RESEARCH SUPPORT FACILITY

I. Facilities

1. Large Spectrograph Laboratory

This laboratory provides, for cooperative use, the Okazaki Large Spectrograph (OLS), which is the largest spectrograph in the world, dedicated to action spectro-scopical studies of various light-controlled biological processes. The spectrograph runs on a 30kW Xenon arc lamp and has a compound grating composed of 36 smaller individual gratings. It projects a spectrum of a wavelength range from 250nm (ultraviolet) to 1,000nm (infrared) onto its focal curve of 10m in length. The fluence rate (intensity) of the monochromatic light at each wavelength is more than twice as much as that of the corresponding monochromatic component of tropical 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, Innova 20) (336.6-528.7 nm, 20W output), two CW dye lasers (Coherent, CR599-01) (420-930nm, 250-1000mW output), A/O modulators (up to 40MHz) to chop the laser beam, a beam expander, and a tracking microbeam irradiator (up to 200mm s^-1 in tracking speed, down to 2 mm in beam diameter) with an infrared phase-contrast observation system.

2. Tissue and Cell Culture Laboratory

Various equipments for tissue and cell culture are provided. This laboratory 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

Computer laboratory maintains several computers to provide computation resources and means of electronic communication in this Institute. Currently, the main system consists of three servers and two terminal workstations: biological information analysis server (SGI Origin 2000), database server (Sun Enterprise 450), file server (Sun Enterprise 3000), data visualization terminal and molecular simulation terminal (both are SGI Octanes). Some personal computers and color/monochrome printers are also equipped. On this system, we provide various biological databases and data retrieval/analysis programs, and support large-scale data analysis and database con-struction for the Institute members.

Computer laboratory also provides network communication services in the Institute. Most of PCs in each laboratory as well as all of the above service machines are connected each other with local area network (LAN), which is linked to the high performance multimedia back-bone network of Okazaki National Research Institute (ORION). Many local services including sequence analysis service, file sharing service and printer service are provided through this LAN. We also maintain a public World Wide Web server that contains the NIBB home pages (http://www.nibb.ac.jp).

4. Plant Culture Laboratory

There are a large number of culture boxes, and a limited number of rooms with environmental control for plant culture. In some of these facilities and rooms, experiments can be carried out at the P1 physical containment level under extraordinary environments such as strong light intensity, low or high temperatures.

5. Experimental Farm

This laboratory consists of two 20 m² glass-houses with precise temperature and humidity control, three green houses (each 6 m²) at the P1 physical containment level, a small farm, two greenhouses (45 and 88 m²) with automatic sprinklers. The laboratory also includes a building with storage and work space.

6. Plant Cell Laboratory

Autotrophic and heterotrophic culture devices and equipment for experimental cultures of plant and microbial cells in this laboratory. A facility for preparation of plant cell cultures including an aseptic room with clean benches, is also provided.

7. Laboratory of Stress-Resistant Plants

This laboratory was found 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 National Institute for basic Biology). The laboratory provides a variety of growth chambers that precisely control the conditions of plant growth and dacilities for molecular biological and physiological enaluations of transgenic plants.

The laboratory is also a base of domestic and international collaborations devorted to the topic of stress resistant 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 unicellular algae are studied action spectroscopically (Watanabe 1995, In “CRC Handbook of Organic Photochemistry and Photobiology”) by measuring computerized-videomicrographs of the motile behavior of the cells at the cellular and subcellular levels. Photoreceptive and signal transduction mechanisms of algal gene expression were also studied by action spectroscopy.

A novel blue-light receptor with an effector role was found from Euglena glacilis (Fig. 1; Iseki et al. 2002, Nature 415, 1047-1051): Euglena gracilis, a unicellular flagellate, shows blue-light type photomovements. The action spectra indicate the involvement of flavoproteins as the photoreceptors mediating them. The paraflagellar body (PFB), a swelling near the base of the flagellum has been considered as a photosensing organelle for the photomovements. To identify the photoreceptors in the PFB, we isolated PFBs and purified the flavoproteins therein. The purified flavoprotein (ca. 400 kDa), with noncovalently bound FAD, seemed to be a heterotetramer of a- and b-subunits. Predicted amino acid sequences of each of the subunits were similar to each other and contained two FAD-binding domains each followed by an adenylyl cyclase catalytic domain. The flavoprotein showed an adenylyl cyclase activity, which was elevated by blue-light irradiation. Thus, the flavoprotein (PAC, photoactivated adenylyl cyclase) can directly transduce a light signal into a change in the intracellular cyclic AMP level without any other signal transduction proteins.

