Molecular cloning and partial characterization of delta-crystallin cDNA sequences in a bacterial plasmid.
Double-stranded cDNA synthesized from delta-crystallin mRNA isolated from lens fiber cells of 15-day-old embryonic chicken was cloned in Escherichia coli chi 1776 in the Pst I site of the plasmid pBR322 by using the oligo(dC) . oligo(dG) joining procedure. Twelve Amps Tetr transformants contained sequences complementary to purified delta-crystallin [32P]cDNA. One of the recombinant clones (p delta Cr-2) had an insert of 1241 +/- 240 base pairs, as judged by R-looping analysis with purified delta-crystallin mRNA. The inserted cDNA represents at least 69% of the delta-crystallin coding sequences. p delta Cr-2 was further characterized by restriction analysis, protection of delta-crystallin [3H]cDNA from digestion by S1 nuclease, and hybrid-mediated arrest of delta-crystallin mRNA translation in vitro. p delta Cr-2 provides an invaluable probe for additional analysis of the primary structure, gene organization, and regulated synthesis of delta-crystallin, the principal protein synthesized during lens differentiation in the chicken embryo.
D000818:Animals; D002642:Chick Embryo; D002874:Chromosome Mapping; D003459:Crystallins / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics
D003459:Crystallins; D004274:DNA, Recombinant; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes