Worm Breeder's Gazette 8(3): 76
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.
I have partially purified class C acetylcholinesterase (AChE) from triton extracts of N2 animals and prepared polyclonal antibodies to the partially purified protein. A rabbit was immunized with a band cut out of an SDS gel, boosted once with another cut out band and then boosted twice more with undenatured samples. I estimate that each injection contained about 10 g of enzyme. Anti-class C antibodies were detected only after the third boost. By ELISA assay the antibodies show no cross-reactivity to forms from the other two known AChE classes, A and B. However, by immulunoblots the antibodies did show cross-reactivity to a number of other C. elegans proteins. Absorption of the antibodies with an extract from ace-3(dc2) animals resulted in the removal of these contaminating antibodies, as shown by immunoblots. (Animals containing the dc2 allele have no detectable class C enzymatic activity and by ELISA have no cross-reactive material, and therefore are apparently null.) I have used the following procedure to determine the localization of class C in whole mounts: 1. Densely settled animals are squashed on 1.0% BSA subbed slides according to the procedure of Albertson et al (WBG 7(1):73). I find that squashing with a cover slip is most convenient. After freezing in liquid nitrogen and prying off the cover slip with a razor blade the slides are passed through methanol, acetone, and a graded series of ethanols to TBS (Tris buffered saline). The following incubations are then performed in a humid chamber, with 0.035 ml reagent placed over the squashed animals. 2. Incubation with 10% normal goat serum (NGS) in TBS for 30 minutes, followed by a brief wash in TBS. 3. Incubation with primary antibody (1:1000) diluted in 1% NGS/TBS overnight, followed by two 10 minute washes in TBS. 4. Incubation with Goat anti-Rabbit IgG antiserum (1:150) in 1% NGS/TBS 30 minutes, followed by a brief wash in TBS. 5. Incubation with PAP reagent (1:100) in 1% NGS/TBS 30 minutes, followed by 2 ten minute washes in TBS. 6. Repeat the two previous steps. 7. Develop the peroxidase reaction with 0.44 mg/ml diaminobenzidine HCl 0.003% H202 in TBS for 5-10 minutes, followed by a brief wash in TBS. 8. Slides are then mounted with 90% glycerin in TBS. The brown reaction product is visible in the nerve ring, in several cell bodies near the nerve ring, and in two cells located near the vulva. In addition to the specific staining seen in these structures there is a diffuse background which is visible. When staining dc2 there is no specific staining, however the background persists. Some non-specific staining is also observed in the pharyngeo-intestinal valve, both in N2 and dc2. The two cell bodies are located under the lateral alae as are their processes which stain and run posteriorly to the level of the anus and anteriorly to the level of the pharyngeo- intestinal valve. I believe that these are the CAN cells. They stain quite well in late stage embryos and L1s, as well as older animals, and are Present in unc-86 animals. As to why the CAN cells should have class C AChE on their surfaces is anyone's guess. While these cells are required for viability as shown by John Sulston, that viability is not dependent upon class C AChE since the ace-3 animals are viable, and in fact show no visible phenotype. In addition to the above mentioned staining I also occasionally see staining in cells which I believe are the PDE cells, based on their location and morphology. Work is in progress to confirm this result as well as to stain animals with altered CAN cells (vab-8).