GENE TRANSFER AMPLIFICATION, AND AN ANALYSIS OF MUTANTS IN THE UMP SYNTHASE GENE: 49

Abstract

To isolate clones of the human UMP synthase gene, we have produced an amplified, human secondary transformant for the human UMP synthase gene using DNA mediated gene transfer and drug resistance to azauridine. The secondary transformant has 35-fold increased levels of ODCase and 10-fold amplification of human Alu sequences. We are presently cloning DNA from an amplified secondary transformant to isolate human genomic clones for the UMP synthase gene using human Alu sequences as markers for the gene. We have isolated a series of CHO mutants in UMP synthase: a) deficient in UMP synthase enzyme activity; b) revertants of the deficient mutants; and c) mutants with amplified UMP synthase enzyme activity. Analysis of a series of CHO mutants deficient in UMP synthase using Southern blots with a rat cDNA probe (kindly provided by D. Parker Suttle) suggests that no gross rearrangements or deletions are responsible for the enzyme deficiencies. CHO cells selected for resistance to azauridine and pyrazofurin have 35-fold elevated levels of UMP synthase enzyme activity with 10-fold UMP synthase gene amplification and 10-fold elevated UMP synthase mRNA levels. We are presently analyzing UMP synthase mRNA levels in the UMP synthase mutants and determining if there is transcriptional regulation of the UMP synthase gene. This work was supported by NIH (1F32 7086-1) and March of Dimes (1-744). This is ERICR contribution #563.