GENETIC CONTROL OF MAMMALIAN CELL SURFACE FUNCTIONS

该研究项目的最终目标是确定遗传和 促癌信号转导机制的分子基础 佛波醇酯TPA在培养的小鼠细胞中的作用。 我们正在接近拟议的 通过使用体细胞遗传学， 分子和生物化学技术。 我们调查的具体目的 是： 1.继续表征TPA非应答性3 T3-L1变体， （a）胶原蛋白（TIE-5）基因表达在促有丝分裂中的新作用 （B）已知原癌基因和生长相关基因的表达 TPA处理后TPA非应答性VT-1细胞中的基因，（c） 鉴定磷酸化增强的新蛋白质组分 对细胞的促有丝分裂反应和cDNA克隆的影响，以及（d） 蛋白激酶C和80 K转位中的细胞骨架成分 TPA刺激后的蛋白质。 2.全面分析迄今为止 未鉴定的TIE（TPA诱导的早期）基因（a）序列 三种TIE基因（TIE-4、-10 B和-44）的分析。（B）表达 TPA刺激后3 T3-L1细胞中三种TIE基因的模式， 生长因子和各种小鼠组织中的功能分析。 使用反义表达载体的三种TIE基因，（d）生物化学 三个TIE基因的特征和亚细胞定位 产品，和（e）TIE基因表达的基因组克隆和调控。 3.用于分离信号转导基因的替代方法 利用变异细胞的基因转移技术。 4.激活DNA 拓扑异构酶I（Topo I）通过蛋白激酶C介导的磷酸化作为一种 信号转导重要核事件。 5.染色体定位 利用细胞杂交DNA板和流式分选技术对生长相关早期基因进行了初步研究 人类染色体 6.未来尝试分离更多受影响的变体， 促分裂反应 从细胞和分子遗传学的角度探讨了 跨膜信号传导机制的解剖是独特的， 所产生的信息和材料应证明对我们的 进一步了解细胞生长和分化的调节， 肿瘤发展过程中的恶性细胞转化。

The ultimate goal of this research project is to determine the genetic and molecular basis of signal transduction mechanisms of the tumor promoting phorbol ester TPA in cultured mouse cells. We are approaching the proposed goals through the use of somatic cell genetics in combination with molecular and biochemical techniques. Specific aims of our investigation are the following: 1. Continued characterization of the TPA-nonresponsive 3T3-L1 variants in terms of (a) A new role of collagen (TIE-5) gene expression in mitogenic response, (b) Expression of the known proto-oncogenes and growth-related genes in TPA-nonresponsive VT-1 cells upon TPA-treatment, (c) Identification of new protein components whose phosphorylation is enhanced upon cell's mitogenic response and cDNA cloning, and (d) Involvement of cytoskeletal components in the translocation of protein kinase C and 80K protein upon TPA stimulation. 2. Comprehensive analysis of the as yet unidentified TIE (TPA Inducible Early) genes in terms of (a) Sequence analysis of the three TIE gene (TIE-4, -10B and -44), (b) Expression patterns of the three TIE genes in 3T3-L1 cells upon stimulation by TPA and growth factors and in various mouse tissues, (c) Functional analysis of the three TIE genes using antisense expression vector, (d) Biochemical characterization and subcellular localization of the three TIE gene products, and (e) Genomic cloning and regulation of TIE gene expression. 3. Alternate approach for isolation of the genes for signal transduction using gene transfer technique for variant cells. 4. Activation of DNA topoisomerase I (Topo I) by protein kinase C-mediated phosphorylation as a significant nuclear event of signal transduction. 5. Chromosomal mapping of growth-related early genes using cell hybrid DNA panel and flow-sorted human chromosomes. 6. Future attempts to isolate more variants affected in mitogenic response. The cell and molecular genetic approaches to the dissection of transmembrane signalling mechanisms are unique, and the information and materials to be generated should prove useful for our further understanding the regulation of cell growth and differentiation and the malignant cell transformation during tumor development.