RETINOBLASTOMA SUSCEPTIBILITY GENE--FUNCTIONAL ANALYSIS

有人提出存在肿瘤抑制基因，或“抑癌基因”。 在先前的实验中，正常细胞与一种 恶性对应物被注意到抑制了恶性表型 后者，以及来自证明非随机损失的研究 来自各种肿瘤的染色体物质。一个这样的基因，Rb1，是 最近进行了克隆和测序。其产品Rb是一种核磷蛋白 其具有细胞周期调节元件的特性。RB是绑定的 被三种DNA病毒癌蛋白(ELA、SV40 Large T和HPV)灭活 E7)，每个包含涉及的短的、同源的、共线的序列 在Rb结合和它们转化细胞的能力方面都是如此。就像这些 病毒在其他方面是无关的，看起来很可能它们的片段 编码该序列的基因组来自于编码一种 以类似的方式与Rb形成复合体的蛋白质。这一假设 通过观察到那些自然产生的RB 到目前为止已测试的突变体在EL A/T/E7结合上存在缺陷 根据初步数据显示，RB的一大片区域 发生这种绑定所必需的。该基因的鉴定和鉴定 它的蛋白质产品可能会提供一个重要的线索，说明 Rb抑制细胞生长。 体外结合试验将被用来定位Rb的最小区域 结合ELA/T/E7所必需的。与Rb结合的突变体的表达 然后，可以检查ELA/T/E7是否具有抑制细胞生长的能力 RB-/-细胞系及其诱导生物学效应的能力 在Rb+/+细胞中与内源性Rb竞争这一推定的 细胞因素。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。艾尔 A/T/E7结合Rb突变体也将被过度生产并在尝试中使用 生化分离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.