Worm Breeder's Gazette 8(2): 34
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.
As a first step in studying the transcriptional machinery of C. elegans, we have purified nematode RNA polymerase II to near homogeneity. Structurally, the enzyme is a typical RNAP II, having two large (205 and 135 kd) and at least eight smaller polypeptides (10- 30 kd). DR 432, an amanitin-resistant strain with a mutation in gene ama-1 (IV), produces an altered RNAP II that is 150 times less sensitive to amanitin than the wildtype enzyme. We have purified RNA polymerase II from DR 432 for structural and functional comparison with wild-type polymerase. DR 432 enzyme is stable during purification, and its specific activity is identical to that of wild- type RNAP II. In a filter-binding assay, DR 432 RNAP II bound [3H]- labelled amanitin with a K at least 100 times higher than that of N2 enzyme. Thus, amanitin resistance in DR 432 is caused by defective binding of the toxin to RNAP II. The nature of the structural alteration of RNAP II in DR 432 is now being studied. Since interesting alleles of the ama-1 locus are expected to encode unstable or inactive polymerase, we have raised antibodies against nematode RNAP II to aid in identifying polymerase polypeptides in crude extracts of worms. We have obtained a high-titer rabbit antiserum against purified RNAP II which detects the 205 kd subunit in Western blots of worm homogenates. Immunofluorescent staining of hamster cells showed that this polyclonal antiserum cross-reacted with RNAP II in mammalian nucleoplasm (nucleoli were not stained, confirming an apparent lack of cross-reactivity with RNAP I). Rabbit anti-RNAP II IgG did not inhibit polymerase activity. Recently, we have obtained hybridomas secreting monoclonal antibodies to nematode RNA polymerases. Of 480 fusions between plasmacytoma cell line 653 and spleen cells from immunized Balb/c mice, about 30 hybridomas were found to make antibody against purified RNAP II by the ELISA test. Other hybridoma lines appear to make antibody against nematode RNAP I; several of these antibodies stain hamster nucleoli. Monoclonal antibodies against RNAP II are being screened for use in functional mapping of wild-type and mutant polymerase subunits.