Organization and structure of an Onchocerca beta-tubulin gene.

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, alpha and beta. Tubulin is particularly important in helminthic parasites as a target for anthelminthic benzimidazoles, which bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in lambda NM1149 (EcoRI and HindIII). Three clones accounted for the entire gene: one from the EcoRI library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3'-ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5'- and 3'-end clones was done by using a series of oligonucleotides. The sequence of the O. gibsoni beta-tubulin gene was completely determined, revealing an exceptionally complex structure as compared to the known beta-tubulin genes: 5,797 base pairs containing 12 exons and 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein.

Organization and structure of an Onchocerca ß-tubulin gene

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, Ó e ß.Tubulin is particulary important in helminthic parasites as a target for anthelminthic benzimidazoles, wich bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in NM1149(EcoRi and HindIII). Three clones accounted for the entire gene: one from the EcoRi library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3' -ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5' - and 3'-end clones was done by ...