Worm Breeder's Gazette 9(1): 19
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.
Hybridization of a probe encoding the large (alpha-amanitin binding) subunit of Drosophila RNA polymerase II (Ingles, et. al., P.N.A.S., 80, 3396, 1983) to C. elegans genomic DNA, under extremely non-stringent conditions, reveals two EcoRI fragments viz., a strong 4.9 Kb band. Genomic clones containing these RI fragments have been isolated from an N2 library (constructed by T. Snutch). To test whether either of these recombinants encode the alpha- amanitin binding subunit of RNA pol II, defined by ama-1 IV (see Rogalski and Riddle, this issue), quantitative blot analysis (using an actin gene probe as a control) was performed on strains heterozygous for the small deficiency mDf4, which spans ana-1. A dosage reduction in hybridization was observed when the 4.9 Kb containing recombinant ( lambda 2b) was used, suggesting that this clone encodes ana-1. Sequences from the 3.5 Kb containing recombinant (lambda 1a) are not included in mDf4, suggesting that the 3.5 Kb and 4.9 Kb fragments are not closely linked. To expand the regions cloned, Alan Coulson and John Sulston kindly fingerprinted lambda 1a and lambda 2b and were able to place lambda 1a in the middle of a contig of 12 independent, overlapping cosmids. Lambda 2b is not in this contig, and no other cosmid spanning lambda 2b was detected. Therefore, to be certain that we have the entire ama- 1 gene and surrounding sequences, we are walking from lambda 2b by conventional means. To further examine lambda 1a and lambda 2b, we have probed them with different regions of the Drosophila pol II probe. A probe encoding the carboxyl-terminus detects sequences in both lambda 1a and lambda 2b, whereas an amino-terminus probe detects only lambda 2b sequences. A similar pattern was observed in yeast (Allison, et. al., Cell, 42, 599, 1985) where the Drosophila probe was found to detect both pol II and pol III subunit genes. Consequently, a yeast pol III probe ( Allison et al, op. cit.) was hybridized to lambda 1a and lambda 2b under non-stringent conditions. Several contiguous EcoRI fragments ( including the 3.5 Kb fragment) were strongly detected in lambda 1a; no hybridization to lambda 2b was observed. Thus, lambda 1a probably encodes the C. elegans pol III large subunit. Prior to sequencing these genes, we are attempting to define the extent of the transcription units of the lambda 1a and lambda 2b genes by Northern blotting.