GENETIC & BIOCHEMICAL REGULATION OF PP60-V-SRC ACTIVITY

这些研究的长期目标是确定独特的生化步骤 到可能受特定干扰的转化细胞 药理制剂。有人提出了实验，这将有助于解开 细胞的遗传和生化作用 病毒癌基因v-src诱导的肿瘤转化。癌症是 一种本质上复杂的现象，在 分子水平。目前在分子水平上缺乏理解 已经阻止了这种直接的药理学方法；常用药物 攻击所有分裂的细胞，限制使用的剂量，并导致严重 副作用。 提出了三组特殊的实验：(1)实验 涉及独特的突变等位基因v-src-L的进一步鉴定，以及 进一步突变对v-src-L的后续操纵；(2)实验 为了测试有丝分裂特异性磷酸化在有丝分裂过程中的作用 V-src的转化；以及(3)(与Harold Varmus博士合作， UCSF)对一组独特的v-src突变体进行了分析。 V-src-L能转化鸡细胞，但不能转化大鼠细胞；它依赖于宿主范围 进行转型。由v-src-L编码的pp60v-src-L蛋白将被 详细表征了固有酪氨酸的Km和Vmax- 对几种底物的特异性蛋白激酶活性；初步 有证据表明，它表现出依赖宿主的底物特异性(a 在迄今已报道的所有癌基因中具有独特的性质)。建议 生化和基因实验将解决这种对宿主的依赖 监管已经完成。进一步的实验将确定相关的 鸡和大鼠的细胞基因参与了这一调控。 定点突变将被用于分析有丝分裂的必要性- Wt pp60v-src的特异性磷酸化：(A)增加了激酶活性 在有丝分裂过程中与pp60v-src相关，以及(B)v-src转化。 该领域的其他人的工作主要集中在pp60c-src上。 最后，自然产生的、生物选择的v-src的集合 等位基因(由H.Varmus产生)将根据序列进行分析 为了搜索pp60v-src的结构域，这些结构域对 转型。这组突变体是独一无二的，因为每个成员 (A)是自然发生的(意味着没有调查员偏见)，以及(B) 突变(意味着相对微小的病变)。

The long-term goal of these studies is to identify biochemical steps unique to the transformed cell which might be amenable to interference by specific pharmacological agents. Experiments are proposed which will help unravel the cell's genetic and biochemical contributions to the process of neoplastic transformation induced by the viral oncogene v-src. Cancer is an intrinsically complex phenomenon which is poorly understood at the molecular level. The current lack of understanding at the molecular level has prevented such a directed pharmacological approach; commonly used drugs attack all dividing cells, limiting the dose employed and inducing serious side effects. Three particular sets of experiments are proposed: (1) experiments involving further characterization of the unique mutant allele v-src-L, and the subsequent manipulation of v-src-L by further mutation; (2) experiments to test the role of mitotic specific phosphorylations in the process of transformation by v-src; and (3) (in collaboration with Dr. Harold Varmus, UCSF) analysis of a unique set of mutant of v-src. v-src-L transforms chicken but not rat cells; it is host-range dependent for transformation. The pp60v-src-L protein encoded by v-src-L will be characterized in detail for the Km and Vmax of the intrinsic tyrosine- specific protein kinase activity against several substrates; preliminary evidence suggests it exhibits a host-dependent substrate specificity (a unique property among all oncogenes reported thus far). Proposed biochemical and genetic experiments will address how this host-dependent regulation is accomplished. Further experiments will identify the relevant chicken and rat cellular genes involved in this regulation. Site-directed mutagenesis will be used to analyze the necessity of mitosis- specific phosphorylation of wt pp60v-src for (a) increased kinase activity associated with pp60v-src during mitosis and (b) transformation by v-src. Work by others in this field has focused on pp60c-src. Finally, a collection of naturally occurring, biologically selected v-src alleles (generated by H. Varmus) will be analyzed with regard to sequence in order to search for domains of pp60v-src which are critical for transformation. This collection of mutants is unique in that each member is (a) naturally occurring (implying no investigator bias), and (b) a point mutant (implying a relatively subtle lesion).