NATIONAL INSTITUTE FOR BASIC BIOLOGY•....... ............. _ ~•1. ... ,.. : ••p...
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ANNUAL REPORT

1992

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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.
Three new professors joined the NIBB
during the past year. Prof. Kimiyuki
Satoh of Okayama University filled theposition of Professor of Biological Regulation (adjunct) to further molecular biological studies on the photochemical reaction centers involved in the primaryprocesses of photosynthesis. Prof. Masatoshi Takeichi of Kyoto University wasappointed to be in charge of Division of
Behavior and Neurobiology (adjunct) toexamine the role of cell recognition
molecules in axon guidance and target
recognition by using the neuro-muscularsystem of Drosophila. Prof. Masayuki
Yamamoto of Tokyo University was
nominated as Professor of Cell Proliferation (adjunct) to explore genes involved
in regulation of meiosis in higher organisms by using homologs cloned from the
Schizosaccharomyces
fissionyeast
pombe. In addition, the Institute continued to be active in exchange of personnel: we appointed five post-doctoral fellows from other universities to research
associates, while two research associates
were promoted to an associate and a full
professor of other universities.
The NIBB is an inter-university research institute and plays many roles as anational and international center for the
study of biology. The Institute is responsible for conducting research projects incooperation with research groups in different universities and institutes. As apart of such cooperative activities, 'the

NIBB carries out Special Programs
which are currently directed to "Biomembrane Research" and "Signal Transduction" . Based on such programs, the
NIBB held the 28th and 29th Conferences in 1992, entitled "Dynamics of thylakoid membrane assembly" (organized by
Prof. Fujita) and "The visual system ofDrosophila, from gene to structure andfunction" (organized by Prof. Hotta).
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 1992.
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

Department of Cell Biology

Department of Developmental Biology

Department of Regulation Biology

Laboratory of Gene Expression
and Regulation

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

1

Divisions

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

Reproductive Biology
Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)
Molecular Neurobiology
Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct). ~ Gene Expression and Regulation I
Gene Expression and Regulation II
Speciation Mechanisms

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FOR BASIC BIOLOGY

Each division has its own research project and is staffed by a professor, anassociate professor and two research associates. A division forms, in principle,
an independent project team. Six of thedivisions are for adjunct professorshipand are under professors who hold joint
appointments with other universities.
The adjunct division has resident research associates. The arrangement aims
to facilitate exchange in research activities in Japan. The Technical Department manages the activities of researchtechniques and helps to promote research activities of each division and also
to maintain the research resources of theInstitute. The Department 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 m 2 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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2. Graduate Student Training Program
Graduate students enrolled in otheruniversities and institutions are trained

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

OFFICE OF DIRECTOR
Publication List:
Tasaka, M., Hasegawa, M., Nakata, M.,
Orii, H., Ozaki, T. and Takeuchi, I.
(1992) Protein binding and DNase-1hypersensitive sites in the cis-acting
regulatory region of the spore-coat
SP96 gene of Dictyostelium. Mechanisms of Development 36, 105-115.

Ozaki, T., Nakao, H., Orii, H., Morio,T., Takeuchi, I. and Tasaka, M. (inpress) Developmental regulation oftranscription of a novel prespore-specific gene (Dp87) in Dictyostelium discoideum. Development 117.

5

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Goro Eguchi

The Department is composed of two regular divisions and three adjunct divisions and conducts researches on the fundamentals of cell structures andfunctions in unicellular eukaryotic organisms, plants
and animals at molecular level utilizing modern technologies including genetic engineering.

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NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor: Mikio Nishimura
Research Associates: Kazuo Ogawa
Makoto Hayashi
Ikuko Hara-Nishimura
Hitoshi MoriNIBB Visiting Scientist: Kazimierz Strzalka 11
Graduate Students: Ryuji Tsugeki
Katunari Tezuka 21
Kaori Inoue
Akira Kato
Tomoo Shimada
Nagako Hiraiwa 31
Yasushi Shigemori41
Technical Staff: Maki Kondo
Katsushi Yamaguchi(nfrom Yagiellonian University)( 21from Nagoya City University)(11from Aichi University of Education)( 4'from Okayama University)

Higher plant cells contain several distinct organelles that play vital roles incellular physiology. During the proliferation and differentiation of these cells, theorganelles often undergo dynamicchanges. The biogenesis of new organelles may occur; existing organelles may
undergo a transformation of function;and other organelles may degenerate.
Since the dynamic transformation of organellar function (differentiation of organelles) is responsible for differentiation- associated events in higher plant cells,
the following two research topics are
currently being studied in this division.
I. Functional transformation of microbodies
1. Microbody transition during the greening and senescence of seedlings
In many seeds, such as those of pumpkin and watermelon, oil is stored as
reserve material in the cotyledons. During the course of germination, thisreserve oil is utilized as the primarysource of energy for the growth of young
seedlings. When cotyledons emerge fromthe soil, the tissues become green andphotosynthetically active upon exposure

to light. Photosynthesis supplies most of
the energy needed for the growth of theseedlings after greening.
During the greening process, a seriesof dramatic metabolic changes which
underlies the shift from heterotrophic toautotrophic growth occur. Accompanying these metabolic changes, many constitutive organelles are functionallytransformed. For example, etioplasts differentiate into chloroplasts and mitochondria acquire the ability to oxidize
glycine. Glyoxysomes, which are microbodies engaged in the degradation ofreserve oil via f)-oxidation and theglyoxylate cycle, are transformed intoleaf peroxisomes that function in several
crucial steps of photorespiration. By
contrast, the reverse transformation of
microbodies from leaf peroxisomes toglyoxysomes occurs in the cotyledons
during senescence. Since cell division
does not occur in the cotyledons of thesefatty seedlings after germination, all ofthese developmental events take place ina fixed number of preexisting cells.
Therefore, these cotyledons are an excellent model system for studies aimed at
elucidating the regulatory mechanisms
responsible for the organelle differentiation.
As a step towards an understanding of
the regulatory mechanisms that operate
during the microbody transition in
pumpkin cotyledons ( Cucurbita sp.,
Amakuri Nankin), nine microbody
enzymes, namely, three glyoxysomal
enzymes,threeleaf-peroxisomal
enzymes and three enzymes present inboth types of microbody have been purified and characterized. A eDNA clone
for the leaf-peroxisomal enzyme, glycolate oxidase, has been screened andcharacterized. An analysis using theeDNA clone revealed that increases in

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FOR BASIC BIOLOGY

activities of leaf-peroxisomal enzymes
during the greening of cotyledons arecaused by the appearance and increases
in levels of the mRNA (Tsugeki et al.
Plant Cell Physiol., in press). With respect to the microbody transition during
the senescence of the cotyledons, labeling experiment with proteinA -goldMSdemonstrated
that
leaf-peroxisomal
enzymes and glyoxysomal enzymes coexist in the microbodies of senescing cotyledons, indicating that leaf peroxisomes are transformed directly to glyoxysomes during senescence of the cotyledons (Fig. 1).

GO

c::

Q)

....Q)Ol=..:

Fig. 1. Localization of glyoxysomal enzymes in senescing pumpkin cotyledons. Immunogold labeling experiments with green, half-green and yellow parts of senescing pumpkin cotyledons were performed using
antibodies against malate synthase (MS ; glyoxysomal enzyme) and against glycolate oxidase (GO; leaf peroxisomal enzyme). MS was detected in the half-green and yellow parts of the senescing cotyledons. Bars represent 0.5,um.

8
2. Molecular chaperones in higher plantcells
As part of our examination of the
microbody transition, we started to study
protein transport in higher plant cells,
focussing on so-called "molecular chaperones". Molecular chaperones are aclass of cellular proteins that function in
the folding and assembly into oligomeric
structures of certain other polypeptides
but that are not components of the final
oligomeric structures. Two different
eDNA clones encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a eDNA libraryof germinating pumpkin cotyledons. The
amino acid sequences deduced fromthese cDNAs include a 32-residue
N-terminal putative mitochondrial presequence that is attached to the each ofmature polypeptides, and the polypeptides are 95.3% identical. From a comparison of the deduced amino acid sequences with Cpn 60 from other sources,
it appears that the mature polypeptidesof pumpkin mitochondrial Cpn 60 are
44-59% identical to other Cpn 60,
namely, GroEL of E. coli, the 60-kDa
heat-shock protein (Hsp 60) of mitochondria in the yeast Saccharomyces
cerevisiae, the P1 protein of mammalian
mitochondria and ribulose-1,5-bisphosphate carboxylase/ oxygenase subunitbinding proteins a and j3 of plastids inhigher plants. The level of mRNA for
mitochondrial Cpn 60 in cotyledons,
hooks and hypocotyls of pumpkin seedlings increased markedly in response toheat stress, as deduced from Northernblot analysis, an indication that pumpkin
mitochondrial Cpn 60 is a heat-induced
stress protein. Chloroplast heat-induced
protein 70 (C-Hsp 70) has also been isolated and characterized. Further investigations on the functions of these molecu-

NATIONAL INSTITUTE

lar chaperones in the transport of protein into organelles are in progress.
II. Biogenesis and transformation ofprotein bodies
1. Processing of precursor proteins invacuoles of developing seeds
Proprotein precursors to vacuolar
components are transported from theendoplasmic reticulum into vacuoles,
where they are proteolytically processedto their mature forms. However, the details of processing mechanisms in plantvacuoles are obscure. We isolated a processing enzyme from castor bean endosperm. The purified enzyme can process
three different proproteins isolated fromeither the endoplasmic reticulum or thetransport vesicles in cotyledon cells to
produce the mature forms of these proteins. The mature proteins are found atdifferent suborganellar locations in thevacuole (protein body): the 2S protein isfound in the soluble matrix; the llS globulin is found in the insoluble crystalloid; and the 51-kDa protein is associated with the membrane. Thus, a single
vacuolar processing enzyme is capable ofconverting several different proprotein
precursors into their mature forms.
Characterization of a purified processing enzyme is required if we are to determine the way in which a single enzyme
can be responsible for the processing ofmany vacuolar proteins with a large variability in molecular structure and theway in which it can recognize the numerous types of processing site. To identify
the processing sites of various vacuolar
proteins, several cDNAs for vacuolar
proteins, which included 11S globulin,
2S albumin, membrane protein MP32
and trypsin inhibitor, were cloned andthe primary structures of their precursors
were deduced. Although processing of

FOR BASIC BIOLOGY

each of these proteins occurs on the
C-terminal side of an asparagine residue,
there is no apparant homology between
the respective primary and secondary
structures of the polipeptides.
2. Transport vesicles for precursors tovacuolar components
We have chosen three proteins, located in different parts of protein bodies,and characterized their biosynthesis andintracellular transport. Although thethree proteins are localized in different
suborganellar parts of protein bodies, allthree are synthesized on the endoplasmic
reticulum as larger precursors and transported via transport vesicles to vacuoles
in a similar manner. Thus, it appears thata single type of intracellular machinery
plays a role in the targeting of these vacuolar proteins.
We have succeeded in isolating thetransport vesicles from maturing pumpkin cotyledons. They contain large
amounts of proprotein precursors.
Double staining of immunoelectron
microscopy demonstrated that the precursors to 11S globulin, 2S albumin and
51k-Da protein are located together ineach vesicle. Further characterization of
the isolated transport vesicles is required
if we are to understand the details of themechanisms for intracellular targeting ofvacuolar proteins and the specific recognition processes in the ER, transport
vesicles and vacuoles at the molecularlevel.
3. Reversible transformation between
vacuoles and protein bodies during seedmaturation and germination
Protein bodies are single membranebound organelles that accumulate seedstorage proteins. The protein bodies areclosely related to vacuoles which are thelytic compartments of plant cells. Protein
bodies are formed by budding from the9vacuoles during maturation of seeds,
while vacuoles result from the fusion ofprotein bodies during germination ofseed. Therefore, the protein body is a
specialized form of vacuole in the dry
seed. The dramatic transformation ofprotein bodies from storage to lytic compartments occurs during the maturationand germination of seeds. In order toclarify the regulatory mechanisms responsible for budding and the fusion of
the membrane, we have focused on one
membrane protein (MP32) of protein
bodies which is rapidly degraded justafter imbibition of seed. We have isolated protein bodies and vacuoles frommaturing and germinating pumpkinseeds at various developmental stages.
Using isolated protein bodies, we haverecently established an in vitro system
for reconstituting the process of fusion of
the organelles. Experiments employing
this in vitro system are currently underway as we attempt to elucidate the molecular mechanisms of membrane fusion
during the germination of seeds.
Publication List:
Hara, T., Hara, R., Hara-Nishimura, I.,
Nishimura, M., Terakita, A., andOzaki, K. (1992) The rhodopsin-retinochrome system in the squid visualcell. In Structures and Functions ofRetinal Proteins, (John Libbey Eurotext Ltd Montroge) Edited by J. L.
Rigaud, pp. 255-258.
Hara-Nishimura, 1., Inoue, K. and Nishimura, M. (1992) Intracellular sorting of protein components of protein
bodies. In Biotechnology for Our
Well-being, (Int. Bio. Symp., Nagoya)
Edited by S. Shimizu, pp. 176-181.
Hara-Nishimura, 1., Kondo, M., Nishimura, M., Hara, R., and Hara, T.
(1992) Structural comparison of

10
retinal photopigments in cephalopods.
In Structures and Functions of Retinal
Proteins. (John Libbey Eurotext Ltd,
Montroge) Edited by J. L. Rigaud,
pp.259-262.
Ogawa, K. (1992) Primary structure andfunction of a dynein motor molecule.
Zoological Science, 9, 265-274.
Tezuka, K., Hayashi, M., Ishihara, H.,
Ak:azawa, T., and Takahashi, N.
(1992) Studies on synthetic pathwayof xylose-containing N-linked oligosaccharides deduced from substratespecificities of the processing enzyme
in sycamore cells (Acer pseudoplatanus L.). Eur. J. Biochem., 203, 401413.
Tsuboi, S., Osafune, T., Tsugeki, R., Ni-

NATIONAL INSTITUTE

shimura, M., and Yamada, M. (1992)
Nonspecific lipid transfer protein incastor bean cotyledon cells: subcellular
localization and a possible role in lipid
metabolism. J. Biochem., 111, 500508.
Tsugeki, R., Mori, H. and Nishimura, M.
(1992) Purification, eDNA cloning
and Northern-blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons. Eur. J. Biochem., 209,453-458.
Tsugeki, R. and Nishimura M. (1992)
Isolation and characterization of
Hsp70 in chloroplasts. In Research in
Photosynthesis. (Kluwer Academic
Publishers, Dordrecht) Edited by N.
Murata., III, 189-192.

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
Professor: Yoshihiko Fujita
Associate Professor: Shigeru Ito
Research Associates: Mamoru Mimuro
Katsunori Aizawa
Visiting Scientists: Kaori Ohki •Anastasios Metis • •Tiang-Qing Gu • • •J~P.s.-Post-doctoral Fellow: Tohru Ikeya
Vzsztmg Fellow: Yuji Nakajima • • • •Technical staff' Akio Murakami(*from College of Marine Sciences and Technology, Tokai University)(**from Department of Plant Biology, Universityof California at Berkeley, U.S.A.)(***from Institute of Botany, Academia Sinica
China)'( • • • *from Advanced Technology Research Center'
Mitsubishi Heavy Industries, LTD)

Flexibility of autotrophic metabolism
in algal organisms in response to growing
environments has been studied. Special
attention has been paid for photosynthesis and nitrogen-fixation. Mechanism
for light-energy harvesting in photosynthesis has also been investigated.
I. Regulation of autotrophic metabolism in response to environments
1. Regulation of PS /IPS II stoichiometry
in cyanophycean thylakoid system
In developing thylakoid system forlight-energy conversion in oxygenic
photosynthesis, PS I/PS II stoichiometryis adjusted in response to light regime soas to balance the two light reactions. We
found that when the light excites preferentially PS I (PS I light) causing electron-poor state of thylakoid electrontransport system (ETS), abandance ofPS I is decreased, and it is increased
under the light which excites preferentially PS II (PS II light) and causes electron-rich state of ETS (cf. Fig. 1). Basedon previous results, we have proposed awork model for mechanism of regulationof PS I/PS II stoichiometry that imbalance of ETS between the two photosystems is monitored, and the signal induces

11

regulation of synthesis of PS I complex
during thylakoid development so as toadjust the stoichiometry best fit for thelight regime.
To evaluate the model, we determined
the rates of synthesis of photosystem
polypeptides, PsaA/B, PsbA, and PsbC
polypeptides. For this determination wedeveloped a pulse-labelling method ~th
Synechcocystis PCC 6714 using immune-precipitation. The developed
method has permitted pulse-labeling as
short as 2 min. Results indicated that (1)PS I, but not PS II, synthesis is regulated
in response to the light regime; the rates
of synthesis of PsaA/B polypeptides is
two-fold faster under PS II light, where
PS I/PS II ratio is higher level than that
under PS I light where PS liPS II ratio islow. However, rates of synthesis of PsbA
and PsbC peptides remains constantunder either PS I and PS II light. The life
time of either PS I or PS II polypeptides
including PsbA polypeptide, is very long:and remains constant under either light
regime, indicating that degradation ofphotosystem complexes is not involved
in PS I/PS II regulation. Changes inrates of synthesis are fast: half time forboth changes is around 4 min. Nateworthy is the effect of rifampicin, the inhibitor of transcription, on the synthesisof PsaA/B polypeptides. Rifampicin did
not suppress the light regulation while
the rates of PS I synthesis and theamount synthesized were decreased withtime when rifampicin was present. Thiseffect suggests that (1) regulation occurs
at a step(s) after transcription though thet~anslation strictly depends on transcription products, and (2) mRNA's of psaAI
B are rather rapidly turing over.
We also found that regulation of Chi asynthesis occurs when PS liPS II stoichiometry is regulated. PS I complex is

12

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Light

D

D

Photosynthetic_::~~P~ie::Y

H~Q -

Light
Photosynthetic
efficiency
-

NAOP•

0.1\

H~O -

- - - NAOP•

H,o -~- NAOP" 1.0

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 -f complex; PC, plastocyanin .

