RESUMEN
Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminoácido Oxidorreductasas/genética , ADN/genética , Proteína-Lisina 6-Oxidasa/genética , Secuencia de Aminoácidos , Animales , Aorta/enzimología , Secuencia de Bases , Bovinos , Clonación Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/ultraestructura , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.