NATIONAL INSTITUTE FOR BASIC BIOLOGYTransgenic tobacco
with(a) vector plasm ids(b) squash eDNA

ANNUAL REPORT
1991(c) Arabidopsis eDNA

1

INTRODUCTION
The National Institute for Basic Biology (NIBB) is a government supported
basic research institute in the field of biology. It aims to stimulate and promotestudies of biology, by conducting firstrate research on its own, and by cooperating with other universities and researchorganizations. The Institute concentrates
on the studies of cellular functions,
growth and development, homeostatic
control, and gene expression and regulation in eukaryotic organisms, to eluciate
the fundamental mechanisms underlying
various living phenomena at the molecular level.
Two new professors joined the NIBB
during the last year. Prof. Masaharu
Noda (former Associate Professor of
Kobe University) filled the position ofProfessor of Molecular Neurobiology topromote molecular biological studies on
the development of the vertebrate central nervous system. Prof. Hitoshi Sakana of the University of California at
Berkeley was appointed to be in chargeof Division of Cell Fusion (adjunct) to
explore the possibility of the somatic
DNA rearrangement in the developmentof the central nervous system.
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" and"Signal Transduction". Based on suchprograms, the NIBB held the 26th and27th Conferences in 1991, entitled"Plant organelle proteins; biosynthesis,targeting and assembly" (organized by
Prof. Nishimura) and "Dynamic aspectsof the cell cycle" (organized by Prof.
Anraku).
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 1991.

Ikuo 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

Research
The Institute conducts its research
programs through three departments andone laboratory organized into 15 divisions.

Divisions

Department of Cell Biology

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

Department of Developmental Biology

Reproductive Biology
Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)

Department of Regulation Biology

Laboratory of Gene Expression
and Regulation{{

Molecular Neurobiology
Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)--[ Gene Expression and Regulation I
Gene Expression and Regulation II

3

FOR BASIC BIOLOGY

Each division has its own research project and is staffed by a professor, anassociate professor and two research associates. A division forms, in principle,
an independent project team. Six of thedivisions are for adjunct professorshipand are under professors who hold joint
appointments with other universities.
The adjunct division has resident research associates. The arrangement aims
to facilitate exchange in research activities in Japan. The Technical Department manages the activities of researchtechniques and helps to promote research activities of each division and also
to maintain the research resources of theInstitute. The Department also undertakes the technical education of its staffs.
Research Support Facility
The research support facility of the
NIBB consists of the Large Spectrograph Laboratory, Tissue and Cell

Culture Laboratory, Computer Facility,
Plant Culture Facility, Plant Cell Culture
Facility, and Experimental Farm. In addition, seven facilities are operated
jointly with the National Institute forPhysiological Sciences (IPS); they consistof the Radioisotope Facility, Electron
Microscope Center, Center for Analytical Instruments, Machine Shop, Laboratory Glassware Facility, Animal Care
Facility, and Low-Temperature Facility.
CampusThe Okazaki National Research Institutes 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 DIRECfOR
Publication List
Morio, T., Ozaki, T., Takeuchi, 1., andTasaka, M. (1991). Transcriptional
regulation of cell-type-enriched genes
during development in Dictyostelium
discoideum analyzed by nuclear runon assays. Develop. Growth & Differ.,
33, 293-298.

Takeuchi, I. (1990). Cell sorting andpattern formation in Dictyostelium
discoideum. In Cell-cell interactions inearly development, J. Gerhart ed.,Wiley-Liss, Inc., pp.249-259.

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.

6

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor: Mikio Nishimura
Research Associates: Kazuo Ogawa
Makoto Hayashi
lkuko Hara-Nishimura
JSPS Post-doctoral Fellow: Luigi De Bellis
Graduate Students: Ryuji Tsugeki
Katunari Tezukan
Kaori Inou£!1
Technical Staff: Maki Kondo( 11from Nagoya City University)(21from Kobe University)

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, which 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 or-ganelles 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 {3-oxidation and the glyoxylate cycle, aretransformed into leaf peroxisomes thatfunction in several crucial steps ofphotorespiration. Since cell division does
not occur in the cotyledons of these fatty
seedlings following germination, all qf
these developmental events take place ina fixed number of preexisting cells.
Therefore, greening cotyledon is an excellent model system for studies to elucidate the regulatory mechanism underlying light-induced organelle differentiation.
As a step to understand the regulatory
mechanisms that operate during microbody transition in pumpkin cotyledons( Cucurbita sp., Amakuri Nankin), nine
microbody enzymes, i.e., three glyoxysomal enzymes, three leaf peroxisomal
enzymes and three enzymes present inboth microbodies have been purified andcharacterized. Immunocytochemical analyses using their specific antibodies demonstrated that glyoxysomes are directlytransformed to leaf peroxisomes during
microbody transition. eDNA clones forthe glyoxysomal enzyme, malate synthase, and the leaf peroxisomal enzyme,
glycolate oxidase, have been screened
and characterized. By the analysis using
these eDNA clones, decrease in glyoxysomal enzymes during microbody transition was shown to be caused not only bydecrease in level of the mRNA but also
by induction of specific degradation system for glyoxysomal enzymes in microbodies.
Reversible transformation of microbodies from leaf peroxisomes to gly-

FOR BASIC BIOLOGY

oxysomes was found to occur in the
cotyledons during the senescence. The
development of glyoxysomal enzymes
was also observed in petals of pumpkin
flower during the senescence, indicatingthat glyoxysomal enzymes may play arole in senescence process.
Concerning protein transport into
microbodies, we have established an invitro translocation system using isolated
microbodies and products of transcription-translation of eDNA for microbody
enzymes. Since the enzyme compositions
and functions of glyoxysomes and leaf
peroxisomes differ from each other, it isof interest that these microbodies possess
different machineries for protein importor not. By the analysis of in vitro translocation system, it was shown that glyoxysomal enzyme, malate synthase can betranslocated in leaf peroxisomes and leaf
peroxisomal enzyme, glycolate oxidase isable to be imported into glyoxysomes.
These results indicate that transport machinery of glyoxysomes is similar to thatof leaf peroxisomes. eDNA for plantmicrobody enzyme, malate synthase wasexpressed in COS 1 cells, a monkey kidney cell line. The expressed malate synthase was shown immunocytochemicallyto be accumulated in microbodies of
COS 1 cells, suggesting that transportmachineries to microbodies are common
in plant and animal cells and are highly
conserved through evolution.
2. Biogenesis and transformation of protein bodies.
i) Biogenesis of protein bodies during
seed maturation.
Protein bodies in pumpkin and castor
bean seeds are separated into threeparts, namely crystalloid, matrix andmembrane. Crystalloid is composed of
crystalline form of a major storage pro-7

tein, US globulin. We have chosen threeproteins located in each part of protein
bodies and characterized their biosynthesis and intracellular transport. Although the three proteins are localized in
the different suborganellar parts of protein bodies, they are synthesized on endoplasmic reticulum (ER) as larger precursors and transported via transport
vesicles to vacuoles in a similar manner,suggesting that a unique machinery plays
a role in intracellular targeting of thesevacuolar proteins.
Proprotein precursors of vacuolar
components are transported from the
ER into vacuoles, where thay are proteolytically processed into their matureforms. Recently the processing enzyme
was purified from castor bean seeds. Our
results show that the purified enzyme
can process three different proproteinsisolated from either ER or transport
vesicles in cotyledon cells to produce themature forms of these proteins which arefound at different suborganellar locations in the vacuole. Thus a single vacuolar processing enzyme is capable of converting several proprotein precursors
into their respective mature forms.
We have succeeded to isolate thetransport vesicles from maturing pumpkin cotyledons (Fig. 1). They contains alarge amount of proprotein precursors.
Further characterization of the isolatedtransport vesicles will be necessary to
clarify the detailed mechanisms for intracellular transport of protein body proteins and specific recognitions between
ER, transport vesicles and vacuoles atmolecular level.
ii) Reversible transformation between
vacuoles and protein bodies during seedmaturation and germination.
Protein bodies are single membranebound organelles which accumulate seed

8

NATIONAL INSTITUTE

,,

-·

.t~
.

Fig. 1. Electron micrograph of dense vesicles isolated from maturing pumpkin cotyledons (A) and immunoelectron micrograph of the vesicles using an antiserum against 2S protein and protein A-gold (B). Bars are
200 nm.

storage proteins. The protein bodies areclosely related to vacuoles which are thelytic compartments of plant cells. Protein
bodies are formed by budding from thevacuoles during seed maturation, while
vacuoles result from the fusion of protein bodies during seed germination.
Therefore, protein body is a specialized
form of vacuoles in dry seed. It must beemphasized that dramatic transformationof protein bodies from a storage to alytic compartment occurs during the maturation and germination of seeds. In
order to clarify the regulatory mechanisms underlying the processes of thebudding and the membrane fusion , wehave especially focused on one membrane protein (MP32) of protein bodywhich was rapidly degraded just after
seed imbibition. Recently eDNA clone
for MP32 was isolated and the primarystructure of its precursor was deduced
from the nucleotide sequence, indicatingthat the precursor protein included an-other membrane protein (MP27) andMP32 on the polypeptide chain. We
have isolated protein bodies and vacuoles from various developmental
stages of maturing and germinating
pumpkin seeds. Using isolated protein
bodies, we have recently established an
in vitro system to reconstitute the fusion
process. Experiments employing this invitro system are currently underway toelucidate the molecular mechanisms ofmembrane fusion during seed germination.
Publication List
De Bellis, L., and Nishimura, M. (1991)
Development of enzymes of the glyoxylate cycle during senescence of
pumpkin cotyledons. Plant Cell
Physiol., 32, 555-561.
De Bellis, L., Tsugeki, R., and Nishimura, M. (1991) Glyoxylate cycle
enzymes in peroxisomes isolated frompetals of pumpkin during senescence.

FOR BASIC BIOLOGY(1991). Plant Cell Physiol., 32,
1227-1235.
Hara-Nishimura, 1., Inoue, K., and Nishimura, M. (1991) A unique vacuolar
processing enzyme responsible forconversion of several proprotein precursors into mature forms. FEES Letters, 294, 89-93.
Hara-Nishimura, 1., Inoue, K., and Nishimura, M. (1991) Reversible transformation between vacuoles and protein bodies in pumpkin cotyledons. In
Molecular Approaches to Compartmentation and Metabolic Regulation,
Huang, A.H.C. and Taiz, L. eds., The
American Society of Plant Physiologists (Rockville), pp.245-246.
Inaba, K., Ogawa, K., and Mohri, H.
(1991) Mapping of ATP-dependenttrypsin-sensitive sites on the B chainof outer arm dynein from sea urchin
sperm flagella. J. Biochem., 110,795-801.
Mori, H. Takeda-Yoshikawa, Y. HaraNishimura, 1., and Nishimura, M.

9(1991) Pumpkin malate synthase.
Cloning and sequencing of the eDNA
and Northern blot analysis. Eur. J.
Biochem., 197, 331-336.
Nishimura, M., and Hara-Nishimura, I.
(1991) Intracellular protein trafficking
in oil seed. In Molecular Approaches
to Compartmentation and Metabolic
Regulation, Huang, A.H.C. and Taiz,L. eds., The American Society of
Plant
Physiologists(Rockville),
pp.206-213.
Ogawa, K. (1991) Cloning of the sea urchin outer arm dynein B-chain eDNA.
In Biology of Echinodermata, Yanagisawa, T. et al. eds., Belkema (Rotterdam), pp.339-346.
Ogawa, K. (1991) ATP-binding site indynein B-chain: Identification bymolecular cloning. Proc. Japan Acad.,
67B, 27-31.
Ogawa, K. (1991) Four ATP-bindingsites in the midregion of the B-heavy
chain of dynein. Nature, 352,
643-645.

10

NATIONAL INSTITUTEDIVISION OF BIOENERGETICS
Professor: Yoshihiko Fujita
Associate Professor: Shigeru ltoh
Research Associate: Mamoru Mimuro
Kaori Ohki
Katsunori Aizawa
NIBB Visiting Scientist: Siba Plasad Adhikary •Fellows: Tohru Ikeya
Jayanti Kumari Sahu
Technical Staffs: Akio Murakami
Masayo Iwaki( •Department of Botany, Utkal University, Orissa,
India)

Light-energy conversion in photosynthesis has been investigated at cell biological and biophysical levels. Cell biological study has been focused on themechanism for regulation of stoichiometry between the two photochemical
reactions in thylakoids, PSI and PSII
complexes, in response to the photosynthetic conditions. Nitrogen-fixation incyanophytes directly coupled withoxygenic photosynthesis has been another subject in the study of this level.
Biophysical study has been focused onmechanisms of the excitation-energy
transfer in light-harvesting pigment-protein complexes and of the electron transfer in the photosynthetic reaction centers.
(1) Regulation of stoichiometric composition ofthylakoids
We have found that the stoichiometry
between PSI and PSII in thylakoid system is not fixed but is changed in response to the photosynthetic conditions.
This regulatory change adjusts the balance between the two light reactions andmaintains a high efficiency of photosynthesis under each condition (cf. Fig. 1
and Fujita 1990). Our previous study forcyanophytes has revealed that this regulatory change is achieved, by control of
PSI formation during thylakoid development, and the signal for the control isclosely related to the redox state of Cyt

b6 -f in the thylakoid ETS. Analysis of
the electron transport within Cyt b6 - f inrelation to the control of PSI formation
was indicated that (1) HQNO-sensitive
Cyt-b6 oxidation is involved in the signal
system and that (2) the signal from ETS
causes stimulation of PSI formation.
Analysis of PSI formation in both processes for apoprotein and Chl a synthesis
has also indicated that (1) the controlledchanges in apoprotein synthesis occurs at
translational level and (2) Chl a synthesis
is also regulated at the step after Pchlide
synthesis. Results suggest a possibility
that the signal from ETS stimulates Chl
a synthesis and resultant larger supply of
Chl a accelerates synthesis and assemblyof PSI complex.
The abundance of Cyt oxidase, another terminal of thylakoid ETS, is alsochanged in parallel with that of PSI suggesting that the abundance is regulated
by a similar mechanism to that for PSI.
However, some of Cyt oxidase has beenreported to be located not in thylakoids
but in cytoplasmic membranes in someparticular strain, so that changes inabundance of Cyt oxidase may be induced by a different signal mechanism
from the control of PSI formation. We
re-examined the location of Cyt oxidasein the same strain as used for the above
observation by a cytochemical electron
microscopy. Results have shown that Cyt
oxidase activity is found only in thylakoids, suggesting that the abundance of
Cyt oxidase is also regulated in responseto the thylakoid signal system.
(2) Day/night oscillation of nitrogenase
activity in the cyanophyte Trichodesmium
The activity for nitrogen-fixation incyanophytes oscillates under day/night
regime; in most cases, high activity atnight and low or insignificant in day

11

FOR BASIC BIOLOGY

JJ,

L i ght

D.
Pho t osynthetic,, _~ ·:'·::'
H,o -~- NAOP"

D.

