibility that the activity of

34

NATIONAL INSTITUTE

A chimeric chicken by microinjection of the dissociated blastoderm cells of a black chicken into the blastoderm cavity of a white leghorn embryo.

transforming~gtowth factor (TGF) is precisely controlled through its binding protein in the course of transdifferentiationis also raised. The role of growth factors
and their mode of action is our next
point of concern.
(2) Gene -regulation in the process oflens transdifferentiation from pigmented
epithelial cells
To investigate the molecular mechanisms of gene-regulation in the course oftransdifferentiation, (i) pigmented epithelial cell- and lens-specific genes wereisolated and the expression of thesegenes was followed in the process oftransdifferentiation: (ii) the genes ofwhich the expression is qualitatively andquantitatively regulated during the transdifferentiation were isolated and thefunction of these genes was investigated.
We have already reported that the lenstransdifferentiation from pigmented epithelial cells proceeds through a bipotent/

intermediate cell state in which the expression of the c-myc gene is activated,but neither the expression of pigmented
epithelial cell- nor lens-specific genes areactivated. Recent analyses of one of thetranscriptionally repressed genes during
the dedifferentiation process showed thata serine protease inhibitor gene (pP344)
is strongly expressed in fully-differentiated pigmented epithelial cells and the
product of this gene may play a role inthe metabolism of extracellular matrix
and the maintenance of the pigmented
epithelial cell phenotype. We have also
succeeded in isolating the pP64 gene ofwhich the expression is qualitatively
regulated at the level of RNA splicing
during transdifferentiation. The deduced
amino acid sequence of the product of
this gene shows high similarity to thehuman TGF binding protein. These data
indicated that the primary structure of
the TGF binding protein is regulated by

FOR BASIC BIOLOGYRNA splicing depending upon cell state
during transdifferentiation. Now, investigation are extending to the role of TGF
and the TGF binding protein in the process of transdifferentiation.
(3) Establishment of a multipotent cellline derived from human pigmented epithelial cells
Adult human pigmented epithelial
cells isolated from an 80-year-old donor
readily transdifferentiated when cultured
in our established medium consisting ofEagle's minimum essential medium, fetal
bovine serum (10%), testicular hyaluronidase (250U/ml medium), andphenylthiourea (0.15 mM). The progenyof completely dedifferentiated pigmented epithelial cells was able to express either lens cell or pigmenCcell
phenotype depending upon given culture
conditions. Seven clones with stable
growth potential from progeny of dedifferentiated cells were maintained forabout one year by repeated subculturing.
In two of these clones cells were foundto preserve their multipotentiality (atleast bipotential) for differentiation.
Cells of one of these two clones, designated as 80HdePEC-C6, synchronously
synthesized melanin as in melanosomes
when maintained in the condition permissive for pigment cell differentiation.
The sister cell population of this clone
could express lens cell phenotype whenreaggregated and maintained in the condition permissive for lens cell differentiation, but not in the monolayer cell
culture condition even with the permissive medium. Forty-six per cent ofcell population in this clone possessed
the normal karyotype as human somatic
cells and expressed stable growth potential. In addition, they have maintained
their morphology as typical epithelial
cells and formed a typical polygonal

35monolayer cell sheet characteristic to theepithelial cell when attained confluency.
This permanent cell line should provide
powerful model systems for studying thedecision mechanism in cell differentiation of multipotent undifferentiated cells.
(4) Production of the blastoderm
chimera of the chicken
We have already succeeded in the introduction of cloned genes into thechicken by the microinjection method inwhich the cloned genes are microinjected
into fertilized eggs and these eggs are incubated in vitro in recipient egg shells tohatching. We have also tried to produce
transgenic chickens by the blastodermal
cell transfer method. We introduced
cloned genes into blastodermal cells
which are supposed to be embryonicstem cells of the chicken and transferredthem into another embryos at the blastoderm stage to produce the blastoderm
chimera. Recently, we have succeeded in
the efficient production of the blastoderm chimera and the F1 progeny
from donor blastodermal cells. Now, weare trying to establish stable embryonicstem cell lines in vitro to introduce
cloned genes into germ lines.
Publication List
Morita, H., Takeuchi, T., Suzuki, S.,
Maeda, K., Yamada, K., Eguchi, G.,
and Kimata, K. (1990) Aortic cells
synthesize a large chondroitin sulphate
proteoglycan capable of binding tohyaluronate. Biochem. J. 265, 61-68.
Kodama, R., Agata, K., Mochii, M., andEguchi, G. (1990) Partial amino acidsequence of the major intrinsic protein(MIP) of the chicken lens deduced
from the nucleotide sequence of aeDNA clone. Exp. Eye Res. 50,
737-741.

36

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL
BIOLOGY ( ADJUNCf)
Professor: Masaki Iwabuchi
Research Associate: Masao Tasaka
Institute Research Fellow: Takefumi Kawata
Visiting Scientist: Michael F. Filosa(from Toronto University)
JSPS Post-doctoral Fellow: Keiko Takemoto
Graduate Students:
Hajime Nakao (from Kyoto University)
Maki Minami (from Kyoto University)
Takahiro Morio (from Kyoto University)

This division is devoted to the study of
the regulatory mechanisms of gene expression involved in cell proliferationand differentiation in higher plants and acellular slime mold. We have focused on
the following two projects:(1) Regulation of cell cycle-dependenttranscription of plant genes.
Histone genes are mostly expressed in
the S phase of the cell cycle, coupled to
DNA synthesis. The molecular mechanisms of S phase-specific gene expression
are unknown. To elucidate the regulatory mechanisms of transcription ofplant histone genes, we have investigatedcis-acting elements and trans-acting factors of wheat (Triticum aestivum) histone H3 and H4 genes. Until now, wehave identified three positively-acting
cis-elements, the hexameric (ACGTCA), octameric (CGCGGATC), andnonameric (CATCCAACG) motifs, inthe proximal region of the H3 promoter.
In vivo transcription experiments withdeletion mutants of the H3 gene suggestthat some of these motifs may contributeto the S phase-specific transcription of
the H3 gene. These motifs are also
shown to be conserved in the regulatory
regions of other plant histone genes. We
have identified three nuclear DNA-binding proteins, HBP-la, HBP-lb and
HBP-2, as candidates of trans-acting
factors. The former two specifically bindto the hexameric motif and the latter

