RESUMEN
Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXvarphi prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S-23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.
Asunto(s)
Vibriosis/microbiología , Vibrio cholerae no O1/aislamiento & purificación , Secuencia de Bases , Toxina del Cólera/genética , ADN Intergénico , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Alineación de Secuencia , Vibrio cholerae no O1/genética , Vibrio cholerae no O1/patogenicidadRESUMEN
The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is 'masked' shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca(2+)-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.
Asunto(s)
Bivalvos/embriología , Bivalvos/metabolismo , Poli A/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bivalvos/genética , Western Blotting , Extractos Celulares , Clonación Molecular , Embrión no Mamífero/metabolismo , Femenino , Fertilización , Regulación del Desarrollo de la Expresión Génica , Guanidina/farmacología , Concentración de Iones de Hidrógeno , Larva/metabolismo , Nucleasa Microcócica/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Oocitos/citología , Oocitos/metabolismo , Óvulo/citología , Óvulo/metabolismo , Poli A/genética , Proteínas de Unión a Poli(A) , Unión Proteica , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Sondas ARN/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Alineación de Secuencia , Rayos UltravioletaRESUMEN
The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.