Uracil Phosphoribosyltransferase Gene Research Articles

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyl-transferase gene sensitizes cancer cells to low concentration of 5-fluorouracil: A new way to overcome the resistance of chemotherapeutic agent

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyl-transferase gene sensitizes cancer cells to low concentration of 5-fluorouracil: A new way to overcome the resistance of chemotherapeutic agent

Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis

The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of ‘hit-and-run’ mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing ≈8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT ‘minigene’, which lacks sufficient continguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, >30% of which were found to have integrated at the UPRT locus as ‘pseudodiploids’ (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of ≈10 –6 per parasite doubling. Applications of ‘hit-and-run’ technology relative to other gene targeting strategies are discussed.

Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii.

The coding region derived from a full-length CDNA spanning the uracil phosphoribosyltransferase (UPRT) gene of Toxoplasma gondii has been ligated into a bacterial expression vector and overexpressed in E. coli. Recombinant UPRT protein migrated with a molecular mass of 27 kDa on SDS polyacrylamide gels and was purified to homogeneity by conventional protein purification techniques. In solution, UPRT behaved as a monomer and exhibited K(m)app values of 3.5 microM for uracil and 243 microM for phosphoribosylpyrophosphate, respectively. Other naturally occurring pyrimidine or purine bases were not recognized as substrates. [14C]Uracil phosphoribosylation was inhibited by 5-fluorouracil with a Ki value of 25 microM and was not activated by GTP. Ample quantities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target.

Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.