Chl a-protein complex. Formation of
stably assembled complexes requires not
only synthesis of apoproteins but alsosynthesis and supply of prosthetic groupsuch as Chl a. Thus, regulation of Chl asynthesis or supply to PS I assembly cancause regulation of PS I synthesis. As
observed by · Myers et al. for Anacystis
nidulans Tx 20, Synechocystis PCC
6714 accumulated large amounts of Chlaprecursors,
protochlorophyllide(Pchlide) and Mg-protoporphyrin, incells for the former and in the medium
for the latter, under PS I light where PSI
synthesis is suppressed, while such precursors were not accumulated under PS
II light, where PS I synthesis is accelerated. A well correspondence between
the two regulations, the regulations of PS
I and Chl a synthesis, was found undervarious conditions. When PS I abundance is increased, levels of Pchlide always became insignificant, and it was accumulated when PS I synthesis is suppressed.
The step(s) in Chl a synthesis regulated was found to be located at the terminal of synthetic path or at the transportation process of Chl a to the site for
PS I assembly, suggesting that regulationof PS I synthesis is achieved by control
of Chl a synthesis or supply to thecomplex assembly. However, the factthat PsaA/ B synthesis is strictly limited
by abundance of mRNA suggests a
possibility that synthesis of apoproteinssuch as PsaA/ B polypeptides is also
regulated in this acclimation. Further
examination is under progress. Nevertheless, all results thus far obtained areconsistent with our work model and cangive more detailed insight view of mechanism for regulation of PS I/ PS II stoichiometry.
2. Mechanism of light-activation of nitrogenase in the cyanophyte Trichodesmium
Nitrogenase in cyanophytes is easily
inactivated by 0 2 as similar to the
enzyme in bacteria. However, nitrogenfixation in Trichodesmium occurs actively in the same cells as those for andsimultaneously with oxygenic photosynthesis. Mechanism of protection of nitrogenase from 0 2 evolved by oxygenic
photosynthesis has been studied, analyzing changes in nitrogenase proteins invivo. Results have revealed that (1) nitrogenase is inactivated by modificationof Fe-protein to inactive form in thedark, and (2) light causes reversed modification to the active form. Inactivation
by 0 2 has been found to be the same
modification of Fe-protein as that in the

FOR BASIC BIOLOGY

dark; phosphorylation/ dephosphorylation of Fe-protein seems to occur while
Fe-Mo protein remains unaltered. Regulation at transcription level seems not to
occur in this case.
Activation by light has been found to
involve protein synthesis. Fe-protein
may be modified to active form by an
enzyme which turns over rapidly; it will
be degradated in the dark and synthesized in light. This light activation of
Fe-protein may enables Trichodesmium
to fix nitrogen simultaneously with andin the same cells as those for oxygenic
photosynthesis.
ll. Energy transfer processes in algal
light-harvesting systems
Energy transfer processes from carotenoids to Chi a were analyzed experimentally and theoretically. We found arequisite for antenna function of carotenoids in terms of molecular structure (8conjugated double-bonds and one ketocarbonyl group) and optical properties(fluorescence from the forbidden S1state with a long life time) which ensure
a high energy transfer efficiency. The absorption band of the forbidden S1 state
was directly found (Mimuro et al., inpress), and the population dynamics inthe excited states can be explained by
the energy gap law of internal conversion. These fundamental properties ofcarotenoids in solution were also investigated in carotenoid-Chi a complexes.
We also started to calculate the energy
transfer matrix element. A most prominent result was the proof for involvement of the S1 state in energy transfer(Mimuro et al., submitted). This will leadto general and extended studies on the
excited state of carotenoids and polyenes.

13
Publication List:
Aizawa, K., Shimizu, T., Hiyama, T.,
Satoh, K., Nakamura, Y., and Fujita,
Y. (1992) Changes in composition of
membrane proteins accompanying theregulation of PS 1/PS II stoichiometry
observed with Synechocystis PCC
6803. Photosyn. Res. 32, 131-138.
Fujita, Y. (1992) Plasticity of biologicalsystem for light-energy conversion inoxygenic photosynthesis. Trans. Mat.
Res. Soc. Japan 10, 25-45.
Itoh, S., and Iwaki, M. (1992) Exchangeof the acceptor phylloquinone by artificial quinones and fluorenones ingreen plant photosystem I photosynthetic reaction center. In Dynamics
and Mechanisms of Photoinduced
Electron Transfer and Related Phenomena. (Eds. Mataga, N., Okada, T.,
and Masuhara, H.), Elsevier, Amsterdam, pp. 527-541.
Iwaki, M., Mimuro, M., and ltoh, S.
(1992) Fluorescence of P700 and antenna chlorophylls in photosystem I
particles containing 11 chlorophylls/
P700. Biochem. Biophys. Acta 1100,278-284.
lwaki, M., Takahashi, M., Shimada, K.,
Takahashi, Y., and ltoh, S. (1992)
Photoaffinity labelling of the phylloquinone-binding
polypeptidesby2-azidoanthraquinoner in photosystem
I particles. FEES Lett. 312, 27-30.
Mimuro, M., Nishimura, Y., Yamazaki,
1., Katoh, T., and Nagashima, U.
(1992) Fluorescence properties of theallenic carotenoid fucoxanthin: Analysis of the effect of keto carbonylgroup by using a model compound. J.
Luminescence 51, 1-10.

14
Mimuro, M., Nagashima, U., Takaichi,
S., Nishimura, Y., Yamazaki, 1., andKatoh, T. (1992) Molecular structure
and optical properties of carotenoids
for the in vivo energy transfer functionin the algal photosynthetic pigment
system. Biochem. Biophys. Acta 1098,
271-274.
Mimuro, M., Nagashima, U., Nagaoka,
S., Nishimura, Y., Takaichi, S., Katoh,T., and Yamazaki, I. (1992) Quantitative analysis of the solvent effect on
the relaxation processes of carotenoids
showing dual emissive characteristics.
Chem. Phys. Lett. 191, 219-224.
Ohki, K., and Fujita, Y. (1992) Photoregulation of phycobilisome structureduring complementary chromatic
adaptation in the marine cyanophyte
Phormidium sp. C86. J. Phyco/. 28,
268-270.
Ohki, K., Zehr, J.P., and Fujita, Y.
(1992) Trichodesmium: Establishmentof culture and characteristics of N2fixation. In Marine Pelagic Cyanobacteria: Trichodesmium and Other Diazotrophs, Carpenter, E.J., Capone,
D.G. and Rueter, J.G. eds., Kluwer
Academic Publishers, Dordrecht,
pp.307-318.

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Ohki, K., Zehr, J.P., and Fujita, Y.
(1992) Regulation of nitrogenase activity in relation to the light-dark
regime in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067. J. Gen. Microbioi. 138, 2679-2683.
Ohashi, K., Murakami, A., Tanaka, A.,
Tsuji, H., and Fujita, Y. (1992) Developmental changes in amounts of thylakoid components in plastids of barley
leaves. Plant Cell Physiol. 33,
371-377.
Shinohara, K., Murakami, A., and Fujita, Y. (1992) Biochemical characteristics of thylakoid membranes in chloroplast of dark-grown pine cotyledons.
Plant Physiol. 98, 39-43.
Tokutomi, S., Sugimoto, T., andMimuro, M. (1992) A model for themolecular structure and orientation of
the chromophore in a dimeric phytochrome molecules.
Photochem.
Photobiol. 56, 545-552.

15

FOR BASIC BIOLOGYDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor: Masayuki Yamamoto
Research Associate: Hidetoshi I ida
Graduate Student: Tomoko Ono (from Nara
Women's University)
Visiting Scientist: Makiko S. Okumura

This division appointed the new Adjunct Professor, M. Yamamoto, in November 1992, and is currently setting up
an experimental system to explore theregulation of meiosis in higher organisms. The major strategy going to betaken is as follows. In the fission yeast
Schizosaccharomyces pombe, which is aunicellular eukaryotic microorganism,
many genes involved in control ofmeiosis have been cloned and characterized. Mutant cells that have a defectin these genes have been obtained byclassical genetics, or by chromosome
manipulation. It is therefore feasible toisolate homologs of these genes fromplants and animals, by using either sequence similarity or functional complementation of the mutants. In preliminary experiments we have shown that
Arabidopsis thaliana has a protein kinase family whose homolog in S. pombe
affects the meiotic process. The search

for the genes regulating meiosis will be
carried out also in nematode, frog, fishand mammals.
Another line of research fulfilled inthis division in 1992 was an extension of
the projects run by the former Adjunct
Professor Y. Anraku. We analyzed themechanism of a rise in the cytosolic free
Ca2+ concentration in response to theaddition of glucose and other metabolizable hexoses in the budding yeast Saccharomyces cerevisiae. We previously
developed a very sensitive system tomeasure the Ca2+ concentration using ajellyfish protein aequorin (Nakajima-Shimada et al., Proc. Natl. Acad. Sci. USA,88, 6878-6882, 1991). Using this system, we could show that ATP is likely to
be a determinant of the rise in the Ca2+
concentration. Furthermore, we isolated
S. cerevisiae mutants apparently defective in the Ca2+ signalling, on the basis of
our recent discovery that Ca2+ is essential for maintaining viability of the cells
destined for mating (Iida et al., J. Biol.
Chern., 265, 13391-13399, 1990).
(No publication to be reported)

16

NATIONAL INSTITUTEDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Hotta
Research Associate: Hitoshi Okamoto
Institute Research Fellows: Keiko Nakao
Akihiro Inoue
Graduate Student: Keita Koizumi(from Univ. of Tokyo)

Brain has an integrated circuitry madeof a complex network of neurons andtheir axons. One of the most intriguingquestions in the contemporary biology is
how this neural network is formed in thedeveloping brain. Our research interest
is currently focused on the two aspects
related to this question, i.e. how neurons
in the brain differentiate and how they
send out their axons to their proper targets.
The latest explosive expansion of ourknowledge in this field can be largely attributed to the recent tide of researchers
to extensively apply genetics in combination with molecular biology to theproper model organisms. Based on our
research experience using fruit fly (Drosophila melanogaster), which has been
established as one of the most suitablematerial for developmental neurogenetics, we have started trying to extend ourknowledge into the realm of vertebrates.
Zebrafish (Brachydanio rerio) has beenchosen as a new material for this purpose.
I. Molecular developmental neurobiology of zebrafish
Embryos of zebrafish stay transparent
throughout the most period of their development, and many of their neurons in
the central nervous system (CNS) are
identifiable with respect to their shapes
and positions. These features have madezebrafish embryos amenable to various
modem approaches in cell biology, e.g.

cell labeling by fluorescent dyes, cell ablation by laser beam and cell transplantation. These experiments have cast muchlight upon the mechanisms underlying
neural differentiation and axonal pathfindings in zebrafish embryos. After such
sophistications in cell biology have been
made, we need to identify the molecules
involved in such developmental eventsand to examine the functions of thesemolecules in vivo by genetic manipulations of embryos. In this context, wehave set out to clone the genes whose
products may control the differentiationof neurons together with the efforts todeveolp new technology for examing
functions of such genes in vivo.
1. Molecular cloning of the Isl-1 gene inzebrafish
Isl-1 is a homeobox-containing DNAbinding protein having a LIM domain, a
Cys-His rich metal binding motif structure. This has been known to be expressed in a subset of motoneurons inthe spinal cord of chick and rat embryos.
We have cloned the eDNA coding forthe zebrafish counterpart of Isl-1 anddetermined the putative amino acid sequence. In situ hybridizations to thewhole mount embryos have revealed
neuronal type specific expressions of thisgene together with a transient early expression in the prechordal mesoderm.
Most intriguingly, this gene is expressed
only in the most anterior one of thethree primary motoneurons in most of
the segments in the embryonic spinal
cord (Fig. 1). We are carrying out theexperiments such as the one described
below to see if this gene is involved in
the process of the final cell subtype specializations by the nascent primary motoneurons.

17

FOR BASIC BIOLOGY

Fig. 1. The Isl-1 mRNA expression in the spinal cord of the zebrafish embryo.

2. A new technique to achieve an ectopic
expression of a foreign gene in zebrafish
embryos
One potent method to analyze thefunction of a cloned eDNA in vivo is tohave it expressed in cells where the geneis normally never expressed and to
examine how the developmental program is affected. To achieve this in thecentral nervous system of zebrafish embryos, we carried out the following seriesof the experiments.
DNA injected into a fertilized egg ispartitioned unevenly in the cell divisions,
resulting in a mosaic embryo with respect to the retention of the foreign geneby individual cells. When examined byusing a recombinant plasmid carrying thelacZ gene under the control of the heatshock promoter, the expression was observed either almost ubiquitously in theentire body (in about 5% of the em-bryos) or only in a small number of thecells (in the rest of the embryos). In thelatter case, the expression was mostly inmuscles and was not observed in thecentral nervous system. Although thereason for this biased expression is notclear, we concluded that it is not feasible
to get an ectopic expression of a foreign
gene only in a subset of neurons in thecentral nervous system by injecting asimple construct containing eDNA under
the direct control of the heat shock promoter.
We have succeeded in overcoming thisproblem by inserting an artificial transposon like element between the heatshock promoter and eDNA. The inserted element consists of the yeast FLP
recombinase gene with the target FRT
sequences on both ends of the element.
In the embryos injected with this construct, the transcription of the eDNA in

18

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Fig. 2. Activation of the injected lacZ gene in a trigeminal neuron of the 24hr zebrafish embryo.

the heat shock condition is interrupted
by the inserted element, and the FLP
recombinase is transcribed instead. The
induced FLP recombinase, then acts
upon the FRT sequences and subsequently causes an excision of the element between the FRT's at a low frequency. In the cells where such an excision takes place, the eDNA becomes inducible by heat shock. Since the excisions happen purely stochastically andhave no predilections to any tissues, we
can expect activations of the injectedgene in a randomly chosen one or agroup of the cells that retain the injected
DNA construct. Fig. 2 shows one of theresults of a model experiment using thelacZ gene instead of eDNA. The expression of the lacZ gene in a single neuron was observed in about 2% of the injected embryos. In addition to the ectopic expression of a foreign gene, thistechnique has a potential for a wider variety of applications including the cell lineage analysis or the replacement of thereporter gene with an arbitrary foreign
gene in an enhancer trapped transgenic
zebrafish.
II. Drosophila neurogenetics
A lethal mutant 1(2)KM13 was isolated on the basis of having excess neurons along the ventral midline in 13hr
embryo of Drosophila melanopgaster.
The primary culture derived from asingle mutant embryo revealed the defects of this mutant in the diffemtiationof both neurons and the mesoderm, especially the muscle pioneers. An enhancer trap line Fz13 was identified as
an allele of 1(2)KM13. In the heterozygotic Fz13, the reporter lacZ gene is expressed in neuroblasts and in the muscle
pioneers during the embryonic period

FOR BASIC BIOLOGY

and in neuroblasts during the larval
period. To identify new genes required
for the de novo neurogenesis during thelarval period, we screened for the mutations that alter the expression of the lacZ
gene in the larva of the heterozygotic
Fz13. A9 is the pupal lethal mutant obtained as a result of this screening. The
larva homozygous for this mutation has asignificantly smaller brain than normal
and a reduced lacZ expression in the
CNS in the heterozygous Fz13 background. To analyze the defects in thelarval neurogenesis in A9, proliferatingneuroblasts in the larva of the A9 homozygotes were labeled with BrdU.
They were abnormal in morphology andshowed almost no sign of proliferation.
No abnormality in the cellular proliferation was observed in the larval discs or in
the embryos of the A9 homozygote including embryonic nervous system.
These data suggest that the A9 gene isrequired specifically for the proliferationof the larval neuroblasts. Molecularcharacterization of this gene is now inprogress.

19
Publication List:
Kobayashi, H., Inoue, A., Mikawa, T.,
Kuwayama, H., Hotta, Y., Masaki, T.,
and Ebashi, S. (1992) Isolation of
eDNA for bovine stomach 155kDa
protein exhibiting myosin light chain
kinase activity. J. Biochem. (Tokyo)
112, 786-791.
Masai, 1., Hosoya, S., Kojima, S., andHotta, Y., (1992) Molecular cloningof a Drosophila diacylglycerol kinase
gene that is expressed in the nervous
system and muscle. Proc. Nat!. Acad.
Sci., 89, 6030-6034.
Go, M., and Hotta, Y. (1992) An
antigen present in Drosophila central
nervous system only during embryonicand metamorphic stages. J. Neurobiol.
23, 890-904.
Ito, K., and Hotta, Y. (1992) Proliferation pattern of embryonic neuroblastin the brain of Drosophila melanogaster. Develop. Bioi. 149, 134-148.

