Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement.

ABSTRACT: URA5 genes encode orotidine-5'-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the

SUBMITTER: Woods JP 

PROVIDER: S-EPMC107550 | biostudies-literature | 1998 Oct

REPOSITORIES: biostudies-literature