title: A PCR-based strategy to generate integrative targeting alleles with large regions of homology.
abstract: Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Jill R
authors.initials: JR
authors.lastname: Blankenship
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Kraus
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Marisol
authors.initials: M
authors.lastname: de Jesus Berrios
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Hull
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Cletus
authors.initials: C
authors.lastname: D'Souza
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1.
authors.forename: Joseph
authors.initials: J
authors.lastname: Heitman
mesh_terms: D000483:Alleles; D003455:Cryptococcus neoformans / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D016681:Genome, Fungal; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D005819:Genetic Markers; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D029701:Saccharomyces cerevisiae Proteins; C083324:TOR1 protein, S cerevisiae