--._.....

---NATIONAL INSTITUTE FOR BASIC BIOLOGY

Wild typeTransformed

0.1 M NaCIANNUAL REPORT

1996

The cover photograph indicates the enhanced tolerance tosalt stress of seedlings of transgenic Arapidopsis, see p. 43.

J

CONTENTSINTRODUCTION........................................................................................................................................... .

ORGANIZATION OF THE INSTITUTE

3

GRADUATE PROGRAMS

4OF ICE OF DIRECTOR

5

volution of photoysnthesis and the mechanism of electron transfer ...................................................

5

rimary processes of photosynthesis were investigated from various points of view;
hylogeny or systematics to ultrafast spectroscopy ...............................................................................

6ole of proteasome in meiotic cell division

6

DEPA TMENT OF CELL BIOROGY

9

D~ISION OF CELL MECHANISMS

10...................................................................................................... .

I. Regulation at the level of protein transport to microbodies duringI
the microbody transition

10

II. Two forms of hydroxypyruvate reductase might be producedby alternative splicing...................................................................................................................... .

12

III. Membrane protein a-TIP of protein-storage vacuoles

12

IV. A rapid increase in the level of binding protein (BiP) is accompanied
by synthesis and degradation of storage proteins in pumpkin cotyledons

12

V. A vacuolar processing enzyme responsible for conversion of proprotein
precursors into their mature forms ....................................................................................................

12

VI. Role of molecular chaperones in organelle differentiation

13

DIVIDION OF BIOENERGETICS

15

DIVISION OF CELL PROLIFERATION (ADJUNCT)18

I. Genes encoding farnesyl cysteine carboxyl methyltransferase in fission
yeast and Xenopus ................................................................................................................................

18

II. Phosphorylation of RNA-binding protein controls cell cycle switch frommitotic to meiotic in fission yeast ... ............................ .. .. ................ ................... ......... ......................

18

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)20

DIVISION OF CELL FUSION (ADJUNCT)

21..............................................................................................

DEPARTMENT OF DEVELOPMENTAL BIOLOGY ......................................................... 23
DIVISION OF REPRODUCTIVE BIOLOGY
I. Endocrine regulation of oocyte differentiation, growth and maturation

24
24

II. Endocrine regulation of male germ cell development and maturation....... ... ... .. .. .. ..... .. .. ..... .. ... .. .. ..

III. Endocrine regulation of gonadal sex differentiation

26
26...............................................................................................

29

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

29

II. Genes associated with abdominal leg development

31

DIVISION OF CELL DIFFERENTIATION32

DIVISION OF MORPHOGENESIS

I. In vitro culture system of iris pigmented epithelial cells for molecularanalysis of lens regeneration ..............................................................................................................

32

II. Role of the mi gene in differentiation and transdifferentiation of thepigmented epithelial cells ... ..... ............................................................. .. .. .. .. ................... .. .................

32

III. Analysis of silver quail mutant

32

IV. Isolation of a novel chick homologue of Serrate and its coexpression
with C-Notch-1 in chick development ..............................................................................................

33

DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)35

I. Regulatory factors involved in the sugar-inducible expression of plant genes

35

II. Mutants of Arabidopsis thaliana with altered patterns of the sugar-inducible
gene expression

35

III. Derepression from negative regulation in the sugar-inducible expression of~-amylase gene of Arabidopsis thaliana ...........................................................................................

35

IV. Screening of mutants of Arabidopsis thaliana with defective
growth and development ... .. .. ... .. ........ ... ....... ... ... ... .. ... ... ..... .. .. .. .. .. .. .. .. ..... ..... .. .. .. .. .. ... ... ... .. .. .. ............

36

DEPARTMENT OF REGULATION BIOLOGY37

DIVISION OF MOLECULAR NEUROBIOLOGY

38

I. Retinotectal projection map and synapse formation

38

II. Receptor-like protein tyrosine phosphatases and brain development

38

Ill. Gene targeting of Na-G, a voltage-gated sodium channel expressed in glial cells

40
42

DIVISION OF CELLULAR REGULATION

I. The temperature-regulated expression of genes for acyl-lipid desaturases
in a cyanobacterium ............ .. ..... .......................... ..................................................... .. ........... .............

42

II. Localization of acyl-lipid desaturases in cyanobacterial cells

42..........................................................

III. The importance of polyunsaturated membrane lipids in photosynthesis........................................

43

IV. Acclimation to heat of the photosynthetic machinery

43

V. Genetic enhancement of stress tolerance in Arabidopsis thaliana and in rice
plants by genetic engineering that allowed the biosynthesis of glycinebetaine

44

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT)......................

45

I. Structural organization of photosystem II reaction center45

II. Dynamic aspects of molecular organization

46

DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)........................................................

48

I. Connectin can function as an attractive target recognition molecule48

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

48

III. msh, a homeobox containing gene essential for neural and muscular development

48.. .. .................

IV. M-spondin and G-spondin: a novel gene family of secreted molecules

48

LABORATORY OF GENE EXPRESSION AND REGULATION51

DIVISION OF GENE EXPRESSION AND REGULATION I

52

I. Identification and characterization of mutable alleles in the Japanese morning glory

52

II. Identification of the mutable alleles in the common morning glory

52

DIVISION OF GENE EXPRESSION AND REGULATION II

53

I. Analysis of E.coli recombinational hot spots

53

II. Atomic structure and functional model of E. coli replication terminatorprotein complexed with Ter DNA ......................................................................................................

53

Ill. The E. coli genome project54

DIVISION OF SPECIATION MECHANISMS I

56

I. Evolution of cytokine receptor families in the immune and nervous systems

56

II. Gene expression and cerebellar long-term plasticity

56

DIVISION OF SPECIATION MECHANISMS II

I. Reproduction of animals... .... .. ...................... ............... .... .......... ................ ..... ................... .. ... ... .... .. .. ..

II. Morphological Evolution in plants

RESEARCH SUPPORT

58
58

59
60

TECHNOLOGY DEPARTMENT.............................................................................................................

61..... .. .. .. ... .. .. ..... .. .. ..... .... .. .. ..... .. ..... .. .. ... .. ... ............ .. .. .. .. .. .. .. ... .. ..... .. ... ........ .. ....... .. ... .. .. .. ... .. .. .. ..

61

RESEARCH SUPPORT FACILITY
I. Facilities

58

II. Research activities... .. ... .. .. .. .. .. .. .... .... ............ ... ..... .. ..... .. .. ... .. .. ..... .. .. ..... .. ... ....... ... ....... .. .. .. ... .... .. ..........

62

RADIOISOTOPE FACILITY (Managed by NIBB)64

CENTER FOR ANALYTICAL INSTRUMENTS (Managed by NIBB)

66

INTRODUCTION
We present here the 1996 Annual Report describing
the research activities of the National Institute for Basic Biology (NIBB) during the past year. The NIBB isa government supported basic research institute established in 1977. It aims to stimulate and promote thebiological sciences, by conducting first-rate research
as a center of excellence, and by cooperating withother universities and research organizations. The Institute concentrates on the study of cellular functions,reproduction and development, neuronal and environmental control, and gene expression and regulation in
eukaryotic organisms at the molecular level.
During the past year, a new division, Speciation
Mechanisms II, was added to the Laboratory of Gene
Expression and Regulation and Drs. Ken-ichirou
Morohashi and M. Hasebe were appointed as Professor and Associate Professor of the new division, respectively. At the same time, the construction of a
new building for the Laboratory of Gene Expression
and Regulation has been made efficiently and wascompleted at the end of 1996. As a result, the Laboratory has been completely established and the number of divisions at the NIBB has now reached 17 in
total.
Including the above-mentioned appointments, theturnover of personnel has been high as usual. In April ,Drs. Shigeru Iida and Yoshinori Ohsumi were appointed as Professor of the Division of Gene Expression and Regulation I and Professor of the Division of
Bioenergetics, respectively. Professor Yoshiki Hotta(University of Tokyo) together with Associate Professor H. Okamoto (Keio University) of the AdjunctDivision of Cellular Communication completed theirterm of office at the end of March and were followed
by Professor Ritsu Kamiya (University of Tokyo).
Professor Goro Eguchi of the Division of Morphogenesis was appointed as the President of Kumamoto
University and left the NIBB in November. Drs. T.
Yoshimori, M. Mimuro, M. Maejima (Nagoya
University), T. Takai (Osaka University), T. Hattori(Mie University) and H. Wada (Kyushu University)
were appointed as Associate Professor of the Divisionof Bioenergetics, the Division of Gene Expression
and Regulation I, the Adjunct Division of Cell Fusion,
the Adjunct Division of Developmental Biology andthe Adjunct Division of Behavior and Neurobiology,
respectively. Associate Professor H. Iida moved toTokyo Gakugei University. Furthermore, we newly
appointed 9 research associates, 6 institute research
fellows and 2 research assistants, while 4 research
associates and 6 institute research fellows moved toother institutions or positions.
The NIBB plays important roles as a national andinternational center for biological research and is responsible for conducting research projects in cooperation with research groups of various universities andinstitutes. As a part of such cooperative activities, the
NIBB carried out Special Programs which are cur-rently directed to "Adaptation and Resistance to Environment" and "Trans-differentiation of Tissue Cells."
Based on these programs, the NIBB held the 36th and37th Conferences entitled as "Stress Signaling andStress Responses in Plants" (organized by Professor
Norio Murata) and "Approaches to Gene Function
Controlling Developmental Processes" (organized byProfessor G. Eguchi), respectively.
In addition, the Institute sponsors symposia andstudy meetings on current topics at the interdisciplinary level by inviting leading scientists in various
related fields, both nationally and internationally. In
1996, an International Symposium "New Prospects of
Photobiology and the Future Plan of the Okazaki
Large Spectrograph" was held at the NIBB , sponsoredby the Ministry of Education, Science, Sports andCulture. The NIBB also provided a training course inbioscience for young investigators and participated in
the International Science School for high school students of Asian-Pacific countries.
At the beginning of October, the Director-General
together with Professor N. Murata visited Canberra tosign a "Memorandum of Understanding between the
NIBB and the Australian National University" in order to promote international cooperation between thetwo institutions. In turn, in October and November,
Professor Howard A. Bern of the University of California, Berkeley, and Professor C. Barry Osmond of
the Australian National University visited Okazaki toconduct a peer review of the activities and the future
plan of the NIBB. We welcome any suggestion and2

NATIONAL INSTITUTE

criticism concerning research activities of the NIBB
from the outside.
Finally, I would like to congratulate Professor I.
Takeuchi, the President of Okazaki National ResearchInstitutes and the former Director-General of the
NIBB, for being awarded the Minakata Kumagusu
Prize for his pioneer work in developmental biologyof the cellular slime mold. Professor (adjunct) M.
Takeichi was awarded the Japan Academy Prize andthe Uehara Prize for his excellent work on cadherins.
In addition, Dr. H. Mohri was awarded a Purple Ribbon Medal for his contribution to reproductive biology.
Hideo Mohri, D. Sc.
Director-General

3

FOR BASIC BIOLOGYORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology, NIBB is apart 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, the Advisory Council and Steering Council. The Advisory
Council is made up of distinguished scholars representing various fields of science and culture, and advises the Director-General on the basic policy of theInstitute. The Steering Council is made up of professors of the Institute and an equal number of leading
biologists in Japan outside NIBB, and advises the
Director-General on the scientific activities of theInstitute. The Council advises on faculty appointments and on the Institute's annual budget as well as
its future prospect.

staffed 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 and helps topromote research activities of each division and also
to maintain the research resources of the Institute. The
Department also undertakes the technical education ofits staff.

Research Support Facility
The research support facility of the NIBB consistsof the Large Spectrograph Laboratory, Tissue and Cell
Culture Laboratory, Computer Laboratory, Plant Culture Laboratory, Plant Cell Culture Laboratory, Experimental Farm, and Laboratory of Stress-Resistant
Plants. In addition, seven facilities are operated jointlywith the NIPS; they consist of the Radioisotope Facility, Electron Microscope Center, Center for Analytical
Instruments, Machine Shop, Laboratory Glassware
Facility, Animal Care Facility, and Low-Temperature
Facility.

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 laboratory organized into 17 divisions.
Each division has its own research project and is

Department/Laboratory

Department of Cell Biology

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 space of
10,930 m2 • Two-thirds of the space was completed in1982 and the remaining third in June, 1983. The
buildings which house the research support facility
were also completed in June, 1983. A building forLaboratory of Gene Expression and Regulation (2,577m2) was newly built in December, 1996.

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

Department of Developmental Biology

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

Department of Regulation Biology

Molecular Neurobiology- { Cellular Regulation
Biological Regulation (adjunct)Behavior and Neurobiology (adjunct)

Gene Expression and Regulation I{
Laboratory of Gene Expression and Regulation
GS en~ E.xpreMssiohn a~d Relgulation II
pectatwn ec amsms
Speciation Mechanisms II

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 a three year Ph.D. course. Those whohave completed a master's course in a university or
are qualified at an equivalent or higher level 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 andinstitutions 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 ltoh
Research Associates: Mamoru Mimuro
Katsunori Aizawa (on leave)
Mika Tokumoto

Evolution of photoysnthesis and the mechanism ofelectron transfer
Shigeru Itoh
We study the evolution of photosyntheis focusing
on the mechanism of solar energy conversion.
Anoxygenic photosynthesis of bacteria seems to have
evolved in the Precambrian Earth just after the evolution of life. Plant-type oxygen-evolving photosynthesis, then, was established in cyanobacteria 3500-2700
million years (Ma) ago and increased atmospheric
oxygen, judging from the microfossils in Stromatolites and the oxidized iron sediments. Symbiosis of
cyanobacteria inside larger cells seems to have produced first plant about 2000 Ma ago.
What can we study to explore the past? We takethree approaches (1) Survey of electron transfer
mechanisms in photosyntheic reaction center (RC)pigment-protein complexes. We replaced quinones
and chlorophylls inside RC to see what happens. We
use the ps-ns laser and ESR spectroscopies at 4-300 K

in the quinone-replaced RCs and found that both theplant and bacterial RCs are highly optimized (lwaki etal , Itoh et al. and Baba et al). The result that was thefirst evidence of the energy-gap-dependent ultra-rapid
electron tunneling in plant, also teaches us the designof prototype RC. (2) Site-directed mutagenesis to construct a prototype of electron transfer protein. We
modified a ferredoxin that functions in N2-fixationand found the conserved motif to be indispensable forthe required extremely strong reducing power (Saeki
et a!). (3) Survey of new photosynthesis. We performed comparative studies of photosynthesis in
purple, green sulfur, hellio-, cyano-bacteria andplants. Our topic in 1966 was the discovery of bacteria that undergo photosynthesis with Zn-bacteriochlorophyll (Wakao et a!). The finding was a surprise by
itself, and also questions for the reason of ubiquitoususe of Mg-chlorophylls in ever known plant and bacterial photosynthesis. We are now studying anotherorganism that undergoes oxygen-evolving photosynthesis with a completely new type of chlorophyll.

Fig. 1. Space-filling (lower) and wire-frame (upper) models of R. capsulata ferredoxin (FdxN). Pink-colored moiety in native
FdxN (left) was altered (right) by site-directed mutagenesis. The mutation exposed the Fe-S cluster (yellow and red) and decreased
its reducing power.

NATIONAL INSTITUTE

6

Primary processes of photosynthesis were
investigated from various points of view;
phylogeny or systematics to ultrafast spectroscopy
Mamoru Mimuro
The origin of oxygenic photosynthetic organisms is
now hypothesized to be symbiosis of two different
types of photosynthetic bacteria. However, it is very
hard to reproduce process(es) of symbiosis in a laboratory. We started to study this process by using marine phytoplankton, dinoflagellates. We think that thefirst step of symbiosis is recognition of counterpart
for symbiosis, thus we tried to find chemicalcompound(s) responsible for recognition. We foundthat some dinoflagellates contain lectin and someshowed the lectin-receptor activity (Hori et al.). These
compounds might be included in the recognition process.
Chi dimer structure is found both in reaction centers
and in antenna. We have already shown that thisdimerization induced a systematic decrease in an intensity of magnetic circular dichroism (MCD). We
extensively analyzed this phenomenon on bacterial
reaction center, green plant reaction center and also
bacterial antenna (Kobayashi et al.). Furthermore, weapplied this method to artificially formed chlorophylla epimer (so called Chi a'). The MCD intensity was
also half of that of monomer, indicating that decreasein the MCD intensity is universal phenomenon (Obaet al.). Spectroscopic properties of Chi a' dimer indicated a stacked face-to-face dimer structure.
Excited state dynamics of carotenoids were analyzed by the femto-second up-conversion method. Relaxation processes from the second excited singlet
state (S 2 state) were shown for the first time to follow
the energy gap law of internal conversion (Mimuro etal., J. Am. Chern. Soc. in press). We applied thismethod to the peridinin-chlorophyll-complex (PCP)
isolated from a dinoflagellate, and determined theenergy transfer pathway from peridinin to Chi a; fromthe S 1 state of peridinin to the S 1 state of Chi a(Akimoto et al.). This is unique in the transfer pathway in algal photosynthesis. We proposed that thistransfer pathway is realized by a unique chemical
structure of peridinin.
Molecular structure of antenna system in a green
photosynthetic bacterium, Chlorojlexus aurantiacus
was proposed; an array of dimer units which are arranged in a spoke-like manner in a rod structure ofchlorosomes (Mimuro et al.).

Role of proteasome in meiotic cell division
Mika Tokumoto
All eukaryotic cells, from yeast to human, containlarge protease complexes called proteasomes, ofwhich the 20S and 26S proteasomes are the two major
types. Recent evidence indicates that proteolysis may
play an important role in the regulation of the meiotic
and mitotic cell cycles. Inhibitor studies suggest that
proteasomes are involved in meiotic maturation ofanimal oocytes. To investigate the roles of proteasomes in meiotic maturation polyclonal antisera
against 20S proteasomes was used to examinechanges of components of proteasome during oocyte
maturation and early development of Xenopus laevis.
Although no significant changes in the proteins common to 20S and 26S proteasomes were observed during oocyte maturation and early development, theamount of a unique 48 kDa protein (p48) componentof the 26S proteasome decreased during oocyte maturation to the low levels seen in unfertilized eggs. p48
levels remained low in fertilized eggs until after the
midblastula transition. These results show that at least
one component of 26S proteasomes changes duringoocyte maturation and early development, and suggestthat alterations in proteasome function may be important for the regulation of developmental events, suchas the rapid cell cycles, of the early embryo. Further
molecular characteristics of p48 will be required todetermine the function of p48.

Publication List:
Akimoto, S. (Hokkaido Univ.), Takaichi, S. (Nippon
Medical School), Ogata, T. (Kitasato Univ.),
Nishimura, Y. (Hokkaido Univ.), Yamazaki, I.
(Hokkaido Univ.) and M. Mimuro (1996) Excitation energy transfer in carotenoid-chlorophyll protein complexes probed by femtosecond fluorescencedecays. Chern. Phy. Lett., 260, 147-152.
Baba, K., Itoh, S., Hastings, G. and Hoshina, S.
(1996) Photoinhibition of photosystem I electrontransfer activity in isolated photosystem I preparations with different chlorophyll contents. Photosynt.
Research 47, 121-130.
Hori, K. (Hiroshima Univ.), Ogata, T. (Kitasato
Univ.), Karniya, H. (Kitasato Univ.) and M.
Mimuro (1996) A preliminary evidence that lectinsugar interaction exists in the initial recognition
process in symbiosis involving dinoflagellates. J.
Phycol., 32, 783-790.
Ishijima, S., Kubo-lrie, M., Mohri, H. andHamaguchi, Y. (1996) Bidirectional sliding of doublet microtubules of sea urchin sperm axonemes. J.
Muscle Res. Cell Motil. 17, 287-288.
Ishijima, S., Kubo-Irie, M., Mohri, H. andHamaguchi, Y. (1996) Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin
sperm axonemes. J. Cell Sci. 109, 2833-2842.
ltoh, S., Iwaki, M., Torno, T. and Satoh, K. (1996)
Dibromo-thyomoquinone (DBMIB) replaces thefunction of QA at 77 K in the Isolated Photosystem
II reaction center (D1-D2- cytochrome b559) complex: Difference spectrum of P680+(DBMIB-) state.
Plant and Cell Physiology 37, 833-839.
Iwaki, M., Kumazaki, S., Yoshihara, K., Erabi, T. andltoh, S. (1996) Delta G0 dependence of the electrontransfer rate in photosynthetic reaction center ofplant photosystem I: Natural optimization of reaction between chlorophyll a (AO) and quinone. J.
Phys. Chern. 100, 10802-10809.

FOR BASIC BIOLOGY

Kawai, H. (Kobe Univ.), Nakamura, S. (Toyama
Univ.), Mimuro, M., Furuya, M. (Hitachi) and
Watanabe, M. (NIBB, Large spectrograph) (1996)
Microspectrofluorometry of autofluorescent flagellum in phototactic brown algal zoids. Protoplasma, ·191, 172-177.
Kobayashi, M. (Tohoku Univ.), Wang, Z.-Y. (Tohoku
Univ.), Yoza, K. (Tohoku Univ.), Umetsu, M.
(Tohoku Univ.), Konami, H. (Tohoku Univ.),
Mimuro, M. and Nozawa, T. (Tohoku Univ.) (1996)
Molecular structures and optical properties of aggregated forms of chlorophylls analyzed by meansof magnetic circular dichroism. Spectrochim. Acta,
51A, 585-598.
Mimuro, M., Nishimura, Y. (Hokkaido Univ.),
Yamazaki, I. (Hokkaido Univ.) Kobayashi, M.
(Tohoku Univ.), Wang, Z.-Y. (Tohoku Univ.),
Nozawa, T. (Tohoku Univ.), Shimada, K. (Tokyo
Metropol. Univ.) and Matsuura, K. (Tokyo
Metropol. Univ.) (1996) Energy transfer processes
in chlorosomes of a green photosynthetic
bacterirum, Chloroflexus aurantiacus. Photosynthe.
Res., 48, 263-270.
Oba, T. (Univ. Tokyo), Watanabe, T. (Univ. Tokyo),
M. Mimuro, Kobayashi, M. (Tsukuba Univ.) andYoshida, S. (Univ. Tokyo) (1996) Aggregation ofchlorophyll a' in aqueous methanol. Photochem.
Photobiol., 63, 639-648.
Ogawa, K. and Mohri, H. (1996) A dynein motor superfamily. Cell Struct. Funct. 21, 343-349.
Ogawa, K., Takai, H., Ogiwara, A., Yokota, E.,
Shimizu, T., Inaba, K. and Mohri, H. (1996) Is
outer arm dynein intermediate chain 1 multifunctional? Mol. Bioi. Cell 7, 1895-1907.
Saeki, K., Tokuda, K.-1., Fukuyama, K., Matsubara,
H., Nadamame, K., Go, M. and Itoh, S. (1996) Sitespecific mutagenesis of Rhodobacter capsulatus
ferredoxin I, FdxN, that functions in nitrogen fixation. J. Biological Chemistry 271, 31399-31406(1996).
Wakao, N., Yokoi, N., Isoyama, N., Hiraishi, A.,
Shimada, K., Kobayashi, M., Kise, H., Takaichi,M., lwaki, M., Itoh, S. and Sakurai, Y. (1996) Discovery of natural photosynthesis using Zn-containing bacteriochlorophyll in an aerobic bacterium
Acidophilium rublum. Plant and Cell Physiology
37, 889-893.

