Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.
Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.
Geary
T G
TG
Animal Health Discovery Research, Pharmacia & Upjohn, Kalamazoo, Michigan 49007, USA.
Nulf
S C
SC
Alexander-Bowman
S J
SJ
Mahmoud
B M
BM
Prichard
R K
RK
Klein
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018509:DNA, Helminth / Q000737:chemistry* / Q000235:genetics; D004183:Dirofilaria immitis / Q000737:chemistry / Q000235:genetics*; D005260:Female; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018510:RNA, Helminth / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014404:Tubulin / Q000737:chemistry / Q000235:genetics*
D018509:DNA, Helminth; D018510:RNA, Helminth; D014404:Tubulin