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Abstract
Against a background of varied and often extravagant sexual cycles, fungi have provided much opportunity for genetic study. Studies involving the basidiomycetes Schizophyllum commune, Coprinus cinereus and Ustilago maydis are noteworthy. More recently, in vitro genetic procedures have extended the potential for detailed genetic study to other species. Two basidiomycetes, the phytosymbiotic Lacearia laccata and the edible mushroom-forming Pleurotus ostreatus, have been investigated as systems for DNA-mediated transformation. Using a standard nonreplicative vector (pAN7-1), transformation of L. laccata occurred at a frequency of 5–50 transformants per pg vector DNA per 107 protoplasts. While transformation of P. ostreatus with the same vector occurred at comparable frequency, Southern blot analysis indicated an unexpected replicative mode of transformation. These P. ostrealus transformants consistently contained in vivo generated recombinant plasmids larger in size than the initial vector. One such plasmid pP01 contained in addition to vectored sequences an insert of chromosomal origin. Sequence analysis of this insert revealed high free-energy hairpin-loop forming subsequences, an associated analogue to centromere-affiliated sequences recognized in other fungi, and at least one putative gyrase recognition site. The pPOl insert does not contain an acceptable match to the ars consensus sequence of Saccharomyces cerevisiae. Autonomous replication of pPOl appears rather to involve a replicon quite different from the yeast ars. Since rather few basidiomycetes have been adapted for conventional genetic study based upon completion of the sexual cycle, transformation protocols are likely to advance the genetic investigation of basidiomycetes more generally. Strategies for transformation involving specific genes for improved symbiosis of the root-colonizing L. laccata and for enhanced nutritional value of P. ostreatus mushrooms are considered.