Worm Breeder's Gazette 14(2): 31 (February 1, 1996)
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.
Department of Biology, Indiana University, Bloomington, IN 47405
It is now clear that eukaryotes are more closely related to archaes than either is to eubacteria. Nevertheless, eubacteria and archaes share the same operons, with the same genes in the same order, whereas eukaryotes lack operons. That means the ancestor to eukaryotes must have also had these same operons, but they subsequently lost them. The discovery of operons in C. elegans was quite surprising, since operons had not previously been found in multicellular eukaryotes, much less in animals. An important question is whether the C. elegans operons are ancient relics of operons found in C. elegans' ancestors or whether they represent an innovation. To begin to answer that question we need to know how widespread operons are within the nematode phylum. It has been shown previously that all nematodes engage in trans-splicing and that they all contain the spliced leader, SL1. However, the trans-splicing that serves to process downstream genes in operons utilizes SL2, and so far SL2 has been reported only in C. elegans and C. briggsae. To address the question of whether operons are ancient or derived, we are searching for gene clusters and for SL2 in other nematodes. However, the search turned out to be more difficult than we first appreciated because the nematode divergences are quite deep. Thus we have begun our search with a free-living nematode, CEW1, brought to our lab from Brazil by Carlos Winter. This nematode, which has previously been called B6/D6 or Dolichorhabditis (Winter, 1992, Comp. Biochem. Physiol. 103B, 189-196) or Oscheius brevesophaga (Carta, Thomas and Sternberg, 1994, wbg 13(3), 108), is currently unclassified. CEW1 is different enough from C. elegans that the vitellogenin genes fail to cross-hybridize (Winter, Penha and Blumenthal, 1996, Mol. Biol. and Evol., in press) and the SL1 RNA genes are arranged differently on the chromosome and are extremely divergent (Winter and Blumenthal, 1991, wbg 11(5), 58). On the other hand, the two species are close enough to give some chance of cloning more highly conserved homologs by hybridization. We began by cloning the CEW1 homolog to the acidic ribosomal protein gene, rp21, from a phage genomic library constructed by Carlos Winter. C. elegans rp21 cDNA was cloned by Andy Fire, who showed that it begins with SL2. The complete sequence of a 2.4 kb CEW1 genomic clone containing rp21 revealed the presence of another gene, for ribosomal protein L27, upstream of rp21. The two genes are present in the same 5' to 3' orientation with only 87 bp of intercistronic sequence, an arrangement quite similar to most C. elegans operons. We have not yet determined whether these same two genes are present in the same operon in C. elegans because so far we have failed to obtain C. elegans rp21 genomic clones. To determine whether the rp21 gene from CEW1 receives SL2, as the C. elegans homolog does, we performed primer extension sequencing using CEW1 RNA and a primer to the rp21 coding region. The sequence demonstrated that the rp21 mRNA was trans-spliced to an SL quite similar to, but not identical to, C. elegans SL2. (We also determined by RT-PCR that L27 mRNA is trans-spliced to SL1.) We are currently cloning the SL2 RNA genes from this species. The sequence of SL2 RNAs from other nematodes should also facilitate comparative phylogenetic analysis, perhaps leading to the identification of sequences necessary for SL2 trans-splicing. These results demonstrate that operons and SL2 trans-splicing are more ancient than the Caenorhabditis genus. It remains to be seen, however, whether this mode of transcription and processing is present throughout the nematode phylum.