GENE DISCOVERY BY FUNCTIONAL PROBING OF GENOME LIBRARIES

第一阶段提议的总体目标是发现完整的名册 信号转导和肿瘤发生的基因。 我们将 集中于人GTP结合蛋白（G蛋白）基因和G- 蛋白偶联受体（GCR）基因，其中只有一小部分是 知道的 这些新基因的发现将使研究 它们在肿瘤转化中的作用。 作为基因家族的成员 G蛋白和GCR具有共同的功能特征， 通过基因组文库的“功能探测”发现。 我们将 衍生简并DNA探针（寡核苷酸，PCR扩增子）， 高度保守的血栓素受体蛋白基序，蛙皮素 受体、凝血酶受体和Gq α蛋白。 我们将优化 这些探针与网格化的人类基因组杂交的条件 大插入片段（80-150 kb）PI和PAC（PI人工）的DNA文库 染色体）克隆。 阳性基因组克隆将通过 使用基序探针进行Southern分析，其特征在于 细胞遗传学作图和基因组测序。 唯一标识符标签 将为每个新基因家族成员推导并用于评估 不同转录本在正常组织和细胞中的表达 来源于小细胞肺癌和甲状腺髓样癌的细胞系。 拟议的商业应用：基因组克隆将被组装 基因家族试剂盒，并出售给基础和应用领域的研究人员。 药物研究。此外，个体特异性的序列 家族成员也将作为探针和引物商业化， 基因表达分析这些资源的商业价值很高， 因为它们构成了药物发现的目标和基础研究的资源， 癌症信号转导紊乱的研究。

The overall goal of this Phase I proposal is to discover full rosters of genes involved in signal transduction and oncogenesis. We will concentrate on human GTP binding protein (G-protein) genes and G- protein coupled receptor (GCR) genes, only a fraction of which are known. Discovery of these novel genes will then permit studies of their role in neoplastic transformation. As members of gene families sharing functional characteristics, G protein and GCRs are amenable to discovery by "functional probing" of genomic libraries. We will derive degenerate DNA probes (oligonucleotides, PCR amplicons) from highly conserved protein motifs of thromboxane receptors, bombesin receptors, thrombin receptors and Gq alpha proteins. We will optimize conditions for hybridization of these probes to gridded human genomic DNA libraries of large-insert (80-150 kb) PI and PAC (PI Artificial Chromosome) clones. The positive genomic clones will be confirmed by Southern analysis with the motif probe and characterized by cytogenetic mapping and genomic sequencing. A unique identifier tag will be derived for each novel gene family member and used to assess expression of different transcripts in normal tissues and in cell lines derived from small cell lung and medullary thyroid cancers. PROPOSED COMMERCIAL APPLICATION: Genomic clones will be assembled into gene family kits and sold to researchers in basic and applied pharmaceutical research. In addition, sequences specific to individual family members will be also commercialized as probes and primers for gene expression analysis. Commercial value of these resources is high, as they constitute targets for drug discovery and resources for basic research on signal transduction derangements in cancer.