Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria
- 1 March 1993
- journal article
- research article
- Published by Springer Nature in Somatic Cell and Molecular Genetics
- Vol. 19 (2), 193-202
- https://doi.org/10.1007/bf01233533
Abstract
Orotate phosphoribosyltransferase (OPRT) and orotidine-5′-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and 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 using 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 without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4–9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.Keywords
This publication has 17 references indexed in Scilit:
- Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesisSomatic Cell and Molecular Genetics, 1992
- Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coliGene, 1991
- Structure and function of the yeast URA3 gene: expression in Escherichia coliGene, 1984
- Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding regionEuropean Journal of Biochemistry, 1983
- Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthaseBiochemistry, 1980
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2-release methodAnalytical Biochemistry, 1975
- Dissociation by elastase digestion of enzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liverBiochemical and Biophysical Research Communications, 1973
- Calcium-dependent bacteriophage DNA infectionJournal of Molecular Biology, 1970
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970