A toolkit for analysing large-scale plant small RNA datasets

Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci. AVAILABILITY: The tools are freely available for use at http://srna-tools.cmp.uea.ac.uk.     

bPE toolkit: toolkit for computational protein engineering

We present a computational toolkit consisting of five utility tools, for performing basic operations on a protein structure file in PDB format. The toolkit consists of five different programs which can be integrated as part of a pipeline for computational protein structure characterization or as a standalone analysis package. The programs include tools for chirality check for amino acids (ProChiral), contact map generation (CoMa), data redundancy (DaRe), hydrogen bond potential energy (HyPE) and electrostatic interaction energy (EsInE). All programs in the toolkit can be accessed and downloaded through the following link: http://www.iitg.ac.in/bpetoolkit/.     

A rapidly evolving genomic toolkit for Drosophila heterochromatin

Heterochromatin is the enigmatic eukaryotic genome compartment found mostly at telomeres and centromeres. Conventional approaches to sequence assembly and genetic manipulation fail in this highly repetitive, gene-sparse, and recombinationally silent DNA. In contrast, genetic and molecular analyses of euchromatin-encoded proteins that bind, remodel, and propagate heterochromatin have revealed its vital role in numerous cellular and evolutionary processes. Utilizing the 12 sequenced Drosophila genomes, Levine et al¹ took a phylogenomic approach to discover new such protein “surrogates” of heterochromatin function and evolution. This paper reported over 20 new members of what was traditionally believed to be a small and static Heterochromatin Protein 1 (HP1) gene family. The newly identified HP1 proteins are structurally diverse, lineage-restricted, and expressed primarily in the male germline. The birth and death of HP1 genes follows a “revolving door” pattern, where new HP1s appear to replace old HP1s. Here, we address alternative evolutionary models that drive this constant innovation.     

A chemical toolkit for proteins--an expanded genetic code

Recently, a method to encode unnatural amino acids with diverse physicochemical and biological properties genetically in bacteria, yeast and mammalian cells was developed. Over 30 unnatural amino acids have been co-translationally incorporated into proteins with high fidelity and efficiency using a unique codon and corresponding transfer-RNA:aminoacyl-tRNA-synthetase pair. This provides a powerful tool for exploring protein structure and function in vitro and in vivo, and for generating proteins with new or enhanced properties.     

A three-dimensional working model for a guide RNA from Trypanosoma brucei.

RNA editing in protozoan parasites is a mitochondrial RNA processing reaction in which exclusively uridylate residues are inserted into, and less frequently deleted from, pre-mRNAs. Molecules central to the process are so-called guide RNAs (gRNAs) which function as templates in the reaction. For a detailed molecular understanding of the mechanism of the editing process knowledge of structural features of gRNAs will be essential. Here we report on a computer-assisted molecular modelling approach to construct the first three-dimensional gRNA model for gND7-506, a ND7-specific gRNA from Trypanosoma brucei. The modelling process relied on chemical modification and enzymatic probing data and was validated by in vitro mutagenesis experiments. The model predicts a reasonably compact structure, where two stem/loop secondary structure elements are brought into close proximity by a triple A tertiary interaction, forming a core element within the centre of the molecule. The model further suggests that the surface of the gRNA is primarily made up of the sugar-phoshate backbone. On the basis of the model, footprinting experiments of gND7-506 in a complex with the gRNA binding protein gBP21 could successfully be interpreted and provide a first picture for the assembly of gRNAs within a ribonucleoprotein complex.     

PipeAlign: A new toolkit for protein family analysis

PipeAlign is a protein family analysis tool integrating a five step process ranging from the search for sequence homologues in protein and 3D structure databases to the definition of the hierarchical relationships within and between subfamilies. The complete, automatic pipeline takes a single sequence or a set of sequences as input and constructs a high-quality, validated MACS (multiple alignment of complete sequences) in which sequences are clustered into potential functional subgroups. For the more experienced user, the PipeAlign server also provides numerous options to run only a part of the analysis, with the possibility to modify the default parameters of each software module. For example, the user can choose to enter an existing multiple sequence alignment for refinement, validation and subsequent clustering of the sequences. The aim is to provide an interactive workbench for the validation, integration and presentation of a protein family, not only at the sequence level, but also at the structural and functional levels. PipeAlign is available at http://igbmc.u-strasbg.fr/PipeAlign/.     

A Gateway MultiSite recombination cloning toolkit

The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org).     

