CLONING AND FUNCTIONAL-ANALYSIS OF A BETA-TUBULIN GENE FROM A BENOMYLRESISTANT MUTANT OF ASPERGILLUS-PARASITICUS

A genomic DNA library prepared from a benomyl resistant strain of Aspe rgillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasilicus genomic DNA fragment, thought to carry a mutant beta-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 be n(r) conferred benomyl resistance (Ben(R)). Ben(R) colonies were obtai ned 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 oc curred most frequently at the chromosomal ben(s) locus, however eviden ce for gene conversion and heterologous recombination was also observe d. The predicted amino acid sequence of ben(r) displayed a high degree of identity (>93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data s uggested that ben(r) encodes a functional mutant beta-tubulin.