one to the nonameric motif. The eDNA
clones encoding HBP-la or HBP-lb
have been isolated from a wheat eDNA
library in A.gtll using South-westernmethod. Sequence analyses of thecDNAs and DNA binding experimentswith bacterially expressed HBP-la and-1 b have revealed that both proteins
have the characteristic properties as thebZIP-type transcription factors. In wheat
seedlings, there is a parallel relationshipbetween the level of the transcripts fromthe HBP-la or -lb and H3 genes. Recently, HBP-la has been shown to function as a repressor in non-proliferating
cells in transient expression experiments
using rice cultured cells. Analyses ofother eDNA clones, cross-hybridizable
to HBP-la or -lb eDNA, have suggested that HBP-la and -lb are members of two distinct protein families, respectively. Since it is demonstrated thatthe hexameric motifs are located in the
promoter regions of genes other than histone genes, and that the hexamer-specific DNA binding proteins are present
in some higher plants, we are now trying
to isolate cDNAs for the protein
counterparts from various plants andhave succeeded in the isolation of some
HBP-lb counterparts.
(2) Regulation of cell-type specific gene
expression during development of Dictyostelium discoideum.
The development of the cellular slime
mold Dictyostelium discoideum is characteristic in that after tissue formation,
two types of cells, the prestalk and prespore cells, differentiate in a fixed proportion at the anterior and posterior
regions of the tissue, respectively. In
these few years, we have concentrated
our effort to understand the regulatory
mechanisms of gene expression in theprocess of prespore cell differentiation.

FOR BASIC BIOLOGY

We have isolated two prespore specificgenes, Sp96 and Dp87. The Sp96 genecodes for one of the main spore coatproteins. Each of these two genes is
unique in the genome and northern-blot
analyses have indicated that theirmRNAs are accumulated only in theprespore cells at the same stage of development. In vitro 'run on' assays haveshown that the expression of the twogenes is mainly controlled at the transcription level and their transcription ismore directly regulated by extracellularcAMP.
To determine the cis-regulatory regions of the Sp96 and Dp87 genes, wehave used a transformation assay
method. In the case of Sp96, we constructed mutant mini-genes with the 5'
upstream, and 3' downstream sequences,then introduced into D.discoideum cells.
Northern analyses indicated that theregion from position -686 to -494 isimportant for temporal and cell typespecific transcription of the Sp96 gene.
This cis-regulatory region coincides with
one of the DNase-1 hypersensitive sites
which appears in the 5' upstream regionof the Dp87 gene after tissue formation.
We have identified the cis-regulatory region of the Dp87 gene in transformation
experiments using chimeric genes whichcontain different length of the 5' upstream sequence and the coding sequence of the CAT gene. The region
from -447 to -356 was found to be indispensable for the stage and cell typespecific transcription. To confirm that
this region really contains the cis-regulatory elements of the Dp87 gene, a DNA
fragment with sequences up to -447 was
jointed upstream of the Escherichia coli
fi-gal gene, introduced into D.discoideum cells, and the expression of thechimeric gene in transformed cells was

37
examined by a histochemical staining
method of fi-galactosidase. Positively
stained cells were randomly distributedin the cell mass during the late aggregation stage of development. The numberof positive cells dramatically increased at
the mount stage at which the accumulation of Dp87 mRNA occurs. The positively stained cells dispersed in the cellmass during the early aggregation stage,
and after the tip formation, no positively
stained cells were observed in the tip andprestalk regions in migrating slugs. Theseobservations indicate that the 5' upstream region up to -44 7 bp is sufficientfor the prespore-specific transcription of
Dp87 gene, and further support that the
distribution pattern of the prestalk andprespore cells in slug is caused by sorting-out of differentiated cells.
We have also identified nuclear factors
which specifically bind to the cis-regulatory regions of these genes by gel
retardation assays. The DNA-binding
proteins, specific to these cis-regulatory
regions, were found only in the nuclear
extract from the slug cells. Competition
experiments using synthetic oligonucleotides were carried out to assign the precise binding sequences. These studies
indicated that the binding sequence in
the Sp96 gene is highly homologous tothat in the Dp87 gene. Competition assays using the Dp87 or Sp96 motifs suggested that the DNA-binding factors
which interact with the homologous cisregulatory regions are probably similar
to each other. It is still unclear whether
the nuclear proteins are trans-regulatory
factors of the two prespore-specificgenes. The purification of these proteins
and the isolation of their eDNA clones
are now in progress.

38
Publication List
Kawata, T., Nakayama, T., Ohtsubo, N.,
Tabata, T. and Iwabuchi, M.
(1990) Cell-cycle regulated gene expression in transformed plant cells.
Developmental Genetics 11, 205-213.
Takemoto, K., Takeuchi, I. and Tasaka,
M. (1990) cAMP regulation of theexpression of prespore-specific genes,
SP96 and Dp87, in disaggregated slug

NATIONAL INSTITUTE

cells of Dictyostelium discoideum. Cell
Differentiation and Development 31,
89-96.
Tasaka, M., Hasegawa, M., Ozaki, T.,
Iwabuchi, M. and Takeuchi, I.
(1990) Isolation and characterizationof spore coat protein (sp96)gene of
Dictyostelium discoideum. Cell Differentiation and Development 31, 1-9.

39

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Norio Murata

Department of Regulation Biology is composed offour divisions. The study of this department is focusedon the molecular mechanisms for the response oforganisms including tissues and cells toward external
and internal environments, such as light, temperature,
hormones, signal-transducing substances.

40
DIVISION OF SENSORY PROCESSING
Professor: To be appointed.!

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDIVISION OF CELLULARREGULATION
Professor: Norio Murata
Associate Professor: Hidenori Hayashi
Research Associates: Takao Kondo
Ikuo Nishida
NIBB Visiting Professor: Antoni R. Slabas•
NIBB Post-doctoral Fellow: Hiroyuki Ohta
JSPS Post-doctoral Fellow: Hajime Wada
Visiting Scientists: Toshio Sakamoto1
Yoshitaka Nishiyama 2
Yasushi Tasaka 3
Zoltan GomboS~
Prasanna S. Mohanty
George C. Papageorgiou6
Csaba Lehef'
Suleyman I. AllakhverdievB
Mahir D. Mamedov 9
Chin B. Lee10
Graduate Student: Hiroyuki Imai(*from University of Durham, England)(from The University of Tokyo)(lfrom The University of Tokyo)(lfrom Green Research Center, Ltd.)( 4from Hungarian Academy Sciences, Hungary)rsfrom Jawaharlal Nehru University, India)(6from National Research Center
Demokritos, Greece)( 7from Hungarian Academy Sciences, Hungary)( 8from USSR Academy of Science, USSR)( 9from Moscow State University, USSR)( 10from Dongeui University, Korea)

Two important objectives of the current research efforts of this division are
to understand (I) the molecular mechanisms of low-temperature tolerance andadaptation in higher plants and microalgae, and (II) the structure and functionof proteins of photosynthetic membranes.
I. The molecular mechanisms of lowtemperature tolerance and adaptation inhigher plants and microalgae.
During this year three major lines ofresearch have been pursued.
(1) Desaturation of fatty acids in microalgae and low-temperature tolerance (i)Understanding of the mechanism of
low-temperature tolerance is one of ourlong-term objectives. A cyanobacterial
low-temperature sensitive mutant has

