Worm Breeder's Gazette 11(5): 38
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.
The C. elegans alpha2(IV) basement membrane collagen gene clb-1 has been shown to be let-2 X. Because let-2 had similar phenotypes to emb- 9, which was previously shown to be the alpha1(IV) gene clb-2, we predicted that it would be clb-1. We sent Craig Mello the clb-1 phage clone and he promptly rescued let-2 with it. The clb-1 gene is approximately 9 kb in length and contains 18 introns. An usual situation was noted in the gene structure. Putative exons 8 and 9 were separated by a supposed intron that was only 30 bp long (figure below). This length is normally too short for a functional intron. Exons 8 and 9 appeared to be duplicates, having the general structure: (Gly-X-Y)(5)-9/10 aa interruption-(Gly-X-Y)(4). The interruption is 9 aa in exon 8, and 10 aa in exon 9. The amino acid sequences of the two exons also have some additional similarity. It seemed possible that these exons would be spliced in a non-standard fashion. Using primers in exons 7 and 10, we performed RT and PCR on mixed population RNA. The PCR product was only large enough to contain either exon 8 or exon 9. Using restriction enzymes that cut specifically in only one or the other exons we found that both exons are present in the PCR product. There was approximately a 1:2 ratio of exon 8:exon 9 in the mixed population RNA PCR product. The two alternately spliced products were cloned and sequenced to confirm the predicted structure. So, the alpha2(IV) collagen mRNA is present in at least two forms, containing either exon 8 or exon 9. The lengths of the triple-helical domains of the alpha1(IV) (1529 aa) and alpha2( IV) (1527 or 1528 aa) chains match closely when only one of the exons is present in clb-1. We are currently attempting in situ hybridization with oligo probes to determine if the two mRNAs are localized to particular tissues. The 5' splice donor sequences of both exon 8 and 9 are unusual. The exon 8 donor differs from consensus at two positions (underlined In table below). Although many donors differ from consensus at one position, only rarely are two non-consensus nucleotides found in a functional donor. The exon 9 donor has a C in place of the normally invariant T at the second position. A few other examples of donors with a C at this position have been identified (see Cell 47:555). In in vitro splicing assays these donors function with greatly reduced efficiency. It is possible that these unusual donors are important for controlling alternative splicing in clb-1.[See Figure 1]