PDGF MODULATED NUCLEAR PROTEIN

血小板衍生生长因子（PDGF）刺激BALB/c-3T3细胞， 快速（在40分钟内）和选择性地合成pI，一个29 K的道尔顿核 蛋白 在发病前很久就注意到pI的优先合成 的DNA合成（12小时），并可能需要最佳的进入PDGF 处理的细胞进入S期。 自发转化BALB/C-3T3 不需要PDGF生长的细胞， 构成时尚。 pI mRNA已使用无细胞 翻译. 它在PDGF治疗后30分钟内积累， 抑制，而是通过添加放线菌酮超诱导。 因此，在本发明中， pI的性质与两种PDGF调节的 原癌基因产物c-myc和c-fos。 c-myc和c-fos mRNA是 在一小时内由PDGF诱导，并通过加入 放线菌酮。 此外，pmyc和pfos是核蛋白。 在 目前，pI不能充分研究，因为适当的 工具-特异性抗血清和重组DNA克隆-并不存在。 这 项目将提供这些工具，以便将来分析pI 功能 将pI纯化至均匀，以制备 单特异性异源血清 这些异源血清将用于定量 在无细胞系统中的pI翻译产物。 将分离mRNA 来自PDGF处理的BALB/c-3T3细胞并基于大小分级; a 将选择富含pI序列的群体并克隆到 表达载体pUC 8/pUC 9。 或者，labmda gt 11表达 可以使用矢量系统。 将通过以下方法选择重组cDNA克隆： 筛选抗原性pI融合蛋白，或通过筛选pI 使用从pI富集的mRNA制备的标记的cDNA探针的cDNA插入物 从多聚核糖体免疫沉淀。 含有pI的克隆的分离 cDNA将允许pI mRNA的表征和定量。 （K）

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)