RESUMEN
Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.
Asunto(s)
Bioensayo , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina Tipo 2/genética , Células Epiteliales/virología , ARN Viral/genética , Animales , Diarrea Mucosa Bovina Viral/diagnóstico , Diarrea Mucosa Bovina Viral/virología , Bovinos , Línea Celular , Coinfección , Virus de la Diarrea Viral Bovina Tipo 1/clasificación , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Virus de la Diarrea Viral Bovina Tipo 1/metabolismo , Virus de la Diarrea Viral Bovina Tipo 2/clasificación , Virus de la Diarrea Viral Bovina Tipo 2/aislamiento & purificación , Virus de la Diarrea Viral Bovina Tipo 2/metabolismo , Perros , Células Epiteliales/patología , Femenino , Células de Riñón Canino Madin Darby , Embarazo , ARN/genética , ARN/metabolismo , Sondas ARN/genética , Sondas ARN/metabolismo , ARN Viral/metabolismo , Tropismo Viral , Replicación ViralRESUMEN
Pre-mRNA splicing is an essential step in the post-transcriptional gene expression control involving protein-splicing factors like U2AF, which is exported to the cytoplasm and implicated in additional cellular functions. Identification of U2AF-associated mRNAs under native conditions was performed by immunoprecipitation and hybridization to Affymetrix GeneChip. Normalization and gene selection methods were performed, but the results were not reliable as they were different for different procedures, mainly because more than 20% of the mRNAs detected are differently enriched and the common normalization methods are based on small differences between them. We implemented a background correction method inspired in a non-specific hybridization method used for pre-processing data from ChIP-Chip technology. In this work, linear regression models are used to model in each array the non-specific hybridization, accounting for interactions between each three consecutive nucleotides into the probe sequence. Every probe intensity on the array was standardized using its predicted intensity and the probes' variance for similar predicted intensities. The standardized probe intensity values showed no need for further normalization and could be directly compared. We propose a probe set score, and a probe set enrichment value (ENRval) and its respective p-value for gene enrichment selection.
Asunto(s)
Biología Computacional/métodos , Modelos Lineales , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos/genética , Sondas ARN , ARN/genética , Algoritmos , Regulación de la Expresión Génica , Inmunoprecipitación/métodos , Sondas de Oligonucleótidos/metabolismo , ARN/metabolismo , Sondas ARN/genética , Sondas ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , G-Cuádruplex , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 5'/química , Regiones no Traducidas 5'/metabolismo , Animales , Secuencia de Bases , Bufo arenarum , Sondas de ADN/química , Sondas de ADN/metabolismo , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Embrión no Mamífero/química , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Guanosina/química , Guanosina/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Biosíntesis de Proteínas , Sondas ARN/química , Sondas ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevis/metabolismo , Dedos de ZincRESUMEN
Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.
Asunto(s)
Proteínas de Unión al ARN/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Inmunohistoquímica , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Ovario/embriología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sondas ARN/genética , Sondas ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/metabolismoRESUMEN
The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.