Ligh tPhotosynthetic
efficiency
-

NAOP·

0.4

1.0

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

time. This oscillation has been interpreted as a time separation of the 0 2sensitive nitrogen-fixation from 0 2 evolving photosynthesis. However, Trichodesmium sp. shows a reverse pattern due
to a direct coupling of nitrogen-fixation
with oxygenic photosynthesis; high in
day time, and insignificant at night. To
know the mechanism for protection of
nitrogenase from 0 2 evolved by photosynthesis, changes in expression andmodification of nitrogenase proteins
were examined in relation to changes in
the activity. Results have indicated thatthe oscillation is due to activation andinactivation of the Fe-protein of nitrogenase; the Fe-protein is modified to theinactive form in the dark, but lightcauses the activating modification. Results suggest that a light-dependent activation of the Fe-protein can compensate
the inactivation by 0 2 and maintain ahigh activity of nitrogenase under simultaneous occurrence of photosynthesis in
this organism.
(3) Excitation energy transfer in lightharvesting pigment-protein complexes
The study in 1991 was focused on theenergy transfer mechanism in fucoxanthin-Chl ale protein complex, an intact
light-harvesting assembly newly isolated
from the brown alga Dictyota dichotoma(FCP A), and in chlorosomes in thegreen bacterium Chloroflexus aurantiacus. Spectroscopic analysis of FCP A has
revealed that (1) fucoxanthin is present
in the protein complex with a specificmolecular geometry, which enables thepigment to transfer efficiently its excitation energy to Chi a. This study also provided the first success in detection of thefluorescence from fucoxanthin and otherketo carotenoids. Analysis of the fluorescence from keto carotenoids has indicated that keto group of carotenoids
induces S1 fluorescence with a long lifetime, both of which are responsible forthe efficient energy transfer to Chl a.
The study for chlorosomes has indicateda heterogeneity of molecular state ofBchl c in chlorosomes and also occurrence of plural pathways of the energy
transfer in chlorosomes. A model formolecular arrangement of two types of
Bchl c oligomers in chlorosomes has
been proposed.
( 4) Electron transfer in photosynthetic reaction center complexes
Mechanism of energy conversion inphotosynthetic reaction center complexes has been investigated by replacing
native quinone with artificial ones. Special attention has been paid for the action of phylloquinone in PSI reaction

12
center of green plants. The reaction
center complex accepted a wide varietyof quinones and analogues and enabled
them to function as the electron acceptor. Results further indicated a uniquestructure of the quinone-binding site in
the aspects of the affinity and reactivityof artificial compounds. A marked difference was observed for the electrontransfer in the reaction center complex
from that in solution. Similar studies arealso undergoing with reaction centers of
PSII and of green-sulfur photosynthetic
bacteria.
Publication List
Ikeya, T., Ohki, K., Takahashi, M., andFujita Y. (1991) Photosynthetic pigment system of picophytoplankton of
cyanophytes isolated from subsurface
water in Kuroshio area. J. Oceanogra.
Soc. Japan., 47, 1-6.
Itoh, S., and Iwaki, M. (1991) Full replacement of the function of the secondary electron acceptor phylloquinone (=vitamin K 1) by non-quinone
carbonyl compounds in green plant
photosystem I photosynthetic reaction
centers. Biochemistry, 30, 53405346.
Iwaki, M., and Itoh, S. (1991) Structureof the phylloquinone-binding (Q~) site
in green plant photosystem I reaction
center: the affinity of quinones andquinoid compounds for the Q~ site.
Biochemistry, 30, 5347-5352.
Iwaki, M., and Itoh, S. (1991) Functionof quinones and quinoids in greenplant photosystem I reaction center. In
Energy Transfer in Inorganic, Organic
and Biological Systems, Bolton, J. ed.,American Chemical Soc. (Washington
DC), pp.163-178.
Katoh, T., Nagashima, U., and Mimuro,M. (1991) Fluorescence properties of

NATIONAL INSTITUTE

the allenic carotenoid fucoxanthin: Its
implication for energy transfer inphotosynthetic
pigment
systems.
Photosyn. Res., 27, 231-236.
Mimuro, M., and Katoh, T. (1991) Carotenoids in photosynthesis: Absorption, transfer and dissipation of lightenergy. Pure & Appl. Chern., 63,
123-130.
Mimuro, M., Nagashima, U., Takaichi,
S., Nishimura, Y., Yamazaki, L, andKatoh, T. (1991) Molecular structure
and optical properties of carotenoids
for the in vivo energy transfer function
in algal photosynthetic pigment system. Biochim. Biophys. Acta, 1098,
271-274.
Murakami, A., and Fujita, Y. (1991)
Steady state of photosynthetic electrontransport in cells of the cyanophyte
Synechocystis PCC6714 having different stoichiometry between PSI and
PSII: Analysis of flash-induced oxidation-reduction of cytochrome f andP700 under steady state of photosynthesis. Plant Cell Physiol., 32,
213-222.
Murakami, A., and Fujita, Y. (1991)
Regulation of photosystem stoichiometry in the photosynthetic systemof the cyanophyte Synechocystis
PCC6714 in response to light-intensity. Plant Cell Physiol., 32, 223-230.
Nakane, H., Iwaki, M., Satoh, S., andItoh, S. (1991) Artificial quinones replace the function of quinone electron
acceptor (QA) in isolated D1-D2-cytochrome b559 photosystem II reaction
center complex. Plant Cell Physiol.,32, 1165-1171.
Ohki, K., and Fujita, Y. (1991) Complementary chromatic adaptation inthecyanobacterium,
Tolypothrix
tenuis: Location of phycoerythrin
newly synthesized by green illumina-

FOR BASIC BIOLOGY

tion. Plant Cell Physiol., 32,
483-488.
Ohki, K., Zehr, J.P., Falkowski, P.G.,
and Fujita, Y. (1991) Regulation of
nitrogen-fixation by different nitrogensources in the marine non-heterocystous cyanobacterium Trichodesmium
sp. NIBB1067. Arch. Microbiol., 156,
335-337.
Ohki, K., Falkowski, P.G., Rueter, J.G.,
and Fujita, Y. (1991) Experimental
study of the marine cyanophyte Trichodesmium sp., a nitrogen-fixing
phytoplankton in tropical and subtropical sea area. In Marin Biology. Its
Accomplishment and Future Prospect,
Proceedings of the 5th Symposium ofInternational Prize for Biology,
Mauchline, J. and Nemoto, T. eds.
Hokusensha (Tokyo), pp.205-216.
Oh-oka, S., Itoh, S., Saeki, K., Takahashi, Y., and Matsubara, H. (1991)
FA/Fs protein from the spinach
photosystem I complex: Isolation in anative state and some properties of theiron-sulfur clusters. Biochim. Biophys.
Acta, 32, 11-17.
Post, A.F., Mimuro, M., and Fujita, Y.
(1991) Light 2 directed changes in theeffective absorption cross-section ofphotosystem II in Synechocystis 27170
are related to modified action on thedonor side of the reaction center. Biochim. Biophys. Acta, 1060, 67-74.
Uehara, K., Mimuro, M., and Tanaka,
M. (1991) Spectroscopic studies ofchlorophyll a aggregates formedby aqueous dimethyl sulfoxide.
Photochem. Photobiol., 63, 123-130.
Yokoyama, E., Murakami, A., Sakurai,
H., and Fujita, Y. (1991) Effect of

13
supra-high irradiation on the photosynthetic system of the cyanophyte
Synechocystis PCC6714. Plant Cell
Physiol., 32, 827-834.
Zehr, J.P., Ohki, K., and Fujita, Y.
(1991) Arrangement of nitrogenase
structural genes in an aerobic filamentous nonheterocystous cyanobacterium. J. Bacteriol., 173, 7055-7058.
Zehr, J.P., Ohki, K., Fujita, Y., andLandry, D. (1991) Unique modifica~tion of adenine in genomic DNA ofthe marine cyanobacterium Trichodesmium sp. strain NIBB 1067. J. Bacteriol., 173, 7059-7062.
Supplements to 1990
Fujita, Y. Regulation of stoichiometry
among thylakoid components in response to light regime: A story of the
dynamic nature of the thylakoid system in cyanophytes. In Regulation of
Photosynthetic Processes, Special Issueof Botanical Mag., Tokyo 2, Miyachi,
S., Kanai, R. and Katoh, S. eds., Jpn.
Soc. Botany (Tokyo), pp.29-42.
Uehara, K., Hioki, Y., and Mimuro, M.
Formation of chlorophyll a-protein
complexes absorbing at 685 nm in thepresence of tetrahydrofuran. In Current Research in Photosynthesis, Baltshefsky, M. ed., Kluwer Academec
Publ. (Dortrecht), pp.241-244.
Zehr, J.P., Limberger, R.J., Ohki, K.,
and Fujita, Y. Antiserum to nitrogenase generated from an amplified
DNA fragment from natural populations of Trichodesmium spp. Appl.
and Environ. Microbiol., 56, 35273531.

14

NATIONAL INSTITUTEDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor: Yasuhiro Anraku
Associate Professor: Souichi Nakamura
Research Associate: Hidetoshi /ida
N/BB Post-doctoral Fellow: Hiro Nakamura
JSPS Post-doctoral Fellow: Junko NakajimaShimada
Graduate Student: Tomoko Ono (from Nara
. ..
.
Women's University)
V1s1tmg Sc1entist: Makiko S. Okumura

Ca2+ is a growth-regulating substancein the cell cycle of eukaryotic cells. For
example, when the availability of caz+becomes limited in the presence of sufficient amount of growth factors, mouse
fibroblastic cells are arrested in the G 1phase of the cell cycle. Most of growth
factors elicit transient increase in the cytosolic free Ca2+ concentration ([Ca2+]).
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+h have beendeveloped, 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 1) therole of Ca2+ in the cell cycle and matingprocess of the yeast Saccharomyces cerevisiae, 2) the establishment of measuringsystems for [Ca2+]i in yeast cells and 3)
the mechanism of regulating Ca2+ homeostasis, with the hope that combination of cell biological and genetic approaches will uncover the molecularmechanisms of Ca2+ signaling.
Establishment and Application of a[Ca2+]i Monitoring System by Using
the Apoaequorin eDNA
The use of fura- 2 in conjunction withdigital image processing enabled us toquantify [Ca2+]i in a single yeast cell,as described in the last issue of the

NIBB Annual Report (also see Iida
et al. (1990) J. Bioi. Chern., 265,
13391-13399). The fura-2 method has
advantages to measure [Ca2+]i in single
cells and spatial distribution of [Ca2+h in
the cytosol and organelles, but has somedrawbacks: 1) fura-2 is sequestered
eventually into an organellar compartment, the vacuole, and 2) fura-2 requires
excitation at 340 and 380 nm. These two
problems make it difficult to monitor[Ca2+]i for a long period. To circumventthese problems, we have chosen a second Ca2+-specific probe, aequorin. It is a
Ca Z+-b"md"mg protein that einits lightupon reacting with Ca2+ and consists ofthree components: apoaequorin (apoprotein), coelenterazine (chromophore)and molecular oxygen. The eDNA for
apoaequorin of jellyfish Aequorea victoria has been cloned and sequenced.
Apoaequorin is composed of 189 aininoacid residues with three EF-hand structures. Although aequorin has been used
as a probe, the use of this protein has
been limited by the difficulty of introducing sufficient amount of it into small
cells, including yeast. To overcome thisdifficulty, we have designed the present
study to express the apoaequorin eDNA
in yeast cells under the control of GALlor the GAP promoter and tested
whether apoaequorin can be accumulated in high enough concentration in thecells to detect a Ca2+ signal. The results
showed that the cells accumulated sufficient amounts of recombinant apoaequorin and that the protein was active andnot toxic to the cells. For an in vivo
method for monitoring of changes in[Ca2+]i, introduction of coelenterazine
into intact yeast cells and regeneration of
aequorin in the cells are essential steps.
We found that aequorin can be effectively regenerated by simply incubating

15

FOR BASIC BIOLOGY

C")

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Changes in [Ca2+L in response to glucose in glucose-starved G 0 /G 1 cells, as revealed by luminescence intensity of aequorin regenerated in intact yeast cells. Twenty-four hours after the start of glucose starvation,
cells expressing the recombinant apoaequorin were harvested, resuspended in Mes/Tris buffer, incubated withcoelenterazine, washed, resuspended in appropriate buffers and monitored for changes in [Ca 2+];· (A) Cells
were resuspended in Mes/Tris buffer containing no CaCI 2 and then supplemented (arrow) with glucose (tracea) or 2-deoxyglucose (trace b). (B) Cells were resuspended in Mes/Tris buffer containing 10 mM CaCI 2 andthen supplemented (arrow) with glucose (trace a), fructose (trace b), mannose (trace c), 2-deoxyglucose(trace d), or 6-deoxyglucose (trace e). This figure shows that a rise in [Ca 2+L is dependent on extracellular
Ca2+, and suggests that the rise is energy-dependent.

intact yeast cells with coelenterazine.
Coelenterazine-incorporated cells responded to extracellular stimuli. A mating pheromone, a-factor, generated extracellular Ca2+-dependent luminescence
specifically in cells of a mating type withmaximal 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 (Fig.

A and B-a). This stimulation corresponded to a change in [Ca2+]i from thebasal level of 180 ± 10 nM to 340 ± 40nM (n=4 ). These results demonstrate theusefulness of the aequorin method inmonitoring [Ca2+L response to extracellular stimuli in yeast cells. Using thismethod, we have investigated mechanisms of Ca2+ mobilization in the initiation of the cell cycle of glucose-starved
G 0 /G 1 cells triggered by glucose. Theresults showed that metabolizable hexoses

16
such as glucose, fructose and mannose,
but not non-metabolizable hexoses suchas 2-deoxyglucose and 6-deoxyglucose,
elicited the [Ca2+h rise in glucosestarved cells. This result implies thepossibility that ATP may be involved inthe [Ca2+]i response. To test this possibility, we added 10 ,uM KCN, a potent
inhibitor of electron transport in mitochondria, with glucose. The [Ca2+]i rise
was dramatically reduced by KCN. This
result is consistent with the notion that
ATP is involved in the [Ca2+]i response.
To clarify this point, several experiments
are in progress.
Ca2+ Homeostasis Maintained by the
Vacuolar H+-ATPase and Vacuolar
Ca2+ fH+ Antiporter
In vitro experiments have shown thatthe vacuolar membrane vesicles of theyeast have a H+-ATPase capable of producing an electrochemical potential difference of protons (~,uH+). The H+-ATPase thus enables the vacuole to incorporate protons from the cytosol. The
vesicles also have a Ca2+fH+ antiporterthat enables the vacuole to take up Ca2+
by extruding protons from the vacuole.
Interestingly, the Ca2+ uptake is inhibited by inhibitors of the vacuolar H+ATPase, such as Cu2+, Zn 2+ and N, N'dicyclohexylcarbodiimide. This observation raises the possibility that the Ca2+
uptake carried out by the Ca2+fH+ antiporter is coupled with the activity of the
H+-ATPase in vivo. If this is the case,the vacuole of mutant cells defective inthe vacuolar H+-ATPase may fail to incorporate Ca2+ and the Ca2+ concentration in the cytosol may become higher inthe mutant than in wild-type cells. To
test this possibility, we measured [Ca2+]iin cells of the cis7, cis8, cis9, cislO andcisll mutants, all of which are defective

NATIONAL INSTITUTE

in H+-ATPase activity, by using thefura- 2 method. The result showed thatthe average [Ca2+]i in wild-type cells was
150 ± 80 nM whereas the mean value inthe five cis mutant cells was 900 ± 100
nM. This observation clearly indicatesthat Ca2+ homeostasis in the cytosol ismaintained by the coupling of Ca2+fH+
antiport with H+-ATPase on the vacuolar membrane.
Publication List
Anraku, Y., Ohya, Y., and Iida, H.
(1991) Cell cycle control by calcium
and calmodulin in Saccharomyces
cerevisiae. Biochim. Biophys. Acta,
1093, 167-177.
Miyamoto, S., Ohya, Y., Sano, Y., Sakaguchi, S., Iida, H., and Anraku, Y.
(1991) A DEL-homologous region of
the yeast CLS4/ CDC24 gene productis important for Ca2+-modulated bud
assembly. Biochem. Biophys. Res.
Commun., 181, 604-610.
Nakajima-Shimada, J., Iida, H., Tsuji,F.l., and Anraku, Y. (1991} Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin eDNA expression system.
Proc. Nat/. Acad. Sci. USA, 88,
6878-6882.
Nakajima-Shimada, J., Iida, H., Tsuji,F.l., and Anraku, Y. (1991) Galactose-dependent expression of the recombinant Ca2+-binding photoprotein
aequorin in yeast. Biochem. Biophys.
Res. Commun., 174, 115-122.
Ohya, Y., Umemoto, N., Tanida, 1.,
Ohta, A., Iida, H., and Anraku, Y.
(1991) Calcium-sensitive cis mutantsof Saccharomyces cerevisiae showing apee phenotype are ascribable to defects of vacuolar membrane H+ATPase activity. J. Bioi. Chern., 266,
13971-13977.

