NATIONAL INSTITUTE FOR BASIC BIOLOGY·.

ANNUAL REPORT1995

The cover photographs indicate distribution of Bombyx
Fkh/SGF-1 protein in embryos. For details, seep. 26.

CONTENTS
INTRODUCTION··································································

1···············································

3

GRADUATE PROGRAMS···························································

4

OFFICE OF DIRECTOR····························································

5

ORGANIZATION OF THE INSTITUTE

Primary processes of light energy conversion
I. Energy transfer processes···········································

5·························································

5

II. Energy conversion processes·····················································

5.....................................

7·················································

8

DEPARTMENT OF CELL BIOLOGYDIVISION OF CELL MECHANISMS

I. Development of microbody membrane proteins during the microbody transition

8

II. Characterization of citrate synthase responsible for the glyoxylate cycle

9III. Membrane proteins of protein bodies and the developmental change duringtransformation between protein bodies and vacuoles · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

IV. A vacuolar processing enzyme responsible for conversion of proprotein
precursors into their mature forms · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·10

DIVISION OF BIOENERGETICS····················································

12

13

DIVISION OF CELL PROLIFERATION (ADJUNCT)I. Animal and plant genes that trans-complement meiotic defects in the fission yeast

13

II. The requirement of cell cycle regulatory genes for meiosis in the fission yeast

13

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)

15

I. Differential expression of islet-1 homo logs during specification of primary motoneuron

15

II. Cloning and expression of AN34/F-Spondin family in zebrafish

15··························

III. Two families of transposon in zebrafish

15

DIVISION OF CELL FUSION (ADJUNCT)17

DEPARTMENT OF DEVELOPMENTAL BIOLOGYDIVISION OF REPRODUCTIVE BIOLOGY···········································

19
20

I. Endocrine regulation of oocyte growth and maturation

20

II. Endocrine regulation of male germ cell development and maturation

22

III. Endocrine regulation of gonadal sex differentiation

22···································DMSION OF CELL DIFFERENTIATION••••••••••••••••••••••••••••••••••••••••••••

25

I. Genes and factors that control the silk gland development and the silk genes transcription

25

II. Other genes of developmental interest· · · •· · · · · · ••· · · · · · · · · · · · · · · •••· · · · · · · · · · •· · · · ·26•••••••••••••••••••••• •••• •••• · · •• •• ••• •• •• •• • •• •• ·28

DIVISION OF MORPHOGENESIS

I. Role of the mi gene in differentiation and transdifferentiation of the pigmented epithelial cells

28

II. The role of integrin in stabilization and instabilization of pigmented epithelial cells

29

III. Comparative analysis of crystallin gene expression between development andregeneration of the lens in newts··················································

29

IV. Comparative analysis of Pax-6 gene expression during eye development in the
newt and Xenopus•· •· · · ••••••· · ••••· · · · · · ••· •· · · ••· · · · •· · •••· ••· ••· · •· •· · ••· · •30

DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)••••••· ••••••••••••••••••••••

31.............................

32•••••• •••••• •••• ••••• •••• ••• ••• •• ••• •• •34

DEPARTMENT OF REGULATION BIOLOGYDIVISION OF MOLECULAR NEUROBIOLOGY

I. Topographic and laminar connection in the chick retinotectal system

34

II. Proteoglycan, protein-tyrosine phosphatase and brain development

34

DIVISION OF CELLULAR REGULATION•· ••· •••· •••••••••••••· ••••· ••· · · •· · · ••••· ••I. Regulation of the expression of genes for desaturases

37···································

37

II. Importance of the unsaturation of membrane lipids in the turnover of the Dl
protein of the photosystem II complex··············································

37

III. Heat stability of the photosynthetic machinery·······································

38

IV. Genetic modification of the tolerance of a cyanobacterium to low-temperature stressby introduction of a gene for the synthesis of glycinebetaine····························

38

V. Genetic modification of the ability ofArabidopsis to tolerate salt and low-temperature stress

38

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT)40

I. Structural organization of photosystem II reaction center40

II. Dynamic aspects of the organization of photosystem II reaction center

40

DMSION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)43

I. Connectin can function as an attractive target recognition molecule

43

II. P750, a novel LRR cell surface molecule expressed on subsets of neurons and muscles

43

III. F-spondin/AN34 family of secreted proteins in Drosophila

43

IV. rH96, a homeobox containing gene essential for neural and muscular development··········LABORATORY OF GENE EXPRESSION AND REGULATIONDIVISION OF GENE EXPRESSION AND REGULATION I•••••· ••· •· ••••••••••· ••••••••

44

47
48

DIVISION OF GENE EXPRESSION AND REGULATION II•••••••••••· •· •· · · · •· •· · •· ••·49

I. Analysis of recombinational hot spot in S. cerevisiae

49

II. Analysis of E. coli recombinational hot spots, Hot

50

DIVISION OF SPECIATION MECHANISMS I· · •· •· ••· · ••••· •· ••••••· ••••· ••••· ••••· ••I. Evolution of cytokine receptor families in the immune and nervous systems

II. Gene expression and cerebellar long-term plasticity

51·················

51····································

51

52

DIVISION OF SPECIATION MECHANISMS IIRESEARCH SUPPORT53

TECHNOLOGY DEPARTMENT54

RESEARCH SUPPORT FACILITY

55

I. Facilities······································································

56

II. Research activities
RADIOISOTOPE FACILITY (Managed by NIBB)

55••••· · · ••· · •••· · ••· •· · •••· ••· · •· · •· ••· •CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB)•· •· · · •••· · •· •· •· · · · · •·

58
60

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¥

INTRODUCTION
We present here the Annual Report 1995
describing the research activities of the National
Institute for Basic Biology (NIBB) during the pastyear.
The NIBB is a government supported basic
research institute in the field of biology. It aims tostimulate and promote studies of biology, byconducting first-rate research as a center of
excellence, and by cooperating with otheruniversities and research organizations. The Instituteconcentrates on the studies of cellular functions,reproduction and development, homeostatic control,and gene expression and regulation in eukaryoticorganisms, to elucidate the fundamental mechanisms
underlying various biological phenomena at themolecular level.
During the past year, the turnover of personnel has
been very high. At the end of March, the former
Director-General, Dr. Ikuo Takeuchi retired from the
NIBB due to his appointment as the President of the
Okazaki National Research Institutes. His position
was taken over by Dr. Hideo Mohri from the
beginning of April. Professor Yoshihiko Fujita of theDivision of Bioenergetics also retired at the end of
March and became Emeritus Professor. His
successor as well as the new professor of theDivision of Gene Expression and Regulation I will
be appointed in the coming April. Professor Masaki
Iwabuchi (Kyoto University) of the Adjunct Divisionof Developmental Biology completed his term ofoffice and was followed by Professor Kenzo
Nakamura of Nagoya University. Two associate
professors, Drs. K. Okada and H. Hayashi, and a
research associate, Dr. T . Kondo, moved as full
professors to Kyoto University, Ehime University,
and Nagoya University, respectively. Drs. M.
Yoshikuni, H. Iida and K. Ogawa were appointed as
Associate Professor of the Division of Reproductive
Biology, the Division of Cellular Regulation and theRadioisotope Facility, respectively. Furthermore, we
newly appointed 6 research associates and 8 instituteresearch fellows, while 10 research associates otherthan Dr. Kondo and two institute research fellows
moved to other institutions.
The NIBB plays important roles as a national andinternational center for the biological research and is
responsible for conducting research projects incooperation with research groups of various
universities and institutes. As a part of suchcooperative activities, the NIBB carried out Special
Programs which are currently directed to"Adaptation and Resistance to Environment" and"Trans-differentiation of Tissue Cells". Based onthese programs, the NIBB held the 34th and 35th
Conferences entitled as "Responses of the
Photosynthetic Apparatus to Environmental Light

Conditions" (organized by Professor Kimiyuki
Satoh) and "Mechanisms of Cell Commitment in
Differentiation" (organized by Professor Goro
Eguchi), respectively.
In addition, the Institute sponsors symposia andstudy meetings on current topics at theinterdisciplinary level by inviting leading scientists invarious related fields, both nationally andinternationally. In 1995, the 11th International
Symposium in conjunction with the Award of theInternational Prize for Biology "Dynamics of theCell" was organized by the NIBB, inviting thelaureate of the 1995 Prize, Professor I. R. Gibbons,and many distinguished investigators in the field ofcell biology. The NIBB also provided a training
course in bioscience for young investigators andparticipated in the International Science School forhigh school students of Asian-Pacific countries.
Fortunate enough, the construction of a newbuilding for the Laboratory of Gene Expression andRegulation (about 2500m 2) has been decided by thegovernment, and institution of the new division,Speciation Mechanisms II, is expected in the coming
year. At the end of the year, the NIBB had asymposium about the future of its own by inviting
authorities in various fields of biology. Discussion at
this symposium was quite stimulative and would be
utilized for making the future plan of the Institute.
Finally, I would like to congratulate that Emeritus

2

NATIONAL INSTITUTE

Professor T. S. Okada, the past President of Okazaki
National Research Institutes and the past DirectorGenaral of NIBB, was honored as Person of Cultural
Merit and Professor G. Eguchi was awarded a Purple
Ribbon Medal and Palmes Academique, Chevarier,from the Government of France.
Hideo Mohri, D.Sc.
Director-General

FOR BASIC BIOLOGY

3

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology, NIBB isa part of the Okazaki National Research Institutes(ONRI) located on a hill overlooking the old town ofOkazaki. The NIBB was established in 1977 and its
activities are supported by Monbu-sho (the Ministryof Education, Science, Sports and Culture) of Japan.
The ONRI are composed of three independent
organizations, National Institute for Basic Biology(NIBB), National Institute for Physiological Sciences(NIPS) and Institute for Molecular Science (IMS).
Policy and Decision Making
The Director-General oversees the operation of theInstitute assisted by two advisory bodies, theAdvisory Council and Steering Council. The
Advisory Council is made up of distinguishedscholars representing various fields of science andculture, and advises the Director-General on thebasic policy of the Institute. The Steering Council ismade up of professors of the Institute and an equal ·number of leading biologists in Japan outside NIBB,
and advises the Director-General on the scientificactivities of the Institute. The Council advises on
faculty appointments and on the Institute's annual
budget as well as its future prospect.
Administration
Administration of the Institute is undertaken by the
Administration Bureau of the Okazaki National
Research Institutes under the direct auspices of the
Ministry of Education, Science, Sports and Culture.
Research
The Institute conducts its research programs
through three departments and one laboratoryorganized into 16 divisions.

Each division has its own research project and isstaffed by a professor, an associate professor and two
research associates. A division forms, in principle, an
independent project team. Six of the divisions are for
adjunct professorship and 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 research techniques andhelps to promote research activities of each division
and also to maintain the research resources of theInstitute. The Department also undertakes thetechnical education of its staff.
Research Support Facility
The research support facility of the NIBB consistsof the Large Spectrograph Laboratory, Tissue andCell Culture Laboratory, Computer Laboratory, Plant
Culture Laboratory, Plant Cell Culture Laboratory,
Experimental Farm, and Laboratory of StressResistant Plants. In addition, seven facilities areoperated jointly with the NIPS; they consist of theRadioisotope Facility, Electron Microscope Center,
Center for Analytical Instruments, Machine Shop,
Laboratory Glassware Facility, Animal Care Facility,and Low-Temperature Facility.
CampusThe Okazaki National Research Institutes covers
an area of 150,000 m2 with four principal buildings.
The NIBB's main research building has a floor spaceof 10,930 m2• Two-thirds of the space was completed
in 1982 and the remaining third in June, 1983. The
buildings which house the research support facility
were also completed in June, 1983.
Divisions

Department/Laboratory

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

Department of Cell Biology

Department of Developmental Biology

Reproductive Biology- { Cell Differentiation
Morphogenesis
Developmental Biology (adjunct)

Department of Regulation Biology

Molecular Neurobiology- { Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)Laboratory of Gene Expression and Regulation-fGene Expression and Regulation I
Gene Expression and Regulation II
Speciation Mechanisms I

4

NATIONAL INSTITUTEGRADUATE PROGRAMSThe NIBB carries out two graduate programs.
1. Graduate University for Advanced Studies
The NIBB constitutes the Department of
Molecular Biomechanics in the School of Life
Science of the Graduate University for Advanced
Studies. The University provides Ph.D. course of
three years duration. Those who have completed
master's course in a university or are qualified
equivalent to or higher are eligible to apply.
The Department consists of the following
Divisions and Fields:
DivisionsFieldsMolecular Cell
Biology

Biomolecular Systems
Cell Dynamics

Developmental
Gene Expression
and Regulation

Gene Expression
Morphogenesis
Transgenic Biology

Regulation
Biology

Biological Regulation
Biological Information

2. Graduate Student Training Program
Graduate students enrolled in other universities
and institutions are trained to conduct research for
fixed periods of time under the supervision of NIBB
professors.

5

FOR BASIC BIOLOGYOFFICE OF DIRECTOR
Director-General: Hideo Mohri
Associate Professor: Shigeru Itoh
Research Associates: Mamoru Mimuro
Katsunori Aizawa

The former members of the Division of
Bioenergetics have been included in the Office of
Director.
Primary processes of light energy conversion
I. Energy transfer processes
Mamoru Mimuro
Primary processes in photosynthesis were studiedin terms of structure-function relationship.
A primary electron donor in the photosystem (PS)II reaction center (RC), P680, was shown to be a
dimer based on the magnetic circular dichroism(MCD) (Nozawa et al., 1995). An MCD signal isinversely proportional to the association state ofpigments, even though its theoretical basis is not yet
known. This relationship was confirmed on RC andlight harvesting II complexes isolated from purpleofbacteria
andartificial
aggregatesbacteriochlorophyll (BChl) c (Kobayashi et al., inpress).
Two types of pheophytin (Pheo) molecules in the
PS II RC was spectrally discriminated. The
photochemically active Pheo molecule has a lower
energy level than that of the photochemicallyinactive Pheo molecule, which is analogous to thebacterial RC (Mimuro et al., 1995). This confirms an
evolutional linkage between bacterial RC and PS II
RC in oxygenic photosynthesis.
An antenna system of green photosynthetic
bacteria, chlorosomes was studied in term of
relationship among morphology, molecular structureand spectral properties (Wang et al., 1995). The
hexanol-induced changes on chlorosomes wereexplained by a direct interaction between BChl c andhexanol, which consequently induced a unidirectional elongation of rod elements and ofchlorosomes. In addition, we found a new component
in chlorosomes which facilitates a rapid energy
migration from chlorosomes to the antenna inmembranes (Mimuro et al., in press).
A new approach for understanding ofphotosynthesis was started by a collaboration entitled"Experimental analyses of the origin of chloroplasts
and phylogeny in algae".
II. Energy conversion processes

Shigeru ltoh
Photosynthesis in plants and bacteria convert solar
energy to electrochemical one in the reaction center(RC) complexes that ar~ the molecular machines

made of proteins and prosthetic groups. RC's seemto be established more than 3 billion years ago in theearly Earth. We study the function of RC's, electrontransfer systems, and their evolution by three
approaches: 1) Exchange of the prosthetic groups byartificial compounds, 2) Genetic modification of theproteins, and 3) Exploration of new electron transfer
systems. We replaced the phylloquinone (vitamin Kr)
in the RC complex of plant photosystem I by
artificial quinones. The method enabled us to change
the driving force of the reaction and clarified the
optimization mechanism of the ultra-rapid electrontransfer with a reaction time of 10-11 second by laser
photolysis. Similar studies were also done in the
newly purified RC of green bacteria that is a
prototype of the plant RC. Mechanism of quinone
reaction was also clarified in the geneticallymodified oxidase of bacterial respiratory chain bycryogenic EPR. Surprisingly, we discovered a neworganism that does not use Mg-chlorophyll but uses
Zn-porphyrin in their photosynthesis. This opened a
new stage in the study of photosynthesis that has
been believed to be obligatory performed by Mgchlorophy ll 's.
Publication List:
Demidov, A A
and Mimuro, M. (1995)
Deconvolution of C-Phycocyanin B-84 and B-155
chromophore absorption and fluorescence spectraof cyanobacterium Mastigocladus laminosus.
Biophys. J. 68, 1500-1506.
Ikegami, 1., ltoh, S.and Iwaki, M. (1995) Photoactive
photosystem I particles with a molar ratio ofChlorophyll a to P700 of 9. Plant Cell Physiol. 36,
857-864.
Mimuro, M., Torno, T., Nishimura, Y., Yamazaki, I.
and Satoh, K. (1995) Identification of aphotochemically inactive pheophytin molecule inthe spinach D 1-D 2 -cyt b 559 complex. Biochim.
Biophys. Acta 1232, 81-88.
Mohri, H., Kubo-Irie, M. and lrie, M. (1995) Outerarm dynein of sperm flagella and cilia in the
animal kingdom. Meme. Mus. natn. Hist. nat. 166,
15-22.
Nozawa, T., Kobayashi, M., ltoh, S., Iwaki, M.,
Mimuro, M. and Satoh, K. (1995) Magnetic
circular dichroism investigation of chlorophylls inreaction centers of photosystems I and II of green
plant photosynthesis. Spectrochimica Acta SlA,
125-134.
Oh-oka, H., Kakutani, S., Kamei, S., Matsubara, H.,
Iwaki, M. and ltoh, S. (1995) Highly purified
photosynthetic reaction center (PscNCytochrome
c551 )2 complex of the green sulfur bacterium
Chlorobium limicola. Biochemistry 34, 13091-

6

13097
Oh-oka, H., Kamei, S., Matsubara, H. and lwaki, M.
Itoh, S. (1995) Two cytochrome c heme function
as the electron donor to P840 in the reaction center
complex of a green sulfur bacterium, Chlorobium
tepidum. FEBS Lett. 365, 30-34.
Sato-Watanabe, M., ltoh, S., Magi, T., Matsuura, K.,
Miyoshi, H. and Anraku, Y. (1995) Stabilization of
a semiquinone radical at the high affinity quinone
binding site of the Escherichia coli bo-type
ubiquinone oxidase. FEBS Lett. 374,265-269.
Sonoike, K., Terashima, 1., Iwaki, M. and Itoh, S.
(1995) Site of destruction of photosystem I ironsulfur centers in leaves of Cucumis sativus L. byweak illumination at chilling temperatures is A1,
the phylloquinone acceptor. FEBS Lett. 362, 235238.
Wang, Z.-Y., Marx, G., Umetsu, M., Kobayashi, M.,
Mimuro, M. and Nozawa, T. (1995) Morphology
andspectroscopyofchlorosomesfrom
Chlorobium tepidum by alcohol treatments.
Biochim. Biophys. Acta 1232, 187-196.

NATIONAL INSTITUTE

7

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairman: Goro Eguchi

This department is composed of two regular divisions andthree adjunct divisions, and conducts researches on thefundamentals of cell structures and functions in plants andanimals at molecular level utilizing modem technologies
including genetic engineering.

8

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor:Mikio Nishimura
Research Associates: Makoto Hayashi
Jkuko Hara-Nishimura
Tomoo Shimada
NJBB Postdoctral Fellow: Akira Kato
Graduate Students: Tetsu Kinoshita
Nagako Hiraiwa
Shouji Mano
Masahiro Aoki I)
Daigo Takemoto 2!
Technical Staffs: Maki Kondo
Katsushi Yamaguchi
Yasuko Koumoto
Visiting Scientists: Kyoko Hatano 3!
from Shinshu University
from Nagoya University! from Kyoto Univerisity

I)
2
)
3

Higher plant cells contain several distinct
organelles that play vital roles in cellular physiology.
During proliferation and differentiation of the cells,
the organelles often undergo dynamic changes. The
biogenesis of new organelles may occur, existingorganelles may undergo a transformation of function,and other organelles may degenerate. Because thedynamic transformation of organellar function(differentiation of organelles) is responsible for theflexibility of differentiation events in higher plant
cells, the elucidation of regulatory mechanisms
underlying organelle transformation are currently
being studied in this division.