20

NATIONAL INSTITUTEDIVISION OF CELL FuSION(ADJUNCT)
Professor: Hitoshi Sakano
Research Associates: Masahiro Ishiura
Fumikiyo Nagawa
Kanae Muraiso
Graduate Student: Setsuyuki Aoki
(from Kyoto University)

In the mammalian immune system, somatic DNA recombination plays key
roles in generating functional immunoglobulin (Ig) and T cell receptor (TCR)
genes. This recombination, known as
V-(D)-J joining, generates a diversity ofreceptor genes by combining multiple
gene segments and by modifying thejunctional sequences. DNA rearrangement is also an effective mechanism foractivating a particular member of amultigene family by bringing the promoter and enhancer elements into close
proximity. It is of interest to study
whether such gene rearrangement is restricted to the immune system, or canoccur in other tissues during development.
I. DNA rearrangement in the immune
systemV-(D)-J type recombination takes
place between two recombination signal
sequences (RSS's) which are each composed of a heptamer (CACAGTG), anda nonamer (ACAAAAACC) (Sakano etal., Nature, 280, 288, 1979). The lengthof the spacer separating the two consensus sequences is always either 12bp or
23bp, and recombination takes place between two heptamer-nonamer pairs only
when one pair is separated by a 12bp
spacer and the other by a 23bp spacer.
Although the heptamer-nonamer signals
are generally well conserved, substantial
deviation is found in many naturally occurring RSS's. Mutation analysis with anartificial substrate has revealed that only

a few nucleotides in the consensus sequences are essential (Nagawa et al.,Mol. Cell. Bioi., submitted). In the heptamer, three nucleotides adjacent to therecombination site have to be conserved,
whereas others can be changed without
loss of substrate activity. In the nonamer,
two residues in the middle of the A -richcore seem to be essential.
The 12/23bp spacer rule is not as
strict as was previously thought. Undercertain circumstances, recombination canoccur between one complete RSS and aheptamer alone. Since the recombinasecannot regulate V -(D)-J joining topreserve the reading frame of the genetic
code, out-of-frame V-(D)-J structures
are generated in two cases out of three.
To rescue out-of-frame V-(D)-J structures, cells undergo V gene replacement,
which is a correction mechanism to replace a non-functional V sequence witha new germline V gene. In collaboration
with Dr. Takemori at the National Institute of Health in Tokyo, we haveidentified circular DNA excised by Vgene replacement (Usuda et al., EMBO
J. 11, 611, 1992) showing that replacement occurs by deletion type recombination. A heptamer-like sequence embedded in the V coding sequence is used
as a truncated RSS, and recombinedwith the 3'-RSS of the gennline V gene.
This variant type of V-(D)-J joining between one complete RSS and a heptamer alone is resposible for some chromosomal abnormalities in lymphocytic
tumors.
For the V-(D)-J recombinase, at least
three activities have been postulated: a
DNA binding activity, an endonucleolytic activity, and a ligase activity. In an
attempt to isolate eDNA clones for RSSbinding proteins, we screened a itgt11
phage library of a pre-B cell line with

FOR BASIC BIOLOGYRSS probes (Shirakata et al., Mol. Cell.
Bioi. 11, 4528, 1991). Among positive
clones, one type of eDNA, T160 appears to be promising. It codes for
81kDa protein that binds to the wildtype 12bp-RSS but not to an RSS in
which the heptamer sequence has beenchanged. The T160 protein contains a
DNA binding domain in the C-terminal
region whose sequence is related to anuclear protein HMG 1. In collaboration
with Dr. Huppi at NIH, we recently
found that a gene coding for the RSS
binding protein T160 (designated as
rsb-1 for recombination signal binding)is tightly linked to the recombination activating genes, rag-1 and rag-2, both in
the mouse (chromosome 2) and in thehuman (chromosome 11) (Huppi et al.,Immunogenetics, 37, 288, 1993).
Both lg and TCR gene rearrangements appear to follow the same 12/
23bp spacer rule probably using thesame recombinase. However, V-(D)-J
joining is strictly regulated in a tissuespecific manner and in a time-orderedfashion. Complete Ig gene rearrangement occurs only in B cells, while TCR
genes rearrange only in T cells. Furthermore, V-(D)-J joining takes place sequentially during lymphocyte development. For example, in B cells, the heavychain gene rearranges before a lightchain gene does. What discriminates between Ig and TCR gene rearrangement
has been a puzzling question. Recently,
in collaboration with Dr. Yamamura at
Kumamoto University, we have obtained
data indicating that the 3'CK enhancer
region may contain DNA element(s)necessary to achieve cell-type specificity
and stage specificity of recombination(Akagi et al., Cell, submitted). When the
1.2kb-3'CK region (Xhoi-EcoRI) was
deleted, V K- J K joining occurred not only21
in B cells, but also in T cells. Even in B
cells, the K gene rearrangement took
place at both the pro-B and the pre-B
stages. In contrast, the substrate with the
3'CK enhancer region underwent rearrangement only in B cells at the pre-B
stage. Thus, the 3'CK region appears tocontain a suppressive DNA element(s)which restricts K gene rearrangement to
the pre-B stage of B cells.
II. Somatic DNA changes in CNS
The brain, like the immune system,
recognizes and memorizes many different external signals. Comparison of theimmune system with the central nervous
system yields interesting parallels. An increasing number of cell-surface markers
and differentiation factors have beenfound to be common to both systems.
In order to examine a possibility of somatic DNA rearrangement in the central
nervous system, we generated transgenic
mice carrying a recombination reporter
gene (Matsuoka et al., Science, 254, 81,1991). Our recombination substrate contained the lacZ gene in a reverse orientation with respect to a ubiquitous promoter from the {:1-actin gene, such thatrearrangement results in {:1-galactosidase({:1-gal) expression. In the construct, sufficient space was maintained in front of
the lacZ gene to allow for flexibility of
the recombination site, because the site
specificity of recombination in the brain
might not be as strict as in V-(D)-J joining in lymphocytes. In the transgenic
mice, we found that not only lymphatic
tissues, but certain areas of the brain
were stained blue with X-gal, a chromogenic substrate of {:1-gal. Since similar
regions in the brain were stained in two
independent founders, detection of {3gal-positive cells was not an accidental
event resulting from transcriptional

22
activity near the integration site of thetransgene (Matsuoka et al., Science, 257,
408, 1992).
To determine whether our substrate
was actually rearranging, we amplified
DNA sequences surrounding the recombination junctions by PCR. Rearrangedtransgenes were not found in DNA fromthe tail or kidney. Brain samples produced multiple bands, each of which differed in size from the 400bp band. In
contrast to the signal joint found in thelymphatic tissues, the recombination
breakpoints we found in brain were notadjacent to RSSs. Recombination junctions were located at various distancesaway from RSSs. In the brain, the joining detected in the reporter gene thus isimprecise in site specificity. At thispoint, the recombination we observed in
the brain appears to be distinct from
V-(D)-J joining. Since the reporter gene
system does not identify rearranging
genes in the brain, a new approachshould be taken when one tries to identify them. For deletion-type recombination, isolation of circular DNA has been
instrumental in the study of antigen receptor gene rearrangement in lymphocytes. Characterization of brain circular
DNA may allow us to identify the rearranging genes in the central nervous system.
Besides DNA recombination, geneconversion is another somatic event
which often occurs in multigene families.
In chicken immunoglobulin genes, geneconversion is exploited to diversify the
primary sequences of variable region
genes. It is of interest to study gene conversion activity in the brain, e.g., in an
odorant receptor multigene family. We
have made transgenic mice carrying asubstrate for detection of the gene conversion activity. The substrate contains a

NATIONAL INSTITUTE

mutated lacZ gene connected to the /3actin promoter/enhancer complex. A
two-base insertion was generated by filling in the Clal site within the lacZ. This
insertion causes frame-shift in the downstream region. We also included a donor
sequence, the Pvuii-Avall fragment of
the wild-type lacZ. From the reporter
construct, {3-gal can be produced whenthe mutation is corrected by the geneconversion mechanism, replacing themutated Clai site with the wild-type sequence from the donor segment. We are
currently analyzing both brain and lymphatic tissues by staining with X-gal.

III. Summary
In the central nervous system, there
are several potential functions for somatic DNA changes. As in the immune
system, recombination and gene conversion could be used to generate diversity
by shuffling gene sequences. DNA rearrangement is also a convenient mechanism for the generation of an active genewhose protein product consists of two
distinct domains, one containing a variable interaction site for potential ligands,
and the other having a fixed biologicaleffector function. Another possibility isthat recombination or gene conversion
could be used to insure the expression of
one member of a multigene family.
Identification and isolation of the rearranging genes in nerve cells should provide a new insight into the role of somatic DNA changes in the development
and assembly of the central nervous system.
Publication List:
Koide, T., Yoshino, M., Niwa, M.,
Ishiura, M., Shiroishi, T., and Moriwaki, K. (1992): The amplified longgenomic sequence (ALGS) located inFOR BASIC BIOLOGY

the centromeric regions of mouse
chromosomes.
Genomics
13,1186-1191.
Supplement to 1991
Ishiura, M. (1991): Direct use of ll phage23
particles for DNA transfection. Methods in Molecular Biology, Vol. 7,
pp.63-80, Humana Press, Clifton,USA.

NATIONAL JNSTITUTE______________]I -FOR BASIC BIOLOGY25

DEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshitaka Nagahama

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

26
LABORATORY OF REPRODUCTIVE
BIOLOGY
Professor: Yoshitaka Nagahama
Research Associates: Michiyasu Yoshikuni
Masakane Yamashita
Minoru Tanaka
JSPS Post-doctoral Fellows: Naoki Shibata
Johannes Kamen
Takeshi Miura
Graduate Students: Akihiko Yamaguchi
Mika Takahashi
Toshinobu Tokumoto
Yoshinao Katsu
Visiting Scientists: Yan Shao- Yjll
Noriyoshi SakaPI
Michiya Matsuyama 31
Tohru Kobayashi
Chiemi Miura
Masamichi Nakashima" I
Jianqiao liang-51
Technical Staff: Hiroko Kajiura
Sachiko Fukada( 11from Institute of Developmental Biology, Beijing, China)
elfrom Fukui Prefectural University)( 31from Mie University)( 4 1from Tohoku University)
tlfrom Wuhan Univeristy)

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 primary study model.
I. Endocrine regulation of oocyte differentiation, growth and maturation
Our research effort in previous years
concentrated on the identification andcharacterization of the molecules (gonadotropin hormones and gonadal steroid
hormones) that stimulate and control
germ cell growth and maturation. It wasin 1985 that we identified, for the firsttime in any vertebrate, 17 a,20{3-dihydroxy-4-pregnen-3-one (17 a,20j3-DP)
as the maturation-inducing hormone ofamago salmon (Oncorhynchus rhodurus). Along with estradiol-17B, which
was identified as the major mediator of
oocyte growth, we now have two known
biologically important mediators of

NATIONAL INSTITUTE

oocyte 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-17j3 to 17a,20j3-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.
As an initial step to address this question, we have isolated and characterized
the eDNA encoding several ovarian steroidogenic enzymes of rainbow trout ( Oncorhynchus mykiss) and medaka ( Oryzias
latipes) which are responsible for estradiol-17 j3 and 17 a,20j3-DP biosynthesis:cholesterol side-chain cleavage cytochrome P450 (P450scc), 3j3-hydroxysteroid
dehydrogenase (3j3-HSD), 17 a-hydroxylase/C17,20-lyase cytochrome P450
(P450c17), P450 aromatase (P-450arom)and 20j3-hydroxysteroid dehydrogenase(20j3-HSD). The deduced amino acid sequences of these fish steroidogenic enzymes
are found to have 45-55% similarity withthose of mammals. The fish eDNA inserts
were confirmed to encode each steroidogenic enzyme by introducing it into nonsteroidogenic mammalian COS-1 cells.
These eDNA clones have been used
for Northern and whole mount in situ
hybridization to investigate the molecular basis of differential production of estradiol-17 j3 and 17 a,20j3-DP duringoocyte growth and maturation in rainbow trout and medaka. In both species,
P450scc and P450c17 (also 3j3-HSD inFOR BASIC BIOLOGY

rainbow trout) mRNA transcripts wereincreased in follicles towards the end of
oocyte growth phase and during oocyte
maturation. Furthermore, incubations ofisolated thecal layers with gonadotropin
resulted in the elevation of P450scc
mRNA. The effect of gonadotropin
becomes more dramatic when the expression of P450scc mRNA is examinedin granulosa cells. P450scc mRNA is notdetected in the absence of gonadotropin,
but markedly expressed in the presenceof gonadotropin. The increase in theamount of P450scc, 3{1-HSD and
P450c17 transcripts provide an explanation for the dramatic increase in
17 a,20{1-DP production in follicles during naturally- and gonadotropin-induced
oocyte maturation.
In contrast, levels of mRNA for
P450arom were high during oocyte
growth, but rapidly decreased duringoocyte maturation. This decrease in
P450arom mRNA levels appears to becorrelated with the decreased ability ofmaturing follicles to produce estradiol17 p. In order to define the molecular
basic of this stage- and gonadotropinspecific regulation of fish P450arom, we
have isolated and characterized the geneencoding medaka P450arom. A fulllength rainbow trout eDNA was used asa probe. The medaka P450arom geneexists as a single copy gene and its coding region is composed of nine exons;
human P450arom gene also contains
nine exons. Although the intron/ exonboundary
sequencesof
medaka
P450arom gene are exactly the same as
those of mammals, introns are much
shorter in medaka than mammals.
Primer extension and S1 mapping experiments identified at least two transcriptional initiation sites. Although wehave previously shown that the gonado-27
tropin-induced increase in P450arom activity in medaka ovarian follicle mediated through the action of cAMP, noclassical cAMP responsive elements(CREs) have been identified in the 5' region of medaka P450arom gene. Instead,
we found two ad4 consensus sequences
located 5' upstream of the putative promoter I.
We have previously shown that
17 a,20{1-DP acts via a receptor on theoocyte surface membrane and not
through cytoplasmic or nuclear receptors. The concentration of 17 a,20{1-DPbinding sites was significantly elevatedduring oocyte maturation. It was also
shown that gonadotropin increased
17a,20{1-DP binding sites; this gonadotropin-induced increase in 17 a,20{1-DPbinding sites coincided with the development of oocyte maturational competence. The early steps following
17a,20{1-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{1-DPinduced oocyte maturation with thehighest activity occurring at the first andsecond meiotic metaphase. Studies fromour laboratories have shown that MPF
activity is not species-specific and can bedetected in both meiotic and mitotic cellsof various organisms, from yeast to
mammals. To understand the molecularmechanisms of MPF formation and activation following the MIH reception on
the oocyte surface, we purified MPF
from mature oocytes of carp ( Cyprinus
carpio) and showed that it consists of
34-kDa cdc2 and 46- and 48-kDa cyclin
B proteins. Immature oocytes contained
a 35 kDa cdc2. Thus, it is concluded thatthe 34-kDa- and 35-kDa cdc2 proteinsare active andinactive forms,

28

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DMAP
H7
H8

.
,_
cdc2 kinasecyclin B

threoninekinaseactivecdc2 kinase

MPF

Fig. 1. Current model of the formation and activation of MPF during fish oocyte maturation.

respectively. The 34-kDa active cdc2 appeared in accordance with the onset ofgerminal vesicle breakdown (GVBD).
Cyclin B was absent in immature oocyte
extracts and appeared when oocytes
underwent GVBD, coinciding with theappearance of the 34-kDa active cdc2.
Cyclin B that appeared during oocyte
maturation was labelled with [35S]methionine, indicating its de novo synthesis.
Precipitation experiments with p 13sucl
and anti-cyclin B antibody revealed thatcyclin B that appeared during oocyte
maturation formed a complex with cdc2,
as soon as it appeared. Therefore, it ismost likely that the 35-kDa inactive
cdc2 preexisting in immature oocytes
forms a complex with de novo synthesized cyclin B at first, then is immediately converted into the 34-kDa active
form, which triggers all changes that accompany oocyte maturation, such as
GVBD, chromosome condensation andspindle formation.
Phosphoamino acid analysis showedthat threonine phosphorylation of the
34-kDa cdc2 and serine phosphorylationof cyclin B were associated with the activation of cdc2. However, it was shownthat cdc2 was activated by mutant cyclin
B that underwent no serine phosphorylation during the activation. These results
indicate that the threonine phosphorylation of cdc2, but not serine phosphorylation of cyclin B, is required for cdc2 activation. Taken together, it is strongly suggested that 17 a,20f3-DP induces oocytes
to synthesize cycline B, which in tum activates preexisting 35-kDa cdc2 throughits threonine (probably Thr161) phosphorylation, produce the 34-kDa active
cdc2. These mechanisms of MPF activation in fish apparently differ from thosein Xenopus and starfishs, in which cyclin
B is present in immature ooctyes andforms a complex with cdc2 (pre-MPF).
II. Endocrine regulation of male germ
cell development and matnration
We have identified two steroidal
mediators of male germ cell development in salmonid fishes (11-ketotestosteroneforspermatogenesis
and17 a,20f3-DP for sperm maturation). A
steroidogenic switch, from 11-ketotestosterone to 17 a,20f3-DP, occurs in salmonid testes around the onset of final
maturation. In vitro incubation studies

FOR BASIC BIOLOGY

using different testicular preparations
have revealed that the site of 17 a,20/3DP 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(Anguilla 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.

