|Budget Amount *help
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
The ras-related small GTP-binding proteins have been implicated in the regulation of various cellular processes including growth, differentiation, cytoskeletal organization, intracellular vesicle transport and secretion.Such genes have been identified in plants but little is known of the mechanisms by which they are regulated or the functins of their encoded proteins.The wheat gene, wgp7, encoding a small GTP-binding protein, was first identified because of its increased transcript levels by SO_2 and NO_2.Over the last 3 years, the following results have been obtained.
1.The wgp7 transcripts are rapidly induced not only by SO_2 and NO_2, in a light-dependant manner, but also by conditions that specifically induce oxidative stress, including the herbicide methyl viologen (paraquat), a catalase inhibitor, and by exposure to cold or UV-C.
2.Transgenic tobacco plants (including T2 and T3 generations) were produced with the wgp7 gene in a sense orientation under control of the CaMV 35S promot
er.Depending on the level of transgene expression, plants with differing morphologies were obtained, including a severe rosette dwarf.High levels of wgp7 transcripts and proteins were expressed in these plants, but no clear differences in response to several oxidative stress conditions have yet been identified.
3.The promoter region of wgp7 has been cloned, and partially sequenced.Current studies are aimed to identify the cis-elements essential for oxidative stress regulated expression.
4.Rocombinant wgp7 protein, synthesized in vitro, was shown to bind GTP.The full length and also highly-variable C-terminal end of the protein were used to raise polyclonal antibodies.Subcellular fractionation of proteins from transgenic tobacco showed that the wgp7 protein was localized to the microsomal fraction, suggesting that it is membrane-bound.In wheat, the wgp7 protein was rapidly induced by oxidative stress treatment.Experiments for subcellular localization of the protein following oxidative stress are underway.
5.Using the wgp7 protein, as well as another small GTP-binding protein, interaction cloning was used to isolate interacting proteins.Consequently, a cDNA clone, encoding a novel protein was identified.Binding of this protein was found to be specific, reversible and priferentially with the small GTP-binding protein in its GDP-bound form.Deletion analysis of this protein showed that an N-terminal specific region of 198 amino acids is sufficient for binding the small GTP-binding protein. Less