Worm Breeder's Gazette 12(2): 56 (January 1, 1992)
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 ama-1 gene encodes the largest subunit of RNA polymerase II (RpoII). It was initially defined by the dominant mutation m118 ,which results in an RpoII that is 150-fold less sensitive to amanitin in vitro than the wild-type enzyme. A common feature of amanitin-resistance mutations affecting RpoII in mammals, flies and worms, is that the resistant enzymes are about 100- to 300-fold less sensitive to amanitin than wild-type enzyme. That is, the mutant RpoII is still the most sensitive Rpo in the animal, and about two orders of magnitude more sensitive than RpoIII. Mutations leading to higher levels of resistance might be lethal, or there might be a second (lower affinity) amanitin binding site on some subunit of RpoII that was not affected by the known mutations. To examine these possibilities, a more resistant RpoII was obtained by EMS mutagenesis of amaI( m118 ),selecting for increased resistance to amanitin (Rogalski et al., Genetics 126: 8 89, 1990). One mutation, m526 ,was obtained, and it was mapped to the ama-1 gene. The double mutant, ama-1 ( m118 m526 ),results in an RpoII that is 150 times more resistant to the toxin than RpoII from the parental allele, ama-1 ( m118 ),and 20,000 times more resistant than the wild-type RpoII. This mutant is the most resistant ever reported; it is sterile at 25°C, and even at 20°C only about 10% of the eggs hatch. Hence, the high level of resistance is correlated with partial loss of function.
In order to determine the mutation sites responsible for resistance to amanitin, regions of the gene were PCR-amplified directly from the mutant worms. The amplified fragments were then cloned and several clones of each were sequenced. As a starting point, the region encompassing exons 6 and 7 was chosen for amplification, since this region is highly conserved among divergent species, and in the mouse, an amanitin-resistance mutation had previously been identified in this region. Also, exons 6 and 7 are in a physical position consistent with the position of m118 on the fine-structure genetic map of ama-1 (Bullerjahn and Riddle, Genetics 120:423, 1988). Genetic data also showed that m526 must be very close to m118 .The m118 mutation is a G to A transition that results in a substitution of tyrosine for cysteine at position 777. This confirms a preliminary observation made by David Bird. The m526 mutation, also a G to A transition, was found only twenty-four bases downstream from m118 ,and results in a substitution of glutamic acid for glycine at position 785. It is interesting that in the mouse RpoII large subunit, the conserved asparagine at position 782 is changed to aspartic acid in an amanitin-resistant mutant (Bartolomei and Corden, Mol. Cell. Biol. 7:586,1987).
To confirm that the m526 mutation was indeed responsible for the increased resistance to amanitin, a rare intragenic recombinant, which was obtained from the double mutant ama-1 ( m118 m526 )and had lost the increased resistance to amanitin, was analyzed in the same manner. The m526 mutation was no longer present in the recombinant, so it must be responsible for the increased resistance to amanitin in the double mutant amaI( m118 m526 ).Therefore, it seems that a relatively small, highly conserved region in the largest subunit of RpoII determines amanitin sensitivity, and is structurally important for polymerase function.
[See Figure 1]