29
Our current research focuses on theisolation and characterization of genes
expressed or suppressed in eel testes
during the HCG-induced spermatogenesis. So far, three eDNA clones (activin
B, 3{3-HSD and an unidentified protein)
have been isolated and characterized.
Northern blot analyses of testes during
the HCG-induced spermatogenesis invivo have shown that activin B mRNA
was nondetectable in testes prior to thetreatment, but could be detected in testes for 1-6 days with HCG. The HCGdependent activin B mRNA expression
was confirmed by in situ hybridization;
the site of activin B mRNA hybridization was restricted only to Sertoli cells intestes treated with HCG for 1-6 days.
HCG treatment also caused a rapid risein the abundance of testicular 3/3- HSD

Fig. 2. In situ hybridization pattern of activin B mRNA in eel testis. A , uninjected testis; B, testis 1 day after
HCG injection showing high levels of activin B mRNA in Sertoli cell cytoplasm.

30mRNA. In contrast, levels of mRNA forthe unidentified protein were abundantin testes prior to HCG treatment, butbecame nondetectable by 1 day after thetreatment.
In salmonid fishes, spermatozoa takenfrom the testes are immotile, but acquire
motility during their passage through thesperm duct. Using male masu salmon(Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17a,20,8-DP is responsible forthe acquisition of sperm motility;
17 a,20,8-DP acts to increase sperm ductpH, which in tum increases the cAMP
content of sperm, allowing the acquisition of motility.
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:
Aoki, F., Choi, T., Mori, M., Yamashita,M., Nagahama, Y., and Kohmoto, K.
(1992) A deficiency in the mechanism
for p34cdc2 protein kinase activation inmouse embryos arrested at 2-cellstage. Develop. Bioi. 154, 66-72.
Choi, T., Aoki, F., Yamashita, M., Nagahama, Y., and Kohmoto, K. (1992)
Direct activation of p34cdc2 protein kinase without preceding phosphorylation during meiotic cell cycle in mouse
oocytes. Biomed. Res. 13, 423-427.
Fujishima, M., Katsu, Y., Ogawa, E., Sakimura, M., Yamashita, M., and Nagahama, Y. (1992) Meiosis-reinitiation-inducing factor of Tetrahymena
functions upstream of M-phase-promoting factor. J. Protozool. 39,
683-690.
Hirai, T., Yamashita, M., Yoshikuni, M.,

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Tokumoto, T., and Nagahama, Y.
(1992) Isolation and characterizationof goldfish cdk2, a cognate variant of
the cell cycle regulator cdc2. Develop.
Bioi. 152, 113-120.
Hirai, T., Yamashita, M., and Nagahama, Y. (1992) Cyclin B in fish
oocytes: Its eDNA and amino acid sequences, appearance during maturation of goldfish oocytes. Mol. Reprod.
Develop. 33, 131-140.
Kamijo, M., Yasuda, H., Yau, P.M.,
Yamashita, M., Nagahama, Y., andOhba, Y. (1992) Preference of human
cdc2 kinase for peptide substrate. Peptide Res. 5, 281-285.
Komen, J., Yamashita, M., and Nagahama, Y. (1992) Testicular development induced by a recessive mutation
during gonadal differentiation of female common carp ( Cyprinus carpio,L.). Develop. Growth Differ. 34,
535-544.
Nagahama, Y. (1992) Endocrine controlof oocyte maturation in teleosts. On
togenez 24, 26-31.
Sakai, N., Tanaka, M., Adachi, S., Miller, W.L., and Nagahama, Y. (1992)
Rainbowtrout
cytochrome
P-450c17(17 a-hydroxylase/17 ,20lyase): eDNA cloning, enzymatic
properties and temporal pattern ofovarian P-450c17mRNA expression
during oogenesis. FEBS Lett. 301,
60-64.
Okada, T.S. and Nagahama, Y. (1992)
Biotechnology in aquatic animal takes
off. Biology International.
Tanaka, M., Telecky, T.M., Fukada, S.,
Adachi, S., Chen, S., and Nagahama,Y. (1992) Cloning and sequence analysis of the eDNA encoding P-450
aromatase (P450arom) from rainbow
trout (Oncorhynchus mykiss) ovary;
relationship between the amount of

FOR BASIC BIOLOGY

P450arom mRNA and the productionof oestradiol-17 {J in the ovary. J. Mol.
Endocrinol8, 53-61.
Tanaka, M., Ohno, S., Adachi, S., Nakajin, S., Shinoda, M., and Nagahama,Y. (1992) Pig testicular 20{3-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity: eDNA cloning of pig testicular
20{J-hydroxysteroid dehydrogenase. J.
Bioi. Chern. 267, 13451-13455.
Yamashita, M., Fukada, S., Yoshikuni,
M., Bulet, P., Yamaguchi, A., Lou,
Y.-H., Zhao, Z., and Nagahama, Y.
(1992) Purification and characterization of maturation-promoting factor infish. Develop. Bioi. 149, 8-15.
Yamashita, M., Fukada, S., Yoshikuni,
M., Bulet, P., Hirai, T., Yamaguchi,

31
A., Yasuda, H., Ohba, Y., and Nagahama, Y. (1992) M-phase-specific histone H1 kinase in fish oocytes: its
purification, components and biochemical properties. Eur. J. Biochem.
205, 537-543.
Yasuda, H., Nakata, T., Kamijo, M.,
Honda, R., Nakamura, M., NinomiyaTsuji, I., Yamashita, M., Nagahama,Y., and Ohba, Y. (1992) Cyclin-dependent Kinase 2 (cdk2) in the murine
cdc2 Kinase TS mutant. Somatic Celland Mol. Genetics. 18, 403-408.
Yoshikuni, M., and Nagahama, Y.
(1992) Endocrine regulation of gametogenesis in fish. Bull. Inst. Zoo!. Aca16,
demia
Sinica,Monograph139-172.

32

NATIONAL INSTITUTEDIVISION OF CELL
DIFFERENTIATION
Professor: Y oshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Shigeharu Takiya
Toshiharu Suzuki
JSPS Postdoctoral Fellow: Kazuhito Amanai
Visiting Scientists: Toshifumi Nagata
Masakazu Fukuta (from Aichi
University of Education)
Graduate Students:
Masakazu Fukuta (Graduate University for Advanced Studies)
Kaoru Ohno (Graduate University for Advanced Studies)
Pin-Xian Xu (Graduate University for Advanced Studies)
Hiroki Kokubo (Graduate University for Advanced Studies)
X in Xu (Graduate University for Advanced
Studies)Kensuke Suzuki (from Nagoya City University)
Technical Staff: 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 developmental regulatory genes
like homeobox genes regulate a set oftarget (structural) genes in specifying theidentities of various regions of the embryos.
I. Genes and factors that control thesilk gland development and the silk
genes transcription
We have been trying to understandthe networks of gene regulation hierarchy that function in the processes ofsilk gland development and differentia-

tion. As a bottom-up type approach forthis project, analyses on the molecularmechanisms that control the differential
transcription of the fibroin and sericin-1
genes in the silk gland should shed lighton the part of networks. In complementing this approach, a top-down type approach referred in the next section
should also help understanding the networks; analyses of regulation hierarchyof the homeobox genes and other regulatory genes, and identification of theirtarget genes expressed in the labial segment, where silk gland is originated, andnearby regions of Bombyx embryos.
Among many factors proposed to bindand control the fibroin and sericin-1
genes, one candidate factor has beencloned and characterized; which is a
POD-domain-containing protein named
as POU-Ml. The POU-M1 accommodates a POU-domain identical to Drosophila Cfl-a, is expressed abundantly inthe larval middle silk gland where thesericin-1 gene is specifically expressed,and binds to the SC region of thesericin-1 gene, a key element for transcription enhancement accommodating
an octamer consensus sequence. To
characterize the transcription regulationof the POU-Ml gene, a genoinic DNA
fragment encompassing the whole coding region and its flanking sequences has
been cloned. This gene does not containany intron. The 5'-flanking region of thegene contains several interesting motifs(see Fig. 1), such as homeo-domainbinding motifs (PB region), sequences
resembling the transcriptional factor Splbinding site (positions -102 to - 38),
and TGTIT motifs (PA and PC regions), but lacks some of typical transcriptional regulatory sequences such as
TATA and CAAT boxes. Transcriptional analysis of a series of deletion

33

FOR BASIC BIOLOGY-984 AATGCCGCTT TCACAAATCA TATCTTTATA TGTATATAAC GTTTTTAAAT CATGATATTT111111111111111111111111-924 CAATTATAAT GTGAAGAAAC GACATTAGCA GAGAGCTGTT TAACTTGCTC TGGGTAACTA-864 GTAGCTGGTA TTCTTATCAA TTTTTGTTTC AAATTCGAGA GTTTCAGAAT TAAACAATTG-804 GAGGAGGAAT TATTTATGTC TACTGCATTA CATTTGTAAA TCTTTCTAAT TGAACGCTTT-744 TAGATCAGTT TTGGATTTAG ATCAGTTTTG TAACGATGTC AAGTCGTTGT TACTTAGTAG-684 AAGA1TI"I I'I' AGTTTATTAG AGTAGATTTG GTAGGTAGGT ACAACGTCTC TGTGCCAGAA-624 TGGTGGACTT CCCAGTTATC GTCTGTATCC CTAGAAGATC TAAAACCTAA TGTAATATCT1111111111111111111111111-564 GCTGAAGTAG AAAATTCCTC TTAATTTGTA GTGAACATTA AAAATATATG TATTAGAAAG
111111111111111111111111-504 CTATTCTATG TGTTTAATGA CGTTCTGTGT ATGTAAAAAT AGTCTAAGAC GTTACAGTGT
PC-444 TTGTAAAAAT GCTCTAATAT ATAATTAGTA CGTTTAATTT TAATTAAATA AAAAAGAGAG..........
PB-384 TCTTAGATCG TGTTCGATTT GTTTATTAAA TTGGGGCCGT GACGTTATCG AAACGCGGTT

1

PA-324 GTAAGCGGTA TATTATTTAA AACGAGTGTA AGTACAGTAA AAGTCGACAA GTCAGTTATA-264 TTCGTCTATA TGACAAGGAA TTGTACTGGG AAGCGCGCCG TCGGTCCTCG GCGGAGCGTC-204 AGGTGCGGGT CGG~G~QT~C GG~G~~~CG G~GAGtaCGG ~GGGCACGGA CGCGAGACGT-144 ACAGACGCGA GAGTTTTCCA GTGCGGGGCG CGAGGGGGCC GAGGGGCGGG GCAACAATTA-84 CACCGTCATT AGCGGGCGGC CGCGCTGGGC GGGGCCGGCC CTGAGAGCGG CGGTCGGCGG+1-24 GCGCGCGCAG CCGCCGAGCC CCGCAGTTCG CTCCGTGCTG CCGTAGCGCA CGCACGCGCA+37

TGTCGCTCGC TGTCGAATCC GGTGATGTGA GGTGGCCCCC GCCGCTACCG CAGCGCAGCG+97

CGACCCCCGC GCAGTTTTCG CGAAAGAATA AGCCAGTTGA AGGCTCGAGA GCCTTCGAGC+157 CGGCAAGACG CCGTCCACCG TGGATCTGAG ATACGGCGCC ATG+197
Met

Fig. 1. DNA sequence of the POU·Ml promoter and its surrounding regions. +1 marks the transcription
initiation site.

mutants of the gene in the nuclear extracts from the middle silk gland of
2-day-old fifth instar larvae revealed thepresence of multiple cis-regulatory elements located both upstream and downstream of the transcription initiation site.
One of these elements, the homeodo-

main-binding element (PB), was identified to mediate negative regulation; deletion of this element resulted in higher levels of transcription (compare lane 3
with lane 5 in Fig. 2). By mobility shift
assay using the POU-Ml specific antibodies, we found that this negative

34

NATIONAL INSTITUTE

I,..
I'

~

N ··•'· A-483'·...r---1·.,,.

%Activity

lfti(IHII--......._..__ _ _ _ __......__ __....._--'=----447~~~~----------~----~~~~-,...5-....._.__. . . ,;__________
J..=.,......,;..._

NormallgG

POU-Miarlfibody

100

210

106

211

240

225

Fig. 2. Effect of immunodepletion of POU-M1 protein on the POU-M1 gene transcription. Affinity purified anti-POU-M1 C1 peptide IgG (lanes 2, 4 and 6) or normal lgG (lanes 1, 3 and 5) was incubated withthe middle silk gland nuclear extract of 2-day-old fifth instar larvae. Protein A-Sepharose was then added andincubated for 1 hr on ice. The mixture was centrifuged and the supernatant was assayed for transcription.

element interacts with the POU-M1 protein. Transcription analysis in vitro using
one of the POU-M1 antibodies (see Fig.
2) indicated that POU-M1 can negatively autoregulate its own gene expression as a consequence of binding to thehomeodomain-binding element with aweaker affinity than the binding of theprotein to the SC site of the sericin-1
gene. Several other elements including
the TGTTT motifs, the possible Splbinding sites and others appear to be activation elements, and an additional negative element as well as a few more activation elements in the downstream of
the intiation site were detected.
The protein FFl in the presence ofanother protein ·FF2 binds to the enhancer element I of the fibroin gene.
FF1 was purified to the homogeniety but
only a partial purification was attainedfor FF2. Use of polyclonal antibody
against the FF1 suggested the FF1 isprobably a transcription factor involvedin the enhancement of the fibroin genetranscription in the posterior silk gland.
Recently, several monoclonal antibodies
against FF1 have been obtained, andcharacterization of FF1 as a transcription
factor and cloning of FF1 are being
planned. Another factor OBF-1 binds tothe enhancer element II in the intron ataround +220 and +290 regions and to
the enhancer element I at around -130

FOR BASIC BIOLOGY

35

Fig. 3. Initiation of the silk gland development by the invagination of a part of the labial segment. The
section was derived fro m stage 218 embryo. The arrow indicates the invagination of the silk gland.

region. The oligonucleotide corresponding to the +290 region competed thetranscription enhancement both by theenhancer I and the enhancer II. Purification and cloning of the OBF-1 are also
being planned.
II. Genes involved in the Bombyx bodyplan
To know the roles of the Bm Ubx, Bm

abd-A, Bm Abd-B, Bm Antp, Bm en,
Bm in, Bm Wnt-1, and Bm cad genes on
the body plan, we have analyzed the expression of these genes in the wild typeand mutant embryos. In situ hybridization to the wild type embryo sections revealed that the expression of the Bm
Antp was detected in the first to thethird thoracic segments. This transcript
was strongly detected in three pairs ofthoracic legs and the caudal legs at stage

20. In the embryos homozygous for the
EN mutation which lacks both the Bm
Ubx and Bm abd-A genes the transcriptof the Bm Antp was detected in the thoracic-type legs and the epidermis fromthe first thoracic segment to the seventh
abdominal segment. The transcript of
the Bm Abd-B was detected from thesixth abdominal segment to the telsonboth in the wild type and ENI EN embryos. From these results we speculatethat functional deficiency of the Bm Ubx
and Bm abd-A genes in the EN/ EN embryos may have caused derepression of
the Bm Antp gene and transformation of
the first to the seventh abdominal segments into the thoracic-type segments.
Development of the silk gland is initiated in the labial segment at the embryonic stage 18 or 19 (see Fig. 3), andmust be under the control of genes that36
specify the labial segment identity. Previously we reported that several homeobox genes like Bm Antp, Bm en, and Bm
in are expressed in the larval silk gland,
and the Nc mutation of the Bm Antp
causes a serious defect on the embryonicsilk gland development. To analyze further the roles of other homeobox genes
on the silk gland development, the cloning and characterization of Bm Dfd, Bm
Scr, and Bm lab have been initiated.
Publication List:
Hui, C.-c., Matsuno, K., Ueno, K., andSuzuki, Y. (1992) Molecular characterization and silk gland expression of
Bombyx engrailed and invected genes.
Proc. Nat!. Acad. Sci. USA 89,
167-171.

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Kuwano, R., Araki, K., Usui, H., Suzuki, Y., and Takahashi, Y. (1992)
Transcription of the rat cholecystokinin gene is initiated at multiple sites:
Verification by an in vitro transcription system. J. Neurochem. 58,
304-310.
Ueno, K., Hui, C.-c., Fukuta, M., andSuzuki, Y. (1992) Molecular analysisof the deletion mutants in the E
homeotic complex of the silkworm
Bombyx mori. Development 114,555-563.

