INVESTIGATION OF DIVERSITY AND ISOTYPES OF THE BETA-TUBULIN cDNA IN SEVERAL SMALL STRONGYLE (CYATHOSTOMINAE) SPECIES

The diversity of the beta-tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-naïve horse. cDNAs encoding beta-tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98–100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3′ Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further beta-tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including 306 bp of coding sequence, was amplified, as was an additional fragment of 600 nucleotides in the case of Cy. pateratum, Cy. coronatum, and Cy. catinatum. Sequencing of the PCR products revealed no evidence for the existence of a second beta-tubulin isotype in cyathostomes. The variation in size was caused by a length polymorphism within the 3′ untranslated region, and 2 functional mRNAs seem to be transcribed from the same gene.