RESUMEN
Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xenovorans, which are common to the genus Burkholderia. We also describe the development and application of a more streamlined version of the PBM technology that significantly reduced the experimental time. Despite the genus containing many pathogenically important species, only a handful of TF operator sites have been experimentally characterized for Burkholderia to date. Our study provides a significant addition to this knowledge base and illustrates some general challenges of discovering operators on a large scale for prokaryotes.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia/genética , Regiones Operadoras Genéticas , Factores de Transcripción/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Burkholderia/química , Burkholderia/clasificación , Burkholderia/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.
Asunto(s)
Clostridium botulinum tipo E/clasificación , Clostridium botulinum tipo E/genética , ADN Bacteriano/genética , Tipificación Molecular/métodos , Polimorfismo Genético , Toxinas Botulínicas/genética , Botulismo/microbiología , Clostridium botulinum tipo E/aislamiento & purificación , Análisis por Conglomerados , Elementos Transponibles de ADN , Microbiología Ambiental , Microbiología de Alimentos , Genotipo , Humanos , Datos de Secuencia Molecular , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. METHODS: Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). RESULTS: For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. CONCLUSIONS: Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Variación Genética , Antígenos de Histocompatibilidad Clase I/genética , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/genética , Polimorfismo de Nucleótido Simple , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Estudios de Casos y Controles , Cisteína Endopeptidasas/genética , Femenino , Humanos , Proteínas de Transporte de Membrana/genética , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Valores de ReferenciaRESUMEN
BACKGROUND: Polymorphisms in the TNF-alpha promoter region have recently been shown to be associated with susceptibility to cervical cancer. Some polymorphisms have been reported to influence transcription for this cytokine. Altered local levels in the cervix may influence an individual's immune response, thereby affecting persistence of human papillomavirus (HPV) 16 infection, a primary etiological factor for cervical cancer. METHODS AND RESULTS: The association of 11 TNF-alpha single-nucleotide polymorphisms (SNPs) with susceptibility to HPV16-associated cervical cancer was investigated. Sequencing of the TNF-alpha promoter region confirmed 10 SNPs, and 1 previously unreported SNP (161 bp upstream of the transcriptional start site) was discovered. Microsphere-array flow cytometry-based genotyping was performed on 787 samples from Hispanic and non-Hispanic white women (241 from randomly selected control subjects, 205 from HPV16-positive control subjects, and 341 from HPV16-positive subjects with cervical cancer). The genotype distribution of 3 SNPs (-572, -857, and -863) was significantly different between case subjects and control subjects. Analysis of haplotypes, which were computationally inferred from genotype data, also revealed statistically significant differences in haplotype distribution between case subjects and control subjects. CONCLUSIONS: We report new associations between several TNF-alpha SNPs and susceptibility to cervical cancer that support the involvement of the TNF- alpha promoter region in development of cervical cancer.