ONCOGENES AND TUMORS IN MARINE INVERTEBRATE LARVAE

描述（改编自申请人的摘要）： 主要研究内容有：（1）鉴定、克隆和鉴定细胞 原癌基因控制分化和生长， 外源形态因子和调节信号在幼虫活动中的作用 这些原癌基因的突变改变的克隆形式， 这些幼虫的转化;和（3）定义的依赖性 肿瘤发生活性和表型的特定序列（等位基因） 克隆的转化癌基因，使用定点突变， 结合转化测定。 申请人建议 通过利用独特的实验来实现这些目标， 软体动物和其他海洋无脊椎动物幼虫的易驾驭性， 他们在分子信号上开发了大量的信息， 受体和传感器控制这些幼虫的变态。 控制正常变态和早期发育的潜在原癌基因 这些幼虫的发育将被鉴定、扩增、克隆， 使用PCR和分子杂交程序测序。 形态发生剂诱导的原癌基因转录将用于 促进原癌基因mRNA的纯化， cDNA的扩增和克隆。 C-ras同源物将被扩增 并使用由申请人最近 从参与调节形态发生的幼虫G蛋白中获得。 定点和随机（多位点）诱变都将用于 将这些基因转化为致瘤转化癌基因的努力。 一 将开发独特的转化系统来筛选转化 癌基因，利用高含量的干细胞在 无脊椎动物的幼虫 定点突变将与 与转化测定一起定义特定 癌基因核苷酸序列（等位基因）和特定的表型 肿瘤转化细胞。 这些实验也可能导致 开发转化细胞组织培养系统， 分析控制这些海洋动物肿瘤发生的过程。 拟议研究的结果应首次界定 形态发生分子机制之间的关系 识别/信号转导控制正常基因激活， 分化和细胞增殖以及那些控制癌症的基因， 贝类和其他海洋无脊椎动物。

DESCRIPTION (Adapted from applicant's abstract): The specific aims of the research proposed are: (1) to identify, clone and characterize cellular proto-oncogenes that control differentiation and growth in response to exogenous morphogens and regulatory signals in the larvae activity of mutationally altered, cloned forms of such proto-oncogenes by transformation of these larvae; and (3) to define the dependence of the tumorigenic activity and phenotype(s) on specific sequences (alleles) of the cloned transforming oncogenes, using site-directed mutagenesis in conjunction with transformation assays. The applicants propose to accomplish these aims by taking advantage of the unique experimental tractability of molluscan and other marine invertebrate larvae, and the extensive information they have developed on the molecular signals, receptors and transducers controlling metamorphosis in these larvae. Potential proto-oncogenes controlling normal metamorphosis and early development in these larvae will be identified, amplified, cloned and sequenced using PCR and molecular hybridization procedures. Morphogen-induced transcription of the proto-oncogenes will be used to facilitate purification of the proto-oncogene mRNAs, and subsequent amplification and cloning of the cDNAs. C-ras homologs will be amplified and cloned using primers constructed from sequences the applicants recently obtained from a larval G protein involved in regulation of morphogenesis. Both site-directed and random (multi-site) mutagenesis will be used in efforts to convert these genes to tumorigenic transforming oncogenes. A unique transformation system will be developed to screen for transforming oncogenes, taking advantage of the high content of stem cells in the invertebrate larvae. Site-directed mutagenesis will be used in conjunction with the transformation assays to define the relationship between specific oncogene nucleotide sequences (alleles) and specific phenotypes of the neoplastic transformed cells. These experiments also may lead to the development of a system for transformed cell tissue culture, for further analyses of the processes controlling tumorigenesis in such marine animals. Results of the proposed studies should for the first time define the relationship between the molecular mechanisms of morphogen recognition/signal transduction controlling normal gene activation, differentiation and cellular proliferation and those controlling cancer in shellfish and other marine invertebrates.