FOR BASIC BIOLOGYDIVISION OF CELL FuSION(ADJUNCT)
Professor: Hitoshi Sakano
Research Associate: Masahiro Ishiura
Fumikiyo Nagawa

We are interested in the somatic DNA
rearrangement in the immune and central nervous systems in the mouse. In the
immune system, V-(D)-J joining generates a vast diversity of antigen receptor
genes by combining multiple gene segments, as well as by modifying the junctional sequences. Somatic DNA rearrangement can also serve as an effective
mechanism for the activation of a particular member of a multigene family. In
the antigen receptor gene, this isachieved by bringing two separate regulatory DNA elements, the promoter andenhancer, into close proximity. This typeof gene activation mechanism may beutilized to determine the irreversible fateof cells during development. It is tempting to ask whether such gene rearrangement is restricted to the immune system,
or whether similar somatic events are exploited by other organ systems, e.g., thecentral nervous system.
Similarities between the immune system and the central nervous system havelong been discussed. Both systems haveto be capable of recognizing and remembering many different external signals. It
is known that some lymphocyte surface
proteins, e.g., CD4, are expressed innerve cells. Furthermore, it has beenshown that RAG-1, a gene involved insite-specific recombination in the immune system, is also transcribed in thebrain. In order to study the developmental control ofthe V-(D)-J type of joining,
we have constructed a recombination reporter gene and generated transgenic

17

animals. The substrate contained two
sets of V-(D)-J joining signals and thebacterial ,8-galactosidase gene, lacZ, inan opposite transcriptional orientation
downstream from the promoterI enhancer complex (Matsuoka et a/.,Science 254: 81-86, 1991). While analyzing transgenic mice, we noticed that
not only lymphatic tissues, but certain
areas of the brain were stained blue with
X-gal. This observation indicates that
DNA recombination activates the transgenic reporter gene during the development of nerve cells. Regions showing ,8gal activity appear around birth, andcontinue to emerge postnatally in newborn mice. While the distribution of the,8-gal activity in the central nervous system is widespread, it is neither diffuse
nor random.
Although the mode of gene rearrangement remains to be studied, it appears
that recombination occurs in certain domains of the brain at specific stages ofdevelopment. The question then arises,
why do brain cells have to undergo generearrangement? Recombination in brain
cells could be acting as switching system
for gene expression by DNA inversion
or by juxtaposing promoters and coding
regions with enhancers by a deletionmechanism. The next task is obviously toidentify the rearranging genes in nerve
cells. For deletion type recombination,
isolation of circular DNA has been instrumental in the study of Ig and TCR
gene rearrangement in lymphocytes(Matsuoka et al., Cell, 62: 135-142,
1990). If the rearrangement in the brain
occurs by a DNA deletion mechanism,
characterization of brain circular DNA
will allow us to identify the rearranging
genes in the central nervous system.

18

NATIONAL INSTITUTEDMSION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Hotta
Research Associate: Hitoshi Okamoto
Institute Research Fellow: Keiko Nakao
Graduate Stuedent: Keita Koizumi(from Univ. of Tokyo)

Our division is currently involved in
the study of molecular mechanisms ofcellular communications and signaling,
especially of the development and differentiation processes of the multicellularorganisms. As model organisms, fruit fly(Drosophila melanogaster) and fish
(Japanese Medaka and zebrafish) arechosen, to study the sensory receptors,
nervous systems and muscles at thegenetic, molecular and cellular levels.
Neurogenetics of Drosophila

For the study of Drosophila, many visual transduction mutants have been isolated by their abnormal visual behavior.
The molecular defects of many visionmutants have been shown to be involvedin the phosphatidyl-inositol metabolisms
in the eye. The results suggest that thevisual signal transduction system islinked to the phospholipid turnover,most likely that of inositol 1,4,5-triphosphate (InsP3). By molecular cloning of
the genes, norpA (no receptor potentialA) and rdgA (receptor degeneration A)
have been identified as structural genes
for eye-specific phosphatidyl inositol
phosphoplipase C and diacylglycerol kinase, respectively.
Other genes of interest are the embryonic lethals of Drosophila which arenormal in embryonic polarities and segmentations, but are causing defects inthe early differentiation of mesoderm,
neuroblasts and glia. One particularly interesting gene, 1(2)KN13 is shown tolack muscle cells both in vivo and invitro (single embryo primary culture)
due to the defects in myoblast fusion
processes in spite of the presence ofmorphologically normal "muscle pioneer
cells".
Molecular Embryology of Fish

After the recent explosive advance inneurobiology and molecular developmental biology of Drosophila, we are at
the stage of extending our knowledge
and molecular technology to vertebrate
systems. It is especially important tostudy how the neurons in the vertebrate
brain acquire their cellular identities andhow they send their axons to proper targets. Embryos of small fresh water fish,
such as zebrafish or Japanese Medaka
fish are useful materials for such study. It
is because the embryos stay transparent
throughout their embryonic development, and many of their early neurons
are identifiable. Many research groups,
including ours, have started exploiting
this advantage to carry out cell manipulations, such as dye-labeling, cell transplantation and cell ablation by laser
beam.
Since April 1991, we have set up thefacility for the fish study. We are currently modifying zebrafish genome by using
gene transfer technique to make transgenic fish, whose embryos are designed
to be amenable to various modem analyses like genetic mosaic method and enhancer-trap technique.
Publication List
Masai, I., and Hotta, Y. (1991)
Genomic organization of a Drosophila
phospholipsase C, norpA, and molecular lesions in two temperature-sensitive mutants. J. Biochem., 109:867-871.

19

FOR BASIC BIOLOGY

A

B

•

(A) Side view of a 24 hr zebrafish embryo. The yolk sac has been removed. (B) A 24 hr zebrafish embryotransformed with the E. coli lacZ gene under the control of the mouse hsp68 promoter. After heat shock andfixation , tissues of a whole mount embryo have been stained blue for {3-galactosidase activity with X-gal.

Okamoto, H., and Kuwada, J.Y. (1991)
Outgrowth by fin motor axons in wildtype and finless mutant of theJapanese Medaka fish. Develop. Bioi.,
146: 49-61.

Okamoto, H. and Kuwada, J.Y. (1991)
Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese Medaka
fish. Develop. Bioi., 146: 62-71.

NATIONAL INS'fiTUTE

I--·---~~----1 .. ··i

L_____ -------

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; differentiation and maturation of the gonad cells, molecular basis of the body plan, and gene regulation in the
differentiated tissues, changes of the differentiatedstates, and different phases of the cell cycle.

22
LABORATORY OF REPRODUCTIVE
BIOLOGY
Professor: Yoshitaka Nagahama
Research Associates: Michiyasu Yoshikuni
Masakane Yamashita
Minoru Tanaka
Post-doctoral Fellows: Noriyoshi Sakai
Naoki Shibata*
Johannes Kamen*
Ian Gleadall*
Christian R. Muller
Toshiaki Hirai
Takeshi Miura
Chiemi Miura
Graduate Students: Akihiko Yamaguchi
Mika Takahashi
Toshinobu Tokumoto
Visiting Scientists: Yan Shao- Yi
Sergei G. Vassetzky*
Devendra N. Saksena *Jennifer Specker
Kazu Haino-Fukushima * *Michiya Matsuyama * * *Technical Staffs: Hiroko Kajiura
Sachiko Fukada(*supported by JSPS)(**from Tokyo Metropolitan University)(***from Mie 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, andoccurs 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 of differentiation,
growth and maturation of germ cells in
multicellular animals, using fish as a pri-

NATIONAL INSTITUTE

mary 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, 20/3-dihydroxy-4-pregnen-3-one (17 a, 20/3-DP)
as the maturation-inducing hormone ofamago salmon (Oncorhynchus rhodurus). Along with estradiol-17 /3, which
was identified as the major mediator of
oocyte growth, we now have two known
biologically important mediators ofoocyte growth and maturation in female
salmonid fishes. It is established that the
granulosa cells are the site of productionof these two mediators, but their production by the ovarian follicle depends on
the provision of precursor steroids by thethecal cell (two-cell type model). A dramatic switch in the steroidogenic pathway from estradiol-17 f3 to 17 a, 20/3-DP
occurs in ovarian follicle cells immediately prior to oocyte maturation. This
switch is a prerequisite step for thegrowing oocyte to enter the maturation
phase, and requires a complex and integrated network of gene regulation involving cell-specificity, hormonal regulation, and developmental patterning.
To investigate the molecular basis forthis switch, our current efforts center on
the cloning and sequencing of the genes
encodings five steroidogenic enzymes responsible for estradiol-17 f3 and 17 a,
20/J-DP biosynthesis: cholesterol sidechain cleavage cytochrome P-450 (P450scc), 3/3-hydroxysteroid dehydrogenase (3/3-HSD), 17a-hydroxylase/

23

FOR BASIC BIOLOGY

Aromase activity(ng/ml)20
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2.6 kb

~
'(

A

B

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Fig. 1. Upper panel, changes in estradiol-17 f3 production by isolated follicular preparations of trout during early (A) and late (B) vitellogenesis and oocyte maturation (migratory nucleus (C) and matured oocyte(D)) and by postovulatory follicles (E). Follicular preparations were incubated in Ringer alone (R) or in thepresence of 100 ng testosterone/mi. Levels of estradiol-17 /3 in the culture medium following incubation were
measured by RIA. The diameters of the follicles used are shown in parentheses (mm). Lower panel, Dectection of 2.6 Kb RNA transcripts that hybridized to the trout aromatase probe from various stages of trout
ovaries. Poly(AtRNAs were extracted from the same ovaries as shown in the upper panel.

C 17,20-lyase cytochrome P-450 (P450c17), P-450 aromatase (P-450arom)and 20 /3-hydroxysteroid dehydrogenase(20/3-HSD). We have already isolated
eDNA clones for these five enzymes; thecomplete nucleotide and deduced aminoacid sequences of P-450c17, P-450arom
and 20,8-HSD cDNAs have been determined. A eDNA clone encoding
P-450c17 was isolated from a rainbow

trout ovarian follicle eDNA library. The
eDNA contained an open reading frameof 1542 nucleotides encoding a proteinof 514 amino acid residues. The trout
P-450c17 expressed in nonsteroidogenic
mammalian COS-1 cells showed both
17 a-hydroxylase and c17,20-lyase activities. The eDNA only hybridized to asingle species of mRNA (2.4 Kb) isolated from rainbow trout ovaries; the24
2.4 Kb transcripts were abundant in
trout ovaries during the later stages ofoogenesis. A eDNA clone encoding
rainbow trout P-450arom was also sequenced and found to contain an openreading frame of 1566 nucleotides predicted to encode a protein of 522 aminoacid residues. Northern blot analysis revealed 2.6 Kb RNA transcripts whichwere present in the trout ovary during
vitellogenesis and hybridized to theeDNA insert. The RNA transcripts were
present only during stages of estradiol17f3 production by the ovarian follicles,
suggesting that estradiol-17 f3 production
is regulated, in part, by the amount of
P-450arom mRNA present (Fig. 1).
Using these P-450c17 and P-450arom
eDNA clones, we are now attempting toclone genomic DNAs for these two
enzymes.
Using synthetic oligonucleotides deduced from the partially determinedamino acid sequence of purified pig
20/3-HSD, we have, for the first time inany species, isolated and sequenced aeDNA encoding 20/3-HSD from a pig
testis eDNA library; this work has been
carried out in collaboration with Drs. S.
Nakajin and S. Ohno (Hoshi University).
The eDNA contains an open-reading
frame of nucleotides predicted to encode
a protein of 289 amino acid residues.
Surprisingly it has 85% amino acid homology with human carbonyl reductase.
The steady state levels of the mRNAtranscripts increased to a maximum intestes from 10-day old pigs but rapidly
declined thereafter to the same levels
found in testes of mature animals. The
pig 20/3-HSD will be used as a probe toclone rainbow trout 20/3-HSD.
We have previously shown that 17 a,
20/3-DP acts via a receptor on the oocyte
surface membrane and not through cyto-

NATIONAL INSTITUTE

plasmic or nuclear receptors. The earlysteps following 17 a, 20/3-DP action involve the formation of the major mediator of this steroid, maturation-promoting
factor or metaphase-promoting factor(MPF). MPF activity cycles during 17 a,
20/3-DP-induced oocyte maturation withthe highest activity occurring at the firstand second meiotic metaphase. Studies.from our laboratories have shown that
MPF activity is not species-specific andcan be detected in both meiotic and mitotic cells of various organisms, fromyeast to mammals. We purified MPF
from mature oocytes of carp ( Cyprinus
carpio). Carp MPF consists of two components; one is homolog of the cdc2+
gene product of fission yeast (Schizosaccharomyces pombe), referred to as
p34cdc2, and the other is cyclin B. Using
monoclonal antibodies against the
PSTAIR sequence of p34cdc2 and E.
coli-produced goldfish cyclin B, we
examined the levels of p34cdc2 and cyclin
B during goldfish oocyte maturation induced in vitro by 17a, 20/3-DP. Protein
p34cdc 2 was found in immature oocyte
extracts and did not remarkably change
during oocyte maturation. Cyclin B was
absent in immature oocyte extracts andappeared when oocytes underwent germinal vesicle breakdown. Cyclin B thatappeared during oocyte maturation waslabeled with [35 S]methionine, indicating
its de novo synthesis. The introduction of
E. coli-produced cyclin B into immature
oocyte extracts induced p34cdc2 activation, which was associated with threonine phosphorylation of p34cdc2 andserine phosphorylation of cyclin B, as
found in oocytes matured by 17 a, 20 f3DP. Cyclin B-induced p34cdc2 activation
was not induced by inhibiting threonine
phosphorylation of p34cdc2 by protein
kinase· inhibitors. These results suggest

