Random integration of transforming DNA is common is Aspergillus, resulting in multiple copies throughout the genome. To study the effects of position, copy number, and promoter character on expression, A. nidulans strains were constructed with single- and multi-copy integrations of a plasmid bearing an expression cassette consisting of an Aspergillus alcA (inducible), or gpdA (constitutive) promoter; the E. coli lacZ gene; and the Aspergillus trpC terminator. Homologous integration to the target loci was selected for by transformation with a truncated or mutated version of the wild-type genes, such that ectopic integration results in a null phenotype. Integration of the lacZ gene was confirmed by PCR amplification of a fragment oflacZ and of the argB gene as control. Genomic locus and copy number were determined by Southern blotting of genomic restriction digests and probing with a32P-labeled lacZ fragment. A rapid method to quantify transgene dosage utilizing real-time PCR was developed. A single-copy native gene (trpC) and lacZ were simultaneously amplified from genomic template from the transformants; by comparing the product curves to a series of standards, the amount of starting template DNA and hence gene copy number was determined. A tentative linear relation appeared for beta-galactosidase expression levels versus lacZ copy numbers, excepting a group of high expressing clones at ~10 copies.
Fungal Genet. Newsl. 50 (Supl):abstract
Full conference title:
22nd Fungal Genetics Conference
- Fungal Genetics Conference 22nd (2001)