Molecular characterization of the 3-phosphoglycerate kinase gene from Aspergillus nidulans

• Main Author: Clements, John Martin
• Published: University of Leicester 1986
• Subjects: 572.8; Genetics
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356628

The Aspergillus nidulans 3-phosphoglycerate kinase (PGK) gene has been isolated from a phage ? genomic library using the equivalent yeast gene as a hybridization probe and the location of the PGK gene physically mapped within the cloned DNA. The nucleotide sequence of the entire PGK gene and its 5' and 3' flanking regions has been determined. The gene was found to contain two 57 base pair introns which have splicing signals similar to those in other filamentous fungi, and codes for a 421 amino acid protein with considerable homology to the Saccharomyces cerevisiae (68%) and mammalian (64%) proteins. Almost total conservation is found in Aspergillus of residues thought to be important for the structure and function of the yeast enzyme. The codon usage shows a similar bias to that found in other filamentous fungi. The PGK gene has been shown to be constitutively expressed at a relatively high level. The transcription start site and polyadenylation sites have been determined. The PGK promoter has both the CAAT and TATA homologies generally found in more complex eukaryotes. Additionally two pyrimidine rich regions of the promoter sequence share similarities to other highly expressed genes in fungi. PGK mRNA exhibts 3' heterogeneity and the major polyadenylation site is positioned 16 bp beyond the eukaryotic consensus polyadenylation signal AAUAAA. Potential hairpin structures are also found in the 3' non-translated region of the PGK mRNA which may be important in transcription termination and polyadenylation. In parallel studies the pyruvate carboxylase protein has been identified by immunoprecipitation from the products of in vitro translations of A. nidulans mRNA as the first step of a strategy to clone a cDNA copy of the PYC mRNA. An attempt to clone the pyruvate carboxylase (PYC) gene from Aspergillus nidulans, by an immunological screen of an Aspergillus genomic library constructed in phage ?, was unproductive.