I. Development of microbody membrane pFoteinsduring the microbody transition
Dramatic metabolic changes which underlie theshift from heterotrophic to autotrophic growth occur
during the in greening of seeds Accompanying thesemetabolic changes, many constitutive organelles arefunctionally transformed. For example, etioplasts
differentiate into chloroplasts and mitochondria
acquire the ability to oxidize glycine. Glyoxysomes,
which are microbodies engaged in the degradation ofreserve oil via b-oxidation and the glyoxylate cycle,
are transformed into leaf peroxisomes that function
in several crucial steps of photorespiration. After thefunctional transitiOn of glyoxysomes to leaf
peroxisomes during the greening of pumpkin
cotyledons, the reverse microbody transition of leaf
peroxisomes to glyoxysomes occurs during
senescence. To clarify the molecular mechanisms
underlying the microbody transition, the change with
development in microbody membrane proteinsduring transformation of glyoxysomal to leaf
peroxisomes was characterized. Two proteins inglyoxysome membranes, with m9lecular masses of
31 and 28 kDa, were purified and characterized. The
31-kDa membrane protein was found to be a novel
isoenzymeof ascorbate
peroxidase.
Intactglyoxysomes and leaf peroxisomes had no latent
peroxidase activity, an indication that the active siteof the ascorbate peroxidase was exposed to thecytosol and that the peroxidase would scavengehydrogen peroxide leaked from microbodies (Fig. 1).

Fig. 1. Possible function of the ascorbate peroxidase localized on microbody membranes. mbAPX, microbody ascorbate
peroxidase; cAPX, cytosolic ascorbate peroxidase; CAT, catalase; AsA, ascorbate; DHA, dehydroascorbate; MDA,
monodehydroascorbate.

FOR BASIC BIOLOGY

Analysis of these membrane proteins during
development revealed that the amounts of thesemembrane proteins decreased during the microbody
transition and that the larger one was retained in leaf
peroxisomes, whereas the smaller one could not befound in leaf peroxisomes after completion of the
microbody transition. These results clearly showedthat membrane proteins in glyoxysomes change
dramatically during the microbody transition, as do
the enzymes in the matrix.
II. Characterization of citrate synthase responsible for the glyoxylate cycle
Citrate synthase catalyzes the conversion of
oxaloacetate to citrate. In plant cells, there are two
isozymes of citrate synthase, one is an enzyme
involved in the glyoxylate cycle in glyoxysomes(gCS) and the other is an enzyme involved in the
TCA cycle in mitochondria. To study the structuralcharacteristics and development of gCS during the
microbody transition at the molecular level, wecloned eDNA for pumpkin gCS. The deduced aminoacid sequence of gCS did not have a typical
peroxisomal targeting signal at its carboxyl terminus.
A study of the in vitro expression of the eDNA andan analysis of the amino-terminal sequence of thecitrate synthase indicated that gCS is synthesized as alarger precursor that has a cleavable amino-terminal
presequence of 43 amino acids. The predicted
amino-terminal sequence of pumpkin gCS was highly
homologous to those of other microbody enzymes,
such as 3-ketoacyl-CoA thiolase of rat and malate
dehydrogenase of watermelon that are alsosynthesized as precursors of higher molecular mass.
An immunoblot analysis showed that the level ofgCS protein increased markedly during germinationand decreased rapidly during the light-induced
transition of microbodies from glyoxysomes to leaf
peroxisomes. By contrast, the level of mRNA forgCS reached a maximum earlier than that of theprotein and declined even in darkness. The level ofthe mRNA was low during the microbody transition.
These results indicate that the accumulation of thegCS protein does not correspond to that of themRNA and that degradation of gCS is induced
during the microbody transition, suggesting that posttranscriptional regulation plays an important role inthe microbody transition.
III. Membrane proteins of protein bodies and thedevelopmental change during transformation
between protein bodies and vacuoles
During seed maturation, protein-storage vacuoles
are converted to protein bodies that are found in dry
seeds. In contrast, during the postgermination growthof seeds, protein bodies fuse with one another andare converted to a central vacuole. To investigate thistransition, we prepared protein-body membranes
from dry seeds of pumpkin (Cucurbita sp.) and9characterized their protein components. Five major
proteins (designated MP23, MP27, MP28, MP32
and MP73) were detected in the protein-bodymembranes. We have isolated cDNAs for MP23,
MP27, MP28 and MP32 and characterized them.
Among the five membrane proteins, MP27 andMP32 disappeared most rapidly during seedling
growth. Both MP27 and MP32 were encoded by asingle eDNA. The deduced precursor polypeptide
was composed of a hydrophobic signal sequence,
MP27 and MP32, in that order. A putative site ofcleavage between MP27 and MP32 was located on
the COOH-terminal side of asparagine 278, an
indication that the post-translational cleavage may
occur by the action of a vacuolar processing enzyme(VPE) that converts proprotein precursors of seed
proteins into the mature forms. Immunoelectron
microscopic analysis showed that MP27 and MP32
was associated with the protein-body membrane ofdry pumpkin seeds. The degradation of MP27 andMP32 starts just after seed germination and proceeds
in parallel with the transformation of the protein
bodies into vacuoles.
Molecular characterization revealed that both
MP28 and MP23 belong to the seed TIP (tonoplast
intrinsic protein) subfamily. TIP is an integral
membrane protein that was originally found in plantseeds and belongs to the MIP (major intrinsic
protein) family, the members of which are widelydistributed in bacteria, animals and plants. The TIPof plant seeds is abundant and is conserved among
both monocots and dicots. The predicted 29-kDa
precursor to MP23 includes six putative membranespanning domains, and the first loop between the firstand second transmembrane domains is larger thanthat of MP28. The N-terminal sequence of themature MP23 starts from residue 66 in the first loop,
indicating that an N-terminal 7-kDa fragment that
contains one transmembrane domain is posttranslationally removed. During maturation of
pumpkin seeds, mRNAs for MP28 and MP23
became detectable in cotyledons at the early stage,
and their levels increased slightly until a rapid
decrease occurred at the late stage. This is consistent
with the accumulation of the 29-kDa precursor and
MP28 in the cotyledons at the early stage. By
contrast, MP23 appeared at the late stage
simultaneously with the disappearance of the 29-kDa
precursor. Thus, it seems possible that the conversionof the 29-kDa precursor to the mature MP23 mightoccur in the vacuoles after the middle stage of seedmaturation.
Bothproteins
werelocalized
immunocytochemically on the membranes of thevacuoles at the middle stage and the protein bodies atthe late stage. These results suggest that both MP28
and the precursor to MP23 accumulate on vacuolar
membranes before the deposition of storage proteins,
and then the precursor is converted to the mature
MP23 at the late stage. These two TIPs might have a
specific function during the maturation of pumpkin

10

seeds.

IV. A vacuolar processing enzyme responsible forconversion of proprotein precursors into theirmature forms
Processing enzymes responsible for the maturationof seed proteins belong to a novel group of cysteine
proteinases with molecular masses of 37 to 39 kDa.
However, the processing enzyme activity can befound not only in seeds but also in vegetative tissues
such as hypocotyls, roots and mature leaves. Thus,
we designated the enzyme as a vacuolar processing
enzyme (VPE). The molecular characterization of all
the members of the VPE family in Arabidopsis isrequired if we are to elucidate the mechanisms ofregulation of genes for VPE homologues and thephysiological functions of these proteins in proteinstorage vacuoles and vegetative vacuoles.
Southern blot analysis showed that a family ofgenes for VPEs in Arabidopsis thaliana wascomposed of three genes, for a- VPE, ~- VPE and yVPE. We isolated the three genes of VPEs from agenomic library of Arabidopsis. The positions ofeight introns were fully conserved among the three
genes, with the exception that the a- VPE gene was
missing the fifth intron found in the ~- VPE and yVPE genes.
Northern blot analysis revealed that a- VPE wasexpressed in rosette leaves, cauline leaves and stemsof Arabidopsis, while ~ - VPE was predominantly
expressed in the flowers and buds. The y- VPE gene
was expressed predominantl y in the stems, with alower level of expression in rosette and cauline
leaves. However, the expression was not detected inroots, flowers plus buds, or green siliques. This result
strongly suggests that the a- VPE and y- VPE genes
encode isozymes of VPE that are specific to

NATIONAL INSTITUTE

vegetative organs. To demonstrate temporal andspacial expression of the promoters of the vegetative
VPE gene, we transformed tobacco plants with areporter gene containing the promoter of the y- VPEgene and the coding region of ~-glucuronidase(GUS). The GUS activity was predominantly
expressed in the senescing tissues (Fig. 2).
Members of the VPE family can be separated into
two subfamilies, one that is specific to seeds andanother that is specific to vegetative organs, such asleaves and stems. The members of the seedsubfamily might function in the protein-storage
vacuoles of seeds, while those of the vegetative
subfamily might function in the lytic vacuoles of
non-storage organs. The VPE cleaved a peptide bondon the carbonyl side of an exposed asparagine
residue on the molecular surface of proprotein
precursors to generate the mature forms of seed
proteins. A similar type of post-translational
processing has been reported in the case of
maturation of vacuolar proteins in vegetative tissues,
such as the proteinase inhibitors of tomato leaves andtobacco stigmas, and the chitinase of tobacco leaves
and cultured cells. These observations suggested that
VPEs are widely distributed in plant tissues and play
crucial roles in the maturation of a variety of proteinsin plant vacuoles. This suggestion is supported by thereport that VPE activity can be detected not only inseeds but also in vegetative organs and, moreover,that two of the VPE genes of Arabidopsis are
specifically expressed in vegetative organs.

Publication List:
De Bellis, L., Hayashi, M., Nishimura, M. and Alpi,
A. (1995) Subcellular distribution of aconitaseisoforms in pumpkin cotyledons. Planta 195, 464-

Fig. 2. Histochemical local~zation of GUS activity in tobacco plants transformed with the y- VPE promoter-gus fusion gene.
Tobacco . was transformed wJth a re~orter gene containing the promoter of the y- VPE gene and the coding region of ~­glucuromda~e (GUS). Transv e r~e sectiOns of. the anthe~s of the transgenic plants were subjected to GUS staining. GUS activity
was fo~nd m the ~~ endothelium, connective and Circular cell cluster in the anther at developmetan l stage 1 (A). The
predommant expressiOn was observed in the circular cell cluster at developmental stage 3 (B).

FOR BASIC BIOLOGY

468.
Hara-Nishimura, 1., Shimada, T., Hiraiwa, N. andNishimura, M. (1995) Vacuolar processing enzyme
responsible for maturation of seed proteins. J.
Plant Physiol. 145, 632-640.
Hayashi, M., De Bellis, L., Alpi, A. and Nishimura,
M. (1995) Cytosolic aconitase is a member forglyoxylate cycle in etiolated pumpkin cotyledons.
Plant Cell Physiol. 36, 669-680.
Inoue, K., Motozaki, A., Takeuchi, Y,. Nishimura, M.
and Hara-Nishimura, I. (1995) Molecularcharacterization of proteins in protein-bodymembrane that disappear most rapidly duringtransformation of protein' bodies into a vacuole.
Plant Journal 7, 235-243.
Inoue, K., Takeuchi, Y., Nishimura, M. and HaraNishimura, I. (1995) Characterization of two
integral membrane proteins located in the protein
bodies of pumpkin seeds. Plant Mol. Bioi. 28,
1089-1101.
Kato, A., Hayashi, M., Mori, H. and Nishimura, M.
(1995)
Molecular
characterizationofaglyoxysomal citrate synthase that is synthesized asa precursor of higher molecular mass in pumpkin.
Plant Mol. Bioi. 27, 377-390.
Kinoshita, T., Nishimura, M. and Hara-Nishimura, I.
(1995) Homologues of a· vacuolar processing
enzyme that are expressed in different organs in
Arabidopsis thaliana. Plant Mol. Bioi. 29, 81-89.
Kinoshita, T., Nishimura, M. and Hara-Nishimura, I.
(1995) A family of the vacuolar processing
enzymes in Arabidopsis thaliana is encoded bythree genes: The sequence and expression of thethird g-VPE gene. Plant Cell Physiol. 36, 15551562.
Nii, N., Yamaguchi, K. and Nishimura, M. (1995)
Effects of fruiting on amylase activity and ribulose
bisphosphate carboxylase-oxygenase content inpeach leaves. J. Jap. Soc. Hort. Sci. 64, 267-273.
Nii, N., Watanabe, T., Yamaguchi, K. and Nishimura,
M. (1995) Changes of anatomical features,photosynthesis
andribulosebisphosphatecarboxylase-oxygenase content of mango leaves.
Annals of Botany 76, 649-656.
Nozue, M., Kubo, H., Nishimura, M. and Yasuda, H.
(1995) Detection and characterization of avacuolar protein (VP24) in anthocyanin-producing
cells of sweet potato suspension cultures. Plant
Cell Physiol. 36, 883-889.
Strzalka, K., Hara-Nishimura, I. and Nishimura, M.
(1995) Physical change in vacuolar membranes
during transformation of protein bodies into
vacuoles in germinating pumpkin seeds. Biochim.
Biophys. Acta 1239, 103-110.
Takeda, S., Kowyama, Y., Takeuchi, Y., Matsuoka,
K., Nishimura, M. and Nakamura, K. (1995)
Spatial patterns of sucrose-inducible andpolygalacturonic acid-inducible expression ofgenes that encode sporamin and b-amylase insweet potato. Plant Cell Physiol. 36, 321-333.

11

Yamaguchi, K., Mori, H. and Nishimura, M. (1995)
A novel isoenzyme of ascorbate peroxidase
localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin. Plant Cell Physiol. 36,
1157-1162.
Yamaguchi, K., Takeuchi, Y., Mori, H. andNishimura, M. (1995) Development of microbody
membrane proteins during the transformation ofglyoxysomes to leaf peroxisomes in pumpkin
cotyledons. Plant Cell Physiol. 36, 455-464.

12

NATIONAL INSTITUTEDIVISION OF BIOENERGETICSThe Division has been closed upon Professor
Yoshihiko Fujita's retirement on March 31, 1995,
and will be re-intiated in 1996 on new projects.

Publication List:
Fujita, Y., Murakami, A., and Aizawa, K. (1995) The
accumulation of protochlorophilide in cells of
Synechocystis PCC 6714 with a low PSI/PSII
stoichiometry. Plant Cell Physiol. 36, 575-582.
Ohki, K. and Fujita, Y. (1995) Intracellular locationof cytochrome oxidase active in vivo in the
cyanophytes, Synechocystis sp. PCC 6714 andAnacystis nidulans Tx20 and R2, grown undervarious conditions. Protoplasma 188, 70-77.

FOR BASIC BIOLOGYDIVISION OF CELL PROLIFERATION(ADJUNCf)
Professor: Masayuki Yamamoto
Research Associate: Masuo Goto
NIBB Postdoctoral Fellow: Takashi Kuromori
Graduate Students: Satsuki Okamoto (from Nara Women's
University)
Fumihiko Hakuno (from The
University of Tokyo)
Chikako Kitayama (from The
University of Tokyo)

This Division aims to explore the regulation ofmeiosis in higher organisms. Meiosis is a crucial stepin gamete formation and is essential for sexual
reproduction. Meiotic steps are highly conserved
among eukaryotic species. The major strategy taken
by us to isolate genes that may be relevant to theregulation of meiosis in animals or plants depends
upon trans-complementation between heterologous
organisms. In the fission yeast Schizosaccharomyces
pombe, which is a unicellular eukaryotic
microorganism, genes involved in control of meiosis
have been well characterized and many of them arecloned (reviewed in Yamamoto (1996) Trends
Biochem. Sci. 21, 18-22). Mutants defective in thesegenes, isolated either by classical genetics or by genedisruption and chromosome manipulation, areavailable. We have thus set out to isolate homologsof these S. pombe genes from animals and a plant,
mainly by using functional complementation of themutants. To facilitate this strategy, we also paid
efforts to elucidation of the regulatory mechanismsof meiosis in the fission yeast.

I. Animal and plant genes that trans-complement
meiotic defects in the fission yeast
Using eDNA libraries prepared from mouse testis,
Xenopus oocyte and Arabidopsis thaliana, we
screened extensively for genes that can rescue loss offunction of the following three genes, which are
involved in the regulation of sexual development in S.
pombe: The sme2 gene, which encodes an RNA
product essential for the promotion of meiosis I; thepdel gene, which encodes cAMP phosphodiesterase;13

and the mesl gene, which is required for thepromotion of meiosis II. Genes encoding putative
kinases, transcription factors, RNA-binding proteins
and others have been isolated in these screenings,
and their possible roles in the regulation of meiosis
are currently under investigation. In particular, wefound that various genes encoding cytoskeletal
proteins can suppress the mesl defect, suggestingthat the regulation of meiosis II is closely related tomodification or reorganization of the cytoskeleton.

II. The requirement of cell cycle regulatory genes
for meiosis in the fission yeast
The cdc2 gene of S. pombe encodes aserine/threonine protein kinase, which plays key
roles in the progression of both G 1 and G2 phases in
the mitotic cell cycle. This kinase regulates G2/M
transition in cooperation with a B-type cyclin
encoded by cdc13, forming a complex called MPF(M-phase promoting factor). The cdc25 geneencodes a tyrosine protein phosphatase that activatescdc2 kinase by dephosphorylation. These three cdc
genes are required for mitosis, and cells carrying atemperature-sensitive mutation in either of thesegenes frequently produce two-spored asci at thesemi-restrictive
temperature,apparently
notundergoing meiosis II. Cells carrying a mutant alleleof cdc2, originally named twsl, have no obvious
defect in the mitotic cell cycle but produce twospored asci skipping meiosis II. These previous
observations suggest that these three genes are
essential for meiosis II. However, the involvement ofthese genes in meiosis I has been left unclear. We
carefully investigated this problem in three different
experimental systems and concluded that the functionof cdc2 is essential for premeiotic DNA synthesis,
and that cdc13 and cdc25 are essential for meiosis I.
We could not conclude whether cdc2 is essential formeiosis I, due to experimental difficulties, although it
appears likely. The necessity of cdc2 function forpremeiotic DNA synthesis in S. pombe contrasts tothe previous report by others that CDC28, the S.
cerevisiae homolog of cdc2, is dispensable for it.

14

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cdc2~
premeiotic DNA
synthesis

cdc 13 , cdc25 expression'(Cdc2 activation)~Ei~meiosis I~ cdc2 , cdc13 , cdc25{!} {!}

meiosis II

Fig. 1. The function of cell cycle genes in the regulation of meiosis in fission yeast. See Iino et al. (1995) for more details.

Publication List:Iino, Y., Hiramine; Y. and Yamamoto, M. (1995)
The role of cdc2 and other genes in meiosis in
Schizosaccharomyces pombe. Genetics 140, 12351245.
Izumiya, H. and Yamamoto, M. (1995) Cloning andfunctional analysis of the ndkl gene encoding
nucleoside diphosphate kinase in Schizosaccharomyces pombe. J. Bioi. Chern. 17, 2785927864.
Kanoh, J., Sugimoto, A. and Yamamoto, M. (1995)
Schizosaccharomyces pombe zfsl + encoding azinc-finger protein functions in the mating
pheromone recognition pathway. Mol. Bioi. Cell 6,1185-1195.

15

FOR BASIC BIOLOGYDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Yoshiki Hotta
Associate Professor: Hitoshi Okamoto
Research Associates: Mika Tokumoto
Shin-ichi Higashijima
Institute Research Fellow: Nobuyoshi Shimada· Brain can be seen as an integrated circuit where
neurons of various identities are interconnected in ahighly ordered manner by their axons. We have beeninterested in how individual neurons aquire their own
identities and how their axons find their own
pathways and finally recognize their proper targets.
Using zebrafish (Dania rerio ), which is suitable forgenetic analysis and gene manipulation, we are trying
to address these questions both at the molecular andcellular levels.

I. Differential expression of islet-1 homologs during specification of primary motoneuron
Islet-1 (Isl-1) is a LIM domain/homeodomain-typetranscription regulator originally identified as an
insulin gene enhancer binding protein. Isl-1 isconsidered to be involved in the differentiation of theneuronal cells. We previously cloned a isl-1 homolog
from zebrafish eDNA library, named it zebrafishis/-1.
Recently, we have isolated two novel is/-1 homologs
from zebrafish eDNA library, named themzebrafishis/-2 and 3.
We examined the mRNA expression pattern ofeach homolog using in situ hybridization to wholemount embryos. All three homologs are expressed in
Rohon-Beard neurons. However the expression inprimary motoneurons diverged. Zebrafishis/-1
mRNA is expressed in the rostral primary
motoneuron (RoP). isl-2 mRNA is expressed in thecaudal primary motoneuron (CaP) and its variant
sibling (YaP). is/-3 mRNA is expressed in the ventral
region of the myotome but not in the primary
motoneurons. The ventral myotome is the region thatthe axon of the CaP extend into. isl-3 mRNA is also
expressed throughout the developing eye and tecta!
region of the midbrain, the target for the retial axons.
These results raise possibilities that the isl-1
homologs may be involved in the specification
and/or target recognition by the primary
motoneurons.

