Way JC (1990) Worm Breeder's Gazette "Sequence of mec-3 from the Caenorhabditis strain WS9- 6"

Way JC

[Worm Breeder's Gazette, 1990]

From Chris Link's lambda library of DNAs from WS9-6, a male-female Caenorhabditis strain, I isolated an apparent mec-3 homologue. A 4kb EcoRI fragment contains exons corresponding to all the exons of mec-3 in C. elegans, as well as four long conserved regions upstream of the start codons. The regions of sequence similarity between the C. elegans and WS9-6 mec-3 genes are very much like the regions of similarity between C. elegans and C. briggsae described by Ding Xue and Marty Chalfie in the last newsletter (including their revision of the coding sequence), except that the overall level of similarity between the C. elegans and WS9-6 genes seems higher. There are four long upstream regions of conservation of 71,30,30 and 24 bp, and a few shorter ones. In the entire coding sequence, there are 29 amino acid substitutions, 19 of which are between the LIM and homeodomains. There are no amino acid changes in the homeodomain. In introns, there is no apparent similarity between the sequences. Also, the WS9-6 introns are generally shorter: the first intron, 395 bp in C. elegans, is only 73 bp in WS9-6. The fact that the intron sequences have completely drifted suggests that the conserved upstream regions have been preserved by selection. The four conserved upstream regions (hereafter termed regions I, II, III and IV) have distinctive features that may relate to how mec-3 is regulated. Regions I and II each have sequences very similar to the POU consensus binding site, and region III contains a close relative to the ISL-1 binding site (ISL-1 is a transcription factor for the insulin gene and is closely related to mec-3.) Region IV contains no obvious sequence features, but can be deleted to cause expression of a mec-3-lacZ fusion in extra cells (Way, WBG 11,2). A provisional organization scheme for this region would thus be that regions I and II mediate unc-86 binding and establishment expression, region III mediates mec-3 binding and maintenance expression, and region IV mediates negative regulation by unidentified proteins. With unc-86 protein from Mike Finney, I have shown that a restriction fragment containing regions I, II and III can be 'band- shifted'. I have not yet done a footprint to identify the actual unc- 86 binding sites. (Since C. elegans and C. briggsae are both hermaphrodite species while WS9-6 is male/female, it would appear that the male/female sex pattern is derived from the male/hermaphrodite pattern. It would be interesting to see which of the sex-determination genes of C. elegans have been altered in WS9-6 to account for this change.)

Files JG et al. (1980) Worm Breeder's Gazette "actin and procollagen genes of C elegans."

Files JG, Hirsh DI, Carson B, Johnson KA, Landel CP, Rosenzweig WD

[Worm Breeder's Gazette, 1980]

We are studying the C. elegans genes coding for actin and procollagen using as probes cDNA clones of Dictyosteleum actin (Firtel) and chicken procollagen (Doty). We have hybridized P-32 labeled Dictyosteleum actin DNA to Southern blots of various restriction digests of whole genome C. elegans DNA. The Dictyosteleum actin DNA hybridized strongly to a small number of bands, four in most restriction digests. From a collection of recombinant charon 10 phage carrying C. elegans DNA, we have isolated several clones complementary to the Dictyosteleum actin DNA. Two of these clones have been characterized. One clone, Act-1, contains a 17 kb insert of worm DNA and has two actin coding regions. As seen by hybridization of Dictyosteleum actin DNA to restriction fragments of the clone. This clone contains a 1000 base inverted repeat sequence, with the two copies of the repeat separated by about 2500 bases. This is visualized in the electron microscope as a snapback of denatured clone Act-1 DNA. Since the repeated sequences are in the same positions as the actin coding regions, they most likely represent two actin genes in a head-to-head ( or tail-to-tail) orientation. R-loop experiments are in progress to verify this conclusion by determining the exact positions of the genes, as well as to check for possible intervening sequences. A second clone, Act-2, with an insert of 14 kb, contains a single actin gene. Thus, three separate actin genes have been identified so far. One additional actin sequence, seen on Southern blots, is yet to be identified on a clone. We have purified actin mRNA from C. ps with clone Act-1 DNA and by DNA-cellulose affinity chromatography using cloned C. A. We have hybridized P-32 labeled cloned chicken procollagen DNA to Southern blots of various digests of C. ding a large number of bands hybridizing to the probe, some strongly and others with decreasing intensity. (Fifteen bands are seen with Eco RI digested C. e cut the cloned procollagen DNA with restriction enzymes into 3 pieces, isolated them, and hybridized each separately to Southern blots of digested C. o of these fragments code for the helical region of collagen. They hybridized to the same bands as the intact procollagen clone. The other fragment, which codes for the telopeptide, did not hybridize to any bands but did hybridize visibly to the procollagen clone itself, present as a control in an amount corresponding to a single copy sequence in the worm DNA. About 50 C. es containing procollagen hybridizing sequences have been isolated after screening recombinant phage with the chicken procollagen probe. Screening with the chicken procollagen probe gave about 25 times the frequency of hybridizing plaques as with the Dictyosteleum actin probe. Hybridization to the procollagen probe showed many plaques with intermediate and weak hybridization, unlike hybridization to the actin probe. We do not know the reason why bands and plaques of weak hybridization are seen using the procollagen probe. There may be a region on that clone that is homologous to a repetitive sequence in C. elegans DNA.