Ectopic integration of transforming DNA is rare among neurospora transformants selected for gene replacement

Abstract

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such 'cotransformants' are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformations selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycin(R)), allowed to conidiate to generate homokaryons and then selected for either Am (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycin(S) (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.