NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT
1993

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INTRODUCfiON
The National Institute for Basic Biology (NIBB) is a government supported
basic research institute in the field of biology. It aims to stimulate and promotestudies of biology, by conducting firstrate research on its own, and by cooperating with other universities and researchorganizations. The Institute concentrates
on the studies of cellular functions,
growth and development, homeostatic
control, and gene expression and regulation in eukaryotic organisms, to elucidate
the fundamental mechanisms underlying
various living phenomena at the molecular level.
In October of 1992, an American
journal published an article "Science inJapan" (Science, 258, 562), in which citation indices of papers published in
1981-1991 by different universities andinstitutes in Japan were shown. The
index of the NIBB was highest in Japanand comparable to those of leading
universities in the United States. We arejustly proud of this and would like tomaintain a high level of research activity
at the Institute.
A new professor is expected to join
the NIBB in early 1994. Dr. Tetsuo
Yamamori, Sub-Teamleader of the
Riken is to fill the position of Professorof Speciation Mechanisms. His researchis aimed at an understanding of themolecular mechanisms for the evolutionof information processing by the nervous
system. During the past year, the Institute continued to be active in exchange of personnel: we newly appointed 14 research associates fromother universities including one foreign
university, while three research associates were promoted to associate professors and a lecturer at 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 "Signal
Transduction" and "Transdifferentiationof tissue cells". Based on such programs,
the NIBB held the 30th and 31st
Conferences in 1993, entitled "Vertebrate germ-line: its development andmaturation" (organized by Prof. Nagahama) and "Japan-France collaborative
workshops on gene manipulation inaves" (organized by Prof. Eguchi), respectively.
In addition, the Institute sponsors
symposia and study meetings on current
topics at the interdisciplinary level by inviting leading scientists in various related
fields, both nationally and internationally. The NIBB also shares its researchresources to make them available to biologists both from Japan and around theworld. Through such activities, the NIBB
is and will continue to be a national andinternational center to promote basic research in the biological sciences.
This report describes an outline of recent research activities of the NIBB andcontains a list of papers published by themembers of the Institute in 1993.
Ikuo Takeuchi, Ph.D. & D.Sc.
Director General

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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)

{

{

-f

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 U niversity provides Ph.D. course of three years
duration. Those who have completed
master's course in a university or are
qualified equivalent to or higher are
eligible to apply.

The Department consists of the following Divisions and Fields:
DivisionsFieldsMolecular Cell
Biology

Biomolecular
Systems
Cell Dynamics

Developmental
Gene Expression
and Regulation

Gene Expression
Morphogenesis
Transgenic Biology

Regulation Biology Biological
Regulation
Biological
Information

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NATIONAL INSTITUTE

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

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

OFFICE OF DIRECTOR
Publication List:

Ozaki, T., Nakao, H., Orii, H., Morio,T., Takeuchi, I. and Tasaka, M.
(1993) Developmental regulation oftranscription of a novel prespore-

specific gene (Dp87) in Dictyostelium
discoideum. Development 117, 12991308.

5

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Yoshihiko Fujita

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

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NATIONA L INSTITUTEDIVISION OF CELL MECHANISMS
Professor: Mikio Nishimura
Research Associates: Kazuo Ogawa
Makoto Hayashi
Ikuko Hara-Nishimura
Hiroshi Mori
JSPS-Post-doctora/ Fellow: Ryuji Tsugeki
Graduate Students: Kaori Inoue
Akira Kato
Tomoo Shimada
Tetsu Kinoshita
Nagako Hiraiwa 1!
Masahiro Aoki2!
Technical Staff: Maki Kondo
Katsushi Yamaguchit Jfrom Aichi University of Education)2( 1from Shinshu University)

Higher plant cells contain several distinct organelles that play vital roles incellular physiology. During proliferationand differentiation of the cells, the organelles often undergo dynamic changes.
The biogenesis of new organelles may
occur; existing organelles may undergo atransformation of function, which otherorganelles may degenerate. In this division, the regulatory mechanisms underlying organelle transformation are currently studied to examine the dynamictransformation of organellar functions(differentiation of organelles) responsiblefor differentiation events in higher plant
cells.
I. Transformation of leaf peroxisomes
to glyo:xysomes in senescing pumpkin
cotyledons.
Dramatic metabolic changes that
underlie the shift from heterotrophic toautotrophic growth occur in greening ofseed germination. Accompanying thesemetabolic changes, many constitutive organelles are functionally transformed.
For example, etioplasts differentiate into
chloroplasts and mitochondria acquire
the ability to oxidize glycine. Glyoxysomes that are microbodies engaged in
the degradation of reserve oil via (3-oxi-

dation and the glyoxylate cycle, aretransformed into leaf peroxisomes thatfunction in several crucial steps ofphotorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during the greening of pumpkin
cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein
A-gold was performed to analyze thereverse microbody transition using antibodies against a leaf-peroxisomal
enzyme, glycolate oxidase, and against
two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase
decreased in the microbodies during senescence whereas in the case of malate
synthase and isocitrate lyase intensities
increased strikingly. Double labeling experiments with protein A-gold particles
of different sizes showed that the leafperoxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies
of senescing pumpkin cotyledons (Fig.
1), indicating that leaf peroxisomes are
directly transformed to glyoxysomes during senescence. These findings indicatethat transformation of microbodies is areversible process and glyoxysomes andthat leaf peroxisomes are directly transformed to other microbodies in greening
and senescing cotyledons, respectively.
II. Characterization of aconitase responsible for glyo:xylate cycle.
As a step to understand the regulatory
mechanisms that operate during microbody transition in pumpkin cotyledons( Cucurbita sp., Amakuri Nankin), nine
microbody enzymes, i.e., three glyoxysomal enzymes, three leaf peroxisomal
enzymes and three enzymes present inboth microbodies have been purified

FOR BASIC BIOLOGY

7

Fig. 1. Double immunogold-labeling of senescing pumpkin cotyledons using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and two glyoxysomal enzymes, malate synthase and isocitrate lyase. The
leaf-peroxisomal enzyme and the glyoxysomal enzymes coexist in the same microbody in senescing pumpkin
cotyledons. Bars represent 0.5 ftm. Mb microbody, Mt mitochondrion, C chloroplast.
(A) Large gold particles (size 15 nm, arrowhead) glycolate oxidase, small gold particles (size 10 nm, arrow)
malate synthase.
(B) Large gold particles (size 15 nm, arrowhead) glycolate oxidase, small gold particles (size 10 nm, arrow)isocitrate lyase.

and characterized. The developmental
changes in the level of mRNA and protein have been analyzed. This year, we
will start analyzing aconitase which catalyzes the reversible interconversion of
citrate, isocitrate, and cis-aconitate.
Aconitase is involved in the Krebs cycle,
localized in mitochondria, and in theglyoxylate cycle in glyoxysomes, but the
enzyme has also been reported to be localized in the cytosol. Aconitase has
been detected in glyoxysomes, but only
less than 0.5% of the total aconitase
activity was recovered in glyoxysomal
fractions after sucrose gradient centrifugation. Whether mitochondria andglyoxysomes contain specific isoenzymesof aconitase remains to be confirmed.

Three isoforms of aconitase (Acol,
Acoii, Acoiii) were found in etiolated
cotyledons of pumpkin and two of them
(Acol, Acoii) were purified. The specificantibody raised against Acol crossreacted with all isoforms of aconitase. Subcellular fractionation and immunoblot
analyses showed that the activity ofaconitase and the immunopositive polypeptide were not detected in glyoxysomes. These findings indicate that
aconitase is not localized in glyoxysomes
although the enzyme is a member of theglyoxylate cycle. eDNA cloning and immunocytochemical analysis are in progress.

8III. Vacuolar processing enzyme responsible for conversion of proprotein
precursors into mature forms.
Proprotein precursors of vacuolar
components are transported from theendoplasmic reticulum to the dense
vesicles, and then targeted to the vacuoles. In the vacuoles, the proproteinsare processed proteolytically to their mature forms by a unique vacuolar processing enzyme. However, the processing
mechanism in plant vacuoles is very obscure. We isolated a processing enzyme,
a 37-kD cysteine proteinase, from castor
bean endosperm (Hara-Nishimura eta!.,
1991, FEBS Lett. 294, 89-93). Our findings show that a single vacuolar processing enzyme is capable of converting several proprotein precursors with a large
variability of molecular structure into
their mature forms.
i) Molecular characterization and localization of a vacuolar processing enzyme.

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Immunocytochemical localization of avacuolar processing enzyme in the endosperm of maturing castor bean (Ricinus communis) seeds places the enzyme
selectively in the dense vesicles as well as
in the vacuolar matrix, where a variety ofproproteins is also present. However, endogenous processing of the proproteins
was not observed in the isolated dense
vesicles. This suggests that the vacuolar
processing enzyme is a latent form in thevesicles (Fig. 2). To characterize themolecular structure of vacuolar processing enzyme, we isolated a eDNA for the
enzyme. The deduced primary structureof a 55-kD precursor is 33% identical toa putative cysteine proteinase of thehuman parasite Schistosoma mansoni.
The precursor is composed of a signal
peptide, a 37-kD active processing
enzyme domain, and a propeptide fragment. Although the precursor expressedin Escherichia coli has no vacuolar processing activity, a 36-kD immunopositive
protein expressed in E. coli. is active.

VacuoleFig. 2. Maturation system for proprotein precursors of various vacuolar proteins. A latent form of the vacuolar processing enzyme is transported into vacuole via dense vesicle along with proprotein precursors. The
precursors are converted into the respective mature forms by the action of the vacuolar processing enzymethat is activated in the vacuole.

FOR BASIC BIOLOGY

active. These findings suggest that theactivation of the vacuolar processing,enzyme requires proteolytic cleavage ofthe 14-kD C-terminal propeptide fragment of the precursor.
ii) Developmental changes of a vacuo-

lar processing enzyme in maturing andgerminating seeds.
During seed maturation of castor
bean, an increase in the activity of thevacuolar processing enzyme in the endosperm precedes the increase in theamount of total protein. The enzymatic
activity reaches a maximum at the latestage of seed maturation and then decreases during seed germination concomitantly with the degradation of seedstorage proteins. We examined the distribution of the enzyme in different tissues of various plants. The processing
enzyme was found in cotyledons of castor bean, pumpkin and soybean, as wellas in endosperm, and a slight processing
activity was also detected in roots, hypocotyls and leaves of castor bean, pumpkin, soybean, mung bean and spinach.
These findings suggest that the proprotein-processing machinery is widely distributed in vacuoles of various planttissues.
IV. Mechanism for biosynthesis and vacuolar processing of 2S albumin, amajor seed protein of pumpkin.
Cell fractionation of pulse-chaselabeled developing pumpkin cotyledonsdemonstrated that the proprotein precursor to 2S albumin is transported fromthe endoplasmic reticulum to dense
vesicles and then to the vacuoles, inwhich pro2S albumin is processed to themature 2S albumin. Immunocytochemical analysis showed that dense vesicles ofabout 300 nm in diameter mediate the9transport of pro2S albumin to the vacuoles. The primary structure of the precursor to 2S albumin has been deduced
from the nucleotide sequence of an isolated eDNA insert. The presence of ahydrophobic signal peptide at the N-terminus indicates that the precursor is a
preproprotein that is converted into
pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of
the pro2S albumin in the isolated
vesicles revealed that the signal peptide
is cleaved off co-translationally on the
C-terminal side of alanine residue 22 ofprepro2S albumin. By contrast, posttranslational cleavage occurs on the
C-terminal side of both asparagine
residues 35 and 74, which are conserved
among precursors to 2S albumin fromdifferent plants. Hydropathy analysis revealed that the two asparagine residues
are located in the hydrophilic regions ofpro2S albumin. These findings suggestthat a vacuolar processing enzyme canrecognize exposed asparagine residues
on the molecular surface of pro2S albumin and cleave the peptide bond on
the C-terminal side of each asparagine
residue to produce mature 2S albumin in
the vacuoles.
V. Role of molecular chaperones ontranslocation of proteins into chloroplasts.
Molecular chaperones are a class ofcellular proteins that function in thefolding and assembly into oligomeric
structures of certain other polypeptides
but that are not components of the final
oligomeric structure. To investigate therole(s) of molecular chaperones in thechloroplasts, we examined whether thehomologues of the 70-kDa heat-shockprotein (Hsp70) and chaperonin 60(Cpn60) interact with newly imported

10proteins to assist in their maturation.
Ferredoxin NADP+ reductase (FNR)
imported into chloroplasts in vitro could
be immunoprecipitated with antisera
raised against the homologue of Hsp70
from pumpkin chloroplasts and against
GroEL from Escherichia coli, which is abacterial homologue of chaperonin 60(Cpn60), in an ATP-dependent manner,an indication that newly imported FNR
interacts physically with homologues ofHsp70 and Cpn60 in chloroplasts. Timecourse analysis of the import of FNR
showed that imported FNR interacts
transiently with the homologue of Hsp70
and that the association of FNR with thehomologue of Hsp70 precedes that withthe homologue of Cpn60. These findings
suggest that homologues of Hsp70 and
Cpn60 in chloroplasts sequentially assist
in the maturation of newly imported
FNR in an ATP-dependent manner.
eDNA for chloroplast Cpn10 has been
isolated and characterized. Further investigation on the function of Cpn10 in
the transport of protein into chloroplasts
is in progress.
Publication List:
De Bellis, L., Tsugeki, R., Alpi, A. andNishimura, M. (1993) Purification andcharacterization of aconitase isoforms
from etiolated pumpkin cotyledons.
Physiol. Plant. 88, 485-492.
Hara-Nishimura, 1., Kondo, M., Nishimura, M., Hara, R. and Hara, T.
(1993) Ooning and nucleotide sequence of eDNA for rhodopsin of thesquid Todarodes pacificus. FEBS Letters 317, 5-11.
Hara-Nishimura, 1., Kondo, M., Nishimura, M., Hara, R. and Hara, T.
(1993) Amino acid sequence surrounding the retinal-binding site in retinochrome of the squid, Todarodes

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pacificus. FEBS Letters 335, 94-98.
Hara-Nishimura, 1., Takeuchi, Y., Inoue,
K. and Nishimura, M. (1993) Vesicletransport and processing of the precursor to 2S albumin in pumpkin.
Plant Journa/4, 793-800.
Hara-Nishimura, 1., Takeuchi, Y. andNishimura, M. (1993) Molecular characterization of a vacuolar processing
enzyme related to a putative cysteine
proteinase of Schistosoma mansoni.
PlantCel/5, 1651-1659,.
Hiraiwa, N., Takeuchi, Y., Nishimura,
M. and Hara-Nishimura, I. (1993) A
vacuolar processing enzyme in maturing and germinating seeds: Its distribution and associated changes during
development. Plant Cell Physiol. 34,
1197-1204.
Nishimura, M., Takeuchi, Y., De Bellis,
L. and Hara-Nishimura, I. (1993)
Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons. Protoplasma 175, 131-137.
Nozue, M., Kubo, H., Nishimura, M.,
Kato, A., Hattori, C., Usuda, N.,
Nagata, T. and Yasuda, H. (1993)
Characterization of intravacuolar pigmented struc;tures in anthocyanin-containing cells of sweet potato suspension cultures. Plant Cell Physiol. 34,
803-808.
Ogawa, K. and Shimizu, T. (1993)
eDNA sequence for mouse caltractin.
Biochim. Biophys. Acta 1216, 126128.
Tezuka, K., Hayashi, M., Ishihara, H.,
Onozaki, K., Nishimura, M. andTakahashi, N. (1993) Occurrence ofheterogeneity of N-linked oligosaccharides attached to sycamore (Acerpseudoplatanus L.) laccase after excretion. Biochem. Mol. Bioi. Internat. 29,
395-402.

FOR BASIC BIOLOGY

Tezuka, K., Hayashi, M., Ishihara, H.,
Nishimura, M., Onozaki, K. and Takahashi, N. (1993) Purification andsubstrate specificity of ,8-xylosidasefrom sycamore cell (Acer pseudoplatanus L.): application for structural analysis of xylose-containing N-linked
oligosaccharides. Anal. Biochem. 211,205-209.
Tsugeki, R., Hara-Nishimura, 1., Mori,
H. and Nishimura, M. (1993) Cloning
and sequencing of eDNA for glycolateoxidase from pumpkin cotyledons andnorthern blot analysis. Plant Cell
Physiol. 34, 51-57.
Tsugeki, R. and Nishimura, M. (1993)
Interaction of homologues of Hsp70
and Cpn60 with ferredoxin-NADP+
reductase upon its import into chloroplasts. FEBS Letters 320, 198-202.

11

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NATIONAL INSTITUTEDIVISION OF BIOENERGETICS
Professor: Y oshihiko Fujita
Associate Professor: Sigeru Itoh
Research Associates: Mamoru Mimuro
Katsunori Aizawa
Visiting Scientist: Sung-Jun Kim •JSPS-Post-doctral Fellow: Tohru Ikeya
Visiting Fellow: Yuji Nakajima • •Technical Staff: Akio Murakami(*from Kyngpook National University)(**from Advanced Technology Research Center'
Mitsubishi Heavy Industries, LTD)

Oxygenic photosynthesis is an energy
yielding metabolism that supports autotrophic life of plants. Primary process of~hotosynthesis, light-energy conversion,m thylakoids is the target for the studyof this research division. The researchhas been conducted for regulatory aspects of thylakoid system in response tophotosynthetic environments and also
for molecular mechanism of light-capturing and conversion in functional molecules.
I. Regulation of PSI/PSII stoichiometry
Light-energy conversion in oxygenic
photosynthesis is driven by the two light
reactions of the two photosystems, PSI
and PSII. Imbalance between the twolight reactions causes lowering the efficiency of energy conversion. Our previous study with cyanophytes has revealed that plants have an ability for
regulating the stoichiometry of the two
photosystems so as to compensate imbalance between the two light reactions.
Further, at least in the cyanophyte Synechocystis PCC 6714, the stoichiometry is
regulated by the control of PSI formation, which occurs in response to thesignal comming from the state of theelectron transport between the two
photosystems.
Controlling mechanism for PSI formation has been conducted in two ways;
determining the target sites for regula-tion (1)in Chl a synthesis and (2)in apoprotein synthesis. Analysis for dynamics
of intermediates of Chi a synthesis
yielded following results (Fujita et al., inpress): (1) Pchlide reduction is regulated
in parallel with regulation of PSI formation. The activity is suppressed underconditions for suppression of PSI formation, such as light regime preferentially
exciting PSI (PSI light). The suppression
is released by the conditions for stimulation of PSI formation, &uch as light
regime exciting preferentially PSII (PSII
light). (2) Changes in the activity of
Pchlide reduction are neither due to acceleration-deceleration of PSI synthesis
nor due to differential occurrence oflight-dependent Pchlide reduction. The
enzyme activity itself is regulated. Analysis for synthesis of PsaA/B peptides,
core peptides of PSI complex, withpulse-labeling method revealed that (1)inhibitors for peptide elongation suppresses PsaA/B synthesis differently inaccelerated and decelerated PSI formations. Inhibition at low inhibitor concentrations was stronger in the former thanthat in the latter, suggesting that pausingof pepetide elongation becomes longer
under conditions for suppression of PSI
formation. (2) The inhibitor for initiationof translation showed similar effect. (3)Effect of chrolamphenicol suggested that
initiation of translation is also regulated;
it is suppressed by PSI light, and PSII
light releases from suppression. Together
with previous results, evidences obtained
show that regulation of PSI formation in
PSI/PSII adjustment occurs at the translation level of PsaA/B synthesis. Since
Chi a stabilizes PsaA/B peptides, we assume that regulation of psaAIB expression at translation level is achieved atleast by Chl a supply.
Changes in thylakoid composition in13

