Worm Breeder's Gazette 10(3): 78
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.
It has been reported that Ascaris RNAs contain a splice leader sequence similar to that of Caenorhabditis tesh et al., WBG 10-1:67). When we first sent Ascaris des RNA samples to the laboratory of D. Hirsh, we also tested them with an oligonucleotide corresponding to the C. elegans spliced leader (SL) and found, as his group did, that a similar splice leader is present in Ascaris. Recently one of us (T. N.) has shown that the same 22 nt leader is present in the parasitic nematode causing human lymphatic filariasis, Brugia malayi (P.N.A.S., in press). Because Ascaris offers potential advantages to do biochemistry not available in Brugia, we have recently collaborated in further characterizing trans-splicing in Ascaris and in cloning the Ascaris leader sequence. As in C. elegans, we have found by Northern blot analysis that a small (~100nt) polyA-RNA species is recognized by the spliced leader, and multiple mRNAs are detected when polyA+ RNAs are used. All RNAs tested by Northern analysis including early embryonic (<30 cell), larval, oocyte and gut were positive, implying, but certainly not proving, no tissue or stage-specificity. By Southern blot at least two enzymes, ScaI and HaeIII, cut genomic DNA to an ~1 kb repeat. The same pattern of digestion was seen using the 5S ribosomal gene probe, indicating the trans-spliced leader is located in the 5S repeat, as found in C. elegans and in Brugia. An Ascaris genomic library constructed in lambda EMBL4 (Bennett and Ward, Dev. Bio. (1986) 118:141) was screened with the 22 mer SL oligonucleotide and with an oligonucleotide from the Brugia 5S genes. Positive lambda clones which contained the 5S gene and the sequence leader have been plaque purified; one has been subcloned and sequenced in part. We have found the Ascaris 22nt sequence leader is exactly the same as the C. elegans and Brugia one, with the rest of the small RNA sequence divergent. To determine the percentage of messages that contain the spliced leader, we have used polyA+ RNA from a mixed C. elegans population and from various Ascaris tissues and developmental stages. With 0.5 g of C. elegans polyA+ RNA, the SL oligonucleotide hybridizes to 10.6% as much RNA as does a labeled oligo dT probe. In the same assay using Ascaris RNAs, only 1.1-2.0% of the messages appear to contain the spliced leader in gut, oocyte and larval RNAs. While this one experiment implies that fewer messages are trans- spliced in Ascaris, the results could also be due to Ascaris messages having much longer polyA+ tails than C. elegans. However, our Northern blots also imply fewer Ascaris messages are trans-spliced when Ascaris and C. elegans polyA+ RNAs are compared.