EFFECTORS AND REGULATORS OF NORMAL AND ONCOGENIC RAS

ras基因对于真核细胞中的细胞生长控制是必需的。 此外，激活的ras基因是最常见的癌基因之一 in human人tumors肿瘤. 在酿酒酵母中， 等位基因（类似于哺乳动物ras中的致癌突变）导致 腺苷酸环化酶的过度刺激。 环化酶活性升高结果 在不受控制的细胞增殖中， 营养缺乏和对热休克的急性敏感性。 虽然做了大量 在理解酵母中ras的生物化学方面取得的进展， 在哺乳动物细胞中的途径不同，仍然不清楚。 这项建议 描述了隔离，通过选择抑制热量的能力， 携带激活ras等位基因的酵母细胞的休克敏感性， 编码ras相互作用蛋白的哺乳动物cDNA。 我们最初的cDNA分离物包括磷酸二酯酶， 降低细胞内cAMP水平。 另外三个克隆人 哺乳动物cDNA表达文库显然编码蛋白质， 通过与光栅直接相互作用干扰光栅功能（两者 野生型和致癌的）。 这些都有可能抑制ras功能 或者通过降低细胞中活性RAS的比例（降低 调节），或者通过与RAS形成非生产性复合体， 其有效浓度（显性负干扰）。 后者 类应包括截短的ras效应物，其结合ras，但 无法传递生长信号。 所有这些分离株都是部分的 cDNA。 为了确定这些基因产物的正常功能， 将分离长度cDNA并在我们的模型系统中表达。 的 干扰RAS功能所需的最小部分也将 通过删除映射确定。 这项工作的一个中心焦点涉及细胞培养实验， 确定正常基因产物在哺乳动物细胞中的作用。 如果 它们通常充当RAS抑制剂或下调剂， 过量生产会干扰哺乳动物细胞中ras的功能， 通过它们阻断或逆转Ras转化的能力来判断。 如果我们的全长克隆编码效应蛋白， 在哺乳动物细胞中的过表达可以提高，而不是拮抗， ras函数 对ras诱导的分化的影响也将在 考察 这些研究的结果应该大大扩展我们对 ras在哺乳动物细胞中的功能。 它们也应该导致一个更好的 对肿瘤发生的一般理解。

ras genes are essential for cellular growth control in eucaryotes. Moreover, activated ras genes are among the most commonly found oncogenes in human tumors. In the yeast Saccharomyces cerevisiae, an activated ras allele (analogous to oncogenic mutations in mammalian ras) causes the overstimulation of adenylyl cyclase. Elevated cyclase activity results in uncontrolled cell proliferation, an inability to arrest in response to nutrient starvation and acute sensitivity to heat shock. Despite great strides in understanding the biochemistry of ras in yeast, the ras pathway in mammalian cells differs and remains unclear. This proposal describes the isolation, by selecting for the ability to suppress heat shock sensitivity in yeast cells harboring an activated ras allele, of mammalian cDNAs that encode ras-interacting proteins. Our initial cDNA isolates include phosphodiesterases that 'suppress' by lowering intracellular cAMP levels. Three additional clones from our mammalian cDNA expression library apparently encode proteins that interfere with ras function through a direct interaction with ras (both wild type and oncogenic). These are likely to suppress ras function either by decreasing the proportion of active ras in the cell (down regulation), or by forming a nonproductive complex with ras and lowering its effective concentration (dominant negative interference). The latter class should include truncated ras effectors that bind to ras but are unable to transmit growth signals. All of these isolates were partial cDNAs. To ascertain the normal functions of these gene products, full length cDNAs will be isolated and expressed in our model system. The minimum portion required for interference with ras function will also be determined by deletion mapping. A central focus of this work involves cell culture experiments to determine the role of the normal gene products in mammalian cells. If they normally act as ras suppressors or down regulators, then overproduction should interfere with ras function in mammalian cells, as judged by their ability to either block or reverse ras transformation. If our full length clones encode effector proteins, then their overexpression in mammalian cells may elevate, rather than antagonize, ras function. Effects on ras-induced differentiation will also be examined. The results of these studies should greatly expand our knowledge of the function of ras in mammalian cells. They should also lead to a better understanding of oncogenesis in general.