A reversible selection system for UMP synthase gene amplification and deamplification.
The bifunctional enzyme UMP synthase provides a unique reversible selection system whereby cells that have amplified the UMP synthase gene can be isolated from a wild-type population and cells that have deleted the extra genes can be selected from a population with amplified copies of the gene. UMP synthase catalyzes the conversion of orotic acid to orotidine 5'-monophosphate (OMP) and then OMP to UMP. In the amplification step, Chinese hamster lung cells are selected for resistance to pyrazofurin and 6-azauridine, two inhibitors of the orotidine 5'-decarboxylase activity that converts OMP to UMP. The resistant cells have increased levels of both activities of UMP synthase as a result of a stable amplification of the UMP synthase gene. The deamplification step depends on 5-fluorouracil (5FU), which is converted to its monophosphate form by the orotate phosphoribosyltransferase activity of UMP synthase. Thus cells with increases in this activity are more sensitive to 5FU cytotoxicity, permitting single-step selection of revertants that have lost their amplified UMP synthase genes. These 5FU-selected cells are similar to the parental cell line in their level of UMP synthase activity and number of UMP synthase gene copies. Reselection in increasing concentrations of pyrazofurin and 6-azauridine allows one to isolate cells that have reamplified the UMP synthase gene. The ability to cycle cells of a single lineage through states of amplification and deamplification will facilitate study of the gene amplification process and the factors that influence the composition and stability of amplified regions.
Suttle
D P
DP
Veterans Administration Medical Center, Memphis, Tennessee.
D000577:Amides; D000818:Animals; D001380:Azauridine / Q000494:pharmacology; D015139:Blotting, Southern; D002262:Carboxy-Lyases / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D005472:Fluorouracil / Q000494:pharmacology; D005784:Gene Amplification; D005796:Genes; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000235:genetics*; D009962:Orotate Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000037:antagonists & inhibitors / Q000235:genetics*; D010430:Pentosyltransferases / Q000235:genetics*; D011720:Pyrazoles; D012263:Ribonucleosides / Q000494:pharmacology; D012266:Ribose; D012641:Selection, Genetic
D000577:Amides; D009097:Multienzyme Complexes; D011720:Pyrazoles; D012263:Ribonucleosides; C002997:pyrazofurin; D012266:Ribose; C027157:uridine 5'-monophosphate synthase; D001380:Azauridine; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase; D005472:Fluorouracil