RETINOBLASTOMA SUSCEPTIBILITY GENE--FUNCTIONAL ANALYSIS

肿瘤抑制基因，或“抑癌基因”的存在， 通过先前的实验，其中正常细胞与 恶性对应物被注意到抑制恶性表型的 后者，以及从研究表明，非随机损失的 各种肿瘤的染色体物质其中一个基因RB 1， 最近克隆并测序的其产物Rb是一种核磷蛋白 其具有细胞周期调节元件的性质。Rb被束缚 (?灭活）由三种DNA病毒癌蛋白（E1 A、SV 40大T和HPV E7），其各自含有短的同源共线序列， Rb结合和转化细胞的能力。因为这些 病毒在其他方面是不相关的，似乎很可能是它们的片段 编码该序列的基因组来源于编码 以类似方式与Rb形成复合物的蛋白质。这一假设 这些天然存在的Rb 迄今为止已经测试的突变体在E1 A/T/E7结合中是缺陷的 初步数据表明，Rb的大区域是 这是发生这种绑定所必需的。该基因的鉴定和 其蛋白质产物可能提供一条重要线索， Rb抑制细胞生长。 体外结合试验将用于绘制Rb的最小区域 结合E1 A/T/E7所必需的。结合至Rb的Rb突变体的表达 然后可以检查E1 A/T/E7抑制细胞生长的能力。 Rb-/-细胞系以及它们诱导生物效应的能力 在Rb+/+细胞中，通过与内源性Rb竞争这种假定的 细胞因子一组抗Rb抗体将用于寻找 与Rb和/或Rb突变体共免疫沉淀的蛋白质。El A/T/E7结合Rb突变体也将被过量生产并用于尝试 以生物化学方法分离Rb结合蛋白。

The existence of tumor suppressor genes, or 'anti-oncogenes', was suggested by previous experiments in which the fusion of a normal cell with a malignant counterpart was noted to suppress the malignant phenotype of the latter, as well as from studies demonstrating non-random loss of chromosomal material from a variety of tumors. One such gene, RB 1, was recently cloned and sequenced. Its product, Rb is a nuclear phosphoprotein which has properties of a cell-cycle regulatory element. Rb is bound (?inactivated) by three DNA viral oncoproteins (ElA, SV40 large T, and HPV E7) which each contain a short, homologous, colinear sequence implicated both in Rb binding and in their ability to transform cells. As these viruses are otherwise unrelated, it seems likely that the segment of their genomes encoding this sequence is derived from a host gene encoding a protein which forms a complex with Rb in a similar manner. This hypothesis is strengthened by the observation that those naturally occurring Rb mutants which have been tested to date are defective in El A/T/E7 binding and by preliminary data which suggest that a large region of Rb is necessary for this binding to occur. The identification of this gene and its protein product might potentially provide an important clue as to how Rb suppresses cell growth. An in vitro binding assay will be used to map the minimum region of Rb necessary for binding ElA/T/E7. Expression of Rb mutants which bind to ElA/T/E7 can then be examined for their ability to suppress cell growth in Rb-/- cell lines as well as for their ability to induce biological effects in Rb+/+ cells through competition with endogenous Rb for this putative cellular factor. A panel of anti-Rb antibodies will be used to look for proteins which co-immunoprecipitate with Rb and/or with Rb mutants. El A/T/E7 binding Rb mutants will also be overproduced and used in an attempt to biochemically isolate Rb binding proteins.