Functional analysis of the expression of the 3′-phosphoglycerate kinasepgk gene inAspergillus nidulans

Summary

A functional analysis of theAspergillus nidulans 3-phosphoglycerate kinasepgk promoter was undertaken using gene fusions to thelacZ gene ofEscherichia coli, and introducing these into a β-galactosidase-deficient strain ofA. nidulans. Expression of a particular gene fusion in transformed strains depends upon the site of integration of the vector into the genome, and when specifically targeted to the catabolic quinate dehydrogenasequtE (selective marker) locus is directly proportional to its copy number. The analysis of transformed strains with single copies ofpgk promoter deletion —lacZ fusions at thequtE locus identified three constitutive, positively acting sequence elements in thepgk gene. Sequence located between − 161 and − 120 nucleotides relative to the transcript start site + 1, and including an element with a seven-out-of-eight nucleotide match (AAGCAAAT; −131 to −124) to the consensus eukaryotic octamer sequence ATGCAAAT, is essential for expression, and deletion of the complete 41-nucleotide sequence abolishes transcription. Sequence encompassing codons 14 to 183 and including the two introns ofpgk contributes approximately one-third of the total activity, and far upstream sequence 5′ to position − 638 contributes approximately a further one-third total activity. In addition, sequence located − 638 to − 488 nucleotides, which includes an apparent consensus feature ofA. nidulans glycolytic genes, affects carbon source-dependent regulation of expression. This region is required for an approximately 50% increase inpgk expression whenA. nidulans is grown on gluconeogenic compared with glycolytic carbon sources.