Worm Breeder's Gazette 7(1): 77

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.

C. elegans RNA Polymerase

T. Sanford, M. Golomb, D. Riddle

Figure 1

The fungal toxin, alpha-amanitin is an effective inhibitor of C.  
elegans RNA polymerase II.  Larvae hatched from eggs into medium 
containing 20 g/ml amanitin do not grow.  Larvae placed in fresh 
medium after 4 days incubation in toxin are unable to resume 
development and eventually die.  In vitro, partially purified RNA 
polymerase II is 50% inhibited by 10 Ng/ml amanitin.  We have isolated 
seven EMS-induced mutants resistant to amanitin.  Preliminary genetic 
crosses indicate that all the mutations are closely linked to dpy-13 
IV, and at least some mutants are semi-dominant.
Partial purification of RNA polymerase involves sonication of the 
worms, ammonium sulfate fractionation of the extract, and heparin 
sepharose and DEAE-cellulose chromatography.  Assays consist of 20-
minute DNA-dependent incorporations of [3H]-UTP into acid-precipitable 
material.  The amount of in vitro activity is comparable to that found 
in Drosophila or in mammalian cells.  The figure below shows the 
results of assays from fractions of a DEAE-cellulose column run on a 
wild-type extract.  Polymerase activity is eluted in the salt gradient 
in two peaks.  Open circles show the results of assays performed in 
the presence of amanitin, while the filled circles represent assays 
done without added toxin.  The first peak contains amanitin-resistant 
activity, presumably representing polymerases I and III.  The smaller 
peak represents amanitin-sensitive activity, and is therefore 
polymerase II.  The chromatographic behavior and stability of the 
enzyme seem quite similar to the mammalian enzyme in all respects.
Assays on extracts from amanitin-resistant mutants are in progress.  
Should a polymerase gene be identified, conditional-lethal alleles 
will be sought, and biochemical studies will be pursued so that 
functional comparisons can be made between normal and mutant enzymes.
[See Figure 1]

Figure 1