41

been isolated which has reduced lowtemperature tolerance. The mutant was
defective in desaturation of fatty acids atthe delta-12 position. A gene (designated as desA) whose product is crucial
for desaturation of fatty acids at thedelta-12 position has been cloned fromthe cyanobacterium, Synechocystis PCC
6803, using the low-temperature sensitive mutant. Restoration of the mutant
with the cloned wild-type gene resulted
in recovered low-temperature tolerance(see Figure). Alteration of the fatty-acidcomposition by genetic manipulation
could play a crucial role in· our understanding of low-temperature tolerance.
Experiment has been performed in thisarea. Transformation of wild type andanother desaturation mutant (Fad6),
which is defective in desaturation of fatty
acids at the delta-6 position, of Synechocystis PCC6803, with a disrupted desA
which had been interrupted by kanamycin-resistance gene cartridge changed
the fatty-acid unsaturation of membrane
lipids of the recipients. This result demonstrates that the fatty-acid unsaturationof membrane lipids can be controlled bygenetic manipulation of desA in Synechocystis PCC6803. Other genes responsible for plant-type desaturation of fatty
acids are being cloned from cyanobacteria and higher plants. (ii) A low-temperature sensitive strain of the cyanobacterium, Anacystis nidulans R2-SPc, wastransformed with desA from Synechocystis PCC6803. The introduction of thisgene increased the tolerance of the recipient to low temperature. This result
demonstrates that the low-temperature
tolerance of cyanobacteria can be enhanced by genetic manipulation of fattyacid unsaturation. (iii) Effects of fattyacid unsaturation on thermal propertiesof photosynthetic activities have been

42
studied with use of cyanobacteria transformed with the disrupted desA gene of
Synechocystis PCC6803 which causes alteration in fatty-acid unsaturation. It was
found that the temperature profile of theheat inactivation of photosystem 1 andphotosystem 2 was not affected by thechange in fatty-acid unsaturation. Tolerance to photoinhibition at low temperature was decreased in transformation
with fatty-acid unsaturation.
(2) Acyltransferase from low-temperature tolerant plant. The enzyme, glycerol3-phosphate acyltransferase, produces
special lipid molecular species that aresuspected to be responsible for the lowtemperature sensitivity of higher plants.
In order to evaluate the role of thisenzyme in low-temperature sensitivity ofhigher plants, the enzyme has been previously purified by our group from a
low-temperature-sensitive plant, squash,
and a eDNA clone encoding its precursor protein has been obtained. In order
to complement and extend our knowledge in acyltransferase and their role in
low-temperature tolerance we have isolated the genomic gene (designated asplsl gene) for the acyltransferase from a
low-temperature-tolerant plant, Arabidopsis. A eDNA clone corresponding to
the mRNA for the plsl gene has also
been isolated and characterized. The
plsl gene (3,397 bp) contains 11 introns(1,412 bp in total), in its open-reading
frame (1,377 bp), and one intron (94 bp)
in the 3'-untranslated region. The openreading frame encodes a polypeptide of
459 amino-acids residues. It is predictedthat the protein has a leader sequence of
90 amino-acid residues which is cleaved
to produce the mature protein of 369
amino-acid residues. In accordance withthis prediction the Arabidopsis mature
protein exhibited enzymatic activity

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when the eDNA gene corresponding to
the 369 amino-acid residues is over-expressed in E. coli as a fusion protein,with a small phage leader peptide.
(3) Plant transformation with acyltransferase gene. Tobacco plants were transformed with Ti-plasmids tqat wereconstructed with the squash and Arabidopsis cDNAs for acyltransferases undercontrol of the 35S CaMV promoter andthe NOS terminator. Evaluation oftransformants with respect to fatty-acidcomposition of their leaf lipids and lowtemperature sensitivity is in progress.
II. The structure and function of proteins

ofphotosynthetic membranes.
Photosynthesis research is focused on
the machinery of the oxygen-evolving
complex of photosystem 2. This complex
is embedded in the chloroplast thylakoid
membrane and consists of approximately
20 different protein components. In
order to elucidate the molecular mechanism of photosynthetic oxygen evolution, the following subjects are being
investigated using techniques of biochemistry, physicochemistry and molecular biology: (i) Purification and elucidation of the properties of the structuralcomponents of the complex. (ii) Threedimensional interaction of the protein
components within the complex. (iii) The
functional role of each component on
oxygen evolution. (iv) Identification of
low-molecular weight components of theoxygen-evolving complex and theirgenes.
In these subject areas, the protective
effect of glycine betaine on the photosystem 2 activity was most extensively investigated. Glycine betaine is a nontoxic
osmolite found in halophilic higher
plants and microbial plants. Spinach
photosystem 2 complex loses its oxygen-

43

FOR BASIC BIOLOGY

0.03 }J90.3 }J9

3.0 }J9

3.0 }J9

3.0 }J9

3.0 }J9

Bluescrlpt

SquashSalmon sperm

rRNA

DNA

Total DNA fromwild typepTZ 19R/8 kbp

Bluescript/ 1.5 kbp(pAV3)

Control

Complementation of a desaturation mutant (Fad12) of Synechocystis PCC6803. The cells of Fadl2 wereapplied to an agar plate and DNA solutions were spotted on the center of agar plates. Then the agar plates
were incubated at low temperature in the light. The cells of Fadl2 were able to grow very slowly at low temperature (see control). When the total DNA from wild type and the recombinant plasmids containing desAgene (pTZ19R/ 8 kbp and pA V3) were applied onto agar plates, the transformants, that recovered growth at
low temperature, appeared on the plates as a spot.