25

FOR BASIC BIOLOGY
17a,20j3-DP

J

~
I
I
I
I
I
I
I
I

~,:~-""?

activecyclln B

34cdc2~MPF?)

cyclln B

Fig. 2. A hypothesis for the formation and activation of MPF during fish oocyte maturation (see text for
details).

that 17 a, 20,8-DP induces oocytes tosynthesize cyclin B, which in turn activates preexisting p34cctcz through threonine phosphorylation of p34cctcz (Fig. 2).
In collaboration with Dr. T. Choi andothers (University of Tokyo), we examined the changes in the phosphorylation
state of p34cctcz and its histone H1 kinase
activity in mouse oocytes and embryos.
The results suggest that changes in phosphorylation states of p34cctcz triggered its
activation at the first metaphase, but notinactivation and reactivation at the firstand second metaphase, respectively.
However, in the mitotic cell cycle, theactivation of p34cctcz protein kinase may
be triggered by its dephosphorylation.
2) Endocrine regulation of male germ
cell development and maturation
We have identified two steroidal
mediators of male germ cell development in salmonid fishes (11-ketotestosterone for spermatogenesis and 17 a,
20j9-DP for sperm maturation). A steroidogenic switch, from 11-ketotestosterone to 17a, 20P-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 cells.
In the cultivated male Japanese eel(Auguilla japonica), spermatogonia are
the only germ cells present in the testis.
A serum-free, chemically defined organ
culture system developed for eel testes
was used to investigate the effect of various steroid hormones on the inductionof spermatogenesis in vitro. We obtained
evidence that 11-ketotestosterone can
induce the entire process of spermatogenesis in vitro from premitotic spermatogonia to spermatozoa within 21
days. We are now attempting to isolate
genes expressed during the hormone-induced spermatogenesis in the eel.
In salmonid fishes, spermatozoa takenfrom the testes are immotile, but acquire
motility during their passage through thesperm duct. Using male masu salmon(Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17a, 20j9-DP is responsible for26
the acqutsthon of sperm motility; 17 a,20{3-DP acts to increase sperm duct pH,
which in turn increases the cAMP content of sperm, allowing the acquisition of
motility. More recently, in collaboration
with Dr. T: Kobayashi (Niigata University), we identified 17 a, 20a-dihydroxy4-pregnen-3-one as the spermiation-inducing hormone in a frog (Rana nigromaculata). It is well established that gonadotropins and androgens are the two
major hormones involved in the regulation of vertebrate spermatogenesis. Thus,
our findings demonstrate, for the firsttime, the distinct role of progestational
steroids in vertebrate spermatogenesis.
Progestational steroids may represent
endocrine effects that have been heretofore unrecognized among the moreclassical hormonal regulators of malegerm cell formation in vertebrates.
Our recently-initiated studies include
work on the molecular mechanisms of(1) sex determination and gonadal differentiation, (2) meiosis initiation, and(3) vitellogenin uptake into the oocytefrom the vascular system.
Publication List
Choi, T., Aoki, F., Mori, M., Yamashita,M., Nagahama, Y., and Kohmoto, K.
(1991) Activation of p34cdcl protein
kinase activity in meiotic and mitotic
cell cycles in mouse oocytes and embryos. Development., 113, 789-795.
Hourigan, T.F., Nakamura, M., Nagahama, Y., Yamauchi, K., and Grau,
E.G. (1991) Histology, ultrastructure,and in vitro steroidogenesis of the testes of two male phenotypes of the
protogynous fish, Thalassoma duperrey. Gen. Camp. Endocrinol., 83,
193-217.
Matsuyama, M., Adachi, S., Nagahama,Y., Kitajima, C., and Matsuura, S.

NATIONAL INSTITUTE(1991) Annual reproductive cycle ofthe captive female Japanese sardine
Sardinops melanostictus: relationshipto ovarian development and serum levels of gonadal steroid hormones.
Marine Bioi., 108, 21-29.
Matsuyama, M., Nagahama, Y., andMatsuura, S. (1991) Observations on
ovarian follicle ultrastructure in themarine teleost, Pagrus major, during
vitellogenesis and oocyte maturation.
Aquaculture, 92, 67-82.
Matsuyama, M., Adachi, S., Nagahama,Y., Kitajima, C., and Matsuura, S.
(1991) Testicular development andserum levels of gonadal steroids during the annual reproductive cycle ofcaptive Japanese sardine. lap. J. Ichthyol., 37, 381-390.
Mineyuki, Y., Yamashita, M., and Nagahama, Y. (1991) p34cdcl kinase homologue in the preprophase band.
Protoplasma, 162, 182-186.
Mita, M., and Nagahama, Y. (1991) Involvement of G-proteins and adenylate cyclase in the action of gonadstimulating substances on starfish ovarian follicle cells. Develop. Bioi., 144,
262-268.
Miura, T., Yamauchi, K., Nagahama, Y.,
and Takahashi, H. (1991) Induction of
spermatogenesis in male Japanese eel,Anguilla japonica, by a single injectionof human chorionic gonadotropin.
Zoo/. Sci., 8, 63-73.
Miura, T., Yamauchi, K., Takahashi, H.,
and Nagahama, Y. (1991) Humanchorionic gonadotropin induces allstages of spermatogenesis in vitro in
the male Japanese eel (Anguillajaponica).
Develop.
Bioi., 146,
258-262.
Miura, T., Yamauchi, K., Takahashi, H.,
and Nagahama, Y. (1991) Hormonal
induction of all stages of spermatogen-FOR BASIC BIOLOGY

esis in vitro in the male Japanese eel(Anguilla japonica). Proc. Nat!. A cad.
Sci. USA, 88, 5774-5778.
Miura, T., Yamauchi, K., Takahashi, H.,
and Nagahama, Y. (1991) Involvement of steroid hormones in gonadotropin-induced testicular maturation inmale Japanese eel (Anguilla japonica).
Biomed. Res., 12, 241-248.
Mori, M., Yamashita, M., Yoshikuni,M., Fukada, S., and Nagahama, Y.
(1991) Maturation-promoting factor
and p34cdc2 kinase during oocyte maturation of the Japanese quail. Develop. Bioi., 146, 246-249.
Nagahama, Y., Matsuhisa, A., Iwamatsu,
T., Sakai, N., and Fukada, S. (1991)
A mechanism for the action of pregnant mare serum gonadotropin on
aromatase activity in the ovarianfollicle of the medaka, Oryzias latipes.
J. Exp. Zoo!., 259, 53-58.

27
Nagahama, Y., Takemura, A., Takano,K., Adachi, S., and Kusakari, M.
(1991) Serum steroid hormone levels
in relation to the reproductive cycle of
Sebastes taczanowskii and S. schlegeli.
Envir. Bioi. Fishes, 30, 31-38.
Yamaguchi,A.,
Yamashita,
M.,
Yoshikuni, M., Hotta, Y., Nurse, P.,
and Nagahama, Y. (1991) Involvement of meiotic prophase of H1 histone kinase and p34cdc2 homologues
in lily (Lilium longiflorum) microsporocytes. Develop. Growth Differ.,
33, 625-632.
Yamashita, M., Yoshikuni, M., Hirai, T.,
Fukada, S., and Nagahama, Y. (1991)
A monoclonal antibody against the
PSTAIR sequence of p34cdc2 , catalytic
subunit of maturation-promoting factor and key regulator of the cell cycle.
Develop. Growth Differ., 33, 617624.

28

NATIONAL INSTITUTEDIVISION OF CELL
DIFFERENTIATION
Professor: Yoshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Shigeharu Takiya
Toshiharu Suzuki
JSPS Postdoctoral Fellows: Chi-chung Hui
Kenji Matsuno
Kazuhito Amanai
Visiting Scientist: Toshifumi Nagata
Graduate Students:
Masakazu Fukuta(Graduate University for Advanced Studies)
Kaoru Ohno
(Graduate University for Advanced Studies)
Pin-XianXu(Graduate University for Advanced Studies)
Hiraki Kokubo(Graduate University for Advanced Studies)
XinXu(Graduate University for Advanced Studies)
Kensuke Suzuki (from Nagoya City University)
Technical Staffs: Miyuki Ohkubo
Chikako Inoue

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 along the developmental programs
and results in transcribing a specific setof genes like the silk fibroin, fibroin
L-chain, P 25 , sericin-1, and sericin-2
genes. The other initiated at the time
when the Division was established in1978 is concerned with how the bodyplan of the silkworm is controlled andhow the homeotic genes regulate a set oftarget (structural) genes in specifying thesegment identities.
Genes and factors that control the silk
gland development and the silk genes
transcription
Development of the silk gland is initiated in the labial segment at the embryonic stage 18 or .19, and must be
under the control of genes that specify
the labial segment identity. Previously
we reported that several homeobox

genes like Bm Antp, Bm en, Bm in, andothers are expressed in the larval silk
gland. The Bm Antp was mapped on theNc mutation locus which had been
mapped about 1.4 eM apart from the Eloci, the Bombyx counterpart of the BXC. The developmental morphology of
the wild type and the Nc/ Nc embryos
has been studied, and abnormal featuresof the Nc/ Nc embryos in the gnathocephalon and thorax regions have beenrecognized (see the following section).
Among the features, a severe disturbance of the silk gland development in the
Nc/ Nc embryos (Fig. 1) has attracted
our attention. This observation triggers
our interest to find target genes of the
Bm Antp in the silk gland development.
Since the Bm en and Bn in are expressed in the middle portion of the silk
gland, we are interested to know which
portion of the silk gland expresses a
Bombyx homolog of Wnt-1. Bm Wnt-1
eDNA has been obtained from an embryonic eDNA library and identified bysequence comparison with the Wnt-1.
Since PCR studies indicated that
Bombyx homologs of cad, lab, Dfd, pb,
and others are expressed in the larval
posterior silk gland, eDNA cloning has
been carried out, and Bm cad eDNA has
been obtained from an embryonic libraryand identified by sequencing.
We have been studying the molecularmechanisms that control the differential
transcription of the fibroin and sericin-1
genes in the silk gland. Several factors(SGF-1 to -4, FBF-A1, and OBF-1 to-5) are proposed to bind and control thesilk genes.
The SGF-2 is specific to the posteriorsilk gland, is presumed to be a key transcription factor binding to the upstream
C, D, and E regions (enhancer I) of the
fibroin gene, and is also presumed to be

29

FOR BASI C BIOLOGY

Fig. 1. Silk glands from the wild type (+) and the N c/ Nc (Nc) embryos at the stage 24.

composed of the FF1 and FF2. The FF1
has a DNA binding ability and is specificto the posterior silk gland while nonfunctional FF1 molecules are present
ubiquitously. The FF2 is present ubiquitously, and has no DNA binding ability
but stabilizes the FF1 binding.
Fibroin gene enhancer II (+ 156/+454) in the intron is composed of 6 octamer-like elements. Footprint experiments and gel shift assays indicated thatthese elements are targets of cell typespecific DNA binding proteins (OBF-1
to -5). The OBF-1 is present in the posterior silk gland and binds to the elements at around + 220 and +290. The
OBF-2 and -3 are present in the middle
silk gland and bind to the elements ataround + 290 and +420, respectively.
The OBF-4 is present in both the posterior and the middle silk glands and binds
to the element at + 370. The OBF-5 ispresent in a cultured cell line derived
from embryos but not in the silk gland,and binds to the element at +250. An
expression eDNA library from the posterior silk gland was screened with oligonucleotide probes covering the elements

at +220 and +290, and a candidate
clone for the OBF-1 was obtained. The
eDNA encodes a zinc-finger type protein, ZP-P1 , having 3 finger domains.
The ZP-P1 mRNA was shown to be expressed preferentially in the posteriorsilk gland.
Previously we have shown that a
POU-domain containing protein (POUM1, a Bombyx homolog of Cfl-a) is
probably identical with the SGF-3 which
is proposed to be a key transcription factor binding to the SC element of thesericin-1 gene. For the definite identification we produced antibodies against
oligopeptides in the POU-M1 protein
sequence. Two antibodies, one against
the N-terminal peptide and the otheragainst the C-terminal peptide, were
found interacting with the POU-M1 protein as well as the SGF-3. Both antibodies gave a specific hypershift of the
POU-Ml/SC probe complex or the
SGF-3 / SC probe complex in gel shift
assays demonstrating that the POU-M1
is identical with the SGF-3.
The SGF-3 gene has been cloned andproteins binding to its presumed pro-

30

NATIONAL INSTITUTE

218

Ubx
head

.

T3

Fig. 2.

In situ hybridization with Bm Ubx probe at the stage 21B.

moter region have been analyzed by
footprints and gel shift assays. Cell-freetranscription experiments of a series ofdeletion mutants of the SGF-3 gene
have indicated the presence of negative
element(s) as well as positive element(s).
Through the characterization of thesefactors and their genes we hope to decipher the control mechanisms at theupper hierarchy as well as at the level ofthe fibroin and sericin-1 genes transcription.
Genes involved in the Bombyx bodyplan
When the Bombyx body plan is compared with the Drosophila, some features are common but unique featuresthat are common to many insets, are also
evident, like ways of blastokinesis anddorsal closure, development of the silk

gland in the labial segment, and formation of the legs in the thorax and abdomen in the embryonic stages. To study
these problems we have been analyzing
the natures of the genes that are presumed to play important roles in Bombyx development. In the past we cloned
and identified Bombyx homologs ofAntp, Scr, Ubx, abd-A, Abd-B, en, in,and Cfl-a. In 1991 Bombyx homologs of
cad and Wnt-1 have been added to thelist. Using these clones as probes wehave analyzed the natures of some mutants and the expressions of these genes
in the wild type embryos.
By Northern blotting and in situ hybridization the expressions of Bm Ubx,
Bm abd-A, and Bm Abd-B have been
analyzed. At and after 1 day of embryonic development 5.4 kb transcript of
the Bm Ubx became detectable, which

31

FOR BASIC BIOLOGY+

Antenn

NeT I

NcT2

NcT3

+TI
Fig. 3. Morphological appearance of the appendages of the antenna, the prothorax (Tl), the mesothorax(T2), and the metathorax (T3) in the wild type (+) and the Nc! Nc (Nc) embryos.

was followed by a strong expression of
5.6 kb transcript peaking at 3 days. Another component of 1.2 kb was expressed at and after 6 days. Also peaking at 3 days 5.3 kb transcript from the
Bm abd-A and 3.2 kb transcript fromthe Bm Abd-B were expressed. An
example of in situ hybrifization for Bm
Ubx expression at 3 days of development is shown in Fig. 2. This stage

corresponds to about the stage 21 when
the katatrepsis and the dorsal closure are
actively taking place. As shown in Fig. 2
a strong expression is localized from themetathorax to the telson at the ventral
side. Expressions of the Bm abd-A andthe Bm-Abd-B were also detected
strongly at the stage 21; the Bm abd-A
from the A1 to the A6 at the ventral
side, and the Bm Abd-B in the whole32
area of the A4 to the telson.
Morphological appearance of the development of the wild type and the N c/
Nc mutant embryos has been analyzed.
Abnormal development of the mutant
embryos was detected from the mandibular segment to the mesothoracic segment. Especially, the prothorax and themesothorax are fused, and the pair ofprothoracic appendages (legs) comescloser to the mid-line and the pair ofmesothoracic appendages (legs) locateswidely apart from each other. As shownin Fig. 3 the prothoracic appendages aretransformed into antenna-like structures,and the mesothoracic appendages partly
lose their thoracic leg characteristics. ·Other abnormality in the labial segment
was described in the previous section(see Fig. 1).

