EXPRESSION OF CATALYTIC DOMAINS OF HUMAN UMP SYNTHASE IN URIDINE AUXOTROPHIC BACTERIA

Orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphat e decarboxylase (ODC), which catalyze the last two steps in de novo UM P biosynthesis, are two distinct monofunctional proteins in bacteria a nd lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains us ing polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC -deficient E. coli, the strains were able to grow on minimal media wit hout uridine. The ODC-transformed bacteria expressed up to 24 times th e level of activity found in a wild-type E. coli line. The OPRT-transf ormed E. coli contained only 4-9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT a nd ODC domains are being produced in the deficient cells by the respec tive vectors. The level of the domain protein approximates the level o f enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human U MP synthase can be separated into active monofunctional domains that w ill function in the bacterial cell environment.