44
evolving activity by dark incubation atroom temperature for a few hours. The
loss of the activity is greatly depressed in
the presence of glycine betaine at highconcentration (e.g. 1 M). Several analyses indicate that glycine betaine protects the Mn cluster, which serves anessential role in oxygen evolution, by
maintaining the structure and interactionof the hydrophilic protein subunits in thephotosystem 2 complex. Similar protective effects have been observed on theactivity of oxygen evolution in thylakoid
membranes isolated from cyanobacteria.
This could be advantageous in understanding the primary photochemical process in the photosystem 2 complex using
techniques of molecular biology.
Publication List
Fork, D.C. and Murata, N. (1990) The
effect of light intensity on the assay ofthe low temperature limit of photosynthesis using msec delayed lightemission. Photosyn. Res. 23, 319323.
Kondo, T. (1990) Shortening of theperiod of the circadian rhythm by aK+ -channel blocker, tetraethylammo-

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nium, in the duckweed, Lemna gibba
G3. J. Bioi. Rhythms 5, 187-194.
Murata, N., Higashi, S. and Fujimura, Y.
(1990) Glycerolipids in various preparations of photosystem II from spinach chloroplasts. Biochim. Biophys.
Acta 1019, 261-268.
Okuyama, H., Sasaki, S., Higashi, S. and
Murata, N. (1990) A trans-unsaturated fatty acid in a psychrophilic bacterium, Vibrio sp. strain ABE-l. J.
Bacteriol. 172, 3515-3518.
Tasaka, Y., Nishida, 1., Higashi, S.,N.
Beppu,
T.
and
Murata,(1990) Fatty acid composition ofphosphatidylglycerols in relation tochilling sensitivity of woody plants.
Plant Cell Physiol. 31, 545-550.
Wada, H. and Murata, N. (1990) Temperature-induced changes in the fatty
acid composition of the cyanobacterium, Synechocystis PCC6803. Plant
Physiol. 92, 1062-1069.
Wada, H., Gombos, Z. and Murata, N.
(1990) Enhancement of chilling
tolerance of a cyanobacterium bygenetic manipulation of fatty acid desaturation. Nature 347, 200-203.

FOR BASIC BIOLOGYDIVISION OF BIOLOGICALREGULATION (ADJUNCT)
Professor: Hidemasa lmaseki
Research Associate: K otaro T. Yamamoto
Satoru Tokutomi
Hitoshi Mori
Graduate Student: Masao Arai*(*Nagoya University)

Growth and development of higher
plants are regulated by plant hormones,
but they are also greatly influenced bychanges in environmental conditions,which provide physical signals to plant
cells. In many cases, the environmental
signals alter endogenous hormonal balance by changing the rates of biosynthesis, inactivation and transport of plant
hormones. To understand the regulatory
mechanisms of plant growth as affectedby the environmental signals, research inthis division has been conducted in three
aspects; (1) development of the sink activities in growing tissues, (2) mechanisms of gene expression regulated byplant hormones and environmental
stress, and (3) acquisition of thermotolerance by non-lethal high temperature.
(1) Development of the sink enzymes in
rapidly growing tissues. Rapid growth ofstem and leaves of seedlings shortly after
seed germination is nutritionally supported by sucrose influx from storage
tissues of the seed and accompanies
massive accumulation of cell wall materials which are mainly various polysaccharides. To maintain the rapid growth,
sucrose must be continuously metabolized. We have found that a dramaticchange of sucrose-metabolizing enzymes,
invertase and sucrose synthase, occurredin stems and leaves which are correlatedwith their growth. However, the contribution of these two enzymes to growthchanges in a tissue-specific manner.

45

Rapid growth of hypocotyl (stem below
the cotyledons) in the dark was correlated with rapid increases in both invertase and sucrose synthase, whereas only
sucrose . synthase increased in slowly
expanding plumule (young etiolated primary leaves). In the light, rapid decreases in both enzyme activities were
followed by cessation of hypocotyl
growth, and invertase but not sucrosesynthase increased concomitant withgrowth of epicotyl (stem above the cotyledons) and expansion of leaves. Bothinvertase and sucrose synthase have been
purified from mung bean seedlings, andtheir respective cDNAs have beencloned (M. Arai, H. Mori and H.
Imaseki, unpublisehd results). Expression of these genes are being examined inrelation to growth capacity of the various
tissues.
(2) Mechanisms of gene expression regulated by plant hormones and tissue
wounding. Auxin induces many physiological processes in plants, including cell
growth and division. Before such physiological processes are induced by auxin,
the synthesis of particular set of proteinsis initiated in the affected tissue. Usingmung bean stem sections, we havecloned 5 different cDNAs for the auxinregulated genes by differential screening.
Sequence analysis revealed that two ofthem were new to the literature, andtheir characterization is under way (K.T.
Yamamoto, H. Mori and H. Imaseki,
unpublished results). Ethylene biosynthesis is variously regulated by otherplant hormones and environmental
stress, and in all cases, the regulatory
step is the formation of 1-aminocyclopropane-1-carboxylic acid (ACC) from
S-adenosylmethionine. However, the
ACC synthase induced by auxin is different from that induced by tissue

46
wounding, and the eDNA for the
wound-induced ACC synthase fromwinter squash fruits has been cloned andsequenced (1). The gene is induced only
by tissue wounding, but not by otherplant hormones. Subsequently, the basic
structure of the gene was determined.
The gene has four introns and the initiation site of transcription is 361 base upstream of the initiation codon (H. Mori,
N. Nakajima and H. Imaseki, unpublished results). To compare genes for the
enzymes induced by different stimuli,
cloning of cDNAs for the ripening-associated enzyme and the auxin-induced
enzyme is under way.
(3) Mechanisms of acquisition of thermotolerance by non-lethal high temperature. When plant seedlings are exposed
to a lethal high temperature (53°) for ashort period (20 min) and returned to a
growth temperature (28°), they are irreversibly damaged and eventually die.
However, a short (1 hour) exposure to a
non-lethal high temperature (40°) provides plants tolerance to the subsequent

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exposure to an otherwise lethal hightemperature. This acquisition of thermal
tolerance was correlated with changes inphysicochemical properties of the membranes. Lethal high temperature caused
the formation of lysophosphatidylethanolamine (PE) in the membrane lipids ofthe control plants but not in the thermotolerant plants. PE was found to increase
membrane permeability in isolated
protoplasts (S. Tokutomi and H. Imaseki, unpublished results), and the thermotolerance acquired by non-lethal hightemperature is related to a mechanism
by which the formation of PE is suppressed at lethal high temperature.
Publication List
1)Nakajima, N., Mori, H., Yamazaki, K.
and Imaseki, H. (1990) Molecularcloning and sequence of a complementary DNA encoding 1-aminocyclopropane-1-carboxylate synthaseinduced by tissue wounding. Plant Cell
Physiol. 31, 1021-1029.

