DETECTION OF PROTEIN-PROTEIN INTERACTIONS DURING GROWTH REGULATORY ACTIVITY

伴随着血管紧张素转换酶基因改变的CAD基因复合体的扩增 多肽在HepG2和中国仓鼠卵巢中的表达 (CHO)细胞用N-膦乙酰L-天冬氨酸(PALA)筛选。在……里面 最小选择压力(3XLD-50)的HepG2细胞，PALA抗性 在没有可观察到的CAD扩增的情况下形成的菌落 吉恩。另一方面，在未经处理的HepG2细胞中，没有代谢 应激时，CAD基因经历了自发扩增并增加 通行号。Southern分析表明，该CAD基因是 与早期相比，传代后期(P160)扩增达30倍 传代(P113)细胞，提示CAD扩增不是 PALA治疗的结果，而不是一个自发的过程。计算机辅助设计 在CHO细胞中扩增需要PALA浓度超过 3XLD-50。双向聚丙烯酰胺凝胶电泳(2D-PAGE) 分析显示，低水平的多肽表达在 选中和未选中CHO单元格，暗示2D-PAGE的用处 在鉴定基因扩增的早期分子产物方面。至 识别和分析潜在的蛋白质-蛋白质相互作用以促进生长 监管活动采用了两种策略。一种体外培养方法 参与了视网膜母细胞瘤(RB)融合蛋白的产生 (Rb6xHis)。利用Rb6xHis亲和层析柱结合十二烷基硫酸钠- PAGE和银染鉴定出8种蛋白质(41~120 kDa) 从HeLa细胞核中提取出与Hela特异性相关的 Rb的羧基末端区域。一种互补的体内遗传分析方法 利用了这两个杂交遗传系统。构造了两个氢化物： 一个由转录激活物的dna结合域组成。 GAL4与Rb羧基末端的cDNA融合，而第二个 由GAL4激活结构域与正常人融合组成的杂交体 肝脏c DNA文库。筛选总文库(2.6×10~(-6)克隆) 获得了1600个阳性克隆(组氨酸选择)，其中146个是 当对β-半乳糖苷酶活性进行筛选时呈阳性。限制 克隆家族特征的酶作图及序列分析 这些克隆中的一个目前正在进行中。

Amplification of the CAD gene complex concomitant with changes in polypeptide expression were determined in HepG2 and Chinese hamster ovary (CHO) cells using N-phosphonacetyl L-aspartate (PALA) selection. In HepG2 cells at minimal selection pressure (3XLD-50), PALA-resistant colonies developed in the absence of observable amplification of the CAD gene. On the other hand, in untreated HepG2 cells under no metabolic stress, the CAD gene underwent spontaneous amplification with increased passage number. Southern analysis indicated that the CAD gene was amplified up to 30-fold in late passage (p160) as compared with early passage (p113) cells, suggesting that CAD amplification is not a consequence of PALA treatment but rather a spontaneous process. CAD amplification in CHO cells required PALA concentrations in excess of 3XLD-50. Two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed altered polypeptide expression between low-level selected and unselected CHO cells, suggesting the usefulness of 2D-PAGE in identifying early molecular products of gene amplification. To identify and analyze potential protein-protein interactions for growth regulatory activity two strategies were employed. An in vitro method involved the generation of a retinoblastoma (Rb) fusion protein (Rb6xHis). Utilizing an Rb6xHis affinity column in combination with SDS- PAGE and silver staining, eight proteins (41 to 120 kDa) were identified from HeLa cell nuclear extracts that associated specifically with the carboxy terminal region of Rb. A complementary in vivo genetic assay utilized the two hybrid genetic system. Two hydrids were constructed: one consisted of the DNA-binding domain of the transcriptional activator GAL4 fused to cDNA of carboxy terminal region of Rb, while the second hybrid consisted of the activating domain of GAL4 fused to a normal human liver cDNA library. Screening the total cDNA library (2.6 x 10-6 clones) yielded 1600 positive clones (histidine selection) of which 146 were positive when screened for beta-galactosidase activity. Restriction enzyme mapping to characterize families of clones and sequence analysis of these clones is currently in progress.