EXPRESSION AND FUNCTION OF PROTO-ONCOGENE C-SRC

原癌基因c-src的表达 Rous肉瘤病毒转化基因v-src的克隆， 组织特异性和发育调节。 而不是 通常观察到的4kb mRNA，2.8至3.3的较小类别， kbc-src mRNA存在于鸡骨骼肌中。 感应 较小的c-src mRNAS的表达与 肌肉中4kb RNA的消失，并在短时间内发生 在孵化之前。 较小的c-src mRNAs缺乏大部分的 酪氨酸激酶结构域，最有可能编码蛋白质， 比酪氨酸激酶。 4kb和更小的c-src mRNA是 显然是由选择性剪接产生的 这个目标 建议是探讨研究这一制度的功能， c-src原癌基因调控的分子机制。 将分离4 kb和2.8 - 3.3 kb RNA的cDNA克隆 并进行测序，以揭示它们的序列内容和潜在的 小的c-src mRNAs的产物。 外显子结构和 将分析c-src DNA的启动子位点以阐明 选择性剪接的模式。 C的表达- 将检查小鼠和大鼠中src以查看是否相似的组织 在哺乳动物中存在特异性和发育调节 物种 针对肌肉特异性的多克隆和单克隆抗体 制备c-src蛋白。 这些抗体将用于 鉴定并定位c-src蛋白在肌肉组织中的表达。 它的功能将通过用抗- 小c-src特有的有义逆转录病毒序列 mRNA。 控制c-src表达的因素将在 细胞和分子水平。 突触肌肉活动的影响 对特定c-src RNA和蛋白质表达的影响将是 通过去神经支配和使用神经毒素。 潜在 与启动子序列相互作用的因子将通过 DNA凝胶阻滞和足迹分析。 dna酶 将绘制超敏和低甲基化位点， 与c-src基因活性相关。 潜在反式作用因子 影响RNA剪接和poly（A）位点选择的方法将是 通过体外RNA加工系统研究。

The expression of proto-oncogene c-src, the cellular counterpart of the transforming gene v-src of Rous sarcoma virus, was found to be tissue specific and developmentally regulated. Instead of the commonly observed 4 kb mRNA, a smaller class of 2.8 to 3.3 kb c-src mRNAs is present in chicken skeletal muscle. Induction of the expression of the smaller c-src mRNAS concurs with the disappearance of the 4 kb RNA in muscle and occurs shortly before hatching. The smaller c-src mRNAs lack most of the tyrosine kinase domain, and most likely code for a protein other than tyrosine kinase. The 4 kb and the smaller c-src mRNAs are apparently generated by alternative splicing. The goal of this proposal is to explore this system to study the function and molecular mechanism of regulation of c-src proto-oncogene. cDNA clones of the 4 kb and 2.8 to 3.3 kb RNAs will be isolated and sequenced to reveal their sequence content and the potential product of the smaller c-src mRNAs. The exon structure and promoter sites of c-src DNA will be analyzed to elucidate the pattern of alternative splicing for those mRNAs. Expression of c- src in mouse and rat will be examined to see if similar tissue specificity and developmental regulation exists in mammalian species. Polyclonal and monoclonal antibodies against the muscle-specific c-src protein will be prepared. These antibodies will be used to identify and then to localize the c-src protein in muscle tissue. Its function will be explored by blocking its expression with anti- sense retrovirus-containing sequences unique to the smaller c-src mRNAs. Factors controlling c-src expression will be investigated at cellular and molecular levels. Effect of synaptic muscle activity on expression of the specific c-src RNAs and proteins(s) will be approached by denervation and by using neurotoxins. Potential factors interacting with promoter sequences will be analyzed by DNA gel retardation and footprinting analyses. DNAse hypersensitive and hypomethylation sites will be mapped and correlated to c-src gene activity. Potential trans-acting factors affecting RNA splicing and poly(A) site selection will be investigated by in vitro RNA processing system.