GENETIC & BIOCHEMICAL REGULATION OF PP60-V-SRC ACTIVITY

基因

• 批准号: 3197637
• 负责人: MICHAEL F VERDERAME
• 金额: $ 12.79万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-10 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3197637
• 关键词: alleles; chickens; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; molecular oncology; mutant; neoplastic transformation; oncogenes; phosphorylation; protein biosynthesis; protein tyrosine kinase; protooncogene; site directed mutagenesis; transposon /insertion element

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).

这些研究的长期目标是确定独特的生化步骤 转化的细胞可能会受到特定的干扰 药理学试剂。 提出了一些实验， 细胞的遗传和生化贡献的过程中， 由病毒致癌基因v-src诱导的肿瘤转化。 癌症是 这是一种内在复杂的现象， 分子水平。 目前在分子水平上缺乏了解 阻止了这种直接的药理学方法;常用药物 攻击所有分裂细胞，限制使用的剂量并诱导严重的 副作用. 提出了三组具体的实验：（1）实验 涉及独特突变等位基因v-src-L的进一步表征，和 通过进一步突变对v-src-L进行后续操作;（2）实验 测试有丝分裂特异性磷酸化在细胞分裂过程中的作用 通过v-src进行转化;以及（3）（与Harold Varmus博士合作， UCSF）分析一组独特的v-src突变体。 v-src-L转化鸡细胞而非大鼠细胞;它是宿主范围依赖性的 进行改造。 由v-src-L编码的pp 60 v-src-L蛋白将被 详细表征了固有酪氨酸的Km和Vmax， 对几种底物特异性蛋白激酶活性;初步 有证据表明，它表现出宿主依赖性底物特异性（a 在迄今为止报道的所有癌基因中具有独特的性质）。 提出 生物化学和遗传实验将解决这种宿主依赖性 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这种调节。 定点突变将用于分析有丝分裂的必要性- 野生型pp 60 v-SRC的特异性磷酸化用于（a）增加的激酶活性 在有丝分裂过程中与pp 60 v-src相关，和（B）通过v-src的转化。 其他人在这一领域的工作主要集中在pp 60 c-src上。 最后，一系列自然产生的，生物选择的v-src 等位基因（由H. Varmus）将进行序列分析 为了寻找对以下关键的PP 60 V-SRC的结构域 转型 这个突变体的集合是独一无二的， 是（a）自然发生（意味着没有研究者偏倚），和（B）一个点 突变体（意味着相对细微的病变）。