NATIONAL INSTITUTE

Publication List
Maeda, T., Usui, H., Araki, K., Kuwano, R., Takahashi, Y., and Suzuki,
Y. (1991) Structure and expression of
rat S-100/3 subunit gene. Mol. BrainRes., 10, 193-202.
Suzuki, T., Matsuno, K., Takiya, S.,
Ohno, K., Ueno, K., and Suzuki, Y.
(1991) Purification and characterization of an enhancer-binding protein of
the fibroin gene. I. Complete purification of fibroin factor 1. J. Bioi. Chern.,
266, 16935-16941.
Suzuki, T., Takiya, S., Matsuno, K.,
Ohno, K., Ueno, K., and Suzuki, Y.
(1991) Purification and characterization of an enhancer-binding protein of
the fibroin gene. II. Functional analyses of fibroin factor 1. J. Bioi.
Chern., 266, 16942-16947.

FOR BASIC BIOLOGYDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Research Associate: Ryuji Kodama
Makoto Mochii
Visiting Scientist: Takamasa S. Yamamoto
Kaichiro Sawada
Graduate Student: Jun Kosaka
Masashi Hyuga
Akio lio
Keiko Ishikawa 1i
Yutaka Imokawa 21
Hisao And<ll
Yuichi Ninomiya 3!
Technical Staffs: Hisae Urai
Tamayo Takagi(/JTsurumi University)
eiNagoya University)( 31Tokyo University of Agriculture)

The lost tissues in the eye of the newt
are completely regenerated through thetransdifferentiation process in which
once-specialized cells lose their phenotypes and redifferentiate into other cell
types. The main source of the regeneration in the urodele eye is the pigmented
epithelium. The in vitro experiments
have revealed that the lens transdifferentiation is observed in the cultures of thepigmented epithelial cells (PECs) not
only from urodeles but also from othervertebrates including birds and human.
So the transdifferentiation of the PEC isa conserved phenomenon among vertebrates, whether or not the lost lens is regenerated in the animal. We are studying
in order to answer the questions why the
PECs can transdifferentiate into the lens
cells and why the pigmented epithelia of
the species other than the newt never regenerate the lost lens in vivo. ltoh andEguchi previously established a uniqueculture system for the lens transdifferentiation using the PECs from the chicken
embryo, in which the differentiated.
states of the cells are well regulated by
the modification of the medium. Thus,
molecular biological approaches havebecome possible using cultured chicken

33

PECs. The phenomena related to thelens regeneration in vivo are also studied
using newts. In addition, we are trying toestablish a transgenic system of birds inwhich the functions of individual genes
can be examined directly. The investigations and the results achieved in 1991 by
the members of this division are as follows.
1. The extracellular factors modulating
the lens transdifferentiation of the
PECs in vitro.
Itoh and Eguchi have revealed that thelens transdifferentiation from the chickembryo PECs is greatly enhanced byphenylthiourea and testicular hyaluronidase in the medium. Recently we revealed that the main activity of the enhancement in the crude hyaluronidase
preparation is not due to the hyaluronidase itself but to a trace of basic fibroblast growth factors (bFGF) contaminating the preparation. In fact the purebFGF enhanced the lens transdifferentiation of the PECs in the presence ofphenylthiourea. Other groups reported
that the bFGF also enhanced the transdifferentiation of the pigmented epithelium into the neural retina. Now we areinvestigating the molecular changes in
the PECs in response to the stimulus by
the bFGF.
Another growth factor which affects
the differentiated states of the cultured
PECs is the transforming growth factor[3 (TGF-[3). As will be described in thenext section, a gene coding for the TGF[3 binding protein is transcriptionallyregulated in the process of the lens transdifferentiation, suggesting an importantrole of the TGF-[3 in the transdifferentiation. The effect of the TGF-[3 on thepigmented epithelial cells was examinedin vitro. The PECs maintain the monolayer cell sheet and exhibit a contact-

34
inhibition of growth in a basic culture
medium, but dramatically change theirmorphology in the presence of the TGF{3. They continue to grow even after theculture has attained the confluency, produce a large amount of extracellular matrix (ECM) components, and result into
multi-cell layers. We revealed that the
TGF-{3 have a strong effect in changing
the stable PECs into a quite different
state of cells. However, the relationshipbetween thus attained cells and the cells
obtained through dedifferentiation by
the effect of dFGF is yet to be clarified.
Whether the TGF-{3 plays a crucial rolein the regeneration process in vivo
should be an important question.
The ECM components also have important roles to control the differentiatedstates of the PECs. It is known that boththe bFGF and the TGF-{3 function interacting with the components of the ECMs
in many experimental systems. Immunocytochemical studies have revealed thatthe major components of the ECM in
the PEC culture are once degraded dramatically and reorganized into a different structure when the TGF-{3 is added
to the medium. The specific adhesion of
the PECs to some ECM components
was demonstrated in a serum-free culture. (Kosaka et al. 1992, Exp. Eye Res.,in press).
2. Gene regulation in the process of thelens transdifferentiation.
To investigate the regulatory mechanisms of the gene expression during thelens transdifferentiation, various eDNA
clones encoding the lens-specific and the
PEC-specific proteins have been isolated
and characterized. Fr example, theeDNA clones coding for a- and {3-crystallins are used to characterize the cellschanging to the lens cells. The eDNA
clones coding for a melanosomal matrix

NATIONAL INSTITUTE

protein and a tyrosinase were characterized to specify the PEC phenotype.
Two eDNA clones were already identified by a differential screening in which
the mRNA levels are compared between
the PEC and the dedifferentiated PEC.
One eDNA clone termed pP64 encodesa polypeptide closely related to the
TGF-{3 binding protein identified in
mammals. Another is termed pP344 andcodes for a polypeptide which has thecommon sequence among the membersof the serine-protease inhibitor family.
The gene of the former clone is transcribed to the two species of the messengers whose sizes are 6.0kb and 5.0kb in
the PEC, but in the dedifferentiatedstate only the 6.0kb one is transcribed.
The translated product from the 5.0kb
messenger lacks theN-terminal region of
the 6.0kb one. Phenotypic changes of
the PECs by the TGF-{3 as mentioned
above leads us to an assumption that the
products of the pP64 gene control thedifferentiated states of the PECs by
modulating the action of the TGF-{3. Weshould reveal whether the two species ofthe pP64 products have different effects
on the action of the TGF-{3. The productof the latter gene, pP344, has an activityof the trypsin-inhibitor, and expected toinhibit some protease in tissues. The
pP344 gene is expressed only by the
fully-differentiated PEC. So the expression of the pP344 gene may stabilize thedifferentiated state of the PEC by protecting the cells from the action of proteases.
We revealed that the chicken connexin
43 is the major component of the gap
junction in the PEC. Now the eDNA
clone and the specific antibodies are available to investigate the molecular change
in the cell-cell communication system
during the lens transdifferentiation.

FOR BASIC BIOLOGY

35

Transient disappearance of the 2NI36 antigens during limb regeneration. Longitudinal sections of thestumps of the amputated forelimbs of the newt 3(a), S(b) and 14 days(c) after the amputation. Upper row
shows indirect immunofluorescent stains and lower shows the corresponding phase-contrast images.

3. An antigen related to the process of
the lens regeneration in newt
We have already reported that an
antigen recognized by a monoclonal
antibody, 2NI36 disappear in the dorsal
region of the newt iris before the dedifferentiation of the region becomes obvious after the Ientectomy, and that apiece of the ventral iris which never regenerate a lens did regenerate a new lens

when it was treated with the monoclonal
antibody. The spatial and temporal distribution of the antigen were investigated
and the antigen molecule was biochemically characterized. The 2NI36 antigen is
widely distributed in various organs of
the adult newt, especially in the tissues
derived from the mesoderm (Imokawa etal., 1992, Int. J. Dev. Biol., in press).
But the antigen was never observed in

36
the embryo before the organogenesis
was completed (Imokawa and Eguchi,
1992, Int. J. Dev. Bioi., in press). The
antigen molecules were purified by theaffinity-chromatographies using the lectin and the antibody, and a group of glycoproteins were identified as the antigen
molecules. We assume that the antigen
molecues may serve for the stabilizationof the differentiated cell states. This assumption was supported by the fact thatthe staining with the 2NI36 in the limb
also becomes undetectable at the stumpafter an amputation (Figure).
4. Development of a transgenic system
in avian.
As described above, the products ofthe pP64 gene and the pP344 gene are
the candidate molecules which have thekey roles in the process of the lens transdifferentiation. The function of the genes
must be examined in vivo by manipulation of the genomes of the animal. We
have already developed an experimental
system to introduce an exogenous DNA
into a fertilized chicken egg and also succeeded in the production of a chimera by
injecting the dissociated blastodermal
cells into the blastoderm. Now we aremaking efforts to enhance the frequencyof the integration of the introduced
DFNAs into the genome. The DNA injection into a fertilized egg and the production of a blastoderm chimera are succeeded also in the quail which havesome advantages in experimental procedures.
Publication List
Hiyoshi, H., Uno, S., Yokota, T.,
Katagiri, C., Nishida, H., Takai, M.,
Agata, K., Eguchi, G., and Abe, S.

NATIONAL INSTITUTE(1991) Isolation of eDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expressionof SP4 mRNA in primary spermatocytes. Exp. Cell Res., 194, 95-99.
ltoi, M., Kodama, R., Takayama, S.,ltoi, M., and Eguchi, G. (1991) Ultrastructural observations of typical gap
junctions in human foetal lens nucleus.
Curr. Eye Res., 10, 1-9.
Kodama, R., Eguchi, G., and Kelley
R.O. (1991) Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle inavian retinal pigmented epithelial cells
in vitro. Cell Tissue Res., 263, 29-40.
Mochii, M., Agata, K., and Eguchi, G.
(1991) Complete sequence and expression of a eDNA encoding achicken 115-kDa melanosomal matrix
protein. Pigment Cell Res., 4, 41-47.
Naito, M., Agata, K., Otsuka, K., Kino,
K., Ohta, M., Hirose, K., Perry M.M.,
and Eguchi G. (1991) Embryonic expression of fi-actin-lacZ hybrid gene
injected into the fertilized ovum of thedomestic fowl. Int. J. Dev. Bioi., 35,
69-75.
Reddy, V.N., Katsura, H., Arita, T., Lin,
L.-R., Eguchi, G., Agata, K., andSawada, K. (1991) Study of crystallin
expression in human lens epithelial
cells during differentiation in culture
and in non-lenticular tissues. Exp. Eye
Res., 53, 367-374.
Takagi, S., Hirata, T., Agata, K., Mochii,M., Eguchi, G., and Fujisawa, H.
(1991) The A5 antigen, a candidate
for the neuronal recognition molecule,has homologies to complement components and coagulation factors. Neuron, 1, 295-307.

FOR BASIC BIOLOGYDNISION OF DEVELOPMENTAL
BIOLOGY (ADJUNCT)
Professor: Masaki Iwabuchi
Associate Professor: Masao Tasaka
Research Associate: Takefumi Kawata
Research Fellow: Hisabumi Takase
JSPS Postdoctoral Fellow: Koji Mikami
Visiting Scientists:
Kiyoshi Kanaya (from University of Tokyo)
Masa-aki Katsura (from Kyoto University)
Takuya Imada (from Kyoto University)
Ken-ichiro Taoka (from Kyoto University)
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 transcription of genes involved in cell proliferation and differentiation in higher plants
and a cellular slime mold. We have carried out the following two projects.
(1) Transcriptional regulation of cell
cycle-dependent genes in plants by transcription factors.
Histone genes are mainly expressed in
the S phase during the cell cycle, coupling with DNA synthesis. The molecularmechanisms of S phase-specific gene expression are unknown. We have chosen
wheat histone genes as a model system
to elucidate the regulatory mechanismsof S phase-specific transcription inhigher plants. Until now, three cis-acting
positive elements,
the hexameric(ACGTCA), octameric (CGCGGATC),and nonameric (CATCCAACG) motifs,
have been identified in the proximal promoter region of the wheat histone H3
gene (TH012). In vivo transcription experiments with deletion and site-directed
mutations of the H3 gene have suggested
that some of these motifs may contributeto the S phase-specific transcription ofthis gene. These motifs are also shown tobe conserved in the regulatory regions ofother plant histone genes. We haveidentified three nuclear DNA-binding

37
proteins, HBP-la, HBP-lb and HBP-2,
as candidates of tarns-acting factors. The
former two proteins specifically bind tothe hexameric motif and the latter one to
the nonameric motif. The eDNA clones
encoding HBP-la, HBP-lb and HBP-2
have been isolated from a wheat eDNA
library in A.gtll using South-western
screening method. Sequence analyses of
the cDNAs and DNA-binding experiments with bacterially expressed HBP-laand HBP-lb have revealed that the bothproteins have the characteristic properties of the bZIP-type transcription factors and HBP-2 has two zinc-finger
structures. In wheat seedlings, there areparallel relationships among the levels oftranscripts from HBP-la, HBP-lb, andfour histone subtype (H2A, H2B, H3
and H4) genes. We also isolated of otherthree eDNA clones which are similar to
HBP-la or HBP-lb eDNA. Analyses ofthe eDNA clones revealed that HBP-laand HBP-lb are classified into the HBPla and HBP-lb protein subfamilies. It isdemonstrated that the hexameric motifs
are located in the promoter regions ofgenes other than histone genes, and thatthe hexamer-specific DNA binding proteins are present in some higher plants.
We isolated a eDNA clone for thecounterpart of HBP-lb from Arabidopsis thaliana.
To analyze the regulation of HBP-laand HBP-lb genes, we have isolated
their cognate genomic clones. Structural
analyses of the clones and DNA-binding
assays using baterially expressed HBP-laand HBP-lb have suggested the possibility of the autoregulation in the expression of genes by HBP-la and HBPlb themselves.
To know how HBP-la, HBP-lb and
HBP-2 interact with a basic transcription
machinery, we isolated a eDNA clone