FOR BASIC BIOLOGY
Light

Light

Photosynthetic

Photosynthetic_::::P~ie::Y

efficiency
H, Q _ _ _ _ _ NADP'

0.<

H~O -

-

NAOP"

H,o -~- NADP'

H,Q -~1.0

NADP'

I.O

~-NAOP'

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

the green alga Chlamydomonas reinhardtii in response to light quality were
determined under collaboration with Dr.
A. Melis (Univ. Cal. , Berkeley). Results
indicated that the pattern of change wassimilar to that in the cyanophyte Synechocystis PCC 6714. Abundance of PSI
was markedly changed while that of PSII
and Cyt b6 - f remained fairly constant,resulting higher PSIIPSII ratio under
PSI light and lower ratio under PSI light.
Changes in PSI/PSII improved photosynthetic efficiency under respective light
regimes. Differently from cyanophyte
system, a marked decrease in LHC, inparallel with PSI decrease, occurredunder PSI light. Results suggest that Chl
supply to assembling of Chi-protein
complexes is a primary determinant foradjustment of PSI/PSII stoichiometry inthis organism also.
II. Mechanism of energy transfer inlight-capturing pigment system
Examination was made for function of
carotenoids in photosynthesis. Relationship between molecular structure andoptical properties in the excited state wasmainly analyzed. (1) The forbidden S1state of neurosporene and spheroidene,
which is responsible for the energy transfer to bacteriochlorophylls a, was di-rect1y measured by the one-photon absorption spectroscopy; the energy level
was higher than and close to that of acceptors. (2) It was proved that the relaxation processes from the excited state ofcarotenoids follow the energy gap law ofinternal conversion. Presence of a ketocarbonyl group induced a rapid internalconversion to the S1 state, which was favorable for energy transfer to chlorophylls a. This corresponds to the case forfucoxanthin in brown algae and diatom.
(3) The S2 lifetime of ~-carotene was directly measured by the up-conversion
method to be 195 ± 10 fs with an isotropy ratio of 0.39 ± 0.02. (4) Theoretical analysis of the energy transfer processes was carried out by calculation of
the energy transfer matrix elements between neurosporene and bacteriochlorophyll a. The electron exchange mechnism is concluded to be less probable,
even if the forbidden singlet state is involved. (5) Based on these results, theenergy transfer through the dipole-multipole interaction from the sl state to(bacteria )chlorophyll a is proposed.
III. Mechnism of electron transfer inreaction center complex
Mechanism of the primary photoenergy conversion in photosynthesis was

14studied in plant and bacterial systems by
the combination of modem spectroscopy, and the biochemical and geneticalmanipulation of the protein complexes.
In the PSI reaction center complex, thetime constant for the primary charge
separation between the electron donor
chlorophyll (P700) and the acceptor
chlorophyll was measured to be 7 X
10-12 s. The following reaction withphylloquinone was shown to take placewith a time constant of 38 X 10-12 s.
The rate varied when various artificial
quinones were introduced into the reaction center complex in place of nativephylloquinone. Functions of quinones invarious reaction center preparations
were also studied. It is shown that thefine adjustment of the energy levels ofnative electron transfer components enable the highly efficient, ultra-fast electron transfer reactions. We isolated andcharacterized the reaction center complex of green bacteria, which seems to beancestry to PSI reaction center. The
complex was shown to have a polypeptide composition simpler than that ofplant PSI but to undergo almost similar
activities. Unique nature of the iron-sulfur centers in this complex was shown bycryogenic EPR studies. The structurefunction relationship of the iron-sulfur
centers of the photosynthetic bacterium
Rhodobacter capsu/atus was also investigated by the site-directed mutagenesis.
Publication List:
lkegami, 1., ltoh, S., Warren, P.G. and
Golbeck, J.H. (1993) Reconstitutionof the photosystem I secondary quinone acceptor (A1) in the P700-Fx
core isolated from Synechococcus
PCC6301. Plant Cell PhysioL 34,
849-853.

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Katoh, T. Tanaka, A. and Mimuro, M.
(1993) Isolation of fucoxanthin-chlorophyll ale protein assembly frombrown algae. In: Methods in Enzymology, Vol. 214, Packer, R. ed., Academic Press, New York, pp. 402-412.
Matsuura, K., Hirota, M., Shimada, K.
and Mimuro, M. (1993) Spectral
forms and orientation of bacteriochlorophylls c and a in chlorosomes of thegreen photosynthetic bacterium Chlorof/ex aurantiacus. Photochem. Photobioi. 57, 92-97.
Mimuro, M. (1993) Identification of theterminal energy donor to the reaction
center in the pigment system of green
plants by time-resolved fluorescence
spectroscopy at -196"C. Plant Cell
Physiol. 34, 321-327.
Mimuro, M., Nagashima, U., Nagaoka,
S., Takaichi, S., Yamazaki, 1., Nishimura, Y. and Katoh, T. (1993) Direct
detection of a low-lying singlet excited(2Ag) state of a linear carotenoid,
neurosporene, in solutions. Chern.
Phys. Lett. 204, 101-105.
Mimuro, M., Takaichi, S., Yamamoto,Y., Ito, M., Nagaoka, S., Nishimura,Y., Yamazaki, 1., Katoh, T. and Nagashima, U. (1993) The effect of molecular structure on the relaxation
processes in carotenoids containing acarbonyl group. Chern. Phys. Lett.
213, 576-580.
Murakami, A. and Fujita, Y. (1993)
Regulation of stoichiometry between
PSI and PSIT in response to light
regime for photosynthesis observedwith Synechocystis PCC 6714: Relationship between redox state of Cyt
b6 - f complex and regulation of PSI
formation. Plant Cell Physio/. 34,
1175-1180.

FOR BASIC BIOLOGY

Nagashima, K.V.P., ltoh, S., Shimada,K. and Matsuura, K. (1993) Photooxidation of reaction center-bound cytochrome c and generation of membrane potential determined by carotenoid band shift in the purple photosynthetic bacterium, Rhodospirillum
molischianum. Biochim. Biophys.
Acta, 1140, 297-303.
Nagae, H., Kakitani, T., Katoh, T. and
Mimuro, M. (1993) A theoretical
study on the interaction between carotenoids and bacteriochlorophyll:
Calculation of the excitation transfer
matrix elements between neurosporene S2 and S1 states and Bchl a S2and S1 states. J. Chern. Phys. 98,
8012-8023.
Nishimura, Y., Shimada, K., Yamazaki,
I. and Mimuro, M. (1993) Energy
transfer processes in Rhodobacter pa/ustris grown under low-light conditions: Heterogenous composition of
LH2 complexes and parallel energy
flow pathways. FEBS Lett. 329,
319-323.
Oh-oka, H., Kakutani, S., Matsubara,
H., Malkin, R. and ltoh, S. (1993)
Isolation of the photoactive reaction
center complex that contains three
types of Fe-S centers and a cytochrome c subunit from the green sulfur
bacterium Chlorobium limico/a f. thiosulfatophilum, strain Larsen. Plant
Cell Physiol. 34, 93-101.
Uehara, K., Hioki, Y. and Mimuro, M.
(1993) The chlorophyll a aggregate
absorbing near 685 nm is selectively
formed in aqueous tetrahydrofuran.
Photochem. Photobiol. 58, 127-132.

Supplements to 1992
Aizawa, K. and Fujita, Y. (1992) Regulation of PSI formation induced bylight quality observed with Synecho-

15

cystis PCC 6714. In Research in
Photosynthesis (Murata, N. ed.,Kluwer Academic Publishers, Dordrecht), Vol. IV, pp. 329-332.
Fujita, Y., Murakami, A. and Aizawa,K. (1992) Light acclimation of thylakoid system in cyanophytes: Regulation of PSI formation in response tolight regime. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. IV,
pp. 301-308.
Hoshina, S., Sue, S., Wada, K., Enami,
I. and ltoh, S. (1992) Stabilization ofiron-sulfur centers A and B in photosystem I particles by chemical crosslinking. In Research in Photosynthesis(Murata, N. ed., Kluwer Academic
Publishers, Dordrecht), Vol. I, pp.
581-584.
Itoh, S., Mimuro, M. and Iwaki, M.
(1992) Organization of chlorophylls inphotosystem I reaction center: Studyby LD and fluorescence measurementsin the ether-extracted particles whichcontain 11 chlorophyll/P700. In Research in Photosynthesis (Murata, N.
ed., Kluwer Academic Publishers,
Dordrecht), Vol. I, pp. 541-544.
Iwaki, M., Taksahashi, M., Shimada, K.
and ltoh, S. (1992) Identification of
the quinone binding site in PSI reaction center complex by photoaffinity
labeling. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I,
pp. 529-532.
Katoh, T. and Mimuro, M. (1992) S1
state of fucoxanthin involved in energy
transfer to chlorophyll a in the lightharvesting protein of brown algae. In
Research in Photosynthesis (Murata,
N. ed., Kluwer Academic Publishers,
Dordrecht) Vol. I, pp. 227-230.

16
Kobayashi, M., Van de Meent, E.J., Ohoka, H., Inoue, K., Itoh, S., Amesz, J.
and Watanabe, T. (1992) Pigmentcomposition of heriobacteria andgreen sulfur bacteria. In Research in
Photosynthesis (Murata, N. ed.,Kluwer Academic Publishers, Dordrecht), Vol. I, pp. 393-396.
Matsuura, K., Hirota, M., Shimada, K.
and Mimuro, M. (1992) Pigment
orientation and energy transfer kinetics in chlorosomes of green photosynthetic bacteria. In Research in Photosynthesis (Murata, N. ed., Kluwer
Academic Publishers, Dordrecht) Vol.
1, pp. 113-116.
Mimuro, M. (1992) A long wavelength
antenna in photosynthetic pigment
system. In Research in Photosynthesis.
(Murata, N. ed., Kluwer Academic
Publishers, Dordrecht), Vol. 1, pp.
259-262.
Mimuro, M., Matsuura, K., Nishimura,Y., Shimada, K. and Yamazaki, I.
(1992) Excitation energy transfer processes in green photosynthetic bacteria: Analysis in picosecond time domain. In Research in Photosynthesis(Murata, N. ed., Kluwer Academic
Publishers, Dordrecht) Vol. I, pp.
17-24.
Murakami, A. and Fujita, Y. (1992) In
vivo electron transport from Cyt b6-f
complex to photosystem I complex in
Synechocystis PCC 6714. In Researchin Photosynthesis (Muratsa, N. ed.,NATIONAL INSTITUTE

Kluwer Academic Publishers, Dordrecht), Vol. II, pp. 503-506.
Nakayama, K., Mimuro, M., Nishimura,Y. and Okada, M. (1992) Chlorophyll
forms and excitation energy transfer
pathways in light-harvesting chlorophyll alb protein complexes from the
Bryopsissiphonous green algamaxima. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I,
pp. 219-222.
Oh-oka, H., Kakutani, S., Itoh, S., Matsubara, H. and Malkin, R. (1992)
Isolation and characterization of thephotoactive reaction center complex
from the green sulfur bacterium Ch/orobium lumico/a. In Research in
Photosynthesis (Murata, N. ed.,Kluwer Acsademic Publishers, Dordrecht) Vol. I, pp. 385-388.
Ohki, K. and Fujita, Y. (1992) Lightdependent maintenance of active nitrogenase in the non-heterogenous
cyanophyte Trichodesmium sp. NIBB
1067. In Research in Photosynthesis(Murata, N. ed., Kluwer Academic
Publishers) Vol. IV, pp. 103-106.
Shimada, K., Nishimura, Y., Yamazaki,
I. and Mimuro, M. (1992) Excitationenergy flow in Roseobacter denitrificance (Erythrobacter sp. OCh 114) at
low temperature. In Research in
Photosynthesis (Murata, N. ed.,Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 137-140.

FOR BASIC BIOLOGYDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor: Masayuki Yamamoto
Research Associate: Hidetoshi /ida
Research Associate: Yoshiyuki ImaiNIBB Post-doctoral Fellow: Masuo Goto
Graduate Student: Tomoko Ono (from The
University of Tokyo)
Visiting Scientist: Makiko S. Okumura

Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiosis is highly conserved
among eukaryotic genera, from yeast to
mammals. This Division aims to explore
the regulation of meiosis in higher organisms. The major strategy currently
taken 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 either
by classical genetics or by chromosome
manipulation. We thus rationalize that it
is possible to isolate homologs of thesegenes from animals and plants, by using
either similarity in nucleotide sequences
or functional complementation of themutants. The main project of this Division has been performed along this line.
We have also carried out two additional
projects. One of them was to characterize the mating pheromone of thefission yeast and examine its potential toaffect cell growth and sexual development of this yeast. The · other was tostudy the effect of calcium on cell proliferation in the budding yeast. The results obtained in these three projects aredescribed below.
I. Screening for mammalian and plant
genes that can replace genes essential
for meiosis in the fission yeast.
We prepared eDNA libraries from17
mouse testis and Arabidopsis thaliana.
The vector carries a promoter functionalin the fission yeast and thus the cloned
genes can be expressed in fission yeast
cells. We transformed two meiotic mutants of S. pombe with these libraries.
One is the sme2 mutant that cannot perform the first" meiotic division. The sme2
gene product has been shown to function
as an RNA molecule. The other mutant
is mes 1, which cannot perform the second meiotic division. We obtained fivemouse genes and four Arabidopsis genesthat can rescue sme2. The Arabidopsis
genes include those encoding a novel
protein kinase family. We isolated afission yeast homolog of this kinase family and have shown that it also has theability to rescue sme2. So far we obtained only one mouse gene that can rescue mesl. This mouse gene generates a
specific transcript in testis that is not detectable in other tissues tested. Further
characterization of the isolated genes isin progress.
II. Characterization of the fission yeast
mating pheromone P-factor.
S. pombe h+ cells secrete a diffusible
mating pheromone called P-factor. Although M-factor secreted by h- cells waspreviously characterized, the identity ofP-factor was not known. We now haveshown that the map2 gene, a defect ofwhich confers mating deficiency only inh+ cells, encodes the precursor of P-factor. We purified P-factor from cells
overexpressing map2 and determined its
amino acid sequence. P-factor is a peptide of 23 residues, with the sequence
Thr-Tyr-Ala-Asp-Phe-Leu-Arg-Ala-TyrGln-Ser-Trp-Asn-Thr-Phe-Val-Asn-ProAsp-Arg-Pro-Asn-Leu. We synthesized
a peptide of this sequence. It has thesame specific activity and chromato-

18

NATIONAL INSTITUTE

graphic profile as the purified P-factor,
suggesting that P-factor is unmodified.
By using mutant cells that are derepressed for sexual development in richmedium, we could show that P-factor
can induce not only responses towards
mating but also arrest of the cell cycle atthe G 1 phase in h- cells (Figure 1). This

proves that the S. pombe mating pheromone has the ability to arrest cell cycle
progression, which has previously been
obscured by the usual requirement formating of nutritional starvation and subsequent growth arrest (Y. Imai and M.
Yamamoto, Genes Dev., 8, 328-338,
1994).

a

b
Oh

6h

Oh

lh

9h

2h

2h

12h

6h

4h

21h

21h

A.

Figure 1. The ability of P-factor to arrest the cell cycle of h- cells at the Gl phase. Flow cytometric analysis of the DNA content was done in S. pombe h- cells (sexually-derepressed mutant) exposed to P-factor.
(a) Cells grown in the presence of 2 t-tg/ml P-factor; and (b) with no P-factor. The main peaks at Oh in (a)and (b) correspond to 2C (G2-equivalent) DNA content, whereas the main peak at 6h in (a) corresponds tolC (Gl-equivalent) DNA content. The Gl arrest is transient and cells gradually resume growth (a, 9-21h).
Without P-factor (b), cells show 2C DNA content because rapidly growing S. pombe cells have only short G 1and S phases and they undergo these phases while the daughter cells are not completely separated.

m. Genetic analysis of the role of calcium during sexual differentiation in
the budding yeast.
We isolated Saccharomyces cerevisiae
mutants apparently defective in the Ca2+
signalling, on the basis of our discovery

that Ca2+ is essential for maintaining
viability of the cells destined for mating.
S. cerevisiae cells do not survive if theycannot take up enough Ca2+ when they
are exposed to the mating pheromone.
The mutants, named mid (mating

FOR BASIC BIOLOGY

pheromone-induced death), die even in
the presence of a sufficient amount of,Ca2+ if they are exposed to the mating
pheromone. The MIDI and MID2 genes
have been cloned. Both genes apparently
encode membrane-associated proteins.
Characterization of these and other MID
genes and the functional relationshipamong them are currently under study.
Publication List:

Gotoh, Y. Nishida, E., Shimanuki, M.,
Toda, T., Imai, Y. and Yamamoto, M.
(1993) Schizosaccharomyces pombe19
Spk1 is a tyrosine-phosphorylated
protein functionally related to Xenopus mitogen-activated protein kinase.
Mol. Cell. Bioi. 13, 6427-6434.
Tabata, S., Sato, S., Watanabe, Y.,
Yamamoto, M. and Hotta, Y. (1993)
Evidence of meiosis-specific regulation of gene expression in lily microsporocytes. Plant Sci. 89, 31-41.
Tanaka, K., Davey, J., Imai, Y. andYamamoto, M. (1993) Schizosaccharomyces pombe mapJ+ encodes theputative M-factor receptor. Mol. Cell.
Bioi. 13, 80-88.

20

NATIONAL INSTITUTEDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Hotta
Associate Professor: Hitoshi Okamoto
Research Associates: Mika Takahashi
Shin-ichi Higashijima
Institute Research Fellow: Akira Chiba
Nobuyoshi Shimada
Graduate Students: Keita Koizumi
Yoko Yasuda

Brain can be seen as an integrated circuit where neurons of various identities
are interconnected in a highly orderedmanner by their axons. We have beeninterested in how individual neurons acquire their own identities and how theiraxons find their own pathways and finally recognize their proper targets. Ourcurrent interest is mainly focused on the
motoneurons because of their accessibility to various cellular manipulations.
Using two different animals, zebrafish(Danio rerio) and fly (Drosophila melanogaster), both of which are suitable forgenetic analysis and gene manipulation,
we are trying to address to these questions both at the molecular and cellularlevels.
I. Molecular Developmental Neurobiology of Zebrafish
Embryos of zebrafish stay transparent
throughout most period of their development. Most neurons in the central nervous system of early embryos are identifiable. In the embryonic brain, a primitive
neuronal network is formed by the earlyborn neurons (primary neurons) andpresents the initial scaffold for the later
extending axons from the secondary
neurons. Our current goal is to identify
the molecules which are involved in the
final determination of the identities orthe axonal pathways by individual primary neurons.

1. ZISH (zebrafish Isl-1 homologues)
genes in the specification of primary
motoneurons
Zebrafish embryos have three subtypes of primary motoneurons (RoP,
MiP and CaP) per hemisegment, each ofwhich extends the axon along the stereotyped pathway and innervates the specific region of the somites. In the last
annual report, we reported the molecularcloning of the eDNA for zebrafish Isl-1.
Its expression pattern in the ventral region of the spinal cord suggested that
Isl-1 may be involved in the specificationof primary motoneurons.
In collaboration with Ziyuan Gong
and Choy Hew at University of Toronto,
we have recently isolated two novel zebrafish eDNA clones which encode theproteins similar to the original lsl-1,
using the salmon pituitary eDNA encoding a novel Isl-1-like protein as a probe.
We named them ZISH (~ebrafish !sl-1
Homologue)-2 and -3, by counting theoriginal zebrafish Isl-1 as ZISH-1.
The expression patterns of the originalIsl-1 (ZISH-1) mRNA and ZISH-2
mRNA are almost complementary.
ZISH-1 mRNA expression is at first observed in randomly distributed ventromedial cells and later restricted to RoP(or RoP and MiP) and the secondary
motoneurons surrounding RoP. And thenumber of ZISH-1 mRNA-positive ventromedial cells increases in the tail region
and in the spaidtail mutant where thesomites surrounding the spinal cord are
missing or defective. These data suggestthat ZISH-1 mRNA expression is downregulated by the influence of the somites.
In contrast, ZISH-2 mRNA is expressed
only by CaP and YaP from the moment
when its expression starts to be detected(around 15 hours after fertilization), justFOR BAS IC BIOLOGY

Fig. 1. The expression patterns of ZISH-2 and
ZISH- 1 mRNA in the spinal cord
Lateral views of the spinal cords of embryos hybridized in situ with ZISH-2 and ZISH-1 cRNAin (A) and (B), respectively. [18 hr embryo in(A), 16 hr embryo in (B)] ZISH-2 and ZISH- 1
mRNA positive cells in the ventral regions of the
spinal cord are indicated by arrowheads in (A)and (B), respectively. Somites are denoted bynumbers, and the somite borders are indicated bywhite lines.

when the nascent CaPs are about to stop
expressing ZISH-1 mRNA. ZISH-2
mRNA is not expressed in the caudal
end of the spinal cord where the somites
are not yet formed. These subtype specific expression patterns suggest that theIsl- 1 (ZISH-1) and ZISH-2 genes mayeither react oppositely to the influence
from the somites or regulate the expression of each other, and together be involved in the determination of cellular
identities (specification) by motoneurons
in embryonic zebrafish.
Although ZISH-3 mRNA is expressed
only by Rohon-Beard cells but not by

21

Fig. 2. The dorsal view of the brain of a 32 hrembryo stained with HNK-1 antibody

motoneurons in the spinal cord, its expression pattern is nonetheless very specific. In the body trunk, it is expressed
intensely only in the ventral region of theaxial muscle which is innervated by CaP.
And in the brain, it is expressed both in
the entire eye (including the retina andthe lens) and in the nascent tectum.
These data suggest that the ZISH-3 gene
may regulate the expression of the molecules that help CaP and the retinal ganglion cells to correctly find their propertargets, the ventral axial muscle and thetectum respectively.
We are currently examining how the22
specification and axonal pathfindings ofprimary motoneurons are affected by theectopic expression of ZISH genes.
2. Search for new cell surface recognition molecules expressed in a subset of
CNS axons.
During neuronal development, growthcones are known to have ability to recognize and extend along specific axonal
pathways. Several lines of evidence suggest that cell surface glycoproteins play
important roles in this process. Immunocytochemical studies have demonstrated
that the monoclonal antibody HNK-1,
originally raised against a human lymphoblastoma, recognizes a subset of
CNS and PNS axons in many vertebrates
including zebrafish by binding to a carbohydrate determinant in several glycoproteins. Thus, we started characterizing
glycoproteins recognized by HNK-1,
hoping to isolate new cell surface recognition molecules expressed in a subset of
CNS and PNS axons during neuronaldevelopment.
Our strategy is as follows: Manybrains of adult zebrafish are homogenized, and from this extract, molecules
which binds to HNK-1 are intensely enriched by using HNK-1 affinity chromatography. The affinity-purified fractions
are used to immunize mice to get a seriesof monoclonal antibodies (MAbs). The
MAbs, thus obtained, are screened withimmunohistochemistry to zebrafish tissues. Hopefully, those MAbs which recognize peptides and not carbohydrates
are used to isolate and characterize newmolecules.
Immunogen which represents several
protein family on Western blotting has
been prepared. Immunization is now inprogress.

NATIONAL INSTITUTE

II. Drosophila neurogenetics
1. Fasciclin m as a synaptic recognition
molecule in Drosophila
The larval neuromuscular system of
the Drosophila consists of uniquely
identified cells, and is a powerful model
system for studying selective synapse
formation. During synaptogenesis, thecell adhesion molecule fasciclin III appears in both motoneuron RP3 and its
targets, muscle 6 and 7. We have tested
whether fasciclin III is necessary and/ or
sufficient for RP3 target selection using
intracellular dye injection.
First, we have found that in the existing fasciclin III null mutant RP3 reliably
formed synapse with its normal targets.
Therefore, fasciclin III is either irrelevant
for the process, or playing a positive role
but its absence can be compensated foranother redundant mechanism ("X").
We now have demonstrated that thelatter is the case. We generated transgenic flies which misexpresses fasciclin
III ectopically on all skeletal muscles
during neuromuscular synaptogenesis.
This was accompanied by creating a construct which placed the fasciclin III gene
under the control of the myosin heavy
chain promoter, and introducing thisconstruct into the fly genome by
P-element mediated genomic transformation. In these flies, PR3 often innervated non-target muscle cells while otheridentified motoneurons innervated targets normally.
Our results provide the single identified-cell level evidence that a cell adhesion molecule functions as a specificsynaptic target recognition molecule invivo.
2. Analysis of A9 gene which affects
larval neurogenesis
P-element insertion line A9 is a pupal

FOR BASIC BIOLOGY

lethal mutant which has a diminishedbrain in the homozygotes. Using BrdU
incorporation analysis, we found larval
neuroblasts of this mutant are morphologically abnormal and showed almost
no sign of proliferation.
We tried to clone this A9 gene andidentified a 0.6 kb transcript around theP-element insertion site which was reduced in A9 homozygotes. The molecular cloning of the eDNA and the subsequent sequencing determination and insitu hybridization indicate that the putative product of this transcript is a secretory glycoprotein expressed in larval
imaginal cells including those in CNS.
These data suggest that the defect in thegene encoding this transcript is responsible for the A9 mutant phenotype. We
are now trying to examine if A9 mutants
can be rescued by P-element mediatedtransformation with this gene.

23
Publication List:
Inoue, A., Hatta, K., Hotta, Y. andOkamoto, H. (1994) Developmental
regulation of Islet-1 mRNA expression during neuronal differentiation inembryonic zebrafish. Devel. Dynam.
199, 1-11.
Masai, 1., Okazaki, A., Hosoya, T. andHotta, Y. (1993) Drosophila retinaldegeneration A (rdgA) gene encodes
an eye-specific diacylglycerol kinasewith cysteine-rich zinc finger motif
and ankyrin repeats. Proc. Nat/. Acad.
Sci. USA 90, 11157-11161.
Okamoto, H. (1993) Neuronal differentiation and specialization in embryoniczebrafish. Neurosci. Res. Suppl. 18,
p. 9.

24

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

Our research interest is focused on theregulatory mechanisms of multigene
families both in the immune and in thecentral nervous systems. In the immune
system, somatic DNA recombinationand gene conversion play importantroles in the expression of antigen receptor genes. Gene rearrangement, known
as V-(D)-J joining, not only generates avast diversity in the receptor genes, butalso activates a particular member of thegene family by bringing enhancer andpromoter elements into close proximity.
For the olfactory system, hundreds ofodorant receptor genes have been reported, although it is yet to be studied
how this multigene family is regulated
for expression. It is known that eachmember of the gene family is expressedin one of the three or four topographically distinct zones, where olfactory neurons expressing one particular kind ofreceptor are randomly distributed. Furthermore, it is assumed that a very
limited number of the receptor genes(possibly one) is activated in each olfactory neuron.
We are interested in knowing howeach neuron select a limited number ofreceptor genes which are to be expressed. One obvious regulatory mechanism is at the level of transcription. In
this mechanism we may need a variety oftranscriptional factors for both positive

and negative regulations. In the mammalian immune system, somatic DNA recombination is utilized to activate one
particular member of the receptor genes,
and to exclude one of the two different
alleles. It is of interest to study whether
such a mechanism is also involved in theregulation of the olfactory system.
In order to study selective expressionof the odorant· receptor genes, we arecharacterizing the genomic structure of
the murine receptor genes. Althoughmany coding sequences have been reported for the odorant receptor genes,
little is known about the 5' and 3' noncoding regions. These regions must contain important DNA elements that regulate the receptor gene expression. We
have isolated genomic clones of theodorant receptor genes from the P 1
phage library. We are currently analyzing the regulatory regions in theseclones. We hope that these studies will
reveal the molecular mechanisms for theselective and zonal expression of theodorant receptor genes.
We have made transgenic mice which
are forced to express one particularmember of the odorant receptors inevery olfactory neuron. It is of interest to
study whether the expression of thetransgene represses the endogenous receptor genes. A plasmid construct contains the mouse receptor # 28 gene
under the control of OMP promoter.
Since the OMP gene is expressed specifically in the olfactory epithelium, the # 28
receptor is expected to be activated in all
mature neurons in the transgenic mouse.
These study will give us a new insight
into the regulatory mechanisms of theodorant receptor genes.
When olfactory neurons form synapses with other neurons, they send theiraxons to the specific region called

FOR BASIC BIOLOGY

glomeruli in the olfactory bulb. Each olfactory neuron sends only one axon to
one of two thousand glomeruli. It isquite amazing that the olfactory neurons
are able to find their right target glomerulus every time when they are regenerated. It is interesting to study how theneuronal activity affects the target specificity and selectivity. We are hoping that
our transgenic approach will become ausuful clue to answer to these questions.
Publication List:
Huppi, K., Siwarski, D., Shaughnessy,Jr., J., Maki, R., Shirakata, M. and
Sakano, H. (1993) Genes associatedwith immunoglobulin V(D)J recombination from a complex locus on mouse
chromosome 2 and human chromosome 11. Immunogenetics 37, 288291.
Kingsbury, L. and Sakano, H. (1993)
DNA recombination in the transgenic

25

mouse brain. In Ataxia-Telangiectasia
Workshop V (Gatti, R.A. and Painter,
R.B., eds., Springer-Verlag, Berlin),
pp. 175-179.
Kondo, T., Strayer, C.A., Kulkarni,
R.D., Taylor, W., Ishiura, M., Golden,
S.S. and Johnson, C.H. (1993) Prokaryotic circadian rhythms in prokaryotes: Luciferase as a reporter ofcircadian gene expression in cyanobacteria. Proc. Nat/. Acad. Sci. U.S.A.
90, 5672-5676.
Sakano, H. (1993) Somatic DNAchanges in the immune and central
nervous system. In Molecular Basis of. Immune Responses (Saito, H. et al.,eds., Academic Press, New York),
pp. 3-13.
Sakano, H. (1993) Recombinase. In The
Encyclopedia of Molecular Immunology (Kendrew, J., ed., Blackwell
Scientific, Oxford), in press.

27

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshitaka Nagahama

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

-28

NATIONAL INSTITUTELABORATORY OF
REPRODUCTIVE BIOLOGY
Professor: Yoshitaka Nagahama
Research Associates: Michiyasu Yoshikuni
Masakane Yamashita
Minoru Tanaka
Tohru Kobayashi
Monbusho Foreign Scientist: Alexander P. Scott
JSPS Post-doctoral Fellows: Naoki Shibata
Takeshi Miura
Chiemi Miura
Graduate Students: Akihiko Yamaguchi
Mika Takahashi
Toshinobu Tokumoto
Yoshinao Katsu
Shinji Onoe
Daisuke Kobayashi
Xiao- Tian Chang
Visiting Scientists: Howard A. Bern 11
Glen Vander Kraak21
Noriyoshi Sakai31
Michiya Matsuyamlfi
Masamichi Nakashima 51
Jian-Quiao Jianlf'l
11from University of California
21from University of Guelph.

31from Fukui Prefectural University
4

1from Mie University

51from Tohoku University

1from Wuhan University

6

The division of reproductive biology
conducts research on the endocrine
regulation of differentiation, growth andmaturation of germ cells in multicellularanimals, using fish as a primary study
model.
I. Endocrine regulation of oocyte differentiation, growth and maturation

Our research effort in previous years
concentrated on the identification andcharacterization of the molecules (gonadotropin hormones and gonadal steroid
hormones) that stimulate and control
germ cell growth and maturation. It wasin 1985 that we identified, for the firsttime in any vertebrate, 17 a, 20 P-dihydro:xy-4-pregnen-3-one (17 a, 20P-DP)
as the maturation-inducing hormone ofamago salmon (Oncorhynchus rhodurus). Along with estradiol-17 p, which
was identified as the major mediator of

oocyte growth, we now have two known
biologically important mediators ofoocyte growth and maturation in female
salmonid fishes. It is established that the
granulosa cells are the site of productionof these two mediators, but their production by the ovarian follicle depends on
the provision of precursor steroids by thethecal cell (two-cell type model). A dramatic switch in the steroidogenic pathway from estradiol-17 P to 17 a, 20P-DP
occurs in ovarian follicle cells immediately prior to oocyte maturation. This
switch is a prerequisite step for thegrowing oocyte to enter the maturation
phase, and requires a complex and integrated network of gene regulation involving cell-specificity, hormonal regulation, and developmental patterning.
We have isolated and characterized
the eDNA encoding several ovarian steroidogenic enzymes of rainbow trout(Oncorhynchus mykiss) and medaka( Oryzias latipes) which are responsible
for estradiol-17P and 17a, 20P-DP biosynthesis: cholesterol side-chain cleavage
cytochrome P450 (P450scc), 3,8-hydroxysteroid dehydrogenase (3P-HSD), 17 ahydroxylase/C17, 20-lyase cytochrome
P450 (P450c17), P450 aromatase (P450arom) and 20P-hydroxysteroid dehydrogenase (20,8-HSD). These eDNA
clones have been used for Northern andwhole mount in situ hybridization to investigate the molecular basis of differential production of estradiol-17 p and17a, 20f1-DP during oocyte growth andmaturation in rainbow trout and medaka. In both species, P450scc andP450c17 (also 3P-HSD in rainbow trout)mRNA transcripts were increased in follicles towards the end of oocyte growthphase and during oocyte maturation.
Furthermore, incubations of isolated thecal layers with gonadotropin resulted in29

FOR BASIC BIOLOGY

the elevation of P450scc mRNA. The effect of gonadotropin becomes more dramatic when the expression of P450scc
mRNA is examined in granulosa cells.
P450scc mRNA is not detected in theabsence of gonadotropin, but markedly
expressed in the presence of gonadotropin. The increase in the amount ofP450scc, 3/3-HSD and P450c17 transcripts provide an explanation for thedramatic increase in 17 a, 20/3-DP production in follicles during naturally- andgonadotropin-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 estradiol17/3.
We have shown that 17a, 20/3-DP
acts via a receptor on the plasma membrane of oocytes. 17 a, 20,8-DP receptor
concentrations increase during oocyte
maturation. Our recent studies suggestthat inhibitory G-proteins are involvedin the signal transduction pathway of thematurational action of 17a, 20{3-DP infish oocytes. The early steps following
17a, 20{3-DP action involve the formation of the major mediator of this steroid, maturation-promoting factor or
metaphase-promoting factor (MPF).
MPF activity cycles during 17 a, 20/3DP-induced oocyte maturation with thehighest activity occurring at the first andsecond meiotic metaphase. Studies fromour laboratory and others have shownthat MPF activity is not species-specificand can be detected in both meiotic andmitotic cells of various organisms, fromyeast to mammals.
Fish MPF, like that of amphibians,
consists of two components, catalytic

cdc2 kinase (34-kDa) and regulatory cyclin B (46- to 48-kDa). Goldfish immature oocytes contain 35-kDa inactive
cdc2 kinase. Although immature oocytes
contain mRNA for cyclin B, they do notcontain cyclin B protein. 17a, 20,8-DP
induces oocytes to synthesize cyclin B.
The preexixting 35-kDa inactive cdc2 kinase binds to de novo synthesized cyclin
B at first, then is rapidly converted into
the 34-kDa active form. Introduction ofa bacterially produced goldfish cyclin B
into immature goldfish oocyte extracts
induces cdc2 kinase activation, concurrent with the shift in apparent molecularweight of cdc2 kinase from 35- to 34kDa, as found in oocytes matured with
17a, 20/3-DP. Phosphoamino acid analysis shows that threonine (Thr) phosphorylation of the 34-kDa cdc2 kinase
and serine phosphorylation of cyclin B
are associated with the activation. The
same phosphorylation is found in
oocytes matured by 17a, 20/3-DP. Cyclin B-induced cdc2 kinase activation is
not observed when threonine phosphorylation of cdc2 kinase and serine phosphorylation of cyclin B are inhibited byprotein kinase inhibitors, although thebinding of the 35-kDa cdc2 kinase to cyclin B occurs even in the presence of theinhibitors. In contrast, cdc2 kinase is activated by mutant cyclins that undergo
no serine phosphorylation during the activation. The site of threonine phosphorylation on cdc2 kinase was mapped toresidue Thr161. These findings indicatethat the Thr161 phosphorylation of cdc2
kinase, but not serine phosphorylation of
cyclin B, is required for cdc2 kinase(MPF) activation in goldfish oocyte (Fig.
1).

MPF activity decreases immediately
after fertilization, coinciding with the degradation of cyclin B protein. Purified

30

NATIONAL INSTITUTE

17a.,20~-Dihydroxy-4-pregnen-3-one
cdc2 kinaseYT•~lasma membrane'

1

Receptor!

Cyclin 8

mANA

t~
I

15

161

~

cdc2 kinase"'~~/L___jiD
L______@

Cyclin B

Cyclin 8 synthesis
Serine kinase(cdc2 kinase)Threonine kinase(p4QM015)!
Fig. 1. Current model of the formation and activation of maturation-promoting factor (MPF) during 17 a,
20,8-DP-induced meiotic maturation of fish oocytes. S, serine; T, tyrosine; Y, threonine; P, phosphorylation.

goldfish 26S proteasome, an ATP-dependent protease, can digest a bacterially
produced goldfish wild hype cyclin B,
producing an intermediate cyclin B (42kDa). In contrast, various cyclin B mutants lacking the first 42, 68, and 96
N-terminal amino acids are not digestedby the proteasome. Amino acid sequence analysis of the 42-kDa intermediate cyclin B reveals that 26S proteasome cleaves the C-terminal piptide
bond of lysine 57. This is the first evidence for the crucial role of 26S proteasome in cyclin B degradation.
II. Endocrine regulation of male germ
cell development and maturation
We have identified two steroidal
mediators of male germ cell develop-

ment in salmonid fishes (11-ketotestosterone for spermatogenesis and 17 a,
20P-DP for sperm maturation). A steroidogenic switch, from 11-ketotestosterone to 17a, 20P-DP, occurs in salmonid testes around the onset of final
maturation. In vitro incubation studies
using different testicular preparations
have revealed that the site of 17 a, 20{JDP production is in the sperm, but its
production depends on the provision of
precursor steroids by somatic cells. The
site of 11-ketotestosterone production isin the testicular somatic cells.
In the cultivated male Japanese eel(Auguilla japonica), spermatogonia are
the only germ cells present in the testis.
A serum-free, chemically defined organ

FOR BASIC BIOLOGY

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.
To isolate the genes that are expressedor suppressed in eel testes during HCGinduced spermatogenesis, we extractedmRNA from control testes and testes
that had been given a single injection of
HCG one day previously. Subtractive
eDNA libraries were constructed toclone specific cDNAs expressed at eachstage. So far, three eDNA clones havebeen isolated and characterized. From its
deduced amino acid sequence, one of
the up-regulated cDNAs was identified
as coding for the activin B subunit. We
used Northern blot analysis and in situ
hybridization techniques to examine sequential changes in transcripts of testicular activin B during HCG-induced spermatogenesis. No transcripts for activin B
were found in testes prior to HCG injection. In contrast, 3.3 kb mRNA transcripts were prominent in testes one day
after the injection. The transcript concentration began to decrease three days
after the injection and there was a further sharp decrease by nine days. The
HCG-dependent activin B mRNA expression in the testes was confirmed byin situ hybridization using a digoxigeninlabelled RNA probe: the signal was restricted to Sertoli cells in testes treatedwith HCG for one to three days. Taken
together, these results suggest that activin acts as a regulator of spermatogonial proliferation.
In salmonid fishes, spermatozoa takenfrom the testes are immotile, but acquire31
motility during their passage through thesperm duct. Using male masu salmon(Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17 a, 20,8-DP is responsible forthe acquisition of sperm motility; 17 a,
20,8-DP acts to increase sperm duct pH,
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:
Shibata, N., Yoshikuni, M. and Nagahama, Y. (1993) Vitellogenin incorporation into oocytes of rainbow trout,
Oncorhynchus mykiss, in vitro: effectof hormones on dunuded oocytes. Develop. Growth Differ. 35, 115-121.
Kamijo, M., Yasuda, H., Yau, P.M.,
Yamashita, M., Nagahama, Y. andOhba, Y. (1993) Preference of human
cdc2 kinase for peptide substrate. Peptide Res. 5, 281-285.
Yasuda, H., Nakata, T., Honda, R.,
Nakamura, M., Yamashita, M., Nagahama, Y. and Ohba, Y. (1993) Involvement of a cdc2-related cell division
kinase in S phase regulation in murine
cdc2 kinase Ts mutant. Somatic Cell
Mole. Genet. 18, 403-408.
Sakai, N., Tanaka, M., Takahashi, M.
and Nagahama, Y. (1993) Isolationand expression of rainbow trout (Oncorhynchus mykiss) ovarian eDNA
encoding 3,8-hydroxysteroid dehydrogenase/ ~ 5 -4 -isomerase.
Fish
Physiol. Biochem. 11, 273-279.