Empirical models for substitution in ribosomal RNA

Empirical models of substitution are often used in protein sequence analysis because the large alphabet of amino acids requires that many parameters be estimated in all but the simplest parametric models. When information about structure is used in the analysis of substitutions in structured RNA, a similar situation occurs. The number of parameters necessary to adequately describe the substitution process increases in order to model the substitution of paired bases. We have developed a method to obtain substitution rate matrices empirically from RNA alignments that include structural information in the form of base pairs. Our data consisted of alignments from the European Ribosomal RNA Database of Bacterial and Eukaryotic Small Subunit and Large Subunit Ribosomal RNA ( Wuyts et al. 2001. Nucleic Acids Res. 29:175-177; Wuyts et al. 2002. Nucleic Acids Res. 30:183-185). Using secondary structural information, we converted each sequence in the alignments into a sequence over a 20-symbol code: one symbol for each of the four individual bases, and one symbol for each of the 16 ordered pairs. Substitutions in the coded sequences are defined in the natural way, as observed changes between two sequences at any particular site. For given ranges (windows) of sequence divergence, we obtained substitution frequency matrices for the coded sequences. Using a technique originally developed for modeling amino acid substitutions ( Veerassamy, Smith, and Tillier. 2003. J. Comput. Biol. 10:997-1010), we were able to estimate the actual evolutionary distance for each window. The actual evolutionary distances were used to derive instantaneous rate matrices, and from these we selected a universal rate matrix. The universal rate matrices were incorporated into the Phylip Software package ( Felsenstein 2002. http://evolution.genetics.washington.edu/phylip.html), and we analyzed the ribosomal RNA alignments using both distance and maximum likelihood methods. The empirical substitution models performed well on simulated data, and produced reasonable evolutionary trees for 16S ribosomal RNA sequences from sequenced Bacterial genomes. Empirical models have the advantage of being easily implemented, and the fact that the code consists of 20 symbols makes the models easily incorporated into existing programs for protein sequence analysis. In addition, the models are useful for simulating the evolution of RNA sequence and structure simultaneously.     

A robust toolkit for functional profiling of the yeast genome

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.     

Genomics and clinical medicine: rationale for creating and effectively evaluating animal models

Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated. In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled. Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine. The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms. Comparative and functional genomics and proteomics provide effective approaches for identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics. By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans. This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation. Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies.     

Digital three-dimensional models of Drosophila development

Digital models of organs, cells and subcellular structures have become important tools in biological and medical research. Reaching far beyond their traditional widespread use as didactic tools, computer-generated models serve as electronic atlases to identify specific elements in complex patterns, and as analytical tools that reveal relationships between such pattern elements that would remain obscure in two-dimensional sections. Digital models also offer the unique opportunity to store and display gene-expression patterns, and pilot studies have been made in several genetic model organisms, including mouse, Drosophila and Caenorhabditis elegans, to construct digital graphic databases intended as repositories for gene-expression data.     

Evaluating mixture models for building RNA knowledge-based potentials

Ribonucleic acid (RNA) molecules play important roles in a variety of biological processes. To properly function, RNA molecules usually have to fold to specific structures, and therefore understanding RNA structure is vital in comprehending how RNA functions. One approach to understanding and predicting biomolecular structure is to use knowledge-based potentials built from experimentally determined structures. These types of potentials have been shown to be effective for predicting both protein and RNA structures, but their utility is limited by their significantly rugged nature. This ruggedness (and hence the potential's usefulness) depends heavily on the choice of bin width to sort structural information (e.g. distances) but the appropriate bin width is not known a priori. To circumvent the binning problem, we compared knowledge-based potentials built from inter-atomic distances in RNA structures using different mixture models (Kernel Density Estimation, Expectation Minimization and Dirichlet Process). We show that the smooth knowledge-based potential built from Dirichlet process is successful in selecting native-like RNA models from different sets of structural decoys with comparable efficacy to a potential developed by spline-fitting - a commonly taken approach - to binned distance histograms. The less rugged nature of our potential suggests its applicability in diverse types of structural modeling.     

Possible three-dimensional models for the polypeptide backbone structure of alamethicin

Based on an empirical procedure of predicting polypeptide backbone structures of proteins, an attempt was made to construct possible three-dimensional models for the secondary structure of alamethicin. Two chemical properties were used to limit the number of possible models: the presence of seven to eight α-aminoisobutyric acid residues whose conformations were quite restricted, and a covalent bond between the proline residue at position 1 and the glutamic acid residue at position 17. The predicted structures appear very compact and show features in agreement with circular dichroism and optical rotatory dispersion results, nuclear magnetic resonance measurements, and compressed monolayer studies.     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.