Molecular analysis of the Septoria nodorum beta-tubulin gene and characterization of a benomyl-resistance mutation

Abstract

The complete nucleotide sequence of a benomyl-resistant allele of the Septoria nodorum beta-tubulin gene (tubAR) has been determined including 745 and 1024 nucleotides 5' and 3' to the tubAR coding region, respectively. tubAR encodes a 447 amino acid polypeptide which shows a high degree of homology with other fungal beta-tubulins. The gene contains three introns at codons 4, 12 and 53, uses 48 of the possible 61 sense codons and has a GC content of 59.1% in its coding region. S1 nuclease mapping has identified two transcriptional start sites 80 bp and 83 bp upstream of the translation start, and a transcriptional termination site 192 bp downstream of the stop codon. The two transcriptional start sites lie just 8 bp and 5 bp downstream of a CT motif consisting of 18 pyrimidine nucleotides interrupted by a single adenine. The wild-type allele tubA+ has been cloned using the polymerase chain reaction and the mutation producing the benomyl-resistant phenotype of tubAR mapped to a C to T transition at the first position of codon 6, resulting in a histidine to tyrosine amino acid substitution.