ELIXIR-UK role in bioinformatics training at the national level and across ELIXIR.

ELIXIR-UK is the UK node of ELIXIR, the European infrastructure for life science data. Since its foundation in 2014, ELIXIR-UK has played a leading role in training both within the UK and in the ELIXIR Training Platform, which coordinates and delivers training across all ELIXIR members. ELIXIR-UK contributes to the Training Platform's coordination and supports the development of training to address key skill gaps amongst UK scientists. As part of this work it acts as a conduit for nationally-important bioinformatics training resources to promote their activities to the ELIXIR community. ELIXIR-UK also leads ELIXIR's flagship Training Portal, TeSS, which collects information about a diverse range of training and makes it easily accessible to the community. ELIXIR-UK also works with others to provide key digital skills training, partnering with the Software Sustainability Institute to provide Software Carpentry training to the ELIXIR community and to establish the Data Carpentry initiative, and taking a lead role amongst national stakeholders to deliver the StaTS project - a coordinated effort to drive engagement with training in statistics.

The mouse genome.

The mouse is a key model organism for the study of mammalian genetics, development, physiology and biochemistry. The determination of the mouse genome sequence was therefore an early priority in the genome project. A draft sequence became available in 2002 and many chromosomes are now close to being finished. Comparative analysis of the mouse genome sequence with that of the human and other genomes has revealed a wealth of information on genome evolution in the mammalian lineage and assisted in the annotation of both genomes. With the availability of a well-annotated mouse genome sequence, mouse geneticists are now poised to undertake the challenge of generating mutations at every gene in the mouse genome. Systematic mutagenesis of the mouse genome will be an important step towards the first comprehensive functional annotation of a mammalian genome and the identification and characterisation of models for the study of human genetic disease.

Building mouse phenotype ontologies.

The structured description of mutant phenotypes presents a major conceptual and practical problem. A general model for generating mouse phenotype ontologies that involves combing a variety of different ontologies to better link and describe phenotypes is presented. This model is based on the Phenotype and Trait Ontology schema proposal and incorporates practical limitations and designing solutions in an attempt to model a testbed for the first phenotype ontology constructed in this manner, namely the mouse behavior phenotype ontology. We propose the application of such a model could provide curators with a powerful mechanism of annotation, mining and knowledge representation as well as achieving some level of free text disassociation.

Ontologies for the description of mouse phenotypes.

Ontologies are becoming increasingly important for the efficient storage, retrieval and mining of biological data. The description of phenotypes using ontologies is a particularly complex problem. We outline a schema that can be used to describe phenotypes by combining orthologous axiomatic ontologies. We also describe tools for storing, browsing and searching such complex ontologies. Central to this approach is that assays (protocols for measuring phenotypic characters) describe what has been measured as well as how this was done, allowing assays to link individual organisms to ontologies describing phenotypes. We have evaluated this approach by automatically annotating data on 600,000 mutant mice phenotypes using the SHIRPA protocol. We believe this approach will enable the flexible, extensible and detailed description of phenotypes from any organism.

A phylogenetic approach to assessing the significance of missense mutations in disease genes.

The identification of deleterious mutations within candidate genes is a crucial step in the elucidation of the genetic bases of human disease. However, the significance of any base or amino acid change within a gene is unknown until detailed structural and functional analysis has been carried out. A potentially rapid way of identifying functionally important sites within a gene is to identify evolutionarily conserved regions. Mutations affecting such sites are assumed to be deleterious for the carrier. In this communication we generalize this approach and present a formal framework to assess whether a specific mutation is deleterious given sequence data from a set of homologues. We propose a score that takes into account the nature of the mutation, the conservation of the affected residue among the different species, and their phylogenetic relationships. Its performance is examined using published TP53 mutations and frequent polymorphic variants.

The comparative genomics of polyglutamine repeats: extreme differences in the codon organization of repeat-encoding regions between mammals and Drosophila.

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes.

A role for selection in regulating the evolutionary emergence of disease-causing and other coding CAG repeats in humans and mice.

