FeatureScan: revealing property-dependent similarity of nucleotide sequences

FeatureScan is a software package aiming to reveal novel types of DNA sequence similarity by comparing physico-chemical properties. Thirty-eight different parameters of DNA double strands such as charge, melting enthalpy, conformational parameters and the like are provided. As input FeatureScan requires two sequences, a pattern sequence and a target sequence, search conditions are set by selecting a specific DNA parameter and a threshold value. Search results are displayed in FASTA format and directly linked to external genome databases/browsers (ENSEMBL, NCBI, UCSC). An Internet version of FeatureScan is accessible at . As part of the HOBIT initiative () FeatureScan is also accessible as a web service at its above home page. Currently, several preloaded genomes are provided at this Internet website (Homo sapiens, Mus musculus, Rattus norvegicus and four strains of Escherichia coli) as target sequences. Standalone executables of FeatureScan are available on request

Weighted Alignment Free Dissimilarity Metric for Promoter Sequence Comparison

Comparative sequence analysis has been a powerful tool in bioinformatics which interprets knowledge about the functionality of a sequence, making use of its structural information. Among the non coding regions of DNA,   the comparison of promoter sequences has received a great deal of attention in medical science as promoter regions play a crucial role in gene regulation. In this work we propose an alignment free sequence comparison metric for comparison of promoter sequences. We use the binary and decimal position specific motif matrices (PSMM) of the promoters which were created for our experiments using the TFSEARCH tool. Simple weighted algorithm is used to compute the dissimilarity between the PSMMs of promoter sequences, thereby analyzing its underlying homology and functionality. The NCBI database was used to obtain the promoter sequences of 500 nucleotides upstream the transcription start site (TSS) of enzyme pyruvate kinase (PKLR) from the glycolysis pathway of different organisms for one experiment and all the enzymes from the glycolysis pathway of organism human for the other. The proposed dissimilarity metric is successful in bringing out differences on both the datasets and the results regarding similarities and differences in promoter sequences could be essential to have a clear knowledge of transcription regulation process in different organisms.The results reveal some useful findings which can be extended for a broader investigation

Mutant analysis of luminescence and autoinduction in a marine bacterium

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution December 1994The marine symbiotic bacterium Vibrio fischeri is striking for its ability both to emit light and to dramatically regulate light emission using a cell-to-cell signalling mechanism called autoinduction. The latter is mediated by a signal molecule called the "autoinducer". The mechanistic bases of both luminescence and autoinduction are well known in V. fischeri, but this knowledge is mostly derived from studies of the cloned luminescence and autoinduction genes expressed in Escherichia coli. In this study, luminescence and autoinduction mutations were systematically generated in V. fischeri to explore aspects of luminescence and autoinduction not addressable in E. coli, such as the adaptive significance of luminescence. Most dramatically, the mutants revealed the presence of multiple autoinducers and autoinducer synthases in V. fischeri. One of the autoinducers (autoinducer-2, or AI-2) was chemically purified and shown to be Noctanoyi- L-homoserine lactone. The genetic locus encoding the AI-2 synthase was cloned and designated ain (autoinducer). Manipulation of ain and AI-2 in V. jischeri demonstrated that the function of AI-2 appears to be to inhibit rather than to promote autoinduction.The WHOI Ocean Ventures Fund and the WHOI Molecular Biology Fund

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. <it>P. fluorescens </it>isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species.</p> <p>Results</p> <p>We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for <it>P. fluorescens </it>WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to <it>P. fluorescens </it>and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with <it>P. fluorescens </it>SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system.</p> <p>Conclusions</p> <p>The improved high-quality draft genome sequence of WH6 contributes towards resolving the <it>P. fluorescens </it>species, providing additional impetus for establishing two separate lineages in <it>P. fluorescens</it>. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of <it>P. fluorescens </it>reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.</p

DNA-Protein interaction: a response regulatory protein associated with Mo homeostasis in Desulfovibrio alaskensis G20.

