NOVEL MITOGENIC REGULATORY GENE

我们发现了一个新的细胞基因322，它可能编码 调节或肿瘤抑制功能。这个基因是从 一个cDNA文库，其代表在转录水平较低的基因， 未转化的细胞，甚至更低（即，抑制） 被激活的癌基因v-src转化我们假设这些基因 可能包括有丝分裂的候选调节因子， 它们的表达可能有助于转化的表型。的 322 RNA的稳态水平在src-、ras-和 与未转化的对照相比，fos转化的成纤维细胞，但 而不是在RAF-neu-或MOS-转化的细胞中。这种下调并不是 322基因等位基因的严重损失或损害的结果，如确定的， Southern blotting。ts-src等位基因的激活或胎儿基因的添加 小牛血清对饥饿细胞的作用导致322 mRNA的快速增加， 水平（1-2小时），然后快速下降（再过4-6小时后） 到抑制水平，表明322转录直接 由v-src和促有丝分裂刺激控制。322的过度表达 cDNA导致细胞增殖率显著降低， 未转化和转化的细胞，并显着抑制src- 和FOS诱导的形态学转化。预备序列 对推定的全长322 cDNA（5.4Kb）的分析表明， 很新奇。尽管最大的开放阅读框编码170 kD的蛋白质， 产物，该cDNA的体外转录/翻译产生207 kD的 多肽，其增加的分子量不能归因于N- 连接糖基化两个富含甘氨酸/精氨酸/赖氨酸的区域， 与Epstein-Barr病毒核抗原同源，在N- 322产物的末端结构域。这些数据强烈表明 322编码一种新的有丝分裂调节因子。我们的总体目标是 描述这个基因产物的功能。具体而言，我们将（i） 表征src和ras诱导转录的机制 322的抑制（起始与平均稳定性），ii）确定 322在转化和未转化细胞中过表达的影响， iii）体外表达322蛋白产物并产生特异性抗- 血清，和iv）鉴定细胞蛋白， 特异性地与322产物相互作用，使用酵母双- 杂交系统或免疫共沉淀分析。 我们的长期目标是 阐明322产品的作用途径或位点。我们设想 这些数据将有助于描述322人参与的情况， 在未转化细胞中的促有丝分裂控制和在 转化细胞这些数据也将增加我们对如何 癌基因在诱导细胞凋亡的过程中破坏细胞通路， 形态转化和肿瘤发生。

We have identified a novel cellular gene, 322, which potentially encodes a regulatory or tumor suppressor function. This gene was isolated from a cDNA library representing genes whose transcription is low in untransformed cells and even lower (i.e.- suppressed) in cells transformed by the activated oncogene, v-src. We assumed that these genes might include candidate regulators of mitogenesis, and that the loss of their expression might contribute to the transformed phenotype. The steady-state level of 322 RNA is depressed >15-fold in src-, ras- and fos-transformed fibroblasts in comparison to untransformed controls, but not in raf- neu- or mos-transformed cells. This down-regulation is not the result of gross loss or damage to the 322 gene allele, as determined by southern blotting. Activation of a ts-src allele or addition of fetal calf serum to starved cells results in a rapid increase in 322 mRNA levels (1-2 hours) followed by a rapid decrease (after another 4-6 hours) to suppressed levels, indicating that 322 transcription is directly controlled by v-src and mitogenic stimuli. Over-expression of the 322 cDNA results in a significant decrease in the proliferation rates of untransformed and transformed cells, and significantly suppresses src- and fos-induced morphological transformation. Preliminary sequence analysis of the putative full-length 322 cDNA (5.4Kb) indicates that it is novel. Although the largest open-reading frame would encode a 170kD product, in vitro transcription/translation of this cDNA yields a 207kD polypeptide whose increased molecular weight cannot be attributed to N- linked glycosylation. Two glycine/arginine/lysine-rich regions, homologous to Epstein-Barr virus nuclear antigens, are found in the N- terminal domain of the putative 322 product. These data strongly suggest that 322 encodes a novel regulator of mitogenesis. Our overall aim is to characterize the function of this gene product. Specifically, we will i) characterize the mechanism of src- and ras-induced transcription suppression of 322 (initiation vs. meassage stability), ii) determine the effects of 322 over-expression in transformed and untransformed cells, iii) express the 322 protein product in vitro and raise specific anti- sera to this product, and iv) identify cellular proteins which specifically interact with the 322 product using either the yeast two- hybrid system or co-immunoprecipitation analysis. Our long-term goal is to elucidate pathways or sites of action of the 322 product. We envision that these data will help characterize the involvement of 322 in mitogenic control in untransformed cells and in tumor suppression in transformed cells. These data would also increase our knowledge of how oncogenes subvert cellular pathways in the process of inducing morphological transformation and tumorigenesis.