Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity.

Abstract

Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta-tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.

Pharmacological intervention in invertebrate aging

“…Commonly researchers have combined drug treatments with genetic mutants to uncover mechanisms. For example, selection for drug resistant mutants has been used to investigate the mechanisms of antinematode compounds (Rand and Russell 1985;Driscoll et al 1989). A number of bio-technology companies, such as DevGen, Exelixis and Divergence have historically incorporated C. elegans as a significant part of their drug discovery strategy.…”

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

“…We designed an sgRNA to target the ben-1 gene ( Figure 1E) and a donor oligo to introduce a nonsense mutation, the Amber stop, at Tyrosine 51 in the first exon of ben-1 ( Figure 1E). ben-1 encodes a β-tubulin that is sensitive to the treatment of benomyl (an anti-microtubule drug) [15], which leads to slow growth and paralysis of animals at 25 °C. As ben-1 loss-of-function mutations are dominant suppressors of the benomyl-induced paralysis or Unc defect [15], we could easily identify mutated F1 heterozygous or homozygous animals placed on 14 mM benomyl plates.…”

“…ben-1 encodes a β-tubulin that is sensitive to the treatment of benomyl (an anti-microtubule drug) [15], which leads to slow growth and paralysis of animals at 25 °C. As ben-1 loss-of-function mutations are dominant suppressors of the benomyl-induced paralysis or Unc defect [15], we could easily identify mutated F1 heterozygous or homozygous animals placed on 14 mM benomyl plates. From 5 wild-type C. elegans animals (N2 strain) injected with the oligo-containing mixture (Supplementary information, Data S1), we identified 16 non-Unc animals from 45 Cas9 transgenic F1 animals (mCherry positive) and 19 non-Unc animals from 219 non-transgenic F1 animals ( Figure 1F).…”

Comparison between the effect of Lawsonia inermis and flubendazole on Strongyloides species using scanning electron microscopy

“…Mechanisms of benzimidazole resistance are well established, and are known to involve the target site mutations. It is associated with the deletion of a benzimidazole-susceptible beta-tubulin gene, decreasing the b-tubulin-benzimidazole interaction (Driscoll et al 1989;Prichard 1994). Analysis of beta-tubulin isotype sequences from susceptible and resistant worms revealed the presence of a phenylalanine-to-tyrosine (PheTyr) mutation at position 200 in all resistant worms examined (Kwa et al 1994).…”