Worm Breeder's Gazette 5(1): 25
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.
Cyclic AMP levels have been assayed in N2 synchronous cultures and starvation-induced dauer larvae as a first step in the analysis of hormonal involvement in dauer larva formation, maintenance, and recovery. N2 worms are synchronized by egg purification (after the method of Ralph Hecht), hatching into M9 buffer, and resuspension in liquid worm medium containing an adenyl cylase deletion mutant of E. coli. The worms are harvested at various time intervals by immediately microwaving to boiling and washing in glass distilled water. This is followed by lyophillization after which the dry weight is determined. The assay uses a cAMP binding protein (Calbiochem) in a competitive binding assay with [3H]cAMP. After equilibrium is reached, the protein is filtered through a nitrocellulose filter and counted in a liquid scintillation counter. Initial results indicate that changes in cAMP concentration are correlated with the life cycle. Assays performed on well fed worms at various times in larval development show a linear decrease in cAMP levels from 25 pmol/mg dry weight in L2's to 15 pmol/mg dry weight in L3's, down to 1 pmol/mg dry weight in L4's. Starvation-induced dauer larvae have a cAMP level of 5 pmol/mg dry weight, or about one-third the level present at the L2 molt. Recovering dauers isolated 6 hours after the addition of food to starvation-induced dauers, show a dramatic drop in cAMP levels to less than 0.1 pmol/mg dry weight. The data collected to this point indicate that dauer larvae have a reduced level of cAMP compared to the L2 an' L3 larval stages and that recovery from the dauer stage results in cAMP levels below that found in L4's. These preliminary studies are to be followed by a more detailed study of cAMP and cGMP levels in N2 worms at all stages of the life cycle and in selected dauer-constitutive mutants. Cyclic GMP assays and pharmacological studies using theophylline as a phosphodiesterase inhibitor are currently being developed.