Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene.

Abstract

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. crasna 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. creasa 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.The qa gene cluster of Neurospora crassa (located on linkage group VII) has been shown to consist of three genes encoding inducible enzymes catalyzing the initial reactions in the catabolism ofquinic acid (1). In addition, the cluster contains a fourth gene, qa-1+, whose protein product positively controls the expression of the other three genes (1). Initial cloning experiments demonstrated that the qa-2+ gene, which encodes catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10), was functionally expressed in Escherichia coli (2-4). More recently, the use of the cosmid packaging procedure (5) has permitted the isolation ofmuch larger segments of.N. crassa DNA containing the qa-2+ gene. One clone (pMSK308) was shown, by transformation of appropriate qa deficient mutants of N. crassa, to carry the qa-3' gene,. which encodes quinate dehydrogenase (quinate:NAD+ 3-oxidoreductase, EC 1.1.1.24), and probably the qa-4+ gene, encoding dehydroshikimate dehydratase (6). Neither the. qa-3' nor the qa-4+ gene was expressed in E. coli but both were functionally active upon reintroduction into N. crassa (6).Evidence is presented in this communication that the complete qa gene cluster from the wild type N. crassa strairr 74A has been cloned as part of a 36.5-kilobase (kb) EcoRI insert in the plasmid pHC79. Results obtained from N. crassa transformation experiments utilizing subclones of this 36.5-kb region of linkage group VII have permitted preliminary localization of the qa-1 regulatory gene as well as the other genes in the cluster (qa-2, qa-4, and qa-3). The use of these clones will now permit the examination of gene organization and regulation at the molecular level in this simple eukaryote. MATERIALS AND METHODS'Materials. Reagents were obtained from the following sources: [a-32P]dATP (400 Ci/mmol; 1 Ci = 3.7 X 1010 becque-

Molecular cloning and analysis of the regulation of cys-14+, a structural gene of the sulfur regulatory circuit of Neurospora crassa.

“…Each sublibrary represented 96 individual clones and was purified by CsCI-ethidium bromide gradient ultracentrifugation (8). Competent spheroplasts were prepared (1,20,26) from conidia of a cys-13 cys-14 al-i strain of N. crassa. cys-13 cys-14 double mutant strains lack both sulfate permease species and cannot transport and thereby utilize inorganic sulfate for growth.…”

Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain.

“…The qa-JF+ gene used in this study was originally obtained from wild-type N. crassa 74-OR23-1A (74A) (36). A plasmid subclone of this gene (kindly provided by Alan Easton) in which all but eight nucleotides of the 5 …”

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

“…An efficient transformation procedure has been developed for Neurospora crassa (6,33), and a number of nuclear genes have been isolated from this organism (e.g., see references 19, 31, 33, and 42). A Neurospora colony filter hybridization procedure has been developed and should facilitate isolation of additional nuclear genes by plasmid or cosmid rescue (37).…”

“…Transformation of N. crassa was carried out essentially as described previously (19,33). Plasmid DNAs used for the transformation were purified by one to three bandings in CsCI-ethidium bromide gradients.…”