Artemia tubulin genes and mRNA

Abstract

623rd MEETING, CANTERBURY 1173 resistance of the mutant strain observed in vivo has been reproduced in vitro using tubulin purified from the mutant.The composition of microtubules formed from the mutant tubulin in the presence of drug was assessed by quantification of stained electrophoretic gels. The ratio of the two 8-tubulin isotypes (81 : 81-210) was determined from several samples at each concentration over a parbendazole concentration range of 0-50 p~. A mean value of 0.86 k 0.08 for the ratio j l : j1-210 was obtained over all parbendazole concentrations and thus there appears to be no enrichment of the mutant tubulin isotype at high drug concentrations. This observation has important consequences for the mechanism of drug resistance in these cells. The result suggests that microtubules are composed of both a181 and alpl-210 protomer, otherwise a loss of alp1 protomers from drugsensitive a l j l microtubules would be expected in high drug concentrations, and the ratio j 1 : j1-210 would change. How resistance is conferred is not known, the pl-210 isotype might be randomly distributed with a certain proportion conferring resistance on the whole microtubule. Alternatively, the j1-210 isotype might be localized, for example, at initiation of assembly so allowing the subsequent addition of a l j l or aIB1-210 protomers during elongation. The threshold at which the mutant isotype ceases to protect the microtubule may be approximated in vitro by mixing tubulin purified from the wild-type and the mutant, and monitoring assembly in the presence of drug.