U037678.pdf (22.77 MB)
Functional analysis of the promoter of the 3-phosphoglycerate kinase gene of Aspergillus nidulans.
journal contributionposted on 2015-11-19, 08:53 authored by Stephen John. Streatfield
Sequence elements contributing to high level expression of the 3-phosphoglycerate kinase (pgk) gene of Aspergillus nidulans have been investigated by the construction of gene fusion vectors using lacZ (?-galactosidase) of Escherichia coli as an assay system, and including the catabolic dehydroquinase (qutE) gene as a selective marker for the transformation of A.nidulans. The importance of targeting these constructs to a specific gene locus has been demonstrated by the analysis of a large number of transformed strains which reveals that the expression of the lacZ reporter gene is dependent upon the site of integration of the vector into the genome, and that when targeted to the qutE locus, is directly proportional to its copy number. The analysis of transformed strains in which single copies of lacZ fusions have been targeted to the qutE locus has identified three constitutive positively acting sequence elements in the pgk gene. Firstly, sequence located between -161 and -120bp (possibly spanning -120) relative to the transcript start site is essential for expression, thus demonstrating that the putative core promoter, including potential TATA and CCAAT boxes, and a pyrimidine rich region, is not alone sufficient to enable expression. A comparison of this sequence to known promoter elements has revealed the presence of an octamer AAGCAAAT (-131 to -124), with a seven out of eight base pair match to the consensus octamer sequence ATGCAAAT characterized as being essential for the expression of several higher eukaryotic genes. Whilst the octamer containing sequence contributes almost a residual 40% of the maximum expression recorded, a second region encompassing codons 14 to 183 and including the two introns of pgk has been shown to account for over 30% of the total activity. However, the full effect of the internal sequence may be greater, since the large 3-galactosidase fusion proteins studied here have been shown to be unstable. Thirdly, an extensive region extending from 0.5 to over 3Kb upstream of the start site has been shown to be required for full expression, accounting for almost 30% of the total recorded activity. Furthermore, sequence located between -638 and -488bp, and including an eight bp consensus element TGAGGTGT common to the four cloned A.nidulans glycolytic genes, has been shown to modulate expression: increasing activity about 1.5-fold on gluconeogenic compared to glycolytic carbon sources. In a separate exercise, the native pgk gene has been replaced with disrupted sequence to generate a pgk mutant strain of A.nidulans. This replacement has been achieved in a diploid strain, from which both pgk+ and pgk-segregants have been isolated, and the analysis of these has allowed the pgk locus to be assigned to chromosome VIII. The pgk- mutant strain that has been isolated is deficient in 3-phosphoglycerate kinase activity, requires both acetate and glycerol for growth, conidiates poorly, and is poisoned by even moderate concentrations of hexoses.