CLONING TUMOR SUPPRESSOR GENES (TSG) FROM HUMAN CHROMOSOME 3P

在过去的十年中，大约有15个TSG被鉴定和克隆。 与 手中的基因，努森的基础理论的预测， 人类致癌作用得到证实，突变的癌症导致TSGs 成为遗传病因学和遗传病发病机制的典范 常见的癌症。 本研究的最终目的是（1）完成分离 和表征的VHL TSG，并开始了解其 生物化学功能;（2）以及从所述细胞中鉴定一种或多种TSG。 3 p21-p22区域，参与肺癌的发生。 我们今年的主要成就是：（1）识别和 VHL基因启动子的分析; 3' UTR和VHL无内含子基因的构建; 不同表达载体中的一组VHL正义和反义小基因 （包括附加型和逆转录病毒）用于离体/体内递送; 高表达VHL肾癌细胞系的建立 转基因和pVHL/vhilin。 (2)SCLC TSG的定位 在700 kb粘粒- P1克隆重叠群的端粒部分内 跨越由重叠缺失定义的候选区域， 等位基因分型 我们和其他人绘制了一些潜在的TSG， 3 p21-p22，即GNAI-2、GNAT-1、Wint 5、β连环蛋白、TGF β RII和 MLH 1从该候选区域中排除。 到目前为止，12个新基因被分离，部分或完整的cDNA 测序，他们的候选人正在评估突变， 分析（外显子的SSCP或RT PCR，然后测序）和 在SCLC细胞系中表达。

In the past decade about 15 TSG(s) were identified and cloned. With the gene in hand the predictions of Knudson's foundational theory of human carcinogenesis were verified and mutated cancer causing TSGs became the paradigm of genetic etiology and pathogenesis of inherited and common cancers. The ultimate goals of our research were (1) to complete the isolation and characterization of the VHL TSG and to begin to understand its biochemical function; (2) and to identify one or more TSGs from the 3p21-p22 region, involved in the causation of lung cancer(s). Our major accomplishments this year are: (1) identification and analysis of the promoter of the VHL gene; identification of the long 3' UTR and construction of a VHL intronless gene; construction of a set of VHL sense and antisense minigenes in diverse expression vectors (including episomal and retroviral) for ex vivo/in vivo delivery; establishment of renal carcinoma (RCA) cell lines overexpressing VHL transgenes and pVHL/vhilin. (2) The positioning of the SCLC TSG within the telomeric part of a 700 kb cosmid - P1 clone contig spanning the candidate region as defined by overlapping deletions and allelotyping. A number of potential TSG mapped by us and others to 3p21-p22 namely, GNAI-2, GNAT-1, Wint 5, beta catenin, TGFbetaRII, and MLH1, were excluded from this candidate region. To date 12 new genes were isolated, partial or complete cDNAs sequenced, and their candidacy is being evaluated by mutational analysis (SSCP of exons or RT PCR followed by sequencing) and expression in SCLC cell lines.