II. Cloning and expression of AN34/F-Spondin
family in zebraflsh
F-Spondin is a secreted protein expressed at highlevels in the floor plate. The C-terminal half of theprotein contains six TSRs (Thrombospondin Type 1
repeats), while the N-terminal half exhibited nohomology to other proteins. Functions of F-Spondinin vivo remain largely unknown. AN34, expressed ina subset of muscle cells in Drosophila, was cloned
and found to encode a secreted protein sharing highdegree of homology in two parts (Hl and H2) with
the N-terminal half of F-Spondin (personalcommunication). This suggests that the homologous
reigions are novel domains important for neuronaldevelopment. Thus, we searched for AN34/FSpondin family from zebrafish (Dania rerio ). By
PCR screening and subsequent eDNA screening, we
identified two novel genes (ANl and AN2) inaddition to zebrafish F-Spondin. ANl and AN2
consist of Hl, H2 and one TSR. The overall structure
are highly similar to AN34 and more related to
AN34 than F-Spondin, suggesing that ANl, AN2 andDrosophila AN34 constitute a novel subfamily. ANl
is expressed weakly in the floor plate and a small
subset of neuronal cells at 22-26 hr. AN2 expression
is first observed broadly around the axial mesoderm(notochord) at 10-12 hr. Then, the expression
becomes restricted to the floor plate and hypochordat 14 hr and continues at high levels up to 26 hr. FSpondin is expressed in the floor plate as seen in rat.
These findings suggest that the AN34/F-Spondin
family play important roles in the midline
development in the vertebrate.

III. Two families of transposon in zebraflsh
We have discovered two families of shortinterspersed repetitive elements (SINEs) in thezebrafish genome. The two families are marked bytheir wide distribution: one family, designatedmermaid, is also present in the genomes of other fish,
amphibian and primates, but absent in the mouse
genome. Some members of the mermaid family were
found in transposon-like repetitive elements
including Tel-like elements which are also
distributed in the genomes of fish and amphibian.
This raises the possibility of horizontal transfer ofthe mermaid family between vertebrates via DNAmediated transposition. The mermaid family is
distinctive in each species except for a conserved
region of approximately 80 bp. The zebrafish
mermaid sequence is about 400 bp long and has atypical SINE structure: split promoter of RNApolymerase III at the 5' end, tandem repeats of shortoligonucleotide (AATG)n at the 3' end and target site
duplications.
The zebrafish mermaid were estimated to be
12,000 copies per haploid genome and highly
polymorphic between AB comp and Darjeelingstrains. We also found that oligonucleotides directedto the conserved region of the mermaid family can beused to recover zebrafish specific DNA fromzebrafish-mouse cell hybrids by PCR (mermaid
PCR). Thus, the mermaid sequence serves as avaluable genetic tool for the zebrafish genome
mapping.
Another SINE family is distributed not only inother Cypriniformes, such as silver carp and grass
carp but also in medaka, at least. The family wasnamed angel since some DNA fragments cloned
from medaka which harbor the entire region of the

16

NATIONAL INSTITUTE

Fig. 1. Expression of AN2 mRNA in the developing zebrafish spinal cord .
.
.
AN2 mRNA is specifically expressed in the floor plate (fp) and hypochord (hy). SC, spmal cord, N, notochord.

SINE migrated anomalously on gels. The anomaly
was temperature-dependent, suggesting a sequenceinduced unusual structure of DNA The angel family
belongs to a new class of elements referred to asminiature inverted repeat transposable elements(MITEs) or inverted repeat SINEs. The angelsequence has relatively long terminal inverted
repeats (TIRs) that flank short non-coding DNA The
TIRs of angel begin with the sequence
TTAAAGGRR, known as the T2 motif first
identified in other inverted repeat SINEs in Xenopus .

Publication List:
Chiba, A, Snow, P., Keshishian, H. and Hotta, Y.
(1995) . Fasciclin III as a synaptic target
recognition molecule in Drosophila. Nature 374,
166-168.
Hosoya, T. , Takizawa, K., Nitta, K. and Hotta, Y.
(1995). glial cells missing: A binary switch
between neuronal and glial determination in
Drosophila. Cell82, 1025-1036.
Tokumoto, M., Gong, Z. , Tsubokawa, T ., Hew, C. L. ,Uyemura, K. , Hotta, Y. and Okamoto, H . (1995).
among
primary
Molecular
heterogeneity
motoneurons and within myotomes revealed by thedifferential mRNA expression of novel Islet-1
homologs in embryonic zebrafish. Dev. Bioi. 171,578-589.

17

FOR BASIC BIOLOGYDIVISION OF CELL FUSION (ADJUNCT)
Professor: Hitoshi Sakano
Research Associate: Masahiro /shiura (until March)
Fumikiyo Nagawa (until April)
Kanae Muraiso (on leave)
Akio Tsuboi
Institute Research Fellow: Hiroaki Kasai
Graduate Student: Setsuyuki Aoki (from Kyoto University)
Nika Yamazaki (from Tokyo Institute ofTechnology)

Our research interest has been focused on somatic
DNA changes in the immune system. In lymphocytes,
DNA recombination and gene conversion play
important roles in the expression of antigen receptor
genes. Gene rearrangement, known as V-(D)-J
joining, not only generates a vast diversity in thereceptor genes, but also insures the activation of aparticular member of the multigene family.
Recently, hundreds of odorant receptor (OR) genes
have been reported in the olfactory system, although
it is yet to be studied how this gene system isregulated for expression. Each OR gene is expressedin one of the four topographically distinct zones,
where olfactory neurons expressing a given OR geneare randomly distributed. It is assumed that only alimited number of the OR genes (possibly one) are
activated in each olfactory neuron.
We have been studying how individual neurons
express a limited number of OR genes keeping therest of the genes silent. In order to study thisselective expression of the OR genes, we have beencharacterizing a Pl clone containing two highly
related genes, MORlO and MOR28. These genes are
92% homologous in the coding regions and linked intandem on mouse chromosome 14. In situ
hybridization has revealed that both genes are
expressed in the same spatial zone within theolfactoryepithelium,
but
never
expressed
simultaneously in the same neuron. Interestingly,
olfactory neurons expressing either MOR10 or
MOR28 project their axons to a distinct, but adjacent
set of glomeruli on the olfactory bulb.
Developmental analyses have revealed that
MOR10-expressing neurons come out earlier than
MOR28-expressing
ones
duringmouseembryogenesis. However, the number of MOR28
neurons overcomes that of MOR10 neurons after
birth; the ratio of cells expressing MOR28 vs.
MOR10 reaches 2.5 : 1 in the adult. These results
suggest that individual olfactory neurons may
activate OR genes through a stochastic mechanism
even between two neighboring and closely related
OR genes; yet this selection appears to be biased inboth the onset and levels of expression.
In order to study whether the projection of axons
to the olfactory bulb exerts influences on the OR
gene expression, we have characterized pdn/pdn
mutant mice in collaboration with Dr. Naruse at

Aichi prefectural colony. The homozygous miceshow a number of developmental abnormalities
including polydactyly and gross malformations of thebrain; they also lack olfactory bulbs. In situ
hybridization has revealed that expression patterns of
the MOR10 and MOR28 genes in the pdn/pdn
embryos are comparable to those found in the wildtype, suggesting that the zonal expression of OR
genes in the olfactory epithelium is regulated
independently of influences from the olfactory bulb.
For the study of the mutually exclusive expressionof OR genes, we have generated transgenic mice
which are devised to express the MOR28 gene inevery olfactory neuron under the control of theolfactory marker protein (OMP) promoter. Since the
OMP gene is expressed in the mature neurons within
the olfactory epithelium, the transgene is expected tobe activated in all olfactory neurons. We believe thatthe study of such transgenic mice will give us new
insight into the molecular mechanisms for OR gene
expression, as well as for neuronal projection to theolfactory bulb.
When the olfactory neurons are regenerated, they
send axons to specific sets of glomeruli in theolfactory bulb. It is amazing that axons expressing agiven OR gene are able to find their target among
two thousand pairs of glomeruli. We hope that ourtransgenic approach mentioned above will become auseful clue to study the target specificity andselectivity in the synapse formation.
Publication List:
Aoki, S., Kondo, T. and Ishiura, M. (1995) Circadian
expression of the dnaK gene in the cyanobacterium
Synechocystis sp. strain PCC 6803. J. Bacterial.
177,5606-5611.
Hiramatsu, R., Akagi, K., Matsuoka, M., Sakumi, K.,
Nakamura, H., Kingsbury, L., David, C., Hardy, R.
R., Yamamura, K. and Sakano, H. (1995) The 3'
enhancer region determines the BIT specificity andpro-B/pre-B specificity of immunoglobulin VK-JK
joining. Cell83, 1113-1123.
Liu, Y., Golden, S. S., Kondo, T., Ishiura, M. andJohnson, C. H. (1995) Bacterial luciferase as areporter of circadian gene expression incyanobacteria. J. Bacterial. 177, 2080-2086.
Liu, Y., Tsinoremas, N. F., Johnson, C. H., Lebedeva,
N. V., Golden, S. S., Ishiura, M. and Kondo, T.
(1995) Circadian orchestration of gene expression
in cyanobacteria. Genes Dev. 9, 1469-1478.
Moriya, H., Kasai, H. and Isono, K. (1995) Cloning
and characterization of the hrpA gene in the terC
region of Escherichia coli that is highly similar to
the DEAR family RNA helicase genes of
Saccharomyces cerevisiae. Nucleic Acids Res., 23,595-598.

18

NATIONAL 1NSTITUTE19

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshiaki Suzuki

Three regular divisions and one adjunct division that has
been reinitiated on new projects during 1995, construct the
Department. The department members conduct molecularanalysis on various aspects of developmental phenomena;differentiation and maturation of the germ cells, molecularbasis of the body plans, and gene regulation in different
phases of the cell cycle, in the differentiating tissues, andduring changes of the differentiated states.

20
DIVISION OF REPRODUCTIVE BIOLOGY
Professor: Yoshitaka Nagahama
Associate Professor: Michiyasu Yoshikuni
Research Associates: Minoru Tanaka
Tohru Kobayashi
JSPS Postdoctoral Fellow: Yoshinao Katsu
Postdoctoral Fellows: Wei Ge
Ahn Ryun-Sap
Kazumasa Suzuki
Takashi Todo
Graduate Students: Shinji Onoe (Graduate University for
Advanced Studies)
Xiao-Tian Chang (Graduate
University for Advanced Studies)
Daisuke Kobayashi (Graduate
University for Advanced Studies)
Yuichi Ohba (Graduate University for
Advanced Studies)
Jun Ding (Graduate University for
Advanced Studies)
Masatada Watanabe (Graduate
University for Advanced Studies)
Shigenari Ijiri (Hokkaido University)
Visiting Scientists: Joel Sohn (California Institute ofTechnology)
Craig Morrey (University of Hawaii)
Jian-Quiao Jiang (University of
Wuhan)The division of reproductive biology conducts
research on the endocrine regulation ofdifferentiation, growth and maturation of germ cells
in multicellular animals, using fish as a primary study
model.

I. Endocrine regulation of oocyte growth andmaturation
Our research effort in previous years concentrated
on the identification and characterization of themolecules (pituitary gonadotropins and gonadal
steroid hormones) that stimulate and control germ
cell growth and maturation. It was in 1985 that we
identified, for the first time in any vertebrate, 17a,20~-dihydroxy-4-pregnen-3-one (17a, 20~-DP) as
the maturation-inducing hormone of amago salmon(Oncorhynchus rhodurus). Along with estradiol-17~,
which was identified as the major mediator of oocyte
growth, we now have two known biologicallyimportant mediators of oocyte growth and maturationin female salmonid fishes. It is established that the
granulosa cells are the site of production of these two
mediators, but their production by the ovarianfollicle depends on the provision of precursor
steroids by the thecal cell (two-cell type model). A
dramatic switch in the steroidogenic pathway from
estradiol-17~ to 17a, 20~-DP occurs in ovarianfollicle cells immediately prior to oocyte maturation.
This switch is a prerequisite step for the growing
oocyte to enter the maturation phase, and requires acomplex and integrated network of gene regulation
involving cell-specificity, hormonal regulation, and

NATIONAL INSTITUTE

developmental patterning.
We have isolated and characterized cDNAs
encoding several ovarian steroidogenic enzymes ofrainbow trout (Oncorhynchus mykiss), medaka(Oryzias latipes), tilapia (Oreochromis niloticus),
and ayu (Plecoglossus altivelis) which areresponsible for estradiol-1713 and 17a, 20j3-DP
biosynthesis: cholesterol side-chaincleavage
cytochrome P450 (P450scc), 3j3-hydroxysteroid
dehydrogenase (3~-HSD), 17a-hydroxylase/C17,20lyase cytochrome P450 (P450c17), cytochrome P450
aromatase (P450arom) and 20j3-hydroxysteroid
dehydrogenase (20j3-HSD). We also isolated thestructural genes encoding P450c17 and P450arom
from medaka. The medaka P450arom gene consistsof nine exons, but spans only 2.6 kb, being muchsmaller than the human P450arom gene (at least 70kb), as the result of extremely small introns.
Northern blots using a tilapia P450arom eDNA
fragment and Western blots using an antiserum
against a tilapia P450arom oligopeptide fragment
revealed a single P450arom mRNA (2.6 kb) and asingle protein (59 kDa) in tilapia ovarian tissue,
respectively. These analyses also reveal that thelevels of both P450arom mRNA and protein are low
in early vitellogenic follicles, increase in midvitellogenic follicles, and decline to non-detectablelevels in post-vitellogenic follicles. Changes in theability of follicles to convert exogenous testosterone
to estrogens (aromatase activity) are similar to thoseof P450arom mRNA and protein. These observations
indicate that the capacity of tilapia ovarian follicles
to synthesize estradiol-17~ is closely related to thecontents of P450arom mRNA and protein withinthem.
In rainbow trout and medaka, P450scc andP450c17 (also 3~-HSD in rainbow trout) mRNAtranscripts increase in follicles towards the end of
oocyte growth phase and during oocyte maturation.
Furthermore, incubations of isolated thecal layerswith gonadotropin result in the elevation of P450scc
mRNA. The effect of gonadotropin becomes more
dramatic when the expression of P450scc mRNA isexamined in granulosa cells. P450scc mRNA is notdetected in the absence of gonadotropin, butmarkedly expresses in the presence of gonadotropin.
The increase in the amount of P450scc, 3~-HSD and
P450c17 transcripts provide an explanation for thedramatic increase in 17a, 20j3-DP production infollicles during naturally- and gonadotropin-induced
oocyte maturation. In contrast, levels of mRNA for
P450arom are high during oocyte growth, but rapidly
decrease during oocyte maturation. This decrease in
P450arom mRNA levels appears to be correlatedwith the decreased ability of maturing follicles to
produce estradiol-17~.

21

FOR BASIC BIOLOGY

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

Q

135kDaI ~:cdc2 kinase
mRNA~

Cyclin B!

1 3 SkDa

Cyclin B synthesis

I
\~kDj

cdc2 kinase~~Qic,·
Translation

Serine kinase(cdc2 kinase)..

Fig. 1. Molecular mechanisms of 17a,
activation in goldfish oocytes.

yc m B.....----r P Threonine kinase(p4QM015)

20~-dihydroxy-4-pregnen-3-one-induced

17a, 20(3-DP acts via a receptor on the plasma
membrane of oocytes. We have identified andcharacterized a specific 17a, 20(3-DP receptor fromdefolliculated oocytes of several fish species. 17a,20(3-DP receptor concentrations increase duringoocyte maturation. Pertussis toxin (PT) catalyzed the
ADP ribosylation of a 40 kDa protein in crude
membranes from rainbow trout oocytes. The 40 kDa
protein is recognized by an antibody against a
subunit of inhibitory G-protein. Treatment of themembrane fraction with 17a, 20(3-DP decreases the
PT-catalyzed ADP ribosylation of the 40 kDa protein.
The specific binding of 17a, 20(3-DP is decreased by
PT. We conclude that the PT-sensitive Gi is involvedin the signal transduction pathway of 17a, 20(3-DP infish oocytes.
The early steps following 17a, 20(3-DP action
involve the formation of the major mediator of thissteroid, maturation-promoing factor or metaphasepromoting factor (MPF). MPF activity cycles during
17a, 20(3-DP. induced oocyte maturation with thehighest activity occurring at the first and second
meiotic metaphase. Studies from our laboratory andothers have shown that MPF activity is not speciesspecific and can be detected in both meiotic andmitotic cells of various organisms, from yeast to
mammals. Fish MPF, like that of amphibians,
consists of two components, catalytic cdc2 kinase(34 kDa) and regulatory cyclin B (46 to 48 kDa).
Immature goldfish oocytes contain only
monomeric 35 kDa cdc2. Although immature
oocytes contain mRNA for cyclin B, they do notcontain cyclin B protein. 17a, 20(3-DP induces
oocytes to synthesize cyclin B. The preexisting 35

maturation-promoting factor (MPF)

kDa inactive cdc2 kinase binds to de novo
synthesized cyclin B at first, and then is rapidly
converted into the 34 kDa active form by a threonine(Thr) kinase, p40M 015 (Fig.l).
Introduction of a bacterially-produced goldfish
cyclin B into immature goldfish oocyte extracts
induces cdc2 kinase activation. Phosphoamino acidanalysis shows that Thr phosphorylation of the 34
kDa cdc2 kinase is associated with the activation.
The site of Thr phosphorylation on cdc2 kinase wasmapped to residue Thr 161. Since goldfish cyclin B
mRNA contains four copies of the usual cytoplasmic
polyadenylation element in the 3'UTR, the initiationof its synthesis during oocyte maturation may becontrolled by the elongation of poly (A) tail, as
suggested in other systems. We exmined thepolyadenylation state of cyclin B mRNA during
goldfish oocyte maturation by means of a PCR poly(A) test, and found that cyclin B mRNA ispolyadenylated during oocyte maturation.
Immediately prior to the transition from metaphaseto anaphase, M-phase-promoting factor (MPF) isinactivated by degradation of cyclin B. We
investigated the role of proteasomes (a nonlysosomal
large protease) in cyclin degradation, using
Eschelicia coli-produced goldfish cyclin B andpurified goldfish proteasomes (20S and 26S). The
purified 26S proteasome, but not 20S proteasome,cleaved both monomeric and cdc2-bound cyclin B atlysine 57 (K57) restrictively in vitro, and produced a
42 kDa N-terminal truncated cyclin B, wnich wastransiently detected at the initial phase of the normal
egg activation. The 42 kDa cyclin B, as well as fulllength one, was degraded in Xenopus egg extracts,
but a mutation on K57 (K57R) inhibited both the22digestion by 26S proteasome and the degradation in
Xenopus egg extracts. These findings strongly
suggest the involvement of 26S proteasome in cyclin
degradation through the first cleave on its Nterminus.

II. Endocrine regulation of male germ cell
development and maturation
We have identified two steroidal mediators of malegerm cell development in salmonid fishes (11ketotestosterone for spermatogenesis and 17a, 20f3DP for sperm maturation). 11-ketotestosterone canalso induce the entire process of spermatogenesis inan organ culture system developed for eel testes frompremitotic spermatogonia to spermatozoa within 21
days. A steroidogenic switch, from
11ketotestosterone to 17a, 20f3-DP, occurs in salmonid
testes around the onset of final maturation. In vitro
incubation studies using different testicularpreparations have revealed that the site of 17a, 20f3DP production is in the sperm, but its production
depends on the provision of precursor steroids bysomatic cells. The site of 11-ketotestosterone
production is in the testicular somatic cells.
In the cultivated male Japanese eel (Auguillajaponica), spermatogonia are the only germ cells
present in the testis. Subtractive hybridization wasused to identify genes that are expressed
differentially in eel testes in the first 24 hr after HCG
treatment in vivo, which ultimately induces
spermatogenesis. One up-regulated eDNA was
isolated from subtractive eDNA libraries derived
from mRNA extracted from control testes and testes
one day after a single injection of HCG. From its
deduced amino acid sequence, this clone was
identified as coding for the activin f3B subunit. The
HCG-dependent activin f3B mRNA expression in thetestes was restricted to Sertoli cells in testes treatedwith HCG for one to three days. A marked
stimulation of activin B production was observed intestes after HCG and 11-ketotestosterone treatment.
Addition of recombinant human activin B induced
spermatogonial proliferation in vitro. Taken together,
these findings suggest the following sequence of thehormonal induction of spermatogenesis in the eel.
Gonadotropin stimulates the Leydig cells to produce
11-ketotestosterone, which, in turn, activates the
Sertoli cells to produce activin B. Activin B then acts
on spermatogonia to induce mitosis leading to theformation of spermatocytes.
In salmonid fishes, spermatozoa taken from thetestes are immotile, but acquire motility during theirpassage through the sperm duct. Using male masu
salmon (Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17a,
20f3-DP is responsible for the acquisition of sperm
motility; 17a, 20f3-DP acts to increase sperm ductpH, which in turn increases the cAMP content ofsperm, allowing the acquisition of motility.

