Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans.
Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genetics and Biology 29, 38-48. Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle and well-developed genetic and molecular approaches. Two different transformation systems have been developed, and a number of genes have been disrupted by homologous recombination. However, the frequency of homologous recombination achieved by these approaches has differed dramatically between strains of the A and D serotypes. Transformation by electroporation in serotype D strains results in homologous recombination at frequencies of 1/1000 to 1/100,000, whereas transformation by the biolistic method has resulted in gene disruption at frequencies between 2 and 50% in serotype A strains. We find that gene disruption by homologous recombination can be achieved in the congenic serotype D strain series by biolistic transformation with frequencies of approximately 1 to 4%. By this approach, we have readily disrupted the genes encoding a MAPK homolog (CPK1), the calcineurin A catalytic subunit (CNA1), and a G protein alpha subunit (GPA1). By physical and genetic methods, we show that these mutations result from targeted recombination events without ectopic integrations. Because genetic approaches can be applied in the congenic serotype D strains, our observations represent a significant advance in molecular approaches to understand the physiology and virulence of this important human pathogen.
Davidson
R C
RC
Departments of Genetics, The Howard Hughes Medical Institute, North Carolina 27710, USA.
Cruz
M C
MC
Sia
R A
RA
Allen
B
B
Alspaugh
J A
JA
Heitman
J
J
D019470:Biolistics / Q000379:methods*; D019703:Calcineurin; D002262:Carboxy-Lyases / Q000235:genetics; D003455:Cryptococcus neoformans / Q000145:classification / Q000235:genetics*; D018274:Electroporation / Q000379:methods; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D017353:Gene Deletion; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D020104:Immunophilins / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D022021:Tacrolimus Binding Proteins; D014170:Transformation, Genetic
D044385:GTP-Binding Protein alpha Subunits; D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases; C070393:CNA1 protein, S cerevisiae; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins