Cloning and functional analysis of a beta-tubulin gene from a benomyl resistant mutant of Aspergillus parasiticus.

A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative benr gene or benr plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if benr conferred benomyl resistance (BenR). BenR colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No BenR colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of BenR and BenS transformants suggested that plasmid integration occurred most frequently at the chromosomal bens locus, however evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of benr displayed a high degree of identity (> 93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that benr encodes a functional mutant beta-tubulin.