Ontology highlight
ABSTRACT:
PROVIDER: PRJNA377408 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5332884_subreads.fastq.gz | Fastqsanger.gz |
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Project description:TRF2 is a paralogue of TATA-box binding protein (TBP) with highest expression in testis. Although Trf2 inactivation in mice leads to arrested spermatogenesis, there is no direct evidence that Trf2 is recruited to chromatin to directly regulate gene expression. We used genetically modified mice where endogenous Trf2 has been modified to carry a TAP-TAG to perform ChIP-reChIP followed by deep sequencing. We found that Trf2 is recruited to all active promoters as a subunit of TFIIA/ALF complex together with TBP.
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Project description:Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known and novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. Keywords: detection, pathogen, virulence mechanism In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.
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Project description:TRF2 is a paralogue of TATA-box binding protein (TBP) with highest expression in testis. Although Trf2 inactivation in mice leads to arrested spermatogenesis, there is no direct evidence that Trf2 is recruited to chromatin to directly regulate gene expression. We used genetically modified mice where endogenous Trf2 has been modified to carry a TAP-TAG to perform ChIP-reChIP followed by deep sequencing. We found that Trf2 is recruited to all active promoters as a subunit of TFIIA/ALF complex together with TBP. To assess the effect of Trf2 inactivation on gene expression we performed RNA-seq on WT and Trf2-/- testes at 21 days of age when haploid cell gene expression is activated.
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