FOR BASIC BIOLOGYDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Visiting Professor:
Volker Schmid (from Zoological Institute,
University of Basel)
Research Associates: Ryuji Kodama
Makoto Mochii
JSPS Postdoctoral Fellows: Keiko Ishikawa
Yutaka Imokawa
Visiting Scientists: Takamasa S. Yamamoto
Kaichiro Sawada
Masashi Hyuga
Graduate Students:
Akio Iio (Graduate University for Advanced
Studies)
Nobuhiko Mizuno (Graduate University for
Advanced Studies)
Yasutaka Matsubara (from Shinshu University)
Atsushi Kishi (from Kumamoto University)
Technical Staff' Chi yo Takagi
Hisae Urai

There is a mode of reparative regeneration, in which the lost tissue or organ
can be compensated by cellular metaplasia (transdifferentiation) of once-specialized tissue cells. In the newt andsome other limited vertebrate species,
the lens and neural retina can be completely regenerated through the transdifferentiation of pigmented epithelial cells(PECs). Such a phenomenon, transdifferentiation, as observed in regenerationof ocular tissues seems to be a highly
useful model for studying stability andinstability in differentiation of tissue
cells. From this view point, lens transdifferentiation of PECs of vertebrates has
been studied in in vivo and in vitro systems, and our in vitro studies have revealed that dormant potential to transdifferentiate into lens cells is widely conserved by the vertebrate species including human.
We are studying in order to answer
the questions why the PECs can transdifferentiate into lens cells and why thepigmented epithelia of the species otherthan the newt and so forth never compensate the lost lens in the in situ eye.
ltoh and Eguchi previously established a37

unique culture system for the lens transdifferentiation using PECs from chickembryos. In this system the differentiated states of PECs are completely
regulated by manipulation of the culture
condition. Thus, molecular biological approaches have become possible using
cultured chick PECs. We are also trying
to establish transgenic systems of birds inorder to examine directly the functionsof individual genes closely related tocontrol of differentiation and transdifferentiation of tissue cells. In addition tothese major projects, the following problems have been recently conducted ascollaborative works with scientists fromthe outside: (1) Pattern formation of
Lepidopteran wing and (2) basic analysisof biocompatibility of hydroxyapatite asa biomaterials. The investigations andthe results achieved in 1992 by themembers of this division are as follows.
I. The extracellular factors modulating
the lens transdifferentiation of the PEC
in vitro
Itoh and Eguchi have established thatthe lens transdifferentiation from PECsof chick embryos in vitro is achieved byphenylthiourea (PTU) and testicular
hyaluronidase, which are added to theculture medium. In 1991, we establishedthat FGF is one of key factors to regulate the lens transdifferentiation of PECs
through the analysis of crude testicular
hyaluronidase. On the basis of this finding, we have studied the mode of actionsof FGF and of PTU in the process of thelens transdifferentiation of PECs in vitro
and the following results were obtained.
FGF can effectively induce collagenase
production by PECs in culture when
added to the culture medium. The zymographic analysis of culture medium using
gelatin as a substrate has revealed that

38
FGF increases production of gelatinase(= latent 75kDa type IV collagenase)and activates gelatinase as well. In addition, the analysis of ECM has revealed
that thick fibrous distributions of tenascin in eXtracellular matrix (ECM) of
PECs cultured without both PTU and
FGF dramatically altered to scatteredfine network by addition of PTU, although distribution of fibronectin is notaffected. Since PECs themselves synthesize type IV collagen to form therather thick ECM consisting mainly of
striated collagen fibers and lens transdifferentiation of this cell type can be suppressed by an artificial collagen substrate, these results are strongly suggesting that FGF might be one of key factors which regulate the differentiatedstate of PECs and the process of thislens transdifferentiation through cooperative functions with ECM in thepresence of PTU.
On the basis of these results, we arenow extending our analysis of roles ofboth FGF and PTU in regulation of thelens transdifferentiation of PECs, particularly focussing on the functions of
ECM.
II. Analysis of functions of pP344 gene
responsible for the lens transdifferentiation of PECs
By 1992, it has been established that
PEC-specific pP344 gene is one of key
genes, whose products must function toregulate the differentiated state and lenstransdifferentiation of PECs in vivo andin vitro. In this year, we have concentrated to analyze the function of pP344
gene and the following results have beenobtained. In situ hybridization has revealed that this gene is only expressed in
the retinal pigmented epithelium butneither in iris pigmented epithelium nor

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ciliary epithelium. Analysis of a molecule
produced by pP344-transfected Cos cells
using an antibody against a peptide representing a distict region of this gene hasrevealed that pP344 gene encodes for asecreted 70kDa protein. We could purify
70kDa protein from the conditioned culture medium of PECs and the amino
and sequencing showed that this protein
was definitely the pP344 gene product.
Moreover, we have confirmed that this
70kDa protein exhibited a strong inhibitory activity on trypsm. Based on thesefindings we are now bending our efforts
on our studies of the regulatory mechanism of the lens transdifferentiation of
PECs.
III. Development of avian- transgenic
systems
In addition to chick transgenic system,
we have tried to establish the transgenic
system of the quail. We firstly established the technique for complete cultivation of quail embryos from single cellstage to hatching. By this technique we
can obtain a high hatchability of 25%.
The ,8-actin-lacZ hybrid gene (Miw Z)
was microinjected to the fertilized ova to
be cultured. Seven out of 17 survived
embryos from 85 to 90 h after injection
exhibited the lacZ gene expression in
their embryonic tissues. Thus, our culture technique will serve as a powerfultool for the production of transgenic
quails.
As to the development of transgenesis
in chick, we have developed a new
method for microinjection of DNA to betransfered to a chick ovum. Although weshould reserve the description of thetechnique in detail at the present stage of
our study, we have obtained dramaticallyhigh efficiency in expression of the transferred gene. In three out of nine survived

39

FOR BASIC BIOLOGY......

... 'tr~ . : --- ­

. '>

...

~·

Fig. 1.

embryos, strong and homogeneous expression of transferred ,8-actin-lacZ hybrid gene (Miw Z) was achieved as Fig 1
shows. In all of the rest six embryos, thetransferred gene was found to be moreor less expressed in the embryonic tissues. Thus, our technique for microinjection of DNA to be transferred to fertilized chick ova is reaching to the goaL
IV. Pattern formation of butterfly
wings through programmed cell death
The wings of butterflies are formed byvigorous extension of the imaginal discepithelium at pupation. However, thepupal wing usually has a different outline
shape from that of an adult butterfly
wing, i.e. the latter often has projections
and indentations at its margin, while theformer has a round outline. This difference was reported to occur throughthe death of wing epithelial cells at themargin during pupal period. We precisely observed this process in Pieris
rapae pupa and found that (1) the exten-

sive cell deaths occur in the period of
about half a day (from 3.5th to 4th day
at 20°C), and that (2) dying cells show aultrastructure characteristic to apoptosis(programmed cell death). Figure 2 showsa crossection of the wing tip on the 4th
day where extensive cell deaths are observed at the very tip. Preliminary study
suggested that cells die after S-phase.
This system is geometrically very simple
since all the cells in a certain area of asimple epithelium die. We believe thissystem is suitable for analyses of themechanisms controlling cell death ingeneraL
V. Cell biological analysis of biocompatibility of hydroxyapatite as a biomaterial
We have firstly established a humangingival fibroblast line, HGF-22 and agingival epithelial cell line, HGE-15.1.
Both of the established cells grow actively and maintain their cellular characteristics with high stability. HGE-15.1

40

NATIONAL INSTITUTE

Fig. 2cells conserve high activity to synthesize
keratin molecules. Using these cell lines,
we have quantitatively analyzed theircellular activities represented by adhesion, spreading, growth and differentiation by culturing cells on the surface of ahydroxyapatite disc, which is thinenough to observe living conditions ofcells by a phase contrast microscope.
Every examined activity expressed byboth HGF- 22 and HGF-15.1 cells cultured on a hydroxyapatite disc was much
reduced in comparison with cells cultured on the surface of commercial plastic cell culture dishes, strongly suggestingthat the biocompatibility of hydroxyapatite is not always so high as thought
thus far.
Publication List:
Eguchi, G. (1992) Transdifferentiationin the vertebrate retinal pigmented
epithelial cell. In Progress in Retinal

Research 12 (N.N. Osborne and G.J.
Chader, eds.). Pergamon Press, Oxford, pp.205-230.
Kosaka, J., Watanabe, K. and Eguchi,
G. (1992) Transdifferentiation ofchicken retinal pigmented epithelial
cells in serum-free culture. Exp. Eye
Res. 55:261-267.
Imokawa, Y., Ono, S. , Takeuchi, T. andEguchi, G. (1992) Analysis of aunique molecule responsible for regeneration and stabilization of differentiated states of tissue cells. Int. J.
Dev. Biol. 36:399-405.
Imokawa, Y. and Eguchi, G. (1992) Expression and distribution of regeneration-responsive molecule during normal development of the newt, Cynops
pyrrhogaster. Int. J. Dev. Biol.
36:407-412.
Sawada, K., Agata, K. and Eguchi, G.
(1992) Crystallin gene expression in
the process of lentoidogenesis in cui-FOR BASIC BIOLOGY

tures of chick lens epithelial cells. Exp.
Eye Res. 55:879-887.
Mochii, M., Iio, A., Yamamoto, H., Takeuchi, T. and Eguchi, G. (1992) Isolation and characterization of achicken tyrosinase eDNA. Pigment
Cell Res. 5:162-167.
Ninomiya, Y., Mochii, M., Eguchi, G.,
Hasegawa, T., Masusige, S. and Kato,
S. (1992) Tissue-specific response of
estrogen receptor gene expression to
estrogen in chick. Biochem. Biophys.
Res. Com. 187:1374-1380.
Oguchi, H., Seto, K., Ishikawa, K., Eguchi, G., Ojima, S. and Hirayama, Y.
(1992) The development of hydroxyapatite coated titanium sticks using ahigh velocity flame spraying technique. Bioceramics 3:199-219.

41

Oguchi, H., Ishikawa, K., Ojima, S., Hirayama, Y., Seto, K. and Eguchi, G.
(1992) Evaluation of a high-velocity
flame spraying technique for hydroxyapatite. Biomaterials 13:471-477.
Supplements to 1991
Oguchi, H., Ishikawa, K., Seto, K. andEguchi, G. (1991) Histological evaluation of titanium alloy sprayed withhydroxy-apatite using a high-velocity
flame-spraying technique. Apatite
1:247-257.
Oguchi, H., Seto, K., Ishikawa, K. andEguchi, G. (1991) Endodontic-endosseos anchoring method using hydroxyapatite sprayed titanium sticks.
Bioceramics 4:363-369.

42

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL
BIOLOGY (ADJUNCT)
Professor: Masaki Iwabuchi
Associate Professor: Masao Tasaka
Research Associates: Koji Mikami
Takefumi Kawata
Research Fellow: Eiki Momotani
Visiting Scientists:
Hisabumi Takase (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)
Masa-aki Katsura (from Kyoto University)
Ayako Sakamoto (from Kyoto University)

This division is devoted to the studies
on transcriptional regulation of genes
whose expression depends on cell proliferation and differentiation in higher
plants and a cellular slime mold. We
have conducted researches on the following two aspects.
I. Trans-acting factors involved in transcriptional regulation of cell cycle-dependent genes in higher plants
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 still unknown. We have focused our attention on transcription ofhistone genes as a model system for thecell cycle-dependent gene expression inhigher plants. The S phase-specific transcription of a wheat histone H3 gene
(TH012) is regulated by a proximal promoter region up to -184 (relative to thetranscriptional initiation site, +1). In
vivo transcription experiments with deletion and site-directed mutations revealed
that a type I element (CCACGTCANCGATCCGCG) (Mikami and Iwabuchi(1993) in: Control of Plant Gene Expression, ed. by D.P.S.Verma, CRC
Press, Boca Raton, pp51-68) may be a

cis-acting element controlling the periodic transcription of the H3 gene. This is
supported by evidence that the type I
element is highly conserved in promotersof many plant histone genes and that nuclear factors specifically to the type I element as wheat HBP (histone promoterbinding protein)-1a and HBP-1b havebeen identified in a variety of plant
species.
Wheat bZIP proteins encoded by different five eDNA clones bound specifically to the type I element as the nuclear
factors HBP-1a and HBP-1b. We concluded that five bZIP proteins constituteone family, designated HBP-1 family. By
differences in overall structure and binding specificity, the HBP-1 family wasgrouped into HBP-1a and HBP-1b subfamilies. All members of the HBP-1a
subfamily had a potential for homo- andhetero-dimerization, and members of the
HBP-1b subfamily were supposed toform heterodimers because of theirstructural similarity. The binding sequences to bZIP proteins are present in promoters of plant genes other than histone
genes. The various combinations amongbZIP proteins in heterodimerization may
generate a relatly expanded repertoire of
their regulatory potential. No heterodimers could be observed between HBP1a and HBP-1b subfamily members. We
proposed that HBP-1a and HBP-1b subfamilies might mediate different regulatory processes.
The heterodimerization between different members of the HBP-1 family
provides the complexity in addressing
the functional properties of the HBP-1
family in the S phase-specific transcription of the H3 gene. From the aboveview-point, we have performed several
experiments. To investigate regulatory
mechanisms of HBP-1a and HBP-1b

FOR BASIC BIOLOGY

gene expression, the promoter regions ofthese genes were fused to a bacterial
GUS gene and resulting constructs werestably introduced into rice cells. The cell
cycle-dependent expression of the fusion
genes in transformed rice cells have been
analyzed. All bZIP proteins of the
HBP-1 family have consensus motifs forphosphorylation by various protein kinases; for example, casein kinase II,
MAP (mitogen-activated protein) kinaseand cdc2 kinase. So, we have started thestudy on relationship between protein kinases and HBP-1a and HBP-1b activities. In addition, we isolated several
eDNA clones encoding proteins analogous to some members of the HBP-1
family from a Arabidopsis eDNA library. Functional analyses of the Arabidopsis bZIP proteins are in progress by agenetical approach. These efforts may
make it possible to demonstrate that atleast some members of the HBP-1 family
are trans-acting factors involved in S
phase-specific transcription of plant histone genes.
II. Function and expression of Dp87
gene in Dictyosterium discoideum
Cells of Dictyosterium discoideum aggrigate to form a multicellular organismand differentiate into prespore and prestalk cells which are the precursors of respective spore and stalk cells in a fully
developed fruiting body. To elucidate
the mechanisms of cell differentiation,
we have studied the regulation of a prespore-specific gene Dp87.
1. Expression of Dp87 protein during development
The Dp87 protein shows a structuralsimilarity to some spore cort proteins.
However, timing of transcription of theDp87 gene was earlier than that of any
other prespore-specific gene so far

43

examined, including spore coat protein
genes. We compared expression profilesof the Dp87 and spore coat proteins immunologically. At late aggrigation stage,a 81kD protein was initially identified inmany small particles, dispite a molecularweight of the protein was estimatedabout 58kD from a primary amino acidsequence. At slug stage, the 83kD protein, slight larger than the product at the
aggrigation stage, was observed in aprespore vacuoles (PSVs) of prespore
cells as is the case for spore coat proteins. In this stage, the Dp87 protein isconcentrated at the center of PSV, while
spore coat proteins were localized nearits inner surface. After formation of amature fruiting body, 83kD protein wasdispersed in the sorus matrix and on the
stalk sheath surface, but not on the spore
surface where spore coat proteins localize. These results suggest that the
Dp87 protein is a novel sorus matrix
protein which is modified post-translationally at different two stages.
2. Function of Dp87 protein
By homologous recombination, we
made a mutant which lack the Dp87
gene. All of independent mutants
showed normal development. This suggest that the Dp87 is not essential for lifeof Dictyosterium or, even if it is important, some other genes can be replaced
its function.
3. Cis-acting regions important for prespore-specific transcription of the Dp87
gene
Chimeric constructs composed by fusion of various parts of the 5' upstream
region to reporter gene were introduced
into Dictyosterium cells. Expression ofthese constructs suggested that at least
three parts of the upstream region affected positively and negatively to the efficient transcription of the Dp87 gene.

44
We speculated that the combinations
among these cis-acting regions may berequired for prespore-specific transcription of the Dp87 gene.
Publication List:

Kawata, T., Imada, T., Shiraishi, H.,
Okada, K., Shimura, Y., and Iwabuchi, M. (1992) A eDNA clone encoding HBP-1b homologue in Arabidopsis thaliana. Nucleic Acids Res. 20,
1141.
Kawata, T., Minami, M., Tamura, T.,
Sumita, K., and Iwabuchi, M. (1992)

NATIONAL INSTITUTE

Isolation and characterization of aeDNA clone encoding the TATA
box-binding protein (TFIID) from
wheat. Plant Mol. Bioi. 19, 867-872.
Tasaka, M., Hasegawa, M., Nakata, M.,
Orii, H., Ozaki, T., and Takeuchi, I.
(1992) Protein binding and DNase-1hypersensitive sites in the cis-acting
regulatory region of the spore-cart
SP96 gene of Dictyosterium. Mech.
Dev. 36, 105-115.

