Affiliation:
1. Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706,1 and
2. Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 631102
Abstract
ABSTRACT
URA5
genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the
Histoplasma capsulatum URA5
gene (
URA5
Hc
) 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 Δ
ura5
Hc
::
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
37 articles.
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