Worm Breeder's Gazette 3(2): 40
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 usual way to radioactively label C. elegans, feeding them bacteria that have been previously labelled with radioactive amino acids, has a number of limitations. It is not feasible to use this method for doing pulse-chase experiments or for labelling worms during periods when they are not eating (i.e., during letharous or as dauer larvae). Because we wish to do pulse-chase experiments in our studies of cuticle synthesis, we have looked for an alternative labelling method. [14C]-carbonate labelling looks very promising. NaH(14)CO3 can be taken up directly by the worms (probably as (14)CO2) and therefore eliminates the necessity of labelling via the food supply. [14C]-carbonate labelling has been used successfully in a variety of systems. It has even been used before on helminths to study their intermediary metabolism. The principal pathway for NaH(14)CO3 labelling of proteins has been fairly well elucidated. It involves the fixation of (14)CO2 into the TCA cycle intermediates oxaloacetate and alpha-ketoglutarate. The labelled intermediates can be transanimated into the amino acids asparate and glutamate respectively. These [14C]-labelled amino acids can then be incorporated into proteins. In ureotelic organisms the amino acid arginine is also significantly labelled through (14)CO2 fixation into the urea cycle. We have used the following labelling procedure: Worms are grown on agar plates with E. coli until one hour before they are to be labelled. The animals are then harvested and washed thoroughly to remove any bacterial contaminants (any whole bacteria in the worm s gut hopefully should be digested in the hour preceeding labelling). The labelling medium consists of M9 buffer and NaH(14)CO3 adjusted to pH 7. We label 10,000-15,000 worms in a 1.5 ml volume of labelling medium (20 Ci/ml) in a small round bottom test tube that is agitated gently throughout the experiment. The labelling period can be relatively short (i.e., 30 minutes) and the test tube should be corked to prevent a significant amount of (14)CO2 from escaping from the labelling medium. The worms are washed free of the labelling medium through a series of washes in a cold M9-NaHCO3 solution (100 mM). The results from our characterization experiments with this labelling procedure have shown that the label enters the worm rapidly ( probably as (14)CO2), that it gets incorporated into macromolecules and that the labelled macromolecules have a molecular weight range (as determined by SDS gels) corresponding to that of proteins. Preliminary results also indicate that the label can be chased out of the worm effectively. We have also done one preliminary experiment to see if the label gets incorporated into the cuticle. It does, and much more effectively if labelling is done during the period prior to L4 molt as compared with the adult. With labelling periods of one- half to three hours we have found that 1-2% of the label gets into macromolecules or the intracellular acid-soluble pool. Dauers take up the label but dead worms do not. The experiments done so far encourage us in the hope that [14C]-carbonate will prove to be useful for pulse-chase and other types of labelling experiments in C. elegans.