NATIONAL INSTITUTE

III. Endocrine regulationdifferentiationof gonadalsex

In tilapia, a pair of gonadal primordia formed on
both sides of the intestine at 15 days after hatching.
The gonadal anlage consists of several roundish
germ cells surrounded by a few stromal cells. At 20
days after hatching, prior to the period of sex
differentiation, positive immunostaining for P450scc,
3f3-HSD, P450c17, and P450arom antibodies
becomes evident for the first time in gonads.
Immunostained stromal cdls are observed in thevicinity of blood vessels (Fig. 2).
From 23-26 days after hatching, morphologicalgonadal sex differentiation begins to be recognized.
Initial ovarian differentiation is marked by theappearance of a narrow space in the stromal tissue,
representing the formatoin of the ovarian cavity. On
the other hand, iniNal testicular differentiation ischaracterized by the appearance of a narrow space inthe stromal tissue, representing the efferent duct
construction. At this stage, germ cells in testes andovaries remain in the gonial stage. In ovaries,
positive immunostaining for four kinds ofsteroidogenic enzymes is recognized in large stromalcells located in the vicinity of blood vessels. In
contrast, no immunoreaction is evident in testes
during sex differentiation. This situation continues
until testes initiate spermatogenesis. Thus, tilapia
ovaries express the steroidogenic enzymes required
for estradiol-17f3 biosynthesis from cholesterol
before sexual differentiation, which is consistent withthe concept that estrogen biosynthesis is essential forsexual differentiation of female phenotype during
early development.

23

FOR BASIC BIOLOGYAc
Fig 2. Immunocytochemical localization of cholesterol side-chain cleavage cytochrome P450 (A),3~-hydroxysteroid

d~hydro~e~ase (B) an~ cytochrome P450 aromatase (C) in tilapia gonads collected at 20 days after hatching (prior to sexualdJfferentJatwn). g, gonmm. X800.

Publication Lists:
Fukada, S., Tanaka, M., Iwaya, M., Nakajima, M.
and Nagahama, Y. (1995) The Sox gene family
and its expression during embryogenesis in the
teleost fish, medaka (Oryzias latipes). Develop.
Growth Differ. 37, 379-385.
Katsu, Y., Yamashita, M., Hirai, T., Tokumoto, T.,
Kajiura, H. and Nagahama, Y. (1995) Molecularcloning and immunological analysis of goldfish
cyclin A during oocyte maturation. Develop. Bioi.
170, 616-625.
Matsuyama, M., Yoneda, M., Takeuchi, H.,
Kagawa,H., Kashiwagi, M., Tabata, K., Nagahama,Y., Ijiri, S., Adachi, S. and Yamauchi, K.(1995)

Diurnal periodicity in testicular activity in the

1apanese flounder Paralichthys olivaceus. Fish.
Sci. 61, 17-23.
Matsuyama, M., Morita, S., Hamaji, N., Kashiwagi,
M. and Nagahama, Y. (1995) Diurnal rhythm intesticular activity in the secondary male ofprogynous wrasse, Pseudolabrus japonicus . In
Proc. of Fifth Int. Symp. on Reproductive
Physiology of Fish (F. W. Goetz and P.Thomaseds.), pp.212-214.
Miura, T., Miura, C., Yamauchi, K. and Nagahama,Y. (1995) Activin B is a major mediator of
hormone-induced spermatogonial proliferation in
the Japanese eel. In Proc. of Fifth Int. Symp. on

24

Reproductive Physiology of Fish (F. W. Goetz and
P. Thomas eds.), pp.284-286.
Miura, T., Miura, C., Yamauchi, K. and Nagahama,Y. (1995) Human recombinant activin induces
proliferation of spermatogonia in vitro in theJapanese eel Anguilla japonica. Fish. Res. 61,
434-437.
Miura, T., Kasugai, T., Nagahama, Y. and Yamauchi,
K. (1995) Acquisition of potential for sperm
motility in vitro in Japanese eel Anguilla japonica.
Fish. Res. 61,533-534.
Nagahama, Y., Yamashita, M., Tokumoto, T. and
Katsu, Y. (1995) Regulation of oocyte maturationin fish. Current Topics in Developmental Biology
30, 103-145.
Nagahama, Y. {1995) Teleost oocyte maturation:
actuality and potentiality. Aquaculture 135, 73-78.
Naito, K., Hawkins, C., Yamashita, M., Nagahama,Y., Aoki, F., Kohmoto, K., Toyoda, Y. and Moor,
D. M. (1995) Association of p34cdc2 and cyclin
B1 during meiotic maturation in porcine oocytes.
Develop. Bioi. 168,627-634.
Nakamura, M. and Nagahama, Y. (1995) Innervation.of testes in the tilapia Oreochromis niloticus.
Aquaculture 135, 41-49.
Saksena, D. N., Miura, T., Jiang, J. and Nagahama,Y. (1995) A rapid activation of immature testis ofJapanese eel (Anguilla japonica) by a single
injection of human chorionic gonadotropin. J.
Biosci. 20, 675-689.
Scott, A. P., Nagahama, Y., Kraak, Van Der andNagler, J. J. (1995) Sulfation and uptake of thematuration-inducing steroid, 17a, 20B-dihydroxy4-pregnen-3-one by rainbow trout ovarian follicles.
Fish Physiol. Biochem. 14, 301-311.
Tanaka, M. {1995) Characteristics of medaka genes
and their promoter regions. The Fish Biology
Journal MEDAKA 1, 11-14.
Tanaka, M., Fukada, S., Matsuyama, M. andNagahama, Y. (1995) Structure and promoter
analysis of the cytochrome P-450 aromatase geneof the teleost fish, medaka (Oryzias latipes). J.
Biochem. 117, 719-725.
Tokumoto, T., Yamashita, M., Yoshikuni, M.,
Kajiura, H. and Nagahama, Y. (1995) Purificationof latent proteasome (20S proteasome) anddemonstration of active proteasome in goldfish(Carassius auratus) oocyte cytosol. Biomed. Res.
16, 173-186.
Tokumoto, T., Yamashita, M., Yoshikuni, M.,
Kajiura, H. and Nagahama, Y. (1995) Purificationand characterization of active proteasome (26S
proteasome) from goldfish ovaries. Biomed. Res.
16, 207-218.
Yamashita, M., Kajiura, H., Tanaka, T., Onoe, S. andNagahama, Y. {1995) Molecular mechanisms of
the activation of maturation-promoting factor
during goldfish oocyte maturation. Develop. Bioi.
168,62-75.
Yoshikuni, M., Oba, Y. and Nagahama, Y. (1995) A

NATIONAL INSTITUTE

pertussis toxin sensitive GTP-binding protein is
involved in the signal transduction pathway of thematuration-induing hormone (17a, 20j3-dihydroxy4-pregnen-3-one) of rainbow trout (Oncorhynchus
mykiss) oocytes. In Proc. of Fifth Int. Symp. on
Reproductive Biology of Fish (F. W. Goetz and
P.Thomas, eds.), pp.348-350.

FOR BASIC BIOLOGYDIVISION OF CELL DIFFERENTIATION
Professor: Yoshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Kaoru Ohno
Hiroki Kokubo
JSPS Postdoctoral Fellow: Vaclav Mach
Graduate Students: Katsuyoshi Matsunami (Graduate
University for Advanced Studies)
Yoshinori Ueno (Graduate
University for Advanced Studies)Technical Staffs: Miyuki Ohkubo (deceased on January 12,
1995)
Chikako Inoue

We conduct two well-associated projects. One is tounderstand how a special tissue like the silk gland of
Bombyx mori differentiates along the developmental
programs and results in transcribing a specific set ofgenes like the silk fibroin and sericin-1 genes. The
other concerns with what the body plan of thesilkworm is and how the developmental regulatory
genes regulate a set of target genes in specifying theidentities of various regions of the embryos.

I. Genes and factors that control the silk glanddevelopment and the silk genes transcription
We have been trying to understand the networks ofregulation hierarchy that function in the processes ofsilk gland development and differentiation. As abottom-up type approach for this project, analyses
on the molecular mechanisms that control thedifferential transcription of the fibroin and sericin-1
genes in the silk gland should provide an information
about a part of the networks. In complementing thisapproach, a top-down type approach should also help
understanding the networks; analyses of regulation
hierarchy of the homeobox and other regulatory
genes, and identification of their target genes
expressed in the labial segment, where the silk glands
originate.
Previously we described cloning and labial
segment-specific expression of the Bombyx Scr. Silk
gland specific transcription factor-1 (SGF-1)
interacts with the SA site of the sericin-1 gene andFA and FB sites of the fibroin gene. Previously, we
purified and sequenced the SGF-1 40 kDa protein,
and found it to be a new member of the fork
head/HNF-3 family. Taking advantage of theimmediate availability of Bombyx fork head (jkh)eDNA in this laboratory, we have analyzed theexpression patterns of SGF-1/fkh mRNA and protein
in developing embryos (see Fig. 1 and H. Kokubo etal. (1996). Development Genes Evolution, in press).
At the time of embryo retraction (stage 20), thetranscripts and protein were detected in theinvaginating silk glands. Interestingly, preceding theappearance of the Bombyx Fkh protein in theinvaginating silk glands, Bombyx Scr disappeared
from the spots. This observation suggests thepossibility that the Bombyx Scr is necessary to

25

determine the nature of the labial segment and induce
the silk gland invagination accompanied by the
Bombyx fkh expression but the Scr protein isprobably not necessary for the direct induction of
Bombyx fkh expression in the invagination spots.
The following observations supported above
suggestions. In the Nc/Nc embryos described byltikawa in 1944 that lack the Bombyx Antp gene (T.
Nagata et al. (1996). Genes Cells, in press), we
observed ectopic expression of Bombyx Scr in thethoracic and abdominal segments (H. Kokubo et al.,in preparation). These ectopic expressions resulted in
inducing ectopic formation of invaginating silk
glands in the prothoracic, mesothoracic, andmetathoracic segments all of which revealed ectopic
expression of Bombyx Fkh.
By the time when the blastokinesis finishes (stage25) and the silk gland fully develops, the Bombyx fkh
transcripts and protein were restricted to the middle
and posterior regions of the silk gland. These results
suggest that besides the role of the transcription
factor for the silk genes the Bombyx fkh!SGF-1 may
play important roles during the silk glanddevelopment.
Among many factors proposed to bind and control
the fibroin and sericin-1 genes, the POU-M1 that
accommodates a POU-domain identical to
Drosophila Cfla was cloned and characterized
previously. The POU-M1 binds to the SC site of thesericin-1 gene and is assumed to enhance thetranscription. A function assay of the POU-M1 isbeing pursued in cell-free transcription system. This
protein also binds to the PB element of the POU-M1
gene and suppresses the transcription. The
expression of the POU-M1 gene has been analyzed
in Bombyx embryos by in situ hybridization andimmunohistochemistry. The gene was expressed
specifically for the first time at stage 18-19 in a pairof restricted sites of the labial segment where a pairof prothoracic glands is going to be formed byinvagination (previously we have mistaken these as
the invagination sites of the silk glands). After thesilk gland invagination, the POU-M1 expression was
detected in the developing silk gland and confined tothe middle portion of the silk gland by lateembryonic stages.
Cloning of a few other transcription factors of the
fibroin gene is being continued.
With an intention to study the regulator-target
relationship between genes, we have begun to use an
antibody-selection method of fixed chromatin inembryonic stages. Screening of one of such
chromatin libraries with a POU-M1 antibody has
revealed a high selection of the sericin-1 promoter
covering SB and SC sites. This result indicates thatthe sericin-1 promoter region is one of the POU-M1
targets and this method should be usefulcharacterizing other regulator-target relationships.

26

NATIONAL INSTITUTE

B

A

c

E

F
·.

Fig. 1. Distribution of Bombyx Fkh/SGF-1 protein in embryos. A A germ anlage at 24 hrs after egg deposition. Two terminal
regions reveal weak signals (arrows). B A germ anlage at 36 hrs. pg indicates the primitive groove. C and D Magnified anterior
and posterior regions of B. Some dots in B and D are the yolk nuclei. E and F Embryos at 48 and 60 hrs. The triangles indicate
primodia of central nervous system. All scale bars represent 100 flm. Revised from H. Kokubo et al. (1996). Develop. Genes
Evol. , in press.

II. Other genes of developmental interest
In continuation of the studies of abdominal
segment identification, we have concentrated on the
study of morphogenesis of embryonic abdominal legs.
Previously we analyzed proteins in the wild typeembryos, and found that two high molecular weightproteins of 260 and 270 kDa (p260/270) are
expressed specifically in a restricted region of theabdominal legs. These proteins are not detectable in
the Eca /Eca embryo that lacks Bombyx abd-A gene asreported previously, suggesting a control under the
Bombyx abd-A.

We have screened an embryonic eDNA librarywith an antibody against the p260/270 and obtained
one type of eDNA This eDNA encodes a long open
reading frame. Amino acid sequences from the Nterminal and two other regions of the p270 matchedwith those of the deduced sequence. Northern blot
analysis revealed the p270 transcripts fromembryonic stage 19-20, whereas the Bombyx abd-A
transcripts were observed from stage 17-18. Fromthese results we speculate that the Bombyx abd-A
may directly regulate the expression of the p270.

FOR BASIC BIOWGY

Publication Ust:
Hui, C.-c. and Suzuki, Y. (1995) Regulation of thesilk protein genes and the homebox genes in silk
gland development. In Molecular model systems in
the Lepidoptera (M. R. Goldsmith and A. S.
Wilkins, eds.), Cambridge University Press,
Cambridge, pp. 249-271.
Mach, V., Takiya, S., Ohno, K., Handa, H., Imai, T.
and Suzuki, Y. (1995) Silk gland factor-1 involvedin the regulation of Bombyx sericin-1 gene contains
Fork head motif. J. Bioi. Chem. 270, 9340-9346.
Ueno, K., Nagata, T. and Suzuki, Y. (1995) Roles ofhomeotic genes in the Bombyx body plan. In
Molecular model systems in the Lepidoptera (M. R.
Goldsmith and A. S. Wilkins, eds.), Cambridge
University Press, Cambridge, pp. 165-180.
Xu, P.-X., Xu, X. and Suzuki, Y. (1995) Molecularcloning of a zinc finger protein belonging to the
GLI-Kriippel family and its expression in the silk
gland. Develop. Growth Differ. 37, 149-155.

27

28

NATIONAL INSTITUTEDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Associate Professor: Ryuji Kodama
Research Associates: Makoto Mochii
Mitsuko Kosaka
Visiting Scientists: Takamasa S. Yamamoto
Akio Iio
Toshiyuki Nagamoto
Graduate Students:Nobuhiko Mizuno (Graduate
University for Advanced Studies)
Yuuichi Mazaki (Graduate University
for Advanced Studies)
Harutoshi Hayashi (jrom School of
Agriculture, University of Tokyo)
Jatupol Kositsawat (jrom School of
Medicine, Universiy of Tokyo)
Technical Staff: Chiyo Takagi

In the newt and some other limited animal species,
the lens and neural retina can be completely
regenerated
throughtransdifferentiationofpigmented epithelial cells (PECs). Such aphenomenon, transdifferentiation, as observed inregeneration of ocular tissues seems to be a highly
powerful model for studying stability and instability
in differentiation of tissue cells. From this view point,
lens transdifferentiation of PECs of vertebrate has
been studied in vivo and in vitro systems, and our invitro studies have revealed that dormant potential totransdifferentiate into lens cells is widely conserved
throughout vertebrate species, and that the cell typespecific genes, are completely inactivated in themultipotent (at least bipotent) dedifferentiated cells
originated from pigmented epithelial cells.
Our studies have been conducted to clarify themolecular
mechanism
controllingthelenstransdifferentiation in vertebrate PECs and also tosearch the reason why the pigmented epithelia ofspecies other than the newt and so forth neverregenerate the lens in the in situ eyes. Based on
findings accumulated up to the last year, we haveconducted analysis of the lens transdifferentiation of
PECs in vivo and in vitro and the following results
have been established.

I. Role of the mi gene in differentiation andtransdifferentiation of the pigmented epithelialcells
The mi gene was first isolated as a mouse gene in
the microphthalmia (mi) locus and shown to encodea basic-helix-loop-helix-leucine zipper (bHLHzip)
protein. The product of the gene was suggested totransactivate the pigment cell specific genes,
tyrosinase and TRP-1, and to have a critical role indevelopment of the melanocytes and the PECs. To
reveal the role of the mi gene in differentiation andtransdifferentiation of the PECs, we isolated theeDNA clones for the chicken mi and characterized
the expression in PECs in vivo and in vitro.
The mi gene was expressed by the embryonic
PECs in tissue specific manner and was inactivatedin the dedifferentiated cells during the in vitrotransdifferentiation process. The mi protein was firstdetected in the whole optic vesicle of 2-day-old
embryo. At later stages the expression wasmaintained only in the outer layer of the optic cup
which was a precursor of the pigmented epithelium(Fig.l).

Fig.l. Expression of the mi protein in the optic cup. The mi protein is specifically localized to the nuclei of the cells
constituting the outerlayer of optic cup of a 3-day-old chicken embryo.

FOR BASIC BIOLOGY

To know the functional role of the mi gene in the
PEC development, the antisense oligonucleotide wasadded to the culture of the optic cups. The
differentiation of the PEC from the optic cup cells
was inhibited by the antisense oligonucleotide
suggesting an essential role of the mi product in theprocess of the PEC differentiation.
A part of the retinal PECs in quail embryos
homozygous in the silver mutation start to proliferate
in the 5-day-old embryo and make an ectopic neuralretina through the transdifferentiation process. The
silver mutant shared some phenotypes with themouse mi mutant. A comparison of the sequences ofthe mi cDNAs from the wild and the silver quails
revealed the presence of a frame shift mutation in thesilver mutant. This result suggests the possibility thatthe mutation in the mi gene may inhibit the default
differentiation of the PECs and may induce thetransdiffereritiation into the neural retina cells.
II. The role of integrin in stabilization and instabilization of pigmented epithelial cells
The extracellular matrix (ECM) has beensuggestedtoplayessential
roles
intransdifferentiation of PECs through extensive cell
culture studies of lens transdifferentiation of PECs.
For
example,collagenmatrixinhibits
transdifferentiation of PECs dissociated from older
chick embryos to lens cells and stabilizes theirdifferentiated phenotype. The pigmented epitheliumin the eye in situ is supported and stabilized by theunderlying ECM consisting of basal lamina andfibrous collagenous matrix as major components. We
examined changes in adhesiveness of PECs tovarious substrates such as fibronectin, collagen type I
and IV, and so forth during dedifferentiation of thesecells in vitro, to approach to the mechanisms
involved in the stabilization and instabilization ofdifferentiated state of PECs.
It has been well demonstrated that thededifferentiated PECs (dePECs) are much lessadhesive to ECM components except fibronectinthan well-differentiated PECs and also that thedifferentiated PECs tightly adhere to ECM by focal
contacts, which has been thought to be mediated byintegrins as receptors of ECM components. Based onthese observations, we isolated cDNAs for a3, a6,
a8, av, ~1 and ~5 integrins from PECs and foundthat all of these classes of integrin genes wereexpressed at similar levels in both PECs and dePECs.
Although the amount of ~ 1 integrin also did notchange during dedifferentiation, focal contact was
not observed in dePECs. When ~1 integrin-mediatedadhesion was blocked by anti-~1 integrin antibody,29

the cell morphology and gene expression patterncame to resemble those of dePECs. In contrast,
dePECs were hardly affected by anti-~1 integrin
antibody. In addition, it was clearly demonstrated
that such a loss of function of ~ 1 integrin in dePECs
was due to phosphorylation of ~1 integrin molecules.
These findings are strongly suggesting that ~ 1
integrin does not function as an adhesion molecule indePECs and plays an essential role in maintenance ofthe differentiated state of the PECs.

m. Comparative analysis of crystallin gene expression between development and regenerationof the lens in newts
The spatio-temporal expression of three major
crystallin genes (aA, ~B1 and y3) in developing andregenerating lenses of the newt were analyzed by insitu hybridization in order to compare the mode ofcell type-specific gene expression in normal lensdevelopment with that in lens regeneration.
In normal lens development, at the stage
immediately before the onset of primary lens fiber
differentiation at the posterior wall of the lens vesicle,
all three crystallin gene transcripts were first detected
simultaneously in the epithelial cells constituting theposterior region of the lens vesicle. The expressionof the crystallin genes was found to continue duringlens fiber differentiation of the posterior cells to theprimarily lens fiber cells. At later stages, theexpression of these three genes was restricted to thesecondary lens fibers. aA- and y3-crystallintranscripts were not detected in lens epithelium at
any stage, while ~B1-crystallin transcript was firstdetected in the lens epithelia at the stage when the
primary lens fiber mass was formed. In lensregeneration, the three crystallin transcripts were firstdetected simultaneously at the ventra-posterior
region of the regenerating lens rudimentcorresponding to the lens vesicle. Although theexpression pattern of ~B1- and y3-crystallintranscripts were almost the same as those in normallens development, aA-crystallin gene was found to
be expressed even by lens epithelial cells in addition
to lens fiber cells, showing clear contrast to the lensdevelopment, in which aA-crystallin w.as never
expressed by the lens epithelial cells.
These results suggest that newts possesses someunique gene expression programme for lensregeneration and also that the regeneration of lensmust not be a simple repetition of the development of
this tissue.

