EXPRESSION AND FUNCTION OF PROTO-ONCOGENE C-SRC

原癌基因c-src在细胞内的表达 发现了Rous肉瘤病毒的转化基因v-src。 具有组织特异性和发育调节性。而不是 通常观察到的4kb的信使核糖核酸，是一个2.8到3.3的较小类别 Kb、c-src mRNAs存在于鸡骨骼肌中。归纳 较小的c-src mRNAs的表达与 肌肉中4kb的RNA消失，并很快发生 在孵化之前。较小的c-src mrna缺乏大多数 酪氨酸激酶结构域，以及最可能的其他蛋白质的编码 而不是酪氨酸激酶。4kb和较小的c-src mRNAs为 显然是由另一种剪接方式产生的。这样做的目的是 建议对该系统进行探索，研究其功能和功能 C-src原癌基因调控的分子机制。 将分离出4kb和2.8kb至3.3kb的RNA的cDNA克隆 并进行测序以揭示它们的序列内容和潜在的 较小c-src mRNAs的产物。外显子结构和 将分析c-src dna的启动子位置，以阐明 这些mRNAs的选择性剪接模式。C-的表达 将对小鼠和大鼠的SRC进行检查，看看相似的组织 哺乳动物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 将制备c-src蛋白。这些抗体将被用来 鉴定并定位肌肉组织中的c-src蛋白。 它的功能将通过用反义寡核苷酸阻断其表达来探索。 检测较小c-src所特有的逆转录病毒序列 MRNAs。 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 关于特定c-src RNA和蛋白的表达(S) 通过去神经和使用神经毒素来接近。潜力 与启动子序列相互作用的因素将通过 DNA凝胶延迟和足迹分析。DNA酶 将绘制过敏和低甲基化部位的地图，并 与c-src基因活性相关。潜在的反式作用因子 影响RNA剪接和Poly(A)位点选择的将是 采用体外RNA处理系统进行研究。

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.