DETECTION OF PROTEIN-PROTEIN INTERACTIONS DURING GROWTH REGULATORY ACTIVITY

生长调节活动期间蛋白质-蛋白质相互作用的检测

• 批准号: 3752779
• 负责人: P J WIRTH
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3752779
• 关键词: CHO cells; DNA binding protein; HeLa cells; N phosphonoacetyl L aspartate; SDS polyacrylamide gel electrophoresis; affinity chromatography; amidohydrolases; aspartate carbamoyltransferase; carbamoylphosphate synthase; cell growth regulation; chimeric proteins; drug resistance; enzyme complex; gene expression; human genetic material tag; intermolecular interaction; natural gene amplification; purine /pyrimidine metabolism; restriction mapping; southern blotting; tissue /cell culture; transcription factor

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.

CAD基因复合体的扩增伴随着 在HepG 2和中国仓鼠卵巢中测定多肽表达 (CHO)使用N-膦酰基乙酰L-天冬氨酸（PALA）选择细胞。 在 最小选择压力（3XLD-50）下的HepG 2细胞，PAA抗性 在没有可观察到的CAD扩增的情况下形成的集落 基因 另一方面，在未处理的HepG 2细胞中， 应激时，CAD基因发生自发扩增， 传代次数。 Southern分析表明，CAD基因是 与早期相比，晚期传代（p160）扩增高达30倍 传代（p113）细胞，这表明CAD扩增不是一个重要的因素。 PALA治疗的结果，而是一个自发的过程。 CAD CHO细胞中的扩增需要超过 3XLD-50 双向聚丙烯酰胺凝胶电泳（2D-PAGE） 分析显示，在低水平表达之间， 选择和筛选CHO细胞，表明2D-PAGE的有用性 鉴定基因扩增的早期分子产物。 到 识别和分析潜在的蛋白质-蛋白质相互作用以促进生长 调节活动采用两种策略。 的体外方法 涉及视网膜母细胞瘤（Rb）融合蛋白的产生 （Rb6xHis）。 利用Rb 6xHis亲和柱与SDS-聚丙烯酰胺凝胶的组合， PAGE和银染鉴定出8种蛋白质（41 ~ 120 kDa 从HeLa细胞核提取物中， Rb的羧基末端区域。 互补的体内遗传测定 利用双杂交遗传系统。 建造了两座桥梁： 一个由转录激活因子的DNA结合域组成 GAL 4与Rb羧基端cDNA融合，而第二个GAL 4与Rb羧基端cDNA融合， 由GAL 4的激活结构域与正常人融合组成的杂交体 肝脏cDNA文库。 筛选总cDNA文库（2.6 x 10-6克隆） 产生1600个阳性克隆（组氨酸选择），其中146个是 当筛选β-半乳糖苷酶活性时为阳性。 限制 酶图谱分析，以表征克隆家族和序列分析 正在进行中