Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The pilus retraction protein PilT has a global influence on the transcriptome of Neisseria gonorrhoeae

ABSTRACT: The ATPase protein PilT mediates retraction of type IV pili. We performed microarrays comparing the transcription profile of the Neisseria gonorrhoeae wild-type strain MS11 and its isogenic pilT mutant. 63 open reading frames were found to be differentially regulated in the pilT mutant. Most interestingly, a loss of function mutation in pilT leads to an upregulation of pilE, which encodes the pilus subunit protein. Ngo arrays were generated as custom 8x15 K microarrays (Agilent Technologies) covering all open reading frames (ORFs) from N. gonorrhoeae strain FA1090 (accession number AE004969) supplemented with all ORFs from the gonoccoccal genetic island of strain MS11 (accession number AY803022) by 6 specific 60-mer oligonucleotides per gene on average. Microarray analysis was performed as dual-color hybridization with dye-reversal color-swaps as technical replicates for Cy-dye specific effect compensation and three independent biological replicates. Microarray features were extracted with the FE 9.5.3.1 software from Agilent Technologies using the GE2-v4_95_Feb07 protocol with default settings. Data were loaded into Resolver (Rosetta Biosoftware) using the MAGE-ML loader and analyzed for differential gene expression. Ratio profiles were combined into ratio experiments and regulated genes were identified with a threshold of 1.75 fold-change and anti-correlation of the color-swapped dye reversals, rendering the analysis highly stringent and robust with P values <0.0001 of the results.

ORGANISM(S): Neisseria gonorrhoeae

SUBMITTER: Hans-Joachim Mollenkopf

PROVIDER: E-GEOD-12258 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:The most widely-used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms and analysis algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. All commercial tiling array platforms performed well, although each platform and analysis algorithm had distinct performance and cost characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. The spike-in DNA samples and the resulting array data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: chip-ChIP simulation For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf For each replicate array, we labeled 1Âµg of spike-in sample and 1Âµg of control sample. The Bioprime Plus Array CGH labeling Module (Invitrogen Catolog number 18095-014) was used. The spike-in was labeled with the red channel dye (Alexa Fluor-647) and the control was labeled with the green channel dye (Alexa Fluor-555) in two of the replicates. A dye swap was performed for the third replicate. These samples were then competitively hybridized to three replicate 244k arrays from Agilent, (AMADID 25150451). We hybridized the samples to the arrays using the Agilent Array CGH protocol, with some modifications. Briefly, labeled DNA was combined with Cot-1 DNA, blocking reagent, and hybridization buffer. The hybridizations were carried out at 60Â°C. The arrays were scanned using an Agilent dual laser scanner, and the images were processed using Agilent Feature Extraction Software.

Project description:The complexity of events associated with age-related memory loss (ARML) cannot be overestimated. The problem is further complicated by the enormous diversity of neurons in the CNS and even synapses of one neuron within a neural circuit. Large-scale single-neuron analysis is not only challenging but mostly impractical for any model currently used in ARML. We simply do not know: do all neurons and synapses age differently or are some neurons (or synapses) more resistant to aging than others? What is happening in any given neuron while it undergoes “normal” aging? What are the genomic changes that make aging apparently irreversible? What would be the balance between neuron-specific vs global genome-wide changes in aging? In the proposed paper we address these questions and develop a new model to study the entire scope of genomic and epigenomic regulation in aging at the resolution of single functionally characterized cells and even cell compartments. In particular, the mollusc Aplysia californica has been implemented as a powerful paradigm in addressing fundamental questions of the neurobiology of aging. The proposed manuscript will consist of four parts. First, we will provide an introduction to Aplysia as a representative of the largest superclade of bilaterian animals (Lophotrochozoa). Aplysia has a short lifespan of 220-300 days with a well-characterized life cycle and characterized phenomenology of aging. Most importantly, Aplysia possess the largest nerve cells in the entire animal kingdom (only eggs are larger); these cells can be uniquely identified and mapped in terms of their well-defined interactions with other neurons forming relatively simpler neural circuits underlying several stereotypic and learned behaviors. Second, we have identified in Aplysia more that a hundred neurological- and age-related genes that were lost in other established invertebrate models (such as Drosophila and C. elegans). The proposed long-term regulatory age-related mechanisms include a high level of conservation among many epigenetic processes known to be lost in nematodes and flies with extremely short lifecycles and particularly derived genomes. We also identify and cloned more than 30 evolutionarily conserved homologs of genes involved in Alzheimer’s, Parkinson’s and Huntington’s diseases as well as age-related hormones. Third, we performed genome-wide analysis of expression patterns of more than 55,000 unique transcripts by comparing two different identified cholinergic neurons (R2 and LPl1) among young and aged animals. This direct single neuron genomic analysis indicates that there are significant cell-specific changes in gene-expression profiles as a function of aging. We estimated that only ~10-20% of genes that are differently expressed in the aging brain are common for all neuronal types - the remaining 80% are neuron-specific (i.e. found in aging neurons of one but not another type). The list of “common aging genes” includes components of insulin growth factor pathways, cell bioenergetics, telomerase-associated proteins, antioxidant enzymes, water channels and estrogen receptors. The rest were neuron-specific gene products (including apoptosis-related proteins, Alzheimer-related genes, growth factors and their receptors, ionic channels, transcription factors and more than 120 identified proteins known to be involved in neurodevelopment and synaptogenesis). Surprisingly, even two different identified cholinergic motoneurons age differently and each of them has a unique subset of genes differentially expressed in older animals. Fourth, we showed that the activity of the entire genome and associated epigenomic modifications (e.g. DNA methylation, histone dynamics) can be efficiently monitored within a single Aplysia neuron and can be modified as a function of aging in a neuron-specific manner including selective histones and histone-modifying enzymes and DNA methylation-related enzymes. This genome-wide analysis of aging allows us to propose novel mechanisms of active DNA demethylation and cell-specific methylation as well as regional relocation of RNAs as three key processes underlying age-related memory loss. These mechanisms tune the dynamics of long-term chromatin remodeling, control weakening and the loss of synaptic connections in aging. At the same time, our genomic tests revealed evolutionarily conserved gene clusters in the Aplysia genome associated with senescence and regeneration (e.g. apoptosis- and redox- dependent processes, insulin signaling, etc.). This is a reference design experiment with all samples being compared to one CNS from Aplysia. Two cholinergic neurons (R2 and LPl1), two ages (young and old), two arrays (AAA and DAA), three biological replicates each sample type. Two direct comparison experiments were also performed. One with young and old abdominal ganglion and the other with young and old R2.

Dataset Information

The pilus retraction protein PilT has a global influence on the transcriptome of Neisseria gonorrhoeae

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure