RESUMEN
P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,ß-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.
Asunto(s)
Intrones/efectos de los fármacos , Intrones/genética , Plexo Mientérico/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores Purinérgicos P2X2/efectos de los fármacos , Receptores Purinérgicos P2X2/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/genética , Fenómenos Electrofisiológicos , Exones/genética , Exones/fisiología , Femenino , Cobayas , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Plexo Mientérico/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Xenopus laevisRESUMEN
Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.
Asunto(s)
Biblioteca de Genes , Opuntia/metabolismo , Proteínas de Plantas/metabolismo , Frío , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Opuntia/genética , Opuntia/fisiología , Proteínas de Plantas/genéticaRESUMEN
Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.