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

Abstract

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.