TRANSFORMATION OF NEUROSPORA CRASSA WITH THE TRP-1 GENE

Previous studies on the transformation of Neurospora crassa showed that homologous integration of transforming DNA is not frequent in Neurospora. Regardless of transformation methods employed, the ga-2 gene was found to be integrated into its resident site in less than 10% of the ga-2('+) transformants. With the am gene the highest proportion of linked type transformants was 30%.;I first obtained trp-1('+) transformants by transforming a trp-1 mutant strain with the wild type trp-1 gene, and then characterized these transformants by genetic and biochemical analyses.;Homokaryotic trp-1('+) transformants were crossed with a trp-1 ad-2 double mutant and trp-1('+) progeny from these crosses were tested for their adenine requirement for growth, in order to determine whether the transforming trp-1 gene integrated into its resident locus. To study possible host effect on the fate of transforming DNA, trp-1('+) transformants obtained from another recipient strain with the same trp-1 gene were also analysed in similar way. Transformants were also tested for their mitotic and meiotic stability.;Sites of integration of the trp-1 gene were also determined by Southern blot analyses and expression of the trp-1('+) gene in transformants was determined by enzyme assays and by Northern blot analyses.;The low frequency of homologous integration of transforming DNA observed in Neurospora is unexpected. In closely-related yeast or Aspergillus integration occurs mainly by homologous recombination. However, it is not certain yet whether the behavior of the ga-2 or the am gene is typical in Neurospora. In my research I have employed the trp-1 gene to examine Neurospora transformation more fully.;Above experiments showed that (1) Trp-1('+) transformants were stable both mitotically and meiotically, (2) the trp-1 gene integrated mainly at the resident site, (3) the host strain was a major factor influencing the fate of transforming DNA, (4) the level of trp-1 gene expression in unlinked transformants varied widely. Thus, my research showed that integration of trp-1 gene by homologous recombination occurred at a high frequency, depending on host strains employed for transformation.