38
encoding a TATA box-binding protein(TFIID) from the wheat seedling eDNA
library. We hope this will help us tomake a reconstitution system of in vitrotranscription in higher plants.
(2) Regulation of cell-type specific gene
expression during development of Dictyostelium discoideum.
After vegetative growth of D. discoideum, cells aggregate to form a multicellular cell mass and differentiate into two
kinds of cells, prespore and prestalkcells. To elucidate the regulatory mechanisms of cell differentiation during development, we have isolated several genes
which were specifically transcribed inprespore and prestalk cells, respectively.
Recently, we have concentrated our effort to understand the regulatory mechanisms of expression of two presporespecific genes (SP96 and Dp87). Promoter region of each gene has beenshown to include the sufficient information for prespore-specific transcription
and we have identified one of the cis-acting regulatory regions of both genes anda short homologous e-rich element'ACACCCA' was found in these cis-acting regions. We have found a nuclear
factor by gel retardation assays, which
specifically bound to these cis-acting regions. The following results and developments have been achieved in 1991.
To obtain detail information for the
Dp87 gene transcription, some parts ofthe promoter region of Dp87 gene weredeleted or replaced by heterologous
DNA fragments, jointed to the E. coli
,8-gal gene, then introduced into D. discoideum cells and the expression of thechimeric gene in transformed cells was
examined by a histochemical staining of/3-galactosidase. The results were summarized into three parts: a) The sequence between -664 and -432 con-

NATIONAL INSTITUTE

tained an element which increased thetranscriptional activity by 10-fold. b) In
addition to the region between -447
and -356, which has been identified
previously, other three regions from-351 to -231, from -231 to -159 andfrom -159 to -64 also included thecis-acting elements essential for the transcription. c) All these cis-acting regions
except for the region between -432 and-356 were functional when they were
jointed to the downstream sequence ofDp87 gene, and were not functional
when they were jointed to the heterologous basal promoter of the actin 15 geneof D. discoideum. However, when theregion from -432 to -356 was jointed
to the actin basal promoter, it increased
the transcriptional activity. These results
indicate that the transcription of Dp87
gene is regulated by the combination ofsome independent cis-acting elements.
As the chimeric gene including the 5'
upstream region up to -664bp and the
j3-gal was expressed only in prespore
cells, histochemical detection of cells expressing j3-galactosidase showed that
prespore cells were randomly appeared
at early aggregating stage on an agar
plate. It was believed that the prespore
cell differentiation was not induced
under submerged condition. Whentransformed cells were incubated in theliquid condition with 1mM of cAMP,some cells were expressed the ,8-galactosidase which indicated to differentiate toprespore cells. The difference between
our result and previous observations wasdue to the different markers; ours is theearliest marker and the other are latemarkers. Our result suggests that there
are two steps of prespore cell differentiation, the first step is induced in the liquidcondition but the next step is not. This
new in vitro method is very useful toFOR BASIC BIOLOGY

study the cell-cell interaction or morphogen(s) which required for the prespore cell differentiation.
Publication List
Morio, T., Ozaki, T., Takeuchi, 1., andTasaka, M. (1991) Transcriptional
regulation of cell-type enriched genes
during development in Dictyostelium
discoideum analyzed by nuclear runon assays. Develop. Growth & Differ.,
33, 293-298.
Tabata, T., Nakayama, T., Mikami, K.,
and Iwabuchi, M. (1991) HBP-1a and
HBP-1b: leucine zipper-type transcription factors of wheat. EMBO J.,
10, 1459-1467.
Takase, H., Minami, M., and Iwabuchi,
M. (1991) Sequence-specific singlestrand DNA-binding proteins that interact with the regulatory regions of39
wheat histone H3 and H4 genes.
Biochem. Biophys. Res. Commun.,
176, 1593-1600.
Takase, H., Tabata, T., Mikami, K., andIwabuchi, M. (1991) Partial purification and characterization of two transcriptional factors, HBP-1a and HBP1b, specific for a cis-acting element,
ACGTCA, of wheat histone genes.
Plant Cell Physiol., 32, 1195-1203.
Tasaka, M. (1991) Developmental regulation of expression of prespore-specific genes in Dictyostelium discoideum. Bot. Mag. Tokyo, 104,359-370.
Yang, P., Katsura, M., Nakayama, T.,
Mikami, K., and Iwabuchi, M. (1991)
Molecular cloning and nucleotide sequence of cDNAs for histone H1 andH2B variants from wheat. Nucl. Acids
Res., 19, 5077.

NATIONAL INSTITUTE41

FOR BASI C BIOLOG Y

DEPARTMENT 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.

42

NATIONAL INSTITUTEDIVISION OF MOLECULARNEUROBIOLOGY
Professor: Masaharu Noda
Technical Staff' Sonoko Ohsawa

The division just started its new projects from September 1991. This divisiondeals with developmental neurobiologyof the vertebrate central nervous system.

The project is aiming to elucidate themechanisms that govern the development of topographic projections in thecentral nervous system. Main subjects
are the molecular analysis of the neuronal migration in corticogenesis and theformation of neural connections in theretinotectal system.

FOR BASIC BIOLOGYDIVISION OF CELLULARREGULATION
Professor: Norio Murata
Associate Professor: Hidenori Hayashi
Research Associates: Takao Kondo
lkuo Nishida
Hajime WadaNIBB Visiting Professors:
Antoni R. Slabas(from University of Durham, England)
Harry Y. Yamamoto(from University of Hawaii, USA)NIBB Post-doctoral Fellow: Hiroyuki Ohta
JSPS Post-doctoral Fellow:
Mahir D. Mamedov(from Moscow State University, Russia)
Visiting Scientists:
Yasushi Tasaka
Mari lwabuchi(from Plantech Research Institute)Dmitry A. Los(from Russian Academy of Sciences, Russia)
Michael P. Malakhov(from Russian Academy of Sciences, Russia)
Zoltan Gombos (fromBiological Research Center Szeged, Hungary)
Eszter S. Kovacs (fromBiological Research Center Szeged, Hungary)
Chin B. Lee(from Dongeui University, Korea)
Graduate Students:
Hiroyuki Imai
Toshio Sakamoto
Yoshitaka Nishiyama(from The University of Tokyo)
Norihiro Satoh (from The University of Tokyo)
Technical Staffs: Shoichi Higashi
Yumiko Makino

The research effort of this division is directed toward the understanding of
tolerance and adaptation of higher plants
and microbial plants to high and low
temperature as well as to high-salt stress.
In particular, our research focuses on themolecular mechanism by which plants
adapt or tolerate such stress conditions.
1. Gene-engineered alteration of thefatty-acid nnsatnration modifies thechilling sensitivity of higher plants.
The chilling sensitivity of plants iscorrelated to the extent of fatty-acid nosaturation of phosphatidylglycerol inchloroplast membranes. Plants with high43
contents of unsaturated fatty acids in
phosphatidylglycerol are resistant tochilling, whereas species with low contents of unsaturated fatty acids in phosphatidylglycerol are sensitive to chilling.
We have suggested that the chloroplastic
enzyme, glycerol-3-phosphate acyltransferase, is an important factor in determining the level of unsaturation of thefatty acids of phosphatidylglycerol.
In order to evaluate its role in the lowtemperature sensitivity of higher plants,
cDNAs encoding the precursor proteinof this enzyme have been isolated from a
low-temperature-sensitive plant, squash,
and from a low-temperature-resistant
plant, Arabidopsis. Tobacco plants weretransformed with Ti plasmids that wereconstructed with the squash and Arabidopsis cDNAs for acyltransferases undercontrol of the 35S CaM promoter andthe NOS terminator. Analysis of the lipids of these transformants demonstrated that only phosphatidylglycerol
was significantly altered in its fatty-acidunsaturation by transformation withthe eDNA for glycerol-3-phosphate
acyltransferase. When squash eDNA wasintroduced, the content of unsaturated
fatty acids in the phosphatidylglycerol
was decreased. In contrast, the introduction of the Arabidopsis eDNA caused asmall but significant increase in thesefatty acids.
Chilling sensitivity of transformed
plants was assayed by two methods: Inactivation of photosynthesis of excised
leaves and visual damage of whole plants
at chilling temperature (Figure 1). With
both methods, the wild-type tobacco
plants showed some sensitivity to chilling
temperature. Upon introduction of thesquash eDNA for glycerol-3-phosphate
acyltransferase, this sensitivity wasmarkedly increased. In contrast, the44

NATIONAL INSTITUTE(a) Control (transgenic(b) Transgenic tobacco(c) Transgenic tobacco

tobacco with vector

with squash eDNA

with Arabidopsis eDNA

plasm ids)
Fig. 1. The low temperature induced symptom of wild-type and transgenic tobacco plants. (a) Control(transformant with vector plasmids). (b) Transformant with the eDNA for glycerol-3-phosphate acyltransferase from squash. (c) Transformant with eDNA for glycerol-3-phosphate acyltransferase from Arabidopsis.

introduction of the Arabidopsis eDNA
for glycerol-3-phosphate acyltransferase
decreased the chilling sensitivity. These
remarkable differences in the chilling
sensitivity were well correlated with theextents of fatty-acid unsaturation ofphosphatidylglycerol.
These results, therefore, demonstratethat it is possible to regulate the chilling
sensitivity of higher plants solely by thegene-engineered alteration of fatty-acidunsaturation of phosphatidylglycerolwith introduction of an appropriate
acyltransferase. Other factors could also
contribute to chilling sensitivity, butthese studies provide the first direct evidence that the fatty-acid unsaturation ofphosphatidylglycerol is a major contributor. Studies on photosynthetic characteristics of the transformants are in progress.
2. The fatty-acid unsaturation affects
thermal properties of cyanobacterial
photosynthesis.

Alteration of the fatty-acid unsaturation of membrane lipids by genetic manipulation could provide an important
information in our understanding of therole of fatty-acid unsaturation in plant
membranes. By combination of techniques of mutation and gene disruption
we could produce three strains of the
cyanobacterium, Synechocystis PCC6803, having distinctly different levels of
fatty-acid unsaturation of membrane lipids. The wild-type strain contains
mono-, di- and triunsaturated fatty acids.
The desaturation mutant (termed Fad6),
defective in fatty-acid desaturation at thedelta-6 position, contains mono- anddiunsaturated fatty acids. The transformant of Fad6 (termed Fad6 / desA: :Kmrin which the gene (desA) for the desaturation at the delta-12 position of fatty
acids has been disrupted, contains only amonounsaturated fatty acid.
The effect of fatty-acid unsaturationon thermal properties of photosynthetic
activities was investigated with these

FOR BASIC BIOLOGY

three strains. It was found that temperature profiles of the photosynthetic activity at room temperature and of the inactivation of photosynthesis at high temperature were not affected by thechanges in fatty-acid unsaturation. In
contrast, exposure to low temperature, inthe liglit, produced distinctly different
degrees of inactivation of photosynthesis
among the strains. Fad6/ desA: :Kmr wasmuch more damaged than the wild type
and Fad6. This observation suggests thatthe presence of the second double bondin the fatty acids is essential for thiscyanobacterium to tolerate the low temperature.
3. Glycinebetaine stabilizes the oxygenevolving complex of higher plants andphotosynthetic microbes.
The protective effect of glycinebetaine
on the photosystem 2 activity was investigated. Glycinebetaine is a nontoxic osmolyte found in halophilic higher plants
and microbial plants. Spinach photosystem 2 complex loses its oxygen-evolving
activity during the incubation at room
temperature for hours. The loss of theactivity was greatly prevented by thepresence of glycinebetaine at high concentration such as 1 M. It was found thatglycinebetaine protects the Mn cluster,which serves an essential role in oxygen
evolution.
We have also discovered that thepresence of glycinebetaine in the medium allows the isolation of cyanobacterial thylakoid membranes that arestable and highly active in the electrontransport and phosphorylation. It must
be remarked that the preparation of
stable and highly active thylakoid membranes from cyanobacteria has been agoal that had been pursued for a longtime in the field of cyanobacteriology.

45
Publication List
Gombos, Z., and Murata, N. (1991)
Lipids and fatty acids of Prochlorothrix hollandica. Plant Cell Physiol.,
32,73-77.
Gombos, Z., Wada, H., and Murata, N.
(1991) Direct evaluation of effects offatty acid unsaturation on the thermal
properties of photosynthetic activities,
as studied by mutation and transformation of Synechocystis PCC6803.
Plant Cell Physiol., 32, 205-211.
Johnson, C.H., Kondo, T., and Hastings,
J.W. (1991) Action spectrum for resetting the circadian rhythm in the
CW15 strain of Chlamydomonas. II.
Illuminated cells. Plant Physiol., 97,1122-1129.
Kondo, T., Johnson, C.H., and Hastings,
J.W. (1991) Action spectrum for resetting the circadian rhythm in the
CW15 strain of Chlamydomonas. I.
Cells in darkness. Plant Physiol., 97,
197-205.
Mamedov, M.D., Hayashi, H., Wada,
H., Mohanty, P.S., Papageorgiou,
G.C., and Murata, N. (1991) Glycinebetaine enhances and stabilizes theevolution of oxygen and the synthesis
of ATP by cyanobacterial thylakoid
membranes.
FEBS Lett.,249,
271-274.
Okuyama, H., Okajima, N., Sasaki, S.,
Higashi, S., and Murata, N. (1991)
The cis/ trans isomerization of the
double bond of a fatty acid as a
strategy for adaptation to changes inambient temperature in the psychrophilic bacterium, Vibrio sp. strain
ABE-l. Biochim. Biophys. Acta.,
1084, 13-20.
Papageorgiou, G.C., Fujimura, Y., and
Murata, N. (1991) Protection ofoxygen-evolving photosystem 2 complex by glycinebetaine. Biochim. Biophys. Acta, 1057, 361-366.

46
Sakamoto, T., Murata, N., and Ohmori,
M. (1991) The concentration of cyclic
AMP and adenylate cyclase activity incyanobacteria. Plant Cell Physiol., 32,
581-584.

NATIONAL INSTITUTE

Sato, N., and Murata, N. (1991) Transition of lipid phase in aqueous dispersions of diacylglyceryltrimethylhomoserine. Biochim. Biophys. Acta, 1082,
108-111.

FOR BASIC BIOLOGYDNISION OF BIOLOGICALREGULATION (ADJUNCT)
Professor: Hidemasa Imaseki
Research Associate: Satoru Tokutomi
Hitoshi Mori
Graduate Student: Masao Arai*(*Graduate Division, Nagoya University)

Growth and development of higher
plants are regulated by plant hormones,
but they are also greatly influenced bychanges in environmental conditions,that 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
topics; (1) development of the sink activities in growing tissues, (2) mechanismsof gene expression regulated by plant
hormones and environmental stress, and(3) acquisition of thermotolerance by
non-lethal high temperature. Great progresses have been achieved on the topics
1 and 2 in 1991.
( 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. To maintain the rapid
growth, sucrose must be continuously
metabolized in growing organs. We havefound that dramatic changes of sucrosemetabolizing enzymes, invertase and sucrose synthase, occurred in growing stems
and leaves and the changes are correlated with the growth rate of individualorgans. The regulatory mechanism
underlying in inductive synthesis of invertase and sucrose synthase was studied
at the molecular level. Both invertase47
and sucrose synthase were purified frommung bean seedlings. We found two
forms of intracellular acid invertase inmung bean seedlings; one is a heterodimer of 30 and 38 kDa polypeptides
and another is a monomer of 70 kDa.
The heterodimeric form is not an artifactual product of the monomeric form produced during or after extraction. The
relative amounts of the two forms incells changed during growth of seedlings(Figure 1). cDNAs for the heterodimeric
form of acid invertase and sucrose synthase have been cloned and their nucleotide sequences were determined. The heterodimeric acid invertase was found tobe synthesized as a single polypeptide
containing a leader sequence of 101
amino acids followed by the 30 kDa and
38 kDa portions. No mRNAs for acidinvertase and sucrose synthase were
found in the embryonal axes in seeds,
but the transcripts for the two genes accumulated to the maximal levels within
15 hours of germination, when the enzymatic activities were still negligible (Araiet al. (1992) Plant Cell Physiol., inpress). The levels of transcripts weremaintained for 3 days, during which
period seedlings grew rapidly and both
enzymatic activities increased markedly.
Presence of mechanisms by which thetranscripts are maintained in translationally inactive states is proposed.
(2) Mechanisms of gene expression regulated by plant hormones and tissue
wounding. Auxin induces many physiological processes of 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

48

NATIONAL INSTITUTE

Days
70 kDa38 kDa2

1

3

4

Fig. 1.