32
Yoshikuni, M., Shibata, N. and Nagahama, Y. (1993) Specific bindings of[ 3 H] 17 a, 20,8-dihydroxy-4-pregnen-3one to oocyte cortices of rainbow trout(Oncorhynchus mykiss). Fish Physiol.
Biochem. 11, 15-24.
Nagahama, Y., Yoshikuni, M., Yamashita, M., Sakai, N., Tanaka, M. andShibata, N. (1993) Molecular endocrinology of oocyte growth and maturation in fish. Fish. Physiol. Biochem.
11, 3-14.
Natsuyama, S., Noda, Y., Yamashita,M., Nagahama, Y. and Mori, T.
(1993) Superoxide dismutase andthioiredoxin restore defective p34cdc2
kinase activation in mouse two-cellblock. Biochem. Biophys. Acta 1176,
90-94.
Nakamura, M., Specker, J.L. and Nagahama, Y. (1993) Ultrastructural analysis of formation of a functional follicle during early vitellogenesis in thetilapia, Oreochromis niloticus. Cell
Tissue Res. 272, 33-39.
Nagahama, Y. (1993) Regulation ofoocyte maturation in aquatic animals:
the comparative and general aspects.
Biology International, Special Issue
28, 27-32.
Specker, J.L., Kishida, M., Huang, L.,
King, D.S., Nagahama, Y., Ueda, H.
and Anderson, T.R. (1993) Immunocytochemical and immunogold localization of two prolactin isoforms in thesame pituitary cells and in the same
granules in ,the tilapia (Oreochromis
mossambicus). Gen. Comp. Endocrinol. 89, 28-38.
Takahashi, M., Tanaka, M., Sakai, N.,
Adachi, S., Miller, W.L. and Nagahama, Y. (1993) Rainbow trout ovarian cholesterol side-chain cleavage
cytochrome P450 (P450scc): eDNA
cloning and mRNA expression during
oogenesis. FEES Letters 319, 45-48.

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Nakamura, M., Tsuchiya, F., Iwasaki, M.
and Nagahama, Y. (1993) Reproductive characteristics of precociously mature triploid male masu salmon, On-·
corhynchus masou. Zoo/. Sci. 10,
117-125.
Hasumi, M., Iwasawa, H. and Nagahama, Y. (1993) Seasonal changes inplasma concentrations of sex steroids
in the salamander Hynobius nigrescens. Gen. Comp. Endocrinol. 90,
51-57.
Lin, D., Black, S.M., Nagahama, Y. andMiller, W.L. (1993) Steroid 17 a-hydroxylase and 17, 20 lyase activities ofP450c17: contributions of serine106
and P450 reductase. Endocrinology
132, 2498-2506.
Kobayashi, T., Sakai, N., Adachi, S.,
Iwasawa, H., Asahina, K. and Nagahama, Y. (1993) 17a, 20,8-Dihydroxy-4-pregnen-3-one is the naturally occurring spermiation-inducing
hormone in the testis of a frog, Rana
nigromaculata. Endocrinology 133,
321-327.
Nakamura, M. and Nagahama, Y.
(1993) Ultrastructural study on the
differentiation and development ofsteroid-producing cells during ovariandifferentiation in the amago salmon,
Oncorhynchus rhodurus. Aquaculture
112, 237-251.
Tokumoto,T.,Yamashita,
M.,
Yoshikuni, M. and Nagahama, Y.
(1993) Changes in the activity andprotein levels of proteasomes duringoocyte maturation in goldfish (Carassius auratus). Biomed. Res. 14,
305-308.
Tokumoto, T., Kajiura, H., Yoshikuni,
M., Yamashita, M. and Nagahama, Y.
(1993) Purification of ubiquitin from
goldfish ( Carassius auratus) oocyte cytosol. Biomed. Res. 14, 309-312.

FOR BASIC BIOLOGY

Iwao, Y., Sakamoto, N., Takahara, K.,
Yamashita, M. and Nagahama, Y.
(1993) The egg nucleus regulates the
behavior of sperm nuclei as well as cycling of MPF in physiologically polyspermic newt eggs. Develop. Bioi.
160, 15-27.
Katsu, Y., Yamashita, M., Kajiura, H.
and Nagahama, Y. (1993) Behavior of
the components of maturation-promoting factor, cdc2 kinase and eyelin
B, during oocyte maturation of goldfish. Develop. Bioi. 160, 99-107.
Kajiura, H., Yamashita, M., Katsu, Y.
and Nagahama, Y. (1993) Isolationand characterization of goldfish cdc2,
a catalytic component of maturationpromoting factor. Develop. GrowthDiffer. 35, 647-654.

33
Yamashita, M., Jiang Jianqiao, Onozato,
H., Nakanishi T. and Nagahama, Y.
(1993) A tripolar spindle formed atmeiosis I assures the retention of theoriginal ploidy in the gynogenetic triploid crusian carp, ginbuna Carassius
auratus langsdorfii. Develop. GrowthDiffer. ~5, 631-636.
Onoe, S., Yamashita, M., Kajiura, H.,
Katsu, Y., Jiang, J. and Nagahama, Y.
(1993) A fish homolog of the cdc2-related protein p40M015 : its eDNA cloning and expression in oocytes.
Biomed. Res. 14,441-444.
Iwamatsu, T., Toya, Y., Sakai, N.,
Terada, Y., Nagata, R. and Nagahama, Y. (1993) Effect of 5-hydroxytryptamine on steroidogenesis andoocyte maturation in preovulatory follicles of the medaka Oryzias latipes.
Develop.
GrowthDiffer.
35,
625-630.

34

NATIONAL INSTITUTEDMSION OF CELL
DIFFERENTIATION
Professor: Y oshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Shigeharu Takiya
Toshiharu Suzuki
Kaoru Ohno
JSPS Postdoctoral Fellows: Kazuhito Amanai
VdclavMach
Visiting Scientists: Masakazu Fukuta (from Aichi
University of Education)
Pin-XianXu
Graduate Students:
Kaoru Ohno (Graduate University for
Advanced Studies)
Pin-Xian Xu (Graduate University for
Advanced Studies)
Hiraki Kokubo (Graduate University for
Advanced Studies)
Xin Xu (Graduate University for Advanced
Studies)
Technical Staffs: Miyuki Ohkubo
Chikako Inoue

Members of the Division have been
involved in two well associated projects.
One, which was initiated in 1968, is tounderstand how a special tissue like thesilk gland of Bombyx mori is differentiated along the developmental programs
and results in transcribing a specific setof genes like the silk fibroin, fibroin
L-chain, P 25 , sericin-1, and sericin-2
genes. The other initiated at the time
when the Division was established in1978 is concerned with how the bodyplan of the silkworm is controlled andhow the developmental regulatory genes
regulate a set of target genes in specifying the identities of various regions of
the embryos.
I. Genes and factors that control thesilk gland development and the silk
genes transcription
We have been trying to understandthe networks of gene regulation hierarchy that function in the processes ofsilk gland development and differentiation. As a bottom-up type approach forthis project, analyses on the molecularmechanisms that control the differential
transcription of the fibroin and sericin-1
genes in the silk gland should shed lighton a part of the networks. In complementing this approach, a top-down typeapproach should also help understanding
the networks; analyses of regulation
hierarchy of the homeobox genes andother regulatory genes, and identificationof their target genes expressed in the labial segment, where the silk gland isoriginated.
Among many factors proposed to bindand control the fibroin and sericin-1
genes, the POU-M1 which accommodates a POD-domain identical to Drosophila Cfl-a was cloned previously andcharacterized. The POU-M1 binds to the
SC region of the sericin-1 gene and is assumed to enhance the transcription. This
protein also binds to the PB element of
the POU-Ml gene and suppreses thetranscription. The expression of the
POU-Ml gene has been analyzed in
Bombyx embryos by in situ hybridization. To our surprise, the gene was expressed specifically for the first time atstage 18-19 (Fig. 1C) in the limited siteof the labial segment where the silk
gland is going to be formed by invagination (Fig. 1A). This location exactly
matches with the site where the Bombyx
Scr expression disappears specifically(Fig. 1B) which was detected in the entire labial segment in the preceding stage(see the section II). The POU-Ml expression continues along with the silk
gland development and is confined tothe middle portion of the silk gland bylate embryonic stages. It was late in thestage 22 when the POU-Ml expression
was also detected in the central nervous
system as expected for a Cfl-a homologue. These observations suggest that
the POU-Ml gene may have multiple

FOR BASIC BIOLOGY

35

Fig. 1 Gene expression at the site of the silk gland invagination at stage 19.
A. A sagittal section stained with hematoxylin-eosine. B. Hybridization with a Bombyx Scr probe. C. Hybridization with a POU-Ml probe. The arrowhead indicates an invagination of the silk gland. The bar indicates 50 urn.

functions; (1) contribution to the commitment of the primordial silk glandcells, (2) roles in maintenance of the silk
gland development, (3) roles in establishing terminally differentiated states as
a positive transcription factor for the
sericin-1 gene and a negative transcription factor for its own gene, and (4) contribution to the commitment of the nerve
cells.

Silk gland specific transcription factor
SGF-1 interacts with the SA, FA andFB sites localized upstream of thesericin-1 and fibroin gene promoters.
Partial purification of this factor wasachieved using SA site-DNA affinity
resin. Competitive gel shift assay using
proteins renatured from SDS-PAGE
showed that SGF-1 corresponds to two
polypeptides migrating on 42 and 43

36

NATIONAL INSTITUTE

B

A

F

R

F

F

R

F

c
ATACATTGTTTGCACAAATG
TATGTAACAAACGTGTTTAC

Fig. 2. SGF-1 interaction with SA site.
Mildly methylated DNA was used as a probe for gel shift assay. DNA extracted from free probe (F) or retarded band (R) was cleaved and resolved in a sequencing gel. A. Footprint of the coding strand using pure
42 kDa protein. B. Footprint of the noncoding strand using protein fraction obtained after DNA affinity
resin. C. SA site in the extend previously identified by DNasel footprinting. G and A residues the methylationof which actually affect SGF-1 binding are indicated in bold. These residues are marked by arrowheads in A
and B.

kDa, respectively. In the next purification step, these two proteins were recovered in a single RPC fraction of morethan 80% purity. The 42 and 43 kDa
proteins were digested directly in SDSp AGE gel, and peptides were eluted,
fractionated and sequenced. Four ofthem yielded useful sequences, whichshould facilitate molecular cloning of
SGF-1 gene. The pure 42 and 43 kDa
proteins were used to study the SA bind-ing site by the methylation interference
assay (Fig. 2). Their footprints appear
identical with each other and with thefootprint obtained from the specific retarded band produced by the SA probe
in a crude extract.
Intronic modulator (enhancer II;+156 / +454) of the fibroin gene is composed of 6 octamer-like elements. The
octamer binding factor-1 (OBF-1) binds
to the elements at around +220 andFOR BASIC BIOLOGY+ 290 in the enhancer II, and also to theelement at around -130 in the upstream
enhancer (enhancer I). The OBF-1 activity was detected only in the posterior silk
gland abundantly at stages only when the
fibroin gene was expressed. Purificationof OBF-1 is in progress and 32 kDa protein recovered from SDS-PAGE showed
the OBF-1 activity. The second octamer
binding factor (OBF-2) binds strongly tothe element at around +420 and weakly
to other elements at around +220, +290
and +370. The OBF-2 binds also to theenhancer I weakly. Gel shift assay using
a POU-M1 specific antibody showedthat the OBF-2 is the POU-Ml. The
third octamer binding factor (OBF-3)
binds to the elements at around +220
and +290, and also to an element ataround -60. An oligonucleotide corresponding to the +290 element competed
the transcription enhancement both by
the enhancer I and II in the posterior silk
gland extracts. Integration of these factors as well as FF1 and 2 which were
purified before will be important for
regulation of the fibroin gene transcription.
II. Genes involved in the Bombyx bodyplan
We have isolated a caudal (cad) homologue from a eDNA library of Bombyx mori embryos. The Bombyx cadtranscripts were firstly accumulated in
the nurse cells and transferred into the
oocyte in a definite period during
oogenesis. The maternal transcripts
formed a concentration gradient spanning anteroposterior axis during the gastrulation stage and were restricted to theanal pad after 2 days of embryogenesis(Development 120, 277-285 (1994)).
This observation gives a sharp contrast
with the Drosophila cad expression pat-37
tern which reveals the corresponding expression profile during the syncytial blastoderm stage. The Bombyx cad protein
was not detected in the ovary and early
9 hrs of eggs, but was first detected
evenly during cellular blastoderm stage.
It was during gastrulation when Bombyx
cad protein concentration gradient
shifted along the anteroposterior axis.
The observed distinct timing and conservation on mRNA as well as protein gradients formation between Drosophila
and Bombyx might contribute to realize
differences in the body plans and givesome clues to elucidate the mechanismand function related to mRNA and protein concentration gradients.
To understand how the labial segment
identity is determined and the silk glanddevelopment is controlled, we havebegun characterizing expression patternsof Bombyx Sex combs reduced (Scr), Deformed (Dfd), fork head (Jkh), and
POU-Ml. The Bombyx Dfd was expressed in the mandibular and maxillar
segments but not in the labial segment
where the Bombyx Scr was specifically
expressed. As described in the previous
section, the Bombyx Scr expression waseliminated in the invagination site where
the silk gland development takes place.
This elimination was complemented withthe specific expression of POU-Ml.
During the silk gland development the
POU-Ml expression was detected in theentire region of the gland in the earlyphase, and restricted to the anterior portion and middle portion of the gland andfinally to the middle portion, while theBombyx fkh expression was detected in
the middle and posterior portions in the
middle phase of development. These observations may outline the framework of
the hierarchy in the silk gland development and differentiation.

38
In continuation of the abdominal segments identification, we have concentrated in the study of morphogenesis ofembryonic abdominal legs. We haveanalyzed proteins in the wild type embryos by SDS-PAGE, and found that a
270 kDa protein (p270) is expressed
specifically in the abdominal legs. We
have purified the p270 from embryos,and prepared a specific antibody against
the p270. Using the antibody no p270
was detected by Western blot analysis in
the homozygous Eca /Eca embryos which
do not carry the Bombyx abd-A gene. It
is likely that the p270 expression isunder the control of the Bombyx abd-A
gene. Immunohistochemical analysis indicated that the p270 is localized in restricted cells of the wild type abdominal

NATIONAL INSTITUTE

legs but not detected in the late embryonic stages. To study a role of the
p270 in morphogenesis of embryonic abdominal legs, molecular cloning of p270
eDNA is being planned.

Publication List:
Fukuta, M., Matsuno, K., Hui, C.-c.,
Nagata, T., Takiya, S., Xu, P.-X.,
Ueno, K. and Suzuki, Y. (1993)
Molecular cloning of a POU domaincontaining factor involved in the regulation of the Bombyx sericin-1 gene. J.
Bioi. Chern. 268, 19471-19475.
Takiya, S. and Suzuki, Y. (1993) Role ofthe core promoter for the preferential
transcription of fibroin gene in theposterior silk gland extract. Develop.
Growth Differ. 35, 311-321.