45

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Yoshiaki Suzuki

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

46

NATIONAL INSTITUTE

DNISION OF MOLECULARNEUROBIOLOGY
Professor: Masaharu Noda
Research Associates: Nobuaki Maeda
Shinji Hirano
Technical Staff: Sonoko Ohsawa
TomokoMori

This division is devoted to the study ofmolecular mechanisms that govern thedevelopment of the vertebrate central
nervous system. We are currently involved in projects to reveal the molecular bases of neuronal cell migration andneuronal network formation.
I. Molecular basis of neuronal cell migration
In the development of laminatedstructures, e.g. the cerebral cortex, of thehigher vertebrate brain, later generated
neurons must pass by the earlier generated ones so as to form an inside-out sequence in the radial dimension. This migration of post-mitotic neurons to theircorrect location within the developing
brain is a key event in the cellular differentiation that leads to the functional
neuronal network.
To elucidate the basic molecularmechanism of neuronal migrations, ourstudy is now focused on the characterization of T61 antigen. Monoclonal antibody T61 was originally isolated with its
inhibitory activity on neurite outgrowthfrom chick retinal explants in vitro. Recently, it turned out that T61 also inhibits the neuroblast migration, which results in aberrant laminar structure of the
optic tectum, when the T61 producing
hybridoma is injected into the ventricleof the developing midbrain of chick embryos. From immunoprecipitation analysis, an antigen molecule of 440 kD was
detected as a major component. T61
antigen is distributed broadly in thedeveloping brain, being concentrated on
the surface of neuronal cells (Figure).
Purification and molecular cloning of
T61 antigen are now in progress.
II. Molecular basis of neuronal network
formation
The development and maintenance ofdiverse neuronal systems in the vertebrate brain depend on various cell-celland cell-substratum interactions. Suchinteractions require varieties of genetic
and epigenetic information. Recently, it
has been recognized that the vertebrate
brain contains a diverse set of proteoglycans, which are supposed to play important roles in the cell-cell interactions in
the nervous system.
Proteoglycans are a group of molecules composed of glycosaminoglycan
chains covalently bound to a proteincore. Glycosaminoglycans are very complex polysaccharides classified into chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate. The structure of glycosaminoglycans is highly variable according to cell
types, the environment of cells, andstages of their differentiation. The coreproteins also show the structural diversity. We presume that this structural variability makes these molecules potent
carriers of the information that is defined
genetically and epigenetically, and accordingly, that proteoglycans can be a
key regulator in the brain development.
In order to examine such possibility,
we first prepared monoclonal antibodies
against brain proteoglycans. One of themonoclonal antibodies we isolated(MAb 6B4) recognized a 600 to 1,000
kD chondroitin sulfate proteoglycan witha core protein of 250 kD (6B4 proteoglycan).
Thisproteoglycan
bears

FOR BASIC BIOLOGY

47

Distribution of T61 antigen in the primary cell culture from ElO chick brain. (A) Phase-contrast photomicrograph. (B) Immunofluorescence of neuronal cells stained with T61 antibody. (B) is the same field as in(A). Note that glial cells are not stained. (C) Higher magnification of growth cones (arrow heads). T61
antigen is concentrated at the tips of growth cones.

chondroitin sulfate, keratan sulfate and
HNK-1 epitope.
The distribution of 6B4 proteoglycan
in the adult rat brain is highly unique. In
the hindbrain, 6B4 proteoglycan is expressed on the cerebellar Purkinje cells
and Golgi cells, and neuronal cell bodies
at particular nuclei, including the pontine nuclei and lateral reticular nucleus.
Almost all of these nuclei are connectedto the cerebellum through the mossy
fiber system.
The expression of 6B4 proteoglycan
shows characteristic features during theformation of the cerebellar mossy fiber
system. The mossy fibers from thepontine nuclei express 6B4 proteoglycantransiently from embryonic day 20 (E20)
to postnatal day 30 (P30), during which
period the axonal outgrowth andglomerular synapse formation occur. The
Purkinje cells, glomeruli, and Golgi cells
start to express 6B4 proteoglycan from
PlO, Pl6, and P20, respectively, in goodaccordance with the onset of their synapse formation. These results suggest thepossibility that 6B4 proteoglycan isclosely involved in the development of
the cerebellar mossy fiber system. Molecular cloning and analysis of the polysaccharide portion of 6B4 proteoglycan are
also in progress.

48

NATIONAL INSTITUTEDIVISION OF CELLULARREGULATION
Professor: Norio Murata
Associate Professor: Hidenori Hayashi
Research Associates: Takao Kondo
Ikuo Nishida
Hajime Wada
Monbusho Foreign Scientists:
Laszlo Vigh(from Biological Research Center Szeged, Hungary)Roland Douce(from University of Grenoble, France)
JSPS Post-doctoral Fellows:
Mahir D. Mamedov(from Moscow State University, Russia)
Dmitry A. Los(from Russian Academy of Sciences, Russia)
Visiting Scientists:
Hiroyuki Imai
Yasushi Tasaka
Hidemi MatsuzakiMari Iwabuchi(from Plantech Research Institute)
Michael P. Malakhov(from Russian Academy of Sciences, Russia)
George C. Papageorgiou(from National Research Center Demokritos,
Greece)Zoltan Gombos(from Biological Research Center Szeged, Hungary)
Ibolya Horvath(from Biological Research Center Szeged, Hungary)Marie-Helene Macherel(EC fellow, France)
Graduate Students:
Toshio Sakamoto
Katsuzo Noguchi
Patcharaporn Deshnium
Yoshitaka Nishiyama (from The University ofTokyo)Norihiro Satoh (from The University of Tokyo)
Technical Staff: Shoichi Higashi
Miki Ida

The research effort of this division is
directed toward the understanding of
tolerance and adaptation of higher plants
and microbial plants to temperature extremes and high-salt stress, with particular emphasis on the molecular mechanisms by which plants acclimate or tolerate these conditions.

I. Genetic manipulation of fatty-aciddesaturation in cyanobacteria
Higher plants, and most cyanobacterial strains, contain high levels of polyunsaturated fatty acids. While polyunsaturated fatty acids are thought to beimportant to the function of biologicalmembranes, their roles are not fully
understood. A fuller understanding ofthese roles will develop as we become
more skilled at modifying the extent of
unsaturation of the fatty acids of membrane lipids. This requires the genetic
manipulation of the genes responsible
for the desaturation of fatty acids.
We have isolated the desA gene of the
Synechocystis
cyanobacterium,
PCC6803. This gene is responsible forthe desaturation of fatty acids at the ~12 position. We constructed a disrupted
desA gene, which was interrupted by the
kanamycin-resistance gene cartridge, inorder to manipulate the extent of desaturation of fatty acids in the membrane lipids of Synechocystis PCC6803. The
wild type contained mono-, di-, andtriunsaturated lipids. The mutant, which
we designated Fad6, contained monoand diunsaturated lipids; and a transformant of Fad6, with a disrupted gene forthe desaturation which we designatedFad6/ desA::Kmr, contained only monounsaturated lipids (Fig. 1).
We used mutants and transformants ofSynechocystis PCC6803 to examine theeffects of the unsaturation of fatty acids
in membrane lipids on the low-temperature photoinhibition of photosynthesis.
The strain most susceptible to low-temperature photoinhibition was Fad6/
desA::Kmr, whereas Fad6 and the wild
type showed the same level of sensitivity
to photoinhibition. This suggests thatdiunsaturated fatty acids play an important role in protection against low-49

FOR BASIC BIOLOGY

(A)

(B)

(C)

(D)

18:0/16:0

18:1/16:0

18:2/16:0

18:3/16:0

Fig. 1. The molecular structure of membrane lipids in the cyanobacterium Synechocystis PCC6803 grown
at 34•c. Arrows indicate the pathway of biosynthesis. As major molecular species wild type contains (B), (C)and (D); Fad6 mutant contains (B) and (C) ; Fad6 / desA: :Km' double mutant contains only (B). By comparing these strains, it is possible to study the biological functions of lipid unsaturation.

temperature photoinhibition. The unsaturation of fatty acids also affected photoinhibition at room temperature, althoughmuch less than at low temperature. By
contrast, photosynthetic electron transport, measured at various temperatures,
was not affected by changes in the extent
of unsaturation of the fatty acids.
II. Temperature-induced desaturationof fatty acids is regulated at the levelof gene expression
Living organisms can maintain themolecular motion, or "fluidity", of membrane lipids by regulating the level of unsaturation in fatty acids. For example,
cyanobacterial cells respond to a decreasein temperature by introducing double
bonds into the fatty acids of membrane lipids, thus compensating the temperatureinduced decrease in the molecular motionof membrane lipids. Desaturases are responsible for the introduction of these specific double bonds.
To study the mechanism of low-temperature induced desaturation of membrane lipids in greater detail, we exam-

ined the effect of low temperature on theexpression of the desA gene from Synechocystis PCC6803. The level of desA
transcript increased 10-fold within 1
hour as temperature decreased from
360C to 22°C. This suggests that the lowtemperature-induced desaturation of
membrane lipid fatty acids is regulated
at the level of the expression of the desaturase genes. The accumulation of thedesA transcript depends on the extent of
temperature change exceeding a threshold level, and not on the absolute temperature.
III. Biochemical and molecular characteristics of enzymes involved in lipid
synthesis
cDNAs and isomeric forms of
enzymes involved in lipid synthesis wereisolated and characterized in order tounderstand the molecular mechanisms oflipid synthesis in higher plants.
Stearoyl-(acyl-carrier-protein) desaturase (SAD) introduces the first doublebond into C18 fatty acids in higher
plants. We isolated a eDNA for this50
enzyme from spinach cotyledons. The
open-reading frame encodes a polypeptide of 399 amino acid residues. It is likelythat a transit peptide, composed of 35
amino acids, is cleaved to produce a mature protein, composed of 364 aminoacids. The deduced amino acid sequenceof the precursor is highly homologous tothose of precursors to SAD from castor
bean, cucumber, and safflower.
The gene (designated ATSJ) and theRNA for the plastid-located glycerol-3phosphate acyltransferase were isolated
from Arabidopsis thaliana. The nucleotide sequences of the gene and theeDNA were determined, and the 5' endof the RNA was mapped by primer extension. The sequences similar to the
TATA box, and the polyadenylation andintron-splicing sequences were found at
the expected locations. The amino acidsequence deduced from the open-reading frame was highly homologous tothose of precursors to plastid-locatedglycerol-3-phosphate
acyltransferase
from squash and pea.
Acyl-(acyl-carrier-protein) hydrolase
releases fatty acids from the end productof fatty acid synthesis in plastids for thesubsequent synthesis of glycerolipids inthe cytoplasm. Isoelectric focusing ofchloroplast stroma proteins from squash
cotyledons suggested that there were atleast three isomeric forms of acyl-(acylcarrier-protein) hydrolase. The pi 4.5
form was isolated after a 100,000-foldpurification from squash cotyledons. The
enzyme is specific for long-chain-acyl(acyl-carrier-protein).
IV. Genetically-engineered alterationof fatty-acid unsaturation modifies thechilling sensitivity of higher plants
The chilling sensitivity of higher plants

NATIONAL INSTITUTE

is correlated with the extent of fatty-acidunsaturation of phosphatidylglycerol inchloroplast membranes. We have suggested that the chloroplastic enzyme,
glycerol-3-phosphate acyltransferase, isan important factor in determining thelevel of unsaturation of the fatty acids ofphosphatidylglycerol.
In order to evaluate the role of glycerol-3-phosphate acyltransferase in the
low-temperature sensitivity of higher
plants, tobacco plants were transformed
with Ti-plasmids. These plasmids wereconstructed with cDNAs encoding the
precursor protein of this enzyme, whichwere isolated from a low-temperaturesensitive plant, squash, and from a lowtemperature-resistant plant, Arabidopsis.
The introduction of squash eDNA decreased the content of unsaturated fatty
acids in the phosphatidylglycerol, while
the introduction of the Arabidopsis
eDNA caused a small but significant increase in these fatty acids.
The chilling sensitivity of transformed
plants was assayed by the inactivation ofphotosynthesis in excised leaves. The
wild-type tobacco plants showed somesensitivity to chilling, but this sensitivity
increased markedly with the introductionof the eDNA for squash glycerol-3phosphate acyltransferase. In contrast,
the introduction of the eDNA for the
Arabidopsis enzyme decreased its chilling sensitivity. These remarkable differences correlated well with the extent
of fatty-acid unsaturation of phosphatidylglycerol. These results demonstratethat it is possible to regulate the chilling
sensitivity of higher plants solely by agenetically-engineered alterationoffatty-acid unsaturation of phosphatidylglycerol, with the introduction of an
appropriate acyltransferase.

FOR BASIC BIOLOGY

V. Heat shock proteins of cyanobacteria
The two most abundant HSPs, HSP70
and HSP64, were recognized in Synechocystis PCC6803 by antibodies raised
against authentic DnaK and GroEL
from Escherichia coli. HSP64 from
Synechocystis PCC6803 was purified tohomogeneity. Two open-reading frames
corresponding to groEL and groES
genes were found in the genome of
Synechocystis PCC6803. Southern blot
analysis indicates that only one groESL
operon is present in the genomic DNA
of Synechocystis PCC6803, whereas theexistence of at least two copies ofgroEL-analogous genes was anticipated.
The level of the transcript of groESL increased 100-fold within 15 min uponheat stress.
VI. Glycinebetaine
stabilizes
theoxygen-evolving complex of higher
plants and cyanobacteria
The photosynthetic oxygen-evolving
activity of the photosystem 2 complex,
prepared from spinach, was labile when
the complex was exposed to high-saltconditions under which the extrinsic proteins were dissociated from the complex.
Glycinebetaine, which is accumulated in
the chloroplast of halophilic higher
plants, prevented the dissociation of the
18-kDa and the 23-kDa extrinsic proteins from the photosystem 2 complex in
the presence of 1 M NaCI. It also
prevented the dissociation of the 33-kDa
extrinsic protein from the complex in thepresence of 1 M MgC1 2 or 1 M CaC12 •We examined the heat stability of various electron-transport and phosphorylation reactions of photosynthesis in thylakoid membranes isolated from Synechocystis PCC6803. The order of sensitivity
to heat was as follows; evolution of51
oxygen> Photosystem 2 = synthesis of
ATP > cytochrome b/f> Photosystem
1. While glycinebetaine was effective inprotecting the evolution of oxygenagainst heat, it did not protect any of theother reactions.
Publication List:
Original papers
Z. Gombos, H. Wada, and N. Murata(1992) Unsaturation of fatty acids inmembrane lipids enhances the tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature
photoinhibition. Proc. Nat/. Acad. Sci.
U.S.A. 89, 9959-9963.
H. Imai, I. Nishida, and N. Murata(1992) Acyl-(acyl-carrier-protein) hydrolase from squash cotyledons specific to long-chain fatty acids: Purification and characterization. Plant Mol.
Bioi. 20, 199-206.
C.H. Johnson (Vanderbilt Univ.) and T.
Kondo (1992) Light induced singularities and period changes of the circadian phototaxis rhythm in the
CW15 strain of Chlamydomonas.
Bioi. Rhythms 7, 313-327.
C. Lehel, H. Wada, E. Kovacs, Z. Torok(Biological Research Center, Szeged),
Z. Gombos, I. Horvath, N. Murata,and L. Vigh (1992) Heat shock protein synthesis of the cyanobacterium
Synechocystis PCC6803: Purificationof the GroEL-related chaperonin.
Plant Mol. Bioi. 18, 327-336.
N. Morita (Hokkaido Univ.), M. Gotoh(Hokkaido Univ.), N. Okajima(Hokkaido Univ.), H. Okuyama(Hokkaido Univ.), H. Hayashi (Higashi Nippon Gakuen Univ.), S. Higashi,
and N. Murata (1992) Both the anaerobic pathway and aerobic desaturation are involved in the synthesis of

52
unsaturated fatty acids m Vibrio sp.
strain ABE-l. FEBS Lett. 297,9-12.
N. Murata, P.S. Mohanty, H. Hayashi,
and G.C. Papageorgiou (1992) Glycinebetaine stabilizes the association of
extrinsic proteins with the photosynthetic
oxygen-evolving
complex.
FEBS Lett. 296, 187-189.
N. Murata, 0. Ishizaki-Nishizawa, Y.
Tasaka, H. Hayashi, S. Higashi, and I.
Nishida (1992) Genetically engineered
alteration in chilling sensitivity ofplants. Nature 356, 710-713.
N. Murata, H. Wada, and Z. Gombos(1992) Modes of fatty acid desaturation in cyanobacteria. Plant Cell
Physiol. 33, 933-941.
I. Nishida, T. Beppu (The Nishi-Tokyo
Univ.), T. Matsuo (Kagoshima Univ.),
and N. Murata (1992) Nucleotide sequence of a eDNA clone encoding aprecursor of stearoyl-acyl carrier protein desaturase from spinach, Spinacia
oleracea. Plant Mol. Bioi. 19,
711-713.
T. Slabas (Durham Univ.), D. Chase

NATIONAL INSTITUTE(Durham Univ.), I. Nishida, N. Murata, C. Sidebottom (Unilever Research,
Bedford),R.
Saffoard(Unilever Reserarch, Bedford), P.S.
Sheldon (Birmingham Univ.), R.G.O.
Kekwick (Birmingham Univ.), D.G.
Hardie (Birmingham Univ.), and R.W.
Mackintosh (Dundee Univ.) (1992)
Molecular cloning of plant /3-ketoacylacyl carrier protein reductase. Homology with nodG gene product of Rhizobium meliloti. Biochem. J. 283,
321-326.
H. Wada, Z. Gombos, T. Sakamoto, andN. Murata (1992) Genetic manipulation of the extent of desaturation of
fatty acids in membrane lipids in the
cyanobacterium
Synechocystis
PCC6803. Plant Cell Physiol. 33,
535-540.
Books
N. Murata, Ed. (1992) Research in
Photosynthesis Vol. I-IV, Kluwer
Academic Publishers, The Netherlands.

