Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA

Abstract

Neurospora trp-1+ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1+ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1+ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1+ transformants obtained with another recipient strain, trp-1+ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.