Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes

Abstract

Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (greater than 10(4) transformants per micrograms DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.