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)并选择性地合成29K道尔顿原子核的PI 蛋白。PI的优先合成早在发病之前就被注意到了。 DNA合成(12小时)，可能是PDGF最佳进入所必需的 处理后的细胞进入S期。自发转化BALB/C-3T3 生长不需要PDGF的细胞在体内合成PI 构成时尚。PI信使核糖核酸的测定采用无细胞方法 翻译。它在PDGF治疗后30分钟内蓄积，而不是 抑制，但更确切地说，是通过添加放线菌亚胺而过诱导的。因此， PI的性质与两种PDGF调制的性质非常相似 原癌基因产物c-myc和c-fos。C-myc和c-fos mRNAs是 在1小时内由PDGF诱导，并通过添加 环己亚胺。此外，pmyc和全氟辛烷磺酸是核蛋白。在… 目前，PI不能得到充分的研究，因为 工具--特定的抗血清和重组DNA克隆--不存在。这 Project将提供这些工具，以便将来对PI进行分析 功能。PI将被提纯至均一，以便制备 单特异性异色酶。这些杂色体将被用于定量 无单元格系统中的PI翻译产品。将分离出mrna 来自PDGF处理的BALB/c-3T3细胞，并根据大小进行分级；a 富含PI序列的群体将被选择并克隆到 表达载体pUC8/pUC9。或者，Labmda GT 11 Expression 可以使用矢量系统。重组cDNA克隆将通过以下方式进行筛选 抗原性PI融合蛋白的筛选或PI的筛选 用从PI富集型mRNA制备的标记的cDNA探针插入 来自多聚体的免疫沉淀。一株含猪的克隆的分离 CDNAs可用于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)