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