Worm Breeder's Gazette 7(2): 58
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.
Partial purification of RNA polymerase II from C. elegans involves sonication of worms in high salt (ammonium sulfate), removal of nucleic acids by DEAE-cellulose chromatography in high salt, and separation of polymerases I and III from polymerase II on a DEAE- cellulose column eluted with a salt gradient. Polymerase assays utilize a calf thymus DNA template and are performed either in the presence or absence of amanitin. Pooled RNA polymerase II fractions from wild-type N2 and the EMS-induced amanitin-resistant mutant, DR432, were assayed with various concentrations of toxin to determine the degree of enzyme sensitivity in vitro. Polymerase II from DR432 (ama- 1 IV) is approximately one hundred fold less sensitive to alpha- amanitin than is the wild-type enzyme. We tentatively conclude that ama-1 IV, closely linked to dpy-13, encodes a polymerase II subunit. RNA polymerase III has been partially resolved from polymerase I by DEAE-sephadex chromatography. A single chromatographic form of polymerase III elutes at 0.18M ammonium sulfate; its activity is stimulated approximately ten-fold by substituting poly-d(AT) for calf thymus DNA template. [See Figure 1] Amanitin sensitivity of RNA polymerase II from wild-type and amanitin-resistant C. elegans. Polymerase II isolated by DEAE- cellulose chromatography was assayed under standard conditions with varying amounts of amanitin. (Dark circles, wild-type; (open circles, enzyme from strain DR432(ama-1 IV).