ONCOGENES AND TUMORS IN MARINE INVERTEBRATE LARVAE

描述(改编自申请人的摘要)： 提出的研究内容如下：(1)鉴定、克隆和鉴定细胞 控制分化和生长的原癌基因 幼虫活动中的外源形态因子和调控信号 这些原癌基因的突变、克隆形式通过 这些幼虫的转化；以及(3)定义对 特定序列(等位基因)的致瘤活性和表型(S) 利用定点突变技术克隆转化癌基因 与转化分析相结合。申请者建议 通过利用独特的实验优势实现这些目标 软体动物和其他海洋无脊椎动物幼虫的可驯性，以及 他们开发了大量关于分子信号的信息， 控制这些幼虫变态的受体和转导。 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、克隆和 用聚合酶链式反应和分子杂交法进行测序。 原癌基因的形态诱导转录将被用于 促进原癌基因mRNAs的纯化，并随后 CDNA的扩增和克隆。C-ras同源物将被扩增 并使用从申请者最近构建的序列构建的引物进行克隆 从一种参与形态发生调节的幼虫G蛋白中获得。 定点突变和随机(多点)突变都将用于 将这些基因转化为致癌转化癌基因的努力。一个 将开发独特的转化系统来筛选转化 癌基因，利用体内干细胞含量高的优势 无脊椎动物幼虫。定点突变将与联合使用 用转化分析法来定义特定的关系 癌基因核苷酸序列(等位基因)和特定表型 肿瘤性转化细胞。这些实验也可能导致 一种用于转化细胞组织培养的系统的开发 这类海洋动物肿瘤发生的控制过程的分析。 拟议研究的结果应首次界定 形态发生的分子机制之间的关系 控制正常基因激活的识别/信号转导， 分化和细胞增殖与癌症的控制者 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.