Cloning of the Trichoderma reesei pyrG gene and its use as a homologous marker for a high-frequency transformation system

Summary The Trichoderma reesei orotidine-5-phosphate decarboxylase gene was isolated by heterologous hybridization with the corresponding Neurospora gene as a probe. A 2.7 kb SalI fragment, which exclusively hybridized to the Neurospora gene, was subcloned in pGEM-5Zf(+). This subclone was termed pFG1 and was used to transform a Trichoderma reesei pyrG^- negative mutant to PYR⁺. The transformation frequency in this homologous system was up to 12000 transformants per g DNA. About one-fifth of the transformants tested were abortive. Perfect mitotic stability was found in half of the non-abortive transformants, correlating with vector integration at homologous and ectopic loci. In the unstable transformants the transforming DNA appears to be present in the form of extrachromosomal elements.