Molecular genetical analysis of the nitrate gene cluster of Aspergillus nidulans

Abstract

Putative DNA clones harbouring the nitrate gene cluster of Aspergillus nidulans was used in a number of transformation experiments to positively identify the clones. The complementation of several deletions within the nitrate gene cluster ruled out the possibility that a suppressor had been cloned and suggested that extensive regions of the nitrate gene cluster were present on the recombinant plasmid. Construction of several subclones and subsequent transformation of various alleles of the nitrate gene cluster allowed the approximate positions of the three genes (crnA, nitrate permease; niiA, nitrite reductase; niaD, nitrate reductase) to be determined relative to the plasmid. These experiments indicated that a part of the niaD gene was missing, although the entire niiA and crnA genes were present on the plasmid. Subsequently, overlapping clones were isolated and shown to contain the remaining niaD sequences. Genetic analysis of many transformants suggested that the plasmids had mainly integrated into the nitrate gene cluster (corresponding site of homology to the plasmid), although occasionally the plasmids had integrated into other chromosomes and here, therefore, complementation was in trans. Reversion studies on chlorate of two niaD transformants, containing plasmid DNA at the nitrate gene cluster, suggested that these transformants were less stable than the wild-type. Construction of promoter-β-galactosidase fusions using DNA from the intergenic region between niiA and niaD suggested that these genes are divergently transcribed. This DNA region had sufficient promoter signals to mediate the nitrate/ammonium regulation of β-galactosidase characteristic to this gene cluster. One transformant, possessing multiple copies of the fusion construct, had elevated levels of β-galactosidase but the maximal nitrate reductase activity was considerably reduced. Evidence obtained, supported the idea that one of the regulatory proteins (nirA) was limiting in this multiple copy transformant.