78NATIONAL INSTITUTE

FOR BASIC BIOLOGY9DEPARTMENT OF CELL BIOLOGY

Chairman: Mikio Nishimura

The department consists of two regular divisions and three
adjunct divisions. One regular division and one adjunct division were started in 1996. The department conducts studies onmolecular dynamics of the cell in higher plants and animals
such as organelle differentiation, autophagy, cell motility andexpression of cell cycle genes and odrant receptor genes.

10

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor: Mikio Nishimura
Associate Professor (adjunct): Masayoshi Maeshima
Research Associates: Makoto Hayashi
Ikuko Hara-Nishimura
Tomoo Shimada
JSPS Postdoctoral Fellow: Tetsu Kinoshita
NIBB Fellow: Akira Kato
Graduate Students: Nagako Hiraiwa
Shoji Mano
Hiroshi Hayashi
Kenji Yamada
Daigo Takemoto 1I
Yuki Tachibe 21
Technical Staffs: Maki Kondo
Katsushi Yamaguchi
Monbusho Foreign Scientist: Luz Marina Melgarejo 31
Visiting Scientists: Yasuko Koumoto
Miwa Kuroyanagi
n from Nagoya University
from Hiroshima University
31 from Colombia National University

21

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, existing organelles
may undergo a transformation of function, while otherorganelles may degenerate. Because the dynamictransformation of organellar function (differentiationof organelles) is responsible for the flexibility of differentiation events in higher plant cells, the elucidation of regulatory mechanisms underlying organelletransformation are currently being studied in this division.

I. Regulation at the level of protein transport tomicrobodies during the microbody transition
Dramatic metabolic changes which underlie theshift from heterotrophic to autotrophic growth occur
in greening associated with seed germination.
Accompanying these metabolic changes, many
constitutive organelles are functionally transformed.
For example, etioplasts differentiate into chloroplasts
and mitochondria acquire the ability to oxidize
glycine. Glyoxysomes which are microbodies
engaged in the degradation of reserve oil via ~­oxidation and the glyoxylate cycle, are transformed
into leaf peroxisomes that function in several crucial
steps of photorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during
the greening of pumpkin cotyledons, the reverse
microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Microbody enzymes
function after their transport to microbodies. Since the
enzyme compositions and functions of glyoxysomes
and leaf peroxisomes differ from each other, it was
likely that the two types of microbodies possess
different machineries for protein import.
Microbody proteins are synthesized in the cytosol

on free polysomes and are transported posttranslationally into microbodies. Two types of targeting signals to microbodies have been reported. One
type of the targeting signal is part of the mature protein. One such signal, the tripeptide Ser-Lys-Leu at
the C-terminal end, has been identified as a targeting
signal. Ser-Lys-Leu and related amino acid sequences
commonly function in mammals, insects, fungi, andplants. Glyoxysomal enzymes, such as malate synthase and isocitrate lyase, and leaf peroxisomal enzymes, such as glycolate oxidase and hydroxypyruvate reductase, contain the targeting signal attheir C-terminal ends.
To characterize the targeting signal, transgenic
Arabidopsis plants that expressed a fusion protein
composed of the C-terminal five amino acids of
pumpkin malate synthase and a bacterial protein, ~­glucuronidase (GUS) were generated. Immunocytochemical analysis of the transgenic plants revealed
that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for the transportof the fusion protein into glyoxysomes in etiolated
cotyledons, into leaf peroxisomes in green cotyledonsand in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was nolonger transported into microbodies when SRL at the
carboxyl terminus was deleted. Transport of proteins
into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of thefusion protein were changed from SRL to SKL, SRM,
ARL or PRL.
A second type of targeting signal involved a cleavable N-terminal sequence. A small group of microbody proteins, such as 3-ketoacyl-coenzyme A (CoA)thiolase, malate dehydrogenase and glyoxysomal citrate synthase, (gCS) are synthesized as precursor proteins with larger molecular masses than those of themature proteins. Each of these proteins has a cleavable presequence at its N-terminal end. Swinkels andcolleagues showed that theN-terminal presequence of
3-ketoacyl-CoA thiolase from rat liver functions as atargeting signal. The N-terminal region of gCS ishighly homologous to those of other microbody proteins that are synthesized as larger precursors. There
are two conserved sequences in their N-terminal regions. One. sequence is RL-X5-HL, first recognized
by de Hoop and Ab. The other sequence,
SXLXXAXCXA, is located at the cleavage site of thepresequence. Transgenic Arabidopsis plants that expressed a fusion protein composed of the N-terminal
region of gCS and GUS, were generated and theirlocalization and processing were characterized by immunological and immunocytochemical analyses (Fig.
1). The fusion protein was transported into functionally different microbodies, such as glyoxysomes, leaf
peroxisomes and unspecialized microbodies, and was
subsequently processed. These observations indicatedthat the transport of gCS is mediated by its aminoterminal presequence and that the transport system isfunctional in all plant microbodies. Therefore, it is11

FOR BASIC BIOLOGY

A

B
24

G

F

A

c
(I)
:::)

Construct

CJI
(I)

0
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Mb

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++

~
CD

......

a:

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~
....
......

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.
• tl

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w

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w

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Mb

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+

+

+

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Mb, microbody; Cyt, cytosol.
Fig. l. Targeting and processing of the chimeric proteins between N-terminal sequence of glyoxysomal citrate synthase (gCS)and ~- g lucuronidase (GUS). (A) Localization of the chimeric proteins in transgenic Arabidopsis plants analyzed by immunoelectron
microscopy. 13-day-old cotyledons were fixed and embedded in LR-white resin. The thin sections were stained with anti-GUS
antibodies. Gold particles indicated the localization of the GUS chimeric proteins in microbodies. Bar in panel ~C42 indicates I
Jlm. (B) Construction of the chimeric proteins. The yellow and green boxes show the consensus sequences which were found in
the N-terminal region of the microbody proteins that contained the cleavable N-terminal presequences. Mutated amino acids areindicated by red letters. (C) Summary of the localization and processing of the chimeric proteins. Arg-16, Leu-17 and Leu-24 in
gCS, which are located in the yellow box, function in the targeting to microbodies, whereas Cys-42 located in the green box
functions in the processing of the presequence.

12

NATIONAL INSTITUTE

unlikely that glyoxysomes and leaf peroxisomes possess different targeting machineries.
A similar analysis of transgenic Arabidopsis plantsthat expressed fusion proteins with substituted aminoacid residues in the two consensus sequences by sitedirected mutagenesis shows that RL-X5-HL functions
in targeting to microbodies and SXLXXAXCXA
plays a role in the processing of the N-terminal region.

lacks the activities of vacuolar proteases. However,
pMP23 and MP28 did not accumulate in the wild-typestrain; the expressed pMP23 and MP28 were degradedin a proteinase A-dependent manner. These results
indicate that pMP23 and MP28 are transported to thevacuolar membrane when expressed in yeast. In vitrotransport assays using the vacuolar membrane vesicles
from the yeast transformants will allow us to further
investigate the function(s) of pumpkin seed TIP.

II. 1\vo forms of hydroxypyruvate reductase might
be produced by alternative splicing

IV. A rapid increase in the level of binding protein(BiP) is accompanied by synthesis and degradationof storage proteins in pumpkin cotyledonsHydroxypyruvate reductase belongs to the leaf-peroxisome-specific enzymes and is induced and accumulated in microbodies during greening. Two different cDNAs encoding hydroxypyruvate reductase wereisolated from a eDNA library of pumpkin cotyledons.
One of the cDNAs, designated HPRl, encodes apolypeptide of 386 amino acids, while the other,
HPR2, encodes a polypeptide of 381 amino acids.
Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, thededuced HPRl protein contains Ser-Lys-Leu at its
carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly
suggests that HPRl and HPR2 are produced by alternative splicing. These findings show that two differenthydroxypyruvate reductases, HPRl and HPR2, are localized in microbodies and in the cytosol, respectively
and accumulation of HPRl in leaf peroxisomes isincreased by light, suggesting that the microbody transition may be regulated by alternative splicing.
III. Membrane protein a- TIP of protein-storage
vacuolesThe vacuole in the cells of plant seeds shows dramatic changes in its morphology and function during
seed maturation through seed germination. To investigate this transition, we prepared protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) andcharacterized their protein components. Five major
membrane proteins, designated MP23, MP27, MP28,
MP32 and MP73, are located in the protein bodymembrane of pumpkin seeds. Both MP28 and MP23
belong to the seed TIP (tonoplast intrinsic protein)subfamily. TIP is an integral membrane protein that isfound in plant seeds and belongs to the MIP (major
intrinsic protein) family. Both MP28 and the 29-kDa
precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, andthen the precursor is converted to the mature MP23 at
the late stage. The two TIPs of pumpkin seeds,
pMP23 and MP28, were expressed in yeast cells under control of the GALl promoter, and the subcellular
localization of the proteins was analyzed. The pMP23
and MP28 proteins stably accumulated in the yeast
vacuolar membrane when the proteins were expressedin the proteinase A-deficient strain (pep4), which

The binding protein (BiP) is a member of the heatshock 70 protein (Hsp70) family that is localized tothe endoplasmic reticulum (ER) of eukaryotic cells,
where it functions as a chaperone and is believed tosupport proper protein folding and protein translocation into the ER lumen. To elucidate the involvementof BiP in the biosynthesis of vacuolar proteins, wehave characterized the protein in pumpkin cotyledons
during seed maturation and seedling growth. Isolated
microsomes from maturing pumpkin cotyledons contained a significant amount of BiP, protein-disulfide
isomerase and calreticulin. We have purified a 70-kDa
protein; sequences of the N-terminus and internalfragments of this protein exhibited a high identity to
the sequence of soybean BiP. Immunoblot analysis
with specific antibodies raised against the purified BiP
showed that the amount of BiP in cotyledons increased markedly at the middle stages and then decreased. The increase was accompanied by the synthesis of storage proteins and the development of theendoplasmic reticulum in the cotyledons at the middle
stage of seed maturation. Most of these storage proteins degraded dramatically between 2 and 5 days
after seed germination, and the degradation was also
accompanied by a rapid increase in the level of BiP.
Subcellular fractionation of the 4-day-old cotyledonsshowed a high accumulation of BiP in the endoplasmic reticulum. It is possible that BiP might be involved in the synthesis of seed storage proteins during
maturation and in the synthesis of hydrolytic enzymes
responsible for the degradation of the storage proteinsduring seed germination.
V. 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). Members of the VPE family can beseparated into two subfamilies, one that is specific toseeds and another that is specific to vegetative organs.

FOR BASIC BIOLOGY

13

B

Fig. 2. Histochemical localization of GUS activity in the hydathode of Arabidopsis transformed with the y-YPE promoter-gus
fusio n gene. Arabidopsis was transformed with a reporter gene composed of the promoter of they-YPE gene and the coding regionof ~-glucuronidase (GUS). GUS activity was observed in the hydathodes at the ends of veins along with the leaf margins, as shownin (A). Hydathodes are structures for that discharge water from the interior of the leaf to its surface. A photograph of the hydathodewith DIC optic of the enclosed region in A is shown in (B).

The molecular characterization of all the members of
the VPE family in Arabidopsis is required if we are to
elucidate the mechanisms of regulation of genes for
VPE homologues and the physiological functions ofthese proteins in protein-storage vacuoles and vegetative vacuoles. We isolated the three genes ofVPEs (aVPE, ~- VPE and y- VPE) from a genomic library of
Arabidopsis. To demonstrate temporal and spatial expression of the promoters of the VPE genes, we transformed Arabidopsis plants with a reporter gene containing the promoter of the VPE genes and the coding
region of ~-glucuronidase (GUS). The ~- VPE gene
was expressed in seeds, but a- VPE and y- VPE were
not. The GUS activity for y- VPE gene was predominatly expressed in the hydathode tissues of leaves(Fig. 2).

VI. Role of molecular chaperones in organelle
differentiation
Molecular chaperones are cellular proteins thatfunction in the folding and assembly of certain otherpolypeptides into oligomeric structures but that are
not themselves, components of the final oligomeric
structure. To clarify the roles of molecular chaperones
on organelle differentiation, we have purified andcharacterized chaperonin and Hsp70s and analyzed
their role in the translocation of proteins into chloroplasts. We isolated a eDNA for the chaperon in I 0 homologues from Arabidopsis thaliana by functional
complementation of the E. coli groES mutant. The
eDNA was 647 bp long and encoded a polypeptide of
98 amino acids. The deduced amino acid sequence
showed approximately 50% identity to mammalian
mitochondrial CpnlOs and 30% identity to GroES. A.
Northern blot analysis revealed that the mRNA for the
Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock
treatment. The CpnlO homologue was constitutively
expressed in transgenic tobaccos. Immunogold andimmunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed

that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic
tobaccos.

Publication List:
Hayashi, M., Aoki, M. , Kato, A., Kondo, M. andNishimura, M. (1996) Transport of chimeric proteins contain a carboxy-terminal targeting signal
into plant microbodies. Plant J. 10: 225-234.
Hayashi, M., Tsugeki, R. , Kondo, M. , Mori, H. andNishimura, M. (1996) Pumpkin hydroxypyruvate
reductases with and without a putative C-terminal
signal for targeting to microbodies may be producedby alternative splicing. Plant Mol. Bioi. 30: 183189.
Higuchi , T., Hisada, H. and Maeshima, M. (1996)
Complete nucleotide sequence of a eDNA for an
intrinsic protein in vacuolar membranes from radish
roots (PGR 96-045). Plant Physiol., 111: 947.
Kato, A., Hayashi, M., Kondo, M. and Nishimura, M.
(1996) Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase. Plant Cell 8:1601-1611.
Kato, A., Hayashi, M. Takeuchi, Y. and Nishimura, M.
(1996) eDNA cloning and expression of a gene for
3-ketoacyl-CoA thiolase in pumpkin cotyledons.
Plant Mol. Bioi. 31: 843-852.
Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H.,
Kondo, M., Hara-Nishimura, I. and Nishimura, M.
( 1996) Isolation and characterization of a eDNA
encoding mitochondrial chaperonin 10 from
Arabidopsis thaliana by functional complementation of an E. coli groES mutant. Plant J. 10: 11191125.
Maeshima, M. , Nakanishi, Y., Matsuura-Endo, C. andTanaka, Y. (1996) Proton pumps of vacuolar membrane in growing plant cell. J. Plant Res., 109: 119125.
Matsumoto, H., Senoo, Y., Kasai, M. and Maeshima,
M. (1996) Response of the plant root to aluminium

14

stress: Analysis of the inhibition of the root elongation and changes in membrane function. J. Plant
Res., 109: 99-105.
Mano, S., Hayashi, M., Kondo, M. and Nishimura, M.
(1996) eDNA cloning and expression of a gene forisocitrate lyase in pumpkin cotyledons. Plant Cell
Physiol. 37: 941-948.
Nishimura, M., Hayashi, M., Kato, A., Yamaguchi, K.
and Mano, S. (1996) Functional Transformation of
microbodies in higher plant cells. Cell Struct.
Funct. 21: 387-393.
Odaira, M., Yoshida, S., and Maeshima, M. (1997)
Accumulation of a glycoprotein homologous toseed storage protein in mung bean hypocotyls in thelate stage of tissue elongation. Plant Cell Physiol.,
38: 290-296.
Robinson, D.G., Haschke, H.-P. Hinz, G., Hoh, B.,
Maeshima, M. and Marty, F. (1996) Immunologicaldetection of tonoplast polypeptides in theplasmamembrane of pea cotyledons. Planta, 198:
95-103.
Yamaguchi, K., Hayashi, M. and Nishimura, M.
(1996) eDNA cloning for thylakoid-bound ascorbate peroxidase in pumpkin and its characterization.
Plant Cell Physiol. 37: 405-409.

NATIONAL INSTITUTE

FOR BASIC BIOLOGY

15

Professor: Yoshinori Ohswni
Associate Professor: Tamolsu Yoshimuri
Research Associates: Takeshi Noda
Yoshiaki Kamada
Graduate Students: Kanae Shirahama (Un iv. of Tokyo)
Tomoko Funakoshi (Univ. of Tokyo)
Satoshi Kame taka ( Univ. of Tokyo)
Chikara Tokunaga (Univ. of Tokyo)
Takayoshi Kirisako (Univ. of Tokyo)
Technical Staff: Yukiko Kabeya
Visiting Professor: Michael Filosa

nance of cell viability. Autophagy is a major route forsequestration of proteins to the lytic compartment.
Vacuole/lysosome is also the destination for endocytic
pathway. Since in 1952 du Duve identified cellular
lytic compartment, lysosome, enzymatic characterization and biogenesis of lysosomal enzymes have beenstudied thoroughly. However, the mechanisms of delivery of proteins to the lysosomes are not known at
a molecular level. Dynamism of lysosomal system has
been studied mostly with electron microscope. Autophagy is one of the most important problems in cellbiology remained to be solved.

This division aims to elucidate the mechanism andregulation of intracellular protein degradation in alytic compartment and its physiological function. Recently it was realized that degradation process plays
essential role for cellular regulation. In the cytoplasm
selective protein degradation takes place by ubiquitin/
proteasome system. Short-lived or abnormal proteins
are selectively eliminated by this pathway. Whilevacuole/lysosome contributes to the bulk turnover ofcytosolic and organelles proteins. However, little is
known about the mechanism of protein degradation incontrast with biosynthesis. Bulk protein degradation is
induced by various nutrient starvation condition,
which is obligatory to cell differentiation and mainte-

Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, Saccharomyces
cerevisiae, induces bulk protein turnover in the vacuoles under various starvation conditions. This wholeprocess corresponds to the process of macroautophagy
in higher eukaryotic cells. By electron microscopic
analyses we succeeded to detect double membrane
structure enclosing a portion of cytosol in the cytoplasm. These yeast autophagosomes immediately fusewith vacuolar membrane, resulting to deliver a single
membrane bound vesicles, autophagic bodies in thevacuoles. When vacuolar proteinase activities areblocked genetically or by specific inhibitor such as
PMSF autophagic bodies are accumulated in the vacu-

DIVISION OF BIOENERGETICS

Fig. I. (a) Freeze-Fracture Image of Yeast under Nitrogen Starvation.
When vacuolar proteinases are blocked, spherical membrane structure, autophagic bodies acc umulate in the vacuole.
(b) Fusion of Autophagosome to the Vacuolar Membrane.
Double membrane structure, autophagosome, encloses a portion of the cy tosol, then fuses with the vacuole to deliver an autophagic
body in the vacuole. These membrane structures show a few intramembrane particles.

16

NATIONAL INSTITUTE

oles. They moves around vigorously in the vacuoles
by Brownian motion, and are easily observed by lightmicroscope. Thus we can follow the progression of
autophagy by the accumulation of autophagic bodies
in real time. Biochemical and immunoelectron microscopic analyses of vacuoles containing autophagic
bodies showed that starvation-induced sequestration is
non-selective, that is, any kind of cytosolic enzymes
and organelles are sequestered to the vacuoles to be
degraded. Freeze-fracture electron microscopy
showed that autophagosomal membrane has characteristic feature and density of intramembrane particles
is quite few. The origin of the autophagosomal membrane and mechanism of formation are key problems
remained to be solved.
Genetical dissection of autophagic process
Yeast, S. cerevisiae, has made great contribution tosolve many fundamental problems in cell biology
because of tractability of genetic and molecularbiological techniques. In order to dissect the complex
process of autophagy to its elementary steps westarted to isolate mutants in the process of autophagy,taking advantage of morphological selection underlight microscope. Total 14 agp mutants, defective in
the induction of autophagy, were isolated. They
cannot induce protein degradation upon shift fromgrowth. medium to the starvation medium. But theygrow normally in a rich medium, but start to die after
2 days in the starvation medium. This suggests thatthe autophagy is necessary for long time maintenanceof cell viability. Homozygous diploid of each apg
mutants is sporulation defective as expected.
Analyses of APG genes
Now we are focusing on characterization of these
APG genes, essential for autophagy. This year wehave finished cloning of twelve APG genes. So far ten
APG genes have been identified. APG 1 codes a novel
Serffhr kinase essential for the induction of autophagy. This provides the first direct evidence for involvement of protein phosphorylation in the process
of autophagy. So far all genes analyzed are novel andnon-essential for vegetative growth. Using the assay
system of autophagy we developed, we are now analyzing genetic interaction among APG genes. Further
molecular biological and biochemical studies on these
APG gene products might provide important clues tounderstand the mechanism of autophagy at a molecular level. Now we obtained antibodies against to these
APG gene products. Intracellular localization of them
may give us clues to elucidate the function of these
proteins.
Diverse pathways of autophagy
Autophagy is one of the fundamental function of all
eukaryotic cells. We found yeast cells induce macro-

Aminopeptidase I
APG5
APG14 APG7
APG13
APG9 APG4
APG6

f

APG12

APGS

... . ..
.
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....
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..
,

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•

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Proteinase

Fig. 2. Scheme of Autophagy in Yeast
All APG genes identified are novel and required at or before the formation of autophagosome.

FOR BASIC BIOLOGY

autophagy under starvation, which is a main route forbulk and non-selective protein degradation. However,
it is also reported that some enzymes are selectively
taken up to vacuoles according to the physiologicaldemands. In methylotrophic yeast, Pichia pastoris degrades peroxisomes by invagination of vacuolar membrane, microautophagic process. Further studies will
uncover more sophisticated pathways for the degradation in the lysome/vacuoles. Recently it was foundthat all APG genes are reuired for targeting of API
from cytosol to the vacuole. It is interest problem how
this selective and constitutive process shares machinery with the autophagy.
Perspective
Autophagy is essential for maintenance of cell viability during starvation. Degradation products may
provide essential nutrients necessary for minimal protein synthesis, or reduction of certain critical activities
in the cytosol may be essential for the maintenance ofviability. Basic molecular devices for the membrane
dynamics are conserved from yeast to higher eukaryotes. We realized some APG genes in yeast show homologues in mammals or higher plants. We are now
developing systems of autophagy in mammalian cultured cell.

Publication List:
Funakoshi, T., Matsuura, A., Noda, T. and Ohsumi, Y.
(1997) Analyses of APG13 gene involved in the autophagy in yeast, Saccharomyces cerevisiae. Gene
179, in press.
Kametaka, S., Matsuura, A., Wada, Y. and Ohsumi, Y.
(1966) Structural and functional analyses of APG5,
a gene involved in autophagy in yeast. Gene 178,
139-143.
Matsuura, A., Wada, Y. and Ohsumi, Y. (1977) Apg1p,
a novel protein kinase required for the autophagic
process in Saccharomyces cerevisiae. Gene 179, inpress.
Moriyasu, Y. and Ohsumi, Y. (1996) Autophagy intabbaco suspension-cultured cell (BY-2) in response
to sucrose starvation. Plant Phsiol. 111, 1233-1241.
Nakamura, N., Matsuura, A., Wada, Y. and Ohsumi, Y.
(1997) Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces
cerevisiae. J. Biochem. 193, in press.
Nakamura, N., Hirata, A., Ohsumi, Y. and Wada, Y.
(1997) VAM6 is required for the vacuolar morphogenesis and maturation of vacuolar membrane protein in the yeast Saccharomyces cerevisiae. J. Biol.
Chern. in press.
Scott, S. V., Hefner-Gravink, A., Morano, K. A., Noda,
T., Ohsumi, Y. and D. J. Klionsky (1996) Cytoplasmto-vacuole targeting and autophagy employ the samemachinery to deliver proteins to the yeast vacuole.
Proc. Natl. Acad. Sci. USA 93, 12304-12308.
Shirahama, K., Yazaki, Y., Sakano, K., Wada, Y. andOhsumi, Y. (1996) Vacuolar function in the phosphate homeostasis of the yeast Saccharomyces17

cerevisiae. Plant Cell Physiol. 37, 1090-1093.
Wada, Y., Ohsumi, Y., Kawai, E. and Ohsumi, M.,(1996) Mutational <yWysis of Vam4/Ypt7p functionin the vacuolar biogenesis and morphogenesis in theyeast, Saccharomyces cerevisiae. Protoplasma 191,
126-135.

18

NATIONAL INSTITUTEDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor;· Masayuki Yamamoto
Research Associate: Masua Goto
Institute Research Fellow: Takashi Kuromori
Visiting Scientist: Satsuki Okamoto
Graduate Students: Chikako Kitayama (from The
University of Tokyo)
Masahiro Fujita (from The Universityof Tokyo)

The major goal of this Division is to elucidate regulatory mechanisms of meiosis. Meiosis is a crucial
step in gamete formation and is essential for sexual
reproduction. Meiotic steps are highly conserved
among eukaryotic species. We have been screening
for genes that may be relevant to the regulation ofmeiosis in animals and plants, by using transcomplementation methods. In the fission yeast Schizosaccharomyces pombe, which is a unicellular eukaryotic
microorganism, genes controlling meiosis have beencloned and characterized extensively, and mutants
defective in these genes are available. We have isolated animal and plant genes that can functionallycomplement such S. pombe mutants. Analysis of thecloned genes is in progress. In addition, efforts arealso paid to in-depth analysis of regulatory mechanisms of meiosis in the fission yeast, to facilitate theabove strategy.

ciency in cells of mating-type M but not in P M cells
defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 aminoacids with several potential membrane-spanning domains, which is 44% identical with famesyl cysteine
carboxyl methyltransferase encoded by STE14 andrequired for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective inmam4 produced famesylated but unmethylated cysteine and small peptides, but no intact M-factor. Theseobservations strongly suggest that the mam4 geneproduct is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore,transcomplementation of S. pombe mam4 allowed us
to isolate an apparent homologue of mam4 from Xenopus laevis (Xmam4). In addition to its sequencesimilarity to S. pombe mam4, the product of Xmam4
was shown to give a famesyl cysteine carboxyl
methyltransferase activity in S. pombe cells. These results will open the way to intensive studies of the role
for methylation in a large body of proteins including
ras-superfamily proteins and its relevance to meiosis.
This work will be published shortly (Y. Imai et a!.
(1997) Mol. Cell. Bioi., in press) .

I. Genes encoding farnesyl

II. Phosphorylation of RNA-binding protein
controls cell cycle switch from mitotic to meiotic in
fission yeast

The mating pheromone signaling is essential for the
induction of meiosis in Schizosaccharomyces pombe.
The S. pombe mam4 mutation causes mating defi-

Little is known about the molecular controls overthe switch from mitotic to meiotic cell cycles. In S.
pombe, the switch from the mitotic to the meiotic cell
cycle is controlled by the antagonistic effects of thecysteine carboxyl
methyltransferase in fission yeast and Xenopus

Fig. 1. A "dot" formed by the RNA-binding protein Mei2 in a meiotic prophase nuclei. Mei2 protein fused to GFP, which emits
green fluorescence, was live-recorded. The yellow spot represents fluorescence of Mei2-GFP, whereas the red region represents
nuclear DNA. The whole cell is faintly visible in red. Prophase nuclei of fission yeast move back and forth, assuming morphology
like a horse tail. Mei2 appears to occupy a fixed position in the meiotic nucleus. See Watanabe et al. (Nature 386, 187-190, 1997)
for more details. We were assisted by Y. Chikashige and Y. Hiraoka (Kansai Advanced Research Center, Communications ResearchLaboratory) in taking this photograph.

FOR BASIC BIOLOGY

Patl protein kinase and the Mei2 RNA-binding protein. We have shown that Mei2 is phosphorylated on
Ser438 and Thr527 by Patl kinase, whose inactivationis necessary to induce meiosis. Mutant Mei2 carrying
alanine in these two positions induces meiosis without
inactivation of Patl kinase, establishing that Mei2 isthe critical target of Pat I. Mei2localizes mainly in thecytoplasm of proliferating cells, but is found in asingle 'dot', closely opposed to the spindle pole body,in prophase nuclei during meiosis I (Fig. 1). Our results emphasize the crucial role of RNA-binding proteins in the initiation and execution of meiosis. This
work has been published lately (Y. Watanabe et al.
(1997) Nature 386, 187-190).

Publication List:
Hakuno, F. and Yamamoto, M. (1996). The Schizosaccharomyces pombe mral gene, which is requiredfor cell growth and mating, can suppress the mating
inefficiency caused by a deficit in the Rasl activity.
Genes Cells 1, 303-315.
Watanabe, Y. and Yamamoto, M. ( 1996). Schizosaccharomyces pombe perJ+ encodes an ATF/CREbinding protein involved in the regulation of gene
expression for sexual development. Mol. Cell. Bioi.
16, 704-711.
Yabana, N. and Yamamoto, M. (1996). Schizosaccharomyces pombe map]+ encodes a MADS-boxfamily protein required for cell-type-specific gene
expression. Mol. Cell. Bioi. 16, 3420-3428.
Yamamoto, M. (1996). The molecular control mechanisms of meiosis in fission yeast. Trends Biochem.
Sci. 21, 18-22.
Yamamoto, M. (1996). Regulation of meiosis in fission yeast. Cell Struct. Funct. 21, 431-436.

19

20

NATIONAL INSTITUTEDIVISION OF CELLULAR
COMMUNICATION (ADJUNCT)
Professor: Ritsu Kamiya
Associate Professor: to be appointed
Research Associate: to be appointed

The research in this laboratory, started in November, 1996, is aimed at an understanding of the molecular mechanisms that regulate the assembly and function of cytoskeletal proteins. Current research effort iscentered on the function of axonemal dyneins, microtubule-based motor proteins that produce force forflagellar beating. The organism employed is Chlamydomonas, a biflagellate green alga particularly suitedfor genetic and molecular biological studies.
A single flagellar axoneme contains at least elevenkinds of dynein heavy chains in inner and outer arms.
To understand the specific function of each heavy
chain, we have been isolating and characterizing mutants that lack different kinds of axonemal dyneins.
Their motility phenotypes have indicated that different dynein species differ in function in a fundamentalmanner. For example, the outer arm heavy chains areimportant for flagellar beating at high frequency,
whereas most of the inner arm heavy chains are important for producing proper waveforms. In addition,
indirect evidence has suggested that the force generation properties differ greatly among different heavy
chains. We are currently trying to measure the force
production by different dyneins using micro-physiological techniques.
The inner dynein arms are known to contain actin as
a subunit. Thus the two independent motility systemsof eukaryotes - the actin-based and microtubule-based
motility systems - should somehow cooperate in thewild type(a~y)

oda4-s7 ( -~)inner arm, although the function of actin in dyneinarms is unknown at present. The mutant ida5 we isolated as a mutant that lacks four subspecies of innerarm dynein was recently found to have a mutation in
the actin-encoding gene. This mutant as well as another independently isolated mutant (ida5-t) has adeletion in the actin gene and express no conventional
actin. The cytoplasm and axonemes of these mutants
lack the conventional actin entirely but contain two
novel proteins which are immunologically distinct
from, but related to, actin. Since Chlamydomonas has
been shown to have only a single copy of actin-encoding gene, it is likely that the two novel proteins inthese mutants originate from other gene(s) that codes
for an actin-like protein which has hitherto been undetected in wild-type cells. The net growth rate of themutants did not differ from that of wild type, but themating efficiency was greatly reduced because of theirdeficient fertilization tubule growth. These results
raise the possibility that Chlamydomonas can live
without conventional actin.
The discovery of the mutants that lack the conventional actin entirely should open the way to studying
the function of actin in the cell as well as in thedynein arm. We have succeeded in transforming the
mutant Chlamydomonas with cloned actin gene andfound that inner dynein arms become restored upontransformation. Studies with artificially mutated actin
gene will enable us to determine whether actin is really essential for cytokinesis or otheJ; fundamental
processes in Chlamydomonas, and what functions arecarried out by actin in the axoneme and cytoplasm. A
most important immediate project will be to characterize the novel proteins found only in the mutants thatlack actin.

, oda11(-a)

oda 11xoda4-s7( -a~) .

Fig. I. Wild-type and mutant axonemes of Chlamydomonas that Jack different heavy chains of outer arm dynein. Wild-type outerarm contains three heavy chains, a , p and y, of which the mutant oda4-s7 Jacks p and odall Jacks a. Lower photographs are theaveraged images of the outer doublet microtubules, showing the structural defect in each mutant (arrowhead).

21

FOR BASIC BIOLOGYDIVISION OF CELL FUSION (ADJUNCT)
Professor: Hitoshi Sakano
Research Associates: Akio Tsuboi (until November)
Kanae Muraiso (on leave)
Institute Research Fellow: Hiroaki Kasai (until March)
Graduate Student: Nika Yamazaki (from Tokyo Institute ofTechnology)

Recently, hundreds of odorant receptor (OR) genes
have been reported in the olfactory system, although
how the expression of this gene system is regulated
has yet to be studied. It is assumed that only a limited
number of the OR genes (possibly one) are activatedin each olfactory neuron. In addition, neurons expressing a given OR gene are known to project theiraxons to a limited number of glomeruli at the fixedsites on the olfactory bulb.
We have been studying how individual neurons express a limited number of OR genes while keeping therest of the genes silent, and how neurons expressing agiven OR gene project their axons to a few subsets
among the 2000 glomeruli within the olfactory bulb.
In order to study the selective expression of the OR
genes, we have been characterizing a Pl clone containing two highly related genes, MORlO andMOR28. These genes are 92% homologous within
their coding regions and linked in tandem on mouse
chromosome 14. In situ hybridization has revealed
that both genes are expressed in the same spatial zone
within the olfactory epithelium, but never expressed
simultaneously in the same neuron.
Developmental analyses have revealed that
MORlO-expressing neurons appear earlier during embryogenesis than those MOR28-expressing. However,the number of MOR28 neurons is greater than that of
MORlO neurons after birth and continues to increase
to a ratio of 2.5: 1 by adulthood. These results suggestthat individual olfactory neurons may activate OR
genes through a stochastic mechanism even between
two closely related OR genes; yet this selection appears to be biased in both the onset and level of expression. Interestingly, olfactory neurons expressing
either MORlO or MOR28 project their axons to two
distinct, but adjacent subsets of glomeruli on the olfactory bulb. We conclude that two highly homologous and tandemly linked OR genes are expressed ina mutually exclusive manner in individual olfactory
neurons, and that neurons expressing either of thesegenes project their axons to two discrete, but neighboring subsets of glomeruli on 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 the olfactory marker protein (OMP) promoter. Since the OMP
gene is expressed in the mature neurons within theolfactory epithelium, the transgene is expected to be
activated in all olfactory neurons. Recently, atransgenic line is found to express the OMP-MOR28
transgene in most of the mature neurons within theolfactory epithelium as well as within the vomeronasal organ. We believe that the study of such transgenic
mice will give us new insight into the molecularmechanisms for OR gene expression, as well as forneuronal projection to the olfactory bulb.
Publication List:
Asai, H., Kasai, H., Matsuda, Y., Yamazaki, N.,
Nagawa, F., Sakano, H. and Tsuboi, A. (1996) Genomic structure and transcription of a murine odorant receptor gene: differential initiation of transcription in the olfactory and testicular cells. Bioche.
Biophys. Res. Comm. 221, 240-247.

22

NATIONAL INSTITUTE

23

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairman: Yoshiaki Suzuki

Three regular divisions and one adjunct division construct
the Department. The department members conduct molecularanalysis on various aspects of developmental phenomena; differentiation and maturation of the germ cells, molecular basisof the body plans, and gene regulation in different phases of
the cell cycle, in the differentiating tissues, and during changesof the differentiated states.

24

NATIONAL INSTITUTEDIVISION OF REPRODUCTIVE BIOLOGY

several fish species which are responsible forcholesterol side-chain cleavage cytochrome P450 (P450scc),
3~-hydroxysteroid dehydrogenase (3~-HSD), 17a.-hydroxylase/C17 ,20-lyase cytochrome P450 (P450c 17)
and P450 aromatase (P450arom). More recently,
eDNA clones encoding 20~-hydroxysteroid dehydrogenase (20~-HSD; a critical enzyme which converts
17a.-hydroxyprogesterone to 17a.,20~-DP) were isolated from eDNA libraries of ayu (Plecoglossus
altivelis). The amino acid sequence deduced from theisolated eDNA had 276 amino acid residues andshared approximately 60% homology with mammalian carbonyl reductase. The clear lysate, which
was prepared from E. coli harboring the eDNA,
catalyzed the production of 17a.,20~-DP. The
identification of 17a.,20~-DP was confirmed by twodimensional thin layer chromatography, followed byrecrystallization. Purification of the E. coli-expressed
eDNA product revealed that it possessed carbonyl
reductase activity and 17a.-hydroxyprogesterone, theendogenous immediate precursor of 17a.,20~-DP, wasa good substrate.
Northern and Western blots revealed a single
P450arom mRNA and a single protein in tilapia ovarian tissue respectively. T1lese analyses also revealed
that the levels of both P450arom mRNA and protein
were low in early vitellogenic follicles, increased in
midvitellogenic follicles, and declined to non-detectable levels in postvitellogenic follicles. Changes in theability of follicles to convert exogenous testosterone
to estrogens (aromatase activity) were similar to thoseof P450arom mRNA and protein. These observations
indicated that the capacity of tilapia ovarian follicles
to synthesize estradiol-17J3 is closely related to thecontents of P450arom mRNA and protein withinthem. The mRNA levels of P450scc, 3~-HSD, andP450c17 are barely detected in ovarian follicles during the midvitellogenic stage, and are abundant infollicles during the postvitellogenic stage and oocyte
maturation. Our preliminary results indicate that
forskolin-induced 17a.,20~-DP production is accompanied by a dramatic decrease in P450arom mRNAlevels in granulosa cells isolated from postvitellogenic
follicles. A 2- to 3-fold increase in P450scc and 3~­
HSD mRNAs and a slight decrease in P450c 17mRNA are also observed during forskolin-induced
17a.,20~-DP production.
Northern hybridization
analysis has revealed that 20~-HSD mRNA transcripts are present in fully vitellogenic follicles andincreased as oocyte maturation proceeded. Timecourse studies further suggest that de novo synthesisof 20J3-HSD in vitro in response to gonadotropin andcAMP occurs, and consists of gene transcriptional
events within the first 6 hr of exposure to gonadotropin and cAMP and translational events 6-9 hr after the
exposure to gonadotropin arid cAMP. Thus, these results suggest that gonadotropin causes the de novo
synthesis of 20~-HSD in the granulosa cell through amechanism dependent on RNA synthesis.
17a.,20~-DP acts via a receptor on the plasma memestradiol-17~ and 17a.,20~-DP biosynthesis:

Professor: Yoshitaka Nagahama
Associate Professor: Michiyasu Yoshikuni
Research Associates: Minoru Tanaka
Tohru Kobayashi
Institute Research Fellow: Akihiko Yamaguchi
JSPS Postdoctoral Fellows: Yoshinao Katsu
Zuxu Yao
NIBB Postdoctoral Fellows: Kunimasa Suzuki
Won-Kyo Lee
Jain-Quiao Jiang
Xiao-Tian Chang
JSPS Research Associates: Mika Takahashi
Takashi Todo
Daisuke Kobayashi
Graduate Students: Yuichi Ohba (Graduate University for
Advanced Studies)
Jun Ding (Graduate University for
Advanced Studies)
Masatada Watanabe (Graduate
University for Advanced Studies)
Guijun Guan (Graduate University
for Advanced Studies)
Monbusho Foreign Scientist: Graham Young (Universityof Otago)
JSPS Visiting Scientist: Allen Schuetz (University of
Maryland)
Visiting Scientist: Craig Morrey (University of Hawaii)

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

I. Endocrine regulation of oocyte differentiation,
growth and maturation
Our research effort in previous years concentrated
on the identification and characterization of the molecules (gonadotropins, gonadal steroid hormones, andcell cycle-regulated molecules) that stimulate andcontrol germ cell growth and maturation. We identified, for the first time in any vertebrate, 17a.,20~­
dihydroxy-4-pregnen-3-one (17a.,20~-DP) as thematuration-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 biologically important mediators of oocyte growth and maturation infemale salmonid fishes. It is established that the
granulosa cells are the site of production of these two
mediators, but their production by the ovarian follicle
depends on the provision of precursor steroids by thethecal cell (two-cell type model). A dramatic switch inthe steroidogenic pathway from estradiol-17~ to17a.,20J3-DP occurs in ovarian follicle cells immediately prior to oocyte maturation. This switch is aprerequisite step for the growing oocyte to enter thematuration phase, and requires a complex and integrated network of gene regulation involving cellspecificity, hormonal regulation, and developmental
patterning.
We have isolated and characterized the eDNA
encoding several ovarian steroidogenic enzymes of

25

FOR BASIC BIOLOGY

brane of oocytes. We have identified and characterized a specific 17a.,20~-DP receptor from defolliculated oocytes of several fish species. Scatchard analysis revealed two different receptors: a high affinity
with a Kd of 18 nM and a Bmax of 0.2 pmoles/mg
protein and a low affinity receptor with a Kd of 0.5
J.1M and a Bmax of 1 pmole/mg protein. 17a.,20~-DP
receptor concentrations increase during oocyte maturation. The interaction between 17a.,20~-DP receptors
and G-proteins was examined. Pertussis toxin (PT)catalyzed the ADP ribosylation of a 40 kDa protein in
crude membranes from rainbow trout oocytes. The 40J4)a protein was recognized by an antibody against
the a. subunit of inhibitory G-protein. Treating themembrane fraction with 17a.,20~-DP decreased the
PT-catalyzed ADP ribosylation of the 40 kDa protein.
The specific binding of 17a.,20~-DP was decreased by
PT. We conclude that the PT-sensitive Gi is involvedin the signal transduction pathway of 17a.,20~-DP infish oocytes. We have cloned two Gia. cDNAs from a
medaka oocyte eDNA library. Specific polyclonal
antibodies against Gia. subunits were generated.
These antibodies were used to examine changes in
Gia. content during 17a.,20~-DP-induced oocyte
maturation and to immunoprecipitate solubilized Gia..
Western blot analysis showed that 17a.,20~-DP receptor concentrations and Gia. content decreased concomitantly in membrane preparations during oocyte
maturation. We also found that significant amounts of
17a.,20~-DP receptors in the immunoprecipitates, indicating that the 17a.,20~-DP membrane receptors are
directly coupled with Gi. This is the first demonstration of direct coupling of the maturation-inducing
hormone (steroid) receptor and heteromeric G-pro-teins (Fig. 1).
The early steps following 17a.,20~-DP action involve the formation of the major mediator of this steroid, maturation-promoting factor or metaphase-promoting factor (MPF). MPF activity cycles during
17a.,20~-DP-induced oocyte maturation with thehighest activity occurring at the first and second meiotic metaphase. Studies from our laboratory and others have shown that MPF activity is not species-specific and can be detected in both meiotic and mitotic
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). Goldfish immature
oocytes contain 35-kDa inactive cdc2 kinase. Although immature oocytes contain mRNA for cyclin B,
they do not contain cyclin B protein. 17a.,20~-DP
induces oocytes to synthesize cyclin B. The preexisting 35-kDa inactive cdc2 kinase binds to de novo synthesized cyclin B at first, then is rapidly converted
into the 34-kDa active form. Introduction of a bacterially produced goldfish cyclin B into immature goldfish oocyte extracts induces cdc2 kinase activation.
Phosphoaminoacid analysis shows that threonine(Thr) phosphorylation of the 34-kDa cdc2 kinase isassociated with the activation. The sites of Thr phosphorylation on cdc2 kinase was mapped to reside Thr161. Since goldfish cyclin B mRNA contains four
copies of the usual cytoplasmic polyadenylation element in the 3'UTR, the initiation of its synthesis
during oocyte maturation may be controlled by theelongation of poly (A) tail. We examined thepolyadenylation state of cyclin B mRNA during goldfish oocyte maturation by means of a PCR poly (A)17a,20~-Dihydroxy-4-pregnen-3-one~(I
Adenylate cyclase

Receptor

OOCYTE~

Cyclin B mANA
Cyclin B synthesis

Polyadenylation-~-~~
Translation

Fig. L Mechanisms of 17a,20~-dihydroxy-4-pregnen-3-one (17a,20~-DP)-induced cyclin B synthesis by fish oocytes. Inhibitory
G-proteins (Gi) is involved in the signal transduction pathway of 17a,20~-DP.

26

test, and found that cyclin B mRNA is polyadenylated
during oocyte matuation. Furthermore, cordycepin
which inhibits poly (A) addition of mRNA, prevented
17a,20~-DP-induced oocyte maturation in goldfi'sh. It
is concluded that elongation of poly (A) tail of cyclin
B mRNA is required for the initiation of its translation(Fig. 1).
Immediately prior to the transition from metaphaseto anaphase, MPF is inactivated by degradation of
cyclin B. We investigated the role of proteasomes (anonlysosomal large protease) in cyclin degradation,using E. coli-produced goldfish cyclin B and purified
goldfish proteasomes (20S and 26S). The purified 26S
proteasome, but not 20S proteasome, cleaved bothmonomeric and cdc2-bound cyclin B at lysine 57(K57) restrictively in vitro, and produced a 42 kDa Nterminal truncated cyclin B, which was transiently
detected at the initial phase of the normal egg activation. The 42 kDa cyclin B, as well as full-length one,was degraded in Xenopus egg extracts, but a mutation
on K57 (K57R) inhibited both the digestion by 26S
proteasome and the degradation in Xenopus egg extracts. These findings strongly suggest that the involvement of 26S proteasome in cyclin degradation
through the first cleave on its N-terminus.

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 l7a,20~-DP
for sperm maturation). A steroidogenic switch, from
11-ketotestosterone to 17a,20~-DP, occurs in salmonid testes around the onset of final maturation. In vitro
incubation studies using different testicular preparations have revealed that the site of 17a,20~-DP production is in the sperm, but its production depends on
the provision of precursor steroids by somatic cells.
The site of ll-ketotestosterone production is in thetesticular somatic cells.
In the cultivated male Japanese eel (Auguillajaponica), spermatogonia are the only germ cells
present in the testis. A serum-free, chemically definedorgan culture system developed for eel testes wasused to investigate the effect of various steroid hormones on the induction of spermatogenesis in vitro.
We obtained evidence that ll-ketotestosterone can
induce the entire process of spermatogenesis in vitro
from premitotic spermatogonia to spermatozoa within
21 days.
We have used subtractive hybridization 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 andtestes one day after a single injection of HCG. From
its deduced amino acid sequence, this clone was identified as coding for the activin ~B subunit. Using
Northern blot analysis and in situ hybridization tech-

NATIONAL INSTITUTE

niques, we examined sequential changes in transcripts
of testicular activin ~B during HCG-induced spermatogenesis. No transcripts for activin ~B were foundin testes prior to HCG injection. In contrast, 3.3 kb
mRNA transcripts were prominent in testes one day
after the injection. The transcript concentration began
to decrease three days after the injection and there
was a further sharp decrease by nine days. The HCGdependent activin ~B mRNA expression in the testes
was confirmed by in situ hybridization using adigoxigenin-labelled RNA probe: the signal was restricted to Sertoli cells in testes treated with HCG for
one to three days. A marked stimulation of activin B
production, but not either activin A or activin AB, wasobserved in testes after HCG and 11-ketotestosterone
treatment. Addition of recombinant human and eel
activin B to the culture medium induced proliferationof spermatogonia, producing mitotic spermatogonia,
within 15 days in the same manner as did llketotestosterone. Taken together, these findings suggest the following sequence of the hormonal inductionof spermatogenesis in the eel. Gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone,
which, in tum, activates the Sertoli cells to produce
activin B. Activin B then acts on spermatogonia to
induce mitosis leading to the formation of spermatocytes (Fig. 2).
To characterize the pathway of activin signal transduction involved in the process of spermatogenesis,
we recently initiated studies on the cloning and expression of activin receptors of eel testes. Three kindsof eDNA clones encoding eel activin type I receptors
were isolated from an eel testis eDNA library. The
predicted proteins of these receptors consist of 510,
493, and 505 amino acids with their deduced aminoacid sequences having a putative signal peptide, acystein-rich extracellular domain containing a potential N-glycosylation site, a putative transmembrane
domain with a well conserved GSGSG motif, and aserine/threonine kinase domain. Northern blot hybridization was performed in testes from eels injectedwith hCG for 1-18 days. The results were that mRNAtranscripts of the three eel activin type I receptors
were expressed in the testes of eels prior to hCG injection, with marked increased in the testes after hCG
injection for 9 days. In situ hybridization revealed thatthese activin type I receptors are localized in germ
cells (spermatogonia and spermatocytes) and Sertoli
cells. To further understand the molecular mechanisms of activin B-induced initiation of mitosis inspermatogonia, we recently isolated eDNA clones
encoding eel homologs of cdc2 kinase, cdk2 kinase,cdk4 kinase, cyclin Al and A2, cyclin Bl and B2,
cyclin Dl, and cyclin E and E' from eDNA libraries
of eel testes. Specific antibodies against some of these
proteins were also raised.

III. Endocrine
differentiation

regulationofgonadalsex

Recently, we, in collaboration with Drs. K. Ozato

27

FOR BASIC BIOLOGY

Testis--Receptor ...__ _ _ _,Leydig cell

l

0

:::::
Q)

11-Ketotestosterone

BM~t~

l

Meiosisinducing substance

Ctl

E__...{W} Receptor

Sertoli celluu......
.....

1 ..J

c.._

0

fJ)

Activin_B_ _ _ __I

Cyclin/cdk
Spermatogonium ~"'-Cell proliferation'-----------------~1

Meiosis initiation"

Spermatocyte/Spermatid/!permatozoon~

Fig. 2. Hormonal regulation of spermatogenesis in the eel testis.

(Nagoya University), M. Nakamura (Teikyo University) and E.G. Grau (University of Hawaii), haveinitiated studies on the roles of sex steroid hormonesin sex determination and differentiation using medaka,tilapia (Oreochromis niloticus), and a sex change fish,
Thalassoma duperrey. In tilapia, a pair of gonadal
primordia formed on both sides of the intestine at 15
days after hatching. The gonadal anlage consists ofseveral roundish germ cells surrounded by a few
stromal cells. At 20 days after hatching, prior to theperiod of sex differentiation, positive immunostaining
for P450scc, 3~-HSD, P450c17, and P450arom
antibodies becomes evident for the first time ingonads. Imrnunostained stromal cells are observed in
the vicinity of blood vessels. From 23-26 days after
hatching, morphological gonadal sex differentiation
begins to be recognized. Initial ovarian differentiation
is marked by the appearance of a narrow space in thestromal tissue, representing the formation of theovarian cavity. On the other hand, initial testiculardifferentiation is characterized by the appearance of anarrow space in the stromal tissue, representing theefferent duct construction. At this stage, germ cells intestes and ovaries 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-17~ biosynthesis from cholesterol before
sexual differentiation, which is consistent with theconcept that estrogen biosynthesis is essential forsexual differentiation of female phenotype during
early development.
In T. duperrey, steroid-producing cells have identified at different stages of sex change using immunocytochemical methods. Immunoreactive P450scc, thefirst enzyme in the steroidogenic pathway, initiallylocalizes in thecal cells. As sex change progresses
towards testis formation, P450scc becomes localized
in interstitial cells. It appears that thecal cells migrate
towards the interstices and become very active androgen-producing cells.
Publication List:

Chang, X.-T., Kobayashi, T., Kajiura, H., Nakamura,
M. and Nagahama, Y. (1996). Isolation and characterization of the eDNA encoding the tilapia(Oreochromis
niloticus)
cytochrome
P450
aromatase (P450arom): changes in P450arom
mRNA, protein and enzyme activity in ovarian follicle during oogenesis. J. Mol. Endocrinol. 18, 5766.
Fukada, S., Tanaka, M., Matsuyama, M., Kobayashi,

28
D. and Nagahama, Y. (1996). Isolation, characterization, and expression of cDNAs encoding the
medaka (Oryzias latipes) ovarian follicle .cytochrome P-450 aromatase. Mol. Reprod. Develop.
45, 285-290.
Jiang, J.-Q., Kobayashi, T., Ge, W., Kobayashi, H.,
Tanaka, M., Okamoto, M., Nonaka, Y. and Nagahama, Y. (1996). Fish testicular 11~-hydroxylase:
eDNA cloning and mRNA expression during spermatogenesis. FEBS Letters 397, 250-252.
Jiang, J.-Q., Yamashita, M., Yoshikuni, M., Fukada,S. and Nagahama, Y. (1996). Organization of cytoplasmic microtubules during maturation of goldfish
oocytes. Zool. Sci. 13, 899-907.
Kobayashi, T., Chang, X.-T., Nakamura, M., Kajiura,
H. and Nagahama, Y. (1996). Fish 3~-hydroxyster­oid dehydrogenase/A5-A4isomerase: antibody production and their use for the immunohistochemical
detection of fish steroidogenic tissues. Zool. Sci. 13,
909-914.
Kobayashi, D., Tanaka, M., Fukada, S. and Nagahama, Y. (1996). Steroidogenesis in the ovarianfollicles of the medaka (Oryzias latipes) during
vitellogenesis and oocyte maturation. Zool. Sci. 13,
921-927.
Mineyuki, Y., Aioi, H., Yamashita, M. and Nagahama,Y. (1996). A comparative study on stainability of
preprophase bands by the PSTAIR antibody. J.
Plant Res. 109, 185-192.
Miura, C., Miura, T., Yamauchi, K. and Nagahama, Y.
(1996). Hormonal induction of all stages of spermatogenesis in germ-somatic cell cultures fromimmature Japanese eel testis. Develop. Growth Differ. 38, 257-262.
Nagahama, Y., Miura, T., Kobayashi, T. and Ding J.
(1996). The role of activin in spermatogenesis infish. Proceedings of Serono Symposium (in press).
Nagahama, Y. (1996). 17a,20~-Dihydroxy-4-pregnen3-one, a maturation-inducing hormone in fish oocytes: Mechanisms of synthesis and action. Steroid
62, 190-196.
Nakamura, M., Specker, J.L. and Nagahama, Y.
(1996). Innervation of steroid-producing cells in theovary of tilapia Oreochromis niloticus. Zool. Sci.
13, 603-608.
Nogarni, A., Suzaki, T., Shigenaka, Y., Nagahama, Y.
and Mineyuki, Y. (1996). Effects of cycloheximide
on preprophase bands and prophase spindles inonion (Allium cepa L.) root tip cells. Protoplasma
192, 109-121.
Sakai, N., Suzuki, N. and Nagahama Y. (1996). Distribution of the site of 17 a,20~-dihydroxy-4pregnen-3-one production in the spermiating amago
salmon (Oncorhynchus rhodurus) testis. Biomed.
Res. 17, 473-477.
Sakai, N., Suzuki, N. and Nagahama, Y. (1996).
Involvement of cyclic AMP in the regulation of
testicular steroidogenesis of amago salmon(Oncorhynchus rhodurus). Biomed. Res. 17, 479486.

NATIONAL INSTITUTE

FOR BASIC BIOLOGYDIVISION OF CELL DIFFERENTIATION
Professor: Yoshiaki Suzuki
Associate Professor: Kohji Ueno
Research Associates: Kaoru Ohno
Hiraki Kokubo
Graduate Students: Katsuyoshi Matsunami (Graduate
University for Advanced Studies)
Yoshinori Ueno (Graduate Uni ve rsity
for Advanced Studies)Technical Staff: 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-! genes. The
other concerns with what the specificity of the
Bombyx body plan is and how the developmental
regulatory genes dictate a set of target genes in specifying the identities of various regions of the embryos.
I. Genes and factors that control the silk glanddevelopment and the silk gene transcription
We have been trying to understand the networks ofregulation hierarchy that function in the processes ofsilk gland development and differentiation. As a bottom-up type approach for this project, analyses on themolecular mechanisms that control the differential
transcription of the fibroin and sericin-1 genes in thesilk gland should provide an information about a partof the networks. In complementing this approach, a
top-down type approach should also help understand-29
ing the networks ; analyses of regulation hierarchy ofthe homeobox and other regulatory genes, and identification of their target genes expressed in the labial
segment, where the silk glands originate.
During the past year we have succeeded in purifying the SGF-2, a key transcription factor for the fibroin gene, which binds to FC, FD, and FE elementsin the enhancer I and enhances transcription specifically in the posterior silk gland (K. Ohno et al. , unpublished). The purification steps include SPSepharose FF, Source 30Q, Dyna beads-FE element
multimers, BioSilect 250, Mini S, and Mini Q. The
SGF-2 is a huge complex revealing an apparent molecular mass of about 1.1 MDa and accommodating
12 components; groups of p30/p32/p33, p36/p47B/
p50B/p55, and p45/p46/p47G/p48/p50G. Through apartial peptide sequencing and PCR analysis, p36 was
identified as a Lim homeodomain family protein, andp47B/p50B/p55 were as nuclear Lim interactor homologues. p30/p32/p33 were identified as variants of p25
which was known as an associating protein to the fibroin heavy chain-light chain complex. This finding
raises the possibility that the p25 variants might be achaperone to the transcription complex. By the use ofantibodies against p45, p47B, p47G, and p50G, it was
found that these components were not or barely detectable in the extract from the middle silk gland
where the fibroin gene is not expressed. Functional
reconstitution experiments as well as developmental
expression pattern analysis on these components arebeing planned.
Previously we reported cloning and labial segment-

Fig. I. Expression of SGF-1/Bm Fkh (A) and SGF-3/POU-M I (B) in embryonic silk gland. (A) At stage 23, signals are seenin the middle and posterior silk gland. Revised from Kokubo et al. (1996) . (B) At stage 21 , signals are weakened in the posterior
region. Revised from Kokubo et al. (1997) Dev. Genes Evol. 206, in press. Arrowheads indicate the borders between the anterior,
middle, and posterior silk gland. All scale bars represent I 00 Jlm.

30

specific expression of the Bombyx Scr. Silk glandspecific transcription factor-1 (SGF-1) is known tointeract with the SA site of the sericin-1 gene and FA
and FB sites of the fibroin gene. Also previously, we
reported the SGF-1 to be a new member of the fork
head/HNF-3{3 family. Expression patterns of SGF-11
jkh mRNA and protein in developing embryos werealso described. At stage 20, the transcripts and protein
were detected in the invaginating silk glands. Interestingly, preceding the appearance of the Bombyx Fkh
protein in the invaginating silk glands, Bombyx Scr
disappeared from the spots. This observation suggests
the possibility that the Bombyx Scr is necessary to
determine the nature of the labial segment and induce
the silk gland invagination accompanied by the
Bombyx jkh expression but the Scr protein is probably
not necessary for the direct induction of Bombyx jkh
expression in the invagination spots (Kokubo et al.,Dev. Bioi., in press).

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The following observations supported above suggestions. In the Nc!Nc embryos described by Itikawa
in 1944 that lack the Bombyx Antp gene (Nagata et al.,1996), we observed ectopic expression of Bombyx Scr
in the thoracic and abdominal segments. These ectopic expressions resulted in inducing ectopic formation of invaginating silk glands in the prothoracic, mesothoracic, and metathoracic segments all of which
revealed ectopic expression of Bombyx Fkh (Kokubo
et al., Dev. Bioi., in press).
By the time when the blastokinesis finishes (stage25) and the silk gland fully develops, the Bombyx jkh
transcripts and protein were restricted to the middle
and posterior regions of the silk gland (Fig. 1A).
These results suggest that besides the role of transcription factor for the silk genes the Bombyx jkh!
SGF-1 may play important roles during the silk glanddevelopment.
We reported that the POU-M1 which binds to theFig. 2. Expression of SGF-3/POU-Ml in embryos. (A) At stage 20, signals are seen in precursor cells of the prothoracic glands(pg), adductor plates (adp), abductor plates (abp), silk glands (silg), subbuccal glands (subg), corpora allata (ca), tracheal system(tr), and in some cells of the central nervous system (CNS). (B) At stage 21, the salivary glands are elongated from the posterior
part of the adp. Both invaginated cells in the anterior mandibular and posterior maxillary segments are fused to form the subg. (C)At stage 23, signals are also seen in the adp, abp, and the pg that reveal mature form. (D) Magnified in posterior region of anembryo at stage 21. Signals are seen in parts of the anus. (E) At stage 25, signals are seen in some cells of the brain (br) and thesuboesophageal ganglion (sg), and in the ca (arrows). (F) At stage 25, signals are seen in the salivary glands (salg) and the adp.
Anterior side is always to the left and dorsal side is up. All scale bars represent 100 J.l.m. Revised from Kokubo et al. (1997) Dev.
Genes Evol. 206, in press.

31

FOR BASIC BIOLOGY

Fig. 3. Expression of p260/p270 in the embryonic abdominal regions at stage 21A. The scale bar represents 100 IJlll.
Revised from Ueno and Suzuki (1997), J. Bioi. Chern. 272, 13519-13526.

SC site of the sericin-1 gene accommodates a PODdomain identical to Drosophila Cfl-a. The expressionof the POU-Ml gene has been analyzed in Bombyx
embryos by in situ hybridization and immunohistochemistry (Fig. 2). The gene was expressed specifically for the first time at stage 18-19 in a pair ofrestricted sites of the labial segment where a pair ofprothoracic glands is going to be formed by invagination. After the silk gland invagination, the POU-Ml
expression was detected in the developing silk glandand confined to the anterior and middle portions of
the silk gland by late embryonic stages (Fig. IB).
A Bombyx homologue of trachealess has beencloned, identified by sequence comparison, andnamed Bm trh (K. Matsunami et al., unpublished).
The Bm trh is expressed first at the invagination sitesof trachea, and continued to be expressed along thetrachea development. Later, it is also expressed in theinvagination sites of the silk glands. The expression in
the silk gland continues along the silk gland development but disappears first in the posterior silk glandand then in the middle silk gland leaving the expression only in the anterior silk gland. This expression
pattern differs from that of trh in the Drosophila salivary gland.

D. Genes associateddevelopment

withabdominal

leg

Through the studies on the mechanisms of
abodominal leg development, we found that a highmolecular weight protein (p260/270) was expressed
specifically in abdominal leg cells (Fig. 3) during
early embryonic stages and disappeared by a latestage. p260/270 consists of two polypeptides withmolecular weights of 260 and 270 kDa. We have established a purification procedure for p260/270 andhave raised an antibody against p260/270. Immunoblot analysis of the Eca;Eca (additional crescent) and£ Nj£N (new additional crescent) mutants (ltikawa,1943), which lack the Bombyx abdominal-A gene
(Ueno et al. , 1992) and therefore do not express abdominal legs, demonstrated that both mutants lacked

p260/270. Therefore we speculate the expression of
p260/270 may be regulated by the Bombyx abdominal-A gene. eDNA cloning and sequencing
demonstrated that p260 and 270 have structures similar to rat fatty acid synthase, which synthesizes
palmitate. Most of the enzymatic domains for palmitate synthesis were well conserved in the amino acidsequences of p260 and p270, while the thioesterase
domains of p260 and p270 were less conserved to thatof rat fatty acid synthase (Ueno and Suzuki (1997),
J. Bioi. Chern. 272, 13519-13526). Purified p260/270
can transfer palmitate to cysteine residues of synthetic
peptides in vitro . We propose that p260/270 may be
involved in protein palmitoylation and may function
in abdominal leg development.

Publication List:
Kokubo, H., Takiya, S., Mach, V. and Suzuki, Y.
( 1996) Spatial and temporal expression pattern ofBombyx fork head/SGF-1 gene in embryogenesis.
Dev. Genes Evol. 206, 80-85.
Mach, V., Ohno, K., Kokubo, H. and Suzuki, Y.
( 1996) The Drosophila Fork head factor directly
controls larval salivary gland-specific expression of
the glue protein gene Sgs3. Nucl. Acids Res. 24,
2387-2394.
Nagata, T., Suzuki, Y., Ueno, K., Kokubo, H., Xu, X.,
Hui, C.-c., Hara, W. and Fukuta, M. (1996) Developmental expression of the Bombyx Antennapedia
homologue and homeotic changes in the Nc mutant.
Genes to Cells 1, 555-568.

32

NATIONAL INSTITUTEDIVISION OF MORPHOGENESIS
Professor: Goro Eguchi
Associate Professor: Ryuji Kodama
Research Associates: Makoto Mochii
Mitsuko Kosaka
Visiting Scientists: Takamasa S. Yamamoto
Akio Iio (from Chubu National
Hospital)
Shin-ichi Higashijima (from Japan
Science and Technology Corporation)
Panagiotis A. Tsonis (from Universityof Dayton)
Katia Del Rio-Tsonis (from Universityof Dayton)
Thomas Reh (from University ofWashington)
Graduate Student: Harutoshi Hayashi (from School of
Agriculture, University of Tokyo)
Technical Staffs: Chiyo Takagi
Sanae OkaIn the newt and some other limited animal species,
the lens and neural retina can be completely regenerated through transdifferentiation of pigmented epithelial cells (PECs). Such a phenomenon, transdifferentiation, as observed in regeneration 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 using in vivo andin vitro systems, and our in vitro studies have revealed
that dormant potential of PECs to transdifferentiate
into lens cells is widely conserved throughout vertebrate species and that such potential becomes evident
when PECs are cultured in vitro. Such transdifferentiation can be accelerated in the presence of phenylthiourea and testicular hyaluronidase and cells passes
through a dedifferentiated status, where expression of
PEC-specific genes, such as mmpll5, tyrosinase,
TRP-1, and pP344 are suppressed. On the other hand,c-myc and pax6 expressions are elevated in the dedifferentiated PECs. We have extended our study to theregulatory mechanisms controlling gene expression
during transdifferentiation and following results are
obtained.

I. In vitro culture system of iris pigmented
epithelial cells for molecular analysis of lensregeneration
We have succeeded in purely isolating and cultivating the iris pigmented epithelial cells of 2 day-old
chick using chemical treatment by dispase and EDTA.
The iris PE cells continued to proliferate very stably
in the control medium and transdifferentiate into lens
cells by the addition of only two defined growth factors to the culture medium. The process of transdifferentiation in this system is very reproducible and thisculture system of the iris PE has made it possible toprepare homogeneous cell population at various
stages during the transdifferentiation process, thus
providing the way for biochemical and molecular bio-

logical analyses. The Northern blot analysis suggeststhat the transcription level of pax6 gene closely correlate with the progression of the transdifferentiation
process of the iris PE cells to the lens.

II. 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 encode abasic-helix-loop-helix-leucine zipper (bHLHzip) transcription factor, which is a possible regulatory genefunctioning in PEC differentiation. We have revealed
that mi is specifically expressed in PECs and precursors of PECs in the embryo, and that mi starts to be
expressed at the optic vesicle stage when no othermarker genes are expressed yet. Expression of mi isalso correlated with PEC differentiation in the culture
condition, where mi expression is down-regulated
under the condition for dedifferentiation but up-regulated under the condition for redifferentiation.
In order to elucidate the role of mi in PEC differentiation, cultured PECs were infected with a retrovirus
construction including mi sequence. The mi overexpression did not cause significant changes in morphology but caused a dramatic change in the pattern ofgene expression. PECs infected with the retrovirus
encoding mi continued to express mmpl15, tyrosinase
and TRP-1 but not pP344 even under a culture condition which promote dedifferentiation. Transfection
and CAT assay revealed that mi transactivates
mmpl15 promoter. These results suggest that mi has acritical role in PEC differentiation by activating
mmpll5, tyrosinase and TRP-1. Moreover, the resultthat pP344 was not transactivated by mi expression
suggested the presence of another regulatory factor in
PEC differentiation which is required to promote
pP344 expression. Our findings revealed that at least
a part of the changes in gene expression pattern accompanying dedifferentiation is regulated by mi inactivation which is induced by dedifferentiation signals.

III. Analysis of silver quail mutant
In the homozygous silver mutant of Japanese quail(Coturnix coturnix japonica ), a part of the outer layerof retina, which normally differentiates into the pigmented epithelium of the retina, forms an ectopic
neural retina tissue (Fig. 1). The ectopic neural retina
was suggested to be formed through transdifferentiation from pigmented epithelium according to morphological observations, but there have been no direct
evidence. We found that the outer layer of retina insilver homozygote once differentiates to pigmented
epithelium judging from the expression of mmp 115 in
4-day embryo. After 5th day of incubation, however,
the outer layer starts to express neural marker genes
which indicates the beginning of the transdifferentiation into the neural retina. The process to form theectopic neural retina in silver embryo is, therefore, a33

FOR BASIC BIOLOGY
- ~

...~,

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.

