The platelet derived growth factor (PDGF) stimulates BALB/c-3T3 cells to rapidly (within 40 min) and selectively synthesize pI, a 29K dalton nuclear protein. The preferential synthesis of pI is noted long before the onset of DNA synthesis (12 hrs) and may be required for optimal entry of PDGF treated cells into the S phase. Spontaneously transformed BALB/C-3T3 cells, which do not require PDGF for growth, synthesize pI in a constitutive fashion. pI mRNA has been assayed using cell-free translation. It accumulates within 30 min of PDGF treatment and is not inhibited, but rather, is superinduced by addition of cycloheximide. Thus, the properties of pI are remarkably similar to two PDGF modulated proto-oncogene products, c-myc and c-fos. The c-myc and c-fos mRNAs are induced by PDGF within one hr and are superinduced by addition of cycloheximide. Furthermore, pmyc and pfos are nuclear proteins. At present, pI cannot be adequately studied because appropriate tools--specific antisera and recombinant DNA clones--do not exist. This project will provide these tools to allow a future analysis of pI function. pI will be purified to homogeneity for the purpose of preparing monospecific heterosera. These heterosera will be utilized for quantifying the pI translation product in a cell-free system. mRNA will be isolated from PDGF-treated BALB/c-3T3 cells and fractionated on the basis of size; a population enriched in pI sequences will be selected and cloned into the experssion vectors pUC8/pUC9. Alternatively the labmda gt 11 experssion vector system may be used. Recombinant cDNA clones will be selected by screening for the antigenic pI fusion protein, or by screening for the pI cDNA insert using a labeled cDNA probe prepared from pI enriched mRNA immunoprecipitated from polysomes. The isolation of a clone containing pI cDNA will allow the characterization and quantitation of pI mRNA. (K)
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