Studies on the Proteins of Thymocytes Induced by X ray and Cause Apoptosis.

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Rat thymocytes irradiated with a low dose of X rays undergo interphase cell death within several hours. This interphase death is known as programmed or physiological cell death, apoptosis, a characteristic and well orchestrated form of selfdestruction, which is prevented by the inhibitors proteinsy nthesis such as cycloheximide or actinomycin D. In this project, we tried to purify and identify the RNA's and proteins which are responsible for the apoptosis of thymocytes irradiated with X ray. We prepared cDNA library from the mRNA of rat thymocytes 4 hours after X ray irradiation. The library was screened for the induced RNA by the subtraction method of X ray irradiated and control thymocyte RNA. 31 clones were obtained. From the cDNA inserted into pGEM plasmid, mRNA was synthesized with RNA polymerase and injected into Xenopus oocytes. 3 RNA's were found to degradate oocytes, but they did not cause interphase death of rat thymocytes. To screen for the RNA which trigers the thymocyte death, we tried to find out the good methods to inject RNA into thymocytes and to decisive feature of apoptosis. The electroporation was found to be most efficient to incorporate radioactive RNA into relatively large number of thymocytes readily. As a criterion the apoptosis, the observation of chromosome destruction on minigel electrophorasis was more reliable than the commonly used dye-exclusion or cell-volume reduction. The screening of RNA by these methods are now in progress. In searching for the induced proteins by two-demensional electrophoresis, we found 5 spots on dye-stained gels and 4 spots on autoradiogram which are stronger for the X ray irradiated samples than in control. Structural analysis of these proteins are now in progress.