Constitutive Mutants in a Regulatory Gene Exerting Positive Control of Quinic Acid Catabolism in Neurospora crassa

Abstract

In Neurospora crassa, evidence has recently been obtained for a cluster of four closely-linked genes controlling the inducible enzymes catalyzing the first three reactions in the catabolism of quinic acid. Three of these genes appear to be the structural genes for the three enzymes. The fourth gene, designated qa-1, has been interpreted as having a regulatory function, since qa-) mutants are pleiotropic types, are noninducible for the three enzymes, and form heterocaryons which complement mutants in the structural genes. The present studies were undertaken to elucidate further the nature of the regulatory role of the qa-) locus. A number of constitutive (qa-1C) mutants (3,4) have reported that the regulatory gene for the galactose system of Saccharomyces cerevisiae is unlinked to the cluster of three structural genes and, furthermore, that no evidence exists for an operator region adjacent to this cluster. Also, although there is evidence that the arom gene cluster in Neurospora crassa has certain operon-like characteristics, such as polarized transcription, again no clear evidence has been obtained for an adjacent operator or promoter region (5). Recent work by Rines (6,8) on the quinic acid catabolic pathway of Neurospora crassa has suggested the occurrence of a regulatory gene, qa-1, controlling the expression of the first three enzymatic activities in the pathway. This qa-1 gene is closely linked to the structural gene, qa-2, which encodes one of the activities, catabolic dehydroquinase (5-dehydroquinate dehydratase, EC 4.2.1.10) (9). Very recent evidence also indicates that the structural genes for the other two enzymes, qa-3, encoding quinate dehydrogenase (quinate: NAD oxidoreductase, EC 1.1.1.24) and qa-4, encoding 5-dehydroshikimate dehydrase, are also closely linked to the qa-1 locus (7). Rines (8) has suggested that the regulatory locus, qa-1, might resemble an operontype system in producing a regulatory protein which controls the synthesis of the three structural gene products. However, alternative models were also considered. For example, the qa-1 gene might encode an enzyme which converts the presumed inducer (dehydroquinic acid) into the actual, as yet unknown, inducer or the locus might produce a polypeptide common to all three structural gene products and necessary for all their activities.Acting on the hypothesis that the wild-type qa-1 + locus produces a regulatory protein necessary to initiate synthesis of the first three enzymes in quinate catabolism, we have sought to recover qa-1 mutants constitutive for the enzymes encoded in the three structural genes. These types have been sought among revertants of various qa-1 mutants (which are pleiotropic mutants, noninducible for all three enzymes) for which revertant selection has occurred on medium containing quinic acid as a sole carbon source. This paper presents evidence for the recovery of such constitutive mutants (qa-1C) in Neurospora and a preliminary characterization of these mutants in terms of the magnitude of their catabolic...

Autogenous regulation of the positive regulatory qa-1F gene in Neurospora crassa.

“…The isolates from these crosses were selected by their ability to grow on sucrose with Fries salts without the aromatic amino acids and to be noninducible in the presence of quinic acid. The constitutive qa-1Sc (M105-R12-1.5) used in this study was a UV-induced revertant of the temperaturesensitive mutant M105 (qa-JSts) that produces a high level of catabolic dehydroquinase at 25°C, but not at 35°C, in the absence of inducer (29). The triple mutant (qa-3-qa4-arom-1-; strain no.…”

Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

“…There is strong genetic evidence that the qa-1 gene encodes a regulatory protein which, in the presence of quinic acid, exerts positive control over the synthesis of the enzymes encoded in the three adjacent structural genes (6,10). The appearance of enzyme activity is very specifically regulated.…”

“…In Neurospora crassa, the catabolism of quinic acid to acetate for use as a carbon source has been amply studied (1)(2)(3). Recent interest has centered on the initial three inducible catabolic enzymes necessary to convert quinic acid to protocatechuic acid (4)(5)(6)(7)(8)(9)(10)(11)(12)(13) Giles, unpublished). This qa gene cluster also includes a fourth gene, qa-1, which apparently regulates the transcription of the three other genes in the cluster.…”

Genetic organization and transcriptional regulation in the qa gene cluster of Neurospora crassa.

“…Prior studies established the presence of four genes in this cluster, of which three (qa-2, qa-3, qa-4) encode inducible enzymes catalyzing the first three reactions in the catabolism of quinic acid to protocatechuic acid (1). The fourth gene in the cluster (qa-1) encodes a regulatory protein which, when combined with the normal inducer quinic acid, appears to act in a positive fashion to control the production of the three qa enzymes (2). The order ofthe four genes in the right arm of linkage group VII has been determined by genetic recombination studies to be qa-1, qa-3, qa-4, qa-2 (3).…”