Figure 1 . Gene replacements at the his-3 locus of Neurospora crassa . Bml r , corresponds to the benomyl resistance allele of the tub-2 gene. HI- Eco RI fragment that will be replaced during the integration. The predicted restriction map generated by the gene replacement event, the approximate location of the sequenced region, and the location of the insert present in pH303 is presented below the downward arrow.
Horizontal arrows indicate direction of transcription.
Conidial spheroplasts preparation and transformation were done by a modification of the combined methods of Schweizer et al. Erlenmeyer flasks, each containing 20 ml of solid medium, were inoculated and allowed to grow for 3 days at 34 C and 2 days at room temperature. Conidia were harvested into 200 ml of sterile water and filtered through Miracloth. Conidial suspensions were centrifuged at 2,000 x g for 15 min and the pellets were suspended in 20 ml of 1 M aqueous sorbitol. Tubes were inverted several times and incubated at room temperature for 30 min.