The long-range goal of this research is to identify and isolate molecules synthesized by Chinese hamster ovary (CHO-Kl) cells during a radiation-induced division delay. Once these delay- related molecules are isolated, molecular probes will be developed and used to determine their induction during the cell cycle. To maximize division-delay related synthesis, proteins, and poly(A)+ will be extracted from irradiated G2-cells AS they recover from the radiation-induced division delay. G2-cells are chosen because: one, all RNA synthesis necessary for division is completed at approximately the S/G2 boundary (1,2,3), making it less likely that mRNAs normally required for division will swamp radiation-induced G2 mRNA synthesis; two, there is evidence indicating that the major portion of delay-related repair-synthesis occurs in G2 (4,5); and three, irradiated G2-cells (>90% G2) can be mitotically selected from an irradiated asynchronous population (67) or re-selected from a synchronous population during a second round of selection (greater than or minus 98% G2). Poly(A) RNAs, extracted from irradiated G2-cells were purified and used as templates to synthesize cDNAs (6,7). The cDNAs were packaged into bacteriophage lambda ZAP-II which in turn were transfected into E. coli XL1-Blue r- to create a cDNA library. A control-cell library will also be constructed. The cDNA libraries will be evaluated by plus or minus screening and the unique, radiation-induced clones identified. Proteins from 35S- and 32P-labeled-control and -irradiated G2-cells will be analyzed by two-dimensional polyacrylamide gel electrophoresis. Proteins with molecular size and iso-electric point differences between the irradiated and control populations will be noted and their physical measurements recorded. The values determine by directly labeling the proteins will be compared to the values obtained from in vitro translation products of the unique radiation-induced cDNA clones; agreement will indicate that the clones represent the unique proteins. Once cDNA clones of the radiation-induced poly(a)+ RNA are established, they will be used to probe for similar species during the cell cycle, to determine whether the response is G2-phase specific, a cell cycle response to radiation, or a repair response that takes place in G2 after irradiation in another cell cycle phase.
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