Functional interpretation of microarray experiments

Over the past few years, due to the popularisation of high-throughput methodologies such as DNA microarrays, the possibility of obtaining experimental data has increased significantly. Nevertheless, the interpretation of the results, which involves translating these data into useful biological knowledge, still remains a challenge. The methods and strategies used for this interpretation are in continuous evolution and new proposals are constantly arising. Initially, a two-step approach was used in which genes of interest were initially selected, based on thresholds that consider only experimental values, and then in a second, independent step the enrichment of these genes in biologically relevant terms, was analysed. For different reasons, these methods are relatively poor in terms of performance and a new generation of procedures, which draw inspiration from systems biology criteria, are currently under development. Such procedures, aim to directly test the behaviour of blocks of functionally related genes, instead of focusing on single genes.     

SNP Chart: an integrated platform for visualization and interpretation of microarray genotyping data

SNP Chart is a Java application for the visualization and interpretation of microarray genotyping data primarily derived from arrayed primer extension-based chemistries. Spot intensity output files from microarray analysis tools are imported into SNP Chart, together with a multi-channel TIFF image of the original array experiment and a list of the actual single nucleotide polymorphisms (SNPs) being tested. Data from different and/or replicate probes that interrogate the same SNP, but that are scattered across the array grid, can be reassembled into a single chart format, specific for the SNP. This allows a quick and very effective 'visualization'/'quality control' of the data from multiple probes for the same SNP that can be easily interpreted and manually scored as a genotype. AVAILABILITY: http://www.snpchart.ca.     

Genome organisation and chromatin structure in Escherichia coli

We have analysed the complete sequence of the Escherichia coli K12 isolate MG1655 genome for chromatin-associated protein binding sites, and compared the predicted location of predicted sites with experimental expression data from 'DNA chip' experiments. Of the dozen proteins associated with chromatin in E. coli, only three have been shown to have significant binding preferences: integration host factor (IHF) has the strongest binding site preference, and FIS sites show a weak consensus, and there is no clear consensus site for binding of the H-NS protein. Using hidden Markov models (HMMs), we predict the location of 608 IHF sites, scattered throughout the genome. A subset of the IHF sites associated with repeats tends to be clustered around the origin of replication. We estimate there could be roughly 6000 FIS sites in E. coli, and the sites tend to be localised in two regions flanking the replication termini. We also show that the regions upstream of genes regulated by H-NS are more curved and have a higher AT content than regions upstream of other genes. These regions in general would also be localised near the replication terminus.     

Genome sequence of enterohemorrhagic Escherichia coli NCCP15658

Enterohemorrhagic Escherichia coli causes severe food-borne disease in the guts of humans and animals. Here, we report the high-quality draft genome sequence of E. coli NCCP15658 isolated from a patient in the Republic of Korea. Its genome size was determined to be 5.46 Mb, and its genomic features, including genes encoding virulence factors, were analyzed.     

Draft Genome Sequence of Escherichia coli LCT-EC106

Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans. Here, we present the complete genome sequence of Escherichia coli LCT-EC106, which was isolated from CGMCC 1.2385.     

MarC-V: a spreadsheet-based tool for analysis, normalization, and visualization of single cDNA microarray experiments

The comprehensive analysis and visualization of data extracted from cDNA microarrays can be a time-consuming and error-prone process that becomes increasingly tedious with increased number of gene elements on a particular microarray. With the increasingly large number of gene elements on today's microarrays, analysis tools must be developed to meet this challenge. Here, we present MarC-V, a Microsoft Excel spreadsheet tool with Visual Basic macros to automate much of the visualization and calculation involved in the analysis process while providing the familiarity and flexibility of Excel. Automated features of this tool include (i) lower-bound thresholding, (ii) data normalization, (iii) generation of ratio frequency distribution plots, (iv) generation of scatter plots color-coded by expression level, (v) ratio scoring based on intensity measurements, (vi) filtering of data based on expression level or specific gene interests, and (vii) exporting data for subsequent multi-array analysis. MarC-V also has an importing function included for GenePix results (GPR) raw data files.     

Letter: Electron microscopic visualization of the folded chromosome of Escherichia coli

The folded Escherichia coli chromosome has been analyzed in the electron microscope following the Kleinschmidt spreading technique. In all cases, the DNA molecule appears intact and supercoiled. In addition, the membrane-associated form of the chromosome shows DNA fibers attached to one or two membrane patches. The DNA molecule is intact, as evidenced by the absence of free ends. Two types of DNA coiling are generally observed: (1) spirals with about four to seven turns per spiral, and (2) stretched plectonemic supercoils, more abundant on the outside of the complex where the spreading has been more extensive.The released folded chromosome appears to be less compact; it extends over a larger area and frequently breaks with the spreading. The intact molecules which can be found show the DNA concentrated in few nueleation areas, with one or a few interconnecting DNA fibers. The folded DNA has very few or no single strand nicks, as evidenced by its extensive supertwisting at high ethidium bromide concentrations.     

