Regulatory regions in the yeast FBP1 and PCK1 genes.

By deletion analysis of the fusion genes FBP1-lacZ and PCK1-lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of beta-galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.     

Yeast MIG1 repressor is related to the mammalian early growth response and Wilms'' tumour finger proteins.

We have cloned a yeast gene, MIG1, which encodes a C2H2 zinc finger protein involved in glucose repression. The fingers of MIG1 are very similar to those present in the mammalian Egr finger proteins, which are induced during the early growth response, and also to the finger protein encoded by a human gene that is deleted in Wilms' tumour cells. MIG1 protein binds to two sites in the upstream region of SUC2, a yeast gene that is repressed by glucose. The MIG1 sites closely resemble the sequence recognized by the Egr proteins. Thus, finger proteins that are similar in both amino acid sequence and DNA specificity are involved in the response of yeast to glucose, and in the mammalian early growth response.     

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Repression and catabolite gene activation in the araBAD operon.

Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.     

Analysis of the Schwanniomyces occidentalis SWA2 gene promoter in Saccharomyces cerevisiae

The effect of different carbon sources on the expression in Saccharomyces cerevisiae of the SWA2 alpha-amylase gene from Schwanniomyces occidentalis was studied from constructs containing its 5' region (-223 to +15), which were fused in-frame to the lacZ gene coding sequence. Maximal expression was achieved with the non-fermentable substrates ethanol and/or glycerol, whereas lower levels were found with maltose or galactose. In contrast, glucose repressed it, even in the presence of any of these other carbon sources. Deletion analyses of the -233 to -85 SWA2 promoter region permitted the identification of two fragments involved in both glucose repression and ethanol activation. A possible region required for cAMP regulation was localised. The SWA2 promoter contains a MIG1-binding GC box whose deletion caused a five-fold increase in the glucose-repressed reporter expression. Despite this, expression of the SWA2 promoter was not MIG1-dependent.     

Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa.

The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and_expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.