A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR.
Recombinant PCR has been used to generate linear fragments for promoter replacement by transformation in Aspergillus nidulans. A cassette vector carrying the pyr-4 non-homologous selectable marker and conditional promoter Pr-alcA was constructed for use as a template for PCR, and is suitable for testing the function of essential genes. Two genes involved in polar growth, cotA and bemA, were used to assess the system. Efficient targeting was possible with both genes using approximately 500bp of flanking homologous sequence. Depending on yield, the linear PCR product could be used directly for transformation, or after first cloning into a suitable vector. bemA, a putative homologue of the Saccharomyces cerevisiae BEM1 gene was identified through sequence comparison. In A. nidulans, this protein appears to have a similar role to the yeast Bem1p, which acts as a scaffold protein involved in the establishment of cell polarity.
Zarrin
Majid
M
Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield S10 2TN, UK.
Leeder
Abigail C
AC
Turner
Geoffrey
G
D001233:Aspergillus nidulans / Q000235:genetics* / Q000254:growth & development; D005240:Feasibility Studies; D020043:Genes, Essential; D005800:Genes, Fungal; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic