Gene disruption methodologies for drug target discovery

Abstract

The present invention provides methods and compositions that enable the experimental determination as to whether any gene in the genome of a diploid pathogenic organism is essential, and whether it is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which one allele of a specific gene is inactivated while the other allele of the gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against such pathogenic organisms. The present invention further provides Candida albicans genes that are demonstrated to be essential and are potential targets for drug screening. The nucleotide sequence of the target genes can be used for various drug discovery purposes, such as expression of the recombinant protein, hybridization assay and construction of nucleic acid arrays. The uses of proteins encoded by the essential genes, and genetically engineered cells comprising modified alleles of essential genes in various screening methods are also encompassed by the invention.

1 . A method for constructing a strain of diploid fungal cells in which both alleles of a gene are modified, the method comprising the steps of: 
 (a) modifying a first allele of a gene in diploid fungal cells by recombination using a gene disruption cassette comprising a first nucleotide sequence encoding an expressible selectable marker, thereby providing heterozygous diploid fungal cells in which the first allele of the gene is inactivated; and    (b) modifying the second allele of the gene in the heterozygous diploid fungal cells by recombination using a promoter replacement fragment comprising a second nucleotide sequence encoding a heterologous promoter, such that expression of the second allele of the gene is regulated by the heterologous promoter.    
     
     
         2 - 75 . (canceled)