Dissertation for a degree in Doctor in Sustainable ChemistryFundação para Ciência e Tecnologia - (SFRH/BD/47040/2008

Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits

A broadly applicable artificial selection system for biomolecule evolution

Biocatalysis offers an attractive alternative to traditional chemical catalysis. However, it is often found that an enzyme with the optimal properties for a specific application is not available within the natural repertoire of enzymes. It is then desirable to obtain an improved variant by altering the sequence of a known enzyme, in a process known as protein engineering. Directed evolution is one of the most powerful tools for protein engineering. In directed evolution, the process of natural evolution is mimicked in the laboratory at a much shorter timescale and selecting for properties that make the enzyme (or any other type of biomolecule) more suitable for an application of human interest. The main bottleneck of directed evolution is the identification of the desired variants amongst a majority of variants without the sought altered or improved property. Selection approaches link the desired activity to an increased survival rate or improved growth. While in principle such methodologies allow for ultra high-throughput analysis of libraries, most selection techniques have a limited scope, and can only be applied to a relatively reduced set of biomolecules or properties. This thesis presents the most broadly-applicable artificial selection system for the evolution of biomolecules ever reported. The selection platform is based on an engineered E. coli strain with impaired regeneration of NAD+, causing a conditional growth defect during anaerobic fermentation. By directly or indirectly linking the activity of the biomolecules of interest to the oxidation of NADH, cells can be rescued from this growth defect. The efficacy of such selection system has been demonstrated by using it to select alcohol dehydrogenase, imine reductase and nitroreductase variants with altered or enhanced catalytic properties, as well as an isopropanol-producing metabolic pathway with optimised regulatory elements leading to a maximised yield of isopropanol. These results confirm the wide scope of the developed selection system, which can replace conventional screening currently used in many cases of direct relevance for industrial processes. Increasing the throughput of the variant search process by many orders of magnitude will lead to the discovery of novel biomolecules and accelerate the implementation of biocatalysis.Open Acces

Genome Holography: Deciphering Function-Form Motifs from Gene Expression Data

DNA chips allow simultaneous measurements of genome-wide response of thousands of genes, i.e. system level monitoring of the gene-network activity. Advanced analysis methods have been developed to extract meaningful information from the vast amount of raw gene-expression data obtained from the microarray measurements. These methods usually aimed to distinguish between groups of subjects (e.g., cancer patients vs. healthy subjects) or identifying marker genes that help to distinguish between those groups. We assumed that motifs related to the internal structure of operons and gene-networks regulation are also embedded in microarray and can be deciphered by using proper analysis.The analysis presented here is based on investigating the gene-gene correlations. We analyze a database of gene expression of Bacillus subtilis exposed to sub-lethal levels of 37 different antibiotics. Using unsupervised analysis (dendrogram) of the matrix of normalized gene-gene correlations, we identified the operons as they form distinct clusters of genes in the sorted correlation matrix. Applying dimension-reduction algorithm (Principal Component Analysis, PCA) to the matrices of normalized correlations reveals functional motifs. The genes are placed in a reduced 3-dimensional space of the three leading PCA eigen-vectors according to their corresponding eigen-values. We found that the organization of the genes in the reduced PCA space recovers motifs of the operon internal structure, such as the order of the genes along the genome, gene separation by non-coding segments, and translational start and end regions. In addition to the intra-operon structure, it is also possible to predict inter-operon relationships, operons sharing functional regulation factors, and more. In particular, we demonstrate the above in the context of the competence and sporulation pathways.We demonstrated that by analyzing gene-gene correlation from gene-expression data it is possible to identify operons and to predict unknown internal structure of operons and gene-networks regulation

Transcription in the Yeast Saccharomyces cerevisiae

The aim of this study was to investigate aspects of transcription of highly expressed yeast genes. To investigate the sequence requirements for transcriptional initiation putative promoters were constructed which contained promoter elements subcloned from the 5'-flanking region of the phosphoglycerate kinase gene (PGK1). The analysis of these promoters was hindered by high levels of transcriptional readthrough from adjacent sequences in the promoter probe vector. A CT-rich block, found upstream of many highly expressed yeast genes has previously been shown to influence the position of the transcriptional initiation site (I-site). Preliminary data presented here is consistent with the existence of a yeast protein which specifically interacts with this region

Bioinformatics

This book is divided into different research areas relevant in Bioinformatics such as biological networks, next generation sequencing, high performance computing, molecular modeling, structural bioinformatics, molecular modeling and intelligent data analysis. Each book section introduces the basic concepts and then explains its application to problems of great relevance, so both novice and expert readers can benefit from the information and research works presented here