Worm Breeder's Gazette 11(4): 39
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.
Of the six blister genes, we have been particularly interested in bli-6 IV because Bob Herman has shown that alleles of sqt-1, dpy-10 and rol-8 can suppress the dominant mutation bli-6(mn4). Although Dpy is often epistatic to Bli, Bob identified non- or very weak Dpy alleles of dpy-2 and dpy-10 that suppress mn4. Since sqt-1, dpy-10 and rol-8 show genetic interactions and all except rol-8 have been shown to encode collagens (Adam Levy has identified a collagen that is likely to be rol-8) there is a good possibility that they may physically interact with the bli-6 gene product. A Bristol/Bergerac polymorphism, identified by hybridizing genomic Southern blots with col-2 at low stringency, had been previously mapped to the vicinity of bli-6, and was named col-4 (Cox et al. Genetics 109:513,1985). Initial three-factor mapping with unc-5 and dpy-20 failed to separate bli-6 and col-4(Berg), so we decided to examine col-4 more closely. Further mapping using unc-44 and unc-24 showed that col-4 is approx. 0.4 mu to the right of bli-6, but concurrent molecular analyses of col-4 produced some unexpected results. The col-4 gene was cloned by identifying a col-2 crosshybridizing clone, in a size selected genomic sublibrary, that showed the expected Br/Bg polymorphism when probed to genomic Southern blots. The first surprise was that col-4 doesn't encode a collagen. The first case, that we know of, where low stringency collagen cross-hybridization has pulled out something other than a collagen. The col-4 sequence predicts a polypeptide consisting primarily of 23 tandem copies of the following 15 amino acid repeat: GAPPSGGPPGPF(D/N)PS (note: NPS is a potential glycosylation site) The only variation in the repeats is that about one-half have D and one-half have N. The repeat could have arisen from a collagen triplet (Gly-X-Y)s by loss of the second and fifth Gly. Note also the high proline content (40%), also indicative of collagen. It seemed likely that col-4 could encode a novel, collagen-related structural protein. High stringency Southern blots showed that the col-4 gene is present in a single copy in the Bristol and Bergerac genomes. The next surprise was that we could not detect any col-4 transcripts. This result was particularly surprising because the codon usage in the repeats (all 6 Pro are CCA, 3/4 Gly are GGA) is that expected for coding sequence. Northern blots and primer extension with several fragments flanking the repeats failed to identify transcripts in N2 RNAs from various stages, including dauer. RNA from males was not examined. No cDNA clones were identified in Bob Barstead's library. The Br/Bg polymorphism is in the region containing the repeats ( Bergerac is approx. 300 bp larger), so we examined Bg RNA but could not identify transcripts. Finally, high-stringency hybridization of the repeat region showed that it is not present in the C. briggsae genome. col-4 has a recognizable 3' structure; 60 amino acids following the repeats is a termination codon followed by AT rich sequence and even a potential polyadenylation signal. On the 5' side of the repeats, however, it is not obvious where an initiator ATG or intron might be. col-4 might not be expressed at all, or only at very low levels, or at some specific time that we did not examine. Possibly it is the remains of a once expressed gene. If col-4 was expressed at some time in the past, as the codon usage suggests, what might it have been? One guess is a cuticle surface protein, like srf-1 which is not present on the surface of certain wild strains of C. elegans (Politz et al. Genetics 117 467, 1987). Surface protein genes might show a high degree of interstrain variability. Indeed, the col-4 repeat has some similarity to repeats in the 22 kD secreted surface protein of Brugia, 5/6 Pro's and 2/4 Gly's align (Mark Blaxter, personal communication). We welcome comments and suggestions.