FOR BASIC BIOLOGY

53

Efficient energy transformation in the
primary process of photosynthesis is ensured by the highly ordered organizationof molecules in the photochemical reaction centers, in a physical, chemical andbiological sense. The project started
from September 1992, in this newly established division, is aiming to elucidate
organization of the reaction center ofoxygenic photosynthesis, the photosystem II reaction center, in two aspects. In
the first approach, the molecular structure of the reaction center will be analyzed by various methods in order toprovide a solid basis for the second approach. In the second approach, the effort will be focused on elucidation of themolecular mechanism of light-regulated
metabolic turnover of a subunit of thereaction center, the D1 protein. The process involves apparently unique, but inessence very general problems in molecular and cell biology; i.e., stability andphotodegradation of proteins in vivo,
light-regulation of gene expression at thetranslational level, post-translationalcleavage of the C-terminal extension ofproteins and incorporation of cofactors
and subunits into multi-component pigment-protein complexes.

mung bean and expression of the geneduring growth of seedlings. Plant Cell
Physiol. 33, 245-252.
Arai, M., Mori, H., and Imaseki, H.
(1992) Expression of the gene for sucrose synthase during growth of mungbean seedlings. Plant Cell Physiol. 33,
503-506.
Kanayama, Y., Mori, H., Imaseki, H.,
and Yamaki, S. (1992) Plant gene register: Nucleotide sequence of a eDNA
encoding NADP-sorbitol-6-phosphate
dehydrogenase from apple. Plant
Physiol. 100, 1607-1608.
Kandori, H., Yoshihara, K., and Tokutomi, S. (1992) Primary process ofphytochrome: Initial step of photomorphogenesis in green plants. J.
Amer.
Chem.
Soc.
114,
10958-10959.
Tokutomi, S., Sugimoto, T., andMimuro, M. (1992) A model for themolecular structure and orientation of
the chromophore in a dimeric phytochrome
molecule.
Photochem.
Photobiol. 56, 545-552.
Yamammoto, K.T., Mori, H., and Imaseki, H. (1992) Novel mRNA sequence induced by indole-3-acetic
acid in sections of elongating hypocotyls of mung bean (Vigna radiata).
Plant Cell Physiol. 33, 13-20.
Yamammoto, K.T., Mori, H., and Imaseki, H. (1992) eDNA cloning ofindol-3-acetic acid-regulated genes:
Aux22 and SAUR from mung bean
(Vigna radiata) hypocotyl tissue. Plant
Cell Physiol. 33, 93-97.

Publication List:(related to previous projects)
Arai, M., Mori, H., and Imaseki, H.
(1992) Cloning and sequence of
cDNAs for an intracellular acid invertase from etiolated hypocotyls of(Suppliments)
Nakasako, M., Wada, M., Tokutomi, S.,
Yamamoto, K.T., Sakai, J., Kataoka,
M., Tokunaga, F., and Furuya, M.
(1990) Quarternary structure of pea
phytochrome I dimer studied with

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY(ADJUNCT)
Professor: Kimiyuki Satoh
Associate Professor: Hirokazu Kobayashi
Research Associate: Satoru Tokutomi
Post-doctoral Fellow: Noritoshi Inagaki
Graduate Student: Tatsuya Torno •(*from Okayama University)

54

small-angle X-ray scattering and rotary-shadowing electron microscopy.
Photochem. Photobiol. 52, 3-12
Tokutomi, S., Mizutani. Y., Anni, H.,
and Kitagawa, T. (1990) ResonanceRaman spectra of large pea phytochrome at ambient temperture: Difference in chromophore protonation
between red- and far red-absorbing

NATIONAL INSTITUTE

forms. FEBS Lett. 269, 341-344.
Mizutani, Y., Tokutomi, S., Aoyagi, K.,
Horitsu, K., and Kitagawa, T. (1991)
Resonance Raman study of· intact pea
phytochrome and its model compounds: Evidence for proton migration during the phototransformation.
Biochemistry 30, 10693-10700.

FOR BASIC BIOLOGYDNISION OF BEHAVIOR AND
NEUROBIOLOGY(ADJUNCT)
Professor: Masatoshi Takeichi
Research Associate: to be appointed

How individual nerve cells find andrecognize their targets during development is one of the central issues in modern biology. The aim of our division is toelucidate the molecular mechanism ofaxon guidance and target recognition byusing the simple and highly accessible
neuro-muscular system of Drosophila.
The musculature of Drosophila embryos consists of 30 identifiable muscle
fibers per hemisegment. Each muscle
fiber is innervated by a few motoneurons
in a highly stereotypic manner. The highdegree of precision and previous cellular
manipulations of neuro-muscular connectivity suggest the presence of recognition molecules on the surface of specificmuscle fibers which guide the growthcones of motoneurons.
By using an enhancer trap method,
several genes have been identified thatare expressed in small subsets of muscle
fibers prior to innervation, and are thus
good candidates for such recognition
molecules. Two of them, connectin andToll, were shown to encode cell recognition molecules which belong to the leucine-rich repeat (LRR) family. In paticu-

55
lar, connectin is expressed not only on asubset of muscle fibers but also on theaxons and growth cones of the very motoneurons which innervate these muscles, strongly suggesting its involvementin the neuro-muscular recognition. We
are currently conducting the following
research projects.
1) Molecular genetic analysis of the roleof connectin and Toll. We are analysing
the mutant animals which lack connectinor Toll to see if there are any defects inthe neuro-muscular connectivity. We arealso generating transgenic lines which ectopically express connectin on muscle
fibers that normally would not express
the molecule.
2) Search for novel connectins. An interesting possibily is that connectins constitute a LRR subfamily which are expressed on different subsets of motoneurons and their target muscles. We aretrying to isolate novel connectins byusing PCR and will study theii- expression pattern.
3) Identification of more genes implicated in neuro-muscular recognition. We
are conducting molecular and genetic
analysis of more enhancer trap lines thatare expressed in specific subsets of muscles and/ or motoneurons to examine
their possible roles in neuro-muscularconnectivity.

57

FOR BASIC BIOLOGYLABOLATORY OF GENE EXPRESSION
AND REGULATION

Head: Yoshihiko Fujita

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

58

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION
AND REGULATION I
Professor: Y oshiro Shimura
Associate Professor: Kiyotaka Okada
Research Associate: Hideaki Shiraishi
Post-doctoral Fellows: Nobuyoshi Mochizuki
Kazuhito A kama
Graduate Students: Koji Sakamotf!
Azusa Yano
Yoichi Ono
Tashiro Ito (from Kyoto
University)
Technical Staff" Hideko Nonaka
AkikoKawai

The principal interest of this laboratory is molecular genetic studies on theregulatory systems of organ development
and on growth control by several environmental stimuli in higher plants. Our
efforts have been focused on flower development and morphogenesis and also
on root responses toward gravity, lightor touching stimuli. For these studies, wehave mainly used a small crusifer, Arabidopsis thaliana. This plant is called "botanical Drosophila", because it has some
remarkable features, such as a small
genome size (1 X 108 base pairs per haploid), short life-cycle (5-6 weeks),
small size (20-30 em 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 markers are mapped on
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 stimulus-response interactions in root.
Mutants with abnormal floral morphology could be divided into the following
types on the basis of the stages of floral
development where the genetic defects

were presumed to occur; namely, stage
1: transition from vegetative to reproductive growth (mutants with delayedtransition or earlier transition), stage 2:
elongation of inflorescence axis (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 floral organs 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. (1988) Development, 104,
195-203; Okada et al. (1989) Cell Differ. Dev., 28, 27-38).
One of the homeotic genes, A GAM 0 US, 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 MCM1 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 et al. (1993)
Plant J., in press). Attempts were also
made to isolate cDNAs which are specifically expressed in floral organs. Two
eDNA clones which are specifically expressed in stamen and pistil were59

FOR BASIC BIOLOGY

8-- f•·· ·--1-------····--,,_· - -,,_,,_,-··-· · -··· -- ,··---·-__ ,,,,-tllt-~tl__ ,,__ ,,____,_,_,-- __,,,,, __,, __ ,,,~ ~------·_____ ,_,· --___ ,···__ -________ ,,- ____-utt--tt -- n----••-• ---•n-- -••-----ttf\-- t--n---•-·--•---•t-H•tt-t--n-•--_ , , , , __ ,, _ _ ,, ___ , __ ,_ ,,,, , __ ___ , , _____ ,,, __ __ , ,_,_,, _ , ___ , ,_ ,,_,, _, __ f\ __ , __--····--··--··---·---·----··----·--··-----·-··---·---·-·-··-··-··-·--··-·---ttlf--u--n----u---•--nt---•n• --u•---n-----ut-t•n-n-n-t--t•-•--_,,,, __ ,, __ ,. ___ ,,, ___,_, ___ ,,_,,, ____ ,,,, ___,,_,_,__,__,,_,,_,,_. __ ,,_, __--····--··--··----·--·-·--··----·-··---t----·-------·---·-··-··-··-·--··-·-______-un - -u -- n --- •--un --~-- • - •·-••------•• --- - -- •----•• -•• - n·H -t-- n--t--_,,,, __ ,, __,, ___ ,, ___ ,,, _____ ,__ ,__ ,,, ____ ,__ ,_, __ ,_____,,_,,_,,_, __ ,,_,__-ulf-•tt--ll---••---••-•--•-•-•••-•-·--•u------•----n-n-n-••-•--n---•-··••••--••--n---•-••---••-----•-••------•••------•------••-••-••-•--••-•-_,,,,__,, __ ,, ____ ,, ______ ,,,,, ___ ,, __ ,_,_, __ ,__,_,,_,,_,,_,,_,__ ,,_, __-nlf--tt--ll-·--nt-t--t-l\1\----•_,,,,__,,__
__ _
,,,,,
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,,,,___, __, ,__,,•----•--•--•-•n•-•-n-ll-ll-t--l\-t-_____ ,,,,,______,,,,_,,,,,.,,_,,_,,_,____
n-t-_,,,,_,,_,.
_,___,__,___,__,_,,,,__,,_,,_,,_,__ ,,_,-ulf-n--n---t-ttn---•-•-•-•--u-•-••---n-tn-n-n-n-•--••-•--tttt-•tt--n----••t-•-_,,,, __ ,, __ ,, ••----•-•---••·-ttt-----••---•·-tt•tt-f\•tt-t--t___ ,,,_,,,,, ___ ,,_, ____ ,, ________ ,,, __
_,,,,
,, __
,,_,,,,, _____ ,,,,_,,, ____,,,,__ ,,____,,,,______,_,_,_, ,, ___________ ,,,__ ,, .. _,___,,,,_____,,_,,_,,_,,_,__ ,,_, __-t\tt-•tt--n---ntt-t•t·-•-••-•-•--••------••---t-t \tt•tt-tt•tt·t·-••-•-••-••-••-•--•t-t-_,,,, __-tut-tt--t•---•--•-•-•---•----•••------•••----tt·,,,,,,__ __,, ___,___ ,,, __,, _____ ,, ___ ,_ ,, _____ ,__ ,_,_,,_,,_,,_,__ ,,_, __,, _______,, ___ ,,__ ,_, ___,,,_,_,_,,,,,_______ ,,_,,, ____,,_,,_,,_, __ ,, ,__,,,,_,, __ ,, ___ ,,,,,__ ,, ,__,,,, __,,,,_____,,,,,,, ______,___,__ ,__ ,, ______,_,,,,,_,,_,,_,___ ,,,,,____ ,, ______ ,,_,,_.,_,__ ,,_, __

_

_

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,,_,,~n - t- - ••-•--

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..

-ttu- -u --fl- --•-• -- •• -•---n--- ntt--·t-tt---u-t-t -n-tt-n - •- - n - • --_,,,,n--••·••-•--•t-t-_,,,,__ __,,__,, __ ___,___•••••-••••---••·--•-•-•---•tttt-tt·,,,, ____ ,, __ ,__ ,, ___,,_,__,__ ,____ _,,_,,_,__ ,,_ ,___,,,, ,, ,, ___ ,____,,, ______ , ,_,_, ,,,, ,_,,_,,_,,_,,_, ,,_, __,,,,_,,_ ___,,,,__ ,_,,,,,_,_, ___ ,__ ,_,,, _____ ,, ___,_,,_,,_,,_, __ ,, __,_,,,,__,, __ ,, ___ ,_,_, __ ,____ ,,, _____ ,, ____ ,_, ___ ,__,,_,_,,_"_,,_, __ ,,_, __,,,,_,, __ ,, ___ ,,, __,,_,,_,_,_,, __ ,, _____,, ______ ,__ ,,_,,_,,_,,_, __ ,,_, __\\-tnt---n-tt-n-t--n-t-_,,,, __ ,, __ -tut--tt--n---•--n----n••-----tu---t-,, ___,_,,_,,,__,______ ,,_, ___ ,,, ___ ,, _________ ,,_,,_,,_,__,,_, __,,,,__,,__ ,, ___,_, __,,,_,___ ,, ____ ,__ ,______ ,,_,,_,,_,__ ,, __ ,__,,,, _,___,,,_,___ ,,____ ,,, ___,____ ,, ______,_, __,, _____ ,,,,,_,_,,_,,_,,_, __,,_, __,,,,__,, __ ____,, __,, ____ ,_,, __ ,, ____,,, _____,, ___ ,_,_,,_,,_,,_,,_, __ ,, __ ,__,,,,__ ,, __ ,,____ ,,,_,,_,__ ,,,,, __ ,,_,,, _____ ,, ___ ,__ ,,,_,,_,,_.,_, __ ,, ___ ,__

_ .._
_
_ _
__ .
-••••--u--n----•-----•-----tllt--n--t t-ttt•\\-\\•\•••1\-tt•••-•--n-t-- utt--u -- u---ntt•-t__ __ - t------t - \\·--- t- u ---t·Ut•Ut-n____ -__n-n-t--n-•--__-ntt--n--n---•--·-•••-----••---•---•u---•-•-n••--·n-n-n-•--n-•--····--··--··----··----·--··-----····------·-·--·--·-1\ll•t\-t\•f\·f·-··-·--

Fig. 1. (A) Flower of the AGAMOUS mutant. (B) Alignment of the nucleotide sequences of the oligonucleotides which are recognized by the AGAMOUS protein. DNA molecules recognized by the AGAMOUS
protein were selected from a mixture of synthetic oligonucleotides containing random sequences in the central
region. Nucleotide sequences of the selected oligonucleotides are shown with the following characters. A , - ;T, =; G, # ; C, %. A sequence motif which is recognized by the protein is visible in the central region of eacholigonucleotide.

isolated, and their expression patterns
were analyzed by in situ hybridization(Ito eta/., in preparation).
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). Using a newly devised system
which provides a constant obstacletouching stimulus to root tips on agar
plate, mutants which show abnormal responses to obstacle-touching stimulus
were isolated (Okada & Shimura (1990)
Science, 250, 274-276). Gravitropic
and phototropic responses were also
analyzed using agar plates. Young seedlings grown on vertical agar plates haveroots which grow straight downward on
the agar surface. When the plates wereput aside, roots bend 90 degrees andgrow to the new direction of gravity. If
the plates were covered with black clothand illuminated from the side, roots
grow to the opposite direction of incoming light. Using these systems, mutants
which show abnormal graviresponse or
photoresponse were isolated (Okada &Shimura (1992) Aust. J. Plant Physiol. ,19, 439-448). Several mutants with abnormal obstacle-escaping response also
show abnormal gravitropism and/ or
phototropism. These results indicate thatroot gravitropic, phototropic and obstacle-escaping responses share at leastin part a common genetic regulatory
mechanism.
Polypeptides induced by gravitropic
stimuli were analyzed using the 2-dimensional gel electrophoresis. Amounts of atleast 11 spots were shown to be increased by the stimuli (Sakamoto et a!.
(1993)
Plant Cell Physiol., 34,

60
297-304.).
Attempts are also being made to
identify and isolate the genes responsible
for the mutants using the transformation
systems mediated by the Ti-plasmid vectors or by a direct gene-tagging system.
For such experiments, it is absolutely
necessary to develop a good, efficientsystem of transformation and transgenic
plant regeneration. We have tested several combinations of A. thaliana ecotypes and Agrobacteria strains and established an efficient system (Akama eta/. (1992) Plant Cell Rep., 12, 7-11).
Publication List:
Okada, K., and Shimura, Y. (1992)

NATIONAL INSTITUTE

Mutational analysis of root gravitropism and phototropism of Arabidopsis
thaliana seedlings. Aust. J. Plant
Physiol., 19, 439-448.
Okada, K., and Shimura, Y. (1992) Aspects of recent developments in mutational studies of plant signalling pathways. Cell, 70, 369-372.
Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K., and Shimura,Y. (1992) Efficient transformation of
Arabidopsis thaliana: comparison of
the efficiencies with various organs,plant ecotypes and Agrobacterium
strains. Plant Cell Rep., 12, 7-11.