FOR BASIC BIOLOGYDIVISION OF BEHAVIOR AND
NEUROBIOLOGY (ADJUNCT)
Professor: Katsuhiko Mikoshiba
Associate Professor: Masaharu Ogawa
Research Associates: Taka-aki Tamura
Teiichi Furuichi
Visiting Scientists: Kousuke Sumita •Ichirou Fujino •Norihiko Yamada*
Yoshimi Ryou*
Masuo Gotoh • •Tetsuichirou Saitoh+
Makoto Nakamura#
Visiting Research Fellow: Yasutaka Makino@
Graduate Students: Atsushi Miyawaki*
Shingo Yoshikawa*
Takafumi Inoue*
Michisuke Yuzaki++(*from Osaka University)(**from Kanazawa University)(+from University of Tokyo)~from Hokkaido University)~from Ono Pharmaceutical Co., Ltd)r++ from Jichi Medical School)

In our research division, we are studying the mechanisms of development,
growth and differentiation of the mammalian nervous system at the molecularand cellular levels. The main themes ofour division are:(1) Second messenger signalling within acell. In the course of our studies on cerebellar ataxic mutant mice, a membrane
glycoprotein P400 was found to be enriched in Purkinje cells from normal
mice but reduced in Purkinje-cell-deficient mutant mice. The eDNA-cloning
and functional expression of the P400
protein show that this protein is a receptor for inositol 1,4,5-trisphosphate(InsP 3), a second messenger that mediates Ca2+ release from intracellular
stores such as the endoplasmic reticulum. We demonstrated that the InsP 3
receptor binds to InsP3 with the N-terminal region and forms a homotetrameric
structure, exhibiting a Ca2+ channel activity in response to lnsP3 molecules.
Our primary goal is to know how the
InsP3-mediated Ca2+ release is involved

47

in the cerebellar neuronal network system, especially in the Purkinje cells. In
addition, we have recently cloned aeDNA of the Drosophila InsP 3 receptor,
which probably allows us to elucidate thefunctional importance of the InsP 3 -mediated Ca2+ release in a wide variety of cell
types.
(2) Studies on the mechanisms of brain
specific gene expression. We have been
studying the structure and function of
the promoters of the myelin basic protein gene by using in vitro transcription
system derived from animal tissues. Our
aim is to understand the molecularmechanisms of tissue-specific gene expression in the nervous system.
Publication List
1) Aoyama, A., Tamura, T. and Mikoshiba, K. (1990) Regulation ofbrain-specific transcription of themouse myelin basic protein gene:
Function of the NF-1-binding site in
the distal promoter. Biochem. Biophys. Res. Commun. 167, 648653.
2) Asaga, H., Inoue, T. and Mikoshiba, K. (1990) Two-dimensionalgel electrophoretic analysis of thecerebella from neuropathologicallymutant weaver, nervous and staggerer mice. Develop. Growth andDiffer. 32, 235-244.
3) Furuichi, T., Shiota, C. and Mikoshiba, K. (1990) Distribution of
inositol 1,4,5-trisphosphate receptor
mRNA in mouse tissues. FEBS
Lett. 267, 85-88.
4) Ikenaka, K., Fujino, 1., Morita, N.,
Iwasaki, Y., Miura, M., Kagawa, T.,
Nakahira, K. and Mikoshiba, K.
(1990) Reliable transient promoter
assay using fluorescein-di-{3-D-galactopyranoside substrate. DNA and48
Cell Bioi. 9, 279-286.
5) Ikenaka, K., Nakahira, K., Takayama, C., Wada, K., Hatanaka, H,
and Mikoshiba, K. (1990) Nerve
growth factor rapidly induces expression of the 68kDa neurofilament gene by posttranscriptional
modification in PC 12h-R cells. J.
Bioi. Chern. 265, 19782-19785.
6) Ino, H., Nakade, S., Niinobe, M.,
Ikenaka, K., Teshima, T., Wakamiya, T., Matsumoto, T., Shiba, T.,
Kawai, N. and Mikoshiba, K.
(1990) Characterization of bindingsites for spider toxin, [3H]NSTX-3,
in the rat brain. Neurosci. Res. 8,
29-39.
7) Inoue, T., Tamura, T., Furuichi, T.
and Mikoshiba, K. (1990) Isolation of complementary DNAs encoding a cerebellum-enriched NF1
family that activate transcription
from the mouse myelin basic protein
promoter. J. Bioi. Chern. 265,
19065-19070.
8) Kondo, T., Ikenaka, K., Kato, H.,
Ito, K., Aimoto, S., Hojo, H. and
Mikoshiba, K. (1990) Long-term
enhancement of synaptic transmission by synthetic mast cell
degranulating peptide and its localization of binding sites in hippocampus. Neurosci. Res. 8, 147-157.
9) Maeda, N., Niinobe, M. and Mikoshiba, K. (1990) A cerebellar Purkinje cell marker P400 protein is an
inositol 1,4,5-trisphosphate (InsP 3)
receptor protein - Purification andcharacterization of InsP3 receptor
complex. EMBOJ. 9, 561-567.

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10) Miura, M., Tamura, T. and Mikoshiba, K. (1990) Cell-specific expression of the mouse glial fibrillary
acidic protein gene: Identification of
the cis-trans-acting promoter elements astrocyte-specific expression.
J. Neurochern. 55, 1180-1188.
11) Mikoshiba, K., Okano, H., Aruga,J., Tamura, T., Miura, M., Ikenaka,
K. and Nakagawa, T. (1990)
Chimeric and molecular genetic
analysis of myelin deficient (shivererand mid) mutant mice. Proc. New
York Acad. Sci. "Myelination anddysmyelination". 605, 166-182.
12) Miyawaki, A., Furuichi, T., Maeda,
N. and Mikoshiba, K. (1990) Expressed cerebellar-type inositol
1,4,5-trisphosphate receptor, P400
has calcium release activity in a fibroblast L cell line. Neuron 5,
11-18.
13) Nakahira, K., Ikenaka, K., Wada,K., Tamura, T., Furuichi, T. and
Mikoshiba, K. (1990) Structure of
the 68 KDa neurofilament gene andregulation of its expression. J. Bioi.
Chern. 265, 19786-19791.
14) Tamura, T., Aoyama, A., Inoue, T.,
Miura, M. and Mikoshiba, K.
(1990) A new transcription element in the JC virus enhancer. J.
Gen. Virol. 71, 1829-1833.
15) Tamura, T., Sumita, K., Hirose, S.
and Mikoshiba, K. (1990) Core
promoter of the mouse myelin basic
protein gene governs brain specifictranscription in vitro. EMBO J. 9,
3101-3108.

49

FOR BASI C BIOLOGYLABOLATORY OF GENE EXPRESSION
AND REGULATION

Head: Yoshihiko Fujita

The Laboratory consists of 2 regular divisions andconducts research into regulatory mechanisms of gene
expression in higher plants and animals.

