RESUMEN
CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Frecuencia de los Genes , Oxigenasas de Función Mixta/genética , Polimorfismo Genético/genética , Alelos , Población Negra , Clonación Molecular , Citocromo P-450 CYP2A6 , ADN/genética , ADN/aislamiento & purificación , Exones/genética , Femenino , Genotipo , Humanos , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Población BlancaRESUMEN
Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-beta genes, we have analysed the potential of Xenopus c-Jun (AP-1) as a mediator of BMP-4 expression during Xenopus development. RNA injection experiments revealed that both heteromeric c-Fos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling was found to activate the Xenopus c-Jun gene only at a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP-1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, we find a spatial overlap of c-Jun and BMP-4 transcripts which suggests that AP-1 might serve as an additional activatory component for the auto-regulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5' flanking region and two in the second intron of the BMP-4 gene. We further demonstrate that AP-1 acts independently of Xvent-2 which has recently been shown to mediate the early expression of BMP-4 in gastrula stage embryos.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Genes jun , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Xenopus/embriología , Xenopus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteína Morfogenética Ósea 4 , Cartilla de ADN/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Activación Transcripcional , Xenopus/metabolismo , Proteínas de XenopusRESUMEN
Like other genes of the transforming growth factor-beta family, the BMP-4 gene is regulated by an autocatalytic loop. In Xenopus embryos this loop can be ectopically induced by injection of BMP-2 RNA. However, cycloheximide treatment subsequent to BMP-2 overexpression revealed that BMP signaling is not direct but requires additional factor(s). As putative mediator we have identified Xvent-2 which is activated by BMP-2/4 signaling and, in turn, activates BMP-4 transcription. Using promoter/reporter assays we have delineated Xvent-2 responsive elements within the BMP-4 gene. We further demonstrate that Xvent-2 which has recently been characterized as a transcriptional repressor can also act, context dependent, as an activator binding two copies of a 5'-CTAATT-3' motif in the second intron of the BMP-4 gene. Replacement of Xvent-2 target sites within the goosecoid (gsc) promoter by the BMP-4 enhancer converts Xvent-2 caused repression of gsc to strong activation. This switch is obviously due to adjacent nucleotides probably binding a transcriptional co-activator interacting with Xvent-2. A model is presented describing the mechanism of BMP-4 gene activation in Xenopus embryos at the early gastrula stage.
