Isolation and characterisation of a beta tubulin isotype 2 gene from two species of cyathostomin.
This study describes the isolation and characterisation of beta tubulin isotype 2 cDNA sequences from two common species of cyathostomin, Cylicocyclus nassatus and Cyathostomum catinatum. The full-length cDNAs for these species were 1709 and 1753 bp in length, respectively, including 1350 bp of sequence inferred to encode 450 amino acids of peptide sequence. They had greatest identity with previously characterised isotype 2 sequences from Trichostrongylus colubriformis, Cooperia oncophora and Haemonchus contortus (96% for C. nassatus and 95% for C. catinatum), and grouped together with these sequences in phylogenetic analysis. Both cyathostomin beta tubulin isotype 2 sequences contained the isotype-specific carboxyl terminal region described previously in other nematode species. Alignment with beta tubulin isotype 1 proteins from other trichostrongyloids, indicated 95 and 94% identity for the isotype 2 sequences of C. nassatus and C. catinatum, respectively. This comparison revealed 14 isotype-specific amino acid substitutions. Also, 2605 bp of beta tubulin isotype 2 genomic DNA sequence were isolated from C. nassatus. Comparison with the previously published isotype 1 gene of C. nassatus indicated differences in genomic organisation between the two isotypes. Reverse transcriptase (RT)-polymerase chain reaction analysis revealed constitutive temporal expression of beta tubulin isotype 1, whilst isotype 2 appeared to be developmentally expressed, with transcripts detected only in RNA derived from adult parasites.
Clark
H J
HJ
Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. hjclark@liverpool.ac.uk
Kaplan
R M
RM
Matthews
J B
JB
Hodgkinson
J E
JE
D000818:Animals; D000871:Anthelmintics; D001483:Base Sequence; D001562:Benzimidazoles; D018509:DNA, Helminth / Q000032:analysis*; D004351:Drug Resistance / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D006736:Horses; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013319:Strongyle Infections, Equine / Q000469:parasitology*; D013320:Strongyloidea / Q000235:genetics*; D014404:Tubulin / Q000235:genetics* / Q000302:isolation & purification
D000871:Anthelmintics; D001562:Benzimidazoles; D018509:DNA, Helminth; D020033:Protein Isoforms; D014404:Tubulin