RESUMEN
Smoking induces a wide range of drug-metabolizing enzymes. Among them, CYP2B6 as well as CYP1A2 is well known to be up-regulated in smokers. Although the induction of CYP1A2 is mediated by the aryl hydrocarbon receptor, the molecular mechanisms of CYP2B6 induction by smoking remain to be fully elucidated. In this study, by preparing cigarette smoke extract (CSE), we addressed the possibility that human constitutive androstane receptor (hCAR) is involved in smoking-mediated induction of CYP2B6. In HepG2 cells, CSE induced CYP1A2 but not CYP2B6, suggesting that CYP2B6 expression is differentially regulated from CYP1A2. Compared with liver in vivo, hCAR expression is dramatically reduced in cultured hepatocytes, such as HepG2. Therefore, to reconstitute hCAR signaling pathways in vitro, we generated adenovirus vector expressing hCAR. Real-time reverse transcription-polymerase chain reaction analyses revealed that the adenoviral transfection of hCAR resulted in the up-regulation of CYP2B6 mRNA, even in the absence of CSE. It is interesting to note that CSE stimulation augmented hCAR-mediated induction of CYP2B6. In contrast, the expression of CYP2B6 was not enhanced by adenovirus vector expressing ß-galactosidase, a control vector, either in the presence or absence of CSE. In summary, hCAR mediated the CYP2B6 induction by CSE in Hep2G cells. These data suggest that smoking up-regulates CYP2B6 through hCAR in vivo.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP1A2/efectos de los fármacos , Nicotiana , Oxidorreductasas N-Desmetilantes/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Humo , Fumar/metabolismo , Células Cultivadas , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Células HEK293 , Células Hep G2 , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , ARN Mensajero/biosíntesis , Receptores de Hidrocarburo de Aril/metabolismo , Regulación hacia ArribaRESUMEN
RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.
Asunto(s)
Briófitas/genética , Complejo de Citocromo b6f/genética , ARN Polimerasas Dirigidas por ADN/genética , Edición de ARN , Secuencia de Bases , Briófitas/enzimología , ADN de Plantas/química , ADN de Plantas/genética , Datos de Secuencia Molecular , Operón/genética , Plastidios/genética , ARN Mensajero/genética , ARN de Planta/química , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Three distinct arginine tRNA genes, trnR-CCG, trnR-ACG, and trnR-UCU, are present in the plastid genome of bryophytes, whereas only the latter two trnR genes are present in the major vascular plants, except for black pine. trnR-CCG is located between rbcL and accD in the moss Physcomitrella patens and it was previously believed to be functional in plastids. However, no trnR-CCG transcript has been detected by Northern hybridization, and the codon usage of CGG is quite low in plastid protein-coding sequences. This raises the possibility that trnR-CCG is non-functional. To investigate this possibility, we integrated a foreign gene into the trnR-CCG coding region via homologous recombination, and constructed stable plastid trnR-CCG knock-out moss transformants. The trnR-CCG knock-out transformants grew normally, indicating that the P. patenstrnR-CCG gene is not essential for plastid function.
Asunto(s)
Bryopsida/genética , Plastidios/metabolismo , ARN de Planta/genética , ARN de Transferencia de Arginina/genética , Bryopsida/metabolismo , Regulación de la Expresión Génica de las Plantas , Genotipo , Conformación de Ácido Nucleico , Fenotipo , Plantas Modificadas Genéticamente/metabolismo , ARN de Planta/metabolismo , ARN de Transferencia de Arginina/metabolismoRESUMEN
Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.
Asunto(s)
Briófitas/genética , Cloroplastos/genética , ADN de Cloroplastos , Evolución Molecular , Datos de Secuencia Molecular , FilogeniaRESUMEN
The complete chloroplast DNA sequence (122 890 bp) of the moss Physcomitrella patens has been determined. The genome contains 83 protein, 31 tRNA and four rRNA genes, and a pseudogene. Four protein genes (rpoA, cysA, cysT and ccsA) found in the liverwort Marchantia polymorpha and the hornwort Anthoceros formosae are absent from P.patens. The overall structure of P.patens chloroplast DNA (cpDNA) differs substantially from that of liverwort and hornwort. Compared with its close relatives, a 71 kb region from petD to rpoB of P.patens is inverted. To investigate whether this large inversion and the loss of rpoA usually occur in moss plants, we analyzed amplified cpDNA fragments from four moss species. Our data indicate that the large inversion occurs only in P.patens, whereas the loss of the rpoA gene occurs in all mosses. Moreover, we have isolated and characterized the nuclear rpoA gene encoding the alpha subunit of RNA polymerase (RNAP) from P.patens and examined its subcellular localization. When fused to green fluorescent protein, RpoA was observed in the chloroplasts of live moss protonemata cells. This indicates that chloroplast RNAP is encoded separately by chloroplast and nuclear genomes in the moss. These data provide new insights into the regulation and evolution of chloroplast transcription.
Asunto(s)
Bryopsida/genética , ADN de Cloroplastos/genética , Secuencia de Aminoácidos , Núcleo Celular/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Mapeo Cromosómico , ADN de Cloroplastos/química , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The rps14 transcript is edited in the moss Physcomitrella patens chloroplast by a C-to-U transition, to create a translation initiation codon, AUG. The efficiency of RNA editing was low, with approximately 20% of rps14 transcripts edited. This suggests that the translation of rps14 mRNA is strictly regulated by RNA editing. This is the first report of RNA editing in P. patens and the creation of a translation initiation codon in rps14 mRNA in chloroplasts.