RESUMO
OBJECTIVE: To develop a microarray platform that allows simultaneous assessment of aneuploidy and quantification of mitochondrial DNA (mtDNA) in human polar bodies and embryos. DESIGN: Optimization and validation applied to cell lines and clinical samples (polar bodies, blastomeres, and trophectoderm biopsies). SETTING: University research laboratory and a preimplantation genetic diagnosis (PGD) reference laboratory. PATIENT(S): Samples from 65 couples who underwent PGD for aneuploidy and/or a single-gene disorder. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 1) Comparison of aneuploidy screening results obtained with the use of the new microarray with those derived from two well established cytogenetic techniques. 2) mtDNA quantification. 3) Analysis of single-nucleotide polymorphisms. RESULT(S): The fully optimized microarray was estimated to have an accuracy of ≥97% for the detection of individual aneuploidies and to detect 99% of chromosomally abnormal embryos. The microarray was shown to accurately determine relative quantities of mtDNA. Information provided from polymorphic loci was sufficient to allow confirmation that an embryo was derived from specific parents. CONCLUSION(S): It is hoped that methods such as those reported here, which provide information on several aspects of oocyte/embryo genetics, could lead to improved strategies for identifying viable embryos, thereby increasing the likelihood of successful implantation. Additionally, the provision of genotyping information has the potential to reveal DNA contaminants and confirm parental origin of embryos.
Assuntos
Aneuploidia , Blastocisto/fisiologia , DNA Mitocondrial/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Corpos Polares/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Diagnóstico Pré-Implantação/instrumentação , Adulto , Análise Mutacional de DNA/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Feminino , HumanosRESUMO
BACKGROUND: DNA microarrays are a key resource for global analysis of genome content, gene expression and the distribution of transcription factor binding sites. We describe the development and application of versatile high density ink-jet in situ-synthesized DNA arrays for the G+C rich bacterium Streptomyces coelicolor. High G+C content DNA probes often perform poorly on arrays, yielding either weak hybridization or non-specific signals. Thus, more than one million 60-mer oligonucleotide probes were experimentally tested for sensitivity and specificity to enable selection of optimal probe sets for the genome microarrays. The heat-shock HspR regulatory system of S. coelicolor, a well-characterized repressor with a small number of known targets, was exploited to test and validate the arrays for use in global chromatin immunoprecipitation-on-chip (ChIP-chip) and gene expression analysis. RESULTS: In addition to confirming dnaK, clpB and lon as in vivo targets of HspR, it was revealed, using a novel ChIP-chip data clustering method, that HspR also apparently interacts with ribosomal RNA (rrnD operon) and specific transfer RNA genes (the tRNAGln/tRNAGlu cluster). It is suggested that enhanced synthesis of Glu-tRNAGlu may reflect increased demand for tetrapyrrole biosynthesis following heat-shock. Moreover, it was found that heat-shock-induced genes are significantly enriched for Gln/Glu codons relative to the whole genome, a finding that would be consistent with HspR-mediated control of the tRNA species. CONCLUSIONS: This study suggests that HspR fulfils a broader, unprecedented role in adaptation to stresses than previously recognized -- influencing expression of key components of the translational apparatus in addition to molecular chaperone and protease-encoding genes. It is envisaged that these experimentally optimized arrays will provide a key resource for systems level studies of Streptomyces biology.
Assuntos
Proteínas de Bactérias/genética , Genômica/métodos , Proteínas de Choque Térmico/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regulon/genética , Proteínas Repressoras/genética , Streptomyces coelicolor/genética , Proteínas de Bactérias/metabolismo , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Genes Bacterianos , Genômica/instrumentação , Proteínas de Choque Térmico/metabolismo , Imunoprecipitação , Ligação Proteica , RNA Ribossômico/metabolismo , RNA de Transferência/genética , Proteínas Repressoras/metabolismoRESUMO
Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.
Assuntos
Acetatos/metabolismo , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Contagem de Colônia Microbiana , Meios de Cultura , Espectroscopia de Ressonância Magnética , Microscopia de Contraste de Fase , Microscopia de Vídeo , Movimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5' end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution.
Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/genética , Proteínas Ferro-Enxofre/análise , Elementos Reguladores de Transcrição , Fatores de Transcrição/análise , Sítios de Ligação , Imunoprecipitação da Cromatina , Cromossomos Bacterianos , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.
Assuntos
DNA Bacteriano/metabolismo , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , Cromossomos Bacterianos , Proteínas de Ligação a DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Fator Proteico para Inversão de Estimulação , Fatores Hospedeiros de Integração/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Histone-like nucleoid structuring protein (H-NS) is a modular protein that is associated with the bacterial nucleoid. We used chromatin immunoprecipitation to determine the binding sites of H-NS and RNA polymerase on the Salmonella enterica serovar Typhimurium chromosome. We found that H-NS does not bind to actively transcribed genes and does not co-localize with RNA polymerase. This shows that H-NS principally silences gene expression by restricting the access of RNA polymerase to the DNA. H-NS had previously been shown to preferentially bind to curved DNA in vitro. In fact, at the genomic level we discovered that the level of H-NS binding correlates better with the AT-content of DNA. This is likely to have evolutionary consequences because we show that H-NS binds to many Salmonella genes acquired by lateral gene transfer, and functions as a gene silencer. The removal of H-NS from the cell causes un-controlled expression of several Salmonella pathogenicity islands, and we demonstrate that this has deleterious consequences for bacterial fitness. Our discovery of this novel role for H-NS may have implications for the acquisition of foreign genes by enteric bacteria.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Transferência Genética Horizontal , Salmonella enterica/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Evolução Biológica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidadeRESUMO
Bacterial core RNA polymerase (RNAP) must associate with a sigma factor to recognize promoter sequences. Escherichia coli encodes seven sigma factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of sigma32, the heat-shock sigma factor, and estimate that there are 120-150 sigma32 promoters in total. Unexpectedly, 25% of these sigma32 targets are located within coding regions, suggesting novel regulatory roles for sigma32. The majority of sigma32 promoter targets overlap with those of sigma70, the housekeeping sigma factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPsigma70 and RNAPsigma32 initiating transcription in vitro with similar efficiency and from identical positions. SigmaE-regulated promoters also overlap extensively with those for sigma70. These results suggest that extensive functional overlap between sigma factors is an important phenomenon.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Choque Térmico/metabolismo , Fator sigma/metabolismo , Sítios de Ligação , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Transcrição GênicaRESUMO
Chromatin immunoprecipitation and high-density microarrays have been used to monitor the distribution of the global transcription regulator Escherichia coli cAMP-receptor protein (CRP) and RNA polymerase along the E. coli chromosome. Our results identify targets occupied by CRP and genes transcribed by RNA polymerase in vivo. Many of the loci of CRP binding are at known CRP regulated promoters. However, our results show that CRP also interacts with thousands of weaker sites across the whole chromosome and that this "background" binding can be used as a probe for organization within the E. coli folded chromosome. In rapidly growing cells, we show that the major sites of RNA polymerase binding are approximately 90 transcription units that include genes needed for protein synthesis. Upon the addition of rifampicin, RNA polymerase is distributed among >500 functional promoters. We show that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects of RNA polymerase behavior and providing an alternative to the "transcriptomics" approach for studying global transcription patterns.