ISOLATION OF THE RETINOBLASTOMA SUSCEPTIBILITY GENE

视网膜母细胞瘤有两种不同的遗传形式，遗传性和 非遗传性。这些形式通常可以根据以下条件进行区分 临床特征、家族史、家系分析(与 多态标记酶(酯酶D)和染色体分析。累计 有证据强烈支持单一视网膜母细胞瘤的存在 两种视网膜母细胞瘤的易感基因。肿瘤发生 似乎涉及到这个基因座上两个等位基因的改变。在……里面 遗传性视网膜母细胞瘤，一个缺陷或缺失的等位基因是遗传的， 而肿瘤的表达取决于额外的突变或缺失 另一个等位基因。我们建议分离视网膜母细胞瘤的易感性 (Rb)基因从酯酶D开始双向“染色体行走” 该基因与13号染色体q14.2带的Rb紧密连锁。 本实验室已成功克隆了酯酶D基因。最好的 Rb基因的候选基因将位于映射到Band的DNA片段中 与正常人胚胎视网膜(HER)基因杂交的13q14 细胞，但不含视网膜母细胞瘤细胞的mRNA。或者，信使核糖核酸 从视网膜母细胞瘤细胞中提取的基因将被用来“减去”HER细胞的cDNAs 其余的克隆将被定位回13q14区域。至 正向鉴定Rb基因，可引入候选克隆 视网膜母细胞瘤细胞观察肿瘤表型逆转 细胞。另一种方法是引入反义链 将这些基因导入正常HER细胞，观察转化为细胞的情况 具有视网膜母细胞瘤的特征。由Rb基因编码的蛋白质(S)将被 从核苷酸序列推导出并通过免疫沉淀鉴定 具有抗体的合成肽的相应蛋白质(S)。 Rb基因的分离是确定分子水平的关键步骤 视网膜母细胞瘤的致癌作用。此外，该基因还可以作为一种 限制性片段长度多态分析在诊断中的应用 遗传性和非遗传性疾病，包括出生前和出生后。

Retinoblastoma occurs in two different genetic forms, hereditary and nonhereditary. These forms can often be distinguished on the basis of clinical features, family history, pedigree analysis (linkage to a polymorphic marker enzyme esterase D) and chromosome analysis. Accumulated evidence strongly favors the existence of a single retinoblastoma susceptibility locus for both types of retinoblastoma. Tumorigenesis appears to involve alterations in both alleles at this locus. In hereditary retinoblastoma, one defective or missing allele is inherited, and expression of the tumor depends on additional mutation or deletion of the other allele. We propose to isolate the retinoblastoma susceptibility (Rb) gene by "chromosome walking" in both directions from the esterase D gene, which is tightly linked to Rb in band q14.2 of chromosome 13. A clone of the esterase D gene is available in our laboratory. The best candidates for the Rb gene will be located in DNA segments mapping to band 13q14 which hybridize with mRNA from normal human embryonic retinal (HER) cells but not with mRNA from retinoblastoma cells. Alternatively, mRNA from retinoblastoma cells will be used to "subtract out" cDNA of HER cells and the remaining cDNA clones will be mapped back to the 13q14 region. To positively identify the Rb gene, candidate clones can be introduced into retinoblastoma cells while observing for phenotypic reversion of tumor cells. An alternative method is to introduce the antisense strands of these genes into normal HER cells and observe for transformation to cells with features of retinoblastoma. Protein(s) encoded by the Rb gene will be deduced from the nucleotide sequence and identified by immunoprecipitation with antibodies to synthetic peptides of the corresponding protein(s). Isolation of the Rb gene is a crucial step in determining the molecular oncogenesis of retinoblastoma. In addition, this gene can be used as a probe in a restriction fragment length polymorphism assay to diagnose hereditary versus non-hereditary disease, both pre- and post-natally.