Worm Breeder's Gazette 14(2): 72 (February 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812-81, Japan|
|2||Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038, USA.|
|3||Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48105, USA|
mek-1 was first identified as a C. elegans homolog of STE-7 in a cDNA project (Waterston et al. 1992) and cloned independently by at least three labs (P. Sternberg, K. Guan and Y. Ohshima). BLAST and FASTA homology search in the Genbank/EMBL database showed that mek-1 has the highest homology to JNK activating kinase/MKK4/SEK1. The JNK/SAPK pathway is known to be involved in stress responses, growth arrest and apoptosis in mammals.
To elucidate the function of mek-1 in vivo, we constructed a cDNA encoding an activated form of mek-1 in which Glu was substituted for both Ser218 and Ser222. The activated mek-1 cDNA was placed under the heat shock promoter of the pPD49.83 expression vector. This construct was introduced together with a dpy-20(+) marker gene into dpy-20 animals by germ line transformation to make FK137 (dpy-20(e2017); ksEx11[hsp-mek-1(act), dpy-20(+)]).
Heat-shock (30 min at 35 degrees C) of FK137 produced the following effects: (1) Egg-laying ceased. (2) Spontaneous movement ceased. The worms continued to respond vigorously to touch, so this was probably a nervous system rather than a muscle effect. (3) Pharyngeal muscle action potentials were suppressed. (4) Pharyngeal muscle contraction was suppressed. (5) Vacuoles appeared in the pharynx and uterus. (This effect was observed about five hours after a 60 min 33 degree C heat shock.)
The effects on action potential were measured by recording EPGs from FK137 at various times after heat shock. Part a of the figure below was recorded from a wild-type control 78 min after the end of a 30 min 35 degree C heat shock. It looks similar to EPGs recorded from untreated wild type or FK137. b was recorded from FK137 worms 61 min after heat shock. The action potentials (bars under traces) have become briefer than normal. 95 min after heat shock muscle action potentials were completely suppressed (c). The brief potentials that remain are probably postsynaptic potentials from the excitatory motor neuron MC (Raizen et al, Genetics 141: 1365-1382). These effects most likely result from suppresion or more rapid than normal decay of the plateau of the action potential, which would be caused by inhibition of Ca++ channels, activation of inhibitory (K+ or Cl-) channels, or both. 235 min after heat shock the worms had recovered somewhat--the action potentials looked like b. 289 min after heat shock electrical activity was normal.
At 95 min after heat shock no pharyngeal muscle motions were visible. At 289 min after heat shock, when electrical activity had returned to normal, no pumping could be seen in the dissecting scope, although a very feeble pharyngeal muscle contraction could be seen at each action potential by Nomarski microscopy. This suppression of contraction persisted for at least a day and probably longer since, with the exception of a few that may have been embryos at the time of heat shock, no heat-shocked transgenic worms grew.