Worm Breeder's Gazette 10(1): 29

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Cloning of the ben-1 gene

M. Driscoll, E. Reilly, E. Bergholz and M. Chalfie

Figure 1

Benomyl and other benzimidazole carbamates interfere with the 
polymerization of microtubules.  In the presence of benomyl, wild-type 
C.  elegans grows slowly, is severely uncoordinated, and has fewer 
neuronal processes in the ventral cord.  Mutations conferring 
resistance to this drug map to one gene, ben-1, on chromosome III.  
Virtually all of the EMS-induced and the TR679-derived alleles result 
in a temperature-dependent dominant drug resistance.  ben-1 animals 
have no visible phenotype when grown in the absence of benomyl.
Since benomyl resistance is conferred by mutations in  -tubulin 
genes in Aspergillus, Saccharomyces, and Physarum, we have performed 
low stringency hybridizations of  -tubulin sequences (provided by 
Linda Gremke and Joe Culotti) to DNA isolated from ben-1 mutants.  DNA 
from two of the TR679 derived strains contains an insertion of 
approximately 2.1 kb in one of the  -tubulin homologous restriction 
fragments.  We have cloned a 7.6 EcoRI fragment from wild-type C.  
elegans that corresponds to this  -tubulin.  The  -tubulin homology 
maps to the right end of the fragment, as depicted below.  The left 
end of this fragment contains a repetitive DNA element (a non-Tc1 
homologous sequence present in approximately 30 copies in C.  elegans).
The two transposon insertions are localized to different sites in 
the 2.1 kb BglII fragment.  Although these are clearly not Tc1 
insertions, we have not yet identified which, if any, of the 
characterized transposons are associated with the ben-1 mutations.
Preliminary transcription analysis suggests that the ben-1 mRNA is 
abundant in wild-type animals.  (Consistent with this observation, we 
have been successful in isolating a highly homologous clone from Julie 
Ahringer's cDNA library.)  The transcript is approximately 3 kb and 
the direction of transcription is from left to right as shown.
Molecular analysis has provided some insights into the mechanism of 
benomyl resistance.  One of our ben-1 alleles (derived from TR679) is 
actually a deletion of the 2.1 and 1.4 kb restriction fragments.  
Since this deletion allele is dominant, it appears that a critical 
level of ben-1 gene product is required for sensitivity to benomyl.  
Genetic and molecular experiments directed towards confirming this 
hypothesis are underway.

Figure 1