50

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION
AND REGULATION I
Professor: Yoshiro Shimura
Associate Professor: Kiyotaka Okada
Research Associate: Hideaki Shiraishi
Post-doctoral Fellows: Makako K. Komaki
Graduate Students: Kazuhito A kama
Hiroyuki Okamoto
Koji Sakamoto

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. Our
efforts have been focused on flower development and morphogenesis and also
on root responses toward gravity, lightor touching stimuli. For these studies, wehave mainly used a small crucifer, Arabidopsis thaliana. This plant is called "botanical Drosophild', because it has some
remarkable features, such as a small
genome size (7 X 107 base pairs per haploid), short life-cycle (5-6 weeks),
small size (20-30 em in height), andease of propagation. These features
make the plant ideally suited for genetic
and molecular biological studies. In addition, more than 150 loci and morethan 250 RFLP fragments are mappedon 5 chromosomes. Experimental techniques such as transformation, regeneration of transgenic plants, and genetagging have been improved. Using thisplant, we have isolated and characterized
many mutants defective in flower development and morphogenesis or in root
growth responsive to the environmental
stimuli. We have also cloned several
genes which might be related to theflower development or to the root
growth response to the stimuli.
Mutants with abnormal floral morphology could be divided into the following
types on the basis of the stages of floral
development where the genetic defects

were presumed to occur; namely, stage
1: transition from vegetative to reproductive growth (mutants with delayedtransition or earlier transition), stage 2:
elongation of inflorescence axis (dwarf:
mutants with short inflorescence axis),
stage 3: formation of floral meristem(mutants lacking floral meristem at the
top of the inflorescence axis), stage 4:
formation of floral organ primordia(mutants with increased or decreased
numbers of floral organs, or with floralorgans at asymmetric or aberrant positions), stage 5: fate determination of thefloral organ primordia (homeotic mutants: mutants where some floral organs
are replaced by other organs), and stage
6: development and morphogenesis offloral organs (mutants with organs ofaberrant structure and function). Most of
the mutants have been shown to havesingle, recessive, nuclear mutations(Komaki et a!., Development, 104,
195-203, 1988; Okada et a!., Cell Differ. Dev., 28, 27-38, 1989; Shiraishi eta!., in preparation). The analysis of
double mutants constructed from thesesingle mutants revealed mostly additive
phenotypes of the parental mutants. In
some cases flowers with novel and highly
complex structures were observed, indicating simultaneous expression and interaction of the two gene products(Komaki eta!., in preparation).
Floral bud formation on the inflorescence axis was studied using the pinformed mutant (Okada et aL, in press).
In some cases this mutant forms no floral
buds at the top of inflorescence axis. But
in other cases flowers with no stamens
and wide petals are formed. Thesephenotypes of the mutant are exactly thesame with wild type plants cultured
in the presence of chemical inhibitors
on auxin polar transport. The polar

FOR BASIC BIOLOGY

51

Fig. 1. Inflorescences of Arabidopsis thaliana wild type (right) and the pin-formed mutant (left). No floral
buds are formed on the inflorescence axis of the mutant.

transport activity and the amount of free
IAA in the mutant inflorescence axis are
decreased to about 10% of wild type.
These observations suggest that normal
level of the polar transport activity in the
inflorescence axis is required in early developmental stages of floral-bud formation. In addition we measured the levelof endogenous free auxin (indole aceticacid) in several parts of a Brassica plant(Ueda et al., in press; J. Ueda: Osaka
Prefectural University).
We have constructed a relatively
simple model which may explain the action of the homeotic genes (Komaki etal., in preparation). The model explains
the flower structure of double mutants
constructed by crossing a series of the
known homeotic mutations.
Roots alter their growth direction
when they encounter obstacles (obstacleescaping response), when their relativeorientation against gravity is changed(gravitropic response), or when they are
illuminated from aside (phototropic response).
Gravitropic and phototropic responses
were analyzed using agar plates. Youngseedlings grown on vertical agar plate
have roots which grow straight downward on the agar surface. When theplates were put aside, roots bend 90 degrees and grow to the new direction ofgravity. If the plates were covered withblack cloth and illuminated from side,roots grow to the opposite direction ofincoming light. Mutants which show abnormal graviresponse or photoresponse
were isolated (Okada et al., in preparation).
In order to analyze the obstacle-escaping mechanisms, a similar system which
provides a constant obstacle-touching
stimulus to root tips was devised. Seedlings were grown on vertical agar plates.
When the angle of the agar plate is

52

NATIONAL INSTITUTE

9lFig. 2. Waving growth of roots of Arabidopsis thaliana wild type on the angled agar plates. Although theroots grow straightly downward on the surface of vertical agar plates, the roots form a wavy pattern on agar
plates angled to 45 degrees. The wavy pattern is due to periodical reversion of root-tip rotation induced by
obstacle-touching stimulus perceived at the root tip. A. Right-hand rotation causes counterclockwise curve.
B. Left-hand rotation causes clockwise curve.

shifted to 45 degrees, the root-tips bend
downward under the influence of gravity
and encounter the agar surface. Due toincapability of penetrating into the agar,
this touching stimulus induces root tip
rotation whose direction is periodicallyreversed. As the result of this response,roots form a wavy growth pattern on the
inclined agar surface. Mutants which
form abnormal wavy patterns were isolated. Genetic analysis has shown that atleast six genes ( wavl-6) are involved inthis growth response (Okada & Shimura,1990). Several mutants also show abnormal gravitropism and/ or phototropism.
These results indicate that root gravi-

tropic, phototropic and obstacle-escaping responses share at least in part a
common genetic regulatory mechanism.
Attempts are being made to identify
and isolate the genes responsible for the
mutants using the transformation systems mediated by the Ti-plasmid vectors
or by a direct gene-tagging system. For
such experiments, it is absolutely necessary to develop a good, efficient system oftransformation and transgenic plant regeneration. We have tested several combinations of A. thaliana ecotypes andAgrobacteria strains and established anefficient system (Akama et a!., in preparation).

FOR BASIC BIOLOGY

Publication List
Okada, K. and Shimura, Y. (1990)
Reversible root tip rotation in Arabi-

53
dopsis seedlings induced by obstacletouching stimulus. Science 250, 274276.

54

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION
AND REGULATION II
Professor: Takashi Horiuchi
Visiting Graduate Students: Masumi Hidaka •Takehiko Kobayashi*(*from Kyushu University)

The laboratory was set up in December 1990. Main subjects of investigation
will be the molecular analysis of DNA
replication, recombination and repair inprocaryotes and eucaryotes. DNA replication termination processes and mechanisms of homologous recombination
enhanced by initiation and termination
events will receive particular attention.
These studies are expected to elucidate
the links between DNA replication,
chromosomal partition and recombination.