The evolutionary expansion of CAG repeats in human triplet expansion disease genes is intriguing because of their deleterious phenotype. In the past, this expansion has been suggested to reflect a broad genomewide expansion of repeats, which would imply that mutational and evolutionary processes acting on repeats differ between species. Here, we tested this hypothesis by analyzing repeat- and flanking-sequence evolution in 28 repeat-containing genes that had been sequenced in humans and mice and by considering overall lengths and distributions of CAG repeats in the two species. We found no evidence that these repeats were longer in humans than in mice. We also found no evidence for preferential accumulation of CAG repeats in the human genome relative to mice from an analysis of the lengths of repeats identified in sequence databases. We then investigated whether sequence properties, such as base and amino acid composition and base substitution rates, showed any relationship to repeat evolution. We found that repeat-containing genes were enriched in certain amino acids, presumably as the result of selection, but that this did not reflect underlying biases in base composition. We also found that regions near repeats showed higher nonsynonymous substitution rates than the remainder of the gene and lower nonsynonymous rates in genes that contained a repeat in both the human and the mouse. Higher rates of nonsynonymous mutation in the neighborhood of repeats presumably reflect weaker purifying selection acting in these regions of the proteins, while the very low rate of nonsynonymous mutation in proteins containing a CAG repeat in both species presumably reflects a high level of purifying selection. Based on these observations, we propose that the mutational processes giving rise to polyglutamine repeats in human and murine proteins do not differ. Instead, we propose that the evolution of polyglutamine repeats in proteins results from an interplay between mutational processes and selection.

Extreme length and length variation in the first ribosomal internal transcribed spacer of ladybird beetles (Coleoptera: Coccinellidae).

DNA sequences of the first ribosomal internal transcribed spacer (ITS1) were isolated from 10 ladybird beetle species (Coleoptera: Coccinellidae) representing four subfamilies (Coccinellinae, Chilocorinae, Scymninae, and Coccidulinae). The spacers ranged in length from 791 to 2,572 bp, thereby including one of the longest ITS1s and exhibiting one of the most extreme cases of ITS1 size variation in eukaryotes recorded to date. The causes of length variation were therefore analyzed. Almost no putatively homologous sequence similarities were identified for the taxa included. The only exception was for the subfamily Coccinellinae, which yielded sequence similarities in six regions of approximately 550 nucleotide positions, primarily at the 5' and 3' ends of ITS1. The majority of differences in ITS1 length between taxa could be attributed to the presence of repetitive elements with comparatively long repeat units. Repetition arose several times independently and was confined to the middle of the spacer which, in contrast to the 5' and 3' ends, had not been inferred in previous studies to be subject to functional constraints. These elements were characterized by high rates of evolutionary change, most likely as a result of high substitution rates in combination with inefficient homogenization across repeats. The repeated origin and subsequent divergence of "long" repetitive elements should thus be assumed to be an important factor in the evolution of coccinellid ITS1.

Rapid restructuring of bicoid-dependent hunchback promoters within and between Dipteran species: implications for molecular coevolution.

Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain-bearing regulator bicoid (bcd) and Bcd-dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd-binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage-like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcdand the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

How slippage-derived sequences are incorporated into rRNA variable-region secondary structure: implications for phylogeny reconstruction.

We analyzed the type and frequency of mutational changes in hypervariable rRNA regions, using the highly length-variable region V4 of the small subunit rRNA locus of tiger beetles (Cicindelidae) as an example. Phylogenetic analysis of indels in closely related species showed that (1) most indels are single nucleotides (usually A or T and sometimes G) or di-nucleotides of A and T. These occur at numerous foci, and they exhibit a strong bias for duplication of 5' single and di-nucleotide motifs but not 3' motifs. (2) Insertions/deletions in stem-forming regions affected paired and unpaired bases with about equal frequency but they did not disrupt the secondary structure. (3) Recurring mutations involving short repeats of the same bases caused parallel evolution of similar sequence motifs in the rRNA of different lineages. The observed types of change are consistent with the propostion that slippage is the main mutational mechanism. Slippage-derived sequences tend to be self-complementary, and therefore the stem-loop structure could be self-organizing as a consequence of the underlying mutational mechanism. Thus, the secondary structure in the cicindelid V4 region may be conserved due to the dynamics of the mutational mechanism rather than to functional constraints. These processes may also have a tendency to produce similar primary sequences irrespective of phylogenetic associations. The findings have implications for sequence alignment in phylogenetic analysis and should caution against the use of secondary structure to improve the determination of positional homology in hypervariable regions.