Nonrandom Mutagenesis of the Escherichia coli Genome by Nitrosoguanidine

The distribution on the genetic map of mutations induced with nitrosoguanidine in stationary-phase cultures of Escherichia coli is nonrandom.     

GandrKB--ontological microarray annotation and visualization

SUMMARY: The Gandr (gene annotation data representation) knowledgebase is an ontological framework for laboratory-specific gene annotation. Gandr uses Protege 2000 for editing, querying and visualizing microarray data and annotations. Genes can be annotated with provided, newly created or imported ontological concepts. Annotated genes can inherit assigned concept properties and can be related to each other. The resulting knowledgebase can be visualized as interactive network of nodes and edges representing genes and their functional relationships. This allows for immediate and associative gene context exploration. Ontological query techniques allow for powerful data access.     

ClutrFree: cluster tree visualization and interpretation

ClutrFree facilitates the visualization and interpretation of clusters or patterns computed from microarray data through a graphical user interface that displays patterns, membership information of the genes and annotation statistics simultaneously. ClutrFree creates a tree linking the patterns based on similarity, permitting the navigation among patterns identified by different algorithms or by the same algorithm with different parameters, and aids the inferring of conclusions from a microarray experiment. AVAILABILITY: The ClutrFree Java source code and compiled bytecode are available as a package under the GNU General Public License at http://bioinformatics.fccc.edu     

Xdigitise: visualization of hybridization experiments

Xdigitise is a software system for visualization of hybridization experiments giving the user facilities to analyze the corresponding images manually or automatically. Images of the high-density DNA arrays are displayed as well as the results of an external image analysis bundled with Xdigitise, e.g. the spot locations are marked and the duplicate correlations are shown by a color scale. AVAILABILITY: Xdigitise can be downloaded from http://www.molgen.mpg.de/~xdigitise.     

Genome sequence of a porcine extraintestinal pathogenic Escherichia coli strain

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen which can infect humans and animals and cause many diseases outside the intestine. Here, we report the first draft genome sequence of a porcine ExPEC strain, PCN033, isolated from a pig with meningitis.     

Red同源重组在大肠杆菌基因组修饰中的应用

Red同源重组技术发展迅速,已广泛应用于大肠杆菌基因组修饰,在点突变、基因敲除、序列整合等方面发挥着重要作用。简要综述了Red同源重组的重组机制和操作策略等研究进展,并介绍了Red同源重组在大肠杆菌基因组减小及多基因代谢途径优化方面的应用情况。     

Design of a seven-genome Escherichia coli microarray for comparative genomic profiling

We describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequenced Escherichia coli genomes in addition to several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Deltastx(2)::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its phi3538 (Deltastx(2)::cat) phage element originated from the E. coli 3538 (Deltastx(2)::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer phi3538 (Deltastx(2)::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis.     

Global identification of CobB interactors by an Escherichia coli proteome microarray

Protein acetylation is one of the most abundant post translational modifications and plays critical roles in many important biological processes. Based on the recent advances in mass spectrometry technology, in bacteria, such as Escherichia coli, tremendous acetylated proteins and acetylation sites have been identified. However, only one protein deacetylase, i.e. CobB, has been identified in E. coli so far. How CobB is regulated is still elusive. One right strategy to study the regulation of CobB is to globally identify its interacting proteins. In this study, we used a proteome microarray containing 4000 affinitypurified E. coli proteins to globally identify CobB interactors, and finally identified 183 binding proteins of high stringency. Bioinformatics analysis showed that these interacting pro teins play a variety of roles in a wide range of cellular func tions and are highly enriched in carboxylic acid metabolic process and hexose catabolic process, and also enriched in transferase and hydrolase. We further used biolayer inter ferometry to analyze the interaction and quantify the kinetic parameters of putative CobB interactors, and clearly showed that Cobb could strongly interact with TopA and AccC. The novel CobB interactors that we identified could serve as a start point for further functional analysis.     

Genome diversity at the serA-linked capsule locus in Escherichia coli

Individual isolates of Escherichia coli synthesize one of more than 70 chemically distinct polysaccharides which form the capsule. In this article we review the genetics of capsule production in E. coli and highlight what this is beginning to reveal in terms of the genetic basis of the structural diversity of polysaccharides. The serA-linked capsule locus can take three different allelic forms. Two of these are associated with capsule genes and are themselves internally variant, whilst the third form has not so far been implicated in capsule biogenesis. Thus the serA-linked region of the E. coli genome is strikingly polymorphic.