Mary E. Case

An efficient transformation system has been developed for Neurospora 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 indicates 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: (i) 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, (ii) linked insertion of a normal qa-2(+) gene accompanied by inactivation of the adjacent qa-4(+) gene, and (iii) insertion of a normal qa-2(+) gene at an unlinked site in the N. crassa genome. This newly integrated qa-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.

A chemical reaction network for the regulation of the quinic acid (qa) gene cluster of Neurospora crassa is proposed. An efficient Monte Carlo method for walking through the parameter space of possible chemical reaction networks is developed to identify an ensemble of deterministic kinetics models with rate constants consistent with RNA and protein profiling data. This method was successful in identifying a model ensemble fitting available RNA profiling data on the qa gene cluster.

The early reactions in the catabolism of quinic acid in Neurospora crassa are controlled by at least four genes which are clustered on linkage group VII. Three of the loci (qa-2, qa-4, and qa-3) encode enzymes that convert quinic acid to protocatechuic acid. The fourth gene (qa-1) encodes a positive regulatory protein which, in the presence of quinic acid, leads to the de novo synthesis of the other proteins in the qa cluster. This communication describes a series of recombinant plasmids that span 36.5 kilobases of linkage group VII and contain the coding sequences for qa-2, qa-4, qa-3, and the qa-1 regulatory protein. The plasmids were obtained by partial digestion of wild-type N. crassa DNA with EcoRI and ligation into the cosmid cloning vehicle pHC79. Two independently derived plasmids (pMSK331 and pMSK335), each containing 36.5-kilobase inserts, were shown by transformation back into N. crassa to contain the entire qa gene cluster. A preliminary physical organization of the gene cluster is presented. An improved procedure for the transformation of N. crassa with plasmid DNA is also described.