Worm Breeder's Gazette 10(3): 83

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.

Mutant RNA Polymerase II is Super-Resistant to à-amanitin

Miriam Golomb

RNA polymerase II (RpoII) from C.  elegans is 50% inhibited by 7 
ng/ml of alpha-amanitin, whereas RpoIII is 50% inhibited by 80  g/ml 
and RNA polymerase is unaffected by 500  g/ml.  Dominant mutations 
have been described in the gene for the large subunit of the enzyme 
from mammals (Lobban et al.  (1976), Cell 8: 65-70), Drosophila (
Greenleaf et al.  (1979), Cell 18: 613-622), and the worm (Sanford et 
al.  (1983), J.  Biol.  Chem.  258: 12804-12809), which render the 
enzyme partially resistant to alpha-amanitin by decreasing binding of 
the drug.  None of these mutations, however, confer the full 
resistance characteristic of animal RpoI.  Using a selection technique 
which increases the permeability of worms to alpha-amanitin, Rogalski 
and Riddle (this issue) have obtained a derivative of a resistant (ama-
1(m118)) strain that is super-resistant to alpha-amanitin.  The new 
mutation (m526) is also at the ama-1 locus and maps very close to m118 
(Rogalski and Riddle, op.  cit.) 
RpoII from the ama-1(m118m526) strain is labile to chromatography 
and cannot be separated in active form from RpoI and III.  However, 
RNA polymerases I, II and III in wild-type nuclei have the same 
sensitivities to alpha-amanitin as the purified enzymes.  These 
enzymes can, therefore, be distinguished in a nuclear run-off assay 
using appropriate DNA probes.  Nuclei were isolated by the method of 
Dixon (WBG 9:3, 73-74) and allowed to incorporate alpha-[32P]-CTP in 
the presence of various concentrations of alpha-amanitin.  Nuclear 
transcripts were hybridized to a Southern blot of restriction 
fragments from cloned genes.  These included genes transcribed by RpoI 
(Ascaris rDNA obtained from K.  Bennett), RpoIII (C.  elegans 5S DNA (
Honda et al.  (1986), Nucl.  Acids Res.  14: 869-881), and RpoII (C.  
elegans splice leader precursor DNA (Honda et.  al., op.cit.; Krause 
and Hirsh (1987), Cell 49: 753-761)).  Splice-leader precursor is 
transcribed in nuclei by RNA polymerase II (M.  Golomb and S.  Bektesh,
In this assay, transcription of splice-leader precursor RNA was 
unaffected by 500  g/ml alpha-amanitin, a concentration at which 5S 
RNA transcription was nearly completely inhibited.  Thus, the m526 
mutation appears to render RpoII fully resistant to alpha-amanitin.