Verification of alternative splicing variants based on domain integrity, truncation length and intrinsic protein disorder

According to current estimations ∼95% of multi-exonic human protein-coding genes undergo alternative splicing (AS). However, for 4000 human proteins in PDB, only 14 human proteins have structures of at least two alternative isoforms. Surveying these structural isoforms revealed that the maximum insertion accommodated by an isoform of a fully ordered protein domain was 5 amino acids, other instances of domain changes involved intrinsic structural disorder. After collecting 505 minor isoforms of human proteins with evidence for their existence we analyzed their length, protein disorder and exposed hydrophobic surface. We found that strict rules govern the selection of alternative splice variants aimed to preserve the integrity of globular domains: alternative splice sites (i) tend to avoid globular domains or (ii) affect them only marginally or (iii) tend to coincide with a location where the exposed hydrophobic surface is minimal or (iv) the protein is disordered. We also observed an inverse correlation between the domain fraction lost and the full length of the minor isoform containing the domain, possibly indicating a buffering effect for the isoform protein counteracting the domain truncation effect. These observations provide the basis for a prediction method (currently under development) to predict the viability of splice variants

Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome

<p>Abstract</p> <p>Background</p> <p>Flax (<it>Linum usitatissimum </it>L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome.</p> <p>Results</p> <p>The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding.</p> <p>Conclusion</p> <p>The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly.</p

Catalytic residues in hydrolases: analysis of methods designed for ligand-binding site prediction

The comparison of eight tools applicable to ligand-binding site prediction is presented. The methods examined cover three types of approaches: the geometrical (CASTp, PASS, Pocket-Finder), the physicochemical (Q-SiteFinder, FOD) and the knowledge-based (ConSurf, SuMo, WebFEATURE). The accuracy of predictions was measured in reference to the catalytic residues documented in the Catalytic Site Atlas. The test was performed on a set comprising selected chains of hydrolases. The results were analysed with regard to size, polarity, secondary structure, accessible solvent area of predicted sites as well as parameters commonly used in machine learning (F-measure, MCC). The relative accuracies of predictions are presented in the ROC space, allowing determination of the optimal methods by means of the ROC convex hull. Additionally the minimum expected cost analysis was performed. Both advantages and disadvantages of the eight methods are presented. Characterization of protein chains in respect to the level of difficulty in the active site prediction is introduced. The main reasons for failures are discussed. Overall, the best performance offers SuMo followed by FOD, while Pocket-Finder is the best method among the geometrical approaches

Multifunctional Basic Motif in the Glycine Receptor Intracellular Domain Induces Subunit-specific Sorting*

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRα1–4 and -β). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3–4 loop). The TM3–4 loops of the subunits GlyRα1 and -α3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and β-galactosidase-tagged domain constructs, the TM3–4 loops of GlyRα1 and -α3, but not of GlyRα2 and -β, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin α3 and α4 were found to interact with the TM3–4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3–4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs