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.