30

NATIONAL INSTITUTE

Fig. 2. Expression of the Pax-6 gene in the newt (A) and Xenopus (B) embryos at the mid-neurula stage of development. In
the newt embryo, the expression of Pax-6 gene is restricted within the neural plate and never observed in the head ectoderm (a),but in the Xenopus embryo (b) Pax-6 gene is strongly expressed in a fairly large area of the head ectoderm including thepresumptive lens region (arrows). The broken line indicates the margin of the anterior half of the closing neural plate (b).

IV. Comparative analysis of Pax-6 gene expression during eye development in the newt and
XenopusAs the first step of comparative analysis of lensdevelopment and regeneration in the newt in whichlens can be regenerated from the iris epithelium, andin the Xenopus, in which lens can be regenerated
from the corneal epithelium in its larval stages, theexpression pattern of Pax-6 gene thought to be an
essential gene for organogenesis of the eye, wasstudied by in situ hybridization using Pax-6 eDNA
homologues cloned in both newt and Xenopus asprobes.
In Xenopus , Pax-6 gene expression can be detectedin the head ectoderm including presumptive lensforming region in addition to eye forming region of
the neural plate as early as the mid-neural plate stage.
The expression of this gene in the head ectoderm isthen gradually restricted to the lens forming region
and eventually concentrated to the lens placodefacing the optic vesicle. Contrary to this, in newts,
Pax-6 gene expression can never be detected in thehead surface ectoderm until the optic vesicle
underlies the ectoderm (Fig. 2).
These observations are strongly suggesting that theregulatory mechanisms of Pax-6 gene expression innewts must be quite different from that in Xenopus
and that the lens development from the ectoderm in
the newt must be much more dependent on the
inductive effect of the optic vesicle than in Xen opus .
Based on these results we are extending our studiesof this line to a more critical analysis of Pax-6 gene
expression and regulation.

Publication List:
Ando, H. , Kodama, R., Tomoda, Y. and Eguchi, G.
Transforming
growthfactor-~1
inducedmorphologicalchangesaccompaniedbyextracellular matrix reconstitution in cultures ofavian retinal pigmented epithelial cells. Japanese
Journal of Ophthalmology 39, 1-11 (1995).

Eguchi, G. Introduction: Transdifferentiation. Semi.
Cell Bioi. 6,105-108 (1995).
Ishida, T., Ando, H., Nomura, S., Ishikawa, K.,
Kurauchi, 0., Mizutani, S., Kobayashi, M ., Eguchi,
G. and Tomoda, Y.
Establishment andcharacterization of human fetal liver epithelial cellline transfected with SV40 T
Antigen.
Immortalized Human Fetal Liver Cell Line 251256 (1995) .
Kageura, H., Eguchi, G. and Yamana, K. The origin
and development of retinal pigment cells andmelanophores analyzed by Xenopus black-white
chimeras. Develop. Growth Differ. 37,157-166(1995).
Kodama, R. and Eguchi, G. From lens regenerationin the newt to in vitro transdifferentiation ofvertebrate pigmented epithelial cells. Semi . Cell
Bioi. 6,143-149 (1995).
Kodama, R., Yoshida, A. and Mitsui, T.
Programmed cell death at the periphery of thepupal wing of the butterfly, Pieris rapae . Raux'sArch Dev. Bioi. 204,418-426 (1995).
Oguchi, H., Ishikawa, K., Mizoue, K., Seto, K. andEguchi, G. Long-term histological evalution ofhydroxyapatite ceramic in humans. Biomaterials
16,33-38 (1995) .
Ono, T. , Muto, S., Matsumoto, T., Mochii, M. andEguchi, G. Gene transfer into circulating primodial
germ cells of quail embryos. Exp. Anim. 44,275278, (1995).
Sawada, K., Agata, K. , Eguchi, G., Quinlan, R. andMaisel, H. The predicted structure of chick lensCP49 and a variant thereof CP49ins, the firstvertebrate cytoplasmic intermediate filament
protein with a laminin-like insertion in helix lB.
Current Eye Res. 14,543-553 (1995).
Schmid, V., Aeschbach, B., Agata, K., Kosaka, J.,
Reber-Miiler, S., Sprenger, N. and Eguchi, G. Cell
adhesion to extracellular matrix is different inmarine hydrozoans compared with vertebrates.
Raux 's Arch Dev. Bioi. 204,465-476 (1995) .

FOR BASIC BIOLOGY31

DIVISION OF DEVEWPMENTAL BIOLOGY(ADJUNCT)
Professor: Kenzo Nakamura

In this laboratory which started in its new form in
October of 1995, research attention will be focusedon the control of plant development by the changes
in metabolic activity of the plant body. In particular,
mechanisms involved in the regulation of gene
expression by the carbohydrate metabolic signal andthe role of sugar-regulation of gene expression in thegrowth and development of plants will be studied.
For this purpose, isolation and characterization ofmutants of Arabidopsis thaliana which show alteredpatterns of the sugar-regulated gene expression anddefects in growth and development will be pursued.
These genetic approaches will be complemented withtheidentification
andcharacterizationoftranscription factors and calcium-dependent protein
kinase which may participate in the sugar-regulated
gene expression.

Publication List:(from previous projects by Professor Iwabuchi'sgroup)
Mikami, K., Katsura, M., Ito, T., Okada, K., Shimura,Y. and Iwabuchi, M. {1995) Developmental andktissue-specific regulation of the gene for thewheat basic/leucine zipper protein HBP-1a (17) intransgenic Arabidopsis plants. Mol. Gen. Genet.
248,573-582.
Mikami, K., Katsura, M., Ito, T. and Iwabuchi, M.
(1995) Potential transcription regulatory sequences
in a promoter region of the wheat basic/leucine
zipper protein HBP-1b (c38) gene. Plant Cell
Physiol. 36(7),1375-1379.

32

NATIONALJNSTITUTE33

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairman: Norio Murata

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

34

DIVISION OF MOLECULAR NEUROBIOLOGY
Professor: Masaharu Noda
Research Associates:Nobuaki Maeda
Masahito Yamagata
Eiji Watanabe
Institute Research Fellow:Masakazu Takahashi
Graduate Students:Hiroki Hamanaka
Junichi Yuasa
Takafumi Shintani
Taeko Nishiwaki
Tatsunori Yamamoto*Technical Staff:Akiko Kawai
Shigemi Ohsugi(*from Nagoya University)

Our efforts have been devoted to studying molecularand cellular mechanisms which underlie thedevelopment of the vertebrate central nervous system.
We are now seeking for the molecules and structuresthat regulate various cellular events in the brain
morphogenesis, such as generation of neuroblasts, theirmigration to form the laminar structure and various
nuclei, the elongation and path-finding of neuralprocesses, and the formation and refinement of specificconnections between neurons. The research has been
conducted using various techniques, including
molecular biology (e.g. eDNA cloning, site-directedmutagenesis), biochemistry (protein, carbohydrate),monoclonal antibodies, neuroanatomy, cell andorganotypic culture, and embryo manipulation(classical embryology, gene transfer with viral vectors,
and gene targeting).
I. Topographic and laminar connection in the chickretinotectal system
Neural connection in the vertebrate brain is selective
in two ways, which we refer to as topographic andlaminar. Topographic specificity determines theorderly maps of connectivity, in which presynaptic
neurons arrayed along spatial axes project tocorresponding arrays of target cells in the target area.
By contrast, laminar specificity determines the local
circuitry, in which presynaptic neurons form synapses
on the particular neurons and portions of the neuron's
surface. We have been studying the retinotectal systemin chickens to reveal the crucial factors that determine
the development of these two classes of neuralconnection.
Historically, most intensively studied in this system
has been the retinotopic map, where retinal ganglion
cells from various parts of the retina project
topographically onto the tecta] surface. Namely, retinal
axons from the temporal (posterior) retina connect tothe anterior part of the optic tectum, and nasal(anterior) retinal axons connect to the posterior part,
thereby establishing a point-to-point projection map.
Many works, since the formulation of 'chemoaffinity

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theory' by Sperry, have supported the involvement ofgradient or position-specific molecules in this
topographically organized connection. Subscribing tothis model, we applied a subtractive hybridization
technique, and found several novel or identified genes
to be topographically expressed along the anteriorposterior axis in the chick retina. Among these
position-specific molecules, two molecules which
belong to a same family of transcription factors arefound to be expressed in a mutually exclusive mannerin either the nasal or temporal part of the retina.
Starting with these molecules, we hope to understandthe molecular mechanism by which retinotectal
topographical connection is established.
Less well studied, but striking, is a laminarselectivity of retinotectal connection in the orthogonal
direction. Retinal axons enter the tectum through themost superficial of its 15 different laminae. Once axons
reach defined loci on the tecta] surface, they branch
inwards and make connections in only 3-4 of the 15
laminae. Moreover, each axon confines most of its
synapses to one of these 'retinoreceptive' laminae, inthat neurochemically-distinguishable subsets of theganglion cells connect to distinct laminae to establish afunctional network. The laminar selectivity in thissystem may require an axon to recognize particulartarget cells (cellular specificity) and particular portionsof the target cell's surface (subcellular specificity). To
identify the cellular and molecular mechanism
underlying these synaptic specificities, we are taking
two interrelated approaches. First, we are trying toestablish culture systems to analyze the recognition
cues between presynaptic axons and their target cells in
vitro. The second approach is to use moleculartechniques, particularly by developing useful probes.
We have already obtained a panel of novel monoclonal
antibodies that label selectively one of 3-4
retinoreceptive layers of the tectum during the onset ofthe retinotectal projection. We hope that further
characterization of these lamina-specific epitopes will
provide a clue to the mechanism that underlies thelamina-specific synapse formation.
II. Proteoglycan, protein-tyrosine phosphatase andbrain development
Developmental processes of the brain requires
various cell-cell and cell-extracellular matrix
interactions. In order to find key molecules in themorphogenesis of the brain, we are focusing our efforts
on brain-specific proteoglycans, because proteoglycans
are major extracellular matrix components in thedeveloping brain. Previously, we identified a PBSsoluble chondroitin sulfate proteoglycan with a 300kDa core protein, named 6B4 proteoglycan.
Expression of 6B4 proteoglycan is dynamicallyregulated during development in various brain regions.

35

FOR BASIC BIOLOGY

Fig 1. 6B4 proteoglycan promotes differentiation of neurons. Cortical neurons were cultured on the coverslips coated (A, B)
or uncoated (C, D) with 6B4 proteoglycan. After 2 days in vitro, the neurons were double-immunostained with dendrite-specific(anti-MAP2; A, C) and axon-specific (anti-NFH; B, D) antibodies. Arrowheads indicate an NFH-positive and MAP2-negativeaxon. Arrows indicate an NFH- and MAP2-positive neuriles. Scale bar; 50f!m.

Recently, we cloned eDNA for 6B4 proteoglycan,
and revealed that the core protein structure
corresponds precisely to the extracellular region of a
receptor-like protein tyrosine phosphatase, PTPs
(RPTP~). 6B4 proteoglycan is characterized by thepresence of the N-terminal carbonic anhydrase-likedomain, a fibronectin type III region, and long serine,
glycine-rich domain which is thought to be thechondroitin sulfate attachment domain. We showedthat the transmembrane form, PTPs (RPTP~), is alsopresent as a membrane-bound chondroitin sulfate
proteoglycan in rat brain. This finding suggests thatthese brain-specific chondroitin sulfate proteoglycans
have a role as cell signaling molecules by binding toneural cell adhesion and extracellular matrix
molecules, in the case of 6B4 proteglycan, possibly
also by competing for ligands of the phosphatase.
In order to examine functional roles of 6B4
proteoglycan, we studied the influence of thisproteoglycan on cell adhesion and neurite outgrowthin vitro using dissociated neurons from the cerebral
cortex and thalamus. 6B4 proteoglycan adsorbed
onto plastic tissue-culture dishes did not support
neuronal cell adhesion, but rather exerted repulsive
effects on the cortical and thalamic neurons. Whenneurons were densely seeded on patterned substrata
consisting of a grid-like structure of alternating polyL-lysine and 6B4 proteoglycan-coated poly-L-lysine
domains, they were concentrated only on the poly-Llysine domains.
However, 6B4 proteoglycan did not retard thedifferentiation of neurons but rather promoted
neurite outgrowth and development of the dendritesof cortical neurons, when neurons were sparsely
seeded on poly-L-lysine-conditioned coverslips
continuously coated with 6B4 proteoglycan (Fig 1.).
This effect of 6B4 proteoglycan on the neurite

outgrowth of cortical neurons was apparent even on
coverslips co-coated with fibronectin or tenascin. By
contrast, the neurite extension of thalamic neurons
wasnotmodifiedby
6B4
proteoglycan.
Chondroitinase ABC or keratanase digestion of 6B4
proteoglycan did not affect its neurite outgrowthpromoting activity, suggesting that a protein moiety
is responsible for the activity. 6B4 proteoglycantransiently promoted tyrosine phosphorylation of an
85-kDa protein in the cortical neurons but not in thethalamic neurons, which correlated with theinduction of neurite outgrowth. These results suggestthat 6B4 proteoglycan modulates morphogenesis anddifferentiationofneuronsspatiotemporally
depending on cell types.

Publication List:
Drescher, U., Kremoser, C. Handwerker, C.,
U:ischinger, J., Noda, M. and Bonhoeffer, F.
(1995) In vitro guidance of retinal ganglion cellaxons by RAGS, a 25 kDa tecta] protein related toligands for Eph receptor tyrosine kinases. Cell 82,
359-370.
Leber, S. M., Yamagata, M. and Sanes, J. R. (1996)
Gene transfer using replication-defective retroviral
and adenoviral vectors. In Methods in Avian
Embryology (Bronner-Fraser, M. ed., Methods in
Cell Biology, Academic Press, Orlando) 51, pp.
161-183.
Maeda,
N.
and
Noda,
M.
(1996) 6B4
proteoglycan/phosphacan is a repulsive substratum
but promotes morphological differentiation ofcortical neurons. Development 122, 647-658.
Maeda, N., Hamanaka, H., Oohira, A and Noda, M.
Purification,
characterization
and(1995)
developmental expression of a brain-specificchondroitin
sulfateproteoglycan,
6B4

36

proteoglycan/phosphacan. Neuroscience 67, 23-35.
Nishizuka, M., Ikeda, S., Arai, Y., Maeda, N. andNoda, M. (1996) Cell surface-associatedextracellular distribution of a neural proteoglycan,
6B4 proteoglycan/phosphacan, in the olfactory
epithelium; olfactory nerve, and cells migrating
along the olfactory nerve in chick embryos.
Neurosci. Res., in press.
Watanabe, E., Maeda, N., Matsui, F., Kushima, Y.,
Noda, M. and Oohira, A. (1995) Neuroglycan C, anovel membrane-spanning chondroitin sulfate
proteoglycan that is restricted to the brain. J. Biol.
Chern. 270, 26876-26882.
Watanabe, E., Matsui, F., Keino, H., Ono, K.,
Kushima, Y., Noda, M. and Oohira, A. (1996) A
membrane-bound heparan sulfate proteoglycan thatis transiently expressed on growing axons in the rat
brain. J. Neurosci. Res., in press.
Yamagata, M. and Sanes, J. R. (1995) Targetindependent diversification and target specificprojections of chemically coded retinal ganglion
cell. Development 121, 3763-3776.
Yamagata, M., Herman, J. -P. and Sanes, J. R.
(1995) Lamina-specific expression of adhesion
molecules in developing chick optic tectum. J.
Neurosci. 15, 4556-4571.

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDIVISION OF CELLULAR REGULATION
Professor: Norio Murata
Associate Professor:Hidetoshi /ida
Research Associates:/kuo Nishida
Dmitry A. Los
Atsushi Sakamoto
Yoshitaka Nishiyama
Monbusho Foreign Scientists: Laszlo Mustard/ 1
Malay Kumar Ra/1
Alia31
JSPS Postdoctoral Fellow: Michael P. Malakhov41
NIBB Fellow:/wane Suzuki
JSPS Visiting Scientists: Rajinder S. Dhindsa 51
Dilley Richard 61
Ding-Ji Sh?1
Eva Hidei 1
P. Pardha Saradhi31
Nelly Tsvetkova 81
Diego de Mendoza 91
Visiting Scientists: Yasushi Tasaka
Makiko S. Okumura
Zoltan Gombo/1
George Papageorgiou 101
Prasanna Mohant/ 11
Ferenc Lajko 11
Julian Eaton-Rye 121
Ashraf S. Haider 131
Estelle Delphin 141
Graduate Students:Katsuzo Noguchi
Patcharaporn Deshnium
Sayamrat Panpoom
Masami /naba
Hiroshi Yamamoto
Technical Staffs:Sho-lchi Higashi
Miki Ida(!from Biological Research Center, Szeged, Hungary)from Centre for Cellular and Molecular Biology,
Hyderabad, India)( 31 from lamia Millia Islamia University, New Delhi,
India)( 41 from Plant Physiology Institute, Moscow, Russia)( 51 from McGill University, Montreal, Canada)( 61 from Purdue University, Lafayette, IN, U.S.A.)( 71 from Institute of Botany, Academia Sinica, Beijing,
China)(B) from Bulgarian Academy of Sciences, Sofia, Bulgaria)( 91 from Universidad Nacional de Rosario, Rosario,
Argentina)( 101 from National Center for Scientific Research
Demokritos, Athens, Greece)( ll) from Jawaharlal Nehru University, New Delhi, India)( 121 from Otago University, Dunedin, New Zealand)( 131 from Agricultural Genetic Engineering ResearchInstitute, Giza, Egypt)( 141 from Laboratoire de Photoregulation et Dynamiquedes Membranes Vegetates, Paris, France)( 21

The research effort of this division is directed
toward a full understanding of the molecularmechanisms that allow plants to acclimate to andtolerate extremes of temperature and salinity. In

37

1995, significant progress was made in the following
areas as a result of studies with cyanobacteria andhigher plants as experimental materials.

