Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement.
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 frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Deltaura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.
Woods
J P
JP
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA jpwoods@facstaff.wisc.edu
Retallack
D M
DM
Heinecke
E L
EL
Goldman
W E
WE
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018390:Gene Targeting / Q000379:methods*; D005800:Genes, Fungal / Q000235:genetics*; D006658:Histoplasma / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D009962:Orotate Phosphoribosyltransferase