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.

A Labelling Technique Using 14C-carbonate

K. DeNevi, M. Kusch

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.