DEVELOPMENT OF A HOMOLOGOUS TRANSFORMATION SYSTEM FOR THE HUMAN PATHOGENIC FUNGUS ASPERGILLUS-FUMIGATUS BASED ON THE PYRG GENE ENCODING OROTIDINE 5'-MONOPHOSPHATE DECARBOXYLASE

A homologous transformation system for the opportunistic fungal pathog en Aspergillus fumigatus was developed. It is based on the A. fumigatu s pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericel la) nidulans and A. fumigatus pyrG mutant strains by the use of protop lasts or electroporation established the functionality of the cloned g ene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed t hat it encodes a truncated, non-functional, PyrG protein. Transformati on of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel p yrG2 allele constructed by in vitro mutagenesis yielded prototrophic t ransformants following recombination between both mutation sites. Anal ysis of transformants carrying the entire plasmid showed that up to 45 % of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fum igatus genome in order to study gene regulation and function.