Fig.1 Photoactivated adenylyl cyclase (PAC): a novel blue-light receptor mediating photoavoidance in Euglena gracilis.

(2) Developmental Biology: Replacement of the ankyrin repeats of mouse Notch2 gene with E.coli b-gactosidase gene induces early embryonic lethality around E10.5. The lethality was suggested due to defects in extraembryonic tissues, because the mutant embryo grew and differentiated further in vitro. Histological examination and in situ hybridization analysis with trophoblast subtype-specific probes revealed that the development of giant and spongiotrophoblast cell layers are normal in the mutant placenta, while vasculogenesis in the labyrinth layer apperaed compromised at E9.5. Since the lethality was circumvented by production of chimeric mice with tetraploidy wild type embryos, we concluded that the embryonic lethality is due to defect in growth and/or differentiation of labyrinthine trophoblast cells. The mutant embryo, however, could not be rescued in the tetraploid chimeras beyond E12.5 because of insurfficient development of umbilical cord, indicating another role of Notch2 signaling in the mouse development. Chimeric analysis with diploid wild type, however, revealed contribution of mutant cells to these affected tissues by E13.5. Thus, Notch2 are not cell autonomously required for the early cell fate determination of labyrinthine trophoblast cells and allantoic mesodermal cells, but plays an indispensable role in the further formation of functional labyrinth layer and umbilical cord.

(3) Computational Biology: Comparative genomics is a useful approach to find clues to understanding complex and diverse biological systems from rapidly growing genome database. We have constructed a workbench system for comparative analysis of microbial genomes named MBGD. Since the number of completed microbial genome sequences has now become more than a hundred, we have enhanced the efficiency of the database greatly to treat such large number of genomes. One of the unique features of MBGD is to allow users to create their own orthologous classification table with specified set of organisms. By this approach, MBGD accommodates comparative analysis for both closely related and distantly related genomes.

We also continue to develop an automated method for orthologous grouping among multiple genomes, which is a key component of MBGD. In addition to splitting fusion genes into orthologous domains, we are also trying to enhance the algorithm to incorporate the information of gene arrangement on each genome.

2. Cooperative Research Program for the Okazaki Large Spectrograph.

The NIBB Cooperative Research Program for the Use of the OLS supports about 20 projects every year conducted by visiting scientists including foreign scien-tists 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". pp, 1276 - 1288).

Publication List:

I. Faculty

Iseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K., Yoshida, K., Sugai, M., Takahashi, T., Hori, T. and Watanabe, M. (2002). A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis. Nature 415, 1047-1051.

Kanesaki, Y., Suzuki, I., Allakhverdiev, Sl., Mikami, K. and Murata, N. (2002) Salt strees and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803. Biochem. Biophys. Res. Commun. 290, 339-48.

Mikami, K., Kanesaki, Y., Suzuki, I. and Murata, N. (2002) The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp.PCC 6803. Mol. Microbiol. 46, 905-15.

Ohmori, M., Terauchi, K., Okamoto, S. and Watanabe, M. (2002). Regulation of cAMP-mediated photosignaling by a phytochrome in the cyanobacterium Anabaena cylindrica. Photochem. Photobiol. 75, 675-679.

Takami, H., Takaki, Y., Uchiyama, I. (2002) Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments. Nucleic Acids Res. 30, 3927-3935.

Uchiyama, I. (2003) MBGD: microbial genome database for comparative analysis. Nucleic Acids Res. 31, 58-62.

II. Cooperative Research Program for the Okazaki Large Spectrograph

Iseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K., Yoshida, K., Sugai, M., Takahashi, T., Hori, T. and Watanabe, M. (2002). A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis. Nature 415, 1047-1051.

Ohmori, M., Terauchi, K., Okamoto, S. and Watanabe, M.(2002). Regulation of cAMP-mediated photosignaling by a phytochrome in the cyanobacterium Anabaena cylindrica. Photochem. Photobiol. 75, 675-679.

Toyoshima, M., Takinami, S., Hieda, K., Furusawa, Y. and Negishi, T.(2002).The involvement of cell cycle checkpoint-mutations in the mutagenesis induced in Drosophila by a longer wavelength light band of solar UV. Photochem. Photobiol. Sci., 1, 178 - 183.