RESUMEN
Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.
Asunto(s)
Apolipoproteínas B/genética , Transcripción Genética , Animales , Composición de Base , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/síntesis química , ADN Polimerasa Dirigida por ADN , Amplificación de Genes , Humanos , Intestinos/análisis , Hígado/análisis , Masculino , Datos de Secuencia Molecular , ARN/aislamiento & purificación , Conejos , Ratas , Ratas Endogámicas , Polimerasa TaqRESUMEN
Two B apolipoproteins (apo) are present in human plasma, designated apoB-100 and apoB-48, and represent translational products from mature apoB mRNAs that differ by a single base. Either the glutamine codon encoded by the single-copy apoB gene at nucleotide 6666 is transcribed and translated to produce apoB-100 or an RNA-editing mechanism substitutes a uracil for cytosine, altering this glutamine codon (CAA) to a stop codon (UAA), prematurely terminating translation to produce apoB-48. In the present report, editing of rat apoB transcripts was evaluated by amplification of RNA with the polymerase chain reaction by use of primers based on the apoB cDNA cloned from a rat liver cDNA library. The combined results of this study show that (i) a single copy of the apoB gene exists in the rat; (ii) the rat apoB gene encodes only the glutamine codon for the synthesis of apoB of higher molecular weight (apoBH); (iii) rat apoB transcripts undergo RNA editing; (iv) apoBH and apoB of lower molecular weight (apoBL) in the rat represent structural equivalents of apoB-100 and apoB-48 in humans, respectively; (v) RNA editing occurs in both the liver and intestine of the rat; (vi) rat hepatic apoB RNA is more extensively edited than is human hepatic apoB RNA, which is consistent with the marked increase in apoBL secretion by the rat liver when compared with human; and (vii) the definitive identification of apoBH mRNA as well as apoBL mRNA in the rat intestine provides a mechanism for the biosynthesis of both apoBH and apoBL by the rat intestine.