I. Regulation of the expression of genes for desaturases
Living organisms are exposed to changes inambient temperatures. However, they are able tomaintain the appropriate fluidity of their membrane
lipids by regulating the extent of unsaturation of theirfatty acids. For example, cyanobacterial cells
respond to a decrease in temperature by introducing
double bonds into the fatty acids of membrane lipids,
thereby compensating for the temperature-induced
decrease in the molecular motion of membrane lipids.
Desaturases catalyze the introduction of these
specific double bonds. We have demonstrated thatthe low temperature-induced desaturation of fatty
acids in membrane lipids is regulated at the level ofexpression of the genes for the desaturases.
We have shown that the levels of the transcripts ofthe desA, desB, and desD genes (for the M2, M5
and il6 desaturases, respectively) increase about 10fold after shift in culture temperature from 34 oc to
22°C. However, the level of the transcript of thedesC gene (for the il9 desaturase) remains constantafter such a temperature shift, suggesting that
expression of this gene is constitutive. Sequencing of
the nucleotides at the 5' termini of the genes for all
four desaturases indicated that the sites of initiationof transcription are quite similar among all fourgenes. Identification of the regulatory elements ofthese genes and cloning of the relevant trans-acting
factors are in progress.

II. Importance of the unsaturation of membrane
lipids in the turnover of the Dl protein of thephotosystem II complex
As part of our efforts to understand the role ofunsaturation of membrane lipids, we disrupted thedesA and desD genes in Synechocystis sp. PCC 6803
by inserting antibiotic-resistance gene cartridges.
This disruption greatly reduced the extent of
unsaturation of the fatty acids of the membrane lipids.
In the wild-type strain, the saturated, monounsaturated, di-unsaturated, and tri-unsaturated lipid
molecules each accounted for 15% of the total
membrane lipids. By contrast, in the desD- mutant,
the triunsaturated lipid molecules were absent, whilein the desA-/desD- mutant, 80% of the total
membrane lipids were monounsaturated and only
15% were fully saturated. These decreases in theunsaturation of membrane lipids greatly reduced thetolerance of the cyanobacterium to low temperaturein the light.
These results, together with other observations,suggest that unsaturated fatty acids play an importantrole in protection of the photosynthetic machinery
against low-temperature photoinhibition. The extent

38of photoinhibition in intact cells reflects a balance
between the light-induced inactivation of thephotosystem II protein complex and the recovery of
the complex from the inactivation. Therefore, wewere able to separate the two processes using
inhibitors of protein synthesis. Our results
demonstrated that the unsaturation of membrane
lipids had no effect on the light-induced inactivation
process but did accelerate the recovery of thephotosystem II complex from the photoinactivatedstate. Further analysis indicated that the unsaturationof membrane lipids accelerated the incorporation of
the precursor to the D1 protein (an important protein
component of the photosystem II complex) or theprocessing of the precursor to the D1 protein to
generate the mature D1 protein.

lll. Heat stability of the photosynthetic machinery
We also focused on the responses of plants tohigh-temperature stress. Photosynthesis is one of thephysiological processes in plants that is mostsusceptible to heat stress. Thus, stabilization of thephotosynthetic machinery against heat helps plants totolerate higher temperatures.
We have studied the molecular mechanism
responsible for the stabilization against heat ofoxygenevolutioninthecyanobacterium
Synechococcus sp. PCC 7002. When thylakoid
membranes isolated from the cyanobacterial cells
were treated with a low concentration of Triton X100, the stability to heating of the evolution ofoxygen decreased markedly. From the extracts
obtained by this treatment with Triton X-100, we
purified two proteins that were able to restore theheat stability of oxygen evolution. One protein was
identified as cytochrome c-550, having a low redox
potential. The other protein of 13 kDa was identified
as a homology of the 9-kDa extrinsic protein of
cyanobacterial photosystem II. These results indicatethat cytochrome c-550 and the 13-kDa protein are
involved in the stabilization against heat of theevolution of oxygen and, therefore, in the stability ofthephotosynthetic
machineryatelevated
temperatures. We isolated the genes for cytochromec-550 and the 13-kDa protein of Synechococcus sp.
PCC 7002. Disruption of these genes in
Synechococcus sp. PCC 7002 is now in progress in
our laboratory. We have also studied the effects ofelevated temperatures on photosynthesis in cultured
soybean cells. The heat stability of oxygen evolution
in these cells was enhanced by 3°C upon an increasein the growth temperature from 25°C to 35°C.

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IV. Genetic modification of the tolerance of acyanobacterium to low-temperature stress byintroduction of a gene for the synthesis of glycinebetaine
When cultured under saline conditions, someplants produce compatible solutes to avoid thedeleterious effects of external salt. Glycinebetaine, aquaternary ammonium compound, is one such solutethat is found in halotolerant plants and bacteria. To
examine the effect of glycinebetaine on photosynthesis in vivo, we transformed the salinitysensitive cyanobacterium Synechococcus sp. PCC
7942 with the codA gene for choline oxidase from
Arthrobacter globiformis, which catalyzes theoxidation of choline to glycinebetaine. The
transformed cells accumulated glycinebetaine at
intracellular levels of 60-80 mM and simultaneously
acquired the ability to tolerate salt stress. We also
observed that the transformed cells could grow at
20°C, a temperature that markedly retarded thegrowth of the wild-type cells. Photosynthesis in thetransformed cells was more resistant tophotoinhibition at low temperatures than that in wildtype cells. Furthermore, the recovery of photosystem
II from photoinhibition at low temperatures wasenhanced in the transformed cells. These findings
suggest that glycinebetaine enhanced the growth of
the transformed cells at 20°C by accelerating therecovery of the photosynthetic machinery fromphotoinhibition.

V. Genetic modification of the ability of Arabidopsis to tolerate salt and low-temperature stress
A higher plant, Arabidopsis thaliana, wastransformed with the codA gene for choline oxidase
under control of the 35S promoter of cauliflower
mosaic virus. Such transformation allowed the plantto accumulate glycinebetaine and enhanced its ability
to tolerate salt and cold stresses. When cultured in
the presence of 100 mM NaCI, transformed plants
grew well whereas wild-type plants failed to grow.
The transformed plants tolerated 200 mM NaCl,
which was lethal to wild-type plants. After plants had
been incubated with 400 mM NaCl for two days, thephotosystem II activity of wild-type plants had
almost completely disappeared, whereas that oftransformed plants remained at more than 50% of theoriginal level. When exposed to low temperatures in
the light, leaves of wild-type plants exhibited
symptoms of chlorosis, whereas those of transformed
plants did not. Thus, the genetic modification of
Arabidopsis thaliana that allowed it to accumulate
glycinebetaine also enhanced its ability to tolerate
both salt and cold stresses.

39

FOR BASIC BIOLOGY

Publications List:(1) Original papers
Avelange-Macherel, M. H., Macherel, D., Wada, H.
and Murata, N. (1995) Site-directed mutagenesisof histidine in the A12 acyl-lipid desaturase of
Synechocystis. FEBS Lett. 361, 111-114.
Deshnium, P., Los, D. A., Hayashi, H., Mustardy, L.
and Murata, N. (1995) Transformation of
Synechococcus with a gene for choline oxidase
enhances tolerance to salt stress. Plant Mol. Bioi.
29, 897-907.
Fukuchi-Mizutani, M., Savin, K., Cornish, E.,
Tanaka, Y., Ashikari, T., Kusumi, T. and Murata,
N. (1995) Senescence-induced expression of ahomologue of A9 desaturase in rose petals. Plant
Mol. Bioi. 29, 627-635.
lida, H., Ohya, Y. and Anraku, Y. (1995)
Calmodulin-dependent protein kinase II andcalmodulin are required for induced thermotolerance in Saccharomyces cerevisiae. Curr.
Genet. 27, 190-193.
Ishizaki-Nishizawa, 0., Azuma, M., Ohtani, T.,
Murata, N. and Toguri, T. (1995) Nucleotide
sequence of eDNA from Spinacia oleracea
encoding plastid glycerol-3-phosphate acyltransferase. Plant Physiol. 108, 1342.
Kanervo, E., Aro, E.-M. and Murata, N. (1995) Low
unsaturation level of thylakoid membrane lipids
limits turnover of the D1 protein of photosystem II
at high irradiance. FEES Lett. 364, 239-242.
Malakhov, M.P., Los, D. A., Wada, H., Semenenko,
V. E. and Murata, N. (1995) Characterization of
the murF gene of the cyanobaterium Synechocystis
sp. PCC 6803. Microbiology 141, 163-169.
Moon, B. Y., Higashi, S., Gombos, Z. and Murata, N.
(1995) Unsaturation of chloroplastic membranelipids protects photosynthesis against lowtemperature photoinhibition in transgenic tobacco
plants. Proc. Nat[. Acad. Sci. U.S.A. 92, 62196223.
Tanida, 1., Hasegawa, A., lida, H., Ohya, Y. andAnraku, Y. (1995) Cooperation of calcineurin andvacuolar H+ -ATPase in intracellular Ca2+
homeostasis of yeast cells. J. Bioi. Chern. 270,
10113-10119.
(2) Review papers
Murata N. and Wada H. (1995) Acyl-lipid
desaturases and their importance in the tolerance
and acclimatization to cold of cyanobacteria.
Biochem. J. 308, 1-8.
Papageorgiou G. C. and Murata N. (1995) The
unusuallystrongstabilizing
effectofglycinebetaine on the structure and function of theoxygen-evolving
photosystem
IIcomplex.

Photosyn. Res. 44,243-252.
(3) Proceedings
Deshnium, P., Los, D. A., Hayashi, H. and Murata, N.
(1995) Glycinebetaine enhances tolerance to salt
stressintransgenic
cyanobacterium.
In
Photosynthesis: From Light to Biosphere (P.
Mathis, ed.), Kluwer Academic Publishers,
Dordrecht, Vol. IV, pp. 637-640.
Murata, N. (1995) Genetic engineering ofphotosynthetic capability under temperature andsalinity stresses. In Photosynthesis: From Light toBiosphere (P. Mathis, ed.), Kluwer Academic
Publishers, Dordrecht, Vol. IV, pp. 763-770.
Papageorgiou, G. C. and Murata, N. (1995)
Thylakoids from salt-stressed Spinacea oleracea
are more active in photosynthetic oxygen evolution
and more tolerant to alkaline pH than thylakoids
from unstressed plants. In Photosynthesis: From
Light to Biosphere (P. Mathis, ed.), Kluwer
Academic Publishers, Dordrecht, Vol. IV, pp. 645648.

40

DIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADJUNCT)
Professor (adjunct): Kimiyuki Satoh
Associate Professor (adjunct): Hirokazu Kobayashi
Research Associate: Noritosi Inagaki
Graduate Students: Kyoichi /sono*
Yoshihiro Narusaka*
Tatsuya Tomo*
Satoshi Matsumoto*
Akihiko Tohri*(*from Okayam a University)

From an energetic point of view, the conversion oflight energy into chemical energy in photosynthesisis the most important biological process on earth.
The highly efficient energy conversion in thisprocess is ensured by the highly ordered organizationof molecules in the photochemical reaction center, ina physical, chemical and biological sense. The
project in this division is aiming to elucidate theorganization of photosystem II reaction center ofoxygenic photosynthesis which has a unique property
to generate a strong oxidant for utilizing the watermolecule as electron donor.
In the first approach, molecular organization of thephotosystem II reaction center, which has been
identified in our study, will be analyzed by several
methods which include crystallographic analysis,
chemical modification & cross-linking analysis andoptical & EPR spectroscopies. Structure-functional
analysis will also be conducted for the reaction
center using random and site-directed mutagenesis
for transformable algae, Synechocystis PCC 6803
and Chlamydomonas reinhardtti. The principal
target of these analyses will be the structure andmolecular environment of P-680, the primary donor,
which determine the redox potential of this uniquephotosystem.
In the second approach, the effort will be focusedon the elucidation of molecular mechanism of lightregulated metabolic turnover of a subunit of thephotosystem II reaction center, D1 protein. Some ofunique steps are involved in this process; i.e., photodamage of the photochemical reaction center,
specific degradation of the impaired protein subunit,
light-regulated gene expression at the translational
level, post-translational cleavage of the C-terminal
extension and the incorporation of cofactors andsubunits into a multi-component pigment-protein
complex.
I. Structural organization of photosystem II reaction center(1) A photochemically inactive pheophytin (Pheo)a molecule in the photosystem II reaction center,
which contains 6 chlorophyll a and two ~-carotene
molecules for every two Pheo a, was identified byfluorescence excitation spectroscopy at 77K, andconfirmed by absorption, linear dichroism, andmagnetic circular dichroism spectra (in collaboration

NATIONAL INSTITUTE

with Dr. M. Mimuro, Div. of Bioenergetics). Whenthe emiSSion was monitored at 740 nm,
photochemically active Pheo a was detected as peaks
at 418, 513, 543, and 681 nm. However, when theemission was monitored at 665 nm, two bands wereobserved at 414 and 537 nm; these bands wereinsensitive to photochemical reduction of the primary
acceptor and thus appear to be due tophotochemically inactive Pheo a.
(2) Solvent extraction at - 4°C in complete
darkness, and subsequent analysis by high-pressureliquid chromatography using an apparatus equippedwith a two-dimensional diode-array detector,
spectroscopically identified 15-cis-~-carotene in thephotosystem II reaction center. The result revises aprevious conclusion that ~-carotene bound to thereaction center takes the all-trans configuration, andgeneralizes the concept established for purple
photosynthetic bacteria that 15-cis-carotenoid isnaturally selected by the photosynthetic reaction
centers (in collaboration with Drs. Y, Koyama et al.,Kwansei Gakuin University).
(3) Site-directed modifications of the environmentof the primary donor of photosystem II reaction
center were attempted, using Synechocystis PCC
6803, in order to elucidate molecular interactions
responsible for the high redox-potential.
II. Dynamic aspects of the organization of photosystem II reaction center(1) The D1 subunit of the photosystem II reaction
center has a C-terminal extension consisting of 8-16
amino acids. Post-translational removal of this partof the protein is essential for the manifestation ofoxygen-evolving function in photosystem II. The
enzyme involved in the C-terminal processing of theD1 precursor protein (pD1) of the photosystem IIreaction center was purified from extracts ofsonicated spinach thylakoids by a method that
included chromatography on quaternary aminoethyl
anion-exchange, hydroxyl-apatite, copper-chelating
affinity and gel-filtration columns. The enzyme was
identified, from its chromatographic behavior, to be amonomeric protein of about 45 kDa. The sequenceof the amino-terminal 27 amino acids of this protein
was determined directly, which exhibited low but
appreciable (37%) homology to that deduced from agene (ctpA) in Synechocystis PCC 6803 that was
proposed recently to encode the processing proteaseform results of genetic complementation analysis.
Based on the amino acid sequence data of thepurified protease, a eDNA clone encoding the
enzyme has identified and sequenced, from a spinachgreen leaf eDNA library. By the analysis, the fulllength transcript was established to consist of 1906
nucleotides and a poly(A) tail, containing an open
reading frame corresponding to a protein with 539
amino acid residues. By comparing the amino acidsequence of the purified protease with that deduced

FOR BASIC BIOl.DGY

form nucleotide sequence of the eDNA clones, theenzyme was shown to be furnished with an extra Nterminal extension characteristic of both a transit
peptide and a signal sequence. This suggests that theprotease is synthesized in the cytosol andtranslocated into the lumenal space of thylakoids.
The mature part of the enzyme consists of 389 aminoacid residues and exhibits a significant sequencehomology with two groups of proteins as
demonstrated by a computer homology search; i.e.,(i) the deduced sequence of a protein proposed to be
the C-terminal processing protease for pD1 in
Synechocystis PCC 6803 and (ii) proteases for Cterminal cleavage identified in Escherichia coli andBartonella bacilliformis.
(2) The synthesis of D1 precursor protein isregulated by light at the stage of translation. The
mechanism of this light regulation was analyzed
using isolated pea chloroplasts. Accumulation of the
precursor and the mature form of the D1 protein of
the photosystem II reaction center in illuminated pea
chloroplasts was prevented by the addition of theinhibitors of photosynthetic electron transport, i.e .,atrazine, 3-(3,4-dichlorophenyl-1,1-dimethylurea andbromoxynil.
Under
such
conditions,thecompensatory accumulation of two translational
intermediates of the D1 protein of 22 and 24 kDa
was induced by the addition of ATP, as also
observed in darkness in the presence of ATP

41(Taniguchi et al., FEBS Lett. 317, 57-61, 1993),
suggesting that the synthesis of the full-length D1protein requires a factor that is generated by theoperation of photosynthetic electron transport. The
accumulation of the full-length D1 protein was
induced in the light, even in the presence of atrazine,
when both 2,6-dichlorophenolindophenol andascorbate were also present and in darkness upon the
addition of dithiothreitol. Moreover, reagents with arelatively low redox potential, namely, duroquinone
and methylviologen, prevented the accumulation.
These observations suggest that the translation of theD1 protein is regulated at specific steps during theelongation of the polypeptide via a redox change in acomponent around photosystem I.
(3) Photo-tolerant mutants of Synechocystis PCC
6803 were obtained by in vitro random mutagenesisof psbAII (gene for D1 protein) by PCR under acondition for reduced fidelity of amplification. A
preliminary characterization has been carried out fora mutant. The mutant strain (G-3) was shown to have
five substitutions in the amino acid in regions otherthan so-called "PEST-like sequence" in the D1
protein, i.e., Phe-260 and Phe-273 to Ser, Gln-304
and Gln-310 to Arg, and Bal-351 to Ala. The mutant
cells were tolerant to high irradiance both in terms ofpigmentation and viability(Fig. 1). The functional
characterization of photosynthesis in the mutant has
been conducted.

Fig. 1. Mixed culture of G-3 mutant and the control (KC) strains. The mixture of cells were plated on BG-11 agar medium .
After colonies were grown under 50 flE·m- 2 ·s- 1 for 10 days, the agar plates were exposed to high photon flux density of 320
flE · m- 2 • s- 1 for 5 days. Pale green colonies, control ; deep green colonies, G-3 mutant (confirmed by mono-culture experiments).

42

Publication List:
Bialek-Bylka, G.E., Torno, T., Satoh, K. andKoyama, Y. (1995) 15-cis-j3-Carotene found in thereaction center of spinach photosystem II. FEBS
Lett. 363, 137-140.
Fujita, S., Inagaki, N., Yamamoto, Y., Taguchi, F.,
Matsumoto, A. and Satoh, K. (1995) Identificationof the carboxyl-terminal processing protease forD1 precursor protein of photosystem II reaction
center of spinach. Plant Cell Physiol. 36, 11691177.
Mimuro, M., Torno, T., Nishimura, Y., Yamazaki, I.
and Satoh, K. (1995) Identification of aphotochemically inactive pheophytin molecule inspinach 0 1-Dz-cyt b559 complex. Biochem. Biophys.
Acta, in press.
Nozawa, T., Kobayashi, M., Wang, Z.-Y., ltoh, S.,
Iwaki, M., Mimuro, M. and Satoh, K. (1995)
Magnetic circular dichroism investigation on
chromophores in reaction centers of photosystem I
and II of green plant photosynthesis.
Spectrochimica Acta 51A, 125-134.
Satoh, K. (1995) Introduction to photosystem IIreaction center -Isolation and some biochemicaland biophysical characterization-. in "Oxygenic
Photosynthesis: The Light Reactions" (D.R. Ort &C.F. Yocum eds.), Kluwer Academic Publishers(Dordrecht), in press.
Taguchi, F., Yamamoto, Y. and Satoh, K. (1995)
Recognition of the structure around the site ofcleavage by the carboxyl-terminal processing
protease for Dl precursor protein of thephotosystem II reaction center. J. Bioi. Chem. 270,
10711-10716.

