Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans

Abstract

The effect of altering the conditions of transformation on the efficiency of gene targeting in filamentous fungi was studied using Aspergillus nidulans as a model organism. The niaD and amdS genes of A. nidulans, which are both involved in nitrogen source utilisation, were selected as target loci. Homologous recombination of transforming DNA at these loci resulted in niaD and amdS mutants with an impaired ability to utilise nitrate or acetamide as the sole nitrogen source, respectively. Vectors were constructed that contained the Neurospora crassa pyr4 gene as a selectable marker and an internal segment of the amdS (0.6-1.27 kb) or niaD (0.9-2.15 kb) genes. The parameters investigated for their effect on gene targeting included (a) length of homologous DNA in the disruption cassette, (b) conformation of the transforming vector (circular or linear), (c) transcriptional status (on/off) of the targeted gene, (d) concentration of DNA in the transformation mix and (e) temperature of incubation of the transformation reaction and of protoplast regeneration on selective media. Parameters shown to have an effect on the targeting frequency at the niaD locus were tested at the amdS locus. The level of gene targeting when circular DNA was used was found to correlate with the size of the homologous segment at both loci. Similarly the level of targeting was shown to increase when vectors were linearised within the region of homology. The level of targeting was unaltered at the niaD locus when transcription was induced at different stages in the transformation procedure. Likewise, targeting was unaffected by altering the amount of DNA in the reaction mix over the concentration range tested. The regeneration temperature did have an effect on targeting, with enhanced targeting observed at 25 degrees compared with 37 degrees C. However, the most dramatic effect was the difference between targeting efficiency at different genetic loci, with targeting of niaD being at least five fold more efficient than amdS under all conditions tested.