Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.
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PMID:Characterization of a sheep elastin cDNA clone containing translated sequences. 632 Aug 24

mRNA was isolated from the thoracic aortas of 16-day chick embryos and used to synthesize blunt-ended heteroduplex molecules consisting of one strand of mRNA and one of cDNA using AMV reverse transcriptase and S1 nuclease. The duplexes were tailed with dCTP and hybridized to the plasmid pBR322 which had been restricted with Pst I and tailed with dGTP. Recombinant plasmids were used to transform E. coli C600 and colonies containing elastin cDNA were selected by in situ hybridization with 32P labeled elastin mRNA and by hybrid selected translation using the nuclease-treated reticulocyte lysate system. mRNA recovered from hybridization to DNA of one clone, pWB1, markedly stimulated incorporation of [3H]valine into a protein which was immunoprecipitable with elastin-specific antibody and had a molecular weight of 72,000, characteristic of tropoelastin. The 230 bp insert of pWB1 was sequenced by the technique of Maxam and Gilbert and found to be derived from a nontranslated region of the 3' end of the mRNA. Nick-translated pWB1 was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single mRNA species of 3.5 kb hybridized to the pWB1 probe and this species increased greatly in amount between day 7 and day 14. This increase was paralleled by an increase in translatable elastin mRNA and by the rate of elastin synthesis of aortas from various age embryos incubated in vivo. The injection of 150 microgram of hydrocortisone 21-phosphate into 8-day eggs produced a significant increase in both the relative rate of tropoelastin synthesized by the isolated aortas and the relative amount of elastin mRNA. These results suggest that the observed changes in elastin synthesis during development and after hydrocortisone administration are governed by the elastin mRNA content of the aortas.
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PMID:Control of elastin synthesis. 708 85