FOR BASIC BIOLOGYDNISION OF MORPHOGENESIS
Professor: Goro Eguchi
Associate Professor: Ryuji Kodama
Research Associate: Makoto Mochii
JEPS Postdoctoral Fellow: Keiko Ishikawa
Visiting Scientists:
Takamasa S. Yamamoto
Kaichiro Sawada
Christine Baader(from Zoological Institute, University of Basel)
Graduate Students:
Akio Iio (Graduate University for Advanced
Studies)
Nobuhiko Mizuno (Graduate University for
Advanced Studies)
Yuuichi Mazaki (Graduate University for Advanced Studies)
Takeshi Kitagawa (from School of Medicine,
Nagoya University)
Jatupol Kosittsawat (from School of Medicine,
University of Tokyo)
Yasutaka Matsubara (from Shinshu University)
Visiting Researcher: Hiroyuki Horiuchi (Research
Associate, Faculty of Agriculture, Hiroshima University)
Technical Staffs: Chiyo 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 species of the vertebrate, the lens and neural retina can becompletely regenerated through thetransdifferentiation of pigmented epithelial cells (PECs). Such a phenomenon,transdifferentiation, as observed in regeneration of ocular tissues seems to bea highly powerful model for studying
stability and instability in differentiationof tissue cells. From this view point, lenstransdifferentiaton of PECs of vertebrates has been studied in in vivo and invitro systems, and our in vitro studies
have revealed that dormant potential totransdifferentiate into lens cells is widelyconserved throughout vertebrate species
including human.
Our studies have been conducted toclarify the molecular mechanism con-39
trolling the lens transdifferentiation of
PECs and also to search the reason why
the pigmented epithelia of species otherthan the newt and so forth never regenerate the lens in the in situ eyes.
Based on findings accumulated up to thelast year, we have conducted analysis of
the lens transdifferentiation, particularly
focussing on genes which have been predicted to have essential roles in regulation of the differentiated state of PECs
and also of lens transdifferentiation ofthem. The followings are abstracts of investigations and results obtained in 1993
through studies of these major projects
and the following additional subjects
which have been conducted as collaborative works with scientists from the outside: (1) Pattern formation of Lepidopteran wing and (2) Basic analysis of biocompatibility of hydroxyapatite as a biomaterial.
I. Analysis of functions of genes responsible for the lens transdifferentiation of PECs
It has been predicted by analysis thus
far that products of two genes, tentatively designated as pP344 and pP64
must function to regulate the differentiated state and lens transdifferentiationof PECs in vivo and in vitro. In this year,
we have extended our study to analyze
functions of these two genes. We analyzed pP344 gene expression during
chicken eye development by RT-PCR
and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. The time course of
expression level showed two peaks; thefirst peak occurred around the lOth day
similarly to the expression of melanosome-related genes, while the second
peak occurred just after hatching when
PECs had completely differentiated, sug-40
gesting that pP344 gene may be related
to the function of fully differentiatedPECs. Anti-synthetic peptide antibodies
detected pP344 protein in the culture
medium of the PECs but not within thecells, strongly suggesting that pP344
gene product is a secreted protein. The
pP64 gene produces two different transcripts, 5.0kb and 6.0kb mRNAs, whose
products are TGFf3-binding proteins,and fully differentiated PECs express
5.0kb m.RNA as a major product in addition to 6.0kb mRNA but both multipotent dedifferentiated PECs and transdifferentiated lens cells express only
6.0kb mRNA. It has been clearly shownthat protein produced by 5.0kb mRNAis secreted by PECs but protein produced by 6.0kb m.RNA is trapped by the
extracellular matrix of PECs in the insitu eye. These results must be the fundamental information for our further
studies on the molecular regulation of
the lens transdifferentiation of PECs.
II. Analysis of transcriptional regulatorin pigmented epithelial cells
The product of mouse microphtalmia(mi) gene is thought to be a class of basic
helix-loop-helix-zip transcriptional factor
which may regulate the directions of differentiation of some types of cells including pigmented epithalial cells, because some mutations in mi gene cause
transformation of pigmented epithelium
to neural retina in mice. To analyze themolecular mechanisms in differentiationand transdifferentiation of cultured pigmented epithelial cell, avian homologs ofmi gene were isolated from eDNA libraries of chicken and quail pigmented
epithelial cells. Nucleotide and aminoacid sequences of mi are well conserved
between aves and mammals. The products of mi genes have a basic helix-

NATIONAL INSTITUTE

loop-helix-zip domain similar to ubiquitous transcriptional regulators TFE3,
TFEB and TFEC, showing possible interactions of mi product and some factors relating the TEFs in gene regulation.
Northern blotting shows activation of mi
gene during differentiation of pigmented
epithelial cells and inactivation of it indedifferentiated state of cells suggesting
the key role of mi gene in differentiationof pigmented epithelial cells. The possibility in which transdifferentiation of pigmented epithelial cells may be regulatedby activity of mi product is now analyzed
by genetic manipulation of mi gene incultured cells.

m. Expression of connexin in the pigmented epithelial cells
The morphological change of the gap
junction in the course of the transdifferentiation of PECs of the chick embryo
was previously studied. In essence, although the gap junction is abundantly
found in the PECs cultured in vitro, it is
temporally lost in the dedifferentiatedPECs but reappear in the lentoids or redifferentiated PECs. Molecular biological analysis showed that the gap junctionin the PECs is made up of chicken connexin 43, which is one of the genes forthe main component of the gap junction.
We are preparing probes for other connexin genes in order to clarify the expression pattern of connexin genes in thecourse of tninsdifferentiation. Here wesummarize some preliminary data on
connexins.
Polyclonal antibodies against connexin
43 were raised against synthetic peptides. One of the antibody against a cytoplasmic loop reacted specifically andcan serve as a good marker. Anotherantibody against an extracellular loop reacted from outside of the cell. This reac-

FOR BASIC BIOLOGY

tion was shown by directly applying theantibody to living cells, and then detecting the bound antibody with fluorescentlabeled second antibody. This result suggests that it may be possible to modify
the gap junctional cell-to-cell communication from outside of the cell, thus
enabling the inhibition of gap junction inlarge population of cultured cells.
The pattern of connexin 43 gene expression was examined in the eyes of the
9-day-old chick embryo by in situ hybridization technique. Although the expression is seen throughout the pigmented epithelium, the expression isconspicuously strong in the iris and theciliary pigmented epithelium. The immunofluorescent staining with anti-connexin
43 peptide antibody also showed highlevel of connexin 43 protein in the irisand ciliary pigmented epithelium. The
expression pattern of a PEC-specificgene, pP344, which was found in our laboratory, showed an opposite gradient ofexpression, i.e. highest expression is seenin the retinal PE. These genes can be excellent markers for the regional specificity of the pigmented epithelium andalso it is possible that they bear someroles in the physiological activity of eachepithelium.
IV. Analyses of the process of programmed cell death in the pupal wingof Pieris rapae
The shape of the wings of butterflies
are formed by an extensive cell death at
the margin of the wings during the pupal
period. We have suggested, throughultrastructural observations of the pupal
wings of Piris rapae, that the cell death
observed here resemble the apoptosis,
which is a characteristic form of programmed cell death reported to occur
throughout the development of verte-

41
brates and invertebrate.
One of the critical symptom of theapoptosis is the fragmentation of the
DNA in the nucleus prior to the death,but examination of such fragmentation
by standard method using the electrophoresis must be difficult because theamount of the tissue is very limited. We
recently adopted the TUNEL method(TdT-mediated dUTP-biotin nick endlabelling) to detect the fragmentation of
DNA in in situ tissue. This procedureadds biotinylated dUTP at existing 3'
ends by an enzyme terminal deoxynucleotidyl transferase. The nuclei containing DNA with many breaks has much
more number of 3' ends than other nuclei, so that much more biotinyl residues
are incorporated. The biotinyl residue isdetected by avidin-biotinyl-horse raddish
peroxidase complex (ABC) yielding
staining on the nuclei with DNA fragmentation. We applied this method to
the whole mount preparation of thepupal wing and showed that nuclei withpositive staining are observed at theperiod and the area of extensive cell
death formerly observed ultrastructurally(Fig.). Although this method provides noinformation on the length of the DNA
fragments, this result strongly suggeststhat there works a mechanism of cell
death including DNA fragmentation resembling the apoptosis in the process ofthe formation of the wing of butterflies.
We also re-examined ultrastructural
data and concluded that cells with amorphology of macrophages are abundantly seen near the area of cell death,suggesting that the dead cells are actively
phagocytosed by these macrophages.
We have concluded at present that theloss of the margin of the pupal wing proceeds by the programmed cell death ofthe epithelial cells at the margin and by

42

NATIONAL INSTITUTE

'
I

f.

) I -;

- }'
•

---

-- y
I

--

I~

--

).

....

_...
·. '

- '·

Fig. The nuclei in which fragmentation of DNA occurrs are stained by TUNEL method (a). They arefound only in the margin of the pupal wing. In the control which were pretreated with DNase I, all the nuclei
are stained (b).

the removal of cell debris through phagocytosis by neighboring epithelial cells
and by macrophages. The molecularmechanism determining the area to be
removed and how this determination isrealized as cell death are two important
aspects of further studies.
V. An attempt to improve biocompatibility of hydroxyapatite as a biomaterial
Based on findings of cell biologicalanalysis conducted thus far, we have attempted to improve biocompatibility ofhydroxyapatite using in vitro culture system of human gingival cells establishedin our laboratory. Adhesion, spreading
and growth of gingival cells, epithelial
cells, and connective tissue fibroblasts,
cultured on the hydroxyapatite can bedramatically enhanced when the hydroxyapatite surface is modified by coatingwith type I collagen molecules after ionetching under condition of 10-2 mmHg
with an ion-sputtering equipment, sug-gesting a strong possibility to improvebiocompatibility of hydroxyapatite materials by biochemical modification of its
surface.
Publication List:
Eguchi, G. and Kodama, R. (1993)
Transdifferentiation. Curr. Opin. Cell
Bioi. 5, 319-325.
Hyuga, M., Kodama, R. and Eguchi, G.
(1993) Basic fibroblast growth factor
as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells. Int. f. Dev.
Bioi. 37, 319-326.
Agata, K., Kobayashi, H., ltoh, Y., Mochii, M., Sawada, K. and Eguchi, G.
(1993) Genetic characterization of themultipotent dedifferentiated state ofpigmented epithelial cells in vitro. Development 118, 1025-1030.
Sawada, K., Agata, K., Yoshiki, A. andEguchi, G. (1993) A set of anti-crystallin monoclonal antibodies for detecting lens specificities: ~-crystallin asFOR BASIC BIOLOGY

a specific marker for detecting lentoidogenesis in cultures of chicken lensepithelial cells. lap. J. Ophthalmol.
37, 355-368.
Orii, H., Agata, K., Sawada, K., Eguchi,
G. and Maisel, H. (1993) Evidence
that the chick lens cytoskeletal protein
CP49 belongs to the family of inter-43
mediate filament proteins. Curr. Eye
Res. 6, 583-588.
Araki, M., Kodama, R., Eguchi, G.,
Yasujima, M., Orii, H. and Watanabe,
K. (1993) Retinal differentiation frommultipotential pineal cells of the embryonic quail. Neurosci. Res. 18,
63-72.

44

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL
BIOLOGY (ADJUNCT)
Professor: Masaki Iwabuchi
Associate Professor: Masao Tasaka
Research Associates: Koji Mikami
Takefumi Kawata
JSPS Postdoctoral Fellow: Hisabumi Takase
Visiting Scientist: Shweta Saran (from Universityof Delhi, India)
Graduate Students: Takuya Ito (from Kyoto Univercity)
Ken-ichiro Taoka (from Kyoto
Univercity)

Cell division is the most fundamental
event in cell growth and cell differentiation during development. One of powerfull approaches for understanding of themolecular mechanisms of cell division is
to investigate the gene expression regulated in a cell cycle-dependent manner.
Representative examples of eukaryotic
cell cycle-dependent genes are histone
genes, expressed mainly in the S phaseduring the cell cycle. We have identified
the cis-acting hexamer (ACGTCA)
motif involved in S phase-specific transcription of the wheat histone H3(TH012) gene, and bZIP-class trans-acting factors specific to the hexamer
motif. Of bZIP proteins, HBP-la(17)and HBP-lb(c38) are known to be essential for controlling periodic transcription of the H3 gene, because genes fortwo proteins are also regulated in a cell
cycle-dependent manner. It is, therefore,
likely that HBP-la(17) and HBPlb(c38) function as regulators of cell division in plants. In 1993, we focused ourattention on the functional significanceof bZIP proteins during plant development (summarized in the sections I andII).

We also have been continuously studying the transcriptional regulation ofwheat histone genes from a different
viewpoint. Based on the function inchromatin formation, histones are classi-

fied into core (H2A, H2B, H3, and H4)
and linker (Hl) proteins. Despite differences in their functions, core and linker histone genes are coordinately expressed in S phase during the cell cycle.
To understand how a coordinate expression of core and linker histone genes
is regulated, we investigated the regulatory mechanisms of the wheat histone
Hl (TH315) gene (surmmarized in thesection III).
I. Regulation of a gene encoding transcription factor HBP-1a(17) in transgenic Arabidopsis
To analyze how the HBP-la(17) and
HBP-lb(c38) genes are regulated invivo, we have produced transgenic
Arabidopsis plants earring chimericgenes which are composed of the HBPla(17) or HBP-lb(c38) promoter andthe p-glucronidase (GUS) coding sequence. The expression patterns of thechimeric genes were histochemocallyanalyzed during development from germination to seed production. Unfortunately, the HBP-lb(c38) promoter/GUS
chimeric gene was not expressed in anytransgenic plants. We, therefore, examined the spacial and temporal expression
pattern of the HBP-la(17) promoterI
GUS fusion gene.
In germinating seedlings, the HBPla(17) promoterI GUS fusion gene wasexpressed in only cotyledons and thereafter in cotyledons and first leaves (Figs.
lA and lB). At eight days after germination, GUS activity was detected
mainly in first and second leaves, although weak GUS activity was still observed in expanded cotyledons (Figs.
lC). The gene expression seemed to decrease as the leaves became older. As
plants progressed from vegitative to productive growth, GUS activity was not

45

FOR BASIC BIOLOGY

detected in floral organs, stems, cauline
leaves and axillary buds (Figs. lD andlE), indicating that the HBP-la(17) promoter is activated in only developing
young leaves in the vegitative growthstage.
We next investigated the effect of lighton the HBP-la(17) expression during
germination. When seven-day-old seedlings grown in light were placed in thedark for a week, GUS activity in young
leaves was decreased (Fig. lF). Sincedark-grown seedlings showed no GUS
activity in any organs (data not shown),
the above result indicated that the expression of the HBP-la(17) gene is controlled by light. This conclusion is

supported by the fact that the HBPla(17) promoter has several characteristic sequences such as boxll, 1-box,
G-box and GATA motif, all of which
are known to be involved in the lightregulated transcnptwn. Since HBPla(17) can specifically interact with the
G-box, we supposed that HBP-la(17) is.implicated in the transcriptional regulation of light-inducible genes in the earlystage of photomorphogenesis as well ascell cycle-dependent gene expression.
This possibility is beeing checked using
plants produced by mating HBPla(17)-expressing transgenic Arabidopsis with several phytochrome-deficent
Arabidopsis mutants.

8

D

F

Fig. 1. Vegitative leaf-specific expression of the HBP-la(17) promoter/ GUS fusion gene in transgenic
Arabidopsis during development.
The spacial and temporal expression pattern of the cheimeric gene was analyzed by histochemical assays
for GUS expression. A , Three-day-old seeding; B, Seven-day-old seeding; C, Eight-day-old seeding; D, Floral organ (mature flower); E, Stem, cauline leaf and axillary bud; F, Plant grown in the dark for a week after
growing in light for a week.

46

NATIONAL INSTITUTE

ll. Functional analysis of HBP-lb(c38)
When the HBP-lb(c38)-expression
plasmid was co-transfected into tobacco
BY-2 cells with reporter plasmids containing the binding sites of HBP-lb(c38),
expression of the reporter gene was repressed. This suggests that HBP-lb(c38)
may act as a transcriptional repressor.
To elucidate the functional roles of
HBP-lb(c38) in plant development, wehave been trying to produce transgenic
Arabidopsis which can overexpress
Arabidopsis HBP-lb(c38)-homologue or
its antisense RNA.

m. Cis-control elements for S phasespecific expression of the wheat histone
Hl geneTo study cell cycle-dependent transcription of the wheat Hl gene, cultured
rice cells were transformed with aAchimeric gene which consists of the GUS
coding sequence and the 5' upstream sequence of the Hl gene spanning from-771 to +74. When cell cycle progression of the stable transformants waspartially synchronized by aphidicolintreatment, the GUS mRNA level was
peaked at 2hr after the removal of drug
and then gradualy decreased (Fig. 2A).
Considering a result of DNA synthesis
rate in aphidicolin-treated cells, we concluded that the wheat Hl gene expression is regulated in an S phase-specificmanner in rice cells.
The Hl promoter that was 5'-deleted
to the position -153 still had the ability
to regulate S phase-specific transcription,although the level of the GUS mRNAtranscribed from the 5' -deleted promoter
appeared to be maximum at lhr after
the removal of aphidicolin (Fig. 2B). The

B
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Time after release from aphidicolin exposure (hr)Fig. 2. S phase-specific expression of the wheat Hl promoter/GUS fusion gene in partially synchronizedrice cells.
Transformed rice cells were partially synchronized by aphidicolin treatment and then released into the Sphase. Accumulation and turnover of the mRNA transcribed from the wheat Hl promoter/GUS fusion gene
was analyzed by the Sl protection assays. Quantity of mRNA was corrected for that of constitutively transcribed HPH(hygromycin phosphotransferase) mRNA. DNA synthesis rates were monitored in parallel bymeasuring the incorporation of [3H)TdR. A, accumulation and turnover of the transcript from the 5' Ll-771(full size) promoter/GUS fusion gene; B, accumulation and turnover of the transcript from the 5'Ll-153 Hlpromoter/GUS fusion gene.

FOR BASIC BIOLOGY

proximal promoter region (-153 to+74) contains several characteristic sequences, conserved in the Arabidopsis
H1 promoters as well. We are now doing
more detailed experiments to identify
the cis-element directing S phase-specifictranscription of the wheat H1 gene.
Publication List:(1) Original articles
Minami, M., Huh, G. -H., Yang, P. andIwabuchi, M. (1993) Coordinate gene
expression of five subclass histones
and the putative transcription factors,
HBP-1a and HBP-1b, of histone
genes in wheat. Plant Mol. Bioi. 23,
429-434.
Nasuda, S., Liu, Y.-G., Sakamoto, A.,
Nakayama, T., Iwabuchi, M. andTsunewaki, K. (1993) Chromosomal
locations of the genes for histones anda histone gene-binding protein family
HBP-1 in common wheat. Plant Mol.
Bioi. 22, 603-614.
Ohtsubo, N., Nakayama, T., Terada, R.,
Shimamoto, K. and Iwabuchi, M(1993) Proximal promoter region of
the wheat histone H3 gene confers S
phase-specific gene expression intransformed rice cells. Plant Mol. Bioi.
23, 553-565.
Ozaki, T., Nakao, H., Takeuchi, I. andTasaka, M. (1993) Developmental
regulation of transcription of a novel
prespore-specific gene (Dp87) in Dictyosterium discoideum. Development
117, 1299-1308.

47
Sakamoto, A., Minami, M., Huh, G. -H.
and Iwabuchi, M. (1993) The putative
zing-finger protein WZF1 interacts
with a cis-acting element of wheat histone genes. Eur. J. Biochem. 217,
1049-1056.
Terada, R., Nakayama, T., Iwabuchi, M.
and Shimamoto, K. (1993) A wheat
histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants. Plant J. 3, 241-252.
(2) Reviews etc.
Mikami, K. and lwabuchi, M. (1993)
Regulation of cell cycle-dependent
gene expression. In Control of Plant
Gene Expression (Verma, D.P.S. ed.),CRC Press, Boca RatonFL''
pp.51-68.
Mikami, K., Takase, H. and Iwabuchi'
M. (1993) Gel mobility shift assay. In
Plant Molecular Biology Manual (G.
Jonker, ed. ), Kluwer Acad. Publ., inpress.
Nakayama, T. and Iwabuchi, M. (1993)
Regulation of wheat histone gene expression. Critical Reviews in Plant
Science 12, 97-110.
Takase, H. and Iwabuchi, M. (1993)
Transcriptional and post-transcriptional regulation of the expression ofwheat histone genes; Cis-acting elements and trans-acting factors. J.
Plant Res. Special Issue 3, 37-SP

49

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Norio Murata

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

50

NATIONAL INSTITUTE

DNISION OF MOLECULARNEUROBIOLOGY
Professor: Masaharu Noda
Research Associates: Nobuaki Maeda
Shinji Hirano
Graduate Students: Hiraki Hamanaka
Jun-ichi Yuasa
Visiting Fellow: Haruyuki Matsunaga*
Technical Staff: Tomoko Mori
Shigemi Ohsugi(*from Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd.)The principal interest of this division ismolecular mechanisms that govern thedevelopment of the vertebrate central
nervous system. Our efforts are currently
focused on projects to reveal the molecular bases of neuronal cell migrationand neuronal network formation.

I. Molecular basis of neuroblast migration
In the developing central nervous system, migration of post-mitotic neurons is
the key process through which laminated
and/ or nucleated structures form out of
the uniform neuroepithelium. Thus, toestablish the functional neuronal network, the course of neuroblast migrationmust be precise with the combination ofradial and tangential movement. Although many adhesion molecules are
known to be expressed in the neuraltissue, the molecular basis of neuroblast
migration remains to be elucidated.
T61 antigen is a good candidate of themolecule which is responsible for theneuroblast migration in the radial direction. Monoclonal antibody T61 was originally characterized by its inhibitory activity on neurite outgrowth from chickretinal explants in vitro. However, itturned out recently that neuroblast
migration was inhibited when the T61
producing hybridoma was injected in theventricle of the developing midbrain ofchick embryos. As far as we know, this is
the first example in which an antibody
inhibited neuroblast migration in vivo.
The expression of T61 antigen is restricted to the neural tissue, and the
distribution within the tissue is rather
uniform. Immunocytechemistry of primary culture cells showed that only
neuronal cells express the antigen. The
finding that T61 antigen is concentrated
at the filopdia of growth cone is consistent with the presumptive function of thismolecule in the cell migration. The T61
detects several bands including a major
440kD band by Western blotting andimmunoprecipitation. These results suggest that T61 antigen is a novel molecule. Screening of Agt 11 library prepared from chick brain with T61 gave
rise to many positive signals with various
intensity. Nucleotide sequence analysis
revealed that these clones are derived
from several different mRNAs. We are
now trying to identify the authentic
eDNA clone from them.