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDIVISION OF BEHAVIOR AND
NEUROBIOLOGY (ADJUNCT)
Professor: Masatoshi Takeichi
Research Associates: Akinao Nose
Kazuaki Tatei
Institute Research Fellow: Emiko Shishido
Post-doctoral Fellow: Takaka Isshiki
Graduate Students: Tatsuo Umeda
Hiraki Taniguchi
Takeshi Umemiya (from Kyoto
University)

How individual nerve cells find and recognize theirtargets during development is one of th~ _c~ntr~lissues in modern biology. The aim of our diVISion IS
to elucidate the molecular mechanism of axon
guidance and target recognition by using the simple
and highly accessible neuromuscular system ofDrosophila.
The musculature of Drosophila embryos consistsof 30 identifiable muscle fibers per hemisegment.
Each muscle fiber is innervated by a few
motoneurons in a highly stereotypic manner. The
high degree of precision and previous cellular
manipulations of neuromuscular connectivity suggest
the presence of recognition molecules on the surfaceof specific muscle fibers which guide the growthcones of motoneurons. We have previously isolated
several enhancer trap lines that express the reporter
gene B-galactosidase (B-gal) in small subsets ofmuscle fibers prior to innervation. By molecularly
characterizing these lines, we are trying to identify
genes that play roles in the specification of themuscles and neuromuscular connectivity. Previous
studies showed that two of the lines are insertions in
the connectin and Toll genes, that encode cellrecognition molecules which belong to the leucinerich repeat (LRR) family. We are currently studying
the function of these genes, and also characterizing
other lines by molecular and genetic analysis.
I. Connectin can function as an attractive target
recognition molecule
Connectin is expressed on a subset of muscle
fibers (primarily lateral muscles) and on the axons,
growth cones of the motoneurons which innervatethese muscles (primarily SNa motoneurons) and on
several associated glial cells. When coupled with its
ability to mediate homophilic cell adhesion in vitro,these results led to the suggestion that Connectinfunctions as an attractive signal for SNa pathfinding
and targeting.
To study the role of Connectin in vivo, we
ectopically expressed Connectin on all muscles byusing MHC (myosin .heavy chain) promoter (MHCconnectin) in the P-element mediated transformants.
In MHC-connectin, SNa nerves were observed tosend extra axon branches that form ectopic nerve
endings on muscles 12, muscles they would neverinnervate in wild type. This phenoype was highly

43penetrant and was observed in over 60% of t~esegments examined. Furthermore, the ectopic
innervation on muscle 12 was dependent on theConnectin expression on SNa. These results showedthat Connectin functions as an attractive andhomophilic guidance molecule for SNa in vivo.
II. P750, a novel LRR cell surface molecule expressed on subsets of neurons and muscles
We are conducting molecular and genetic analysis
of other enhancer trap lines that are expressed insmall subsets of muscles and/or neurons. One ofthem, P750 expresses B-gal in subsets of ventral anddorsal muscles and in a small number of neurons in
the central nervous system (CNS). The eDNA
cloning and sequencing revealed that P750 encodes anovel transmembrane protein that belongs to LRR
family. It is interesting that three of the five muscle
enhancer trap lines that we have thus far
characterized contain LRRs. It may point to theimportant role of this motif in the development ofmuscles and/or neuromuscular specificity. Within the
LRR family, P750 was found to be most related to
the Drosophila tartan gene that have been implicatedin neural and muscular development. To study thefunction of P750, we have isolated the loss-offunction mutants, and are currently characterizing thephenotype.
III. F -spondin/AN34 family of secreted proteins in
Drosophila
Another enhancer trap line, AN34 expresses B-gal
in a single muscle fiber (#18) per hemisegment(Fig.1A). The remarkable specificity in its expression
pattern (one out of 30 muscle fibers) makes it a goodcandidate for the muscle target recognition molecule.
AN34 was found to encode a secreted protein withextensive amino acid similarity to rat F-spondin. Fspondin is a secreted molecule expressed at highlevels in the floor plate and has been shown topromote neural cell adhesion and neurite extension invitro. These results strongly suggest that AN34 is
involed in motoneuronal gidance and/or targeting.
There are three regions that are highly conserved
between AN34 and F-spondin. One of them (domain
III) is a known repeating motif called
thrombospondin type I repeats (TSRs). The othertwo (domain I and II) are novel conserved sequencesthat we identified. The amino acid identity in domain
II is particularly high suggesting its functional
importance. We thus searched for novel genes in
Drosophila that contain this domain by using PCR,
and succeeded in cloning two new genes. Similarly,
Higashijima et al. (Div. of Cellular Communication)cloned two novel genes in addition to F-spondin in
the zebrafish. All of these newly cloned genes sharesimilar overall structure with AN34 and F-spondin inthat they possessed domain I, II and one to six TSRs.

44

NATIONAL INSTITUTE

Fig. 1. Expression of the members of AN34/F-spondin family in Drosophila .
(A) AN34 enhancer trap line express B-galactosidase (B-gal) in the nuclei of single muscle fiber #18 (arrows) in eachhemisegment. Anti B-gal antibody staining in blue and general muscle staining (as visualized with a monoclonal antibody 270)in brown.
(B) #8 in situ hybridization. #8 mRNA is expressed in a small subset of glia along the longitudinal connectives.
They thus constitute a novel gene family of secreted
protein with potential roles in neural adhesion.
One of the newly identified genes in Drosophila(tentatively called #3) is expressed in a subset ofhemocytes, secretory cells of the extracellular matrix(ECM) proteins. Another (called #8) is expressed ina subset of glia that sit along the longitudinal axon
tracts in the CNS (Fig.lB).
The specificity of their expression suggest thatthey might be involved in the guidance of specificaxons. We are currently trying to isolate the loss-offunction mutants of these three genes (AN34, #3, #8)
as well as the transgenic flies that ectopically express
these molecules (as described for Connectin) to study
their roles in neural development.

homeobox. Analysis of the loss-of-function mutants
revealed severe defects in the formation of muscles
and the CNS. The dorsal muscles that normally
express the molecule were often missing totally or
failed to form properly. The overall structure of the
CNS axon tract was also seriously disrupted. Close
examination of the development of the LGB leneage
using specific markers showed that the glioblasts failto divide and migrate properly in the mutants. These
results suggested that rH96 play essential roles in thespecification and/or differentiation of precursors forneurons, glia and muscles. We are currently trying toectopically express rH96 to characterize more indetail the function of rH96 in the development ofmuscles and the nervous system.

IV. rH96, a homeobox containing gene essential
for neural and muscular development

Publication List:

Another line rH96 is expressed in the precursors of
dorsal muscles. It is also expressed in a subset ofneuroblasts and glioblasts in the CNS. In particular,
it is expressed in the longitudinal glioblasts (LGB),
precursor cells for the longitudinal glia whose
lineage is well characterized. rH96 was shown to
encode a putative transcription factor with a

Kimura, Y., Matsunami, H., Inoue, T., Shimamura,K., Uchida, N., Ueno, T., Miyazaki, T. andTakeichi, M. (1995) Cadherin-11 expressed inassociation with mesenchymal morphogenesis in
the head, somite, and limb bud of early mouseembryos. Develop. Bioi. 169, 347-358.
Matsunami, H. and Takeichi, M. (1995) Fetal brain
subdivisions defined by R- and E-cadherin

FOR BASIC BIOLOGY

expressions: evidence for the role of cadherin
activity in region-specific, cell-cell adhesion.
Develop. Bioi. 172,466-478.
Nakagawa, S. and Takeichi, M. (1995) Neural crest
cell-cell adhesion controlled by sequential andsubpopulation-specific expression of novel
cadherins. Development 121, 1321-1332.
Overduin, M., Harvey, T.S., Bagby, S., Tong, K.l.,
Yau, P., Takeichi, M. and Ikura, M. (1995) Threedimensional solution structure and calcium
interaction of the epithelial cadherin domain
responsible for selective adhesion. Science 267,
386-389.
Shibamoto, S., Hayakawa, M., Takeuchi, K., Hori, T.,
Miyazawa, K., Kitamura, N., Johnson, K.R.,
Wheelock, M.J., Matsuyoshi, N., Takeichi, M. andIto, F. (1995) Association of p120, a tyrosine
kinasesubstrate,with
E-cadherin/catenincomplexes. J. Cell Bioi. 128, 949-957.
Shibuya, Y., Mizoguchi, A., Takeichi, M., Shimada,K. and Ide, C. (1995) Localization of N-cadherin
in the normal and regenerating nerve fibers of thechicken peripheral nervous system. Neuroscience67, 253-261.
Shiozaki, H., Kadowaki, T., Doki, Y., Inoue, M.,
Tamura, S., Oka, H., lwazawa, T., Matsui, S.,
Shimaya, K., Takeichi, M. and Mori, T. (1995)
Effect of epidermal growth factor on cadherinmediated adhesion in a human oesophageal cancer
cell line. British J. Cancer 71, 250-258.
Takeichi, M. (1995) Morphogenetic roles of classic
cadherins. Current Opinion Cell Bioi. 1, 619-627.

45

46

NAUQNALINS'IlTUTE/

47

FOR BASIC BIOLOGYLABORATORY OF GENE EXPRESSION AND REGULATION

Head: Yoshitaka Nagahama

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

48

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGUlATION I
Closed during 1995 and will be reinitiated in 1996
on new projects.

Publication List: (From the previous projects)
Ito, T., Hirano, Y., Shimura, Y. and Okada, K.
(1995) Two touch-inducible calmodulin andcalmodulin-related genes tandemly located on
chromosome of Arabidopsis thaliana. Plant Cell
Physiol. 36, 1369-1373.
Mikami, K., Katsura, M., Okada, K., Shimura, Y.
and lwabuchi, M. (1995) Developmental andtissue-specific regulation of the gene for the wheat
basic/leucine zipper protein HBP-1a(17) intransgenic Arabidopsis plants. Mol. Gen. Genet.
248, 573-582.
Okada, K., Ito, T., Sawa, S., Yano, A., lshiguro, S.
and Shimura, Y. (1995) Mutational analysis offlower development in Arabidopsis thaliana. In"Modification of gene expression and nonMendelian inheritance" (eds. Oono, K. andTakaiwa, H.) NIAR Japan, pp. 145-156.

49

FOR BASIC BIOLOGY

ends of 35S and 5S rRNA genes, and the latter is 5'
ends of these two genes. The HOT1 DNA fragment
contains the NTS1, 5SRNA gene and NTS2 region
but it was later found to be composed of two noncontiguous cis-elements, E and I, located in NTS1
and NTS2, respectively. Because E and I positionallyand functionally overlapped with the enhancer andinitiator of the 35S rRNA transcription, respectively,
Roeder's group suggested that transcription by RNA
polymerase I, initiated at the 35S rRNA promoter
site may stimulate recombination of the downstream
region, thereby revealing Hotl activity.
The NTS1 has a site at which the replication forkis blocked and we termed this site SOG, but latercalled RFB (replication fork hlock). By assaying Rfb
activity for various DNA fragments derived from the
NTS1 and cloned on plasmids, we determined theminimal region, about 100 bp long, located near theenhancer region of the 35S rRNA transcription andessential for blocking replication fork advancing in a
direction opposite that for transcription. The RFB
sequence has no homology to any other known
sequence and has no characteristic structure such 2fold symmetry, repeated structure, etc.; hence, transfactor(s) may have a role in blocking the fork.
Interestingly, this region is included in one of two
cis-elements required for a recombinational hotspot,
Hotl, activity.
To investigate functional relationships between thefork blocking activity in RFB and the hotspot activity
in HOT1, we first isolated mutants defective in Hotlactivity and examined whether these mutations
would also affect Rfb activity. Using a colony color
sectoring assay method, we isolated 23 Hot defective
mutants from approximately 40,000 mutagenizedcolonies. Among these Hot- mutants, one proved to
be a rad52 mutant; the other 4 mutants lose fork
blocking activity (Fig. 1).

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

Homologous recombination may occur m allorganisms. While related functions apparently
involve exchange between two parent-derived
chromatids and repair of DNA damage incurred byphysical and chemical reagents, many questions
remain unanswered. As deduced from our analyses ofrecombinational hotspots of E. coli & S. cerevisiae,in particular the activity related to DNA replication
fork blocking events, the physiological function ofhomologous recombination, especially in normallygrowing cells is better understood.
I. Analysis of recombinational hot spot in S. cerevisiae

HOT1 is a mitotic recombinational hotspot in theyeast S. cerevisiae and was first identified by Keiland Roeder. HOT1 stimulates both intra- and interchromosomal recombination, and for a precise
analysis enhancement of excisional recombination
between directly repeated DNAs at its nearby site
was investigated. HOT1 was originally cloned on a
4.6 kb Bgiii fragment which locates in rRNA
repeated genes (about 140 copies) on chromosome
XII. A single rRNA unit consists of two transcribed
35S and 5S rRNA genes and two non-transcribedregions, NTS1 and NTS2, the former is between 3'-

A

B

c

D

E

F

Fig. 1. Correlated relationship between Hotl and Rfb activities of various yeast strain . Upper panels are colonies and lower
panels are the 20 gel analysis pattern . In the upper panels the sectored and the uniform red colonies show Hotl active andinactive phenotype, respectively. In the lower panels presence (indicated by arrow) and absence of spot on the arc in 20 agarose
gel electrophoretic pattern show Rfb active and inactive property, respectively. A; diploid (FOBJ /fobl-4), B; wild haploid(FOB!), C; fobl-4 mutant, 0; fobl-4 carrying a plasmid with the FOB! gene, E; disruptant of fob! gene (jobl: :LEU2), F;
diploid (jobl-4/fobl ::LEU2).

50

Genetic analysis of these mutants revealed that all
four were recessive for the Rfb phenotype anddefined one complementation group. This mutation
was designated fobl ( fork .!!locking function) and
one ofthefobl mutants,fobl-4, was further analyzed.
First, from yeast eDNA bank, we cloned FOBl geneby selecting a DNA fragment which had suppressive
activity for Hot deficiency of the mutant. The
minimal FOBl plasmid was shown to complement
both the Hof and Rfb- phenotype of the fobl-4
mutant, suggesting that both phenotypes are caused
by a mutation in the FOBl gene (Fig. 1). DNA
sequencing of the FOBl gene revealed that theputative Fob1 protein consists of 566 amino acids
and has a molecular mass of 65,000 daltons. We
have overproduced and purified a protein with thismolecular weight and the expected amino acidsequence of N-terminus of Fob1 protein. We are
testing whether the Fob1 protein has binding activity
to RFB sequence or not. We found the same
sequence as that of the FOBl gene in sequence of thechromosome IV cosmid 9727. Thus, the FOBl geneis apparently not linked with the rRNA gene cluster
present in chromosome XII. Sequencing of the fob 14 mutant gene revealed two mutational changes in
the open reading frame, one is non-sense (amber)and other is a miss-sense mutation. The amber
mutation may account for the two defective
phenotypes of the fobl-4 mutant and why it is nonleakyness.
Other workers showed that transcription of therRNA gene is involved in the Hotl activity, while thetranscription appears not to be responsible for the
Rfb activity. Our finding indicates that fork blocking
event is required for Hotl activity and seems not tobe responsible for rRNA transcription. Thus, in yeast,
both two independent events, one is fork blocking at
RFB site and other is transcription of the rRNA gene,are required for activation of Hotl.
IT. Analysis of E. coli recombinational hot spots,
Hot
In E. coli RNase H defective (rnh -) mutants, wefound 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 (HotA-H) of such Hot DNA, 7of which (HotA-G) were clustered in a narrow region,
called replication terminus region (about 280 kb) on
the circular E. coli genome. Analysis of the HotA, B
and C revealed that blocking of replication fork at
the Ter, replication terminus, sites is responsible forthese Hot activities. Further analysis of these Hot led
to design of a putative model, in which the ds(double
stranded)-break occurs at the fork arrested at the
DNA replication fork blocking (Ter) site, throughwhich the RecBCD recombinational enzyme enters
the ds-DNA molecule and enhances recombination

NATIONAL INSTITUTE

between directly repeated Hot DNA, when the
enzyme meets an appropriately oriented Chi
sequence.
To know how to activate other termination eventindependent Hots, especially HotG, D and H, DNA
sequences of these Hot fragments were determined
and found that all Hot DNAs contained Chi sequence,
thereby suggesting that RecBCD enzyme may be
involved in these Hot activation such as in HotA. We
now examine what event occurs near the Hot DNA
sites, which probably provides an entrance site for
RecBCD enzyme.
Publication List:
Horiuchi,T.
andFujimura,Y.
(1995)
Recombinational rescue of the stalled DNA
replication fork: a model based on analysis of an
Escherichia coli strain with a chromosome regiondifficult to replicate. J. Bacterial. 177, 783-791.
Horiuchi, T., Nishitani, H. and Kobayashi, T. (1995)
A new type of E. coli recombinational hotspot
which requires for activity both DNA replication
termination events and the Chi sequence. In"Molecular
Mechanismof
Genetic
Recombination". Advance Biophysics vol. 31, 133147.

51

FOR BASIC BIOWGYDIVISION OF SPECIATIONMECHANISMS I
Professor: Tetsuo Yamamori
Research Associates: Satoshi Koike
Yuriko Komine
Technical Staffs: Hideko Utsumi

Our research goal is to understand mechanisms
underlying evolution of the nervous system. In order
to approach this question, we are currently focusing
on two systems.
I. Evolution of cytokine receptor families in the
immune and nervous systems
Recently, it has been recognized that cytokines,
defined as intercellular mediators in the immune
system, have a variety of roles in the nervous system
as well. One such a factor, LIF (leukemia inhibitory
factor) known also as CDF (Cholinergic
Differentiation Factor), is a pleiotropic factor whichshows a remarkable repertoire of activities fromembryonic stem cells to neurons (Yamamori, T. In
Chemical Factors in Neuronal Growth, Degeneration
and Regeneration (Ed. by C. Bell), Elsevier, in press).
Recent study have revealed that CDF/LIF and its
receptors belong to the IL-6 family and the receptor
family.
Based on Bazan's model which predicted thecytokine receptor family as a member of
immunoglobulin super gene family (1990) and themodel of the interaction among the members of the
IL-6 family (ligand) and the IL-6 receptor family(Taga and Kishimoto, 1992; Stahl and Yancopoulos,
1993), we proposed that the evolution of the IL6/class IB receptor family may have occurred in atleast two major steps (Yamamori and Sarai, 1994).
Firstly, binding subunits of an IL-6 receptor and for a
CDF/LIF receptor evolved and secondly, a thirdbinding subunits of a CNTF receptor evolved. Our
evolutional consideration predicts that the binding
subunits generally determine the specificity of thereceptors and it is possible that novel members of thecytokine family and their receptors exist in the
nervous system.
II. Gene expression and cerebellar long-term plasticity
In order to know roles of the genes involved inlong-term memory, we choose the cerebellum as amodel system. In the cerebellum the conjunctive
stimuli of parallel fibers and a climbing fiber to a
Purkinje cell induce prolonged reduction of asynaptic efficacy between the paralleled fiber to the
Purkinje cell (LTD; long-term depression, Ito et al.,1982).
Previously, we examined the expression of 10
immediate early genes (lEGs) including all the
known Fos and Jun family in cerebellar slices under
the pharmacological condition that cause long-term

desensitization of the Purkinje cell to AMPA (aglutamate analogue). Among the lEGs examined, Fosand Jun-B were predominantly induced under the
conjunctive condition (Nakazawa et al., 1993).
Recently, we have examined Jun-B expression invivo under a conjunctive protocol of AMPA, a
pharmacological substitute for parallel fiber
stimulation, and climbing fiber stimulation via
electric stimulation of Inferior Olive. June-B are
predominantly induced around the local area where
the AMPA and climbing fiber stimulation wereconjunct. These results suggest that the coincidence
mechanism may exist at gene expression level andlead to a cerebellar long-term plasticity.
We are currently working to identify the moleculesthat are induced after Jun-B induction and playing
roles in cerebellar long-term plasticity.
Publication List:
Yamamori, T. Mikawa, S. and Kado, R. (1995) JunB expression in Purkinje cells by conjunctive
stimulation of climbing fibre and AMPA.
NeuroReport 6, 793-796.

52

NATIONAL INSTITUTEDIVISION OF SPECIATIONMECHANISMS II

The Division will be initiated in 1996.

FOR BASIC BIOLOGY

53

RESEARCH SUPPORT54

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head:Hiroyuki Hattori
Common Facility Group
Chief:Kazuhiko Furukawa
Research Support Facilities
Mamoru Kubota(Subunit Chief)
Chieko Nanba
Toshiki Ohkawa
Masayo lwaki
Kaoru Sawada
TomokiMiwa
Hideko Nonaka
Radioisotope Facility
Yousuke Kato(Subunit Chief)
Yoshimi Matsuda
Center for Analytical Instruments
Akio Murakami(Unit Chief)
Sonoko Ohsawa
Yukiko Kabeya
Yumiko Makino
Takeshi Mizutani
Glassware Washing Facility(Kazuhiko Furukawa)(Toshiki Ohkawa)Research Support Group
Cell Biology Group
MakiKondo
Katsushi Yamaguchi
Developmental Biology Group
Hiroko Kobayashi(Unit Chief)
Sachiko Fukada
Chikako Inoue
Chiyo Takagi
Regulation Biology Group
Shoichi Higashi
Miki Ida
AkikoKawai
Shigemi Ohsugi
Gene Expression and Regulation GroupKoji Hayashi
TomokoMori
Yasushi Takeuchi
Hideko Utsumi

The Technology Department is a supportingorganization for researchers and research organization within the NIBB. The Department develops
and promotes the institute's research activities and at
the same time, maintains the research functions of
the institute.
The Department is organized into two groups: one,
the Common Facility Group, which supports andmaintains the institute's common research facilities
and the other, the Research Support Group, whichassists the research activities as described inindividual reports.
Technical staffs participate, through the department, in mutual enlightenment and education, andincrease their capability in technical area. Eachtechnical staff is proceeded to the fixed divisionusually and they support the various research with
their special biological and biophysical techniques.
The Department hosts an annual meeting fortechnical engineers who work in various fields of
biology at universities and research institutes
throughout Japan. At this meeting, the participantspresent their own activities and discuss technicalproblems. The Proceedings are published soon after
the meeting.

