Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in...

The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2+ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2+) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmids isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.