EFFICIENT TRANSFORMATION OF NEUROSPORA-CRASSA BY UTILIZING HYBRID PLASMID DNA

An efficient transformation system has been developed for N. crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2+ gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2- mutation and an arom-9- mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2+ and had normal levels of catabolic dehydroquinase. DNA-DNA hybridization evidence with appropriate labeled probes indicate clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2+ gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: replacement of the qa-2- gene by the qa-2+ gene without any effect on the expression of the other genes in the qa cluster, linked insertion of a normal qa-2+ gene accompanied by inactivation of the adjacent qa-4+ gene, and insertion of an a normal gene at an unlinked site in the N. crassa genome. This newly integrated qua-2+ genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2+ transformants are unlinked to the qa-2- gene of the recipient.