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. ·~
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•

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Fig. l. Ectopic neural retina in silver mutant quail.
Retinal section of 7-day-old embryo from sil ver homozygote was stained with anti-neurofilament antibod y. Ectopic neural retina
funned via transdifferentiation of pigmented epithelium is positively stained (*).
NR: original neural retinatransdifferentiation process. Furthermore, we analysed
the sequence of mi gene in the silver homozygote andfound that there is a two-base deletion which causes
a production of truncated mi protein. We have confirmed using an in vitro assay that the truncated miprotein has less activity in activating the mmpl15
promoter. These results suggest that the mutation in
the mi gene induces transdifferentiation of PECs toneural retina cells in silver homozygote.

IV. Isolation of a novel chick homologue of Serrate
and its coexpression with C-Notch-1 in chickdevelopment
Intercellular signaling mediated by the transmembrane proteins, Notch as receptor and its ligands,
Delta and Serrate, plays essential roles in thedevelopmental fate decision of many cell types in
Drosophila. The Notch genes are highly conserved
both in invertebrates and in vertebrates, suggestingthat Notch pathway regulates cell fate decisions
during vertebrates development.
As a starting point of our study on the role of the
Notch pathway in the control of the differentiation ofocular tissues, we cloned homologues of Notch , Delta

and Serrate in the chicken. Among them , there was anovel chick homologue of Drosophila Serrate, named
as C-Serrate-2. We examined the expression patternsof C-Serrate-2 and other homologs during the earlychick development using whole-mount in situ hybridization . Tissues with conspicuous expression of Notch
receptors and ligands included the forebrain , the myotome and the apical ectodermal ridge (AER) of thelimb bud of a 4-day-old chick embryo.
In the early development of ocular tissues, C-Serrate-1 expression was observed in the thickened ectoderm of the lens primordium at stage 13 (Fig. 2). At
stage 14, C-Notch-1 transcripts were coexpressed with
C-Serrate-1 in the lens placode. When the lens placode invaginates to form a lens vesicle in 3-day-embryos (stage 18-21), C-Serrate-1 expression wasrestricted to the posterior region of the lens containing
differentiating lens fiber cells. On the other hand, CNotch-1 was restricted to the anterior region of thelens containing proliferating cells.
These combination of expression patterns during
developmental processes is useful in predicting possible roles and mode of functions of intercellular signalling through the Notch pathway.

34

NATIONAL INSTITUTE

Sl

Sl

Sl

a

Fig. 2. Expression of C-Serrate-1 and C-Notch-1 in the eye primordium of the chick embryo.
Embryos at stage 13 (a), stage 14 (b, d) and stage 18-21 (c, e) were hybridized with C-Serrate-1 (S1) or C-Notch-1 (N1) probes .
LP: lens placode, L: lens, OC: optic cup, the scale bar: 100 ~m

Publication List:
Mazaki, Y., Mochii, M., Kodama, R. and Eguchi, G.
(1996) Role of integrins in differentiation of chickretinal pigmented epithelial cells in vitro. Develop.
Growth Differ. 38, 429-437.
Ono, T. , Murakami, T., Tanabe, Y., Mizutani, M.,
Mochii, M. and Eguchi, G. (1996) Culture of naked
quail (Coturnix coturnix japonica) ova in vitro foravian transgenesis: culture from the single-cellstage to hatching with pH-adjusted chicken thickalbumen. Camp. Biochem. Physiol. 113A(3), 287292.
Sawada, K., Agata, K. and Eguchi, G. (1996) Characterization of terminally differentiated cell state by
categorizing eDNA clones derived from chicken
lens fibers. Int. J. Dev. Bioi. 40, 531-535.
Furukawa, K., Yamamoto, T., Takamune, K.,
Sugimoto, Y., Eguchi, G. and Abe, S.-1. (1996)
Genesis of newt sperm axial fiber: eDNA cloning
and expression of a 29 kDa protein, a major component of the axial fiber, during spermatogenesis. Int.
J. Dev. Bioi. 40(6), 1109-1118.
Hayashi, H., Mochii, M., Kodama, R., Hamada, Y.,
Mizuno, N., Eguchi, G. and Tachi, C. (1996) Isolation of a novel chick homolog of Serrate and its
coexpression with C-Notch-1 in chick development.
Int. J. Dev. Bioi. 40(6), 1089-1096.

FOR BASIC BIOLOGYDIVISION OF DEVELOPMENTAL BIOLOGY(ADJUNCT)
Professor: Kenzo Nakamura (Nagoya University)
Associate Professor: Tsukaho Hattori (Mie University)
Research Associate: Masa-aki Ohto
Graduate Students: Yasuhiro Koide (Nagoya University)
Hideyuki Hirano (Nagoya University)
Masamitsu Kuriyama (Nagoya
University)
Yukiko Iwata (Nagoya University)

Our research attention has been focused in the areaof developmental control of gene expression and cellfunction related to the storage tissues and organs inplants, in particular mechanisms involved in the regulation of gene expression in response to the level ofsugars and mechanisms involved in the targeting ofstorage proteins to the vacuole are studied.

I. Regulatory factors involved in the sugarinducible expression of plant genes
Availability of sugars and inter-organ transport ofsugars are essential in the growth and development ofplants. Expression of a variety of genes is now known
to be regulated, either positively or negatively depending on the gene, by the level of sugars at thesugar-importing sink sites as well as at the sugar-exporting source sites of the plant body. Thus sugars are
not only important as sources for cellular energy andmacromolecules but also as signalling molecules controlling the growth and development by changing thepattern of gene expression in plants.
Although genes coding for sporamin and ~-amy­lase, two major proteins of the tuberous root, are expressed specifically in tuberous roots of sweet potato
under the normal growth conditions, their expression
is inducible by high levels of sugars in other vegetative tissues. Expression of GUS reporter genes under
the control of promoters of the sporamin gene or the~-amylase gene is inducible by sugars in leaves oftransgenic tobacco plants, and these fusion genes are
expressed in tubers of transgenic potato plants. It issuggested that massive transport of sugars to developing tuberous roots plays an important role in maintaining the high-level expression of sporamin and ~­amylase genes. The sugar-inducible expression of
sporamin and ~-amylase genes requires synergistic
action of two separate cis regulatory elements in eachcase, and one of the element is shared between thetwo genes. The activity to bind this conserved sequence motif in the nuclear extract increased upontreatment of the tissue witb sucrose. A bZIP-type
DNA binding protein and a novel DNA binding protein, which contains unique Zn-finger motif conserved
among several plant DNA binding proteins, bind toother regulatory elements.
Experiments with various inhibitors suggested thatthe sugar-inducible expression of sporamin and ~­amylase genes requires continuous dephosphorylationof proteins, Ca2+-signalling and the activity of protein

35

kinase. It was also found that in leaves of tobacco, thelevel of 54 kDa-protein with autophosphorylation
acitivity in the plasma membrane increases upontreatment of the tissue with sucrose. The 54 kDa
autophosphorylation protein was solubilized from theplasma membrane by 1% sodium deoxycholate(DOC), and it was purified to about 1,000-fold compared to the crude extract. The 54 kDa-protein phosphorylated histone IllS in a Ca2+-dependent mannerand it cross-reacted with antibody raised against calcium-dependent protein kinase (CDPK) of Arabidopsis thaliana. By RT-PCR reaction with primers
designed from sequence motifs highly conserved
among plant CDPKs, we identified 8 eDNA fragmentsfrom leaves of tobacco which codes for different
isoforms of CDPKs. Antibodies raised against kinasedomains of two of these isoforms did not cross-reacted with the 54 kDa-protein. These results suggestthat the 54 kDa-protein is a novel isoform of tobacco
CDPK which is tightly associated with the plasma
membrane.

II. Mutants of Arabidopsis thaliana with alteredpatterns of the sugar-inducible gene expression
Similar to the ~-amylase gene of sweet potato, expression of the ~-amylase gene of Arabidopsis
thaliana (AT/3-Amy) occurs in response to high levelsof sugars. We identified several mutants ofArabidopsis which showed altered response of theexpression of AT/3-Amy to sugars in leaves. A recessive mutation, lbal, significantly reduced the level ofexpression of AT/3-Amy under high levels of sugars.
By contrast, a recessive mutation, hbal, caused increased levels of expression of AT{3-Amy in response
to lower levels of sugars, the results suggesting that
HBAJ might function to maintain low level expression of AT/3-Amy until the level of sugars reaches
some high levels. It is suggested that the expression of
AT/3-Amy is regulated by a combination of both positive and negative regulation depending on the level ofsugars.

III. Derepression from negative regulation in thesugar-inducible expression of ~-amylase gene of
Arabidopsis thaliana
In addition to the AT/3-Amy:GUS fusion gene, inwhich the sequence from -1,587 to +136 of AT/3-Amy
was fused with gus, the AT{3-Amy[il-172}:GUS fusion
gene where the 5' -upstream of the promoter was truncated to -172, showed sugar-inducible expression inleaves of transgenic Arabidopsis plants in a manner
similar to the endogenous AT/3-Amy. A sequence between -172 and -61 of AT/3-Amy was essential for thesugar-inducible expression and it conferred sugar-inducibility on the -89 core promoter of CaMV 35S.
Linker scanning of the sequence between -172 and-62 in the AT{3-Amy[il-172]:GUS fusion gene identified two cis-regulatory elements. When sequence substitutions were made in the negative regulatory region36

A, levels of GUS actiVIty in non-treated leaves increased to those in sugar-treated leaves. On the other
hand, substitutions in the positive regulatory region B
abolished the expression. The region A contained asequence which is highly homologous to the sequencemotif that is conserved between sporamin and ~-amy­lase genes of sweet potato and required for theirsugar-inducible expression.
In contrast to AT/3-Amy:GUS, AT/3-Amy[-1-172]:
GUS did not show increased sensitivity to low levelsof sugars in hbal mutant plants. In the wild type
plants, the 5' -truncation of the promoter to -172, but
not to -310, caused increased sensitivity to low levelsof sugars similar to the pattern of expression of AT/3Amy in hbal plants. These results suggest that thenegative regulation by HBAJ might require ciselement(s) located between -310 and -173, and further support that the sugar-inducible expression of
AT/3-Amy might involve derepression from negative
regulations.
IV. Screening of mutants of Arabidopsis thalianawith defective growth and development
Neither lbal nor hbal mutation affected the sugarregulated gene expression in general suggesting thatsugar-regulated expression of a variety of plant genes
is mediated by multiple mechanisms. Nevertheless,lbal and hbal mutant plants showed pleiotropic effects on the sugar-inducible accumulation of anthocyanin. Furthermore, these mutant plants showed
several characteristic growth properties. In particular,
several growth and developmental properties of
Arabidopsis plants, which in wild type plants occurs
in response to changing levels of sugars, are affectedin these mutant plants. To obtain further insights into
the role of the sugar-regulated gene expression in thegrowth and development of plants, research attention
will be focused on screening of mutants ofArabidopsis thaliana with defects or anomalies in thegrowth and organ development that are affected by
the availability of sugars in the wild-type plants. To
aid this purpose, we are constructing increasing number of mutagenized plants after transformation withmultiple copies of enhancer fragments in the T-DNA.

NATIONAL INSTITUTE37

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 development ofcentral nervous systems in mice, chickens and Drosophila, andalso on the molecular mechanisms for the response of plants
toward external environments, such as light, temperature andsalinity.

38

DIVISION OF MOLECULAR NEUROBIOLOGY
Professor: Masaharu Noda
Research Associates: Nobuaki Maeda
Masahito Yamagata
Eiji Watanabe
NIBB Fellow: Masakazu Takahashi
Graduate Students: Hiraki Hamanaka (-Sept. 30, 1996)
Junichi Yuasa
Takafumi Shintani
Taeko Nishiwaki
Chika Saegusa (Oct. 1, 1996 -)Visiting Scientists: 1kuko Watakabe (Oct. 1, 1996 -)Tatsunori Yamamoto* (-Aug. 31,
1996)
Technical Staffs: Akiko Kawai
Shigemi Ohsugi(*from Nagoya University)

We study the molecular and cellular mechanisms
underlying the development of the vertebrate central
nervous system. Our experiments are designed to analyze molecules and structures involved in regulationof various cellular events in brain morphogenesis suchas generation of neuroblasts, their migration to form
laminar structures and various nuclei, elongation andpath-finding of neural processes, and the formation
and refinement of specific connections between neurons. These studies utilize various techniques, including molecular biological (e.g. eDNA cloning,site-directed mutagenesis), biochemical (protein, carbohydrate) and immunological methods (monoclonalantibody production), in addition to neuroanatomy,
cell and organotypic culture (immortalized-cell production), and embryo manipulation (classical embryology, gene transfer with viral vectors, and genetargeting).

I. Retinotectal projection map and synapse
formation
Topographic maps of neuronal connectivity havebeen reported for various parts of the nervous system.
In the visual system of birds, retinal ganglion cellaxons from the nasal (anterior) retina connect to a
caudal (posterior) part of the midbrain visual center,the optic tectum, and temporal (posterior) retinal
axons connect to the rostral (anterior) part, thereby
establishing a point-to-point projection map. To understand the development of the retinotectal projection map, Sperry formulated the chemoaffinity theory
in 1963, which is generally accepted today. His notionof chemoaffinity consists of five elements: (1) neurons are intrinsically different from each other, (2)these differences are position-dependent, (3) the differences are acquired very early, independently of
their connection partners, (4) the differences are biochemical in nature, and (5) presynaptic and postsynaptic cells with matching biochemical labels connect
with one another in a specific manner. Subscribing tothis hypothesis, we employed a subtractive hybridization technique to identify molecules that display

NATIONAL INSTITUTE

asymmetrical distributions between the nasal and thetemporal retina. We discovered several distinct transcripts which are topographically expressed in theretina. Among these position-specific molecules, twowinged-helix transcriptional regulators named CBF-1
and CBF-2 were expressed in the nasal and temporal
retina, respectively. The winged-helix domain is a
DNA-binding motif found in a family of transcription
factors such as Drosophila forkhead and vertebrate
HNF-3. By in situ hybridization, it was revealed thatCBF-1 and CBF-2 transcripts began to be detected
topographically in the primordial retina by embryonicday (E) 2, before birth of the retinal ganglion cells.
Their topographic expression in the retina ceased by
E10, prior to the onset of retinotectal connections.
Misexpression of each factor in the retina using a
replication-competent retroviral vector caused
misprojection on the tectum along the rostro-caudal
axis, suggesting that they determine the naso-temporalaxis of the retina, and consequently specify the topographic projection of the retinal ganglion cell axons to
the tectum by controlling expression of their target
genes (Fig. 1). Our results provide evidence for thechemoaffinity theory, and this approach is promisingfor systematic discovery of a series of topographic
molecules, determination of their functional hierarchy,and elucidation of the molecular and cellular mechanisms that generate the topographic map.
After reaching their appropriate sites along therostro-caudal and dorso-ventral axes of the tectum
retinal axons begin to seek their appropriate lamin~
termination sites among 15 distinct laminae within thetectum. Our second aim is to identify molecular andcellular cues for ingrowing axons to arborize in appropriate laminae, to recognize particular postsynaptic
partners, and finally to establish functional synaptic
connections. Molecular and cellular studies for thispurpose are currently underway.

II. Receptor-like protein tyrosine phosphatases andbrain development
Protein tyrosine phosphorylation is implicated in
the various aspects of brain development. The pattern
and level of tyrosine phosphorylation ofthe cellular
proteins are controlled by a series of protein tyrosine
kinases and protein tyrosine phosphatases (PTPs). In
recent years, many classes of receptor-like PTPs
(RPTPs) have been cloned and characterized. In 1994,
we identified a brain-specific extracellular chondroitin
sulfate proteoglycan, 6B4 proteoglycan/phosphacan
as a splice variant of a proteoglycan-type RPTP,
PTPt;/RPTP~. PTP~ and 6B4 proteoglycan have beensuggested to play some roles in neurite outgrowth,
neuronal cell migration and synapse formation. To
understand the functions of these molecules, it is crucial to dissect the molecular bases of the signal transductions. Therefore, we attempted firstly to identify
the ligands of PTP~.
Since 6B4 proteoglycan was considered to be identical to the extracellular portion of PTP~ including

FOR BASIC BIOLOGY

39

a)RetinaOptic Tectum

Fig. 1. CBF-1 and CBF-2 specify the retinotectal projection map(a) CBF-1 and CBF-2 are expressed in the nasal and temporal retina, respectively. (b, c) Recombinant retrovirus-mediatedmisexpression of each factor changed the retinotectal projection map.

carbohydrate modifications, we used this molecule foraffinity chromatography to purify PTPs-binding proteins. From the CHAPS extract of rat brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose.
N-terminal amino acid sequencing identified the 18kDa protein as pleiotrophin/heparin-binding growthassociated molecule (HB-GAM), which was originally isolated from the brain as a mitogenic andneurite-promoting factor. Scatchard analysis of 6B4
proteoglycan-pleiotrophin binding revealed low (Kd =3 nM) and high (Kd = 0.25 nM) affinity binding sites.
Chondroitinase ABC digestion of the proteoglycan
reduced the binding affinities to a single value (Kd =13 nM) without affecting the number of binding sites.
This suggested the presence of two subpopulations of
the proteoglycan with different chondroitin sulfate
structures. The binding of 6B4 proteoglycan to pleiotrophin was inhibited differently by various chondroitin sulfates. Interestingly, in contrast to chondroitin
sulfate C which strongly inhibited binding of 6B4
proteoglycan to pleiotrophin (IC 50 = 400 ng/ml), chondroitin sulfate A had almost no effect. These results

suggested that chondroitin sulfate chains participate in
the ligand-receptor interaction.
Immunofluorescence analysis indicated that both
6B4 proteoglycan and PTPs are localized on corticalneurons especially at the growth cones of their extending neurites. Anti-6B4 proteoglycan antibody
added to the culture medium strongly suppressed
pleiotrophin-induced neurite outgrowth of corticalneurons . Chondroitin sulfate C, but not chondroitin
sulfate A, potently inhibited pleiotrophin-induced
neurite outgrowth, consistently with observations regarding inhibition of the ligand-receptor interaction.
These results suggested that pleiotrophin is a functional ligand for PTPs.
PTPs has another family member, RPTPy, whichshows a high degree of structural similarity to PTPs.
Pleiotrophin also has another family member,
midkine, which also has mitogenic and neurite-outgrowth promoting activities. Therefore, the ligand-receptor relationship between pleiotrophin, midkine andPTPs, RPTPy is an important question which should
be addressed. As the first step, we cloned RPTPy
cDNAs to reveal its molecular diversity. eDNA clones

40

NATIONAL INSTITUTE

CAH

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PTP
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-DII

RPTPy-A

RPTPy-B

RPTPy-C

RPTPy-S

Fig. 2. Schematic representation of RPTPy isoforms
RPTPy consists of a carbonic anhydrase-like domain (CAH; open circles), a fibronectin type III-like domain (FN III; hatchedboxes), a transmembrane segment (TM; open boxes) and two tyrosi ne phosphatase domains (PTP-DI and -DII; shaded boxes). The
dashed line indicates the deleted sequence in RPTPy-B. RPTPy-C possesses only a single tyrosine phosphatase domain, and RPTPyS is an extracellular variant of RPTPy.

for the four splicing variants of RPTPy were isolated
from rat brain eDNA libraries (Fig. 2). We designatedthese molecules as RPTPy-A, -B, -C and -S. RPTPyA was the longest form and had a similar structure tohuman and mouse RPTPy. RPTPy-B was devoid of
the intracellular juxtamembrane 29 amino acids of
RPTPy-A. Recently, this type was independently
found in a human kidney-derived cell line, ACHN, bySorio et a!. The other two variants were novel:
RPTPy-C had a single phosphatase domain, andRPTPy-S was a secretory type of RPTPy. mRNAs ofthese four species were expressed in the brain, kidney,lung and heart. Transfection of RPTPy-A and -S expression plasmids into COS7 cells resulted in the expression of membrane-bound 190- and secretory 120kDa proteins, respectively. RPTPy is thus comparable
to PTPs with regard not only to structure but also to
the presence of both secretory and transmembrane
forms. However, any RPTPy variants ectopically expressed in these cells did not exist as proteoglycans
contrasting to PTPs variants.

III. Gene targeting of Na-G, a voltage-gatedsodium channel expressed in glial cells
Glial cells have been considered to be inexcitable.

Despite the lack of electrical excitability, they express
voltage-gated sodium channels with properties similar
to the sodium channels in excitable cells. The cellularfunction of these voltage-gated sodium channels is notclear. An in vitro study raised the possibility that glial
sodium channels serve as a pathway for sodium-ion
entry to fuel Na-K ATPase, which requires three sodium ions for transport of two potassium ions. A dropin the intracellular concentration of sodium ion concentration through these channels may hamper
ATPase activity. To test this hypothesis and clarify theroles of glial sodium channels in vivo, we are currently attempting to generate knock-out mice deficientin the glial voltage-gated sodium channel, Na-G, theeDNA of which was recently cloned. We have successfully produced chimeric mice which contain thetargeted embryonic stem-cells contribution.
By applying gene targeting technology to othernovel genes expressed in the brain, we intend to investigate the molecular mechanisms underlying thedevelopment and function of the brain.