II. Proteoglycan and brain development
Proteoglycans have been recognized toplay important roles in the regulation ofcell growth, differentiation and adhesion.
The vertebrate brain also contains manykinds of proteoglycans, which are considered to be involved in the neuronalmigration, axon guidance and axonal
outgrowth.
6B4 proteoglycan is a brain specificlarge chondroitin sulfate proteoglycan
with a 300-kDa core protein. This proteoglycan has the soluble and membrane-bound forms, and its expression is
dynamically regulated during development of the brain. In the adult rat hindbrain, 6B4 proteoglycan is selectively expressed around the neurons constituting
the cerebellar mossy fiber system. In the

FOR BASIC BIOLOG Y

51

Fig. 1. Cocultures of slices from cerebellar cortex and pontine nuclei. Fluorescent micrographs to illustrate
Purkinje cells (A) and fibers from pontine nuclei (B) in cerebellar slices. PC, Purkinje cell; DN, deep cerebellar nuclei; W, white matter.

early postnatal animals, the expression of
6B4 proteoglycan is highly correlatedwith the synapse formation of this system, suggesting that 6B4 proteoglycan
plays important roles in the cerebellar
circuit formation.
To investigate this possibility, we developed an in vitro reconstitution systemof cerebellar circuits, in which slices of
cerebellum and pontine nuclei were cocultured. The latter is a major precerebellar nuclei from which many mossy
fibers project to the cerebellum in vivo.
During long-term culture (2-3 weeks),
Purkinje cells developed well-arboreddendrites and projected to the deep
cerebellar nuclei as revealed by antiInsP3 receptor monoclonal antibody
staining (Fig. lA). On the other hand,the pontine fibers projected to the cerebellar granule cell layer as shown by Dii
staining (Fig. lB). Such a projection pattern is very similar to that observed invivo. We are now studying the functionof 6B4 proteoglycan in the fiber projection and the neural circuit formation
using this system.
Publication List:
Noda, M. (1993) Structure and functionof sodium channels. In Molecular
Basis of Ion channels and Receptors
Involved in Nerve Exitation, Synaptic
Transmission and Muscle Contractions. Annals of The New York
Academy of Sciences. Vol. 707, pp.
20- 37.

52

NATIONAL INSTITUTEDIVISION OF CELLULARREGULATION
Professor: Norio Murata
Associate Professor: Hidenori Hayashi
Research Associates: Takao Kondo
Ikuo Nishida
Dmitry A. Los
Monbusho Foreign Scientist:
George C. Papageorgiou1'
JSPS Fellow: Wei-Ai Su 2l
Visiting Scientists: Yasushi Tasaka
Michael P. Ma/akhov3J
Zo/tan Gombosfl
Marie-Helene Machere[5J
Ding-Ji Shi6l
Byoung Yong Moon 7l
Ana Maria Otero Casaf!J
Tomoko Shinomura 9l
Masaya Ishikawa 10!
Graduate Students: Toshio Sakamoto
Katsuzo Noguchi
Patcharaporn Deshnium
Yoshitaka Nishiyama 11 i
Technical staffs: Sho-Ichi Higashi
Mikiida(1J from National Research Center Demokritos,
Greece)( 2! from Shanghai Institute of Plant Physiology,
P.R. China)( 31 from Plant Physiology Institute, Moscow,
Russia)( 41 from Biological Research Center Szeged,
Hungary)
ECfellow, France)~J from Institute of Botany, Academia Sinica, P.R.
China)(! K OSEF fellow, from Inje University, Korea)( 8' from University of Santiago, Spain)
fl from Advanced Research Laboratory, HitachiLtd.)( 0! from National Institute of Agrobiological
Resources)( 11' from the University of Tokyo)t'

The research effort of this division is
directed toward the understanding of
tolerance and adaptation of plants totemperature extremes, with particularemphasis on the molecular mechanisms
by which plants acclimate or toleratethese temperature conditions. In 1993,
several significant advances were madein the area of temperature response andrelated areas in the study of cyanobacteria.

I. Cyanobacterial desaturases.
Higher plants, and most cyanobacterial strains, contain high levels ofpolyunsaturated fatty acids, which areimportant in their response to ambient
temperature.
We isolated the desA genes for ~12
desaturases of the acyl-lipid type from
Synechocystis
thecyanobacteria,
PCC6803, Synechocystis PCC6714,
Synechococcus PCC7002, and Anabaena variabilis and found four conserved sequence domains. We over-expressed the desA gene of Synechocystis
PCC6803 in Escherichia coli using thebacteriophage T7 polymerase system.
The ~12 desaturase, thus over-expressedin E. coli, was active in vitro when reduced ferredoxin was added as an electron donor. This result indicates that the
cyanobacterial desaturase is similar tothe plastidial desaturases in terms of theelectron-donating system, but is dissimilar to the cytoplasmic desaturases thatuse cytochrome b5 as an electron donor.
We isolated the desB and desC genes
of Synechocystis PCC6803, which encode the w3 and ~9 desaturases, respectively, of the acyl-lipid type. We transformed another cyanobacterial strain,
Synechococcus PCC7942, with the desAand desB genes. This strain contains only
the ~9 desaturase. The mode of fatty
acid desaturation in the transformed
cells demonstrates that the w3 desaturase can introduce a double bond at the
w3 position of fatty acids that contain an
unsaturated bond at the ~12 position.
II. Importance of membrane-lipid onsaturation in tolerance to low-temperature photoinhibition.
To understand the roles of unsaturation of membrane lipids, we transformed
Synechococcus PCC7942 with the desAFOR BASIC BIOLOGY

gene. This transformation greatly modified the extent of unsaturation of thefatty acids of membrane lipids. In thewild-type strain, only monounsaturated
lipid molecules existed; in the transformant, each of the monounsaturated anddiunsaturated lipid molecules contributes
to about 50% of the total membrane lipids. This change in the unsaturation of
membrane lipids greatly reduced susceptibility to low temperature.
These results suggest that diunsaturated fatty acids play an important rolein protection against damage to photosynthetic processes by low temperature.
By contrast, photosynthetic electrontransport, measured at various temperatures, and susceptibility to high temperature were not affected by changes in theextent of unsaturation of the fatty acids.
III. Regulation of the expression of thedesA gene by changes in the fluidity ofthe plasma membrane.
Living organisms can maintain themolecular motion, or "fluidity", of membrane lipids by regulating the level of onsaturation in fatty acids. For example,
cyanobacterial cells respond to a decrease in temperature by introducing
double bonds into the fatty acids ofmembrane lipids, thus compensating forthe temperature-induced decrease in themolecular motion of membrane lipids.
Desaturases are responsible for the introduction of these specific double
bonds. We have demonstrated that the
low temperature-induced desaturation of
fatty acids of membrane lipids is regulated at the level of the expression of thedesaturase genes.
To study the mechanism of the lowtemperature-induced regulation of theexpression of the desA gene in greater
detail, we examined the effect of mem-53
brane fluidity on the gene expression bycatalytic hydrogenation of the unsaturation bonds of fatty acids of membrane lipids. A 4-min hydrogenation of the
Synechocystis PCC6803 cell increased
the level of fully saturated lipids at theexpenses of diunsaturated lipids inplasma membrane, but had no such effect in thylakoid membranes. After thishydrogenation, the level of mRNA ofthe desA gene increased 10-fold within
30 min. This increase in the mRNA level
after hydrogenation resembles that following a decrease in ambient temperature. These results suggest that the decease in fluidity of plasma membrane is
the first response to temperature change.
IV. Heat stability of photosynthesis.
We also focused on the response ofplants to high-temperature stress. Since
photosynthesis is the physiological process most susceptible to heat stress inplants, its heat stability is an importantfactor in the tolerance of plants to hightemperature.
We studied a component responsible
for the heat stability of photosynthesis in
the cyanobacterium, Synechococcus
PCC7002. When thylakoid membranes
isolated from the cyanobacterial cells
were treated with a low concentration of
Triton X -100, the heat stability ofoxygen evolution was decreased by 4°C.
From the extracts with Triton X-100, we
purified a protein that increased the heatstability of oxygen evolution by 4°C. The
protein was identified as a low redox
potential cytochrome c-550 having amolecular mass of 16 kDa. We isolated
the gene encoding this cytochrome from
Synechococcus PCC7002, and determined its nucleotide sequence. The deduced amino-acid sequence revealed
that the gene product consists of a transit

54

NATIONAL INSTITUTE

peptide of 34 residues and a mature protein of 136 residues. These results indicate that cytochrome c-550 is involvedin the mechanism of heat stability ofoxygen evolution and, therefore, in theheat stability of photosynthesis.
V. Two groEL-homologous genes incyanobacterial cells.
There are a wide variety of heat shockproteins, most of which are highly conserved in both prokaryotes and eukaryotes. Cellular levels of these proteinsdramatically increase upon heat shock orother environmental stresses. Some heatshock proteins act as molecular chaperones that assist in the folding and assembly of other proteins.

To examine the role of heat shockproteins in acclimation to high-temperature stress, we attempted to isolate thegenes for the heat shock protein groELfrom Synechococcus PCC7002. We discovered that there are two groEL-homologous genes. One of the groEL-homologous genes is accompanied by thegroES gene, and the groES and groEL
genes constitute the groESL operon as
the groESL operon in E. coli. The othergroEL-homologous gene is not accompanied by the groES homologous gene.
However, this groEL-homologous generetains the carboxyl-terminal repeat of
Gly-Gly-Met, which is typical in thegroEL gene of E. coli. Heat shock increased the levels of mRNAs of bothc 1 2 3c 1 2 3(A) Temperature shift(B) Hydrogenation

Fig. 1. Regulation of the desA gene expression in Synechocystis PCC6803 analyzed by RNA-DNA blot
hybridization. (A) Temperature-induced regulation. Cells grown at 36'C were subjected to a temperature shift
to 22'C, and incubated in the light for 15 minutes (1), 30 minutes (2), and 60 minutes (3). Control (c) corresponds to cells before the temperature shift. (B) Hydrogenation-induced regulation. Cells grown at 36'C were
subjected to hydrogenation for 4 min to partially hydrogenate the lipids of plasma membrane, and incubated
at 36'C for 15 minutes (1), 30 minutes (2), and 60 minutes (3); Control (c) corresponds to cells which werechydrogenated but before the incubation.

55

FOR BASIC BIOLOGY(A)

groEL-1-35 -10

IR

RB

(B)

groEL-2-35 -10

IR

RB

Fig. 2. Organization of two groEL-homo!ogous genes in Synechococcus PCC7002. (A) The groESL
operon consisting of a groEL-homologous gene (groEL-1) and a groES gene. (B) The other groEL-homoiogous gene (groEL-2) which is not accompanied by the groES gene. RB, the ribosome binding site; IR, a 9-bp
inverted repeat found in the promoter region; -10 and -35, -10 and -35-like sequences, respectively.

groEL-homologous genes. To understand the function of the product of thegroEL-homologous genes, we haveknocked out one of the groEL homologous genes by insertional disruption witha kanamycin resistance gene cartridge.
We are currently investigating the responses of these mutants to high-temperature stress.
Publication List:(1) Original papers
Allakhverdiev, S.l., Hayashi, H., Fujimura, Y., Klimov, V.V. and Murata,
N. (1993) Inactivation of photosynthetic oxygen evolution by o-phenanthroline and LiC10 4 in photosystem 2of pea. Photosynth. Res. 35, 345349.
Hayashi, H., Fujimura, Y., Mohanty,
P.S. and Murata, N. (1993) The roleof CP 47 in the evolution of oxygen
and the binding of the extrinsic 33kDa protein to the core complex of
Photosystem II as determined bylimited proteolysis. Photosynth. Res.
36,35-42.
Higashi, S. and Murata, N. (1993) An invivo study of substrate specificities ofacyl-lipid desaturases and acyltransferases in lipid synthesis in Synechocystis

PCC6803. Plant Physiol. 102, 12751278.
Kondo, T., Strayer, C.A., Kulkarni,
R.D., Taylor, W., Ishiura, M. Golden,
S.S. and Johnson, C.H. (1993) Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc. Nat/.
Acad. Sci. USA 90, 5672-5676.
Lehel, C., Los, D., Wada, H., Gyorgyei,J., Horvath, 1., Kovacs, E., Murata, N.
and Vigh, L. (1993) A second
groEL-like gene, organized in a
groESL operon is present in the
genomeofSynechocystis
sp.
PCC6803. J. Bioi. Chem. 268,
1799-1804.
Los, D., Horvath, 1., Vigh, L. and Murata, N. (1993) The temperature-dependent expression of the desaturase
gene desA in Synechocystis PCC6803.
FEES Lett. 318, 57-60.
Malakhov, M.P., Wada, H., Los, D.A.,
Sakamoto, T. and Murata, N. (1993)
Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9.
Plant Mol. Bioi. 21, 913-918.
Mamedov, M., Hayashi, H. and Murata,
N. (1993) Effects of glycinebetaine
and unsaturation of membrane lipids
on heat stability of photosynthetic electron-transport
andphosphoryla-

56

Synechocystis
tion reactions in
PCC6803. Biochim. Biophys. Acta
1142, 1-5.
Mohanty, P., Hayashi, H., Papageorgiou,
G.C. and Murata, N. (1993) Stabilization of the Mn-cluster of the oxygenevolving complex by glycinebetaine.
Biochim. Biophys. Acta 1144, 9296.
Nishida, 1., Tasaka, Y., Shiraishi, H. and
Murata, N. (1993) The gene and theRNA for the precursor to the plastidlocated glycerol-3-phosphate acyltransferase of Arabidopsis thaliana.
Plant Mol. Bioi. 21, 267-277.
Nishiyama, Y., Kovacs, E., Lee, C.B.,
Hayashi, H., Watanabe, T. and Murata, N. (1993) Photosynthetic adaptation to high temperature associatedwith thylakoid membranes of Synechococcus PCC7002. Plant Cell
Physiol. 34, 337-343.
Vigh, L., Los, D.A., Horvath, I. and
Murata, N. (1993) The primary signal
in the biological perception of temperature: Pd-catalyzed hydrogenationof membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc. Nat!. A cad. Sci.
USA 90, 9090-9094.
Wada, H., Schmidt, H., Heinz, E. and
Murata, N. (1993) In vitro ferredoxindependent desaturation of fatty acids
in cyanobacterial thylakoid membranes. J. Bacteriol. 175, 544-547.
Wada, H., Macherel, M.-H. and Murata,
N. (1993) The desA gene of the
cyanobacterium Synechocystis sp.
strain PCC6803 is the structural genefor .6.12 desaturase. J. Bacteriol. 175,
6056-6058.
(2) Reviews
Murata, N., Wada, H., Gombos, Z. andNishida, I. (1993) The molecular

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mechanism of the low-temperature
tolerance of plants studied by genetechnology of membrane lipids. In Interacting Stresses on Plants in a
Changing Climate (M.B. Jackson and
C.R. Black, eds. ). Springer-Verlag,
Berlin, pp. 715-723.
Nishida, 1., Imai, H., Ishizaki-Nishizawa, 0., Tasaka, Y., Shiraishi, H.,
Higashi, S., Hayashi, H., Beppu, T.,
Matsuo, T. and Murata, N. (1993)
Molecular and physiological studies ofglycerol-3-phosphate acyltransferase,
acyl-ACP hydrolase and stearoyl-ACP
desaturase. In Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants (N. Murata andC.R. Somerville, eds. ). The American
Society of Plant Physiologists, Rockville, Maryland, pp. 79-88.
Wada, H., Gombos, Z., Sakamoto, T.,
Higashi, S., Los, D.A., Heinz, E.,
Schmidt, H. and Murata, N. (1993)
Fatty acid desaturation in cyanobacteria. In Biochemistry and Molecular
Biology of Membrane and Storage Lipids of Plants (N. Murata and C.R.
Somerville, eds. ). The American Society of Plant Physiologists, Rockville,
Maryland, pp. 67-78.
Wada, H., Gombos, Z., Sakamoto, T.
and Murata, N. (1993) Role of lipids
in low-temperature adaptation. In
Photosynthetic Responses to the Environment (H.Y. Yamamoto and C.M.
SJ:JPth, eds. ). The American Society for
Plant Physiologists, Rockville, Maryland, pp. 78-87.
(3) Book

Murata, N. and Somerville, C., Eds.
(1993) Biochemistry and Molecular
Biology of Membrane and Storage Lipids of Plants, the American Society ofPlant Physiologists, Rockville, Maryland.

FOR BASIC BIOLOGYDIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY(ADJUNCT)
Professor: Kimiyuki Satoh
Associate Professor: Hirokazu Kobayashi
Research Associates: Satoru Tokutomi
Noritoshi Inagaki
Graduate Students: Kyoichi lsono*
Y oshihiro N arusaka •Tatsuya Torno •(*from Okayama University)

The efficient energy transformation in
the primary processes of photosynthesis
is ensured by the highly ordered organization of molecules in the photochemical
reaction centers, in a physical, chemicaland biological sense. The project in thisdivision is aiming to elucidate the organization of photosystem II reaction
center of oxygenic photosynthesis which
has a unique property to generate astrong oxidant for utilizing water molecules as electron donor.
In the first approach, the basic molecular structure of the reaction center,
which has been isolated in our study, will
be analyzed by several methods which
include crystallographic analysis, chemical modification and cross-linking analysis and physical methods. Structurefunctional analysis will also be conductedon the reaction center proteins using
random and site-directed mutagenesis
for transformable algae, Synechocystis
PCC 6803 and Chlamydomonas reinhardtti. The target of these analyses is
the structure and molecular environmentof P-680, the primary donor, which
determine the redox potential of this system.
In the second approach, the effort will
be focused on the elucidation of molecular mechanism of light-regulated metabolic turnover of a subunit of the reaction center, the D1 protein. The process
involves some of apparently unique57
steps; i. e., light-regulated gene expression at the translational level, post-translational cleavage of the C-terminal extension of protein and the incorporationof cofactors and subunits into multicomponent pigment-protein complexes.
I. Structural· organization of photosystem D reaction center.
The structure and molecular interactions of the primary donor (P-680) in
the photosystem II reaction center havebeen investigated by detecting light-induced Ff-IR difference spectra upon theformation of its triplet state. From theband positions of the keto and carbomethoxy C=O stretches, the hydrogenbonding properties of the two chlorophylls of P-680 were found to be asymmetrical; in one chlorophyll both theketo and carbomethoxy C=O groups
form hydrogen bonds, while in the otherchlorophyll the keto C=O is not hydrogen-bonded whereas the carbomethoxy C=O probably is hydrogenbonded. Considering the orientation of
P-680 analyzed by EPR and the structure of bacterial reaction center determined by X-ray crystallography together
with the sequence homology between theD1 and D2 subunits of photosystem II
and the L and M subunits of purple bacteria, a model of the P-680 structure andits interactions with apoproteins has
been proposed (collaborative researchwith Drs Noguchi and Inoue, RIKEN).
Site-directed modification of the protein
subunits responsible for this hydrogenbonding interaction (Ser-191 and Thr192) is now in progress in order to prove
this hypothesis.
Chemical cross-linking analysis has
also been conducted for the isolated
photosystem II reaction center to analyze
the gross structure; i. e., the nearest

58

neighbors of the constituent subunits andamino acid residues in cross-contact in
the reaction center complex.

