Exploiting single-molecule transcript sequencing for eukaryotic gene prediction

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0729-7) contains supplementary material, which is available to authorized users.     

Marsupials and monotremes possess a novel family of MHC class I genes that is lost from the eutherian lineage

Background

Major histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes.

Results

Application of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT, which has not previously been characterized. These class I genes are found in both American and Australian marsupials, and in monotremes, at an evolutionary chromosomal breakpoint, but are not present in non-mammalian genomes and have been lost from the eutherian lineage. We show that UT family members are expressed in the thymus of the gray short-tailed opossum and in other immune tissues of several Australian marsupials. Structural homology modelling shows that the proteins encoded by this family are predicted to have an open, though short, antigen-binding groove.

Conclusions

We have identified a novel sub-family of putatively non-classical MHC class I genes that are specific to marsupials and monotremes. This family was present in the ancestral mammal and is found in extant marsupials and monotremes, but has been lost from the eutherian lineage. The function of this family is as yet unknown, however, their predicted structure may be consistent with presentation of antigens to T-cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1745-4) contains supplementary material, which is available to authorized users.     

Galectins as tools for glycan mapping in histology: comparison of their binding profiles to the bovine zona pellucida by confocal laser scanning microscopy

Gene divergence has given rise to the galectin family of mammalian lectins. Since selective binding to distinct β-galactosides underlies the known bioactivities of galectins, they could find application in cyto- and histochemistry. The pertinent question on the characteristics of their individual reactivity profiles therefore needs to be answered. Toward this end, comparative studies of a panel of galectins in defined systems are required. We here characterise the staining profiles of seven human lectins as well as five natural derivatives originating from proteolytic truncation and serine phosphorylation and one engineered variant. As test system, bovine germinal vesicle oocytes with their glycoprotein envelope (zona pellucida), which presents bi- to tetraantennary complex-type N-glycans with N-acetyllactosamine repeats and core fucosylation, were processed. Technically, confocal laser scanning microscopy was used, first with plant lectins to map the sialylation status. Hereby, α2,3/6-sialylation was detected in the superficial filamentous meshwork of the zona pellucida, while sialic acid-free glycan chains were found to characterise the main inner part of the compact layer of the zona pellucida. Galectin staining was specific and non-uniform. Significant differences in reactivity were detected for the superficial filamentous meshwork and the compact layer of the zona pellucida between galectins-1 to -4 versus galectins-8 and -9. The typical staining profiles intimate a spatially organised display of N-glycans in the different layers of the zona pellucida, underscoring the potential of galectins as cyto- and histochemical tools. Our results encourage further comparative analysis and research to trace the underlying structural and/or topological properties.     

The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability

The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.