(Yamamoto et a!. (1992) Plant Cell
Physiol., in press). Sequence determination revealed that two of them were new
to the literature, and their characterization is under way.
Ethylene biosynthesis is variously
regulated by other plant hormones andenvironmental stress, and in all cases, theregulatory step is formation of 1-aminocyclopropane-1-carboxylic acid (ACC)from S-adenosylmethionine catalyzed by
ACC synthase. Thus, ACC synthase is .
induced by various stimuli. We havefound that ACC synthases induced by
auxin, wounding and fruit ripening aredifferent molecules. We have cloned
cDNAs for the wound- and auxin-induced enzymes from winter squash.
Their nucleotide sequences are significantly different, and the expression ofrespective gene is regulated in a stimulus-specific manner. Tomato was foundto contain three different genes for ACC
synthases induced by wounding, ripening
and auxin, and their genes are expressed
differentially by types of stimuli (Figure

49

FO R BASIC BIOLOGY

II

stage

Ill

R-typeW-type
N

w

N

w

N

IV

-w

N

w

Fig. 2.

2; Mori, H. and Imaseki, H . unpublished). Comparative studies on the deduced primary structure of ACC synthases of the wound-, ripening- andauxin-type from different plant sources
revealed that there are seven conserved
regions among them. An effort to determine the specific sequences in the genesthat regulate expression by the specificstimulus is being made.
Publication List
Arai, M., Mori, H . and Imaseki, H.
(1991) Roles of sucrose-metabolizing
enzymes in growth of seedlings. Purification of acid invertase from growing
hypocotyls of mung bean seedlings.
Plant Cell Physiol., 32, 1291-1302.
Ishige, H., Yamazaki, K., Mori, H . andImaseki, H. (1991) The effects ofethylene on coordinated synthesis ofmultiple proteins: accumulation of anacidic chitinase and a basic glycoprotein induced by ethylene in leaves of
azuki bean, Vigna angularis. Plant
Cell Physiol., 32, 681-690.
lshizuka, M. , Yamada, F., Tanaka, Y.,
Takeuchi, Y. and Imaseki, H . (1991)
Sequential induction of mRNAs forphenylalanine ammonia-lyase in slices
of potato tuber. Plant Cell Physiol. ,32,57-64.
Nakagawa, N., Mori, H., Yamazaki, K.
and Imaseki, H. (1991) Cloning of acomplementary DNA for auxininduced 1-aminocyclopropane-1-carboxylate synthase and differential expression of the gene by auxin andwounding. Plant Cell Physiol. , 32,
1153-1154.

50

NATIONAL INSTITUTEDMSION OF BEHAVIOR AND
NEUROBIOLOGY (ADJUNCf)
Professor: Katsuhiko Mikoshiba
Associate Professor: Masaharu Ogawa
Research Associate: Teiichi Furuichi
Visiting Scientists: Atsushi Miyawaki*
Shingo Yoshikawa*
Kousuke Sumita •Ichirou Fujino •Norihiko Yamada*Y oshimi Ryou •Taka yuki Michikawa •Shinobu Shimada* •Makoto Nakamura+
Visiting Research Fellow: Yasutaka Makino++(*from Osaka University)(**from The University of Tokyo)(+from Hokkaido University)(++from Ono Pharmaceutical Co., Ltd.)In our research division, we are studying the mechanisms of development,
growth and differentiation of the mammalian nervous system at the molecularand cellular levels.
Our studies in last year mainly focusedon the second messenger signalling withina cell. In the course of our studies on
cerebellar ataxic mutant mice, a membrane glycoprotein P400 was found to beenriched 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), asecond messenger that mediates Ca2+ release from intracellular stores such as theendoplasmic reticulum. The increase intlie cytoplasmic Ca2+ content probably
modulates the functions of various Ca2+associated proteins, such as calmodulin,
Ca2+I calmodulin dependent protein kinase, etc, leading to a variety of cellularresponses, including metabolism, secretion, contraction, neural excitation andcellular proliferation. We demonstrated
that the InsP 3 receptor binds to InsP3 with
the N-terminal region and forms ahomotetrameric structure, exhibiting a
Ca2+ channel activity in response to InsP3
molecules. Our primary goal is to know
how the InsP3-mediated Ca2+ release isinvolved in the neuronal network system,
especially in the cerebellar Purkinje cells.
In addition, we have recently cloned aeDNA of the Drosophila InsP3 receptor,
which probably allows us to elucidate thefunctional importance of the InsP 3-mediated Ca2+ release in a wide variety of cell
types.
Publication List
Mikoshiba, K., Furuichi, T., Maeda, N.,
Yoshikawa, S., Miyawaki, A., Ninobe,M., and Wada, K. (1991) Primarystructure and functional expression of
the inositol 1,4,5-trisphosphate receptor, P400 • In Neuroreceptor Mechanisms
in Brain, Kito, S., ed., Plenum Press,
New York, pp.84-95.
Miyawaki, A., Furuichi, T., Ryou, Y.,
Yoshikawa, S., Nakagawa, T., Saitoh,T., and Mikoshiba, K. (1991) Structure-function relationships of themouse inositol 1,4,5-trisphosphate receptor. Proc. Nat!. A cad. Sci. USA, 88,4911-4915.
Mori, Y., Friedrich, T., Kim, M-S., Mikami, A., Nakai, J., Ruth, P., Bosse, E.,
Hofmann, F., Flockerzi, V., Furuichi,T., Mikoshiba, K., Imoto, K., Tanabe,T., and Numa, S. (1991) Primary structure and functional expression fromcomplementary DNA of a brain calcium channel. Nature, 350, 398-402.
Nakagawa, T., Okano, H., Furuichi, T.,
and Mikoshiba, K: (1991) The subtypes of the mouse inositol 1,4,5-trisphosphate receptor are expressed in atissue-specific and developmentally
specific manner. Proc. Nat!. Acad. Sci.
USA, 88, 2644-2648.

51

FOR BASIC 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.

52
DIVISION I OF GENE EXPRESSION
AND REGULATION
Professor: Yoshiro Shimura
Associate Professor: Kiyotaka Okada
Research Associate: Hideaki Shiraishi
Post-doctoral Fellow: Masako K. Komaki
Graduate Students: Kazuhito A kama
Hiroyuki Okamoto
Koji Sakamoto
Azusa Yano
Tashiro Ito(from Kyoto University)
Technical Staff: Hideko Nonaka

The principal interest of this laboratory
is molecular genetic studies on the regulatory systems of organ development andon 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, light or
touching stimuli. For these studies, wehave mainly used a small crucifer, Arabidopsis thaliana. This plant is called "botanical Drosophila", 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 gene tagging 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 would be related to theflower development or to the root
growth response to the stimuli.
Mutants with abnormal floral morpho-

NATIONAL INSTITUTE

logy 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 (mutants
with short inflorescence axis, dwarfs),
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 al., Development, 104,
195-203, 1988; Okada et al., Cell Differ. Dev., 28, 27-38, 1989). The analysis of double mutants constructed fromthese single 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 et al., in preparation).
Floral bud formation on the inflorescence axis was studied using the pinformed mutant (Okada et al., 1991). 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FOR BASIC BIOLOGY

the presence of chemcal inhibitors on
auxin polar transport. The polar 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 a!., J. Plant Physiol. 137,
628-630; J. Ueda: Osaka Prefectural
University).
We have constructed a relatively
simple model which may explain the action of the homeotic genes (Komaki eta!., in preparation). The model explains
the flower structure of double mutants
constructed by crossing a series of the
known homeotic mutations.
One of the homeotic genes, AGAMOUS, which is involved in the development of stamens and carpels is known
to encode a putative DNA-binding protein which share a homologous region
with the DNA-binding domains of transcription factors, yeast MCM 1 andhuman SRF. Using the AGAMOUS
protein overproduced in E. coli, we haveshown that it binds to specific DNA
sequences in vitro. The consensus sequence for the binding of the AGAMOUS protein was similar to that ofyeast MCM1 (Shiraishi eta!., in preparation). Attempts have also been made toisolate Arabidopsis eDNA and genomic
DNA clones which have homologous sequences with DEF A, a homeotic geneof Antirrhinum majus. The homology
between the DEF A and the coding region of the isolated homolog of Arabidopsis was about 60% (Okamoto et a!.,in preparation). The map position of the53
isolated gene is being investigated to
examine whether the gene corresponds
to any known mutants of A. thaliana.
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 srainght 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 a!., 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 isshifted 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 ( wav1-6) are involved inthis growth response (Okada & Shimura,
Science, 250, 274-276, 1990). Several
mutants also show abnormal gravitro-

54

NATIONAL INST ITUTE

Flowers of a homeotic mutant, ap2-1 , of Arabidopsis thaliana. A single mutation of the apetala2 genecauses temperature-dependent abnormalities on flowers. Sepals are converted to leaves, and petals carry pollen sacs at lower temperature (left). At higher temperature, sepals and petals are converted to carpels and stamens respectively (right).

pism and / or phototropism. These results
indicate that root gravitropic, phototropic and obstacle-escaping responses
share at least in part a common genetic
regulatory mechanism.
Polypeptides induced by gravitropic
stimuli were analyzed using the 2-dimentional gel electrophoresis. Amounts of atleast 11 spots were shown to be increased by the stimuli (Sakamoto et a!.,in preparation).
Attempts are being made to idenify
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 systemof transformation and transgenic plantregeneration. We have tested several
combinations of A. thaliana ecotypes
and Agrobacteria strains and establishedan efficient system (Akama et a!., in
preparation).
Publication List
Okada, K., Ueda, J. , Komaki , M.K.,
Bell, C.J. , and Shimura, Y. (1991) Requirement of the auxin polar transportsystem in early stages of Arabidopsis
floral bud formation . Plant Cell, 3,677-684.
Ueda, J., Komaki, M.K., Okada, K. , andShimura, Y. (1991) Identification andquantitative distribution of indole-3acetic acid in Brassica juncea Czern. J.
Plant Physiol., 137, 628-630.

FOR BASIC BIOLOGYDIVISION OF GENE EXPRESSION
AND REGULATION II
Professor: Takashi Horiuchi
Research Associate: Hidaka Masumi
JSPS Post-doctoral Fellow: Takehiko Kobayashi
Technical Staff: Yoko Fujimura

We are attempting to examine mechanisms involved in chromosomal replication, recombination and repair. Homologous recombination and replication,
especially initiation and termination process, have been given special attention.
On procaryotic and eucaryotic
genomes, there is a site, named termination ( ter) site or fork blocking site, atwhich the DNA replication fork isseverly impeded. We directed attention
to the entire DNA replication termination system in Escherichia coli and clarified the following: To block progress of
the DNA replication fork in E. coli, two
factors are required, one factor is the ter
sequence in the E. coli system and theconsensus 22 bp sequence is 5'-(A/T)(G /T)TAGTTACAACAPy(A/TI C)C(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)-3'.
The sequence is represented by ([) andhas activity which inhibits travel of thereplication fork in a specific direction. In
the above orientation, only the fork,
traveling from the right, is inhibited. The
six ter sites in the E. coli genome, are arranged as shown in Figure 1. The terbinding protein (Ter protein or TBP),
another factor essential for termination
reaction, is coded by a gene called tau(tus), the map position of which is at
35.5 min on the E. coli linkage map. At
the promoter region of this gene locates
one of the five Ter sites, TerB. Ter protein, with a molecular mass of 36k daltons, is a basic protein and binds specifically to the ter sequence. Using an invitro oriC reconstructed system, the Ter
protein dependent termination reaction

55
can occur in vitro at the ter site, in apolar fashion, on the genome of theoriC-terC chimera plasmid. Ter protein
bound at the ter sequence blocks the replication fork by impeding the action ofhelicase, with polarity in vitro.
The following progress was made bymembers of this division in 1991.
(1) E. coli ter-Ter protein complex canfunction in eucaryotic DNA replication
systems in vitro (Hidaka et al. (1992) J.
Bioi. Chern. in press).
We found that the E. coli ter-TBP
complex impedes action of the three E.
coli helicases, DnaB, Rep and UvrD
proteins. To generalize the intrinsic nature of the orientated ter-TBP complex
against eucaryotic helicases, we tested
the potential of the complex to inhibit
the action of two helicases, Simian Virus
40 (SV40) large tumor (T) antigen, andHelicase B, derived from SV40 andmouse FM3A cells, respectively. The
complex impedes the unwinding of thetested helicases in a specific orientation,with the same polarity observed in caseof blockage of a replication fork, and as
a result, there was a block of SV40
DNA replication in both crude and purified enzyme systems, in vitro. As the
specificity in polarity of inhibition extends to heterologous systems, there may
be common structure/mechanism features in helicases.
(2) Structural analysis of eucaryotic replication fork blocking site (Kobayashi etal. (1992) Mol. Gen Genet. in press).
In addition to procaryotes, eucaryotic(yeast, higher plants and humans) cells
were found to have the DNA replication
fork blocking site. All these sites heretofore identified are located at the region
within the rRNA gene cluster and theblocking activity exhibits polarity. To
compare eucaryotic and procaryotic

56

NATIONAL INSTITUTE

thr

min TerE

25 26 TerD TerA

41 (200kb)I I[ 1[1loci pyre

TerF

31

(65kb)(270kb)

nar

trg

pyrF

(75kb)

tau

I~

manA

----------->~---><r-----

Fig. 1. Arrangement of DNA replication terminus (Ter) sites and tau gene, a structural gene for Ter binding protein, on the E. coli genome. Upper part shows circular and expanded E. coli chromosome map andlower arrows mean direction of the DNA replication fork blocked at the Ter sites.
·

blocking systems, it is essential to elucidate structure of the site and the molecular mechanism of fork blocking rection.
We attempted to localize more
precisely Saccaromyces cerevisiae fork
blocking site. We named this "sog" site,which was identified using two dimensional (20) agarose gel electrophoresis.
First we cloned the sog site into a YEp-

type plasmid, a derivative of 2 f.-liD plasmid, and analyzed the replication intermediates using 20 gel electrophoresis(see Fig. 2). The blocking activity of thesog site remained even on the plasmid
not involved in any rRNA gene and inhibited fork movement in the same polar
fashion as on the genome. We named
this method "sog assay". Using thisassay, we narrowed the region required

57

FOR BASIC BIOLOGY

~
1 Sl

2nd

--<
... . ...
2-fold linear position

2-fold linear pos ition
Linear spot • ,A . Non-barrier

Linear spot•

B _Barrier

Fig. 2. Detecti on of DNA replication intermediate molecules using 2-D(dimensional) agarose gel electrophoresis.
A. A DNA fragment without replicati on fork barrier. A large spot and an arc, shown in the upper gel pattern , correspond to a linear DNA fragment and its replication interm ediates, molecular structure of whi ch are
shown schematically along the spot and the arc in the lower fi gure.
B. A DNA fragment with replicati on fork barrier. In addition to the pattern of A , a new spot on the arc anda line startin g from the spot are seen. The new spot corresponds a specific Y-shaped molecule, at a specificsite (barri er) on which the DNA replication fo rk is blocked. The molecular structures of this and other intermediates are also shown schematically in the lower fi gure.

for sog activity to a 109 bp fragment
which locates at an interspacer region
placed between the transcriptional termination sites of 35S and SS rRNA genes.
It is also close to the enhancer region for
35S transcription and overlaps with thesite essential for recombinational hotspots, HOTJ. These results suggest thatin addition to fork blocking function , sagsite might be link to control of 35S transcription and trigger homologous recombination.
(3) Homologous recombinational hotspot activity near the replication termination sites in E. coli; termination event

and Chi sequence dependency.
In wild type E. coli cells, an uniqueorigin, oriC and initiator protein, DnaA
protein, are essential for DNA initiationof replication. When either of these isdefective, the cells die. However in thesemutants, when a ribonuclease H defective mutation (rnh- ) is introduced, thecells survive. The replication system,
which is oriC- and dnaA-independent, isapparently activated under rnh- conditions. de Massy et al. reported that in
rnh- conditions, DNA replication initiates at several new origins in which
two most active origins were located in

58
the DNA replication termination region.
However the- precise locations of themwere not determined. We intended toclone the new replication origin(s) activated in rnh-·condition. Whole chromosomal DNA digested with the EcoRI
enzyme was ligated with the Kmr DNA
fragment, transformed to the rnh- derivative host and from the Kmr transformants, we obtained 8 kinds of plasmid-like DNA, each of which contains a
specific DNA fragment, termed "HotAH", derived from the E. coli geno~e.
Because Hot DNA could not be transformed into a mutant strain in which the
Hot corresponding region on the chromosome was deleted, the Hot DNA, obtained as ccc DNA, was formed throughexcision from the host chromosome into
which Hot DNA was once integrated,rather than through an autonomous replication, thus the Hot fragments are
hyper-recombinogenic. All but one(RotH) of the Hot DNA fragments locates at the DNA replication termination
region on the E. coli genome. All Hot
fragments so far tested have Chi activity.
In 3 Hot (A, B and C) groups, introduction of tau- mutation (tus-; termination-

NATIONAL INSTITUTEless) into the rnh- host genome or Chimutation (at least RotA) both into thehost genome and into the plasmid-like
RotA DNA reduces the recovery of Hot
plasmid-like DNA to less than 1/10.
Furthermore even in wild type cells,
RotA activity, which was dependent ontermination event and Chi sequence, wasobserved albeit at reduced level. These
data suggest that the Y -shaped molecule
formed by blockage of the DNA replication fork at the ter site might be entrance
for RecBCD enzyme, through which the
enzyme passes, then travels along the
double helical DNA, meet with the Chi
sequence, is modified and activates the
nearby region to recombine efficiently.
Indeed this interpretation is consistent
with our previous observation that the
Y-shaped molecule was accumulated inlarger amounts in rnh- cells than in wild
type cells, because in rnh- cells, DNA
replication initiating at unidentified replication origins, which are located at
termination region, would be immediately blocked at the Ter sites and thus
these Y -form DNA molecules would beaccumulated.

59

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

Research Support Group

Research Support Facilities
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
KaoruKatou
TomokiMiwa
Mariko Saitoh
Kimiko Yamamiya

Cell Biology Group
Akio Murakami (Unit Chief)
Masayo Iwaki
Maki Kondo

Radioisotope Facility
Kazuhiko Furukawa (Unit Chief)
Yoshimi Matsuda
Mitsuaki Higashi
Center for Analytical Instruments
Hisashi Kojima
Yukiko Koyama
Eri Ueno
Glassware Washing Facility(Hiroyuki Hattori)(Toshiki Ohkawa)

The Technology Department is a supporting organization for researchers andresearch organization within the NIBB.
The Department develops and promotes
the institute's research activities and, atthe same time, maintains the researchfunctions of the institute.
The Department is organized into twogroups: one, the Common Facility. Group, which supports and maintains
the institute's common research facilities

Developmental Biology Group
Hiroko Kajiura (Unit Chief)
Miyuki Ohkubo
Sachiko Fukada
Chikako Inoue
Tomoyo Takagi
Hisae UraiRegulation Biology Group
Shoichi Higashi
Yumiko Makino
Sonoko Ohsawa
Gene Expression and Regulation Group
Yoko Fujimura
Hideko Nonaka
Koji Hayashi

and the other, the Research Support
Group, which assists the research activities as described in individual reports.
Technical staffs participate, throughthe department, jn 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.

60

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY
Head of Facility: Yoshitaka Nagahama
Research Associates:
Masakatsu Watanabe (Spectrograph)
Yoshio Hamada (Tissue and Cell Culture)
Kenta Nakai (Computer Facility)

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 gratingsurface composed of 36 smaller individual gratings. It projects a spectrum of awavelength range from 250 nm to 1,000
nm onto its focal curve of 10 m inlength. The fluency rate (intensity) of themonochromatic light is more than twice
as much 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-, timeand spatial-resolutions. It is composed ofa high-power Ar-ion laser (Coherent, Innova 20) (336.6-528.7 nm, 20 W output), two CW dye lasers (Coherent, CR599-01) (420-930 nm, 250-1000 mW
output), A/0 modulators (up to 40
MHz) to chop the laser beam, a beam
expander, and a tracking micro-beam 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 and damaging actionsof light on living organisms have been
conducted.
Publication List
Andrady, A. Fueki, K., and Torikai, A.
(1991) Photoyellowing of mechanicalpulp. Part 1: Examining the wavelength sensitivity of light-induced yellowing using monochromatic radiation. Tappi J., 74, 162-168.
Fujiwara, A., Tazawa, E., and Yasumasu, I. (1991) Activating effect oflight irradiation at various wavelengths
on the respiration in sperm of theechiuroid, Urechis unicinctus, in thepresence of carbon monoxide. J.
Biochem., 109, 486-491.
Fukuda, Y., and Osawa, Z. (1991)
Wavelength effect on the photodegradation of polycarbonate andpoly(methyl methacrylate)-confirmation of the photo-degradation mechanism of PC/PMMA blends. Polym.
Deg. Stab., 34, 75-84.
Hasunuma, K. (1991) Molecular mechanism of light signal perception inplants and fungi. Trends. Photochem.
Photobiol., 1, 311-319.
Johnson, C.H., Kondo, T., and Hastings,

FOR BASIC BIOLOGY

J.W. (1991) Action spectrum for resetting the circadian photoaxis rhythmin the CW 15 strain of Chlamydomonas. II. Illuminated cells. Plant.
Physiol., 97, 1122-1129.
Kawai, H., Kubota, M., Kondo, T., and
Watanabe, M. (1991) Action spectra
for phototaxis in zoospores of thebrown alga Pseudochorda gracilis.
Protoplasma, 161, 17-22.
Kondo, T., Johnson, C.H., and Hastings,
J.W. (1991) Action spectrum for resetting the circadian phototaxis
rhythm in the CW 15 strain of Chlamydomonas I. Cells in darkness. Plant
Physiol., 95, 197-205.
Matsunaga, T., Hieda, K., and Nikaido,
0. (1991) Wavelength dependent formation of thymine dimers and (6-4)
photoproducts in DNA by monochromatic ultraviolet light ranging from150 to 365nm. Photochem. Photobioi., 54, 403-410.
Mori, T., Nakane, M., Hattori, T., Matsunaga, T., lhara, M., and Nikaido,
0. (1991) Simultaneous establishmentof monoclonal antibodies specific foreither cyclobutane pyrimidine dimeror (6-4) photoproduct from the same
mouse immunized with ultraviolet-irradiated DNA. Photochem. Photobioi., 54, 225-232.
Nakaoka, Y., Tokioka, R., Shinozawa,T., Fujita, J., and Usukura, J. (1991)
Photoreception of Paramecium cilia:
localization of photosensitivity andbinding with anti-frog-rhodopsin IgG.
J. Cell. Sci., 99, 67-72.
Srivastava, P.K., Mori, Y., and Hanazaki, I. (1991) Wavelength-dependent
photo-inhibition of chemical oscillators: uncatalyzed oscillators with phenol and aniline as substrates. Chern.
Phys. Lett., 177, 213-218.
Takahashi, T., Yoshihara, K., Watanabe,

61
M., Kubota, M., Johnson, R., Derguini, F., and Nakanishi, K. (1991)
Photoisomerization of retinal at 13ene is important for phototaxis ofChlamydomonas reinhardtii: simultaneous measurements of phototactic
and photophobic responses. Biochem.
Biophys. Res. Commun., 178, 12731279.
Tazawa, E., Fujiwara, A., and Yasumasu, I. (1991) Activation of COinsensitive respiration in echiuroid
eggs by light irradiation at various
wavelengths.
Comp.
Biochem.
Physiol., 99B, 207-211.
Tokioka, R., Matsuoka, K., Nakaoka,
Y., and Kito, Y. (1991) Extraction ofretinal from Paramecium bursaria.
Photochem. Photobiol., 53, 149-151.
Torikai, A., and Fueki, K. (1991)
Photodegradation mechanism of polymers. Polym. Appl., 35, 10-15.
W~tanabe, M. (1991) High-fluence ratemonochromatic light sources, computerized analysis of cell movements,and microbeam irradiation of a moving cell - current experimental methodology at the Okazaki Large Spectrograph. In Biophysics of Photoreceptors and Photomovements of Microorganisms, Colombetti, G., Lenci, F.,
Raeder, D.-P., and Song, P.-S. eds.,
Plenum Publ. Corp. (New York),
pp.327-337.
Wayne, R., Kadota, A., Watanabe, M.,
and Furuya, M. (1991) Photomovement in Dunaliella salina: Fluence
rate-response curves and action spectra. Planta., 184, 515-524.
Yasumasu, 1., Tazawa, E., and Fujiwara,
A. (1991) Photo-activation of respiration in the presence of CO in sperm of
several marine invertebrates. Comp.
Biochem. Physiol., 100B, 141-147.
Yoshida, K., Takimoto, A., and Sasaki,

62
Y. (1991) The shape of fluence-response curves for the light-induced
transcript accumulation in pea seedlings. Photochem. Photobiol., 54,
495-497.
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 analysing 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
Kawai, H., Kubota, M., Kondo, T., and
Watanabe, M. (1991) Action spectra
for phototaxis in zoospores of thebrown alga Pseudochorda gracilis.
Protoplasma, 161, 17-22.
Kuroiwa, T., Kawano, S., Watanabe, M.,
and Hori, T. (1991) Preferential digestion of chloroplast DNA in male gametangia of Bryopsis maxima. Protoplasma, 163, 102-113.
Takahashi, T., Yoshihara, K., Watanabe,M., Kubota, M., Johnson, R., Derguini, F., and Nakanishi, K. (1991)
Photoisomerization of retinal at 13ene is important for phototaxis ofChlamydomonas reinhardtii: simultaneous measurements of phototactic
and photophobic responses. Biochem.
Biophys. Res. Commun., 178, 12731279.
Watanabe, M. (1991) High-fluence ratemonochromatic light sources, computerized analysis of cell movements,and microbeam irradiation of a moving cell - current experimental meth-

NATIONAL INSTITUTE

odology at the Okazaki Large Spectrograph. In Biophysics of Photoreceptors and Photomovements of Microorganisms, Colombetti, G., Lenci, F.,
Raeder, D.-P., and Song, P.-S. eds.,
Plenum Publ. Corp. (New York),
pp.327-337.
Wayne, R., Kadota, A., Watanabe, M.,
and Furuya, M. (1991) Photomovement in Dunaliella salina: Fluence
rate-response curves and action spectra. Planta., 184, 515-524.
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
the myosin heavy chain in vertebrate
non-muscle cells by interrupting its synthesis with the introduction of antisense
RNA molecules.
Computer Facility: In order to meet various computational needs in the NIBB,
various kinds of computers are maintained in this laboratory. They are:
VAX/VMS machines (VAX11/780 andmicroVAX II), UNIX workstations(SPARCstation IPX and IPC and DECstation2100), and personal computers(PC-9801 and Macintosh machines).

FOR BASIC BIOLOGYAll of them are linked to the ethernet
LAN of Okazaki National Research Institutes as well as many personal computers within this institute. Thus, from eachlaboratory, computers of this laboratory
can be accessed. In addition, a NetWare
server machine (QuarterL) is used to integrate PCs in the NIBB and it enables
the sharing of PostScript printers. Several softwares for DNA and amino acidsequence analyses are installed in our
computers.
As for its own work, efforts to develop
new methodology for sequence analysis
have been initiated. For example, an expert system has been constructed inorder to predict various protein localization sites from amino acid sequence
information only. Now, a software to63
predict splice sites in mRNA precursors
is also under development.
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 glasshouses with precision
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.

64

NATIONAL INSTITUTE

RADIOISOTOPE FACILITY(managed by NIBB)Head of Facility: Yoshihiko Fujita
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, 35S, 45 Ca, 1251 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, 14C, 32P and 35 S in the NIBB, and3H, t4C, 32p, 35S, 45Ca and 1251 in the
NIPS, are processed. The subcenter in
the NIBB is also equipped with a recombinant DNA research laboratory. The
members of tl}.e Radioisotope Facility
maintain and control the center and subcenter, and provide users appropriate
guidance for radioisotope handling.

NIBB
8UE>Center
3H, 14C, 32P, 358

Radioisotope Center
3H , 14c , 22J:\Ia, 2aMg , 32P , 358 , 36c1 , 42__
. -K, 45ca, 898r

1251 ,133Ba

65

FOR BASIC BIOLOGYCENTER OF FACILITY FOR ANALYTICAL INSTRUMENTS(managed by NIBB)
Head: Norio Murata

The Center for Analytical Instruments
consists of the following five sections. (1)Chemical Analysis, (2) Preparation of
Biological Materials, (3) Spectroscopic
Analysis, (4) Physical Analysis, and (5)Microscopic Analysis. Each section isequipped with instruments for generaluse 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-II
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
SIMADZU RF-5000
Spectrophotometer
HITACHI 330
Spectropolarimeter
JASCO J-40S
4. Section for Physical Analysis
Analytical Ultracentrifuge
HITACHI282
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-1 & II
Microsope Photometer
CARL ZEISS MPM 03-FL
Two-Dimension Microdensitometer
JOYCE LOEBL 3CS