FOR BASIC BIOLOGY

Publication List:
1. Original Papers
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, 161-183.
Maeda, N. and Noda, M. (1996) 6B4 proteoglycan/
phosphacan is a repulsive substratum but promotes
morphological differentiation of cortical neurons.
Development 122, 647-658.
Nishizuka, M., Ikeda, S., Arai, Y., Maeda, N. andNoda, M. (1996) Cell surface-associated extracellular distribution of a neural proteoglycan, 6B4 proteoglycanlphosphacan, in the olfactory epithelium,
olfactory nerve, and cells migrating along the olfactory nerve in chick embryos. Neurosci. Res. 24,
345-355.
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. 44, 84-96.
Watanabe, E., Kushima, Y. and Oohira, A. (1996)
Heparan sulfate proteoglycan associated with growing parallel fibers of granule cells in the rat cerebellar cortex. J. Brain Sci. 22, 67-75.
Maeda, N., Nishiwaki, T., Shintani, T., Hamanaka, H.
and Noda, M. (1996) 6B4 proteoglycan/phosphacan, an extracellular variant of receptor-likeprotein tyrosine phosphatase 1;/RPTP~, binds pleiotrophin/HB-GAM. J. Bioi. Chem. 271, 21446-21452.
Yuasa, J., Hirano, S., Yamagata, M. and Noda, M.
(1996) Visual projection map specified by expression of transcription factors in the retina. Nature
382, 632-635.
Shintani, T., Maeda, N., Nishiwaki, T. and Noda, M.
(1997) Characterization of rat receptor-like protein
tyrosine phosphatase y isoforms. Biochem. Biophys.
Res. Comm., 230, 419-425.
2. Proceedings
Maeda, N. and Noda, M. (1996) 6B4 proteoglycan/
phosphacan and development of the brain. In "Generation of Neuronal Diversity and Specificity in the
Brain", pp. 66-70. (Taniguchi International Symposium)
Yamagata, M. and Noda, M. (1996) Layer-specificneuronal connections in the retinotectal system. In"Generation of Neuronal Diversity and Specificity
in the Brain", pp. 101-103. (Taniguchi International
Symposium)
Yamagata, M., Takahashi, M. and Noda, M. (1996)
Expression of ezrin in a subset of the chick retinotectal projection. (Society for Neuroscience Abstract)

41

42

NATIONAL INSTITUTEDIVISION OF CELLULAR REGULATION
Professor: Norio Murata
Associate Professor: Hajime Wada 11 (adjunct)
Research Associates: Atsushi Sakamoto
Yoshitaka Nishiyama/wane Suzuki
Monbusho Foreign Scientists: Malay Kumar Ray 2i
Zoltan Gombos 3i
NIBB Fellow: Akira Katoh
JSPS Visiting Scientists: Ding-Ji Shi 4i
Eva Hideg 3i
P. Pardha Saradhi 5i
Diego de Mendoza 61
Richard A. Dilley 7!
Peddisetty Sharmila 5 i
JSPS Postdoctoral Fellows: Alia 5 i
Michael P. Malakhov 8i
EC Fellow: Ana Maria Otero Casal 9i
NIBB Visiting Scientists: Yasushi Tasaka
Oxana Malakhov 10i
Monika Debreczeny 31
Prasanna Mohanty 11 i
Byong Yang Moon 12!
Leonid Kislov 81
Kostas Stamatakis 13!
Silvia Franceschelli 14 i
Laszlo Mustardy 3i
Nelly Tsvetkova 15!
Tony H. H. Chen 16i
Otto Zsiros 31
Dmitry A. Los 8i
Julian Eaton-Rye 171
Suleyman I. Allakhverdiev 18i
Graduate Students: Sayamrat Panpoom
Masami Inaba
Hiroshi Yamamoto
Yuji Tanaka
Fumiyasu Yamaguchi
Technical Staffs: Sho-lchi Higashi
Hideko Nonaka
n from Kyushu University, Fukuoka, Japan
from Centre for Cellular and Molecular Biology,
Hyderabad, India
3i from Biological Research Center, Szeged, Hungary
41 from Institute of Botany, Academia Sinica, Beijing,China
5 i from lamia Millia /slamia University, New Delhi, India
61 from Universidad Nacional de Rosario, Rosario,
Argentina
71 from Purdue University, Lafayette, IN, USA
8i from Plant Physiology Institute, Moscow, Russia
91 from Universidad de Santiago, Santiago de Compostela,Spain
101 from Institute of General Genetics, Moscow, Russia
11 i from Jawaharlal Nehru University, New Delhi, India
12i from Inje University, Kimhae, Korea
13i from National Centre for Scientific Research"Demokritos", Athens, Greece
141 from International Institute for Genetics andBiophysics, Naples, Italy
151 from Bulgarian Academy of Sciences, Sofia, Bulgaria
161 from Oregon State University, Corvallis, OR, USA
17i from Otago University, Dunedin, New Zealand
18i from Institute of Soil Science and Photosynthesis,
Moscow, Russia
2i

The research effort of this division is aimed at establishing a full understanding of the molecularmechanisms by which plants can acclimate to andtolerate stresses that arise from changes in environment conditions, with particular emphasis on temperature stress and salt stress. In 1996, significant progress
was made in research on the following topics in studies with cyanobacteria and higher plants as experimental materials.
I. The temperature-regulated expression of genes
for acyl-lipid desaturases in a cyanobacterium

Most living organisms are exposed to changes inthe temperature of their environment. In response to adecrease in temperature, an increase occurs in theextent of unsaturation of membrane lipids, which enhances the fluidity of the membranes. This mechanism provides compensation for the decrease in themolecular motion of membrane lipids, which is
caused by the decrease in ambient temperature. Individual acyl-lipid desaturases introduce a double bondinto fatty acids that are esterified to the glycerol
moiety of membrane lipids. The cyanobacterium
Synechocystis sp. PCC 6803 has four genes, desA,
desB, desC and desD, that encode the M2, ro3, ~9
and ~6 acyl-lipid desaturases, respectively. We investigated the regulation of the expression of the genes
for these desaturases in response to changes in temperature. We found that low temperatures enhance thesteady-state levels of mRNAs for all the desaturases
with the exception of the ~9 desaturase. Functional
analysis of promoters, using a bacterial gene for luciferase as a reporter, revealed that, to some extent, itis the activation of the promoters of the genes for the~12 and ro3 desaturases that is responsible for theincreases in the levels of their mRNAs after a downward shift in temperature. In addition, the lifetimes ofthe mRNAs for the ~6. ~12, and ro3 desaturases wereextended at lower temperatures, indicating that thestability of mRNAs might also be involved in thecontrol of the levels of the desaturases. Western blotting analysis demonstrated that the levels of the ~6,~12 and ro3 desaturases increase at low temperatures,
while the level of the ~9 desaturase remains constant.
Thes~ results indicate that the expression of the genefor the ~9 desaturase is basically independent of temperature, while the expression of the genes for the ~6.
~12, and ro3 desaturases is regulated by temperature.

II. Localization of acyl-lipid desaturases incyanobacterial cells
An immunocytochemical study was performed in
an attempt to localize the four acyl-lipid desaturases
in Synechocystis sp. PCC 6803 using antibodies raised
against synthetic oligopeptides that corresponded tothe carboxyl-termini of the individual desaturases.
The individual preparations of antibodies were specific to the respective desaturases. Immunogold labeling and electron microscopy revealed the specific

43

FOR BASIC BIOLOGY

distribution of the desaturases in regions that corresponded to both the cytoplasmic and the thylakoid
membranes, suggesting that all four desaturases arelocated in both types of membrane. Localization of
the desaturases in thylakoid membranes was further
confirmed by Western blotting of proteins from isolated membranes. These findings indicate that thedesaturation of the fatty acids of membrane lipids
occurs within the thylakoid membranes as well as
within the cytoplasmic membranes in the cyanobacterial cells.

III. The importance of polyunsaturated membrane
lipids in photosynthesis
We demonstrated previously that polyunsaturated
membrane lipids are important in the ability of thephotosynthetic machinery to tolerate low temperatures
by targeted disruption of genes for desaturases in
Synechocystis sp. PCC 6803, in which all polyunsaturated fatty acids of membrane lipids were
replaced, as a result, by monounsaturated fatty acids.
To confirm this finding using an alternative approach,
we introduced the desA gene for the .0.12 desaturase of
Synechocystis sp. PCC 6803 into cells of
Synechococcus sp. PCC 7942 that contained monounsaturated fatty acids but no polyunsaturated fatty
acids. As the result of this transformation, half of themonounsaturated fatty acids in the membrane lipids
were replaced by diunsaturated fatty acids . Comparison of the transformed cells with the wild-type
cells revealed that the increase in the number ofdouble bonds in the membrane lipids enhanced theability of the cells to resist photoinhibition at low
temperatures by accelerating the recovery of thephotosystem II complex from photoinhibitory damage. These findings indicate that polyunsaturated fatty
acids are important in the protection of the photosynthetic machinery from damage by strong light at
low temperatures.

IV. Acclimation to heat of the photosynthetic
machinery
The evolution of oxygen is one of the reactions ofphotosynthesis that is most susceptible to high temperature. The molecular mechanism underlying thestabilization of the photosynthetic machinery against
heat-induced inactivation has been studied in the
cyanobacterium Synechococcus sp. PCC 7002. We
demonstrated previously that cytochrome c550 , locatedin the lumen of thylakoids, is involved in the stabilityof the oxygen-evolving machinery at high temperatures . We searched for another factor that mightenhance the heat stability and recently identified aprotein of 13 kDa as an important factor. The geneTransformed

Wild type

0.1 M NaCI
Fig. I. Effects of salt stress on the growth of seedlings after germination of seeds of wild-type and transformed Arabidopsis
thaliana. Seeds from wild-type and transformed plants were germinated on a plate of Murashige-Skoog medium supplemented with
0.1 M NaCI and solidified with Gellan gum, and incubated at 22°C for 20 days .

44encoding the 13-kDa protein was cloned from
Synechococcus, and the deduced amino-acid sequence
revealed that this protein is homologous to the PsbU
protein, an extrinsic protein of photosystem II, whichhas previously been found in thermophilic species of
cyanobacteria. Inactivation of the gene in
Synechococcus sp. PCC 7942, by insertion of an
antibiotic-resistance gene cartridge, had no significant
effect on the oxygen-evolving activity or on thephotoautotrophic growth at temperatures within thephysiological range. However, when mutant cells andwild-type cells that had been grown at a physiologicaltemperature were exposed to a higher temperature, theoxygen-evolving activity was lost much more rapidly
from the mutant cells than from the wild type. These
results indicate that the PsbU protein plays an important role in stabilizing the oxygen-evolving machineryat high temperatures.

V. Genetic enhancement of stress tolerance inArabidopsis thaliana and in rice plants by genetic
engineering that allowed the biosynthesis ofglycinebetaine
Glycinebetaine (hereafter abbreviated as betaine) is
a compatible solute that is found in a number of halotolerant species of plants and bacteria. It has been
implicated in the protection of cellular functions
against salt and other types of environmental stress.
To examine the effect of betaine on the protection of
the photosynthetic machinery in vivo against salt
stress, we transformed Arabidopsis thaliana, which
does not normally accumulate betaine, with the codA
gene for choline oxidase (which catalyzes theconversion of choline to betaine) from Arthrobacter
globiformis (H. Hayashi et al. (1997) Plant 1., inpress). Transgenic Arabidopsis plants acquired theability to synthesize betaine and to tolerate salt stressthroughout their life cycle. Figure I shows theenhanced tolerance to salt stress (100 mM NaCl) ofseedlings of transgenic Arabidopsis. Furthermore, thetransformation enhanced the protection against lowtemperature photoinhibition and accelerated therecovery from photo-induced damage. Thus, accumulation of betaine both enhanced salt tolerance andcontributed to resistance to photoinhibition at low
temperature.
We also introduced the codA gene of A. globiformis
into rice, one of the most important crops worldwide,
aiming to enhance the salt tolerance of this cereal
crop. Transgenic rice plants expressed the codA gene
and accumulated betaine. Furthermore, the oxygenevolving machinery of the transgenic plants was moretolerant to salt stress than that of the wild-type plants.
These results demonstrate the potential usefulness ofthe codA gene in the engineering of stress tolerance ina wide variety of agronomically important crops.

NATIONAL INSTITUTE

Publication List:(1) Original articles
Allakhverdiev, S. 1., Feyziev, Ya. M., Ahmed, A.,
Hayashi, H., Aliev, Ja. A., Klimov, V. V., Murata, N.
and Carpentier, R. (1996) Stabilization of oxygen
evolution and primary electron transport reactions
in photosystem II against heat stress with glycinebetaine and sucrose. J. Photochem. Photobiol. 34,
149-157.
Gombos, Z., Wada, H., Varkonyi, Z., Los, D. A. and
Murata, N. (1996) Characterization of the Fad12
mutant of Synechocystis that is defective in 1112
acyl-lipid desaturase activity. Biochim. Biophys.
Acta 1299, 117-123.
Ishizaki-Nishizawa, 0., Fujii, T., Azuma, M., Sekiguchi, K., Murata, N., Ohtani, T. and Toguri, T.
(1996) Low-temperature resistance of higher plants
is significantly enhanced by a nonspecific cyanobacterial desaturase. Nature Biotech. 14, 10031006.
Malakhov, M., Wada, H., Los, D. A. and Murata, N.
(1996) The coxD gene for heme 0 synthase in
Synechocystis. Biochim. Biophys. Acta 1273, 8486.
Mustardy, L., Los, D. A., Gombos, Z. and Murata, N.
(1996) Immunocytochemical localization of acyllipid desaturases in cyanobacterial cells: Evidence
that both thylakoid membranes and cytoplasmic
membranes are sites of lipid desaturation. Proc.
Natl. Acad. Sci. U.S.A. 93, 10524-10527.
Nishida, 1., Swinhoe, R., Slabas, A. and Murata, N.
( 1996) Cloning of Brassica nap us CTP:
phosphocholine cytidylyltransferase cDNAs bycomplementation in a yeast cct mutant. Plant Mol.
Bioi. 31, 205-211.
Tasaka, Y., Gombos, Z., Nishiyama, Y., Mohanty, P.,
Ohba, T., Ohki, K. and Murata, N. (1996) Targetedofacyl-lipid
desaturasesinmutagenesis
Synechocystis: Evidence for the important roles ofpolyunsaturated membrane lipids in growth, respiration and photosynthesis. EMBO J. 15, 6416-6425.

(2) Review articles
Murata, N., Deshnium, P. and Tasaka, Y. (1996) Biosynthesis of y-linolenic acid in the cyanobacterium
Spirulina platensis. In y-Linolenic Acid (Y.-S.
Huang and D. E. Mills, eds.) pp. 22-32. AOCS
Press, Champaign, Illinois.
Nishida, I. and Murata, N. (1996) Chilling sensitivity
in plants and cyanobacteria: The crucial contribution of membrane lipids. Annu. Rev. Plant Physiol.
Plant Mol. Bioi. 47, 541-568.

FOR BASIC BIOLOGYDIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADJUNCT)
Professor (adjunct): Kimiyuki Satoh
Associate Professor (adjunct): Hirokazu Kobayashi
Research Associate: Noritoshi Inagaki
NIBB Fellow: Hiroshi Kuroda
Graduate Students: Yoshihiro Narusaka*
Tatsuya Torno*
Akihiko Tohri*(*from Okayama University)

From an energetic point of view, the conversion ofradiant energy into chemical energy in photosynthesisis the most important biological process on our planet.
The highly efficient energy conversion in this process
is ensured by the highly ordered organization of molecules in the photochemical reaction center, in aphysical, chemical and biological sense. The projectin this division is aiming to elucidate the dynamic aswell as static organization of molecules in the photosystem II (PSII) reaction center (RC) of oxygenic
photosynthesis which has a unique property to generate a strong oxidant leading to the extraction ofelectrons from water for the synthesis of organic compounds.
In the first approach, molecular organization of the
PSII RC, which has been identified in our study, will
be analyzed by biochemical and biophysical techniques which include crystallographic analysis,
chemical modification & cross-linking analysis andoptical & EPR spectroscopies. Structure-functional
analysis for the constituent subunits will also be conducted using random and site-directed mutants oftransformable algae, Synechocystis PCC 6803 andChlamydomonas reinhardtii. The principal target ofthese analyses is the structure and molecular environment of the RC chlorophylls, P-680, which determine
the redox potential of this photosystem.
In the second approach, the effort will be focusedon the elucidation of molecular mechanism of lightregulated metabolic turnover of a subunit of the PSII
RC, D 1 protein. Some of unique phenomena are
involved in this metabolic process; i.e., photo-damageof the function of photochemical RC, specific degradation by proteases of a photo-damaged protein
subunit, light-regulated gene expression at the translational level, post-translational cleavage of the Cterminal extension and incorporation of cofactors andsubunits into multi-component pigment-protein complexes.

I. Structural organization of photosystem IIreaction center(1) The purified PSII RC contains D1 and D2 proteins, a. and ~ subunits of cytochrome b-559 (Cytb559) and the psbl gene product. It also contains a
number of cofactors important in the primary processes of PSII, such as chlorophyll a (Chi a),pheophytin a (Pheo a) and ~-carotene (~-Car), as well45

as cytochrome b-559 heme. Taking advantage of biased distributions of amino acids in the constituent
protein subunits, we have conclusively demonstrated
that they are present in an equimolar ratio in the isolated complex. We also have re-evaluated the pigment
stoichiometry in the isolated PSII RC, by HPLC and
ICP emission spectroscopy, to be 6/2/2/1 for Chi a/
Pheo a/~-Car/Cytb-559.
(2) Pheo a molecule on the inactive branch of photochemical electron transport system in the isolated
PSII RC was identified by fluorescence excitation
spectroscopy at 77K, and confirmed by absorption,
linear dichroism (LD) and magnetic circular dichroism spectra (in collaboration with Dr. Mimuro ). Whenthe emission was monitored at 740 nm, photochemically active Pheo a was detected as peaks at 418, 513,
543, and 681 nm, as described in previous reports.
However, when the emission was monitored at 665
nm, two bands were observed at 414 and 537 nm·
these bands were insensitive to photochemical reduc~tion of the primary acceptor and thus appear to be dueto photochemically inactive Pheo a. Two ~-Car molecules in the complex were also discriminated byfluorescence excitation and LD spectra. These observations were discussed in the light of current understanding of the molecular organization of pigments
and the relationship of pigment arrangement to theoptical properties in the PSII RC.
(3) Pigments in the purified PSII RC were extractedwith diethyl ether containing varied amount of water.
As mentioned above, the preparation originally contained approximately six Chi a two ~-Car and two
Pheo a per one photochemically active Pheo a. The
treatment with 30 - 50% water-saturated ether solution extracted one ~-Car, as well as one Chi a absorbing at 677 nm, remaining 60% of the photochemical
activity to reduce Pheo a. With 60 - 80% water-saturated ether, almost all of the ~-Car in the RC was
extracted. However, nearly 50% of the photochemical
activity was retained in the complex, after the ether
treatment. The absorption, fluorescence excitation and
LD spectra demonstrated the presence of two spectral
forms of ~-Car in the PSII RC. The short-wavelength
form of ~-Car, with absorption peaks at 429, 458 and489 nm, was selectively extracted with ether at the
low water content, whereas the long-wavelength
form, with peaks at 443, 473 and 507 nm, was extracted only at the higher water content. The extraction of carotenoid enhanced the photobleaching ofChi. Based on the photobleaching experiments it wassuggested that Chi a forms with peaks at 667 and 675
nm are located close to the short-wavelength form of~-Car that can transfer excitation energy to thephotoactive Pheo a on the D1 protein.· Based on theseobservations, the topology of pigments in the PSII RC
was discussed.
(4) Isolated PSII RC contains no functional plastoquinone, QA and Q8. However, externally added
quinones, such as dibromomethylisopropyl benzoquinone (DBMIB), serve as the electron acceptor. The

46

NATIONAL INSTITUTE

present study provided evidence that DBMIB fully
replaces the function of QA and rapidly oxidized Pheoeven at 77 K (in collaboration with Dr. Itoh). The useof DBMIB in the nanosecond spectroscopy at Ti K
enabled us to inspect the spectrum of theP680+(DBMIB-) state that is fully reversible and contains neither the absorption change of the excitedstates nor the irreversible bleaching of the other pigments. The feature of the spectrum was discussed inrelation to the difference spectra of 3P680/P680 andPheo-JPheo couples measured at 77-280 K.

observation was that these translational intermediates
accumulated in illuminated chloroplasts by the combined addition of ATP and inhibitors of photosynthetic
electron transport, such as atrazine, 3-(3,4-dichlorophen y1)-1, 1-dimeth y1urea and3,5 -dibrome-4-h ydroxybenzonitrile. This indicates that the synthesis of
the full-length D1 protein requires a factor(s) generated by the operation of photosynthetic electrontransport. The accumulation of the full-length D1 protein, however, was induced even in the presence of
inhibitory concentration of atrazine in the light whenboth 2,6-dichlorophenolindophenol and ascorbate, asystem donating electrons to the photosystem I (PSI),
were also present, and in darkness as well upon the
addition of a reductant, dithiothreitol (DTT), suggesting that the translation of D1 protein is regulated via
redox change of a component(s) around the PSI.
The accumulation of these two intermediates was
also observed by pulse-labeling in in vitro run-off
translation system in darkness using thylakoid-boundpolysomes and oxidized S100 fraction prepared frompea chloroplasts. However, the synthesis of full-length
D1 protein was achieved in this system by the addition of DTT-reduced stroma fraction. The fractionationof DTT-reduced stroma by a gel-filtration column

II. Dynamic aspects of molecular organization(1) The light-regulated synthesis of the D1 protein
in PSII RC was analyzed using isolated pea chloroplasts. In the presence of externally added ATP in
darkness, isolated chloroplasts accumulated two proteins of about 22 and 24 kDa which precipitate withspecific antibodies raised against the D1 protein. By
chasing in the light, these proteins disappeared concomitant with the appearance of the precursor and themature forms of D1 protein. The pulse-chase experiment in the light and the polysome analysis unequivocally concluded that these two components were thepaused intermediates of D1 protein. An unexpected

nONA

4

~

><

mRNA

"pCt A/,:osit

sequeoce

c:pA gnal se//e(S)c(~Chloroplast

-8 2
.E
~
..... 0IV

ec. -2~
T:I: _ 4 ~----+-+---------------------~
S
100

200
Sequence

300

400

500

positionFig. 1. A schematic drawing of maturation steps and function of C-terminal processing protease (CtpA) for the precursor D1
protein. The CtpA is synthesized in cytosol as a precursor with the N-terminal extension, consisting .of a transit sequence (blue;T) and a signal sequence (red; S), for translocation into thylakoid lumen. The signals on the pCtpA are excised in two steps during
the import, as shown in the figure. The mature CtpA cleaves off C-terminal extension of the pD 1 protein as an integral event forthe organization of oxygen evolving machinery in the PSII. Inset shows a hydropathy plot of the spinach pCtpA by a program of
Kyte and Doolittle (1982). Two arrows indicate putative cleavage sites for the maturation of pCtpA.

FOR BASIC BIOLOGY

demonstrated that the major activity corresponded tothe molecular mass of about 40 kDa. The fractionation
by DEAE anion-exchange chromatography and byammonium sulfate precipitation of the active fraction
indicated that the purification of this redox-responding regulatory factor is possible by the standard biochemical procedures, although further steps of
purification is evidently required at present.
(2) The D1 protein of PSII RC is synthesized as aprecursor that is processed by cleavage at the C-terminus. The C-terminal processing is necessary for theassembly of water-oxidizing machinery (Mn-cluster).
The enzyme involved in this process was purified
from sonicated extracts of thylakoids, by a methodthat includes chromatography on QAE-anion exchange, hydroxylapatite, Cu-chelating affinity andgel-filtration columns. Based on the amino acid sequence data of the purified protease, eDNA clones
encoding the enzyme was identified and sequenced,
from a spinach green leaf eDNA library. By theseanalysis, the full-length transcript was established toconsist of 1906 nucleotides and a poly (A) tail, containing an open reading frame (ORF) corresponding
to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified proteasewith that deduced from nucleotide sequence of theeDNA clones, the enzyme was shown to be furnishedwith an extra amino-terminal extension consisted of
150 amino acids which is characteristic of both a transit peptide and a signal sequence. This suggests thatthe protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids (Fig. 1).
The enzyme can be expressed in E. coli in its active
form and purified in homogeneity.
Three sorts of compounds were used as substrate
for the analysis of enzymatic activity, i.e., (i) synthetic
oligopeptides corresponding to the C-terminal sequence of the precursor D1 protein, (ii) in vitro transcribed/translated precursor D1 protein, and (iii) in
vivo synthesized precursor D1 protein in thylakoid
membranes. A series of substitutions at Ala-345, forexample, were shown to have marked effects on thevalue of Vmax, in the previous analysis. Mixed culture
experiments using genetically-engineered Chlamydomonas mutants substituted at this position (fromSer to Val or Gly) demonstrated that these substitutions seriously affect the viability of this organism. No
specific inhibitor, except for substituted C-terminal
oligopeptides of precursor D 1 protein, could be found
for the enzyme. The identification of the catalytic
center of this unique enzyme is now in progress.
Publication List:

Inagaki, N., Yamamoto, Y., Mori, H. and Satoh, K.
(1996) Carboxyl-terminal processing protease forthe D 1 precursor protein: cloning and sequencing of
the spinach eDNA. Plant Mol. Bioi. 30, 39-50.
Itoh, S., Iwaki, M., Torno, T. and Satoh, K. (1996)
Dibromothymoquinone (DBMIB) replaces the function of QA at 77 K in the isolated photosystem II

47

reaction center (D1-D2-cytochrome b559 ) complex:
Difference spectrum of the P680+(DBMm-) state.
Plant Cell Physiol. 37, 833-839.
Kuroda, H., Kobashi, K., Kaseyama, H. and Satoh, K.
(1996) Possible involvement of a low redox potential component(s) downstream of photosystem I in
the translational regulation of the D 1 subunit of thephotosystem II reaction center in isolated pea chloroplasts. Plant Cell Physiol. 37, 754-761.
Narusaka, Y., Murakami, A., Saeki, M., Kobayashi,
H. and Satoh, K. (1996) Preliminary characterization of a photo-tolerant mutant of Synechocystis sp.
PCC 6803 obtained by in vitro random mutagenesisof psbA2. Plant Sci. 115, 361-366.
Satoh, K. (1996) Introduction to the photosystem IIreaction center -Isolation and biochemical and biophysical characterization-. in "Oxygenic Photosynthesis: The Light Reactions" (D.R. Ort & C.F.
Yocum eds), Kluwer Academic Publishers(Dordrecht), Vol. 4, pp.193-211.
Takahashi, Y., Utsumi, K., Yamamoto, Y., Hatano, A.
and Satoh K. (1996) Genetic engineering of theprocessing site of D1 precursor protein of photosystem II reaction center in Chlamydomonas
reinhardtii. Plant Cell Physiol. 37, 161-168.

