Expression of a dominant active form of the MAP kinase kinase
mek-1 under a heat shock promoter leads to paralysis, egg laying defects, cessation of pharyngeal pumping and eventually death (WBG 14#2,
p72). However, the heat shock produces high levels of an activated kinase in all the cells, whereas
mek-1 is normally expressed only in a small number of cells. We are particularly interested in the pharyngeal phenotype, since
mek-1 is expressed in the pharynx and pharyngeal pumping can be further analyzed electrophysiologically. To study the pharyngeal defect, we want to express
mek-1(dom) in the pharynx only and, since this might lead to suppression of feeding and thus growth arrest, we want to be able to induce expression in these cells at any given time. To accomplish this, we used the temperature sensitive
smg-7(
r1131ts) mutant to stabilize messages engineered with a specific, long
unc-54 tail', driven by a cell-specific promoter (with thanks to Phil Anderson; a similar scheme was described recently by Getz et al., WBG 14#5,
p26). Injected at 4 ng/ul, a
myo-2::GFP construct with the
unc-54 tail was not expressed in wild-type animals but was expressed at considerable levels in
smg-7(ts) at both 15 and 25 degrees. Introducing
mek-1(dom) with the same promoter and tail fragments in
smg-7(ts) often led to a starved appearance and growth delay even at 15 degrees at very low concentrations. This is consistent with significant levels of
mek-1(dom) present in the pharyngeal muscle cells, the presumed site of action for
mek-1 to affect pharyngeal pumping. We are currently using Andy Fire's
smg-1(
cc546ts) mutant for further analysis.