Worm Breeder's Gazette 5(1): 34
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.
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.