55

FOR BASIC BIOLOGYTECHNOLOGY DEPARTMENT
Head: Hachiro Honda
Common Facility Group
Chief: Hiroyuki Hattori

Research Support Group

Research Support Facilities
Chieko Nanba
Masahiro Kawaguchi
Mamoru Kubota
Takashi Yamamoto
TomokiMiwa

Cell Biology Group
Akio Murakami (Unit Sub-chief)
Masayo lwaki
MakiKondo

Radioisotope Facility
Kazuhiko Furukawa (Unit Chief)
Yoshimi Matsuda
Mitsuaki Higashi
Center for Analytical Instruments
Hiroko Kajiura (Unit Sub-chief)
Hisashi Kojima
Yukiko Koyama

Developmental Biology Group
Shinji Adachi (Unit Sub-chief)
Sachiko Fukada
KaoruKatou
Miyuki Ohkubo
Regulation Biology Group
Yoko Fujimura
Shoichi Higashi
Gene Expression and Regulation Group
Hideko Nonaka

Glassware Washing Facility
Toshio Ohkawa
Hisaya Uemura

The Technology Department is a supporting organization for reseachers andresearch organization within the NIBB.
The Department develops and promotes
the institute's research activities and, atthe 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
and education to increase their capability
in technical area. Each technical staff isproceeded to the fixed division usuallyand they support the various researchwith their special biological, biochemicaland biophysical techniques.

56

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY

Head of Facility: Yoshitaka Nagahama
Research Associates:
Masakatsu Watanabe (Large Spectrograph)
Yoshio Hamada (Tissue and Cell Culture)

The facility maintains large-scale experimental equipment and facilities for
growing and maintaining biological specimens. The facility is shared among the
research members, and has five laboratories.

The Large Spectrograph Laboratory:
This laboratory has the largest spectrograph in the world, dedicated mainly toaction spectroscopical studies of various
light-controlled biological process (theOkazaki Large Spectrograph, OLS). The
spectrograph runs on a 30 kW-xenon arc
lamp and has a compound grating-surface composed of 36 smaller individual
gratings. It projects a spectrum of wavelength ranged from 250 nm to 1,000 nm
onto its focal curve of 10 m in length.
The fluence rate (intensity) of the monochromatic light is more than twice asmuch as that of the tropical sunlight atnoon at each wavelength.
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-,time-, and spatial-resolutions. It is composed of a high-power Ar-ion laser(Coherent, Innova 20) (336.6-528.7
nm, 20 W output), two CW dye lasers(Coherent, CR-599-01) (420-930 nm,
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 ,urn s- 1
in tracking speed, down to 2 ,urn in beam

diameter) with an infrared phase-contrast observation system.

COOPERATIVE RESEARCH
PROGRAM FOR THE OKAZAKI
LARGE SPECTROGRAPH

More than 30 projects every year are
conducted by visiting scientists including
foreign scientists as well as those in theinstitute under the NIBB Cooperative
Research Program for the use of the
OLS.
Action spectroscopical studies forvarious regulatory actions of light on living organisms have been conducted.
C>

Publication List
Furusawa, Y., Suzuki, K., and Sasaki, M.
(1990) Biological and physical dosimeters for monitoring solar UV-B light. J.
Radial. Res. 31, 189-206.
Iino, M. (1990) Phototroposm: mechanisms and ecological implications. Plant
Cell Environ. 13, 633-650.
Matsunaga, T., Mori, T., and Nikaido,
0. (1990) Base sequence specificity ofa monoclonal antibody binding to (6-4)
photoproducts.
Mutat.
Res.
235,
187-194.
Mori, T., Matsunaga, T., Chang, C.-C.,
Trosko, J. E., and Nikaido, 0. (1990)
In situ (6-4) photoproduct determination
by laser cytometry and autoradiography.
Mutat. Res. 236, 99-105.
Nakagawa, T., Oda, J., Koizumi, H., Furukawa, T., Yasui, C., and Ueda, T.
(1990) Ultraviolet action spectrum for
intracellular free Ca2+ increase in humanepidermal keratinocytes. Cell Struct.
Funct. 15, 175-179.

FOR BASIC BIOLOGY

Nakamura, S., Watanabe, M., Hatase,K., and Kojima, M. K. (1990) Light
inhibits flagellation in a Chlamydomonas mutant. Plant Cell Physiol. 31,
399-401.
Nakahigashi, K., Komine, Y., Watanabe,M., and Inokuchi, H. (1990) An E.
coli promoter that is sensitive tovisible light. Jpn. J. Genet. 65, 381386.
Ogura, T., and Takahashi, S. (1990)
Observation of the Fen-02 stretching
raman band for cytochrome oxidase
compound A at ambient temperature.
J. Amer. Chern. Soc. 112, 56305631.
Ogura, T., Takahashi, S., Shinzawa-lto,
K., Yoshikawa, S., and Kitagawa, T.
(1990) Observation of the Fe4+=ostretching Raman band for cytochrome oxidase compound B at ambient
temperature. J. Bioi. Chern. 265,
14721-14723.
Sugai, M., and Furuya, M. (1990)
Photo-inhibition of red-light induced
spore germination in Pteris vittata:cyanide, azide and ethanol counteracts
restorable inhibitory action of near
UV and blue-light but not that of far
UV. Plant Cell Physiol. 31, 415-418.
Tada, M., Watanabe, M., and Tada, Y.
(1990) Mechanism of photoregulated carotenogenesis in Rhodotorula
minuta. VII. Action spectrum forphotoinduced carotenogenesis. Plant
Cell Physiol. 31, 241-246.
Tokutomi, S., Mizutani, Y., Anni, H.,
and Kitagawa, T. (1990) ResonanceRaman spectra of large pea phytochrome at ambient temperature: difference in chromophore protonation
between red- and far red-absorbing
forms. FEBS Lett., 269, 341-344.
Torikai, A., Ohno, M, and Fueki, K.
(1990) Photodegradation of poly57
(methyl methacrylate) by monochromatic light: Quantum yeild, effect ofwavelength and light intensity. J. Appl.
Polym. Sci. 41, 1023-1032.
Ueda, T. (1990) Photosensory transduction in the slime mold Physarum
polycephalum. Trends Photochem.
Photobiol. 1, 25-29.
Ueda, T., Nakagaki, T., and Yamada, T.
(1990) Dynamic organization of
ATP and birefringent fibrils during
free locomotion and galvanotaxis in
the plasmodium of Physarum polycephalum. J. Cell Bioi. 110, 10971102.
Van Volkenburgh, E., Cleland, R. E.,
and Watanabe, M. (1990) Lightstimulated cell expansion in bean
(Phaseolus vulgaris L.) leaves. II.
Quantity and quality of light required.
Planta 182, 77-80.
The faculty of the Large Spectrograph
Laboratory conducts its own work.
Photoreceptive and signal transduction
mechanisms of phototaxis of singlecelled, flagellate algae are studied actionspectroscopically by measuring computerized-videomicrographs of the motile
behavior of the cells at the cellular andsubcellular levels. Photoreceptive andsignal transduction mechanisms of algal
gametogenesis are also studied by action
spectroscopy.
Publication List
Tada, M., Watanabe, M., and Tada, Y.
(1990) Mechanism of photoregulated carotenogenesis in Rhodotorula
minuta. VII. Action spectrum forphotoinduced carotenogenesis. Plant
Cell Physiol. 31, 241-246.
Nakamura, S., Watanabe, M., Hatase,K., and Kojima, M. K. (1990) Light
inhibits flagellation in a Chlamydo-