48

NATIONAL INSTITUTEDIVISION OF BEHAVIOR AND
NEUROBIOLOGY (ADJUNCT)
Professor: Masatoshi Takeichi
Research Associates: Akinao Nose
Kazuaki Tatei
Institute Research Fellow: Emiko Shishido
JSPS Postdoctoral Fellow: Takaka /sshiki
Graduate Students: Hiraki Taniguchi
Takeshi Umemiya (from Kyoto
University)

How individual nerve cells find and recognize theirtargets during development is one of the central issues
in modem biology. The aim of our division is to elucidate the molecular mechanism of axon guidance andtarget recognition by using the simple and highly accessible neuromuscular system of Drosophila.
The musculature of Drosophila embryos consists of
30 identifiable muscle fibers per hemisegment. Eachmuscle fiber is innervated by a few motoneurons in ahighly stereotypic manner. The high degree of precision and previous cellular manipulations of neuromuscular connectivity suggest the presence of recognition
molecules on the surface of specific muscle fibers
which guide the growth cones of motoneurons. We
have previously isolated several enhancer trap linesthat express the reporter gene p-galactosidase <P-gal)in small subsets of muscle fibers prior to innervation.
By molecularly characterizing these lines, we are trying to identify genes that play roles in the specification of the muscles and neuromuscular connectivity.
Previous studies showed that two of the lines are insertions in the connectin and Toll genes, that encode
cell recognition molecules which belong to the leucine-rich repeat (LRR) family. We have been 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, growthcones of the motoneurons which innervate thesemuscles (primarily SNa motoneurons) and on several
associated glial cells. When coupled with its ability tomediate homophilic cell adhesion in vitro, these results led to the suggestion that Connectin functions 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 to send
extra axon branches that form ectopic nerve endings
on muscles 12, muscles they would never innervate inwild type. This phenoype was highly penetrant andwas observed in over 60% of the segments examined.
Furthermore, the ectopic innervation on muscle 12
was dependent on the Connectin expression on SNa.
These results showed that Connectin functions as an

attractive and homophilic guidance molecule for SNa
in vivo.
II. P750, a novel LRR cell surface molecule
expressed on subsets of neurons and muscles
We have been conducting molecular and genetic
analysis of other muscle enhancer trap lines. One ofthem, P750 expresses p-gal in subsets of neurons andmuscles, including the RP5 motoneuron and its target,
muscle 12. The eDNA cloning and sequencing revealed that P750 encodes a novel transmembrane protein that belongs to LRR family. It is interesting thatthree of the five muscle enhancer trap lines that wehave thus far characterized contain LRRs. Within the
LRR family, P750 was found to be most related to the
Drosophila tartan gene that have been implicated inneural and muscular development. We have recently
found that in the loss-of-function mutant of P750, thesynaptic arborization pattern on muscle 12, a P750expressing muscle, is abnormal, suggesting that thismolecule play some roles in neuromuscular target
recognition and/or stabilizaion of the synapses.
III. msh, a homeobox containing gene essential forneural and muscular development
Another line rH96 was found to be a P-element
insertion in the muscle segment homeobox (msh)gene, that was previously cloned as a homeobox containing gene. By generating and analysing both lossof-function and gain-of-function (ectopic expression)
mutants, we showed that msh is essential for neuraland muscular development. During CNS development, msh is specifically expressed in the dorsal neuroectoderm (Fig. 1A) and subsequently in manyneuroblasts and their progeny derived from this region(Fig. 1B). We found that the loss of msh results in the
failure of the proper differentiation of many neuraland glial progeny derived from the dorsal neuroectoderm. Conversely, ectopic expression of msh in theentire neuroectoderm severely disrupts the formationof midline structure and differentiation of neuroblasts
located in the ventral neuroectoderm. These results
suggest that msh plays crucial roles in the dorso-ventral (DV) specification of the CNS. The vertebratehomologues of msh, Msxs are also know to be expressed in the dorsal portion of the spinal cord. Our
work on msh raises a possiblity that this family ofgenes may plays a conserved role during DV patterning of the CNS.

IV. M-spondin and G-spondin: a novel gene familyof secreted molecules
By molecularly characterizing another enhancer
trap line, AN34 which is also expressed in a subset ofmuscles and neurons, we identified a novel secreted
protein, termed M-spondin, that is highly homologous
to rat F-spondin. F-spondin is a secreted molecule expressed at high levels in the floor plate and has beenshown to promote neural cell adhesion and neurite

49

FOR BASIC BIOLOGY

A

B

Fig. 1. The expression pattern of msh mRNA. Stage 8 (A) and stage 11 (B) whole mount Drosophila embryos hybridized in situ
with msh riboprobe.

extension in vitro. We found three regions that arehighly conserved between M-spondin and F-spondin.
One of them is a known repeating motif called
thrombospondin type I repeats (TSRs). The other two
domains (termed FS 1 and FS2) are novel conserved
sequences that we identified. By using PCR, wecloned two more genes that share similar overall
structure with M-spondin and F-spondin in that they
possessed FS1 , FS2 and one to six TSRs. The
idintification of these genes thus defines a novel genefamily of secreted protein with potential roles in celladhesion. One of the newly cloned genes, termed Gspondin, is expressed in a subset of glia that sit along
the longitudinal axon tracts in the CNS . The specificexpression pattern of G-spondin suggests that it may
play a role in the guidance of specific axons.

Publication List:
Nose, A. , Takeichi, M. and Goodman, C.S . (1996)
Molecular genetic analysis of the role of Connectinin neuromuscular recognition in Drosophila. In
Basic Neuroscience in Invertebrates (Japan Sci.
Soc. Press), pp49-64.
Uemura, T. , Oda, H., Kraut, R. , Hayashi , S., Kataoka,
Y. and Takeichi, T. (1996) Zygotic DE-cadherin
expression is required for processes of dynamic
epithelial cell rearrangement in the Drosophila embryo . Genes & Develop. 10, 659-671.

Kimura, Y., Matsunami, H. and Takeichi, M . (1996)
Expression of cadherin-11 delineates boundaries,
neuromeres and nuclei in the developing mousebrain. Develop. Dynamics 206, 455-462.
Uchida, N., Honjo, Y. , Johnson, K.R., Wheelock, M.J.
and Takeichi, M. (1996) The catenin/cadherin adhesion system is localized in synaptic junctions, bordering the active zone. J. Cell Bioi. 135, 767-779.
Tanaka-Matakatsu, M. , Uemura, T. , Oda, H. , Takeichi,
M. and Hayashi, S. (1996) Cadherin-mediated celladhesion and cell motility in Drosophila trachea
regulated by the transcription factor Escargot. Development 122, 3697-3705.
Redies, C. and Takeichi, M. (1996) Cadherins in thedeveloping central nervous system: An adhesivecode for segmental and functional subdivisions.
Develop. Bioi. 180, 413-423.
Pai, L. , Kirkpatrick, C., Blanton, J., Oda, H.,
Takeichi, M . and Peifer, M. (1996) a-Catenin andDE-cadherin bind to distinct regions of Drosophila
Armadillo. J. Bioi. Chemistry, in press .

so

NATIONAL INSTITUTE

51

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.

52

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION I
Professor: Shigeru /ida
Research Associates: Rie Terada
Yoshiki Habu
Graduate Students: Yasuyo Hisatomi
Atsushi Hoshino
Technical Staff: Sachiko Tanaka-Fukada

This group's move along the Tokaido line from
Department of Biological Science and Technology,
Science University of Tokyo, begun in April and its
formation continued throughout the year. The maininterest of the group is in understanding the biology ofdynamic genome, namely, genome organization andreorganization and their impact on gene expression
and regulation.
Although there are many elements affecting organization and reorganization of the genomes, we havecurrently focused on mobile genetic elements in general and plant transposable controlling elements in particular.
I. Identification and characterization of mutablealleles in the Japanese morning glory
The Japanese morning glory (Pharbitis nil or Ipomoea 11il) is a traditional horticultural plant in Japan,
and extensive physiological and genetical studies on
the plant have been conducted. Among several mutable loci that condition variegated flower phenotypes,
two mutable alleles, flecked and speckled have beenchosen for molecular elucidation of the variegatedphenotypes.
The flecked mutant bearing white flowers with colored flecks and sectors (Fig. lA). The flecking was
regarded as recurrent somatic mutation from the recessive white to the dominant pigmented allele, accompanied by changes in genotypes from thehomozygous recessive to the heterozygous condition.
We have molecularly characterized the flecked mutation in one of the anthocyanin genes, A-3, and foundthat the mutable a-Jfiecked (flecked) allele carries atransposable element of 6412 bp, termed Tpnl , inthe anthocyanin biosynthesis gene for dihydroflavonol
4-reductase (DFR). Among the three copies of the

DFR gene in the genome of the Japanese morning
glory, Tpnl resides within the second intron 9 bp upstream of the third exon of the DFR-B gene. The
flower variegation is shown to be due to somatic reversion of the DFR-B gene by Tpnl excision (seeInagaki et a!. (1996) Theor. Appl. Genet. 92, 499504). Structural characteristics revealed that Tpnl
belongs to En!Spm family of elements and that it islikely to be a non-autonomous element deficient toproduce active transposases.
It is known that the frequency and the timing of the
flecking phenotypes in the mutable a-Jflecked line are
generally heritable by their progeny but sometimes
conversion of these phenotypes is also observed. Thus
the flecking phenotypes caused by the excision of
Tpnl from the DFR-B gene may be determined either
by trans-acting activities of its related autonomous
element or by heritable modifications of Tpnl itself.
We are currently focusing on these lines of investigations.
Fig. lB shows another flower variegation caused by
the mutable speckled allele. Our genetic studies indicate that another element termed speckled-activator
acting in trans on the speckled allele is necessary toconfer the speckled variegation phenotype. Our current hypothesis is that the mutable speckled allele
carries a non-autonomous transposable element andspeckled-activator must be a related autonomous element. To test this hypothesis, we are trying to identify
the mutable speckled allele.

II. Identification of the mutable alleles in thecommon morning glory
The mutable afiaked line of the common morning
glory (Pharbitis purpurea or Ipomoea purpurea) alsobears white flowers with colored flakes and sectors(Fig. I C). The mutable afiaked allele is known to exhibit
incomplete dominance. Interestingly, not only intensely colored flakes but also white spots and sectors
were often observed in lightly colored flowers ofmorning glory with the heterozygous state A/afi"k'". To
understand these allelic interactions in molecular
terms, we are trying to identify the mutable afiaked
allele. Although several new mobile genetic elements
have been found in the mutable line, we are still inprocess of identifying the mutable afiaked allele.

Fig.l Flower variegation. (A) a-Jfhked; (B) speckled; (C) afiaked.

FOR BASIC BIOLOGYDIVISION OF GENE EXPRESSION AND
REGULATION II
Professor: Takashi Horiuchi
Research Associates: Masumi Hidaka
Takehiko Kobayashi
Ken-ichi Kodama
Katsuki Johzuka
Graduate Students: Katufumi Ohsumi
Keiko Taki
Hiroko Urawa
Technical Staffs: Kohji Hayashi
Yasushi Takeuchi

Homologous recombination which may occur in allorganisms apparently involves exchange between twoparent-derived chromatids plus the repair of DNA
damage incurred by physical and chemical reagents.
As deduced from our analyses of recombination
hotspots of E. coli and S. cerevisiae, in particular theactivity related to DNA replication fork blocking
events, the physiological function of homologous recombination, especially in normally growing cells is
better understood. In 1996, work on the three following interrelated subjects has advanced our knowledgeof related factors.

I. Analysis of E. coli recombinational hot spots
In E. coli RNase H defective (mh-) 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 and a strikingly enhanced
excisional recombination occurs between the repeats.
We identified 8 groups (HotA-H) of such Hot DNA,
7 of which (HotA-G) were clustered in a narrow region, known as replication terminus region (about 280kb) located on the circular E. coli genome. Analysis of
HotA, B and C revealed that blocking of replication
fork at the Ter, replication terminus, sites is responsible for these Hot activities. Further analysis led todesign of a putative model, in which the ds (double
stranded)-break occurs at the fork arrested at the DNA
replication fork blocking (Ter) site. Through this site
the RecBCD recombinational enzyme complex enters
the ds-DNA molecule and enhances recombination
between directly repeated Hot DNA when the enzyme
complex meets an appropriately oriented recombinational hotspot sequence, Chi.
To determine how other termination event-independent Hots can be activated, we chose HotH, because
it locates only outside of replication terminus region.
HotH is a 11.2 kb EcoRI DNA fragment and locatesat about 91 minutes on the E. coli genetic map. We
found that it contains the 3' end region of an rRNA
operon, rmD, and also a Chi sequence at the downstream region of rmD. HotH activity was abolished,when a mutation was introduced into the Chi sequence to destroy its activity, which means that Chi isprobably required for HotH activity. We are now investigating events which occur near the HotH site and53
which probably provide an entrance site for RecBCD
enzymes.

ll. Atomic structure and functional model of E. coli
replication terminator protein complexed with Ter
DNA
Replication of prokaryotic chromosomes is terminated in a defined terminus region. In E. coli, thistermination is mediated by a site-specific DNA-binding protein designated as Tus (or tau). The Tus protein(MW 36 kD) is a monomeric molecule that binds tosix specific sequences (Ter) within the replication terminus region. The Tus- Ter complex halts passage of
replication machinery in only one direction and allows passage in the other direction. This complex
controls the passage of DnaB helicase, a constituentof the replication machinery. It is imperative to know
the three-dimensional (3D-) structure to gain insight
into the mechanism, by which the Tus protein recognizes Ter sites and blocks the replication fork, in apolar manner. Toward this end, we are doing crystallographic studies of the Tus protein in a complex withTer DNA in collaboration with Drs. K. Kamada andK. Morikawa (Protein Engineering Research Institute,
Osaka).
The most suitable crystals for X-ray analysis weregrown from a mixture of the Tus protein and a 16 baselong DNA duplex, by microdialysis against PEG solution. The crystal structure was determined at 2. 7 A
resolution by the multiple isomorphous replacement
method and anomalous scattering (MIRAS) and has
been refined to provide an R-factor of 17.0%.
Tus protein adopts an unique structure, as comparedwith other DNA binding proteins (Figure 1). The
structure is divided in two domains, both of which areclassified as a+ ~ type. These domains form a positively charged central cleft which mainly accommodates the Ter DNA elements, as if two protruded
a-helical regions on both sites and a central ~-sheet
region would embrace the DNA duplex. The mostremarkable feature of the complex is an extensive
protein-DNA interface which involves many direct
and indirect polar interactions. The two ~-sheets ofboth domains and the interdomain antiparallel ~­strands are jointly responsible for recognition of the
DNA in the major groove. This structure motif, while
unique in detail, is similar to those of MetJ and Arc.
The Ter DNA substantially deviates from the canonical B-DNA in the vicinity of central ~-bulges of theinterdomain ~-strands inserted into the major groove.
This local deformation of the Ter DNA makes for an
overall bend of 20•.
Genetic approaches facilitate the isolation of mutant
Tus proteins and most single mutations are mapped tothe interdomain region. These mutants that affect efficiency of replication arrest partially or completely
impair binding of the Tus protein with DNA. Therefore, the replication arrest and Tus- Ter DNA binding
seem to be inseparable.
The overall structural features of the complex favor

54

NATIONAL INSTITUTE

Figure I. (a) Tus complexed with a putative Y-forked DNA, as viewed from the fork-block site. The single stranded DNAs near
the terminus were hypothetically constructed to realize inhibition of fork progression. (b) A view from the free-passage side. Arrow(pink) denotes the putative unwinding motion of DNA. The unwinding of the helicase from the lower right would displace theyellow DNA strand.

the interpretation that the a-helical regions of both
domains on the fork-blocking side act as a directional
DNA clamp and prevent the passage of replication
machinery along DNA (Figure 1). Presumably, thepassing mechanism from the opposite side relies upon
the structural disturbance of the ~-sheet region, as
induced by the unwinding motion of helicases. This
interpretation fits the physical collision model that the
intrinsic structure of the complex dominates the polararrest of the replication fork.

III. The E. coli genome project
E. coli genome project in Japan was started in 1989,
the objective being to analyze, in an independently
living organism, nucleotide sequences from 0 min ina clockwise direction. In April 1995, the DNA sequences of the 0-12.7 mjn region in the genetic map
were determined and they were published or regis-

tered. At that time I (T. H) was appointed leader of theJapan E. coli genome project and a new group of researchers was reorganized to analyze DNA sequencesof the region from 12.7 min to 70 min, using Kohara
lambda clones and a new protocol , including a shotgun sequence technique.
In January 1997, we:; determined about a 2.2 mega
nucleotide sequence of a region corresponding to 12.7
-69.0 min (including DNA replication terminus region). Combining these data with those (69.0-100min) provided by Fred Blattner in the USA, the entire
E. coli genome was expressed in a single, continuous
nucleotide sequence (4,636,552 bp), in which 3803
putative open reading frames were identified. Analysis of the genomic structure by computer is inprogress. At this same time, Blattner's group determjned independently the entire E. coli DNA sequence.
Our sequence data, registered in DNA Data Bank ofJapan (DDBJ) are also available through Web sites

FOR BASIC BIOLOGY(http://mol.genes.nig.ac.jp/ecoli/, http://www.ddbj.nig.
ac.jp/, http://bsw3.aist-nara.ac.jp/).

Publication List:
Kamada, K., Ohsumi, K., Horiuchi, T., Shimamoto,N. and Morikawa, K. (1996) Crystallization andpreliminary X-ray analysis of the Escherichia coli
replication terminator protein complexed with
DNA. Proteins 24, 402-403.
Ohshima, T., Aiba, H., et al. (1996) A 718-kb DNA
sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map. DNA Research 3, 1-19.
Kobayashi, T., and Horiuchi, T. (1996) A yeast geneproduct, Fob 1 protein, required for both replication
fork blocking and recombinational hotspot activities. Genes to Cells 1, 465-474.
Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto,N. and Morikawa, K. (1996) Structure of a replication terminator protein complexed with DNA. Nature 383, 598-602.
Aiba, H., Baba, T., et al. (1996) A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage
map. DNA Research 3, 363-377.
ltoh, T., Aiba, H., et al. (1996) A 460-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 40.1-50.0 min region on the linkage
map. DNA Research 3, 379-392.

55

56

NATIONAL INSTITUTEDIVISION OF SPECIATIONMECHANISMS I
Professor: Tetsuo Yamamori
Research Associates: Satoshi Koike
Yuriko Komine
Akiya Watakabe
Institute Research Fellow: Takashi Kitsukawa
Visiting Scientist: Kimiko Yamamori
Graduate student: Kotarou Torii (Hokuriku Advanced
Technology and Science University)
Technical Staffs: Hideko Utsumi
Miki Ida

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
It has been recognized that cytokines, defined asinter-cellular mediators in the immune system, have avariety of roles in the nervous system as well. One
such a factor, LIF (leukemia inhibitory factor) known
also as CDF (Cholinergic Differentiation Factor), is apleiotropic factor which shows a remarkable repertoire of activities from embryonic stem cells to neurons (Yamamori, T., 1996). Recent study haverevealed that CDF/LIF and its receptors belong to theIL-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 the model of theinteraction among the members of the IL-6 family(ligand) and the IL-6 receptor family (Taga andKishimoto, 1992; Stahl and Yancopoulos, 1993), we
proposed that the evolution of the IL-6/class IB receptor family may have occurred in at least 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 third binding subunits ofa CNTF receptor evolved. Our model predicts that thebinding subunits generally determine the specificity of
the receptors and it is possible that novel members of
the cytokine 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 a synaptic
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 (a glutamate
analogue). Among the lEGs examined, Fos and 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 Inferior
Olive stimulation. June-B are predominantly induced
around the local area where the AMPA and climbing
fiber stimulation were conjunct (Yamamori et al.,1995). These results suggest that the coincidence
mechanism may exist at gene expression level andlead to a cerebellar long-term plasticity (Fig. 1).
Toward further confirming this hypothesis, we are
currently characterizing several molecules which are
induced in Purkinje cells 3 hours after conjunction
and following Jun-B induction in collaboration with
Dr. Ryoji Yano's group (Frontier Research program,
RIKEN). Most of these genes are unknown genes andwe expect that the characterization will bring us new
information on the late process of cerebellar longterm plasticity.

57

FOR BASIC BIOLOGY

Climbing Fiberlong-term syna
plasticity

Purkinje Cell

Publication List
Yamamori, T. (1996) Immediately early gene expression. In "Coincidence detection in the nervous system" Eds by A. Konnerth, R.Y. Tsien, K. Mikoshiba
and J. Altman, Human Frontier Science Program,
Strasbourg, 118-121.
Yano, R, Nakazawa, K., Kado, R., Karachot, L.,
Mikawa, S., Ito, M., Komine, Y. and Yamamori, T.
(1996) Cerebellar long-term plasticity and gene expression. In "Integrative and molecular approach tobrain function" Proceeding of the Uehara Memorial
Foundation Symposium (Eds by M. Ito, and Y.
Miyashita), Elsevier, 35-44.
Yamamori, T. (1996) Leukemia inhibitory factor andphenotypic specialization. In "Chemical Factors in
Neuronal Growth, Degeneration and Repair" (Ed by
C. Bell), Elsevier, 265-292.