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factor(s) caused by illmnination, in addition to energy supply by ATP.

m. Biophysical analyses on the moleII. Dynamic aspects of the organizationof photosystem II reaction center
The D 1 subunit of photosystem II reaction center has a C-terminal extension
consisting of 9-16 amino acid residues.
Post-translational removal of this extension is absolutely required for constituting the machinery of oxygen evolution inphotosystem II. The enzyme involved inthis processing has been purified and the
N-terminal partial amino acid sequence
was determined.
The recognition signal was analyzed
for the enzyme using substituted synthetic oligopeptides corresponding to the
C-terminal sequence of precursor protein. A series of systematic substitutions
around the cleavage site (between Ala344 and Ala-345) were synthesized toanalyze the function of specific aminoacid and the sequence in the recognition.
The efficiency of these oligopeptides as a
substrate and their effectiveness as an inhibitor were examined. The conclusionfrom this analysis is that the secondary
structure formed by the presence of specific amino acids around the cleavage
site, i. e., Asp-342 and Ile-343, is important in the recognition.
The synthesis of D 1 precursor protein
is regulated by light at the stage of translation. The mechanism of this light regulation was analyzed using isolated pea
chloroplasts. The isolated chloroplasts
was shown to accumulate translation intermediate(s) of D1 protein in thepresence of externally added ATP in thedark. The result of analysis suggested
that the translation and/ or stable accumulation of D1 protein require(s)cular event during photo-transformation of phytochrome.
Phytochrome is a photoreceptor ingreen plants responsible for a variety ofmorphogenetic responses including lightregulated gene expression. Molecular
event during its phototransformationfrom an inactive to an active forms, Pr
and Pfr, respectively, was studied byusing three different biophysical techniques. (1) Primary event on absorptionof quanta is proposed to be isomerization of the chromophore composed of an
open-tetrapyrrol. We had detected a
photoproduct from Pr with rise time of
24ps by sub-picosecond flash photolysis,
which has been proven to be a novel one
formed directly from the excited stateand probably resulted from isomerization (collaborative research with Drs
Kandori and Yoshihara, IMS). (2) As
the second event, we are proposing
proton migration from the chromophore
to the protein moiety based on resonance Raman scattering, which may be a
trigger of the conformational change.
We found that a proton associates with
the C-ring nitrogen of the chromophorein Pr dissociates prior to the formationof a meta-intermediate by low-temperature resonance Raman scattering (collaborative research with Drs Mizutani andKitagawa, IMS. Mizutani et al., 1994,
published). (3) Ultraviolet resonanceRaman scattering was measured to figureout the details in the conformationalchange of the protein moiety. Hydrophobicity around (a) Trp(s) in the chromophoric domain, possibly aroundTrp365 and (or) Trp567 , increases onphototransformation. The content of

FOR BASIC BIOLOGY

a-helices and nonregular structure which
are less populated in the chromophoric
domain than the other domains, are almost unchanged by the phototransformation (collaborative research with Drs
Mizutani, Kaminaka and Kitagawa,
IMS).
Publication List:
Mizutani, Y., Tokutomi, S., Kaminaka,
S. and Kitagawa, T. (1993) Ultraviolet
resonance Raman spectra of pea intact, large and small phytochromes:
Difference in molecular topography of
the red- and far-red-absorbing forms.
Biochemistry 32, 6916-6922.
Noguchi, T., Inoue, Y. and Satoh, K.
(1993) Ff-IR studies on the triplet
state of P680 in the photosystem II reaction center: Triplet equilibrium
within a chlorophyll dimer. Biochemistry 32, 7186-7195.
Satoh, K. (1993) Isolation and properties of the photosystem II reaction
center. In The Photosynthetic Reaction Center (J. Norris and J. Deisenhofer, eds.). Academic Press, New
York, pp. 289-318.

59
Satoh, K. (1993) Molecular organizationof the photochemical apparatus ofoxygenic photosynthesis. In Frontiersof Photo-biology (A. Shima et al.,eds. ). Elsevier Sciences Publishers,
Amsterdam, pp. 3-11.
Taguchi, F., Yamamoto, Y., Inagaki, N.
and Satoh, K. (1993) Recognition signal for the C-terminal processing protease of D 1 precursor protein in thephotosystem II reaction center: An
analysis using synthetic oligopeptides.
FEES Lett. 326, 227-231.
Taniguchi, M., Kuroda, H. and Satoh, K.
(1993) ATP-dependent protein synthesis in isolated pea chloroplasts:
Evidence for accumulation of a translation intermediate of the D1 protein.
FEES Lett. 317, 57-61.
Torno, T., Enami, I. and Satoh, K.
(1993) Orientation and nearest neighbor analysis of psbi gene product in
the photosystem II reaction center
complex using bifunctional cross-linkers. FEES Lett. 323, 15-18.

60

NATIONAL INSTITUTE

tion molecules on the surface of specificmuscle fibers which guide the growthcones of motoneurons.
By using an enhancer trap method,
several genes have previously been
identified that are expressed in small
subsets of muscle fibers prior to innervation, and are thus good candidates forsuch recognition molecules. Two ofthem, connectin and Toll, were shown to
encode cell recognition molecules which
belong to the leucine-rich repeat (LRR)
family. In paticular, connectin is expressed not only on a subset of muscle
fibers but also on the axons and growthcones of the very motoneurons which innervate these muscles (Figure 1). Its specific expression both in presynaptic motoneurons and postsynaptic muscles, andits function as a homophilic cell adhesion
molecule in vitro strongly suggested thatconnectin play a role in the neuro-muscular specificity. We are currently study-

DIVISION OF BEHAVIOR AND
NEUROBIOLOGY(ADJUNCT)
Professor: Masatoshi Takeichi
Research Associate: Akinao Nose
Institute Research Fellow: Tomoko Tominaga
Visiting Scientist: Tatsuo Umeda

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 recogni-

connectin

Toll

24

...sq*~:r t:-L::::tlet:lll

23
2 2--..,..::;jll-l21

Fig. 1. Expression pattern of connectin and Toll. Schematic diagrams showing expression of connectin
and Toll (yellow) on the surface of subsets of muscle fibers and expression of connectin on the subset of motoneurons that innervate the connectin-positive muscles (red). (from Nose et a!. , Cell 70, 1992)

FOR BASIC BIOLOGY

ing the function of connectin by molecular genetic methods and also trying toclone novel genes implicated in theneuro-muscular connectivity.
I. Molecular genetic analysis of thefunction of connectin.
To study the _role of connectin in vivo,
we misexpressed connectin on muscle
fibers that normally do not express themolecule by using Toll promoter. As
shown in Fig. 1, Toll is expressed onventral muscles #6, 7, 14-17 and 28, adifferent subset of muscle fibers fromthose expressing connectin. We used a 7
kb Toll upstream sequence sufficient forthe muscle expression to misexpress connectin on these muscle fibers in P-element mediated transgenic flies.
The analysis of the transgenic flies(Toll-connectin) showed that the development of a motor nerve (SNb) is abnormal. After leaving the CNS, SNb
normally grows directly into the ventral
muscles and makes synaptic contact withthe target muscles by st17. In Toll-connectin, the SNb axons often take abnormal trajectory and fail to make the right
synaptic contact by this stage. They often
grow dorsally along another nerve (ISN)or take an independent path below theventral muscles. The results suggest that
ectopically expressed connectin on theventral muscles (#6, 7, 14 and 28) prevent the SNb from growing into thesemuscle fibers, pointing to connectin's
role as a inhibitory signalling molecule in
the formation of neuro-muscular connectivity. Thus in addition to its possible
role as an attractive homophilic recognition molecule (for the motoneurons andtheir target muscles both expressing themolecule), connectin may serve as an inhibitory recognition molecule for othermotoneurons that do not express the61molecule (probably via heterophilic interaction).
We are currently trying to misexpress
connectin in yet different subsets ofmuscle fibers by using GAL4 system, tofurther analyse connectin's role in vivo.
D. Cloning of novel genes implicated in
the neuro-muscular connectivity in
Drosophila
1. Search for novel connectins. An interesting possibily is that connectins constitute a LRR subfamily which are expressed on different subsets of motoneurons and muscles. We are trying to isolate novel connectins by using PCR andwill study their expression pattern andfunction.
2. Cloning and characterization of otherenhancer trap lines

We are conducting molecular andgenetic analysis of two other enhancer
trap lines that are expressed in specificsubsets of muscles and/ or motoneurons.
rQ224 expresses the reporter gene (Bgal) in a small subset of neurons including a motoneuron RP3 but not RP1.
These two motoneurons take the same
peripheral pathway as they exit the CNS
and send axons via motor nerve SNb.
However, despite the similarlity of theirtrajectories, once they reach the target
region, they show distinct behaviors: theRP3 growth cone projects onto the cleft
region between muscle #6&7 while theRP1 growth cone goes past 6&7 to innervate · muscle # 13. Expression ofrQ224 only in RP3 but not in RP1 suggests its possible role in such specific aspects of target recognition. The eDNA
cloning and partial sequence analysis
showed that the ORF contains a signal
sequence. rQ224 product thus is probably a surface or secreted molecule with62
potential roles in recognition. The sequencing of the complete eDNA is now
in progress.
The other line AN34 expresses B-gal
in a single muscle fiber ( # 18) per hemisegment. The remarkable specificity in
its expression pattern (one out of 30
muscle fibers) makes it a good candidate
for the muscle target recognition molecule. The eDNA cloning and sequencing
revealed that AN34 protein shows extensive amino acid similarity to rat
F-spondin, a secreted molecule expressed at high levels in the floor platethat has been shown to promote neuralcell adhesion and neurite extension invitro.
We are currently trying to isolate theloss-of-function mutants of these twolines as well as the transgenic flies that
ectopically express these molecules (asdescribed for connectin) to study theirroles in the neuro-muscular development.
Publication List:
Redies, C., Engelhart, K. and Takeichi,
M. (1993) Differential expression ofN- and R-cadherin in functional neuronal systems and other structures ofthe developing chicken brain. J. Comparative Neurology 333, 398-416.
Redies, C. and Takeichi, M. (1993) Nand R -cadherin expression in the optic
nerve of the chicken embryo. Glia 8,
161-171.
Hamaguchi, M., Matsuyoshi, M., Ohnishi, Y., Gotoh, B., Takeichi, M. andNagai, Y. (1993) p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin celladhesion system. EMBO J. 12, 307314.
Takeichi, M., Hirano, S., Matsuyoshi, N.
and Fujimori, T. (1993) Cytoplasmic

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control of cadherin-mediated cell-celladhesion. Cold Spring Harbor Quant.
Bioi. LVll, 327-334.
Uemura, T., Shiomi, K., Togashi, S. andTakeichi, M. (1993) Mutation of twins
encoding a regulator of protein phosphatase 2A leads to pattern duplication in Drosophila imaginal discs.
Genes and Develop. 7, 429-440.
Fujimori, T. and Takeichi, M. (1993)
Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated non-functional N-cadherin. Mol.
Bioi. Cell4, 37-47.
Oda, H., Uemura, T., Shiomi, K., Nagafuchi, A., Tsukita, S. and Takeichi, M.
(1993) Identification of a Drosophila
homologue of a-catenin and its association with the armadillo protein. J.
Cell Bioi. 121, 1133-1140.
Redies, C. and Takeichi, M. (1993) Expression of N-cadherin mRNA during
development of the mouse brain. Develop. Dynamics 197, 26-39.
Matsunami, H., Miyatani, S., Inoue, T.,
Copeland, N.G., Gilbert, D.J., Jenkins, N.A. and Takeichi, M. (1993)
Cell binding specificity of mouse
R -cadherin and chromosomal mapping of the gene. J. Cell Science 106,401-409.
Watabe, M., Matsumoto, K., Nakamura,T. and Takeichi, M. (1993) Effect ofhepatocyte growth factor on cadherinmediated cell-cell adhesion. Cell
Struct. Funct. 181, 117-124.
Oka, H., Shiozaki, H., Kobayashi, K.,
Inoue, M., Tahara, H., Kobayashi, T.,
Takatsuka, Y., Matsuyoshi, N., Hirano, S., Takeichi, M., and Mori, T.
(1993) Expression of E-cadherin celladhesion molecules in human breast
cancer tissues and its relationship tometastasis. Cancer Res. 53, 16961701.

FOR BASIC BIOLOGY

Doki, Y., Shiozaki, H., Tahara, H.,
Inoue, M., Oka, H., Iihara, K., Kadowaki, T., Takeichi, M. and Mori, T.
(1993) Correlation between E-cadherin expression and invasiveness invitro ih a human esophageal cancer
cell line. Cancer Res. 53, 3421-3426.
Takeichi, M. (1993) Cadherins incancer: Implication for invasion andmetastasis. Curr. Opinion Cell Bioi. 5,
806-811.
Takeichi, M., Watabe, M., Sibamoto, S.,
Ito, F., Oda, H., Uemura, T. and Shi-

63
mamura, K. (1993) Dynamic control
of cell-cell adhesion for multicellularorganization. C. R. Acad. Sci. Paris,
Sciences de Ia vie/Life sciences 316,
818-821.
Steinberg, M.S. and Takeichi, M. (1993)
Experimental specification of cell sorting, tissue spreading and specific patterning by quantitative differences incadherin expression. Proc. Nat! Acad.
Sci. USA 91, 206-209.

65

FOR BASIC BIOLOGYLABOLATORY OF GENE EXPRESSION
AND REGULATION

Head: Goro Eguchi

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

66

NATIONAL INSTITUTEDMSION OF GENE EXPRESSION
AND REGULATION I
Professor: Y oshiro Shimura
Associate Professor: Kiyotaka Okada
Research Associate: Sumie Ishiguro
JSPS Post-doctoral Fellow: Nobuyoshi Mochizuki
Graduate Students: Azusa Yano
Yoichi Ono
Toshiro ltol)
Takuji Wada
Tokitaka Oyama1!
Technical Staff: Hideko Nonaka
AkikoKawai(l)from Kyoto University)

The principal interest of this laboratory is molecular genetic studies on theregulatory systems of organ development
and on growth control by several environmental stimuli in higher plants. For
these studies, we have mainly used asmall crusifer, Arabidopsis thaliana. This
plant is called "botanical 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 inheight), and ease of propagation. Thesefeatures make the plant ideally suited forgenetic and molecular biological studies.
In addition, more than 360 loci andmore than 500 RFLP and RAPD markers are mapped on 5 chromosomes.
Experimental techniques such as transformation, regeneration of transgenic
plants and gene tagging have been improved. Using this plant, we have isolated and characterized many mutants
defective in flower development andmorphogenesis or in root responses toward physical stimuli such as gravity,
light or touching.
I. Development and morphogenesis offlowers
Mutants with abnormal floral morphology could be divided into the following
categories on the basis of the stages offloral 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 aberrant positions), stage 5: fate determination of thefloral organ primordia (homeotic mutants: mutants where some floral organs
are replaced by other organs), and stage
6: development and morphogenesis offloral organs (mutants with organs ofaberrant structure and function). Most of
the mutants. have been shown to havesingle, recessive, nuclear mutations (Komaki et a/. (1988) Development, 104,
195-203; Okada et a/. (1989) Cell Differ. Dev., 28, 27-38).
The AGAMOUS gene of Arabidopsis
thaliana is a homeotic gene involved in
the development of stamens and carpels.
This gene encodes a putative DNAbinding protein sharing a homologous
region with the DNA-binding domains,
MADS boxes, of yeast MCM1 andmammalian SRF. Using the MADS domain of AGAMOUS protein overproduced in E. coli, we have shown that theconsensus sequence of the high-affinity
binding sites of the AGAMOUS MADS
domain was 5'-TT(A/T/G)CC(A/T)6 GG(A/T/C)AA-3'.
Comparisonswith the binding-site sequences of other
MADS-box proteins revealed that theMCM1 binding-sites show similarities
with the binding-site sequence of the
AGAMOUS MADS domain (Shiraishi
etal (1993) PlantJ., 4, 385-398).

67

FOR BASIC BIOLOGY

The mutant apeta/a3 of A. thaliana
and the mutant deficiens of Antirrhinum
majus have a homeotic conversion of petals to sepals and stamens to carpels. We
have isolated a homologous gene to the
DEFICIENS from A. thaliana andshown complete complementation of
apetala3 mutation by introducing theisolated gene. These results show that
the APETALA3 is a homologue of
DEFICIENS structurally and functionally. The 5'-upstream region of APETALA3 gene contains three SRE-likesequence, where MADS box-containing
proteins are assumed to bind and regulate expression in tissue- and stage-specific manner (Okamoto et a/. (1994)
Plant Mol. Bioi., in press). Now we aretrying to isolate the target genes, ofwhich the expression is regulated by the
MADS-box proteins, AGAMOUS,
APETALA3 or PISTILLATA.
Attempts were also made to isolate
cDNAs which are specifically expressedin floral organs. From a eDNA library ofyoung floral buds of A. tha/iana, we isolated two eDNA clones whose aminoacid sequences are highly homologous
with the known amidophosphoribosyltransferase cDNAs. Northern blot analysis showed that one gene is expressedin flowers and roots, while the othergene is mainly expressed in leaves (Ito eta/., submitted).
II. Stimulus-response interactions inroot
Roots alter their growth direction
when their relative orientation against
gravity is changed (gravitropic response),
when they are illuminated from aside(phototropic response), or when they encounter obstacles (obstacle-escaping response). Using a newly devised system
which provides a constant obstacle-

touching 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 (Okada & Shimura (1992)
Cell, 70, 369-372).
Physiological and biochemical aspectsof stimulus-response reaction in roots
were also analyzed. Proteins newly synthesized in cells of root tips of Arabidopsis seedlings after gravistimulation andphoto-induced tactile stimulation wereseparated by two-dimensional gel electrophoresis. Intensities of 14 protein
spots were shown to increase after continuous rocking treatment for 24 hours.
Analysis of 2P]-labeled proteins revealed that the continuous rocking enhanced the phosphorylation of proteinsin two spots. When the seedlings inflasks were illuminated from the front,
and the roots bent towards the back wallof the flasks, total of 12 spots wereP

68
newly appeared or enhanced (Sakamoto
et al. (1993) Plant Cell Physiol., 34,
297-304).
III. Technical improvement for molecular cloning
In order to isolate the genes responsible for the mutants, some technical improvements for gene tagging systems in
A. thaliana were attempted. For such experiments, it is absolutely necessary todevelop a good, efficient system of transformation mediated by Agrobacterium
and of transgenic plant regeneration. We
have tested several combinations of A.
thaliana ecotypes and Agrobacteria
strains and established an efficient system (Akama et al. (1992) Plant Cell
Rep., 12, 7-11). We also carried out socalled in planta transformation, in which

NATIONAL INSTITUTE

adult A. thaliana plants are directly infected with Agrobacterium and genetransfer occurs during floral development. In this system, it is thought thatproblems caused by somaclonal variation
are avoided. About four hundred oftransformants were generated andscreened.
Publication List:
Sakamoto, K., Shiraishi, H., Okada, K.
and Shimura, Y. (1993) Proteins induced by physical stimuli in root tipsof Arabidopsis thaliana seedlings.
Plant Cell Physiol. 34, 297-304.
Shiraishi, H., Okada, K. and Shimura,Y. (1993) Nucleotide sequences recognized by the AGAMOUS MADS
domain of Arabidopsis thaliana invitro. Plant J. 4, 385-398.

Complementation of apetala3 mutation. A homeotic mutant apetala3 of A. thaliana (right) was completely
complemented (left) by introducing the wild type APETALAJ gene.

