GENETIC CONTROL OF MAMMALIAN CELL SURFACE FUNCTIONS

这项研究项目的最终目标是确定基因和 促肿瘤信号转导机制的分子基础 佛波酯TPA在培养的小鼠细胞中的表达。我们正在接近拟议的 通过结合使用体细胞遗传学和 分子和生化技术。我们调查的具体目标 以下是： 1.对TPA无反应的3T3-L1变异体的继续鉴定 关于(A)胶原(Tie-5)基因表达在有丝分裂中的新作用 反应，(B)已知的原癌基因和生长相关基因的表达 TPA处理后对TPA无反应的VT-1细胞中的基因，(C) 磷酸化增强的新蛋白质组分的鉴定 关于细胞的有丝分裂反应和cdna克隆，以及(D)参与 蛋白激酶C和80K转位中的细胞骨架成分 蛋白质对TPA刺激的影响。2.对目前为止的全面分析 (A)序列中未识别的Tie(TPA可诱导早期)基因 三个Tie基因(Tie-4、-10B和-44)的分析(B)表达 TPA和TPA刺激3T3-L1细胞后三种Tie基因的表达谱 生长因子和小鼠不同组织中，(C)功能分析 使用反义表达载体的三个Tie基因，(D)生化 三个TiE基因的特性及其亚细胞定位 以及(E)TIE基因表达的基因组克隆和调控。 3.分离信号转导基因的替代方法 利用基因转移技术获得变异细胞。4.DNA的激活 拓扑异构酶I(Topo I)在蛋白激酶C介导的磷酸化过程中的作用 信号转导的重大核事件。5.染色体定位 应用细胞杂交DNA拼板和流式分选技术筛选生长相关早期基因 人类的染色体。6.未来尝试分离更多受影响的变种 有丝分裂反应。细胞和分子遗传学研究进展 对跨膜信号机制的剖析是独一无二的，并且 将生成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控以及 肿瘤发生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.