Worm Breeder's Gazette 12(4): 67 (October 1, 1992)
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.
We are interested in two genes that are important for cholinergic function, cha-1 and unc-17 .These genes share a portion of their 5' untranslated regions, but the two encoded proteins have no sequence in common. cha-1 encodes choline acetyltransferase (ChAT, the acetylcholine synthetic enzyme, while the unc- l7 protein product ( unc-17 )is a unique protein of unknown function. We cloned the full length coding sequences from cha-1 and unc-17 cDNAs into two E. coli expression systems: pMal from New England Biolabs and QIA from Qiagen. The pMal vector had an inducible promoter followed by a gene encoding a maltose binding protein (MBP) and a sequence coding for a factor Xa cleavage site. We inserted cha-1 or unc-17 downstream of this site. Induction caused production of MBP fused to ChAT or unc-17 :we purified these proteins with amylose affinity column chromatography. The yield for both proteins was approximately 4-5 mg/1 culture. We separated pure ChAT after factor Xa cleavage, but we were unable to recover pure unc-17 ,possibly due to protein instability. Using the Qiagen system, we inserted the cDNAs downstream of a sequence encoding six histidines. This system should maximize the likelihood of recovering active protein after purification. We purified the his-ChAT protein using nickel-agarose column chromatography. We are still in the process of purifying the his- unc-17 protein. The yield of ChAT is lower than with the MBP system, approximately 1-2 mg/1.
We identified four potentially immunogenic regions from the predicted sequence of each of the two proteins and synthesized peptides of 11-19 amino acids in length. We injected these peptides into mice, rabbits, or chickens. Both rabbits and chickens produced polyclonal antibodies that recognized the fusion proteins on immunoblots. Antibodies to three different ChAT peptides recognize both ChAT fusion proteins, while antibodies to two different unc-17 peptides recognize MBP- unc-17 .All the sera exhibit very high nonspecific staining in fixed worms. Therefore, we purified these sera with the ChAT and unc-17 fusion proteins. Affinity purified ChAT serum still showed considerable background staining and both sera only stain lightly-fixed tissue. Therefore, we are trying to make antibodies to the fusion proteins, as well as testing a variety of purification procedures.
We have just begun to identify cells that stain with the sera. Two non-neuronal regions, near the pharyngeal-intestinal valve and the anal muscles, show diffuse staining with anti-ChAT sera but not with anti- unc-17 sera. With both sera, there is extensive staining in similar cells in the nerve ring and ventral and dorsal nerve cords. The staining is not readily localized to specific cell bodies in the ventral cord. The distribution of the staining within cells is different for the two sera. Anti-ChAT staining is fairly uniform within the nerve cords and includes labeling of most of the commissures. Anti- unc-17 staining is very punctate within individual cells and is absent from virtually all commissures. The distribution of anti- unc-17 staining superficially resembles the distribution of synaptic regions. Interestingly, in unc- 104, a mutant with defective synaptic vesicle transport, anti- unc-17 staining is different. There is an increased amount of punctuate anti- unc-17 staining around a subset (approximately 3/4) of the nuclei in the ventral cord and very little staining in the dorsal cord. Anti-ChAT staining in unc-104 is relatively unchanged from the wild-type pattern.
With the kind assistance of Pam Hoppe, we rescued both unc-17 and cha-1 mutants with the same cosmid ( F5G7 ).The staining patterns in the rescued worms were similar to wild type worms. However, we saw an increased level of anti- unc-17 staining and a small amount of staining in some commissures and around particular cell bodies in the nervous system. By using mutants, over-expression, and with increased antibody specificity, we hope to identify the majority of putative cholinergic neurons, i.e., ChAT and unc-17 containing neurons, in the next months.