RESUMO
Genome-wide mapping of transcription factor binding is generally performed by chemical protein-DNA crosslinking, followed by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Here we present the ChIP-seq technique based on photochemical crosslinking of protein-DNA interactions by high-intensity ultraviolet (UV) laser irradiation in living mammalian cells (UV-ChIP-seq). UV laser irradiation induces an efficient and instant formation of covalent "zero-length" crosslinks exclusively between nucleic acids and proteins that are in immediate contact, thus resulting in a "snapshot" of direct protein-DNA interactions in their natural environment. Here we show that UV-ChIP-seq, applied for genome-wide profiling of the sequence-specific transcriptional repressor B-cell lymphoma 6 (BCL6) in human diffuse large B-cell lymphoma (DLBCL) cells, produces sensitive and precise protein-DNA binding profiles, highly enriched with canonical BCL6 DNA sequence motifs. Using this technique, we also found numerous previously undetectable direct BCL6 binding sites, particularly in condensed, inaccessible areas of chromatin.
Assuntos
Imunoprecipitação da Cromatina/métodos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Processos Fotoquímicos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Análise de Sequência de DNA , Raios UltravioletaRESUMO
Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lacticaseibacillus rhamnosus/genética , Lacticaseibacillus rhamnosus/isolamento & purificação , Feminino , Genes Bacterianos , Genoma Bacteriano , Humanos , Lactente , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Óperon , Regiões Promotoras Genéticas , Estabilidade de RNA , Saliva/microbiologia , Estresse Fisiológico , Vagina/microbiologiaRESUMO
This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 degrees C denaturation and 60 degrees C annealing/extension) PCRs were performed in 5-10 microl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25-30 min. Rapid high-resolution agarose gel electrophoresis of 70-150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50-100 pg) was obtained using ultra sensitive dyes such as GelStar and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan real-time PCR counterparts.
Assuntos
Baculoviridae/isolamento & purificação , Densovirinae/isolamento & purificação , Penaeidae/virologia , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Baculoviridae/genética , Densovirinae/genética , Eletroforese em Gel de Ágar , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Temperatura , Fatores de Tempo , Vírus da Síndrome da Mancha Branca 1/genéticaRESUMO
We describe the development and operation of a two-laser, large-field hyperspectral scanner for analysis of multicolor genotyping microarrays. In contrast to confocal microarray scanners, in which wavelength selectivity is obtained by positioning band-pass filters in front of a photomultiplier detector, hyperspectral microarray scanners collect the complete visible emission spectrum from the labeled microarrays. Hyperspectral scanning permits discrimination of multiple spectrally overlapping fluorescent labels with minimal use of optical filters, thus offering important advantages over standard filter-based multicolor microarray scanners. The scanner uses two-sided oblique line illumination of microarrays. Two lasers are used for the excitation of dyes in the visible and near-infrared spectral regions. The hyperspectral scanner was evaluated with commercially available two-color calibration slides and with in-house-printed four-color microarrays containing dyes with spectral properties similar to their commercial genotyping array counterparts.
