Worm Breeder's Gazette 10(3): 84

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 Novel ama-1 Mutation That May Eliminate RNA Polymerase II Sensitivity to à-amanitin

Teresa M. Rogalski and Donald L. Riddle

Figure 1

The ama-1 gene encodes the largest subunit of RNA polymerase II, a 
protein of approximately 200,000 MW.  Rare, dominant mutations in the 
ama-1 gene result in an RNA polymerase II that is more resistant to 
alpha-amanitin than the wild-type enzyme (Sanford, Golomb and Riddle, 
J.  Biol.  Chem.  258:12804-12809,1983; Rogalski, Bullerjahn and 
Riddle, Genetics 120:409-422, 1988).   Although these mutations 
decrease the amanitin sensitivity of RNA polymerase II, presumably by 
affecting the site where amanitin binds, they do not eliminate it 
completely.  For example, RNA polymerase II from the resistant ama-1(
m118) strain requires 1.0  g/ml amanitin for 50% inhibition in vitro 
compared to 7 ng/ml for the wild-type enzyme (Sanford, Golomb and 
Riddle, op.  cit.).  
To determine whether it was possible to obtain a completely 
resistant enzyme, we devised a selection for a super-resistant 
derivative of the resistant ama-1(m118) parent.  Resistant 
hermaphrodites are unaffected in vivo by amanitin concentrations up to 
and including 800  g/ml.  However, the addition of 0.5% Triton X-100 
to the growth medium increases the sensitivity of the ama-1(m118) 
strain, such that these animals are unable to grow at a concentration 
of about 80  g/ml amanitin.  This has allowed us to select for mutants 
that will grow under conditions where ama-1(m118) hermaphrodites will 
One mutant has been found among 4x10+E6 F1 progeny of EMS-
mutagenized worms.  In contrast to the resistant parent, which 
exhibits near normal growth and fertility, the super-resistant mutant 
develops slowly, has a much reduced brood size at 20 C, and is sterile 
at 25 C.  The initial genetic analysis indicated that this new 
mutation, m526, was closely linked to ama-1, and further 3-factor 
mapping placed it at the ama-1 locus.  Strains carrying various 
combinations of the wild-type (+), resistance (R) and super-resistance 
(SR) ama-1 alleles have been tested for their level of amanitin 
resistance, and the genotypes are listed below in order of increasing 
+/+ < +/SR < +/R < R/R < R/SR < 
These data show that resistance is dominant to wild-type as in other 
organisms.  Surprisingly, the +/SR worms are much less resistant than 
the +/R worms.  In fact, +/SR hermaphrodites are only marginally more 
resistant than +/+ animals, suggesting that the super-resistant 
subunit may not function well in the presence of the wild-type subunit.
Preliminary results indicate that RNA polymerase II from the ama-1(
m118m526) strain is more resistant to amanitin in vitro than RNA 
polymerase III, which is 10,000-fold more resistant than the wild-type 
polII and 100-fold more resistant than polII from ama-1(m118) 
homozygotes (M.  Golomb, this issue).  Thus, in the super-resistant 
strain amanitin sensitivity is presumably due to inhibition of RNA 
polymerase III.  We plan to select for amanitin-resistant polIII 
mutants in the super-resistant polII genetic background.  
To aid in finding the m526 site in the 8.7 kb ama-1 gene, we have 
attempted to position m526 relative to m118 on the ama-1 fine-
structure map (Bullerjahn and Riddle, Genetics 120:423-434, 1988).  
One putative recombinant has been obtained and is currently being 
analyzed.  The data thus far suggest that m526 is very close to m118, 
so we are cloning and sequencing this region of the super-resistant 
[See Figure 

Figure 1