55

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Head of Facility: Mikio Nishimura
Associate Professor: Masakatsu Watanabe
Research Associates:Yoshio Hamada(Tissue and Cell Culture)
Kenta Nakai (Computer ;-April 30,
1995)

Technical Staff: Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
TomokiMiwa
Hideko Nonaka
Kimiko Yamamiya (-March 31, 1995)
Masayo /waki (June 1, 1995-)

The Facility provides large-and medium-scale
instruments and facilities for biophysical, molecularbiological, and computational analyses as well as for
growing and maintaining biological specimens. The
facility is shared among the research members, andhas seven laboratories, among which the Large
Spectrograph Laboratory and the Laboratory ofStress-Resistant Plants are dedicated to cooperative
use under the NIBB Cooperative Research Programs.

I. Facilities
1. The Large Spectrograph Laboratory

This laboratory provides, for cooperative use, theOkazaki Large Spectrograph (OLS), which is thelargest 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 grating
composed of 36 smaller individual gratings. It
projects a spectrum of a wavelength 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 ismore than twice as much as that of the corresponding
monochromatic component of tropial sunlight atnoon (Watanabe et al., 1982, Photochem. Photobiol.,36, 491-498).
A tunable two-wavelength CW laser irradiation
system is also available as a complementary lightsource to OLS to be used in irradiation experiments
which specifically require ultra-high fluence rates aswell as ultra-high spectral-, time-and spatialresolutions. It is composed of a high-power Ar-ion
laser (Coherent, Innova 20) (336.6-528.7 nm, 20 W
output), two CW dye lasers (Coherent, CR599-01)(420-930 nm, 250-1000 mW output),
NO modulators (up to 40 MHz) to chop the laser
beam, a beam expander, and a tracking microbeam
irradiator (up to 200!lms· 1 in tracking speed, down to211m in beam diameter) with an infrared phasecontrast observation system.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely

rooms which satisfy the P2/P3 physical containment
level. This facility is routinely used for DNA
recombination experiments.
3. Computer Laboratory
To meet various computational needs and to provide
means of electronic communication in this Institute,
many kind of computers are equipped: VAX/VMS
UNIX
engineering
workstations
machines,(SPARCstations, NEWS machines, DECstations),
and some personal computers (Macintosh's andWindows machines). All of these machines areconnected each other through the Ethernet, the Fast
Ethernet, or the CDDI, which are also linked by
optical fibers to the high performance multimedia
backbone network of Okazaki National ResearchInstitutes. Since this backbone network, called
ORION, is joined to the Internet, the users of thesemachines can access various services and databaseson the Internet. Each laboratory has several
computers that are also connected to the network of
the Institute. The Computer Laboratory provides
various computational services to the Institutemembers: file servers for Macintosh and NetWare
users, some print servers that accept printing requests
from PC and UNIX users, a computational serverthat provides sequence analyses and databaseretrievals, communication servers to the Internet, andso on. The laboratory also provides an information
dispatching service to the Internet using the World
Wide Web (URL is http://www.nibb.ac.jp).
4. Plant Culture Laboratory
There are a large number of culture boxes, and alimited number of rooms with environmental control
for plant culture. In some of these facilities androoms, experiments can be carried out at the P1
physical containment level.
5. Experimental FarmThis laboratory consists of two 20 m2 glass-houses
with precise temperature and humidity control, asmall farm, two greenhouses (45 and 88 m2 ) with
automatic sprinklers, two open aquariums (30 and 50t) and several smaller tanks. The laboratory also
includes a building with office, storage and workspace.
6. Plant Cell Laboratory
Autotrophic and heterotrophic culture devices andare equipped for experimental cultures of plant andmicrobial cells. A facility for preparation of plant
cell cultures including an aseptic room with cleanbenches, is also provided.
7. Laboratory of Stress-Resistant Plants
This laboratory was founded to study transgenic
plants with respect to tolerance toward various
environmental stresses. It is located in the
Agricultural Experimental Station of Nagoya

56

University (30 km from the National Institute for
Basic Biology). The laboratory provides a variety ofgrowth chambers that precisely control theconditions of plant growth and facilities formolecular biological, and physiological evaluationsof transgenic plants. The laboratory is also a base ofdomestic and international collaborations devoted tothe topic of stress-resistant transgenic plants.

IT. Research activities
1. Faculty
The faculty of the Research Support Facility
conducts its own research as well as dcientific andadministrative public services.
(1) Photobiology: Photoreceptive and signal transduction mechanisms of phototaxis of single-celled,
flagellate algae are studied action spectroscopically(Watanabe 1995, In CRC Handbook of Organic
Photochemistry and Photobiology) by measuringcomputerized-videomiceographs of the motile
behavior of the cells at the cellular and subcellularlevels (Erata et al. 1995, Protoplasma). Photoreceptive and signal tranduction mechanisms of algal
gametogenesis are also studied by action
spectroscopy.
(2) Developmental Biology: Notch is an integral cellsurface membrane protein that is known to play a keyrole in developmental cell-cell interactions in
Drosophila, particularly in lateral specification ofneural versus epidermal cell fates, a processdescribed thus far only in invertebrates. It is thoughtto act by a direct signaling pathway rather than
through one of the classical signal transduction
cascades. The mammalian genome is known tocontain three Notch homologues but theirdevelopmental significance is not clear. To
investigate their role in mammalian development, wehave sequenced the murine Notch 2 eDNA,
determined the primary sequence of its protein, andhave investigated its genomic organization. We are
now attempting to produce a mutant in which theankyrin repeat region of Notch 2 is replaced by lacZ.
Analysis of the mutant phenotype will provide us
with insights about the significance of the repeat in
Notch 2 signal transduction in relation to its
developmental importance.
(3) Computational Biology: Efforts to develop new
methodology for sequence analysis have been
continued. A system to predict protein localizationsites from the sequences had been developed and is
now open to the public through the Internet(http://psort.nibb.ac.jp). Sequence motifs are thought
to be good indices of biological functions.
Methodology of protein sequence analysis based onsequence motifs has been studied and is implemented
as a computer program. The product of computerassistance for sequence analysis is being constructedusing the World Wide Web.

NATIONAL INSTITUTE

Many kind of databases become available in recent
years, but almost all of them are those of molecular
information such as nucleic acids or amino acids
sequences. It is also desired to construct a databasefor biological functions like networks of theregulation of gene expression. As a model case,
Bacillus subtilis genome database is constructed torepresent the categorized classification of geneproducts in collaboration with Dr. N. Ogasawara(NAIST) and Human Genome Center, University ofTokyo. Other kind of biological functional databases
including image data are planned to represent theaccumulated knowledge of development, differentiation, and morphogenesis.

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 conducted by visiting scientists including
foreign scientists as well as those in the Institute.
Action spectroscopical studies for various
regulatory and damaging actions of light on livingorganisms, biological molecules, and organic
molecules have been conducted (Watanabe, 1995, In
CRC Handbook of Organic Photochemistry andPhotobiology).

Publication List:
I. Faculty

Erata, M., Kubota, M., Takahashi, T., Inouye, I. and
Watanabe, M. (1995) Ultrastructure andphototactic action spectra of two genera ofcryptophyte flagellate algae, Cryptomonas andChroomonas. Protoplasma, 188,258-266.
Higuchi, M., Kiyama, H., Hayakawa, T., Hamada, Y.
and Tsujimoto, Y. (1995) Differential expressionof Notch1 and Notch 2 in developing and adultmouse brain, Mol. Brain Research, 29, 263-272.
Nakamura, S., Kawanishi, E., Nakamura, S.,
Watanabe, M., and Kojima, M. K. (1995) A newparalyzed
flagellamutant,OC-10,
in
Chlamydomonas
reinhardtii thatcan bereactivated with ATP. Phycol. Res., 43, 65-69.
Watanabe, M. (1995) Action spectroscopy
photomovement and photomorphogenesis spectra.
In "CRC Handbook of Organic Photochemistry
and Photobiology", (Edited by B. Horspool and P.S. Song), CRC Press, Boca Raton, pp. 1276-1288.
II. Cooperative Research Program for the Okazaki
Large Spectrograph
Erata, M., Kubota, M., Takahashi, T., Inouye, I. and
Watanabe, M. (1995) Ultrastructure andphototactic action spectra of two genera ofcryptophyte flagellate algae, Cryptomonas andChroomonas. Protoplasma, 188,258-266.
lseki, M. and Wada, S. (1995) Action spectrum in
ultraviolet region for phototropism of Bryosis
rhizoids. Plant Cell Physiol. 36, 1033-1040.

FOR BASIC BIOWGY

Miyoshi, N., Kondo, T., Kubota, M., lshiguro, K.,
Watanabe, M. and Fukuda, M. (1995) Effect ofmolecular formation of photosensitization forgeneration of active oxygen species. Photomed.
Photobiol. 16, 79-82.
Nakamura, S., Kawanishi, E., Nakamura, S.,
Watanabe, M., and Kojima, M. K. (1995) A newparalyzed
flagellamutant,OC-10,
in
Chlamydomonas reinhardtii that can bereactivated with ATP. Phycol. Res., 43, 65-69,
Takano, J.-1., Koizumi, H., Ohkawara, A., Kamo, N.
and Ueda, T. (1995) Ultraviolet action spectra for
peroxide generation in human and pig epidermail
keratinocytes loaded with dihydrorhodamine 123.
Arch Dermatol. Res. 287,321-325.
Torikai, A., Chigita, K., Okisaki, F. and Nagata, M.
(1995)
Photo-oxidative
degradationofpolyethylene containing flame-retardant agents bymonochromatic light. J. Appl. Polym. Sci. 58, 685690.
Torikai, A., Hattori, A. and Eguchi, T. (1995)
Wavelength effect on the photoinduced reaction ofpolymethylmethacrylate. J. Polymer Sci. :Part A:
Polymer Chem. 33, 1867-1871.
Torikai, A., Kobatake, T., Okisaki, F. and Shuyama,
H. (1995) Photodegradation of polystyrene
containing flame-retardants: wavelength sensitivity
and efficiency of degradation. Polymer Degradn.
Stab. 50, 261-267.
Torikai, A. and Mitsuoka, T. (1995) Electron spinresonance studies of poly(methyl methacrylate)irradiated with monochromatie light. J. Appl.
Polym. Sci. 55, 1703-1706.
Ueda, T. (1995). Spatiotemporal dynamics ofglycolysis and cellular metabolism: toward
intelligence by nonlinear chemical processes.
Sensors Materials 7, 147-157.
Watanabe,M. (1995). Action spectroscopy
photomovement and photomorphogenesis spectra.
In "CRC Handbook of Organic Photochemistry
and Photobiology", (Edited by B. Horspool and P.S. Song), CRC Press, Boca Raton, pp. 1276-1288.

57

58

NATIONAL INSTITUTE

RADIOISOTOPE FACILITY(Managed by NIBB)
Head: Takashi Horiuchi
Associate Professor: Kazuo Ogawa
NIBB Postdoctoral Fellow: Hiroyuki Takai
Technical Staffs: Kazuhiko Furukawa (Radiation
Protection Supervisor)
Yosuke Kato (Radiation Protection
Supervisor)
Yoshimi Matsuda (Radiation Protection
Supervisor)

This Facility consists of a main center where avariety of radioisotopes such as 22 Na, 125 I, 32 P, 3H and
14
C are handled as well as various species of y-ray
emitting nuclides and two subcenters, one placed in
the NIBB and the other in the NIPS. At thesubcenters, only a limited variety of radioisotopes
such as 35 S and 32 P are processed. The members of
the Radioisotope Facility are engaged in maintaining
and controlling both the center and subcenters, andprovide users an appropriate guidance forradioisotope handling.
In 1995, the new system for the going in and out
the controlled area was introduced. ID card has been
issued to each registered individual and nonregistered persons are allowed to enter the controlled
area. In this system, when he or she goes in and out
the controlled area, his or her names, times, andplaces are automatically recorded by a computer.
Furthermore, the renewal of the radiation monitoring
system and the radioactive contamination assessment
system in this Facility has been supported by thesecondary supplementary budget of the Ministry of
Education, Science and Culture and is going on until
the end of Mach, 1996. In these refreshed systems,
the radioactivities of air, and draining in thecontrolled area of the three places will be monitored
by a computer for 24 hours. The third subcenter ofthis Facility will open in the next spring (1997) in thebuilding of the Laboratory of Gene Expression andRegulation that is now under construction.
The teaching staffs are also engaged in their own

Aresearch on the structure and function of dynein
motor protein. Dyneins are a group of microtubuleactivated ATPases that serve to convert chemicalenergy into mechanical energy. They have been
divided into two large subgroups, namely, theaxonemal and cytoplasmic dyneins. Figure 1 shows
the localization of two dyneins in the outer arms
(Ogawa et a!., 1977) and the mitotic apparatus(Mohri et a!., 1976) that have been visualized by thesame antibodies directed against the motor domain ofaxonemal dynein (fragment A).
The native dyneins are very large. They range inmolecular mass up to 1 to 2x10 6 daltons and they arecomplex proteins. Each dynein contains two or three
heavy chains (HCs) with ATPase activity, whichrange in molecular mass up to 500 kDa. The motor
activity of dynein is associated with these chains.
Some functional differences have been reported
between HCs of outer-arm dynein. Sea urchin outerarm dynein is a heterodimer of HCs (a and f3) and at
least the f3-HC is able to induce gliding of
microtubules in vitro. The a-HC might amplify thefunction of f3-HC and it has been reported to have no
motile activity. After the first cloning of f3-HC fromsea urchin ciliary axonemes (Gibbons et a!., 1991;
Ogawa, 1991), the sequences of HCs of axonemal
and cytoplasmic dyneins from a variety of organisms
were determined in their entirety. Without exception,
all the HCs cloned to date contain four P-loop (ATPbinding) sequences in the midregion of the molecule.
Thus, they can be classified as a four P-loop family.
The outer-arm dyneins contain two or threeproteins that range in molecular mass from 70 to 120
kDa and copurify with HCs. ICs of sea urchin outerarm dynein are abbreviated as IC1, IC2, and IC3.
Those of Chlamydomonas are called IC78 and IC69,
and ICs of cytoplasmic dynein are called IC74.
Chlamydomonas IC78 and IC69 were cloned by
Wilkerson et a!. (1995) and Mitchell and Kang
(1991),respectively. The sequences of sea urchin IC2
and IC3 were determined by Ogawa et a!. (1995).

B

Fig. I. Localization of dynein in sea urchin sperm axoneme (A) and a cleaving egg (B).

FOR BASIC BIOLOGY

59

IC3
IC69
IC2
IC74
Fig .2. Relative positions of WD repeats (filled boxes) in sea urchin outer-arm dynein IC2 and IC3, Chlamydomonas outerarm dynein IC78 and IC69, and rat cytoplasmic dynein IC74. Colours mean subclass of ICs.
IC1 has recently been cloned (Ogawa, accession
number D63884). Rat brain IC74 was cloned by
Paschal et a!. (1992). Thus, all the ICs found in theaxonemal and cytoplasmic dyneins of the modelorganisms used for studies of dynein function havebeen completely sequenced. Comparison of aminoacid sequences of IC2 and IC3 with those of IC78
and IC69 and with that of IC74 showed that,
although all five ICs are homologous, IC2 is much
more closely related to IC78, and IC3 is much moreclosely related to IC69, than either sea urchin chainor either Chlamydomonas chain is related to eachother. Regions of similarity between all five ICs arelimited to the carboxy-terminal halves of themolecules. Similarity are due primarily toconservation of the WD repeats in all of these chains.
The WD repeats are involved in protein-protein
interactions in a large family of regulatory molecules(Neer et al. , 1994). The relative positions of WD
repeats in these chains are shown schematically in
Fig. 2.
A parsimony tree for these chains (Ogawa et a!. ,1995) shows that, although the carboxy-terminal
halves of all of these chains contain WD repeats, thechains can be divided into three distinct subclasses(IC3 plus IC69, IC2 plus IC78, and IC78). By
contrast, sea urchin IC1 is not a member of the WD
family . Sequence analysis showed that IC1 consistsof a thioredoxin-like (TRXL) sequence and three
nucleoside
diphosphatekinase-like(NDKL)
sequences. IC1 might play a dynamic role in flagellar
bending and/or wave propagation.

Publication List:
Ogawa, K., Kamiya, R., Wilkerson, C. G. andWitman, G. B. (1995) Interspecies conservation ofouter arm dynein intermediate chain sequences
defines two intermediate chain subclasses. Mol.
Bioi. Cell 6, 685-696.

Shimizu, H., Majima, T., Takai, H., Inaba, K. andTomie, T. (1995) Morphological changes ofwrasse sperm axoneme after their motility
initiation observed with use of Atomic ForceMicroscopy. SPIE 2384, 45-51.
Takai, H. and Morisawa, M. (1995) Change inintracellular K concentration caused by external
osmolarity change regulates sperm motility ofmarine and freshwater teleosts. J. Cell Sci. 108,1175-1181.

60

NATIONAL INSTITUTECENTER FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head of Center: Masaharu Noda
Technical Staffs: Akio Murakami
Yukiko Kabeya
Yumiko Makino
Sonoko Ohsawa
Takeshi Mizutani

The center provides analytical instruments forbiological and biophysical studies. These
instruments consist of the following four groups andare supported by experienced technical staffs. Eachgroup is equipped with instruments for general use
as listed below.
·
Instruments for Protein and Gene Analysis
Amino Acid Analyzer
HITACHI 835
Automatic Plasmid Isolation System
KURABO PI-10m:
DNA Sequencers
ABI 370A, 373A-36
DNNRNA Synthesizers
ABI 381A, 392
Nucleic Acid Extractor
ABI340A
Peptide Synthesizers
ABI 430A, 431A, 432A
Protein Sequencers
ABI 470A, 473A, 492
Instruments for Chemical Analysis
Capillary Electrophoresis
ABI270A
Gas Chromatographs
SHIMADZU GC-7APTF, GC-14APFSC
Glycoprotein Analysis System
TAKARA Glyco-Tag
High Performance Liquid Chromatographs
SHIMADZU LC-lOAD, 6AD
Preparative Electrophoresis System
ABI 230A
Preparative Ultracentrifuges
BECKMAN LS-80, L5-75
Table-top Ultracentrifuges
BECKMAN TL-100
Micro Preparative System
PHARMACIA SMART System

Instruments for Physical and Spectroscopic
Analysis
Atomic Absorption Spectrophotometer
PERKIN-ELMER 603
Dual Wavelength Spectrophotometer
HITACHI557
EPR Spectrometer
BRUKER ER-200D
GC/Mass Spectrometer
JEOLDX-300
Inductively Coupled Plasma Spectrometer
SEIKO SPS 1200A
Infrared Spectrophotometer .
JASCOA-302
Laser Raman Spectrophotometer
JASCO R-800
Light Scattering Photometer
CHROMATIX KMX-6DCNMR Spectrometer
BRUKER AMX-360wb
Spectrofluorometers
HITACHI 850
SIMADZU RF-5000
Spectrophotometers
HITACHI 330
PERKIN ELMER Lambda Bio
Spectropolarimeter
JASCOJ-40S
DELFIA Research Fluorometer
PHARMACIA
Insttuments for Microscopic and Image Analysis
Bio Imaging Analyzers
FUJIFILM BAS 2000
Imaging Analysing Systems
KONTRON IBAS-1 & II
Electrophoresis Imaging System
PDI The Discovery Series
Microscopes
CARL ZEISS Axiophot, Axiovert
Microscope Photometer
CARL ZEISS MPM 03-FL
Microdensitometer
JOYCE LOEBL 3CS

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