ABSTRACT
BACKGROUND: Here we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. RESULTS: In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resulting functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. CONCLUSIONS: PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequence-based genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.
Subject(s)
Genome, Bacterial , Herbaspirillum/genetics , High-Throughput Nucleotide Sequencing/methods , Internet , Molecular Sequence Annotation/methods , Software , Computational Biology/methods , Computers , Water MicrobiologyABSTRACT
Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.
Subject(s)
Gene Expression Regulation/physiology , Genome , RNA Editing/physiology , Transcription Factors/metabolism , Animals , Base Sequence , DNA/genetics , Enzyme-Linked Immunosorbent Assay , Genetic Markers , Transcription Factors/geneticsABSTRACT
Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.
Subject(s)
Caenorhabditis elegans/genetics , Caenorhabditis/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Genetic Techniques , Genome, Helminth , Regulatory Elements, Transcriptional/genetics , Zinc Fingers , Animals , Deoxyribonucleases, Type II Site-Specific/genetics , Gene Targeting , Genes, Helminth , INDEL Mutation , Mutagenesis , Transcription Factors/chemistry , TransgenesABSTRACT
Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
Subject(s)
Combinatorial Chemistry Techniques/methods , Genetic Engineering , Mutagenesis, Site-Directed/methods , Transcription Factors/genetics , Transcription Factors/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , DNA/genetics , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Genome , Humans , K562 Cells , Molecular Sequence Data , Receptors, CCR5/genetics , Vascular Endothelial Growth Factor A/genetics , XanthomonasABSTRACT
Grouping of gene expression patterns across biological experiments, treatments and time-series data is performed in q-intervals of measurements using phase-shifted analysis of gene expression (PAGE); a Java-based tool to find clusters of genes that share trends of expression profiles within the dataset. The patterns and genes within q-Clusters are visualized in trend plots and compared to determine biological relevance from the gene annotations.
