ISOLATION OF THE RETINOBLASTOMA SUSCEPTIBILITY GENE

视网膜母细胞瘤发生在两种不同的遗传形式，遗传和 非遗传性的。 这些形式往往可以区分的基础上， 临床特征、家族史、家系分析（与 多态性标记酶酯酶D）和染色体分析。 积累 证据强烈支持单个视网膜母细胞瘤的存在 两种类型视网膜母细胞瘤的易感基因位点。 肿瘤发生 似乎涉及该基因座上两个等位基因的改变。 在 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 我们建议将视网膜母细胞瘤易感性 (Rb)基因的“染色体步行”在两个方向从酯酶D 该基因与13号染色体q14.2带的Rb紧密连锁。 一 本实验室已克隆出酯酶D基因。 最好的 Rb基因的候选者将位于DNA片段中， 13q14与正常人胚胎视网膜（HER）mRNA杂交 细胞，但不与视网膜母细胞瘤细胞的mRNA。 或者，mRNA 将用于“减去”HER细胞的cDNA 剩余的cDNA克隆将被定位回13q14区域。 到 阳性鉴定Rb基因，候选克隆可以被引入到 视网膜母细胞瘤细胞，同时观察肿瘤的表型逆转 细胞 另一种方法是引入反义链的 将这些基因导入正常HER细胞中，并观察细胞转化 有视网膜母细胞瘤的特征 由Rb基因编码的蛋白质将是 从核苷酸序列推断并通过免疫沉淀鉴定 与相应蛋白质的合成肽的抗体结合。 Rb基因的分离是确定分子生物学的关键步骤。 视网膜母细胞瘤的肿瘤发生 此外，该基因可用作 限制性片段长度多态性分析中的探针来诊断 遗传性与非遗传性疾病，包括产前和产后。

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.