Worm Breeder's Gazette 4(1): 18
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.
The major cuticle proteins of C. elegans are cross-linked by disulfide bonds which facilitates their purification. We expect, however, that other non-disulfide cross-linked proteins exist which are integral components of the cuticle and necessary for its proper functioning. We hope to be able to identify some of these proteins by finding buffer soluble or SDS soluble proteins which follow similar synthetic patterns as the major cuticle proteins. Enzymes associated with cuticle synthesis and possible cuticle protein precursors should also be amenable to study once the synthetic patterns of the major cuticle proteins become known. Towards this end, we have used radioactive NaH(14C)O3 (DeNevi and Kusch, W.B.G. vol.3, no.2) to follow cuticle synthesis in C. elegans during its development from a dauer to an adult. We start with dauer populations purified by two rounds of SDS resistance and centrifugation through 15% ficoll. To avoid possible complications caused by eggshells which copurify with cuticles, we now use fer-1 (hc1) in all our experiments, a temperature sensitive sperm defective which lays shell-less oocytes at 25 C. As expected, we find the peak periods of incorporation of label into the L4 and adult cuticles coincide with each of the peaks of lethargus, at about 10 and 19 hours post-dauer for the L4 and adult molts respectively at 25 C. No significant incorporation into the L4 cuticle occurs before 6 hours, nor into the adult cuticle before 16 hours post dauer. In both cases, incorporation of label into the new cuticle begins 2-3 hours before the peak of lethargus, reaches its highest level at the lethargus peak, and then gradually falls off. Some incorporation into the cuticle fraction apparently continues even after the population has completed lethargus, suggesting that new material is synthesized and added to the cuticle as the worm grows. In contrast to the fluctuation observed with the cuticle fraction, incorporation into TCA precipitable material increases steadily over these time periods. At the peak of lethargus the adult cuticle accounts for 10-15% (5-10% for the L4) of the total macromolecules labelled by the carbonate. Approximately 60% of the label in the L4 and adult cuticles is soluble in SDS plus a reducing agent, - mercaptoethanol; the rest is insoluble. 90% of the label incorporated into the L4 cuticle fraction during a 30 minute pulse at the L4 lethargus is subsequently lost at the next lethargus period, consistent with this fraction being cuticle. Little or no change is observed in the TCA precipitable fraction over this time period. Fluorograms of the SDS + ME soluble adult cuticle fraction separated by SDS-PAGE have been prepared. The pattern obtained is similar to coomassie blue stained gel patterns revealing two major bands at about 90,000 and 100,000 and a limited number of other bands in the 60,000-200,000 MW range. These results encourage us in our belief that the bulk of the carbonate is getting incorporated into amino acids rather than sugar groups, etc. Other experiments are in progress to further characterize the types of molecules labeled by NaH( 14C)O3. Comparison of the SDS + ME soluble adult cuticle proteins of N2 and HCl reveal no differences, indicating that none of these proteins are eggshell components.