Amino acid reiterations in yeast are overrepresented in particular classes of proteins and show evidence of a slippage-like mutational process.

Long amino acid repeats are often observed in eukaryotic proteins. In humans, several neurological disorders are caused by proteins containing abnormally long polyglutamines. However, no systematic analysis has attempted to investigate the relationship between reiterations of particular amino acids and protein function, the possible mechanisms involved in the generation of these regions, or the contribution of selection in restricting their genomic distribution, in a large collection of wild-type proteins. We have used baker's yeast open reading frames to study these questions. The most abundant amino acid repeats found in yeast proteins are repeats of glutamine, asparagine, aspartic acid, glutamic acid, and serine. Different amino acid repeats are concentrated in different classes of proteins. Acidic and polar amino acid repeats are significantly associated with transcription factors and protein kinases, while serine repeats are significantly associated with membrane transporter proteins. In most cases the codon structures encoding the repeats at the gene level show a significant bias toward long tracts of one of the possible codons, suggesting that trinucleotide slippage has played an important role in generating these reiterations. However, many, particularly those encoding serine repeats, do not show evidence of slippage. The distributions of codon repeats within proteins and between coding and noncoding regions of the genome, and of amino acids between proteins with different functions, suggest that repeats of these kinds are subject to strong selection.

Analysis of the primary sequence and secondary structure of the unusually long SSU rRNA of the soil bug, Armadillidium vulgare.

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions.

High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.

Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species.

The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase.

The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI). In the MTP complex, the amino-terminal region of MTP (residues 22-303) interacts with the amino-terminal region of apoB (residues 1-264). Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517-603 of MTP. PDI binds in close proximity to this apoB binding site on MTP. The proximity of these binding sites on MTP for PDI and amino acids 512-721 of apoB was evident from studies carried out in a yeast two-hybrid system and by co-immunoprecipitation. The expression of PDI with MTP and apoB16 (residues 1-721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73%. The interaction of residues 512-721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.

Modelling the secondary structures of slippage-prone hypervariable RNA regions: the example of the tiger beetle 18S rRNA variable region V4.

Variable regions within ribosomal RNAs frequently vary in length as a result of incorporating products of slippage. This makes constructing secondary structure models problematic because base homology is difficult or impossible to establish between species. Here, we model such a region by comparing the results of the MFOLD suboptimal folding algorithm for different species to identify conserved structures. Based on the reconstruction of base change on a phylogenetic tree of the species and comparison against null models of character change, we devise a statistical analysis to assess support of these structures from compensatory and semi-compensatory (i.e. G.C to G.U or A.U to G.U) mutations. As a model system we have used variable region V4 from cicindelid (tiger beetle) small subunit ribosomal RNAs (SSU rRNAs). This consists of a mixture of conserved and highly variable subregions and has been subject to extensive comparative analysis in the past. The model that results is similar to a previously described model of this variable region derived from a different set of species and contains a novel structure in the central, highly variable part. The method we describe may be useful in modelling other RNA regions that are subject to slippage.

Phylogenetic analysis of slippage-like sequence variation in the V4 rRNA expansion segment in tiger beetles (Cicindelidae).

Sequence variation in the middle part of the small-subunit rRNA was studied for representatives of the major groups in the family Cicindelidae (Coleoptera). All taxa exhibited a much expanded segment in variable region V4 compared to D. melanogaster. This expanded segment was not found in other groups of beetles, including three taxa in the closely related Carabidae. Secondary structure predictions indicate that the expanded segment folds into a single stem-loop structure in all taxa. Despite its structural conservation, the fragment differs strongly in primary sequence, even between closely related sister taxa. Several features of these sequences are consistent with slippage replication as the mechanism that has generated this sequence variation: the level of internal sequence repetition as measured by the relative simplicity factor (RSF), its variation in length between close relatives, and the strong nucleotide bias compared to the remainder of the gene. With few exceptions, there was also a correlation between sequence length and the level of sequence repetition, frequently interpreted as the result of slippage. Phylogenies inferred from the expansion segment were not consistent with existing hypotheses from other molecular data for the group. This indicates that DNA sequences in this region are not homologous throughout the entire Cicindelidae, but it leaves open the possibility that this expansion segment can be used for phylogeny reconstruction within subgroups. The implications of a phylogenetic approach to the understanding of slippage-like evolution are discussed.