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

We are examining mechanisms related
to chromosomal replication, recombination and repair. Homologous recombination, replication, especially initiation andtermination processes, and coupled
events have been given special attention.
I. DNA replication terminus ( Ter) sitein E. coli is an entrance for recombination enzyme (RecBCD)
From the E. coli chromosome, we isolated EcoRI DNA fragments with higher
homologous recombinational hotspot(Hot) activity, as follows: whole E. coli
chromosomal DNA was digested with
EcoRI enzyme, ligated with the 7 kb
Kmr fragment and introduced into the
RNase H-defective (rnh-) host, then anumber of Kmr transformants were collected. From these transformants, plasmid-like ccc (covalently closed circular)
DNA was extracted by the alkaline miniprep method. Some clones contained theplasmid-like ccc DNA in higher amounts
than in other clones. These DNA fragments were classified into 8 groups,
termed HotA to H DNA. The Hot DNA
was formed through excision from thehost chromosome into which Hot DNA
was once integrated rather than throughautonomous replication, because Hot
DNA was unable to be transformed into
a mutant in which the Hot corresponding region on the chromosome wasdeleted. Thus, the amount of ccc Hot
DNA recovered from the transformant
indicates the recombinational Hot activity. We, therefore, called this "Hotassay". Hot DNA shared the following

61
properties; (1) Hot activity was extraordinarily enhanced in the rnh- strain, (2)all Hot DNA, so far tested has Chi activity. As shown in Fig. 1, seven groups
were clustered into a narrow DNA replication terminus region (about 280 kb).
In particular, two Hot activities, HotA
and C, were located on EcoRI fragments
just next to the two EcoRI fragments
carrying the DNA terminus sites, TerB
and C, respectively. To examine the relationship between Hot and Ter activity,
we carried out a Hot assay using the
Ter-binding protein less (tau-) mutant.
The Hot activities of A, B and C groups
disappeared when the tau- strain wasused as a host, hence the Hot activities
are termination event-dependent. In addition, at least HotA activity proved torequire a Chi sequence present on the
HotA fragment, because mutational destruction of the Chi sequence resulted indisappearance of the Hot activity. Fromthese results, locations of the Hot andthe Chi sites and their orientations,
we proposed the following models:
RecBCD, a Chi responsible enzyme, has
good access to the DNA replication fork
blocked at the Ter site, the molecule ofwhich is known to be accumulated extensively under rnh- conditions. It enters
duplex DNA, travels on a newly synthesized (branched) portion of duplex
DNA and triggers recombination of the
nearby region when it meets a Chi sequence. Other Hot DNA groups, Hot
activity of which is termination event-independent, might be activated by otherevents induced under rnh- conditions. In
rnh- cells, the two most active replication origins are located at the terminus
region. Thus, termination independent
Hot activities might require the initiation
event occurring under rnh- conditions.
In the T4 phage, there are hotspots

62

NATIONAL INSTITUTE

HotH
metB

0/100

recA

~

@
HolE
HotA
Q) .. _an u li:i@ifu¥84 §f Mi +TerB
dif

HotG HolD

@'

HotC

t

ii&W

HotF

wei •
Terc

HotB

Ill

Ill *

TerA

Figure 1. Location of Hot DNA fragments of the £ . coli chromosome.
The upper part of the Figure shows locations of Hot DNA fragments on the £ . coli circular map and lower
one represents the three expanded £caRl restriction maps, each of which contains a Ter site indicated by ( ~)and on which 7 kinds of Hot DNA are clustered. Relevant markers are also shown.

closely linked to replication origins. Furthermore, HOTJ, a recombination hotspot in yeast, might express activity
through a mechanism similar to that forE. coli Hot DNA, as described below.

II. Molecular analysis of HOTI, a recombinational hotspot in Saccharomyces cerevisiae
H 0 T1 is an active recombinational

hotspot present at the transcriptional enhancer-initiator region of repeatedrRNA genes in S. cerevisiae. Data obtained from analysis of the HOTJ phenomenon by other workers suggest thatseveral properties of the HOTJ are similar to those of the E. coli Hot-Chi system described above. (1) Both systems
enhance homologous recombination (seeFig. 2), (2) both require two distinct

FOR BASIC BIOLOGY

63

Figure 2. Colony color sectoring assay for yeast HOTI (developed by Lin and Keil, 1991).
Left; colonies of the strain containing no HOTJ element produce very few white sectors. Right; colonies ofthe strain carrying the HOTJ elements, E and I, produce numerous white sectors. The elements enhance excisive recombination between repeated leu2 genes, a AD£5, 7 gene located between which is eliminated frequently and white sectors are produced. Using this assay we are examining whether the sog site (E element)in yeast is functionally exchangeable with the E. coli analogous site, Ter.

elements for activities; In Yeast, E (enhancer) and I (initiator), in E. coli, Ter(terminus site) and Chi are essential. (3)One of the two elements in both cases,
has DNA replication fork blocking activity. In E. coli, the Ter site has this activity. Similarly, in yeast, Fangman's andour groups found that the E element has
fork blocking (we named "sog") activity.
Thus, the replication fork stopped at theE element to which yeast recombination
enzyme(s) such as RecBCD enzyme
might access and stimulate recombination. To test this hypothesis, the following experiments are ongoing. (1) In
crude extract of yeast, we are searchingfor protein(s) that specifically bind to thesog (E element) site, using gel retardation assay. (2) We are investigating
whether the sog site (E element) in yeast
is functionally exchangeable with the E.
coli analogous site "Ter" (Fig. 2).
III. DNA fragments that delay DNA
replication fork movement on the plasmid carrying them
During the course of experiments to

identify DNA fragment carrying terminus ( Ter) activity, we found two DNA
plasmid clones, on which progress of the
DNA replication fork is significantly anduniformly delayed, but not stopped at adefinite site such as Ter. In prokaryotes,
the rate of replication fork is rapid(about 1 kb/sec). Thus, the replicationintermediate of a plasmid in E. coli cells,
which appears as an arc in this 2D gel,cannot be recovered to any great extent.
Southern hybridization using radiolabeled DNA as a probe is essential for
detection. However, in the case of thisparticular plasmid, because of the delay
in replication, the intermediate molecules greatly accumulated and EtBr
staining facilitated visibility.
Both DNA fragments were derived
from the terminus region of the E. coli
chromosome and had activities that delayed replication. They shared the following properties.
(1) The activity has polarity. Rate of
DNA replication in a clone was delayed,while that of an orientation isomericclone (with the same fragment but in an

64
opposite orientation) was normal. (2)Cells carrying either plasmid were elongated. We are examining mechanisms
through which the two DNA fragments
delay replication of the plasmid carrying
them.
Publication List:
Hidaka, M., Kobayashi, T., Ishimi, Y.,
Seki, M., Enomoto, T., AbdelMonem, M., and Horiuchi, T. (1992)
Termination complex in Escherichia

NATIONAL INSTITUTE

coli inhibits SV40 DNA replication invitro by impeding the action of T
antigen helicase. J. Bioi. Chern., 267,
5361-5365.
Kobayashi, T., Hidaka, M., Nishizawa,
M. and Horiuchi, T. (1992) Identification of a site required for DNA replication fork blocking activity in therRNA gene cluster in Saccharomyces
cerevisiae. Mol. Gen. Genet. 233,
355-362.

FOR BASIC BIOLOGYDIVISION OF SPECIATIONMECHANISMS
Professor: To be appointed

65

66

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head: Hiroyuki Hattori
Common Facility Group

Research Support Group

Research Support Facilities
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
TomokiMiwa
Mariko Saitoh
Kimiko Yamamiya
Takeshi Mizutani

Cell Biology Group
Akio Murakami (Unit Chief)
Masayo /waki
MakiKondo
Katsushi Yamaguchi

Radioisotope Facility
Kazuhiko Furukawa( Unit Chief)
Yoshimi Matsuda
Nobuya Sugimoto
Center for Analytical Instruments
Hisashi Kojima
Yukiko Kabeya
Yumiko Makino
Glassware Washing Facility(Kazuhiko Furukawa)(Toshiki Ohkawa)

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

Developmental Biology Group
Hiroko Kajiura (Unit Chief)
Miyuki Ohkubo
Sachiko Fukada
Chikako Inoue
Tomoyo Takagi
Hisae UraiRegulation Biology Group
Shoichi Higashi
Sonoko Ohsawa
Miki Ida
TomokoMori
Gene Expression and Regulation Group
Yoko Fujimura
Hideko Nonaka
Koji Hayashi
AkikoKawaiand the other, the Research Support
Group, which assists the research activities as described in individual reports.
Technical staffs participate, throughthe department, in mutual enlightenment
and education to increase their capability
in technicalarea. Each technical staff isproceeded to the fixed division usuallyand they support the various researchwith their special biological, biochemicaland biophysical techniques.

67

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

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

tropical sunlight at noon (Watanabe etal.,1982, Photochem. Photobiol., 36,
491-498).
A tunable two-wavelength CW laser
irradiation system is also available as acomplementary light source to OLS tobe used in irradiation experiments which
specifically require ultra-high fluence
rates as well as ultra-high spectral-, timeand spatial-resolutions. It is composed ofa high-power Ar-ion laser (Coherent, Innova 20) (336.6-528.7 nm, 20 W output), two CW dye lasers (Coherent, CR599-01) (420-930 nm, 250-1000 mW
output), A/0 modulators (up to 40
MHz) to chop the laser beam, a beam
expander, and a tracking microbeam irradiator (up to 200 1-lm s- 1 in tracking
speed, down to 2 f-tm in beam diameter)with an infrared phase-contrast observation system.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell
culture are provided. This laboratory isequipped with safety rooms which satisfy
the P2/P3 physical containment level.
This facility is routinely used for DNA
recombination experiments.
3. Computer Laboratory
To meet various computational needsin the Institute, various kinds of computers are provided: VAX/VMS machines(VAX11/780 and micro VAX II),
UNIX workstations (SPARC stations
IPC, IPX, and 10 and DEC station
2100),
andpersonalcomputers(PC9801, IBM compatible, and Macintosh machines). All of them are linked to
the FDDI loop LAN of the Okazaki
National Research Institutes (ORION)
via ethernet. All laboratories in the Institute have at least one microcomputer68connected to the ethemet and thus they
can access to this facility or to any siteconnected by internet. In addition, a
NetWare server machine (Quarter L) is
used to integrate PCs and it enables reference searches and sharing of Post
Script printers from any laboratory.
Various databases and software formolecular biological researches are also
provided.
4. Plant Culture Laboratory
There are a large number of cultureboxes, cubicles, and a limited number ofrooms with environmental control forplant culture.
5. Experimental FarmThis laboratory consists of two 20 m2
glass-houses with precision temperatureand humidity control, a limited farm,
two greenhouses (45 m2, 88 m2) with
automatic sprinklers and window control, two open aquariums (30 t, 50 t) andseveral smaller tanks. The laboratory
also includes a building with office, storage and work-space.
6. Plant Cell Culture Laboratory
Autotrophic and heterotrophic culture
devices are equipped for experimental
cultures of plant and microbial cells.
7. Molecular Biological Analysis Laboratory
In order to facilitate molecular biological analyses, high performance equipments such as DNA sequencer (ABI
370A), peptide synthesizers (ABI 430A
and 431A), nucleotide synthesizer (ABI
381A), imaging analyzers (Fuji BAS
2000-3060 and -3080; PDI Discovery
Series) are provided.
II. Research Activities
1. Faculty
The faculty of the Research Support
Facility conducts its own research as well
as scientific and administrative public

NATIONAL INSTITUTE

services.
(1) Photobiology: Photoreceptive andsignal transduction mechanisms ofphototaxis of single-celled, flagellate
algae are studied action spectroscopicallyby measuring computerized-videomicrographs of the motile behavior of the cells
at the cellular and subcellular levels.
Photoreceptive and signal transduction
mechanisms of algal gametogenesis arealso studied by action spectroscopy.
(2) Developmental Biology: Myosin is aubiquitous protein in eukaryotes. It is
presumed that this molecule plays a keyrole in cell motility as well as other functions. The role of myosin in non-muscle
cells might be better understood if wildtype cells could be compared with mutant cells with altered myosin molecules,
though such mutants have not been isolated. The faculty intends to examine thefunction of the myosin heavy chain invertebrate non-muscle cells by interrupting its synthesis with the introduction ofantisense RNA molecules.
(3) Computational Biology: Efforts todevelop new methodology for sequence
analysis have been continued. An expert
system to predict various protein localization sites from amino acid sequence
only has been constructed, and used toanalyze unknown ORFs determined byan European yeast genome project, in
collaboration with A. Goffau et al (Univ.
Catholique Louvain, Belgium). Now,
main efforts are concentrated on finding
some rules for splice-site selection frommRNA precursor sequences in collaboration with H. Sakamoto (Kobe Univ.).
For this purpose, an aberrant splicing
database was constructed. Some hypothetical rules extracted from the database
are now being tested for their predictability by incorporation into a computer
program.

FOR BASIC BIOLOGY

2. Cooperative Research Program for the
Okazaki Large Spectrograph
The NIBB Cooperative Research Program for the Use of the OLS supports
about 30 projects every year conductedby visiting scientists including foreignscientists as well as those in the Institute.
Action spectroscopical studies forvarious regulatory and damaging actionsof light on living organisms, biologicalmolecules, and organic molecules havebeen conducted.
Publication List:

I. Faculty
Nakai, K., and Kanehisa, M. (1992) A
knowledge base for predicting protein
localization sites in eukaryotic cells.
Genomics 14, 897-911.
Takahashi, T., Kubota, M., Watanabe,M., Yoshihara, K., Derguini, F., andNakanishi, K. (1992) Diversion of thesign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs.
FEBS Lett. 314, 275-279.

69

II. Cooperative Research Program for
the Okazaki Large Spectrograph
Johnson, C.H., and Kondo, T. (1992)
Light pulses induce "singular" behavior and shorten the period of the circadian phototaxis rhythm in the CW
15 strain of Chlamydomonas. J. Bioi.
Rhythms, 7, 313-327.
Kawai, H. (1992) Green flagellar autofluorescence in brown algal swarmers
and their phototactic responses. Bot.
Mag. Tokyo 105, 171-184.
Srivastava, P.K., Mori, Y., and Hanazaki, I. (1992) Photo-inhibition ofchemical oscillation in the Ru(bpy)/+catalyzed Belousov-Zhabotinskii reacChern. Phys. Lett. 190,
tion.
279-284.
Takahashi, T., Kubota, M., Watanabe,M., Yoshihara, K., Derguini, F., andNakanishi, K. (1992) Diversion of thesign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs.
FEBS Lett. 314, 275-279.
Takeda, J., and Abe, S. (1992) Light-induced synthesis of anthocyanin in carrot cells in suspension. IV. Action
spectrum. Photochem. Photobiol. 56,
69-74.

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NATIONAL INSTITUTE

RADIOISOTOPE FACILITY(managed by NIBB)Head of Facility: Yoshihiko Fujita
Technical Staffs: Kazuhiko Furukawa
Yoshimi Matsuda
Nobuya Sugimoto

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, 125 I and various species ofbeta and gamma-ray emitting nuclides

are handled. A laboratory facility forrecombinant DNA research is installedin the center. At the subcenters, only alimited number of radioisotopes such as
3H, 14 C, 32p and 35 S in the NIBB, and3H, 14C, 32p, 35S, 45Ca and 125I in the
NIPS, are processed. The subcenter in
the NIBB is also equipped with a recombinant DNA research laboratory. The
members of the Radioisotope Facility
maintain and control the center and subcenter, and provide users appropriate
guidance for radioisotope handling.

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FOR BASIC BIOLOGYCENTER OF FACILITY FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head of Facility: Mikio Nishimura
Technical Staffs: Hisashi Kojima
Yukiko Kabeya
Yumiko Makino
The Center of facility for Analytical Instruments consists of the following fivesections. (1) Chemical Analysis, (2) Preparation of Biological Materials, (3) Spectroscopic Analysis, (4) Physical Analysis,and (5) Microscopic Analysis. Each section is equipped with instruments forgeneral use as listed below.

1. Section for Chemical Analysis
Amino-Acid Analyzer
HITACHI 835
Gas Chromatograph
SHIMADZU GC-7 APTFSHIMADZU GC-14APFscHPLC
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
LI<B 2127 TACHOPHOR
Preparative Ultracentrifuge
BECKMAN L8-80
Two-Parameter Cell Sorter
BECTON-DICKINSON FACS11
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
Microplate Reader
CORONA MTP-120
CORONA MTP-100F
4. Section for Physical Analysis
Analytical Ultracentrifuge
HITACHI 282
Differential-Scanning Calorimeter
SEIKO DSC100
EPR Spectrometer
BRUKER ER 200D
GC-Mass Spectrometer
HITACHI M-80
GC/LC-Mass Spectrometer
JEOLDX-300
NMR Spectrometer
BRUKER AMX 360wb
Viscometer
CONTRAVES RM-30
5. Section for Microscopic Analysis
Film Data Analysis System
NAC MOVIAS GP-2000
Image Analyzer
KONTRON IBAS-1 & II
Microscope Photometer
CARL ZEISS MPM 03-FL
Two-Dimension Microdensitometer
JOYCE LOEBL 3CS

RouteNo .24K

National Institute for Basic Biology
Okazaki 444, Japan