58
monas mutant. Plant Cell Physiol. 31,
399-401.
Van Volkenburgh, E., Cleland, R. E.,
and Watanabe, M. (1990) Lightstimulated cell expansion in bean
(Phaseolus vulgaris L.) leaves. II.
Quantity and quality of light required.
Planta 182, 77-80.
Nakahigashi, K., Komine, Y., Watanabe,M., and Inokuchi, H. (1990) An E.
coli promoter that is sensitive tovisible light. Jpn. J. Genet. 65, 381386.
Tissue and Cell Culture Laboratory: Thisis a facility for tissue and cell culture.
This laboratory is equipped with safety
rooms which satisfy the P2/P3 physicalcontainment level. This facility is routinely used for DNA recombination experiments.
The faculty of the Tissue and Cell
Culture Laboratory also conducts its
own work. Myosin is a ubiquitous protein in eukaryotes. It is presumed that
this molecule plays a key role in cell motility as well as other functions. The roleof myosin in non-muscle cells might bebetter understood if wild-type cells could
be compared with mutant cells with altered myosin molecules, though suchmutants have not been isolated. The faculty intends to examine the function of

NATIONAL INSTITUTE

the myosin heavy chain in vertebrate
non-muscle cells by interrupting its synthesis with the introduction of antisense
RNA molecules.
Laboratory Computer Facility: The
NIBB's computing is handled by a network of Digital Equipment Corporation's VAX 11/780 and VAX 2,000with Floating Point System's AP120B
and 5310 array processors. An Ethernet
interfaces the network with a number oflaboratory PC's. An extensive softwaresystem for time-series analysis developed
in-house has been operating for the last
several years. A limited number of image
processing routines are also available.
Plant Culture Facility: There are a large
number of culture boxes, cubicles, and alimited number of rooms with environmental control for plant culture.
Experimental Farm: This facility consistsof two 20 m2 glass-houses with precise
temperature and humidity control, alimited farm, two greenhouses (45 m2 ,88 m2) with automatic sprinklers andwindow control, two open aquariums(30 t, 50 t) and several smaller tanks.
The facility also includes a building with
office, storage and work -space.
Plant Cell Culture Facility: Autotrophic
and heterotrophic culture devices areequipped for experimental cultures ofplant and microbial cells.

FOR BASIC BIOLOGYRADIOISOTOPE FACILITY(managed by NIBB)Head of Facility: Yoshihiko Fujita
Associate Professor: Kohji Hasunuma (moved)
Technical Staffs: Kazuhiko Furukawa
Yoshimi Matsuda
Mitsuaki Higashi

The facility is composed of a main
center and two subcenters, one in the
NIBB and the other in the NIPS. The facility is being used for molecular analysesof organisms. At the center, a variety ofradioisotopes including 3H, 14 C, 22Na,
32p, 35 S, 45 Ca, 125 I and various species ofbeta and gamma-ray emitting nucleides
are handled. A laboratory facility forrecombinant DNA research is installedin the center. At the subcenters, only alimited number of radioisotopes such as
3H, 14 C, 32P and 35 S in the NIBB, and3H, t4c, 32p, 35S, 45Ca and t25I in the
NIPS, are processed. The subcenter in
the NIBB is also equipped with a recombinant DNA research laboratory. The

59
members of the Radioisotope Facility
maintain and control the center, andprovide users appropriate guidance forradioisotope handling.
The faculty of the facility conducted
his own research on the analysis of signaltransduction system in plants and fungi.
The study in 1990 was mostly focusedon higher plants. Analysis of proteins in
the plasma membrane of etiolated pea
seelings indicated that (1) red lightstimulates ATP-GTP binding to 4 peptides in the membranes, (2) among them,a 37kDa peptide was found to react withthe antibody against the a-subunit oftransducin, and red light causes loss of
this peptide from the membrane, and (3)ADP-ribosylation of this peptide by theaction of pertussis toxin is stimulated by
red light. These features suggest thattransduction of light signal in pea seedlings involves the action of ATP-GTP
binding proteins.

60

NATIONAL INSTITUTECENTER OF FACILITY FOR ANALYTICAL INSTRUMENTS(managed by NIBB)
Head: Norio Murata

The Center of facility for Analytical
Instruments consists of the following fivesections. (1) Chemical Analysis, (2)Preparation of Biological Materials, (3)Spectroscopic Analysis, (4) PhysicalAnalysis, and (5) Microscopic Analysis.
Each section is equipped with instruments for general use as listed below.
1. Section for Chemical Analysis
Amino-Acid Analyzer
HITACHI 835
Gas Chromatograph
SHIMADZU GC-7 APTFHPLC
SHIMADZU LC-6AD
SPECTRA-PHYSICS SP-8700
Ion Chromatograph
DIONEXQIC
Peptide Synthesizer
BECKMAN 990C
2. Section for the Preparation of Biological Materials
Coulter Counter
COULTERZB
Isotachophoresis System
LKB 2127 TACHOPHOR
Preparative Ultracentrifuge
BECKMAN L8-80
Two-Parameter Cell Sorter
BECTON-DICKINSON FACS-11
3. Section for Spectroscopic Analysis
Atomic-Absorption Spectrophotometer
PERKIN-ELMER 603
Differential Refractometer
CHROMATIX KMX-16
Dual-Wavelength Spectrophotometer
HITACHI 557
Inductively Coupled Plasma Spectrometer

SEIKO SPS 1200A
Infrared Spectrophotometer
JASCO A-302
Laser-Raman Spectrophotometer
JASCO R-800
Light-Scattering Photometer
CHROMATIX KMX-6DC
Spectrofluorometer
HITACHI 850
HITACHI MPF-4
SHIMADZU RF-5000
Spectrophotometer
HITACHI 330
Spectropolarimeter
JASCO J-40S
4. Section for Physical Analysis
Analytical Ultracentrifuge
HITACHI 282
Differential-Scanning Calorimeter
SEIKO DSC100
EPR Spectrometer
BRUKER ER 200D
GC-Mass Spectrometer
HITACHI M-80
GC/LC-Mass Spectrometer
JEOLDX-300
NMR Spectrometer
BRUKER AMX 360wb
Viscometer
CONTRAYES RM-30
5. Section for Microscopic Analysis
Film Data Analysis System
NAC MOVIAS GP-2000
Image Analyzer
KONTRON IBAS-I & II
Microscope Photometer
CARL ZEISS MPM 03-FL
Two-Dimension Microdensitometer
JOYCE LOEBL 3CS

RouteNo.24X

National Institute for
Basic Biology
C>kazaki444,Japan,I/