Adding functional entities to plasmids

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.     

Adding some SPICE to DAS

The distributed annotation system (DAS) defines a communication protocol used to exchange biological annotations. It is motivated by the idea that annotations should not be provided by single centralized databases but instead be spread over multiple sites. Data distribution, performed by DAS servers, is separated from visualization, which is carried out by DAS clients. The original DAS protocol was designed to serve annotation of genomic sequences. We have extended the protocol to be applicable to macromolecular structures. Here we present SPICE, a new DAS client that can be used to visualize protein sequence and structure annotations. AVAILABILITY: http://www.efamily.org.uk/software/dasclients/spice/     

Adding confidence to gene expression clustering

It has been well established that gene expression data contain large amounts of random variation that affects both the analysis and the results of microarray experiments. Typically, microarray data are either tested for differential expression between conditions or grouped on the basis of profiles that are assessed temporally or across genetic or environmental conditions. While testing differential expression relies on levels of certainty to evaluate the relative worth of various analyses, cluster analysis is exploratory in nature and has not had the benefit of any judgment of statistical inference. By using a novel dissimilarity function to ascertain gene expression clusters and conditional randomization of the data space to illuminate distinctions between statistically significant clusters of gene expression patterns, we aim to provide a level of confidence to inferred clusters of gene expression data. We apply both permutation and convex hull approaches for randomization of the data space and show that both methods can provide an effective assessment of gene expression profiles whose coregulation is statistically different from that expected by random chance alone.     

Adding amino acids to the genetic repertoire

Considerable progress has been made in expanding the number and nature of genetically encoded amino acids in Escherichia coli, yeast and mammalian cells in the past four years. To date, over 30 unnatural amino acids have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding orthogonal tRNA-aminoacyl-tRNA synthetase pair. The incorporated amino acids contain spectroscopic probes, post-translational modifications, metal chelators, photoaffinity labels and unique functional groups. The ability to genetically encode additional amino acids, beyond the common 20, provides a powerful approach for probing protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.     

Adding time-calibrated branch lengths to the Asteraceae supertree

New inference techniques,such as supertrees,have improved the construction of large phylogenies,helping to reveal the tree of life.In addition,these large phylogenies have enhanced the study of other evolutionary questions,such as whether traits have evolved in a neutral or adaptive way,or what factors have influenced diversification.However,supertrees usually lack branch lengths,which are necessary for all these issues to be investigated.Here,divergence times within the largest family of flowering plants,namely the Asteraceae,are reviewed to estimate time-calibrated branch lengths in the supertree of this lineage.An inconsistency between estimated dates of basal branching events and the earliest asteraceous fossil pollen record was detected.In addition,the impact of different methods of branch length assignment on the total number of transitions between states in the reconstruction of sexual system evolution in Asteraceae was investigated.At least for this dataset,different branch length assignation approaches influenced maximum likelihood(ML)reconstructions only and not Bayesian ones.Therefore,the selection of different branch length information is not arbitrary and should be carefully assessed,at least when ML approaches are being used.The reviewed divergence times and the estimated time-calibrated branch lengths provide a useful tool for future phylogenetic comparative and macroevolutionary studies of Asteraceae.     

Adding pyrrolysine to the Escherichia coli genetic code

Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context. We show that PYLIS has no effect on the level of neither Pyl nor Cyc incorporation. Exogenously supplying our reporter system with d-ornithine significantly increases suppression efficiency, indicating that d-ornithine is a direct precursor to Pyl.     

Adding value to fusion proteins through covalent labelling

Recombinant proteins are often expressed with an additional polypeptide the role of which is to aid purification, detection or functional studies. Recently, the role of these tags has been extended to mediate the labelling of the protein of interest with chemically diverse compounds. These approaches are of importance for protein science as they allow proteins to be equipped with properties that cannot be genetically encoded and also enable the use of a single fusion protein for several different applications. Several new approaches have been developed for the covalent labelling of fusion proteins both in living cells and in vitro.     

Adding facets to TNF signaling. The JNK angle

In the October 3 issue of Cell, Deng et al. (2003) report that a JNK-dependent mitochondrial signal, the release of Smac/DIABLO, is required for TNF-induced caspase 8 activation and apoptosis. This process is interceded by a novel processed form of the BH3 only protein Bid.     

Adding Value to Ecological Risk Assessment with Population Modeling

Current measures used to estimate the risks of toxic chemicals are not relevant to the goals of the environmental protection process, and thus ecological risk assessment (ERA) is not used as extensively as it should be as a basis for cost-effective management of environmental resources. Appropriate population models can provide a powerful basis for expressing ecological risks that better inform the environmental management process and thus that are more likely to be used by managers. Here we provide at least five reasons why population modeling should play an important role in bridging the gap between what we measure and what we want to protect. We then describe six actions needed for its implementation into management-relevant ERA.