FOR BASIC BIOLOGYDIVISION OF GENE EXPRESSION
AND REGULATION II
Professor: Takashi Horiuchi . .
Research Associates: Masum1 Hzdaka
Takehiko Kobayashi
Graduate Student: Katufumi Ohsumi
Technical Staff' Yasushi Takeuchi

Homologous recombination on thechromosome is often uniform, however,in both procaryotes and eucaryotes,
there are specific regions or sites, named"hotspots", where homologous recombination occurs at a higher rate. DNA replication origin in procaryotes (phage) isone example. Another example is the"HOTJ" site in yeast which has activity
to stimulate recombination, homologously, in adjacent regions. As molecularmechanisms involved in enhancing homologous recombination are not fully
characterized, related studies, in an attempt at elucidation, are ongoing in ourlaboratory. Special focus has been directed to termination processes, and homologous recombinations.
I. Analysis of E. coli recombinational
hotspots, Hot.
In E. coli, there are replication fork
blocking event-dependent recombinational hotspots. In E. coli RNase H defective (rnh-) mutants, we found specific
DNA fragments, termed Hot DNA,
when DNA in the ccc form is integrated
into the E. coli genome by homologous
recombination to form a directly repeated structure, a strikingly enhanced
excisional recombination between the repeats occurs. We obtained 8 groups ofsuch Hot DNA, 7 of which were clustered in a narrow region, called replication terminus region (about 280 kb) on
the circular E. coli genome. A terminussite ( Ter) can impede the replication fork
in a polar fashion. The six Ter sites are

69
located approximately symmetrically interminus and its surrounding region. To
block the fork at the Ter site, anotherprotein factor, Ter binding protei~ encoded in the tau (or tus) gene ts required. In tau- cells, Hot activity of
HotA, B and C DNAs disappears,
thereby indicating that the Hot activity isfork arrest-dependent. In rnh- cells, analternative new replication origin(s)other than an ordinary replication origin(oriC), located at the terminus region is
activated so that a newly initiated fork isimmediately blocked at one of the Ter
sites and consequently, there is an accumulation of stalled forks. It seems fairly
certain that the rnh- specific accumulation of the fork (which we confirmed) isthe cause of the Hot activity at the
nearby site. In addition, at least for
HotA activity, the presence of a Chi, an
E. coli recombinational hotspot sequence, properly oriented on HotA
DNA itself or somewhere between HotA
and Ter site, is required. We prepared aputative model (Figure 1), in which thefollowing events may occur; (a) Chromosomal structure of a HotA DNA
transformant, in which repeated HotA
DNAs flanks a Kmr fragment. (b) When
the DNA replication fork proceeds fromleft to right (this can occur under rnhconditions), there is an efficient blockagainst the fork at the TerB site and theresulting Y-shaped molecules accumulate most in the rnh- strain, less in wild
type and not at all in the tau- strain. (c)A ds-break is introduced, probably bynicking at a single stranded DNA complementary to the newly synthesized lagging strand. (d) A Chi responsible
enzyme, RecBCD, enters the duplex
DNA through the ds-break, and travels
to the Chi site with concomitant degradation of the newly synthesized

70

NATIONAL INSTITUTE

HotA

Kmr

HotA

TerB

a

b

c

DNA fork blocking ....... ~-~~~m~VM~m~WA~W
·~
....

r

"ds-break"

==~~~
.y

......

Wff/LW4

•

d

e

f

~
======:::::JiiiQ::::::::::::::::::::~

0g

ccc HotA DNA

X======:::::~-•:::::::::::::::::::::::

Figure 1. A model for enhancement of excisional recombination between RotA DNAs (see text).

ds-DNA molecules, by exonucleolytic
activity. (e) The Chi sequence modulates
the exonucleolytic activity. (f) The resulting enzyme stimulates excisional homologous recombination between repeated RotA DNAs, resulting in production of the ccc Hot-Kmr DNA mole-cule.
II. Analysis of a yeast recombinational
hotspot, HOTJ.
In yeast, Saccharomyces cerevisiae,
there are also DNA replication fork
blocking sites in rRNA repeated genes

71

FOR BASIC BIOLOGY

9.08kb

Bgiii-B-~>~~o~~~~~~E---Bgiii-A--~::. !-element;--:35S
18S

5.8S

....
..
..
....·

--;r----1.. :......268'.

..·

bi-=:=Ec:::tb~U• •••St..

": 1"! ........

:...:

,

,,··

on / .·:~:........ ~:NTS! NTS2 '·················•.•....

...·····3;s~sHindlll

5S ··..

Pv~II ~ Sph;··..

'[ilincll

E-element
Enhancer

- sog
0

500

1000

1500 (bp)

Figure 2. Physical map of the rRNA gene repeat and location of the replication fork block ( SOG) site.
The direction of transcription of the 35S and SS rRNA genes (open arrow), the mature 18S, 5.8S, 26S and SS
rRNAs (closed bar), the introns (open bar), the two non-transcribed spacer regions (NTSl and NTS2), ori(replication origin) and 1-element (a component of HOTJ DNA) are indicated. BgliiA and B DNA fragments are produced when rDNA is digested with Bglll restriction enzyme. NTSl and its surrounding region
are expanded (Bell eta!., 1977), under which three solid bars represent theE-element (another HOTJ component), enhancer (enhancer element of 35S rRNA transcription) and SOG (replication fork blocking site; in-this case the SOG can inhibit the fork traveling from the right side only). The relevant restriction sites are also
shown.

(about 140 copies) on chromosome XII.
A single repeat unit consists of two transcribed 35S and 5S rRNA genes and two
non-transcribed regions, NTS1 andNTS2 (Figure 2). The 35S rRNA gene istranscribed by RNA polymerase I, apolymerase specific for 35S rRNA transcription, and transcription of the 5S
RNA gene, the direction of which is opposite that of the 35S rRNA, is carriedout by RNA polymerase III, anotherpolymerase specific for 5S rRNA andtRNA production. The NTS1 has a site,termed SOG, at which the replication

fork is blocked. We obtained evidence
that fork blocking activity at the SOG
site, termed SOG activity, is expressed
not only on the genome but also on theplasmid, suggesting that the SOG site
functions in any context (Kobayashi etal., 1992). By assaying SOG activity forvarious DNA fragments, derived fromthe NTS 1 and cloned on plasmids, we
determined the minimal region (about
100 bp long), located near the enhancer
region of the 35S rRNA transcription
and essential for blocking the replication
fork advancing in a direction opposite

72that for transcription. The SOG sequence is unique and has no characteristic structure, such as 2-fold symmetry,
repeated structure etc., hence, a transfactor(s) may have a role in blocking thefork. Interestingly, this region is included
in one of two cis-elements required for a
recombinational hotspot, HOTJ, activity.
HOTJ is a yeast mitotic homologous
recombinational hotspot, identified by
Keil and Roeder (1984). HOT! stimulates both intra- and inter-chromosomal
recombination, and for a precise analysis, enhancement of excisional recombination between directly repeatedDNAs at its nearby site was investigated.
HOT! was originally cloned on a 4.6 kb
Bgffi fragment (Fig. 2) and it was later
found to be composed of two non-contiguous cis-elements, E and I, located in
NTS1 and NTS2, respectively. Because
E and I positionally and functionallyoverlapped the enhancer and initiator of
the 35S rRNA transcription, respectively, they suggested that transcription

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by RNA polymerase I, initiated at the
35S rRNA promoter site may stimulate
recombination of the downstream region, thereby reveal HOTJ activity.
However, our finding that this E region
contains the SOG site may give anotherinterpretation.
To examine the functional relationshipbetween SOG and HOTJ activities,
HOTJ defective mutants were isolated
and their fork blocking activities were
subjected to 2D agarose gel electrophoresis. One was rad52 mutant defective in a gene included in homologous
recombination. The remaining H 0 T1
mutants are being examined to determined whether or not their fork blocking
activity is active.

Publication List:
Nishitani, H., Hidaka, M. and Horiuchi,
T. (1993) Specific chromosomal sites
enhancing homologous recombinationin Escherichia coli mutants defective
in RNase H. Mol. Gen. Genet. 240,
307-314.

FOR BASIC BIOLOGYDIVISION OF SPECIATIONMECHANISMS
Professor: Te:tsuo Yamamori (from early 1994)

73

74

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head: Hiroyuki Hattori
Common Facility Group
Chief' Kazuhiko Furukawa
Research Support Facilities
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
TomokiMiwa
Kimiko Yamamiya
Sonoko Ohsawa
Takeshi MizutaniRadioisotope Facility
Yoshimi Matsuda
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
and the other, the Research Support
Group, which assists the research activities as described in individual reports.
Technical staffs participate, throughthe department, in mutual enlightenment

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

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

75

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Head of Facility: Yoshiaki Suzuki
Associate Professor: Masakatsu Watanabe
Research Associates:
Yoshio Hamada (Tissue and Cell Culture)
Kenta Nakai (Computer)
Technical Staff: Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
TomokiMiwa
Mariko Saitoh (-May 30)
Kimiko Yamamiya
Takeshi Mizutani
Sonoko Ohsawa (Aug. 4-)

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 componentof 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 f.im s- 1 in tracking
speed, down to 2 f.im 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 levels.
This facility is routinely used for DNA
recombination experiments.
3. Computer Laboratory
To meet various computational needes
in this Institute, various computers
are equipped: VAX/VMS machines(VAX11/780 and micro VAX II),
UNIX workstations (SPARC stations
IPC, IPX, and 10; DEC station 2100),and personal computers (PC9801, IBM
compatible machines, and Macintosh).
Through Ethernet or CDDI, all of them
are linked to the backbone FDDI of ourinstitute, which is further linked to the
new hyper multimedia network ofOkazaki National Research Institutes

76(the ORION network). Each laboratory
has at least two computers connected to
the network and all sites linked to theInternet are accessible from there. In addition, NetWare server machines (Quarter L and DECpc) works as file servers
and printer servers. Latest databases andsoftwares of various kinds are also maintained.
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 m 2
glass-houses with precision temperatureand humidity control, a limited farm,
two greenhouses (45 m 2, 88 m 2) with
automatic sprinklers and window control
two open aquariums (30 t, 50 t) and several smaller tanks. The laboratory also
includes a building with office, storage
and work-space.
6. Plant Cell Culture Laboratory
Autotrophic and heterotrophic culture
devices and equipped for experimenatal
cultures of plant and microbial cells.
7. Molecular Biological Analysis Laboratory
In order to facilitate molecular biological analyses, high performance equipments such as DNA sequencers (ABI
370A; 373A-18), peptide synthesizers(ABI 430A and 431A), a nucleotide
synthesizer (ABI 381A), imaging analyzers (Fuji BAS 2000-3060 and -3080;
PDI Discovery Series) and a glycoprotein analysis system (Takara Glyco-Tag)are provided.
ll. Research Activities
1. Faculty
The faculty of the Research Support
Facility conducts its own research as well

NATIONAL INSTITUTE

as scientific and administrative public
services.
(1) Photobiology: Photoreceptive andsignal transduction mechanisms ofphototaxis of single-celled, flagellate
algae are studied action spectroscopicallyby measuring computerized-videomicroscopically the motile behavior of the cells
at the cellular and subcellular levels.
Photoreceptive and signal tranduction
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 myosis in non-muscle
cells might be better understood if wildtype cell could be compared with mutant
cells with altered myosin molecules,
though such mutants have not been isolated. The faculty intends to examine thefunction of the myosin heavy chain invertebrate non-muscle cells by interrupting its synthesis with the introduction ofantisense RNA molecules.
(3) Computational Biology: Efforts todevelop new methodology for sequence
analysis have been continued. Currently,
there are two projects. One is to develop
an expert system to predict various protein localization sites from amino acidsequence data. The other is to constructa prediction system of mature mRNA
sequence from their precursors. The former work is done in cooperation with
Dr. A. Goffeau (Univ. Catholique
Louvain, Belgium) et al. of yeast genome
sequencing group and Dr. T. Shimizu(Hirosaki Univ.). Currently, we are trying to find a reliable way to predict
transmembrane segments of membraneproteins correctly. The latter work is acooperation with Dr. H. Sakamoto(Kobe Univ.) and we are now focusing

FOR BASIC BIOLOGY

on the study of aberrant and alternative
splicing events: we have constructed anovel databese containing aberrant
splicing mutations of mammalian genes(Nakai and Sakamoto, Gene, in press).
2. Cooperative Research Program for the
Okazaki Large Spectrograph
The NIBB Cooperative Research Program for the Use of the OLS supports
about 30 projects every year conductedby visiting scientists including foreignscientists as well as those in the Institute.
Action spectroscopical studies forvarious regulatory and damaging actionsof light on living organisms, biologicalmolecules, and organic molecules havebeen conducted.
Publication List:
I. Faculty
Goffeau, A., Slonimski, P., Nakai, K.,
and Risler, J. L. (1993) How many
yeast genes encode for membrane
spanning proteins? Yeast9, 691-702.
Goffeau, A., Nakai, K., Slonimski, P.,
and Risler, J. L. (1993) The membrane proteins encoded by yeast chromosome III genes. FEBS Lett. 325,
112-117.
Goto, N., Yamamoto, K. T. and
Watanabe, M. (1993) Action spectra
for inhibition of hypocotyl growth ofwild-type plants and of the hy2 longhypocotyl mutant of Arabidopsis
thaliana L. Photochem. Photobiol. 57,
867-871.
Kataoka, H. and Watanabe, M. (1993)
Negative phototropism in Vaucheria
terrestris regulated by calcium Ill. The
role of calcium characterized by use ofa high-power argon-ion laser as thesource of unilateral blue light. Plant
Cell Physiol. 34, 737-744.
Saitou, T., Tachikawa, Y., Kamada, H.,
Watanabe, M. and Harada, H. (1993)

77
Action spectrum for light-induced formation of adventitious shoots in hairyroots of horseradish. Planta 189,590-592.
Takahashi, T. and Watanabe, M. (1993)
Photosynthesis modulates the sign ofphototaxis of wild-type Chlamydomonas reinhardtii. Effects of red background illumination and 3-(3',4'dichlorophenyl)-1, 1-dimethylurea.
FEBS Lett. 336, 516-520.
II. Cooperative Research Program for
the Okazaki Large Spectrograph
Goto, N., Yamamoto, K. T. and
Watanabe, M. (1993) Action spectra
for inhibition of hypocotyl growth ofwild-type plants and of the hy2 longhypocotyl mutant of Arabidopsis
thaliana L. Photochem. Photobiol. 57,
867-871.
Kataoka, H. and Watanabe, M. (1993)
Negative phototropism in Vaucheria
terrestris regulated by calcium III. The
role of calcium characterized by use ofa high-power argon-ion laser as thesource of unilateral blue light. Plant
Cell Physiol. 34, 737-744.
Matsunaga, T., Hatakeyama, Y., Ohta,
M., Mori, T. and Nikaido, 0. (1993)
Establishment and characterization ofa monoclonai antibody recognizing the
Dewar isomers of (6-4)photoproducts.
Photochem. Photobiol. 57, 934-940.
Mitsuoka, T., Torikai, A. and Fueki, K.
(1993) Wavelength sensitvity of thephotodegradation of poly(methyl
methacrylate). J. Appl. Polym. Sci. 47,
1027-1032.
Oishi, T. and Ohashi, K. (1993) Effectsof wavelengths of light on the photoperiodic gonadal response of blindedpinealectomized Japanese quail. Zoo/.
Sci. 10,757-762.
Okazaki, N., Mori, Y. and Hanazaki, I.

78(1993) Effect of adding starch on thephoto-inhibition of oscillation in the"Briggs-Rauscher" reaction. Chem.
Lett. 1135-1138.
Saitou, T., Tachikawa, Y., Kamada, H.,
Watanabe, M. and Harada, H. (1993)
Action spectrum for light-induced
formation of adventitious shoots inhairy roots of horseradish. Planta 189,590-592.
Sasaki, M., Takeshita, S., Sugiura, M.,
Sudo, N., Miyake, Y., Furusawa, Y.,
and Sakata, T. (1993) Ground-based
observation of biologically active solar
ultraviolet-B irradiance at 35 N latitude in Japan. J. Geomag. Geoelectr.
45, 473-485.
Sekiguchi, T., Mori, Y. and Hanazaki, I.
(1993)
Photo-responseof the[Ru(bpy)3]2+/Br0 3- /H+ system in acontinuous-flow stirred tank reactor.
Chem. Lett. 1309-1312.
Shichijo, C., Hamada, T., Hiraoka, M.,
Johnson, C. B. and Hashimoto, T.
(1993) Enhancement of red lightinduced anthocyanin synthesis in

NATIONAL INSTITUTE

sorghum first internodes by moderate
low temperature given in the preirradiation culture period. Planta 191,
238-245.
Tada, M. (1993) Methods for investigating photoregulated carotenogenesis. In Methods in Enzymology 214(L. Packer ed.). Academic Press, San
Diego, pp.269-267.
Takahashi, T. and Watanabe, M. (1993)
Photosynthesis modulates the sign ofphototaxis of wild-type Chlamydomonas reinhardtii. Effects of red background illumination and 3-(3',4'dichlorophenyl)-1, 1-dimethylurea.
FEBS Lett. 336, 516-520.
Torikai, A., Kato, H., Fueki, K., Suzuki,
Y., Okisaki, F., and Nagata, M.
(1993) Photodegradation of polymer
materials containing flame-cut agents.
J. Appl. Polym. Sci. 50, 2185-2190.
Torikai, A., Mitsuoka, T. and Fueki, K.
(1993) Wavelength sensitivity of thephotoinduced reaction in polycabonate. J. Polym. Sci. 31, 2785-2788.

FOR BASIC BIOLOGYRADIOISOTOPE FACILITY(managed by NIBB)Head of Facility: Takashi Horiuchi
Techinical Staffs: Kazuhiko Furukawa
Yoshimi Matsuda

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, 14C, 22Na,
32P, 35 S, 45 Ca, 1251 and various species ofbeta and gamma-ray emitting nucleides

79

are handled. A laboratory facilty for recombinant DNA research is installed in
the center. At the subcenters, only alimited number of radioisotopes such as
3H, 14C, 32P and 35 S in the NIBB, and3H, 14C, 32p' 3ss, 45Ca and 125J 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.

80

NATIONAL INSTITUTECENTER 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) PhysicalAnalysis, and (5) Microscopic Analysis.
Each section is equipped with instruments for general use as listed below.
1. Section for Chemical Analysis
Amino-Acid Analyzer
HITACHI 835
Gas Chromatograph
SHIMADZU GC-7APTF
SHIMADZU GC-14APFSCHPLC
SHIMADZU LC-6AD
SPECTRA-PHYSICS SP-8700
Ion Chromatograph
DIONEX QIC
Spectrophotometric HPLC Detector
SHIMADZU SPD-M10AV
Peptide Synthesizer
BECKMAN 990C
2. Section for the Preparation of Biological Materials
Coulter Counter
COULTER ZB
Isotachophoresis System
LKB 2127 TACHOPHOR
Preparative Ultracentrifuge
BECKMAN L8-80
Two-Parameter Cell Sorter
BECTON-DICKINSON FACSII
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
Microplate Reader
CORONA MTP-120
CORONA MTP-100F
Spectrofluorometer
HITACHI 850
HITACHI MPF-4
SIMADZU RF-5000
Spectrophotometer
HITACHI 330
Spectropolarimeter
JASCO J-40S
4. Section for Physical Analysis
Analytical Ultracentrifuge
HITACHI 282
Differential-Scanning Calorimeter
SEIKO DSC100
EPR Spectrometer
BRUKER ER 200D
GC-Mass Spectrometer
HITACHI M-80
GC/LC-Mass Spectrometer
JEOL DX-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 ffiAS-I & II
Microscope Photometer
CARL ZEISS MPM 03-FL
Two-Dimension Microdensitometer
JOYCE LOEBL 3CS

RouteN o.24H

National Institute for Basic Biology
Okazaki 444, Japan,•