58

NATIONAL INSTITUTEDIVISION OF SPECIATIONMECHANISMS II
Professor: Ken-ichirou Morohashi
Associate Professor: Mitsuyasu Hasebe
Reserch Associate:
Graduate Students:
Technical Staff:

This division was newly established in 1996 with
two research groups. These two groups managed by
the professor, Ken-ichirou Morohashi (1), and the associate professor, Mitsuyasu Hasebe (II), haveadopted animals and plants, respectively, as the materials for the research. Our attention has been and will
be directed to understanding mechanisms underlying
evolution of the reproductive system of animals andevolution of the morphology in plants. These efforts
with a variety of the materials are expected to give us
deeper insights into speciation mechanisms of organism.

I. Reproduction of animals
Because of the fundamental and pivotal function of
the gonads, extensive efforts have been made to characterize the differentiation processes including
sex-dependent differentiation. By focusing on the steroidogenesis as one of the gonad specific functions,
we indentified a steroidogenic tissue specific transcription factor designated Ad4BP or SF-1. Recent
studies with an antiserum to Ad4BP/SF-l and a genedisrupted mice clearly demonstrated that the transcription factor is essential for differentiation of thesteroidogenic tissues, the gonads and the adrenal cortex. Showing a good correlation with the observation
above, a particular cell population identified as
Ad4BP/SF-1 immunoreactive cells (AGP, adrenogenital primordium) was revealed to give rise to the
gonads and adrenal cortex. These observations gave
us many interesting issues as follows. What is thesignal for the onset of the Ad4BPISF-l gene transcription in the particular cells comprising AGP? What is
the mechanism underlying the separation of the AGP
into the adrenal and gonadal primordia? How is themechanism employed for sex differentiation of the
gonads? Why dose such sex dependent differentiation
not occur in the adrenal cortex? To address the issues,
the nuclear factors, WTl, SRY, SOX9, and DAXl as
well as Ad4BP/SF-l, critically implicated in gonadal
and adrenocortical differentiation will be investigatedfrom the molecular and morphological aspects.

II. Morphological Evolution in plants
Morphological diversity among taxa is the mostprominent characteristics in the biodiversity. Ourgroup aims to explore genetical and molecularchanges which caused renovative morphologicalchanges during the course of plant evolution. We seArabidopsis(angiosperm),
Gnetumlected(gymnosperm), Ginkgo (gymnosperm), Ceratopteris(pteridophyte), Physcomitrella (bryophyte), andChlamydomonas (green algae) as model plants tocompare the genes related to morphogenesis. We arefocusing on the following three topics; (1) Evolutionof floral homeotic genes (MADS gene family and
LEAFY gene homeologue, which is a regulator of the
MADS genes), (2) Comparison of homeobox genefunctions between sporophyte and gametophyte generations to reveal the evolution of the bothgenerations, and (3) establishment of mutant library ofPhyscomitrella patens to get new morphological mutants which have not been obtained from the study ofthe higher plants having only reduced gametophytic
generations. Some MADS, LEAFY and homeobox
genes have been cloned from the gymnosperms,
Ceratopteris, Physcomitrella and the analyses of theexpression pattern are in progress. Some of thosegenes were observed to be expressed in both gametophyte and sporophyte generations and the regulation
systems are studied.

59

FOR BASIC BIOLOGYRESEARCH SUPPORTJ60

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head: Hiroyuki Hattori
Common Facility Group
Chief Kazuhiko Furukawa
Research Support Facilities
Mamoru Kubota (Subunit Chief)
Chieko Nanba
Toshiki Ohkawa
Masayo Iwaki
Kaoru Sawada
Tomoki Miwa
Radioisotope Facility
Yousuke Kato (Subunit Chief)
Yoshimi Matsuda
Center for Analytical Instruments
Akio Murakami (Unit Chief)
Sonoko Ohsawa
Yumiko Makino
Takeshi Mizutani
Yuki Nakashima (Technical Assistant)
Glassware Washing Facility(Kazuhiko Furukawa)(Toshiki Ohkawa)Research Support Group
Cell Biology Group
Maki Kondo (Subunit Chief)
Yukiko Kabeya
Katsushi Yamaguchi
Developmental Biology Group
Hiroko Kobayashi (Unit Chief)
Chikako Inoue
Chiyo Takagi
Sanae Oka
Naomi Sumikawa
Regulation Biology Group
Shoichi Higashi (Subunit Chief)
Hedeko Nonaka
Akiko Kawai
Shigemi Ohsugi
Gene Expression and Regulation Group
Sachiko Tanaka
Tomoko Mori
Koji Hayashi
Miki Ida
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 increase
theircapability
intechnical
area.
Each
technical 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.

FOR BASIC BIOLOGYRESEARCH SUPPORT FACILITY
Head of Facility: Takashi Horiuchi (April 1, 1996 -)Associate Professor: Masakatsu Watanabe
Research Associates: Yoshio Hamada, (Tissue and Cell
Culture)Atsushi Ogiwara (Computer)
NIBB Fellow: Mineo lseki (Large Spectrograph)
Technical Staffs: Masayo 1waki
Mamoru Kubota
Chieko Nanba
Toshiki Ohkawa
Kaoru Sawada
Tomoki Miwa
Hideko Nonaka (-April 30, 1996)

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 of StressResistant Plants are dedicated to cooperative use under the NIBB Cooperative Research Programs.
On Nov. 12-14, 1996, the Research Support Facility
organized an international symposium at NIBB on"New Prospects of Photobiology and the Future Planof the Okazaki Large Spectrograph" participated byabout 40 foreign and about 120 Japanese active scientists, and then got an overwhelming support for theidea of constructing a new generation spectrograph(Okazaki Super Sectrograph) providing about 2 ordersof magnitude higher fluence rates (intensities) andversatile combinations of monochromatic irradiations
by the use of over 50 tunable laser systems as the
major light sources.

I. Facilities
1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, theOkazaki Large Spectrograph (OLS), which is the largest spectrograph in the world, dedicated to action
spectroscopical studies of various light-controlled biological processes. The spectrograph runs on a 30 kWXenon 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 at noon
(Watanabe et al., 1982, Photochem. Photobiol., 36,
491-498).
A tunable two-wavelength CW laser irradiation system is also available as a complementary light source
to OLS to be used in irradiation experiments which
specifically require ultra-high fluence rates as well as
ultra-high spectral-, time-and spatial resolutions. It is
composed of a high-power Ar-ion laser (Coherent,

61

Innova 20) (336.6-528.7 nm, 20 W output), two CW
dye lasers (Coherent, CR599-01) (420-930 nm, 2501000 mW output), A/0 modulators (up to 40 MHz) tochop the laser beam, a beam expander, and a tracking
microbeam irradiator (up to 200 J.tm s- 1 in tracking
speed, down to 2 Jlm in beam diameter) with an infrared phase-contrast 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: UNIX servers
and engineering workstations (SPARCstations, IRIS
machines, NEWS machines, etc.), VAX minicomputer system, and some personal computers(Macintosh's and Windows machines). All of thesemachines are connected each other with local area
networks, which are also linked to the highperformance multimedia backbone network ofOkazaki National Research Institutes. Since thisbackbone network, called ORION, is joined to theInternet, the users can access various services anddatabases on the Internet.
The Computer Laboratory provides various computational services to the institute members: file servers
for Macintosh and NetWare users, print servers for PC
and UNIX users, computational servers that provides
sequence analyses and database retrievals, communication servers to the Internet, and so on.
A new computational server with large scale disk
system was introduced at the end of this year andstarted to provide database search service. 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 m 2 glass-houses
with precise temperature and humidity control, asmall farm, two greenhouses (45 and 88 m 2) with
automatic sprinklers, two open aquariums (30 and 50t) and several smaller tanks. The laboratory also includes a building with office, storage and work-space.
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 clean-

62
benches, 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 University (30 km
from the National Institute for Basic Biology). The
laboratory provides a variety of growth chambers thatprecisely control the conditions of plant growth andfacilities for molecular biological, and physiologicalevaluations of transgenic plants.
The laboratory is also a base of domestic and international collaborations devoted to the topic of stressresistant transgenic plants.

II. Research activities
1. Faculty

The faculty of the Research Support Facility conducts its own research as well as scientific and administrative 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 measuring computerized-videomiceographs of the motile behavior of
the cells at the cellular and subcellular levels (Erata etal. 1995, Protoplasma). Photo-receptive and signal
tranduction mechanisms of algal gametogenesis arealso 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 of neuralversus epidermal cell fates, a process described thus
far only in invertebrates. It is thought to act by adirect signaling pathway rather than through one of
the classical signal transduction cascades. The mammalian genome is known to contain three Notch homologues but their developmental significance is notclear. To investigate their role in mammalian development, we have 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: One of the main theme ofcomputational biology is to predict biological functions from amino acids or nucleic acids sequences.
Homology search is a popular method that assists topredict biological roles by inference. However, it isvery time consuming process as the rapid growing ofsequence libraries. Use of sequence motifs and signa-

NATIONAL INSTITUTE

ture patterns is a convenient and speedy method topredict directly the biological functions. A new
method to extract sequence motifs from groups ofrelated proteins has been studied and the methodology
was applied to analyze unknown protein sequences.
The method was implemented as a computer tool on
a UNIX workstation.
Another important theme of bioinformatics is the
problem of databases. Recently, many bacteria genomes have been completely sequenced. To store andto publish the results of sequencing projects, databasesystems are indispensable. Since the purpose of genome sequencing is to understand biological functions
from genetic information, the genome sequence database must also oriented to the biological function. As
a model case of such database, Bacillus subtilis open
reading frames database is constructed in collaboration with Prof. N. Ogasawara (NAIST) and Human
Genome Center of the University of Tokyo. As thecompletion of sequencing of 1.3 Mbase regions by
Japanese researchers groups, the first step of database
construction has been finished and is released using

www.
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 living organisms, biological molecules, and organic molecules
have been conducted (Watanabe, 1995, In CRC Handbook of Organic Photochemistry and Photobiology).

Publication List:
I. Faculty

Gualdi, R., Pascale, B., Zheng, M., Hamada, Y.,
Coleman J.R. and Zaret, K.S. (1996). Hepatic specification of the gut endoderm in vitro: cell signalling
and transcriptional control. Gene Dev. 10, 16701682.
Hayashi, H., Mochii, M., Kodama, R., Hamada, Y.,
Mizuno, N., Eguchi, E. and Tachi, C. (1996). Isolation of a novel chick homolog of serrate and its
coexpression with C-Notch1 in chick development.
Int. J. Dev. Bioi. 40, 1089-1096.
Higuchi, H., Kiyama, H., Hayakawa, T., Hamada, Y
and Tsujimoto, Y.(1995). Differential Expression of
Notch1 and Notch2 in developing and adult mousebrain. Mol. Brain Res. 29, 263-272.
Kato, H., Sakai, T., Tamura, K., Minoguchi, S.,
Shirayoshi, Y., Hamada, Y., Tsujimoto, Y. andHonjo, T. (1996). Functional conservation of mouse
Notch receptor family members. FEBS leu. 395,
221-224.
Kawai, H., Nakamura, S., Mimuro, M., Furuya, M.,
Watanabe, M. (1996). Microspectrofluorometry of

FOR BASIC BIOLOGY

the autofluorescent flagellum in phototactic brown
algal zoids. Protoprasma 191, 172-177.
Ogiwara, A., Ogasawara, N., Watanabe, M. and Takagi,
T. (1996) Construction of the Bacillus subtilis ORF
database (BSORF DB). Proc. Genome Informatics
1996, 228-229.
Ogiwara, A., Uchiyama, 1., Takagi, T. and Kanehisa,
M. (1996) Construction and analysis of a profile
library characterizing groups of structurally known
proteins. Protein Science, 5, 1991-1999.
Shinomura, T., Nagatani, A., Hanzawa, H., Kubota,
M., Watanabe, M., Furuya, M. (1996). Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.
Proc. Natl. Acad. Sci. USA 93, 8129-8133.
Yamazaki. Y., Kataoka, H., Miyazaki, A., Watanabe,
M., Ootaki, T. (1996) Action spectra for photoinhibition of sexual development in Phycomyces
blakesleeanus. Photochem. Photobiol. 64, 387-395.
II. Cooperative Research Program for the Okazaki
Large Spectrograph
Andrady, A. L., Torikai, A. and Kobatake, T. (1996).
Spectral sensitivity of chitosan photodegradation. J.
Appl. Polym. Sci. 62, 1465-1471.
Kawai, H., Nakamura, S., Mimuro, M., Furuya, M.
and Watanabe, M. (1996). Microspectrofluorometryof the autoflulrescent flagellum in phototactic
brown algal zoids. Protoplasma 191, 172-177.
Munakata, N., Morohashi, F., Hieda, K., Suzuki, K.,
Furusawa, Y., Shimura H. and Ito, T. (1996). Experimental correspondence between spore dosimetry and spectral photometry of solar ultraviolet radiation. Photochem. Photobiol. 63, 74-78.
Nakagawa, T., Umemura, S., Kakiuchi Y. and Ueda,
T. (1996). Action spectrum for sporulation andphotoavoidance in the plasmodium of Physarum
polycephalum, as modified differentially by temperature and starvation. Photochem. Photobiol. 64,
859-862.
Saitow, F. and Nakaoka, Y. (1996). Photodynamic
action of methylene blue on the Paramecium membrane. Photochem. Photobiol. 63, 868-873.
Shibata, H., Noda, T., Ogura, T., Suginaka, K., Matui,Y., Ozoe, Y., Sawa, Y. and Kono, Y. (1996). A
soluble-form pf pro-oxidant lumazine from cyanobacterial cells generates superoxide anion undernear-UV irradiation. Biochim. Biophys. Acta 1274,
129-134
Shichijo C., Hamada, T., Johnson, C. B. andHashimoto, T. (1996). Effects of moderately low
temperature on phytochrome responses during
preirradiation: anthocyanin synthesis in Sorghum
bicolor at high-end low-Pr/P101 ratios. Photochem.
Photobiol. 63, 328-335.
Shinomura, T., Nagatani, A., Hanzawa, H., Kubota
M., Watanabe, M. and Furuya, M. (1996). Action
spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thalia-

63

na. Proc. Nat!. Acad. Sci. USA 93, 8129-8133.
Takeuchi Y., Murakami, M., Nakajima, N., Kondo, N.
and Nikaido, 0. (1996). Induction and repair ofdamage to DNA in cucumber cotyledons irradiatedwith UV-B. Plant Cell Physiol. 37, 181-187.
Tazawa, E., Fujiwara, A., Ishida, 0. and Yasumasu, I.
(1996). Photoactivation of NADH cytochrome creductase in mitochondria islated form sperm, eggs
and viscera of sea urchin, oyster, abalone and echiuroid. Comp. Biochem. Physiol. 114B, 245-250.
Yamazaki, Y., Kataoka, H., Miyazaki, M., Watanabe,
M. and Ootaki, T. (1996). Action spectra for photoinhibition of sexual development in Phycomyces
blakesleeanus. Photochem. Photobiol. 64, 387-392.
Yasumasu, 1., Tazawa, E., Asami, K. and Fujiwara, A.
(1996). Does the low respiratory rate in unfertilized
eggs result mainly from depression of the redox
reaction catalyzed by flavoproteins? Analysis of therespiratory system by light-induced release of COmediated inhibiton. Develop. Growth Differ. 38,
359-371.

64

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 and two
branch offices. The third branch office will open in
the laboratory of Gene Expression and Regulation in
May 1, 1977. The members of the Radioisotope Facility are engaged in maintaining and controlling boththe center and two branch offices, and providing users
an appropriate guidance for radioisotope handling.
The teaching staffs are also engaged in their own
research. They are interested in clarifying the structure and function of dynein motor protein. Dyneins
are a group of microtubule-activated ATPases thatserve to convert chemical energy into mechanicalenergy. They have been divided into two large subgroups, namely, the axonemal and cytoplasmic
dyneins. Figure I shows the localization of two
dyneins in the outer arms (Ogawa et al. , 1977) and themitotic apparatus (Mohri et al., 1976) that have beenvisualized by the same antibodies directed against the
motor domain of axonemal dynein (fragment A) .
The native dyneins are very large. They range inmolecular mass up to I to 2 mega daltons and they arecomplex proteins as shown in Figure 2. Each dynein
contains two or three heavy chains (HCs) with ATPase activity, which range in molecular mass up to
500 kDa. The motor activity of dynein is associatedwith these chains. Some functional differences havebeen reported between HCs of outer arm dynein . Sea
urchin outer arm dynein is a heterodimer of HCs (alpha and beta) and at least the beta-HC is able to induce gliding of microtubules in vitro. The Alpha-HC
might amplify the function of beta-HC and it has beenreported to have no motile activity. After the firstAcloning of beta-HC from sea urchin ciliary axonemes(Gibbons eta!., 1991; Ogawa, 1991), the sequences of
HCs of axonemal and cytoplasmic dyneins from avariety of organisms were determined in their entirety.
Without exception, all the HCs cloned to date containfour P-loop (ATP-binding) sequences in themidregion of the molecule. Thus, they can be classified as a four P-loop family.
The outer arm dyneins contain two or three proteinsthat range in molecular mass from 70 to 120 kDa andcopurify with HCs. ICs of sea urchin outer-arm
dynein are abbreviated as IC1, IC2, and IC3. Those ofChlamydomonas are called IC78 and IC69, and ICs ofcytoplasmic dynein are called IC74. Chlamydomonas
IC78 and IC69 were cloned by Wilkerson et al. (1995)
and Mitchell and Kang (1991), respectively. The sequences of sea urchin IC2 and IC3 were determinedby Ogawa eta!. (1995). Finally, the sequence of ICI
was determined this year by Ogawa et a!. ( 1996).
Thus, all the ICs found in the axonemal and cytoplasmic dyneins of the model organisms used for studies
of dynein function have been completely sequenced.
Comparison of amino acid 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 ismuch more closely related to IC69, than either sea
urchin chain or either Chlamydomonas chain is related to each other. Regions of similarity between all
five ICs are limited to the carboxy-terminal halves ofthe molecules. Similarity are due primarily to conservation 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 (Neeret a!., 1994). A parsimony tree for these chains (Ogawa
et al., 1995) shows that, although the carboxy-terminal halves of all of these chains contain WD repeats,
the chains 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. ICI has a unique primary structure, theN-terminal part is homologous to the sequence ofthioredoxin, the middle part consists of three repetitive

B

Figure 1. Localization of dynein in sea urchin sperm axoneme (A) and a cleaving egg (B).

65

FOR BASIC BIOLOGY

Outer Arm Dyneins
Chlamydomonas

Sea Urchin

Heavy Chains

Intermediate Chains••••

Light Chains

••

••

••
••

Fig . 2. Substructures of outer arm dyneins from sea urchin sperm flagella and Chlamydomonas flagella.

sequences homologous to the sequence of nucleoside
diphosphate kinase, and the C-terminal part contains ahigh proportion of negatively charged glutamic acidresidues. Thus, ICI is a novel dynein intermediatechain distinct from IC2 and IC3 and may be a multifunctional protein. Then, a question arises as whether
Chlamydomonas outer arm dynein contains ICl. The
answer is "no". Because it consists of just two intermediate chains. Alternatively, the answer is "yes".
Because the sequences of two light chains (LC 16 andLC14) from Chlamydomonas outer arm dynein showsthat they are members of novel family of thioredoxin(Patei-King et al. 1996). The thioredoxin-related partof ICI is more closely related to those of two redoxactive Chlamydomonas light chains than thioredoxin.
Antibodies were prepared against the N-terminal andmiddle domains of ICl expressed as His-tagged proteins in bacteria. These antibodies cross-reacted withsome dynein polypeptides (potential homologues of
ICI) from distantly related species. We propose herethat the three intermediate chains are the basic coreunits of sperm outer arm dynein because of their ubiquitous existence.

Publication List:
Kazuo Ogawa, Hiroyuki Takai, Atsushj Ogiwara,
Etsuo Yokota, Takashi Shimizu, Kazuo Inaba, andHideo Mohri (1996) Is outer arm dynein intermediate chain 1 multifunctional? Mol. Biol. Cell 7,
1895-1907.
Kazuo Ogawa and Hideo Mohri (1996) A dynein
motor superfamily. Cell Struct. Funct. 21, 343-349.

66

CENTER FOR ANALYTICAL INSTRUMENTS(Managed by NIBB)Head of Facility: Masaharu Noda
Technical Staffs: Akio Murakami
Yumiko Makino
Sonoko Ohsawa
Takeshi Mizutani

This center provides analytical instruments for biological and biophysical studies. These instruments
consist of the following four groups and are supported
by experienced technical staffs. Each group isequipped with instruments for general use as listed
below.
Instruments for Protein and Gene Analysis
Amino Acid Analyzer
HITACHI L 8500A
Automatic Plasmid Isolation System
KURABO PI-IOm;
DNA Sequencers
ABI 373S, 310
DNA/RNA Synthesizers
ABI 392, 394
Nucleic Acid Extractor
ABI 340A
Peptide Synthesizers
ABI 431A, 432A
Portein Sequencer
ABI 473A, 492
Biomolecular Interaction Analysis System
PHARMACIA BIAcore 2000
AFFINITY SENSORS IAsys
Instruments for Chemical Analysis
Capillary Electrophoresis
ABI 270A
Gas Chromatographs
SHIMADZU GC-7A, GC-14A
Glycoprotein Analysis System
TAKARA Glyco-Tag
High Performance Liquid Chromatographs
SHIMADZU LC-lOAD, 6AD
Preparative Ultracentrifuges
BECKMAN L8-80, L5-75
Table-top Ultracentrifuges
BECKMAN TL-100
Micro Preparative System
PHARMACIA SMART System
Flow Cytometer
COULTER EPICS XL

Instruments
AnalysisforPhysicalandSpectroscopic

Atomic Absorption Spectrophotometer
PERKIN-ELMER 603
Dual Wavelength Spectrophotometer
HITACHI 557
EPR Spectrometer
BRUKER ER-200D
GC/Mass Spectrometer
JEOL DX-300
Inductively Coupled Plasma Spectrometer
SEIKO SPS 1200A
Infrared Spectrophotometer
JASCO A-302
Laser Raman Spectrophotometer
JASCO R-800
Light Scattering Photometer
CHROMATIX KMX-6DCNMR Spectrometer
BRUKER AMX-360wb
Spectrofluorometers
HITACHI 850
SHIMADZU RF-5000
Spectrophotometers
HITACHI 330
VARIAN Cary5G
PERKIN ELMER Lambda Bio
Spectropolarimeter
JASCO J-40S
DELFIA Research Fluorometer
PHARMACIA
Microplate Luminometer
BERTHOLD MicroLumat LB96P
Microplate Reader
Corona MTP-120, -IOOF
Instruments 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
BIOIMAGE
Microscope
CARL ZEISS Axiophot, Axiovert
Microscope Photometer
CARL ZEISS MPM 03-FL
Microdensitometer
JOYCE LOEBL 3CS

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