110 MOLECULAR GENETICS OF MAMMAL I AN UMP SYNTHESIS

Abstract

To study regulation of pyrimidine biosynthesis in mammalian cells we isolated a series of Chinese hamster ovary(CHO) cell mutants defective in the enzymatic steps required for UMP synthesis. Urd-A mutants are deficient in CAD, the multifunctional protein catalvzing the first 3 activities of the pathway. Urd-B mutants are deficient in the 4th step of the pathway, DHO dehydrogenase, and Urd-C mutants are deficient in UMPS, the multifunctional protein catalyzing the last two steps of UMP synthesis. Urd-C mutants are functionally equivalent to cells from human natients with orotic-aciduria. The human Urd-A locus nans to human chromosome 2 and Urd-C to chromosome 3. To analyze mutations in these genes we isolated separate CHO cell mutants in which the CAD and UMPS qenes are amplified by ca. 25-fold, We fused these two mutants to create a hybrid cell line in which both CAD and UMPS are amplified. We used these amplified lines to construct both cDNA (in lambda gt 10) and genomic (in lambda EMBL-3) libraries. From these libraries we isolated a clone which contains a 1.9 kb UMPS cDNA, a clone of qreater than 3.0 kb which appears to be a CAD cDNA, and in addition several genonic clones which probably contain at least part of the CHO UMPS gene. We have initiated experiments using these clones to study requlation of UMP synthesis and the nature of the defects in our various mutants. In one Urd-A mutant, mRNA levels are markedly reduced, but transcriptional elongation rate as measured by nuclear run on is not reduced.