RESUMO
Efficiency and specificity of PCR amplification is dependent on several parameters, such as amplicon length, as well as hybridization specificity and melting temperature of primer oligonucleotides. Primer design is thus of critical importance for the success of PCR experiments, but can be a time-consuming and repetitive task, for example when large genomic regions are to be scanned for the presence of a protein of interest by chromatin immunoprecipitation experiments. We present here a webserver that allows the automated design of tiled primer pairs for any number of genomic loci. PCRTiler splits the target DNA sequences into smaller regions, and identifies candidate primers for each sub-region by running the well-known program Primer3 followed by the elimination of primers with a high cross-hybridization potential via BLAST. Tiling density and primer characteristics are specified by the user via a simple and user-friendly interface. The webserver can be accessed at http://pcrtiler.alaingervais.org:8080/PCRTiler. Additionally, users may download a standalone Java-based implementation of this software. Experimental validation of PCRTiler has demonstrated that it produces correct results. We have tiled a region of the human genome, in which 96 of 123 primer pairs worked in the first attempt, and 105 of 123 (85%) could be made to work by optimizing the conditions of the PCR assay.
Assuntos
Primers do DNA/química , Reação em Cadeia da Polimerase , Software , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Humanos , InternetRESUMO
BACKGROUND: Nucleosomes are nucleoproteic complexes, formed of eight histone molecules and DNA, and they are responsible for the compaction of the eukaryotic genome. Their presence on DNA influences many cellular processes, such as transcription, DNA replication, and DNA repair. The evolutionarily conserved histone variant H2A.Z alters nucleosome stability and is highly enriched at gene promoters. Its localization to specific genomic loci in human cells is presumed to depend either on the underlying DNA sequence or on a certain epigenetic modification pattern. RESULTS: We analyzed the differences in histone post-translational modifications and DNA sequences near nucleosomes that do or do not contain H2A.Z. We show that both the epigenetic context and underlying sequences can be used to classify nucleosomal regions, with highly significant accuracy, as likely to either contain H2A.Z or canonical histone H2A. Furthermore, our models accurately recapitulate the observed nucleosome occupancy near the transcriptional start sites of human promoters. CONCLUSION: We conclude that both genetic and epigenetic features are likely to participate in targeting H2A.Z to distinct chromatin loci.
Assuntos
Histonas/análise , Nucleossomos/química , Sequência de Bases , Cromatina , Epigênese Genética , Histonas/classificação , Humanos , Processamento de Proteína Pós-Traducional , Sítio de Iniciação de TranscriçãoRESUMO
Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Staphylococcus aureus/genética , Transcrição Gênica , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cultura em Câmaras de Difusão , Feminino , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/metabolismo , Regulação para CimaRESUMO
The incorporation of variant histone H2A.Z within chromatin is important for proper gene expression and genome stability. H2A.Z is inserted at discrete loci by the Swr1 or Swr1-like remodeling complexes, although very little is known about the nature of the targeting mechanism involved. Replacement of canonical histone H2A for H2A.Z has been shown to modify nucleosome dynamics, although discrepancies still exist in the literature regarding the mechanisms. Recent experiments have shown that H2A.Z can allow nucleosomes to adopt stable translational positions as compared to H2A, which could influence the accessibility to DNA regulatory proteins. This review provides a brief overview of H2A.Z biology and presents hypotheses that could reconcile contradictory reports that are found in the literature regarding the influence of H2A.Z on nucleosome stability.
Assuntos
Histonas/metabolismo , Transcrição Gênica , Animais , Metilação de DNA/genética , Humanos , Nucleossomos/metabolismoRESUMO
Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial sigma factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for sigma(F), sigma(C), and sigma(K), and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of sigma factor genes. Our results revealed that many sigma factors are expressed from autoregulated promoters.
Assuntos
Proteínas de Bactérias/metabolismo , Imunoprecipitação da Cromatina/métodos , Regulação Bacteriana da Expressão Gênica , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Fator sigma/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Fator sigma/genéticaRESUMO
UNLABELLED: MtbRegList is a database dedicated to the analysis of gene expression and regulation data in Mycobacterium tuberculosis. It is designed to contain predicted and characterized regulatory DNA motifs cross-referenced with corresponding transcription factor(s), and experimentally identified transcription start sites. MtbRegList can also handle flexible and complex genomic search requests, besides having a noteworthy browsing capability. AVAILABILITY: MtbRegList is freely available at http://www.USherbrooke.ca/vers/MtbRegList